13th NUCLEAR MAGNETIC RESONANCE USERS MEETING
th MAY 2nd TO 6™, 2011
HOTEL DO FRADE, ANGRA DOS REIS, RJ, BRAZIL
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M EXTENDED m ABSTRACTS BOOK Contents THE STICKY FINGERS OF INFLUENZA VISUALIZED BY MODERN SOLUTION NMR 01 Ad Bax (National Institute of Health, NIH, USA)
SOLID STATE NMR METHODS FOR STUDYING FUNCTIONAL SUPRAMOLECULAR MATERIALS 02 Hans Spiess (Max Planck Institute for Polymer Research, Germany) /
DYNAMIC NUCLEAR POLARIZATION NMR AT HIGH MAGNETIC FIELDS 03 WHY TWO ELECTRONS ARE BETTER THAN ONE Robert Griffin (Massachussets Institute of Technology, MIT, USA)
NMR STUDIES OF THE INTERACTIONS OF SMALL GD3+ - BASED MRI CONTRAST AGENTS WITH TARGET PROTEINS 05 Carlos Geraldes (University of Coimbra, Portugal)
RESIDUAL CHEMICAL SHIFT ANISOTROPY (RCSA): A TOOL FOR THE CONFIGURATIONAL ANALYSIS OF SMALL MOLECULES 06 Fernando Hallwass (Federal University of Pernambuco, Brazil)
RESIDUAL DIPOLAR COUPLINGS IN ORGANIC STRUCTURE DETERMINATION 07 Christina Thiele (Technical University of Darmstadt, Germany)
RECENT PROGRESS IN MAGNETIC RESONANCE TECHNIQUES FOR POROUS MEDIA RESEARCH 08 Yi-Qiao Song (Schlumberger-Doll, USA)
SINGLE-SCAN MULTIDIMENSIONAL NMR AND MRI BY SPATIOTEMPORAL ENCODING: PRINCIPLES, OPPORTUNITIES AND CHALLENGES 09 Lucio Frydman (Weizmann Institute of Sciences, Israel)
BIOPHYSICS OF PROTEINS AND MEMBRANES: CAN WE LEARN SOMETHING FROM THE ELECTRON SPIN? 10 Antonio José da Costa Filho (USP/Ribeirão Preto, Brazil)
ADVANCES IN DOSY AND PURE SHIFT TECHNIQUES AND APPLICATIONS 11 Mathias Nilsson (University of Manchester, England, UK)
NMR STUDIES OF BACTERIAL NUCLEOID ASSOCIATED PROTEINS OF THE H-NS FAMILY 12 Miquel Pons (University of Barcelona, Spain)
xii ELECTRON SPIN RESONANCE SPECTROSCOPY: A RENAISSANCE 13 Jack Freed (Cornell University, USA)
HIGH-RESOLUTION SOLID-STATE NMR STUDIES OF DEEP-EARTH MINERALS 14 STEPHEN WIMPERIS (UNIVERSITY OF GLASGOW, SCOTLAND, UK)
HIGH PRESSURE NMR SPECTROSCOPY: EXCITED STATES OF PROTEINS AND THEIR ROLE IN PROTEIN-PROTEIN RECOGNITION 15 Hans Kalbitzer (University of Regensburg, Germany)
PROBING MICELLES AND REVERSE MICELLES BY NMR 16 Anita Marsaioli (State University of Campinas, Brazil)
INVISIBLE STATES IN PARAMAGNETIC COPPER PROTEINS 17 ALEJANDRO VILA (UNIVERSITY OF ROSÁRIO, ARGENTINA)
ULTRAFAST 2D NMR: PRINCIPLES, RECENT DEVELOPMENTS AND NEW APPLICATIONS IN ANALYTICAL CHEMISTRY 18 Patrick Giraudeau (University of Nantes, France)
PO 01 - STRUCTURE-FUNCTION CORRELATION IN HUMAN GALECTIN-4 20 P.S. Kumagai, F.H. Dyszy, M.C. Nonato, M.Dias-Baruffi, A.J. Costa-Filho
PO 02 - CARBOHYDRATE-BINDING MODULES: STRUCTURE AND INTERACTION BY NMR OF THE BACILLUS SUBTILIS CELLULASE CEL5A 22 M.L.Sforça, R. Z. Navarro, J.L. Neves , F. M. Squina, R. Ruller, M. T. Murakami, A. C. M. Zeri
PO 03 - SOLID-STATE 13C AND 31P NMR STUDIES OF POROUS CHARS PREPARED BY CHEMICAL AND THERMAL TREATMENT OF PEAT AND A LIGNOCELLULOSIC PRECURSOR 24 H. D. A. Honorato, J. C. C. Freitas, M. A. Schettino Jr., A. G. Cunha, F. G. Emmerich, E. V. R. de Castro.
PO 04 - EFFECT OF PARAMAGNETIC IONS ON THE T2 DISTRIBUTION CURVES OF PETROLEUM 26 D. Ker, G. F. Carneiro, R. C. Silva, E. V. R. Castro, V. Lacerda Jr, L. A. Colnago, L. L. Barbosa
xiii PO 05 - DETERMINATION OF OIL CONTENT IN SEEDS WITH POTENTIAL FOR BIODIESEL PRODUCTION BY LOW-FIELD NMR 28 G. F. Carneiro, A. F. Constantino, R. B. dos Santos, V. Lacerda Júnior, R. C. Silva, S. J. Greco, J. C.C. Freitas, E. V. R. de Castro
PO 06 - DIFFUSIOMETRY STUDIES OF WATER AND OIL MIXTURES BY LOW-FIELD 1H NMR 30 G. F. Carneiro, V. Lacerda Júnior, R. C. Silva, L. L. Barbosa, J. C.C. Freitas, E. V. R. de Castro.
PO 07 - UNEQUIVOCAL ASSIGNMENT OF THREE SYNTHETIC INTERMEDIATES OF GUAIANES AND NOR-GUAIANES: AN EXPERIMENTAL AND THEORETICAL APPROACH 32 L. R. Barbosa,Y. W. Vieira, V. Lacerda Jr., K. T. de Oliveira, R. B. dos Santos,S. J. Greco, T. J. Brocksom, E. V. R. de Castro
PO 08 - ARE HYDROGEN BONDS SECONDARY INTRAMOLECULAR INTERACTIONS RESPONSIBLE FOR PHENYLALANINE CONFORMATIONAL PREFERENCES? 34 R.A. Cormanich, F.P. dos Santos, L.C. Ducati, R. Rittner
PO 09 - STERIC/HYPERCONJUGATIVE EFFECTS BALANCE GOVERNS THE VALINE CONFORMATIONAL EQUILIBRIUM 36 R.A. Cormanich, F.P. dos Santos, L.C. Ducati, R. Rittner
PO 10 - NMR FOR PHOSPHORUS DETERMINATION DURING DECOMPOSITION OF TYPHA ANGUSTIFOLIA 38 C.S. Oliveira, L.M. Lião, G.B. Alcântara, F. Petacci, S.S. Freitas
PO 11 - 29Si and 13C NMR SPECTROSCOPY OF COMMERCIAL ALUMINA IONIC LIQUIDS 40 Naira M.S. Ruiz, Fábio L.L. Farias, Sandra S.X. Chiaro, Sonia M.C. de Menezes, Leandro Luza and Jairton Dupont
PO 12 - THE INFLUENCE OF SOLVENT ON THE METHYLENE HYDROGENS 1H NMR SIGNALS FOR THE 2-PHENYLPROPYL HALOACETATES 42 R.B. Sousa, C.F. Tormena
PO 13 - NMR ASSIGNMENTS OF NEOMYCIN B AND PAROMOMYCIN 44 Julyana Rosa Machado, Elizabethe Gomes Sanches, Roselêne Ribeiro Riente, Jochen Junker
PO 14 - NMR ANALYSIS OF JATROPHA CURCAS EXTRACTS 46 Quézia da Silva Sant'Anna, Raquel Pantoja Rodrigues, Nicolas Carel, Jochen Junker
xiv PO 15 - NMR IDENTIFICATION OF A CHALCONE AND A STILBENE FROM ROOTS OF DEGUELIA DUCKEANA A.M.G. AZEVEDO (FABACEAE) N. M. Lima, A. C. Oliveira, C.V. Nunez
PO 16 - WITH NMR TOWARDS NEW DIAGNOSTIC METHODS FOR DENGUE Camilla do Nascimento Bernardo, Marcela Cristina Oliveira Nogueira, Érika Pereira de Aquino, Sina Schmidtke, Elizandes L Azeredo, Claire F Kubelka, Jochen Junker
PO 17 - DISCRIMINATION OF BIODIESEL BLENDS WITH 1H NMR AND CHEMOMETRICS I.S. Flores, L.M. Lião, G.B. Alcantara, S.M. Cabral, M.R. Monteiro
PO 18 - INFLUENCE OF THE SOME 1H NMR PARAMETERS ON THE ACCURACY OF MULTIVARIATED CALIBRATION MODELS - PLS I.S. Flores, L.M. Lião, G.B. Alcantara, M.R. Monteiro
PO 19 - ASSESSMENT OF S180 METABOLIC PROFILING AFTER ANTITUMOR DRUGS USING HIGH-RESOLUTION MAGIC-ANGLE-SPINNING NMR SPECTROSCOPY £ B.C.B. Martinelli, G.B. Alcantara, L.M. Lião, A.L. Oliveira, E.P. Silveira-Lacerda, F.C. Pereira.
3 PO 20 - THROUGH SPACE CONTRIBUTION FOR THE JFH COUPLINGS IN SOME UNSATURATED COMPOUNDS L.C. Ducati, R.H. Contreras, C.F. Tormena
PO 21 - CONFORMATIONAL STUDY OF 17-a-ETHYNYLESTRADIOL USING RDC José Adonias Alves de França; Fernando Hallwass
PO 22 - PH-DEPENDENT AXIAL LIGANDS OF COPPER-SUBSTITUTED CYTOCHROME C REVISITED T.Prieto, J.F.Lima, I.L. Nantes, O.R.Nascimento,
PO 23 - SYNTHESIS, PURIFICATION, CHARACTERIZATION AND NMR STRUCTURAL ELAVUATION OF DISINTEGRIN-LIKE PEPTIDES D. A. T. Pires, L. G. M. Arake, C. J. Nascimento, C. Bloch Jr
PO 24 - HETEROGENEOUS CLUSTER SIZE DISTRIBUTION OF COHERENCES IN THE QUANTUM DYNAMICS OF AN INFINITE SPIN % NETWORK C.M. Sánchez, A.K. Chattah, R.H. Acosta, P.R. Levstein
PO 25 - QUANTIFICATION OF LIPIDS COMPOSITION IN COMMON BEANS THROUGH 1H HR-MAS NMR SPECTROSCOPY E.G. Alves Filho; L.M.A. Silva; G.B. Alcantara; P.Z. Bassinello; L.M Lião
xv PO 26 - EVALUATION OF NEW INHIBITORS OF ACETYLCHOLINESTERASE BY NMR: 1 -METHYLPYRIDINE-2-HYDRAZONE AND 1-METHYLPYRIDINE-2-GUANYL HYDRAZONE 70 E. C. Petronilho, N. G. de Castro, A. C. Pinto, J. D. Figueroa-Viliar
PO 27 - CONFORMATIONAL EFFECT STUDY OF NEW TRANQUILIZERS BY NMR AND MOLECULAR MODELING 72 A. A. Vieira and J. D. Figueroa-Viliar
PO 28 - FIRST IMPLEMENTATION OF ULTRAFAST 2D NMR IN THE SOUTHERN HEMISPHERE 74 L.H.K. Queiroz Jr., A.G. Ferreira, P. Giraudeau, D.P.K. Queiroz
PO 29 - APPLICATION OF MODIFIED CONTINUOUS WAVE FREE PRECESSION METHOD FOR WATER SUPPRESSION IN 2D EXPERIMENTS 76 C. J. Duarte, L. A. Colnago, T. Venâncio
PO 30 - NMR STRUCTURAL STUDIES OF A NOVEL OCELLATIN P1 ISOFORM ISOLATED FROM THE SKIN SECRETION OF LEPTODACTYLUS LABYRINTHICUS 78 Eliane S. Fernandes, Carolina O. Matos, Cesar A. Prías-Márquez, Eduardo M. Cilli, Osmindo R. Pires Júnior, Wagner Fontes, Mariana S. Castro, Luciano M. Lião, Aline L. Oliveira
PO 31 - NMR CHARACTERIZATION OF A NEW POLYCYCLIC PHENAZINE FROM 1,4-NAPHTHOQUINONE 80 J. D. de Souza Filho, E. N. da Silva Júnior, M. J. da Silva, M. C. F. R. Pinto, C. A. de Simone, J. G. Soares, J. R. M. Reys, W. T. A. Harrison, Carlos E. M. Carvalho, M. O. F. Goulart and Antonio V. Pinto
PO 32 - NMR STUDIES ON A D-GLUCOSAMINE DERIVED MACROCYCLE 82 J. D. de Souza Filho, R. J. Alves and M. C. Pires
PO 33 - UNDERSTANDING ENZYME PROMISCUITY AND REVERSE MICELLAR SYSTEM BY 1H NMR 84 B.Z. Costa, A.J. Marsaioli
PO 34 - NMR STRUCTURAL STUDIES OF PHYLLOSEPTIN-2 PEPTIDE AT DIFFERENT PH VALUES 86 Naira de Oliveira Torres, Rodrigo Moreira Verly, Dorila Piló-Veloso, Jarbas Magalhães Resende.
PO 35 - USE OF FILTER DIAGONALIZATION METHOD TO PROCESS 13C NMR STEADY STATE FREE PRECESSION SIGNAL OBTAINED DURING IN SITU ELETROCHEMICAL REACTION 88 L.M.S. Nunes, T.B. Moraes, C. J. Magon, L.A. Colnago PO 36 - USE OF FILTER DIAGONALIZATION METHOD TO SUPPRESS BROAD LINE IN 1H NMR SPECTRUM OF BREAST CANCER CELLS 90 R.M.Maria,T.B.Moraes,C.J. Magon,W.F.AItei,A. D.Andricopulo.L.A.Colnago
PO 37 - THE APPLICATION OF SOLID STATE NMR TO EVALUATE THE QUALITY OF COMMERCIAL DRUGS 92 L.A.M. Magalhães, A.G. Ferreira, J. Ellena, T. Venâncio
PO 38 - INTERNAL GRADIENT MAPPING WITH DISTANT DIPOLAR FIELD CONTRAST 94 E.V. Silletta, M.B. Franzoni, R.H. Acosta
PO 39 - PENDANT CHAIN DYNAMICS IN MODEL PDMS NETWORKS PROBED BY SPIN DIFFUSION EXPERIMENTS 96 R.H. Acosta, M.B. Franzoni, M.A. Villar, E.M. Vallés, D.A. Vega, G.A. Monti
PO 40 - MULTINUCLEAR SOLID STATE NMR INVESTIGATION OF TWO POLYMORPHIC FORMS OF CIPROFLOXACIN-SACCHARINATE 98 Y. Garro Linck, A. K. Chattah, R. Graf, C. B. Romanuk, M. E. Olivera, R. H. Manzo, G. A. Monti, H. W. Spiess
PO 41 - SSNMR AND DIELECTRIC STUDY OF VULCANIZED POLYBUTADIENE RUBBER 100 A.L. Rodriguez Garraza, P. Sorichetti , A.J.Marzocca, C.L.Matteo , G.A.Monti
PO 42 - WHY STUDY THE SIGN OF THE GEMINAL COUPLING CONSTANT? 102 Denize C. Favaro, Cláudio F. Tormena
PO 43 - STRUCTURAL STUDIES OF YTTRIUM ALUMINOBORATE LASER GLASSES USING SOLID STATE NMR AND ELECTRON SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY 104 H. Deters, J.F. Lima, C.J. Magon, C.N. Santos, A.S.S. de Camargo, A. C. Hernandez, C.R. Ferrari, H. Eckert
PO 44 - STUDY OF TRANSVERSE RELAXATION IN LIQUID COW'S MILK DURING FERMENTATION PROCESS 106 P.M. dos Santos, A.A. Souza, T. Venâncio, L.A. Colnago, E.R. Pereira-Filho
PO 45 - STUDY OF METABOLIC PROFILE BY LC-SPE-NMR OF SPILANTHES A CMELLA 108 D.S.Santos; A.G.Ferreira, P.C.Nogueira, A.F.Blank
PO 46 - NMR CHARACTERIZATION OF RESINOUS MATERIAL OCURRED IN VALVES CONTROLED BY HYDRAULIC FLUIDS IN OIL PRODUCTION 110 Sonia Maria Cabral de Menezes, Luiz Silvino Chinelatto Júnior, ítalo José Rigotti, Bruno Charles do Couto, Flavio Alves Zuim, Thiago Aquino Damasceno, Adriana Santos Mendes
xvii PO 47 - STRUCTURAL AND ORIENTATIONAL DETERMINATION OF THE ANTIMICROBIAL PEPTIDE PHENYLSEPTIN IN MEMBRANE-MIMICKING ENVIRONMENT 112 V.H.O. Munhoz, M.T.Q. de Magalhães, R.M. Verly, S.F.C. de Paula, J.M. Resende, C. Aisenbery, D. Piló-Veloso, C. Bloch Jr., B. Bechinger
PO 48 - METABOLIC STUDIES WITH BOVINE BLOOD PLASMA EMPLOYING HIGH-RESOLUTION NMR AND CHEMOMETRICS 114 Matheus P. Postigo, Ana Carolina de Souza Chagas, Márcia Cristina de Sena Oliveira, Luiz Alberto Colnago
PO 49 - IS A PLANAR W ARRANGEMENT THE ONLY ONE EFFICIENT 4 TRANSMISSION OF PATHWAY JHH? 116 Denize C. Favaro, Karen Canto, Cláudio F. Tormena
PO 50 - NOVEL ALKALOID FROM PILOCARPUS MICROPHYLLUS 118 A. C. H. F. Sawaya, Y. D. Costa , P. Mazzafera , L. G. Martins
PO 51 - PROBING CH-71 INTERACTIONS IN CYCLOHEXANOL, TRANS-1,2- CYCLOHEXANEDIOL AND a-D-GLUCOSE WITH AROMATIC ANISOTROPY EXPERIMENTS AND THEORETICAL CALCULATIONS 120 E.A. Basso, R.M. Pontes, A. A. Cândido
PO 52 - MECHANISTIC INSIGHTS ON THE REACTIONS OF A PHOSPHOTRIESTER WITH NUCLEOPHILES: NUCLEOPHILIC VS GENERAL-BASE PATHS 122 R. Moreira, M. Medeiros, E.H. Wanderlind, E.S. Orth, P.S.M. Oliveira, T.A. S. Brandão, F. Nome.
PO 53 - A LOW-COST, PORTABLE HALBACH MAGNET FOR LRNMR 124 M.G.A. Carosio, L.F. Cabeça, L.A. Colnago
PO 54 - USES OF CP SEQUENCE WITH LOW REFOCUSING FLIP ANGLE (CP-CWFP) TO MEASURE TEMPERATURE AND THERMAL DIFUSIVITY IN OILSEEDS 126 M.G.A. Carosio, F.D. Andrade, L.A. Colnago
PO 55 - TOTAL ASSIGNMENT OF THE 1H AND 13C NMR AND STEREOCHEMISTRY OF TWO NEW CHALCONE DIMERS 128 M. Fernanda M. Villari, João B. Fernandes, Paulo C. Vieira, M. Fátima G.F. da Silva, Antonio G. Ferreira
PO 56 - IDENTIFICATION OF TRITERPENE BY NMR FROM MINQUARTIA GUIANENSIS BRANCHES 130 L. M. C. Cursino, C. V. Nunez
PO 57 - UNILATERAL NMR: CONSTRUCTION AND APPLICATIONS TO MONITOR THE TRANSESTERIFICATION REACTION 132 L. F. Cabeça, L. V. Marconsini, L. A. Colnago
xviii PO 58 - NMR STUDIES ON A PLATINUM(II) COMPLEX DERIVED FROM PYRAZINAMIDE PRESENTING IN VITRO ANTITUMORAL PROPERTIES 134 T. S. Ribeiro, L. Sartori, L. B. Borré, R. A. S. San Gil, J. D. Figueroa-Viliar, N. A. Rey
PO 59 - qHNMR OF ANTIFUNGAL ANTIBIOTIC GRISEOFULVIN IN POLYMERIC MICELLES 136 M.E.N.P.Ribeiro, M.G.S.Vieira, N.M.P.S.Ricardo, N.V.Gramosa
PO 60 - STRUCTURE ELUCIDATION OF THE NEW DITERPENE E/VV-TRACHYLOBAN-18,19 - DIOL BY 1H AND 13C NMR 138 M.G.S. Vieira, E.R. Silveira, N.V. Gramosa
PO 61 - DETERMINATION OF BIODIESEL IN DIESEL USING 1H AND 13C NMR WHIT MULTIVARIATE DATA ANALYSIS 140 Francisco F. Gambarra-Neto, Clayton R. de Oliveira, Marcos R. Monteiro, Antonio Gilberto Ferreira
PO 62 - SOLID STATE NMR AS STRUCTURAL PROBE FOR LUMINESCENT INORGANIC-ORGANIC HYBRID MATERIALS 142 T. B. Queiroz, M. Botelho, R. M. Ilibi, J. Fernandez-Hernandez, M. D. G. López, H. Eckert, L. de Cola, A. S. S. de Camargo
PO 63 - MULTI-BAND AUTOMATICALLY TUNABLE HIGH-SENSITIVE NUCLEAR QUADRUPOLE RESONANCE SPECTROMETER 144 L.M.C. Cerioni, F. Picco, D.J. Pusiol
PO 64 - USE OF T1 AND T2 TO MEASURE THE SOLUBILITY PRODUCT OF PARAMAGNETIC IONS IN SOLUTION 146 P.F. Cobra, L. L. Barbosa, L.V. Marconcini, L.A. Colnago
PO 65 - NMR STUDIES OF THE ABSOLUTE STEREOCHEMISTRY ASSIGNMENT OF AMINES ACHIEVED BY CYCLOHEXYL-BASED CHIRAL AUXILIARIES 148 P.F. Bertotti, I.S. Resck, A.H.L. Machado
PO 66 - INTRODUCING 170 NMR IN STERIC EFFECT STUDIES USING NMR AND MOLECULAR MODELING 150 J.D. Yoneda, K.Z. Leal, M.H.R. Veiloso, E.B. Lindgren, P.R. Seidl
PO 67 - COMPARATIVE STUDY OF BRAZILIAN HEAVY OIL BY PROTON NUCLEAR MAGNETIC RESONANCE 152 F.B. da Silva, P.G.P.Fiorio, P.R.Seidl, M.J.O.C. Guimarães, K.Z. Leal
PO 68 - ELECTRONIC PARAMAGNETIC RESONANCE AND MAGNETIC CIRCULAR DICHROISM STUDIES OF NITRIC OXIDE-PROMOTED CHANGES OF THE AXIAL LIGANDS OF CYTOCHROME C HEME IRON 154 S. M. S. Pinto, K. C. U. Mugnol, T. Prieto, O. R. Nascimento, I. L. Nantes
xix PO 69 - STRUCTURAL AND ORIENTATIONAL DETERMINATION OF THE ANTIMICROBIAL PEPTIDE HYLASEPTIN P2 IN MEMBRANE-MIMICKING ENVIRONMENT V.H.O. Munhoz, S.F.C. de Paula, J.M. Resende, D. Piló-Veloso, B. Bechinger
PO 70 - DETERMINATION OF THE ABSOLUTE CONFIGURATION OF CARBOXYLIC ACIDS BY 77Se NMR SPECTROSCOPY J.G. Ferreira, S.M.C. Gonçalves
PO 71 - CHIRALITY RECOGNITION OF CARBOXYLIC ACIDS BY 77Se NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY S.M.C. Gonçalves, J.G. Ferreira
PO 72 - SIMULATION OF TRANSVERSE RELAXATION TIME IN POROUS MEDIA BASED ON PHASE SPACE RANDOM WALK M. N. d Eurydice, T.J. Bonagamba
PO 73 - THIOREDOXINS VISIT AN OPEN EXCITED CONFORMATIONAL STATE DURING THE CATALYTIC TURNOVER F. Gomes-Neto, C Cruzeiro-Silva, N. L Rodrigues, C. A. Miyamoto, A. S. Pinheiro, L. E. S. Netto, A. P. Valente and F. C. L Almeida
PO 74 - A NEW DIMENSION IN PULSED NMR SPECTROSCOPY OF PROTEINS ... Munte, C. E., Arnold. M., Kremer, W., Hartl, R., Beck Erlach, M., Kõhler, J., Meier, A., Kalbitzer, H. R.
PO 75 - QUANTITATIVE DETERMINATION AND VALIDATION OF EXCIPIENT USING QNMR SPECTROSCOPY C.M.G.de Souza, M.F.P.S. Mota, C. Matteucci, J.M.A. Bispo, P.C. Leal
PO 76 - STRUCTURAL CHARACTERISATION OF THE CYTOCHROME P450-ORF10 FROM THE STREPTOMYCES CLAVULIGERUS .. BY PULSED EPR J F Lima, L S Goto, C O Hokka, A P U Araújo, O R Nascimento.
PO 77 - ON THE QUANTUMNESS OF NUCLEAR MAGNETIC RESONANCE D.O. Soares-Pinto, L.C. Celeri, R. Auccaise, J. Maziero, R.S. Sarthour, I.S. Oliveira, M.H.Y. Moussa, E.L.G. Vidoto, E.R. de Azevedo, R.M. Serra, T.J. Bonagamba
PO 78 - MULTI-QUANTUM ECHOES AND COHERENCE SELECTION IN
GdAI2 WITH ZERO-FIELD NMR R. Oliveira-Silva, C. Rivera-Ascona, J.R. Tozoni, E.L.G. Vidoto, J. Teles, T.J. Bonagamba
PO 79 - CONSTRUCTION OF AN NMR POROUS MEDIA ANALYZER E.L.G. Vidoto, M.B. Andreeta, M.N. d' Eurydice, E.L. Oliveira, J.G. da Silva, A.D.F. Amorim, R. Oliveira-Silva, T.J. Bonagamba PO 80 - STUDY OF ORGANOTIN COMPOUNDS IN SOLUTION BY 119Sn NMR 178 Rubens R. Teles, Ivani Malvestiti and Fernando Hallwass
PO 81 - AMPLIFING THE EFFECTS OF MOLECULAR MOTIONS IN DIPSHIFT-LIKE EXPERIMENTS 180 M.F. Cobo, A.Achilles, D. Reichert, K.Saawaechter, E.R. deAzevedo
PO 82 - INTERMOLECULAR INTERACTIONS BETWEEN SOLIDS AMOXICILLIN AND OMEPRAZOLE: A SOLID STATE NMR STUDY 182 Lorena Mara Alexandre e Silva, Marcos Guillermo Russo, Griselda Edith Narda, Javier Alcides Ellena, Antonio Gilberto Ferreira, Tiago Venâncio
PO 83 - ELUCIDATING THE C-TERMINAL DOMAIN OF HUMAN SEPTIN 2 184 E. Crusca, C.E. Munte, R.C. Garratt
PO 84 - A NEW PROGRAM FOR THE SIMULATION OF NMR PULSE SEQUENCES 186 Clara Luz S. Santos, Claudia J. Nascimento, and José Daniel Figueroa Villar
PO 85 - STRUCTURAL CHANGES IN HYBRID PHYTOCYSTATINS ANALYZED BY HIGH-RESOLUTION NUCLEAR MAGNETIC RESONANCE 188 I.A.Cavini, R.C. Garratt, F. Henrique-Silva, H.R. Kalbitzer, C.E. Munte
PO 86 - QUANTUM COMPUTATION IN SOLID CRYSTALS BY LOW FIELD NUCLEAR QUADRUPOLE RESONANCE 190 J. Teles, R. Oliveira-Silva, R.S. Polli, E.L.G. Vidoto, E.L. Oliveira, D.O. Soares-Pinto, T.J. Bonagamba
PO 87 - PURE T2 - T2 EXCHANGE: AN ENHANCEMENT 192 M. N. d' Eurydice, T.J. Bonagamba
PO 88 - HIGH-PRESSURE NMR STUDIES ON THE PLASMODIUM FALCIPARUM THIOREDOXIN 194 E. C. Azevedo, H. R. Kalbitzer, C. E. Munte,
PO 89 - MAGNETIC SUSCEPTIBILITY CHARACTERIZATION OF SEDIMENTARY ROCK CORES BY NUCLEAR MAGNETIC RESONANCE 196 A.A. Souza, L. Zielinski, A. Boyd, M.D. Hurlimann, T.J. Bonagamba
PO 90 - PROBING NATURAL POROUS MEDIA WITH 2D LOW FIELD NMR RELAXOMETRY AND DIFUSIOMETRY 198 E.H. Rios, G.C. Stael, R.B.V. Azeredo
PO 91 - PROBING THERMAL BEHAVIOUR OF CEFALEXIN MONOHYDRATE 200 BY13C SOLID STATE NMR Daniel L M. Aguiar, Rosane A S. San Gil
xxi PO 92 -APPLICATION OF NMR ANALYSIS TO THE STUDYOF ASPHALTENES .. 202 Camila Pedroso Silveira, Peter Rudolf Seidl, Fernanda Barbosa da Silva, Fábio Henrique S. Rodrigues, Ljubica Tasic, Sônia Maria Cabral de Menezes, Maria José O.C. Guimarães
PO 93 - CHARACTERIZATION OF HDT CATALYSTS USING 170 AND 27AI MAS NMR TECHNIQUES 204 H.R.X. Pimentel, R.A.S. San Gil, S.M.C. Menezes, S.S.X. Chiaro
PO 94 - A METHOD TO DETERMINE THE FRACTION OF COMPONENTS IN HETEROGENEOUS SAMPLES BY TIME-DOMAIN NUCLEAR MAGNETIC RESONANCE 206 L.M.C. Cerioni, T.M. Osan, M. Medina, G. Albert, D.J. Pusiol
PO 95 - NUCLEAR MAGNETIC RESONANCE (NMR) STUDY OF NECROSIS AND ETHYLENE-INDUCING PROTEIN 2 (NEP2) 208 E.G. Pereira, G.A.P. de Oliveira, A.P. Valente, V.S. de Paula, J.L. da Silva, F.C.L. Almeida, J.C.M. Cascardo, C.V. Dias.
xxii THE STICKY FINGERS OF INFLUENZA VISUALIZED BY MODERN SOLUTION NMR
Justin Lorieau, Alex Grishaev, John Louis, and Ad Bax Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, MD 20892, USA
All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp^-Tyr^-Gly23 and have been carried out without isotopic labeling. Our heteronuclear triple resonance NMR study is carried out on full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline (DPC) and a range of small bicelles, and reveals a remarkably tight helical hairpin structure, with its N-terminal a-helix (Gly1- Glu11) packed tightly against its second a-helix (Trp14-Gly23), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive
Hans Wolfgang Spiess Max-Planck-lnstitute for Polymer Research, P. O. Box 3148, D-55021 Mainz, Germany
Functional nanostructures are in the focus of current materials science. They occur in advanced synthetic as well as in biological systems through self-assembly of carefully chosen building blocks. Secondary interactions such as hydrogen bonding, aromatic pi-interactions, and electrostatic forces are of central importance. Here, high resolution solid state NMR provides unique and highly selective information on structure and dynamics of such systems1, e.g., on hydrogen bond networks in the solid state,2 stacking, and cooperative molecular motions of discotics3 and macrocycles.4 Solid state NMR is also able to elucidate self-assembly, conformation and dynamics of polypeptides.5 For full structural and dynamic elucidation, the spectroscopic data have to be combined with other techniques, in particular X-ray scattering, microscopy, dielectric spectroscopy and last, but not least, quantum chemical calculations. The findings will be related to the function of such materials, such as proton- and photo- conductivity.
References
1. H. W. Spiess, Macromolecules 2010, 43, 5479-5491 2. Y. J. Lcc, T. Murakhtina, D. Scbastiani, H. W. Spiess, J. Am. Chem. Soc. 2007, 129, 12406. 3. M. R. Hansen, T. Schnitzler, W. Pisula, R. Graf, K. Miillen, H.W. Spicss, Angew. Chem. Int. Ed. 2009, 48, 4621. 4. M. Fritzsche, A. Bohle, D. Dudenko, U. Baumeister, D. Sebastiani, G. Richardt, H. W. Spiess, M. R. Hansen, S. Hoeger, Angew. Chem. Int. Ed. 2011, 50, in press 5. G. Floudas and H.W. Spiess, Macromol. Rapid Commun. 2009, 30, 278.
^ 4- 2 DYNAMIC NUCLEAR POLARIZATION NMR AT HIGH MAGNETIC FIELDS WHY TWO ELECTRONS ARE BETTER THAN ONE
R. G. Griffin Francis Bitter Magnet Laboratory and Department of Chemistry Massachusetts Institute of Technology, Cambridge, MA 02139
Nuclear magnetic resonance (NMR) is probably the most versatile analytical technique available to chemistry and biochemistry because it is non-perturbing and offers site-specific atomic resolution available with few other approaches. It is very forgiving as to the physical state of the sample, being applicable to gases, solutions and to amorphous and crystalline and microcrystalline solids. In addition, for similar reasons NMR (or MRI) is widely used in many other areas of science ranging from basic nuclear physics to medical imaging. Despite its enormous versatility, the sensitivity of the NMR experiments is relatively low because it is based on observation of low energy spectroscopic transitions between nuclear Zeeman levels. As a consequence, there are continuing efforts to develop new NMR methods and instrumentation that improve the signal-to- noise of the experiments. Some of the most successful of these involve transfer experiments that move polarization from a highly polarized spin reservoir to a weakly polarized one, leading to an enhancement in the NMR signal intensities proportional to the ratio of the magnetic moments of the two spin species. It is now appreciated that the largest gains in signal intensities in these sorts of experiments can be achieved by transferring polarization from an electron spin(s) to a nuclear spin system. This is generally accomplished via microwave irradiation of the electron paramagnetic resonance (EPR) spectrum, an experiment known as dynamic nuclear polarization (DNP) NMR. Since contemporary NMR experiments are performed at magnetic fields of ~5-23 T, the required microwave radiation falls into the frequency range 140-660 GHz, or the millimeter wave regime. This presentation discusses the implementation of DNP/NMR experiments in high magnetic fields.
\ E = 250 w "fc t V ir
n . A .
bTbk 300 200 100 0 ,3C Chemical Shift [ppm]
Figure 1: A new polarizing agent with superior performance in DNP experiments is introduced that utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro-tether. The observed NMR signal intensities are enhanced by a factor 250 when compared to that achieved via Boltzmann polarization. Over the last few years we have developed cyclotron resonance maser (a.k.a. gyrotron) microwave sources that operate at frequencies of 140-460 GHz that permit DNP enhanced NMR (DNP/NMR) experiments in magnetic fields of 5-16.4 T (1H NMR frequencies of 211-700 MHz, respectively). We review the instrumentation used for these experiments, which include new NMR probe designs and tunable gyrotron sources. In addition, we discuss two mechanisms that are currently used for DNP
3 h experiments in solids at high fields - the solid effect and cross effect — and the polarizing agents appropriate for each. These include biradicals that enable increased enhancements at reduced concentrations of the paramagnetic center. Figure 1 depicts recent results obtained from the rigid biradical bis-TEMPO-bis-ketal (bTbk) where we observe an enhancement of -250, or a reduction in signal averaging time of 62,500. In addition, we discuss applications of DNP/NMR that illustrate its utility in enhancing signal-to-noise in MAS NMR spectra of a variety of biological systems including membrane and amyloid proteins whose structures are of considerable scientific interest. Presently, enhancements that are routinely available and range from 40-250 depending on experimental variables such as temperature, magnetic field, microwave B^ polarizing agent, etc. Finally, we describe extensions of these experiments that permit observation of 13C liquid state spectra where we have observed enhancements of 140-400 in small molecules and a protein.
k + 4 NMR STUDIES OF THE INTERACTIONS OF SMALL GD3+ - BASED MRI CONTRAST AGENTS WITH TARGET PROTEINS
Carlos F.G.C. Geraldes Department of Life Sciences and Center of Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal
An high specificity of targeted MRI contrast agents is a current important objective in the development of these diagnostic imaging tools. A study using combined NMR and modeling techniques to study the interaction of small Gd3+-based chelates with target proteins is described. The systems studied include: a) Gd3+ complexes of DOTA mono(amide) and DTPA bis(amide) glycoconjugates bearing one or two terminal sugar (galactosyl or lactosyl) moieties with the lectin Ricinus Communis agglutinin (RCA12o) as a model for the hepatocyte asialoglycoprotein receptor (ASGPR); b) the angiographic MRI agents Gd(BOPTA), Gd(DTPA-cholate) and Gd(NAPHTO-EGTA) interacting with human serum albumin (HSA); c) the two enantiomers of a Gd3+ tetraaza complex of a ligand bearing a triphenylene chromophore and three optically active phenylmethyl amide pendant arms, SSS-(A)-[Gd.L]3+ and RRR-(A)-[Gd.lf+, in their binding to HSA. 1H saturation transfer difference (STD) NMR techniques using the diamagnetic analogous La3+ or Y3+ complexes were combined with water proton relaxometric (NMRD) studies and compared with the results of computational docking for the Gd3+ complexes. Competitive assays with known HSA specific site binding ligands (eg. warfarin, ibuprofen, A/-dansyl sarcosine) were performed to identify the preferences of the individual complexes for HSA drug site I or II. As an example, preferential binding of the SSS-(A)-[Y.L]3+ isomer, relative to the to the RRR-(A) form, to the most enantioselective drug site II of HSA was found.
5 h RESIDUAL CHEMICAL SHIFT ANISOTROPY (RCSA): A TOOL FOR THE CONFIGURATION ANALYSIS OF SMALL MOLECULES
Fernando Hallwass Departamento de Química Fundamental, CCEN, Universidade Federal de Pernambuco, Av. Luiz Freire s/n, CEP 50.740-540, Recife - PE, Brazil ([email protected])
In recent years Nuclear Magnetic Resonance (NMR) has been enhanced by weakly orienting molecules in anisotropic solution and thus recovering anisotropic NMR parameters such as residual dipolar couplings (RDCs) and residual chemical shifts anisotropies (RCSAs). In this talk will be described an approach to measure RCSAs reliably, using the Kuchel scalable alignment device1, and to interpret RCSAs structurally, using CSA tensors calculated from Density Functional Theory (DFT) combined with the Gauge Independent Atomic Orbital (GIAO) methodology. RCSAs deliver orientation information that can be used to determine conformation and configuration of molecules. For instance, using RDCs and RCSAs together, but not individually, allowed a clear differentiation of estrone from 13-epi-estrone. We expect that RCSAs will be measured and used in the near future whenever RDCs are measured to improve the determination of stereochemistry of small molecules.
References
1. Kuchel P.W., Champman B.E., Müller N., Bubb W.A., Philp D.J.Jorres A.M. J. Magn. Reson. 180, 256-265 (2006).
f 4 6 RESIDUAL DIPOLAR COUPLINGS IN ORGANIC STRUCTURE DETERMINATION
Christina M. Thiele Technische Universitãt Darmstadt, Clemens Schõpf Institut für Organische Chemie und Biochemie, Petersenstr. 22, 64287 Darmstadt, Germany, email: [email protected]
The determination of the three dimensional structure of organic compounds by high-resolution solution state NMR spectroscopy usually involves the measurement of 3J couplings, NOEs and more seldomly cross correlated relaxation to obtain information about dihedral angles, distances and projection angles, respectively. If the determination of the relative configuration of stereogenic elements is the goal of the structural investigation the elucidation of conformation and configuration superimpose each other entailing the necessity to determine both structural aspects simultaneously. Especially in cases of conformational flexibility, these conventional NMR restraints often do not lead to unambiguous configurational assignments. Residual dipolar couplings (RDCs), which belong to the class of anisotropic NMR-parameters, can yield information complementary to 3J couplings and NOE parameters and allow the assignment of diastereotopic protons and the determination of relative configurations even in the presence of (a limited degree of) motion. In contrast to conventional NMR-parameters RDCs contain global information.111 An overview of the use of RDCs for organic structure determination will be given and the determination of the relative configuration and conformer population in an a-methylen- y-butyrolactone I-21 will be described in detail. The determination of the solution conformation of catalytically active species'31 will also be discussed.
References
1. Reviews: C. M. Thiele, Eur. J. Org. Chem., 2008, 5673-5685; C. M. Thiele, Cone. Magn. Res. 2007, 30A, 65-80. 2. C. M. Thiele, A. Marx, R. Berger, J. Fischer, M. Biel, A. Giannis, Angew. Chem. 2006, 118, 4566-4571, Angew. Chem. Int. Ed. 2006, 45, 4455-4460; C. M. Thiele, V. Schmidts, B. Bóttcher, I. Louzao, R. Berger, A. Maliniak, B. Stevensson, Angew. Chem. 2009, 121, 6836-6840, Angew. Chem. Int. Ed. 2009, 48, 6708-6712. 3. R. S. Stoll, M. V. Peters, A. Kühn, S. Heiles, R. Goddard, M. Bühl, C. M. Thiele, S. Hecht, J. Am. Chem. Soc. 2009, 131, 357-367; B. Bóttcher, V. Schmidts, J. A. Raskatov, C. M. Thiele, Angew. Chem. 2010, 122, 210-214, Angew. Chem. Int. Ed. 2010, 49, 205-209.
7 I- RECENT PROGRESS IN MAGNETIC RESONANCE TECHNIQUES FOR POROUS MEDIA RESEARCH
Yi-Qiao Song, Schlumberger-Doll Research, Cambridge MA 02139
NMR has become an important technique for characterization of porous materials in recent years. In particular, its importance in petroleum exploration has been increased by the recent progress in NMR well-logging techniques for the characterization of both rocks and the natural fluids. Such advanced techniques are increasing being accepted as a valuable logging service especially in the technically challenging areas, such as deep-sea exploration. The continuous rise of global demand for energy and the difficulty of significantly increasing production capacity have made such oil reservoirs much more attractive. As a result, there is an urgent need of advanced technologies to better understand porous structures and fluid composition in oil and gas reservoirs. This talk will outline the MR technical development and their applications in the study of pore structure, rock heterogeneity, composition and molecular dynamics of crude oils, and biological materials. Specific topics include two- dimensional NMR, internal magnetic fields, MRI of rocks, and the diffusion physics.
I-+8 SINGLE-SCAN MULTIDIMENSIONAL NMR AND MRI BY SPATIOTEMPORAL ENCODING: PRINCIPLES, OPPORTUNITIES AND CHALLENGES
Lucio Frydman Department of Chemical Physics, Weizmann Institute, Rehovot, Israel
We have recently developed a scheme enabling the acquisition of arbitrary multidimensional NMR spectra and/or NMR images (MRI), within a single scan. This is by contrast to the hundreds or thousands of scans that are usually needed to collect this kind of data. Provided that the target molecule's signal is sufficiently strong, the acquisition time of multidimensional NMR experiments can thus be shortened by several orders of magnitude. This new "ultrafast" methodology is compatible with existing multidimensional pulse sequences and can be implemented using conventional hardware. The manner by which the spatial encoding of the NMR interactions —which is the new principle underlying these new protocols— proceeds in these experiments, will be summarized. The protocol's performance will be exemplified with a variety of homonuclear and heteronuclear 2D and 3D NMR/MRI acquisitions on chemical, biochemical and biological systems, carried out within a =1 sec time scale. The incorporation into these experiments of nuclear hyperpolarization procedures capable of increasing the single-scan sensitivity of liquid state NMR by factors ranging from 103-106, will also be assessed. Time permitting, the main challenges faced by these methodologies and options to solve these that are currently being explored, will be described.
9 I- + BIOPHYSICS OF PROTEINS AND MEMBRANES: CAN WE LEARN SOMETHING FROM THE ELECTRON SPIN?
Antonio J. Costa-Filho 1 Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de SãoPaulo, São Carlos, SP, Brazil 2 Departamento de Física, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
One of the major goals when studying biological macromolecules resides on the understanding of the interactions that determine structure and/or function. Several approaches are available to help obtaining both structural and dynamical information about the processes under investigation. In recent years one of those approaches has been resurging due to its successful application to problems such as probing structural changes related to protein function. This approach is based on the joint use of an unpaired spin, which is attached to a selected site in a biomolecule (protein or phospholipids), and the electron spin resonance (ESR) phenomenon. In this presentation, we will firstly cover some basic aspects of spin label ESR and then discuss a series of problems studied in our group: a calcium binding protein of the S100 family, a fatty acid binding protein from human brain, and a dehydrogenase involved in nucleotide biosynthesis. These results include spectra obtained from proteins containing a specifically labeled non-native cysteine residue, whose use constitutes the basis of the so-called site-directed spin labeling ESR (SDSL-ESR) methodology, and that are, to the best of our knowledge, the first SDSL-ESR experiments totally performed in a Brazilian group. Financial support: FAPESP, CNPq, CAPES.
10 h ADVANCES IN DOSY AND PURE SHIFT TECHNIQUES AND APPLICATIONS
Mathias Nilsson School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK
Mixture analysis by diffusion NMR is a powerful technique that is steadily gaining ground. The standard way to resolve individual component NMR spectra is by diffusion-ordered spectroscopy (DOSY1), and hence the terms "DOSY data" and "DOSY experiment" are in common use. Normally this requires that the molecular species have different size and with well separated (proton) NMR signals. The default criterion for signal separation (size) can be extended by allowing mixture components to interact with a matrix1. Differential association with the matrix allows separation of spectra from molecules that have very similar diffusion rates in free solution (e.g. isomers ). The whole spectrum of a molecular species generally shows identical diffusion behaviour, and this covariance can be effectively exploited to defeat problems with spectral overlap by multivariate methods such as SCORE where whole spectra are fitted simultaneously. When diffusion is complemented by a third independent dimension (such as relaxation2 or concentration change during a reaction3) the data can become trilinear, allowing the use of powerful multi-way methods, such as PARAFAC. Finally, pure shift methods5,6 suppress the effects of homonuclear couplings to give spectra without multiplet structure (i.e. only one peak per chemical shift), in 1D and 2D spectra such as DOSY and TOCSY, improving resolution by an order of magnitude or more.
References
1. Evans R, Haiber S, Nilsson M, Morris GA. Anal. Chem. 2009, 81, 4548-4550. 2. Nilsson M, Botana A, Morris GA. Anal. Chem. 2009, 81, 8119-8125. 3. Nilsson M, Khajeh M, Botana A, Bernstein MA, Morris GA. Chem. Commun. 2009, 1252-1254. 4. Khajeh M, Botana A, Bernstein MA, Nilsson M, Morris GA. Anal. Chem. 2010, 82, 2102-2108. 5. Nilsson, M. & Morris, G.A. Chem. Commun. 2007, 933-935. 6. Aguilar, J.A., Faulkner, S., Nilsson, M. & Morris, G. Angew. Chem. Int. Ed. 2010, 49, 3901-3903.
11 )• NMR STUDIES OF BACTERIAL NUCLEOID ASSOCIATED PROTEINS OF THE H-NS FAMILY
Tiago Cordeiro, Carles Fernández de Alba, Jesús Garcia, and Miquel Pons* Institute for Research in Biomedicine (IRB Barcelona). Baldiri Reixac, 10-12. 08028-Barcelona. Spain and Departament de Química Orgânica. Universitãt de Barcelona (UB). Marti i Franquès, 1-11. 08028-Barcelona. Spain e-mail: mpons&.ub.edu
Nucleoid associated proteins (NAP) are responsible for packaging the bacterial chromosome and actively regulating the expression of large number of genes to provide a coordinated global response to environmental changes, including the manifestation of a pathogenic phenotype upon guest colonization. One of the most abundant proteins in Gram negative bacteria is H-NS. Members of the H-NS protein family are involved in the regulation of horizontally acquired genes that include most genes involved in pathogenicity and antibiotic resistance. H-NS family members bind to DNA as oligomers. Hha, a co-regulator, binds to the oligomerization domain of H-NS and changes its specificity. We shall present ongoing work in our group on the characterization of the H-NS based regulatory system including a) the determination of the three dimensional structure of a DNA complex of the DNA binding region of Ler, a nucleoid associated protein that competes with H-NS for the activation of the LEE operon encoding for a type III secretion system in enterohaemorragic E. coli (EHEC), b*) the solid-state NMR characterization of full length H-NS oligomers (in collaboration with Marc Baldus and Marie Renault, Utrecht), c) the characterization of a plasmidic variant of H-NS specifically repressing horizontally transferred genes and, d) the role of conformational dynamics of an H-NS co-regulator in the discrimination of different gene pools in enterobacteria.
We thank C. Griesinger and H. Schmidt (Góttingen) for their help in the Ler project and O. Millet for his help in the dynamic studies of the H-NS co-regulator. This work has been supported by funds from Spanish MICINN and from the EC projects Bio-NMR and EAST-NMR. We thank CAPES (Brazil) and the Spanish Ministry of Education for the award of an interuniversity cooperation program between the universities of Barcelona and Campinas. ELECTRON SPIN RESONANCE SPECTROSCOPY: A RENAISSANCE
Jack Freed Cornell University, USA
Recent developments in instrumentation, theoretical methodologies, and sitedirected spin labeling have greatly enhanced the capabilities and applications of ESR. 1) ESR has been extended to high frequencies utilizing quasi-optical methods. High frequency ESR provides a faster "snapshot" of motional dynamics and enables a multi-frequency ESR approach to study complex molecular modes of motion. 2) 2D- ESR (analogous to 2D-NMR) frequently leads to dramatic spectral effects from molecular motions, which are particularly sensitive to their microscopic details, extracted by powerful methods of analysis. 3) Modern pulse ESR methods provide powerful means of determining large distances ranging from 10 - 90 Â. It is now possible to determine structure and function in large protein complexes in solution and in membranes. 4) ESR microscopy permits 3D micro-imaging with micron resolution. ESRM (vs. NMRM) is more sensitive per spin, its resolution is not limited by molecular diffusion, and it utilizes significantly less expensive magnet technology.
i3 y HIGH-RESOLUTION SOLID-STATE NMR STUDIES OF DEEP-EARTH MINERALS
Stephen Wimperis School of Chemistry and WestCHEM, University of Glasgow, Glasgow G12 8QQ, UK e-mail:stephen. wimperis&.glasgow. ac. uk
Keywords: solid-state NMR, quadrupolar nuclei, hydrous minerals, dynamics
Over the past 20 years, the development of new NMR methodologies has revolutionised the way in which quadrupolar nuclei (ie, those with spin quantum number I > 1) are studied in the solid state. At the start of this period, the focus of the research effort was on the design and improvement of experimental methods for obtaining high-resolution NMR spectra of nuclei such as 170,23Na and 27AI under magic angle spinning (MAS) conditions. However, the field has now matured and recent interest has been centred on obtaining structural information from disordered and amorphous materials and on performing studies of dynamics. In both these areas, the assistance of modern, efficient codes for the "first-principles" calculation of solid-state NMR parameters is proving invaluable. In this talk, I will review the main developments in this field in the last decade. To provide a framework, examples will mostly be drawn from our work since 1999 on synthetic silicate minerals that serve as models for those found in the "deep Earth", ie, in the mantle at depths in excess of 40 km. Originally, this work_utilised mainly high- resolution 170 (I = 5/2) NMR but, more recently, 1H, 2H, 19F, 25Mg and 29Si experiments have all proven useful, together with the associated two-dimensional heteronuclear correlations. The studies presented in the talk will include ones looking at (i) crystal structure, (ii) the structural chemistry of hydrogen, (iii) positional disorder and (iv) dynamics in a variety of anhydrous, nominally anhydrous and hydrous mantle silicates.
References
1. Ashbrook S. E.; Berry A. J. and Wimperis S. Am. Mineral. 1999, 84, 1191-1194. 2. Ashbrook S. E.; Berry A. J. and Wimperis S. J. Am. Chem. Soc. 2001, 123, 6360-6366. 3. Ashbrook S. E.; Berry A. J. and Wimperis S. J. Phys. Chem. B 2002, 106, 773-778. 4. Ashbrook S. E.; Antonijevic S.; Berry A. J. and Wimperis S. Chem. Phys. Lett. 2002, 364, 634-642. 5. Ashbrook S. E.; Berry A. J.; Hibberson W. O.; Steuernagel S. and Wimperis S. J. Am. Chem. Soc. 2003, 125, 11824-11825. 6. Ashbrook S. E.; Berry A. J.; Frost D. J.; Gregorovic A.; Pickard C. J.; Readman J. E. and Wimperis S. J. Am. Chem. Soc. 2007, 129, 13213-13224. 7. Griffin J. M; Wimperis S.; Berry A. J.; Pickard C. J. and Ashbrook S. E. J. Phys. Chem. C 2009, 113, 465-471. 8. Griffin J. M; Yates J. R.; Berry A. J.; Wimperis S. and Ashbrook S. E. J. Am. Chem. Soc. 2010, 132, 15651-15660.
EPSRC, LEVERHULME TRUST
• •» 14 HIGH PRESSURE NMR SPECTROSCOPY: EXCITED STATES OF PROTEINS AND THEIR ROLE IN PROTEIN-PROTEIN RECOGNITION
H. R. Kalbitzer, W. Kremer, M. Arnold, T. Ernst, J. Koehler, M. Beck Erlach, C. E. Munte, M. Spoerner Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany
In addition to anisotropic compression high hydrostatic pressure can induce multiple effects in proteins including denaturation, depolymerisation, and changes of side chain protonation state. Most important, it can be used to stabilize structural intermediates occurring only at low concentrations at ambient pressure. Folding and function of proteins are two aspects of proteins which are usually considered as basically unrelated phenomena that are optimized by evolution independently. High pressure experiments are usually performed in quartz or sapphire cells but ceramics is the most promising material. For using ceramic cells in cryoprobes, a new safety system was devised that prevents damage of the probe when the cells explode [1], Most of the high-pressure NMR spectroscopy is performed using static pressures. We have developed a microprocessor controlled in-line pressure system where well- defined, large pressure jumps (of the order of 100 MPa) can be performed in the ms- time scale. Pressure jumps can be performed in the pressure range up to 200 MPa. Since in contrast to temperature effects pressure effects in protein are in general completely reversible, pressure jumps can be incorporated in multi-dimensional NMR pulse sequences that can correlate different structural state and can be used to investigate their dynamic behaviour. A number of different pulse sequences incorporating pressure jumps are presented. We present two different applications of high pressure NMR spectroscopy to protein science: *(1) experiments on the protooncogene product Ras confirm he paradigm that the minimum number of folding intermediates is determined by the number of all functional states of a protein ("essential" folding intermediates) that can be inferred from the funnel model of folding/unfolding and the associated energy landscape [2] Here, we demonstrate the supposed fundamental link using the Ras- protein complexed with the GTP analogue GppNHp that occurs in two structural states coexisting in solution. State 1(T) and state 2(T) represent the GEF- and effector interacting states of Ras, respectively. Application of high pressure represents a perturbation of the energy landscape, leading to an increased population of the state 1(T) as observed by NMR spectroscopy. (2) High pressure NMR spectroscopy was used for the study of the early association/dissociation steps of the Alzheimer peptide Ap(1-40).
References
1. Erlach, M., Munte, C. E., Kremer, W., Hartl, R., Rochelt, D., Niesner, D. and Kalbitzer, H. R. (2010) Ceramic cells for high pressure NMR spectroscopy on proteins. J. Magn. Reson. 204, 196-199.
2. Kalbitzer, H. R., Spoerner, M., Ganser, P., Hosza, C. and Kremer, W. (2009) Fundamental link between folding states and functional states of proteins. J. Am. Chem. Soc., 131, 16714-16719.
15 h PROBING MICELLES AND REVERSE MICELLES BY NMR
Anita J. Marsaioli State University of Campinas, Brazil
Micelles and reverse micelles are self-assembled molecules of nano meter dimension, these particules are formed in polar and apoiar media respectively and can be detected by light scaterring. Some aspects of the self-assembled particles in solutions containing water, organic solvent and encapsulated small molecules can be better revealed by applying diffusion 1H NMR (DOSY), NOESY, ROESY, STD NMR providing access to the dynamic and architechtural aspects of these assembling at the molecular level. This approach was successfully applied to solve membrane mimetic, cyclodextrin and local anesthetic interactions as well as grounds to the understanding of quorum sensing and quorum quenching phenomena of pathogenic Gram-negative bacteria. More recently reverse micellles were involved in enzymatic promiscuity. Reverse micelles are discrete nanoscale particles composed of a water core surrounded by surfactant. The amount of water within the core of reverse micelles can be easily manipulated to directly affect the size of the reverse micelle particle. In our case study lipases mediating Bayer Villiger oxydation of ketones was the topic of interest and the change in performance of encapsulated proteins within reverse micelles was evaluated.
f 4 16 INVISIBLE STATES IN PARAMAGNETIC COPPER PROTEINS STUDIED BY NMR
A.J. Vila*1, L.A. Abriata1 and M.E. Zaballa1 institute for Molecular and Celular Biology (IBR), University of Rosário, Rosário, Argentina e-mail :vila(a)jbr. gov. ar
Keywords: paramagnetic proteins, NMR, Cu(ll)
NMR of oxidized copper proteins has been largely unexplored due to the slow electron relaxation times of Cu2+. However, T1, T3 and CuA centers display relatively fast electron relaxation rates which make them amenable to NMR studies. In the binuclear copper sites CuA and T3, this is due to low-lying excited states that are populated at room temperature and contribute to the reactivity of the metal site. NMR can shed light on these invisible, partially populated, electronic states. The engineering of different axial ligands in CuA tunes the energy gap between the ground state and the invisible excited state, thus providing a mechanism to regulate the electronic structure of the metal site at room temperature. This information nicely complements the picture provided by ground state magnetic techniques such as EPR and ENDOR.
References
1. Abriata L.A., Ledesma G.N., Pierattelli R. and Vila A.J. J.Am.Chem.Soc. 2009, 131, 1939-46 2. Zaballa M.E., Ziegler L., Kosman D.J. and Vila A.J. J.Am.Chem.Soc. 2010, 132, 11191-6. *
HHMI, CONICET, ANPCyT
17 h ULTRAFAST 2D NMR: PRINCIPLES, RECENT DEVELOPMENTS AND NEW APPLICATIONS IN ANALYTICAL CHEMISTRY
Patrick Giraudeau and Serge Akoka Université de Nantes, CNRS, CEISAM UMR6230, Nantes, France
Two-dimensional Nuclear Magnetic Resonance (2D NMR) is a powerful tool for non-destructive analysis of complex mixtures. However, it is hampered by long acquisition durations due to the numerous U increments required to record the whole 2D FID. Beyond the timetable constraints generated by this time limitation, 2D NMR is ill-suited for studying irreversible processes such as hyphenated NMR, or dynamic phenomena occurring on a short timescale, such as kinetics of fast chemical reactions. Moreover, long experiment durations make 2D experiments more sensitive to temporal instabilities which highly affect the precision of quantitative 2D NMR measurements. Recently, L. Frydman and co-workers proposed an ultrafast 2D NMR approach \ where a complete 2D NMR correlation can be recorded in a single scan, ie in a fraction of a second. In order to deal with the limitations of this promising technique, we performed several methodological developments 2 5 that highly improved the performances of ultrafast 2D NMR in terms of sensitivity and resolution. Thanks to these recent advances, we successfully showed the potentialities of this methodology for fast and precise quantitative analysis 6. Very recenlty, the ufo-qNMR (Ultrafast Optimized Quantitative NMR) was applied successfully to kinetic studies of fast occuring processes. Moreover, we developed a novel methodology to measure specific isotopic enrichments in complex biological mixtures. The principles of ultrafast 2D NMR will be presented, as well as the recent methodological developments in the field. Examples of applications involving a variety of samples and pulse sequences will be described.
References
1.Frydman, L.; Scherf, T.; Lupulescu, A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15858. 2.Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, 190, 339-345. 3.Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, 192, 151-158. 4.Giraudeau, P.; Akoka, S. J. Magn. Reson. 2008, 195, 9-16. 5.Giraudeau, P.; Akoka, S. J. Magn. Reson. 2010, 205, 171-176. 6.Giraudeau, P.; Remaud, G.S. ; Akoka, S. Anal. Chem. 2009, 81, 479-484.
Y 18 ISA 1 JSiN LJs2jD ABSTRACTS PO 02 STRUCTURE-FUNCTION CORRELATION IN HUMAN GALECTIN-4 P.S. Kumagai*1, F.H. Dyszy1, M.C. Nonato2, M.Dias-Baruffi2, A.J. Costa-Filho1'3 1 Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil. 2 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. 3 Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. patriciasuemy@ ursa. if sc. usp. br
Keywords: galectin-4;CRDs; EPR.
Galectins are a family of animal lectins characterized by their specific affinity for (3- galactosides and have gained much attention in the last few years mainly due to their involvement in inflammatory and neoplasic processes1. In such processes, several issues are still not clear such as the mechanisms of interaction with different carbohydrates, the specificity of these interactions and the particular roles played by galectins in inflammation, cell adhesion, tumor progression and metastasis. In structural terms, galectin-4 is a 36 kDa monomer that contains two carbohydrate recognition domains (CRD-I in N-terminal and CRD-II in C-terminal) connected by linking peptide2. In this work, we studied structural alterations in the carbohydrate-recognition domains (CRD-I and CRD-II) from human galectin-4 (Gal-4). Our goals are two-fold: (1) to monitor conformational changes in each domain upon binding of specific ligands (lactose, galactose, and glucose) and then to correlate the observed changes with structural differences between the two CRDs; (2) to investigate the interaction between the CRDs and lipid model membranes. To achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism (CD) and Electron Spin Resonance (ESR). The CRD domains were expressed and purified using previously described protocols3. CD experiments were carried out in a Jasco J-815 spectropolarimeter in the 195-250 nm range and using 7-10 |nM protein concentration in the absence and in the presence of lactose, galactose, and glucose (1:4 protein:carbohydrate). EPR measurements were performed in a Varian E109 spectrometer, operating at 9.4 GHz. Figure 1a shows the CD spectra of CRD-I during thermal unfolding in temperature range of 10 to 80°C. The general appearance of CRD-I spectrum is dominated by 13- sheet secondary structure, with a negative peak around 216 nm. Conversion of the molar elipticity at 216 nm to fraction of unfolded protein led to the thermal transition patterns shown in Figure 1b. Similar CD spectra were observed for the CRD-II domain (data not shown) and resulted in thermal transitions in Figure 1c. From these data, we can see that the domains respond differently to the presence of carbohydrates. CRD-I does not have its structural stability affected by any of the ligands, whereas CRD-II shows a clear increase in thermal stability, which in turn is also sugar-dependent. All transitions ended up being irreversible and a fitting to an irreversible two-state model yielded the activation energy and transition temperature for each case. To gain further insight on the structural changes upon carbohydrate binding, we labeled the native cysteine residues present in the CRD-II structure with the spin label MTSL. The EPR spectra of labeled CRD-II in the presence of lactose are shown in Figure 2. The major feature to be observed is the appearance of a second more- immobilized component (broad peak in the low field region) superimposed on the three sharp lines assigned to cysteine residues with high mobility. This indicates that lactose stabilizes a second and more rigid conformation of the protein at the specific cysteine positions.
y 4 20 ' (nm> Temperature ("C) Temperature CC)
Figure 1: a) CD spectra of CRD-I in the range 10-80°C; b) thermal unfolding of CRD-I in the absence and presence of ligands; c) thermal unfolding of CRD-II in the absence and presence
of ligands. In panels b and c, fD refers to the fraction of unfolded protein that is calculated from the molar elipticity at 216 nm.
i
3320 3340 3360 3380 3400 3420 3440 3460 3480 3500 Magnetic Field (G)
Figure 2: EPR spectra of labeled CRD-II measured in several different conditions: protein in its apo form, in the presence of lactose, in the apo form and in the presence of ficoll (to reduce buffer viscosity), and in the presence of lactose and ficoll.
Gal-4 recoginzes carbohydrates present in the surface of cell membranes. To investigate possible mechanims of interactions between the CRD-I domain and model membranes, we measured the EPR spectra of labeled phospholipids containing a spin probe at different positions along the lipid acyl chain (n-PC) or at the headgroup region (DOPTC) and incorporated in DMPC or DMPC/Lac-PE vesicles. Subtle lineshape changes were observed after addition of Gal-4. To quantitatively analyze such differences we will perform non-linear least-squares simulations of the spectra. Overall the results obtained so far show that CRD-I and CRD-II have distinct behaviors in terms of carbohydrate recognition. This may be due to specific differences in their structures and certainly suggests an unequal role in protein function.
REFERENCES:
1. Cooper, D.N.W., Biochim. Biophys. Acta., 2002, 1572, 209-231. 2. Oda, Y. et al„ J. Biol. Chem., 1993, 268, 5929-5939. 3. Zimbardi, A.L.R.; Pinheiro, M.P.; Dias-Baruffi, M.; Nonato, M.C., Acta Cryst., 2010, 66, 542-545. 4. Mchaourab, H. S. et al., Biochem., 1999, 38, 2947-2955.
FAPESP, CNPq, CAPES.
21 h AURKMN- MAY 2nd In 6th, 2011 IIOT1-L DO FRADE. ANGRA DOS Hl ls. K F. lilt \/ll
PO 02
CARBOHYDRATE-BINDING MODULES: STRUCTURE AND INTERACTION BY NMR OF THE BACILLUS SUBTILIS CELLULASE CEL5A M.L.Sforça*1, R. Z. Navarro1'2, J.L. Neves1 , F. M. Squina3, R. Ruller3, M. T. Murakami1, A. C. M. Zeri**1 1. Laboratório Nacional de Biociências - Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brasil 2. Departamento de Biologia - Pontifícia Universidade Católica de Campinas, Campinas, SP, Brasil 3. Laboratório Nacional de Ciência e Tecnologia do Bioetanol - Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brasil e-mail: *mauricio. sforca@lnbio. org. br, **ana.zeri@lnbio. org. br
Keywords: nmr structure; cellulase; interaction.
Carbohydrate-binding modules (CBM) are non-catalytic domains of enzymes that hydrolyze carbohydrates such as cellulose and xylan, and play important roles promoting the association of the enzyme with substrates. Understanding the mechanism by which the enzyme recognizes their target carbohydrates, strategies can be designed to manipulate these interactions, with considerable biotechnological importance. CBMs are divided into families based on amino acid sequence similarity (http://www.cazy.org/Carbohydrate-Binding-Modules.html). The CBM from Bacillus subtilis cellulase cel5A, studied in this project, belongs to family 3. The pET28a-CBM plasmid, expressing the protein with a Histidine tag, was transformed into BL21 (DE3) Escherichia coli expression cells* and grown in M9 minimal medium supplemented with kanamycin, 13C-glucose and 15N-ammonium chloride (Sigma Aldrich and Cambridge Isotopes). Protein expression was induced by addition of IPTG. After addition of PMSF and sonication, the supernatant fraction was isolated by centrifugation and purified by Ni-affinity and size exclusion chromatography. NMR experiments were performed using a Varian Inova spectrometer at the National Biosciences Laboratory (LNBio/CNPEM), operating at a 1H Larmor frequency of 599.887 MHz and temperature of 293 K. The spectrometer is equipped with a triple resonance cryogenic probe and a Z pulse-field gradient unit. The 15N-13C labeled CBM sample was dissolved in 300.0 pL phosphate buffer pH 7.2 containing 5% D20, at a final concentration of approximately 0.3 mM. Protein backbone resonance peaks were assigned using 1H-15N HSQC and standard triple resonance experiments including HNCACB, CBCACONH, HNCO, HNCACO (1) with a 15N-13C labeled CBM sample. In order to assign the side chain resonances, the 15N-13C labeled CBM sample was lyophilized and dissolved in 100% D20, followed by acquisition of HCCH-TOCSY and hCCH-TOCSY spectra. NOE-derived distance restraints were obtained from the 15N- HSQC-NOESY and 13C-HSQC-NOESY (separately optimized for aliphatics and aromatics) both collected with 80 ms mixing times. The structure of the CBM protein was calculated in a semi-automated iterative manner with the program CYANA version 2.1 (2). The structures obtained were further refined by restrained minimization and molecular dynamic studies using the CNS software (3). For cello-oligosaccharide binding experiments, the 2D 1H-15N HSQC spectra were acquired on uniformly 15N- labeled samples with different molar equivalent ratios of 1,4a D-cellopentaose or 1,4a D-cellotetraose. A total of 1960 distance restraints, containing 701 medium and long range NOEs, were used for structure calculations. The Ramachandran plot analysis shows that 97.4% of PHI and PSI angles are in the most favorable and allowed regions, with no angles in the disallowed region. The r.m.s.d for the backbone and for heavy atoms are quite low (0.91 and 1.66 A respectively), confirming the good definition of the structure. The 20 structures with the lowest energy were selected, Figure 1A, to represent the
y 4 22 ensemble of protein structures. The tertiary structure of the CBM from Bacillus subtilis cellulase cel5A is predominantly a p-sandwich with a small helix region, and was deposited in the Protein Data Bank under accession code 2L8A. Chemical shift perturbation measurements reveal that residues Q369, Q453, R455 and N463 participate in the interaction with the oligosaccharides tested. The weak binding detected can be advantageous for the function of these CBM because this interaction should only guide de cellulose toward the Catalytic Domain (CD) of the enzymes and not bind to it strongly. The superposition of the carbohydrate-binding domain of the Bacillus subtilis cellulase cel5A (2L8A.pdb) and the crystallographic structure of the Endoglucanase Cel9G from Clostridium cellulolyticum (1G87.pdb) (4) containing the Catalytic Domain (CD) and the CBM domain are shown in Figure 1B, and agree with Small angle X-ray scattering (SAXs) data from our collaborators (unpublished data). Highlighted residues in sphere representation from the 2L8A CBM domain appear as an extended flat surface that could bind and guide cellulose strands towards the active aminoacids in the CD also highlighted in sphere representation. The structural analyses contribute to the establishment of a rational framework for future functional and biochemical studies of enzyme targets of biotechnological importance.
Figure 1: (A)Superposition of the 20 lowest energy tertiary structures of the CBM from Bacillus subtilis cellulase cel5A (2L8A.pdb). (B) Superposition of CBM from Bacillus subtilis cellulase cel5A and the crystalografic structure of the Endoglucanase Cel9G from Clostridium cellulolyticum containing the Catalytic Domain (CD) and the CBM domain (1G87.pdb). Highlighted residues in sphere representation correspond to the active aminoacids located in the CD of the Endoglucanase Cel9G from Clostridium cellulolyticum (1G87) and aminoacids of the CBM from Bacillus subtilis cellulase cel5A (2L8A) that interacted with the cello-oligosaccharids.
REFERENCES 1. Sattler M.; Schleucher J. & Griesinger C. Prog Nucl Mag Res Sp 1999, 34:93. 2. Guntert P.; Mumenthaler C. e Wuthrich K. J Mol Biol. 1997, 273:283. 3. Brunger A.T.; Adams P.D.; Clore G.M.; DeLano W.L.; Gros P.; Grosse-Kunstleve R.W.; Jiang J-S.; Kuszewski J.; Nilges M.; Panuu N.S.; Read R.J.; Rice L.M.; Simonson T. e Warren L.G. Acta Crystallogr D Biol Crystallogr 1998, 54:905. 4. Mandelman D.; Belaich A.; Belainch J. P.; Aghajari N.; Drigues H. e Haser R. Journal of Bacteriology 2003, 185:4127.
LNBio/CNPEM, CTBE/CNPEM, CNPq, CAPES
23 h \ I rREMN - MAY 2nd lo 6th, 201! HO'I I I. l)i)I H.\ni:. -Will! \ 1)P*> HHS.HJ.HK \/il
PO 03 SOLID-STATE 13C AND 31P NMR STUDIES OF POROUS CHARS PREPARED BY CHEMICAL AND THERMAL TREATMENT OF PEAT AND A LIGNOCELLULOSIC PRECURSOR H. D. A. Honorato1*, J. C. C. Freitas1'2, M. A. Schettino Jr.2, A. G. Cunha2, F. G. Emmerich2, E. V. R. de Castro1. 1LABPETRO, Departamento de Química, UFES, Vitória, ES, Brazil. 2LMC/LPT, Departamento de Física, UFES, Vitória, ES, Brazil. *herciliodeangeli@yahoo. com. br
Keywords: 13C NMR; 31P NMR; porous carbons.
The surface properties of porous carbons and biochars are known to be determinant for most of their applications. Oxygenated functional groups present at the edges of aromatic lamellae in pore walls influence decisively the surface chemistry of these materials [1,2]. In this work, solid-state 13C and 31P nuclear magnetic resonance (NMR) spectroscopy was used for the analysis of porous carbons prepared from two precursors - a lignocellulosic material and peat -, using H3P04 as the chemical activating agent. The lignocellulosic precursor chosen for the preparation of the activated carbons was the testa of Mesua Ferrea oil seed, which is found extensively in North East India and is used in the production of biodiesel. The peat used in this work was collected from a boghead in the north region of state of Espírito Santo, Brazil. The lignocellulosic precursor was first charred at 450 °C before activation, under N2 flow. The porous carbons were prepared by chemical activation with H3P04, using an impregnation procedure. The activation heat-treatments were carried out at different final temperatures (in the ranges 500-950 °C for the lignocellulosic precursor and 350- 650 °C for peat) under N2 flow. Some samples were prepared by carbonization of the precursors without any activating agent, for comparison. The products were initially washed repeatedly with hot distilled water. Some selected samples were also washed by refluxing with 25% HN03 for 2h at the water boiling point and then filtered and washed again with hot distilled water for several times. Here we discuss only the NMR results referring to the activated carbons prepared from the lignocellulosic precursor; the results corresponding to the peat-derived samples will be detailed elsewhere. Solid-state NMR spectra were recorded at room temperature using different NMR spectrometers. Experiments with 1H-13C cross-polarization (CP) were conducted in a Varian 400 spectrometer at a frequency of 100.5 MHz (magnetic field of 9.4 T), whereas NMR spectra with single pulse excitation (SPE) were recorded using a Chemagnetics-Varian 200 spectrometer at 51.4 MHz for 13C (4.7 T) or a Chemagnetics-Varian 360 spectrometer at 145.8 MHz for 31P (8.45 T). The chemical shifts were externally referred to tetramethylsilane (TMS) for C and to an aqueous H3PO4 solution for P. All experiments were conducted with magic angle spinning (MAS) in different probes suitable for each spectrometer. Recycle delays varied in the range 1-10 s, being adjusted to avoid saturation problems. 13 The C SPE-MAS NMR spectra (recorded with 5 s recycle delay) of the H3P04- activated carbons prepared at different temperatures are shown in Fig. 1. The spectrum corresponding to the non-activated char, also shown in Fig. 1 for comparison, shows the presence of a sizeable aliphatic contribution around 25 ppm. All activated samples are predominantly aromatic. The presence of resonances due to oxygenated functional groups is observed in the range 160-210 ppm.
f- 4 24 250 200 150 100 50 0 -50 Chemical shift (ppm TMS) Figure 1.13C SPE-MAS NMR of the charred lignocellulosic precursor and the activated carbons (AC) prepared from the lignocellulosic precursor at different activation temperatures using H3P04 as the activating agent.
The chemical shift of the strong aromatic peak in the 13C NMR spectra decreases with the increase in the activation temperature, as a consequence of the increase in the diamagnetic susceptibility of the graphene-like planes [3], It is interesting to mention that this trend is also observed for the chemical shifts of the main resonance found in the 31P NMR spectra (Fig. 2), which indicates the proximity of the phosphorous species and the aromatic planes in the porous porous carbons. Future work is in progress to correlate the NMR results with the chemical characteristics of the prepared materials.
600 650 700 Temperature (°C) Figure 2. Comparison between the changes in the isotropic chemical shifts of the main resonances detected in C and P NMR spectra of the activated carbons prepared from the lignocellulosic precursor at different activation temperatures.
REFERENCES 1. Leon Y Leon, C. A.; Radovic, L. R. Chemistry and physics of carbon, vol. 24, P. A. Thrower (ed.), 1994. 2. Chia, C. H.; Munroe, P.; Joseph, S.; Lin, Y. Aust. J. Soil Res. 2010, 48. 3. Freitas, J. C. C.; Emmerich, F. G.; Cernicchiaro, G. R. C.; Sampaio, L. C.; Bonagamba, T. J. Solid State Nucl. Magn. Reson. 2001, 20.
PETROBRAS, FINEP, CNPq, CAPES, FAPES, LABPETRO, Univ. of Warwick, UFES
25 h PO 13
EFFECT OF PARAMAGNETIC IONS ON THE T2 DISTRIBUTION CURVES OF PETROLEUM D. Ker1, G. F. Carneiro1, R. C. Silva1, E. V. R. Castro1, V. Lacerda Jr1, L. A. Colnago2, L. L. Barbosa1 1 Research and Methodology Development Laboratory for Crude Oil Analysis - LabPetro, Department of Chemistry, Federal University of Espírito Santo, Vitória, Brazil. 2Embrapa Instrumentação Agropecuária, São Carlos-SP, Brasil e-mail: luciolbarbosa@yahoo. com. br
Keywords: low field NMR; paramagnetic ion, petroleum.
The presence of metals in crude oil is inevitable and undesirable under several aspect. Even traces of metals can lead to corrosion of ducts or affect the refining. The main metallic constituent of petroleum are nickel, vanadium, iron and copper. In the last decade low-field NMR (LF-NMR) technique has been widely applied in the petroliferous sector to predict crude oil properties. NMR signal is associated to some physical and chemical properties of oils and reservoir, such as viscosity, density, porosity and relative hydrogen index (RHI)1. The knowledge of the viscosity is critical for the technical and economical success of petroleum production. For instance, the high viscosity is a great challenge for oil recovery. Recently, one of the low-field 1H NMR application in the petroleum industry is predicting fluids viscosity, specially from analysis in situ, as in profiling proceeding2. The models frequently correlate the viscosity of oils to the 1H transverse relaxation times (T2), however, the experiments do not take into account the possible effects associated with the presence of paramagnetic ions (PMI)3,4. The present of PMI reduce the longitudinal relaxation time (T^ of water protons water as previously demonstrated by Block, Hansen and Packard5. The aim of the present work was to analyze the influence of paramagnetic ions 2+ 2+ 2+ 3+ Cu ,Ni , Mn and Fe on the T2 distributions curves. One crude oil sample with 53 wt.% water and kinematic viscosity of 45.0829 mm2/s at 27.5°C was choosen for the analysis. The low-field 1H NMR experiments were conducted in a MAFRAN Ultra spectrometer, 1 from Oxford Instruments, operating at 2.2 MHz for H. The T2 measurements were performed in triplicate using the CPMG pulse sequence with 16 scans, time between echoes of 200 ps, 8192 echoes and recycle delay of 3s. T2 distributions curves were obtained by the Inverse Laplace Transform method.
The T2 distributions obtained for the crude oil and the doped oil samples with PMI are shown in Figure 1. As can be observed the "bulk" water has one peak well defined in 2.62s. Moreover, it can be verified that the crude oil T2 distribution curve present four peaks, being the first three peaks are associated to oil fractions and the last peak, at 670 ms, refers to the water contained in the crude oil. Such oil present distinct peaks due to its multi-component nature (paraffinic, aromatics, resins and asphaltene)6. The distribution curves showed that the doping of oil with PMI lead to the suppression of the water peak. The reduction in T2 of solvent nucleus in the presence of PMI is due to the strong interaction between the protons of water and the unpaired electron spins of PMI. This result clearly illustrates the influences of paramagnetic ions on the T2 distribution profiles for oil samples, which can lead to erroneous models for the correlation between T2 values and oil viscosity.
26 T2/US
Figure 1: T2 distribution curve for crude oil and doped oil samples with 1% w/w PMI.
It can be verified in Table 1 that the T2 value is reduced with increse in concentration for crude oil doped with Ni2+, but constant for Cu2+ and Fe3+ ions. However, the sample with the addition of Mn2+ ion present three peaks for 1.2% wt. PMI that are assigned to overlapping of signals from water/oil and oil fraction for low T2 values..
Table 1: T2 values for two concentration of salt PMI
Percentage of ion (w/w) T2 (ms) Ion 42 Ni2+ 32 Cu2+ 1.0% 1 ; 55 Mn2+ 3.6 ; 65 Fe3+
20 Ni2+ 1.2% 33 Cu2+ 0.8 ; 21 ; 160 Mn2+ 3.6 ; 65 Fe3+
In conclusion, the dopping of crude oil with PMI produced shift of water peak in the T2 distribution curve. The strong interation between the PMI and crude oil provoked supression of water peak, leading to the overlapping of peaks, which can lead to erroneous models for the correlation between T2 values and oil viscosity.
REFERENCES:
1. Bryan, J.; Kantzaz, A. and Bellehumeur C. SPE Reservoir Evaluation & Engineering 2005, 8. 2. Coates, G.R.; XIAO, L.; Manfred, G.P. NMR Logging - Principles and Applications. Houston: Halliburton Energy Services, 1999. 3. Kantzas A. J. Cand. Petroleum Techn.2009, 48. 4. Bryan J. and Kantzas A., SPE Reservouir Evaluation & Engineering. 2005, 44. 5. Block F.; Hansen W. W. e Packard M. E., Phys Rev. 1946, 70 6. Ramos P. F. O et ai. Chemometrics and Intelligent Laboratory Systems. 2009, 99
PETROBRAS, FINEP, SCHLUMBERGER, LABPETRO DQUI/UFES
27 h PO 05
DETERMINATION OF OIL CONTENT IN SEEDS WITH POTENTIAL FOR BIODIESEL PRODUCTION BY LOW-FIELD NMR G. F. Carneiro*1, A. F. Constantino2, R. B. dos Santos"!,2, V. Lacerda Júnior1,2, R. C. Silvai, S. J. Greco2, J. C.C. Freitas3, E. V. R. de Castrol. 1LabPetro - DQUI - UFES, Vitoria - Brazil. 2LPQO - DQUI - UFES, Vitoria - Brazil. 3DFIS - UFES, Vitoria - Brazil, e-mail: giovannafcticb.qmail. com
Keywords: low-field NMR; oil content.
Biodiesel appeared in the last decade as an alternative fuel, produced from renewable resources and less harmful to the environment. Assessing the oil content of seeds and nuts is essential when selecting which ones can be used as a possible oil source. The usual method for determining oil content is solvent extraction, which suffers from the drawbacks of being a slow and sample destructive1. Low field 1H NMR constitutes an alternative method, predicting oil content with good accuracy in a fast and non-destructive way2 This work aims at determining the oil contents of different oil matrixes by low field NMR. Munguba (Pachira aquatic Aubl.), nogueira-de-iguape (Aleurites moluccana L. Wild.), cutieira (Joannesia princeps Veil), linseed (Linum usitatissimun), peanut (Arachis hipogaea L.) and sunflower (Helianthus annuus L.) seeds were used in this study. They all present a high lipids content, being, therefore, possibly good oil sources for biodiesel production. A Maran-2 Ultra NMR spectrometer from Oxford Instruments, operating at 52mT (2.2MHz for 1H), was used in all experiments, which were conducted in a 51mm diameter probe. At first, a calibration curve was built for each of the oil-rich seeds with samples of its own vegetable oils. In order to build the curve, single-pulse experiments were carried out, with 16 points recorded for each transient, a dwell time of 2ps, a recycle time of 10s and a total of 128 added transients for the nogueira seed and 32 transients for the other seeds. The oil mass was related to the amplitude of the free induction decay (FID) recorded in these experiments, yielding the curves shown in Figure 1. The correlation coefficients of the calibration curves were well above 0.97, which is usually taken as the minimum value for this kind of analysis2.
y 2 y R -0 9932 Rs=0 9994 o X CD X E « X ra X" 5 5 a Munguba X • Nogueira o Cutieira X itensity fa.u.; MM R Signantgnsityía.l u • 'ÍÍ-.ÍP, signal int&fisij^ ts u \
2 X "o R =0.9996 x R'=0 9998 / X vn li- y A ft x' E - y y/ 5 / X y • Peanut a Linseed a Sunflower X' Tli.tR slip's! irter>sit/ia"u./ Bmr »ijr»l inter.su» , N M R signal intensity fa.u.i Figure 1: Calibration curves for the vegetable oils studied.
4 28 For each seed type, 3 groups with 20-30g, previously crushed and dried in an oven at 100°C for 1h, were analyzed before and after oil extraction at room temperature with hexane. This procedure allowed the determination of extraction yields by NMR. A comparison between these values and the ones obtained by the usual method is shown in Figure 2.
Figure 2: Pairing of values obtained by the solvent extraction method and by low-field NMR. The line corresponding to the identity function is shown, as a guide to the eyes.
The results obtained by both methods were slightly divergent, with an average error of ca. 9 % in the NMR predictions. However, these values can be used as they are, since the pairing of values obtained by both methods indicates a reasonably high correlation coefficient of ca. 0.953. The average oil contents of the seeds calculated by NMR are presented in Table 1 The fact that most NMR-calculated yields were higher than the expected values is probably due to experimental loss in the extraction process. On the other hand, lower values (obtained" especially for linseed) may be explained by the extraction of substances with short values of T2 that are possibly NMR-invisible due to the dead time of the equipment (70ps).
Table 1: Average oil content for the studied seeds.
Oil Average Oil .. . Average Oil Matrix Content (wt.%) Content (wt.%) Munguba 62.4 Linseed 45.7 Nogueira 69.2 Peanut 43.0 Cutieira 49.0 Sunflower 41.9
Such experiments show that low-field 1H NMR analysis can produce reliable results regarding the prediction of oil content in seeds and, therefore, can be used for predicting expected extraction yields with good accuracy.
REFERENCES: 1. Lima, J. R. O.; Silva, R. B.;Silva, C. C. M.; Santos Jr., J. R.; Moura, E. M.; Moura, C. V. R. Química Nova, 2007, 30,.600-603. 2. Colnago, L. A. Análise do Teor de Óleo em Sementes por RMN. Circular Técnica, Embrapa, 1996. 3. Godoy, I.J; Teixeira, J. P. F.; Nagai, V.; Rettori, C. Bragantia, 1986, 45 (1), 161-169.
FAPES; FINEP; PRPPG-PIVIC; LABPETRO-DQUI/UFES.
29 h PO 06
DIFFUSIOMETRY STUDIES OF WATER AND OIL MIXTURES BY LOW-FIELD 1H NMR G. F. Carneiro*1, V. Lacerda Júnior1, R. C. Silva1, L. L. Barbosa1, J. C.C. Freitas2, E. V. R. de Castro1. 1LabPetro - DQUI - UFES, Vitoria - Brazil. 2DFIS - UFES, Vitoria - Brazil, e-mail: qiovannafc(p).Qmail. com
Keywords: low-field NMR; crude oil; diffusion coefficient.
Water is found with oil and gas in wells and can also be injected for the control of reservoir pressure in extraction processes. The presence of water in crude oil production chain affects the efficiency of the process as a whole. Therefore, knowing water content is important in the moment of the oil extraction, but it is also important in transporting and selling stages.1 Low-field 1H NMR is a technique that has been successfully applied in reservoir and produced fluids characterization. The non-destructiveness of the technique and the easy-to-do measurements are big advantages. In addition, it is possible to develop in- situ measurements, producing instant results.2
In this work mixtures of a crude oil (°API 29,1) and a 50 ppm MnCI2 aqueous solution were used in order to study the quantification of water in oil by diffusion coefficient. Three mixtures were prepared with the crude oil and the solution, with 25, 50 and 75 wt.% of the solution in each. The MnCI2 was added to water in an attempt to simulate a situation where water and oil have similar T2, which would make a water content analysis by 1H NMR relaxometry more complicated. A Maran-2 Ultra NMR spectrometer from Oxford Instruments, operating at 52 mT (2.2 MHz for 1H) together with an accessory for experiments with pulsed field gradient (PFG) was used in all experiments, which were conducted in a 51mm diameter probe. CPMG experiments were performed with 2048 echoes recorded for each transient and a total of 16 added transients. The T2 distribution curves were computed by the Inverse Laplace Transform (ILT) of the time-domain signals, using the WinDXP® software and are shown in Figure 1. The curves show that oil and MnCI2 solution have overlapping T2, as expected.
50ppm
MnCI2 solution Crude Oil
E < 1000 -
Figure 1: T2 distribution curves for the crude oil and the MnCI2 solution studied.
Diffusion coefficient measurements were made according to the Tanner sequence,3 with use of a list of 10 different values of the PFG duration (x2). For each x2 value, 20 points were recorded, with a recycle time of 1s, a total of 32 transients for the crude oil and 16 transients for the MnCI2 solution and the mixtures. The magnetic field gradient applied (g) was of 12.60 G/cm for the MnCI2 solution, 25.20 G/cm for the mixtures and 39.06 G/cm for the oil.
Y 4- 30 Tli t2 T When ln(M/M0) is plotted as a function of ( ~ ^ i)a straight line is expected, according to Tanner's equation:
M Ct2 - rt) In- ln2 - f Ofl'Tj' r (1)
where M/M0 is the signal attenuation due to the applied PFG, x2 is the duration of the pulse in ms, n is the gap between the application of the gradient pulses in ms, y the 1H magnetogyric ratio and D is the self-diffusion coefficient of hydrogen-containing molecules in the sample.3
The plots for crude oil (Figure 2a) and MnCI2 solution (not shown) do have a trend for a straight line. But for the water-oil biphasic mixtures (Figure 2b) the same trend is not apparently observed. An analysis of the curves and of the mean values of diffusion coefficient (Table 1) shows that mixtures exhibit a behavior that is intermediate between pure crude oil and pure MnCI2 solution. Also, the diffusion coefficient for crude oil and MnCI2 solution are very distinct. Moreover, when the MnCI2 solution is predominant in the mixture, plotting only the first points result in a diffusion coefficient (1.9x10~9 m2/s) very similar to the one obtained for the solution itself. Similarly, when crude oil is predominant, plotting only the last points yield a diffusion coefficient (7x10"11 m2/s) very close to the one measured for the oil itself.
(a) (b)
0.3 0.4 I 0.2 0.3 2 1 T, (V1/3.X,) t, (v /3t,)
Figure 2: Plots for obtaining diffusion coefficients for (a) crude oil and for (b) mixture of crude oil and MnCI2 aqueous solution with 50 wt. % each.
Table 1: Diffusion coefficients measured for the samples.
Water content (wt. %) in Sample D (m2/s) D (m2/s) biphasic mixtures 9 10 50 ppm MnCI2 2.75x10' 25 2.5x10" Crude oil 7.7x10~11 50 5.1x10"10 75 9.5x10"10
These findings indicate that diffusion coefficient measurements by low-field 1H NMR can be useful for the identification of water in oil.
REFERENCES: 1. Becker, J. R. Crude Oil: Waxes, Emulsions and Asphaltenes. Tulsa: PennWell Books, 1997. 2. Metz, H.; Màder, K.. Int J Pharm, 2008, 364, 170-175. 3. Tanner, J.E.. J Chem Phys, 1970, 52, 2523 - 2526.
FAPES; FINEP; LABPETRO-DQUI/UFES; SCHLUMBERGER.
31 h PO 07 UNEQUIVOCAL ASSIGNMENT OF THREE SYNTHETIC INTERMEDIATES OF GUAIANES AND NOR-GUAIANES: AN EXPERIMENTAL AND THEORETICAL APPROACH L. R. Barbosa1,Y. W. Vieira2, V. Lacerda Jr.*1, K. T. de Oliveira2, R. B. dos Santos1, S. J. Greco1, T. J. Brocksom2, E. V. R. de Castro1 1 Department of Chemistry-CCE/UFES, Vitória-ES, Brazil department of Chemistry-CCET/UFSCAR, São Carlos-SP, Brazil [email protected]
Keywords: NMR; guaianes; assignment
Fused five- and seven-membered ring systems show peculiar structural features and have stimulated a number of research projects in both theoretical and synthetic fields.1 In addition, these carbon building blocks can be considered as key structures for the syntheses of a large number biologically active compounds, such as sesquiterpenes, diterpenes and sesterterpenes; also, the wide spectrum of biological activities allied with structural complexity make these compounds interesting targets for the synthetic and NMR studies.2 In recent years a significant number of works involving structural assignments by using 1D and 2D-NMR analysis and sophisticated theoretical calculations have been performed.3,4 The theoretical calculations are based on the application of molecular models in order to obtain mathematical solutions, which provide data and physico- chemical parameters. By using calculations, we can predict spectroscopic properties to assist the analysis of experimental spectra such as infrared, Ramafi, NMR and others. Particularly, the use of theoretical calculations has been considered as an excellent support for structural assignments and providing a good correlation between experimental and theoretical results.3,4 This work has as main objective the unequivocal assignment of the NMR signals of 1H and 13C NMR of three synthetical intermediates of guaianes and nor-guaianes These studies were performed by using 1D and 2D NMR techniques and compared to the theoretical calculations of chemical shift, õ (DFT calculations).
Figure 1. Synthetical intermediates of guaianes and nor-guaianes.
The structures of compounds (a, b and c) were drawn with the GaussView 4.15 program and then a conformational search was performed, the structures found in the search were optimized by using Gaussian 036 program and the calculations were carried out in different levels of theory. The calculations of chemical shifts (8) were performed at the B3LYP/cc-pVTZ level, using GIAO7 method. The solvent effect was included (chloroform) in the calculations. The values of chemical shifts were obtained considering the difference between the values obtained in the calculations (tensor of blindage) and the reference (TMS). In order to obtain an unambiguous assignment of the NMR signals of compounds (a, b and c) the spectra 1D NMR of 1H, 13C{1H}, DEPT-135 and 2D NMR of COSY, HSQC, HMBC and NOESY were analyzed. The main assignments are listed in Table 1 below.
f + 32 Table 1. Chemical shifts of 1H and 13C, S (ppm). Compound (a) Compound (b) Compound (c) c S(13C) 5(1H) 8(13C) S(1H) S(13C) 5(1H) 1a 3.01 1 48.2 62.5 — 213.4 — 1b 3.31 2a 2.28 2 130.3 — 141.2 — 40.7 2b 2.35 3 124.5 5.57 121.2 5.45 44.6 2.67 5 43.2 2.74 44.8 2.04 123.9 5.55 8 26.1 1.79 19.6 1.74 27.5 1.73 9a 4.75 9 110.1 4.75 109.7 20.8 1.64 9b 4.78 10 20.5 1.73 21.2 1.75 147.7 — 11a 4.65 11 148.2 — 148.0 — 109.8 11b 4.70 12a 2.52 12a 2.56 12 39.6 38.4 12b 2.68 12b 2.08 12'a 2.71 12'a 2.65 12' 41.0 40.6 12'b 2.80 12'b 2.36 13 — — 129.6 5.53 133.4 5.62
13' — — 127.0 5.66 137.3 5.65
The experimental data of compounds (a, b and c) were confronted with the theoretical data, which showed better results when the solvent effect was considered. The data generated for compounds (a, b and c) are listed in Table 2 below.
Table 2.Comparison between theoretical and experimental values of chemical shifts, 5. a b c A5* A5* A5* SD** 0.12 0.90 0.33 1H NMR ~ MD*** 0.15 0.53 1.17 SD** 3.96 1.49 2.90 13C NMR MD*** 5.99 3.26 6.80 A8* = | §TEOR - 5EXP.| ** SD = Standard deviation *** MD = Mean deviation ((XA8)/n, n = number of compared chemical shifts) A complete NMR study of three synthetical intermediates of guaianes and nor- guaianes compounds was performed using a combination of 1D and 2D NMR experiments and theoretical calculations of chemical shifts (8). Most of the signals of 1H and 13C NMR were assigned. The calculations with solvent effect introduced better results and the theoretical model used, B3LYP/cc-pVTZ was adequate to describe the values of 1H and 13C NMR chemical shifts (S).
REFERENCES 1. B. M. Fraga, Nat. Prod. Rep. 2004, 21, 669. 2. K. T. de Oliveira, Spectrochim. Acta Part A 2006, 63, 709. 3. L. H. K. Queiroz Jr., et. al., Magn. Reson. Chem. 2011, 49,140. 4. M. G. Constantino, Spectrochim. Acta Part A 2004, 61, 171. 5. GaussView, Version 4.1, Dennington II, R.; Keith, T.; and Millam, J.; Semichem, Inc., Shawnee Mission, KS, 2007. 6. Gaussian 03 Revision C.02, Gaussian, Inc., Wallingford CT, 2004. 7. T. Helgaker, et. al., Chem. Rev., 1999, 99, 293. FAPES/FUNCITEC, PPGQUI-UFES, CAPES, LABPETRO-DQUI/UFES
33 h PO 13
ARE HYDROGEN BONDS SECONDARY INTRAMOLECULAR INTERACTIONS RESPONSIBLE FOR PHENYLALANINE CONFORMATIONAL PREFERENCES? R.A. Cormanich*, F.P. dos Santos, L.C. Ducati, R. Rittner State University of Campinas, Campinas, Brazil e-maii:[email protected]
Keywords: NMR spectroscopy, conformational analysis, amino acids
The amino acids conformational preferences have been most explained as due to the formation of intramolecular hydrogen bonding (HB),1 despite of steric and hyperconjugative effects, which stabilize even simpler systems.2 In special, phenylalanine conformers stability has been arbitrarily attributed to possible N- H—7t (aromatic n electrons) main chain/side chain interactions.3 Also, solid and solvated + amino acids exhibit the zwitterionic form ( H3N-CHR-COO~), which is a structure far from the neutral one present in the polypeptide chains, being prohibitive the studies in solutions and, hence, the NMR studies. In this way, in order to understand the interactions that govern the phenylalanine rotational isomerism and to bypass the experimental difficulties, the study of phenylalanine methyl ester (phe-OCH3) - a compound that is soluble in organic solvents and not presents the zwitterionic structure - is proposed here by NMR spectroscopy and theoretical calculations. The observed signals in the NMR experiments of a particular molecular system represents an average of all conformers present in such system. Table 1 shows the 3JHH values in several solvents, which indicate a variation in Jhe conformational populations relative to the side chain of the phe-OCH3, from low to high dielectrical constant solvents.
Table 1: Chemical shifts (8) in ppm and coupling constants (J) in Hz to the phe-OCH3 conformers in solvents of different dielectric constants (e).
He NH20 / I I II (' '—c—c—c—0-CH3 II d3 Hb Ha
3 3 2 Solvent e 5Ha 5Hb ÔHc ÔHd JHaHb J»mc Jh bHc
CDCI3 4,8 4,10 3,09 3,25 3,72 7,9 4,8 14,0 CD2CI2 9,8 4,13 3,13 3,27 3,78 7,6 4,9 14,1 Piridine-d5 12,3 3,90 2,99 3,15 3,61 7,4 5,7 13,4 Acetone-d6 20,7 4,15 3,21 3,28 3,70 6,5 5,2 13,8 MeOD* 32,7 3,95 3,11 3,11 3,37 6,6 6,6 — CD3CN 37,5 4,00 3,08 3,15 3,67 6,1 6,1 13,9 DMSO-d6* 46,7 3,66 2,88 2,88 3,57 6,7 6,7 ...
* Hb e He are indistinguishable in CD3OD and DMSO-d6 solutions.
Three side chain conformers relative to the main chain are possible (Figure 1). From 3 3 the JHaHb and JHaHC approximation values in solvents of higher £ values in Table 1, two hypothesis emerge: one is that the conformational equilibrium would shift to the "c" arrangement and another is that the "a" and "b" arrangements would be in equivalent proportions. a b C Hb
H'VL/'Hc Ha
Figure 1: Three possible energy minima side chain arrangements of the phe-OCH3.
34 In order to give an interpretation to the NMR results, many theoretical potential energy surfaces at B3LYP/cc-pVDZ level were built for the phe-OCH3 compound, resulting in 16 conformers, which were optimized at B3LYP/aug-cc-pVDZ in chloroform, acetonitrile and DMSO by the IEFPCM implicit model (Table 2).
31 1 Table 2: Relative energy' (kcal mol" ) and population (%) of phe-OCH3 conformers.
Isolated CHCI3 CH3CN DMSO Conformer E %P E %P E %P E %P la 0,0 20,6 0,0 25,8 0,0 15,1 0,0 21,0 lb 0,2 15,8 0,2 18,4 0,0 15,1 0,0 21,0 Ic 0,3 13,3 0,7 7,9 0,7 7,3 0,9 4,6 lllb 1,4 1,9 1,1 4,0 0,8 6,5 0,8 5,4 lllc 1,0 4,1 1,4 2,4 1,5 3,1 1,6 1,4 IV1a 0,9 4,3 0,7 7,9 0,6 8,1 0,5 9,0 IV1b 0,1 18,0 0,3 15,5 0,2 12,3 0,2 14,9 IV1c 1,9 0,9 1,7 1,5 1,3 3,9 1,2 2,8 IV2a 2,7 0,2 2,8 0,2 1,5 3,1 1,5 1,7 IV2b 0,4 11,2 0,8 6,7 0,8 6,5 0,7 6,4 Via 2,3 0,5 2,3 0,5 1,8 2,3 1,5 1,7 V1b 0,8 5,1 1,4 2,4 1,5 3,1 1,5 1,7 V1c 3,0 0,1 3,3 0,1 3,1 0,6 3,1 0,1 V2a 2,6 0,3 1,8 1,2 1,1 4,8 1,1 3,3 V2b 1,1 3,2 1,0 4,8 0,8 6,5 0,9 4,6 V2c 2,3 0,4 2,2 0,6 2,1 1,7 2,1 0,6 [aJZero point energy correction included.
The most stable la and lb conformers (Table 2) are the ones which show larger population changes on going from CHCI3 to DMSO solutions. This suggests that the second hypothesis is correct, i.e., the "a" and "b" arrangements are equal in energy and that the theoretical calculations are in agreement with the experimental data. Indeed, QTAIM calculations predict that some conformers exhibit an C-H - 0 intramolecular HB. However, it can be concluded, based in Popelier criteria,4 that such interactions are not true HB and, indeed, just occur in unstable conformers (Via, V1c and V2c). Also, N-H - rcinteraction s were not shown in QTAIM calculations. NBO analysis corroborates with QTAIM results. It suggests that both hyper- conjugative and steric effects play an important role in the phe-OCH3 conformational preferences and cannot be ignored (Table 3). Thus, the arbitrary assumptions in the literature are divergent of the present work, which shows that hyperconjugation and steric effects, and not HB, rule the phenylalanine conformational preferences.
Table 3: Total energies of the real system (AEFULL), energy of the hypothetical case where hyperconjugation is removed (AELewis) and hyperconjugative energies (AEhyper) 1 for the most stable and some unstable phe-OCH3 conformers in kcal mol" . la lb IV1b IV2a V1c V2a
AET 0,00 0,16 0,08 2,74 2,97 2,55 AHl_ewis 5,87 3,34 4,97 4,77 6,40 4,47 AEhiper 8,03 5,27 7,06 4,02 5,44 4,19
REFERENCES: 1. Blanco S.; López J. C.; Mata S.; Alonso J. L. Angew. Chem. Int. Ed. 2010, 49, 9187. 2. Cormanich R. A.; Freitas M.P. J. Org. Chem., 2009, 74, 8384. 3. Lee K. T.; Sung J.; Lee K. J.; Kim S. K.; Park Y. D., J. Chem. Phys., 2002, 116, 8251. 4. U. Koch, P. L. A. Popelier, J. Phys. Chem., 1995, 99, 9747.
FAPESP, CNPQ
35 f- AUREMN - MAY 2nd to 66,2011." HOTEL DO FRADE, ANGRA DOS RI IS. RJ, BRAZO,
PO 13
STERIC/HYPERCONJUGATIVE EFFECTS BALANCE GOVERNS THE VALINE CONFORMATIONAL EQUILIBRIUM R.A. Cormanich, F.P. dos Santos, L.C. Ducati, R. Rittner* State University of Campinas, Campinas, Brazil e-mail:rittner(p).iqm.unicamp.br
Keywords: Conformational analysis, NMR spectroscopy, valine
Amino acids conformational analysis has gained special attention in the literature, since the several folding pathways of protein molecules must be primarily restricted by the total conformational space determined by the individual amino acid residues.1 Although much effort has been made to understand glycine - the simpler amino acid - conformers, such studies are scarce to valine and to higher amino acids.2 Indeed, as well as to glycine, conformational preferences of valine have been attributed to possible intramolecular hydrogen bonding, while hyperconjugation and steric hindrance are ignored. Also, solution studies have been prohibitive and, hence, NMR studies cannot be encountered in the literature.2 3 The amino ester has the advantage of not + presenting the H3N-CHR-CO~ bipolar form and, therefore, is a good approximation of an amino acid in a polypeptide chain, which can be used as model to study the effects that govern peptide folding. In this way, valine methyl ester (val-OCH3) study is proposed here by NMR spectroscopy and theoretical calculations.
3 The JHH values obtained in solvents of different dieletric constants shows that there are no meaningful conformational changes in respect of val-OCH3 side chain (Table 1).
Table 1: Chemical shifts (8) in ppm and coupling constants (J) in Hz to the val-OCH3 conformers in solvents of different dielectric constants (e). H c d 3 NH2 O
t* HoC C -c— -c—0— -CH3e i I I I Hb Ha 3 3 Solvente E 5Ha 5Hb 5Hc ÔHd SHe «•HaH1 b JHbH1 c CDCI3 4,8 3,95 2,39 1,10 1,10 3,83 4,4 6,9 CD2CI2 9,8 3,94 2,39 1,10 1,10 3,84 4,4 7,0 Pyridine-d5* 12,3 4,16 2,49 1,11 1,14 3,72 4,9 6,9 Acetone-d6* 20,7 4,18 2,50 1,12 1,14 3,83 4,6 6,9 MeOD 32,7 3,75 2,22 1,02 1,02 3,81 4,7 6,9 CD3CN 37,5 3,77 2,23 1,00 1,00 3,78 4,5 6,9 DMSO-d6 46,7 3,68 2,07 0,92 0,92 3,72 5,0 7,0 * He and Hd are not equivalent, in acetone and pyridine, but they exhibit the same JHH value with Hb.
Natural bond orbital (NBO) analysis and the quantum theory of atoms in molecules (QTAIM) calculations can help us to understand the conformer stabilities (see below). NBO analysis gives a structure with maximum electron localization, i.e., a Lewis structure with doubly occupied orbitais, where only classic effects are present. NBO analysis (Table 2) shows that both hyperconjugation and steric effects are important to val-OCH3 conformational equilibrium.
Table 2: Total energies of the real system (AEFULL), energy of the hypothetical case where hyperconjugation is removed (AELeWis) and hyperconjugative energies (AEHYPER) 1 for some representative val-OCH3 conformers in kcal mol" . la lb Ic I Via IV2a Via V1b V2a
A£T 1,07 0,25 0,00 1,58 1,27 3,87 3,19 2,22 AH[_ewis 7,55 7,77 6,28 9,13 8,79 4,19 0,00 6,61 AHhiper 9,50 10,47 9,21 9,99 9,42 3,94 0,00 6,70
36 In accordance with the NMR outcomes, the 17 conformers found by B3LYP/aug-cc- pVDZ calculations, optimized by the IEFPCM implicit model in chloroform, acetonitrile and DMSO, show that there is no significant conformer population changes (Table 3).
[a] 1 Table 3: Relative energy (kcal mol" ) and population (%) of val-OCH3 conformers.
Isolated CHCI3 CH3CN DMSO Conformer E %P E %P E %P E %P la 1,1 6,6 0,9 6,6 0,8 6,1 0,9 6,0 lb 0,3 26,2 0,0 28,6 0,0 24,2 0,0 25,6 Ic 0,0 39,9 0,0 28,4 0,0 24,3 0,0 23,7 Ilia 2,1 1,1 1,8 1,4 1,7 1,4 1,7 1,5 lllb 0,9 8,2 0,6 10,7 0,4 11,8 0,5 10,5 lllc 1,6 2,8 1,2 4,0 1,1 4,1 1,2 3,3 IV1a 1,8 1,8 1,5 2,1 1,2 3,1 1,3 3,1 IV1b 1,4 4,0 0,9 6,4 0,6 8,4 0,7 8,4 IV1c 1,3 4,7 1,0 5,6 0,8 6,4 0,8 7,2 IV2a 2,1 1,1 2,2 0,7 1,8 1,1 1,9 1,1 IV2b 3,9 0,1 4,2 0,0 6,2 0,0 1,9 1,1 Via 3,2 0,2 3,1 0,2 2,7 0,3 2,7 0,3 V1b 2,9 0,3 2,7 0,3 1,8 1,2 1,9 1,1 V1c 2,2 0,9 2,2 0,7 2,1 0,7 2,1 0,7 V2a 2,8 0,4 2,3 0,6 2,1 0,7 2,0 0,8 V2b 2,1 1,2 1,4 2,7 1,0 4,9 1,1 4,3 V2c 2,4 0,7 2,0 1,0 1,7 1,3 1,7 1,4 [a|Zero point energy correction included.
Moreover, hydrogen bonds are not important in the conformational preferences according to QTAIM calculations, due to its instability and that they exist only in higher energy conformers (Figure 1). Thus, Steric/Hyperconjugative interactions are the governing effects in val-OCH3 rotational isomerism.
la * lb Ic Ilia lllb lllc
# *« ,-XX Jk* L 4 1 » «. ** ; & • í \ ,, » » » • f-4 x .-«r v - » I ' * »
IV1a IV1b IV1c IV2a IV2b Via 4 1 \ • "»' e * » • v X , 0-- c -f »- - „ * - * * V . • t * •,»*" • *«- - •w-* ' * "V" ' V V • V1b V1c V2a V2b V2c / / —v * ! + - • . * \ » «. • - * " " ; .: wt • 4r «, * » * * • -
Figure 1: Val-OCH3 molecular graphs obtained by QTAIM.
REFERENCES: 1. Masman, M.F.; Lovas, S.; Murphy, R.F.; Enriz, R.D.; J. Phys. Chem., 2007, 126, 10682. 2. Alonso, J.L.; Cocinero, E.J.; Lessarri, A.; Angew. Chem. Int. Ed., 2006, 45, 3471. 3. Alonso, J.L.; Pena, I.; López, J.C.; Vaquero, V., Angew. Chem., 2009, 121, 6257.
FAPESP, CNPq
37 h PO 10
NMR FOR PHOSPHORUS DETERMINATION DURING DECOMPOSITION OF TYPHA ANGUSTIFOLIA C.S. Oliveira*1, L.M. Lião1, G.B. Alcântara1, F. Petacci2, S.S. Freitas2 11nstituto de Química, Universidade Federal de Goiás, Goiânia-GO, Brazil 2Departamento de Química, Universidade Federal de Goiás - CAC, Catalão-GO, Brazil e-mail: karolsilvaoliveira@ vahoo. com.br
Keywords: Typha angustifolia; decomposition;31P NMR
Typha angustifolia L. is a perennial lignocellulosic vascular plant of the Thyphaceae family largely distributed in Brazil, and popularly known as "Taboa". Due to its high rate of growth is frequently associated with depletion of oxygen dissolved, fish death and terrestrialization of wetlands1 and has been considered a weed in Brazil and other countries. Decomposition of macrophytes plays an important role in nutrient cycling, carbon and energy flows in aquatic ecosystems2. In several ecosystems the availability of nutrient resources depends on microbial decomposition of detritus 3'4. Procedures for extraction of phosphorus are frequently used to- known the distribution and mobility of this chemical element on sediment5. However, the study of forms of phosphorus in aquatic plants and, its main source in lakes, sediments are scarce. In this context, the objective of this work was to describe the speciation of phosphorus by 31P NMR during decomposition process of T. angustifolia, a dominant aquatic macrophyte in Goiás State, Brazil, under laboratory controlled conditions in respect to the presence and absence of bacteria during 120 days. Samples of T. angustifolia were collected on February of 2008 in a park lake located at Catalão, Goiás, Brazil. In laboratory, plants were washed, dried (40 °C) and fragmented (size ca. 0.3-0.5 cm) to the study. Only aerial parts were used to decomposition experiment at room temperature (27.3 ± 2.6 °C). The experiment was divided in two sets: in the first one the samples were treated with the chloramphenicol antibiotic (NeoFenicol®, 500 mg, Neoquímica, Anápolis, Brazil), and in the last the experiment was conduced without antibiotics. A solution containing 300 mL of water and 18 mg of antibiotic per week was used. After 90 days of treatment each sample received 234 mg of chloramphenicol to suppress bacteria growth 3,e. The studies of dynamic species of phosphorus during decomposition by 31P NMR were based on previously procedures described by Cade-Menun (2005). Detritus in each time sampling were extracted with 50 mL of a 0.25 M NaOH by 20 minutes. After filtration, the solution was immediately lyophilized (Freeze Dry System/Freezone® 4,5 , Labconco) and 100 mg of residue was dissolved in D20 and analyzed by a Brüker Avance III 500 spectrometer (11.75T of magnetic field) at 298 K, using a 5 mm direct probehead. The 31P spectra were obtained at 202.45 MHz with 90° rf pulses, spectral widths 40760 Hz, 3072 transients with 64k data points, acquisition time 0.8 s, receiver gain 203 and relaxation delays 2 s. It was processed with 32k data points and multiplied by an exponential weight function corresponding to a linebroadening of 10.0 Hz. The phase and baseline correction were manually corrected using the Bruker software. The 31P NMR spectra obtained from in natura, 15, 60, 90 and 120 days of degradation, in the absence (A) and presence (B) of the chloramphenicol antibiotic, are showed in Figure 1. The signals at 6.11, 4.50, -0.80 and -4.10 ppm were respectively attributed to inorganic orthophosphate (Pi), monoester phosphate (PMN), nucleotide phosphate (NP), and to pyrophosphate (PPi)5'7 During decomposition process of T. angustifolia significant differences between the two treatments were observed. In experiment conduced without chloramphenicol antibiotic the signal of inorganic orthophosphate (Pi) decreased and the signal of monoester phosphate (NP) increased. Important factors
* 38 can influence the vegetable decomposition like as temperature, pH, nutrient content in vegetal tissue and the presence of microorganisms.
i PMN A Pl i PMN B
NP 120 dteys Pi
^"'«"VvVi./ "V^sA»'. » (Wv?TVv-
90 days -MfigA
60 days
15 days
m natum
PPi PPi
7 6 5 4 3 2 1 0 -1 -2 -3 ppm 7 6 5 4 3 2 1 0 -1 -2 -3 -4 -5 pprn
Figure 1: 31P NMR spectra of T. angustifolia samples at different times in absence (A) and presence (B) of chloramphenicol. Legend: Pi, orthophosphate; PMN, monoester phosphate; NP, nucleotide phosphate ; PPi, pyrophosphate.
The inhibition'of the microorganism proliferation resulted in the alteration of macrophyte decomposition dynamic. Thus, there was not a regular degradation of the present compounds, like observed in experiments conduced without chloramphenicol. This suggests that bacteria present in the samples control the decomposition, which inorganic phosphate (orthophosphate - Pi) was especially consumed. On the other hand the content of monoester phosphate (PMN) increased. This study represents the first report about consumption of specific forms of phosphorus by microorganisms during decomposition process in macrophytes.
REFERENCES: 1. Alvarez, J.A. & Bécares, E. Ecological Engineering, v. 28, 2006, 99-105. 2. Masifiwa W.F., Okello W., Ochieng H. and Ganda E. Uganda Journal of Agricultural Sciences, v.9, 2004, 389-395. 3. Gamage, N. P. D. & Asaeda, T. Hydrobiologia, v. 541, 2005, 13-27. 4. Alvarez, S., Guerrero, M.C., Soil Biology & Biochemistry, v. 32, 2000, 1941- 1951. 5. Ahlgren, J., L. Tranvik, A. Gogoll, M. Waldeba" CK, K. Markides, and E. Rydin. Environmental Science Technology, v. 39, 2005, 867-872. 6. Gaur, SPK Singhal & S. K. Hasija. Aquatic Botany v. 43, 1992, 1-15. 7. Cade-menun, B.J., Liu, C.W., Nunlist, R., McColl, J.G. Journal of Environmental Quality, v. 31, 2005, 457-465.
UFG, DQ-UFG-CAC, CAPES, CNPQ, FINEP
39 h AtTfKMN iMAY 2nd in 6th, 2011 I lull-1 UUIHADI . \\C;R.\ I?OS HM.S.RI HRA/II.
PO 11
29SI AND 13C NMR SPECTROSCOPY OF COMMERCIAL ALUMINA IONIC LIQUIDS Naira M.S. Ruiz1*, Fábio L.L. Farias2, Sandra S.X. Chiaro2, Sônia M.C. de Menezes2, Leandro Luza3 and Jairton Dupont3 1 Pontifícia Universidade Católica do Rio de Janeiro (PUC-RIO), Rua Marquês de São Vicente, 225, Rio de Janeiro, RJ, CEP 22451-041 2 CENPES-PETROBRAS, Avenida Horácio Macedo, 950, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, CEP 21941-915 3IQ-UFRGS, Av Bento Gonçalves, 9500, Agronomia, Porto Alegre, RS, CEP 91501-970
Keywords: 13C CP/MAS ;29Si MAS NMR; ionic liquids supported on alumina;
ionic liquids are salts of an organic cation (ammonium, phosphonium, imidazolium, pyridinium, etc.).1,2 Their chemical and physical properties may be quite varied, as they can be tuned by choosing a specific combination of cation and anion among numerous possibilities. Because of their ability to be recovered and recycled and their good thermal stability and nonflammability properties, many applications were found to some specific areas. For application as electrolyte membranes and catalysis, there is a need for immobilizing ionic liquids on solid supports or within a solid matrix. The confinement of ionic liquids within alumina and silica-derived networks is being considering promising in catalysis research3,4. The present work has used liquid and solid state NMR experiments to investigate the influence of distinct ionic liquids immobilized on commercial alumina and the effect on their mobility under confinejnent in such support in order to propose a higher performance catalyst system suitable for the oil refining industry. For solid supported samples 29Si MAS and 13C CP/MAS NMR experiments were performed on a spectrometer (VARIAN INFINITY plus - 9.4 T) at 79.2 MHz and 100.26 MHz of observing frequencies, respectively. The spectrometer was equipped with a 7.5 mm CP/MAS probe and the samples were packed in zirconia rotors and spun at the magic angle at 5 kHz for 29Si and 13C. 29Si MAS spectra were acquired using a spectral width of 100 kHz, single pulse duration of 5.5 (is (tt/2), a recycle delay of 20 s, acquisition time of 10.2ms and 500 scans. The spectra were referenced to kaolin (-91.5ppm) and a line broadening of 200 Hz were used for processing. For the acquisition of 13C CP/MAS spectra, a spectral width of 50 kHz, acquisition time of 20.48ms, a single pulse duration of 4.2|is (n/2), a recycle delay of 1s with 6ms contact time and 50000 scans were accumulated. They were referenced to hexamethylbenzene (methyl at 17.3 ppm) and 300 Hz of line broadening was used for processing the spectra. Liquid state 29Si and 13C NMR spectra were obtained using CDCI3 with TMS as internal reference on a Varian MR400 spectrometer. 45° pulses (7.7ps for 29Si and 5.2ps for 13C) were applied for both nuclei as well as pulse delays of 1s. The four IL used in this study have the same cation: 1-butyl-3-methylimidazolium (IMI) combined to different anionic species (BF4 , NTF2j PF6 j.The fourth IL sample presented a modified silil-alkyl imidazolium cation and CI anion. Table 1 presents the comparison between chemical shifts of carbon atoms in the pure silanized IL sample and of this same IL supported on alumina. One can notice a significant change of õ of the methylene carbon directly attached to the silicon atom (2). Figures 1 and 2 show the 13C NMR spectra of the pure and immobilized silanized IL, respectively. This suggests that a stronger interaction between the IL and alumina may occur close to the Si atom. The same behavior was observed at the 29Si MAS NMR spectra represented by 6 of the silicon resonance of the same samples (Table 2). No significant variations on õ were observed for the other IL studied but the enlargement of the lines of the spectra due to the mobility restrictions of the ionic liquid inside alumina porous.
I- * 40 13di NUCLEAR MAGNETIC RI'SONANCE USER* Mi l I I\G
Table 1 13C MMR chemical shifts of the silanized IL 5 (ppm) Carbon atom at IL õ (ppm) IL supported in alumina silanized structure IL liquid state (solid state) CH3-O-SÍ (1) 50.0 51.3
(CH2 - Si) (2) 5.5 11.4 (CH2-CH2-Si) (3) 23.7 25.8 CH2-N (4) 39.5 36.1 CH3-N (5) 38.0 36.1 ÇH (8) (IMI ring) 136.0 138.2 ÇHs (6 e 7) (IMI ring) 123.7 and 122.3 123.0
Figure 1 C NMR Spectrum of silanized IL
(C6 + C7) (C4 + C5)
(C8) (C1) •r n j ! /I 220 2 00 180 160 140 120 100 80 60 40 20 0 - 20 40 Qiemcal Shift (ppm) Figure 2 13C CP/MAS NMR Spectrum of the silanized IL. Table 2 29Si chemical shifts of the silanized IL õ (ppm) 29Si õ (ppm) 29Si Sample (Pure IL) (IL supported in alumina) (Liquid State) (Solid State) Silanized IL -42,6 - 48,4 and -56,8 This study has shown that NMR is a powerful analytical tool for the development of new catalysts containing ionic liquids immobilized in solid matrices, as well as to understand their interaction with the matrix. References 1 Dupont, J., R.F. de Souza, and P.A.Z. Suarez, Chemical Reviews, 2002, 102, 3667-3692. 2 Hólderich, W.F., C. de Castro, and M.H. Valkenberg. Green Chem., 2002, 4, 88-93. 3 Mehnert, C.P., R.A. Cook, and E. Mozeleski, Chem. Commun., 2002, 24, 3010-3011. 4 Karout, A., Pierre, C.A., Journal of Non-Crystalline Solids, 2007, 353, 2900-2909. 41 b + PO 11 THE INFLUENCE OF SOLVENT ON THE METHYLENE HYDROGENS 1H NMR SIGNALS FOR THE 2-PHENYLPROPYL HALOACETATES R.B. Sousa*, C.F. Tormena Instituto de Química - UNICAMP - CP 6154 CEP 13083-971 - Campinas - SP - Brazil e-mail:bellischemistry(p).Qmail.com Keywords: NMR; chemical equivalence; haloacetate. It is known that in systems of the type X-CH2-Y, where X is an atom or group of atoms and Y is a group which has no symmetry plane, the methylene hydrogens (CH2) are in the most cases chemically nonequivalent'11, which means that the hydrogens present different chemical shifts.121 For the interpretation of a 1H NMR spectrum several factors are considered,131 but among them we will focus the chemical equivalence or nonequivalence of the methylene hydrogens geminal to the halogen in 2-phenylpropyl 471 haloacetates' (Fig. 1), taking into account that in this type of compounds the CH2 group is far away from the asymmetric center. Figure 1. Studied compounds, where X = F, CI, Br, and, I. The unequivocal structural assignment was performed by NMRjH and 13C spectra. As can be observed from Fig. 2 for F, CI and Br derivatives, signals for hydrogen atoms from CH2-X group present a second order AB spin system. In these cases the coupling constants and chemical shifts were obtained after spectral simulation using SpinWorks program'81. The results obtained after spectra simulations are listed in Table 1. F CI CD3CN CDCI3 CD3CN CDCI3 a) b) C) d) 1 \ ^M / N h I . .J V • i " 4.8 4.8 ppm 4.8 ppm 4.10 ppm 440 ppm Br I CD3CN CDCI3 CD3CN CDCI3 aso ppm 3.80 PP"l 3.70 ppm 3.70 &S6 ppm 1 Figure 2. H NMR signals for CH2-X group (X = F, CI, Br and I) in polar and non polar solvents. I- * 42 2 Table 1. Experimental JHH coupling constant (Hz) for the studied compounds. Solvent £ F CI Br 1 Benzene 2.3 -15.0 -14.6 -12.0 * Chloroform 4.8 -15.3 -15.0 -12.4 * Acetone 20.7 -15.2 -15.2 -12.6 * Acetonitrile 37.5 -15.2 -15.2 -12.8 * DMSO 46.7 -15.5 -15.4 -12.8 * "hydrogen atoms from CH2-I group are chemically equivalent. The methylene hydrogen atoms from CH2-X (X = F, CI and Br) group exhibit different chemical shifts, showing second order AB spin system pattern, which may have central peaks partially separated (Fig. 2a and 2c) or coalesced (Fig. 2b, 2d, 2e and 2f). However, for iodine derivatives (Fig. 2g and 2h) in polar and non polar solvents methylene hydrogen atoms exhibit identical chemical shifts as a A2 spin 1 system. The pattern of the H NMR signals for CH2-X group changes considerable with solvent and with halogen bonded to CH2 group. For the iodine derivative, the signal for the methylene hydrogens from CH2-I fragment are chemically equivalent, in all solvent used in this study. It is well known that the methylene hydrogen atoms neighboring asymmetric centers in most cases are chemically nonequivalent.191 However, in the compounds under study, the CH2-X group is far away from the asymmetric center and this should resulting in the equivalence of methylene hydrogen atoms, but this is not the case as described early. The chemically nonequivalence phenomenon observed for CH2-X group, for studied compounds (except for the iodine derivate), possesses strong influence of solvent, which can be due to change in the conformation. These results highlight the importance of understand properly the effects responsible for the observed patterns on the 1H NMR signals to avoid misinterpretation which could lead to equivocal in the structural assignment. REFERENCES: 1. Snyder, E. I.; J. Am. Chem. 1963, 85, 2624. 2. Sanders, J. K. L.; Modern NMR Spectroscopy - A Guide for Chemists. 2nd ed.,1993. 3. Ault, A.; J. Chem. Educ. 1970, 47, 812. 4. Tormena, C. F.; et. a/.; J. Chem. Soc., Perkin Trans. 2. 2001, 815. 5. Ftéhberg, C. E.; Fischer, C. H.; J. Am. Chem. Soc. 1944, 66, 1203. 6. Furniss, B. S., et. ai.; Vogei's Textbook of Practical Organic Chemistry. 5th ed., 1989, 705. 7. Vankar, Y. D.; et. air, Tetrahedron Lett. 1984, 25, 233. 8. Marat, K.; SpinWorks 3.1.7, Copyright 2010, University of Manitoba. 9. Tormena, C. F.; et air, Magn. Reson. Chem. 2002, 40, 279. FAPESP, CNPq, CAPES and Bruker do Brasil 43 h PO 13 NMR ASSIGNMENTS OF NEOMYCIN B AND PAROMOMYCIN Julyana Rosa Machado*1, Elizabethe Gomes Sanches2, Roselêne Ribeiro Riente2, Jochen Junker1 11NCT-IDN e CDTS/FioCruz, Rio de Janeiro, Brazil 2FarManguinhos, Rio de Janeiro, Brazil * [email protected] Keywords: Aminoglicoside; Paromomycin; NMR assignment Aminoglycosides are important antibiotics used in the treatment of many serious bacterial infections. Paromomycin (PA) or Amynosidin is an aminoglycoside which has not only antibacterial activity but also anti-protozoan. It's a powerful oral agent for the treatment of infections, for example, Leishmaniosis and Tuberculosis. Most aminoglycosides are produced by microorganisms, while another part is semi- synthetic. PA is produced by Streptomyces riomosus var. Paramomycinus and inhibits both gram-positive and gram-negative bacteria. However, it is potentially toxic in the presence of other aminoglycosides. As all family members of Neomycin, PA is chemically composed of a cyclohexane, which is linked to a hexose and a pentose by the anomeric hydroxyl, and in turn, the pentose is linked to the anomeric hydroxyl of the hexose, as depicted in figure 1. PA does not have a defined NMR assignment or x-ray structure. But, REID & GAJJAR (1987) have published an assignment for Neomycin B, a molecule very similar to PA. R HC Figure 1 - Constitution of Paromomycin (a) and Neomycin B (b). Due to the pharmacological potential, this study aims to use 1H and 13C 1-D and 2-D techniques of Nuclear Magnetic Resonance spectroscopy (NMR) for the complete assignment of Paromomycin, in an attempt to perform a 3D structure elucidation in the end. Our assignment of PA will be assisted by a revisited assignment of Neomycin B published in the literature in 1987. We have decided to revisit that assignment because of bogus proton assignments in the original publication. The proton spectra show intense overlap, especially in the region of NH2 and OH. 44 In contrast, all carbons can be distinguished nicely in the carbon spectra; hence we believe we can obtain the complete assignment. PA shows some difficulties in the assignment, specially the very similar carbon chemical shifts in the hexoses, that cause superposition. The HSQC spectra show the complete assignments, obtained with the help of the superposition of PA and Neomycin B. Precise proton chemical shifts were obtained using the RESET-HSQC (SAKHAII et al., 2009), which results in narrow lines and an almost like proton decoupled 1D projection. •4» C2/H2 C3/N3 cr/Krê '«teei/Hi ©aw , C6"/H6" C6VH6 e armr „ .C4-/H4- C4/H4. • '® • C17H1' C17HJ* O » i < > i nm Figure 2 - Overlay of the HSQC NMR spectra of Neomycin B (red) and PA (blue/cyan) with respective assignments (black = no difference between the two). REFERENCES 1. Davidson, R.N.; Den Boer, M.; Ritmeijer, K.; Transactions of the Royal Society of Tropical Medicine and Hygiene, 2009, 103, 653—660. 2. Kudyba, I.; Fernandez, D.P.; Bõttger, E.C.; Vasella, A.; Carbohydrate Research, 2007, 342, 499-519. 3. Reid, G.D.; Gajjar, K.; The Journal Biological Chemistry, 1987. 262, 7967-7972. 4. Sundar, S.; Chafravarty, J.; Expert Opinion Investigation Drugs, 2008, 17(5), 787- 794. 5. Sakhaii, P.; Haase, B.; Bermel, W.; Journal of Magnetic Resonance, 2009, 199, 192- 198 INCT-IDN, CDTS/FioCruz, FarManguinhos/FioCruz 45 h PO 10 NMR ANALYSIS OF JATROPHA CURCAS EXTRACTS Quézia da Silva Sant'Anna, Raquel Pantoja Rodrigues, Nicolas Carel, Jochen Junker CDTS/FioCruz, Rio de Janeiro, Brazil *queziasantanna@yahoo. com.br Keywords: Jatropha curcas; forbol; NMR analysis Jatropha curcas, popularly known as jatropha, is considered one of the most suitable plants to be used in the production of biodiesel. Additionally, there are several reports in the literature regarding jatropha biological activity, especially the tumor promoting effect of phorbol esters has been observed. But, anti-malarial activity of the extract has been reported as well, although not very well explained. Our aim is to find the different compounds contained in jatropha that are responsible for the different activities. Complications start with the literature searches. Whilst the literature agrees that phorbol esters are found in jathropa, there is disagreement about whether to find them in the extracts or in "the cake. Furthermore, the anti malarial activity is reported several times, but without detailed description of the extract methodology or active compounds. Hence we started our work with our own extracts. Extracts from Jatropha curcas seeds (Son et al, 2010) were prepared using different solvents and analyzed using NMR and chromatography coupled to mass spectrometry methods (GC-MS). The 1H-NMR spectrum is very clean, showing only a small number of well-defined peaks. The 13C-NMR spectrum shows only a few peaks, as expected for fatty acids with long CH2 chains (figure 3). «soce eoox 35»5 1,2,4-Trimethytbertz«ne 1,2,3-Trimethyiberizenô JU- i ÍOOG 1S90 2600 26.00 3&SQ «ao som s&te esse «.» Figure 2 - GC-MS spectra of the hexane extract and major components. * 46 According to the GC-MS spectra (figure 2), the major compounds found in the extracts are: Oleic and Linoleic acids (both fatty acids), 1,2,3/4-Trimethylbenzene and Squalene. None of them are reported as having biological activity. Hence, our first extracts did not reveal any compounds that could be responsible for the biological activity reported. tW 1SÇ 5© * Figure 3 - Proton (left) and Carbon (right) NMR spectra of the hexane extract. The Proton spectra shows some aromatics (from Trimethylbenzene) and aliphatics from the fatty acids. REFERENCES 1. Adam,S.E.I.; Magzoub, M.; 1975. 2. Ankrah et al; Phytother. Res. 2003, 17, 697-701. 3. Berchmans; Hirata; Bioresource Technology 2008, 99, 1716-1721; 4. Devappa et al; Journal of the American Oil Chemical Society 2010, 87, 697-704; 5. Gbeassor et al; Journal of Ethnopharmacology 1989, 25, 115- 118; CDTS/FioCruz, INCT-IDN, Embrapa 47 h Al'RI:MN MAY 2nd lo 6th, 2011 HOTl'.l. FX) FRADE. ANGRA DOS REIS, RJ. BRAZIL PO 15 NMR IDENTIFICATION OF A CHALCONE AND A STILBENE FROM ROOTS OF DEGUELIA DUCKEANA A.M.G. AZEVEDO (FABACEAE) N. M. Lima, A. C. Oliveira, C.V. Nunez Laboratório de Bioprospecção, Instituto Nacional de Pesquisas da Amazônia - INPA. e-mail: [email protected] Keywords: stilbene; chalcone; Fabaceae Deguelia duckeana (Fabaceae)1 was collected in Manaus (AM) and identified in the Herbarium of the INPA. The roots dichloromethane extract phytochemical analysis resulted in the isolation of two substances which were identified as the stilbene 3,5,4'- trimethoxy-4-prenylstilbene (1) and the chalcone 4-hydroxylonchocarpin (2). The molecular structures of the substances were determined by NMR spectral data analysis (1H, 13C, DEPT 135°) and 2D NMR techniques (COSY, HSQC and HMBC) and compared with the literature available.2,3 The spectral data were obtained in Varian lnova-500 instrument and were recorded at 125 MHz for 13C and 500 MHz for 1H. The experiments were realized at 28 °C, using chloroform-deuterated (CDCI3) as solvent and internal standard. The 1H RMN spectral data obtainment was using the pulse s2pul sequence, pulse 45.0 degrees, relaxation delay of 1000 seconds, acquisition time of 2.049 seconds, line broadening of 0.2 Hz and 16 repetitions were made. The 13C RMN spectral data obtainment was utilized the pulse s2pul sequence, pulse 45.0 degrees, relaxation delay of 1000 seconds, acquisition time of 1300 seconds, line broadening of 0.5 Hz and 1088 repetitions were made. The DEPT spectral data obtainment was similar to the 13C NMR, with the difference in pulse 90.0 degrees, acquisition time was 1000 seconds, line broadening of 1.0 Hz and 256 repetitions. Figure 1: 3, 5, 4'- trimethoxy-4-prenylstilbene (1) 4-hidroxylonchocarpin (2) In the 1H NMR spectrum of substance 1 there were observed the characteristic signs of trans-ethylenic chain of a trans-stilbene [õH 7.08 (1H; d; J = 16.0 Hz; H-8); 7.00 (1H; d, J = 16.0 Hz; H-7)]; of aromatic ring p-substituted [õH 7.51 (2H; dt, J = 8.5 and 2.5 Hz; H-2' e H-6') e 6.96 (2H; dt, J = 8.5 e 2.5 Hz; H-3' e H-5')] of 3,3-dimethylallyl group [ÕH 3.37 (2H; d; J = 7.5 Hz; H-7'); 5.25 (1H; m); 1.80 (3H, s, CH3-10') and 1.65 (3H, s, CH3-H')]. We observed the presence of a singlet in õH 6.75 (2H), which was attributed to two equivalent aromatic hydrogens H-2 and H-6, as well as two singlets, one at õH 3.88 (6H) and other at 3.74 (3H), assigned respectively to two methoxyl in A ring and one in B ring (Table 1). The NMR spectrum of 13C is compatible with the proposed structure as it shows the presence of 5CH3, 1CH2, 9CH and 7C, in addition to typical signs of 3,3-dimethylallyl group [õc 22.4 (CH2, C-7 '), 123.0 (CH, C-8 '), 25.9 (CH3, C-11'), 17.8 (CH3, C-10')], the aromatic p-substituted [õc 130.2 (C, C-1'), 127.7 (2CH, C-2' and C-6') 114.2 (2CH, C-3 and C-5') and 159.3 (C, C-4')]; carbons C-2 and C-6 equivalent [õc 102.2 (2CH)], C-7 and C-8 [õc 127.2 (CH) and 127.6 (CH)], the two aromatic methoxyl equivalent õc 55.8 and a third methoxyl group at õc 55. f + 48 1 Table 1: H NMR data of substance 1 (CDCI3, 500 MHz) H 5 (ppm) MULTIPLICITY and J (Hz) 2' e 6' 7.51 2H, dt, J = 8.5 e 2.5 8 7.08 1H, d, J = 16.0 7 7.00 1H, d, J = 16.0 3' e 5' 6.96 2H, dt, J = 8.5 e 2.5 2 e 6 6.75 2H, s 8' 5.25 1H, m 4'- OCH3 3.74 3H, s 3,5- OCH3 3.88 6H, s 7' 3.37 2H, d, J =7.5 10' 1.80 3H, s 11' 1.69 3H, s The substance 2 was recognized as a chalcone via proton trans-olefins õH 7.85 and 7.54 (1H, d, J = 15.5 Hz) in positions a and p. The duplets õH 5.57 e 6.74 (J = 10 Hz) and the singlets at 1.45 (6H), characterized the ring gem-dimethylpyrane. The chemical shift of the carbonyl at õc 192.4 and the high signal deprotection of õH 13.75 was observed as strong evidence of /3-hydroxy carbonyl coupled with consistent with the chain 2'-hydroxychalcone. The signs at õH 6.38 and 7.72 (d, J = 8.5 Hz) integrating for 1H each, were assigned to H-5 and H-6', respectively. The duplets in ÕH 6.89 and 7.56 (J = 8.5 Hz) for the four aromatic protons confirmed the presence of B ring p- substituted. The existence of eight methinic carbons and two carbons sp2 nature characteristic of gem-dimethylpyrane ring observed in the spectrum of DEPT supports this proposition (Table 2). 1 13 Table 2: H (CDCI3, 500 MHz) and C (CDCI3, 125 MHz) NMR data of substance 2 H õ MULTIPLICITY and J (Hz) C 5 2 e 6 7.56 2H, d, J = 8.5 1 128.4 (C) 3 e 5 6.89 2H, d, J = 8.5 2 e 6 130.9 (CH) a 7.45 1H, d, J = 15.5 3 e 5 116.3 (CH) P 7.84 1H, d, J= 15.5 4 158.3 (C) 5' 6.38 1H, d, J = 8.5 a 118.3 (CH) 6' 7.72 1H, of, J = 8.5 P 144.4 (CH) 3" 5.60 1H, d, J = 10.0 C=0 192.4 4" 6.76 1H, d, J= 10.0 1' 114.4 (C) OH (C-2') 13.75 1H, s 2' 161.2 (C) OH (C-4') 5.36 1H, si 3' 109.8 (C) 2CH3 (C-2") 1.46 6H, s 4' 160.0 (C) 5' 108.5 (CH) 6' 130.9 (CH) 2" 78.1 (C) 3" 128.1 (CH) 4" 116.2 (CH) 2CH3 28.7 (CH3) REFERENCES: 1. Missouri Botanical Garden (MOBOT), 2011 (accessed on February 25, 2011). 2. Ngadjui, B. T.; et al.. Phytochemistry, 1998, 48, 2, 349-354. 3. Braz Filho, R.; et al.. Phytochemistry, 1975, 14, 1454-1456. CT-AGRO/CNPq, PPBio/CNPq/MCT, CENBAM/CAPES/CNPq, FAPEAM 49 f- IMS— PO 16 WITH NMR TOWARDS NEW DIAGNOSTIC METHODS FOR DENGUE Camilla do Nascimento Bernardo*1, Marcela Cristina Oliveira Nogueira1, Érika Pereira de Aquino1, Sina Schmidtke1, Elizandes L Azeredo2, Claire F Kubelka2, Jochen Junker1 1INCT-IDN & CDTS/FioCruz, Rio de Janeiro, Brazil 2IOC/FioCruz, Rio de Janeiro, Brazil *camilla_nb@yahoo. com. br Keywords: Dengue; blood plasma; PCA Dengue is a viral disease that is quite common in tropical countries. The diagnosis ;of Dengue is done by eliminating other possible causes of the observed symptoms, a direct antibody diagnosis becomes possible only after several days into the disease. Hence, for the first days, a patient is either not treated (not strong enough symptoms) or treated "on suspicion". A method for early identification of Dengue is desired. The blood is the main means of transport inside the human body, not only for proteins (like antibodies), but also for small molecules. Due to changes in their metabolism, infected cell could be producing different secondary metabolites, which will show up in the blood plasma. This changes should appear quickly, faster then antibodies. Blood plasma has been extensively studied by NMR [1, 2] and many small molecules can be associated to certain NMR signals. In our study we have analyzed blood plasma from health and infected subjects using proton NMR. The samples were all filtered with 3kDa filters* freeze-dried and re- suspended in D20. The resulting 1D NOE spectra [3] were then subjected to an extensive Principal Components Analysis (PCA) [4,5,6], in order to obtain a differentiation. Signal ranges that showed significant changes within the same group were excluded for the PCA, as well as the glucose signals were excluded. Figure 1 - Application of the PCA model (shown in purple) for the differentiation between control (blue) and infected (green). We observe one false negative (light green), but that one is also identified as "outside" of our model in the model statistics. Upper row from left to right: model statistics, model distribution, variation explained. Lower row from left to right: 4 Plots of different PC combinations, 4 plots of chemical shift contributions. Figure 1 shows the result of our PCA model applied to the classification of additional samples. With the exception of one sample, all of them were correctly classified; the wrong one was actually marked as being "out of the model" meaning that a proper classification was impossible. It turns out that PC2 is the most important component for the differentiation between healthy and infected subjects, whereas PC1, PC3 and PC4 do not contribute 4 50 to the differentiation. From the PCA model we can identify the most important regions responsible for that result, mainly 1.92 ppm and 3.38 ppm. The later is shown in figure 2, together with a region that does not differ. According to assignments published in the literature for blood plasma we can associate the varying signals to a series of compounds: proline, lysine, arginine, acetate, treonine, p-glucose, pyruvate, citroline, glutamine, isoleucine and other smaller ones. Currently we are trying to establish a connection between the observed signal changes and known body reactions. Figure 2 - Superposition of the infected (green and purple) and control (red and blue) 1H NMR spectra. Whilst the superimposed spectra on the left show no difference between the samples, a clear difference is visible in the superposition on the right (red star). The first change that we can assign is the change in the arginine signal. Arginine is associated with the normal immune response of the body[7]. We hope to be able to identify further signals and to refine the list of compounds. REFERENCES 1 AlaKorpela, M; et al.; Prog Nucl Mag Res Sp. 1995, 27, 475. 2. Zhang, GQ; Hirasaki, GJ; J. Magn. Reson. 2003, 163, 81. 3. Lucas et al.; J. Pharmaceut. Biomed. 2005, 39, 156. 4. Ramadan et al.; Talanta 2006, 68, 1683. 5. Le Moyec et al.; NMR Biomed. 2005, 18, 421. 6. Lenz et al.; J. Pharmaceut. Biomed. 2003, 33, 1103. 7. Bronte, V and Zanovello, P.; Nat. Rev. Immunology 2005, 5, 641. INCT-IDN, CDTS/FioCruz, IOC/FioCruz, FAPERJ si y - MAY 2nd l»6(h.20Il [X)l-K \l)l . W PO 17 DISCRIMINATION OF BIODIESEL BLENDS WITH 1H NMR AND CHEMOMETRICS I.S. Flores*1, L.M. Lião1, G.B. Alcantara1, S.M. Cabral2, M.R. Monteiro3 1 Universidade Federal de Goiás, Instituto de Química, Goiânia-GO, Brazil 2Petrobras/Cenpes/QM, Ilha do Fundão, Rio de Janeiro/RJ, Brazil 3Universidade Federal de São Carlos, CCDM, São Carlos-SP, Brazil e-mail: iqor. sa violi&.pmail. com Keywords: Biodiesel; 1H NMR; PCA Biodiesel is the major substitute for fossil diesel as its physical chemistry properties are very similar to those of diesel. This biofuel can be used either pure or blended into fossil diesel1. A large variety of vegetable oils such as soybean, babassu coconut, corn, and castor oil could be used for the production of biodiesel, and depending on the vegetable oil and alcohol chosen, the ester mixture produced will present very different properties. The length of the fatty acid chain was shown to have a marked influence on the viscosity and crystallization temperature of the systems, whereas branching affected only the crystallization temperature to a significant extent. High viscosity at lower temperatures could be a result of micro-crystal formation and would cause serious problems in fuel lines and in engine filters. In this work we propose, for the first time, the use of 1H NMR and principal components analysis (PCA) to determine blend levels of methyl biodiesel from different feedstocks. To this end, eight biodiesel samples from babassu, castor, cotton, peanut, pinion, soybeans, sunflower, and tallow were chosen. The samples were analyzed either pure or combined into binary blends of 20/80, 40/60, 60/40, and 80/20% (v/v). The NMR-PCA methodology has successfully discriminated castor, cotton soybean, and tallow biodiesel blends (Figure 1). On the other hand, pinion and peanut biodiesels in B100 form were only categorized after the identification of all the other biodiesels. O to ' ' O O O O 5 o o° CO o o o . o <$> oo „ o 0 0 CD V400 o -0 05 • . V'". VV4 • p s* • o tfa a 0 C • 0 I • Castor BwdKset Blends • Soybean Biodiesel Blends {O Otttev Blends O Otter Blends IA' . . ° . (B) _ é i -0.3 -0.2 -0.1 Q D D o o0 • ' • • • »V3 o O • o o a = J" • O °0 00 • ° oO o • • O % O 8> 0 9 c o . o o D Tallow Biodiesel Blends O Other Blends Figure 1. PCA score plots obtained from 1H NMR data of pure and blended biodiesel samples, according to the regions A: 5 2.1-2.3, B: S 0.9-1.1 and 2.8-2.9, C: 5 1.2-1.4 |> + 52 1 ">!i' Mill \K \1\fi\l- lie KJ-SiiS Wl. I ShR and D: S 1.4-1.6. Samples V1 (castor/tallow-20/80), V2 (pinion/cotton-20/80), V3 (sunflower/soybean-50/50) and V4 (cotton/soybean-80/20) were used for validation. A complementary method for PCA is the integration of specific regions of the 1H NMR spectrum (Table 1) which can be used in the identification of biodiesel from babassu, sunflower, pinion and peanut in blends. Table 1. 1H NMR signal integration of the selected regions for biodiesel discrimination. Biodiesel lt(„c=0)+ DP "(CHS) + DP l>>(A»yiic) ±DP % total unsaturated ">(Bis- Allylic) ± DP % esters B100 62.27-2.317 50.86 -0.91 51.976-2.08 esters 5 2.74 - 2.8 polyunsaturated Cotton 0,68±0,00 1,03+0,00 0,97±0,00 70,64 0,36±0,00 52,64 Peanut 0,68±0,01 1,02±0,01 1,10±0,01 81,07 0,27±0,00 39,56 Babassu 0,68±0,01 1,02±0,01 0,02±0,00 16,07 0,36±0,00 3,10 Sunflower 0,68±0,01 1,02±0,01 1,23+0,01 90,17 0,34±0,00 49,65 Castor 0,69±0,01 1,04 ±0,02 0,83 ±0,01 59,54 0,03±0,00 4,59 Pinion 0,68±0,01 1,02±0,01 1,07±0,01 78,19 0,23±0,00 34,21 Tallow 0,68±0,00 1,02±0,01 0,53±0,00 38,84 0,02+0,00 2,83 Soybean 0,69±0,01 0,97±0,02 1,14+0,02 85,43 0,38±0,01 56,43 It - value of integration refers to all chemical species lb - value of integration was restricted to esters of biodiesel DP - standard deviation of triplicates Through this strategy biodiesel from babassu and sunflower could be identified due to extreme values of the integration, 16.07 and 90.17%, respectively, according the total of unsaturated esters characteristic of each biodiesel. Through the levels of esters is also possible to identify peanut biodiesel and jatropha. This is possible crossing information obtained by PCA and relative integration. Another possibility generated by integration of these signals is the distinction between B100 and blends of castor, tallow, soybean, and cotton biodiesel (Figure 2). BIODIESEL UNKNOWN I INTEGRATION SIGNALS BABASSU OR PRINCIPAL COMPONENTS SUNFLOWER BIODIESEL ANALYSIS - PCA INTEGRATION COTTON, SOYBEAN, TALLOW SIGNALS OR CASTOR BIODIESEL PINION OR PEANUT BLENDS INTEGRATION SIGNALS BIODIESEL J= BIODIESEL (B100) BLENDS Figure 2. Methodology for feedstock identification used on biodiesel of castor, tallow, soybean, and cotton production. Therefore, the principal components analysis and integration of the characteristic signal regions in 1H NMR spectra proved to be a powerful tool for determining the biodiesel present in binary blends. Consequently, it was very useful in identifying biodiesel according to their sources. Through this strategy was possible to discriminate biodiesel from cotton, soybean, and tallow, which represents 98% of the feedstocks used in Brazilian biodiesel production, and others with great potentiality of use. REFERENCES: 1. Knothe G.; Gerpen J. V. and Krahl J. The Biodiesel Handbook, 5a ed., 2005. MCT/FINEP/CT-INFRA, FUNAPE/UFG, CNPQ, CAPES 53 I- + Al'Rl-MN MAY 2nd to 6(h, 2011 HOTEL DO FRADli. ANGRA DOS RF.1S. RJ. BRAZIL PO 18 INFLUENCE OF THE SOME 1H NMR PARAMETERS ON THE ACCURACY OF MULTIVARIATED CALIBRATION MODELS - PLS I.S. Flores*1, L.M. Lião1, G.B. Alcantara1, M.R. Monteiro2 1 Universidade Federal de Goiás, Instituto de Química, Goiânia-GO, Brazil 2Universidade Federal de São Carlos, CCDM, São Carlos-SP, Brazil e-mail: igor. sa violi&.gmail. com Keywords: Biodiesel; RMN-PLS; Factorial design The use of NMR as a quantitative tool (qNMR) has many advantages like the ability to detect small structural differences and the need for little or no pretreatment of the sample. In addition, provides an overview of all types of compounds contained in the sample. However, there is a need for determining a large number of parameters relate to the spectral characteristics. The acquisition parameters and processing of NMR data are responsible, among other characteristics, for their signal to noise ratio and digital resolution. In addition, there are mathematical manipulations that allow you to change the FID and obtain a NMR spectrum with better signal resolution or higher sensitivity. In this context, this work proposes the evaluation of the influence of some parameters on the accuracy of multivariate calibration models - Partial Least Square (PLS) through factorial design. For this purpose, PLS models were built from the ternary blends of biodiesel from babassu, castor and sunflower for determination of their contents according the feedstock. The factorial design is performed by selecting a fixed number of levels (values) for each of the variables (factors). Then tests that consider all the possible combinations are performed. Significant information about effects can be obtained with a smaller number of experiments, producing the same conclusions that a greater number of tests. Thus, is possible to quantify the effect of factors in evaluating a particular response. The factorial design is a method that assesses the influences of variables on the response simultaneously. Therefore, is possible to verify the effects of interactions between variables. In this way was performed a 25 factorial design, analyzing the interaction and/or effects of the individual parameters Time Domain (TD), Data Size (SI), and Line- Broadening (LB), besides the selection of variables and Savitzky-Golay smoothing. All spectra were acquired with 20 scans at 298 K and receiver gain equal to 36 using a Bruker Biospin Avance III 500 spectrometer. The results presented in Table 1 shows that the use of all spectral information and the Savitzky-Golay smoothing produced the lowest predicting errors, especially the 2nd test in condition 3. This demonstrates that the processing of spectral data with this type of smoothing is well appropriated to minimize the effect of noise. Furthermore, the use of all spectral information takes significant advantage over the selection of variables. This allows for better correlation of data with their concentration when there is great spectra similarity. The mathematical resource LB has great influence on the accuracy of the models when an inadequate treatment of the spectral noise is observed. In general, increasing the LB value from 0.0 to 0.5 causes a decreasing on RMSEP value, which makes it more appropriate. The use of "zero filing" with TD and SI 64k, on the 7th test in condition 2, generated the worse predictive model. This demonstrates that this resource may not be suitable for some specific conditions. The selected region for investigation of the variable selection effects corresponds to the region of olefinic hydrogens. This spectral range was chosen because it has remarkable spectral differences for biodiesel evaluated. f 4 54 Table 1. Influence of some parameters on the accuracy of PLS models Condition Tests LB TD SI Variable Savitzky- RMSEP Selection Golay 1(6°) 0.0 32k 32k n n 2.67 2 (3°) 0.5 32k 32k n n 1.44 3(4°) 0.0 64k 32k n n 2.98 1 4(8°) 0.5 64k 32k n n 1.73 5(7°) 0.0 32k 64k n n 2.04 6(1°) 0.5 32k 64k n n 1.13 7(2°) 0.0 64k 64k n n 2.33 8(5°) 0.5 64k 64k n n 1.64 1(3°) 0.0 32k 32k y n 2.96 2(7°) 0.5 32k 32k y n 2.88 3(5°) 0.0 64k 32k y n 2.82 2 4(4°) 0.5 64k 32k y n 2.99 5(1°) 0.0 32k 64k y n 2.04 6(8°) 0.5 32k 64k y n 2.91 7(2°) 0.0 64k 64k y n 4.07 8(6°) 0.5 64k 64k y n 2.32 1(3°) 0.0 32k 32k n y 0.92 2(8°) 0.5 32k 32k n y 0.64 3(1°) 0.0 64k 32k n y 0.82 3 4(6°) 0.5 64k 32k n y 0.65 5(7°) 0.0 32k 64k n y 0.81 6(5°) 0.5 32k 64k n y 0.65 7(2°) 0.0 64k 64k n y 0.72 8(4°) 0.5 64k 64k n y 0.67 1(4°) 0.0 32k 32k y y 2.73 2(5°) 0.5 32k 32k y y 1.78 3(2°) 0.0 64k 32k y y 2.56 4 4(8°) 0.5 64k 32k y y 1.97 5(7°) 0.0 32k 64k y y 2.13 6(1°) 0.5 32k 64k y y 1.42 7(6°) 0.0 64k 64k y y 1.39 8(3°) 0.5 64k 64k v y 2.20 The table 2 shows that the third condition is less impacted by NMR parameters when compared to the others. Both main effects and interaction effects are not as significant when using Savitzky-Golay smoothing and full spectral information. Table 2. Main effects and interaction calculated Conditions Main Effects 1 2 3 4 1) LB -1.0 -0.2 -0.2 -0.4 2) TD 0.4 0.4 -0.0 0.0 3) SI -0.4 -0.1 -0.0 -0.5 2nd order interactions 1 x 2 0.0 -0.6 0.0 0.5 1 x 3 1.0 0.2 0.2 0.4 2x3 0.0 -0.8 0.0 0.0 3rd order interactions 1x2x3 0.1 -0.7 0.0 0.3 The obtained results show that the evaluated parameters have great influence on the prediction error values (RMSEP) in the built models. Therefore, the preliminary study of these parameters to optimize the conditions for qNMR analysis as pre- processing of data used for quantification via multivariate calibration (PLS) are need. MCT/FINEP/CT-INFRA, FUNAPE/UFG, CNPQ, CAPES 55 y PO 18 ASSESSMENT OF S180 METABOLIC PROFILING AFTER ANTITUMOR DRUGS USING HIGH-RESOLUTION MAGIC-ANGLE-SPINNING NMR SPECTROSCOPY B.C.B. Martinelli*1, G.B. Alcantara1, L.M. Lião1, A.L. Oliveira1, E.P. Silveira-Lacerda2, F.C. Pereira2. 1 NMR Laboratory, Institute of Chemistry, Federal University of Goias, Brazil 2 Laboratory of Molecular Genetic and Cytogenetic, Institute of Biological Sciences, Federal University of Goias, Brazil e-mail: martinellibcb&.qmail. com Keywords: HR-MAS NMR; Metabolism; Sarcoma 180 (S180) The biochemical reactions, like synthesis of cell membrane constituents and energy turnover, may also be altered in cancerous cells. Magnetic Resonance Spectroscopy (MRS) of biological tissues, in vivo and in vitro, reflects tissue metabolites, and thus aberrant biochemical reactions can be observed by MRS1. In this context, this research aims to evaluate the metabolic profile of S180 (Sarcoma 180) mouse tumor cells' line, well as their metabolic profile after the treatments with six new antitumor drugs being developed, which will be called Au 08, Au 10, Au 14, Au 16, Au 18 and Au 21. For this, fresh S-180 tumor cells maintained in mice were used. The 1H NMR measurements were performed on Bruker Avance III spectrometer (operanting at 500,13 Hz to 1H), that was equipped with HR-MAS probe for 4mm. The samples were placed in a zirconium rotor of 50 |uL followed by adding a D20/2,2,3,3-d4-3- trimethylsilyl sodium propionate solution. The metabolism of S180 cells before and after drug treatments was assessed using CPMGPR pulse sequence, at a temperature of 23°C, with 512 scans, 4.0 s of relaxation delay, 64 k data points, 3.27 s of acquisition time and T=1 .0 ms for 128 loop cycles. The data set from S180 cells before and after drug treatment was obtained in triplicates. The metabolism of S-180 cells before drug treatments was monitored about approximately 22 hours, for comparing the normal behavior Thus, it was possible to monitor the some metabolic changes, as we can see to peak area in table 1 and figure 1. Table 1. Metabolic profiling os S180 before drug treatments, with their chemical shifts and areas. INTEGRATION TMSP 1.0000 1.0000 1.0000 10000 1.0000 1.0000 1.0000 ETHANOl 1.4452 1.3425 1.2797 1.2072 1.1545 1.0751 1.0320 1.0125 1.0141 ALANINE 3.2744 3.2249 3.1249 3 1026 7 0.370 2.8613 2.7750 2.7440 2.7347 2.7431 2.7122 ACETATE 0.378? 0.4029 0.4026 0.4059 0.4019 0.3991 0.40? 7 0.4036 0.4083 CRf ATINE 3.9402 3 9270 3.7208 3. .5458 3.4704 3.3359 3 2783 3 2709 3.2653 CHOLINE 2.4239 2.7395 3.3442 4.0517 4.2459 4 552? 4.6523 PHOSPHOCHOLINE S 0079 4.8341 4.6099 10.6642" 3.8183 .3.6512 3.4820 3.4509 GLYCFROPHOSPHOCOIJNF 8 3066 7.6745 6.64S5 5.0026 4.3083 3.8614 3.7474 TAURINE 3 5853 3 4725 3.3180 3.2291 2.9938 2.8320 2 777S 2.7730 CREATINE 3.4710 3.3624 3.1835 3.0759 3.0139 2.7704 2 7514 2.S753 2.4998 2 4784 2.4634 2 4128 0.0201 URACII 0.0217 0.0303 0.0325 0.0310 0.0365 0.0.303 0.0392 0.0403 0.0403 0.0417 0 0437 0 0457 0.04.30 0.0442 0.0489 0.0474 Tl ROSIN E 0.0502 0.0474 0.0475 0.0471 0 0500 0.0491 0.0486 0.0519 0.0482 0.0536 0.0484 0.0475 0 0549 0.0524 TIROSINE 0 0532 0.0531 0.0518 0.0534 0 0541 0.054S 0.0548 0.0539 0.0537 0.0291 0.0304 0.04665 0.0439 URACII 0 0365 0 0399 0 0454 0.0472 0.0462 0.0480 0.0498 0 0510 0.0395 0.0701 0.0704 0.0768 HISTIDINF 0.0502 0.0670 0.0697 0.0739 0.0732 0.0759 0.0777 0.0466 0.0461 0.0511 INOSINT 0.0447 0.04665 0.0513 0.0509 0.0514 0.0495 0.0310 0.0319 INOSINE 0.0312 0.0303 0.0319 0 0.317 0.0336 ULWL 1 T T" ' 1 ' ' Figure 1. Metabolic variation of S180 mouse tumor cells' line before drug treatment. f 4 56 The signals metabolites assignments of 1H HR-MAS NMR spectra were performed with the aid of standards and by TOCSY technique which has confirmed the presence of the metabolic uracil, tyrosine and taurine correlation. All assignments were compared with already published data2. Figure 2 shows some metabolite variations in the S180 after drug treatments. The most significant changes were observed in the following metabolites: uracil in the Au 16, 18 and 21 drugs, phosphocholine in the Au 08, 16 and 18 drugs, glycine in the Au 16, 18 and 21, alanine in the Au 16 and 21 and acetate in Au 16 and 10 drugs. S-180 + AU JMt. S-180 + AU 10 . I.U., . lilt. , I. .1 .^.I.Jx-J S-180 + AU 14 ..I LjUJU 'hi X 7 6 5 4 3 2 ppm Figure 2. 1H HR-MAS NMR spectra after treatment with Au 08, Au 10, Au 14, Au 16, Au 18 and Au 21 drugs. Therefore, HR-MAS NMR technique showed to be a powerful tool to monitoring the cellular metabolism of S180 after and before treatments with antitumor drugs. The comprehension of the cellular metabolism of these antitumor drugs can be useful to future action mechanism against some tumor types. REFERENCES: 1. B.D. Ross, NMR Biomed. 5 (1992) 215. 2. Beathe Sitter, Tone F. Bathen, May-Britt Tessem, Ingrid S. Gribbestad; Prog. NMR Spectr. 54 (2009) 239-254 UFG, CAPES, CNPq, FINEP 57 I- + PO 20 3 THROUGH SPACE CONTRIBUTION FOR THE JFH COUPLINGS IN SOME UNSATURATED COMPOUNDS L.C. Ducati,3 R.H. Contreras,b C.F. Tormena3* a) Chemistry Institute - State University of Campinas, Campinas, Brazil- CP 6154 CEP 13083- 970. e-mail: tormenatçbjqm.unicamp.br b>Department of Physics, FCEyN, University of Buenos Aires and IFIBA-CONICET, Buenos Aires, Argentina. 3 Keywords: Through Space, Theoretical Calculations, JFH coupling constant It has long been known that compounds containing fluorine atoms that are crowded against one another exhibit unusually large FF nuclear spin-spin coupling constants, 1 SSCCs, JFF due to through space (TS) contributions. For this reason, theoretical and experimental studies were applied to elucidate its Spin-Spin Coupling Constant (SSCC) transmission mechanisms, but not much attention was given for through space (TS) contributions to unusual JFX and particularly JFH. 3 We report new insights on the unexpected behaviour of JHF SSCCs observed for simple unsaturated four-, five-, six- and seven-membered rings containing a fluorine atom. All geometries and SSCC were calculated at BHandH/EPR-lll level of theory 3 using the Gaussian 03 package. Experimental JHF coupling constant were taken from literature.2 For compounds 1-4 three different types of coupling pathways are envisaged, namely: a) through the o-framework; b) through the n electronic system; AN and c) through-space, involving F lone pairs as well as the CTC-F D ^C-H orbitais. As shown in Table 1, theoretical (Jcalc) and experimental (Jexp) are in good 3 agreement and also an inverse relation between JHF and through space distance between the two coupled nuclei [r(F H)] was observed. Based on the classic approach of sum of van der Waals radii (19F = 1.47 A and 1H = 1.20 A; Sum = 2.67 A) it would be expected that a TS contribution component is operating in compounds 1 and 2 which possess the F—H distance shorter than sum of van der Waals radii. For compounds 1 and 2 a positive contribution to the FC term is observed while the PSO term has a negative contribution. For compound 3 the experimental value is nearly zero apparently due to the cancellation between FC and PSO contributions. For 3 compound 4 the experimental JHF coupling is negative and FC and PSO terms contribute with the same weight to describe the observed value. 3 Table 1. Experimental and theoretical JHF (HZ) coupling constant at the BHandH/EPR- lll level. 12 3 4 r(F—H) / A 2.429 2.504 2.694 2.978 jExp 21.5 18.4 - -8.8 JFC 26.5 22.2 5.4 -4.9 ajFC-n -5.3 -6.1 -4.4 -4.8 bjFC-TS 31.8 28.3 9.8 -0.1 jSD -0.9 -0.9 -0.5 -0.3 jPSO -3.5 -3.4 -3.2 -4.5 jDSO 0.1 0.0 -0.5 -1.1 cjcalc 22.0 17.9 1.2 -10.8 58 13th'NUCLEAR MA(,M IK KI-.SONAVIi USERS MEETING a)7r contribution obtained from methyl group replacement rule 3. b) JFC-TS contribution is the difference between J|F^C - J^; c)Jcalc = JFC + JSD + Jpso + JDS0 While PSO terms are almost equal along the series (1 to 4), FC terms decrease exponentially (Fig. 1) when [r(F—H)] distance between coupled nuclei increases. 2.4 2.5 r(F—H)/Angstron Figure 1. Correlation between J FC TS contribution and r(F—H) distance. 3 To evaluate the a-contribution involving in the JHF transmission coupling pathway, FCCP-CMO approach, which has recently been developed in our group, was applied. In this analysis Canonical Molecular Orbital (CMO) are expanded in terms of NBO orbital contributions.4 For compounds 1 and 2 no virtual CMO orbital containing the 3 coupling nuclei were found, suggesting that for these compounds the JHF coupling is transmitted TS asjt were a "first order property". For 1 and 2, the TS component is the 3 most important contribution involved in the JHF SSCC transmission. It is well known that the Fermi Contact (FC) term is transmitted like the "Fermi Hole", which spans the whole spatial region covered by each canonical molecular orbital (CMO). This takes place through exchange interactions. For compounds 3 and 4 are observed two virtual CMO orbitais that are participating 3 in the a-framework transmission component of the JHF coupling. For compounds 3 and 3 4 FC term for the JHF SSCC is transmitted through three possible mechanisms, described above, while for compounds 1 and 2, the through space coupling is the most important contribution for the FC term. The most important conclusion from results discussed above is that the FC term of 3 JHF coupling can be transmitted using different contribution such as through the o- framework, through the n electronic system and through-space and balance between them is responsible for the FC term calculated for studied compounds. REFERENCES: 1. Peralta, J. E.; Barone, V.; Contreras, R. H. J. Am. Chem. Soc. 2001, 123, 9162. 2. Berger, S.; Braun, S.; Kalinowski, H. O. NMR Spectroscopy Of The Non-Metallic Elements; John Wiley & Sons: Bonn, 1997. 3. Rae, I; Weigold, J; Contreras, R. H.; Yamamoto, G. Mag, Reson Chem, 1992, 30, 1047. 4. Contreras, R. H.; Gotelli, G.; Ducati, L. C.; Barbosa, T. M.; Tormena, C. F. J. Phys. Chem. A 2010, 114, 1044. FAPESP, CNPq, CAPES and IFIBA-CONICET 59 I- + ALHEMN- MAY 2nd 10 6th, 2(111 110T1-L DO I-RADK. ANGRA DOS RMS. RJ. BRAZIL PO 21 CONFORMATIONAL STUDY OF 17-o-ETHYNYLESTRADIOL USING RDC José Adonias Alves de França*; Fernando Hallwass Departamento de Química Fundamental, UFPE *adonias. if&.gmail. com Keywords: RDC; ethynylestradiol Recently, the application of partially aligning samples in anisotropic solution to determine the configuration and conformation of small organic molecules has been increased in NMR1. Besides isotropic chemical shift, coupling constant and NOE measurements, anisotropic parameters, such as residual dipolar coupling (RDCs), appear to provide additional information to investigate chemical structures. Dipolar coupling between two nuclei (/ and j) contain important structural information since their values depend on the internuclear distance {r^ and on the angle between the internuclear vector and the axis of the external magnetic field. In isotropic solution, dipolar coupling are averaged to zero because of molecular tumbling. However in anisotropic solution it is possible to recover partially the dipolar coupling to obtain new structural information. The RDCs can be easily extracted as the difference between the total observed couplings in aligned and non-aligned conditions. The aim of this work was to investigate the possibility to use RDCs as an additional parameter to determine the conformation of a test molecule: 17-a-ethinylestradiol (Figure 1). This molecule is a hormone and has pharmacological activity as contraceptive2. Figure 1. Structure of 17-a- ethinylestradiol. NMR spectra were acquired in a Varian VNMRS-400 spectrometer, at 300K, operating at 399,7 MHz for 1H, 100,5 MHz for 13C. 2D experiments (HSQC 1H-13C and HMBC 1H-13C) were carried out in order to assign the signals correctly and to measure the RDCs. As alignment medium was employed the DMSO compatible (S)-2- acrylamido-1-propanesulfonic acid gel (APS)3. 100 mg of 17-a-ethinylestradiol were dissolved in DMSO and poured in a NMR tube with APS gel. Before the NMR experiments were performed, the gel was swollen during 15 days. The geometries were optimized using D FT at the GIAO//B3LYP/6-311+G(d) level. The quality of the fit was calculated by back computation of RDCs, using Cornilescu's quality factor Q values4. These computations were performed using a modified version of the MSpin program5. Table 1.15 RDCs obtained experimentally for 17-a- ethinylestradiol. RDC (Hz) RDC(Hz) C1-H1 6.4 C11-H11 p 3.6 C2-H2 -2.4 C12-H12„ -0.8 C4-H4 4.0 C12-H12;, 9.6 C7-H7a -3.6 C14-H14 7.6 Í» + 60 C8-H8 6.4 C16-H16* -0.8 C9-H9 7.2 C16-H16/? 5.6 C11-H11 a 8.8 C20-H20 19.2 CH3 -0.8 Table 1 shows the values of 15 RDCs extracted experimentally and their corresponding carbon-hydrogen pair. Beyond the correct isomer, three more isomers were drawn, alternating the stereochemistry of carbons 13 and 17 (Figure 2). The experimental values were compared with back-calculated values for these four isomers. As expected, the smaller Q was observed by isomer 1, which is the correct structure. lá i * j 4 m j jl * 4 9 jr j. j* j fj à w^m j v j j j ^ * Isomer 1: Q = 0.280 Isomer 2: Q = 0.568 ^ # J * 7 J J ^j f j j j •* Isomer 3: Q = 0.454 Isomer 4: Q = 0.458 Figure 2. Four isomers of ethinylestradiol and the Q factor. In conclusion, it was possible to distinguish the correct isomer of ethinylestradiol between four isomers based on the comparison of the Q-factors. REFERENCES 1. Kummerlówer, G.; Luy, B. Trends in Analytical Chemistry, 2009, 28, 483-493. 2. Andrew, M. N.; O'Connor, W. A.; Dunstan, R. H.; MacFarlane, G. R. Ecotoxicology. 2010, 19, 1440-1451. 3. Braghiroli, A.; Mussati, E.; Bella M. Di Tetrahedron:Assymetry , 1996, 7, 831-836. 4. Cornilescu, G.; Marquardt, J. L.; Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1998, 120, 6836- 6837. 5. MSpin. MESTRELAB RESEARCH SL, Santiago de Compostela, SPAIN. http://www.mestrelab.com. PRONEX / FACEPE-CNPq (Brazilian Agencies) and Alexander von Humboldt Foundation 61 I- + AUREMN - MAY 2nd hi 6th, 2011 lioll I [>i)l-R-\l)h AM,HA DfiS Hi 1-i HI. Ull\/ll PO 22 PH-DEPENDENT AXIAL LIGANDS OF COPPER-SUBSTITUTED CYTOCHROME C REVISITED T.Prieto*1,2, J.F.Lima1, I.L. Nantes3, O.R.Nascimento1'3 1 Departamento de Biofísica, IFSC-USP, São Carlos, SP, Brazil 2 Centro Interdisciplinar de Investigação Bioquímica, UMC, Mogi das Cruzes, SP, Brazil 3Centro de Ciências Naturais e Humanas, UFABC, Santo André, SP, Brazil e-mail:prieto(p).ursa.ifsc. usp.br Keywords: copper-substituted cytochrome c; axial ligands; spectroscopy study. Cytochrome c is a hemeprotein located on the external side of the inner mitochondrial membrane (IMM). Associated to the IMM, cytochrome c acts as a mobile electron carrier between Complexes III and IV of the respiratory chain as well as an antioxidant trapping of superoxide ion. These functions are totally dependent of the protein redox center, the heme iron. In specific conditions cytochrome c detaches from the IMM and attains the cytosol where it binds to a protein complex, the apoptosome and triggers the cell apoptosis. For the apoptosis process, the role played by cytochrome c heme iron is not well established. In addition, cytochrome c heme iron, is crucial for a diversity of nanotechnological applications, such as the development of new catalysts and biosensing. Considering the high biological and nanotechnological relevance of cytochrome c, it is important put efforts to better understand and modulate the activity of the protein redox center. In the present study it was revisited the pH- dependent coordination sphere and reactivity of copper-substituted cytochrome c by using the following spectroscopy techniques: UV-Vis, EPR, CD ancJ MCD. As previous described by Flinday et al [1], Cu" substituted cytochrome c presents pH-dependent conformational changes that are evidenced by changes of Soret, p and a bands. At both extreme pH values (3 and 13) the Soret band is blue shifted (from 422 to 400 nm) and the p/a ratio presented a significant decrease changing from 1.26 to 0.57. These differences are consistent with pH-induced axial ligands via protonation/deprotonation of ionizable amino acid side chains. To better characterize Flinday's interpretation, we measured the EPR and UV-Vis experiments at the pH range 3 to 13 (at 1 pH unit intervals) and the EPR were measured at the temperature 10 K instead of 77 K. Figure 1 shows the Cu" cytc experimental and simulated EPR spectra at pH: 3, 7 and 13. The EPR spectral characteristics of Cu"cytc at low pH values correspond to a distorted pentacoordinated symmetry of copper ion suggesting a strong coordination with the cyt c methionine 80 sulfur atom. The increase of pH promotes deprotonation of histidine 18 allowing its imidazol group to occupy the sixth coordination position. The acid transition led the protein to assume an axial configuration. Similarly to the observed at highly acid condition, at high pH values, the EPR spectrum corresponds to a distorted coordinated symmetry, suggesting the occupation of imidazol nitrogen and the disruption of methionine sulfur, producing a less pentacoordinated distortion symmetry, as shown by EPR parameters in Table I. Table I - EPR parameters pH 3.0 pH 7.0 pH 13.0 g values 1.9477 2.0640 2.1710 2.0380 2.0720 2.2130 1.9250 2.0523 2.1917 An (mT) 1.85 1.74 1.66 1.48 1.49 0.33 1.87 1.75 1.64 ACu (mT) - 0.35 0.35 17.23 - Lw (mT) 8.86 3.49 6.63 0.35 0.35 5.20 1.5 0.42 2.63 + 62 HihNU.I i.\K M\t,M IH KI S«)\AS("l I SI R- Mi l I IN«1 Magnetic Field (mT) Figure 1 - Experimental and simulated EPR spectra of Cu"cytc at different pH values: pH 3.0, pH 7.0 and pH 13.0 as described in graph. To corroborate on these interpretation, UV-Vis and MCD measurements were done at the same pH values (see Figure 2). Different to that was observed at pH 7, at high and low extreme pH values, the Soret band was blue shifted and exhibited a shoulder at 391 nm. The MCD spectra at these two extreme pH values exhibited a more intense optical activity on the shoulder than in the Soret band consistent with the partial overlap of the n-n* transitions (Faraday B term) to the n-d one associated to a Faraday A term (charge transfer band). These results suggest a charge transfer band to the fifth ligand at these two pH resulted from the distorted pentacoordinated symmetry produced by the two postulated axial ligands at extreme pH values. Wavelength (nm) Wavelenght (nm) Figure 2 - MCD and UV-Vis spectra of Cu"cytc at different pH values: pH 3.0, pH 7.0 and pH 13.0 as described in graph. The reaction of Cu"cytc with HOOH at neutral pH followed by EPR, showed an EPR signal decreasing during time reaction (20 % signal reduction in first reaction minute), indicating the reaction mechanism of Cu" to Cu'", as occurs to native cytochrome c Compound II. The peroxide cleavage mechanism will be determined by EPR spin trap technique in future. REFERENCES: 1. Mugnol K.C.U., Ando R. Nagayasu R.Y., Faljoni-Alário A., Brochstain S., Santos P.S., Nascimento O.R., Nantes I.L. Biophysical Journal, 2008,4066. 2. Findlay M.C., Dickinson L.C., Chien J.C.W. J. Am. Chem. Soc. 1977, 5168. Supported by FAPESP, CNPq and CAPES. 63 I- + AÜR1-.MN- MAY 2nd 10 6th, 2011 HOTEL DO FRADli, ANGRA DOS Rl.ilS, RJ. BRAZIL PO 21 SYNTHESIS, PURIFICATION, CHARACTERIZATION AND NMR STRUCTURAL ELAVUATION OF DISINTEGRIN-LIKE PEPTIDES D. A. T. Pires*1'2, L. G. M. Arake2, C. J. Nascimento1'3, C. Bloch Jr2 1 Laboratório de RMN, Instituto de Química, Universidade de Brasília, Brasília, Brasil 2Laboratório de Espectrometria de Massa, Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brasil 3Instituto de Biociências - Univ. Federal do Estado do Rio de Janeiro, RJ, Brasil dieqo.pires.88(Q).qnriail.com Keywords: Disintegrin, NMR and Mass Spectrometry. Disintegrins are cystein/rich polypeptides involved in the inhibition of the blood platelets aggregation. They are known to have the tripeptide region RGD which binds to the integrin receptors, modifying its function, and thus competitively inhibits normal integrin-ligand interactions1. When a blood vessel wall is broken, the aggregation of platelets has a tendency to seal the opening and so it facilitates to stop bleeding. However, in some cases, platelets aggregation can block blood flow, which can cause thrombosis2. Integrins can regulate and influence migration as well as the growth of the cells and apoptosis. Cyclic peptides containing the KGD sequence can also bind to the integrin receptors3. For those peptides, it has been shown that cyclization is important to provide conformational restrictions, increasing the peptide affinity and selectivity for the receptor3. From the cDNA data bank containing antimicrobial peptides sequences that were transcripted on Hypsiboas punctatus skin dermal glands on the dorsal region we found a particular one that encodes a peptide possessing a KGD site which composes a 15 amino acid primary structure with two cystein residues on the positions 4 and 14. With the objective to study the folding behavior of this peptide and the potential biological activity (inhibition of the blood platelets aggregation), we designed two new peptides, analogous to the first, replacing the lysine residue for arginine (RGD) and histidine (HGD). These changes were mainly due to the structural similarity of these amino acids to lysine residue. Also, as stated above, cyclic peptides containing the RGD sequence are the ones that bind to integrins1. Peptides were synthesized by solid-phase methodology (Fmoc/t-butil strategy), wherein a polymer was used to anchor the peptide during all stages of the synthesis3. They were purified by Reversed Phase High Performance Liquid Chromatography (RP- HPLC Ci8), using a gradient elution with milliq water (0,1% of TFA) and acetonitrile (0,1% of TFA)5. Chromatographic analysis indicated the presence of oxidized and reduced forms which were characterized using Mass Spectrometry (Maldi-TOF). Both the reduced and the oxidized forms of the peptides were sequenced by MS/MS experiments (MALDI-TOF/TOF) using b and y series. The disulfide bond between cys- 4 and cys-14 occurs naturally at room temperature in the presence of water and air oxygen, without the need of additional oxidative process. This is observed during the purification by the increase of the signal correlated to the oxidized form (disulfide bond) accompanied by the decrease of the signal due to the reduced form (the two cysteins without bonding). Moreover, the 3D structures of both forms (reduced and oxidized) of each peptide were analyzed by Ion Mobility (Synapt HDMS) and we were able to detect the difference between their drifting times. In addition, the drifting time of the isolated oxidized form compels us to state the existence of one stable structure. 13 1 TOCSY, HSQC ( C- H) and NOESY spectra were acquired using H20/ D20 (60%/40%) as solvent. All experiments were performed on a Bruker Avance III 800 MHz spectrometer using 60 ms and 130 ms mixing time for the TOCSY and NOESY, respectively. Y + 64 By the assignment it was possible to observe that the RGD peptide shows significantly difference between carbon chemical shifts when compared to the observed values for the KGD and HGD peptides. A comparison of some carbon chemical shifts for the three peptides is shown in Table 1. Table 1: Comparison of some carbon chemical shifts for the three peptides KGD peptide RGD peptide HGD peptide (Carbon chemical (Carbon chemical (Carbon chemical Shift - ppm) Shift - ppm) Shift - ppm) CQ (thr-2) 62,047 53,846 62,012 Ca (giy-3) 45,558 43,980 45,502 Ca (cys-4) 56,242 50,411 56,255 Ca (tyr-4) 57,128 50,942 57,199 Ca (ser-8) 61,061 49,490 60,731 ca (giy-10) 45,277 43,835 45,481 Ca (leu-12) 54,428 49,336 54,533 Ca (ile-13) 59,211 52,170 59,234 Ca (ser-15) 58,047 51,450 58,014 Calculations for the determination of the tridimensional structure were performed for the KGD peptide using CNSSolve software6,7 and first results show the presence of an antiparallel beta sheet with the KGD sequence at the fold of the sheet. Cys4 and cys-14 form a disulfide bond that seems to stabilize the tridimensional structure exposing the active KGD fragment. For the RGD and HGD peptides we are still calculating the structures for comparing them to the KGD structure. Biological tests (inhibition of the blood platelets aggregation) are being performed for all samples and results will allow correlating structure of each peptide and the possible biological activity. REFERENCES: 1. Ruoslahti, E; Pierschbacher, A. D; Science, 1987, 238, 491. 2. Born, G. V. R; Cross, M. J; J. Physol, 1963, 168, 178. 3. Rouslahti, E; Annu. Rev. Cell Dev. Biol; 12, 1996, 697. 4. Chan, W. C; White, P. D; Fmoc Solid Phase Peptide Synthesis, 1th ed, Oxford 2000, ch. 2. 5. Leite, J. R. S. A; Silva, L. P; Rodrigues, M. I. S; Prates, M. V; Brand, G. D; Laçava, B. M; Azevedo, R. B; Bocca, A. L; Albuquerque, S; Bloch Jr, C; Peptides, 2005, 26, 565. 6. Brunger, A.T. et at. Acta Crystallogr. D Biol. Crystallogr. 1998, 54, 905. 7. Brunger, A.T. Nat. Protoc. 2007, 2, 2728. CAPES, CNRMN, Embrapa Recursos Genéticos e Biotecnologia 65 I- + PO 20 HETEROGENEOUS CLUSTER SIZE DISTRIBUTION OF COHERENCES IN 1 THE QUANTUM DYNAMICS OF AN INFINITE SPIN /2 NETWORK C.M. Sánchez*, A.K. Chattah, R.H. Acosta, P.R. Levstein FaMAF-Universidad Nacional de Córdoba & IFEG-CONICET, Córdoba, Argentina e-mail:claudia@famaf. une. edu. ar Keywords: decoherence; multiple quantum NMR The study of generation, evolution, and control of coherent quantum dynamics in large Hilbert spaces has drawn much theoretical and experimental attention over the last years due to the direct application on, for instance, quantum information processing1. The main drawback on the implementation of quantum registers arises from the degradation of the correlation between states due to interactions with the environment. This process is generally known as decoherence and it determines how much information can be transmitted from one quantum manipulation to the next. In previous works, we studied the decoherence that occurs during the evolution of dipolar coupled nuclear spin systems using solid state NMR2,3. In particular, we measured spin systems in an infinite network with only intermolecular interactions (adamantane), other with only intramolecular interactions (5CB) and one with both intra and intermolecular interactions (ferrocene). The loss of information, or decoherence, can be quantified through the Loschmidt echo (LE) which is generated by reverting the time evolution of the system. For this work, we implemented a + + sequence to make the systems evolve under HDQ OC(IJ lk + Ij" LK~). * After the experimental observation of a striking distribution of coherence orders found in adamantane shown in Figure 1, we analyzed in detail the formation of different sized clusters and their evolutions under HDQ. Order of coherence Figure 1: Distribution of coherence orders after 1080 ^s of evolution under HDQ in adamantane: a) linear scale and b) logarithmic scale. In Fig. 1a) the linear scale seems to respond to the conventional gaussian distribution of coherence orders in infinite systems. However, a logarithmic scale reveals a behaviour that a single gaussian is not able to fit. In Fig.1b), we show the accurate fitting obtained with two gaussians (two free parameters each: width and area, A) suggesting the existence of well differenciated cluster sizes. The zero order 66 i'lnVi II M< \1.\fi\l IIC KI-sOV\M"l I SI RSMTI I IN.ii coherence is not considered because it accumulates the polarization arising from the decoherence processes. By using the spin counting model introduced by Baum et al.4 an estimation of the mean cluster size (N=co2/2) represented by each gaussian and the corresponding fraction of spins involved can be obtained from the fittings. Fig.2a) displays the mean number of spins in each cluster as a function of the evolution time. Note that there is a group of spins that stabilizes in clusters of around 20, which can be associated to the nearest neighbours, after approximately 240 (is while the other one grows exponentially reaching cluster sizes above 2000 spins. 0 2 4 6 8 10 12 2 4 6 8 10 12 Number of loops L (Time fcs] = L* 120 ) Figure 2: a) Number of spins correlated in each cluster size, b) Areas of gaussian fittings representing the fraction of spins involved in each cluster size. Fig.2b) shows the decay of the LE, i.e. the forward and backward evolution of the system, and the evolution of the proportion of spins involved in each kind of cluster arising from the A parameter. As can be seen at very short times all spins are forming small clusters which very soon correlate with other spins giving rise to larger clusters. Note that -around 400 |is the number of spins involved in large clusters overcome the group forming the clusters with N~20. After that, the larger clusters dominate the dynamics up to times where its signal equals the noise level (around 10~4) becoming under the detection limit. Consequently, at very long times only the few small clusters are still detectable (see inset). A Markovian model in the space order of coherence vs number of correlated spins was used to simulate the dynamics of cluster evolutions. Selection rules are incorporated to characterize the Hamiltonian and jump probabilities take into account the coupling network. Within the spins effectively moving in this space a leaking probability is introduced to simulate decoherence. Comparisons with experimental data shows very good agreements. REFERENCES: 1. C.H. Bennett; D.P. DiVicenzo; Nature (London), 2000, 404, 247. 2. Sánchez, C.M.; Pastawski, H.M.; Levstein, P.R.; Physica B, 2007, 398, 472 3. C.M. Sánchez, P.R. Levstein, R.H. Acosta, A.K. Chattah, Phys. Rev. A, 2009, 80, 012328. 4. Baum, J.; Munowitz, M.; Garroway, A. N.; Pines, A.; J. of Chem. Phys., 1985, 83, 2015. CONICET, ANCYPT, MPIP-MPG, FONCYT, SECYT-UNC 67 I- + AÜKi-MN - MAY 2nd 10 6rh, 2011 HOTEL DO TRADE. ANGRA DOS REIS, RJ. BRAZIL PO 21 QUANTIFICATION OF LIPIDS COMPOSITION IN COMMON BEANS THROUGH 1H HR-MAS NMR SPECTROSCOPY E.G. Alves Filho*1; L.M.A. Silva1; G.B. Alcantara1; P.Z. Bassinello2; L.M Lião1 1 Chemistry Institute, Federal University of Goiás, Goiânia, GO, Brazil 2Embrapa Rice and Beans, Santo Antônio, GO, Brazil *elenilson. qodoytfl) yahoo, com, br Keywords: Common beans, lipids, 1H HR-MAS NMR Oils and fats from edible seeds of legumes are constituents of many foods and play important organolepetic and nutritional roles. A balanced proportion of fatty acids in the diet is very important for human health, as beneficial and prejudicial properties have been attributed to certain fatty acids, depending mainly on their degree of unsaturation. Thus, the determination of fatty acid composition of seeds is essential for evaluating their suitability for different uses in the diet and in the food industry, as well as for the quality control of foods.1 The lipid content in common beans (Phaseolus vulgaris) is generally low when compared to other macronutrients and provides diversity in the fatty acid composition, with a substantial amount of unsaturated fatty acids. Oleic (7-10%), linoleic (21-28%), and a-linolenic (37-54%) are the most common unsaturated fatty acids, representing 65 to 87% of total lipids. Consequently, there is a great incentive to produce foods with good proportions of fatty acids. Therefore, the determination of their compositions in edible seeds of legumes is essential for assessing the nutritional quality.2 In this context, increased efforts have been employed to develop more attractive procedures for determining of fatty acids composition in edible seeds. Thus, this work presents a new methodology by 1H HR-MAS NMR. This technique combines the typical advantages of solid and liquid-state NMR analyses and has recently been reported as an analytical tool in study for solid food product investigations without any pre- treatment. To develop this research, five cultivars of common beans from different commercial groups acquired under the same conditions and planting season (September/2009) were obtained from the Embrapa Rice and Beans Research Center (Santo Antonio de Goiás, GO, Brazil). The analyzed cultivars were: BRSMG Tesouro, BRS7762 Supremo, Jalo Precoce, Pérola and Radiante. For 1H HR-MAS NMR all the beans were peeled, powdered using liquid nitrogen, and 30 mg suspended in 110 mg of D20/TMSP-d4 (sodium-3-trimethylsilylpropionate-2,2,3,3-d4) solution, used as intern standard. For extracts evaluation 100 g of each peeled bean grains were soaked in 600 mL of water during 16 hours in room temperature, and the soak water was removed. Others 600 mL of water was added and bean grains were submitted to pressure cook process during the time established on Mattson cooker apparatus (25 weighted plungers). The soaking and cooking water extracts were lyophilized, and 20 mg 1 suspended in 600 ^L of D20/TMSP-d4 for NMR analyses. One dimensional H NMR experiments were recorded at 28 °C using a Bruker Avance III 500 spectrometer operating at 500.13 MHz (1H), and equipped with a HR-MAS probehead. The analyses were made in triplicate using Composite Pulse Presaturation Sequence (CPPR) to solvent signal presaturation. The spectra were recorded using 128 scans, 64 k data points, spectral widths 8012.8 Hz, acquisition time 4.01 s and relaxation delay (d1) 2 s, sufficient for the proton with the longest relaxation time in the sample. A 50 rotor spinning at the magic angle at 5 kHz was used. The fatty acid quantities were calculated according to the characteristic signals from each compound. In this attempt, TMSP-d4 was calibrated to 9 1H and related with the alkyl chains for each fatty acid: linolenic (0.98 ppm to 3 1H); linoleic and linolenic (2.74 ppm to 3 1H for each); oleic, linoleic and linolenic (2.02 ppm to 12 1H). J. + 68 13th NUCLEAR MAGNETIC RESONANCL USERS MEETING Comparing each one of these areas with the area of the isolated signal from linolenic acid (0.98 ppm) was possible to identify the contents of each fatty acid in mixture, as used by Barison et al.3 The spectra was resolved through a mathematical deconvolution process to elimination of overlapping peaks related to material impurities, being conducted using the Lorentzian function of the TOPSPIN® 2.1 program. The deconvolution linked simplicity and accurate determination of the lipids amount based on 1H NMR spectroscopy. Special attention was given to the signals integral limits, in order to avoid the inclusion of side bands, which could affect the accuracy of the results, being also very important to perform a good phase adjustment. The results obtained by this proposed technique are presented in Table 1 and were consistent with those described in the literature (0.8 to 1.5% for the raw beans).4 Table 1 - Composition of fatty acids (mg.g1) of common beans. Fatty OLEIC LINOLEIC LINOLENIC Acids CommonBeansr-— Raw 20.1 ±0.5 25.4 ± 0.6 32.1 ±0.8 JALO After Soaking 4.9 ±0.2 5.2 ±0.1 8.2 ±0.1 PRECOCE After Cooking 32.8 ±0.6 49.7 ±0.9 39.1 ±0.8 Raw 29.4 ± 0.5 33.2 ±0.7 31.1 ± 0.6 BRS7762 After Soaking 12.7 ±0.4 15.1 ±0.4 9.9 ±0.1 SUPREMO After Cooking 18.1 ±0.4 27.8 ±0.6 13.4 ±0.7 Raw 14.5 ±0.4 16.5 ±0.4 16.8 ±0.4 BRSMG After Soaking 11.8 ±0.3 15.5 ±0.3 9.7 ±0.2 TESOURO After Cooking 27.7 ±0.6 40.7 ±0.8 32.1 ± 0.8 Raw 26.2 ± 0.6 29.5 ± 0.7 31.5 ±0.7 PÉROLA After Soaking 2.0 ±0.2 5.3 ±0.1 0.5 ±0.1 After Cooking 39.9± 0.8 40.6 ± 0.8 44.3 ± 1.0 Raw 20.7 ±0.5 22.0 ± 0.5 26.2 ± 0.7 RADIANTE^ After Soaking 9.3 ±0.3 9.5 ± 0.2 11.3 ±0.3 After Cooking 34.1 ± 0.6 48.7 ± 0.8 37.1 ±0.8 The presented results show that the lipids content in raw beans ranged from 1.45 to 3.32%. After 16 hours soaking period, a medium decrease of 61.4% for all cultivars was observed. It is known that through leaching, the protein contents are reduced when the beans are submitted to the soaking procedures5 and a lipid-protein complex could be formed. Thus, the protein may serve as a carrier lipid.6 Paradoxically, after the cooking an increase in the concentration of lipids was observed, except to BRS7762 Supremo bean which concentration was reduced. This may be attributed to water loss that generates a reduction in the percentage of bean mass or loss of minerals to the ambient.7 In conclusion, the described method was very efficient in determining the fatty acids composition in beans. REFERENCES: 1. Woollett A.L.; Spady K.D.; Dietschy M.J., J. Lipids Res., 1992, 33. 2. Chiaradia A.C.N. & Gomes J.C., Qui. Nut. Tec., 1997, 180. 3. Barison A.; Silva C.W.P.; Campos F.R.; Simonelli F.; Lenz C.A.; Ferreira A.G., Mag., Res. Chem., 2010, 48. 4. Geil P.B.; Anderson J.W.; J. Am. College Nutr., 1994, 13. 5. Kataria A.; Chauhan B.M.; Punia D. Plant Foods for Human Nutrition, 1992, 42. 6. Barampama Z. & Simard R.E., Plant Foods for Human Nutrition, 1995, 48. 7. Delfini R.A.; Canniatti-Brazaca S.G., Alim. Nutr., 2008, 19. MCT/FINEP/CT-INFRA, CNPq, CAPES and EMBRAPA 69 I- + PO 26 EVALUATION OF NEW INHIBITORS OF ACETYLCHOLINESTERASE BY NMR: 1-METHYLPYRIDINE-2-HYDRAZONE AND 1 -METHYLPYRIDINE-2- GUANYL HYDRAZONE E. C. Petronilho1, N. G. de Castro2 A. C. Pinto3, J. D. Figueroa-Villar*1 'Medicinal Chemistry Group, Department of Chemistry, Military Institute of Engineering, Rio de Janeiro, Brazil institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 3Laboratory of Molecular Pharmacology, Biomedicas Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. e-mail:figueroa(a).ime. eb. br Keywords: Acetylcholinestarse, hidrazones and guanylhydrazones, Alzheimer. The most important dementia problem of mankind in the 21st Century is the Alzheimer disease (AD), which today affects about 23 million people in the whole world. This disease is present in 12 to 15% of the population older than 65, but the early-onset Alzheimer disease version can occur in young people.1 One of the main problems of AD is the reduction of activity of cholinergic neurons, a problem that can be combated by increasing the amount of the neurotransmitter acetylcholine (ACh).2 The best procedure to increase the concentration of ACh is the inhibition of the enzyme acetylcholinesterase (AChE) which is responsible for the control of nerve pulse transmission by hydrolysis of ACh. The active site of AChE possesses a great affinity for cationic compounds, simply because ACh contains the positive -N(CH3)3 group. According to this, the AChE inhibitors used for the treatment of AD are all cationic compounds, like donepezil, tacrine, galantamine and rivastigmine. AChE is strongly inhibited by organophosphorus (OP) compounds, and the enzyme can be reactivated by cationic oximes, like pralidoxime (3), a compound with great affinity for the AChE active site.3 According to these data, we planned, synthesized and evaluated a new family of cationic compounds, analogues of pralidoxime, as potential agents for the treatment of AD. The new products were prepared by methylation of the nitrogen atom o 2- pyrididecarboxyaldehyde (1) with methyl iodide, to afford the cationic form 2, which was reacted with hydroxylamine hydrochloride to prepare pralidoxime (3), with hydrazine hydrochloride to obtain 1-methylpyridine hydrazone (4) and with aminoguanidine hydrochloride to afford 1-methylpyridine guanylhydrazone (5), as shown in Figure 1. NH2OH.HCI ^OH EtOH 25 °C I© CH3 3 CH3I N2H4.H2O EtOH, Reflux "N© "NH2 N CHO "N®, CHO I© l ® I© l ® CH3 4 CH3 2 H2NNHC(NH2)2.HCI EtOH, Reflux 'N® ^ N NH.HCI I© l I CH3 5 H Figure 1: Synthesis of cationic inhibitors of AChE 70 M(ll \K\1.\Í,M lICRI-SUSANCI.rSMíSMI I I1\l> Pralidoxime (1) was prepared to be used as the positive reference for the inhibition of AChE. The inhibition of AChE was carried out by NMR and using micro plates. The NMR tests were performed by monitoring the methyl signal intensity of ACh (S 1.96) and the formed acetate (8 1.72) along the reaction time. The samples were prepared in the 5 mm tubes with a complete volume of 600 ju.L and concentration of AChE from Electrophorus electricus of 0.2 |uM, ACh 2.5 mM and the inhibitors (3, 4 or 5) at 5.0 nM, and using as solvent a phosphate buffer solution (0.1 M, pH 7.4) in D20. The AChE solution contained 1% of bovine serum albumin (BSA) as a stabilizer. The experiments were conducted um a Varian UNITY-300 NMR spectrometer with the temperature controlled as 25.0±0.1 °C. The results are shown in Table 1. Table 1: Time for hydrolysis of 50% of ACh with AChE with and without inhibitors by NMR Inhibitor Time (min.) None 51 Pralidoxime (3) 59 1-Methylpyridine-2-hydrazone (4) 70 1-Methylpyridine-2-guanylhydrazone (5) 56 The Ellman test was conducted in micro plates with 96 small wells using AChE from Electrophorus electricus (0.05 U/ML), DTNB (0.25 mM, 5 jiL), acetylthiocholine (0.5 mM) and the inhibitors at concentrations 0.003, 0.010, 0.100, 0.300 and 1.000 mM, and using the phosphate buffer as solvent. The results are shown in Figure 2. 100 an 80 70 60 50 40 30 20 10 0 AChE AChE+3 AChE+4 AChE+5 Figure 2: Inhibition of AChE by compounds 3, 4 and 5 by Ellman test. The results obtained by NMR are in agreement with the tests by the Ellman method, indicating the cationic hydrazone is a very effective AChE inhibitor, as it was concluded by a previous work developed by ab initio molecular modeling.4 REFERENCES: 1. F.G. de Felice, M.N.N. Vieira, L.M. Saraiva, J.D. Figueroa-Villar, J. Garcia-Abreu, R. Liu, L. Chang, W.L. Klein & S. T. Ferreira FASEB Journal, 2004, 18:1366-1372. 2. S. M. Stahl J. Clin. Psychiatry. 2000. 61: 813-814. 3. R.T. Delfino, T. S. Bibeiro & J. D. Figueroa-Villar, J. Braz. Chem. Soc. 2009, 20:407- 428. 4. R.T. Delfino & J.D. Figueroa-Villar J. Phys.Chem.B. 2009, 113: 8402-8411. CNPq, CAPES, BRAZILIAN MINISTRY OF DEFENSE, INBEB 71 I- + PO 22 CONFORMATIONAL EFFECT STUDY OF NEW TRANQUILIZERS BY NMR AND MOLECULAR MODELING A. A. Vieira arid J. D. Figueroa-Villar* Medicinal Chemistry Group, Department of Chemistry, Military Institute of Engineering, Rio de Janeiro, Brazil e-mail:fiaueroa(d).ime.eb. br Keywords: molecular modeling and NMR, 5-chlorobarbiturate tranquilizers, conformational effects The chlorination of 5-benzylbarbiturates (1) with trichloroisocyanuric acid lead to the discovery of a new family of efficient tranquilizers without sleeping effects, the 5-chloro- 5-benzylbarbiturates (2).1 In order to study the correlation between the tranquilizing activity and molecular structure, it was used the combination of NMR with molecular modeling for the conformational analysis of compounds with CI at the ortho and para position of the aromatic ring in these compounds (Figure 1). 1a RI=R2=H 2ARI=R2=H 1 b R1 =CI, R2=H 2b R1 =ci, R2=H C 1 R-|=H, R2=CI 2C R1=H, R2=CI Figure 1: New tranquilizers and their precursors In compounds 1 and 2, C5 is not in conjugation with the aromatic ring, and it could be expected that the type and position of the substituents on the benzyl ring would not have an important impact on the chemical shift of C5. However, the comparison of the 13C NMR chemical shifts for all products (1 and 2) (Table 1) indicated that C5 has significant differences in 5 values depending on the position of the chlorine atom at the aromatic ring. These differences are greater of 5-chloro-5-benzylbarbituric acids (2, A5max 2.4) than for the non-chlorinated compounds (1, ASmax 1.9). In the tranquilizers, 8C5 is clearly affected by the substituent position on the aromatic ring, where for the ortho position (2c) its value is 64.3 ppm while 2a (without substituents) and 2b (with CI at the para position) have 5C5 at 61.9 ppm. A similar condition is observed for the precursors, where 1a and 1b have SC5 at 49.3 and 50.0 ppm, and the compound with substituent at the ortho position (1c) has the lower value (48.1 ppm). Table 1: 13C Chemical Shifts of C5 and C7 of compounds 1 and 2 1 2 R 8C5 6C7 A5C5-C7 ÔC5 SC7 AÔC5-C7 a(H) 49.3 33.3 16.0 61.9 43.5 18.4 b (11 -CI) 50.0 33.0 17.0 61.9 42.2 19.7 c (9-CI) 48.1 30.6 17.5 64.3 40.8 23.5 + 72 Hill Ml 1 I.M< MACiSI-IKKISOYWl. I SMi>i \1I-I ll\<. The major value of ASC5-C7 for the C5 chlorinated compounds indicates that the chlorine atom at C5 has influence on the polarization of the C5-C7 bond. The increase of AÔC5-C7 is 15.0 and 15.9 % for compounds 2a and 2b, but more that twice (34.3 %) for the ortho compound (2c). This is a clear indication that the conformational changes in these barbiturates are due to the position of the substituents on the aromatic ring. These apparently small NMR chemical shift differences are important information regarding the conformation of the barbiturates, which explanation needs a molecular modeling study. The calculations were conducted with the Spartan 06 program using the B3LYP method with the 6-311G(d) basis set. The conformational analysis shows that compounds with or without CI at C5 have the most stable conformation with a C8-C7-C5-H angle close to 180°, leading to a n- type interaction between the aromatic and pyrimidine rings. On the other hand, compounds with chlorine at the ortho position have the most stable conformer with a dihedral angle close to 142° and without rc-type interaction between the rings. Figure 2 shows the most stable conformer for the para and o/?fro-chlorinated compounds with CI at C5. A B C Figure 2: Conformers for compounds 2b and 2c: (A) the most stable of 2a; (B) the second of 2a and (C) the most stable of 2c. The conformer structures show that, for compounds without substituent at the ortho position, the chlorine atom can interact, via molecular orbitais, with the aromatic ring and, with greater intensity, the aromatic ring performs a n-interaction with the barbituric ring. However, the presence of groups at the ortho position leads to conformations that decrease the possible interaction between the aromatic and the pyrimidine rings and also between the C5 chlorine atom and the aromatic ring via molecular orbitais. These conformational differences support the chemical shift differences observed between the para and ortho-substituted compounds. The biological in vivo tests with these compounds showed that the 0rf/70-substituted products are the most effective tranquilizers,1 indicating that their conformation leads to a better affinity for the receptors, a process that is now under study. REFERENCES: 1. A. A. Vieira, N. M. Gomes, M. E. Matheus, P. D. Fernandes and J. D. Figueroa-Villar, J. Braz. Chem. Soc. 2011, 22: 364-371. CAPES, CNPq, BRAZILIAN MINISTRY OF DEFENSE, INBEB 73 I- + PO 20 FIRST IMPLEMENTATION OF ULTRAFAST 2D NMR IN THE SOUTHERN HEMISPHERE L.H.K. Queiroz Jr.*1, A.G. Ferreira1, P. Giraudeau2, D.P.K. Queiroz1 'Department of Chemistry - Federal University of São Carlos, São Carlos, Brazil 2 University of Nantes, CNRS, CEISAM UMR6230, Nantes, France e-mail:professorkenq(çb.Qmail.com Keywords: single-scan; ultrafast NMR; 2D NMR. Nuclear Magnetic Resonance (NMR) is a powerful investigative tool with applications in biochemical, organic and pharmacological analysis, among others. In this context, multidimensional NMR spectroscopy is of fundamental importance. However, these experiments are associated with very long acquisition durations due to the necessary U incrementation along the indirect domain. Many approaches have been proposed to bypass this drawback and allow the acquisition of nD NMR spectra in a reduced time1,2. A few years ago, Frydman and co-workers3 proposed an approach that allows the acquisition of nD NMR spectra with only one scan, also known as "ultrafast NMR". This method uses spatial encoding instead of parametric U incrementation. The information is first spatially encoded, then followed by a position-independent mixing period, and finally, the information is spatially decoded via an Echo Planar Imaging-based scheme. Ultrafast NMR represents a new frontier, and allows the study of short timescale phenomena. However, despite all the advantages of this method, it presents some limitations in terms of resolution, spectral width and sensitivity4.'Moreover, ultrafast 2D NMR pulse sequences are not commercially available, and they involve the adjustment of a number of non-standard parameters (gradient amplitudes and durations, chirp pulse parameters, etc.) In this context, a major problem is the implementation of ultrafast NMR as routine experiments, which is indispensable towards their use together with hyphenated techniques such as LC-NMR. This work aims at studying the implementation of ultrafast NMR for routine experiments. To our knowledge, this is the first study about ultrafast NMR developed and reported in Brazil. n/2 1H LuJ _FGe Figure 1: Ultrafast COSY pulse sequence We performed the experiments on a Bruker Avance III 400 MHz including z-axis gradients. The pulse sequence used for the ultrafast COSY experiment is shown above in Figure 1. The spatial encoding pulses were set as smoothed chirps, the pulse length was 15 ms and the total sweep width 60 kHz. The encoding gradients amplitude was set to Ge = 6,8 G/cm, while 128 gradient pairs with Ga = 35,5 G/cm and Ta = 256 74 13th NUCLEAR MAGNETIC RESONANCE USERS MEETING were applied for the acquisition . The conventional COSY spectrum was recorded with a routine pulse sequence avaliable within the commercial software Bruker TopSpin 3.0, 256 t1 increments and 6 scans. To implement the ultrafast methodology we first carefully calibrated the spatial encoding gradient parameters and the excitation pulses, to ensure that the frequency dispersion induced by the gradients match the frequency widths of encoding pulses. The amplitude and duration of the pre-acquisition gradient were adjusted to correctly position the signals in the ultrafast dimension. Acquisition parameters were also adjusted to optimize the observable spectral width, and the equilibrium between acquisition gradients was adjusted to compensate for shearing problems. Moreover, peaks were initially characterized by an asymmetric sine shape along the ultrafast dimension, thus highly affecting the 2D peaks. To reduce this asymmetry we performed an apodization in the ultrafast domain5, while preserving the resolution and improving the S/N ratio. All spectra were processed using a special routine in Bruker program TopSpin 3.0, including zero-filling and apodization. We obtained ultrafast COSY spectra with a good resolution and sensitivity, with a 2D pattern identical to the one of a conventional COSY but in a much shorter time (Figure 2). a) Conventional b) Ultrafast 0 § « O if ® # ê 0 @ Q % O Total experiment time ~ 63 min Total experiment time ~ 0.17 s 5.5 5.0 4.0 3.5 F2 [ppm] 5.5 5.0 3.5 F2 [ppm] Figure 2: Comparison between (a) Conventional COSY spectrum and (b) Ultrafast COSY spectrum obtained on a Levamisol sample in methanol. As can be seen, the implementation of ultrafast NMR as routine experiments faces some hurdles, some of which have already been overcome. Currently, we are working on some tools that facilitate the implementation of these experiments, such as developing a new algorithm enabling the real-time processing of the ultrafast NMR data on the commercial software. We are also working on the implementation of heteronuclear 2D experiments. Finally, we are focusing on the implementation of these experiments in LC-NMR, to allow the real-time identification of analytes undergoing continuous chromatographic separation. REFERENCES: 1. Kupce, E.; Freeman, R.; J. Magn. Reson. 2003, 162, 300-310. 2. Schanda, P.; Brutscher, B.; J. Am. Chem. Soc. 2005, 127, 8014-8015. 3. Frydman, L.; Scherf, T.; Lupulescu, A.; Proc. Natl. Acad. Sci. USA 2002, 99(25), 15858- 15862. 4. Giraudeau, P.; Akoka, S.; J. Magn. Reson. 2008, 192, 151-158. 5. Giraudeau, P.; Akoka, S.; Magn. Reson. Chem. 2011 accepted. CAPES, CNPq, FAPESP 75 I- + PO 20 APPLICATION OF MODIFIED CONTINUOUS WAVE FREE PRECESSION METHOD FOR WATER SUPPRESSION IN 2D EXPERIMENTS C. J. Duarte*1, L. A. Colnago2, T. Venâncio1 1 Universidade Federal de São Carlos, Brazil; 2Embrapa Instrumentação, Brazil junkerduarte(a). yahoo, com, br Keywords: Water suppression; CWFP; COSY SPECTRUM Most of the molecules of biochemical interest must be studied in non-deuterated water, in order to observe the exchangeable protons. As the solvent concentration is much higher than that of the solute, it composes most of the NMR signal. Therefore, it is necessary to suppress the intense solvent signal in order to observe the solute signals close to solvent and to avoid other spectral complications such as baseline and phase distortions. Several methods have been designed to suppress solvent signals1 and practically all the schemes involve some drawbacks such as suppression of exchangeable protons by the process of saturation transfer and discrepancies in signal amplitude and phase. A variation of the steady state free precession (SSFP) method, termed continuous wave free precession (CWFP), has been proposed for various analytical applications, including solvent suppression2,3. In the conventional CWFP technique, the spin system is submitted to a train of n pulses with same phase, intensity and duration, and separated by time Tp « T2* (effective transverse relaxation time). This sequence strong suppresses the water signal but it introduces some phase and intensity distortions. To eliminate this anomalies a modified CWFP pulse sequence with phase alternation is present and it uses in a 2D experiment (gCOSY) was demonstrated. The modified CWFP sequence for solvent suppression uses a train of pulses with phase alternation (0X-TP- 9y-Tp- 0-x-Tp- 9.y)n 0.y, where 8 is the flip angle of pulse and Tp is the time interval between pulses. The experiments were carried out in a 9.4T Bruker DRX-400 instrument equipped with a 5mm probe with indirect detection. The sample used for water suppression was 2mM sucrose dissolved in 90:10 H20/D20. The modified CWFP 1D pulse sequence was written in the programming module of the TOPSPIN software. The CWFP train of 2048 pulses was inserted in a gCOSY pulse sequence just before the first 7t/2 pulse. a) 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4,0 3.8 3.6 3.4 ppm Figure 1: Partial 1H spectra of 2mM sucrose: a) without water suppression, b) using conventional CWFP and c) using modified CWFP. 0=71/2, 16 scans, Tp=500(is 76 The CWFP signal is formed by FID and echo-like components and the reason for phase and intensity anomalies is exactly the interference between these components5. Several procedures have been used for eliminating the echo component of the signal5,6, but in this work we used phase alternation of the pulses, as proposed by Rudakov4. The fig. 1 compares 1H NMR spectra for a sucrose sample without water suppression (a), by using the conventional CWFP (b) and modified CWFP pulse sequence (c). For both b and c spectra, the pulse sequences provide solvent suppression, but the conventional CWFP (b) causes phase and intensity anomalies. This effect can be seen in the doublet of the anomeric proton around 5.4 ppm, which has an inverted phase and reduced intensity. These anomalies do not occur when the water signal is suppressed using the modified CWFP with phase alternation, figure 1(c). In addition to 1D application, it is important to consider the potential implementation of CWFP in multidimensional experiments. To show this possibility we have included the modified CWFP, with phase alternation to a g-COSY experiment to suppress the solvent signal. Fig. 2 shows the results for the same 2mM solution of sucrose of figure 1. The COSY experiment with the modified CWFP pulse sequence, which was named mCWFP-gCOSY, has shown an excellent suppression of water signal, as can be observed in the fig. 2. ,.JJ„JL.J* SJLJUIL ppm 3.0 3.5 4.5 5,0 55 5.5 Figure 2: COSY spectrum of sucrose 2mM using pulse sequence mCWFP-gCOSY. For the CWFP train of pulses increment: 0=TT/2, Tp=500^S. Considering the success of the modified CWFP in order to suppress solvent signal with minimum problems of phase and intensity anomalies, the modified pulse sequence can also be used for solvent suppression in others multidimensional experiments. REFERENCES 1. Zheng, G.; Price, W.S., Progress in Magnetic Resonance Spectroscopy, 2010, 56. 2. Azeredo, R. B. V.; Colnago, L. A.; Engelsberg, M., Analytical Chemistry. 2000, 72. 3. Venâncio, T.; Azeredo, R.B.V.; Engelsberg, M., Colnago, L.A., Annals of Magnetic Resonance, 2003, 2. 4. Rudakov, T. N.; Belyakov, A. V., Journal of physics D: Applied Physics, 1998, 31 5. Ernst, R.R.; Anderson, W., The Review of Scientific Instrumentation, 1966, 37 6. Freeman, R.; Hill, H. D. W., Journal of Magnetic Resonance, 1971, 4. CNPQ, CAPES and FAPESP 77 I- + PO 20 NMR STRUCTURAL STUDIES OF A NOVEL OCELLATIN-P1 ISOFORM ISOLATED FROM THE SKIN SECRETION OF LEPTODACTYLUS LAB YRINTHICUS Eliane S. Fernandes*1, Carolina O. Matos1, Cesar A. Prías-Márquez2; Eduardo M. Cilli3; Osmindo R. Pires Júnior2, Wagner Fontes2; Mariana S. Castro2, Luciano M. Lião1, Aline L. Oliveira1. 11nstituto de Química, Universidade Federal de Goiás, Goiânia, Brasil. 2Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, Brasil. 3Instituto de Química, Universidade Estadual Paulista, Araraquara, Brasil *eliane ufq&.yahoo.com.br Keywords: peptide; three-dimensional structure; SD$ micelles The skin of amphibians has been the source of a wide variety of biologically active substances. Currently, the emergence of fungi, bacteria and viruses resistant to multiple drugs has stimulated interests in the development of antimicrobial peptides as human therapeutics. In general, potential antimicrobial drugs should provide a selective toxicity, rapid action, a broad spectrum antimicrobial and no selective resistance mechanisms in microorganisms. That is exactly what happens with antimicrobial peptides isolated from skin secretion of anurans. In fact, they are quite different from antibiotics used in therapy today, both in its structure and mechanism of action1,2. The major driving force for the action of antimicrobial peptides is its ability to lyse cell membranes, which rapidly kill a broad spectrum of microorganisms. In this sense, the 3D structure identifies where each residue resides and highlights those that are important for activity or those that could potentially be mutated to increase antimicrobial activity1,2. This study reports the structural characterization of a novel Ocellatin-P1 isoform isolated from the skin secretion of the pepper-frog Leptodactylus labyrinthicus. The skin secretions were obtained by electrical stimulation and then lyophilized. The freeze-dried secretion was fractioned by RP-HPLC on a C8 column. All eluted fractions were tested against pathogenic Escherichia coii and Staphylococcus aureus. One bioactive peptide with 2510.41 Da was purified by C18-RP-HPLC and sequenced by Edman degradation, yielding the N-terminal sequence: 1GLLDTLKGAAKNWGGLASKVMEKL25 (Ocellatin-P1G16). Analysis by Fischer's esterification demonstrated an amidation at the C-terminus. This isoform differs from Ocellatin-P1 in only one residue (a glycine instead of a serine at position 16th) and was active against both pathogenic bacteria. With the aim to understand the conformation properties and mechanism of action of the peptide Ocellatin-P1G16, we have determined its 3D structure by 1H NMR. Recognizing that its activity involves membrane interaction, the studies were carried out in the presence of SDS micelles. A NMR sample was prepared by dissolving the synthetic peptide to the final concentration of 1 mM in 500pL of a 100 mM SDS-d25 solution, 10% of D20 and pH 4.3. Homonuclear 2D TOCSY, NOESY and COSY NMR experiments were recorded at 36 °C on a BRUKER AVANCE III 500 spectrometer operating at 11.75 T. The complete assignment of the backbone and side-chains 1H resonances of Ocellatin-P1G16 was performed using standard sequential assignment procedures. NOE-derived distance restraints were obtained from 2D 1H-NOESY collected with 60 ms mixing times. All NMR data were processed using the nmrPIPE/nmrVIEW software. A quick analysis of the type and location of the secondary structure was performed using the Chemical Shift Index (CSI). This method is based on the difference of the 1H chemical shift of a-CH of each residue with respect to tabulated values. A group of 78 I'III SI I II.AK M.V.MIK" Kl-sovwn I SIR1; Ml I.I INÍ.I three or more values of CSI less than -0.1 indicates the presence of a-helix structure. The CSI values (Figure 1) for Ala9-Lys24 indicated the presence of helical segment in this region. The structure calculation was performed with XPLOR-NIH, using a simulated annealing protocol. A set of 186 distance constraints, of which 133 was medium-range constrains, were used to determine the Ocellatin-P1G16 structure, resulting in a final average of 7.44 constraints per residue. The 20 lower energy structures (from a total of 100 calculated structures) were chosen to represent the peptide solution 3D structure (Figure 2). -N -N residue -C -C Figure 2. 20 final structures (left) and ribbon Figure 1. Chemical Shift Index of the representation of the lowest energy structure peptide leptodace. (right) of Ocellatin-P1G16. As expected, the NMR-derived structure of Ocellatin-P1G16 in the presence of SDS micelles (Fiture 2) is predominantly helical, with a regular a-helix spanning the region from residues Leu2-Lys24.The peptide exhibited a well defined structure as evidenced by the superposition of the 20 lower energy models (Figure 2, left) and the low root mean square deviation (RMSD) values over all the backbone atoms (0.24 ± 0.13 Â). In addition, the good quality of the NMR models is highlighted by the fact that the majority of the (/> and ip dihedral angles are in the most favored or in the additionally allowed regions. This diagram showed that 76.5% of the peptide angles are in favored regions of a-helix, ,18.8% are in additional allowed regions and 3.75% are in generously allowed regions. Figure 2 (right) also highlights the hydrophobic residues in black and the hydrophilic residues in gray, showing that this peptide has amphipathic nature. The helical surface includes four positively charged amino acids residues (Lys7, Lys11, Lys20, Lys24) and two negatively charged amino acids residues (Asp4, Glu23) and they could be the key for a selective interaction with the biological membranes. As perspective to analyze the molecular details of how this peptide interacts with SDS micelles, H/D exchange experiments will be realized. Dissection of the structural features of this peptide will be very important to unveil the molecular mechanism associated to its biological function and will provide new insights into the biological activity of peptides. REFERENCES 1. Bhutia S. K. and Maiti T. K„ Trends Biotechnol. 2008, v. 26, 210. 2. Libério M. S. etal., Amino Acids. 2011, v. 40, 51. FUNAPE, CAPES, CNPq, and FINEP 79 I- + AUREMN MAY 2nd In 6ih, 2011 HOTEL DO FRADE, ANGRA 1XK REIS, RJ. BRAZIL PO 31 NMR CHARACTERIZATION OF A NEW POLYCYCLIC PHENAZINE FROM 1,4-NAPHTHOQUINONE J. D. de Souza Filho*1, E. N. da Silva Júnior*1, M. J. da Silva2, M. C. F. R. Pinto2, C. A. de Simone34, J. G. Soares3, J. R. M. Reys3, W. T. A. Harrison5, Carlos E. M. Carvalho6, M. O. F. Goulart3 and Antonio V. Pinto2t 11nstituto de Ciências Exatas, Departamento de Química, UFMG, Belo Horizonte, MG, Brazil 2Núcleo de Pesquisas de Produtos Naturais, Rio de Janeiro, Brazil 3lnstituto de Química e Biotecnologia, UFAL, Maceió, Alagoas, Brazil 4Instituto de Física, Departamento de Física e Informática, USP, São Carlos, SP, Brazil 5Department of Chemistry, University of Aberdeen, Aberdeen, Scotland 6lnstituto de Química, UFRJ, Ilha do Fundão, Rio de Janeiro, RJ, Brazil e-mail:[email protected] fin memoriam Keywords: NMR; naphthoquinones; phenazines. Phenazines derived from lapachol (1) and lapachone have been shown to possess potential activities against causative agents of neglected diseases, for instance, Mycobacterium tuberculosis1 and Plasmodium falciparum2 Lapachol (1) furnishes a complex mixture of products when submited to the conditions showed in Figure 1. After fractionation using silica gel column chromatography, three main compounds were isolated. Spectroscopic data for substances 2 and 3 are in accordance with literature.3'4 The structure of compound 4 was determined based on 1D and 2D NMR experiments and elem&ntal analyses. 2, 35% 3, 30% 4, 10% Figure 1. Reaction of lapachol (1) with orffro-phenylenediamine. Numbering of the phenazine 4 according to IUPAC: 6,6-dimethyl-7,8-dihydro-6H,9H- benzo[a]pyrido[3,2,1-de]phenazin-9-one. The NMR experiments were performed at 9.4 T. For measuring the J couplings, the proton NMR 64K fid was Gaussian Fourier transformed (lb -1 and gb 0.5), without zero filling, and its values were calculated from the best digitized resonance lines. The very first assignment is related to H-15 (S7.79, dd, J 1.12 and 8.84 Hz) that showed a correlation to the geminal methyl protons at C-6 (£ 1.89, s) in the NOESY contour plot (d8 400 ms and 200 ms)(Figure 2a). The COSY contour plot well defined the three different spin systems showing cross peaks between H-15 and H-14 (<5 7.49, J 1.78, 7.14 and 8.84 Hz), H-14 and H-13 (£7.32, J 1.12, 7.14 and 7.94 Hz), and H-13 and H- 12 (S 7.96, dd, J 1.78 and 7.94 Hz) in the upper aromatic moiety. In the left aromatic spin system, the more deshielded H-1 was registered as a complex multiplet at 58.87- 8.92 due to its second order splittings. As well for H-4, the same feature is observed at £8.34-8.40 range. The signals of H-2 an H-3 are registered in a complex envelope at £7.68-7.74 range. Surprisingly, the shape of this multiplet seems like a AA' moiety of a AA'XX' spin system and the ones from H-1 and H-4 seems also with the shape of a BB' part of a AA'BB'spin system, with inversion in three resonance lines. As the chemical shifts of H-2 and H-4 are not the same, this spin system must be named as \ + 80 HiliM'CI r.AR MAGNETIC RESONANCE I SI RS MITTING AA'BC. The third spin system composed by the two methylene groups stays on two complex multiplets at £2.89-2.95 and £2.00-2.05 ranges, assigned to H-7a,b and H- 8a,b, respectively. These previous assignments promptly allowed the carbon-13 ones as CH3(£28.2), C-1(£124.89), C-4(£125.08), C-7 (£16.4), C-8(£39.9), C-12(£131.38), C-13(£ 123.01), C-14(£129.11) and C-15 (£117.97). The two AA' protons H-2 and H-3 showed a overlapped correlation with two carbons namely C-2 and C-3 at £130.33 and £130.86, respectively. This assignment were supported by the HMBC experiments (d6 70ms and 140ms) that showed correlations due to three bond couplings between H-1 to C-3 and H-4 to C-2. The three non-protonated carbons C-6, C-9 and C-10 showed its chemical shifts at £ 59.6, £179.23 and £ 147.38, respectively. The HMBC experiments showed the expected correlations between the geminal methyl hydrogens and the two methylene hydrogens, H-7 and H-8 to C-6. Moreover, H-8 exhibited a three bond long range couplings to C-9 and C-10a(£ 136.69). The chemical shift of C-8a(£ 109.97) was assigned by the simultaneous correlations with H-7 (3J) and H-8 (2J). The chemical shift of C-11 a was assigned to the line at £ 136.25 because of the correlations to H-13 and H-15 in a three bond mechanism. The same feature was observed in the assignment of C-15a at £131.17 due to the correlations with H-12 and H-14 chemical shifts. Finally for the left aromatic ring, the NMR signals of C-4a and C-9a were assigned to £ 132.06 and £ 131.67 by the three bond correlations to H-1 and H-4, respectively. Despite the values of the delays for long range evolution couplings in the HMBC experiment (d6 70ms and 140ms), it was possible to observe correlations due to four bonds (H-8 to C-10 and H-4 to H-9) and a very weak one by five bonds (H-3 to C-9). These facts are due to highly conjugated 7r-system. Very impressively, it was possible also to observe a five bond correlation between the methyl hydrogens with C- 15 (Figure 2b). (a) (b) Figure 2. a) NOE correlation of H-15 (F2) to methyl protons (F1), b) Five bonds long range correlation of methyl protons (F2) to C-15 (F1). REFERENCES: 1. Coelho T. S.; Silva R. S. F.; Pinto A. V.; Pinto M. C. F. R.; Scaini C. J.; de Moura K. C. G.; Silva P. E. A. Tuberculosis. 2010, 90, 293. 2. Andrade-Neto V. F.; Goulart M. O. F.; da Silva Filho J. F.; da Silva M. J.; Pinto M. C. F. R.; Pinto A. V.; Zalis M. G.; Carvalho L. H.; Krettli A. U. Bioorg. Med. Chem. Lett. 2004, 14, 1145. 3. Benedetti-Doctorovic V.; Escola M.; Burton G. Magnetic Res. Chem. 1998, 36, 529. 4. Carvalho C. E. M.; Brinn I. M.; Pinto A. V.; Pinto M. C. F. R., J. Photochem. Photobiol. A: Chem. 2000, 126, 25. FAPEMIG, CNPq, CAPES 81 I- + AURI-MN" MAY 2nii lo 6th,2011 HOTEL DO FRADE, ANGRA DOS REIS. RJ, BRAZIL PO 31 NMR STUDIES ON A D-GLUCOSAMINE DERIVED MACROCYCLE J. D. de Souza Filho*1, R. J. Alves2 and M. C. Pires2 11nstituto de Ciências Exatas, Departamento de Química, UFMG, Belo Horizonte, MG, Brazil 2Faculdade de Farmácia, UFMG, Belo Horizonte, MG, Brazil e-mail: peixe@ ufmg. br Keywords: NMR; sugar; macrocycle. The macrocycles form an important class of compounds that, by having various biological activities and many drugs available, are targets of intense research.1 This work reports the preparation of a 12-membered macrocycle (1) by Bu3SnH/AIBN- mediated radical carbocyclization2 of a suitable iodo-D-glucosamine intermediate (Figure 1)3. C02Me Bu3SnH /AIBN benzene, reflux 59.7% Figure 1 - Radical carbocyclisation conditions to the synthesis of macrocycle 1. The NMR experiments for the macrocicle 1 were performed in two different field strengths, 9.4 T and 14.1 T, and solutions, DMSO and CDCI3. As expected we observed changes in the chemical shifts, in the scalar couplings and in the nOe's results. The total assignment of the proton and carbon spectra were supported by the whole set of 2D contour plots (HSQC, HMBC, COSY and NOESY). The DMSO solution and the 14.1 T field (600 MHz) were choosen for the NMR analysis. The main signals in the proton NMR spectrum of 1 that were critical to the stablishment of the cyclisation were two double doublets at 3.24 ppm and 3.57 ppm (H-1"a and H-1"b - Figure 1) and a centre overlapped double doublet at 5.35 ppm (H-9') composing an ABX spin system. Moreover, the C-13 NMR spectra (C13CPD and DEPT135) showed two signals at 34.7 ppm and 47.9 ppm that were promptly assigned to C-1" and C-9', respectively. Until here the regioselectivity were still unresolved. Only the values of the chemical shifts for these presented nuclei for the two possible cyclisation pathways i. e. 12-exo-trig and 13-endo-trig4 that will lead to different macrocycles would not be an unequivocal statement. So, the HMBC experiment was fundamental for this stablishment. In the HMBC (d6, 65 ms) contour plot the correlations due to 3JH-T,C-IO', 34H-9\C-I5', 3^H-9\C-H'. 2Jh- 9',C-IO' and 3JH-II\C-9' certifies the linkage between the ortho carbon of benzamidic aromatic ring to the ancient olefinic carbon at malonic moiety. The main correlations that confirms the 12-exo-trig cyclisation process are those ones between the signals due to H-1"a,b and the ortho carbons C-7",3" of the benzilic moiety, due to a three bond long range coupling. Conversely, the correlations due to 3J between H-3",7" signals and C-1" are also registered (Figure 2a). Furthermore, the benzylic stereogenic center at position 9' could generate two diastereomers. Nevertheless, radical reactions are highly stereoselectives5 and because of the purity of the compound and the high quality of the NMR spectra, without duplicated signals, one can expected for only one diastereomer. So the facial diastereoselectivity should be inspected by nOe's experiments. Then, the solution of the macrocycle in DMSO were submitted to NOESY (d8, 400 ms and 800 ms) and ROESY (p15, 600 ms) experiments at 9.4 T (400 MHz \ + 82 iJdi NUULLAR MAC.M.; IC RF.SONANCL LbLUS MliETINC for 1H). The NOESY experiment showed negative nOe's for the bulky molecule and positive nOe's for the more mobile benzyl group. In there, H-9' exhibited no nOe's and only cosy artifacts with the vicinal H-1"a,b protons. In the ROESY experiment H-9' showed roe with H-1"a (3.24 ppm) and TOCSY with H-1"b (3.57 ppm). This results were not conclusive about the facial diastereoselectivity because the lack of nOe's does not mean that the nuclei are far apart. The same experiments were done for the compound dissolved in CDCI3. Again, no conclusive nOe's were observed for H-9'. Fortunatly we measured qualitative nOe's at an another different NMR field. So, DMSO and CDCI3 solutions of the macrociclic compound were evaluated by NOESY at 14.1 T (600 MHz for 1H) with mixing times of 120 ms and 400 ms. The experiment with the CDCI3 solution also did not showed any conclusive nOe's related to H-9'. On the other hand, and very lucky, the NOESY experiment performed on the DMSO sample with the mixing time 400 ms, showed a very discrete noe between the benzylic H-9' and H-2 from the sugar moiety (Figure 2b). These results exemplifies the dependence of Overhauser effects on the magnetic field strength and the molecular tumbling rates.6 For this molecule all these dependences were verified, it means, the very mobile system in CDCI3 with positive nOe's, the highly viscous DMSO solutions with negative nOe's and the field strength dependence, 9.4 T or 14.1 T. Another impressive observation was the positive nOe's for the benzylic group even in DMSO solution that implies in its faster mobility than the rest of the molecule. (a) (b) Figure 2 - a) HMBC correlations of H-1"a,b to H-7", H-3", H-2" and H10'; b) NOESY correlation of H-9' to H-2 REFERENCES: 5. Kohli R. M.; Walsh C. T. Chem. Comm., 2003, 3, 297. 6. Beckwith A. L. J.; Bowry V. W.; Bowman W. R.; Mann E.; Parr, J.; Storey J. M. D. Angewandte Chemie, International Edition, 2004, 43, 1, 95. 7. Pires M. C. Dissertação de Mestrado, 2009, Faculdade de Farmácia, UFMG 182p. 8. Baldwin J. E.; Adlington R. M.; Mitchell M. B.; Robertson J. Tetrahedron, 1991, 47, 30, 5901. 9. Srikanth G. S. C.; Castle, S. L. Tetrahedron, 2005, 61, 44, 10377. 10. Claridge T. D. W. Tetrahedron Organic Chemistry Series, 1999, 19, 382p. BRUKER-BIOSPIN, FAPEMIG, CNPq, CAPES 83 I- + ALfRfcMN MAY 2nd to 6th, 2011 HOTEL DO I'RADli. ANGRA DOS PO 21 UNDERSTANDING ENZYME PROMISCUITY AND REVERSE MICELLAR SYSTEM BY 1H NMR B.Z. Costa*1, A.J. Marsaioli1 1 Universidade Estadual de Campinas, Campinas, Brazil e-mail:[email protected] Keywords: STD, lipase promiscuity, reverse micelles. Enzyme reaction versatility is a phenomenon known as enzymatic promiscuity and indicates that the same enzyme may act on different reaction conditions, different functional groups and substrates, and/or performing different chemical transformations.1 Lipases are the most extensively described enzymes in the literature that present promiscuous performances. It is well established that lipases, in organic medium or in the presence of specific substrates can catalyze several organic reactions. Herein we considered the action of two lipases from Candida genus (C. antarctica and C. cylindracea) in Baeyer-Villiger oxidation2 of 2-pentylcyclopentanone (1), in the presence of hydrogen peroxide and octanoic acid in toluene (Fig. 1) o O t: II Lipase (5 mg) "O Octanoic acid (6.3 mmol; 100 uL) I hCU 30 % (12.7 mmoU 1,0 mL} 1 Toluene 110 inLl 58 (ittTOl; 10 pL Figure 1: Baeyer-Villiger oxidation of 2-pentylcyclopentanone (1) mediated by a lipase. To study this reaction at the molecular level, 1H-NMR was selected for having an arsenal of techniques to assess the dynamics of interactions between large and small molecules. In this work we used saturation transfer difference (STD)3 experiments and measurements of longitudinal (7?) and transverse (T2) relaxation times. NMR experiments were performed simulating reactions conditions in deuterated organic solvents, in presence and absence of the enzyme. The STD experiments were carried saturating the enzyme signal at -0.5 ppm, and the signal of the water present in the organic phase at 5.05 ppm. The tests conducted by saturating the water signal, employed saturated water molecules to report intermolecular interactions in the system, by transferring saturation for specific chemical species that participate in the supramolecular system formed. Experiments carried out by saturating the water signal indicated the formation of reverse micelles of octanoic acid in toluene. The saturation transfer for the alpha carbonyl hydrogen in octanoic acid (Fig 2), suggested that the hydroxyl group and bulk water are one signal both undergoing saturation. This also might indicate that this portion of the fatty acid is closest to the aqueous interior of the reverse micelle, which is compatible with that type of aggregation. The STD experiments irradiated at -0.5 ppm, in the presence and absence of enzyme did not cause any saturation transfer. The hydroxyl chemical shift changed from 12 to 6 ppm in presence of lipase indicating that a reverse micelle system organization of the octanoic acid molecules was induced under these conditions. j. 4. 84 Figure V. A) Off-resonance STD spectra (irradiation at 30 ppm). B) On-resonance STD spectra (irradiation at 5.05 ppm) of C. cylindracea system. C). On-resonance STD spectra (irradiation at 5.05 ppm) of C. antarctica system. The experimental results of T? and T2 indicated that both evaluated lipases participate in the proposed micellar system, as the relaxation times of octanoic acid decrease in the presence of the enzyme (Table 1). Additionally the 2- pentylcyclopentanone relaxation times indicated a participation in the micellar system, and its most likely location is near the nonpolar tail of octanoic acid. Table 1: Relaxation times variation of 2-pentylcyclopentanone in the absence and presence of lipase in the oxidation reaction conditions. Relaxation Time3 Lipase Composto AT* (s) AT2(S) Octanoic acid 0,187 0,058 C. cylindracea 2-pentylcyclopentanone 0,065 0,004 Octanoic acid 0,439 0,186 C. antarctica 2-pentylcyclopentanone 0,663 0,142 A T Tabsence Tpresence On going additional experiments, include DOSY and light scattering measurements for a better understanding of this supramolecular system. This micelle system was confirmed by testing with methylene blue. The dye was transferred together with water molecules to the organic solvent, only when the enzyme was present, ie, the lipase induced the micelle formation. Therefore, confinement of the enzyme in a restricted aqueous environment, limited by a narrow molecular barrier of fatty acids in an organic medium, is the necessary condition for this enzymatic reaction to occur, simulating a native environment for lipase and avoiding any possible denaturation due to contact with organic solvents. REFERENCES: 1- Hult, K.; Berglund, P. Trends Biotechnol., 2007, 231. 2- Rios, M.Y.; Salazar, E.; Olivo, H.F. J. Mol. Catai B: Enzym. 2008, 61. 3- Mayer, M.; Meyer, B. Angew. Chem. Int. Ed. Engl. 1999, 38, 1784-1788. CNPq, CAPES, FAPESP, ANP 85 I- + PO 20 NMR STRUCTURAL STUDIES OF PHYLLOSEPTIN-2 PEPTIDE AT DIFFERENT PH VALUES Naira de Oliveira Torres*, Rodrigo Moreira Verly, Dorila Piló-Veloso, Jarbas Magalhães Resende Departamento de Quimica/ICEx/UFMG *[email protected] Keywords: peptide antibiotics; peptide structure; amphipathic a-helix Several antimicrobial peptides have been found to be essential for the innate immune defense of different organisms. Anurans are valuable sources of these peptides. Many studies demonstrate that different anuran species store different classes of biologically active peptides 1 which are alternative agents against pathogenic bacteria and fungi. The peptide Phylloseptin-2 (PS-2) has been isolated from the skin secretion of the tree frog Phyllomedusa hypochondrialis that inhabits Brazilian tropical forests.2 Antimicrobial assays demonstrate that PS-2 exhibts marked antimicrobial activity against both Gram-positive and Gram-negative bacteria2'3. This cationic peptide is naturally amidated at C-terminus and it is composed of nineteen amino acid residues, of which three possibly charged histidine residues are found at positions 7, 17 and 18. Peptide primary sequence PS2 FLSLI PHAIN AVSTL VHHF-NH2 Multidimensional Nuclear Magnetic Resonance Spectroscopy is a powerful tool used in structural investigations of peptides and proteins, either in aqueous medium or in membrane mimetic environments. We have previously described the solution NMR structure-of PS-2 in a mixture of TFE-d2:H20 (60:40, v/v) at pH 7.0 (phosphate buffer). The peptide structure shows a well-defined amphipathic a-helical segment which is specially stabilized close to C-terminus due to electrostatic, aromatic and capping interactions.3 The obtained results indicate a partially charged state for the His-17 and His-18 side chains at pH 7.0. In order to further investigate the effect of these charged residues in the structural stability of the peptide, we have performed CD and NMR measurements of PS-2 in TFE:H20 mixtures (60:40, v/v) at different pH values. PS-2 was prepared by solid-phase peptide synthesis, by using Fmoc (9- fluorenylmethyloxycarbonyl) chemistry. The obtained peptide was purified by phase reverse High Performance Liquid Chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. CD spectra of the peptide in different TFE:H20 ratios and at different pH (acid, neutral and basic media) have been recorded. The data shows predominantly helical conformations for all TFE:H20 60:40 solutions, however the different spectral profiles suggest structural differences, which can be correlated to the variation of the effective charge of the three histidine side-chains. Two samples containing PS-2 at 2 mM in TFE-d2:H20 (60:40, v/v) were prepared at pH 5.6 and 8.6. In both samples 2,2-dimethyl-2-silapentane sulfonate (DSS) was used as internal reference standard. TOCSY, NOESY, 1H-13C HSQC and 1H-15N HMQC experiments were acquired at 20°C on a Bruker Avance-lll 800 MHz spectrometer equipped with a triple resonance (1H/13C/15N) 5 mm gradient probe. The assignment of backbone atoms was performed by the standard methodology proposed by Würthrich4 in combination with the heteronuclear experiments. The NMR spectra were processed with NMRPipe and then analysed using NMRVIEW. The obtained NOE intensities were converted into semi-quantitative 86 distance restrains and then structure calculations were performed using Xplor-NIH. The stereochemical quality of the lowest energy structures was validated by PROCHECK- NMR. The display, analysis, and manipulation of the three-dimensional structures were performed with the MOLMOL. The spin systems of all residues have been recognized on the TOCSY spectra. The more frequent amino acid residues in PS-2 sequence as Leu and His were identified by the combination of TOCSY, NOESY and heteronuclear experiments. These spin systems were assigned to each amino acid type and were connected by using the NOESY spectra. Many of the sequential connections were completed mainly on the basis of the strong dww connections (Fig. 1). The high number of cross peaks d«N(i,i+3), d a/5 (i,i+3), and doN (i,i+4) suggested that the peptide shows a significant helical content at both pH values. 200 NOE based structures of PS-2 have been generated from the restrains available in the NOESY spectra obtained from the samples at pH 5.6 and 8.6. These most stable structures have then been compared with the already published results obtained from a pH 7.0 sample.3 8.9 8.7 8.5 8 3 8*.l 7.9 7 7 7.5 7.3 7.1 6.9 6.7 6.5 1H chemical shift (ppm) Figure 1 - Amide-amide region of the NOESY spectra of Phylloseptin-2 in TFE-d2/H20 (60:40, v/v) at pH 5.6 (purple), 7.0 (turquoise) and 8.6 (black). The most stable structures obtained from Xplor-NIH calculations indicate that PS-2 shows an amphipathic distribution of its residue side chains within the helical segment, however the presence or absence of a positive charge on the His residues lead to important structural differences. REFERENCES 1. Rinaldi, A. C.; Curr. Opin. Chem. Biol., 2002, 6, 799-804. 2. Leite, J.R.S.A.; Peptides 2005, 26, 565. 3. Resende, JM et al. Peptides 2008, 29, 1633. 4. Würthrich, K.; NMR of Proteins and Nucleic Acids, Wiley, New York, 1986. CAPES, FAPEMIG, PRPq-UFMG, CNRMN 87 I- + PO 21 USE OF FILTER DIAGONALIZATION METHOD TO PROCESS 13C NMR STEADY STATE FREE PRECESSION SIGNAL OBTAINED DURING IN SITU ELETROCHEMICAL REACTION L.M.S. Nunes*1, T.B. Moraes2, C. J. Magon3, L.A. Colnago4 11nstituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil 2,3lnstituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil 4EMBRAPA Instrumentação Agropecuária, São Carlos, Brazil e-mail:nunes. luizafcb.gmail. com Keywords: FDM; 13C SSFP, organochlorine The coupling between high resolution NMR and electrochemistry techniques (EC- NMR) has gain importance in recent years with the improvements in cell design for in situ measurements1. All papers using EC-NMR involve the application for simultaneous in situ detection of electrogenerated species using 1H NMR, due to high sensitivity of the nuclei allowing fast data aquisition. Webster1 proposed the use of 13C NMR due to the well resolved spectra. However he did not implemented the 13C NMR-EC in situ analysis due to low the receptivity of the nucleus. The experiment with a high concentrated solution, with 25mM of the substrate took about 12 h using standard 13C NMR sequence. This long time did not allow the use of 13C NMR-EC in situ. To improve the 13C NMR signal to noise ratio (S/N) we have been using steady state free precession sequence (SSFP)2. This sequence uses., a train of pulses with same phase and amplitude separated by a time interval x< T2. This sequence can improve the S/N of 13C spectra by more than 5-fold when compared with standard sequence which is equivalent to a reduction in 20-fold in total scanning time3. In this paper we are showing that 13C NMR obtained with SSFP sequence can be used in EC-NMR experiments allowing a short total scanning time in approximately 10 minutes. However, this technique introduces several phase and truncation anomalies in the Fourier transform spectra. To solve this problem the SSFP time domain signal have been processed with filterdiagonalization method (FDM)4 The FDM is a nonlinear, parametric method for fitting time domain signals with summation of sinusoids. The FDM resolution is not limited by the Fourier Transform Uncertainty Principle. It solves the original large and ill-conditioned nonlinear fitting problem by converting it into pure linear algebra problems of diagonalizing some small data matrices in the frequency domain. Electrochemical measurements were performed using a potentiostat PalmSens (Palm Instruments BV) with a conventional three electrode system comprising carbon fiber filament as the working (WE) and the counter (CE) electrode. A silver wire was used as a pseudo reference electrode (RE). NMR measurements were performed with a Varian INOVA 400 MHz 1H NMR spectrometer utilizing a 10-mm probe. The 13C NMR measurements were obtained by SSFP sequence (figure 1) using a kI4 pulse with 30 ms acquisition time (at), 0.3 ms recycle delay (D), 8.000 scans (n). The sample used in the study was a 50mM solution of 9-choroantracene in CD3CN containing 0.5M Bu4NPF6, as the supporting electrolyte. The in situ EC-NMR experiments were performed with potentiostat coupled in the cell placed inside the high-field NMR spectroscopy, the potentiostat was located about 3 m from the superconducting magnet. The 13C SSFP measurements were performed for 10 minutes during electrochemical reaction. H 88 at D Figure 1: Representation of the 13C SSFP sequence, a TT/4 flip angle, at=0.1s and d1=0.3 ms and n=8000 The 9 chloroantracene SSFP spectra obtained with SSFP sequence are shown in figure 2. Figure 2a show the Fourier transform spectrum and figure 2b, the same spectrum, but processed with FDM. Figure 2: 13C SSFP NMR spectra obtained (a) Fourier transform and (b) FDM processing in the solution 50mM 9-choroantracene in CD3CN containing 0.5M Bu4NPF6 as the supporting electrolyte in a 10-mm o.d NMR tube. The figure 2(b) presented a spectrum with higher spectral resolution than the FT spectrum, figure 2(a). This diference is related to the limitation of Fourier transform method to the Uncertainty Principle. FDM is not limited but the number of acquisition points. Therefore, the FDM spectrum shows high resolution and no phase or trucation problem and can be a useful method to process 13C SSFP signal acquired during eletrochemical reactions. REFERENCES 1. Webster R.D.; Anal. Chem. 2004, 76, 1603-1610. 2. Carr, H.Y.; Physical Review, 1958, 112, 1693-1701. 3. Santos, P. M., Sousa, A.A. Colnago, L.A.; Appl. Magn. Res. 2011( accepted). 4. Mandelshtam V.A.; Progr. NMR Spectrosc, 2001, 38, 159-196. FAPESP, EMBRAPA Agricultural Instrumentation PO 21 USE OF FILTER DIAGONALIZATION METHOD TO SUPPRESS BROAD LINE IN 1H NMR SPECTRUM OF BREAST CANCER CELLS R.M.Maria*1,T.B.Moraes2,C.J. Magon2,W.F.AItei2,A. D.Andricopulo2,L.A.Colnago3 11nstituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil 2Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil 3Embrapa Instrumentação, São Carlos, Brazil rommaria(q)jqsc. usp. br Keywords: HR MAS; FDM; cell culture Nuclear magnetic resonance spectroscopy (NMR) has been a powerful technique for metabolites analysis in cultured cells, providing information about their biochemical status both in vitro and in vivo.1 The NMR analysis of biological tissues and intact cells which are heterogeneous samples is complicated due to two principal factors: restricted mobility of molecules and the difference in magnetic susceptibility.2 The molecular mobility in heterogeneous samples is restrict, making the dipolar coupling perceptible. In these samples there is a difference in the magnetic susceptibility, which, added to the dipolar coupling, contributes to the broadening of NMR resonances. However, these two factors can be reduced or eliminated by fast rotation of the sample in the magic angle spinning, 54.7°, in the static magnetic field direction. Normally, the sample spin rates are in the range of 2 to 5 kHz.3 In this paper we used High Resolution Magic Angle Spinning (HR-MAS) NMR to analyze breast cancer cells metabolites and the use of Filter Diagonalization Method (FDM) as an alternative method to Fourier transform to process the time domain NMR signal to frequency domain. FDM is a nonlinear, parametric method for fitting time domain signals through sum of sinusoids. The method resolution is not limited by the Fourier Transform Uncertainty Principle and so it solves the original large and ill- conditioned nonlinear fitting problem by converting it into pure linear algebra problems of diagonalizing some small data matrices in the frequency domain. The 1H NMR spectra of the cells were acquired using an INOVA 400 spectrometer with a 9.4 T magnet (Varian, Palo Alto, CA) and high resolution MAS probe. The spectra were acquired with presaturation (1.5 s), 90° flip angle, 3 s acquisition time, recycle delay of 3 s, 256 scans and spin rate of 2800 Hz. Human breast cell line, MCF-7, was maintained in DMEM (modified Dulbecco's Minimum Medium - Cultilab) enhanced with 10% fetal calf serum (Cultilab). The cells were incubated at 37°C in a humidified atmosphere with 5% of C02. Subcultures were obtained by treating cells with trypsin in PBS (phosphate buffered saline) for 2 minutes and so, 2 mL of culture medium was added and the cells were centrifuged in 1.000 rpm for 5 minutes. After that, the medium was carefully extracted to not destroy the pellet formed. The medium was discarded and the cells were washed with phosphate buffered saline (PBS). Figure 1 shows a typical Fourier Transform HR-MAS spectrum of MCF-7 cells. In this spectrum it is possible to observe tenth of sharp signals. Although the MAS technique improves the spectral resolution of small molecules in the cells, the spinning is not fast enough to reduce the line width of the cell membranes components and others immobile structures. Therefore, it was necessary to eliminate these broad signals of the immobile molecules to obtain high resolution spectra. Currently, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used as a filter to eliminate these broad signals.4 As larger molecules have shorter transverse relaxation time, T2, they can be suppressed by collecting the FID approximately after 3T2. 13th NUi II.Ml M.\(iM riCRKSONANCKl SI RS MEETING J lljÁlI ID • « « 2 D Elffnt Figure 1: HR-MAS spectrum of tumoral cell culture (MCF-7). Figure 2 shows the culture cell (MCF-7) expanded HR-MAS spectra for breast cancer cells processed with FT (black) and FDM (red). The FDM can easily suppress the broad lines from the sharp ones and can be an alternative to the CPMG filter. FDM can also be used to suppress the water peak (data not shown) without the use of presaturation. j I ia J1 )á j Mil i IM •*sÀ ÉL „tai —i 1 1 1 > 1— I 2 0 6(ppm) Figure 2: Real component of the Fourier Transformed spectrum of breast cancer cell line (MCF-7) (black) and the FDM spectrum of the same NMR data (red). With these results we can conclude that FDM can be a powerful technique to process the HR-MAS spectra of heterogeneous materials, such as breast cancer cell, without using CPMG filter and water presaturation method. REFERENCES: 1. Merz A.L. e Serkova N.J. Biomark medicine. 2009, 3 2. Cuperlovic-Culf M.; Barnett D.A; Culf A.S. e Chute I. Drug discovery today. 2010,15 3. Sitter B.; Bathen T.F.; Tessem M. e Gribbestad I.S. Progress in nuclear magnetic resonance spectroscopy. 2009, 54 4. Claridge T.D.W, High-resolution NMR techniques in organic chemistry, 1999. FAPESP, Embrapa Instrumentação 91 I- + PO 21 THE APPLICATION OF SOLID STATE NMR TO EVALUATE THE QUALITY OF COMMERCIAL DRUGS L.A.M. Magalhães1, A.G. Ferreira1, J. Ellena2, T. Venâncio1 1 Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil 2lnstituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil lyepemapalhaes&.yahoo.com.br Keywords:mebendazole; solid state NMR; polymorphism Most of the drugs in solid state can be crystallized in different forms, in a behavior known as polymorphism. This is a serious problem for the pharmaceutical industry, because the bioavailability of the drug can be dependent of the properties of the different crystals. The polymorphism is dependent of the temperature, /humidity, light incidence, synthetic pathway, type of the additives used in the formulation, etc. Nowadays several techniques have been used to evaluate commercial drugs, such as X-Ray diffraction, Raman and IR spectroscopy, thermal analysis, solid state NMR, etc. As the problem is not so trivial to be solved, it is recommended to use a pool of techniques, because all of them have at least one limitation. In Brazil the agency that controls the registration of new medicines and the quality of them is named ANVISA, and nowadays there is no regulation to control the polymorphism in terms of quality of the product, but only the registration of new polymorphs. Several pharmaceutical industries, especially those ones that produce generic medicines, receive raw material from other countries. Regarding the long journey to be delivered in Brazil, any problems during the trip can be detected after a judicious analysis of this raw material. The analysis of the formulated drug is also important to ensure a good quality of the medicine offered to the population. Hence, our aim with this work is to try to include the solid state NMR in this pool of techniques used to characterize the polymorphism in Brazil. To show that possibility we have used mebendazole (Fig 1) as a model, because it is easier to acquire it from the local commerce. Mebendazole can exist in three different forms: A, B and C. The form A is more stable and had no anti-helmintic activity when alone or when present above 30% in polymorphic mixtures, the form B is more soluble but it is more toxic to the organism, and form C is the pharmaceutically preferred because its solubility is sufficient to guarantee optimal bioavailability.1,2 o Figure 1: Chemical structure of Mebendazole. In this study we used standard samples of mebendazole that were identified as form A and C by using X-Ray Powder Diffraction and Raman.3 Five commercial samples available in the Brazilian market from three distinct manufacturers were analyzed: MAN A (batch 1), MAN B (batches 1 and 2) and MAN C (batches 1 and 2). The experiments were performed in a Bruker Avance III NMR equipment by employing a 9.4T Oxford Magnet. The samples were packed in a 4mm diameter cylindrical zirconia rotor and spun at 5 kHz in a solid state probehead. The pulse sequence used was CP-TOSS (Cross Polarization - Total Suppression of Sidebands), with 2ms of contact time and 5s of recovering time. f % 92 •'ill Ml II \i< \I.\fiM- NC RI-SOV\N(l. I MU'-MirilM. Figure 2 shows the CPTOSS 13C NMR spectra of the polymorphs A and C of mebendazole and of the five commercial samples analyzed in this work. The main difference observed in the spectra appears in the aromatic region between 110 and 145 ppm. The signals at approximately 35 and between 60 and 110 ppm refers to samples excipients (as polysaccharides). and C of Mebendazole and of the five commercial samples Other signs that show the difference between the polymorphs are the methyl groups at 51.4 and 54.5 ppm for the polymorphic A and C respectively, and the signal of the carbonyl of the benzoyl group at 195.0 for form A and 197.0 for form C. It is important to note that the samples MAN B/ Batches 1 and 2 are manufactured by the same industry, however showed different polymorphs for different batches, demonstrating the importance of conducting an effective quality control of the formulated drug available in the market. No mixture of polymorphs was identified in this work. The results show that solid state NMR is able to discriminate the mebendazole polymorphs, as well as commercial samples. The minimum time required for the analysis is 5 min by considering 100% of one of the polymorphs. In a mixture, depending on the amount of both polymorphs, it is possible to spend approximately 30 min. The solid state NMR has the advantage of presenting a spectrum much clearer and easier to interpret in relation to IR and Raman spectroscopy, in a relatively short time, and the X-ray powder diffraction is effective in detection of relatively simple structures, but ineffective for the analysis of commercial products, where there are several other compounds in addition to the possible other polymorphic forms of the drug in question. REFERENCES: 1. Ayala, A. P.; Siesler, H. W.; Cuffini, S. L. J. Raman Spectrosc. 2008, 39, 1150-1157. 2. Froehlich, P. E.; Gasparotto, F. S. Rev. Ciênc. Farm. Básica Apt. 2005, 26(3), 205-210. 3. Martins, F. T.; Neves, P. P.; Ellena, J.; Cami, G. E.; Brusau, E. V.; Narda, G. E. J. Pharm. Sci. 2009, 98(7), 2336-2344. FAPESP (09/13860-2), CNPq (475903/2009-9, 142384/2010-0) 93 I- + PO 38 INTERNAL GRADIENT MAPPING WITH DISTANT DIPOLAR FIELD CONTRAST E.V. Silletta, M.B. Franzoni, R.H. Acosta* FaMAF-Universidad Nacional de Córdoba & IFEG-CONICET, Cordoba, Argentina racosta&famaf. une, edu. ar Keywords: porous media, internal gradients, distant dipolar field A particle diffusing in a confining medium is a general model for a number of physical, chemical, biological, and industrial processes. It may describe organic molecules or metabolites in biological cells or brain tissue, reactive species near porous catalysts, ions near rough electrodes or cellular membranes, oxygen in human lungs, water molecules in cements or rocks, etc. When such a particle encounters an interface, they may interact in different ways depending on their physical and chemical properties. The interaction at the microscopic level can often be represented in terms of "reflection" and "absorption" at the interface. In the former case, the particle does not change its state and continues to diffuse in the bulk. In the latter case, the motion of the particle is terminated, either by absorption on or transfer through the interface, by chemical transformation into another particle, or by surface relaxation in a NMR experiment. NMR is of particular interest as being a method to "label" or "encode" Brownian trajectories of spin-bearing particles by using magnetic fields. The heterogeneous nature of the samples broadens the spectrum of the NMR measurement due to the existence of magnetic susceptibility differences between materials in the samples, for example, between a porous matrix with saturating fluid local magnetic field gradients develop at the interfaces. These local magnetic fields with pronounced spatial variations are commonly referred to as "internal gradients" and the main facts governing their strength depend on the susceptibility difference between the materials, the applied magnetic field and the pore size, shape and the geometry of the pore network. The internal gradients scale roughly with the applied magnetic field as: 9 °c Ax B0 where Ax is the magnetic susceptibility difference experienced between the pore surface and the detected fluid, and B0 is the applied field. The presence of the internal gradient often interferes with NMR relaxation and diffusion measurements and great effort has been made in the design of pulse sequences that can cope with this effect [1,2], On the other hand, decay rates due to susceptibility differences can readily be exploited to obtain characteristics of porous media by using the information provided by the internal gradients [3], An alternative method to probe the influence of internal gradients relies on the use of the Distant Dipolar Field (DDF). In liquids at high magnetic fields the dipolar interaction that is normally averaged out can be reintroduced by the application of sequences like the Cosy Revamped by Asymmetric Z-Gradient Echo Detection (CRAZED) [4], If the symmetry on the sample is broken, as for instance by the application of a magnetic field gradient, intermolecular Multiple Quantum Coherences (iMQC) are converted into observable signal by intermolecular dipolar couplings. The area of the gradients (GT) defines a characteristic length scale, referred to as the correlation distance dc = 7t/yGT that is typically between 10 - 10 mm, over which the magnetization is highly modulated. In macroscopic homogeneous samples the correlation distance plays an unimportant role, unless it is comparable to the diffusion occurring during the time of the sequence [5], However, in heterogeneous samples, the contrast in an imaging ^ + 94 experiment can be greatly influenced, since the iMQC signal provides a direct measure of the dipolar field at a selected correlation length [6], Figure 1: Spin echo images with echo time TE =3 ms, 256 x 256 acquired pixels and slice thikness of 1 mm. a) Reference image with T2 contrast, b) Difference of iDQ weighted image with reference. The influence of internal gradients is clear near the capillaries coated with iron oxide. In this work we explore the use of double quantum intermolecular coherences (iDQC) as a contrast agent to probe the internal gradients of a model sample. Recently a set of capillaries arranged in a standard NMR tube was introduced as a suitable model sample to probe the internal fields in a direct visualization [7], here we use a set of capillaries of outer diameter OD = 1.4 mm contained in a 10 mm NMR tube which is filled with distilled water. The exterior walls of the capillaries are coated with a solution of iron oxide diluted in varnish at 0.1%; four of the capillaries are left untreated and are used for control purposes. Figure 1a shows a spin-warp spin echo (TE = 3 ms) 2D image with a 1 mm slice selection along the long axis of the sample. Experiments were performed in a 7T Bruker Biospin magnet using an Avancell console. In order to generate the iDQC contrast, a CRAZED sequence is applied prior to the imaging sequence, in this way only signals arising from distant dipolar couplings contribute to the image formation. Figure 1b shows the difference between a reference image (Fig. 1a) and an iDQC image acquired with a correlation distance dc = 80 |am. The iDQC signal generation in the presence of the internal gradients can be observed to be greatly influenced. From a pixel by pixel fitting the values of the internal gradients can be obtained. Images acquired as a function of the correlation distance will be shown. REFERENCES: 1. M.D. Hurlimann; J. Magn. Reson. 1998 131, 232. 2. R.M. Cotts; M.J.R. Hoch; T. Sun; J.T. Markert; J. Magn. Reson. 1989 83, 252. 3. Y.-Q. Song; S. Tyu; P.N. Sen; Nature 2000 406, 178. 4. W.S. Warren; W. Richter; A.H. Andreotti; B.T. Farmer, II; Science 1993, 262, 2005. 5. P.P. Zãnker; J. Schmiedeskamp; H.W. Spiess; R.H. Acosta; Phys. Rev. Lett. 2008, 100, 213001. 6. L.S. Bouchard; W.S. Warren; J. Magn. Reson. Med. 2005 177, 9. 7. H. Cho; S. Ryu; J.L. Ackerman; Y.-Q. Song; J. Magn. Reson. 2009 198, 88. CONICET, FONCYT, SECYT-UNC, MPIP-MPG 95 I- + PO 20 PENDANT CHAIN DYNAMICS IN MODEL PDMS NETWORKS PROBED BY SPIN DIFFUSION EXPERIMENTS R.H. Acosta*1, M.B. Franzoni1, M.A. Villar2, E.M. Vallés2, D.A. Vega3, G.A. Monti1 1FaMAF-Universidad Nacional de Córdoba & IFEG-CONICET, Córdoba, Argentina 2PLAPIQUI-Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina 3Departamento de Física, Universidad Nacional del Sur, Bahia Blanca, Argentina racosta&.famaf. une, edu. ar Keywords: polymer networks, chain dynamics, spin diffusion Over the last 5 decades the dynamic response of entangled polymer melts has been an outstanding problem in polymer science. Most of the viscoelastic and diffusive properties of polymer melts and concentrated polymer solutions are profoundly influenced by topological interactions. The most successful model to deal with topological constrains is the tube model. According to this model, the topological confinement exerted on a given molecule by the surrounding media can be modeled as a hypothetical tube that severely suppress the motion perpendicular to the tube's local axis, but permits the diffusion along the tube. Polydimethylsiloxane (PDMS) networks are ideal systems to probe chain dynamics, however, in the context of spin diffusion experiments no work has been carried out to our knowledge to the moment. The usual approach for determination of domain size probed by interaction between different structures does not apply as the whole network, entangled and dangling chains have the same chemical structure. This means that the whole sample has the same spectroscopic components. In this work we propose to use the differences between transverse relaxation times to generate the both the generation of the magnetization gradient and the determination of the spin diffusion. Two different experiments are proposed in order to obtain information on the dynamics of the pendant chains, that is, chains that are chemically attached to the network by one end while the second end is free or physically entangled with the network [1,2], In the first experiment the pendant chains are selected by the use of a dipolar filter (DF) [3] and left to equilibrate with the network. The second approach is to select the network chains by the application of a Double Quantum Filter (DQF) [3-5] that will leave magnetization only on those chains that have a very well defined residual dipolar coupling, which arises from the restricted mobility of the chains that form the network. Detection is performed with a CPMG sequence at 0.5 T with a Bruker Minispec mq20. 2i (ms) 2i [ms] Figure 1: a) Hahn eco evolution for a PDMS network, b) Evolution with diffusion time from a DQF signal. Magnetization diffusion from elastic to pendant chains is observed. 96 13th NUCLEAR MAGNETIC RESONANCE USERS MEETING Relaxation of transverse magnetization is mainly determined by the dipole- dipole magnetic interaction between protons. This interaction is modulated at different extents by molecular motions and is sensitive to differences in the motion of the chains that form the polymer network. This technique is very precise to measure the ratio between the entangled chains that form the network and the pendant material [6]. Figure 1a shows a typical Hahn Echo evolution, two different contributions are clearly observed, for short evolution times a Gaussian decay is given by the chain that form the network while at longer times a liquid-like (exponential) decay is obtained for the pendant material. By fitting of this decay curve a very well determined ratio of both types of chains can be obtained. Figure 1b shows the CPMG data as a function of the diffusion time, for short times the rigid-like chains present the greatest contribution while as time increases a raise in the liquid-like ones is observed (DQF). We performed a series of experiments for different networks with very distinctive characteristics. The cross-linker used was of two types, trifunctional and tetrafunctional [1,2,6] with PDMS chains of molecular weight Mw = 23900 g/mol. This means that the networks have very different structural properties, being the tetrafunctional ones more rigid. The length of the pendant material was also used as a variable, we chose to add very short chains that will on the average not entangle physically to the network (Mw = 26400 g/mol), and longer ones that present one physical entanglement in average (Mw = 60600 g/mol). b) Figure 2: a) Evolution of pendant fraction for DF signals, b) Evolution of pendant fraction for DQF signals. In the first case the evolution is independent of the chain length or network structure: triangles: trifunctional; squares: tetrafunctional. Figure 2a shows the pendant material fraction with magnetization as a function of diffusion time (DF), it can be observed that all networks present the same decay, independent of the network type or chain length. This is an indication that the monitored process corresponds to a Nuclear Overhausser Effect (NOE) rather than to spin diffusion [7], On the other hand, Fig. 2b shows that diffusion from the network to the pendant chains (DQF) present a dependence on network structure. REFERENCES: 1. R.H. Acosta; D.A. Vega; M.A. Villar; G.A. Monti; E.M. Valles; Macrom. 2006 39, 4793. 2. R.H. Acosta; G.A. Monti; M.A. Villar; E.M. Valles; D.A. Vega; Macrom. 2009 42, 4674. 3. M. Mauri; Y. Thomann; H. Schneider; K. Saalwàchter; SSNMR 2008 34, 125. 4. Saalwachter, K.; Prog. NMR Spectrosc. 2007, 51,1. 5. Baum, J.; Pines, A.; J. Am. Chem. Soc. 1986, 108, 7447. 6. D.A. Vega; M.A. Villar; E.M. Valles; C.A. Steren; G.A. Monti; Macrom. 2001 34, 283. 7. M. Gaborieau; R. Graf; H.W. Spiess; SSNMR 2005 28 160. CONICET, FONCYT, SECYT-UNC, MPIP-MPG 97 I- + PO 21 MULTINUCLEAR SOLID STATE NMR INVESTIGATION OF TWO POLYMORPHIC FORMS OF CIPROFLOXACIN-SACCHARINATE Y. Garro Linck1, A. K. Chattah1, R. Graf2, C. B. Romanuk3, M. E. Olivera3, R. H. Manzo3, G. A. Monti*1 and H. W. Spiess2 1 FaMAF- Universidad Nacional de Córdoba & IFEG-CONICET, Cordoba, Argentina. 2Max-Planck-lnstitut für Polymerforschung, Postfach 3148, D-55021 Mainz, Germany. 3Departamento de Farmacia, Facultad de Ciências Químicas, Universidad Nacional de Córdoba, Ciudad Universitária, 5016, Córdoba , Argentina monti&.famaf. unc.edu. ar Keywords: NMR Spectroscopy, DQMAS, Polymorphism. Multicomponent crystalline pharmaceutical solids, as for example complexes or salts, are usually developed to improve the pharmaceutical performance of a single organic molecule in terms of solubility, stability, bioavailability and/or organoleptic properties.(1,2) On the other hand, the phenomenon of polymorphism and its influence on the chemical and physical properties of molecular crystals is well known.(3) This is especially true for pharmaceutical compounds, where polymorphic changes in the drugs can lead to significant effects on bioavailability. The present multicomponent compound is a new ciprofloxacin saccharinate recently obtained.(4) Ciprofloxacin (CIP), is a widely prescribed, broad-spectrum oral fluoroquinolone antibiotic approved for the treatment of several types of infections. Interestingly, ciprofloxacin saccharinate (CIP-SAC) can exist in two different polymorphic forms, CIP-SAC (I) and CIP-SAC (ll).(5) 1H NMR spectra under very fast MAS were recorded for the three samples. 20 15 10 5 0 -5 S1H/ppm Figure 3. Chemical structure of CIP-SAC showing the labels used in this work and 1H MAS spectra at 60 kHz of (a) CIP, (b) CIP-SAC (I) and (c) CIP-SAC (II). Figure 1 shows the chemical structure of CIP-SAC and the labels used in this work and the 1H NMR spectra for (a) CIP, (b) CIP-SAC (I) and (c) CIP-SAC (II). In the spectrum of CIP, aromatic protons (H(2), H(5) and H(8)) appear unresolved. Signals from methylene groups (H(1b) and H(1c)) are resolved but are observed at negative ppm. These negative chemical shift values can be attributed to strong interactions between methylene groups and neighboring aromatic moieties/6' In the 1H solid state spectra of CIP-SAC (I) we can observe three signals in the range from 10-15 ppm, two of them correspond to the carboxyl proton, a clear evidence of the existence of at least two molecules in the asymmetric unit in this polymorph.(4) Methylene protons in CIP-SAC (I) are detected at negative ppm values under the presence of ring currents from aromatic moieties in close spatial proximity. CIP-SAC (II) presents fewer resonance than the former sample. No shifts to negative ppm values can be seen in this compound, indicating a substantial If- t 98 difference in the crystal structure compared to the other polymorph. Note that the NH(14)+ signals in the two polymorphs are shifted towards lower frequency values relative to that of CIP. This is indicative of the salt formation. In contrast, because of the zwitterionic character of CIP, on formation of the salt, protons from the piperazine (Pip) group do not exhibit significant changes. 18 1S 10 5 0 -5 18 15 ' 10 * 5 ' 0 -5 18 15 10 5 0 -5 Single quantum dimension Singie quantum dimension Single quanlum dimension Figure 2.1H-1H DQMAS NMR spectrum of: (a) CIP-SAC (II), (b) CIP , (c) CIP-SAC (I). The 1H-1H DQ MAS correlation spectrum of CIP-SAC (II) (Figure 2 (a)) shows strong autocorrelation peaks at A>DQ= 6.6 ppm and CODQ= 15.4 ppm, corresponding to Pip-Pip and saccharine-saccharine correlations respectively. The close spatial proximity of aromatic proton sites from neighboring molecules can be directly observed in the 1H-1H DQ correlation spectrum of CIP-SAC (II). The DQ signal observed at CODQ = 13.8 ppm = 7.2 + 6.6 ppm results from a DQ coherence between the aromatic proton sites H(5) and H(2). The intramolecular distance, however, is by far too large to excite a DQ coherence between these two sites, providing direct evidence for a molecular packing with close proximities between aromatic moieties of neighboring molecules. This kind of interactions is a key element of the complex molecular organization in CIP-SAC (II). Figure 2 (b) shows the 1H-1H DQ NMR correlation spectrum of CIP. A strong autocorrelation signal is present ODQ= 3.3 + 3.3 = 6.6 ppm between piperazine protons, allowing the assignment of these proton sites. The cyclopropyl proton sites are well resolved and show correlation signals with protons of the aromatic groups. The 1H-1H DQ MAS spectrum of CIP-SAC (I) (Figure 2 (c)) shows multiplicity of sites. In spite of the complexity, the DQ correlation pattern of CIP-SAC (I) yields features of both, the CIP-SAC (II) DQ correlation spectrum as well as the CIP DQ correlation spectrum . To conclude, CIP-SAC (I) and CIP-SAC (II) exhibit similarities in the molecular conformation. The unknown crystal packing of CIP-SAC (I) presents a molecular site with a packing similar to CIP-SAC (II) and a site with an arrangement closer to that in the pure drug. REFERENCES: 1 R. D. B. Walsh et al.; J. Chem. Commun. 2003, 186. 2 J. F. Remenar et al.; J. Am. Chem. Soc. 2003, 125, 8456. 3 Polymorphism in the Pharmaceutical Industry, Edited by R. Hilfiker, Wiley-VCH, 2006 4 C. B. Romanuk et al.; J. Pharm. Sci. 2009, 98, 3788. 5 C.B. Romanuk et al.; Int. J. of Pharmaceutics 2010, 391, 197. 6 R.M. Gomila, et al. Journal of Molecular Structure (Theochem) 2000, 531, 381. ANPCYT, CONICET, MINCYT CORDOBA, SECYT UNC, MPIP-MPG 99 I- + PO 20 SSNMR AND DIELECTRIC STUDY OF VULCANIZED POLYBUTADIENE RUBBER A.L. Rodriguez Garraza1, P. Sorichetti2, A.J.Marzocca1, C.L.Matteo 2, G.A.Monti*3 1LPyMC, Departamento de Física, Facuitad de Ciências Exactas y Naturaies, Universidad de Buenos Aires, Ciudad Universitária, Pabeilón I, C1428EGA, Buenos Aires, Argentina. 2Facuitad de Ingeniería, Departamento de Física, LSL, Universidad de Buenos Aires, Av. Paseo Colon 850, C1063ACV, Buenos Aires, Argentina. 3FaMAF- Universidad Nacional de Córdoba & IFEG-CONICET, Cordoba, Argentina. e-mail:monti(p).famaf. unc.edu.ar Keywords: polybutadiene;13C SSNMR; dielectric. Polybutadiene rubber, BR, is one of the most popular synthetic rubbers when combined to form blends, due to its excellent mechanical properties. One of the most extended uses of BR is in the tread and sidewalls of tyres where it is blended with other rubbers such as natural rubber NR and styrene butadiene rubber SBR This work continues our researches of the influences of the molecular structure and the cure level in the physical properties of polybutadiene rubber [1, 2], The cure reaction of two compounds of high- and low-cis BR prepared with the systems TBBS (n-t-butyl-2-benzothiazole sulfenamide)/sulfur was characterized by means of rheometric analysis. The correlation between dielectric properties and cure level was studied by broadband dielectric spectroscopy. The correlation between microstructural changes and cure level was detected by 13C solid state nucelar magnetic resonance (SSNMR) experiments. The samples studied this work were prepared with two types of commercial polybutadienes provided by Lanxess: Buna CB-55 NF (BR2) (produced by neodymium catalysis), and Buna CB-25 (BR1) (lithium grade BR). Table 1: Microstructure and density (pp) of the polymers (as provided by the supplier) cis 1,4 trans 1,4 vinyl Mn Degree of Polymer content content content (g/mol) Polydispersity branching PP 33 [g/cm ] (%) (%) (%) BR1 97 2 1 130000 2.6 15 0.91 1.3 < 5 0.91 11 BR2 38 51 125000 These compounds were characterized at 433 K by means of torque curves obtained with an Alpha MDR 2000 rheometer [2], Solid state NMR expermiment were conducted on a BRUKER AVANCE II spectrometer operating at 300.13 MHz for protons and 75.4 MHz for 13C., equiped with a 4 mm CP/MAS probe. The 13C signals were acquired using direct polarization, n/2 pulse duration was 5 jus, under MAS condition (3 kHz) and inverse gated broad band proton decoupling. Dielectric relaxation measurements in the range from in the range from 10 Hz to 1GHz at room temperature (295 ± 1 K). The dielectric relaxation of polymers was anlyzed using the Havriliak and Negami a (HN) dielectric function s(co) — (co) = stF+Azj l + (icor0) '(1) [3]. The relevant parameter are the relaxation strength as, the limiting value of the permittivity at low frequencies elf and the relaxation time r0 . Figure 1 shows the change in the amount of cis and trans molecular structures during the vulcanization of the compound with high cis content obtained in our NMR measurements. In this plot an over cured sample was 100 also included. In these samples the content of vynil structure was not detected. It is evident that not only the change in the network structure due to the formation of crosslinks could affect the parameters involved in equation (1) but also the molecular microstructure. The isomerisation c/'s to trans once the vulcanization process advances is evident from these data. These results agree with that obtained in other researches of sulphur-cure high cis BR by means of NMR [4, 5]. This change necessary will produce a decrease in the dielectric response once the vulcanization advances because the structure 1,4-trans has no dipole moment. While Ae decreases, eLF is almost constant as the curing reaction progresses. On the contrary, in the BR2 compound, with a significantly lower c/s-(38%) and higher vinyl-(10%) content, the change in the molecular microstructure of this compound during curing, as measured by NRM, are not significant. Therefore, the variation in Ae, s/_Fand r01 I; ; are associated with the modifications of the crosslinked network, with the consequent change in the crosslink density during the advance of the vulcanization reaction. Table 1: Crosslink density, and dielectric relaxation parameters BR1 BR2 Cure time Hcs(10"4) Ae Cure time HCS(10^) As £LF x [ps] £LF T [ps] [s] [mol/ml] 0 [s] [mol/ml] 0 555 0.300 0.54 2.58 75 775 0,655 0,21 2,42 1561 690 1.084 0.49 2.62 121 825 0,867 0,20 2,45 1181 780 1.188 0.46 2.59 271 950 0,961 0,23 2,47 541 960 1.199 0.36 2.61 321 1130 0,970 0,33 2,49 377 1800 0.977 0.34 2.59 358 1850 0,739 0,41 2,53 229 85. 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 3500 cure time [sj aire time [s] Figure 1: Polymers microstructure, obtained by NMR experiments, as a function of the cure time REFERENCES 1. A.J.Marzocca; A.L. Rodriguez Garraza; P.Sorichetti; H.O.Mosca; Polymer Testing, 2010,29, 477. 2. A.J.Marzocca; A.L. Rodriguez Garraza; M.A.Mansilla; Polymer Testing 2010, 29, 119. 3. S.Havriliak; S.Negami; J.Polym.Sci., Polym. Symp. 1966, 14, 89. 4. R.S.CIough; J.L.Koenig; Rubber Chem. Techno!. 1989, 62, 908 5. S.R.Smith; J.L.Koenig; Rubber Chem. Technol. 1992, 65, 176. MINCyT CORDOBA, SECyT UNC, CONICET, ANPCyT ioi H PO 22 WHY STUDY THE SIGN OF THE GEMINAL COUPLING CONSTANT? Denize C. Favaro*, Cláudio F. Tormena Institute of Chemistry, UNICAMP - Caixa Postal 6154 CEP-13084-862 e-mail: den fa varo(3).iqm. unicamp.br Keywords: geminal coupling constant, HSQC-TOCSY-IPAP, hyperconjugative interactions. Long-range heteronuclear coupling constant are extensively used for determination of constitution, configuration and conformation of molecules, and provide powerful tools in structural studies of small organic molecules, biomolecules and elucidation of natural products.1 Although, vicinal spin-spin coupling constant (SSCC) continue to be the most commonly used NMR parameter for stereochemical analyses, during the last decades it has been observed a notable increase in the use of heteronuclear "JCH (n = 1 and 2) 2 1 3 SSCC for studying conformations and configurations. While JCH and JCH are positive, 2 JCH coupling constant are characterized by changing their sign from positive to negative. It should be highlighted that determination of relative signs of couplings constants is important not only for theoretical aspects, but also in practical structure 3 1 3 2 determination. As was observed for JCH and JCH coupling constants, the geminal JCH couplings is transmitted mainly by the Fermi contact (FC) contribution,2,4 which means 2 that hyperconjugative interactions should affect considerably JCH in a fragment X-C-Y, where C is a carbon atom. In this way, two rules have been applied to explain the experimental "anomalous" results. First, lM where interactions transferring charge into any of the antibonding orbitais belonging to the coupling pathway yield a positive 2 increase to KXY (Reduced SSCC); while the second, llM, involving interactions which transfer charge from any of the bonding orbitais belonging to the coupling pathway 2 2 yield a negative increase to KXY In this work we will describe the influence of the 2 hyperconjugative interactions in the coupling constant JCH in 2-F-4-Í- butylcyclohexanols, see scheme 1. (i) (H) (ill) (IV) Sheme 1. 2-F-4-f-butylcyclohexanols 2 5,6 JCH couplings were measured using the HSQC-TOCSY-IPAP pulse sequence. Considering hyperconjugative interactions plays an important role for the Fermi contact transmission mechanism of the long-range couplings, the energies involving into hyperconjugative interactions were evaluated using the Natural Bond Orbital (NBO) analysis.7 Table 1. Experimental and theoretical geminal coupling constants (Hz) for the 2-F-4-Í- butylcyclohexanols l-IV. Couplings pathway (I) (II) (III) (IV) JciH2 3.0 (4.09) -2.6 (-2.56) -2.8 (-2.46) -3.1 (-5.48) Jc2H1 -4.6 (-2.92) -4.4 (-4.75) 4.2 (4.79) -4.5 (-3.51) Jc2H3a -7.7 (-6.97) <1 (2.05) 1.6 (3.47) -6.9 (-6.60) Jc2H3e -6.5 (-6.18) -7.5 (-6.57) -7.2 (-6.55) -6.7 (-6.24) + 102 Theoretical values. From data listed in Table 1, it can be observed that there is a strictly correlation between sign of the geminal coupling constant (2Jcpw) and orientation of the X substituint (X = F or O) in relation to couple hydrogen. For example, for the compound 2 2 2 2 III Jc2hi and Jc2H3a are positive but JCih2 and Jc2H3e are negative. For positive values the dihedral angle between H-C-C-X is around 180 degree, while for negative values dihedral angles are around 60 degree. This behavior can be assigned to hyperconjugative interactions (Table 2) involving the H-C-C-X fragment. The Diff values from Table 2 can be used as indicative of electronic derealization (donation and back- donation) along of geminal coupling constants pathway. Table 2. Main hyperconjugative interactions (kcal.mol1) affecting the 2Jc2hi, 2Jc2H3a, 2Jc2H3e and 2Jçih2 in compound III. *JC2H1= 4.2 Hz *Jc2H3a= 1.6 Hz D A HI Back Diff. D A HI Back Diff. /T* OC1H1 o*C2F 4.77 1.02 3.75 Oc3H3a O C2F 5.32 1.12 4.20 JciH2 = -2.8 Hz Jc2H3e = -6.9 Hz D A HI Back Diff. D A HI Back Diff. OC2H2 o*cic6 3.23 1.71 1.52 OC3H3e 0*C1C2 3.42 1.60 1.82 D - donor bonding orbital; A - acceptor antibonding orbital; HI - hyperconjugative interactions; Back - back donation; Diff. = HI - Back. Similar analysis can be performed for remaining compounds. For all studied compounds a relationship between the positive values of the geminal coupling constants and the balance of the main hyperconjugative interactions (Table 3) involving the coupling pathway was established. 1 2 2 Table 3. Main hyperconjugative interaction (kcal.mol" ) affecting the JCiH2and Jc2H3ain compound I and II, respectively. (1)- "JC1H2= 3.0 Hz (II) - 'Jc2H3a < 1 HZ D A , HI Back Diff. D A HI Back Diff. OC2H2 o*CIOH 3.91 0.95 2.96 Oc3H3a a*c2F 5.19 1.07 4.12 The results presented in this study shown that orientation of the heteroatom (F and O) is crucial to the sign of the geminal coupling constant. These results together 2 2 with previous one reinforce the applicability of JCH coupling constant as an important tools for stereochemical determination of small organic molecules. REFERENCES 1. K.E. Kõvér, G. Batta, K. Fehér, J. Magn. Reson. 2006, 181, 89. 2. R. H. Contreras, P. F. Provasi, F. P. dos Santos, C. F. Tormena, Magn. Reson. Chem. 2009, 47, 113. 3. V. Blechta, J. Schraml, Magn. Reson. Chem. 2011, 49, 111. 4. R. H. Contreras, J. E. Peralta, Prog. NMR Spectrosc. 2000, 37, 321. 5. P. Nolis, "Disseny i aplicado denous mètodes de RMN" Tese de Doutorado, Universitat Autônoma de Barcelona, 2007. 6. a) L. Lerner, A. Bax, J. Magn. Reson. 1986, 69, 375. b) D. G. Davix, J. Magn. Reson. 1989, 84, 417. 7. NBO 5.G. E. D. Glendening, J. K. Badenhoop, A. E. Reed, J. E. Carpenter, J. A. Bohmann, C. M.Morales, F. Weinhold (Theoretical Chemistry Institute, University of Wisconsin, Madison, Wl, 2001). 103 I- + PO 43 STRUCTURAL STUDIES OF YTTRIUM ALUMINOBORATE LASER GLASSES USING SOLID STATE NMR AND ELECTRON SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY H. Deters,*1, J.F. Lima,2 C.J. Magon2, C.N. Santos2, A.S.S. de Camargo,2 A. C. Hernandez,2 C.R. Ferrari,2 and H. Eckert1 Instituí für Physikaiische Chemie, WWU Münster, Corrensstrasse 30, D48149 Münster, Germany 2IFSC, Universidade de Sao Paulo, Sao Carlos, S.P. Brazil e-mail:eckerth(a).uni-muenster. de, magon(g).if. sc. usp. br Keywords: borate glasses, solid state NMR, pulsed EPR In the search for new and efficient rare-earth (RE) doped laser glasses that are insensitive to fluorescence quenching effects, the spectroscopic and photophysical properties of both the glassy frameworks as well as of the RE dopants in such materials are presently in the focus of much attention. As radiation properties are well- known to be related to the environment of the RE ions and their extent of clustering, detailed structural information is required in order to facilitate the design of composite materials with optimized emission properties. Glasses of the ternary system Y2O3-AI2O3-B2O3 have been introduced as excellent alternatives to single crystalline host materials for special laser applications involving self-frequency doubling or self-sum frequency mixing.'11 The present contribution deals with detailed structural studies of samples within the composition ranges 60 B203 - (40-x) Al203 -x Y203 and (80-x) B203 - x Al203 - 20 Y203, using modern solid state nuclear magnetic resonance (NMR) methods. Detailed information on the organization of the framework is obtained by 11B and 27AI single-and double resonance NMR techniques under magic-angle sample spinning (MAS) conditions. The majority of the boron atoms are three-coordinated, whereas the alumina species are present in the coordination states four, five and six in comparable amounts. All of them are in intimate contact with both the three- and the four-coordinate boron species and vice versa, as indicated by 11B/27AI rotational echo double resonance (REDOR) data.'21 These results are consistent with the formation of a homogeneous, non-segregated glass structure. Based on charge balance considerations as well as 11B NMR lineshape analyses, the dominant borate species are concluded to be meta- and pyroborate anions. Unfortunately, the RE ions themselves cannot be studied by NMR due to their paramagnetism. To study the local environments of these fluorescent ions we have developed a comprehensive examination strategy consisting of three approaches. The first approach is based on the solid state NMR analysis of diamagnetic mimic species such as 89Y. As a matter of fact, this study is the first application of 89Y solid state NMR to a glass-forming system. Both the 89Y chemical shifts and the Y-3d core level binding energies are found to be rather sensitive to the yttrium bonding state and reveal a clear preference of the rare-earth species to be covalently linked to boron rather than aluminium atoms of the framework.'21 In the second approach, the concentration-dependent influences of the paramagnetic rare-earth ions upon the NMR observables of the constituent nuclei of the glassy network (11B and 27AI) are examined. Paramagnetic broadening effects observed on the 11B resonances have been found to depend linearly on the RE ion concentration, suggesting a statistical distribution of these species and the absence of clustering phenomena. In the third approach, the paramagnetic ions are being studied directly by pulsed electron paramagnetic resonance (EPR) techniques, and dipolar and contact interactions with nearby nuclei are probed by electron spin echo envelope modulation J. + 104 (ESEEM) spectroscopy. In case of an anisotropic hyperfine interaction of the observed electron spins with nearby nuclei, forbidden EPR transitions with concomitant changes in the nuclear orientational spin quantum numbers become weakly allowed, and as a result, the electron spin echo amplitude is modulated - in the weak-coupling limit - by the corresponding nuclear Zeeman frequencies. Thus, the experiment can identify the types of nuclei present in the second or third coordination sphere of the rare-earth ions. In yttrrium aluminoborate glasses the data reveal significant hyperfine interactions of the unpaired electron spins with both the 27AI and the 11B nuclei. Figure 1 shows that the ratio of the corresponding modulation depths depend systematically on the Al/B ratio in the framework, which is again consistent with the formation of homogeneous rather than a segregated atomic distributions. Quantitative distance information has been extracted from these data on the basis of spectral simulations carried out for crystalline model compounds as well as for simple spin cluster geometries. ioB 27a| B = 9 kG ~i—>—i—•—i—1—i—•—i—1—i—'—i—1—i 5 10 15 20 25 30 35 40 v / MHz Figure T: Fourier Transform of the ESEEM of glasses along the series (B203)o.8- x(AI203)x)(Y203)o.i95Ybo.oo5 (0< x < 0.40) measured by the three-pulse sequence using an echo delay value of 136 ns enabling the observation of all the hyperfine couplings with comparable sensitivity. REFERENCES 1. Mohr, D.; Silva, W.F.; Santos, C.N.; de Camargo, A.S.S.; Vermelho, M.V.D.; Li, M.S.; Hernandes, A.C.; Ibanez, A.; Eckert, H. e Jacinto, C. J. Appl. Phys. 106, 023512 (2009) 2. Deters, H.; A. S. S. de Camargo, A.S.S.; Santos, C. N.; Ferrari, C. R.; Hernandes, A.C.; A. Ibanez, A.; Rinke, M. T. e Eckert, H. J. Phys. Chem. C 113(36), 16216-16225 (2009). This work was funded by FAPESP and CNPq. H.D acknowledges support by the NRW Forschungsschule "Molecules and Materials". A.S.S.C. acknowledges support by the Alexander-von-Humboldt-Foundation. 105 I- + PO 21 STUDY OF TRANSVERSE RELAXATION IN LIQUID COW'S MILK DURING FERMENTATION PROCESS P.M. dos Santos1*, A.A. Souza2, T. Venâncio3, L.A. Colnago4 e E.R. Pereira-Filho1 1 Grupo de Análise Instrumental Aplicada, Departamento de Química, Universidade Federal de São Carlos, São Carlos, Brazil; 2lnstituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil; 3Laboratório de Ressonância Magnética Nuclear, Departamento de Química, Universidade Federal de São Carlos, São Carlos, Brazil; 4Embrapa - Instrumentação Agropecuária, São Carlos, Brazil. e-mail: polianamacedos&hotmail. com Keywords: milk; low-field NMR; transverse relaxation. Milk can be considered a heterogeneous fluid, composed by a complex mixture of specific proteins, fats, minerals, vitamins and other components in appropriated concentration. This fluid contains all nutrients required for growth and development of newborns. World cow's milk production in 2008 stood at over 578 million tonnes, with the top ten producing countries accounting for 55.4% of production. Brazil is the 6th largest producer in the world, with over 27 million tonnes and accounting for 4.67% of world cow's milk production1. Recently, there were several cases of milk adulteration in the world2,3, with severe consequences to child healthy. Therefore, there is a demand for fast, non-destructive and green methods to measure the quality of milk and milk products. Low-Resolution NMR (LRNMR) has been successfully used'in analysis of a wide range of complex food products. In comparison with wet chemical analytical methods, LRNMR is faster, non-destructive and does not produce chemical waste. Also, it can be applied in packed samples, in contrast with other spectroscopic methods used for milk and dairy industry analyses, like infrared spectroscopy. In this case, using LRNMR the analysis can be performed direct in the final product to be acquired by the consumer. Therefore, the aim of this study was to investigate the use of transverse relaxation time (T2) using LRNMR spectrometer to monitor the quality of milk during the fermentation. The fermentation can be an indication of poor hygienic storage and processing, age of milk, and also has been used in the processing of several milk products, such as cheeses and yogurt. The relaxation measurements were performed on a SLK-SG-100 spectrometer (Spin Lock Magnetic Resonance Solutions, Córdoba, Argentina) with a resonance frequency of 9 MHz for protons. The T2 was measured using the Carr-Purcell- Meiboom-Gill sequence, with an echo time (2x) of 1 ms, a repetition delay of 1.5 s, a train of 5000 echoes and 4 scans. The measurements were performed at 25°C with raw liquid cow milk. The pH varied from 6.68 (fresh milk) to 4.46 (sour milk) during approximately 33 hours. Figure 1 shows the evolution of pH (black line) and T2 (blue line) during the fermentation time, indicating three different T2 behaviors. In the first phase, which corresponds to the first 27 hours, the T2 values were almost constant (0.17 - 0.19 s). After this period, the T2 values increase fast (0.21 to 0.6 s), reaching a maximum (1.3 s) at about 33 hours. These results can be explained by the influence of fermentation in the reduction of pH and also by the solubility of caseins, both causing the pronounced increase of T2 values. The caseins, the major proteins in milk, occur as micelles made up of four major components: a, p, y and K. a and p caseins comprise 50% and 30%, respectively of the whole casein content of milk, while the K and y caseins are present in only 12% and 5%, respectively. 'f 4- 106 13th M l.11 <\R MAGNETIC RESONANCE I Sl-R<í Ml I-1 INC. 7.0 -pW/ 1.4 4.o I- o.o 0 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Time (hours) Figure 1: Evolution of T2 (blue line) and pH (black line) with the fermentation time. Figure 2 shows the correlation between pH and T2 variation. Three phases (1-3) can be clearly distinguished, considering a linear behavior for each phase during the whole fermentation process. During the first phase, the pH changes from 6.67 to 5.92, indicating that T2 values are not a good indicator of the beginning of fermentation process. In the second phase T2 values show large variation from 0.22 to 0.61 s while the pH changes from 5.67 to 4.70. In the third period, the T2 shows a sharp increase from 0.66 to 1.28, while the pH varies only from 4.62 to 4.46. 7.0- I 6.5- * • 6.0- • (1) , Isoelectric Point of Casein y x ••Í / i • 5.5- Isoelectric Point of Casein p •• i 5.0- 4.5- 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 T2 (S) Figure 2: Correlation between pH and T2 during the fermentation process. Three (1-3) regimes can be clearly distinguished, whose inflection points are strongly related to the precipitation of casein protein. It is also possible to see two inflection points separating the three described phases. The equivalent point at pH = 5.7 corresponds to the isoelectric point of casein y (pH = 5.8), which led to the formation of solid casein protein in the milk. The second variation of T2 occurs at an equivalent pH point of about 4.7, which corresponds to the isoelectric point of casein p.These results indicate that LRNMR is potentially useful form monitoring fermentation and consequently the quality of raw milk. REFERENCES: 1. http://www.fao.org/ 2. http://en.wikipedia.org/wiki/2008_Chinese_milk_scandal 3. http://g1.globo.com/Noticias/Brasil/0,,MUL161409-5598,00.html FAPESP and CNPq 107 I- + PO 21 STUDY OF METABOLIC PROFILE BY LC-SPE-NMR OF SPILANTHES ACMELLA D.S.Santos*1; A.G.Ferreira1, P.C.Nogueira,2 A.F.Blank3 1 Universidade Federal de São Carlos, Departamento de Química, São Carlos, SP, Brazil 2Universidade Federal de Sergipe, Departamento de Química, São Cristóvão, Sergipe, Brazil 3Universidade Federal de Sergipe, Departamento de Agronomia, São Cristóvão Sergipe, Brazil. e-mail:dannvauimica23(a). yahoo. com.br Keywords: LC-SPE-NMR, Spilanthes acmella, hyphenated techniques. The recent techonological development in analytical techniques, especially in hyphenated techniques such as GC-MS, LC-MS and LC-NMR have played an important role in the elucidation of products of plant extract, which are a complex matrix containing hundreds or thousands of secondary metabolites. Nowadays, these techniques can achieve levels of sensitivity and selectivity that were unthinkable until a few years ago without the exhaustive work of isolation that often leads to compounds already known. In the present work, the hyphenated LC-SPE-NMR technique was used for the first time in Brazil and South America, in the analysis of a methanolic extract from S. acmella, a native species of South America commonly named as "jambu", which presents several biological activities and has been used by cosmetic industry in preparations for skin care1,2. The aim of this study was to characterize and elucidate the structure of the greatest possible number of substances using minimal manipulation of the extract. The chromatographic method was developed in an Agilent system, equipped with a quaternary pump, an autosampler and a diode array detector (DAD) interface coupled to the Prospekt II SPE unit and coupled to NMR equipment, Bruker AVANCE III, 14.1 T (600 MHz for hydrogen frequency), equipped with a 5 mm cryo-probe® with ATMA®, and a cryo-fit® unit for measuring volumes of up to 60 pL, allowing the LC- SPE-NMR coupling. Initially, a chromatographic method for selective separation of all compounds in the methanolic extract of dry leaf at a concentration of 25 mg/mL was utilized with a 10- 60% (Me0H/H20) gradient elution for 50 min (Figure 1). Also, a specific method at isocratic mode with mobile phase of 85% (Me0H/H20) was used for 6 min. (Figure 2) separating only the compound in the retention time at 4.8 min. Both methods were developed with C18 (150x4,0 mm) stationary phase with 5 pm particle diameter, injection volume of 25 pL and flow rate of 1 mL/min., and were monitored at the wavelength of 230 nm. The chromatographic band of interest with 1.18 pg/mL or 5,9 mM was concentrated by on-line SPE, in C18 cartridges. At each chromatographic run, 29.5 ng of the compound was concentrated yielding a total of 205.6 ng after seven runs. Afterwards, the cartridge was dried with N2 gas in order to remove any solvent present and the compound was transferred to the spectrometer with 60pL of deuterated acetonitrile. The concentration of this compound present on cryo-fit® cell was of 3.44 ng/pL. With this amount of sample the 1H NMR spectrum was obtained with a Presaturation Pulse Sequence (Iclpncwps) for solvent saturation, using 64 scans, 4K data points, spectral widths 12019 Hz, acquisition time 2.7s and relaxation delay (d1) 1s. Moreover, the COSY spectrum was also obtained with a Presaturation Pulse Sequence (cosygpprqf) for solvent saturation, using 16 scans, 4K data points, spectral widths 9014 Hz, acquisition time 0.22s and relaxation delay (d1) 1s. Figure 1: Chromatographic profile of the methanolic extract in gradient elution. The compound was identified as (2E,6Z,8E)-N-(2-metilbutil)-2,6,8- decatrienamida, a alkamide through analysis of the 1H and COSY spectra (Table 1). Table 1: NMR data obtained for the alkamide found in the methanolic extract of Spilanthes acmella dry leaf. Posição 5 1H (mult.,Jem Hz) SCOSY Va 3.12 (dt 12.91, 6.23) - 1'b 3.03-3.33 (m) - 2' 2.33-2.28 (m) - 3' 1.10 (dq 13.83, 7.49) - 4' 0.98 (d 6.34) - 5' 0.95 (t 15.10, 7.61) - 2 2.3 (dt 11.99, 7.26) - 3 6.66 (dt 14.41, 7.03) - 4 1.55-1.48 (m) - 5 1.40-1.33 (m) - 7 5.98 (t (AB) 11.30, 7.15) - 8 6.34-6.40 (m) 5.98 9 5.70 (dq 14.76, 6.92) 6.34 10 1.77 (dd 7.38, 6.46) - This paper presents a contribution to the quality control of S. acmella species and a possibility of establishing an analysis protocol using the LC-SPE-NMR mentioned for other plant extracts. REFERENCES: 1. R. S. Ramsewak etal., Phytochemistry, 1999, 51 2. Nakatani, N.; Nagashima, M. Pungent Alkamides from Spilanthes acmella L. var oleracea Clarke. Bioscience, Biotechnology, Biochemistry, 1992, 56. FAPESP, CAPES, CNPq. 109 I- + PO 21 NMR CHARACTERIZATION OF RESINOUS MATERIAL OCURRED IN VALVES CONTROLED BY HYDRAULIC FLUIDS IN OIL PRODUCTION Sonia Maria Cabral de Menezes1*, Luiz Silvino Chinelatto Júnior1, ítalo José Rigotti1, Bruno Charles do Couto1, Flavio Alves Zuim1,Thiago Aquino Damasceno2, Adriana Santos Mendes2 1PETROBRAS/CENPES/QM, Rio de Janeiro, Brazil 2Pontificia Universidade Católica do Rio de Janeiro (PUC/RJ), Rio de Janeiro, Brazil *soniac(a).petrobras. com.br Keywords: hydraulic fluid; metal salts; NMR. Hydraulic fluids are used in valve activation and deactivation systems in offshore oil platforms. PETROBRAS has recently detected a problem in a directional control valve which was ascribed to a resinous material formed on the surface of a nickel plated solenoid, normally kept immersed in the hydraulic fluid. Some preliminary analysis of this material indicated the presence of a Ni salt that was probably being formed by contact of the solenoid with the fluid. In order to investigate this problem, precipitation was induced by individually adding different salts (Ni, Mn, Cu and Zn nitrate) to a commercial hydraulic fluid. The precipitated materials (Ni, Mn, Cu and Zn ppt) were isolated and analysed by NMR. 1H and 13C NMR experiments were performed on a Varian INOVA-300 spectrometer (7.05T), at 27°C in a 5mm direct detection probe. The 1H NMR spectra of 5% v/v solutions were acquired at 299,98MHz using 7.45ps (45°) rf pulses, 1.0s of pulse delay and 128 transients. The 13C NMR spectra of 5 to 40% v/v solutions were acquired at 75,44MHz using 7.70 ps (45°) rf pulses, 1s of pulse delay and 7000 to 25000 transients were accumulated. The induced precipitates isolated from the hydraulic fluid and the resinous material removed directly from the solenoid were dissolved in CDCI3 containing tetramethylsilane (TMS) 0.05% v/v as internal reference. The liofilization residue of the commercial hydraulic fluid studied was dissolved in D20 containing 4,4- dimethyl-4-silapentane-1 -sulfonic acid (DSS) 0.5% wt/wt as internal reference. The spectra obtained for the precipitates are presented in figure 1. The Zn2+ ppt spectra are the only ones with well-resolved signals. Line broadening and/or disappearance of lines were observed in all other metals precipitate spectra and were attributed to the paramagnetic character of those metal ions1. The Zn2+ ppt spectra were then used for structure elucidation. ! 1 j 11 ,v./, , ,1 : ) 2 2 — — ,1 1 i 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 7.5 7.Ü 1.5 1.0 0.5 0.0 220 200 180 160 140 120 100 80 60 40 20 0 fl (ppm) fl (ppm) Figure 1 - 1H (left) and 13C (right) NMR spectra of the induced precipitates of the hydraulic fluids studied. From top to bottom: Zn2+ ppt, Mn2+ ppt, Cu2+ ppt and Ni2+ ppt y + 110 I'Mi'NLi II M< \I\(,MTI«1 KI-SDNAsn 1 SI R-MI I IIN(i The 1H NMR spectrum has (a) two multiplets at 7.78 and 7.56ppm with a 2:3 integration indicating a mono-substituted aromatic ring; (b) a triplet close to 3.01 ppm (2H) and a singlet at 2.70ppm (3H), (c) a triplet around 2.37ppm (2H), attributed to an a-carbonyl CH2 group, (d) and three multiplets at 1.64, 1.54 and 1.35ppm (2H:2H:2H), which were attributed to a —CH2CH2CH2- group. The 13C NMR spectrum has a signal at 183.9ppm (carboxylic acid salt carbonyl), signals at 137.0, 132.5, 129.0 and 127.3 (mono-substituted aromatic ring carbons); 50, 37 and 34ppm (carbons next to heteroatoms) and three signals between 27-25ppm (aliphatic carbons). These data and elemental analysis, which indicated also the presence of S and N lead, to the following structure: Figure 2 - Structure of the induced precipitates of the hydraulic fluid studied. M=Metals Such structure is compatible with the line broadening and disappearance of the 13 signals attributed to the carbonyl and CH2 carbons in the C NMR spectrum of the Cu2+, Mn2+ and Ni2+ ppt as these are closer to the paramagnetic center. Furthermore, a patent related to hydraulic fluid composition indicates this class of compounds as a possible constituent2. In order to confirm that the proposed structure corresponded to the resinous material observed on the solenoid, it was washed with acetone and the solvent free residue analyzed by NMR. The resulting 1H and 13C NMR spectra (Figure 3) were compatible with those of the nickel precipitate (first spectrum from bottom to top in figure 1). Figure 3 - 1H (left) and 13C (right) NMR spectra of acetone-extracted residue. NMR spectra of the liofilization residue of the commercial hydraulic fluid used in this study (not presented) also confirmed the presence of the structure shown in figure 2. REFERENCES 1. Lamar, G. N.; Chem. Phys. Lett. 1971, 10, 230. 2. Smith, I. D. And Kennedy, J. C.; Patent application 20100016187. 111 I- + PO 38 STRUCTURAL AND ORIENTATIONAL DETERMINATION OF THE ANTIMICROBIAL PEPTIDE PHENYLSEPTIN IN MEMBRANE-MIMICKING ENVIRONMENT V.H.O. Munhoz"12 M.T.Q. de Magalhães3'4, R.M. Verly1, S.F.C. de Paula1, J.M. Resende1, C. Aisenbery2, D. Piló-Veloso1, C. Bloch Jr.3, B. Bechinger2 1 Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos, 6627, 31270090, Belo Horizonte-MG, Brazil ( [email protected]) 2Université de Strasbourg / CNRS UMR7177, Instituí de Chimie, 4, rue Blaise Pascal, F-67070 Strasbourg, France 3Embrapa Recursos Genéticos e Biotecnologia, PqEB- Final W5, Brasília - DF, Brazil 4Pós-graduação em Biologia Molecular, Instituto de Biologia, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, Brazil e-mail:victor. munhoz&.gmail. com Keywords: antimicrobial peptides, solid-state NMR Phenylseptin (Phes) is a new class of antimicrobial peptides isolated from the anuran Hypsiboas punctatus. They are known to be phenylalanine-rich peptides on their AMerminus. There are two knowing forms belonging to this class which share, to some degree, the same amino acid sequence, except for an enantiomerization at one phenylalanine residue. Both peptides show highly a- helical and amphipathic structures when in contact with surfactant assemblies, such as micelles and bilayers. In vitro assays have shown that both peptides have mild antimicrobial activity against Gram-positive and Gram-negative bacteria1. The Phenylseptin containing D-amino acid is considerably more active than its homologue, suggesting that the AMerminus region plays an important role on the interaction between the peptide and the bacterial membrane. Nonetheless, the exact mechanism which describes the antimicrobial activity of these peptides is still not fully elucidated, even though there are some models for this biological process which are well accepted albeit not yet completely proven. In order to shed a light on some aspects of this mechanism, static cross- polarization 15N and 2H solid-echo solid-state NMR were performed in samples with the peptide in contact with mechanically oriented lipid bilayers. By combining these two experiments, it will be possible to determine the most probable orientation of the peptide when it interacts with membrane-like media. This orientation is treated as a combination between two angles: the tilt angle, which is defined as the angle between the helix axis vector and the bilayer normal; and the rotational pitch angle, which is related to the rotation of the helix along its axis. The tilt angle can be determined by the 15N chemical shift anisotropy and is related to the overall insertion of the helix on the bilayer, while the rotational pitch angle is obtained through the quadrupolar splitting on the 2H experiment and is mostly related to the anchoring of some regions of the helix on hydrophobic core of the bilayer2'3. In this work, the samples consisted on a mixture containing the peptide and POPC on a 3:100 molar rate spread on around 18 very thin glass plates with the dimensions of 8x22 mm, stacked one over the other, and hydrated at 93% of relative humidity. The 15N experiment was performed on Bruker Avance AMX400 wide-bore with a commercial double-resonance E-free probe and the 2H experiment, on a Bruker Avance 300 with a static commercial triple-resonance probe. The peptides were synthesized with 15N label at L9 and 2H label at A10. The resulting spectra for the peptides show similar values for the chemical shift (76 ppm for L-Phes and 74 ppm for D-Phes), suggesting that there isn't a significant variation on the tilt angle showed by both peptides. There is, however, a marked ^ + 112 difference between the deuterium quadrupolar splittings (35 kHz for L-Phes against 27 kHz for D-Phes). By calculating the tilt and rotational pitch angle values with the quadrupolar splitting and chemical shift anisotropy as restraints, one can build the plots shown in Figure 1, in which the red curve represents the possible angle values derived from 15N chemical shift anisotropy restraints and the blue ones, the likely angle values calculated with deuterium quadrupolar splitting restraints. The intersections between both curves show the most likely orientation based on both restraints used. These angular values are applied to helical structures and by analysis of the hydrophobic groups inserted into the bilayer, it was possible to choose the angle combinations IV, for L-Phes and I, for D-Phes as the most feasible orientations. With these results, it was possible to observe that both peptides, although similar in amino acid composition, interact very differently with membrane-like environments and thus exert different activity. The cause of the difference between their activities probably is the differential anchoring, given the variation on the 2H quadrupolar splitting, which is quite sensitive to strong interactions between the side chains and the bilayer. a) b) J ii \ VI j i lit I!) IV Rotation around z axis Rotation around z axis Figure 1: Possible alignments for L-Phes (a) and D-Phes (b) based on 15N chemical shift anisotropy and 2H quadrupolar splitting orientational restraints (red and blue lines, respectively). The intersection points define orientations of the helices which are coherent with both restraints. REFERENCES: 1. De Magalhães et al., article in preparation. 2. Bechinger, B.; Sizun, C.; Con. Magn. Res. 2003, 18A, 130 3. Aisenbrey, C.; Bechinger, B.; J. Am. Chem. Soc. 2004, 126, 16676 CNPq, FAPEMIG, CNRS 113 I- + PO 48 METABOLIC STUDIES WITH BOVINE BLOOD PLASMA EMPLOYING HIGH- RESOLUTION NMR AND CHEMOMETRICS Matheus P. Postigoa,b*; Ana Carolina de Souza Chagas0; Márcia Cristina de Sena Oliveirac; Luiz Alberto Colnagob aChemistry Institute of São Carlos, IQSC-USP bEmbrapa Agricultural Instrumentation, CNPDIA cEmbrapa Southeast Livestock, CPPSE [email protected] Keywords: bovine metabolomics, 1H-NMR,13C-SSFP-NMR, Ivermectin, food safety. Despite the constant advances in food science and quality control methods, which gathers Analytical Chemistry, Veterinary and Farmacology, there are several issues related to misuse of veterinarian drugs, such as Ivermectin, a powerful and effective anti-helmintic drug. On the other hand, parasitic diseases are a real problem for farmers, once they lead to body mass loss, reduced milk production and in some advanced cases, death, representing a thread to this economic sector. The balance between avoiding diseases and respecting the recommended dosis^ and drug withdrawals period is the major focus of cattle producers, which is often unobtained. One of the most difficult problems when analyzing animal tissues for drug trace detection is their small concentration, besides their fast metabolism rate. However, molecules such as Ivermectin disturb animal natural metabolism. In this context, this study presents a novel method for detecting the consequences of misadministration of Ivermectin in blood plasma composition, in order to provide a new way to understand bovine metabonomics. Furthermore, it can be applied for several animal and human diseases, as cancer, diabetes and gout, which are characterized by metabolites level disturbances. Blood plasma samples were provided by Embrapa Southeast Livestock, from both male and female adult oxen. Samples were semi-lyophilized for concentration increase and added of 20% of D20 for the lock signal, followed by addition of 100 mM of phosphate buffer, at pH = 7.4.1 NMR analysis were carried out in a Varian INOVA 400 spectrometer, with magnetic field of 9.4 T, which provides a frequency of 400 MHz for 1H. 1H data were obtained by summing 32 spectra obtained with a conventional PRESAT sequence, in order to suppress water signal, much more intense than metabolites signals. 13C spectra were obtained by both traditional2 and SSFP pulse sequences. Table 1 shows the parameters for all the sequences employed. Table 1: NMR acquisition parameters 13C 13C Parameter 1H Conventional Technique SSFP Technique d1 (delay time) 3 s 900 ms 50 ms pw (pulse width) 6.8 ps (90°) 6.0 ps (30°) 6.0 ps (30°) at (acquisition time) 1.2 s 480 ms 100 ps Number of Scans 32 8192 « 120,000 Total Time 5 min 3h15 1h20 As noted, SSFP pulse sequence allows a much higher number of acquisitions in a shorter period of time. However it demands a more careful spectra treatment, due to phase distortions caused by the fast pulsing. Despite this, the gain in signal-to-noise ratio is evident as shown in Figure 1. + 114 These results show that SSFP-NMR can be a valuable technique for fast 13C data acquisition, which is a powerful complementary tool for metabolite identification, made via 1H-NMR. Furthermore, 1H-NMR is applied to exploratory analysis such as PCA and HCA, what result in models capable to provide some new information about metabolism behavior. After chemometric studies, we are able to identify the regions in 1H spectra responsible for distinguishing samples. Inside these regions, several peaks are found to vary between different animals and for the same animal before and after Ivermectin administration. Frequently, the large amount of peaks demands some tools for aiding this identification, so we employed both Human Metabolome Database (HMDB)3 and Metabolomics Database4 which allows identifying taurine, creatine, creatinine, hippurate, citrate and urea as main metabolites, and a modest variation for acetate signals, what can be related to a light ketosis condition, to be confirmed in future experiments! Finally, NMR analysis for both 1H and 13C represent a fast metabolic data acquisition method and also a first step for comprehension of metabolism behavior when this is externally disturbed, as by Ivermectin administration. REFERENCES 1. Beckonert, O., Keun; H. C., Ebbels; T. M. D.; Bundy, J.; Holmes, E., Lindon, J. C.; Nicholson, J. K. Nature Protocols, 2007, 2(11), 2692. 2. Braun, S.; Kalinowski, H. O.; Berger, S. 150 and More Basic NMR Experiments: A Practical Course. Weinheim: Wyley-VCH, 1998, 596 p. 3. Wishart, D. S. et al., Nucleic Acids Res., 2007, 35, D521-6. 4. Lundberg, P.; Vogel, T.; Malusek, A.; Lundquist P. O.; Cohen, L.; Dahlqvist, O., 2005, MDL - The Magnetic Resonance Metabolomics Database, ESMRMB, Basel, Switzerland. FAPES, Embrapa (CNPDIA and CPPSE) 115 h * PO 48 IS A PLANAR W ARRANGEMENT THE ONLY ONE EFFICIENT 4 TRANSMISSION OF PATHWAY JHh? Denize C. Favaro, Karen Canto*, Cláudio F. Tormena Chemistry Institute, UNICAMP - P.O. Box 6154 CEP-13084-862 e-mail: karencanto&jom. unicamp.br 4 Keywords: JH2aH6a, hyperconjugative interactions, coupling pathway. The pathways of nuclear spin-spin coupling are of substantial interest for the interpretation of NMR spectra. Stereochemical properties of NMR spin-spin coupling constants (SSCCs) are mostly based on the ability of their Fermi Contact (FC) for being 1 n transmitted through the electronic molecular structure. For JXy coupling with n < 3 as well as for through-space SSCCs, exchange interactions play a key role in their transmission. On the other hand, for n > 3, charge transfer interactions are excellent carriers for the spin information associated to the FC interaction.2 In this context, the 3 most important sources of stereochemical information were, for many years, JHH SSCCs, which are strongly dependent on the torsion angle of the respective vicinal 3 3 hydrogen atoms, as has been shown by Karplus. While the JHH couplings are widely applied to assign conformational preferences, less attention is dedicated to long-range 4 3 JHH SSCCs, probably due to its usually smaller absolute values than those of JHH 4 SSCCs. Long-range JHH coupling constants were measured in rigid tert- butylcyclohexanes, with W-type (diequatorial) coupling constant being significantly larger than diaxial and equatorial-axial couplings.4 It is well-known for cyclohexanes systems, the long-range diequatorial 4jh2,h6 coupling is operated'according to the W- type coupling pathway. Whilst for cyclohexanones and their derivatives the long-range couplings is observed between hydrogen atoms in the axial orientation, according studies performed by Freitas et al5, which showed that in 2-bromocyclohexanone the transmission of 4JH2aH6a is governed by orbital interactions, especially those involving 1 the 7T*c=o orbital. To corroborate with the H NMR findings, the present work has as the main goal the investigation the influence of the carbonyl and hydroxyl groups on the coupling transmission efficiency for 2-X-4-f-butylcyclohexanones and 2-X-4-t- butylcyclohexanols (X = F, CI and Br), see scheme below. a) (2) 13) 14) = . »<) (a)X l .(t>, X definido, guj majn questjon js: |s y\j arrangement the unique coupling pathway for 4 the JHH transmission? 4 Table 1. Experimental and theoretical JHH (HZ) for 2-X-4-f-butylcyclohexanones and 2-X-4-Í- butylcyclohexanols, (X = F, CI and Br). Compounds Couplings pathway Compounds Couplings pathway **H2aH6a Jht2eH6c 1a 1.70 (0.9) 4a 1.20 1b 1.80 (1.1) 4b 1.50 1c 2.06 (1.2) 4c 1.70 2a 0.65 5a 1.33 2b 0.77 5b 1.67 2c 1.03 5c 1.90 3a 0.20 6a 1.07 3b 0.27 6b 1.33 3c 0.50 6c 1.60 Experimental values are in parentheses. + 116 As shown in Table 1, experimental JH2aH6a were observed for studied compounds containing a carbonyl group belonging to the coupling pathway. A similar behaviour was observed recently for the oxide 1,3-dithiane derivatives studied by Tormena et al.5 It must be highlighted that in the alcohols, even when the hydroxyl group is in the axial orientation, leading to strong hyperconjugative interactions (Table 2), such as > 4 Oc2H2a cr*c-o and Oc6H6a^CT*c-o, the JH2aH6a was not observed experimentally. However, when the carbonyl group is present along the coupling pathway, the 4Jn2aH6a couplings are transmitted efficiently and their experimental values were measured. As can be observed from Table 2, hyperconjugative interactions along the coupling pathway is 2 kcal.mol"1 higher in energy for ketones than for alcohols, increasing the electron derealization along the coupling pathway leading to the transmission of 4JH2aH6a more effectively. Table 2. Mainly hyperconjugative interactions (kcal.mol1) for the transmission of 4JH2aH6a SSCCs in (1-3). . . .. Compounds „ . .. Compounds . . .. Compounds nteractions —-z !rr Interactions ^r =— Interactions —r —r— 1a 1b 1c 2a 2b 2c 3a 3b 3c 3 9 4 2 > CTC2H2a^ 5 7 5 4 5 5 CTc2H2a^ CT*C.0 4.3CTc2H2a— 0*C1-H 1 2.4 2.6 2.7 TT C=0 4-2 CTC6H6a^ 6 2 6 0 6 1 ac6H6a^CT*c_o ^ 4.0 CJc6H6a^CT*C1-H1 2. 8 2.7 2.7 TT oo The effect of the C=0 group on the 4JH2aH6a coupling pathway can be easily observed directly from 1H NMR spectra for 1a, 1b and 1c, shown in Figure 1, where for simplicity only signals for hydrogen atoms at position 2 are displayed. The unequivocal assignments were obtained using COSY and HSQC spectra. ras-2-F-4-t-butylcyctohexanone c/s-2-CI-4+butylcyclohexanone cis-2-Br-4-t-butylcyclohexanone 5,20 5.18 5.16 5.14 5.12 5.10 5.08 5.09 5.04 ppm 4-58 4.57 4.56 4.55 4.54 ppm 4.72 4.71 4.70 4.69 4.68 4.67 4.66 ppm 1 Figure 1. H NMR signals for H2a for c/s-2-X-4-f-butylcyclohexanone (X = F, CI and Br). In the current work, the rationalizations for the transmission mechanisms involving the long-range 4JH2aH6a coupling constant for 2-X-4-f-butylcyclohexanones and 2-X-4-f-butylcyclohexanols, (X = F, CI and Br) were presented; as well as the observation that the coupling pathways for the transmission of 4JH2aH6a in these compounds are similar with those observed for homoallylic couplings.6 REFERENCES 1. Castillo, N.; Matta, C. F.; Boyd, R. J. J. Chem. Inf. Model. 2005, 45, 354. 2. Krivdin, L. B.; Contreras, R. H. Annu. Rep. NMR Spectrosc. 2007, 61, 133. 3. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870. 4. Haddon, V. R.; Jackman, L. M. Org. Magn. Reson. 1973, 5, 333. 5. Gauze, G. F.; Basso, E. A.; Contreras, R. H.; Tormena, C. F. J. Phys. Chem. A 2009, 113, 2647. 6. Barfield, M.; Chakrabarti, B. Chem. Rev. 1969, 69, 757. CNPq , FAPESP, BRUKER do Brasil and IQ-UNICAMP 117 h * PO 50 NOVEL ALKALOID FROM PILOCARPUS MICROPHYLLUS A. C. H. F. Sawaya1, Y. D. Costa \ P. Mazzafera \ and L. G. Martins2 1 Department of Plant Biology, Institute of Biology, UNICAMP, Campinas, Brazil 2Department of Organic Chemistry, Institute of Chemistry, UNICAMP, campinas, Brazil lucasmartins&.icim. unicamp. br Keywords: Pilocarpus microphyllus; imidazole alkaloids Pilocarpine and other related imidazole alkaloids are characteristic of several species of the Pilocarpus genus. Pilocarpine is the best known alkaloid, and the only one being economically exploited for the treatment of glaucoma and as a stimulant of sweat and lachrymal glands. This alkaloid is still obtained by extraction from Pilocarpus microphyllus leaves however the extracted residue still contains the alkaloids shown in Figure 1. Some of these alkaloids may also be found in other species, such as P. carajaensis, P spicatus, P. trachyllophus and P. racemosus. (1) Of these only pilocarpine and pilosine have been tested for pharmacological activity. C16H18N2O3 C16H16N2O2 trachyllophiline (1) anhydropilosine (2) 3-hydroxumethyl-4-(3-methyl-3H-imidazol -4-yl)-1-phenylbutan-1-one Figure 1: Structures of alkaloids found in the extract of Pilocarpus microphyllus. The industrial alkaloidical residue was evaluated by LC-MS-MS and by NMR applying the metabolomics principle detecting several previously unreported alkaloids, many of which had their structure proposed based on their fragmentation pathway. Consequently it became necessary to determine their molecular structures requiring isolation of the novel alkaloids by applying several crhomatographic techniques. Compound 1 was isolated by RP preparative HPLC and data analyses (Table 1) of the 1H and 13C 1D and 2D NMR spectra (HSQC, HMBC, COSY, DEPT 135 and DEPT 90) were consistent with the proposed structure 1 a novel alkaloid here named Trachyllophiline. Finally searching for a methodology to provide an overview of the alkaloidic residue by NMR we applied pulsed gradient diffusion spectroscopy (DOSY) in and aqueous solution containing p-cyclodextrin revealing that pilocarpine and it isomer changed it diffusion rate from 5.48 to 3.87 10~10 m2 s"1 while the novel alkaloid 1 changed it diffusion rate from 4.61 to 3.09 10~10 m2s"1, indicating that both alkaloids have similar association constants to the p-cyclodextrin. The topology of the complexes are on going investigation. The 1H NMR spectrum of the mixture of alkaloids and p- cyclodextrin is shown in the Figure 2 with the diffusion rate. 118 ; -111 M i I l.-\l< M M'.M I !C KISUSWI I M-Rt1 M 1 Table 1: NMR spectrum assignments to Trachyllophiline (1) in CDCI3 ( H NMR at 499.8828 MHz and 13C NMR at 124.9797 MHz). Structure Position § 1H NMR (multiplicity) >13C NMR 2 7.25 (s) 137.4 4 6.65 (s) 126.9 5 29.5 6 2.55 (m) 25.1 7 2.39 (m) 36.2 8 2.93 (dd), 3.15 (dd) 39.9 9 199.8 11 3.45 (dd), 3.55 (dd) 63.4 14 3.50 (s) 31.3 15 136.9 16,20 7.83 (d) 128.5 17,19 7.34 (t) 127.9 18 7.45 (t) 133.0 Chemical shift /ppm Figure 2. 1H NMR spectrum of the mixture of alkaloids and p-cyclodextrin with the diffusion rate of each compound. REFERENCES: 1. Sawaya A. C. H. F., et al., Genet. Resour. Crop Evol. 2011, 471. FAPESP, CNPq, CAPES/DGU, ANP 119 h * PO 50 PROBING CH-ti INTERACTIONS IN CYCLOHEXANOL, TRANS-1,2- CYCLOHEXANEDIOL AND a-D-GLUCOSE WITH AROMATIC ANISOTROPY EXPERIMENTS AND THEORETICAL CALCULATIONS E.A. Basso*, R.M. Pontes, A. A. Cândido 'Departamento de Química, Universidade Estadual de Maringá, Maringá, Brasil e-mail:eabasso(çb.uem.br Keywords: aromatic induced shift; carbohydrates; shielding tensor calculations. INTRODUCTION The elucidation of the role of enzyme-substrate interactions in the innumerous biological processes is among the greatest challenges of chemistry.1 Recognition of carbohydrates, for instance, is accomplished with the aid of interaction with aromatic moieties, like those found in Trp, Phe and Tyr amino acid residues.2,3 There are at least two difficulties to be faced while investigating such systems, namely, the experimental assessment of these interactions and the application of computational quantum chemistry to such large molecular aggregates. In this work, we tried to further the knowledge on this field by analyzing the aromatic-substrate interactions over a hierarchy of hydroxyl arrangements, Figure 1. HO (1) (2) (3) Figure 1: Studied compounds: cyclohexanol (1), trans-1,2-cyclohexanediol (2), a-D- Glucose (3). EXPERIMENTAL Trans-1,2-cyclohexanediol was prepared by the oxidation of cyclohexene under HOOH/HCOOH. Cyclohexanol was obtained commercially and distilled prior usage while a-D-Glucose, also from a commercial source, was used as received. gHSQC experiments were performed on a Varian Mercury Plus BB operating at 300 MHz for 1H. The theoretical calculations were carried out using Gaussian 03 and GAMESS suite of packages. RESULTS AND DISCUSSION Initially, we conducted NMR measurements on cyclohexanol solutions on pure acetone, 10-90 % benzene, and pure benzene, Table 1. The hydrogens at C3 and C4 are shielded relative to that of C1. Based on this, the benzene ring was supposed to be positioned somewhere near these atoms. Since H3eq has a somewhat larger shielding, we proposed the structure labeled as A in Fig. 2 for the cyclohexanol-benzene complex. To further support this conclusion, we submitted structure A and also and alternative structure, B in Fig. 2, to geometry optimization using the B3LYP functional with corrections to dispersion interactions (B3LYP-D/6-31+G(d,p)). The structure without the 120 benzene molecule was also optimized to serve as reference. NMR shielding tensors were then calculated at the mPW1PW91//6-31+G(d,p) level of theory. The NMR results calculated to structure B are incompatible with the experimental measurements; whereas the pattern obtained with structure A closely matches the experimental one. Table 1: Chemical Shift Displacements Induced by Benzene (in ppm)a 10% bz-d6 50% bz-d6 90% bz-d6 100% bz-d6 H2 -0,01 0,00 0,03 0,04 H2eq -0,01 -0,01 0,00 0,01 H3 -0,01 -0,08 -0,09 -0,03 H3eq -0,03 -0,12 -0,14 -0,06 H4 -0,01 -0,09 -0,11 -0,05 H4ea -0,02 -0,09 -0,09 -0,02 a These values are referred to the pure acetone solution. Negative values mean shielding relative to H1. Figure 2: Possible structures for the benzene-cyclohexanol complexes. A similar analysis was conducted for frans-1,2-cyclohexanediol. The results resemble those of cyclohexanol since the complexation site for the benzene ring is also in a 1,3-arrangement relative to one of the hydroxyl groups. In other words, the benzene ring avoids the close contact with the hydroxyl region, probably by some kind of steric repulsion with the lone pairs or the acetone molecules. Complexation to the acetone molecule is an important factor. Without this complexation, the benzene ring has free access to all the regions around the molecules and the shifts displacement become homogeneous (last column of Table 1). In a-D-glucose, the benzene ring necessarily complexes to a hydrogen vicinal to an OH group; H2 was identified as the site of complexation of the benzene ring from NMR measurements. The experiments for a-D-glucose were conducted in methanol/water/benzene solutions because of its low solubility in organic solvents. The theoretical analysis of such system is a bit more complex is will be presented latter. REFERENCES: 1. Bertozzi, C. R.; Kiessling, L. L. Science, 2001, 297(5512), 2357-2364. 2. Raju, R. K.; Ramraj, A.; Vincent, M. A.; Hillier, I. H.; Burton, N. A.; Phys. Chem. Chem. Phys. 2008, 70(43), 6500-6508. 3. Ramírez-Gualito, K.; Alonso-Ríos, R.; Quiroz-Garcia, B.; Rojas-Aguilar, A.; Diaz, D.; Jimenez-Barbero, J.; Cuevas, G.; J. Am. Chem. Soc. 2010, 737(50), 18129-18138. CNPq, FUNDAÇÃO ARAUCÁRIA 121 h * PO 52 MECHANISTIC INSIGHTS ON THE REACTIONS OF A PHOSPHOTRIESTER WITH NUCLEOPHILES: NUCLEOPHILIC VS GENERAL-BASE PATHS R. Moreira*1, M. Medeiros1, E.H. Wanderlind1, E.S. Orth1, P.S.M. Oliveira1, T.A. S. Brandão2, F. Nome1. 1 Laboratório de Catálise e Fenômenos interfaciais, INCT-Catáiise, Departamento de Química, Universidade Federal de Santa Catarina, 88040-90, Florianópolis/SC.Brazil 2Departamento de Química, ICEX, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte/MG, Brazil -mail: moreira. raphaell&.pmail. com Keywords: imidazole, hydroxylamine, phosphate ester. Reactions of phosphate esters have been of increasing interest due to their important role in many biological processes, such as cellular signaling, which are ruled by multiple catalyses by different enzymes. Thus, it is not unprecedented the interest in mimicking these dephosphorylation reactions and evaluate different types of assistances, such as nucleophilic, general-base, general-acid, etc.1 A typical phosphate ester model that has drawn our attention is the triester tripyrid- 2-yl phosphate (TPP), which shows impressive rate enhancements in reactions with nucleophiles such as hydroxylamine (103-fold) and imidazole (100-fold), compared to the spontaneous reaction.2 These enhancements, along with isotope solvent effects, indicate that the reaction of TPP with hydroxylamine is nucleophilic and is general-base catalyzed by imidazole. Usually hydroxylamine and imidazole act similarly as efficient nucleophiles in reactions with phosphate esters, although with TPP there seems to occur by two different mechanisms. We report a detailed 1H NMR study for the reactions of TPP with hydroxylamine and imidazole, where we follow the appearance and disappearance of several important species which are shown in Figure 1, which allow a better understanding of the mechanisms of these reactions. Hydroxylamine Nucleophilic pathway General base pathway + NH3Q- /=N INT. 2-Pyridone H J IMZ fr°i » cO k^N O rr°A jQ rr 1 1 NH^ TPP CiT" - NH2OH TPP I ^ -HOO + ^FNY O ° HONHNH2 DPP 2-Pyridone "fishing intermediate1 O HO" O • ex Fumaric acid Succinic acid O DPP Reaction of TPP with Hydroxylamine Reaction of TPP with Imidazole Figure 1: Reaction schemes for reactions of TPP with imidazole and hydroxylamine Reactions of TPP with the nucleophiles hydroxylamine (0.5M) and imidazole 1 (0.36M) were followed by H NMR in D20 at pH 9, and all spectrum assignments are f, 4 122 13th NUCLEAR MAGNETIC RESONANCE USERS MBi-.TING given in Table 1. Results agree with previous hypothesis, where the reaction of TPP with hydroxylamine undergoes a nucleophilic pathway, while with imidazole, undergoes a general-base pathway, assisting the water attack on the phosphorus atom, Figure 1. Table 1: NMR spectrum assignments for the species given in Figure 1. NMR spectrum assignments TPP 6.61 ppm (ddd, 1H. J= 7.2, 6.6, 1.2 Hz); 6.66 ppm (ddd, 1H. J= 9.2, 1.1,1.1 Hz); 7.55 ppm (ddd, 1H. J= 6.6, 2.0, 0.8 Hz); 7.74 ppm (ddd, 1H, J=9.4, 7.4, 2.3 Hz) 8,24 ppm (dd, 1H, J=5,0, 2,0 Hz); 7,91 ppm (ddd, 1H, J= 8,2, 8,2, DPP 2,0 Hz); 7,29 ppm (ddd, 1H, J=7,4, 5,1, 0,8 Hz); 7,24 ppm (ddd, 1H, J= 8,2, 2,0, 0,8 Hz) 7,74ppm (ddd, 1H, J= 9,4, 7,4, 2,3 Hz); 7,55 ppm (ddd, 1H, J= 6,6, 2-hydroxy 2,0, 0,8 Hz); 6,66 ppm (ddd, 1H, J=9,2, 1,1, 1,1 Hz); 6,61 ppm pyridine (ddd, 1H, J= 7,2, 6,6, 1,2 Hz) Fumaric acid 6,53 (s, 2H) Succinic acid 2,40 (s, 4H) For the reaction of hydroxylamine, the species TPP, 2-pyridone, DPP, fumaric acid and succinic acid were detected, and the nucleophilic attack was corroborated by "fishing" the intermediate diimine with fumaric acid, which is easly reduced to succinic acid by the reative diimine. Indeed, hydroxylamine attacks the phosphorus atom leading to 2-hydroxy pyridine (2-pyridone) and the intermediate INT, which is attacked by another molecule of hydroxylamine to generate di-2-pyridyl phosphate (DPP) and hydroxyhydrazine that then dehydrates giving the corresponding diimine. Normally, diimine decomposes forming hydrazine and nitrogen gas, but fumaric acid has been shown to be an important and reliable tool to capture (fish) the diimine. On the other hand, for the reaction of TPP with imidazole, only TPP, DPP, IMZ and 2-pyridone were detected in the NMR experiments, which indicates that in fact, imidazole acts as a general base catalyst (Figure 1). Tthe nucleophilic pathway can be ruled out since phosphorylated intermediates were not detected (as found for other nucleophrlic reactions with imidazole).3 Interestly, the intermolecular general-base reaction with imidazole seems to be more eficient than an intramolecular general base catalysis by the pyridone group, probably due to the higher basicity of imidazole. In conclusion, this study gives insights on the possible pathways for the reaction of TPP with nucleophiles, and shows that depending on the substrate's nature, nucleophiles such as imidazole prefer to act as efficient general base catalyst. Conversely, the less hindered hydroxylamine acts as an efficient nucleophile, followed by decomposition of the phosphorylated intermediate with formation of diimine, which was captured by addition of fumaric acid. REFERENCES 1. Orth E.S.; Brandão T.A.S.; Souza B.S.; Pliego J.R.; Vaz B.G.; Eberlin M.N.; Kirby A.J.; Nome F. J. Am. Chem. Soc. 2010,132,8513. 2. Kirby A.J.; Medeiros M.; Oliveira P.S.M.; Brandão T.A.S.; Nome F. Chemistry-A European Journal. 2011, inpress. 3. Orth E.S.; Brandão, T.A.S.; Wanderlind E.H.; Medeiros M.; Nome F. Estudos cinéticos de reações intra- e intermoleculares de desfosforilação com imidazol usando 1H e 31P RMN; Presented in XI Jornada Brasileira de Ressonância Magnética, Curitiba, 2010. INCT-Catálise, CNPq, CAPES, FAPESC, UFSC 123 h * PO 53 A LOW-COST, PORTABLE HALBACH MAGNET FOR LRNMR M.G.A. Carosio*1'2, L.F. Cabeça2, L.A. Colnago2 1 Instituto de Química de São Carlos - USP, São Carlos, Brasil 2Embrapa Instrumentação, São Carlos, Brasil m. qabbiiii&.qmail. com Keywords: halbach magnet; CPMG; LRNMR. The need for portable analysis of some important analytes, including food, toxins, agrifood products, and drugs have increased. Demand for portable analytical equipments is growing and, as a result, a number of field deployable analytical methods are being developed, including mass spectrometric and infrared based systems. Nuclear Magnetic Resonance (NMR), however, offers a non-destructive, reagentless analytical method for this proposal. NMR is one of the more important techniques for quantitative and qualitative analysis.1 Conventional NMR spectrometers have the sample enclosed by the magnet, shim and RF coils, which typically prevent the sample from being readily studied by other, non-NMR spectroscopic. However, one-sided magnet systems, such as the unilateral NMR offer easy access to the sample, but the NMR signal itself arises only from the surface of the sample and not the bulk, which is inadequate for many applications.2 The Halbach dipole magnets, originally proposed by Klaus Halbach as focusing magnets for particle accelerators, are permanent magnets consisting of segments joined together in such way as to create a dipole magnet transverse to the long axis of the magnet. A number of Halbach dipoles can be combined in an array so as to create a homogeneous magnetic field transverse to the long axis of the array, in an arrangement that is convenient for NMR because a solenoid can be more easily used for the NMR RF coil rather than a saddle coil.3 The Halbach magnet has some advantages: the relative ease of construction and low cost of the magnet array, the portability of the magnet and the homogeneity of the magnetic field around the center of the magnet compared with unilateral NMR. Therefore, the goal of this study was the construction of a low-cost, portable and open- acess Halbach magnet for LRNMR applications in agrifood products.2,3 The home-made Halbach array was costructed with eight NdFeB permanent magnets, with 50 x 50 x 30 mm each. The magnetic axis of each magnet is parallel to the 30 mm dimension. The magnets were assembled in a 200x200 mm plastic container, shown in Figure 1B). The magnetic field in the center of the magnet was 0.28 T equivalent permanent magnet (11 MHz to H1). The Halbach sensor was driven by a CAT-100 Tecmag console, a power amplifier 3205 AMT and a preamplifier of Miteq AU 1114. The analysis were performed with a CPMG pulse sequence using pulse widths of 7 ps, x = 400 ps, acquisition time of 64 ps at the center of the 800 echoes, a recycle delay of 500 ms and 250 scans. In Figure 1A) is shown a scale drawing of the magnet designed with dimensions of 200x200 mm. The magnet is composed of eight NdFeB rectangular prism and Figure 1B) is the photograph of the physical magnet. The home-made single coil probe tuned at 11 MHz used a solenoidal coil with 30 mm diameter and height of 15 mm, equivalent to a sample volume of 45 ml_. The application of the sensor in agrifood samples was demonstrated using banana, castor bean seeds and latex samples (Figure 2). Figure 2 shows three CPMG decays of latex, castor bean seeds and banana, and the T2 of each sample was 41.5 ms, 37.2 ms and 140.0 ms, respectively. J. * 124 r>'li\U 1 l.\K MAC |M-1 n1 Kl-VJVVSCI I St-RS Mil-1 IMi Figure 1: A) Diagram of the Halbach magnetic. The arrows show the direction of the magnetic field. B) View of a simple open-access home-made Halbach magnet array with RF coil. The position of the coil can be mechanically adjusted to locate the field centre. 1,0- 0,8- IÜ 0,6-) >to 10 ) 0,4-| 0,2- 0,0- -0,2- -50 50 100 150 200 250 300 350 Time(ms) Figure 2: CPMG decays for latex, castor bean seeds and ripe gold banana. In conclusion, the low cost, portable and open-access Halbach magnet is capable of being used in LRNMR analysis using CPMG sequence. Above all, the open-access nature of the array allows the envisage of a wide-variety of experimental scenarios in which NMR is coupled with other techniques.2 REFERENCES: 1. Demas, V.; Herberg, J.L.; Malba, V.; Bernhardt, A.; Evans, L.; Harvey, C.; Chinn, S.C.; Maxwell, R.S.; Reimer, J.; Journal of Magnetic Resonance. 2007, 189, 121-129. 2. Hills, B.P.; Wright, K.M.; Gillies, D.G.; Journal of Magnetic Resonance. 2005, 175, 336- 339. 3. Dogan, N.; Topkaya, R., Subasi, H.; Yerli, Y.; Rameev, B.; Journal of Physics. 2009, 153,012047. FAPESP, EMBRAPA INSTRUMENTAÇÃO 125 h * PO 48 USES OF CP SEQUENCE WITH LOW REFOCUSING FLIP ANGLE (CP-CWFP) TO MEASURE TEMPERATURE AND THERMAL DIFUSIVITY IN OILSEEDS M.G.A. Carosio*1'2, F.D. Andrade1'2, L.A. Colnago2 11nstituto de Química de São Carlos - USP, São Carlos, Brasil 2Embrapa Instrumentação, São Carlos, Brasil m.pabbiiii&.gmail.com Keywords: CWFP, seed, temperature. Tropical agriculture and particularly the Brazilian will be one of the economic activities that will suffer an impact with the increase in global temperature. The need of food in an increasing population often threatens natural resources as people strive to obtain the most out of lands already in production or push into virgin territory for new agricultural land.1 The damage is increasingly evident, for example: erosion of arable lands, salinity, desertification, and threats to biodiversity.2 The situation is likely to be further worsened by the potential impacts of global warming and climate change on the growing conditions of crops.3 Soil temperature is an important factor for-plant growth because the soil is responsible for transporting and storing water, solutes, gases and heat.4 The current methods to measure soil temperature in function of dept are based on conventional thermometry but they don't provide information of seed's temperature. We propose in this paper that it's possible to use low resolution NMR to measure oilseeds temperature, using oilseeds as a sensor and Carr-Purcell (CP) sequences with low refocusing flip angles, named CP-CWFP. CP-CWFP is better than CWFP to measure the longitudinal and transverse relaxation times in a single scan, when TI~T2. Therefore, CP-CWFP sequence can be a useful method in low resolution NMR, widely used in agriculture, food and petrochemical industry, because the samples tend to have similar relaxation times in low magnetic field.5 The measurements were carried out in a SLK-100 Spin Lock spectrometer, (Córdoba, Argentine), model SL.IM.01 with 0.23T permanent magnet and a 33mm probe. The T* measurements were carried with the CP-CWFP pulse sequence. The parameters used were: TT/2 pulses = 6.2ps, tau = 141.48 ps and acquisition time 10.6 ps. The seed temperature in the soils samples were checked using an infrared thermometer. The oilseed used was Macadamia integrifoli. In Figure 1 is ilustrated a CP-CWFP signal. The signal decays to a minimum value then increase to a steady state with a constant T* value. Using T*, the magnetization after the first pulse, M0, and at the steady state, MCPCFP, it is possible to calculate T^ and T2, through Equation 1. Time (s) Figure 1: CPCWFP signal in different temperatures. With T*, M0 and (MCPCFP), T^ and T2, can be calculate using equations 1. + 126 '.lihNU.I l.-\M \J.\CiM IIC RI^OVXSCl.l SI RS Mi l-1 INC T12 T'/2 t2 = Mqpcwfp fW0 t — M cpcwfp / M0 Equation 1 Figure 2A shows the increase of T*, T-i and T2 with increase of temperature. Therefore, with this information, it was possible to predict how the heat was transferred inside oilseed in function of time (Figure 2B). This figure shows that the three time constant values decay with time, with a time constant (t) equal to 7.3 minutes. This constant is used to calculate the oilseed thermal diffusivity, according to Equation 2, where A is thermal diffusivity, t is the constant obtained at Figure 2B, and a is the diameter of macadamia nut. A = (a / TT) 2. 1 /1 Equation 2 B) 40 45 Temperature(°C) Time (min) Figure 2: A) T^ T2 and T* of a macadamia nut as a function of temperature obtained with CP-ÇWFP sequence. B) The decay of T1; T2 and T* with time. According to Equation 2, Á=3.12.10~6 m2s-1 for macadamia nut. This property is important to store and dry seeds. This study shows that with CP-CWFP is possible to calculate Ti and T2 in a single experiment and calculate the thermal diffusivity of oilseeds. The non-invasive nature of the measurement makes it possible to obtain information in vivo and quickly, compared with other techniques. REFERENCES: 1. FAO. 31st Session of the Committee on World Food Security, 2005. 2. Foley J.A.; Defries R.; Asner G.P.; Barford C.; Bonan G.; Carpenter S.R.; Chapin F.S.; Coe M.T.; Daily G.; Gibbs H.K.; Helkowski J.H.; Holloway T; Howard E.A.; Kucharik C.J.; Monfreda C.; Patz J.A.; Prentice I.C.; Ramankutty N.; Snyder P.K. Science, 2005, 309, 570-574. 3. Intergovernmental Panel on Climate Change. Kanagawa, 2003, 675. 4. Prevedello C.L.; Física do solo. 1996. 446. 5. Andrade F.D.; Netto A.M.; Colnago L.A.; XI Jornada Brasileira de Ressonância Magnética, 2010, 65. FAPESP, EMBRAPA INSTRUMENTAÇÃO 127 h * PO 55 TOTAL ASSIGNMENT OF THE 1H AND 13C NMR AND STEREOCHEMISTRY OF TWO NEW CHALCONE DIMERS M. Fernanda M. Villari, João B. Fernandes*, Paulo C. Vieira, M. Fátima G.F. da Silva, Antonio G. Ferreira Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luiz, Km 235, 13565-905 São Carlos, SP, Brazil. *e-mail:[email protected] Keywords: 1D and 2D NMR; Chalcone dimers; structures assignment. Leaf-cutting ants are considered agricultural pests due to large amount of plant material to maintain the nest and the symbiotic fungi Leucoagaricus gongylophorus growth. The main method to control ants is using bait, which consists of toxic compound in an inert support containing an attractant for ants. Current baits contain synthetic insecticides with large residual effects. Natural products are potential compounds to replace them, since they have faster degradation than synthetic compounds, and consequently do not increasing the amount of waste in the environment1. In this context, this study evaluated the toxic effects of extracts and fractions from leaves, stems, roots and branches of the plant Astronium graveolens against the symbiotic fungus of Atta. The genus Astronium (Anacardiaceae) is described in the literature having fungicidal and insecticidal activities. Extracts of Astronium fraxinifolium Schott showed activity against the larvae of Aedes aegypti, Astronium balanseae extracts showed activity against the microorganism Klebsiella penumoniae and volatiles from Astronium graveolens were repellent to Atta laevigata. Unusual chalcone dimers are present in species of Anacardiaceae. For structural elucidation and total assinment of the NMR data of new chalcone dimers are used different NMR techniques, since they present several asymmetric center and different substitutions in the structure. This communication reports the application of 1D (1H and 13C NMR) and 2D (COSY, HSQC, HSBC and gNOESY) spectral techniques to establish the complete structures of two new chalcone dimers 1-2 obtained from Astronium graveolens and the stereochemistry of the chalcone dimers 3, previous obtained by BANDEIRA et al.1 from Myracrodruon urundeuva. 1D and 2D spectra were measured on a Bruker DRX 400 spectrometer operating at 400 1 13 MHz for H and 100 MHz for C, using CD3COCD3 (2) or CD3OD (1, 3) as solvents. The plant A. graveolens (AG) was obtained in the Greenhouse in Ibaté-SP. The roots, stems and branches were separately dried in an oven with air circulation at 50°C and powdered in Willey mill. They were extracted with ethanol. After resting for three days, the solvent was filtered and the extracts concentrated in rotary evaporator. This procedure was repeated three times. The extract of the root (RAG) was submitted to a liquid-liquid partition with solvents of increasing polarity (hexane, ethyl acetate (RAGACT) and hydroalcoholic (RAGAQ). Ethyl acetate fraction (RAGACT, 1g) was fractionated using exclusion chromatography Sephadex LH-20, this procedure was repeated 3 times. Fractions were compared by thin layer chromatography and NMR. The compound 1 (17,1 mg) was obtained by junction of four fractions (153.3 mg) and refractioned in Sephadex LH-20 chromatographic column (h = 69 cm and c|> = 2.3 cm, 100% methanol) resulting in 5 fractions, the 4th fraction (106,1 mg) was submitted to High Performance Liquid Chromatography [HPLC, C-18, H20: MeOH (4.5:5.5), flow: 0.8 mL/min (analytical) and 4.53 mL/min (preparative), generating four peaks, the second corresponds to 1. The compound 2 (17.8 mg) was obtained from the junction of four fractions (56.9 mg), these were separated by HPLC (C-18, H20: MeOH (1:1), flow: 0.8 mL/min (analytical) and 4.53 mL/min (preparative). It was obtained three peaks, the latter corresponding to the 2. The compound 3 (20.4 mg) was obtained by junction of two fractions (25.4 mg) and it was again applied to Sephadex LH-20 (h = 69 cm and <]> = 2,3 cm, 100% MeOH). The 10th fraction corresponded to 3. *• 128 I'lhMl.Il •\KM\(,MníKI><(>\\\n.l Sl-RV Mi I II\(, On base of analysis of the NMR spectra data of compounds 1-3 and those related to matoside (3) and urundeuvine C (4)1 was possible establish the structures of 1 and 2 that differ of 4 in the stereochemistry of the carbons 8 and 8". These structures presented different 1H and 13C NMR data only for those of positions 7, 8, 7" and 8" (Table 1). In order to determine the relative stereochemistry were used the coupling constant (J) between the hydrogen 7, 8, 7" and 8" and 1H-1H gNOESY experiments, thus all carbons 7 and 7" have relative configuration R in 1-4 and the stereochemistry for carbons 8 and 8" are 8S,8"R in 1, 8R,8"R in 2, 8R,8"S in 4 (maioside) and 8"S in 3 (urundeuvine C). OH HO 1 H8 = P; H8" = a 2 H8 = a; H8" = a 4 H8 = a; H8" = p OH Table 1 1H NMR data of 1-4 and g-NOESY of 1 and 3 for hydrogen 7, 8, 7" and 8" 1 g-NOESY 2 g-NOESY 4 3 3 g-NOESY CD3OD Acetone CD3OD Acetone CD3OD 500 MHz 400 500 H 400 MHz 400 MHz MHz MHz 5.40 7.40 (C-6); 5.55 5.38 7.33 7.34 7.83 (C-6'); (d, 13.2) 4.35 (C-8"); (d, 14.8) (brs) (s) (s) 6.99 (C-6) 7 3.68 (C8) 8 3.68 6.56 3.62 4.13 (C- 3.08 - (dd, 12.0; (C- (dd, 10.8; 7"); (brd, 11.4) 13.2) 2"/6"/3"/5") 14.8) 5.55 (C-7) 7" 4.45 6.40 (C-3); 4.13 6.84 4.43 4.39 4.40 5.00 (C-8"); (d, 5.6) 6.56 (C- (d, 10.8) (C2"/6") (d, 10.6) (d, 8.0) (d, 6.1) 6.48 (C-3); 2" .6".3".5"); 6.19 (C-3) 7.15 (C- 8.18 (C-6'") 3.62 (C-8) 2".6"); 7.95 (C-6'") 8" 4.35 5.40 (C-7) 4.02 7.20 (C-6"') 4.34 5.00 5.01 4.39 (C-7"); (dd, 5.6; (t, 10.8) 6.84 (C- (dd, 10.6; (d, 8.0) (d, 6.1) 6.75 (C- 12.0) 276") 11.4) 3".5"); 7.95 5.55 (C-7) (C-6'") Table 1 13C NMR data of 1-4 for carbons 7, 8, 7" and 8" c 1 2 41 3 31 Acetone CD3OD CD3OD Acetone CD3OD 400 MHz 400 MHz 500 MHz 400 MHz 500 MHz 7 79.4 79.7 75.7 141.5 141.6 8 44.9 52.3 48.6 124.7 124.8 7" 47 52.2 46.7 48.3 48.4 8" 43.8 47.7 43.2 51.0 51.1 REFERENCES: 1. Bandeira, M.A.M.; Matos, F.J.A. and Braz Fo., R. Magn. Res. Chem. 41:1009-1014,2003. FAPESP, INCT - Controle Biorracional de Insetos Pragas, CNPq. 129 h * PO 56 IDENTIFICATION OF TRITERPENE BY NMR FROM MINQUARTIA GUIANENSIS BRANCHES L. M. C. Cursino*; C. V. Nunez Coordenação em Pesquisas de Produtos Naturais, Instituto Nacional de Pesquisas da Amazônia - INPA. cecilia&.inpa. gov, br Keywords: 3-/3-methoxy-lup-20(29)-ene; NMR; Olacaceae. M. guianensis (Olacaceae) is found in Amazon region, Nicaragua, Panama and Costa Rica1. This species showed the acetylenic acid2,3; xantone3and terpenes3'4. The aim of this study was the phytochemical study of M. guianensis branches extracts. The plant collection was realized in Reserva Florestal Ducke, Manaus, AM. The dichloromethane extract was submitted to column chromatographic on silica gel obtaining several fractions, among these a fraction showed crystals and they were analyzed by Nuclear Magnetic Resonance (NMR) of 1H and 13C. The spectral data were obtained in Varian lnova-500 instrument and were recorded at 125 MHz for 13C and 500 MHz for 1H. The experiments were realized at 28 °C, using 1 chloroform (CDCI3) as solvent and internal standard. To obtain the H NMR spectra it was utilized the sequence of pulse s2pul, pulse 45.0 degrees, relaxation delay of 1.000 seconds, acquisition time of 2.049 seconds, line broadening of 0.2 Hz and there was made 16 repetitions. To obtain the 13C NMR spectra it was utilized the sequence of pulse s2pul, pulse 45.0 degrees, relaxation delay of 1.000 seconds, acquisition time of 1.300 seconds, line broadening of 0.5 Hz and there was made 1000 repetitions. To spectral data of DEPT was similar as NMR of 13C, the difference was observed in pulse 90.0 degrees, acquisition time of 1000 seconds, line broadening of 1.0 Hz and there were 256 repetitions. 1 The H NMR spectral data analysis showed the õH 4.57 (1H; d; J = 2.4 Hz;H-29) and 2 4.69 (1H; q; J = 1.5 and 1.0 Hz; H-29) of a hydrogen linked to a sp carbon, in õH 2.63 (1H; dd; J = 4.4 and 2.2 Hz; H-3) of a hydrogen linked to carbon connected to a methoxyl group, in õH3.35 (3H; s) related to 3-/3-methoxyl group. 13 The C NMR spectral data analysis showed õc 150.99 (C; C-20) and 5C 109.29 (CH2; C-29) characteristic signs of lupane skeleton. There were observed signs as õc 88.99 (CH; C-3) of carbon connected methoxyl group and õc 57.51 (OCH3; C31) of a methoxyl group. As the most common triterpene with this skeleton is the lupeol, in order to determine if the proposition of 3-p-methoxyl group it was correct, first we performed calculations taking off the a, p and y OH effects and then putting on the respective OMe effects. Then they were also compared with the literature available5 (Table 1). Table 1: Calculated a, P and y effects on lupeol chemical shifts 3^ + Calculated (3-/3- Lupeol -OH Calculated OMe methoxy-lup- Data6 Effect (Lupene) H.CO' Effect 20(29)-ene) 1 38.7 +5 43.7 -4 39.7 2 27.4 -8 19.4 +5 24.4 3 79.9 -41 38.9 +51 89.9 4 38.8 -8 30.8 +5 35.8 5 55.3 +5 60.3 -4 56.3 Other 13C NMR chemical shifts are shown in table 2. f- + 130 l.íihVl 1 I \K M.\(iM I IC KI-NOWNCI I SI1<>< M! [ IIV, Table 2:13C NMR chemical shift data of 3-/3-methoxy-lup-20(29)-ene Mahato and Mahato and Carbon Observed Carbon Carbon Observed Carbon Kundu,1994 Kundu, 1994 1 38.7 40.02 CH2 17 43.0 43.01 C 2 27.4 25.21 CH2 18 48.0 47.99 CH 3 78,9 88.69 CH 19 47.9 48,35 CH 4 38.8 38.08 C 20 150.9 150.99 C 5 55.3 55.89 CH 21 29.8 31.92 CH2 6 18.3 20.97 CH2 22 40.0 40.89 CH2 7 34.2 34.32 CH2 23 28.0 28.02 CH3 8 40.8 38.62 C 24 15.4 16.00 CH3 9 50.4 50.47 CH 25 16.1 16.14 CH3 10 37.1 37.22 C 26 15.9 16.09 CH3 11 20.9 22.26 CH2 27 14.5 14.53 CH3 12 25.1 29.36 CH2 28 18.0 18.00 CH3 13 38.0 38.81 CH 29 109.3 109.29 CH2 14 42.8 42.83 C 30 19.3 19.33 CH3 15 27,4 27.45 CH2 31 - 57.51 OCH3 16 35.5 35.61 CH2 4.57 H H 4.69 Figure 1: NMR coupling of COSY (<£>) and HMBC (<£-) to 3-/3-methoxy-lup-20(29)-ene. This is the first report of 3-/3-methoxy-lup-20(29)-ene in Olacaceae family. REFERÊNCIAS: 1Mobot, 2011 (Accessed on 03/02/2011). 2Marles, R. J. ; Farnsworth, N. R.; Neill, D. A. Jour, of Nat. Prod. 1989, 52(2), 261-266. 3EI-Seedi, H. R.; Hazell, A. C.; Torssell, K. B. G. Phytochem. 1994, 35(5), 1297-1299. 4Cursino, L. M. C.; Mesquita, A. S. S.; Mesquita, D. W. O; Fernandes, C. C.; Pereira Junior, O. L.; Amaral, I. L. A.; Nunez, C. V. Acta Amazônica. 2009, 39(1), 181-186. 5Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric identification of organic compounds; Wiley, J and Sons, INC, Ed.; United States, 2005. 6Mahato, S. B.; Kundu, A. P. Phytochem. 1994, 37(6), 1517-1575. CNPq/CT-Agro, PPBio/INPA/CNPq/MCT, FAPEAM, CENBAM/CNPq/MCT. 131 h * PO 57 UNILATERAL NMR: CONSTRUCTION AND APPLICATIONS TO MONITOR THE TRANSESTERIFICATION REACTION L. F. Cabeça1, L. V. Marconsini1, L. A. Colnago*1 1Embrapa - Instrumentação Agropecuária, R. XV de Novembro, 1452, São Carlos - SP colnago@cnpdia. embrapa. br Keywords: Biodiesel; transesterification reaction; CPMG. The magnet of the unilateral NMR (UNMR) sensor was constructed in a classical "U-shaped" geometry, as described by Blümich and co-workers [1]. The static magnetic field is generated by two permanent magnets with anti-parallel magnetization. The magnetic pole was built with two pieces of NdFeB alloy (5.08 x 5.08 x 2.54 cm). Surface coil (constructed with a single flat radio frequency coil etched from a standard printed circuit board and tune at 14.3 MHz) is positioned in the gap between the two magnets so that the polarized static magnetic field B0 and radio frequency B1 are approximately orthogonal to each other, within a fairly large volume above the probe surface. The aim of this work was to evaluate the potential of constructed UNMR sensor to monitor the transesterification reaction in situ and the effect of methanol contamination in biodiesel quantification. The transesterification reaction, catalyzed by NaOH, was prepared according to Murugesan, 2009 [2], The reaction was performed in a 20 ml_ beaker containing 6 g of soybean oil, 6 g of anhydrous methanol (1:1 oil-to-alcohol mole ratio) and 0.06 g NaOH (1% w/w of oil). The system was stirred during the reaction at room temperature (Figure 1A) and it was stopped for date acquisition. The Unilateral sensor was driven by a CAT-100 Tecmag console, a power amplifier 3205 AMT and a preamplifier of Miteq AU 1114. The analysis were performed with a CPMG pulse sequence using pulse widths of 3 ps, x = 500 ps, acquisition time of 64 ps at the center of the 1000 echoes, and a recycle delay of 500 ms and 500 scans. The effects of methanol contamination on biodiesel measurements using the LRNMR (bench top and UNMR) and UNMR were performed with a CPMG pulse sequence. Mixtures containing 5%, 10%, 20%, 30% and 50% MeOH in biodiesel were prepared for analysis. To calculate methanol influence on the intensity of the CPMG signals of the biodiesel/methanol mixtures, average values of intensities for the first one hundred echoes were used. The Figure 1A illustrates the experimental assembly to monitor the transesterification reaction in situ. It consists of stirrer and a becker with the reaction media on the top of UNMR sensor. Reaction Time (min.) Figure 1: A) UNMR sensor monitoring the transesterification reaction. B) Variation of the effective time constante (T2eff) of CPMG signal in function of reaction time. Figure 1B shows the variation of effective T2 (T2eff) with the transesterification reaction time, monitored in situ. This figure shows a reduction of T2eff with reaction time. The decay of the CPMG signal of the soybean oil was longer (110 ms) than the one observed for biodiesel (29 ms), which had an inverse result when compared to the j. • 132 I MliNl'C I I:\R MAGNETIC RLISONANCU L'SERN ML:L 'I INC. conventional LRNMR experiment. A longer CPMG decay value for more viscous samples is a well-known behavior of NMR signals acquired with UNMR sensors [3], and can be understand trough the equation bellow. f t y2G2DT2t^ Mt (echo at t) oc exp V 1T2 J 3 J where the Mt is magnetization, y is gyromagnetic ratio, G is gradient and D is the diffusion coefficient. A calibration curve prepared between T2eff and biodiesel concentration in the mixture was used to quantify the biodiesel in the transesterification reaction. The effect of methanol residue in the T2eff of biodiesel/oil mixture was also verified. When using bench top NMR the T2 values increase with the methanol concentration in the mixture (Figure 2A). Figure 2A shows T2 measurements were very sensitive to the presence of methanol contamination in biodiesel. This sensitivity could be explained by the increase of the contribution of methanol in the CPMG signal. The same measurements were made using the UNMR and the T2eff were 45 ms ± 3 ms for all methanol concentration in biodiesel. This indicates that UNMR measurements are not sensible to methanol contamination. This property can be explained by the equation above. The methanol is much smaller molecule than oil and biodiesel and it diffuses is very fast. Therefore, its magnetization loses coherency very quickly in the presence of strong G and it is not fully refocused by the CPMG sequence. The presence of the methanol in the biodiesel using UNMR can be monitored by the reduction of the intensity of the biodiesel CPMG signal (Figure 2B). 0 20 40 60 80 100 0 10 20 30 40 50 MeOH in Biodiesel (%) MeOH in Biodiesel (%) Figure 2 - Variation in the average intensity of the first 100 echoes of the CPMG signal for biodiesel/methanol mixture. A) Bench top NMR and B) UNMR. Therefore, this results shows that UNMR can be a useful sensor to monitor transesterification reaction in situ, direct the the reactor. One of the problems observed when using UNMR to monitor the transesterification reaction was its strong dependence on the gradient, which depended on the size of the magnets, its construction and the distance of the sensitive volume from the sensor. Therefore, the sensitivity of the sensor to diffusion should always be tested to obtain similar values. REFERENCES: 1. Blümich B.; Perlo J.; Casanova F. Prog. Nucl. Magn. Reson. Spectrosc. 2008, 52, 197. 2. Murugesan A.; Umarani C.; Chinnusamy T. R.; Krishnan M.; Subramanian R.; Neduzchezhain N. Renew. Sustain. Energy Rev. 2009, 13, 825. 3. Pedersen H. T.; Ablett S.; Martin D. R.; Mallett M. J. D.; Engelsen S. B. J. Magn. Reson. 2003, 165, 49. FAPESP, CNPq, FINEP 133 h * PO 58 NMR STUDIES ON A PLATINUM(II) COMPLEX DERIVED FROM PYRAZINAMIDE PRESENTING IN VITRO ANTITUMORAL PROPERTIES T. S. Ribeiro*1, L. Sartori1, L. B. Borré2, R. A. S. San Gil2, J. D. Figueroa-Villar3, N. A. Rey1 1 Pontifical Catholic University of Rio de Janeiro, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 3Military Institute of Engineering, Rio de Janeiro, Brazil tsantanaribeiroda). yahoo, com, br Keywords: platinum(ll) complex, solution and solid-state NMR studies, anticancer activity Among the transition metal complexes with antitumor activity, platinum(ll) compounds are the most active.1 However, the toxic side effects associated with these compounds and the emergence of resistant tumors have stimulated the search for novel analogues. Studies showed that, in general, all platinum(ll) complexes with a given size and shape, and with a certain polarity and capable of binding to biomolecules will likely have anticancer activity.2,3 4 Pyrazinamide (PZA, C5H5ON3) is a drug widely used for tuberculosis treatment. Recently, we synthesized a platinum(ll) complex of PZA, namely, cis-[PtCI2(PZA)2]-1.5 5 H20, which displayed a potent in vitro activity against K562 human cancer cells. With the aim of improve our knowledge concerning the solution chemistry of cis- 1 [PtCI2(PZA)2]-1.5 H20, it is reported herein a H NMR study of thjs complex in DMSO- 1 13 d6. H and C spectra of free PZA were obtained in the same conditions for the sake of comparison. Additionally, 13C CPMAS solid-state NMR experiments were performed for ligand and platinum(ll) complex to elucidate univocally the coordination mode of PZA. Table 1 summarizes all the obtained data and the respective assignments. Table 1: NMR spectra assignments for the PZA ligand and its platinum(ll) complex Position PZA c/s-[PtCI2(PZA)2]-1.5 H20 N-Ha 7,79 (s; 1 H)b 8,07 (s; 1H) N-Hb 8,26 (s; 1H)b 8,40 (s; 1H) £ _ 2 — — 3 9,18 (d, 4J= 1,5; 1H) 9,43 (d, 1H) jr 5 8,63 (dd, *J= 1,5 and 4J= 2,5; 1H) 8,72 (dd, 1H) to 6 8,85 (d, JJ= 2,5; 1H) 9,07 (d, 1H) C=0 — — 2 145,1 Could not do the 13C NMR of the O 3 143,6 CO 5 147,4 complex due to solubility problems. to 6 143,4 C=0 165,0 2 148,6 153,9 cn o < 3 143,5 147,1 2 ^ 5 148,6 153,9 to CL O 6 143,5 147,1 C=0 167,3 165,3 The presence of two absorptions related to -NH2 hydrogen atoms rules out the possibility of PZA coordination through this group.5 However, the most relevant information come out from the 13C CPMAS spectra. It can be clearly seen that all the carbon nuclei were de-shielded upon coordination: a a-charge donation through the f- + 134 amide carbonyi O-donor to the platinum center removes electron density from PZA and produces this result. The only exception is precisely the carbonyl carbon, whose signal is shifted from 167.3 (in free PZA) to 165.3 ppm. It is suggested that the effect could be related to the % back-donation from the platinum into the C=0 group, confirming thus the previously proposed structure. An 1H NMR kinetic experiment was conducted by measuring the spectra of a DMSO-d6 solution of the complex after 0.5 and 36 h of its preparation. It was observed that, over time, DMSO is able to replace PZA in the platinum(ll) coordination sphere, giving rise to mono (I) and bi-substituted (II) species and releasing free PZA (Figure 1). At the time of 36 h, signals relative to the cis- [PtCI2(PZA)2] species could not be more observed. This result is of great interest, since DMSO was the solvent used to prepare the stock solutions employed in the biological assays. These solutions were then diluted with water to desired concentrations. Hb A. CO, .a JGI DHS&Í, \ / . < Pf 0: c D,c' "'CCS cd» PtPZA (36 h) * A J«tvJi * eis-[ PtCIjiPZA), free PZA V1 /\) \ PtPZA (0.5 h) PZA n*„ ft 4 n f 8 ? ? ? 6 Figure 1: 1H NMR spectra of PZA and the PtPZA system (after 0.5 and 36 h of dissolution) in DMSO-d6. REFERENCES 1. Guerra, W.; Fontes, A. P. S.; Almeida, M. V.; Silva, H. Química Nova 2005, 809. 2. Fiorentino, M. V.; Ghiotto, C. Inorganica Chimica Acta 1987,13. 3. Barnad, C. F. J.; Cleare, M. J.; Hydes, P. C. Chemistry in Britain 1986, 1001. 4. Ozols, R. F. Seminars in Oncology 1989, 22. 5. Sartori, L.; Freitas, M. C. R.; Diniz, R.; Rey, N. A. Síntese e Caracterização de um Complexo de Pt(ll) da Pirazinamida. XXXIII RASBQ 2010. 6. Woon, T. C.; Fairlie, D. P. Inorganic Chemistry 1992, 4069. CAPES/CNPq/FAPERJ 135 h * PO 50 qHNMR OF ANTIFUNGAL ANTIBIOTIC GRISEOFULVIN IN POLYMERIC MICELLES M.E.N.P.Ribeiro1, M.G.S.Vieira1, N.M.P.S.Ricardo1, N.V.Gramosa*1 1 Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, Fortaleza,CE, Brazil *e-mail:nilce@dqoi. ufc. br Keywords: griseofuivin, polymeric micelles, qHNMR. Polymeric micelles usually consist of several block copolymers and may serve as nanoscopic drug carriers. Due to their nanoscopic size, they penetrate into pathological sites such as solid tumors and may release specific drugs [1]. Solubilization of water- insoluble drugs into solution is one of the main problems found in formulating such drugs in liquid dosage forms [2], Griseofuivin (1) is an antifungal antibiotic widely used against several filamentous fungi. It is a poorly soluble drug in water and is used as standard in the study of solubilization of drugs in polymeric micelles. The polymeric surfactants comercially known as Brijs (EOmCnH2n+i, m and n are the number of units), become interesting because they are non-ionic, decreasing the toxicity of these types of carriers. They contain a hydrophilic head, with varying number of polyoxyethylene (POE) groups and a distinct hydrophobic tail consisting of a polymethylene chain [3]. Quantitative NMR spectroscopy (qNMR) has become increasingly important for natural products analysis and is also used in drug analysis, agriculture, material science, etc.. Advances in the development of qNMR methods in drug analysis is mainly due to the simple preparation of samples, the speed of analysis and the possibility of obtaining structural information in a complex matrix [4,5], In recent years qHNMR has been used to quantify chemical constituents in mixtures because it is one of the most reliable and precise methods [6]. The aim of this work is to demonstrate the application of qHNMR as a tool in the quantification of the antifungal antibiotic griseofuivin incorporated into the copolymer micelle of polyoxyethylene-(20)-stearyl-ether (brij 78) (2) (Figure 1). [CH3(CH2)15CH2CH2][0CH2CH20(CH2CH20)18CH2CH20H] AB CD EE FF GG 2 Figure 1: Chemical structures of griseofuivin (1) and copolymer Brij 78 (2) The samples were prepared using a portion (10 cm3) of a stock 1 wt. % copolymer water solution added to a finely-ground (1 mm2 mesh) griseofuivin powder (0.02 g). The mixture was stirred at 37 °C for 72 h and filtered (0.45 pm Millipore). A solution with solubilized drug was then freeze dried (24 h, <103 mmHg) to remove the water. Two samples (MGC-1 and MGC-2) were prepared. The samples MGC-1 (15.2 mg) and MGC-2 (16.0 mg) were dissolved in 600 pL of CDCI3 and placed into 5 mm NMR tubes. The 1H NMR spectra were recorded on a Bruker DRX 500 (11.7 Tesla; 499.80 MHz for 1H) spectrometer using a 5 mm inverse detection z-gradient probe at 298K. The 136 spectra were obtained using the 90° rf pulse (9.20 ps), a spectral width of 12019 Hz, 256 transients with 64K data points, an acquisition time of 2.73 s and relaxation delay of 10 s. Each spectrum was processed with LB 0.3 Hz and zero-filled. The phase was corrected manually for each sample using the Bruker Software and polynomial baseline correction was applied over the spectral range. The chemical shifts were reported in parts per million (ppm) and all the spectra were referenced to the residual proton signal 1 of the CDCI3(<5h 7.27). The H NMR experiments were repeated two times for each tube. The amount of griseofulvin solubilized per gram of polymer was determined by 1H NMR through the peak integrals of the methyl protons signal at 5H 0.95 (CH3-5') of griseofulvin and at § 0.87 (H-A) of Brij 78 (Figure 2). The molar ratio of griseofulvin and Brij 78 was calculated according to Malz and Jancke (2005) [4] considering the integrated signal areas and the relative number of spins which cause the signal. The amount of the griseofulvin in the mixture with Brij 78 was calculated as 8.2 and 8.5 mg per 1g of polymer for MGC-1 and 9,3 mg per 1g of polymer for MGC-2. These data were similar to those found by the UVA/is method 8.9 and 8.5 mg per 1g of polymer for MGC-1 and MGC-2, respectively. In conclusion, gHNMR can be used as method to quantification of the griseofulvin encapsulated by polymer micelles. 1 H NMR data for griseofulvin (500MHz, CDCI3, S): 6.13 (H-5, 1H, s), 5.52 (H-2', 1H, s), 4.16 (4-OCH3,3H, S), 3.91 (6-OCH3, 3H, s), 3.63 (2'-OCH3, 3H, s), 3.00 (H-4', 1H, dd, J = 16.0, 3.5 Hz), 2.41 (H-5', 1H, dd, J= 16.7, 4.0 Hz), 2.86 (H-6\ 1H, m), 0.95 (5'-CH3, 3H, d, J = 6.9 Hz). 1 H NMR data for Brij 78 (500MHz, CDCI3, 8): 0.87 (H-A, t, J = 6,7 Hz), 1.27 (H-B, m), 1.56 (H-C, m), 3.44 (H-D, m), 3.57 (H-E, m), 3.61 (H-F, m), 3.71 (H-G, m). H-A 5'-CH LuJ UJ -J WjJ SB N ÉS « h w 5 $ 3 0 4.S 4 O JO 2.5 1 Figure 2: H NMR spectrum in CDCI3of mixture of griseofulvin and Brij 78 polymer REFERENCES 1. Tyrrell Z. L., Shena, Y., Radosza, M. Progress in Polymer Science 2010, 1128. 2. Ahmed, M. O. European Journal of Pharmaceutics and Biopharmaceutics 2001, 221. 3. Sowmiya, M.,Tiwari, A. K„ Saha, S. K. J. Colloid Interface Sci. 2010, 97. 4. Malz, F.; Jancke, H.J. Journal of Pharmaceutical and Biomedical Analysis 2005, 813. 5. Pauli, G.F., Jaki, B.U., Lankin, D.C. J. Nat. Prod. 2005, 133. 6. Jianga, Y., David, B., Tua, P., Barbin, Y., Anal. Chim. Acta 2010, 9. CAPES (PRODOC), CNPq, PRONEX (CNPq/FUNCAP), CENAUREMN 137 y PO 60 STRUCTURE ELUCIDATION OF THE NEW DITERPENE EA/T-TRACHYLOBAN-18,19 - DIOL BY 1H AND 13C NMR M.G.S. Vieira1, E.R. Silveira1, N.V. Gramosa*1 1 Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, Fortaleza,CE, Brazil *e-mail:niice@dqoi. ufc. br Keywords: trachylobane, Xylopia nítida, 1H & 13C NMR Xyiopia nitida is a plant of the family Annonaceae widely distributed in the Araripe Plateau, Ceará State, Northeast, Brazil, where it is popularly known as "embiriba branca" [1]. Previous phytochemical studies with Xylopia species showed mainly diterpenes and alkaloids. The principal types of diterpenes found in this genus were atisane, kaurane, labdane and trachylobane, while the aporphine alkaloids were predominant [2,3], The aim of this work is to show the application of 1D and 2D NMR techniques for the structural elucidation of enf-trachyloban-18,19-diol (1) (Figure 1), a new diterpene isolated from the roots of X. nitida Dunal. The hexane extract of the roots of Xylopia nitida was chromatographed over a silica gel column yielding (1) as colorless crystals. The NMR experiments were recorded on a Bruker DRX 500 (11.7 Tesla; 499.80 MHz for 1H) spectrometer using a 5 mm inverse detection z-gradient probe at 298 K. The 1H NMR spectra were obtained using the 90° rf pulse (9.20 ps), relaxation delay D1 of 1s, spectral width of 12019 Hz, 8 transients with 64k data points. The phase was corrected manually using the Bruker Software. The 13C NMR spectra were obtained using a spectral width of 32679 Hz, a 14.25 ps (30°) pulse, and 8k scans with 65 K data points. The 1H NMR spectrum was calibrated using the residual 13 proton signal of the solvent CDCI3 at <5H 7.27 and for C NMR spectrum at <5C 77.3 (central peak). 2 The HRESIMS of 1, {mp = 127.0-128.3°C; [a] o° = -41* (c 0.1; CHCI3)}, showed a + molecular ion adduct of m/z 327.2349 (M + Na) suggesting a MF: C2oH3202 (Calc. 327.2295). The 1H NMR spectrum showed characteristic signals for a cyclopropane moiety: 8H 0.56 (1H, d, J= 7.7 Hz, H-12) and 0.80 (1H, dt, J = 7.5; 2.4 Hz, H-13). Two methyl singlets at §H 0.90 (3H, H-20) and 1.12 (3H, H-17), and four dublets at ÔH 3.91 (1H, J= 10.5 Hz, H-19a), 3.88 (1H, d, J = 10.5 Hz, H-18a), 3.71 (1H, d, J= 10.5 Hz, H- 19b) and 3.33 (1H, d, J = 10.5 Hz, H-18b) characteristic of two diastereotopic hydrogens of two oxymethylenes. Trachylobanes are well known diterpenes possessing a cyclopropane ring which the standard skeleton presents four methyl groups. Thus, the data above suggested that two of the methyl groups were oxydized. The sites of oxydation determination as the carbons 18 and 19 was accomplished through the long-range heteronuclear correlations observed in the HMBC spectrum of 1 for the hydrogens at SH 3.71 & 3.91 with the carbinolic carbon at Sc 74.0 (C-18) and of the hydrogens at SH 3.33 & 3.88 with the other carbinolic carbon at 8C 65.0 (C-19). All of those hydrogens also showed long-range correlations with the carbons at 6C 53.55 (C-5), 41.8 (C-4) and 30.5 (C-3). All the other assignments are depicted in Table 1. This is the first report about the isolation of eni-trachylobane-18,19-diol (1) in the literature. y f 138 (1) TABLE 1 - NMR assignments of e/i£-trachyloban-18,19-diol C 5c ÕH m* J(Hz) HMBC 1 39.1 1.52 d 13.2 H-3; H-20 0.78 dt 13.2; 3.9 2 17.4 1.55 m - H-1 1.35 m - 3 30.5 2.00 d 11.6 H-18; H-19 0.96 m - 4 41.8 - - - H-5; H-18; H-19 5 53.5 0.94 m - H-1; H-3; H-9; H-18; H- 19; H-20 6 20.8 1.60 m - H-5 7 39.3 1.35 m - H-15 8 40.7 - - - H-6; H-9; H-11; H-13; H-14; H-15 9 53.6 1.14 m - H-1; H-11; H-12; H-15; H-20 10 38.3 - - - H-1; H-5; H-20 11 21*1 1.88 dt 14.3; 2.6 H-12 1.63 dd 14.3; 6.9 12 20.8 0.56 d 7.7 H-13; H-14; H-15; H-17 13 24.4 0.80 dt 7.5; 2.4 H-14; H-15; H-17 14 33.6 2.02 m - H-9; H-12; H-15 1.14 m - 15 50.5 1.36 d 11.2 H-17 1.23 d 11.2 16 22.6 - - - H-14; H-15; H-17 17 20.7 1.12 s - H-12; H 13; H-15 18 74.0 3.88 d 10.5 H-5; H-19 3.33 d 10.5 19 65.0 3.91 d 10.5 H-3; H-5; H-18 3.71 d 10.5 20 15.1 0.90 s - H-1; H-5; H-9 *Multiplicity REFERENCES 1. Martins, D. Tese de doutorado, Universidade de São Paulo, 1996, Brazil. 2. Martins, D., Alvarenga, M. A., Roque, N. F., Felício, J. D. Quim. Nova, 1995, 14. 3. Harrigan, G. G., Bolzani, V. S., Gunatilaka, A. A. L. Kingston, D. G. Phytochemistry 1994,109. FUNCAP, CNPq, CAPES, CENAUREMN 139 b I li'I mu i mi I1— PO 61 DETERMINATION OF BIODIESEL IN DIESEL USING 1H AND 13C NMR WHIT MULTIVARIATE DATA ANALYSIS Francisco F. Gambarra-Netoa*, Clayton R. de Oliveiraa, Marcos R. Monteirob, Antonio Gilberto Ferreiraa aUFSCar/DQ/Laboratório de RMN, bUFSCar/DEMA/CCDM/Laboratório de Combustíveis chicoqambarra(çò.Qmail. com Keywords: biodiesel quality control; chemometrics; 1H NMR/13C NMR In order to sell biodiesel as a fuel (B5) in diesel, it must meet a set of requirements defined in CNPE N° 6, which specify the minimum allowable concentrations of biodiesel in diesel. The increase in biodiesel has a positive effect on the national energy1. The amount of B5 in diesel allowed by CNPE N° 6 is 5.0% (v/v). Generally this parameter of biodiesel is obtained by infrared2, but this technique needs preparation of sample in KBr, which is unnecessary with 1H and 13C NMR spectroscopy. The amount of information available in an NMR spectrum and the easy sample preparation turns this spectroscopic technique very attractive for the assessment of biodiesel control quality. As an analytical tool, 1H and 13C NMR spectroscopy has many advantages, but its main limitation is the low sensitivity comparing to other analytical techniques3. To overcome this problem, multivariate calibration methods have been used directly on NMR spectral data. The most straightforward advantage of multivariate calibration is a noise reduction obtained by using more measurements of the same«phenomenon when compared to univariate calibration4. In this work we have developed a model to predict the amount of B5 in diesel by using 1H and 13C NMR spectroscopy and Successive Projection Algorithm-Multiple Linear Regression5 (SPA-MLR), which corresponds to the combination of a variable selection and multivariate calibration algorithms, respectively. The samples consist at B5 obtained of UFSCar-CCDM combustible laboratory, in the concentration range from 4.5 to 5.6% (v/v). A 0.470 ml aliquot of each sample was 1 mixed with a capillary contained 0.200 ml of CDCI3 and the H NMR spectra were recorded on a Bruker DRX400 - 9.4 Tesla spectrometer, in a 5 mm inverse probe at 303K, using quantitative experimental conditions with accumulation of 128 transients and TMS as external reference. The spectrum of each sample was acquired three times to take into account the variations in adjustment of magnetic field homogeneity. A 2.000 ml aliquot of each sample was mixed with a capillary contained 0.600 ml of D20 with TSPD-d4 and the 13C NMR spectra were recorded on a Bruker Avance III 400 - 9.4 Tesla spectrometer, in a 10 mm probe at 303K, using quantitative experimental conditions with accumulation of 4096 transients. The spectrum of each sample was acquired once time. The regions signals of solvents and external references were discarded for all the 18 1H and 13C spectra. The number of data points (variables) used in the FID was 64K and 32K for 1H and 13C respectively. The plus remaining 57,000 variables were considered as working spectral range for chemometrics applications. Moreover the spectra were separated into two groups, which are 12 spectra to calibration set and 6 to validation set. Among the 57,000 variables, SPA algorithm selected only 5, 4 in 1H and 1 in 13C RMN, which correspond to more informative signals associated to biodiesel (figure 1). These five variables generated an MLR equation to calculate the amount of B5 in diesel from NMR data. The validation regression analysis has shown a correlation coefficient R2 of 0.997 and a root mean square error of validation (RMSEV) of 0.027% (v/v) (figure 2). f 140 Till M l í l.-\K M.\(IM-1 Jt' Ki-^OVWfl I Si RN Ml I I INCJ V ÍÍAIIjuL "I I "I1 7 6 5 Chemical shift (pprn) Figure 1.1H and 13C NMR spectrum of B5 showing selected variables. 4 5 Variabel number Figure 2. Variable number versus RMSEV values plots for B5 in diesel. The study has shown that 1H and 13C NMR and selection variable algorithm together with multivariate calibration are enough to generate an efficient model to predict the composition of B5 in diesel samples. This will be important to build robust multi- parameter quality control method in order to supply the demand of Brazilian biodiesel trade. REFERENCES 1. Resolução CNPE N° 6; Conselho Nacional de Política Energética; 19-09-2009; DOU 26-10-2009. 2. Liquid petroleum products; Infrared spectrometry method; EN 14078: 2009. 3. Field, L. D., Sternhell, S.; Analytical NMR\ John Wiley & Sons, 1989. 4. Bro, R.; Anal. Chim. Acta, 2003, 500, 185-194. 5. Araújo, M. C. U.; Saldanha, T. C. B.; Galvão, R. K. H.; Yoneyama, T; Chame, H. C.; Visani, V.; Chem. Intell. Lab. Syst., 2001, 57, 65-73. CNPq 141 h * PO 50 SOLID STATE NMR AS STRUCTURAL PROBE FOR LUMINESCENT INORGANIC-ORGANIC HYBRID MATERIALS T. B. Queiroz,1* M. Botelho,1 R. M. Ilibi,2 J. Fernandez-Hernandez,3 M. D. G. López,3 H. Eckert,2 L. de Cola,3 A. S. S. de Camargo1 11nstituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sancarlense 400, São Carlos - SP, CEP 13560-970, Brazil. 2lnstitut für Physikalische Chemie.WWU Münster, Corrensstr. 30, D-48149 Münster, Germany. 3Physikalisches Instituí, WWU Münster, Mendelstrasse 7, D-48149, Münster, Germany. * corresponding author: branguinho&.ursa. ifsc. usp. br Keywords: luminescent hybrid materials; local bonding effects; solid state NMR. The design of efficient luminescent hybrid materials for applications as sensors, OLEDs or solid state lasers is an important area of materials science research. An important strategy for generating optical materials is to disperse molecular emitters in inorganic crystalline or glassy solid host matrices, resulting in mechanically stable inorganic-organic hybrid materials with higher chemical stability and improved photophysical properties [1,2]. As the emission wavelengths, excited state lifetimes and quantum yields are strongly influenced by both the intermolecular interactions among the guest molecules and the interactions between guest molecules and the host matrices, a fundamental understanding of these structural issues is an important requirement for developing improved design strategies. In principle, such information can be obtained by solid-state NMR spectroscopy, owing to its element-selectivity, its inherently quantitative character, and its great sensitivity to local bonding effects. We are currently developing new solid state NMR approaches for characterizing a range of different luminescent inorganic-organic hybrid materials [3], The host matrices studied include ordered mesoporous silica (MCM-41), mesoporous sodium aluminosilicate glasses, as well as layered clay materials, while the luminescent guest species are rhodamine, iridium coordination compounds, and europium complexes. The materials are prepared using three distinct host-guest interaction principles: a) covalent attachment via grafting reactions, b) Coulombic interactions driving topotactic ion exchange, and c) dipolar and van-der-Waals interactions directing templated sol-gel co-assembly. Multinuclear solid state NMR techniques using magic angle spinning with and without cross polarization (CP and SP) are useful for monitoring the various steps of synthesis, assembly and processing. This can be exemplified through a series of 29Si MAS and 13C{1H} CP-MAS experiments (fig. 2) proving the functionalization and ligand grafting in MCM-41 (scheme in fig. 1) prior to formation of the Europium complex. Guest-host and guest-guest interactions can be probed efficiently via chemical shift or dipolar spectroscopy and spin-lattice relaxation time measurements. Finally, spin counting studies lend themselves to absolute and relative quantifications of the luminescent species incorporated. 142 ililiM'il l.-M< M\fiM IHRWASVI I Sl-K«i Mil 'I i\< i C=Q Figure 4: Scheme of the bicarboxylic acid-bipyridine ligand grafting in MCM-41 (2 -> 3) via amino-propyl-triethoxysilane (APTES) functionalization (1 -> 2). The labels of the coordinated atoms in blue (for 29Si NMR) and in red (for 13C NMR) are assigned in the spectra of the fig. 2. M 1 1 Si SPMAS (4kHz) O C{ H} CPMAS - H decoupling Q / I 2 T 3 MCM-41-30-Si-(CH2)3-NH2 Q \ © MCM-41 40 20 -20 -40 -60 -80 -100 -120 -140 -160 200 100 50 -100 5 (ppm) S(ppm) Figure 5: 29Si NMR of MCM-41 (left) before (in black) and after APTES functionalization (blue). The observation of T3 and T2 groups indicates successful APTES condensation on the MCM-41's surface. Also 13C{1H} CP-MAS NMR (right) of the MCM-41 after functionalization and ligand grafting (in black) and of the crystalline form of the ligand (red). The peaks assignments are shown in fig. 1, based on semi- empirical shift calculations.. One can note that the peak 1 in the free ligand splits into peaks 1 and 1' after the grafting, indicating the nucleophilic substitution at one of the carboxyl groups of the ligand. References: 1. D. Zhao, S-J. Seo, B-S. Bae, Advanced Materials 19 (2007) 3473 2. F. del Monte, D. Levy, Journal of Physical Chemistry. B 102 (1998) 8036. 3. R. Li, L. Zhang, J. Ren, T. B. de Queiroz, A. S. S. de Camargo, H. Eckert, J. of Non- Crystalline Solids 356 (2010) 2089 143 h * PO 61 MULTI-BAND AUTOMATICALLY TUNABLE HIGH-SENSITIVE NUCLEAR QUADRUPOLE RESONANCE SPECTROMETER L.M.C. Cerioni12, F. Picco1, D.J. Pusiol*1'2 1 Spinlock S.R.L., Av. Sabattini 5337, X5020DVD, Córdoba, Argentina 2CONICET (Argentinian National Research Council) e-mail: dpusiol&.nmr-spectrometers. com Keywords: nuclear quadrupole resonance; pharmaceuticals; polymorphisms Nuclear quadrupole resonance is a nondestructive, highly specific, noninvasive spectroscopic technique, and unlike nuclear magnetic resonance (NMR) it requires no static external magnetic field. NQR can be used to detect signals from solids containing 1 14 35 17 23 79 127 nuclei with spin quantum number I > /2 ( N, CI, 0, Na, Br, l). NQR frequency depends on the spin /, the quadrupole coupling constant (QCC) and the asymmetry parameter 77, the last both related to the electric field gradient tensor (EFG); which makes NQR highly sensitive to local environment changes. These features, as it has been shown, make NQR a powerful technique for identifying and study the properties of different polymorphs as well different hydrates forms in pharmaceutical agents, providing effective assistance at the main steps of drug development, manufacturing process and quality control.1"4 Vast majority of solid pharmaceutical forms have unreported NQR frequencies. Furthermore, the frequency range of the transition frequencies for a given nucleus can spread several MHz. As the intensity of the NQR signals Js very low, the implementation of a broadband probe would make signal detection very difficult. However, the implementation of a narrow-band has the drawback that the frequency scanning range of a single experiment is not large (-10-100 kHz), depending on the quality factor of the resonant tank circuit. Thus looking for the unknown NQR frequencies can be a very time consuming procedure. The aim of this work was the development and implementation of a high sensitive pulse NQR spectrometer, capable of measuring in different frequency bands, allowing the scan of new NQR signals in a fast, automatic and unattended way. Due to the great number of pharmaceutical agents containing 14N and 35CI, the spectrometer was developed for measuring in two different ranges corresponding to the intervals in which most of the reported frequencies are: 1.9-4.1 MHz (14N) and 34- 36 MHz (35CI).4 In order to make the spectrometer capable of measuring in both bands, a two capacitively coupled high-Q coils probe-head was built. To cover the whole bandwidth, the high-Q tuning and matching capacitors are mechanically adjustable. The spectrometer also includes a full bandwidth Q-damper system for reduction of the dead time between RF transmission and NQR signal acquisition; a switchable transmitter filters for elimination of the power amplifier distortion; and a sample temperature control for avoiding frequency drifts due to the temperature dependency of the transition frequencies. A block diagram of the spectrometer is shown in Figure 1. The tuning and matching capacitors are adjusted by a fast auto-tuning algorithm, feedbacked by reflected power. Q-damper frequency band and power amplifier filters are also fast and automatically adjusted. This allows making a full frequency sweep, performing measurements on the whole range of interest in a complete automatic way. For operating the spectrometer, a dedicated software was developed. This software allows the selection of parameters as initial, final and step frequency for the NQR signal scan, as well as pulse sequence and other experiment parameters. The software also allows to measure with several pulse sequences in each frequency step. Once the parameters have been set, the software automatically performs every necessary task to scan the defined spectrum. In addition, the software is capable of making spectra reconstruction implementing the spin-echo Fourier transform mapping spectroscopy.5 PC The spectrometer sensibility was tested using the standard samples hexamethylenetetramine (hmt) for 14N, and p-diclorobenzene (pdb) for 35CI. The spectrometer allows to detect 19 mg of hmt in 30 minutes using 16 averages of a 100 pulses SSFP sequence, and 7 mg of pdb in 1 minute using 80 averages of a Spin-Echo sequence. The spectrometer was tested reconstructing the previously reported spectra of pharmaceutical samples including carbamazepine, furosemide and hydrochlorothyazide. Nowadays, the spectrometer is been used for finding new NQR signals. Unreported lines were found in diclofenac sodium, aripiprazole and clopidigrel bisulfate. No NQR signal of these last two samples had been reported before. REFERENCES: 1. Pérez, S.C.; Cerioni, L.; Wolfenson, A.E.; Faudone, S.; Cuffini, S.L.; Int. J. Pharm. 2005, 298, 143-152. 2. Balchin, E. et al\ Anal. Chem. 2005, 77, 3925-3918. 3. Latosinsnska, J.N.; Expert Opin. Drug Discov. 2007, 2(2), 225-248. 4. Limandri, S. et al, 2011, 83(5), 1773-1776. 5. Busandri, A.; Zuriaga, M. J. Magn. Reson. 1998, 131, 224-231 145 h * PO 61 USE OF T1 AND T2 TO MEASURE THE SOLUBILITY PRODUCT OF PARAMAGNETIC IONS IN SOLUTION. P.F. Cobra*1, L. L. Barbosa2, L.V. Marconcini3, L.A. Colnago3 11nstituto de Química de São Carlos, São Carlos, Brasil 2Universidade Federal do Espírito Santo, Vitória, Brasil 3 Embrapa Instrumentação, São Carlos, Brasil e-mail:paulofcobra(p).amail.com Keywords: low-field NMR, CP-CWFP, paramagnetic ions. Nuclear magnetic resonance (NMR) relaxometry has been used in analytical chemistry to quantify the paramagnetic ions concentration in solution for more than 50 years. The method is based on the determination of spin-lattice, T-i, or transverse, T2, relaxation time of the solvent, which depends on the concentration of the paramagnetic species. Both Ti and T2 of the solvent are shortened by interactions with paramagnetic particles, which contain one or more unpaired electrons. Therefore, the paramagnetic ions contribution to the total relaxation rate is the dominant part in the relaxation mechanism of the hydrogen of the solvent.1'2 Bloembergen shows that T-\ and T2 in aqueous solution of paramagnetic ions are identical. Solomon confirmed this relation experimentally for Fe+3 aqueous solutions but not for Cr3+, Mn+2 and Gd+3. Bloembergen explains that this phenomenon is due to the spin exchange between the paramagnetic ions and the hydrogen in the neighboring water molecules.1'2 3+ 2+ 2+ 2+ Nole and Morgan studied T-i and T2 of hydrogen in Cr , Mn , Co and Nd aqueous solution in the frequency range of 2.7 to 28.7 MHz. They observed a decrease of Ti with the decrease of the frequency for Cr3+ and Mn2+ but not for Co2+ and Nd2+, while T2 was constant for each ion. They also reported large values for T-|/T2 as observed elsewhere as a result of the effect of high frequency.3 In this paper, the Carr-Purcell (CP) sequence with low refocusing flip angle, also known as CP-CWFP was used to measure the hydroxides solubility products (Ksp) of Fe(OH)3, Mn(OH)2 and Cu(OH)2 aqueous solutions in function of pH. The CP-CWFP sequence is ~ and can be used to measure T, and T2 in a single experiment.4 4 1 Fifteen solutions of each compound, FeCI3.6H20 3.6x10" mol.L , CuS04.5H20 4 1 -4 1 7.2x10" mol.L" and MnS04.5H20 1.2x1o mol.L" , were prepared with pH from 1 to 12. The T-i and T2 measurements with CP-CWFP were carried out on a SLK-SG-100 (Spin Lock Magnetic Resonance Solutions, ARG) 0.23 Tesla (9 MHz for 1H) bench-top spectrometer. The pulse sequence used was the CP-CWFP, using n/2 pulse width of 6.45 ps, echo time (r) of 141.47 ms, acquisition time of 10.6 ps, using 2,000 to 60,000 pulses and 4 scans. It was employed 1 mL of solution at 25 °C in each measurement. The ^^ and T2 were calculated by monoexponential curve fitting T* (time constant of steady state build up) and the intensity after the first pulse (M0) and in the steady state (MCP-CWFP). Figure 1 shows the relaxometric titration curves of the paramagnetic ions (Fe3+, 2+ 2+ Cu and Mn ). This figure illustrates that all ions have a Ti and T2 titration curve with a very sharp transition at pH 3.25, 6.50 and 9.36 for Fe+3, Cu+2 and Mn+2, respectively. Figure 1 also shows a subtle increase of the relaxation time, which is directly related to the increase of pH. In the turning point region, however, where the precipitation of the paramagnetic ions occurs in the hydroxide form, the pH variation is abrupt. Based on this figure it was possible to calculate the T^fT2 ratio which is 1.10, 1.20 and 2.93 for Fe+3, Cu+2 and Mn+2 respectively, at low pH values, before the equivalent point. 13thNUCLUAR MAGNETIC RESONASC 1.1 SI RS MEETING The titration curves were fitted with a sigmoid function and its first derivative gives the end point that can be used to calculate the Ksp. For these solutions, both relaxation times showed the same end point, indicating that Ti and T2 can be used to calculate Ksp. The values obtained for the -logKsp by NMR were 41.7 for Fe(OH)3, 21.9 for CU(OH)2 and 13.3 for Mn(OH)2,. According to literature, the -logKsp found for these 5 hydroxides were 38.7 (FeOH)3), 19.3 (Cu(OH)2) and 12.7 (Mn(OH)2). The differences encountered in the -logKsp values obtained through NMR measures in comparison with those found in literature are attributed to the difference in the temperature of the measurements. PH Figure 1: (•) Ti and (0)T2 values for milli-Q water containing FeCI3.6H20, CuS04.5H20 and MnS04.H20 in function of pH. In conclusion, it was demonstrated that both T-i and T2 can be used to determine the Fe(OH)3, Cu(OH)2 and Mn(OH)2 Ksp based in the influence of the magnetic moment change, caused by interaction of the paramagnetic ions on water hydrogens. We propose some advantages for the NMR method in comparison with the traditional method: the technique can be used at high viscosity solvents; relaxometric titration dispense chemical indicators and ion-selective electrodes; the use of electrolyte supports is not needed to eliminate currents migrations that interfere in the analysis; and the NMR measures don't depend of colored components like the spectrophotometers one. REFERENCES 1. Bloembergen, N.; J. Chem. Phys. 1957, 27, 572-573. 2. Solomon, I.; Phys. Rev. 1955, 99, 559-565 3. Nole, A.W.; Morgan, L.O.; J. Chem. Phys. 1957, 26, 642-648. 4. Andrade F.D.; Netto A.M.; Colnago L.A.; XI Jornada Brasileira de Ressonância Magnética, 2010, 65. 5. Skoog, A.D.; Holler, F. J.; Nieman, T. A.; Principles of Instrumental Analysis; Harcourt Brace & Company, New York, 1998. FAPESP, CNPq, EMBRAPA Instrumentação 147 h * PO 38 NMR STUDIES OF THE ABSOLUTE STEREOCHEMISTRY ASSIGNMENT OF AMINES ACHIEVED BY CYCLOHEXYL-BASED CHIRAL AUXILIARIES P.F. Bertotti, I.S. Resck, A.H.L. Machado* LITMO - Laboratory of Isolation and Transformation of Organic Molecules. New Technologies in Organic Synthesis Research Group. Universidade de Brasília - UnB, Brasília, Brazil e-mail:nagelo@ unb.br Keywords: NMR; sterochemistry assignment; chiral auxiliaries. Absolute stereochemistry assignment is a timeless challenge in natural product and synthetic organic chemistry. The most wildly used tool to assign the absolute stereochemistry of organic compounds is the NMR analysis of the derivative product of unknown absolute stereochemistry chiral compound prepared with chiral auxiliaries. Mosher's acid (a-methoxy-a-trifluoromethylphenylacetic acid or MTPA) and its analogs have a special position among the chiral auxiliaries used for this purpose. However, the reported differences between chemical shifts of MTPA derivatives have often been too small and the use of both enantiomers of the Mosher's acid or both enantiomers of the compound with unknown stereochemistry is necessary.13 Cyclohexyl-based chiral auxiliaries (CCA) have been used in organic synthesis since the 1970's and they have found widespread use as chiral auxiliaries. In 1975, E. J. Corey and co-worker introduced 8-phenylmenthol. This compound was designed to take advantage of the phenyl moiety to shield one of the two possible pro-chiral faces during an organic transformation. Some CCA'S are illustrated in Figtire 1.4"7 (-)-8-phenylmenthol 2 3 Figure 1: Cyclohexyl-based Chiral Auxiliaries (CCA's) This ability of the CCA's to block part of an organic molecule was envisioned as an structural feature in order to develop a new NMR tool for assigning the absolute stereochemistry of amines. The synthesis of the diastereomeric carbamates 5 and 6, respectively from /.-proline methyl ester and D-proline methyl ester, is presented below. Et3N, CH2CI2 r.t. OH CI Triphosgene 89 % yield 5 Ph Pyridine, CH2CI2 r.t. 94 % yield Et3N, CH2CI2 r.t. 92 % yield 6 Figure 2: Synthesis of the distereomeric carbamates 5 and 6. ^ + 148 Both diastereomeric carbamates had their structures confirmed by spectroscopic analysis and the observed chemical shifts for hydrogens Ha, Hb and H° are shown in Table 1. Table 1: 1H NMR assignments for Ha, Hb and Hc of 5 and 6 shown in Figure 3. Compound Ha Hb Hc £(ppm) 4.29, dd, £(ppm) 3.47-3.40, m, J(ppm) 3.29, dt, J = 8.6, 3.9 Hz (major rotamer) J= 10.3, 7.1 Hz (minor rotamer) + + (major +rotamer ) J(ppm) 2.82, dd, £(ppm) 2.57, ddd, £(ppm) 2.98, dt, J= 8.5, 3.2 Hz J= 10.4,7.6,4.8 Hz J= 10.3, 7.4 Hz (major rotamer) (minor rotamer) (minor rotamer) £(ppm) 3.65-3.50, £(ppm) 4.44, dd, 8(ppm) 3.65-3.50, m, m, J = 8.9, 1.9 Hz (minor rotamer) (minor rotamer) (minor rotamer) + 6 + + i>(ppm) 4.20, dd, £(ppm) 2.24, dt, £(ppm) 3.03, ddd, J = 8.5,4.3 Hz J= 10.2, 7.1 Hz J= 10.2, 7.8, 5.4 Hz (major rotamer) (major rotamer) (major rotamer) Despite the presence of two rotational conformers, a surprisingly large 1.38 ppm shielding for Ha of the most populated rotamer of the carbamate 5, when compared to 6, was observed. The observed results could be understood by the model presented below, where an out of phase stacking of the aromatic and the pyrrolidine moieties approximates only half of the cyclic amine to the aromatic shielding cone. observed shielding for Ha H3CO2C~ N H", n H\ . . cc o^o /^•N"—'K „ A H3co2c0=^ H H not observed shielding for Ha C H3CO2C. H -jjb HA H" Figure 3: Suggested model to comprehend the observed 1H NMR data of 5 and 6. The synthesis of new diastereomeric carbamates and carbonates are ongoing in our research group to verify the generality in the use of CCA's as an useful NMR tool to assign the absolute stereochemistry of organic compounds. REFERENCES 1. Wenzel, T. J.; Chisholm, C. D. Chirality, 2011, 23, 190. 2. Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem.1969, 34, 2543. 3. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512. 4. Corey, E. J.; Ensley, H. E. J. Am. Chem. Soc. 1975, 97, 6908. 5. Whitesell, J. K. Chem. Rev. 1992, 92, 953. 6. Gnas, Y.; Glorius, F. Synthesis 2006, 1899. 7. Sarakinos, G.; Corey, E. J. Org. Lett. 1999, 7, 1741. IQ-UnB, CAPES, CNPq 149 h * PO 50 INTRODUCING 170 NMR IN STERIC EFFECT STUDIES USING NMR AND MOLECULAR MODELING J.D. Yoneda1, K.Z. Leal*2, M.H.R. Velloso3, E.B. Lindgren2, P.R. Seidl4 'instituto de Ciências Exatas, Universidade Federal Fluminense, Volta Redonda, Brazil instituto de Química, Universidade Federal Fluminense, Niterói, Brazil 3Centro Universitário Norte do Espírito Santo, Universidade Federal do Espírito Santo, São Mateus, Brazil 4Escola de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil e-mail:kzleal(çb.uol. com, br Keywords: Adamantanes;17O NMR; steric effects. The separation of steric and electronic contributions have been very important to analyze structural influences on molecular properties. Although these interactions have been studied for a long time and applied to many systems, it is still very difficult to trace a line to separate the influence of these two contributions. The steric effect is a result of the orbital interpenetration of groups spatially close. Thus, a geometrical rearrangement occurs, where nuclei change their positions and the charges are redistributed, changing interatomic distances, bond lengths, angles and dihedrals. In this work we will associate these changes to the steric effect resulted from the spatial action of the substituent, using NMR and molecular modeling. This is a continuation of our work on adamantane derivatives1"4, now introducing 170 NMR data in our studies, since that 170 NMR chemical shifts are strongly affected by electronic and steric effects of the substituent, intramolecular hydrogen bonding and torsion bond angles5. The compounds studied are showed in Figure 1. Compound R 1 CH2COOH 2 COOH 3 CONH2 Figure 1: Adamantane derivatives with carboxamide and carboxylic acid substituents at position 1. The natural abundance 170 NMR spectra were obtained on a VNMRS (Varian, USA) spectrometer operating at 67.77MHz with a magnetic field of 11.75T. The instrument was equipped with a 5 mm probe. The pulse width was 30ms (90°). An acquisition time of 50 ms. Measurements were made without sample spinning. Chemical shifts were determined directly from the spectra using the threshold on the respective signal and were expressed in ppm relative to the oxygen of a deuterated water external reference placed in a concentric capillary tube (5D20 = 0:0 ppm). The deuterium signal from D20 was used to lock the system. Compounds were studied at 60 °C in C2CI4solutions. Theoretical calculations were done on the Gaussian 03W package of molecular orbital programs6. The geometry was optimized using the B3LYP/6-31G(d,p) basis set. Magnetic shielding tensors were calculated using the GIAO method. Chemical shifts 150 values for 1H and 13C were obtained relative to the isotropic shielding of TMS (31.76 ppm for 1H and 191.8 ppm for 13C) as calculated by that method at the same level. 17 Chemical shifts values for 0 were obtained relative to isotropic shielding of H20 (328.73 ppm for 170). This reference was chosen according to the work of Costa et al5 and the calculated value is inside the range mentioned by them. The experimental and calculated chemical shifts for 170 are in Table 1. The experimental and calculated values are in relatively good agreement for the compound 3, however they are not good for compounds 1 and 2. The experimental data for these last ones are in according to the expected range of chemical shifts for carboxylic acids according to Boykin7. The point is that calculations were carried out in vacuum while carboxylic acids display only one 170 resonance, suggesting that the two oxygen atoms are equivalent due to fast proton exchange in dimeric or polymeric forms7. Table 1: 170 NMR chemical shifts (ppm) for compounds 1, 2 and 3. Compound Experimental Calculated 1 263.8 (C=0) 397.5 (C=0) 202.2 (OH) 2 245.2 (C=0) 388.1 (CO) 183.6 (OH) 3 322.2 378.6 It is interesting to notice that the experimental values for compounds 2 and 3 are approximately the mean value of the calculated chemical shifts for the carbonyl and the hydroxil groups of the carboxylic acid. Concerning on the steric effects, the same approach which we have been using1"4 can be applied here. The effect can be noticed by a bond polarization in the directions in which the substittient points out, C-H bonds are shortened, and hydrogen atoms are deshielded. When the alkyl group of the substituent points in the direction of the adamantane system, an inverse bond polarization is observed and the hydrogens are shielded in according to our work on alkyl adamantanes3. REFERENCES: 1. Yoneda J.D.; Seidl P.R.; Leal K.Z. Ann. Magn. Reson. 2008, 7(1), 32. 2. Yoneda J.D.; Leal K.Z.; Seidl P.R. Ann. Magn. Reson. 2005, 4 (1), 9. 3. Seidl P.R.; Yoneda J.D.; Leal K.Z. J. Phys. Org. Chem. 2005, 18 (2), 162. 4. Seidl P.R.; Leal K.Z.; Yoneda J.D. J. Phys. Org. Chem. 2002, 15, 801. 5. Costa V.E.U.; Nichele A.G.; Carneiro J.W.M. J. Mol. Struct. 2004, 702, 71. 6. Frisch, M.J. et al. Gaussian 98, Gaussian Inc. Pittsburg, PA, 1998. 7. Boykin, D.W. 170 NMR Spectroscopy in Organic Chemistry, 1st ed., 2000. PIBIC/CNPq, CNPq, FAPERJ PO 50 COMPARATIVE STUDY OF BRAZILIAN HEAVY OIL BY PROTON NUCLEAR MAGNETIC RESONANCE F.B. da Silva1, P.G.P.Fiorio1, P.R.Seidl*1, M.J.O.C. Guimarães1 K.Z. Leal2 1 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 2Fluminense Federal University , Niterói, Brazil e-mail:pseidl(p).eQ. ufrj. br Keywords: Asphaltenes; solvent mixtures; 1H-RMN Currently, there is a trend in oil production to increase reserves, mainly of heavy and extra heavy oils. The need to use these crudes efficiently in order to transform them into lighter products has also encouraged the study of their heavier fractions, among which their constituents known as asphaltenes stand out. Petroleum produced from Brazil's largest fields contains appreciable amounts of asphaltenes, thus research in this area has been focused on the development of processing technologies and efficient use of waste materials1. Asphaltenes are very complex macromolecules containing condensed aromatic and saturated rings, aliphatic chains and heteroatoms. They correspond to fractions insoluble in hydrocarbons such as heptane, but soluble in aromatics such as toluene. They have a tendency to aggregate and precipitate causing major damage in the petroleum industry2. The solvent mixture technique has been developed with the aim of greater understanding of the tendency of aggregation of asphaltenes. It was employed to separate asphaltenes from a vacuum residues (VR) obtained from-two off-shore fields, A and B,4,5 and here we report its application to a very heavy oil C. This oil is completely soluble in naphthenic hydrocarbons, so N1 was used in combinations with paraffinic solvents P1 and P2. Results are compared to those from the IP-143 Standard Method (Table 1 and 2). Table 1: Percentage of Asphaltene vs Type of Blend Fraction of Fraction of Samples Blends S/MPa1'2* asphaltene (%) soluble (%) N1/P1 14.9 12.5 A 87.5 N1/P2 15.5 5.4 94.6 N1/P1 14.9 7.0 93.0 B N1/P2 15,5 1.8 98.2 N1/P1 14.9 5.2 c 94.8 N1/P2 15.5 1.9 98.1 * 8 = Solubility parameter determined by: f>- = (<|) = volume fraction of solvent). Table 2: Percentage of Asphaltene by the IP Standard Method Fraction of Fraction of Samples asphaltene (%) Soluble (%) A 11.9 88.1 B 9.3 90.7 C 9.7 90.3 152 Tables 1 and 2 indicate that VR A contains a larger asphaltene fraction and that the N1P1 solvent mixture is considerably more efficient in separating asphaltenes (insolubles) than N1P2 for the three samples. Oil C showed lower levels of extraction compared to residues A and B. This may be due to the fact that the blends techniques have been optimized for vacuum residues. The N1P1 mixture has a lower solubility parameter compared to N1P2, giving a larger amount of asphaltenes [4], Table 1 also shows that selectivity of asphaltene extraction depends on the type of paraffinic hydrocarbon that is mixed with the. naphthenic solvent, [4,5] indicating that further fractionation is possible varying the proportions of P1 and P2. Constituents of asphaltenes that were precipitated were monitored by 1H- NMR (Table 3). Table 3:1H NMR Analyses of asphaltenes Percentage* Percentage* Percentage* Percentage* (%) (%) A (%) B (%) C IP -143 Types of Hidrogen N1/P1 N1/P2 N1/P1 N1/P2 N1/P1 N1/P2 A B Aromatic 21.9 30.7 22.9 22.5 21.4 19.0 27.8 24.6 Ha 17.6 17.2 17.4 20.7 15.7 16.5 20.3 22.7 HP 31 38 28.7 29.4 27.5 26.1 33.1 34.2 HY 29.5 14.1 31.0 27.4 35.4 38.4 18.8 18.6 Total Saturated 78.1 69.3 77.1 77.5 78.6 81.0 72.2 75.4 *Percentage of peak área. Table 3 reflects the relative composition of the asphaltene fraction. [5] A higher percentage of Ha indicates that the molecules separated by this method have a higher degree of substitution on aromatic rings, of Hp indicates longer chains and Hy indicates, terminal or branching methyl hydrogens. In contrast to VRs there does not appear to be a large discrepancy between structures of constituent of C separated by different blends or IP methods. This observation may be related to the fact that C corresponds to a crude oil in which asphaltenes are stabilized by other components. REFERENCES: 1. LEITE, L.F, TN Petróleo, v 14, p 60, 2000. 2. SILVA, F.B., et al., Petro & Química, v 327, 64-66, 2010. 3. QUINTERO, L.C.N. Submitted to Energy & Fuels, 2011. 4. MOURA, M.B.R, Petro & Química, v 316, 30-32, 2009. 5. SILVA F.B., et al., Petrophase, Abstract P2-43, 2010. CAPES, PETROBRAS, CNPq 153 h * PO 50 ELECTRONIC PARAMAGNETIC RESONANCE AND MAGNETIC CIRCULAR DICHROISM STUDIES OF NITRIC OXIDE-PROMOTED CHANGES OF THE AXIAL LIGANDS OF CYTOCHROME C HEME IRON S. M. S. Pinto1, K. C. U. Mugnol1, T. Prieto2, O. R. Nascimento12, I. L. Nantes1* 'Universidade Federal do ABC, Santo André, Brazil 2Universidade de São Paulo, São Carlos, Brazil e-mail:ilnantes(çb.ufabc.edu.br Keywords: cytochrome c, EPR, nitric oxide. From previous studies about the effects of nitrosative species on cyt c structure in which nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, it was found a new cytochrome c species.1 The EPR spectra of the new cytochrome c species revealed the formation of a low-spin cyt c form (S=1/2) with gi=2.736, g2=2.465, and g3=2.058 after incubation with SIN1. These data suggested that the concomitant presence of NO» and 02«- generated from dissolved oxygen, in a system containing cytochrome c, promotes chemical and conformational modifications in the protein, resulting in a hypothetical bis-histidine hexacoordinated heme iron. To investigate the axial ligands of the less rhombic cytochrome c heme iron generated by NO', a recombinant mutated H26N and H33N cytochrome c was incubated with SN1 in the same conditions used to generate the less distorted wild type cytochrome c. The recombinant mutated cytochrome c was obtained using the plasmid pJRhrsN constructed by Rumbley, Hoand, and Englanderf carrying the two substitutions H26N and H33N, and yeast heme lyase. In the following, magnetic circular dichroism (MCD) and Electronic Paramagnetic Resonance (EPR) were used to compare wild type and recombinant cytochrome c challenged by SIN 1-treatment. Figure 1 panels A, and B, shows comparatively the MCD spectra of wild type cytochrome c and the recombinant H26N H33N form before and after 82 min incubation with SIN1. The MCD spectra of all cytochrome c species are not limited to the one electron transitions and results from the overlap of n-n* and n-d transitions. Also the small energy separation of vibrational bands contributes to the complexity of MCD spectra. All the studied cytochrome c species presented a derivative Soret band feature with the zero crossing matching with the UV-visible Soret band peak consistent no significant contribution of Faraday B-term. For wild type cytochrome c, despite the partial SIN1-induced reduction of heme iron form characterized by the derivative band with zero crossing at 550 nm, the Soret band did not exhibited the contribution of Faraday B-term present in the MCD spectrum of wild type Fe" cytochrome c.3 Wavelength (nm) Wavelength (nm) Figure 1: MCD spectra of (A) wild type and (B) recombinant H26N H33N cytochrome c before (black lines) and after (gray lines) 82 min incubation with equimolar SIN1. The spectra were run at room temperature with 150 mM cytochrome c and magnetic field = 860 mT. 154 Figure 2 A shows the EPR spectra of 150 pM wild cytochrome c before (black line) and after (gray line) 82 min incubation with an equimolar amount of SIN1. The EPR spectrum of the wild type cytochrome c corresponds to the well-known Fe(lll) low-spin form with a rhombic symmetry that displays signals at g1=3.07 and g2=2.23 and g3 = 1.35. After 82 min incubation with SIN1, the EPR signal of the Fe(lll) low-spin form decreased concomitantly with the appearance of a low spin form signal (S=1/2, g1 =2.736, g2=2.465 and g3=2.058). The significant decline of changes in g1, g2, and g3 values suggests the conversion of cyt c to a low-spin species with a much lower rhombic symmetry. The remarkable approximation among g values observed for SIN1- treated cyt c reinforces the supposition of a histidine residue as the amino acid that replaces Met80 in the sixth coordination position of the heme iron, since a bis-histidine coordinated heme iron displays a more symmetric structure. Consistent with the above proposal recombinant H26N H33N cytochrome c was not converted to the more symmetrical form after incubation with SIN1 as shown in Fig. 2 B, black and gray lines. 100 200 300 400 100 200 300 400 Magnetic Field (mT) Magnetic Field (mT) Figure 1: EPR spectra of (A) wild type and (B) recombinant H26N H33N cytochrome c before (black lines) and after (gray lines) 82 min incubation with equimolar SIN1. The spectra were ruq with 150 mM cytochrome c and magnetic field = 860 mT. Experimental measurement conditions are microwave frequency 9.4715 GHz, microwave power 5.05 mW, modulation frequency 100 kHz, field modulation amplitude I.0 mT, conversion time 81.92 ms, time constant 20.48 ms, amplifier gain 45 dB, at II.0 K. REFERENCES: 1. Mano C. M., Barras; M. P., Faria, P. A.; Prieto, T.; Dyszy, F. H.; Nascimento, O. R. Nantes, I. L.; Bechara, E. J. H. Free Radio. Biol & Med. 2009, 841. 2. Kawai, C.; Pessoto, F. S.; Rodrigues, T.; Mugnol, K. C. U.; Tortora, V.; Castro, L.; Milicchio.V. A.; Tersariol, I. L. S.; Di Mascio, P.; Radi, R.; Carmona-Ribeiro, A. M.; Nantes, I. L. Biochemistry, 2009, 8335. 3. Mugnol, K. C. U.; Ando, R. A.; Nagayasu, R. Y.; Faljoni-Alario, A.; Santos, P. S.; Nascimento, O. R.; Nantes, I. L. Biophysical J. 2008, 4066. FAPESP, CNPq, CAPES 155 h * PO 69 STRUCTURAL AND ORIENTATIONAL DETERMINATION OF THE ANTIMICROBIAL PEPTIDE HYLASEPTIN P2 IN MEMBRANE-MIMICKING ENVIRONMENT V.H.O. Munhoz*1', S.F.C. de Paula1, J.M. Resende1, D. Piló-Veloso1, B. Bechinger2 1 Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos, 6627, 31270090, Belo Horizonte-MG, Brazil.( vmunhoz@ufmg. br) 2Université de Strasbourg / CNRS UMR7177, Institut de Chimie, 4, rue Blaise Pascal, F-67070 Strasbourg, France e-mail:victor. munhoz(p).gmaíl. com Keywords: antimicrobial peptides, solid-state NMR The Hylaseptin P2 (HSP-2, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial, highly a-helical peptide present on Hypsiboas punctatus anurans, mainly found at the Amazon rainforest. It has considerable activity against patogens such as bacteria (both Gram-positive and Gram-negative) and fungi. Like most of the antimicrobial peptides, it is believed that its mechanism of action is guided by its affinity with the bacterial membrane, involving steps of interaction with the membrane surface followed by the lysis of the cell. Although the outlines of the mechanism are well consolidated, its full pathway is not yet completely unveiled, hence the need to perform different studies to elucidate it better. One of the techniques that have been very useful to study peptide-phospholipid interactions is the solid-state NMR of locally isotopically-labelled*peptides in oriented bilayers. Through this methodology, it is possible to elucidate some aspects of the mechanism of action of these membrane-binding peptides, by deriving information on the orientation of the helix when the interaction takes place. This is possible due to the anisotropic character of properties such as the chemical shift and the deuterium quadrupolar splitting for solid samples1. For this work, different experiments were done in order to get complementary information regarding the peptide lipid interaction. The 31P experiment was made in order to assess the overall orientation of the membrane, the 15N cross-polarization 15 gives information on the helix tilt angle, due to the unique size of the N SJ3, although it is insensitive to the rotation of the helix along its axis because of the 5U and S22 values, which are very close one to the other2. The quadrupolar splitting measured on 2 2 H of peptides containing a H3-Ala labelled residue furnishes such information since the splitting depends on the angle between the Ca-Cp bond vector and the external magnetic field vector. The information derived from this experiment is thus the rotational pitch angle which, alongside with the tilt angle, describes satisfactorily the orientation of the helix1. In this work, the samples consisted on a mixture containing the peptide and POPC on a 1.5:100 molar rate spread on around 18 very thin glass plates with the dimensions of 8x22 mm, stacked one over the other, and hydrated at 93% of relative humidity. The 15N experiment was performed on Bruker AVANCE AMX400 wide-bore with a commercial double-resonance E-free probe and the 2H experiment, on a Bruker AVANCE 300 with a static commercial triple-resonance probe. The peptides were synthesized with 15N label at L18 and 2H label at A16. The resulting 31P spectrum is shown in Figure 1a and it shows a main sharp signal, followed just by another one with much lesser intensity, indicating that the bilayer is very oriented. The 15N spectrum (Figure 1b) shows a single relevant peak at 514 ppm and the 2H spectra, depicted in Figure 1c, gives a quadrupolar splitting of 27 kHz. j. + 156 By calculating the tilt and rotational pitch angle values with the quadrupolar splitting and chemical shift anisotropy as restraints, one can build the plots shown in Figure 2a, in which the red curve represents the possible angle values derived from 15N chemical shift anisotropy restraints and the blue ones, the likely angle values calculated with deuterium quadrupolar splitting restraints. The intersections between both curves show the most likely orientation based on both restraints used. Figure 2b shows these angular values are applied to theoretical a-helical structures of HSP-2, and by analyzing it, the alignment I shows up as the most probable, since it best allows the distribution of hydrophobic sidechains inside the hydrophobic core and the hydrophilic ones on the interface between the bilayer surface and the aqueous medium. a) b) C) W V/W1aM| ^ Vivvvywwi' Figure 1: HSP-2 at 1.5 mol% in oriented lipid bilayers: (a) 31P, (b) 15N, and (c) 2H spectra7 ^ a) to sRi C) i\ O s o ill cr Rotation around z axis Figure 2: (a) Simulation of ^ and Av0 for pairs of pitch & tilt angles and the resulting possible alignments for an helix in front view (b) and side view (c) REFERENCES: 1. Aisenbrey, C.; Bechinger, B.; J. Am. Chem. Soc. 2004, 126, 16676. 2. Bechinger, B.; Sizun, C.; Con. Magn. Res. 2003, 18A, 130. CNPq, FAPEMIG, CNRS 157 h * PO 50 DETERMINATION OF THE ABSOLUTE CONFIGURATION OF CARBOXYLIC ACIDS BY 77Se NMR SPECTROSCOPY J.G. Ferreira*, S.M.C. Gonçalves Departamento de Química Fundamental, UFPE, 50.670-901, Recife, Pernambuco, Brasil. e-mail:ieielyferreir(çb.hotmail.com Key-Words:77Se NMR; Absolute configuration; Chiral carboxylic acids. The spatial arrangement of substituent groups in a molecule has a fundamental role in its physico-chemical and biological properties.1 For this reason, there is a continuous demand for the asymmetric synthesis of several classes of compounds and, consequently, for the absolute stereochemistry assignment of the chiral products - an indispensable task. Recently, methods based in NMR spectroscopy for the determination of absolute configuration have been reported, and several articles appeared describing attempts to develop chiral auxiliaries, capable of assigning the absolute configuration of different compounds.2 In this work, we propose an empirical model for the assignment of the absolute configuration of «-substituted carboxylic acids by 77Se NMR. The methodology employed was the following: in the NMR tube, the derivatization was carried out by mixing and shaking the enantiopure carboxylic acid, commercially obtained, with the chiral auxiliary, (f?)-3-Phenyl-2-(phenylselanyl)propanol,1, synthesized in our laboratory. Then, the 77Se NMR spectra acquisitions were registered. The experiments with the carboxylic acids 2-7 (Figure 1) were repeated for both enantiomers, one at a time. o HO RI H DCC/DMAP/CDCI3 (mix and shake) DCC/DMAP/CDCI3 O R? HO H R, Figure 1: General scheme of the methodology applied for the determination of the absolute configuration of carboxylic acids by 77Se NMR. Examining the 77Se NMR spectra of the diastereomeric esters, obtained from the (R)- and (S)- «-substituted carboxylic acids, it was possible to propose an empirical model for the determination of the absolute configuration of these compounds, based solely in these experimental data. The NMR spectra showed, for each pair of diastereomeric esters, some shielding/unshielding effect over the selenium nucleus. Let us consider Figure 2. This effect could be explained by considering the influence caused by the magnetic cone exerted by the carbonyl group3. Thus, it can be associated to the conformational equilibrium led by steric interactions and/or electronic effects generated by different spatial arrangements of the substituents connected to the stereocenter of the carboxylic acid. Consequently, the predominant conformer in the equilibrium, is the one that contributes the most of the chemical shift of the 77Se nucleus (causing the 77Se nucleus to be either shielded or unshielded). 158 i'lliM I.II \l< MAf.^l IIC KI SO\AN( l I Sl ltN MI TI l\Ci Ph-^V^o- M SePh L H ^ Ph—'® SePh L h Steric and/our electronic interaction between the SePh and L groups. Ph Y SePh HW LI ^ Ph—'OI $ SePo-nkh H.. L More stable conformer: deshielding 77Se nucleus L = Large group (larger steric and/our electronic effect) Signal + : shielding region M = Medium group. Signal -: deshielding region Figure 2: Empirical model for the determination of the absolute configuration of a- substituted carboxylic acids by 77Se NMR. All the absolute configurations of the carboxylic acid stereocenters (2-7) were proved when the proposed model, based in the 77Se chemical shifts of each diastereomeric derivative (Figure 3), was applied - demonstrating the efficiency of the model in the assignment of absolute stereochemistry. Figure 3: 8 (ppm) values of 77Se for the diastereomeric ester derivatives of the alcohol (ft)-3-Phenyl-2-(phenylselanyl)propanol 1. REFERENCES: 1. Nguyen, L. A.; Int. J. Biomed. Sci., 2006, 2, 85. 2. (a) Wenzel, T. J.; Chisholm, C. D.; Chirality, 2011, 23, 190. (b) Seco, J. M.; Quinoá, E.; Riguera, R.; Chem. Rev., 2004, 104, 17. 3. Sullivan, G. R.; Dale, J. A.; Mosher, H. S. ; J. Org. Chem., 1973, 38, 2143. FACEPE, CNPQ, PRONEX/FACEPE/CNPQ 159 h * PO 50 CHIRALITY RECOGNITION OF CARBOXYLIC ACIDS BY 77Se NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY S.M.C. Gonçalves*, J.G. Ferreira Departamento de Química Fundamental, UFPE, 50.670-901, Recife, Pernambuco, Brasil. e-mail:simone(jd).ufpe. br Keywords:77Se RMN; chiral derivatizing agent; enantiomeric excess. The significant growth of asymmetric synthesis, and the development of new drugs, requires fast and trustworthy new methods to determine the enantiomeric purity of chiral substrates. Non-chiroptical methods based on GC, in the stationary chiral phase13, and in HPLC1b, are generally used in the determination of the enantiomeric excess (ee) of chiral substances. On the other hand, these methods present some restrictions. Indeed, in the case of direct GC analysis, the analytes may racemize due to the use of high temperatures.2 Nevertheless, in the last 40 years, a relevant escalation in the development of methods based on NMR spectroscopy to control ee, mainly through the observation of 1H, 19F, 31P nuclei3, has ocurred. Reports of the literature, also describe4 applications of 77Se NMR to the determination of ee. In this work, we show results that point to the efficiency of chiral y9-arylselanyl alcohols, when employed as chiral derivatizing agents (CDA), in the enantiodiscrimination of a-, ft-, ^substituted chiral carboxylic acids through 77Se NMR. The synthesis of the /?-arylselanylalcohols (1, 2, 3 and 4) was carried out as described in the literature5, and started from the corresponding .a-bromo-substituted carboxylic acids, as in the reaction scheme below (Figure 1), ^ ,C02H H2S04(aq), KBr R .C02H ArSeSeAr R. (1) - R-CH2Ph, Ar-Ph OH Y M MO Y M PU TUP* (2) - R=CH2Ph; Ar=1-naphtyl | NaN02(aq) | NaBH4, THF 1| (3) - R=Ph Ar=Ph NH 2h C Br 2h 2 '°° .°°C SeAr |4) - R^Phi Ar=1-naphtyl Yields: 40-55% Figure 1: General reaction scheme for the synthesis of /?-arylselanylalcohols. The 77Se NMR experiments were carried out by using the /?-arylselanylalcohols as CDAs, and by making them react with racemic samples of carboxylic acids, directly in the NMR tube, in the presence of DCC and DMAP. After shaking for one minute, the corresponding diastereomeric esters were produced, and that was followed by the acquisition of the77Se NMR spectra (Figure 2). OR' ° R' > JL X ., DCC, DMAP 77 R. DCC.DMAP^ R^^o^^rv. SeNMR> J. a CDCI3, 25° I . SeAr J SeAr Diasteriomeric Mixture ^yLiyV1 n = 0,1,2. Figura 2: Reaction scheme for the preparation of the diastereomeric esters, as well as an example of the 77Se NMR resolved signals. Table 1 presents the spectroscopic 77Se NMR experiment data. 160 Table 1: Chemical shifts of 77Se (ppm) and the differences in chemical shields (Hz) for the diastereomeric ester derivatives, in CDCI3. Ph Ph^j^OH "Y^OH Carboxilic acids Se-(l-naphtyi) SePh Se-(l-naphtyl) (3) (2) (4) 5, = 354,25 8, = 280,17 8, = 424,17 S| = 350,18 S2 = 355,02 62 = 282,17 S2 = 425,97 82= 351,07 A8 = 44 Br A5 = 114 AS = 102 AS = 63 8,= 355,29 8, = 281,72 8, = 425,12 8, = 350,33 82 = 356,52 S2 = 282,57 8, = 427,00 S2= 352,01 AS = 70 AS = 48,5 AS = 107 AS = 96 Br O 8, = 353,67 S, = 280,20 8, = 422,33 5, = 349,10 6, = 356,95 S2= 285,45 82 = 424,59 5, = 350,73 V^c A8 = 187 À8 = 299 AS = 129 AS = 93 Ph O 8, = 356,83 8, = 282,87 S| = 424,52 8, = 350,60 8, = 357,21 S, = 283,37 S2= 424,92 S2= 351,02 AS = 22 AS = 28,5 AS = 23 A8 = 24 S = 284,50 Me O 8= 358,09 8, = 426,57 8, = 352,59 A8 = 0 AS = 0 S2 = 426,89 S2= 352,86 OH AS =18 AS = 16 O 8= 358,12 8 = 284,26 8, = 426,03 6, = 352,14 A8 = 0 A8 = 0 8, = 426,38 5, = 352,51 A8 = 20 AS = 21 Me 5, = 352,28 8, = 278,99 5, = 422,51 8, = 349,15 8, = 357,78 OH 8, = 286,60 St = 424,32 S2= 350,63 A8 = 314 OMe AS = 434 AS = 103 AS = 84 8, = 353,47 8, = 279,78 S, = 422,48 8, = 349,42 62 = 357,21 8, = 285,34 S2 = 424,67 8,= 350,91 OH A8 = 213 AS = 317 A5= 125 A8 = 85 Ph O 5= 357,29 S, = 283,67 8, = 425,47 8, = 351,66 AS = 0 82 = 284,02 S2 = 427,80 8, = 353,37 A8 = 20 AS =133 A8 = 97 8= 358,26 S, = 284,57 8, = 426,78 5, = 352,75 10 AS = 0 A8 = 0 S2 = 427,29 52 = 353,31 OH AS = 29 A5 = 32 Examining the results of the ten experiments, as shown in Table 1, one can at most see two selenium signals (A8). Furthermore, the larger anisocrony of the two signals was observed when alcohol (2) was used - the exhibited difference in chemical shift was AS = 434Hz (Table 1 - entry 7). Moreover, alcohols (3) and (4) led to the larger enantiodiscrimination potential, capable of distinguishing between alkyl substituents, up to seven bonds distant from the selenium nucleus, with a 77Se signal separation, AS of 20Hz and 21 Hz, respectively (Table 1 - entry 6). REFERENCES: 1 - (a) Subramanian, G.; A Practical Approach to Chiral Separations by Liquid Chromatography. VCH: Weinheim, 1994. (b) Schurig, V.; J. Chromatogr. A, 2001, 906, 275. 2 - Trapp, O.; Schurig, V.; Chem. Eur. J.; 2001, 7, 1495. 3 - (a) Wenzel, T. J.; Chirality, 2003,15, 256.(b) Parker, D.,Chem. Rev., 1991, 91, 1441. 4 - (a) Menezes, P. H.; Gonçalves, S. M. C.; Hallwass, F.; Silva, R. O.; Bieber, L. W.; Simas, A. M.; Org. Lett., 2003, 5, 1601. (b) Ferreira, J. G.; Gonçalves, S. M. C., J. Braz. Chem.Soc., 2010, 21, 2023. (c) Orlov, N. V.; Chem. Commun., 2010, 46, 3212. 5 - (a) Frick, J. A.; Synthesis, 1992, 621. (b) Sharpless, K. B.; Lauer, R. F. J. Am. Chem. Soc. 1973, 95, 6137. FACEPE, CNPQ, PRONEX/FACEPE/CNPQ 161 h * PO 52 SIMULATION OF TRANSVERSE RELAXATION TIME IN POROUS MEDIA BASED ON PHASE SPACE RANDOM WALK M. N. d' Eurydice*1, T.J. Bonagamba1 11nstituto de Física de São Carlos - USP (IFSC-USP) e-mail:mrcl@ifsc. usp. br Keywords: porous media, random walk, transverse relaxation. NMR transverse relaxation time (T2) distribution of fluids imbibed in porous media such as rock cores, ceramics, bones, cements, etc, is considered one of the most important information to characterize such materials allowing understanding its permeability, wettability and porosity distribution. Lots of efforts have been done to simulate these properties and associate them with T2 distributions obtained from CPMG magnetization decays in a more detailed way[1_3]. Usually, based on Monte Carlo random walk to reproduce a Brownian motion of a fluid, it is possible to track and classify the interaction of the particles with the walls of as much complex systems as porous media can be. This work caries out a novel propose which combines two random walk approaches (over the space[3] and phase-space[4]) to simulate a fluid imbibed in porous media. Taking the BPP model into account, the nuclear spin relaxation of a spin 1/4 fluids can be described by the fluctuations in the vicinity local field due to molecules relative random walk over the space, which produces a perturbation on the dipole- dipole Hamiltonian. This perturbation is responsible to generate a coherence loss in the spin phase-space. Furthermore, by including the boundary conditions described by the wall of a porous media, the spin relaxation of the fluid enclosed within this space is enhanced by collisions with the pore surfaces due the presence of a sort of possible interactions like dipole-dipole, cross relaxation, paramagnetic/ferromagnetic ions and free electrons. The relationship between transverse relaxation time distribution and pore properties, e.g. morphology and S/V ratio distribution, is well-established and can be divided in two regimes known as fast and slow exchange describing the fluid dynamics inside such materials; both can be obtained through the simulation. In these simulations the pore space is represented by a three-dimensional voxel-based description where both real and virtual idealized models can be used as boundary conditions input. The most realistic models are reproduced by a set of binary images obtained throughout X-ray microtomography (micro-CT scanning)'31 repre- senting transverse slices of a sample, then assembled as a computer 3D matrix representation, as shown in Figure 1. Micro-CT scanning Slice Binarization Slice stacking 3D Matrix representation Figure 1: Procedure to build the 3D Sandstone rock core representaion. The resolution of the 3D image is 1.573 pm3/voxel. Figure 2 shows a selected region which presents a high level of connectivity and the respective collision counting map, which can be related to the transverse f, 4 162 I.ill- Mill M< \1 \(,\1- lie KI-WN Wl. I SHJN \1l"l 11\(. relaxation time distribution, once the higher the number of collisions the shorter will be T2. Regions with different collision rates are marked with different colors. a) [ b) Figure 2: a) Selected region of the Sandstone rock core micro-CT 3D image, b) Simulation collision counting map along 2.5 s. The CPMG decay obtained in this simulation is shown in Figure 3a and its respective relaxation time distribution in Figure 3b. Furthermore, the number of collisions affects the displacement of the particles during the simulation and their correlation histogram is shown in Figure 3c. a) 0 o'cT 1.0 1.5 2.5 Time (,s) 10' 101 10" 20 40 60 80 100 120 Relaxation Time (*) Displacement (/i.s) Figure 3: a) CPMG Signal decay, b) Transverse relaxation time distribution, c) Collision x Displacement correlation histogram. The model is under development and other noise sources dependencies must be taken into account: gradients, background gradients, material impurities and other interactions can be simulated helping to improve the characterization through NMR techniques once more detailed information can be extracted. REFERENCES: 1. E. Vergés, D. Tost, D. Ayala, E. Ramos, e S. Grau, S. Geology v.234, 2011, 109. 2. Gunasekaran S.; Kumar T. R. e Ponnusamy S. Spectrochimica Acta Part A. 2006, 104. 3. O. Talabi, S. Alsayari, S. Iglauer, e M.J. Blunt, J.P.S. and Engineering v.67, 2009, 168. 4. A.C. Olivieri, Concepts In Magnetic Resonance v.9, 1997, 337. CAPES, FAPESP, CNPq e IFSC-USP 163 h * PO 61 THIOREDOXINS VISIT AN OPEN EXCITED CONFORMATIONAL STATE DURING THE CATALYTIC TURNOVER F. Gomes-Netol, C .Cruzeiro-Silva,1, N. L Rodriguesl, C. A. Miyamoto"!, A. S. Pinheiro"!, L. E. S. Netto2, A. P. Valente"! and F. C. L Almeida"!* 1- Centro Nacional de Ressonância Magnética Nuclear - Instituto de Bioquímica Médica - Universidade Federal do Rio de Janeiro - Rio de Janeiro - Brazil; 2 - Instituto de Biociências - Universidade de São Paulo - São Paulo - Brazil. *falmeida@ cnrmn. bioqmed. ufrj. br. Keywords: Thioredoxin, NMR and Protein Dynamics, Molecular Dynamic Simulation. Thioredoxins are proteins that function as disulfide reductases, through the oxidation of two cysteine residues, in a conserved active site CXXC. The Saccharomyces cerevisiae contains two different cytoplasmic isoforms: Trx1 and Trx2. Trx1 and Trx2 specifically interact with different cellular targets. To establish a structure-function relationship between these proteins, we have determined the three-dimensional structure of Trx1 and 2 by NMR. Comparison of the Trx's structures show that they mainly differ in the active site (Pinheiro, et al., 2008 and Amorim,et al., 2007). We measured the backbone dynamics of both reduced and oxidized forms of Trx1 and the mutant D24N. We showed that the dynamics of yeast thioredoxin 1 (Trx1) reveal features of the catalytic cycle that cannot be understood based solely on the structure. The efficiency of the enzyme depends on a minor open excited conformation that is visited during the catalytic cycle. The residues D24 (Trx1), acts as an important proton acceptor, essential for the catalytic reduction mechanism, bOt we showed that it also modulates the slow dynamics of Trx1. According to the dynamics for native Trx1 and the mutant in different oxidation states, we observe that this residue undergoes one of the most important dynamic differences between the reduced and oxidized forms. In the oxidized state this residue displays motions in millisecond timescale. Trx1 exhibits multiple conformational states. The millisecond dynamics of the interacting loops are correlated with the proton transfer to D24. We also investigated the residence time of water in reduced and oxidized Trx1. There is a water channel that connects the interacting loops of Trx1 with residue D24, which showed the longest water residence time (Figure 1). The residues that showed slow dynamics correlate with the water channel, suggesting that there is a major closed conformational state in equilibrium with an open state. We also constructed an ensemble of structures using tCOONCORD (Seeliger, et al., 2007) to evaluate the millisecond timescale equilibrium. We could demonstrate theoretically the existence of conformational states with tightly bound water at D24, which we interpreted as the closed states, and conformations in which there is only fast exchange water molecules at D24 (open states). f + 164 Figure 1: Residence time of water in Thioredoxin 1: (A) oxidized and (B) reduced form. The residues colored in red show a water molecule with a long time of residence and the residues colored in gray have a water molecule short residence time. REFERENCES: 1. G. C. Amorim, A. S. Pinheiro, L. E. Netto, A. P. Valente, F. C. L. Almeida H. P.; Dale M. J. Biomol NMR, 2007, 38, 99. 2. A. S. Pinheiro,G. C. Amorim, L. E. Netto, A. P. Valente, F. C. L. Almeida H. P.; Dale M. Proteins, 2008, 70(2):584. 3. Seeliger, D., J. Haas, etal. (2007). Structure 15(11): 1482-1492. FAPERJ, CNPq, CAPES, INBEB 165 h * PO 50 A NEW DIMENSION IN PULSED NMR SPECTROSCOPY OF PROTEINS Munte, C. E.*1'2, Arnold, M.1, Kremer, W.1, Hartl, R.1, Beck Erlach, M.1, Kõhler, J.1, Meier, A.1, and Kalbitzer, H. R.1 11nstitute of Biophysics and physical Biochemistry, University of Regensburg, Regensburg, Germany 2Physics Institute of São Carlos, University of São Paulo, São Carlos, Brazil e-mail:claudia. [email protected] Keywords: high-pressure NMR; pressure-jump; HPr. The success of the NMR spectroscopy in biological science is closely coupled on the invention of multidimensional spectroscopy. The combination of different HF- and gradient pulses in appropriate acquisition schemes allowed the correlation of properties of the nuclear spins of the system, such as J-couplings and dipolar couplings, and to deduce structural information of the macromolecule. An interesting concept that expands the possibilities of the pure HF- and magnetic gradient based multidimensional NMR spectroscopy is the introduction of an additional pulsed perturbation in the sequence, creating a new physical dimension. For protein structural studies, the application of high hydrostatic pressures up to 350 MPa inside an NMR spectrometer leads to a perturbation of the thermodynamic equilibrium and can be used for the study of excited conformational states of proteins, protein folding, protein aggregation and ligand interaction at atomic resolution [1], A time dependent pressure perturbation can be introduced in NMR spectroscopy by manually changing the static pressure of the system. Here, only slow processes in the time range of minutes to days can be observed and the pressure-jump cannot be inserted in a pulse sequence [2], The only method to obtain a better time resolution is the use of a spectrometer-controlled fast pressure-jump system. We have developed a microprocessor controlled pressure-jump unit that is able to perform average pressure changes of more than 25 GPa/s at the sample and can be fully integrated in the NMR pulse programs (Fig. 1). It consists of a high pressure cell inserted into the NMR probe that is connected via high pressure tubes to the high pressure pump. A pressurized liquid is transmitted via a polyethylene membrane to the sample. Valve 3 is a safety valve that is closed automatically when the high pressure cell is exploding. When valve 1 is opened and valve 2 is closed, the pressure created by the pump is transmitted to the sample; closing of valve 1 and opening of valve 2 resets the pressure to atmospheric pressure. Since opening and closing of the valves is microprocessor controlled, it can be inserted at any point in the standard NMR pulse programs. The pressure response measured with sensor 2 (Fig. 1) is mainly limited by the switching time of the high pressure valves, for a pressure step of 80 MPa about 30 ms are required. p-sensor 1 p-sensor 2 30 ms valve 3 valve 1 pump % s ipillary Figure 1: Schematic view and performance of the pressure-jump apparatus, (left) The pressure-jump system, (right) the pressure response measured at sensor 2. 166 BlhN UCLI-AR MAGNETIC RES'a s In general, two different types of dynamic pressure perturbation experiments can be performed. In the first type the pressure change is performed before starting the pulse sequence (Pressure Perturbation Transient State Spectroscopy, PPTSS); in the second type the pressure change is performed during the NMR-pulse sequence (Pressure Perturbation State Correlation Spectroscopy, PPSCS). The PPTSS experiment measures the time dependent pressure response of a biological system after a periodic perturbation. In contrast, to normal NMR experiments a non-equilibrium system is established here. Possible applications of PPTSS are the determination of rate constants in folding/unfolding experiments, of free activation energies and activation volumes for conformational changes, ligand binding and polymerization reactions. The incorporation of a pressure change into the NMR pulse sequence in the PPSCS experiment allows the correlation of structure dependent NMR parameters at different pressures. Typical applications here would be the transfer of spectral assignments performed in one state (e.g. the well-folded ground state of a protein at ambient pressure) to the spectrum obtained from a different pressure induced state (e.g. the denatured state or an excited conformational state in slow exchange). Exemplifying a PPTSS experiment, the time course of the pressure induced denaturation/refolding of an active centre mutant of S. carnosus HPr was observed by one dimensional NMR spectroscopy, after increasing the pressure from 0.1 to 80 MPa and vice versa. The H£ resonances lines of Tyr64 in the folded state and of all three tyrosine residues in the unfolded state are shown when the system is in its thermodynamic equilibrium at 80 MPa and at 0.1 MPa, and 100 ms after a pressure-jump from 80 MPa to 0.1 MPa (Fig. 2). An example for an PPSCS experiment is the 1 15 application of a pressure-jump in a H, N-HSQC at the interface between U and t2 time evolution, allowing the correlation of chemical shifts at one pressure with the corresponding chemical shifts at another pressure. A small part of such a spectrum is shown in figure 2 with the correlation peaks that connect the resonances of Gly54 and Thr87 of the HPr from S. aureus at low and high pressures. 80 MPa 0,1 MPa Pressure Jump (80 MPa - •0.1 MPa| Gly 54 Ttir 87 p ~ ambient P = 88 MPa pressure jump 6.90 6.85 6.75 6.70 [ppm] 7,55 7.50 7,45 7.40 f2 Eppm) Figure 2: Examples of PPTSS (left) and PPSCS (right) experiments. REFERENCES 1. Akasaka, K. Chem. Rev. 2006, 106, 1814-1835. 2. Kitahara, R.; Royer, C.; Yamada, H.; Boyer, M; Saldana, J.-L.; Akasaka, K.; Roumestand, C. J. Mol. Biol. 2002, 320, 609-628. FAPESP, DFG, BFS, FCI 167 h * PO 61 QUANTITATIVE DETERMINATION AND VALIDATION OF EXCIPIENT USING QNMR SPECTROSCOPY C.M.G.de Souza*1, M.F.P.S. Mota1, C. Matteucci2, J.M.A. Bispo1, P.C. Leal3 1 Center for Metrology in Chemistry, IPT, São Paulo, Brazil 2Quality Control, IPT, São Paulo, Brazil 3Quimsar Química Fina Ltda., Palhoça, Brazil e-mail: [email protected] Keywords: Quantitative NMR specstroscopy, validation, metrology. NMR is by definition a quantitative spectroscopic tool because the intensity of a resonance line is directly proportional to the number of resonant nuclei. Quantitative NMR spectroscopic methods are widely used nowadays because they can be considered primary method of measurement considering the criteria in the CCQM (Comitê Consultatif pour la Quantité de Matière - the committee for chemical measurements) definition1"5. Traditional chromatographic methods are well established and documented in analytical standards however qNMR is cited in international pharmacopoeias for evaluation of compositions of polymers, excipients and impurities in drugs4. According to the DIN EN ISO/IEC 17025 the definition for the validation of analytical method is "confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled". The validation process requires the testing of linearity, robustness, parameters of accuracy, specificity, and selectivity1,2. Taking advantage of metrological properties of quantitative NMR, in the present work experiments were performed for the determination of a known component in a mixture of fatty acids in commercial products which have castor oil as veterinary pharmaceuticals excipient. The sample was used without previous sample preparation, fatty acid separation, purification or derivatization by traditional chromatographic methods. The methodology was validated and accuracy, linearity, limits of detection and quantification, and ruggedness were established. 1 The H NMR spectra of 10% (v/v) of the sample in CDCI3 (99.8% with 0.05% of TMS, Cambridge Isotopes) were acquired in a Bruker DRX400 and in an Agilent VNMR400 (9.4 T) spectrometers at 300K. The experiments were measured with the following optimized parameters: 90° pulse (9.13ps), relaxation delay of 30s, 32k data points corresponding to a 6.5s of acquisition time and 32 scans. TMS was used as internal reference standard and the same naphthalene solution in CDCI3 was used as quantitative external standard in coaxial insert tube for every measurements. The integral areas were normalized and the quantification of ricinoleate was performed using the specific methinic integral at ô=3.6ppm. For statistical proposes each measurement was repeated 5 times. For the evaluation of the specificity and the selectivity the spectrum of 2D NMR (homo- and heteronuclear) had confirmed that the referring methinic signal is exclusive and do not show overlapping. A calibration curve was prepared using known concentration of amount castor oil and ethyl oleate mixture (10 to 90%, v/v). Also control samples of the mixture castor oil and ethyl oleate (with known concentration of 15 and 75%, v/v) were used to confirm measurement precision, linearity, reproducibility, uncertainty. Commercial veterinary pharmaceuticals samples (1, 2, and 3) with unknown amount of castor oil were prepared in triplicate. The calculations for each analytical stage used Microsoft Excel software and were validated by using appointed MathCad software. The methodology was validated and accuracy, linearity (> 0.999%), limits of detection (0.13 %), quantification (0.34%), and ruggedness were established. The intermediate control samples, prepared as the calibration curve, were used for recovery ^ 4- 168 :?iii\nil \K MAÍiM-11<" Kh^f >V\\n I •»! R-. Mln measurements. The results showed a range from 98.4% to 102.9% which indicate a good recovery for this analytical method. The repeatability represents the agreement degree between the results of successive measurements of the method under the same conditions. Table 1 shows the results of determined average concentration, standard deviation (SD), relative SD and the combined uncertainty of the commercial samples. Table 1: Results for measurement uncertainty of commercial samples Determined Relative Commercial Standard Combined average standard sample deviation uncertainty concentration (%) deviation (%) 1 48,11 0,748 1,55 1,727 2 48,06 0,318 0,66 1,671 3 48,02 0,313 0,65 1,698 The final result for each analyzed commercial sample (castor oil content) is showed in Table 2. Table 2: Determined amount of castor oil in commercial samples Sample Determined average Concentration (%) Commercial Product 1 48,11 ±3,45 Commercial Product 2 48,06 ± 3,34 Commercial Product 3 48,02 ± 3,40 In was showed that 1H NMR technique can be used as a metrological tool to determine a validated method for quantitative determination of concentration of components of mixtures with advantages of sample preparation and rather quick and easy analysis when compared to traditional chromatographic methods. According to the method developed it was found that the maximum combined measurement uncertainty was 1.7% for a confidence interval of 95%. The methodology makes possible wide-spread application especially for complex mixtures of fatty acids in a better way than traditional analytical methods. No separation or sample preparation is necessary, in a short time of analysis with high precision and accuracy. REFERENCES: 1. Malz F.; Jancke H., J Pharm. Anal. 2005, 38, 813 - 823. 2. Gadape H.H.; Parish K.S, J. Chem. Pharm. Res. 2011, 3(1), 649-664. 3. Mackenzie I. S.; Anal. Proc. 1984, 21, 500-506. 4. Jancke H.; Malz, F.; Haesselbarth, W., Accred Qual. Assur. 2005, 10, 421 - 429. 5. Diehl, B.; Malz, F.; Holzgrabe, U., Spetroscopy Europe 2007, 19, 15- 19. 169 h * PO 48 STRUCTURAL CHARACTERISATION OF THE CYTOCHROME P450-ORFIO FROM THE STREPTOMYCES CLAVULIGERUS BY PULSED EPR J F Lima1, L S Goto2, C O Hokka2, A P U Araújo1, O R Nascimento1. 1 Grupo de Biofísica Molecular "Sérgio Mascarenhas", Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil. 2Grupo de Engenharia Bioquímica, Departamento de Engenharia Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil; e-mail:jflima@gmaii. com Keywords: cytochrome P450, clavams, pulsed EPR. Streptomyces clavuligerus produces the clinically-important /^-lactamase inhibitor clavulanic acid, and its biosynthesis is of great interest. The penultimate step in clavulanic acid biosynthesis remains unclear. The transformation required for this step involves at least two events: oxidative deamination and double epimerisation of (3S, 5S)-clavaminic acid into (3R, 5/?)-clavaldehyde. Downstream of the enzymatically- known part of the clavulanic acid gene cluster lies orf10, a putative gene that possibly encodes a cytochrome P450-like protein [1], Spectroscopic measurements of the purified protein indicated several cytochrome P450 features, including spectra that were consistent with the structure of P450 and the presence of catalytically-relevant heme redox transitions and states, including the related to binding of some substrate analogues used to characterise features of the protein binding site. The protein substrate-free hydroperoxide reaction was also described in detail using EPR. Furthermore, peroxide shifnt reaction adducts were captured and characterised using spin trapping. The results prove that the orf10- encoded protein is a functional cytochrome P450 capable of binding ciavam sub- structure replacements and supporting a substrate free homolytic peroxide scission mechanism [2]. This work describes the investigation of the orf10 by advanced EPR techniques with the aim to compare its structure with other cytochrome P450-like protein. Pulsed EPR is a powerful spectroscopic technique that probes the environment surrounding paramagnetic centers [3], Here, the pulsed EPR techniques echo detected field sweep (EDFS) and hyperfine sublevel correlation spectroscopy (HYSCORE) were used to get structural information through the interaction between the paramagnetic center (Fe3+) with the surrounding nuclei (14N and 1H). These experiments were conducted with an X-Band Bruker Elexsys E-580 at a temperature of 16K. Figure 1A shows the EDFS spectrum, which is typical for a low-spin complex with orthorhombic symmetry and g values: gx= 1.912, gy= 2.242, gz= 2.400. This spectrum is equivalent to an absorption mode of a continuous wave EPR spectrum. Figure 1B and 1C shows the nitrogen HYSCORE spectra at magnetic field positions corresponding to g = gy (i.e., 3110 Gauss) and g = gz (i.e., 2901 Gauss). The spectra shows strong double quantum cross-peaks in the (-, +) quadrant, which are indicative of strong coupling case (|a/2| > |vN|, with a, the hyperfine coupling, at this position and vN the nitrogen Zeeman interaction).These cross-peaks are attributed to the porphyrin nitrogens based on DFT computations of low spin iron porphyrins [4], Simulations of the porphyrin nitrogen contributions to the HYSCORE spectra allow a determination of the interaction parameters and porphyrin symmetry. No nitrogen was detected in the apical position as expected for a P450-like protein. From these preliminary results, one can conclude that orf10-encoded protein has structural characteristics compatible with a cytochrome P450-like protein. + 170 l3íhNUCUi,M< \!\f,M I If KISn\,\S( li USERS MBK- 03 T3 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 Magnetic Field N I 3<1> Frequency (MHz) Frequency (MHz) Figure 1: A) EDFS spectrum, B) nitrogen HYSCORE spectrum of orf10-encoded taken at position g = gy and C) B) nitrogen HYSCORE spectrum of orf10-encoded taken at position g = gy. REFERENCES 1. Jensen S.E.; Paradkar, A.S., Antonie Van Leeuwenhoek, 1999, 75 (1-2):125. 2. Goto L. S.; Hokka C. O.; Lima J. F., Nascimento O. R.; Araújo A. P. U., submitted to publication. 3. Schwiger A.; Jeschke G., Principles of Pulsed Electron Paramagnetic Resonance, Oxford University Press, New York, 2001. 4. Vinck E.; Van Doorslaer S., Phys. Chem. Chem. Phys., 2004, 6, 5324. FAPESP and CNPq. 171 h * PO 48 ON THE QUANTUMNESS OF NUCLEAR MAGNETIC RESONANCE D.O. Soares-Pinto*1, L.C. Celeri2, R. Auccaise3, J. Maziero2, R.S. Sarthour4, I.S. Oliveira4, M.H.Y. Moussa1, E.L.G. Vidoto1, E.R. deAzevedo1, R.M. Serra2, T.J. Bonagamba1 11nstituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil. 2Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Paulo, Brazil. 3Empresa Brasileira de Pesquisa Agropecuária, Rio de Janeiro, Brazil. Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, Brazil, e-mail: diogo.osp@ursa. if sc. usp.br Keywords: quantum information; nonclassical correlations; decoherence. Nowadays it is widely accepted the idea that it is possible to implement a macroscopic (or ensemble) analog of a quantum information processor using Nuclear Magnetic Resonance (NMR) spectroscopy [1], From the creation of pseudopure states, many quantum protocols and algorithms for quantum information processing (QIP) were implemented in such system [1], NMR also allowed the experimental test of different proposals connected to QIP with a precision rarely achieved by other experimental technique. Thus, NMR has been successfully used as a test bench for many QIP implementations, although it has also been continuously criticized for not presenting entanglement in most of the systems used so far [1]. The nonexistence of entangled states become a problem when it is shown the necessity of such states to make possible the quantum computation and quantum information processing (QCQIP) [2], The states used as base for the implementation quantum protocols in NMR, known as pseudopure states, do not present entanglement, except in specific situations. This fact raised the question about the quantum nature of NMR QIP implementations [3], On the other hand, it was shown that the existence of entangled states is a necessary but not sufficient condition to QCQIP [4]. There are other important characteristics, as for example, the efficient implementation and manipulation of the quantum states. Thus, considering that the main feature of NMR is the excellent control of the unitary transformations coming from the radiofrequency pulses, one can see that this spectroscopic technique allows obtaining unique and very efficient methods for manipulating quantum states and the generation of protocols for quantum information processing. This can be seen as part of the justification of the successful experiments On the other hand, as suggested in Refs. [3,5], the existence of quantum correlations different from entanglement can be another motif for the success of the NMR implementations. It is possible to measure such correlations in a bipartite system using a quantity called quantum discord [6]. The most interesting is that some mixed separable states, or unentangled, can present non-null quantum discord, indicating the presence of nonclassical correlations more general then entanglement. The algorithms that use such correlations for QIP present advantage over their classical counterpart. The models based in such kind of correlations are denominated as nonuniversal quantum computational models. Thus, this sort of correlation has a significant role in quantum information protocols. With that in mind and that the NMR states are mixed separable states, recently we have demonstrated theoretically and experimentally, first for quadrupolar I = 3/2 nuclear spins (equivalent to two logical qubits), and then for two coupled I = 1/2 spins (equivalent to two physical qubits), through quantum state tomography and computation of the quantum discord, the existence of such correlations in a NMR system as well as the effects of decoherence over them [7,8,9]. In the context of the study of decoherence processes, we present a derivation of the Redfield formalism for treating the dissipative dynamics of a time-dependent quantum system coupled to a classical environment [10]. We compare such formalism with the + 172 master equation approach when the environments are treated quantum mechanically. Focusing on a time-dependent spin-1/2 system we demonstrate the equivalence between both approaches by showing that they lead to the same Bloch equations and, as a consequence, to the same characteristic times T1 and T2 (associated with the longitudinal and transverse relaxations, respectively). These characteristic times are shown to be related to the operator-sum representation and the equivalent phenomenological-operator approach. We apply these equivalent formulations to the problem of state protection, noting that it is a crucial problem in literature where several distinct techniques have been proposed to control the effects of decoherence on quantum states, aiming to enlarge the fidelity of quantum information protocols. Finally, we present a protocol to circumvent the decoherence processes due to the loss of energy (and thus, associated with T-,). To this end, we simply associate the time- dependence of the quantum system to an easily achieved modulated frequency. A possible implementation of the protocol is also proposed in the context of NMR [10]. All these studies, carried at IFSC - USP and CBPF laboratories, shows that although the entanglement is not usually present in the NMR system, there exists classical and quantum correlations that could be useful for some quantum information protocols, and also came to corroborate the idea that there is a real quantum nature in the NMR QIP implementations. REFERENCES: 1. Oliveira I. S.; Bonagamba T. J.; Sarthour R. S.; Freitas J. C. C. e deAzevedo E. R., NMR Quantum Information Processing. 1a ed, 2007. 2. Braunstein S. L.; Caves C. M.; Jozsa R.; Linden N.; Popescu S. e Schack R., Physical Review Letters 1999, 83 1054. 3. Laflamme R.; Cory D. G,; Negrevergne C. e Viola L., Quantum Information & Computation 2002, 2 166. 4. Linden N.~e Popescu S., Physical Review Letters 2001, 87 047901. 5. Vedral V., Foundations of Physics 2010, 40 1141. 6. Ollivier H. e Zurek W. H„ Physical Review Letters 2001, 88 017901. 7. Soares-Pinto D. O.; Céleri L. C.; Auccaise R.; Fanchini F. F.; deAzevedo E. R.; Maziero J.; Bonagamba T. J. e Serra R. M., Physical Review A 2010, 81 062118. 8. Auccaise R.; Céleri L. C.; Soares-Pinto D. O.; deAzevedo E. R.; Maziero J.; Souza A. M.; Bonagamba T. J.; Sarthour R. S.; Oliveira I. S. e Serra R. M., submitted 2011. 9. Auccaise R.; Maziero J.; Céleri L. C.; Soares-Pinto D. O.; deAzevedo E. R.; Bonagamba T. J.; Sarthour R. S.; Oliveira I. S. e Serra R. M., submitted 2011. 10. Soares-Pinto D. O.; Moussa M. H. Y.; Maziero J.; deAzevedo E. R.; Bonagamba T. J.; Serra R. M. e Céleri L. C., submitted 2011. FAPESP, CAPES, CNPq, FAPERJ and INCT - Infoquant/MCT. 173 h * assail PO 78 MULTI-QUANTUM ECHOES AND COHERENCE SELECTION IN GDAL2 WITH ZERO-FIELD NMR R. Oliveira-Silva1, C. Rivera-Ascona1, J.R. Tozoni2, E.L.G. Vídoto1, J. Teles3, T.J. Bonagamba1 11nstituto de Física de São Carlos - Universidade de São Paulo Caixa Postal 369, 13560-970 - São Carlos - SP - Brazil 2Instituto de Física de Uberlândia, Universidade Federal de Uberlândia Uberlândia, Minas Gerais, Brazil 3Centro de Ciências Agrárias - Universidade Federal de São Carlos Rodovia Anhanguera, km 174, Araras -SP- Brazil e-mail: rsilv&.ursa. if sc. usp. br i Keywords: zero-field NMR; coherence selection; GdAI2 The zero-field NMR experiments in magnetic materials are a source of information about the nuclear environment, perturbed by the hyperfine interactions between different nuclei and electrons in their vicinity. To study such systems we used the two radiofrequency (rf) pulses sequence sketched in Figure 1. t preparation j acquisition Figure 1: Basic pulse-sequence used in the zero-field NMR experiments. With this pulse-sequence we performed two basic 27Al NMR experiments using the sample GdAI2: i) keeping constant the delay time between the two rf pulses, x, and sweeping the excitation frequency [1] we obtained the broadband spectra, and ii) keeping the excitation frequency constant, we obtained the quadrupole oscillations by varying x. Figure 2a shows the 27AI FID and the five multi-quantum echoes detected [2] and Figure 2b the quadrupole oscillations measured for the first echo [3]. 1st echo a) 0.2-, b) 2nd echo 3rd echo i! ftP* 2> -0.2 - 4th echo 5th echo w •HW -0.3- 0 20 40 60 80 100 120 140 160 30 80 90 120 Time Ej-ts) Time (us) Figure 2: a) 27AI FID and five multi-quantum echoes and b) first-echo quadrupole oscillation measured at 48.9 MHz and 4.2 K. From FID and multi-quantum echoes we obtained the respective spectra shown in Figure 3a. From the quadrupole oscillations of the first, second and third echo we got the quadrupole couplings at 48.9 MHz and 62.1 MHz. In the case of GdAI2, due to the relatively high spectral resolution, the quadrupole couplings can be measured directly from the spectra, see insert of Figure 3a. f + 174 a) -FID b) • 1st Echo -2nd Echo A 1st echo at 48.9 MHz - 3rd Echn 'Mr 2nd echo at 48.9 MHz 3rd echo at 48.9 MHz 1st echo at 62 1 MHz ^ 2nd echo at 62 1 MHz J 1 \ 3rd echo at 62.1 MHz 48 CU 86 0.0 1.0 2.5 Frequency (MHz) Frequency (MHz) Figure 3: a) 27AI broadband from FID, first, second and third echo and b) quadrupole coupling spectra obtained from the Fourier transform of the quadrupole oscillations at 4.2 K. In order to observe individually each echo and better explore the physical information contained therein with good signal-to-noise ratio, we implemented coherence selection of the multi-quantum states by phase cycling the two RF pulses. Figure 4 shows the results of the selection of all coherences from the 27Al multi- quantum states. Comparison with Figure 2a shows that the coherence selection was very well performed, indicating that the nth order coherence contributes only to the formation of the nth echo. The reported results indicate that the study of other magnetic materials that show multi-quantum echoes could also benefit from the methodology presented here [4,5], list 60 80 100 Time (us) Figure 4: Multi-quantum echoes selection. Similar results were obtained for 155Gd e 157Gd using the same methodologies. REFERENCES 1. Lord, J.S. and P.C. Riedi, Measurement Science & Technology 1995, 6(2), 149-155. 2. Bauer, M.; Dormann, E.; Physics Letters A 1990, 146, 55-59. 3. Abe, H.; Yasuoka, H.; Hirai, A.; Journal of the Physical Society of Japan 1966, 21, 77- 88. 4. Butterworth, J; Proceedings of the Physical Society of London 1965, 86, 297-304. 5. Golub, V. O.; Kotov, V. V.; Podyelets, Y. A.; Pogorely, A. N.; Hyperfine Interactions 1990, 59, 293-296. CNPq, CAPES, FAPESP, IFSC-USP 175 h * PO 50 CONSTRUCTION OF AN NMR POROUS MEDIA ANALYZER E.L.G. Vidoto, M.B. Andreeta, M.N. d' Eurydice, E.L. Oliveira, J.G. da Silva, A.D.F. Amorim, R. Oliveira-Silva, T.J. Bonagamba Instituto de Física de São Carlos - Universidade de São Paulo Caixa Postal 369, 13560-970 - São Carlos - SP - Brazil e-mail: evidoto&.ifsc. usp.br Keywords: porous media; low field spectrometer; low-cost NMR equipment During the last three decades, the NMR group of IFSC-USP has been dedicated to the improvement of several research areas, such as material and quantum information sciences, being one of the most important contributions of this group the development of NMR instruments. Since the study of porous media requires a low-field magnet to avoid problems associated with magnetic susceptibility [1], a project was designed to construct a low-cost spectrometer. The aim on designing this NMR porous media analyzer was based on making it as low-cost as possible. The following components were designed and constructed by the group: resistive magnet, magnet power supply, radiofrequency (rf) components (probes, amplifiers, filters, duplexers, etc.), shielded gradient coils and necessary software (pulse programming, signal acquisition, basic signal processing and inverse Laplace transform). The commercial equipments which take part of the spectrometer are general purpose broadband radio frequency (RF) excitation/detection and data acquisition systems controlled by a personal computer. Although the primarily objective of the equipment was the characterization of porous media at a 1H frequency of 2 MHz, it was possible to extend its application for higher 1H frequencies and execute other NMR experiments, such as Nuclear Quadrupole Resonance (NQR) and Zero-Field NMR experiments. To show the capabilities of this general purpose spectrometer, we present some representative experimental data obtained with it. Fig. 1a shows the 1H CPMG [2] signal and Fig. 1b its inverse Laplace transform of a Berea sandstone rock. Time (s) Tj (ms) Figure 1: a) 2 MHz 1H NMR CPMG signal of Berea sandstone rock measured at room temperature and b) its inverse Laplace transform. Fig. 2a shows the 157Gd zero-field FID and echo time signals, acquired at the frequency of 27.1 MHz and 4.2 K, for the intermetallic GdAI2 and Fig. 2b shows the spectra obtained from Fourier transform of both signals, resulting in a measured quadrupole coupling of 680 kHz [3]. 176 a) 1.0 b) "^0.8- TO. -2*0.6 '55 FID Echo fflc: I 04 15 L "I c I i u>0.2 - \ w' \ / \ CO \ / V, '•v. y v Echo 0.0 0 2Q 40 60 80 25.0 25,5 26,0 26.5 27 0 27.5 28.0 28.5 29.0 Time (us) Frequency (MHz) Figure 2: a) 157Gd FID and echo time signals acquired at the frequency of 27.1 MHz and 4.2 K and b) their respective spectra obtained by Fourier transforms. 35 Fig. 3 illustrates the NQR spectrum obtained for CI using a KCI03 single crystal sample, in a presence of a perturbative magnetic field of approximately 10 G [4]. -is -12 " 3 e » ^ ^ - Frequency (kHz) 35 Figure 3: NQR s'pectra of CI of KCI03 single crystal under a perturbative magnetic field at room temperature. All these experiments are being successfully explored in the study of porous media and on proposing new methods to characterize these materials at different NMR frequencies (ranging from 2 to 400 MHz), in the study of magnetic materials, by employing more advanced pulse sequences developed by our group, and, finally, in the implementation of methods for quantum information processing using NQR. All these studies will be discussed in more detail in other posters of our group to be presented during this AUREMN meeting. REFERENCES: 1. Dunn, K.J.; Bergman, D.J.; LaTorraca, G.A. Nuclear Magnetic Resonance: Petrophysical and Logging Applications] Pergamon: Elsevier Science, 2002. 2. Meiboom, S. and Gill, D. Rev. Sci. Instrum 1958, 29, 688-691. 3. Dormann, E.; et al., Journal of Magnetism and Magnetic Materials 1984, 45(2-3), 207-218. 4. Bain, A.D. and Khasawneh, M. Concepts in Magnetic Resonance Part A 2004, 22A(2), 69- 78. CNPq, CAPES, FAPESP, IFSC-USP. 177 h * PO 50 STUDY OF ORGANOTIN COMPOUNDS IN SOLUTION BY 119SN NMR Rubens R. Teles1, Ivani Malvestiti1 and Fernando Hallwass*1 1 Departamento de Química Fundamental, UFPE *hallwass(p).ufpe.br Keywords: 119Sn NMR; organotin. Organotin(IV) compounds present a wide range of pharmacological1 and industrial applications2,3 as well as intermediates in organic synthesis. The general molecular formula of these compounds are RnSnX^n (n= 1-4), R can be a alkyl or aryl group and X a nucleophilic species, such as: halide, carboxylate, alcohol, etc. However, due to the vacant 5d orbital, tin atom can have different states of hybridization, forming compounds with a variety of coordination numbers, ranging from four to eight. Particularly in solution, the intermolecular interactions produce species with higher coordination numbers and associated structures, such as dimers, trimers, and even polymeric structures. In this way, 119Sn NMR arises as an important tool to investigate the structure and dynamic of these species in solution. Moreover, the great advantage to use 119Sn NMR is its natural abundance (8,58%) and the large spectral width (5.500 ppm). In this work were investigated the species of dioxastanolanes formed by the reaction of diols and diorganotin oxide (Scheme 1), and studied the dynamics between these species. The analyses of 119Sn NMR spectra brought additional information about these compounds that were not described before using 1H and 13C I^MR spectroscopy4" Scheme 1 OH 2 AU R 2SnO .Sn' Kdim. v Kpol. Aggregates -H,0 OH VI „ l.V R[= Me or Ph Product 1A= Ri methyl, R2 phenyl R2= BU or Ph Product 1B= R-| phenyl, R2 phenyl Product 2A= Ri methyl, R2 butyl Product 2B= R-| phenyl, R2 butyl The dioxastanolanes were prepared from diols (0,33 mmol) and diorganotin oxide (0,33 mmol) in benzene by azeotropical distillation using a dean-stark system. The product was dissolved in CDCI3 for obtaining NMR spectra. The spectra were acquired in a Varian VNMRS-400 spectrometer, at 300K, operating at 399,7 MHz for 1H, 100,5 MHz for 13C and 149 MHz for 119Sn. Tetramethyltin neat was used as external reference for the calibration of 119Sn NMR spectra (6=0ppm)7. In order to assign the signals correctly 2D experiments (HMQC 1H-13C and HMQC 1H-119Sn) were carried out. 119 Figure 1 shows four Sn NMR spectra with different Ri and R2 groups. In Figure 1A and 1B, where R2 is a phenyl group, were observed well-defined signals, indicating the formation of discrete structures. Analyzing the chemical shifts were possible to determine the presence of one tetracoordenated structure (near - 40 ppm), pentacoordenated structures (- 80 to -130 ppm) and hexacoordenated structure (near - 240 ppm). The formation of these species is independent of diol used. However, if R2 178 is a butyl group the spectra were more complex to analyze due to the broad signal, characteristic of aggregates formation. The chemical shifts indicated pentacoordinated structure. In this system, there is a dynamic process of chemical exchange. Therefore the signals in the spectra are an average from tetra-, penta- and hexacoordinated species. In conclusion, we observed that 119Sn NMR spectra are helpful to determine the structure and to understand the dynamic process of organotin compound in solution. Ri= Methyl Ri= Methyl R2= Phenyl R2= Butyl . / ! VI \ Ri= Phenyl Ri= Phenyl R2= Phenyl R2= Butyl J/ A vV W,yWtv^^wW^w^M^^^'' Figure 1 /^Sn NMR spectra of organotin compounds. REFERENCES . 1. Gielen, M.; J. Braz. Chem. Soc., 2003, 14, 870-877. 2. Hoch, M.; Appl. Geochem.; 2001, 16, 719-743. 3. Wuest, J. D.; Zacharie; J. Org. Chem., 1984, 49(1), 166-168. 4. Shanzer, A.; Libman, J.; Gottlieb, H.E.; J. Org. Chem., 1983, 48(24), 4612-4617. 5. Luchinat, C.; Roelens, S.; J. Am. Chem. Soc., 1986, 108(16), 4873-4878. 6. Roelens, S.; Taddei, M.; J. Chem. Soc. Perkin Trans II, 1985, 799-804. 7. Blunden, S.J.; Frangou A. e Gillies G. Org. Magn. Reson., 1982, 20, 170. PRONEXI FACEPE-CNPq (Brazilian Agencies) 179 h * PO 81 AMPLIFING THE EFFECTS OF MOLECULAR MOTIONS IN DIPSHIFT-LIKE EXPERIMENTS M.F. Cobo1, A.Achilles2, D. Reichert2, K.Saawaechter2, E.R. deAzevedo*1 'Universidade de São Paulo, São Carlos, Brazil 2 Martin-Luther-University Halle-Wittenberg, Halle, Germany e-mail:azevedo(iS).ifsc. usp. br Keywords: Dipolar Chemical Shift Correlation; molecular motion; coupling amplification Molecular dynamics is related with many properties of solid organic materials, such as optical activity, mechanical resistance, conductivity, etc. Among the NMR techniques used to probe molecular motions, the Dipolar Chemical Shift Correlation method (DIPSHIFT)1 can be used to study motions with rates ranging from tens of Hz to few kHz, the so called intermediate regime.2 In this method, the heteronuclear dipolar coupling between a rare (X) and abundant (H) nuclei is used to probe molecular motions. This is achieved by the pulse sequence shown in Figure 1.a). The spins interact through the heteronuclear dipolar coupling during a variable time U, which ranges from zero to one rotor period (tr). During /1t homonuclear decoupling is applied to the abundant nuclei to guarantee the local nature of the heteronuclear dipolar interaction. After the t1 evolution, heteronuclear decoupling is turned on and a n pulse applied at tr is followed by another evolution period of duration tr. This produces an echo at 2tr, which has the amplitude dependent on the accumulated phase due to the dipolar coupling evolution during tp tr/2 1,12 tr/2 tr/ 2 • k=1 10® Hz —• k = 1 10" HZ i o.s // k = 510s Hz . . ii'iinr* 06 13 «C \\N // c I , I jyyvw J L 2tr (•I»'* •- N-1 N-1 E 0.2 % / k-110'Hz (, 0.0i::- ! 1 Homo „ i.. u.4 ••J.b U.S 1u H Dea„c MeuriunuuedHeteronuoleair UBDec, tl/t„f " Homo DEC Het DEC Figure 1: a) DIPSHIFT pulse sequence, b) Spin dynamics simulations of the DIPSHIFT signal obtained at 2fras function of i7/fr for several motion rates (k). c) Recoupled - DIPSHIFT pulse sequence. The effects of molecular motions in the DIPSHIFT curve can be seen in the spin dynamics simulations3 displayed in the Figure 1 .b). For motions with rates in the order of tens of Hz, the DIPSHIFT curves loose intensity at = tr as a result of the decreasing of T2 due to the molecular motion. Increasing the motion rate, the T2 effect decreases, but the minimum of the DIPSHIFT curve becomes shallower, being this behavior related to the reduction of the effective heteronuclear dipolar interaction. These two features provide information about molecular motions. In systems where the heteronuclear coupling is weak or the motion reorientation angle is small, the use of DIPSHIFT becomes difficult because the curves become rather insensitive to the dipolar couplings and, consequently, to the modulations introduced by the motion. This can be partially overcome using an implementation of the DIPSHIFT pulse sequence proposed by Hong et al.,4 where the accumulated phase is amplified by a REDOR like train of k pulses.5 However, in this pulse sequence the time in which the system interact through 4 heteronuclear dipolar coupling is constant and, as result, the motion induced effect on T2 is always the same, i.e., the intensity reduction due T2 along the U is not observed and motion effects on T2 are not probed in this sequence. As a result, this experiment has a reduced dynamic window (100 kHz to 1MHz) as compared with the standard DIPSHIFT method. f + 180 13th NUCLEAR MAGNETIC RESONANCE USFRS MITTTNG Keeping in mind the necessity of amplifying the phase accumulated and the need to have an experiment which is not time constant we proposed another implementation of the DIPSHIFT pulse sequence based on the REDOR pulse sequence. The pulse sequence is shown in Figure 1.c). In this implementation, which will be named as recDIPSHIFT, two trains of (N-1) n pulses separated by half rotor period are face to each other with no n pulse in the middle. When the homonuclear decoupling is applied during the full period, the accumulated phase due to the evolution under the heteronuclear dipolar coupling is amplified N times. Nevertheless, as t-i runs from zero to Ntr, the evolution time under heteronuclear dipolar coupling is not constant, producing a T2 modulation in the same fashion as the standard (not recoupled) DIPSHIFT experiment. a) c) v 0 0 Figure 2: a) Ntr- recDIPSHIFT simulations for a rigid C-H group separated by 1.5 Á. b) DIPSHIFT curves for TMSI (Trimetyl Sulfoxonium Iodide) at several temperatures, c) 4tr- recDIPSHIFT curves for TMSI at several temperatures. The dipolar coupling amplification effect is demonstrated in Figure 2.a), which shows a set of simulations for the recDIPSHIFT for a weak (2 kHz) CH dipolar coupling for several values of Ntr. Figure 2.b) shows the DIPSHIFT experiment performed in a model sample, the Tri-Methyl Sulfoxoniun Iodide, a molecular crystal which has three CH3 groups (effective dipolar coupling of -12 kHz) executing permutations between three positions (three site jumps) of a cone of 109° aperture2. Increasing the temperature, the motion rate increases, and one can observe the motion induced decrease in T2 and the averaging of the dipolar coupling. In the Figure 2.c) it is shown the curves obtained using 4frrecDIPSHIFT for the same sample and temperatures. Comparing Figures 2.b) and 2.c) it is observed that the 4tr- recDIPSHIFT experiment retains the T2 modulation and the motion effects are amplified. Further simulations and experiments were performed using different homonuclear decoupling sequences, MAS-frequencies, dipolar couplings and decoupling powers in order to find out the best experimental conditions and identify possible imperfections that could compromise the performance of the experiment. These analyses showed that is crucial to establish a compromise between the RF power, MAS frequency and dipolar coupling, in order to avoid intensity loose in the amplitude of DIPSHIFT echo at U=Ntr. This kind of imperfection have been observed in many DIPSHIFT results in the literature, but no clear explanation neither ways to avoid it were given before. Using the Average Hamiltonian Theory we identified the exact origin of the artifacts and establish the conditions to avoid them. Furthermore, we also clearly show that in recDIPSHIFT like experiments the simple Lee-Goldburg (LG) homonuclear decoupling performs as good as, or even better than, its more intricate variants, such as Frequency Switched and Phase Modulated Lee-Goldburg (FSLG and PMLG) pulse sequences. The reasons for that will be discussed. REFERENCES: 1. MunowitzM.; Griffin R. Et al; J. Am. Chem. Soc. 1981, 103, 2529 2. deAzevedo E.R.; Saalwaechter K. et al; J. Chem. Phys. 2008, 104505 3. Veshtort M.; Griffin R.G., J. Magn. Reson., 2006, 178, 248 4. Hong M.; Gross J.D. et al; J. Mag. Res. 1997, 129(1), 85 5. Gullion T.; Schaefer J.; J. Mag. Res. 1989, 81(1), 196 FAPESP, CNPq, CAPES PROBRAL 181 h * PO 50 INTERMOLECULAR INTERACTIONS BETWEEN SOLIDS AMOXICILLIN AND OMEPRAZOLE: A SOLID STATE NMR STUDY Lorena Mara Alexandre e Silva1, Marcos Guillermo Russo2, Griselda Edith Narda2, Javier Alcides Ellena3, Antonio Gilberto Ferreira1, Tiago Venâncio1 1 Universidade Federal de São Carlos, São Carlos, Brazil 2Universidad Nacional de San Luis, San Luis, Argentina 3Universidade de São Paulo, São Carlos, Brazil venancio@ufscar. br Keywords: amoxicillin, omeprazole, solid state NMR. Amoxicillin (AMX, fig. 1a) is one of the most employed antibiotics to treat infections caused by different types of bacteria [1], Omeprazole (OMZ, fig.1b) is widely employed in the treatment of gastric disorders (and also gastric ulcer) which can be one of the most frequent side effects of several drugs [2], The association of these two drugs also promotes the elimination of Helicobater pylori, which is a bacterium that causes the majority of gastric ulcer, being a factor to develop stomach tumors [3], Fig. 1. Chemical structure of: amoxicillin (a) and an omeprazole tautomer (b). Recently, binary systems have been widely studied [4] in order to understand the mechanisms of the interactions drug-drug and drug-additives. It has been demonstrated that solid state NMR can be a very useful tool to elucidate these mechanisms [5]. Therefore, the aim of this work is to use the solid state NMR for studying the interactions between different forms of AMX and OMZ in a binary complex (AMX-OMZ), in order to have some information to estimate the stability of the complex. In this work we analyzed five different samples: OMZ, AMX.3H20 (AMHh), anhydrous AMX (AMXa), co-grinding AMXa-OMZ and AMXh-OMZ mixtures in 5:5 mass ratio. The isolated drugs were used as received and the AMX-OMZ binary complex was prepared by co-grinding. All of them were packed in a 4mm zirconia rotor. A Bruker Avance III NMR equipment was used by employing a 9.4T Oxford Magnet and a 4mm solid state probehead. For each sample it was performed a pool of solid state NMR techniques including 13C-CPTOSS (cross polarization with total sideband suppression), (1Hx13C)-FSLG-HETCOR (frequency switched Lee-Goldburg heteronuclear correlation). For CPTOSS experiment we have worked with a 5KHz spinning speed, 2ms of contact time and 5s of recovering time. In fig. 2 it is shown the FSLG-HETCOR contour maps for all the samples. When we compare both figures 2c and 2a, all the correlation signals of the carboxylic proton (~10.5ppm) from the AMXa (fig.2a) shift to ~8.5ppm as the binary complex is formed (fig.2c). No changes were revealed by analyzing OMZ (fig.2e). It can be concluded that AMXa and OMZ molecules probably interacts through the amino group from AMXa and the unprotonated nitrogen from five-member ring because it is possible to observe a weak correlation signal between C2 (159ppm) and a proton in ~5.0ppm. 182 a/ "MZ-AMXa 5.5 co-grinding t .fciOMZíAMVh'5í,c: sr',3'^ t I» ! ! tf-» i I V » " .1 idi AM«i ! t? f Ch Fig. 2. FSLG-HETCOR maps for: (a) AMXa-OMZ, (b) AMXh-OMZ, (c) AMXa, (d) AMOXh and (e) OMZ. Experimental parameters: contact time of 200ps, 10KHz of spinning speed. CPTOSS experiments revealed a higher crystallinity of AMXh, when compared to AMXa. A low crystallinity was also observed for OMZ. Fig.2b presents the FSLG-HETCOR obtained for the AMXh-OMZ binary complex. Few differences can be seen in the AMXh signals in the complex (fig.2b) by comparing to fig.2d. It is only observed some change in the 5H from ~11 to 11.5ppm. Most of the differences come from the OMZ signals when we analyze both figures 2e and 2b. The main difference is in the 8H from the signals correlated to proton in 8.5ppm, which shifted to 11.5ppm in comparison to fig.2e. The interaction between AMXh and OMZ must be occurring through the unprotonated nitrogen from 5-member ring and the proton of the phenolic group from AMXh because it is also observed a signal correlated to a proton in ~8ppm. The presence of water molecules seems to preserve the crystallinity of AMXh, leading to conclude that water is not involved in the interaction between OMZ-AMXh, which should be weaker than in the AMXa-OMZ binary complex. References 1. A. Ghassempour, H. Rafati, A.L. Inasab; Y. Bashour, H. Ebrahimzadeh, M. Erfan AAPS Pharm. Sci. Tech., 2007, 8(4). 2. R. M. Claramunt, C. López, J. Elguero ARKIVOC, 2006, 5. 3. R.J. Adamek, M. Wegener, J. Labenz, M. Freitag, W. Opferkuch, G.H. Ruhl, Am. J. Gastroenterol, 1994, 89(1). 4. A. Krupa, M. Majda, R. Jachowicz, W. Mozgawa, Thermochimica Acta, 2010, 509. 5. H. W. Spiess, Macromolecules, 2010, 43. FAPESP (2009/13860-2), CNPq (475903/2009-9), CONICET, UNSL 183 I- + PO 83 ELUCIDATING THE C-TERMINAL DOMAIN OF HUMAN SEPTIN 2 E. Crusca*, C.E. Munte, R.C. Garratt Physics Institute of São Carlos, University of São Paulo, São Carlos, Brazil e-mail: [email protected]. usp.br Keywords: Neuropathology; Septins; Coiled-coil Septins are a conserved family of binding proteins to guanine nucleotide which are involved in many cellular processes. The functions of these proteins are probably a result from their inherent ability to assemble into complexes and form highly ordered polymers. These complexes can act as diffusion barriers for the compartmentalization of the cell membrane, vesicle trafficking and ways of protein interaction to specific intracelular locations [1], The sequences of all members of this family can be divided into three distinct domains: a N-terminal variable domain, a central GTPase domain and a C-terminal domain that typically includes sequences characteristic of coiled-coils. Some septins are related to pathological states, for example, septins SEPT1, SEPT2 and SEPT4 appear to accumulate in filamentous deposits (Lewy bodies) in Alzheimer's disease; SEPT4 was also co-located with the protein alpha-synuclein in Parkinson disease [2], Specific combinations of septins form hetero-oligomeric complexes that polymerize into non-polar filaments in vivo and in vitro. The complex formed by the human septins SEPT2, SEPT6 and SEPT7 was solved by Sirajuddin et al. (2007) by X-ray crystallography [3] (Fig. 1). However, the C-terminal domains do not presented electron density in the crystal structure, providing no information about these parts of the complex which may be critical for determining the way that different septins interact in the assembly of hetero-oligomeric complexes. a Figure 1: Graphical representation of the complex formed by septins 2/6/7: crystallographic structure (a) the schematic representation indicating the predicted formation of coiled-coils by the C-terminal regions (b). It is expected that the SEPT6 SEPT7 and form a hetero-coiled-coil and SEPT2 a homo coiled-coil [3,4], f * 184 I?i1: NLTLEAR MAGNETIC RESONANCE t'SFRS MITTTNTi According to previous results obtained by sets of Nuclear Magnetic Resonance (NMR) experiments of SEPT2C, the spectra obtained showed two dynamically different regions. The chemical shift index indicated that the central region of the domain is in an alpha helical conformation, in agreement with the predicted coiled-coil structure. i SOO ^ -Í 00 1 1 3 S 7 9 11 13 16 Í7 19 2' 25 25 27 2S 31 33 55 3? 39 41 43 45 47 49 5 53 55 57 59 Residue Figure 2: The Ca chemical shifts of residues in SEPT-2C compared with the Ca chemical shifts corresponding to a random coil conformation. To remove the flexible N- and C-terminal regions, a peptide SEPT2CC comprising the residue Asn15-Gln44 of Sept2C has been engineered (Fig. 3) and synthesized (GeneScript, EUA). Previous circular dichroism analysis clearly showed that SEPT2CC is structurated in a coiled coil. SEPT2CC -NKDQILLEKEAELRRMQEMIARMQAQMQMQ- Figure 3: Construction of SEPT2CC with two possibilities to the coiled-coil phase. We carried out a set of 2D-homonuclear NMR experiments COSY, TOCSY and NOESY of SEPT2CC. The spectra are being analyzed and the preliminary results indicate a high content of a-helix. REFERENCES 1. Neufeld, T. P.; Rubin, G. M. Ce//1994, 77, 371-379. 2. Kinoshita, A.; Kinoshita, M.; Akiyama, H.; etal.. Am. J. Pathol. 1998,153,1551-1560. 3. Sirajuddin, M.; Farkasovsky, M.; Hauer, F.; etal.. Nature 2007, 449, 311-315. 4. Weirich, C.S.; Erzberger, J.P.; Barrai, Y. Nature Rev. 2008, 9, 478-489. FAPESP, CNPq and CAPES 185 h * PO 52 A NEW PROGRAM FOR THE SIMULATION OF NMR PULSE SEQUENCES Clara Luz S. Santos1; Claudia J. Nascimento2,3 and José Daniel Figueroa Villar1* 1 Seção de Química - Instituto Militar de Engenharia -RJ- Brazil figueroa&.ime. eb.br Instituto de Química - Universidade de Brasília (UnB) - Brasília - DF - Brazil 3Instituto de Biociências - UNIRIO - Rio de Janeiro - Brazil Keywords: pulse sequences; quantum operators; MATLAB The comprehension of NMR pulse sequences is a fundamental step for the design of new pulse sequences. Differently from other forms of spectroscopy, NMR cannot be well described in terms of energy levels. One important tool for describing NMR experiments is the use of the vector model.1 Despite of being useful for the description of some NMR experiments in a simple way, it cannot be applied to all coupled spin systems, which prevents from the explanation of simple experiments (COSY, for example). Most of the more recent NMR pulse sequences must be described by quantum mechanical using density matrixes2 which are the basis for the description using spin product operator formalism. Quantum operators allow the description of spins after radiofrequency pulses, and during free precession and coupling evolution giving us a complete description of the experiment. This is very important not only for the understanding of an experiment, but also for the development and implementation of new experiments. Application of product operator formalism to NMR experiments is extensively described in the literature.3,4 However, the extensive calculations, even considering simplifications, like spin-echo sequences and 180° pulse in the middle of t-i evolution time, are very exhausting, mainly for beginning students. In this work we propose a simple mathematical program using the MATLAB application for the description of simple NMR experiments for two spin 1/4 nuclei. Also a quick guide was developed allowing people that are not familiarized to the software perform some simulations. MATLAB is a simple software that allows the description of symbols, graphics and calculations in a very similar way to the written algebraic expressions. The interface can be seen in Figure 1. -jii >-',', ÍUJ ;ç pj%»it- iíí H>- £dít De|«g Psialle! O'"-"* -;' ii^'lc-,.- = Et»t"|. Ini-idta. ibM r. . i • o a •' • - •1*1 ;>m - - ./r: • : . - ' 16*1 double* <16x1 dcjbfe* fflh- - dc.iN?> , ffilv <15*1 H: • 11 r.: - oJi <15 d ' , - • Figure 1: MATLAB's interface showing the programmed matrix f, 4 186 • iNETIC RES The spin system for two nuclei I and S is represented by alx + bly + clz + dSx + eSy + fSz + glxSx + hlxSy + ilxSz + jlySx + klySy + llySz + mlzSx + nlzSy + olzSz + pll, where II is the identity and the small letters are the corresponding state coefficients. The system state is described by a vector in which the coordinates are the coefficients of the operators. For example, the (lz + Sz) equilibrium state is 0IX + 0ly + 1lz + 0Sx + 0Sy + 1SZ + OlxSx + OlxSy + 0IXSZ + OlySx + OlySy + OlySz + 0IZSX + OlzSy + 0IZSZ + Oil which is represented by the vector [1,0,0,0,0,1,0,0,0,0,0,0,0,0,0,0]'. On the basis of product operators theory and the equation of motion it is possible to build matrixes that are multiplied by a vector that describes a state and this gives as a result the new system state. The new system states occur after time evolution or due to the effect of applied radiofrequency pulses. The spin-echo and COSY pulse sequences have already been tested in this program. The HSQC sequence is now being simulated and some new improvements for simplification of the results will be implemented on the program. Also we are going to implement the use of magnetic field gradients for testing with the corresponding g pulse sequences. MATLAB is a simple and useful software for the necessary calculation for the complete description of pulse sequences. We believe that it will be very useful for NMR beginners: the result is easily obtained and the user can evaluate the results correlating them with the information observed in the experimental spectra. REFERENCES 1. Freeman, R. Spin Choreography: basic steps in high resolution NMR. 1997. Oxford University Press, Oxford. 2. Farrar, T.C.; Harriman, J.E. Density matrix theory and its application in NMR spectroscopy: an introduction to the theory and applications. 1998, Farragut Press. 3. Sorensen, O.W.; Eich, G.W.; Levitt, M.H.; Bodenhausen, G.; Ernst, R.R. Progress in NMR Spectroscopy 1983, 16, 163. 4. Kessler, H.; Gehrke, M.; Griesinger, C. Angewandte Chemie International Ed. Engl. 1988, 27, 490. FAPERJ; FINEP; CNPq, INBEB, FUNDAÇÃO RICARDO FRANCO 187 h * PO 50 STRUCTURAL CHANGES IN HYBRID PHYTOCYSTATINS ANALYZED BY HIGH-RESOLUTION NUCLEAR MAGNETIC RESONANCE I.A.Cavini*1, R.C. Garratt1, F. Henrique-Silva2, H.R. Kalbitzer3, C.E. Munte1 1 Physics Institute of São Carlos, University of São Paulo, Brazil 2Department of Genetic and Evolution, Federal University of São Carlos, Brazil 3Institute for Biophysics and Physical Biochemistry, University of Regensburg, Germany e-mail: [email protected] Keywords: cysteine proteinase inhibitor; canecystatin; triple resonance assignment. Proteolytic enzymes have a variety of functions essential for all living organisms. In particular, some cathepsins form a group of cysteine proteases of the papain subfamily, / which primarily function as endopeptidases within lysosomes. The human cathepsins B and L are involved in a large number of illnesses, including, muscular dystrophy, arthritis, cancer and neurodegenerative diseases. Due to the participation of these enzymes in different physiological events, their action is regulated by protein inhibitors. The cystatin superfamily of proteins is composed of specific inhibitors of cysteine proteinases that exhibit tight competitive inhibition. Phytocystatins or plant cystatins are proteins characterized by the absence of disulfide bonds and the presence of an N- terminal a-helix consensus sequence. In plants they control germination process, apoptosis and also defense mechanisms against insects and pathogens [1], The best studied phytocystatin is oryzacystatin-1 (from rice), whose three-dimensional structure was solved by solution NMR [2], Its fold can be described as a five-stranded antiparallel p-sheet wrapped around a central five-turn a-helix, being stabilized by a hydrophobic cluster formed between the two which contains a specific L-A-R-F-A-V-like sequence (Fig. 1). Figure 1: Solution NMR structure of orizacystatin-1 (pdb number 1EQK) [2], The inhibitory activity is caused by a wedge-shaped hydrophobic edge of the cystatin molecule being inserted into the proteinase active-site cleft, blocking access of substrates to the active site of the proteases. This wedge is formed by three structural elements: the N-terminal region, a loop connecting the second and third P-strands and a second binding loop between P-4 and P-5 strands. Both loops physically interact with the active site of the cysteine protease, the first through its Q-X-V-X-G motif and the second via residues P83 and W84 (in oryzacystatin-1). The N-terminal region does not directly interact with the active site, but makes extensive contacts with the protease, playing an important role in the binding process. 188 The sugarcane (Saccharum officinarum) cystatins, also known by canecystatins, were biological characterized and was shown the inhibitory effect of canecystatin CaneCPI-4 in human cathepsins B and L and, in particular, canecystatin-1 was efficient in inhibition of several classes of peptidases [3]. In a recently published paper [4], new phytocystatins were synthesized starting from the sequences of oryzacystatin-1 and canecystatin-1. A shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity. This clone, named A10, presented two unanticipated point mutations (I30T e L97Q) as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously totally abolishes the inhibitory activity. The authors conclude that mutations disrupt the hydrophobic core of phytocystatins, increasing the flexibility of the N-terminal region, and leading to the detected increase in inhibitory activity. The current work aims to provide information about the structural changes due to mutations and hybridization occurred in the A10 hybrid. For this, high-resolution NMR measurements of the isotopically labeled (13C/15N) native protein canecystatin-1 was obtained and analyzed to afford the complete sequential assignment of the protein. We used the triple resonance spectra HNCA, CBCA(CO)NH and HNCO to assign the protein backbone (Fig. 2) and H(C)CH-TOCSY to assign the side-chain signals. E28 A2S L32 A33 F35 A36 V37 A38 H40 N41 S42 K43 Figure 2: Strip plot displaying part of the sequential connectivities of canecystatin-1. New 2D heteronuclear NMR measures (1H,15N-HSQC) of the A10 canecystatin-1 hybrid and its reverse mutant (T30I e Q97L) will be acquired and the spectra will be assigned and compared. The information obtained may be related with stability or solubility changes occurred with the protein. A better understanding about the mechanism of inhibition used by phytocystatins is necessary, given the importance of cysteine proteases in physiological processes. REFERENCES 1. Gianotti, A.; Rios, W. M.; Soares-Costa, A.; et al.. Protein Expr. Purif. 2006, 47, 483-489. 2. Nagata, K.; Kudo, N.; Abe, K.; etal.. Biochem. 2000, 39, 14753-14760. 3. Oliva, M. L. V.; Carmona, A. K.; Andrade, S. S.; et al.. Biochem. Biophys. Res. Commun. 2004, 320, 1082-1086. 4. Valadares, N. F.; Dellamano, M.; Soares-Costa, A.; etal.. BMC Struc. Biol. 2010, 10, 30. CNPq 189 h * PO 52 QUANTUM COMPUTATION IN SOLID CRYSTALS BY LOW FIELD NUCLEAR QUADRUPOLE RESONANCE J. Teles1, R. Oliveira-Silva2, R.S. Polli*2, E.L.G. Vidoto2, E.L. Oliveira2, D.O. Soares-Pinto2, T.J. Bonagamba2 1 Centro de Ciências Agrárias - Universidade Federal de São Carlos Rodovia Anhanguera, km 174, Araras - SP - Brazil 2lnstituto de Física de São Carlos - Universidade de São Paulo Caixa Postal 369, 13560-970 - São Carlos - SP - Brazil e-mail:dioQo.osp(S).ursa. ifsc.usp.br Keywords: nuclear quadrupole resonance; quantum computation; quantum information. Nuclear Magnetic Resonance (NMR) is one of the most extensively studied and applied techniques in the demonstration and characterization of quantum states of spin systems [1,2]. NMR achieves success in Quantum Computation (QC) studies mainly due to its great ability to control nuclear spin dynamics through radio frequency pulses (RF). Current technology in radiofrequency electronics enable pulse design with high temporal resolution compared with the evolution times related to the main interactions that take place in NMR studies. The control over the dynamics and the relatively large relaxation times of the nuclear magnetic states enable operations before the non- unitary transformations act on the system quantum state. These characteristics make NMR a suitable choice in preparing initial states, implementing logic gates, and reading of quantum states [3-6]. Although the use of spin 1/2 nuclei is the most common chtfice to implement the quantum bits (q-bits) in NMR, there are other possibilities using higher spin nuclei. We can associate logic states of a system with N q-bits to magnetic states of a nucleus with spin / For example, a 2 q-bits system could be implement with a 3/2 nucleus. Nuclei with spin higher than 1/2, besides the dipolar magnetic interaction also have a quadrupole electric interaction, presenting two terms in their Hamiltonian: one corresponding to Zeeman interaction with external magnetic field and the other corresponding to quadrupole interaction. In case of low field NMR, the Zeeman interaction works as a perturbation and the quadrupole interaction as the main interaction. This technique is usually named Nuclear Quadrupole Resonance (NQR). Crystals formed by ions CI03 - such like NaCI03 and KCI03 are compounds quite interesting for the processing of quantum information using NQR. The nucleus of 35CI presents spin 3/2, making possible the study of systems of 2 q-bits. Moreover, the quadrupole coupling in those crystals is of the order of dozens of MHz, enabling magnetic fields of the order of hundreds of Gauss to unfold the spectral lines above the spectral widths and still to maintain the magnetic interaction as a perturbative first order term. The Figure 1 illustrates a spectrum of 35CI (resonance around 28.1 MHz) in a crystal of KCI03 under a weak magnetic field generated in the proximities of a superconductor magnet. For the RF pulse sequence design that implement the quantum logic gates, we adapted the methodology of the strongly modulated pulses (SMP) already broadly used in studies of QC through RMN in liquid and solid samples [7-9], This technique is based on the numeric optimization of pulses that implement rotation operations in specific spins of homonuclear systems. At this time, we have successfully optimized five quantum gates for the spin 3/2, including the CNOT^ and CNOTB gates, and we are 35 applying them to the quantum states of the CI nucleus in our KCI03 sample. Besides the logic gates, the success in the implementation of those sequences will make possible the preparation of the initial pseudo-pure states, which can be obtained by the procedure of temporal average. Methods of quantum state tomography will be built f, 4 190 •,'lliM (II M< \I.\fiM 111' HI M)\ \M"I. I ^I-R». MI11INC, using the characteristic symmetries of the Hamiltonian, in a similar way already developed to NMR systems. Procedures of phase cycling of the RF pulses and the evolution of the irreducible tensors that expand the operator density will be also studied to reach those objectives [7,10]. 1.0 i i -1f i . ,...., i ... i • , ft 0.8 £ w 0,2 / Vf 1 1 0.Q 1 1 "!' •*• ' "I > ' I' ' ' ' I ' ' ' I ! ' '1 >""< ' 1 • -50 -40 -30 -20 -10 0 10 20 30 40 50 Frequency (kHz) Figure 1: NQR Spectrum of the 35CI nucleus in the presence of a weak magnetic field, measured in the frequency of 28,1 MHz. That spectrum was obtained with the sample at a distance of approximately 2 m of a superconductor magnet of 2 T, where the magnetic field has the intensity of some mT. REFERENCES: 1. I. S. Oliveira, T. J. Bonagamba, R. S. Sarthour, J. C. C. Freitas, E. R. deAzevedo. NMR Quantum Information Processing. Elsevier, 2007. 2. L. M. K. Vandersypen, I. L. Chuang. NMR Techniques for Control and Computation. Reviews of Modern Physics, 76:1037-1069, 2004. 3. N. A. Gershenfeld, I. L. Chuang. Bulk Spin-Resonance Quantum Computation. Science, 275(5298):350-356, 1997. 4. D. G. Cory, A. F. Fahmy, T. F. Havel. Ensemble quantum computing by NMR spectroscopy. Proceedings of the National Academy of Sciences of the United States of America, 94(5): 1634-1639, 1997. 5. L. M. K. Vandersypen, M. Steffen, G. Breyta, C. S. Yannoni, M. H. Sherwood, I. L. Chuang. Experimental realization of Shor's quantum factoring algorithm using Nuclear Magnetic Resonance. Nature, 414(6866):883-887, 2001. 6. G. L. Long, H. Y. Yan, Y. Sun. Analysis of density matrix reconstruction in NMR quantum computing. Journal of Optics B-Quantum and Semiclassical Optics, 3(6):376- 381, 2001. 7. J. Teles, E. R. deAzevedo, R. Auccaise, R. S. Sarthour, I. S. Oliveira, T. J. Bonagamba. Quantum state tomography for quadrupolar nuclei using global rotations of the spin system. Journal of Chemical Physics, 126:154506, 2007. 8. A. D. Bain, M. Khasawneh. From NQR to NMR: The complete range of quadrupole interactions. Concepts in Magnetic Resonance Part A, 22A (2): 69-78, 2004. 9. E. M. Fortunato, M. A. Pravia, N. Boulant, G. Teklemariam, T. F. Havel, D. G. Cory. Design of strongly modulating pulses to implement precise effective Hamiltonians for quantum information processing. Journal of Chemical Physics, 116(17):7599-7606, 2002. 10. J. Baugh, O. Moussa, C. A. Ryan, A. Nayak, R. Laflamme. Experimental implementation of heat-bath algorithmic cooling using solid-state nuclear magnetic resonance. Nature, 438(7067):470^173, 2005. FAPESP, CNPq, CAPES, IFSC-USP 191 h * PO 87 PURE T2 - T2 EXCHANGE: AN ENHANCEMENT M. N. d' Eurydice*1, T.J. Bonagamba1 11nstituto de Física de São Carlos - Universidade de São Paulo Caixa Postal 369, 13560-970 - São Carlos - SP - Brazil e-mail:mrcl(q).ifsc. usp. br Keywords: porous media, relaxometry, exchange. 111 2D T2-T2 Exchange experiments are useful for identifying spins migrating amongst sites with different physical-chemical environments'21, leading them to have different transverse relaxation times. The transverse relaxation times presented by the migrating spins in the initial and final sites can be detected by an experiment comprised of two CPMG periods separated by a mixing time, both used for measuring the transverse relaxation times in the sample. This mixing time has the order of necessary time for the spins to move through different sites, although never longer than T1p Despite measuring the same relaxation time distribution before and after the mixing time, the resulting 2D patterns are composed of diagonal and off-diagonal peaks. Whilst the diagonal peaks represent spins that did not migrate or migrated to sites with similar physical-chemical environments and presenting comparable relaxation times, the off- diagonal peaks are associated with spins migrating to sites with different physical- chemical environments, showing different relaxation times before and after the mixing time. Once the experiments are carried out under highly homogeneous magnetic fields and the sample displays well-separated relaxation time distributions, the resulting 2D patterns are much easier to interpret, with clearly identifiable off-diagonal peaks. However, when the experiments are performed under inhomogeneous magnetic fields, with the spins presenting broad and overlapping relaxation time distributions, the 2D patterns are more difficult to understand. This problem is increased in cases where there is a huge spin population of non-migrating spins, resulting in a very intense and broad diagonal ridge compared with the off-diagonal peaks. This issue is further complicated when the off-diagonal peaks are too close to the diagonal ones as they become inseparable. An additional problem associated with the presence of a huge diagonal ridge is the 2D inverse Laplace transform'3,41 sensitivity, which makes it much harder to determine the accurate positions of the smaller off-diagonal peaks. In this work we propose a novel and efficient method to overcome the problems associated with the intense diagonal ridges discussed above, entitled: "Pure T2-T2 Exchange". This method is comprised of two associated measurements. The first consists of the introduction of an initial preparation followed by a very short mixing time, much shorter than the typical spin migration times at the beginning of the standard 2D T2-T2 sequence. As a result the 2D signal decay pattern, S, is identical to those obtained with the standard T2-T2 exchange method as shown in Figure 1a. The second experiment is performed with the same timings of the first one; however the short and long mixing times are swapped. In this second case, there is no time for the spins to migrate between different sites, resulting in a pure diagonal ridge, denominated reference signal S0, as can be seen in Figure 1b. Figurei shows the S and S0 T2-T2 Exchange distributions from water saturated Berea rock core, which was enclosed in a special sample holder to avoid water evaporation during the experiment. In order to remove the intense diagonal, an appropriate filter is set comparing S and S0 distributions, where the region enclosed by the intersection between them is chosen to select the values along the diagonal of the distribution S. Then a new S'0 decay is calculated and used to subtract the undesirable non-migrating spins information from >• + 192 1 • 1 "«ill M l.11 \I< \1\tiM- I K KI-KiAASfl. I SI RS MM the Exchange decay (S) over the time domain (As = S-S'0) and finally perform the inverse Laplace transform on As. gj Distributions Distribution 50 Figure 1: T2-T2 a) Exchange (S), and b) reference (So), patterns. The Pure T2-T2 Exchange pattern resulting from this procedure is shown in Figure 2, where non-diagonal exchange peaks can be observed very close to the diagonal, which were not clearly visible in the standard T2-T2 Exchange experiment. 10° 10"' 10 a 10u 101 lst Stage Relaxation Time (s) Figure 2: Pure T2-T2 Exchange pattern. The procedures for obtaining the best Pure Exchange are still under development and additional successful experiments are being carried out for several porous media. REFERENCES: 1. J.H. Lee, C. Labadie, C.S. Springer e G.S. Harbison, J.A.C. Society v. 115, 1993, 7761. 2. Y. Song, L. Zielinski e S. Ryu, Physical Review Letters v. 100, 2008, 20. 3. L. Venkataramanan e M.D. Hurlimann, IEEE T.S. Processing v.50, 2002, 1017. 4. K. Washburn e P. Callaghan, Physical Review Letters v.97, 2006, 25. CAPES, FAPESP, CNPq and IFSC-USP 193 h * PO 50 HIGH-PRESSURE NMR STUDIES ON THE PLASMODIUM FALCIPARUM THIOREDOXIN E. C. Azevedo*1, H. R. Kalbitzer2, C. E. Munte1'2 1 Physics Institute of São Carlos, University of São Paulo, Brazil 2Institute for Biophysics and Physical Biochemistry, University of Regensburg, Germany e-mail: [email protected] Keywords: Thioredoxin, High-pressure NMR, Plasmodium falciparum The malaria parasite Plasmodium falciparum spends part of its developmental life on human erythrocytes. When in the human cell, it is challenged with enhanced stress oxidative. The Plasmodium falciparum thioredoxin (P/Trx) is part of this effective regulatory redox system being a promising target for drugs development. It has already been isolated and sequenced thioredoxin to a variety of prokaryotcs and eukaryotes. The P/Trx has 104 residues with molecular weight around 12 KDa and active-site Cys-Gly-Pro-Cys. The 3D model structure protein reveals a (3-strand region formed by five strands and surrounded by four a-helices (Fig. 1). This structure gives the protein a very compact form and a very stable behavior. Figure 1: Reduced P/Trx showing the active-site cysteins. High-pressure NMR experiment is one of the most effective ways to evaluate local changes on the behavior of the protein. The aplication of high hydrostatic pressures increases the population in a excited state, so that the parcial molar volume difference can be spectroscopicaly detected and enable the determination of the structures, at least qualitatively [1,2]. The dependence between the chemical shift values and the pressure from the amide protons and nitrogen atoms can be analyzed as a second order Taylor expansion: ,2 6 (p, To) = õo (po, To) + B, (PO, T0) (p - p0) + B2 (p0, T0) (p - p0)' which õo is the chemical shift in a environment whose pressure is p0and temperature is T0. The first order coeficient (B^ gives information about the hydrogen bonds, i.e., if it is compressed or not. The information arises from the signal analysis: if the signal is positive, it generaly indicates that the hydrogen bond is more compressed than the model peptide. On the other hand, if the signal is negative, the hydrogen bond is less compressed. For the second order coeficient (B2), high values are related to a global conformation transition [1], The proton chemical shift need to be corrected [3,4], while no corrections are available for the 15N. 194 In this work, we are analyzing the two high pressure NMR data sets from the reduced and the oxidized P/Trx. Our aim is to find the similarities and differences in the pressure response of both proteins and correlate it to the conformational changes that occur after oxidation. Samples were isotopically enriched with 15N and produced in an identical manner. For both proteins a complete set of 1H-15N-HSQC were aquired in the pressure range of 30 to 2000 bar (intervals of 200 bar). All spectra were analyzed and the 1H chemical shift dependence with pressure leads to the 15 coefficients BT and B2 (Fig. 2). The N analysis is still in progress. Figure 2: Plots of the proton first (top) and the second (bottom) order coeficients for the reduced (red) and oxidyzed (blue) P/Trx. REFERENCES 1. Kremer, W.; Kachel, N.; Kuwata, K.; etal.. J. Biol. Chem. 2007, 31, 22689. 2. Foguel, D.; Suarez, M. C.; Ferrao-Gonzales, A. D.; et al.. Proc. Natl. Acad. Sci. USA 2003, 100, 9831. 3. Kremer, W.; Arnold, M. R.; Kachel, N.; Kalbitzer, H. R. Spectroscopy 2004, 18, 271. 4. Arnold, M.R.; Kremer, W.; Lüdemann, H. D.; Kalbitzer, H. R. Biophys. Chem. 2002, 96, 129. CNPq 195 h * PO 89 MAGNETIC SUSCEPTIBILITY CHARACTERIZATION OF SEDIMENTARY ROCK CORES BY NUCLEAR MAGNETIC RESONANCE A.A. Souza*1'2, L. Zielinski3, A. Boyd2, M.D. Hurlimann3, T.J. Bonagamba1 11instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil 2 Schlumberger Brazil Research and GeoEngineering Center, Rio de Janeiro, Brazil 3Schlumberger-Doll Research Center, Cambridge, United States of America e-mail:[email protected] Keywords: low-field NMR; magnetic susceptibility; rock core. The determination and correlation of the magnetic susceptibility effects in the nuclear magnetic relaxation time distributions contain valuable informations about the pore geometry in sedimentary rock cores.1 Many studies have been published on the subject, whose results have helped the oil exploration and production industry to optimize their methods and strategies, thereby reducing production costs and increasing the useful lifetime of their reservoirs. More specifically, the magnetic susceptibility difference (Ax) between the rock matrix and the fluid that fills the pore space, causes distortions in the local magnetic fields of the pores. Such effect scales with their distribution of pore sizes, matrix composition and geometries. The larger the pore, the smaller the distortion of the field; and the more complex the pore shapes, the greater the distortion effect. This property is of fundamental importance in the study of porous media by NMR, because any contrast in magnetic susceptibility will alter the relaxation time distjribution of nuclear magnetization, thereby producing a "fingerprint" of the porous media.1 1 Sun et al. studied the effect of magnetic susceptibility in T2 relaxation spectra for two samples of sandstone rocks. They developed a new pulse sequence, in which a distribution of internal gradients could be obtained, showing that the effect is pronounced for small pores. Wilson et al.2 studied the effect of internal gradients in the spectra of the diffusion coefficient (D) and two-dimensional diffusion-T2 relaxation. They have established the degree of interference in those measurements. The presence of induced internal gradients decreases the accuracy of pulsed magnetic gradients that encodes the diffusive space. Leu et al.3 examined the extention of this effect and its features in different NMR techniques, commonly used to measure D. Rocks with high susceptibility showed large deviations in the measured values. The work presented here has as a main objective to study and establish correlations between magnetic susceptibility and magnetic relaxation phenomena and diffusion by NMR in sedimentary rocks. These types of rocks are very similar to oil reservoir rocks, since both were formed by similar geological process. Two samples of cylindrical cores (1.5" x 1.5"), a Red Massillon Sandstone and an Indiana Limestone were studied. Both samples were saturated with KCI brine of 50,000 ppm concentration, under application of vacuum. The NMR equipments used were: an electro-magnet built in the laboratories of the IFSC-USP, with magnetic field (B0) of 0.05 T (resonance frequency of 2 MHz for 1H), operated by a Discovery (Tecmag Inc., USA) digital console; and a superconducting magnetic of 2 T (resonance frequency of 85 MHz for 1H) (Oxford, UK), also operated by the Discovery console. The technique employed was CPMG (Carr- Purcell-Gill-Meiboon) for T2 measurements. Assuming the so-called "fast-diffusion regime" for the 1H spins inside each pore, the CPMG decay will be multiexponential, reflecting in great aproximation all the pore sizes filled by the spins. The distribution of relaxation times was performed by applying the Inverse Laplace Transform, stabilized via the regularization method,4 using an inversion algorithm developed in Matlab environment by Schlumberger-Doll Research. Figure 1 shows the distributions of T2 obtained by applying the inverse Laplace transform in the CPMG decays measured in magnetic fields of 0.05 T (Figures A and K + 196 B) and 2 T (Figures C and D), acquired with several echo-times (TeCho): 0.4, 0.7 1,1.6, 2, 2.6, 3.2 and 4 ms, for Indiana Limestone (Figures A and C) and Red Massilon Sandstone (Figures B and D). ^ Indiana Limestone W Rod Massiion Sandstone Mult T K -> MHz (1) Figure 1: Multi-TeCho T2 spectra for several echo times CPMG experiments: (a) Indiana Limestone, (b) Red Massilon Sandstone, both at 2 MHz; (c) Indiana Limestone and (d) Red Massilon Sandstone, both at 85 MHz. The results show the presence and contribution of internal gradients induced by the magnetic field B0, in the T2 relaxation spectra. Figures 1A and B, the low-field results, show, as expected, a small displacement effect of T2 due to diffusion in internal gradients. This behaviour was confirmed for all echo times applied, confirming that spectra acquired in low magnetic fields reflects in great approximation the pore size distribution. Figures 1C and D show the results for B0 = 2 T, where it is possible to see a pronounced shift to lower T2 values with the increasing echo time. This effect indicates the role of internal field gradients, mediated by Brownian molecular diffusion, with consequent loss of information about the sizes of the pores. This effect is considerably pronounced for the Red Massilon sample, since sandstones generally present higher amounts of paramagnetic impurities in its matrix. Both samples present two clear peaks, and also an interesting observed feature shown in Figures 1C and D is the difference between the shifts of each peak. Each peak represents different sites inside the rocks, and the study of this difference should reveal details about the pore sites chemistry and geometry. For this purpose, quantification methods is being developed and will be confronted with imaging techniques, like Thin-Section analysis and X-Ray CT-Scan. Subsequently, two-dimensional NMR experiments TrT2 and D-T2, capable of investigating molecular dynamics details of the saturating fluids, differences in the chemistry of pore sites, and to measure the restricted diffusion due to collisions of the fluid molecules with the pore walls,4 will be performed and correlated with the magnetic susceptibility findings. REFERENCES: 1. Sun B.; Dunn K. Physical Review E. 2002, 051309. 2. Wilson R.C.; Hurlimann M.D. Journal of Magnetic Resonance. 2006, 183. 3. Leu G.; Fordham E.J.; Hurlimann M.D.; Frulla P. Magnetic Resonance Imaging. 2005, 305. 4. Sunn B.; Dunn K. Magnetic Resonance Imaging. 2005, 259. FAPESP, CNPq, CAPES, Schlumberger Ltda 197 h * PO 50 PROBING NATURAL POROUS MEDIA WITH 2D LOW FIELD NMR RELAXOMETRY AND DIFUSIOMETRY E.H. Rios*1, G.C. Stael1, R.B.V. Azeredo2 1 Geophysics Department, National Observatory, Rio de Janeiro, Brazil 2 Chemistry Institute, Fluminense Federal University, Niterói, Brazil e-mail: [email protected] Keywords: low field, petrophysics; 2D maps. The development of 2D low field NMR techniques that correlate relaxation times and diffusion, such as TrT2 and D-T2 maps, was impelled by the necessity of improving the interpretation of biphasic petrophysical systems (e.g. water-oil saturating porous rocks), where frequently it is observed a high level of superposition of each fluid signal in the common 1D spectra.1,2 However, as it will be shown, even with just one fluid, they can be very informative. In this study, 12 water saturated cylindrical reservoir rock samples (1.5" diameter x 1.8" length) were measured in a MARAN 2MHz benchtop NMR spectrometer (Oxford Instruments, UK). The 2D pulse sequences used in this work combine the CPMG (Carr-Purcell-Meiboom-Gill) sequence and the following techniques: InvRec (Inversion Recovery) for T^T^ Figure 1a; and PFG-STE (Pulsed 2,3 Field Gradient Stimulated-Echo) for D-T2, Figure 1b. The data was inverted with fast 2D inverse Laplace transformation, with Matlab routines developed by Schlumberger researchers.2 (a) 180% 90°;, 180°„ 180%.*, 180V». 180",. | InvRec (T.) | ~ CPMG (T,i (b) 90% 90%.,, 90%. 180%,, 180%.., 180%,, PFG-STE (D) | CPMG (T-J Figure 1: Pulse sequences for obtaining the 2D maps: TrT2 (a) and D-T2 (b). Figure 2a presents the D-T2 map for sample B-02 and its respective 1D projections. Two distinct domains can be clearly identified: the more intense, with T2 distribution centered on 450ms and diffusion coefficient distribution centered on the water theoretical D value, 2x10"9m2s"1, corresponds to the predominantly larger pores; the second domain, with shorter T2 (relaxation distribution centered on 70ms) and smaller D value, is related to small pores, revealing a signature of the translational diffusion movement restricted by the pore walls.1,3 Varying the diffusion time parameter A, different pore-size scales can be investigated, data not shown. From the restricted diffusion extension, it is possible to evaluate important petrophysical quantities, such as tortuosity and permeability.4 Figure 2b shows the TrT2 map for the same sample. It shows a narrow range for 7^2 ratio parameter, between 1 and 2, typical values for sandstones. When just one fluid saturates a porous media, the TI/T2 ratio depends on differences in the surface chemical composition1,2. This result suggests that, at least in the grains surface, this sample has a homogeneous composition. Although the majority samples 198 had this behavior, some individuals curiously presented TI/T2 ratios larger than 2, mainly for small pores, as showed in Figure 2c for sample B-06. This is not an expected result mainly because all the samples are from the same rock formation, with similar and homogeneous mineralogy. Lately, it Was informed by Petrobras (CENPES) that those samples were previously submitted tp wettability inversion tests, where the samples were coated with a chemical agent that, even with subsequent cleaning steps, cannot be completely removed. Therefore, this behavior can be attributed to the chemical agent residues that accumulate mostly on the small pores surface, modulating the relaxation times differently, according to the homogeneity and thickness of the layer. (a) B-02 D*= 2X10 N. restricted diffusion pseudo porestze . 2000 0 10' (C) B-06 100001 50001 T2(S) 10 10 10 10' 10 10' I 50'* 10" 10 10 Figure 2: 2D maps. D-T2 map (a), with its respective 1D projections, and TrT2 map (b) of a completely cleaned sample, B-02. An Irregular TrT2 map, sample B-06. These results show the application of the D-T2 and TrT2 maps even for a one saturating fluid rock system. While the first one gives information about restrict diffusion, that can be related with important petrophysical properties, the second one informs qualitatively about chemical heterogeneities, been also a useful instrument for quality control. REFERENCES: 1. Sun B.Q. and Dunn K.J. Magnetic Resonance Imaging. 2005, 23. 2. Song Y.-Q.; Hurlimann M.D., Flaum M., Frulla P. and Straley, C. Journal of Magnetic Resonance. 2002,154. 3. Dunn K.J.; Bergman D.J. and Latorraca G.A. Nuclear Magnetic Resonance Petrophysical and Logging Applications, 1° ed„ 2002, 293. 4. Sen P.N; Concepts in Magnetic Resonance Part A, 2004, 23A. CNPq, FINEP, PETROBRAS AND SCHLUMBERGER 199 y PO 91 PROBING THERMAL BEHAVIOUR OF CEFALEXIN MONOHYDRATE BY 13C SOLID STATE NMR Daniel L.M. Aguiar1, Rosane A.S. San Gil*1 1 Universidade Federal do Rio de Janeiro, Instituto de Quimica, Laboratório Multiusuário de RMN de Sólidos Profa. Adelina Costa Neto, Rio de Janeiro, Brazil e-mail:rsangil@iq. ufrj. br Keywords: Solid State NMR; Cephalexin monohydrate; DSC; solid thermal behaviour Cephalexin (Figure 1) is an oral first generation cephalosporin which is widely used in Gram positive infections. It is reported that cephalexin can show more than one crystalline arrangement, i.e., this drug has distinct polymorphic forms1. We can expect that since they have different crystal packings, they also present distinct solubilities and hence bioavailability.2 Just one polymorphic form is thermodynamically stable, therefore transitions between different meta-stable forms are possible, and these conversions depend on the temperature, pressure and relative humidity during storage.2,3 The stability relationship between crystalline solid phases can be classified as monotropic (irreversible) or enantiotropic (reversible)2,3 and is useful to predict the kinetic features of medicines. In this communication the solid state thermal behaviour of cephalexin monohydrate (Sigma Aldrich) was evaluated by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and 13C CPMAS NMR. H,NT 1I4 2' 13 NH ^ 0 O ^o Figure 1. Chemical structure of cephalexin 13C CPMAS (cross polarization magic angle spinning) spectra were recorded on a BRUKER AVANCE III 400 WB 9.4T (100.6 MHz to 13C) spectrometer, by using a 4 mm CPMAS probehead and Zr02 rotors spinning at 6.3 KHz at the magic angle. The spectra were obtained at temperatures in the range 298-425K. The contact time (optimized) was 2000 ps, di was 3s (up to 400K) and 1s (from 400 to 425K and down to 300K), after cooling to room temperature. HMB (CH3 at 17.3ppm) was used as external chemical shift reference and the 79Br chemical shifts was used to adjust the temperature according Thurber.4 The TGA curve (Figure 2, left) shows that the first mass loss (5.4%) was compatible with 1 mol of water and that around 190°C the decomposition starts. DSC curve (Figure 2, right) was carried out below the decomposition temperature with two heating cycles alternating with a cooling cycle. In the first heating cycle the cephalexin monohydrate showed one endothermic peak at 127°C that cannot be reproduced in the subsequent heating cycle. Figure 2. Thermal analyses of cephalexin mononyarate: TGA (left; ana DSC (right). J. * 200 Those data suggests that cephalexin monohydrate exhibit a monotropic polymorphic transition when submitted to heating process. 13C solid state NMR studies (Figure 3) were carried out according to DSC data, i.e., the spectra were acquired to follow the molecular modifications induced by heating. The spectrum obtained at ambient temperature showed the resonances readily assigned by comparison with solution spectrum, although there are some ambiguities in the aromatic and carbonyl region, where the great number of signals precludes making the assignments with confidence. The monohydrate dehydration in situ could blow the rotor cap and damage the probe. In order to avoid water evolution the amount of sample inside the rotor was reduced to the major sensivity5 area (50pl_) and surrounded by Zr02 (powder) as shown in Figure 3 (right). 300k t2? r . ' I. 426K ' ,, 152*C ' ' 400K . • " . ' ; (127*0) ii»*ft . t jf r, * v^fc^^^wt^^/^^y*'' '* " »'-.«,«. , 4 ifltot+t- 350 K err- o Hsgrxftr 1 C arorn 6 ? « 30GK (27 C) Figure 3. Variable temperature 1JC CPMAS solid state NMR spectra of cephalexin monohydrate (left); rotor sampling packing scheme is also shown (right) Solid state NMR data furnished information on the orientations of the benzyl ring and of the cephem group. At 350K part of aromatic signals coalesced, whereas the cephem group are practically motionless. At the transition temperature following DSC (400 K) all the signals are affected and exhibit very different molecular packing in comparison with the cephalexin monohydrate at 300K. Finally at 425K the polymorphic transition was complete (DSC) confirmed by the distinct resonances, compared with the first spectrum at 300K. At 400 and 425K the motional frequency moves from the site exchange regime to the dipolar broadening regime and we have a broadening of the linewidth. Finally after cooling the sample the spectrum obtained was not similar to that of original cephalexin monohydrate at 300 K, confirming that the polymorphic system under study had an irreversible (monotropic) transition. REFERENCES 1. STEPHENSON, G.A.; GROLEAU, E.G.; KLEEMANN, R.L.; XU, W.; RIGSBEE, D.R. J. Pharm. Sci. 1998, 8/(5), 536-542 2. SAIFEE, M. ;INAMDAR, N.; DHAMECHA, D.L.; RATHI.A.A. International Journal of Health Research. 2009, 2, 291-306 3. KATRINCIC, L.M.; SUN, Y.T.; CARLTON, R.A.DIEDERICH, A.M.; MULLER, R.L.; VOGT, F.G. Int. J. Pharm. 2009, 366, 1-13 4. THURBER, K.R.; TYCKO, R. J. Magn. Reson. 2008,196, 84-87 5. ZIARELLI, F.; VIEL, S.; SANCHEZ, S.; CROSS, D.; CALDARELLI, S. J. Magn. Reson. 2007,188, 260-266. CNPq 201 h * PO 92 APPLICATION OF NMR ANALYSIS TO THE STUDY OF ASPHALTENES Camila Pedroso Silveira1, Peter Rudolf Seidl2*, Fernanda Barbosa da Silva2, Fábio Henrique S. Rodrigues1, Ljubica Tasic1, Sônia Maria Cabral de Menezes3 and Maria José O.C. Guimarães2 11nstituto de Química, UNICAMP, Campinas, SP, 2 Escola de Química, Universidade Federal do Rio de Janeiro, 3 Cenpes, Petrobras, Rio de Janeiro, RJ, Brazil. * pseidl@eq. ufrj. br Keywords: Asphaltenes; 1H NMR; 13C NMR. Asphaltenes correspond to a class of aromatic compounds, which are the main constituents of the heaviest1 and most polar2 fraction of petroleum, and are defined as being soluble in toluene and insoluble in heptane1. They play an important role in determining the quality of a crude oil, so a high concentration of these compounds brings lower prices when it is marketed2,3. Asphaltene molecular structures have not been completely elucidated, so their respective contributions to many of the physical and chemical characteristics attributed to this class of compounds are still not well understood. However, it is known that the main structural features of asphaltenes are the hydrocarbon skeleton composed of aromatic and naphthenic cores and aliphatic chains4 and some heteroatoms which are usually part of the aromatic rings1"4. Asphaltenes have a high tendency to aggregate and form coke, and may represent one of the most serious problems in production and storage of crude oil1 .For these reasons the study of molecular structures of asphaltenes and their properties is of great interest, since it could enable the development of new exploration and/or oil processing techniques. Asphaltene samples were extracted from Brazilian crude oils and residues and fractionated by two different methods. The traditional procedure, IP143, uses n- heptane as precipitation solvent, however it is time-consuming and energetically unfavorable. Therefore an alternative method, based on solvent blends made up of mixtures of naphthenic and paraffinic compounds, was applied so as to verify if it is possible to substitute the IP143 method without losing information on its asphaltene composition. 1H Nuclear Magnetic Resonance (NMR), 13C NMR, elemental analysis and X-ray fluorescence data were collected for each asphaltene sample. Elemental analyses were acquired on a Perkin-Elmer 2400 Series II - CHNS/O Analyzer in CHN operating mode in order to quantify Carbon, Hydrogen and Nitrogen percentages. Sulfur and Oxygen percentages were obtained by difference as this was possible due to X-ray fluorescence data. NMR spectra were obtained in a Varian INOVA 500 MHz spectrometer equipped with 5 mm probes (bbsw) at 25°C (298K) and SpinWorks software was used to process the data. The samples were prepared using 15 mg of asphaltenes, 5 mg of chromium acetylacetonate (Cr(AcAc)3), used as relaxation 13 reagent, and 500 ^L of CDCI3. To obtain C data the decoupler was adjusted in the gated mode to avoid NOE in order to obtain quantitative information. DEPT spectra were also acquired to identify CHn groups. The relaxation delays were set to be 5 times longer than the longest ^ values of either carbon (13C NMR) or hydrogen signals (DEPT) and were 15 s and 2 s long, respectively. At least 36 h were required to complete the 13C NMR experiments. f 4- 202 IP-N Figure 1. DEPT spectra from BNP (green, upper panel) and IP-N samples (black, lower panel). Figure 1 illustrates typical DEPT spectra of the same crude oil sample (N) obtained by applying two different extraction methods, referred to IP-N for IP143 and BNP for blends. These spectra are remarkably similar, only differing in the intensities of certain absorptions. The 13C and DEPT NMR spectra are in good agreement with those of other asphaltenes5, exhibiting signals in the 115 to 132 ppm spectral region, typical for aromatic and heteroaromatic carbons. The DEPT spectra showed several absorptions in the 22 to 32 ppm region, reflecting a high proportion of secondary carbons, and at 18 ppm indicating primary carbons in long chains. Intense signals in the 30 ppm region, which are characteristic of aliphatic secondary carbons in long chains with more than 5 carbons, were also present. Thus, by NMR, we were able to show that asphaltenes from the same sample, but extracted by two distinct procedures contain the same functional groups in different proportions. The 13C NMR spectra, in particular, reveal that the new extraction procedure could be applied in fractionation of petroleum and vacuum residues. Our study provided important data on asphaltene structures enabling the construction of 3D models that can simulate properties of interest and will be used to refine our work on solvent effects5. REFERENCES 1. Avid, B.; Sato, S.; Takanohashi, T.; Saito, I.; Energy & Fuels, 2004, 18, 1792-1797. 2. Speight, J. G.; Oil & Gas Science and Technology- REV.IFP 2004, 59(5), 467-477. 3. Yen, T. F.; Chilingarian, G. V.; Asphaltenes and Asphalts. Vol. 2 Developments in Petroleum Science, 2009. 4. Artok, L.; Su, Y.; Hirose, Y.; Hosokawa, M.; Murata, S.; Nomura, M.; Energy & Fuels 1999, 13, 287-296. 5. Carauta, A.; Seidl, P.; Chrisman, E.; Correia, J.; Menechinin, P.; Silva, D.; Leal, K.; Menezes, S.; Souza, W.;Teixeira, M.; Energy & Fuels 2005, 19, 1245-1251. PETROBRAS, CNPq, NALCO 203 h * PO 52 CHARACTERIZATION OF HDT CATALYSTS USING 170 AND 27AI MAS NMR TECHNIQUES H.R.X. Pimentel1'2, R.A.S. San Gil*1, S.M.C. Menezes3, S.S.X. Chiaro3 1 Universidade Federal do Rio de Janeiro, Instituto de Quimica, Lab. Multiusuário de RMN de Sólidos Profa. Adelina Costa Neto, Rio de Janeiro, Brazil 2 Universidade Federal Fluminense, Niterói, Brazil 3CENPES/PETROBRAS, Rio de Janeiro, Brazil e-mail:[email protected] Keywords: HDT catalysts, 170 MAS NMR, 27AI MAS NMR Alumina-supported NiMo, CoMo and NiW catalysts have been widely used in the oil refining industry. Combinations of Ni (or Co) and Mo (or W) in hydrodesulfurization (HDS) catalysts have been more active than Mo or W alone. Mo and W are generally described as the active components and Co and Ni as the promoters.1 The effect of metal-support interactions on the catalyst activity is a matter of extensive research. In this communication preliminary results using 170 solid state NMR are presented in the first time in the literature. The objective of this research is to address possible interactions between the support and the metallic phases, through the use of MAS NMR as a tool, observing 27AI and 170 sites. Samples of synthesized (from Pural SB y-AI203) named B1 Ni, B1Mo, BINiMo and BINiMoP resp., and commercial catalysts named ComlNiMoP, Com2NiMoP, Com3NiMo and Com4NiMoP were provided by Cenpes/PETROBRAS. Those materials were enriched by exchanging with 17 2 27 H2 0 40%, following the procedure described by Walter et al. The analyses of AI MAS NMR were performed by using a Bruker Avance DRX 300 spectrometer (7.05 T), at 78.2 MHz, 4mm zirconia rotors, rotation of 10 kHz and one pulse (Bloch decay) pulse sequence. The interval between pulses was 0.5 s, and 1024 scans were +3 acquired. Pulse of 1 ps (71/6), spectral width of 93.9 kHz and [AI(H20)6] to 0 ppm was used as reference. 170 MAS NMR spectra were performed by using a Bruker AVANCE III 400 WB spectrometer (9.5 T), at 54.2 MHz, with 4 mm probe and zirconia rotors. Rotation of 10 kHz, interval between pulses 10 s and number of scans of 50 k were employed. Spectral width of 50 kHz was used for one pulse spectrum (sample B1), whereas for the other samples Hahnecho acquisitions (P1-X-P2) with P1 of 1 ps, rotation at 12.5 KHz, interval between pulses of 1s, 60 k scans and spectral width of 17 250 KHz were used. The reference was the standard solution of H2 0 10% at 0 ppm. Figure 1 shows the 27AI MAS spectra of synthesized (B1 series) and commercial (Com series) samples resp. The positions of the maxima are listed in Tables 1 and 2. BlltoNP CCUMP vm* Jy OQMMto j\ ! JV BIN! ,~JV Figure 1.27AI MAS NMR of the B1 series (left) and Commercial series (right). All samples presented signals with maxima in the region of AIIV (-60 ppm) and AIVI (~0 ppm). A slight reduction in the amount of tetrahedral sites was observed in the B1 series, suggesting that part of this site on the alumina surface was coordinated with f, 4 204 the impregnated metal after calcination. A signal with maxima at 41 ppm was detected in commercial sample ComlNiMoP, but the low concentration precludes the confirmation of its nature (AIIV or Alv) by MQMAS experiment. Figure 2 shows the 170 MAS NMR spectra obtained for the two series of samples. Distinct lineshapes were observed, depending on the impregnated and calcined metal precursor present. Table 1- Chemical shifts and percentage of the sites of the B1 series "Sample 8, ppm Al'v(%) (5) ppm AIVI (%) B1 62.0 23.2 4.7 76.8 B1 Ni 62.9 20.0 5.1 80.0 B1Mo 63.0 19.7 5.4 80.3 BINiMo 60.8 21.0 5.3 79.0 BiNiMoP 62.1 19.6 1.3 80.4 Table 2- Chemical shifts and percentage of sites of the commercial series Sample 5, ppm Al'v(%) (5) ppm Al'v/v(%) (S) ppm AIVI (%) ComlNiMoP 57.1 20.3 41.1 - 2.5 79.7 Com2NiMoP 66.6 21.9 - - 0.8 78.1 Com3NiMo 63.1 21.3 - - 3.6 78.7 Com4NiMoP 63.7 18.6 - - 0.7 81.4 Figure 2.170 MAS NMR spectra of the B1 series (left) and Commercial series (right). Based on the literature data3'4,5 it was possible to suggest the presence of O-Mo and also of Mo-O-Mo sites at 273 ppm and 376.1 ppm in BINiMoP and Com3NiMo samples. Besides that, the AI-0 sites (10 to 100 ppm) in metal supported samples with distinct lineshapes compared with the alumina precursor (B1 series) is a strong indication that metal-support interactions are present in the materials under study. REFERENCES 1. Hong,S.T.; Park, D. R.; Yoo, S.J.; Kim, J.D.; H. S. Park; Res. Chem. Intermed., 2006,32(9), 857-870. 2. Walter,T.H.; Oldfield, E.; J. Phys. Chem., 1989,93,6744-6751. 3. Klimov,O.V.;Pashigreva, A.V; Bukhtiyarova, G.A.;Budukva,S.V.;Fedotov,M.A.;Kochube, D.I.; Chesalov, Y. A.; Zaikosvskii, V.I.; NosKov, A. S.; Catai Today, 2010,750,196-206. 4. Gerothanassis, I. P.; Prog. Nucl. Mag. Res. Sp., 2010, 56, 95-197. 5. Maksimovskaya.R.I.; Maksimov.G.M.; Inorg. Chem.,2007,46, 3688-3 205 h * PO 48 A METHOD TO DETERMINE THE FRACTION OF COMPONENTS IN HETEROGENEOUS SAMPLES BY TIME-DOMAIN NUCLEAR MAGNETIC RESONANCE L.M.C. Cerioni1'2, T.M. Osan1'2, M. Medina1, G. Albert1, D.J. Pusiol*1'2 1 Spinlock S.R.L., Av. Sabattini 5337, X5020DVD, Córdoba, Argentina 2CONICET (Argentinian National Research Council) e-mail: [email protected] Keywords: Food, Time Domain Magnetic Resonance, Heterogeneous Molecular Systems. It is often necessary to quantitatively determine the fraction of components in complex systems containing different kinds of chemical substances which give origin to different contributions to the NMR signal. In particular, determining the oil and water content by means of a low-resolution NMR pulse spectrometer with a magnetic field strength up to about 1 Tesla and an average homogeneity of about 500 ppm over the entire sample volume, and thus a proton resonant frequency of less than about 50 MHz, is made more difficult in products or samples containing water and oil with a relatively high water content due to the superposition of water signal and oil signal, since the contribution of the water exceeds that of the oil precluding an accurate determination of the oil content. Determination of oil and water content is possible in a simple way only in high-resolution NMR spectrometers which are generally much expensive for this kind of applications than low-resolution NMR spectrometers, complicating their use for quality control chemistry in the industry. Õne way to obtain acceptable results for the oil content of samples with a water content of more than about 12% by means of low-resolution NMR consists in pre-drying the samples before taking the NMR measurements in order to get rid or at least to reduce the disturbing water component. However, the disadvantages of this method are that is more or less labor-intensive, only allows for the determination by NMR of the oil (or fat) content and the measurement process requires too much time which is unacceptable when a large number of measurements are required. Thus, as in many applications is necessary to determine the oil as well as the water contents of a sample in very short measurement times, the predrying method turns out to be of scarce practical applicability.1 The International Standard ISO 10565 specifies a method for the determination of the oil and water contents of commercial oil seeds using pulsed nuclear magnetic resonance. It is applicable to rapeseeds, soya beans, linseeds and sunflower seeds but it is required the seeds to have a water content less than 10%. For seeds with higher water contents, drying is necessary before the oil content can be determined by pulsed NMR and therefore this method is of scarce practical applicability in industry.2 The aim of this work was to develop a method for determining the fraction of components in heterogeneous samples with the aid of Time Domain Nuclear Magnetic Resonance (TD-NMR). The method comprises two procedures, the first one is used to calibrate a Low Resolution TD-NMR spectrometer and the second one is used to determine the fraction of components, particulary water and oil, in heterogeneous samples, without pre-drying the samples. The proposed method can be used to determine the fraction of components in systems where at least two of the components of interest have longitudinal-relaxation times (Tj) profiles having no overlap. It is based on the use of inversion-recovery type pulse sequences, choosing proper time intervals between pulses in order to cancel out the signal of the component with larger mean value of T}.3 Calibration and measurement processes are performed by a combination of spin- echo and inversion recovery pulse sequences, and require the measurement of 7? recovery curves. In order to obtain practical measurement times, the proposed method + 206 makes use of fast Ti measurement procedures.4 The fraction of components is calculated from the NMR signals originated by the different pulse sequences taking into account corrections due to relaxation processes. A typical case where the method can be applied is olive pastes. As an example, in Figure 1 are showed 7"? profiles for an olive paste. In red is showed the profile of the sample before a drying process and, in blue, after this process. In the red Ti profile, the peak with a larger mean T1 value is associated to the water component. The blue T1 profile is associated to the oil component. Figures 2 and 3 show typical calibration curves for water and oil contents. The method proposed in this work allowed for the determination of fractions of oil and water in olive pastes with errors within 2.6 % for water and 0.4 % for oil. T, (ms) Figure 1: Longitudinal relaxation profiles (7?) of a olive paste before and after drying 5 6 5.8 NMR signal amplitude (a.u.) NMR signal amplitude (a.u) Figure 2: Calibration curve for water Figure 3: Calibration curve for oil REFERENCES: 1. G. Rubel, Journal of the American Oil Chemists Society, 1994, 71 {10), 1057. 2. International Standard ISO 10565, "Oilseeds - Simultaneous determination of oil and water contents - Method using pulsed nuclear magnetic resonance spectroscopy" (1998-08-15) 3. Bernstein, M. et al\ "Handbook of MRI pulse sequences", Elsevier-Academic Press, 2004, 625. 4. Sigmund, E. E. et at, Sol. State Nuc. Mag. Reson. 2006, 29, 232. CONICET, FONTAR, SPINLOCK 207 h * PO 50 NUCLEAR MAGNETIC RESONANCE (NMR) STUDY OF NECROSIS AND ETHYLENE-INDUCING PROTEIN 2 (NEP2) E.G. Pereira*1, G.A.P. de Oliveira1, A.P. Valente1, V.S. de Paula1, J.L. da Silva1, F.C.L. Almeida1, J.C.M. Cascardo2, C.V. Dias2. 11nstituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 Bloco K, 21941-902, Rio de Janeiro, Brasil 2 Departamento de Biologia, Universidade Federal de Santa Cruz, Rodovia Ilhéus Itabuna, Km 16, 45662-900 - Bahia, Brasil email:elenqp(p).bioqmed. ufrj. br Keywords: NEP2, NMR, CSI Witches' broom disease (WBD) of cacao (Theobroma cacao) is one of the most important phytopathological problems to afflict the Southern Hemisphere in recent decades.1 In Brazil, the disease is endemic in the Amazon region, and in 1989 was introduced into southern Bahia, the largest area of cacao production in the country.2 The gene enconding necrosis and ethylene-inducing proteins (NEPs) are apparently located at the same chromosome and the study of M. perniciosa genome led to identification of similar NEPs: NEP1 and NEP2. Those proteins are involved in crucial steps in the plant disease and may be a target for drug design4. In this work, we assigned the resonances of NEP2, a 22 kDa protein, using triple resonance strategy with a 2H, 13C and 15N sample. Then we compared the NEP2 secondary structure by chemical shift index (CSI) with Nep1-like protein (NLP) from the phytopathogenic oomycete Pythium aphanidermatum with 1.35 Á. The analysis of CSI indicates that its secondary structure is majority in beta-sheet with two alpha-helix regions in the N- and C- terminals, in agreement with its homologous NLP deposited crystal structure. In addition, we observed that around 22 residues that we did not found assignments are localized in loop regions. We are now analysing the observed NOEs and we will pursue the structure calculation. With the information about NEP2 structure and dynamics, we hope to understand its membrane interaction process and therefore its pathogenesis. REFERENCES 1. Griffith G.W. and Nurnberger, T., Phytochemistry, 2003, 67. 2. Pereira J.L., deAlmeida L.C.C., Santos S.M., Crop Protection, 1996, 15. 3. Garcia O., Macedo J.A.N., Tibúrcio R., Zaparoli G., Rincones J., Bittencourt L.M.C., Ceita G.O., Micheli F., Gesteira A., Mariano A.C., Schiavinato M.A., Medrano F.J., Meinhardt L.W., Pereira G.A.G. and Cascardo J.C.M., Mycological Research III, 2007. 4. Ottmann C., Luberacki B., Küfner I., Koch W., Brunner F., Weyand M., Mattinen L., Pirhonen M., Anderluh G., Seitz H.U., NürnbergerT. and Oecking C., PNAS, 2009, 106. CAPES, CNPq 208