Synthesis and Characterization of Some Oxovanadium(IV) Complexes Involving

Indi an Journal of Chemistry Vol. 41A, January 2002, pp. 98-142

Posters

Oxovanadium(IV) complexes of bioinorganic relevance: Synthesis and characterization of some oxovanadium(IV) complexes involving schiff bases derived from biologically active 4-benzoyl-3-methyl-l-phenyl-2-pyrazolin-5-one and certain aromatic amines

R C Mauryu*, H Singh, T Singh & A K Singh Coordination Chcmistry Laboratory, Dcpartlllent of PG Studics and Research in ChcmiSll"y. RD Univcrsity, Jabalpur 482 001 (MP). India

Recognition of vanadium as an essential trace element found in living organisms i has generated increasing interest in the structure and function of its complexes. Tunicates accumulate vanadi um(V) from the marine environment and store it in their blood cells in the reduced tetra- and tri-valent states. The mushroom 2 accumulates vanadium to produce amavadin . Spectroscopic investigati ons have shown a distinct preference of 3 this metal center for N-and/or O-coordinated environments . There is increasing interest in the biological chemistry of vanadium due to the discovery of two types of vanadium dependent enzymes: vanadium nitrogenase and vanadium bromoperoxidase. Although in recent years a number of vanadium complexes have been synthesized and characterized as models of amavadin and vanadium dependent metalloenzymes, the biological role of vanadium in li ving organisms and also in both reductive and oxidative catalytic transformations is still debatable, and not c1 ear3. Since the seventies, vanadium has been associated with insulin and its role in the body. The recently reported4 and structurally documented bis(maltolato)oxovanadium(IV) (BMOV) has been found to be effective to regulate glucose levels in the plasma in di abetic rats with no significant toxic effects on hepatic and kidney functions. In view of the above, the spectral and magnetic behaviour of some oxovanadium(lV) complexes in N,O and N20 2- type schiff base environments have been studied. The schiff bases were prepared by the condensation of 4-benzoyl, -3-methyl-l-phenyl-2-pyrazolin-5-one with certain aromatic amines, viz, p-anisidine, /11- phenitidine, p-toluidine, a-phenylenediamine, m-phenylenediamine, p-phenylenediamine or benzidine. The bi and tetra-dentate schiff bases were reacted with vandal sulphate in 2: 1 and 1: 1 mole ratio to obtain mononuclear and ligand bridged binuclear complexes. All the complexes have been characterized by analytical data, molar conductance and magnetic measurements, IR, UV -vis and EPR spectral studies.

References I Rehder D, Angew Chern Jnt Ed Engl, 30 (1991) 148; Butler A & Walker J Y, Chern Rev, 93 (1993) 1937. 2 Kneifel H & Bayer E, Angew Chern lnt Ed Engl, 12 (1973) 508. 3 Dai J, Wang H & Mikuria M, Polyhedron, 15 (1996) 1801 and references cited therein. 4 Burgess J, De Castro B, Oliveira C, Ramgel M & Schlidwein W, Polyhedron, 16 (1997) 789 and references cited therein.

A model study for vanadium protein interactions: Synthesis and characterization of oxovanadium (IV and V) complexes with L-carnosine

Asit R Sarkar* & Shipra Mandai Department of Chemistry, University of Kalyani, Kalyani 741 235, WB, India

The binding of a metal ion by a protein may principally involve ionisable side chains and the -NHCO group of the peptide chain backbone'. The axial coordination of histidine to the vanadium center in haloperoxidases has increased the interest in the interactions of vanadium with ligands containing the imidazole moietl. Most of the POSTERS 99 reported investigations in this field are on mixed ligand vanadium (IV and V) complexes with amino acids or its adducts in solution3 The reports on the interactions of vanadium with peptides containing the imidazole moiety 4 in the solid state are scanty. Till date there is no report on the interaction of vanadium (IV and V) with the ligand L-carnosine ( ~-al a nyl-L-hi st idin e), which itself is a ligand of biochemical importance5 In the present work, potential metal binding sites of L-carnosine wi th respect to vanadium have been investigated. Two novel oxovanadium complexes containing dideprotonated L-carn osine anion were isolated havin g the composition

NH4[V02L]. 2H20 and VOL. 2H20, where H2L =L-carno sin e. The infrared spectra of th e complexes indicate th at the dideprotonated L-carnosin e coordin ates to va nadium in both the complexes through the three donor atoms, namely the oxygen of th e carboxylate group, two nitrogens one each from the primary amine and the other from the amide group of the ligand. The pattern of shi ft of the I3C NMR signal for the C(2), C(4) and C(S) 6 signals for the complex NH4[V02L]. 2H]O, with respect to the free li gand indicates that the imidazole moiety of L-carnosine has, undergone tautomeric change from N(3)-H to N(l) - H. The nitrogen donor atom of the imidazole moiety coordinates to vanadium through N(3). The EPR spectrum of the complex VOL. 2H20, ex hibits eight resonance li nes in accordance with the presence of vanadiu m(IV) species in the complex, whi ch is consistent with the observed magnetic moment (l 74 BM). The cyclic voltammogram of th e complex shows the presence of a reversible redox couple involvi ng one electron transfer.

References I Cams DC. Holst H, Keramidas A D & Rehder D, In org Chelll. 34 ( 1995) 2524. 2 Mukherjee J, Gangul y S & Bhattaeharjee M, Indian J Ch el1l, 35A (1996) 47 1. 3 Elvin gson K, Keramidas A D, Crans D C & Pelt ersson L, Inorg Ch ell7 , 37 (1 998) 6153. 4 Keramidas A, Miller S M, Anderson 0 P & Crans DC, J AI1I chelll Soc, 11 9 (1997) 8901. 5 Kochen R, Yamamoto Y, Cundy K C & Ames B N, Proc Natl Acad Sci, USA, 85 (1988) 3 175. 6 Sarkar A R & Sarkar M, J chem. Research (5), (1997) 304.

Chromium(III) induced long range ordering of collagen: A step in exploring a unified theory for tanning

Raghuraman Gayatri, Rama R ajaram & Thirumalachari Ramasami* Central Leather Research Institute, Adyar, Chennai 600020, India

Stability against enzymatic degradation of collagen and dimensional changes of skin forms the basic principle of tanning. A mixture of Il-hydroxy species bearing dimeric, trimeric, tetrameric and higher oligomeric structures of chromium(III) is used in commercial tanning process. We have recently demonstrated that the different chromium(III) complexes, viz, [(H20)4Cr(OHhCr(H20)4t+ 1 (dimer), [Cr3(OHMH20)9]5+ 2 (trimer) and [Cr4(OHMOhCH20)u]4+ 3 (tetramer) present in commercial tanning salts are capable of inhibiting collagenase activity employing different mechanisms. An explanation for the stability of chrome tanned rat tail tendon (RTT) collagen against enzymatic attach has been offered. In the present work, an attempt has been made to investigate the ultra- and micro-structural changes in collagen induced by different chromium(III) complexes of relevance to tanning by employing circular dichroism (CD) and atomic force microscopy (AFM) techniques. Significant changes in CD spectra are observed when collagen solution is treated with dimer, 1 and trimer, 2. On the other hand, the interaction of collagen with tetramer, 3 does not influence the CD spectra of the protein sample. The influence of the secondary structural changes on the quaternary structure of the protein has been addressed using AFM technique. The studies reveal changes in the D-periodicity of RTT fibre treated with different species of chromium(III). Differences in topological distribution as well as the nature of reaction of various chromium(III) complexes with collagen may be responsible for such observations. Studies with monomeric collagen in solution provide convincing evidence for the chromium induced self-assembly of the protein into supramolecular aggregates with an axial repeat of 64±2 nm in the case of dimer, 1 and 72±1 in the case of trimer, 2. The studies indicate that a unified theory of tanning may include the level of conformational changes and induced long range ordering in the protein, collagen. 100 INDIAN J CHEM, SEC. A, JANUARY 2002

Influence of ligand environment in the induction of apoptosis by Cr(Ill) compounds

K Balamurugan, Rama Rajaram & T Ramasami* Chemical Laboratory, Central Leather Research Institute, Chennai 600020, India

The redox state and the speciation of chromium involved in generating mutagenicity and carcinogenicity in living systems have attracted wide interest. The biotoxicity of chromium is in focus; but the underlying mechanisms for the observed toxicity is not well-understood. In one of our earlier studies, we have demonstrated the influence of Cr(lll) to cause apoptosis in human lymphocytes and afforded the species specific explanation. In the present study the influence of trisphenanthrolinechromium(III) 1, trisbipyridinechromium(III) 2 and aquapentamminechromium(III) 3, has been evaluated, for their effect on cell culture systems involving human peripheral blood lymphocytes. Complexes 1 and 2 bring about inhibition of DNA synthesis as assessed by eH] thymidine incorporation and decrease in viability of cells in a dose-dependent manner (5-50 MM). It is found that apoptosis is brought about by these Cr(lll) complexes as verified through DNA fragmentation and by morphological studies. Complex 3 is inefficient in inducing apoptosis. The complexes 1 and 2 can, in principle, participate in redox processes generating radical species, which can induce mutagenicity of Salmonella typhymurium. Whether redox pathways are responsible for switching on the genes up-regUlating apoptosis is being addressed in this work. Studies to experimentally investigate the formation of oxygen radicals in vivo in cell culture systems are being carried out.

The interaction of chromium(III) schiff base complexes with DNA

R Vijayalakshmi, M Kanthimathi, V Subramanian & Balachandran Unni Nair* Chemical Laboratory, Central Leather Research Institute, Chennai 600020, India

The binding of schiff base complexes of chromium(III) of the type [Cr(salen)(H20)2t and [Cr(salpm)(H20ht [where salen denotes 1,2 bis(salicylideneamino)ethane and salpm denotes 1,3 bis(salicylideneamino)propane] to calf thymus DNA has been investigated by absorption and emission spectra, circular dichroism, melting temperature and viscosity measurements. These chromium(III) complexes show absorption hyperchromicity accompanied by red shift in charge transfer band, fluorescence enhancement, increase in melting temperature, some structural changes in CD spectra and changes in specific viscosity when bound to calf thymus DNA. The binding constant Kb has been determined from absorption measurements for both the complexes and found to be (2.5±0.4)x103 Ml for [Cr(salen)(H20ht and (1.7±0.3)x104 Ml for [Cr(salpm)(H20ht. From the binding stoichiometry of DNA - [Cr(salpm)(H20ht, the number of binding site size has been determined and found to be ten base pairs per bound complex molecule. The chromium(III) complexes also bring about single strand cleavage in plasmid DNA. The experimental results show that the chromium(III) complexes bind to DNA by non-intercalative mode. Major groove binding is the preferred mode of interaction for these schiff base complexes of chromium(III).

Synthesis of manganese complexes with different bidentate ligands

B Adhikary*', K Mitra', S Biswas', C R Lucasb & S K Chattopadhyay" 'Department of Chemistry, Bengal Engineering College (Deemed University), Botanic Garden, Howrah 711 103, WB, India bDepartment of Chemistry, Memorial University of Newfoundland St John's, Newfoundland, AlB 3X7, Canada

There has been considerable interest in the chemistry of manganese complexes largely as a result of the l 3 importance of various manganese species in biological systems . • We have synthesized a few manganese POSTERS 101

, ' ,\ .' '

.~ ,t' /' ...... "'\ i' " -

, .:. :' \1 \/ !' .J- ___ I ,. '/ \ \ I \. .,,1. I '" I .... , . , . \ 1 : '

ORTEP diagrams of Mn(NeLhCNCSh (Above) and Mn(CILhCNCSh (Below). complexes of different N - N* donors like Mn (N-N*) X2(X =N3, SCN, N-N* = N - aryl pyridine 2-aldimine) and Mn (N - N*) X2 (X = bpy and phen), Crystal structure determination of two of the complexes, Mn (CILh (NCS)2 and Mn (MeLh (NCS)2 (CIL=N-(4-chloro)pyridine 2-aldimine and MeL=N-(4-methyl)pyridine 2-aldimine) shows them to have trans (pyridine), cis, cis geometry with two cryatallographically distinct units in each of them, Electron transfer of these complexes has been studied by cyclic voltammetry and other electrochemical 4 techniques. Chemical oxidation of some of these complexes using various oxidizing agents like Ce+ and H20 2 has been studied.

References 1 Weighardt K, Angew Chern Int Ed Engl, 28 (1989) 1153. 2 Pecoraro V L, Baldwin M J & Gelasco A, Chern Rev, 94 (1994) 807. 3 Kaneko M, Chern Rev, 10 1 (2001) 21.

Synthesis of two novel benzimidazole derivatives mediated by MnCIII)

b b Shyamal K Chattopadhyay", Kamala Mitra", Collin R Lucas , David a Miller & Bibhutosh Adhikari*" "Department of Chemistry, Bengal Engineering College (Deemed University), Howrah 711 103, WB , India bDepartment of Chemistry, Memorial University of Newfoundland St John's, Newfoundland, AlB 3X7, Canada

Formation of carbon-carbon and carbon-nitrogen bonds mediated by transition metal complexes is now a well studied field. However, efficient methods for carbon-nitrogen bond formation, possibly through the intermediary l 2 of transition metal complexes, are less developed, though much sought-after processes . of importance in both 3 biological and medicinal chemistry. In this paper, we describe Mn(III) mediated -4 C-N bond formation in the synthesis of two novel benzimidazole derivatives, 1-(4-tolyl) 2-(2-pyridyl) 5-(aminophenyl) 6-(methyl) benzimidazole (2a) and 1-(4-chlorophenyl) 2-(2-pyridyl) 5-(aminophenyl) 6-(chloro) benzimidazole (2b) starting from simple schiff bases 1a and lb. Structure of the perchlorate salt of 2a, i.e, (2aH) (CI04) has been determined by X -ray crystallography.

Mn(OAc)2 + KMn04 + HOAc + 1 ~ 2 + other products ... (1) 102 INDIAN J CHEM, SEC. A, JANUARY 2002

l'vln(OAc~ ~¢ N :-U- X Nfl ¢ J(

2a)X=Mc 1a X=Me 5 21>, X=CI til, X=Q

ORTEP diagram of (2aH)(CI04 )

Acknowledgement: SKC and BA thank UGC, CSIR and AICTE for financial helps.

References I Hart wig J F, Angel'v Chelll /nl Ed Eng, 37 ( 1998) 2046. 2 Saha A, Ghosh A K, Majumdar P, Mitra K N, Mondal S, Rajak K K, Falvello L R & Goswamy S, Organomelallics, 18 ( 1998) 3772. 3 Sinder B B, Chelll Rev, 96 (1996) 339, 4 Melikyan G G, in Organic reaclions Vol. 49, edited by L A Paquette, (John Wiley, New York) 1997, pp 427,

Reactivity of binuclear manganese complexes with dioxygen

Urmila Pal Chaudhuri & Narasimha N Murthy* Department of Chemistry, Indian Institute of Technology, Madras 600 036, India

The redox-active class of Mn-enzymes process reduces dioxygen at their active-site as part of their biological function. These include catalase which disproportionates hydrogen peroxide to less toxic dioxygen (2H20 1 --7 O2

+ 2H20) and photosynthetic water oxidase which oxidizes two molecules of water to dioxygen (2H20 --7 O2 + 4e' + 4H+) during photosynthesis. The active-sites of these enzymes are believed to contain binuclear or tetran uclear manganese centers. During their function, various manganese-(di)oxygen intermediates have been proposed to l form. The exact nature and properti es of these intermediates are still unclear . Hence, study of model manganese 2 complexes, to gain insight into their active-site structure and function, is of immense value . We have designed and synthesized a variety of binuclear Mn(II) complexes containing various ligands with o and N donor atoms and explored their reactivity with O2. Among them, complexes ([Mn2(Ll-0-hf+ (1), (Ll-O- = 2-(2-pyridylethy)amino-phenolate) and ([Mn2(L2-0'hf+ (2), (L2-0- = 2-[bis(2-pyridylethy)amino phenolate] show contrasting reactivity. The results of these investigations are discussed.

References Li ang W, Roelots T R, Cinco R M, Rompel A, Latimer M J, Yu W, Sauer K, Klein M P & Yachandra V K, JAm chem Soc, 122 (2000) 3399. 2 Limburg J, Vrettos J S, Liable-Sands L M, Rheingold A L, Crabtree R H & Brudvig, G W et ai. Science, 253 (1999) 1524, POSTERS 103

Synthesis and characterization of dinuclear Mn(H) tri(J.!-carboxylato) complexes p with the hindered hydrotris(3,5-diisopropyl-l-pyrazolyl)borate (=Tpi r2) ligand

Udai P Singh*t, Rajender Singht, Shiro Hikichi* & Yoshihiko Moro-oka* tDepartment of Chemistry, University of Roorkee, Roorkee 247 667, India tResearch Laboratory of Resources Utilization, Tokyo Institute of Technology 4259 Nagatsuta. Midori-ku. Yokohama 226-8503. Japan

Coordination chemistry of metal-carboxylato species is an interesting subject in bioinorganic chemistry stand point, because the carboxylate group of glutamate and aspartate works as supporting ligand for the metal centers in various metalloproteins I . The carboxy late groups are also thought to play an important role in structural bonding and protein transfer via hydrogen-bonding interaction in proteins2. Here we report the synthesis and characterization of unsymmetrical dimanganese(ll) complexes with a structurally unique (/J.-carboxylatoh unit. A dimanganese(lI) complex, TpiPr2Mn-(/J.-Obzh-Mn(Pzh (pz =3,5-diisopropylpyrazole) has been synthesized from a mixture of Mn(CI)(Pz)Tpipr2 and sodium benzoate. X-ray analysis establishes the unsymmetric coordination environment of each manganese ion with a structurally unique bridging unit consisting of three carboxylate groups. The reaction of di(/J.-hydroxo) dimanganese (II, II) complex with benzoic acid results in the formation of intramolecular hydrogen bonded Mn tri(/J.-carboxylato) complex, TpiPr2Mn-(/J.-Obzh-Mn(TpiPr2H). X-ray crystallography reveals the unsymmetrical coordination environments for manganese centers. One of the two TpiPr2 ligands, which is bound to five-coordinated Mn center, is protonated by the action of the third carboxylic acid, and the resulting non-Mn-binding N-H moiety forms intramolecular hydrogen bond with the oxygen donor of carboxylate ligand. In this complex, N61 acts as a proton-acceptor during the third carboxylate-bridge formation. Such ligand exchange process is suggested to be involved in some enzymatic reactions, e.g, binding of protic substrate. Steric congestion in the bimetallic core results in the large separation of the manganese centers bridged by the syn-anti carboxylate ligand.

References 1 Lippard S J & Berg J M, Principles of bioinorganic chemistry, (University Science Books, California, USA) 1994. 2 Holm R H, Kennepohl P & Solomon E I, Chem Rev, 96 (1996) 2237.

Concentration-jump relaxations study on the dimerization of tetra sodium salt of transition metal (II)-4, 4', 4", 4'" -tetra sulfophthalocyanine in surfactant microheterogeneous aqueous media

N Rajendiran* & J Santhanalakshmi Department of Physical Chemistry, University of Madras, Guindy Campus, Chennai 600025, India \ Tetrasulfonated metal(lI) phthalocyanines (MPCTS) are water soluble, and they aggregate extensively in water. l 2 7 Extent of aggregation is composition dependent . . At concentration around 10- M monomers exist while above 10-5 M dimers and higher aggregates exist. Presence of aggregate and pure monomer is indicated in the UV - visible spectroscopy as two major bands one at 660 to 700 nm and others 340 to 450 nm for dimers and for 4 monomers3. Distinctly red shifted Sand Q bands occur in presence of surfactant . Dimerization is the initial stage of aggregation. Aggregation behaviour masks vital properties of MPCTS such as photoactivity, paramagnetism, electro chemical redox potential, etc. Addition of surfactant supra molecular assemblies containing aqueous medium to MPCTS in J x 10-5 M condition alters the dimerization equilibrium by transforming dimers to monomers and vice-versa. The monomer-dimer equilibrium D ... 2M of MPCTS has been investigated by spectrophotometric measurement in the stopped-flow relaxation method at 25°C. In aqueous solution both in the absence and in the presence of SDS, CTAB and TXlOO micellar solutions at 104 INDIAN J CHEM, SEC. A, JANUARY 2002

characteristics Amax of monomer, monomerization (k1) rate constant was determined for M(II) : Mn, Co, Ni and

Cu MPCTS solutions. From the relaxation time data for each systems dimerization rate constant (k. 1) values were determined. Ko=k/k.l values were determined in presence of various composition of the anionic, cationic, 5 and neutral micellar solutions . For constant MPCTS, Ko varies as SDS > TX100 > NaCI> CTAB for Mn(II). For constant SDS, Ko varies as Mn(II) > Co(ll) > NiCII) > Cu(II). The intercalation parameter of MPCTS per micelles were also evaluated. Implication results are discussed.

References I Yu-Chuyang, Richard W J & Seiders R P, Inorg Chern, 24 (1985) 1765. 2 Schelly Z A & Eyring EM, J phy Chern, 74 (1970) 617. 3 Blagrove R J & Gruen L C, Aust J Chern, 25 (1972) 2552. 4 Zelina J P & Rusting J F, J Porphyr Phthal, 3 (1999) 188. 5 Valduga G & Jori G, J inorg Biochern, 29 (1987) 59.

Evidence of topoisomerase type functions at metal constellations in spPAP radical enzyme

a b b S Y Rane*a, S P Dagade , D Srinivas , V S Gupta & Khursheed Aha "Department of Chemistry, University of Pune, Pune 411 007, India bNational Chemical Laboratory, Pune 411 008, India

Recent structures of Il-phosphato mammalian purple acid phosphatases, viz, PAP's from Uf1(l .sSA) and from 2 3 rat (2.7 A) suggest a diiron (III, III) active site. But its physiological functions still remain to be proven . Our "metal constellation" studies in PAP of plant origin, viz, sweet potato (spPAP), quantitize prime catalytic diiron site together with co-catalytic manganese and zinc sites with trace copper. Resonance and electronic spectral signatures in resting spPAP enzyme and its activity assay establish redox states of metal ions and Tyr-residue4 in both resting and Il-phosphato active forms. Evidence for topoisomerase-type functionality in nucleic acid interactions relates to its physiological functions. This leads us to its future scope as anti-tumour agents. A unique redox mechanism is proposed for such physiological functions of spPAP.

References I Guddat L W, McAlpine AS, Hume D, Hamilton S, Jersey J de & Martin J L, Structure, 7 (1999) 757. 2 Uppenberg J, Lindquist F, Sevensson C, Ek-Rylander B & Anderson G, J rnoLec Bioi, 290 (1999) 201. 3 Twichett Mark B & Sykes A G, Eur J inorg Chern, (1999) 2105. 4 Rane S Y, Dagade S P, Bakare P P, Date S K, Lagu M S, Gupta V S & Srinivas D, Proc MTIC(VIII,) (2000), 59 (lISe, Bangalore).

Spectroscopic!characterization of the superoxide reductase from Pyrococcus furiosus

Michael D Clay, Francis E Jenney Jr, Peter L Hagedoorn, Michael W W Adams, & Michael K Johnson* Departments of Chemistry, Biochemistry and Molecular Biology, and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602

Evidence has recently emerged for a novel mechanism of oxygen detoxification in anaerobic microorganisms involving superoxide reductase (SOR)l. SOR is proposed to catalyze the reduction of superoxide to hydrogen peroxide utilizing electrons from NADPH, by way of rubredoxin and a rubredoxin:NADPH oxodoreductase1. SOR activity was first identified in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus and associated with a protein known by the trivial name of neelaredoxin. The enzyme is a horhotetramer of 14.3-kDa subunits and the X-ray crystal structure indicates a novel mononuclear iron active site coordinated by the POSTERS 105 imidazole nitrogens of four histidines (three N and one N*) in a planar arrangement, with a cysteinyl sulfur occupying one axial position2. In two of the four subunits of oxidized P. furiosus SOR, a conserved glutamate carboxylate serves as an axial ligand to form a six-coordinate, octahedral environment. In the other two subunits of oxidized P. furiosus SOR and in ascorbate-reduced P. furiosus SOR, the sixth coordination site is either vacant or occupied by a solvent molecule2. In this work, oxidized and reduced forms of P. furiosus SOR have been investigated by absorption, VTMCD, and EPR spectroscopy. EPR studies of oxidized SOR reveal two types of high-spin ferric S =5/2 species. Resonances at g =10.05, 7.22, and 5.S3 arise from a species with axial zero-field splitting (ZFS), EID=0.06 and D=-0.S6 cm-'. In addition, there is a minor component « 5% of total Fe) with resonances at g =9 .65 and 4.31 that arises from a 3 rhombic S =5/2 species, EID - 0.33, and may correspond to adventitiously bound Fe + ion. Cyanide and azide binding results in homogeneous low-spin S = 112 (g =2.29, 2.25, 1.94) and high-spin S =5/2 (EID - 0.33) species, respectively. The blue colour of SOR results from a broad sulfur-to-ferric charge transfer band centered near 660 3 nm. VTMCD studies on this band resolve three transitions and these are assigned to S( 0-) ~ Fe + and Sen) ~ 3 Fe + charge transfer bands based on the polarizations deduced from analysis of MCD magnetization data. The ground and excited states properties of the predominant S =5/2 species in the oxidized SOR are indicative of an axially symmetric six-coordinate high spin ferric center and are accordingly assigned to the crystallographically defined glutamate-bound species. The pentacoordinate iron sites that are also observed in the oxidized crystal 3 /2 structure are, therefore, attributed to partial reduction of the high potential Fe + + site (Em - + 250 m V versus NHE), either during the crystallization or in the synchrotron beam during data collection. Reduced SOR has no X-band EPR resonances in perpendicular or parallel mode. UV/vis/NIR-MCD, and saturation magnetization data show that reduced SOR is high spin ferrous S=2 with a positive axial ZFS (D>O). NIR-MCD also provides evidence for a square pyramidal ferrous site based on the energies of ligand field excited states. The vacant Fe coordination site of reduced SOR is, therefore, proposed to be the site of superoxide binding and reduction. A reaction cycle is proposed involving a ferric hydroperoxide intermediate that is destabilized by the trans cysteinate ligand in order to promote dissociation of hydrogen peroxide.

References I Jenney FE, Verhagen M F 1 M, Cui X & Adams M W W, Science, 286 (1999) 306. 2 Yeh A P, Hu Y, Jenney F E, Adams M W W & Rees D C, Biochemistry, 39 (2000) 2499.

Iron(III) complexes of tripodal phenolate ligands as models for non-heme iron enzymes

Marappan Velusamy & Mallayan Palaniandavar* Department of Chemistry, Bharathidasan University, Tiruchirappalli, 620024, India

A large number of mononuclear and binuclear non-heme iron enzymes in microorganisms perform a variety of important biological functions. Dioxygenase enzymes like catechol 1,2-dioxygenase (CTD) catalyse the oxidative cleavage of catechols to cis, cis-muconic acids with the incorporation of two atoms of molecular oxygen. The crystal structure' of the CTD enzyme reveals that the coordination geometry of mononuclear iron(III) is trigonal bipyramidal with four endogenous protein ligands (Tyr40S, Tyr447, His460, and His462) and a solvent derived ligand. To date several complexes involving the iron-phenolate coordination environment as models2 for the active site of CTD enzymes have been studied. We have already isolated and investigated a series of mononuclear iron(III) complexes of linear tridentate and linear/tripodal tetradendate phenolate ligands 3 as functional models for catechol 1,2-dioxygenase . However, no closely mimicking structural model has been isolated to date to study its ability to catalyse the dioxygenase reactions. We describe here the results from our studies on the structures and spectral and redox properties of a few novel models for CTD enzymes. The iron(III) bis-phenolate complexes of the type [Fe(L)CI] [H2L' =N,N 2 dimethy I-N', N'-bis(2-hydroxy-4-nitrobenzy I)ethy lenediarnine, H2L =N,N -dime thy I-N' ,N'-bis(2-hydroxy- 3 ,5-dimethy 1- 3 benzyl)ethylenediamine, and H2L =N,N-dimethyl-N',N' -bis(2-hydroxy-3,5-t-butylbenzyl)ethylenediamine] have 106 INDIAN 1 CHEM, SEC. A,1ANUARY 2002 been isolated and characterized using UV -vis and EPR spectral techniques. The X-ray crystal structure of the 1 bis-phenolato complex [Fe(L )(H20)CI] possesses a distorted octahedral geometry around iron(III) with trans disposition of the coordinated phenolato moieties. In contrast, the bis-phenolato complex, [Fe(e)CI], adopts an unusual five-coordinated distorted trigonal bipyramidal geometry. The trigonal plane comprises one tertiary amine and two phenolate ions and the chloride ion and the tertiary amine nitrogen are coordinated axially. The unique reactivities observed for the complexes when treated with catechols and peroxides have been discussed.

References I Ohlendrof D H, Orville A M & Lipscome 1 D, J rnolec Bioi, 244 (1994) 586. 2 Solomon E I, Bumold T C, Davis M I, Kemsley J N, Lee S K, Lehnert N, Neese F, Skulan A 1, Yang Y S & Zhou 1, Chern Rev, 100 (2000) 235, Que L 1r & Ho R Y N, Chern Rev, 96 (1996) 2607. 3 Viswanathan R & Palaniandavar M, J chern Soc Dalton Trans, (1995)1259; Viswanathan R, Palaniandavar M, Balasubramanian T & Muthiah P T, J chern Soc Dalton Trans, (1996) 2519; Viswanathan R, Palaniandavar M. Balasubramanian T & Muthiah P T, Inorg Chern , 37 (1998) 2943.

Novel aromatic 30 1t core modified expanded porphyrins

V G Anand, S K Pushpan, A Srinivasan, B Sridevi, S J Narayanan & T K Chandrashekar* Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India

Expanded porphyrins have become an interesting class of compounds because of their diverse applications in molecular recognition, drug delivery, as MRI contrasting agents and as photosensitizers for photodynamic therapy in treatment of cancer. These properties of expanded porphyrins have instigated many research groups across the globe in hot pursuit of these macrocycles. The last decade of the millennium witnessed a flood of activity on the synthesis of 22 1t and 26 1t systems. Increasing the number of 1t electrons in these systems leads to novel macrocycles, with altered structural and electronic properties. So far, all attempts to synthesize the higher analogues of these macrocycles have yielded only non-aromatic systems even though they follow the 4n+2 rule. We present here the first synthesis of aromatic 30 1t meso phenyl expanded porphyrins and its different isomers 1,2 and 3. We have developed easy and efficient synthetic methods either through [4+3] acid catalysed condensation, or through an oxidative coupling reaction involving appropriate precursors in high yield. Spectral analyses reveal inversion of one or more rings in these systems.

X =S.OnrSe 2 x = SIS. Ar =Ph nr I\ll"S v = SIS.

References I Narayanan S 1, Sridevi B, Chandrashekar T K, Vij Ashwani & Roy Raja, J Arn chern Soc, 33 (1999) 9053. 2 Srinivasan A, Anand V G, Narayanan S 1, Pushpan S K, Chandrashekar T K, Sugiura K I & Sakata Y, J org Chern, 64 (1999) 8693. POSTERS 107

'Axial-bonding' type hybrid porphyrin arrays: Synthesis, electrochemistry and photophysical properties

P Prashanth Kumar & Bhaskar G Maiya* School of Chemistry, University of Hyderabad, Hyderabad 500 046. India

Axial substituted porphyrin-based arrays are being constructed for the use in biomimitic photosynthesis, molecular electronic, molecular catalysis etc. Recently, we have reported 'axial-bonding' type hybrid arrays based on phosphorus(V), tin(IV) and germanium(lV) porphyrins 1.2. Here, the axial bonding capability of aluminium(III) porphyrin has been utilized for the construction of arrays 1 and 2 shown below. This paper deals with the design, synthesis, spectral characterization, electrochemistry and photochemical properties of these novel arrays. 0,0 0

/\ ~ - ~0 0 O1() 010 2

M = 2H (a). Zn (b), Cu (c).

References 1 Rao T A & Maiya B G, Inorg Chern, 35 (1996) 4829. 2 Giribabu L, Rao T A & Maiya B G, Inorg Chern, 38 (1999) 4971.

Synthesis, spectroscopy and photophysical studies of a series of phenylene ethynylene bridged porphyrin donor-bridge-diimide acceptor arrays

N PRedmore, I V Rubtsov & M J Therien* Department of Chemistry, University of Pennsylvania-Philadelphia Pennsylvania 19104 USA

We are interested in exploring the effect of extended ~-conjugation on the photophysical behaviour of porphyrin donor-bridge-acceptor systems. The syntheses, electronic spectroscopy, photophysics, and electron transfer dynamics of a series of porphyrin donor-phenylene ethynylene bridge-diimide acceptor arrays will be presented. 108 INDIAN J CHEM, SEC. A, JANUARY 2002 ...... ::..,-....-...,.:::;:-==--o-W-octYI Ph 0 0

~~ ~ o-~-octYI~~~ Ph -- OCtYI-~-o o-W-oCO/I o 0 Ph 0 0

o~~0 octyl-N - N ,>----:=--n ~ -octyl o ~ b 0 Ph ~

Metalloporphyrins initiated polymerization in microemulsions

K A Srinivas, Chetna Angarish & S M S Chauhan* Department of Chemistry, University of Delhi. Delhi 110 007, India

Selected metalloporphyrins such as aluminium, cobalt, zinc, manganese, and titanium metalloporphyrins have served as excellent initiators for polymerization of different monomers 1. Microemulsion polymerization has been widely studied and is effective in producing nanosized stable polymer latexes. We report the microemulsion polymerization of various monomers using different water soluble metalloporphyrins to understand the molecular mechanism of polymerization in microemulsions. The required metalloporphyrins have been prepared by the metallation of water soluble and organic soluble 2 porphyrins with corresponding metal (II) salts . In a typical case the polymerization of methyl methacrylate was carried out in n-hexane solution of 0.1 M AOT with CIsTPPS4Co(lI) as an initiator at 60°C under nitrogen atmosphere for 18 hrs. The formation of poly(methyl methacrylate) (PMMA) was confirmed by IR, NMR, and other spectroscopic data. Similarly other polymers were prepared from the corresponding monomers initiated by water soluble and organic soluble metalloporphyrins in microemulsions. The detailed mechanism and product formation in microemulsion polymerization has been presented.

References I Aida T & Inoue S, Acc chern Res, 29 (1996) 39. 2 Chauhan S M S & Sahoo B B, Bioorg Med Chem, 7 (1999) 2629.

Spectroscopic studies on human serum albumin and methemalbumin

Amisha Kamal J K & Digambar V Behere* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Navy Nagar. Colaba. Mumbai, 400 005 India

Interaction of human serum albumin (HSA) with monomeric hemin to form methemalbumin has been followed by UV-visible, fluorescence and circular dichroism spectroscopy. Using optical difference spectroscopy of HSA titration with hemin, equilibrium dissociation constant for 1: 1 complex has been deduced as, KD = (4.0±0.I)xlO·5 M. POSTERS 109

Methemalbumin shows peroxidase activity. Analysis of the data from steady-state assay with 22'-azino-bis-(3- ethylbezthiazoline-6-sulphonate) (ABTS) as reducing substrate shows considerably reduced peroxidase activity as compared to horseradish peroxidase (KM =433 ± 40 11M, specific activity 8 )lMlminlmg). Circular dichroism of HSA in the 250-300 nm region, on sequential addition of hemin, shows gain in circular dichroic intensity due to the coupled oscillator interaction of the hemin absorption dipoles and the emission dipoles of the aromatic residues located near the bound hemin. Fluorescence intensity of the single intrinsic tryptophan (Trp214) of HSA is quenched by the bound heme. From this Trp214 -7 heme separation of 22 A has been deduced. The studies indicate that hemin binds His146 on domain I of HSA.

Stability and spectroscopic properties of soybean peroxidase

Amjsha Kamal J K & Djgambar V Behere* Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India

Peroxidases (donor: H20 2 oxidoreductase, E.C. 1. 1 1.1.7) are a class of heme-containing enzymes whose primary function is to catalyze oxidation of a variety of hydrogen donors at the expense of hydrogen peroxide. Soybean peroxidase (SBP) isolated from the hulls of soybean seeds is a class III plant peroxidase that shows thermal stability and hence is particularly promising as a biocatalyst and biosensor. We have undertaken the study of spectroscopic and physicochemical properties of soybean peroxidase under various conditions of pH, temperature and denaturants like urea and guanidine hydrochloride to investigate the nature of the stability of the protein. SBP is l.6 times more active than HRP under physiological conditions. We observed that the enzyme undergoes backbone melting with T m =86 °C at neutral pH as measured by far UVCD. The far UVCD measurements also show that the native enzyme has -71 % total helical content. The secondary structure elements computed from the far UVCD of SBP at pH values from 4.6 - 8.0 indicate that pH dependent conformational change involves primarily the ~-sheet random coil transition. At neutral pH the protein denatures in presence of guanidine hydrochloride with Cm= 6.5 M. The Gibbs free energy, L1Gu(D) of unfolding determined assuming two state F"",U model of solvent denaturation deduced at neutral pH is 43.3 kllmol.

Reactions of ferricytochrome C - Kinetics and mechanism of oxidation of vanadate by ferricytochrome C in phosphate buffers

N R Suja, N Sridevi & K K Mohammed Yusuff* Department of Applied Chemistry, Cochin University of Science and Technology, Kochi, Kerala 682 022, India

Cytochrome c is an iron containing metalloprotein that functions as an electron transfer agent through cyclic oxidation and reduction stages in the respiratory chain of all vertebrate species. Vanadate is known to cause lung disease in mammals'. We found that vanadate ion is oxidized by ferricytochrome c and therefore found it worthwhile to elucidate the mechanism of the reaction kinetically. The kinetics of the reaction were studied at 30°C in the pH range 7-8 (phosphate buffers), by initial rate method employing [ferricytochrome c] « [vanadate]. The initial rate of the reaction increased with increase in [ferricytochrome c] and a plot of initial rate versus [ferricytochrome c] was linear passing through origin indicating first order with respect to the latter. A similar observation was made in the case of vanadate ion, showing that the reaction is first order with respect to vanadate ion also. The initial rate decreased with increase in ionic strength and a plot of log(initial rate) against ...J)l was a straightline with negative slope indicating that the reactive species are oppositely charged. The initial rate was found to increase with increase in pH and a plot of 1IInitial rate versus [H+] was linear with an intercept on rate axis. In the pH range 7-8, the vanadate ion is known 2 to exist as a single species , V 3094-. It is also known that ferricytochrome c exists in a deprotonation equilibrium 110 INDIAN J CHEM, SEC. A, JANUARY 2002

3 in this pH range . Therefore, from the effect of pH on the present reaction rate it may be concluded that deprotonated ferricytochrome c is the reactive species. The spectra of the reaction mixtures indicated absence of complex formation between the reactants. On the basis of these observations we propose the following mechanism for the reaction: K HctyC(IIIt <=> CytC(III) +H+ (1)

CytC(III) + V (IV) ~ CytC(II) + V (V) (2) (slow) From the above mechanism the rate of the reaction may be given as, Rate =k[ cytC(III)] [V (IV)] ... (3)

kK [cytC(III)], [V (IV)] Rate = ------... (4) {K +[H+]}

where [cytc(IIIn represents the total concentration of all the species of ferricytochrome c. As may be seen from Eq. (4), the ratio of the intercept to slope of the plot of llInitial rate versus [H+] corresponds to K and the value obtained from our experimental plot is of the order of 10-7 mol dm-3 in the pH range 7-8. The second order rate constant of the reaction, k at pH 8 and 30°C has also been determined from this 2 3 l plot to be about 1 x 10- dm mor S-I.

References I Waters M D, Gardner D E & Earnshaw A, Taxieal appl Pharmacal, 28 (1974) 253. 2 Greenwood N N & Earnshaw A, Chemistry of elements, (Maxwell & Macmillan International Ed, Pergamon Press, Oxford) 1989. 3 Hugo T & Akesson A, Science, 90 (1939) 67.

Peroxidase activity of carboxymethylated cytochrome c: Spectroscopic and kinetic studies

Swati Prasad, Shyamalava Mazumdar & Samaresh Mitra*

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Col aba, Mumbai 400 ~OS, India

Carboxymethylation of horse heart cytochrome c has been shown to modify its sixth axial ligand (Met 80), which ruptures the Fe-S (Met 80) bond, causing structural changes including change in the axial ligation around the heme iron. We have .investigated the peroxidase activity of carboxymethylated cytochrome c (ccytc), reaction of ccytc with hydrogen peroxide and its characterization by spectroscopic and kinetic techniques. The optical spectrum suggests that the reaction of ccytc with H20 2 proceeds through only one intermediate, compound I. The apparent rate constant (kapp) for the reaction of ccytc with H20 2 was found to be l.4x 101, 1 2 7.2x10 and 2.1x10 MI S· I at pH 7.0,5.0 and 3.5 respectively. These values are lower by 5-6 orders as 7 I 7 l compared to classical peroxidases- horseradish peroxidase (l.8 x 10 Mls· ), lactoperoxidase (1.67 x 10 Mls· ) 7 I 2 I and cytochrome c peroxidase (4.5 x 10 Mls· ), but comparable to wild- type myoglobin (2.67 x 10 Mls· ), 3 l I MnHRP (l.7 x 10 Mls· ) and MnMb (4.5 Mls· ). Ccytc was found to catalyze the oxidation of organic and inorganic substrates- 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and potassium iodide (KI). The specific activity of ccytc measured spectrophotometrically using ABTS as substrate, was found to be 288, 1 473 and 872 ~ min·lmg· at pH 7.0, 5.0 and 3.5 respectively, as compared to the reported specific activity for I HRP (1150 11M min·lmg· ). Resonance Raman spectra were retorded between pH 3.5-7.0, which indicated the existence of a population distribution of different ligation states- bis-histidine, histidine-water and five coordinate forms of ccytc at each pH. The results obtained could explain the pH dependence of peroxidase activity of ccytc and its reaction with hydrogen peroxide. POSTERS 111

Binding of camphor and its analogs to Pseudomonas putida cytochrome P450cam: Steady-state and picosecond time-resolved fluorescence studies

Swati Prasad, Shyamalava Mazumdar & Samaresh Mitra* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Col aba, Mumbai 400005, India

The binding of camphor and its analogs to cytochrome P450cam has been investigated by steady-state and time resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The steady-state fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The Stern Volmer quenching constant (Ksv ) with acrylamide was found to be larger for camphor-free P450cam (1.45 MI) than for camphor-bound P450cam (0.72 MI), indicating greater fluctuations in the camphor-free enzyme I . The high bimolecular rate constant for P450cam supports dynamic fluctuation in the enzyme. The time-resolved fluorescence decay profile for camphor bound and camphor-free P450cam gives four lifetime components in the range from 99ps to 4.5ns. The shortest lifetime component is assigned to W42, and its fluorescence is greatly affected on binding of camphor. It is suggested that the camphor access channel is close to W42 residue, which provides experimental support to the dynamic fluctuations involved in the passage of camphor through the access channel next to the flexible F-G helix-Ioop l helix segment . We have also used several camphor analogs and the site-directed mutant Y96A of P450cam to probe the effect of active-site protein-substrate interactions on the structural fluctuations involving entry/passage of the substrate through the access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo significant changes in their local environment upon removal of protein-substrate interactions in P450cam . The time-resolved fluorescence decay follows a four-exponential model, giving four lifetime components in the range from 45ps to 4.5ns. The results indicate that the fluorescence of W42 and dynamic fluctuations involved in the passage of the substrate through the access channel is greatly affected by the removal of protein-substrate interactions in P450cam.

Reference 1 Prasad S, Mazumdar S & Mitra S, FEBS Letters, 477 (2000) 157.

Studies on electron transfer pathway and conformational change in cytochrome P450cam-putidaredoxin complex

l Takehiko Tosha , Shiro Yoshioka!, Satoshi Takahashi!, Hiroshi Hori2, Koichiro Ishimori! & Isao Morishima! * 'Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan 2Divi sion of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Osaka, Japan

Electron transfer (ET) from putidaredoxin (Pd) to cytochrome P450cam is one of the crucial steps for the hydroxylation of camphor. Both · specific recognition to form the effective ET pathway and conformational change induced by the formation of ET complex. are supposed to be essential for the ET reaction between P450cam and Pd. First, to identify amino acid residues responsible for the ET reaction, we focused on Gln360 in P450cam, which is located at the putative Pd binding site. We have replaced Gln360 by Glu (Q360E), Leu (Q360L) and Lys (Q360K) and measured their ET rates and redox potentials. In the ET from reduced Pd to ferric P450cam (first ET), the intracomplex ET rates for the Q360E and Q360L mutants were suppressed with the decrease in their redox potentials for the Fe3+lFe2+ couple. In contrast to these mutants, the Q360K mutant exhibited the accelerated ET rate which was 1.5-fold of the wild type enzyme. Since its redox potential was still lower than that of the wild type enzyme, the improved ET rate for the Q360K mutant cannot be explained from the alteration of the redox potential. One of the possible reasons for the enhanced ET rate in the Q360K mutant is that the Lys residue at position 360 forms a more effective ET pathway by its longer and flexible side chain to 112 INDIAN J CHEM, SEC. A, JANUARY 2002 facilitate the ET from Pd to P450cam. Thus, we propose that the amino acid residue at position 360 is the key residue for the first ET. We also investigated the reduction rate of oxy-P450cam by reduced Pd (second ET). The rates of the second ET were decelerated for all mutants compared with that for the wild type enzyme. Considering the decrease of the redox potential for the Fe3+lFe2+ couple, the slower ET rates in all mutants would be attributed to the decrease in the redox potential of oxy-P450cam. In spite of the improved first ET rate, the Q360K mutant exhibited the decelerated second ET rate. These results allow us to suggest that the ET pathway for the second ET is different from that for the first ET. Next, we also examined the conformational change by the formation of P450cam-Pd complex. In order to clarify the conformational change of P450cam upon binding Pd in detail, we measured the IH-NMR spectra of the ferrous-CO form of P450cam in the absence or presence of reduced Pd. We detected a ring current shifted signal in the presence of Pd, which was not observed in the free P450cam or Pd. This result indicates that conformational change is induced around the heme upon binding Pd. The relationship between the conformational change and the ET reaction has been discussed.

The role of water molecules in the association between cytochrome P450cam and putidaredoxin

Yoshiaki Furukawa, Koichiro Ishimori & Isao Morishima* Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501 , Japan Email: [email protected]

Cytochrome P450cam is one of the hemoproteins for the electron transfer reactions, which catalyzes the hydroxylation of d-camphor at the 5-exo position. The reaction cycle requires two electrons, which are sequentially transferred by a [2Fe-2S] cluster protein, putidaredoxin (Pd), from Pd reductase to P450cam. Previously, our group has reported 1 that the association of ferric P450cam with oxidized Pd is characterized by negative entropy of binding (-93.2 JmorlK\ On the typical protein associations, about 10 water molecules are excluded from the interface of the complex, resulting in increase of entropy. Negative entropy change on the ferric P450camJoxidized Pd association would not, therefore, be explained by the water release from the interface. Alternatively, uptake of water molecules at the complex interface can well be considered. However, in the physiological condition, the electron transfer reaction cannot be observed between ferric P450cam and oxidized Pd. To gain an insight into the water uptake upon the physiological association between P450cam and Pd, we examined the osmotic pressure (Posm) effects on the association process in the ferric P450camJreduced Pd

(physiological pair). The elevated P os m by addition of polyols or sugars promotes the dehydration of the protein surface, and would decrease the binding affinity when uptake of water is essential for the complexation. As shown in Fig. 1, the association constant, Ka, between ferric P450cam and reduced Pd was decreased by the increase of Posm. The use of P osm represents a powerful and unique method for an estimation of the number of water molecules involved in the association process. The volume change of water molecules upon the association (~V) was estimated from the data in Fig. 1 by using (8InKa/8Posmh= -~VIRT. In this study, ~V was found to be 457 mL/mol, which implies the uptake of 25 water molecules during the ferric P450camJreduced Pd complexation. The association was also reduced in the association between ferric P450cam and oxidized Pd, but ~ V was relatively smaller (ca. 290 InL/mol) than that of the ferric P450camJreduced Pd complexation. Although these results are consistent with the water uptake upon the P450camlPd association, it is interesting to note that the 6,. V is dependent on the redox-state of Pd. It has been well known that reduced Pd exhibits high affinity with ferric P450cam, while oxidized Pd does not. At the presentation, we will discuss the possibility that the interfacial water molecules regulate the redox-dependent difference in the affinity between P450cam and Pdx. POSTERS 113

-0.5

Ol ~ -l.0 E -l.5

-20 . 0 2 4 6 8 Osmotic Pressure (MPa) Fig. I-The dependence of the association constant on osmotic pressure. [(e) without any co-solvents C- ) glycerol C+) ethylene glycol C.A.) glucose) Reference I Aoki M, Ishimori K, Fukada H, Takahashi K & Morishima I, Biochim Biophys Acta, 1384 (1998) 180.

Selective CO recombination dynamics on a and ~ subunits of human carbonmonoxy hemoglobin: A time resolved resonance Raman study

G Balakrishnan', X Zhao', E Podstawka2, J R Kincaid2. & T G Spiro'* 'Department of Chemistry, Princeton University, Princeton, NJ 08544, USA 2Marquette University , Department of Chemistry, Milwaukee, WI 53233 USA tE-mail: spiro@princeton edu

The molecular mechanism of hemoglobin (Hb) cooperativity is of great importance to the current thinking about 2 3 protein dynamics and allostery. Recently our groupl and others • have studied the allosteric R-T transition pathway of Hb using resonance Raman spectroscopy. In light of our continuing interest in understanding the various intermediates and their structural implications on the allosteric transition, herein we report the CO 3 recombination kinetics obtained from time resolved resonance Raman (TR ) spectral measurements. From our earlier studies and other transient absorption studies4.5, Hb had been proposed to have similar CO recombination kinetics for both a and ~ subunits with subtle differences, however, there is no direct experimental evidence available to date. Here we report the individual CO recombination kinetics of a and ~ subunits of HbCO based on selective l5N isotopic labeling of heme in either the a or ~ subunits. Isotopically labeled HbCO ('5al4~ and or l4al5~, 25 mM in heme, pH 7.4 phosphate buffer) has been photolyzed with 419 nm (Q-switched Nd:YLF-pumped Ti:shapphire, 70 mW, 1 KHz, 20 ns) pulse, and the resonance enhanced Raman bands at various time delays have been obtained using a 425 nm (0.5 mW) probe pulse. As both CO binding and l5N labeling induce shifts, the V4 Raman band of the heme is an ideal probe to follow the CO recombination kinetics. Specific l5N labeling, (e.g. labeling heme at the subunit) results in the splitting of the V4 band into a doublet. Deconvoluting this doublet at various time delays, and plotting the intensities as a function of time delays yields the individual CO recombination kinetics for a and ~ subunits separately. Control experiments on unlabeled HbA lead to a geminate recombination (GR) yield of -50% which is in good agreement with both step scan FT-IR and transient absorption studies. Our new results on isotope hybrids establish that there is no significant difference in either GR yield or CO recombination kinetics between a and ~ subunits. References I Jayaraman V, Rogers I, Mukerji I & Sprio T, Science, 269 (1995) 1843. 2 Rousseau D L & Freidman J M, Biological applications of Raman spectroscopy, edited by T G Spiro, Vol. 3, (1987) Ch. 4, pp. 133 and references therein . 114 INDIAN J CHEM, SEC. A, JANUARY 2002

3 Nakashima S, Kitagawa T & Olson J S, Chem Phys, 228 (1998) 323. 4 Scott T W, Friedman J M, Ikeda-Saito M & Yonetani T, FEBS Lett, 158 (1983) 68. 5 Esquerra R M, Goldbeck R A, Reaney S H, Batchelder A M, Wen Y, Lewis J W & Kliger D S, Biophys J,78 (2000) 3227.

Conformational changes monitored by fluorescence study on reconstituted hemoglobins

V Swarnalatha, S Ramasamy & P T Manoharant* Department of Chemistry and Regional Sophisticated Instrumentation Centre, Indian Institute of Technology - Madras, Chennai 600036, India. tE-mail: [email protected] (or) [email protected]

The intrinsic fluorescence of proteins containing tryptophan and tyrosine has been used as an important probe in understanding the dynamics and conformational charactersitics of biological molecules. Hemoglobin, (Hb) one such macromolecule is a tetrameric protein with two alpha and two beta subunits. The reversible oxygenation reaction involved in the respiratory cycle is associated with a definite conformation change of T H R in Hb. This transition from the deoxy (T) to oxy (R) and vice-versa is accompanied by the rupture of various specific salt bridges and H-bonds, at the interfacial regions of the subunits. The tryptophan residue B-37 located at the cx,FG-B2C (cx2FG-B,C) interface in human hemoglobin is well known· to monitor the conformational events of the macromolecule. The fluorescence emission of this tryptophan residue allows one to detect the changes in the micro - environment at the interfacial "switch - region" which is associated with the quaternary transition of the macromolecule. Thus, the intrinsic fluorescence originating from the B-37 tryptophan in human hemoglobin serves as a valuable "spectroscopic marker" for monitoring the conformational changes during the oxygenation mechanism. We report herein a detailed study on some of the metal ion reconstituted hybrid hemoglobins by fluorescence spectroscopy.

References I Mizukoshi H et ai, Biochimica et Biophysica Acta, 700 (1982) 143. 2 Hirsch R E et ai, Biochim Biophy Res Comm, 93 (1980) 432.

Spin labelled studies on NiHb-A correlation to the mixed metal ion environment in Hb

S Ramasamy & P T Manoharan* Department of Chemistry and Regional Sophisticated Instrumentation Centre, Indian Institute of Technology Madras, Chennai 600036, India. *E-mail: ptm @rsic.iitm.ernet.in (or) [email protected]

The study of biomolecules at the molecular level is of great importance not only to decipher the structure and function but also to understand the dynamical alterations involved in the biological process. The spin label which is a synthetic, site-directed, paramagnetic organic molecule with a nitroxide free radical can be used as a probe or reporter group since it has a molecular structure and/or chemical reactivity that results in its attachments or incorporation at some particular target site in a biological macromolecule so that the macromolecule can be studied by EPR spectroscopy. The denaturation pathway of NiHb due to pH variation was monitored by the use of EPR spectroscopy by attaching a spin label, 3-(maleimidomethyl)pROXYL. The EPR spectrum of the spin labelled NiHb shows a spectrum, due to three different motions of the spin label attached to the carrier. On varying the pH to acidic and alkaline conditions, a sequential change in the EPR lines were observed, which may be attributed to the change POSTERS 115 in all the three different kinds of spin label motions which in tum can be related with the metal ion coordination of Ni(II) in the heme pocket. A careful simulation of the experimental EPR spectra was done to get an idea about the rotational correlation time, angle of the orientation of label relative to the carrier molecule and the relative weights with which they represent the total spectrum. The results were well correlated to the dynamics between the 4- and 5-coordinated metal ion environments.

References 1 Manoharan P T, Wang J T, Alston K & Rifkind J M, Hemoglobin, 14 (1990) 41. 2 Timofeev V P & Samarianov B, Appl magn Reson, 4 (1993) 523. 3 Timofeev V P & Samarianov B, J chem Soc Perkin Trans, 2 (1995) 2175.

The development of mode of action toxicity biosensors

James Hudson & A E G Cass* Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2A Y and Peter Whalley The Water Research Centre. Medmenham

A toxic mode of action is the specific interaction between a chemical and a biochemical system that leads to perturbation in an organism. As this interaction is inherently chemical, compounds that are structurally or otherwise related will often stress the same mode of action. This phenomenon creates a system of classifying compounds in accordance with their toxicity and this is the source of the analytical power of the mode of action biosensor. That is to say a mode of action biosensor will respond to or detect all compounds that exert their toxicity through a specific mechanism. One such biosensor uses the enzyme cytochrome c oxidase to detect respiratory poisons such as cyanide and azide. To produce this biosensor an electrochemical cell incorporating cytochrome c and cytochrome c oxidase is used. The catalytic effect produced by the enzyme is collapsed in the presence of respiratory poisons and this serves as a means of detection. Another biosensor uses liposomes to detect respiratory uncouplers. These liposomes contain the pH sensitive fluorophore, pyranine and a pH gradient relative to the bathing media. In the presence of respiratory uncouplers this proton gradient is dissipated and the effect observed by changes in the excitation spectra of the fluorophore.

pH induced conformational transition in the binuclear euA domain of Paracoccus denitrificans cytochrome oxidase

l 2 2 l Sayan Gupta , A Wame , Matti Saraste & Shyamalava Mazumdar *

1Department of Chemical Sciences, Tata Institute of Fundamental Research Homi Bhabha Road, Col aba, Mumbai 400 005, India 2European Molecular Biology Laboratory, Postfach 10,2209, Meyerhofstrasse I 6900 Heidelberg, Germany pH induced conformational transition in the CuA domain of the subunit II of cytochrome oxidase of Paracoccus denitrificans has been investigated using various spectroscopic and stopped flow kinetic methods. UV -visible absorption and circular dichroism studies showed that increase in pH from pH 6-10 leads to a biphasic conformation change with pKa = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was however shown to remain unchanged in these two conformational states. Thermal and urea induced unfolding studies showed that the 'low pH' conformation is more stable compared to the 'high pH' conformation 116 INDIAN J CHEM, SEC. A, JANUARY 2002 of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the 'low pH' form while oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the 'high pH' form. Steady state fluorescence studies showed that the ' low pH' form consists of two tryptophan residues exposed to the solvent and the pH induced conformational change is associated with movement of one tryptophan residue from the surface to the core of the protein at high pH. Time-resolved tryptophan fluorescence decay of the protein was best fitted to a three exponential model, which was also verified with maximum entropy method analysis. The fast picosecond lifetime component was assigned to Trp123 residue, which is buried in the core of the ~ barrel structure of the protein. Conformational transition from the 'low pH' to the 'high pH' form was found to be associated with drastic decrease in the magnitude of the lifetime of this residue. Stopped flow pH jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step a followed by fast conformational equilibrium. Redox titration of the protein with reduced cytochrome c indicated that the 'low pH' form has lower redox potential compared to the 'high pH' form. The results are discussed in the light of understanding the pH induced gating mechanism of the CuA site in the intact subunit II of cytochrome oxidase.

A rational design approach to enzyme engineering and immobilisation: The design of dehydrogenase enzymes for use in a biofuel cell

C M Halliwell & A E G Cass* Department of Biochemistry, Imperial College of Science, Technology and Medicine, London. SW7 2A Y and E Simon, C S Toh & P N Bartlett Department of Chemistry, University of Southampton, Southampton SO 15 5RR

Biofuel cells use biocatalysts, such as enzymes or whole cells, to convert a chemical fuel into electrical energy 1. The extractable power of a biofuel cell, as with a conventional fuel cell, is the product of the cell voltage and cell current. At the bioanode the half cell current is derived from the oxidation of NADH, which is produced by the enzymatic reduction of NAD+ and oxidation of a suitable substrate (fuel). Therefore, the anodic current is greatly influenced by the behaviour of the biocatalyst, which for the sake of maximizing the surface concentration of both and therefore the enzymatically reduced cofactor, is usually immobilized on the electrode surface. We have investigated a number of dehydrogenase enzymes for their stability as potential anodic biocatalysts in a biofuel cell. The enzymes were genetically modified such that a number of different immobilization strategies that were compatible with polyaniline (PANI) film doped with a suitable poly(anion) could be investigated. In particular, Bacillus stearothermophilusL-Lactate dehydrogenase and zinc dependent 2 alcohol dehydrogenase 2.3 were labeled with a histidine tag for immobilization on Ni +doped films. References I Willner I, Arad G & Katz E, Bioelectrochemistry and Bioenergetics, 44 (1998) 209. 2 Wigley D Bet. ai, J moiec Bioi, 223 (1992) 317. 3 Sakoda H & Imanaka T. J Bacteriol, 174(4) (1992) 1397.

An optical biosensor for measuring freshness of fruit and canned juices using tiron-polypyrrole sensing film

Sabari Dutta, Zahra Afrasiabi, Pallavi Kulkarni & Subhash Padhye* Department of Chemistry, University of Pune, Pune 411 007, India A polypyrrole film incorporating water soluble 1,2 dihydroxybenzene 3,5 disulphonic acid disodium salt monohydrate (tiron) reagent is used for the first time for determining the tyrosinase activity in fruits using both POSTERS 117

ITO-Glass+

Polypyrrole film

tiron Inunobilised substrate o-quinone

~9/ Solution

NaS0 OH tyrosina\e 3yI~ OH S03Na Tiron a-quinone

Fig. I-Sensing scheme of the tiron optical sensor for the detection of tyrosinase activity potentiometric as well as optical methods. The enzyme activity using the polypyrrole-tiron film was confirmed by catalytic conversion of the substance to the a-quonine by the enzyme (Fig 1). The immobilization of substrate instead of the enzyme increases the stability and shelf life of the sensor under laboratory conditions. The sensor is reversible and can be used for atleast 6 cycles without any loss in activity.

Metal complexes of carboxamidrazone analogs as antitubercular agents: Synthesis, X-ray crystal-structure, spectroscopic properties and antimycobacterial activity against H37R v cell line

Uday Sandbhor, Prasad Kulkarni, Jayendra Patole & Subhash Padhye* Department of Chemistry, University of Pune, Pune 411007, India

Tuberculosis is the leading cause of death from a single infectious pathogen in the world. Twenty-five percent of the global TB cases are found in India, many of which are multi-drug resistance. The HIV epidemic and socio-

() ,N, ~ o N Ar ll.. ~ .... N~ .... Ar 'N~/ N N """ NH2 H (I) (3)

(4) 118 INDIAN J CHEM, SEC. A, JANUARY 2002 economic factors have further contributed to this increase. Hence there is an urgent need to explore and evaluate new anti mycobacterial agents as well as novel targets for chemotherapeutic interventions. In the present work we have investigated a new class of antitubercular agents viz. the pyridine -2 carboxamidrazones,first described by Mamlol et aZ'and further pursued by Billington and coworkers2 which can serve as basis of building large combinatorial libraries of potential anti mycobacterial compounds. The prototype of these carboxamidrazones is represented by 1 in which the amino group serves three functions, viz. hydrogen bond donation to target molecule, maintenance of planarity via intramolecular N-H---N hydrogen bonding and supply of electron density to other heteroatoms thereby enhancing their ability to chelate metal or accept hydrogen bonds. The contribution of this side-chain amino group to the antimycobacterial activity of these compounds was examined by its deletion as in 2 and 3. The condensed hydroxy aldehydes are further appended with biologicallt relevant metal ion as in 4 when their antimycobacterial activities are found to be enhanced several folds, which suggest their role in copper transport mechanism in this bacteria. References I Bafini E, Mamolo M. Vio L & Predominanto. J Chemotherapy, 5 (1993) 164; Mamolo M, Vio L. Farmaco, 51 (1996) 65. 2 Billington D, Coleman M, Ibi abuo J, Lambert P, Rathbone D & Toms K. Drug design and discovery, 15 (1995) 269.

Derivatives of a naturally occuring hydroxynaphthoquinone and their copper complexes as antitumor agents

Nikhil Gokhale, Dilip Saha & Subhash Padhye* Department of Chemistry, University of Pune, Pune 411 007, India

Quinonoidal ligands when used as therapeutic agents can exercise their cytotoxic action by various mechanisms which include redox cycling, arylation, intercalation, induction of DNA strand breaks, generation of free radicals and alkylation via quinone methide formation. The ability of 1,4 naphthoquinone to produce oxidative stress by redox cycling and to act preferentially as alkylating agents is well established. The representative examples of this class of compounds include well-known anticancer drugs of the anthracycline and naphthazarin series. Lawsone (2-hydroxy l,4-naphthoquinone) is a monohydroxy naphthoquinone pigment of henna plant possessing various biological properties, including antitumor activity.

0 OH

I I 2' ~ N~N:lC*''-'::: 3' I CH 3 oJ=° 6' h 4' 5' Fig. I-Schematic representation of C-3 phenylazo substituted lawsone. The present work describes synthesis and characterization of new C-3 substituted phenylazo derivates of lawsone (Fig 1) and their copper complexes and their in vitro anticancer activities against the human breast cancer cell line NCF- 7. The enhanced antitumor activities observed for the copper compounds are probably due to inhibition of estradiol binding to its receptor preventing dimerisation of estrogen and subsequent DNA binding.

DNA binding study of Co (III) polypyridyl complexes

Pitchumony Tamil Selvi & Mallayan Palaniandavar* Department of Chemistry, Bharathidasan University, Tiruchirappalli 620024, India

The use of transition metal chelates is central in the effort to determine the principles governing site-specific recognition of DNA. The interaction of several copper(II) and ruthenium(II) complexes with DNA has been POSTERS 119 studied as models for the site specific reactions. But, though Co(IIl) systems possess interesting properties of metallointercalation and DNA cleavage properties in addition to binding selectivity, they have not been extensively studied. The DNA binding and photocleavage studies involving cobalt(1I) complexes of a family of 1,1O-phenanthroline ligands have been undertaken I. We have undertaken a spectral and electrochemical 2 3 investigation of Cu(II)-bis(phenanthroline) complexes bound to calf thymus DNA. We have demonstrated that Cu(n) bis-complex of 5,6-dimethylphenanthroline (dmp) effects the novel conversion of right-handed B-DNA to left-handed Z-DNA. So the present work aims to study the ability of Co (III) complexes and mixed ligand complexes of variously substituted phenanthrolines to induce such trans conformational changes. Further, the application of mixed ligand complexes permits variation in the geometry, size and hydrophobicity by systematically adjusting complex ligands and their substituents, and gives the opportunity to determine how these factors contribute to its affinity towards DNA binding. The present work reports the results of our investigation on the DNA interaction of Co(III) mixed ligand systems containing the most commonly studied ligands 1 ,10-phenanthroline and 2,2'-bipyridine to study the effect of the coligand on the mode and selectivity in binding to DNA. The complexes [Co(bipyhCphen)]Cb, [Co(bipyh(imp)]CI3, [Co(bipyh (dppz)]Cl3, [Co(phenh(bipy)]CI3, and [Co(phenhCimp )]Ch (imp = imidazo-l,1 O-phenanthroline) have been' used for the present investigation. The UV -Vis titration of the complexes with CT DNA has been performed and the binding constant of the complexes bound to DNA have been calculated. Competitive binding studies with ethidium bromide, viscometry and circular dichroism spectra have been also used to study the interaction of the complexes with DNA. The effectiveness of the complexes [Co(bipyhCdmp)]3+ and [Co(dmp)3]3+ in inducing B ~ Z transition has been investigated by circular dichroism spectra.

References I Arounagiri S & Maiya B G, Inorg Chem, 35 (1996) 4267 2 Mahadevan S & Palaniandavar M, II10rg Chelll, 37 ( 1998) 693 ; Mahadevan S & Palaniandaver M, Biocol1jugate Chem, 7 (1996) 138. 3 Mahadcvan S & Palaniandavar M, 1110rg Chem , 37 (1998) 3927; Mahadevan S & Palaniandavar M, J chem Soc Chem C0111111UI1 , (1996) 2547.

Copper(II) complexes of pentadentate bis(benzimidazolyl)-dithioether ligands as models for blue copper proteins: Synthesis, structure, spectra and redox properties

R Balamurugan", M Palaniandavar"*, R Srinivasa Gopalanb & G U Kulkarnib "Department of Chemistry, Bharathidasan University, Tiruchirappalli, 620024, India; bChemistry and Physics of Materials Unit, lawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India.

Blue copper proteins, in their oxidized form, exhibit a number of specific macroscopic properties like an intense blue colour with high extinction coefficient (> 3000 L morl cm-I around 600 nm), distinctive electron paramagnetic resonance spectra with low A II (30 - 60 x 10-4 cm- \) and unusually high reduction potentials (+ 184 to 680 mY vs NHE) that distinguish them from small molecule cupric complexes. These extraordinary properties are related to the specific coordination geometry as well as the nature of the coordinated donor atoms cysteine sulphur and methionine sulphur. A considerable research effort has been devoted to the synthesis of model compounds exhibiting the typical properties of type 1 copper proteins, for understanding the spectral features and the associated electronic structure of the blue copper si te, with the goal of relating the electronic structure properties to the active site geometry and reactivity. Several Cu(II) complexes containing benzimidazole and thioether donors have been synthesized as models for the active sites in the proteins and their structures and spectral and redox behaviour have been investigated. In the present work the copper(II) complexes of a series pentadentate ligands having N3S2 donor set have been isolated and studied using EPR and electronic spectral methods. The molecular structures of the complexes [Cu(Ll)](BF4h] [Ll =N, N'-bis{4'-(s"-benzimidazolyl) (methyl-3' thiabutyl }amine] and [Cu(L2)(BF4h] [L2 == N,N' -bis{ 4' -(2"-benzimidazolyl) (methyl)-3' -thiabutyl }amine] have been successfully determined by X-ray crystallography. Interestingly, one of the thioether sulphurs in the ligands occupy the axial position in the complexes exhibit positive redox potentials similar to proteins. The present work 120 INDIAN J CHEM, SEC. A, JANUARY 2002

c. el3 "',··t .... _-"

CD ... -. C14 C I. c'"

ICu(Ll )I(BF-lh also provides an opportunity to gain insight into the relative importance of the contributions from the coordination of thioether sulphur and the benzimidazole nitrogen, both of which are know to contribute to the positive CUll/CUI redox potential.

Synthesis and characterization of [Ru(NTB)Cht [NTB = tris(benzimidazol- 2-ylmethyl)amine] and its reactivity toward alkane functionalization

Mariappan Murali & MaIIayan Palaniandavar* Department of Chemistry, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.

Ruthenium-catalyzed oxidation of organic compounds has gained much attention in the light of bioinorganic interest, due to their relevance to heme and non-heme iron enzymes such as cytochrome P450 and methane monooxygenase (MMO) respectively. In the catalytic cycles of these enzymes, high-valent Fe =0 intermediates have been postulated and recently some intermediates in the MMO catalysis have been detected. Contrary to the Fe = 0 species, the stability of high-valent Ru = 0 complexes could enable us to study the properties and reactivities of putative and unstable active species and throw light on the iron oxidation chemistry. As an attempt to gain mechanistic insights into Ru-catalyzed oxidation reactions, which are related to those of iron compounds, we report herein the synthesis and characterization of a new mononuclear Ru(III) complex

C13 POSTERS 121 of the tripodal ligand tris(benzimidazole-2-ylmethyl)amine (NTB), capable of catalyzing alkane functionalization. The coordinated NTB ligand is expected to be robust against oxidative degradation. The structure of the title compound has been successfully determined by X-ray crystallography. The octahedral coordination geometry of the complex around ruthenium(III) is slightly distorted as seen from the N2-Ru-N6 bond angle of 162.1 (2)° which is less than the id~al angle of 180°. The Ru-Nbzim bond distances are shorted than the Ru-Npy distances found in its pyridine analogue [Ru(TPA)Cht [TPA = tris(2-pyridylmethyl)amine] and the Ru-Nam ine distance of 2.122(4) A is longer than that in the pyridine analogue [2.068(5) A], obviously due to the presence of bulky benzimidazole moieties. The complex shows an axial EPR spectrum and two redox waves corresponding to RuIl/RullI and RullI/Ru iV couples in MeCN solution. The title compound has been revealed to catalyze alkane functi.onalization . The reactivity of the complex will be discussed in comparison with that of its iron(III) analogue [Fe(NTB)Clzt (ref 1). Reference 1 Vi swanathan R, Palaniandavar M, Bal asubramani an T & Muthiah T P, illorg Chem, 37 (1998) 2943.

DNA recognation by simple metal complexes-Relevant to zinc fingers in TF III A

P Rabindra Reddy* & M Radhika Department of Chemi stry, Osmani a Uni versity, Hyderabad-500 0007, Indi a.

The interaction of DNA with simple model systems Zn-Cys-His, Zn-Cysme-Hisme, and Zn-CysGly-HisGly are probed by absorption spectroscopy. The decrease in absorbance, which is an index of the interaction between the DNA and zinc complexes was gradual and reached saturation at - 1:1. The findings tend to show two types of associations,first of these is a non-specific recognition of the sugar-phosphate backbone, by one of the imidazoles present, and the second, a specific interaction involving the appropriately placed amino group of zinc complex with the guanine base of DNA. The demonstration that relatively small structures of the type Zn Cys-His, Zn-Cysme-Hisme and Zn-CysGly-HisGly can interact with DNA would be of potential use in the design of diverse recognition elements. The interactions of other similar metal complexes are addressed.

IH-NMR studies on weakly spin-coupled paramagnetic binuclear metal complexes

Gnana Sutha S, Moni S & Narasimha N Murthy* Department of Chemistry , Indian In stitue of Technology, Madras 600 036, India.

Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to the chemist for the elucidation of the structure of diamagnetic molecules. Application of this technique to paramagnetic metal systems particularly with single metal-ion center is limited because of unfavourable electronic relaxations. Lately, these studies in binuclear systems have become increasingly useful in probing metalloprotein active-site 2 structure (e.g. SOC)I and of model complexes • However, fundamental studies on such systems are still largely unexplored. Besides, a new class of redox-active metalloproteins with binuclear transition metal-ion centers bridged by oxo, hydroxo or carboxylate groups, is emerging. So, study of synthetic (model) metal complexes to understand the structure and magnetic properties of such spin coupled binuclear centers is of immense value. We have designed and synthesized a variety of new weakly spin-coupled paramagnetic binuclear metal ligand complexes, [M2(L-O')(X)f+, having M =Cu(II), Ni(II), Co(ll), VO(II); L-O- =binucleating ligand with 0 and N donor atoms and X =three-atom bridging ligands. Studies on their structure and magnetic properties using a combination of ID and 2D IH-NMR spectroscopic techniques, along with synthesis and X-ray structural investigations will be discussed.

References 1 Clementi C & Luchinat C, Acc chern Res, 31 (1998) 351. 2 Murthy N N, Karlin K D, Bertini I & Luchinat C, JAm chem Soc, 119 (1997) 2156. 122 INDIAN J CHEM, SEC. A, JANUARY 2002

Electronic excited state structure and dynamics of donor-acceptor diarylbutadienes as probed by fluorescence and photoisomerization studies

D Manjula, S Kanvah, G R Mahalaxmi & Anil K Singh* Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India

Linear polyenes play important role in biological light-induced energy and sensory transduction. Consequently, the excited state properties and related photobiological processes of linear polyenes have attracted a great deal of attention in recent yearsI.2. However, complete understanding of the excited state electronic structure and dynamics of linear polyenes is not yet available. One of the problem relates to the involvement of conformationally/configurationally twisted dipolar excited states in the photoprocesses of these polyenes. In this context, a,w-diphenylpolyenes [C6Hs(CH=CH)nC6Hs] have been chosen as model system to study the nature of 3 si nglet excited state structure and potential energy surface of linear polyenes .4. In this study, several donor-acceptor diarylbutadienes have been synthesized and their UY -vis absorption, fluorescence excitation and emission, and photoisomerization properties examined in organic solvents. It has been found that the ground state of this dienes is largely insensitive to solvent polarity. In contras t, th e fluorescent singlet excited state is significantly influenced by solvent polarity, substituents and temperature. The donor-acceptor dienes exhibit pronounced solvatochromic fluorescence with re latively low fluorescence quantum yield in polar solvents. The dienes substituted with strong acceptor groups like -N02 and -COOR are found to exhibit unusually red-shifted fluorescence emission with high change in dipole moments of the singlet excited states. Further, the solvatochrornic fluorescence of these dienes linearly correlates with solvent parameters like ET(30) and n*. At 77 K, the fluorescence band undergoes blue shift. The time-resolved fluorescence of some of the dienes is characterized by single exponential fluorescence decay with generally increasing lifetimes in polar solvents. Irradiation of the dienes results in isomerization of one of the C=C double bond of the diene moiety. Only one-photon-one-bond isomerization is observed. It has been suggested that the fluorescence in non-polar solvent originates from the locally excited state. In polar solvent, there can be intramolecular charge transfer leading to a polarized but planar excited singlet state emitting at a longer wavelength. In some dienes (e.g. with strong acceptor groups line -N02 and -COOR) there can be a conformational relaxation across one of the single bond leading to a dipolar, non-planar intramolecular charge transfer excited state, which is highly stabilized in a polar environment, and hence fluorescence at much red-shifted wavelength. Such non-planar polar excited singlet state can be formed due to twisting of a single bond, preferably the bond connecting the acceptor (e.g .. -N02/-C02Me) and the donor arylidene group. Such molecular motions are inhibited at low temperature causing the seines in rigid environment to fluorescence at shorter wavelengths as compared to fluorescence at ambient temperature. These results can also be accounted in terms of state switching mechanism. The energy difference between the excited states of such compounds in rather low and changes in substituent pattern or in polarity of the media can alter the state order giving different photophysical and photochemical properties. The photoisomerization can occur from a C = C double bond twisted dipolar excited state. Such dipolar excited state can exhibit selective photoisomerization, depending on the substituent and solvent. Thus, it can be inferred that conformationally as well as configurationally relaxed 5 8 dipolar excited states can be involved in photoprocesses of these compounds - . Mechanism of formation and involvement of non-polar polar excited states in the photoprocesses responsible for biological photo-sensory and energy transductions is also discussed. These studies have brought out interesting features of the structure and potential energy surface of the singlet excited state of linear polyenes of biological significance. Financial assistance from BRNS (37/7 /95-R&D II1559) is gratefully acknowledged.

References 1 Birge R R, Biochim Biophys Acta, 10 I 6 (1990) 293. 2 Pepe J, J Photochem Photobiol B Biology, 48 (1999) I. 3 Allen M T & Whitten D G, Chem Rev, 89 (1989) 1691. 4 Arai T & Tokumaru K, Adv Potochem, 20 (1995) I. 5 Singh A K, Manjula D & Kanvah S, New J Chern, 23 (1999) 1075. POSTERS 123

6 Singh A K, Manjula D & Kanvah S, J phys Chem A, 104 (2000) 464. 7 Singh A K. & Mahalaxmi G R, Ph%chel11 PIIO/obiol, 71 (2000) 387. 8 Singh A K & Kanvah S, New J Chem. 24 (2000) 639.

Chemistry of some glycosylamines derived from protected and free monosaccharides

G Rajsekhar & Chebrolu Pulla Rao* Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology, Powai, Mumbai 400 076, India.

The results of research on nucleic acids, vitamins, co-enzymes, and inhibitors of enzymes have prompted us to investigate the glycosylamine reactions '. A few glycosylamines of 4,6-0-butylidene-a-D-glucopyranose have been synthesised using different aryl amines involving various donor atoms, in order to facilitate the synthesis of various model molecules suitable for metalloenzymes. The both mono and diglycosylamines exist in the ~ anomeric form and known to rearrange in solution. Since the glycosylamines are considered as active-site 2 directed, reversible inhibitors of glycosidases , these glycosidic products will be screened for the inhibition of glycosidases. The glycosidic products are characterized by analytical and spectral techniques and in a few cases by single crystal XRD. We thank Prof. E Kolehmainen, Prof. K Rissanen and Mr. P K Saarenketo of University of Jyvaskyla, Finland for NMR and single crystal XRD data.

Rreferences 1 Lalegerie B, Legler G, & Yon J, Biochimie, 64 (1982) 977. 2 Lai H Y L, & Axelord, Biochem Biophys Res COl11mlll1 , 54 (1973) 463.

Synthesis and characterization of 4,6-0-ethylidene-D-glucopyranosyl-amine based schiff base ligands and their metallochemistry

Ajay Kumar Sah & Chebrolu Pulla Rao'" Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology, Powai, Mumbai 400076, India.

Sugars are one of the most important building blocks of the biosphere, which assembles to result into the biopolymers of the cells such as polysaccharides and nuCleic acids. Complex carbohydrates play not only a structural role in the cell (Cellulose the major structural component of plant cell wall and peptidoglycans of bacterial cell walls) but may serve as a reservoir of chemical energy to be enlarged and depleted as the organism wishes. It has a number of metal binding sites but its interaction with the transition metal ions has not been well explored. Only during the past 10-12 years, some work on the structures of interaction of saccharides with transition metal ions has been appearing in the literature.' We have recently reported the results of interaction of alkali and alkaline-earth metal ions with a glycosylamine derived from the condensation of 4,6-0-ethylidene-D glucopyranose with anthranilic acid.2 Herein we are presenting the synthesis and crystal structures of a novel saccharide-schiff base ligand suitable for transition metal binding, derived from the condensation of 4,6-0- ethylidene-~-D -glu copyranosylamine with a series of substituted salicylaldehydes. A series of transition metal 2 2 2 2 ion complexes of Ni +, Cu +, Zn +, YO/, Mool+ and U02 + have also been synthesised using these ligands. In most 'of the cases the ligand and the metal complexes have been characterised based on single crystal XRD 2 study. Details of the ligands and their complexes with Cu + will be presented in the poster. We thank Prof. E. Kolehmainen, Prof. K Rissanen and Mr. P K. Saarenketo of University of Jyvaskyla, Finland for NMR and X-ray data. 124 INDIAN J CHEM, SEC. A, JANUARY 2002

References 1 Gyurcsik B & Nagy L, Coord chern Rev, 203 (2000) 81 and references cited therein. 2 Sah A K, Rao C P, Saarenketo P K, Wegelius E K, Rissanen K & Kolehmainen E, J chern Soc, Dalton Trails, (2000) 3681 and references cited therein.

Functionalization of calix[4]arene and herni-calix-naphthalenes

P Venkateswara Rao & Chebrolu P Rao* Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400 076, India

Calixarenes are versatile molecules possessing both hydrophilic and hydrophobic groups. They provide structural framework to develop model molecules upon introducing with appropriate functional groups. In this direction, the lower rim modification of calixarenes with pendant amine (2-aminomethylpyridine & 2- aminomethylbenzimidazole), amino acids and ester (Gly, Ala, Phe, Asp and Glu) have been synthesized and thoroughly characterized by techniques such as FfIR, NMR, MS. The structure of one of the amido-calixarene derived from 2-aminomethylpyridine was established by single crystal X-ray diffraction studies. The amino acid derived molecules further possess the side chain functional groups (Asp and Glu) and hence act as good candidates for metal ion binding. Such developments are expected to result in bio-mimicking of metalloenzymes. These molecules possess not only the metal ion binding groups from pendant amino acids but also have additional cation binding site (unprotected phenol groups and ether oxygens) and an arene cavity for inclusion of small molecules. The molecules have further potential to extend as dendrimers. Another set of molecules are developed from 2-naphthol known as hemi-calixnaphthalene, and these partly resembles one of the isomer of calix[4]naphthalene. Further the hemi-calixnaphthalene was derivatized with various groups and the resultant products were characterized both by spectral and crystallographic techniques.

a b

R' R" R'

2a H CH 3 3a H Gly (a) H2NCH(R')C02R" , Et3N, THF 3bCH Ala 2b CH3 CH 3 3 (b) LiOH, THF/H 0 3c CH Ph Phe 2 2c CH 2Ph CH 3 2 3d CH C0 H Asp 2d CH 2C0 2CH 2Ph CH 2Ph 2 2 3e CH CH C0 H Glu 2e CH2CH2C0 2Et Et 2 2 2

e ~~{I~

{I~ {I~ ~-- C>" 7 QNb POSTERS 125

Benzothiazoline and other glycosylarnine derivatives of saccharides: Interactions with metal ions

T Mohan Das & Chebrolu P Rao* Department of Chemistry , Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India

Glycobiology deals with different types of saccharide containing molecules: The roles played by saccharides in biology is largely unexplored when compared with the plethora of information available with proteins and nucleic acids I. Interaction of metal ions with saccharide molecules is inevitable in biological systems and this would certainly affect the activities of biomolecules such as glycoproteins or lipopolysaccharides2. Thio glycosides of different saccharides are prepared and the products are characterized using different spectral techniques. Interaction between D-glucobenzothiazoline and some transition metal ions has been studied and the isolated products have been characterized using analytical, spectral and electrochemical techniques and the corresponding results will be presented. N-Glycosides of 4,6-0-benzylidene D-glucopyranose were synthesized using different aromatic amines and the products were well characterized. The amines include both mono- and diamine types. Metal ions, such as, alkali, alkaline earth, main group and transition group elements are used to synthesise complexes with an N glycoside derived from anthranilic acid. The corresponding products are also characterized using different spectral techniques. Metal ion complexes of the N-glycosides derived from the benzimidazole derivative were also synthesized and characterized. In all the cases, the glycosides are found in /3-anomeric form, even if these were synthesized from an a-anomeric precursor. We thank Dr. E Kolehmainen, University of Jyvaskyla, for NMR data

References 1 Whitefield D M, Stojkovski S & Sarkar B, Coord Chem Rev, 122 (1993) 171; Angyal S J, Adv Carbohydr Chem Biochem, 47 (1989) 1. 2 Kennedy J F & White C A, Bioactive carbohydrates in chemistry, biochemistry and biology; (John Wiley & Sons, New York) 1983 .

Mono-, di- and tetranuc1ear complexes of eo(HI), Ni(H), eu(H) and Zn(H) with hydroxy( -OH)-rich molecules: Syntheses and crystal structures and catecholase activity of eu(H) complexes

Mishtu Dey & Chebrolu Pulla Rao* Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology, Powai, Mumbai 400076, India

The chemistry of transition metal ion complexes of hydroxy (aryl-OH and alkyl-OH) containing molecules is of great importance in the context of the biomimetic studies of metalloenzymes, where the active centers are associated through such groups via coordination. The coordination chemistry and biomimetic aspects of molecules containing -OH groups have taken a lead position in the recent literature. Our efforts pertaining to 2 2 oxo-metal centres of vanadium (cis- V02+) I , molybdenum (cis-Mo02 +)2, uranium (trans-U02 +)3 and non-oxo Ti(IV)4 resulted in understanding the coordination chemistry aspects of a number of -OH containing molecules including their structural diversities and reactivity aspects. However, such aspects are not well addressed in the literature in the case of Co(III), Ni(II), Cu(II) and Zn(II), while these are essential trace elements of biological systems. Therefore, we have focused our attention in developing the coordination chemistry aspects of -OH containing ligands of these metal ions. These complexes have been characterized by analytical, spectral, magnetic and electrochemical methods including single crystal X-ray diffraction studies. The Cu(II) and Ni(II) complexes have exhibited characteristic core conversion reactions. Further, the Cu(II) complexes have shown substantial catecholase activity. 126 INDIAN J CHEM, SEC. A. JANUARY 2002

References I Asgcdom G, Srccdhara A, Kivikos ki J, Kolehmainen E & Ra o C P, Illorg Ch elll , 35 (1996) 5674. 2 Ran C P, Sreedh ara A, Venkatcswara Rao P. Vcrghcse M Bindu, Riss an en K. Kol chm ain cn E. Lokanath N K, Srid har M A. & Prasad J Sashidhara , J chelll Soc. Daltoll TrCIIIS. ( 1998) 2383. 3 Vc nka tcswara Ran P, Rao C P, Srccdh ara A, Wcgclius E K. Rissancn K & Kol ehmai nc n E. J chelll Soc, Daltoll Trail s, (2000) 12 ! 3 . .:I Vcnkatcswara Rao P, Rao C P, Wege li us E K, Rissancn K & Kolch maincn E, J chelll Soc, Daltoll Trails, ( 1999) 4469.

Antitumor, anti-HIV and antibacterial activities of some Ru(H) and Ru(Hl) polypyridyl complexes

Lal lan Mishra*, Ajay K Yadaw, Ragini Sinha & Ashok K Singh Department of Chemistry, Facu lty of Science Banaras Hindu University, Varanasi 221005, Indi a

So me ruthenium (IT/III) polypyridyl complexes have been synthesized and characterized using spectroscopic tec hniques (IR, IH/ DC, IH_IH COSY, TOCSY (Total correlation) NMR, UVIVis} along with molar conductance, elemental analysis and FAB -mass data. The interaction of these complexes with buffered calf thymus DNA solution has also been monitored usin g UYIVis, IH NMR and luminescence techniques. Hyperchromism observed in the MLCT regions (420A80 nm) on addition of calf-thymus DNA solution (citrate buffered 0.30 mg/mL, pH 8.6) indicated that complex bind with DNA electrostatically. However, a plot of DNA base pairs/complex vs. luminescence anisotropy for a representative dinuclear complex [Ru2L1(phen)4]

(PF6h.3f-h) (LIH2 obtained by coupling of diazonium chl oride of 3-amino-l,2,4-triazole with acetyl acetone and L3 = 4-amino-3-hydrazino-5-marcapto-l ,2,4-triazole) have been found very active especially against A549 (lCso, 1.14 each) and IA9 (ICso , 0.80 and 0.90) respectively. Same complexes also showed significant anti -HIY activity. Corresponding IC so values 0.33 and 10.60 ~g/mL) for these complexes were found many fo ld lower than that of standard compound AZT (azido thymidine) (lCso, 500 ~g/mL). In a different class of drugs two categories of Ru(II)/Ru(lII) complexes containing 2,6-bi s(2' benzimidazolyl) pyridine and chalcones have also been synthesized, characterized and evaluated fo r their 4 6 antitumor and anti-HIY activities . . Additionally, some mononuclear ruthenium polypyridy I complexes containing 3'-hydroxyA'-substituted f1 avones as co-ligands have also been developed7 which showed interesting antibacterial activities against E.coli however these complexes did not show their interaction with DNA as monitored by the electrophoretic mobility of DNA in the presence of these complexes. References Mi shra L, Yadaw A K, Choi C S & Araki K, Indian J Chel1l , 38A (1999) 339. 2 Mi shra L, Yadaw A K, Srivastava S & Patel A B, New J Chem, 24 (2000) 505. 3 Mi shra L & Yadaw A K, Illdiall J Chelll, 39A (2000) 660. 4 Mishra L & Sinha R, Indiall J Ch elll . 39A (2000) 1295. 5 Mi shra L & Sinha R, Illdian J Chel1l. 39A (2000) 1131. 6 Mi shra L, Sinha R, Itokawa H, Bastow K F, Tachibana y , Nakanishi N, Kilgore N & Lce K H, J Bio·organic alld lIl edicin al Chemistry, (2001) [acceptedj. 7 Mi shra L & Singh A K, Indian J Chelll, (2001) [acceptcd].

Spin quantitation in electronic structural analogue of active form of galactose oxidase

S Y Rane*"', D R Thube", S K Ingalea & D Srinivasb "Department of Chemistry, Univcrsity of Pune, Pune 4 11 007, India bNationa l Chemical Laboratory, Pune 411 008, Indi a

Reactivity of free radicals like Tyr-272, topasemiquinone is utilized by nature as motive power for transformation of alcohol, amine to aldehyde in respective metalloenzymes viz. galactose oxidase (GO's), amine POSTERS 127

oxidase (AO' s) I. Inactive form of apoenzyme signifies crucial role of CUll site in quinoenzymes. Although recent 2 2 2 reports ,4 on structural/functional models with phenoxyl radical coordinating Cu +/Zn + suggest mechanism of catalytic activity in GO's,class-II type interaction between Cu2+- Tyr radical and prime role of Cu2+ site is still remained obscure. Our former efforts to establish symbiotic relation in functional molecular assembly5.6 of quinoenzymes in hydrophobic and hydrophilic environments provoke us to report electronic structural analogue of GO's in the present paper. Redox interactions between copper ion and spin carrier orthofunctionalised p-naphthoquinoneoxime ligand result in naphthosemiquinoneoxime (NSQox) radical coordination subject to electronic control of Cu2+ environment. Mimics of native enzyme met with spectroscopic characteristics of Cu2+ NSQox interactions, established from spin quantitation and charge transfer studies. Thermal and electrochemical studies of model compound set its thermodynamic behaviour in solid and solutions states. Electronic structure of model compound is confirmed such as [Cu2+(2-oxido-1,4-NQox)(4-0H-2-oxido-1,2-NSQox)], Its catalytic activity investigations are in progress which will provide insight in its mechanistic aspect.

References 1 Fontecave M & Pierre J L, Coord Chem Rev, 170 (1998) 125. 2 Chaudhari P, Hess M, Florke U & Wieghardt K, Angew Chem Int Ed Eng, 37 (1999) 2217. 3 Itoh S, Taki M, Takayama S, Nagatoma S; Kitagawa T, Sakurada N, Arakawa R & Fukuzumi S, Angew Chem Int Ed Eng, 38 (1999) 2774. 4 Yamato K, Takanori I, Doe M, Akio I, Takeji T, Kojima Y, Kikunga T, Nakamura S, Naka T & Yano S, Bull chem Soc Japan, 73 (2000) 903. 5 a) Rane S Y, Karacan M.S, Somer G, Karcan N M, Khan E M, Allkhverdiev S I, Padhye S, Kilmov V V & Rengar G, Biochemistry, 33 (1994) 12210; b) Rane S Y, Khan E M, Thube D R, Padhye S B, Bakare P P & Date S K, Indian J Chem Sec- A, (2000) in press. 6 Rane S Y, Gawali S D, Kumbhar A S, Padhye S B & Bakare P p, J Thermal Anal Cal, 55 (1999) 249.

Synthesis, crystal structure, spectral studies and catechol oxidase activity of trigonal bipyramidal eu(II) complexes derived from a tetradentate diamide bis-benzimidazole ligand

Manisha Gupta, Sanjib Kumar Das & Pavan Mathur* Department of Chemistry, University of Delhi, Delhi 110 007, India and Ray J Butcher Department of Chemistry, Howard University, Washingtoh DC-20059, USA

A new benzimidazole based diamide ligand, N,N'-bis(glycine-2-benzimidazolyl) hexa-nediamide, [GBHA], has been synthesized and utilized to prepare Cu(II) complexes of general composition [Cu(GBHA)X]X, where X is an exogenous anionic ligand (X=C1', N03', SCN'). X-ray structure of one of the complex [Cu(GBHA)Cl] Cl.H2).CH30H has been obtained. The compound crystallizes in monoclinic space group C2/c with unit cell dimensions, a=26.464(3)A, b= 1O.221O(8)A, c=20.444(2)A, a=90°, 13= 106.554(7)°, y=90°, V=5300.7(9)N, Z=8. To the best of our knowledge [Cu(GBHA) C1] Cl.H20 . CH30H complex is the first structurally characterized mononuclear TBP Cu(II) bisbenzimidazole diamidecomplex having coordinated amide carbonyl oxygen. The coordination geometry around the Cu(II) ion is distorted trigonal bipyramidal ('t = 0.59). Two carbonyl oxygen atoms and a chlorine atom form the equatorial plane, while the two benzimidazole imine nitrogen atoms occupy the axial positions. The geometry of the Cu(II) centre in the solid state is not preserved in DMSO solution, changing to squara pyramidal 1, as suggested by the low temperature EPR data gil> g.L > 2.0023. All the complexes display a quasi-reversible redox wave due to the Cu(II)/Cu(l) reduction process. EV2 values shift anodically from Cl- < N03- < SCN', indicating that bound Cl- ion stabilizes Cu(II) ion while N bonded SCN' ion destabilizes Cu(II) state in the complex. When calculated against NHE, the redox potentials tum out to 2 be quite positive as compared to other Cu(II) benzimidazole bound c0Il1plexes . It is therefore concluded that ll binding of amide carbonyl oxygen destabilizes Cu(II) state. The complex [Cu (GBHA)(N03)](N03) could be 128 INDIAN J CHEM, SEC. A, JANUARY 2002

I successfully reduced by the addition of dihydroxy-benzenes to the corresponding [Cu (GBHA)](N03). IH NMR of the reduced complex shows slightly broadened and shifted IH signals. The reduction of Cu(II) complex presumably occurs with the corresponding 2eo oxidation of the quinol to quinone. Such a conversion is reminiscent of the functioning of a copper containing catechol oxidase from sweet potatoes3 and met form of the enzyme tyrosInase. 4 .

References I Patra A K, Ray M & Mukherjee R, J chern Soc, Dalton Trans, (I 999) 2461. 2 (a) Nakao Y, Onoda M, Sakurai T, Nakahara A, Kinoshita L & Ooi S, lnorg Chirn Acta, 151 (1988) 55; (b) Addison A W, Hendricks H M J, Reedijk J & Thompson L K, lnorg Chern, 20 (1981) 103; (c) Palaniandavar M, Pandiyan T, Laxminarayan M & Manohar H, J chern Soc, Dalton Trans, (1995) 457; (d) Sakurai T, Oi H & Nakahara A, lnorg Chirn Acta, 92 (1984) 131. 3 Kalbunde T, Eicken C, Sacchettini J & Krebs B, Nat Struct Bioi, 5 (1988) 1084. 4 Solomon E I, Baldwin M J & Lowery M D, Chern Rev, 92 (1992) 521.

Synthesis, characterization and DNA binding studies of ruthenium(II) complexes of modified 1,10 phenanthroline ligands

C V Sastri, D Easwaramoorthy'& Bhaskar G Maiya*t School of Chemistry, University of Hyderabad, Hyderabad 500 046, India 'Crescent Engineering College, Vandalur, Chennai 600048, India tEmail : [email protected]

Metal complexes of polypyridyl ligands of the type [M(phenhLL] n+, where LL is either 1,10 phenanthroline (ph en) or modified phen, are increasingly being employed in studies related to the development of DNA binding and cleaving agents for use in various biochemical and biomedical applications. Recently we have reported ruthenium(II) complexes of ligands derived from appropriate modification of 1,1O-phenanthroline (phen) and 103 their DNA interactions • In this paper, results of analogous studies carried out with ruthenium(II) complexes of new modified phen ligands are presented (structure I). POSTERS 129

References 1 Arounaguiri S & Maiya B G, lnorg Chern, 38 (1999) 842. 2 Ambaroise A & Maiya B G, lnorg Chern, 39 (2000) 4256. 3 Ambaroise A & Maiya B G, lnorg Chern, 39 (2000) 4264 ..

Physico-chemical studies on chiral complexes and their reactivity towards DNA

Fareeda Athar, Farukh Arjmand* & Sartaj Tabassum Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India

New asymmetric ligands have been synthesized by condensing Q-phenylene diamine with CS2 and PhCHOlMeCHO, and their complexes with Mnll, COlI, Nill, CUll and ZnlI were prepared and characterized by elemental analyses, conductivity measurements, IR, UV-vis, EPR and NMR spectra. The transition metals in the complexes show squ~re planar geometry and are ionic. Photokinetic studies of the DNA-metal complexes [CIOHIOS4N2CU](N03)2 and [CIOHIOS4N2Ni](N03h. were carried out and the rate constants K'(DNA-complex) were calculated. The results indicate that DNA reacts with the metal complex in two steps. DNA first undergoes structural degradation and is then completely hydrolysed as indicated by spectral changes consistent with earlier results. The symmetric N2S2 macrocyclic metal complexes show a strong propensity for DNA inhibition and can be used as an intercalating binding model.

Mechanism of reduction of a vitamin B 12 model compound by vitamin C

Bishnu Prasad Panda & Nirod Kumar Mohanty* Post Graduate Department of Chemistry, Sambalpur University, Jyotivihar 768 019, India

It is known that vitamin C reduces vitamin B 12. It is further known that regular intake of large doses of vitamin C may lead to BI2 deficiency. Studies on the kinetics and mechanism of the reaction between vitamin C and B I2 is therefore of interest. In this investigation wehave chosen ~ model impound of B 12 and studied its reaction with vitamin c. .Such study may be of some consequence in understanding the mechanism of reaction between B 12 and vitamin C. In this communication, therefore, we report the kinetics and mechanism of reduction of chloropyridinecobaloxime(III), a vitamin B12 model compound by ascorbic acid (vitamin C). The reaction has been studies under second order condition at pH range 4 to 5.6 and temperature range 35 to 45°C. The second order rate constant increased with increasing pH. Determination of stoichiometry shows that one mole of oxidant combines per mole of reductant. Kinetic results indicate that the reaction proceeds through two pathways. Co(I) and dehydroascorbic acid have been visualized as products of reaction. The rate and activation parameters have been computed and a suitable mechanism has been proposed.

Ultrafast photophysics and photochemistry in solution

D Goswami* Tala Institute of Fundamental Research, Mumbai 400 005, India and E W Castner, Jr. Department of Chemistry, Rudgers University, New Jersey, USA

In solution, many chemical processes and reactions occur on ultrafast timescales ranging from 10-14 and 10-10 seconds . . Because of the rapid intermolecular fluctuations that occur, this time scale is more rapid in the liquid phase than in the gas phase. Femtosecond light pulses can thus play an important role in inducing and probing 130 INDIAN J CHEM, SEC. A, JANUARY 2002 transient photophysical and photochemical processes in solution. A chirped pulse regeneratively amplified Ti:sapphire laser system has been used to make a frequency-doubled (-400 nm) excitation pulse and a broad band white-light continuum (-425-1100 nm) as the probe pulse. The results on the singlet-to-triplet intersystem crossing (ISC) dynamics in tris-bipyridyl-ruthenium ion [(bpYhRu2+] in water and the excited state photophysics of cobalt (II) octaethylporphyrin (OEP) in toluene and penta-amine ruthenium pyridine ion [(NH3)5Ru(py)f+ in water are presented herein. Though the excitation of (bpYhRu2+ is a strongly allowed singlet transition, the widely studied photochemistry of this species occurs from the MLCT state. This state is characterized by an emission lifetime of -1 /-ls at 620 nm. On excitation of aqueous (bpy hRu2+ with a femtosecond laser pulse, a pulse limited drop in the ground-state recovery signal at 450 nm (Fig. 1) has been observed. These observations indicate that intersystem crossing from the singlet to the triplet manifold occurs in less than 150 fs.

o Cl -5 -1 0 § o o o

00 0_5 , _0 15 2.0 10 20 30 Time (picoseconds) Time Delay (ps) Time (ps) Fig. 1-Evidence of fast ISC Fig. 2-Fast decay of excited Fig. 3-Absorption transient for in aqueous [(bpYhRu)2+ ion. [(NH3hRu(py)f+ in water. CoIlOEP.

Typically, ruthenium(lI) octahedral complexes are characterized by well-characterized MLCT excited states, with lifetimes in tens of nanoseconds I. However, we show that the excited state decay of the octahedral ruthenium(lI) ion can also be very fast (2.75 ps) as seen from our transient absorption recovery of excited [(NH3)5Ru(py)]2+ in water at 450 nm (Fig. 2). Similarly, by probing the absorption spectrum of Coll(OEP) before, during and after photoexcitation (Fig. 3) results in resolving a 6 ps exponential decay time. Such fast dynamics might provide new insights to the current understandings of the charge-transfer dynamics of transition metal complexes.

Reference 1 Holten D &Gouterrnan M, Optical properties of polypyridine and porphyrin complexes, (Academic Press, New York), 1992, pp. 626.

Co-ordination chemistry of eu & Zn: Models of superoxide dismutase

LaBan Mishra* & Kumari Bindu Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

Dinucleating ligands having two metal binding sites bridged by a pyrazine moiety have been designed and synthesized as model ligands for copper and zinc superoxide dismutase (Cu, Zn, Sod); the corresponding mono nucleating complexes have also been synthesized for comparison. The above complexes have been characterized by spectral (lR, NMR and UV/visible) data along with their elemental analysis and FAB mass data. The ESR spectra of complexes exhibited signals due to the spin-spin interaction between two copper ions while the ESR spectrum of the Cu - Zn heterodinuclear complex shows a signal which is characteristic of mononuclear copper complexes. Model studies using the reported procedure have also been carried out in addition to the electrochemical properties Cu(II) complexes. POSTERS 131

Self-activating nuclease activity of helix-like chain copper (II) complexes of hydroxyl-rich ligands

A vinash S Kumbhar, Sunil S Tonde & Subhash Padhye* Department of Chemistry, University ofPune, Pune 411 007, India · and Ray J Butcher Chemistry Department, Howard University, Washington D.C. 20059, USA

Copper(II) complexes of Schiff bases derived from [1+1] condensation of salicylaldehyde, 2,3- dihydroxybenzaldehyde and 2,3,4-trihydroxybenzaldehyde with anthranilic acid have been synthesized and characterized by elemental analysis, IR, UV -visible spectra, room temperature magnetic susceptibility, EPR and cyclic voltammetry. The X-ray structure of [CULl]" where Ll stands for the tridentate Schiff base derived from the condensation of salicylaldehyde and anthranilic acid has been solved and refined to R=O.0314. The crystals are monoclinic of space group P2 1 with cell constants a=9.6820(13), b=7.1446(11), c=9.9315(13)A, ~ = 98.385(8)°, Z = 2. The copper(II) ions are in a square planar environment sequentially bridged by carboxylate groups in the syn-anti conformation giving rise to a helix-like chain. All the complexes cleave plasmid pBR322 DNA without any oxidizing or reducing agent by a self-activating mechanism probably by the localized generation of singlet oxygen or singlet oxygen-like entity.

References 1 Rardin R L, Tolman W B & Lippard S J, New J Chern, 15 (1991 ) 417. 2 Colacio E, Dominguez-VeraJ, Costes J-P, Kiveks R, LaurentJ-P & Sundberg M, Inorg Chern, 31 (1992) 774. 3 Kumbhar A S, Damle S G, Dasgupta S T & Rane S Y, J chern Res, 98 (1999).

Molecular modeling of protein phosphatases 1 and 2a with three various dications 2 2 2 2Zn+ , 2Mn+2 and Zn+ -Fe+ , and selected bioligands in the active centers

Edyta Wozniak-Celmer*, Stanislaw Oldziej & Jerzy Ciarkowski Faculty of Chemistry, University of Gdansk, Sobieskiego 18,80-952 Gdansk, Poland E-mail: [email protected]

The oligomeric metalloenzymes protein phosphatases dephosphorylate OH groups in SerfThr or Tyr residues of proteins. The catalytic units of SerfThr protein phosphatases 1, 2A and 2B (PP1c, PP2Ac and PP2Bc, respectively) while retaining -45% sequence similarity, have their active centers practically identical. This feature gives a strong argument that the unknown structure of PP2Ac could be successfully homology-modeled 132 INDIAN J CHEM, SEC. A, JANUARY 2002 from PPlc and/or PP2Bc, whose structures are known. A theoretical model of PPlc has been built, implementing a phosphate and a metal dication in its catalytic site. The latter has been modeled, together with a structural hydroxyl anion, as a triangular pseudo-molecule (ZNO, MNO or ZFO), composed of two metal cations (double Zn+2 or Mn+2 or Zn+2_Fe+2, respectively) and the OH- group. To the free catalytic unit two PP1c 29 4o 30 inhibitor sequences R RRRPpTPAMLFR of DARPP-32 and R RRRPpTPATLVLr2 of Inhibitor-I, and two putative substrate sequences LRRApSVA (PP2A substrate) and QRRQRKpRRTI (PPI substrate) have been subsequently docked. In the next step, a free PP2Ac model has been built via homology re-modeling of the PPlc template and four of the same sequences have been e docked to it. Thus, together, 30 starting model complexes have been built, allowing for the combination of the ZNO, MNO and ZFO pseudo-molecules, free enzymes and the peptide ligands docked in the catalytic sites of PP1c and PP2Ac. All models have been subsequently submitted to 250-300 ps molecular dynamics (MD) using the Amber5.0 program and the final 50 ps of equilibrated trajectories have been taken for further analyses. The theoretical models of PPlc complexes, notwithstanding the dication type, exhibit increased mobilities in the following residue ranges: 195-200, 273- 278, 287-209 for the inhibitors sequences and 21-25, 194-200, 222-227, 261, 299-302 for the substrate sequences. Paradoxically, the analogical models appear much more stable in similar simulations for PP2Ac, where only residues 6-10 and 14-18 exhibit slightly increased mobility in the inhibitor complexes while no areas of increased mobility have been found in the substrate complexes. Another general observation is that the complexes with Mn dications are more stable than those with Zn and Zn2+_Fe2+ dications for both PPlc and PP2Ac units. The PPl/PP2A theoretical models incorporating the ZFO and the ZNO dications appear to be the least and the most labile, respectively. Depending on the bioligand, the PPIcIPP2Ac appear to be most flexible in the following areas (PP1c numbering): DARPP-32: the residues 20-26; 210, 287-290 (ZNO, MNO dications), Inh-l: 196-201,276-277,301-304, PP2A substr: 22-27,194-198,273-276,298-304 (ZFO, MNO dications), PP1 substr: 20-27, 97-98, 132-135 (ZFO,MNO dications), 184-190, 198-201, Free enzymes: 6-26, 130, 196-216,223-226,262-263,274-276. The results suggest that the homology-modeled PP2Ac, both with and without the selected bioligands, is stable in the MD time-scales typical of this work. An increased mobility of the PP2Ac prosegment - the only part having no sequence homology with PPlc - suggests the PP2Ac pro segment ultimate geometry to be different than that of its PPlc template.

Acknowledgement This work was supported by Grant 3 T09A 045 19 from the Polish State Committee for Scientific Research.

Quantum chemical studies on the interaction of cisplatin with guanine

Prasad V Bharatam*, Rajnish Moudgil, Kavita Sharma & Damanjit Kaur Department of Chemistry, Guru Nanak Dev University, Amritsar' 143 005, India

Cisplatin cis-[Pt(NH3hCh], 1 is an anticancer agent with relatively high toxicity'. It is highly effective in treating testicular, ovarian, cervical, bronchogenic, oropharyngeal and bladder cancers and is also active against lymphoma, osteosarcoma and melanoma neuroblastma'-3. DNA is the major target of cisplatin in the cancer cells (forming 2) and this interaction is responsible for its antitumor activitl. Ab initio calculations using effective core potentials (ECPs) have been carried out on cis-[Pt(NH3hCh], [Pt(NH3)4]2+, [cis-(NH3hPt(HNCH2h]2+, [cis (NH3hPt(pyh]2+ and [cis-(NH3)2Pt(guanineh]2+ etc. as models for the Pt-DNA interactions to quantitatively estimate electron distribution, energy of stabilizations, etc. Comparison of stabilization energies indicate that there is a gradual increase in the stabilization in the order of increasing Lewis basicity of the replacing ligands. Replacement of N'H3 in [(NH3hPt(N'H3h]2+ by guanine leads to a stabilization of about 71.2 kcallmol. POSTERS 133

1 2

References 1 Wong & Giandomenico, Chern Rev, 99 (1999) 2451. 2 Jamieson & Lippard, Chern Rev, 99 (1999) 2467. 3 Pavankumar, et ai., J cornp Chern, 20 (1999) 365.

Hydroxylation of phenethylamine by a copper(II) complex modeling the CUB site of dopamine ~-hydroxylase

Arindam Mukherjee, Manas K Saha & Akhil R Chakravarty* Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India

Dopamine ~-hydroxylase(D~H)1 is a copper containing metalloenzyme. The active CUB site of the enzyme is known to catalyze the hydroxylation of dopamine at the ~-carbon position. Using a mononuclear copper(ll) complex CullL[ L = bis(salicylaldirninato)-N,N' -(2-hydroxy-propane-2-yl)]2, we have been able to hydroxylate

A B [QJILl Ascorbic acid

. J I -118T~ "122I

B "167

Cntaly1ic cycle I I I. . "160 .,70 rn/z ~ the aromatic and aliphatic C-H bonds in the phenethylarnine thus modeling3 the CUB site of D~H. Complex CullL on reaction with ascorbic acid forms a copper(l) intermediate which in the presence of oxygen reacts with phenethylamine(A) to give a trihydroxylated species. The product has been tentatively analyzed as

(OHhC6H3CH(OH)CH2NH2 (B) from the GC mass spectral studies (Fig. I). The catalytic c0pper(II) species has been successfully functionalized at the aliphatic -OH position with a variety of carboxylic acid viz. propionic acid and acrylic acid. Further studies are on to heterogenize the homogeneous catalyst on a polymer matrix.

References 1 Abolmali B, Taylor H V &Weser U, Struct bond, 91 (1998) 91. 2 Kitajima N, Whang K, Uchida A & Sasada Y, Chern Cornrnun, (1986) 1504. 3 Reddy PAN, Datta R & Chakravarty A R, Inorg Chern Cornrnun, 3 (2000) 322. 134 INDIAN J CHEM, SEC. A, JANUARY 2002

Reactivity of binuclear copper complexes with O2: The first example of aromatic ring hydroxylation by copper(II)

Narasimha N Murthy Department of Chemistry, Indian Institute of Technology, Madras 600 036, India

Binuclear transition metal-ion centers are quite prevalent at the active-sites of metalloproteins processing dioxygen. For example, hemerythrin (Rr) and methane-monooxygenase (MMOH) involved in O2 transport and methane (CR4) hydroxylation reaction, respectively, contain non-heme diiron centers. Dimanganese catalase enzyme is involved in the disproportionation of hydrogen peroxide (H20 z) to less toxic dioxygen (Oz), while the photo-induced water oxidation catalyst (WOC) in plants which oxidizes two molecules of water to dioxygen, as part of photosynthesis, possess two interconnected dimanganese centers. Similarly, binuclear copper containing hemocyanin (Hc) transports dioxygen in lower organisms, while tyrosinase (Tr) incorporates oxygen atom from dioxygen into phenolic substrates. Therefore, studies on the interaction of dioxygen (Oz) with transition metal ion (Cu, Fe and Mn) model complexes are of current interest not only to gain better understanding of the structure and function of the above metalloproteins but also to get insights into industrially important metal catalyzed oxidation processes. As part of our continued investigations on the interaction of binuclear copper(l) centers with dioxygen', we have synthesized binuclear copper(II) complexes [Cuz(L)(OAc')z]2+, where L is a binucleating ligand derived from 1,3-phenylenediamine in which the amine hydrogens are replaced by 2-pyridylethyl donor groups. To our surprise, even these copper(II) complexes react with dioxygen resulting in [Cuz(L-O-) (Oac-)f+, in which the phenyl ring which is part of the ligand system has been hydroxylated. This is the first example of such aromatic C-H hydroxylation brought out by dioxygen and copper(II). Results of these investigations have been discussed.

Reference 1 Murthy N N & Karlin K D, Mechanistic bioinorganic chemistry, Advances in chemistry series, edited by H H Thorp and V L Pecoraro, (American Chemical Society) (1995), Ch.6, pp. 165.

Peroxynitrite reactioris with nucleotides and antioxidants

2 K Indira Priyadarsini'*, S Santosh Kumar , Harimohan' & K B Sainis2 IRC & CD Division, 2Cell Biology Division Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

Peroxynitrite (ONOO-) is produced in vivo by diffusion controlled reaction of nitric oxide (NO) with superoxide (Oz} Under physiological conditions, it exists in equilibrium with its protonated form. While the anionic form is stable, the protonated form isomerises to nitrate with a rate constant of 1.3 s-' (ref. I). It has been found to cause irreversible damage to several important biomolecules like cell membrane, DNA, proteins and enzymes. Its formation has been implicated in several diseases like atherosclerosis, inflammatory conditions, neurological 2 disorders, etc . Peroxynitrite (PON) can react with different endogenous antioxidants such as ascorbate, (X tocopherol and thiol containing molecules which form the primary defense against oxidative stress leading to their depletion and reduced availability. Hence exogenous compounds, specially of dietary origin that are capable of scavenging reactive nitrogen species may playa pivotal role in preventing/controlling degenerative 3 diseases . It reacts with substrates in different ways such as one and two electron oxidations, nitration, nitrosylation and oxygen transfer. Earlier, we have tested the antioxidant properties of two natural phenols, ellagic acid and vanillin against reactive oxygen species. Ellagic acid, found in fruits and nuts has been shown4 to have better antioxidant properties than vitamin E. Vanillin, a natural plant phenol is used as a food-flavouring agent in chocolates, cakes 5 and ice creams . In our laboratory experiments, these antioxidants have prevented peroxynitrite induced single strand breaks formation in plasmid pBR322 DNA. To understand the mechanisms involved in the observed POSTERS 135 protection, stopped-flow spectrometer has been employed. Using this technique kinetics of reaction of peroxynitrite with individual components like nucleotides, sugars and antioxidants have been studied at pH 7 by monitoring the absorbance of peroxynitrite at 302 nm. In case of ellagic acid and vanillin, in addition to the decay of peroxynitrite, formation of the new absorption signals in 400 nm region due to nitrated products have also been followed. Rates of the reactions have been monitored by exponential fitting of either the decay or formation signal. Based on the rate constants, the most probable site of attack on DNA could be deoxyguanosine and only a minor fraction goes to sugar and different bases. Both ellagic acid and vanillin show very high reactivity towards peroxynitrite by oxidation and nitration reactions, which may be responsible for their DNA protecting activity. The rate constants of peroxynitrite with ellagic acid and vanillin are in the order of 3700 ± 350 and 5627 ±485 M's·' respectively, whereas rate constants of nucleotides and sugar vary from 1 to 30 M's·'.

References 1 Koppenol W H J, Moreno J J, Pryor W A, Ischiiropulos H & Beckman J S, Chern Res Toxieol, 5 (1992) 834. 2 Smith M A, Richey H P, Sayre L M, Beckman J S & Perry G, J Neurosci, 17 (1997) 2653. 3 Ames N B, Shigenaga M K & Hagen T M, Proc Natl Acad Sci USA , 90 (1993) 7915. 4 Hassoun E A, Walter A C, Alsharif N Z & Stohs S J, Toxicology, 124 (1997) 27 . 5 Prince R C & Gunson D E, Trends Biochern Sci, 19 (1994) 521.

Metabolism, maturation and capacitation in Spermatozoa: Role of calcium ion

Sudha Srivastava National Facility for High Field NMR, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India

During recent years, the presence of compounds related to sperm motility and the effects of various chemical and physical agents on sperm motility in both in vivo and in vitro systems have been studied extensively to reveal control mechanisms or causative factors of sperm motility more precisely. Elements and ions are known to influence sperm motility directly or indirectly. Calcium ions are well known to play both a critical and multifaceted role in the process of mammalian fertilization. These are essential for the acrosome reaction in spermatozoa for all species. Here we present the effect of various external factors such as UV radiation, cold shock, metabolic inhibitors, metabolic activators, on metabolism and maturation of spermatozoa. Role of Ca+2 ion in sperm capacitation and its mechanism of action has been discussed.

Characterization of a cell lytic enzyme from Pseudomonas sp

Suresh Shastryt* & M S Prasad Central Food Technological Research Institute, Mysore, 570 013, India tpresent address :Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India E-mail: suri @tifr.res. in

Microbial gums confer viscosity in aqueous systems fecilitating their applications in food and non-food industry. Xanthan gum from Xanthomonas campestris has wide applications in food, pharmaceutical, agricultural, pesticides, printing, textiles and oil recovery operations.The presence of bacterial cell biomass in xanthan poses a major problem during recovery of cell-free microbial gums due to the high viscosity of gum. Therefore, there is a need to identify an enzyme that is active at high viscosity for cell digestion in viscous gum solutions. An extracellular cell lytic activity against Xanthomonas campestris has been isolated from Pseudomonas sp. The lytic action of crude culture filterate on X. campestris has been observed by scanning electron microscopy. The enzymatic digestion of X. campestris cells cause loss of cell shape and lead to an aggregated debris. The enzymatic action has been uniformly distributed on cell surface. Its action on membrane proteins is monitored 136 INDIAN J CHEM, SEC. A, JANUARY 2002 by SDS-PAGE. The decline in absorbance recorded during cell digestion and lysis of SDS-soluble membrane proteins could be correlated as membrane proteins have a possible role in maintenance of cell integrity. The cell lytic enzyme has been purified by fractionation on Biogel P-lOO column chromatography.The enzyme has a 35kDa alkaline serine protease associated with a l5kDa protein. The observation of peptides and amino acids in HPLC profile of cell hydrolysate at A280nm confirms the cell lytic aCtion of the enzyme. TEM of X. campestris cells treated with the enzyme show an intial removal of outer cell membrane leading to cell disintegration. SDS PAGE shows that the purified enzyme degraded both the 29kDa and 26kDa fractions of casein.

Acknowledgement This work was supported by fellowship from the CSIR, New Delhi, India.

Regiospecific and quantitative oxidation of palladium-carbon bonds using hydrogen peroxide catalyzed by iron (III) porphyrins

Parvesh Wadhwani & Debkumar Bandyopadhyay* Department of Chemistry, Indian Institute of Technology, New Delhi 110 016, India

Metalloporphyrins are often used as catalysts to mimic a variety of oxidation reactions carried in vivo by various metalloenzymes I. These reactions become more biologically/industrially relevant if iron(III) porphyrins are used in combination with hydrogen peroxide. It has been established that electronegatively substituted iron(III) porphyrins are oxidatively robust and act as very efficient catalyst where MCPBA, C6HsIO and C6FsIO are used as terminal oxidants. Previous reports from our laboratory have shown that the regiospecific oxygenation of a series of C-Pd bonded azo-compounds of type 1 has been achieved by oxidants such as

(1)

R = H; Mc; R '= Me; CH2-Ph #I)rescnt work I a : R= 4-Me, R'= Me 23 : R= 4-Me, R'= Mc Ib:R=H R'=Mc 2b : R= H R'= Mc

MCPBA and C6FsIO[Eq. (1)] 2.3. In this activation of the C-H bonds the selective oxidation of C-M to C-OM bond of compound 1 has remained unsuccessful where hydrogen peroxide was used as terminal oxidant. In this poster we wish to present results of our first success in achieving the selective and quantitative oxygenation of C-Pd bonds of 1 by H20 2 where meso-tetrakis (pentafluorophenyl) porphyrinatoiron(III) chloride (F2oTPPFeCl) 4 is used as catalyst . The effect of variable electronegative iron(III) centers and the enigmatic role played by solvent has also been discussed.

References 1 Cytochrome P450, structure. mechanism and biochemistry, edited by PRO Montellano, (Plenum Press, New York). 1986. 2 Kamaraj K, Bandyopadhyay D, J Am chern Soc, 119 (1997) 8099. 3 Kamaraj K, Bandyopadhyay D, OrganometaLLics, 18 (1999) 438. 4 Wadhwani P, Bandyopadhyay D, Organometallics, 19 (2000). POSTERS 137

Role of metal ions in carcinogenesis

G M Peerzada Department of Chemistry, University of Kashmir, Srinagar 190006, Jammu & Kashmir, India

The role of metals in cancer development is a very complex problem. The carcinogenic activity depends not only on the nature of the metals, but also on their oxidation states, nature of salts, mode of administration, nature of species, sex and age, types of cancer etc. Because of these parameters, the problem of metal carcinogenesis is perplexing e.g, elemental lead is not carcinogenic, while lead acetate supplemented diets produce tumors in rats. Iron dextran injection induces tumors in rats, while injection of simple iron compound and dextran separately does not produce any tumor in the experimental animals. In metal-ion carcinogenesis, the metal-metal detoxification is very important e.g, nickel subsulphide is well established as a potential carcinogen, but when it is administered along with manganese powder, its carcinogenic effect is drastically reduced. There are different schools of thought regarding the probable mechanism of metal-ion promoted 2 2 carcinogenesis. According to H J Issaq (Chemical Review, 1980) the carcinogenic metals like Cd +, Ni + etc. being strongly electrophilic in nature can combine covalently with the nucleophilic sites of macromolecules. Such interaction may be a route of carcinogenic interaction. According to Furst, after the metal ions penetrate into the cell, they may either retard or accelerate the kinetics of the metabolic processes (anabolic and catabolic) conducted by different enzymes by inciting a competition between the invading and the biometals. Such interferences with the kinetics of the cell metabolic processes may induce malignancy. From the comparative analysis of metal concentrations in cancerous and non-cancerous tissues of the same species, one can say that the concentration of different trace and bulk metals depends upon the type of cancer and each type of cancer should be regarded as a separate entity. Further, from the analysis of metal content of DNA and RNA, isolated from different tumor tissues of rat liver, trace metals like zinc, cobalt, iron etc. are found considerably at higher concentrations than those found in DNA and RNA of normal ones. Lastly the variation of metal levels in serum, plasma, blood and tissues, during tumor growth and its regression demands some role of metal ions in carcinogenesis.

Meso ferrocenyl porphyrins

S Venkataraman, S J Narayanan, S Ranjan Dey, V G Anand & T K Chandrashekar* Department of Chemistry, Indian Institute of Techno[ogy, Kanpur 208016, India

Ferrocene functionalized porphyrins are receiving attention because of their possible application in molecular electronic devices, their attraction as multi electron redox catalysts and models for photosynthetic electron transfer reaction. Many porphyrins containing ferrocene groups linked to the periphery of the porphyrins through a spacer are known, but example of ferrocene attached directly to the meso carbons are limited. Direct linkage of 138 INDIAN J CHEM, SEC. A, JANUARY 2002 electroactive groups to porphyrin n-system not only induces strong electronic coupling, but also promotes the communication between the electroactive groups and the porphyrin ring, which is essential for application in molecular devices. Synthesis of porphyrins containing one to four ferrocenyl groups attached to meso carbon using dipyrromethane unit, the orientation of ferrocenyl ring is fixed and this prevents the formation of inseparable atropisomers. Preliminary spectroscopic studies on 3 suggest stronK electronic coupling between the ferrocenyl group and the porphyrin n-system. Furthermore cyclic voltammetry studies reveal a simultaneous four-electron transfer at slightly more positive potential than ferrocene. More importantly, formation of beautiful molecular rings of 12IlM diameter and O.5IlM thickness is observed in SEM photographs of thin films of 3. The details of these results are discussed in this poster.

Reference 1 Narayanan Seenichamy Jeyaprakash, Venkatraman Sundararaman, Dey Suhas Ranjan, Sridevi Bashyam, Rao Venkataramana, Anand G & Chandrashekar Tavarekere K, Synlett, 12 (2000) 1834.

Metal-drug interactions and crystal structure of tristhiabendazole cadmium(II) nitrate

a a b b P Thomas Muthiah *, B Umadevi , Xiugi Shui & Drake S Eggleston * aDepartment of Chemistry, Bharathidasan University, Tiruchirappalli 620024, India bSmithKlineBeecham Pharmaceuticals, King of Prussia, PA 19406, USA

Many drugs offer multiple metal-binding sites and metal ions may modulate drug activities. In this context it will be of interest to investigate the molecular aspects of metal-drug interactions. We have already reported the 1 2 crystal structures of a cadmium complex of trimethoprim and a cobalt complex of thiabendazole . Cadmium complex3 of thiabendazole has been discussed herein. The three bidentate thiabendazole ligands are bonded to the central cadmium ion, which occupies the special position in the lattice. In this context, a brief review of metal complexes of small drugs has also been presented.

References 1 Muthiah P Thomas & Robert J Justin, J chern Cryst, 29 (1999) 223. 2 Umadevi B et al.,lnorg chim Acta, 234 (1995) 149. 3 Umadevi B, Ph.D. Thesis, June 1997, Bharathidasan University, Tiruchirapalli, India.

Cleavage of DNA by novel copper(II) Schiff base complexes

a b b Pattubala A N Reddy", Bidyut K Santra , Girish Neelakanta , Subramony Mahadevan & Akhil R Chakravarty"* aDepartment of Inorganic and Physical Chemistry, bDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India

Artificial metallonucleases have proven to be efficient tools for the foot-printing and sequence-specific targeting of nucleic acids. A complete understanding of how to target DNA sites with specificity will lead to novel chemotherapeutic methods and to the development of highly sensitive diagnostic agents. Herein, we report a series of novel copper complexes, [Cu(salgly)LJ, where salgly is a tridentate Schiff base ligand and L = bpy(la), phen(lb), dpq(lc), dppz(ld). The complexes, on reduction to copper(l) species by ascorbic acid, cleave DNA in the presence of dioxygen. The mechanistic aspects of these oxidative cleavages are similar to those observed for [Cu(phenht (2). The present results supplement the ongoing research in the development of new chemical nucleases using redox and/or photoactive transition-metal complexes. POSTERS 139

DNA 2 1~lblclu l\'ickcd D N;\

(r ei clcclrophoresi\ ~ C'lI(I) complex lI ' lng p lCI <) 1)'\.\. T ris-I Ie 10iaCI blliler Scheme I : CatalYllc ReacllOll al 37"('. II,;\-

References I Sigman D S, Acc chem Res, 19 (1986) 180. 2 Erkkila K E, adorn D T & Barton J K, Chem Rev, 99 (1999) 2777. 3 Reddy PAN, Datta R & Chakravarty A R, lnorg Chem Comm, 3 (2000) 322.

Heterogeneity in the folding intermediates of barstar detected by time-resolved fluorescence resonance energy transfer

G S Lakshmikanth, Jayant Udgaonkar & G Krishnamoorthy* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India

Heterogeneity of folding intermediates has been proposed as a hallmark of protein folding process. A triple mutant of barstar having a single tryptophan (W53) in its core and a single cysteine sidechain has been prepared. The sulphhydral sidechain of the cysteine has been coupled to 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB). The TNB group quenches the fluorescence of W53 to the extent of -95% by fluorescence resonance energy transfer (FRET) in the native state. The quenching gets relieved in the denatured state. Fluorescence decay kinetics of W53 has been analyzed as a distribution of lifetime by the unbiased maximum entropy method (MEM). The distribution of lifetimes has been translated into a distribution of distance between the W53 (the donor) and the TNB group (acceptor). Such distance distributions could reveal the structural heterogeneity of the intermediate species involved in the folding process. This FRET-based method has also been used in detecting small changes in the structure under various native-like and denaturing conditions.

Reaction of a model sulphite oxidase with dimethyl sulphite

P K Chaudhur/, K Nagarajan±, B R Srinivasan+ & S Sarka~ * #Department of Chemistry, University of Pune, Pune;±Department of Chemistry , Indian Institute of Technology, Kanpur; Indi a +Department of Chemistry ,Goa University, Goa, India

Sulphite oxidase catalyzes the oxidation of sulphite to sulphate, the last step of degradation of sulphur containing amino acids. The fundamental chemistry in relevance to the initial reaction of sulphite with sulphite oxidase has been rigorously pursued with the help of model compound using phosphine as model oxo acceptor species. The only model compound {(BU4NhMo02(mnth)(rnnt = maleonitriledithiolate) (1) has been found to be able to 140 INDIAN J CHEM, SEC. A, JANUARY 2002 perform biologically relevant reaction of oxidizing hydrogen sulphite to hydrogen sulphate both in terms of saturation kinetics as well as anionic inhibition. Nordlaner and co-workers have proposed a direct attack of the sulphur atom of sulphite on one of the oxo groups of molybdenum based on rate of phosphine oxidation towards 1 correlated to the relative basicity of phosphine I . An alternative proposal which involves direct co-ordination of oxoanion of bisulphite to the molybdenum center in the case model compound has been put forward by us2 based on the work by Bray and co-workers. The feasibility of direct co-ordination of sulphite to the 3 molybdenum centre has also been suggested recently by Rajagopalan and co-workers . A very important experiment performed by Brody and Hille with the native enzyme utilizing dimethylsulphite as the substrate laid 4 the importance of lone pair in such oxo-transfer reaction . In view of the fact that our model complex(l) functionally mimicked the native sulphite oxidase is of interest its reactivity towards dimethylsulphite to gain further insight into the mechanism of the reduction of molybdenum centre of this enzyme. The reactivity of anionic bisulphite and of dimethylsulphite towards 1 is consistent with direct co-ordination of bisulphite to the molybdenum site of 1 as proposed earlier2 which is Schematically shown below:

o 0- o •• S-OH IV •• _11 VI -/11 VIMo S-OH 0 -Mo + o 0 V\\o j

[VI](~\ OH 1-*-- IVnO + HSO,

References 1 Lorber C, Plutino M R, Elding L I & Nordlaner E, J chem Soc Dalton Trans, (1997) 3997. 2 Das S K, Chaudhury P K, Biswas Dulali & Sarkar S, JAm chem Soc, 116 (1994) 9061. 3 George G N, Garrett R M, Graf T, Prince R C & Rajagopalan K V, JAm chem Soc, 120 (1998) 4522. 4 Brody M S & Hille R, Biochim Biophys Acta , (1995) 133.

Preliminary voltammetric characterization of Clostridium pasteurianum Fe-only hydrogenase I

Sean J Elliout*, Brian J Lemon#, John W Peters# & Fraser A Armstrong*t tInorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, England

# Department of Biochemistry and Chemistry, Utah State University, UT, USA Email: [email protected]

The iron only hydrogenase from the anaerobic microorganism Clostridium pasteurianum (CpI) is a soluble enzyme responsible for the interconversion of molecular hydrogen and protons. Hydrogenase enzymes of several types are known in a variety of organisms, though the function of hydrogenase activity may be tuned toward either hydrogen oxidation or proton reduction. CpI is a complex metalloenzyme, containing several iron sulphur clusters, including a multi-nuclear active site, termed the H-cluster. The crystal structure of CpI has recently been solved, as has a homologous protein from Desulfovibrio desuljuricansl . POSTERS 141

When applied to a graphite electrode, a solution of CpI yields an electroactive layer that demonstrates catalytic cyclic voltammetry, showing features indicating both the reduction of protons as well as the oxidation of hydrogen. Utilizing protein film voltammetry over a range of pHs, both in the presence and absence of 1 bar hydrogen, a pH dependence of £;so is observed which follows the Nernst equation. Further, a preference for proton reduction is revealed. And finally, protein film voltammetry of the CO-inhibited form of CpI2 shows that CO-inhibition can be removed by reductive activation of the protein.

References 1 Peters J W, Current Opinion in structural Biology, 9 (1999) 670 and references therein. 2 Lemon B J & Peters J W, Biochemistry, 38 (1999) 12969.

Amino acid selective "unlabeling" for stereospecific NMR assignments of CH3 groups of Val and Leu residues in proteins

H S Atreya & K V R Chary* Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India

A novel methodology for stereospecific NMR assignments of CH3 groups of Val and Leu residues in proteins, using amino acid selective "unlabeling" is presented. The strategy is based on selective unlabeling of amino acid residues in uniformly or fractionally l3C and/or 15N labeled proteins, which simplify the multidimensional heteronuclear NMR spectra. This further aids in sequence specific resonance assignments of both backbone and side-chain nuclei. The methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP). In this case, simultaneous unlabeling of Leu, Ile, Lys and Thr residues in a fractionally (15 %) l3C-Iabeled protein has helped in resolving the CG-HG correlations arising from all the Val residues of Eh-CaBP in the [l3C_ 1H] HSQC spectrum. This in turn, has aided in their stereospecific resonance assignments. In another preparation, simultaneous unlabeling of Ile, Lys and Thr has helped in resolving CD-HD and CG-HG correlations arising from all the Leu and Val residues in Eh-CaBP, respectively.

Automated protein NMR assignments in seconds

H S Atreya, K V R Chary*, S C Sahu & Girjesh Govil Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India

A novel approach for automated NMR assignments in proteins, which takes few seconds to complete the assignments, is presented. The algorithm, T ATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra, which correlate IHN and 15N chemical shifts with that of 13Ca, l3 C f3 and l3C'/IHa. Such a spectral data is used to create a "master-list", consisting of all possible sets of IHNj, 15Nj, l3Cajll.1, 13Cf3j/I.1 and !3C' jlI./Haj/I.1 chemical shifts. Subsequently, based on a statistical analysis of !3Ca and 13Cf3 chemical shifts of 100 proteins derived from BioMagResBank, it is shown that the 20 amino acid residues can be grouped into 8 distinct categories, each of which is assigned a unique 2-digit code. With the help of such a code, the program uses the master-list to search for neighbouring partners of given residue along the polypeptide chain and identifies a maximum possible stretch of residues. This results in the sequence specific resonance assignment of that stretch of residues. The procedure is repeated till all the residues are assigned. The program has been tested using experimental data on a calcium binding protein (15 kDa) which has substantial internal homology and using published data on four other proteins in the molecular weight range of 18-42 kDa. In all these cases nearly complete sequence specific resonance assignments are achieved. 142 INDIAN J CHEM, SEC. A, JANUARY 2002

Cation dependent conformational switches in d-TGGCGGC containing two triplet repeats of fragile X-syndrome: NMR observations

P K Patel, Neel S Bhavesh & R V Hosur* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India

Higher ordered structures formed by different DNA sequences have been widely investigated in recent years because of their implications in a variety of biological functions. Among these, G-quadruplexes have exhibited a great variety depending on the exact sequence, the lengths of the G-stretches, interception by other nucleotides and environmental conditions such as pH, temperature, salt type and its concentration. We report here interesting conformational switches observed by NMR in the sequence d-TGGCGGC containing two GGC triplet repeats related to the disease Fragile X-Syndrome. At neutral pH, the solution structure is a parallel stranded quadruplex in presence of K+ ions. Lowering the pH does not cause a major change in the structure, however, the chemical shift patterns of the C4 and G3 base protons suggest protonation of the C-tetrad in the center of the quadrup1ex. In contrast, the sequence forms an antiparallel duplex in Na+ containing solutions. As the pH of the Na+ sample is lowered, an equilibrium mixture of a duplex and a quadruplex appears and at pH 2.2, the molecule exists entirely as a quadruplex. These results would be of significance from the point of view of recognition and regulation by different helicase enzymes, which have been found to discriminate between different types of quadruplex structures.