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Synthesis of Guanidine Amino Acids and Investigation of Their Interactions with Nucleic Acid

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Synthesis of Guanidine Amino Acids and Investigation of Their Interactions with Nucleic Acid Synthesis of Guanidine Amino Acids and Investigation of their Interactions with Nucleic Acid Thesis submitted for the degree of Doctor of Philosophy by Vicki Doreen Möschwitzer Supervisor: Dr. James E. Redman School of Chemistry Cardiff University July 2011 Declaration This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed . (candidate) Date . Statement 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Signed . (candidate) Date . Statement 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed . (candidate) Date . Statement 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for interlibrary loan, and for the title and summary to be made available to outside organisations. Signed . (candidate) Date . I Acknowledgements First I would like to thank Doctor James E. Redman very much for offering me this interesting topic and for the opportunity to work in his research group. I am particularly grateful for his constant support, his constructive suggestions and proofreading of this thesis. I thank Doctor Robert Richardson for his expert advice, his helpfulness and for permitting the use of the high pressure reactor of the POC lab. Professor Gerald Richter and Doctor Nicholas Tomkinson are thanked for helpful discussions in the viva and revising the reports. They provided invaluable contributions to the progress of the project. I would like to thank Steven Hill for many discussions regarding our synthetic problems and for the fun working atmosphere in the lab. I would like to thank Doctor Anne-Sophie Rouziere for her instrumental biochemical expertise and for the enjoyable tea breaks in the office. I thank all technical and administrative staff from Cardiff University for their tireless support, especially Alun Davies. Last but not least I would like to thank my beloved family and Johan Brandt, which stood all the time by my side and were always an immense support for me. EPSRC and Cardiff University are thanked for financial support. II Abbreviations AAA 2-amino-2-(2-aminopyrimidin-5-yl)-acetic acid AAP 2-amino-3-(2-aminopyrimidin-5-yl)-propanoic acid Ac acetyl AIA 2-amino-2-(2-iminohexahydro-pyrimidin-5-yl)acetic acid AIP 2-amino-3-(2-iminohexa-hydropyrimidin-5-yl)propanoic acid Ala, A alanine Alloc allyloxycarbonyl aq. aqueous Ar aromat Arg, R arginine Asn, N asparagine Asp, D aspartic acid BINAP 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl Bn benzyl Boc tert-butyl carbonate BOP benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate tBu tert-butyl br broad Br2 bromine Bu4NCl tetrabutylammonium chloride Cat. catalyst Cys, C cysteine cys, c D-cysteine CDCl3 deuterated chloroform CD3OD deuterated methanol CO2 carbon dioxide Cs2CO3 caesium carbonate DCM dichloromethane DIOP 4,5-bis(diphenylphosphinomethyl)-2,2-dimethyl-1,3-dioxolane DIPAMP ethylenebis[(2-methoxyphenyl)phenylphosphine] III DMF dimethylformamide DNA deoxyribonucleic acid Me-DUPHOS 2,2′,5,5′-Tetramethyl-1,1′-(o-phenylene)diphospholane EI MS electron ionisation mass spectrometry eq. equivalent ESI MS electrospray ionisation mass spectrometry Et3N triethylamine Fe iron FDAA N-(2,4)-dinitro-5-fluorophenyl)-L-alanine amide Fmoc fluorenylmethyloxycarbonyl Gln, Q glutamine Glu, E glutamic acid Gly, G glycine H2 hydrogen gas HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium HCl hydrochloric acid HCN hydrogen cyanide His, H histidine HIV-1 human immunodeficiency virus 1 HMPA hexamethylphophoramide H2O water + H3O hydronium ion HOBt hydroxybenzotriazole HPLC high performance liquid chromatography or high pressure liquid chromatography HRMS high resolution mass spectrometry Hyp hydroxyproline IEP isoelectric point Ile, I isoleucine IR infrared KBr potassium bromide KCl potassium chloride KH2PO4 monopotassium phosphate IV K2HPO4 dipotassium phosphate Leu, L leucine LiBr lithium bromide Lys, K lysine M molar MALDI-ToF matrix-assisted laser desorption/ionization - time-of-flight Me-DUPHOS 1,2-bis(2,5-dimethylphospholano)benzene MeOH methanol Met, M methionine MgSO4 magnesium sulfate mM millimolar mp melting point mRNA messenger ribonucleic acid N2 nitrogen gas NAG N-acetylglucosamine NaH sodium hydride NaHCO3 sodium hydrogen carbonate or sodium bicarbonate NAM N-acetylmuramic acid NaNO2 sodium nitrite NaOH sodium hydroxide Na2SO4 sodium sulfate Negative-Peptide (H2N-EEEEGGG)2KGGGX-NH2 NH3 ammonia NH4Cl ammonium chloride NMR nuclear magnetic resonance O2 oxygen org. organic PAM Phenylacetamidomethyl Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PdCl2 palladium chloride Pd(OH)2 palladium hydroxide Phe, F phenylalanine pm picometre Pmc pentamethylchromansulfonyl V Positive-Peptide (H2N-RKRKGGG)2KGGGX-NH2 PPh3 triphenylphosphine PPh3O triphenylphosphine oxide ppm parts per million i-PrMgCl isopropylmagnesium chloride Pro, P proline PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Pyl, O pyrrolysine Rev regulator of expression of virion RNA ribonucleic acid rpm rotation per minute Rre Rev response element sat. saturated Sec, U selenocysteine Ser, S serine SPPS solid phase peptide synthesis Tar trans-activating response element Tat trans-activator of transcription TEAP triethylammonium phosphate buffer TFA trifluoroacetic acid TGFβ-1 transforming growth factor beta 1 THF tetrahydrofurane Thr, T threonine TLC thin layer chromatography Trp, W tryptophane Trt trityl Tyr, Y thyrosine Val, V valin X Fmoc-Glu(biotinyl-PEG)-OH or N-α-Fmoc-N-γ-(N-biotinyl-3- (2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl)-L-glutamine YB-1 Y-box binding protein-1 VI Abstract Certain amino acids have a high propensity to be found at the intermolecular contact regions in protein-nucleic acid complexes. One of them is arginine whose side chain commonly makes contact through electrostatic interactions with phosphates and hydrogen bonding to nucleobases. Rigidified arginine analogues in which the side chain is cyclised would reduce torsional entropy loss occurring on binding. In this project amino acids were synthesised with cyclic guanidine side chains as modular arginine analogue building blocks for preparation of nucleic acid binding peptides. The synthetic strategies to prepare the amino acids involve Grignard reactions and Heck coupling reactions with iodo-pyrimidines. The Heck coupling is followed by a catalytic asymmetric hydrogenation to generate the stereo centre. An enzymatic kinetic resolution is essential after the Grignard coupling to provide stereoselectivity. Small molecule interactions with nucleic acids can be used as a tool for modulating processes like transcription and translation. Ultimately peptide binding screens against the mRNA of TGF-β1 may be undertaken to study the role of protein binding in regulation of translation. Within this thesis a peptide library was prepared via the split and recombine method using macro-beads which provides valuable groundwork for the aim of continuing research. This would be the synthesis of peptides containing rigidified arginine mimetics that interact with mRNA of TGF-β1 or other nucleic acids. VII Table of Contents Declaration ............................................................................................................................................... I Acknowledgements .................................................................................................................................. II Abbreviations.......................................................................................................................................... III Abstract ................................................................................................................................................ VII 1. Introduction ...................................................................................................................... 1 1.1 Amino acids ............................................................................................................................. 1 1.2 Arginine Derivatives ................................................................................................................ 6 1.3 Peptides/Proteins ................................................................................................................. 10 1.4 Solid Phase Peptide Synthesis (SPPS) .................................................................................... 13 1.5 Protein/Peptide Binding to Nucleic Acids .............................................................................. 16 1.6 α-Helix Stabilisation in Oligopeptides ................................................................................... 18 1.7 Transforming Growth Factor beta 1 (TGF-β1) ........................................................................ 19 1.8 Scientific Goals ...................................................................................................................... 21 2. Results and Discussions ................................................................................................. 22 2.1 Synthesis of Arginine Mimetics ............................................................................................. 22 2.1.1 Introduction .................................................................................................................................
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