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Chemoenzymatic Synthesis of Isotopically Labelled CHEMO ENZYMATIC SYNTHESIS OF ISOTOPICALLY LABELLED FOLATES A thesis submitted to Cardiff University for the degree of Doctor of Philosophy by Antonio Angelastro Supervisor: Prof. Rudolf K. Allemann 2017 DECLARATION This work has not been submi tted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. 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 , and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter -library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter -library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ………………………………………… (candidate) Date ………………………… i ABSTRACT Dihydrofolate reductase (DHFR) is a key enzy me in cellular anabolism . I t catalyses the reduction of 7,8 -dihydrofolate (H 2F) to 5,6,7,8 -tetrahydrofolate (H 4F) via hydride transfer from the C4 position of NADPH to the C6 position of H2F accompanied with protonation at the N5 position of H 2F. Due to th e importance of DHFR as a n anticancer and antimicrobial target , the catalytic mechanism of DHFR has long been the focus of intense research. Kinetic isotope effect (KIE) measurements can provide insight into the mechanism of DHFR catalysis and guide the ra tional design of novel anti -DHFR drugs . However, because of a lack of a practical strateg y to introduce heavy atoms ( 15 N, 13 C) into H2F, current research is mostly restrained to the study of hydrogen isotope effects. In this thesis , a fourteen step, one -po t chemoenzymatic synthesis of labelled H2F is reported . The flexibility of this synthetic approach enable s the production of various isotopically enriched H2Fs from simple starting materials such as D-glucose. The labelled substrates were used to measure , for the first time , heavy atom KIEs and to derive information about the transition state of the chemical step during DHFR catalysis. This methodology is widely applicable to other biochemically important substrates and cofactors and it can be used for a wi de variety of in vitro and in vivo investigations. ii ACKNOWLEDGEMENTS This PhD thesis marks the realisation of a dream, the end of a journey started decades ago inside a small chemistry lab from a small town in southern Italy , Solofra . My dream. My jou rney. In every journey you make in life, you meet people along the way. People who are fundamental for making your dreams come true. This is one such case, and I would like to express my deep est gratitude to all who helped, supported and guided me during my PhD studies. Thank you Prof. Rudolf Allemann for having trusted me. It was you who granted me the honour to be your student, to mature as a scientist within your research group , and the support that shape d my passion for science. Thank you Dr Louis Lu k for the training , help, support and the endless discussions we have had during these years. Your teachings and friendship have been precious and fundamental to me. Thank you to Dr Rob J enkins and Thomas Williams for your extensive help with my favourite analytical technique: mass spectrometry. Thank you to all my colleagues from the Allemann group with whom I have shared my passion for science. To Dr Enas Behiry for her help with the pre -steady state kinetics, to Dr Luke Johnson for his fundamental help with NMR spectroscopy (and checking my English!!) , to Dr Veronica Gonzalez for her help with cloning, to Dr Juan Faraldos for the enjoyable discussions on terpenes and organic chemistry, and to Dr Melody Demiray with whom I share the passion for coffee and cigarettes. Thank you to Marianna Loizzi, a beloved friend, co lleague and best -housemate - ever with whom I shared both the joy and pain of the PhD life. Thank you to all the people from the Luk, Casini and Tsai group s with whom I have enjoyed evenings at t he pub . Thank you to my family . My dad, mum , sister , brother and brother -in -law who always granted me unconditional support in every choice I made in my life, including to pursue a PhD in Wales . iii Thank you to my extraordinary fiancée, Cinzia. Last but not least , I would like to thank God and the Divine Mercy of Jesus from whom I have been given the strength and comfort for realising this dream. iv TABLE OF CONTENTS DECLARATION ................................ ................................ ................................ ............................. i ABSTRAC T................................ ................................ ................................ ................................ ... ii ACKNOWLEDGEMENTS ................................ ................................ ................................ ............ iii LIST OF FIGURES ................................ ................................ ................................ ....................... x LIST OF TABLES ................................ ................................ ................................ ....................... xv LIST OF ABBREVIATIONS ................................ ................................ ................................ ....... xvi 1. INTRODUCTION ................................ ................................ ................................ ................... 1 1.1 The role of enzymes in biology and drug discovery ................................ ............... 2 1.2 Kinetic isotope effect: a powerful tool to dissect transition -states on an atomic scale ... ................................ ................................ ................................ ................................ .... 10 1.3 The role of dihydrofolate reductase (DHFR) in pharmacotherapy and drug design. ................................. ................................ ................................ ................................ ... 13 1.4 The catalytic mechanism of DHFR from Escherichia coli ................................ .... 19 1.5 Synthesis of isotopically labelled folates ................................ ............................... 25 1.5.1 Isotopic labelling of folate’s pterin moiety ................................ ..................... 26 1.6 How folates are made by nature: the folate de novo biosynthetic pathway ....... 29 1.7 Aims ................................ ................................ ................................ ............................ 31 2. A DISULFIDE -BASED NADP + RECYCLING SYS TEM WITH A HIGH TURNOVER NUMBER ................................ ................................ ................................ ................................ ..... 33 2.1 Preface ................................ ................................ ................................ ........................... 34 2.1.1 ATP recycling systems ................................ ................................ ..................... 35 2.1.2 NADP + recycling systems ................................ ................................ ................ 36 2.1.3 The glutaredoxin (GRX) system: an ideal recycling scheme for NADP + ..... 38 2.2 Construction of the glutaredoxin system for NADP + recycling ........................... 39 2.2.1 Gene expression, purification and characterisation of recombinant glutaredoxin 2 (GRX2) and 6 -phosphogluconate dehydroge nase (6PGDH) ............... 40 2.2.1.1 Characterisation and activity assay of 6PGDH ................................ ...... 40 2.2.1.2 Characterisation and activity assay of EcGRX2 ................................ .... 41 2.2.2 In vitro assembly of the GRX sy stem and determination of the maximal TTN …….. ................................ ................................ ................................ ............................ 43 2.3 Conclusion ................................ ................................ ................................ ................. 47 3. CHEMO -ENZYMATIC SYNTHESIS OF ISOTOPICALLY LABELLED GUANOSINE NUCLEOTIDES ................................ ................................ ................................ .......................... 48 3.1 Preface ................................ ................................ ................................ ............................. 49 3.1.1 Background: the de novo and salvage pathway of guanosine nucleotides ...... 50 3.2 Chemo -enzymatic synthesis of guanosine nucleotides ................................ ....... 53 3.3 Synthesis of isotopically label led guanine ................................ ............................. 55 v 3.3.1 Traube purine synthesis of guanine ................................ ..............................
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