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Novel Derivatives of Nicotinamide Adenine Dinucleotide (NAD) and Their Biological Evaluation Against NAD- Consuming Enzymes

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Novel Derivatives of Nicotinamide Adenine Dinucleotide (NAD) and Their Biological Evaluation Against NAD- Consuming Enzymes Novel derivatives of nicotinamide adenine dinucleotide (NAD) and their biological evaluation against NAD- Consuming Enzymes Giulia Pergolizzi University of East Anglia School of Pharmacy Thesis submitted for the degree of Doctor of Philosophy July, 2012 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. ABSTRACT Nicotinamide adenine dinucleotide (β-NAD+) is a primary metabolite involved in fundamental biological processes. Its molecular structure with characteristic functional groups, such as the quaternary nitrogen of the nicotinamide ring, and the two high- energy pyrophosphate and nicotinamide N-glycosidic bonds, allows it to undergo different reactions depending on the reactive moiety. Well known as a redox substrate owing to the redox properties of the nicotinamide ring, β-NAD+ is also fundamental as a substrate of NAD+-consuming enzymes that cleave either high-energy bonds to catalyse their reactions. In this study, a panel of novel adenine-modified NAD+ derivatives was synthesized and biologically evaluated against different NAD+-consuming enzymes. The synthesis of NAD+ derivatives, modified in position 2, 6 or 8 of the adenine ring with aryl/heteroaryl groups, was accomplished by Suzuki-Miyaura cross-couplings. Their biological activity as inhibitors and/or non-natural substrates was assessed against a selected range of NAD+-consuming enzymes. The fluorescence of 8-aryl/heteroaryl NAD+ derivatives allowed their use as biochemical probes for the development of continuous biochemical assays to monitor NAD+-consuming enzyme activities. The introduction of different substituents in position 8 on the adenine ring allowed the modulation of their fluorescence, resulting in the development of more sensitive and alternative probes compared to the known fluorophore ε-NAD+. The different substitutions introduced on the adenine ring also allowed us to probe the active site of an NAD+-dependent bacterial DNA ligase. The selective activity of 8-aryl/heteroaryl NAD+ derivatives against different NAD+-consuming enzymes offers excellent opportunities for their application as tool compounds in in-vitro/in-vivo studies, and as inhibitor templates for drug discovery. i Parts of this thesis have already been published in scientific journals, and presented at scientific conferences. A novel fluorescent probe for NAD-consuming enzymes Giulia Pergolizzi, Julea N. Butt, Richard P. Bowater and Gerd K. Wagner Chemical Communications, 2011, 47, 12655-12657. Two-Step Synthesis of Novel, Bioactive Derivatives of the Ubiquitous Cofactor Nicotinamide Adenine Dinucleotide (NAD) Thomas Pesnot, Julia Kempter, Jörg Schemies, Giulia Pergolizzi, Urszula Uciechowska, Tobias Rumpf, Wolfgang Sippl, Manfred Jung, and Gerd K. Wagner Journal of Medicinal Chemistry, 2011, 54, 3492-3499. 3th – 8th July 2011: Synthesis and biological evaluation of novel fluorescent NAD+ analogues. Giulia Pergolizzi, oral communication, European School of Medicinal Chemistry (ESMEC) (Urbino, Italy). April 2011: Base-modified NAD+ derivatives – Novel Chemical Tools for the Investigation of NAD+-consuming Enzymes. Giulia Pergolizzi, Julea N. Butt, Richard P. Bowater and Gerd K. Wagner, poster presentation, RSC Carbohydrate and Bioorganic Meeting (5th April) & RSC Bioorganic Postgraduate Symposium (15th April) (London, UK). July – September 2010: Novel NAD+ derivatives modified at the adenine base: synthesis and biological applications. Giulia Pergolizzi, Julea N. Butt, Richard P. Bowater and Gerd K. Wagner, poster presentation, 6th Nucleic Acids Forum Meeting (2nd July, Liverpool, UK) & 8th NACON (Nucleic Acid Conference) (12th – 16th September, Sheffield, UK). ii TABLE OF CONTENTS List of Figures List of Schemes List of Tables List of Figures and Tables in Appendix List of Abbreviations Acknowledgements 1 Β-NAD+: AN ESSENTIAL BIOLOGICAL MOLECULE 1 1.1 β-NAD+ structure 1 1.1.1 Redox properties of β-NAD+ 4 1.1.2 β-NAD+ catabolism and NAD+-consuming enzymes 6 1.1.3 β-NAD+ biosynthesis 10 1.2 NAD+-consuming enzymes 13 1.2.1 NAD+-consuming enzymes: similarities and relationships 14 1.2.2 Intracellular and extracellular β-NAD+ balance 20 1.3 NAD+ derivatives as mechanistic probes for NAD+-dependent enzymes 23 1.3.1 NAD+ derivatives as inhibitors of NAD+-dependent enzymes 26 1.3.1.1 Nicotinamide modification 26 1.3.1.2 Nicotinamide ribose modification 28 1.3.1.3 Pyrophosphate bond modification 29 1.3.1.4 Adenine modification 31 1.3.2 NAD+ derivatives as non-natural substrates of NAD+-dependent enzymes 32 1.4 Assays for NAD+-consuming enzymes 35 1.4.1 Cyanide and dehydrogenase assays 36 1.4.2 -NAD+ and its application for NAD+-consuming enzyme assays 37 1.4.3 NGD+, NHD+ and NXD+ for ADPRC fluorescence-based assays 40 iii 1.4.4 6 and 8-alkyne NAD+ for NADase activity assays 41 1.4.5 β-NAD+ quantification by reaction with acetophenone 43 1.4.6 A continuous colourimetric assay for the detection of PARP activity 43 1.4.7 Alternative assays for the detection of sirtuins activity 44 1.4.8 Assays for the detection of DNA ligase activity 45 1.5 Project Objectives 46 2 SYNTHESIS OF ARYL/HETEROARYL ADENINE-MODIFIED NAD+ DERIVATIVES 50 2.1 Synthetic strategies to aryl/heteroaryl adenine-modified NAD+ derivatives 50 2.2 Substitution of the adenine ring by palladium-mediated cross-couplings 52 2.3 Synthetic route to 2-aryl/heteroaryl AMP derivatives 56 2.3.1 Iodination of C-2 and 2′,3′-O-isopropylidene protection of adenosine 57 2.3.2 Suzuki-Miyaura and Stille cross-couplings on C-2 of 2′,3′-O-isopropylidene-2- iodo adenosine 59 2.3.3 Phosphorylation of 5′-OH of 2′,3′-O-isopropylidene-2-substituted adenosine and subsequent deprotection to 2-aryl/heteroaryl AMP derivatives 61 2.4 Synthetic route to 6-aryl/heteroaryl AMP derivatives 63 2.4.1 Suzuki-Miyaura cross-coupling on C-6 of 6-chloro purine nucleoside 64 2.4.2 2′,3′-O-isopropylidene protection and phosphorylation of 5′-OH of 6- substituted purine nucleosides, with subsequent deprotection to 6-aryl/heteroaryl AMP derivatives 66 2.5 Synthetic route to 8-aryl/heteroaryl AMP derivatives 67 2.5.1 Bromination of C-8 of adenosine and adenosine monophosphate 69 2.5.2 Suzuki-Miyaura cross-coupling on C-8 of 8-bromo adenosine monophosphate to 8-aryl/heteroaryl AMP derivatives 70 2.6 Synthesis of aryl/heteroaryl adenine-modified NAD+ derivatives (pathway a) 76 2.6.1 AMP activation via phosphoromorpholidate intermediate 76 2.6.2 Coupling with β-NMN 77 iv 2.7 Alternative synthetic strategies to aryl/heteroaryl adenine-modified NAD+ derivatives 79 2.7.1 Aryl/heteroaryl adenine-modified NAD+ derivatives via phosphoramidite method (pathway b) 79 2.7.2 Aryl/heteroaryl adenine-modified NAD+ derivatives via one-pot, two-step procedure (pathway c) 80 2.8 Conclusions 82 3 CONFORMATIONAL AND SPECTROSCOPIC ANALYSIS OF ARYL/HETEROARYL ADENINE-MODIFIED AMP AND NAD+ DERIVATIVES 83 3.1 Conformational analysis of nucleosides and nucleotides 84 3.1.1 Conformation of aryl/heteroaryl adenine-modified AMP derivatives 86 3.1.2 Conformation of aryl/heteroaryl adenine-modified NAD+ derivatives 87 3.2 Nucleobases and their photochemical properties 88 3.3 Design of emissive nucleosides 90 3.3.1 Fluorophore features 90 3.3.2 Families of existing nucleobase-modified emissive nucleosides 91 3.4 Measurements of quantum yield for aryl/heteroaryl adenine-modified AMP and NAD+ derivatives 94 3.5 Spectroscopic analysis of aryl/heteroaryl adenine-modified AMP derivatives 95 3.6 Spectroscopic analysis of aryl/heteroaryl adenine-modified NAD+ derivatives 101 3.7 Fluorescence quenching in 8-aryl/heteroaryl NAD+ derivatives 106 3.7.1 Fluorescence basis for internal quenching in NAD+ derivatives 106 3.7.2 NMR basis for internal quenching in NAD+ derivatives 111 3.8 Conclusions 117 v 4 EVALUATION OF 8-ARYL/HETEROARYL NAD+ DERIVATIVES AS FLUORESCENT PROBES FOR NAD+-CONSUMING ENZYMES 120 4.1 Nucleotide pyrophosphatases (NPPs) 121 4.1.1 Fluorescence-based assay for nucleotide pyrophosphatase 123 4.1.2 Kinetic analysis of NPP activity 129 4.2 NAD+-glycohydrolases (NADases) and ADP-ribosyl cyclases (ADPRCs) 131 4.2.1 Fluorescence-based assay for NAD+-glycohydrolase 133 4.2.2 Fluorescence-based assay for ADP-ribosyl cyclase 135 4.2.3 Mechanistic aspects of ADPRC activity 139 5 EVALUATION OF ARYL/HETEROARYL ADENINE-MODIFIED NAD+ DERIVATIVES AS INHIBITORS AND NON-NATURAL SUBSTRATES FOR NAD+-CONSUMING ENZYMES 145 5.1 NAD+-dependent DNA ligases 145 5.1.1 Expression, purification and activity of Escherichia coli DNA ligase 153 5.1.2 Biological testing of 2-substituted AMP and NAD+ derivatives 155 5.1.2.1 Biological activities of 2-substituted AMP and NAD+ derivatives 160 5.1.3 Biological testing of 6-substituted AMP and NAD+ derivatives 162 5.1.3.1 Biological activities of 6-substituted AMP and NAD+ derivatives 163 5.1.4 Biological testing of 8-substituted NAD+ derivatives 164 5.1.4.1 Biological activities of 8-substituted NAD+ derivatives 166 5.1.5 Summary 168 5.2 Sirtuins 170 5.2.1 Biological testing of 8-substituted NAD+ derivatives 172 5.2.2 Biological activities of 8-substituted NAD+ derivatives 174 6 CONCLUSIONS AND FUTURE WORK 176 vi 7 EXPERIMENTAL 184 7.1 General 184 7.1.1 Synthesis and characterization 184 7.1.2 Chromatography conditions 185 7.1.3 Enzymology 186 7.2 Synthesis 186 7.2.1 General synthetic procedures 186 7.2.2
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