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DNA-inspired Janus AT and GC heterocycles : synthesis, structural analysis and self-organization Asadi, Ali 2009

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DNA-Inspired Janus AT and GC Heterocycles: Synthesis, Structural Analysis and Self-Organization by ALT ASADI A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2009 © Au Asadi 2009 Abstract Inspired by the significance of hydrogen bond driven self-organization, especially from the base-pairing interactions of double helical DNA, this dissertation discusses the synthesis and characterization of a number of DNA-inspired self-complementary heterocycles and the supramolecular ensembles derived from them. Specifically, two projects have been completed. Each of these projects addresses the high yielding syntheses of heterocycles with defined hydrogen bond accepting and donating capabilities designed to self-assemble under the general purview of base pairing. The first chapter provides an introduction to general concepts such as base-pairing as well as an outline of the diverse synthetic supramolecular ensembles that have been prepared by utilizing such interactions. Chapter 2 focuses on the syntheses and solid-state structures of three self-complementary DNA-inspired heterocycles which contain ADA-DAD hydrogen bond acceptor-donor patterns (Janus AT 1-3). These novel heterocycles represent diaminopurine thymine hybrids that, in two of the three cases, relate to previously reported heterocyclic hybrids of guanine and cytosine. All three heterocycles crystallized and afforded the first X-ray crystal structures of such heterocycles and revealed their extended H-bonded arrays. This chapter also introduces the synthetic development to build Janus AT deoxynucleosides, capable of being oligomerized, as the current trend of this project. The potential use of Janus AT heterocycles in DNA and RNA recognition is briefly discussed as well. Chapter 3 will disclose the synthesis and characterization of a DNA-inspired self-complementary heterocycle capable of AAD-DAA hydrogen bond pairing, which self-organizes to a tetrameric rosette, that unlike a 0-quartet, 900 A D D 7’NN\ 4x ‘NGj/ DMSON N N or H DMF III needs no metal binding or peripheral component for pre-organization (Janus GC 1). Notably, ESI-MS, variable temperature ‘H-NMR, 2D-NOESY and DOSY ‘H-NMR have been exploited to validate the tetrameric stoichiometry in this non-covalent rosette comprising twelve H-bonds. At the end of each of these chapters, a section pertaining to ongoing efforts and proposed future research is included. HNH A c:, HNNN A HH D ON AIII A HNNN D N X N I I R H Janus AT I R= Butyl (I, 2a, 3a), Heptyl (2b) Hexyl (3b) Janus AT 2 (X=N), Jan us AT 3 (X=CH) R H -R Janus GC I D=Donor A=Acceptor R=Butyl iv Table of Contents Abstract.ii List of tables x List of figures xi List of schemes xv List of abbreviations xvi Acknowledgments xviii Dedication xix 1. Chapter 1: Background and Significance 1 1.1 Introduction 1 1.2 Major terms and definitions 3 1.2.1 Hydrogen bonding; a definition 3 1.2.2 Jorgensen’s secondary interactions 6 1.3 Hydrogen bonding in nature 8 1.3.1 DNA/RNA base pairing 9 1.3.2 G-quartets in nature and self-assembly of guanosine derivatives 13 1.3.3 The i-Motif; self-assembly of cytosine in nature 18 1.4 Supramolecular chemistry 19 1.4.1 Supramolecular synthons 19 1.4.2 Crystal engineering 20 1.5 One and two component self-complementary hydrogen bonding systems 21 1.5.1 Two component self-complementary hydrogen bonding systems 22 1.5.2 One component self-complementary hydrogen bonding systems 26 1.5.2.1 Tectons and molecular tectonics 26 1.5.2.2 Janus molecules 27 1.5.2.2.1 Janus type heterocycles; formation of rosette architectures 29 1.5.2.2.2 Rosettes; Janus type heterocycles containing a (DAA-ADD) hydrogen bonding motif 31 1.5.2.2.3 New Janus type DNA-inspired heterocycles presented in this dissertation 35 1.6 Objective of this dissertation 36 2. Chapter 2: Novel DNA-Inspired Janus-AT Heterocycles: Synthesis, Self-Assembly, and Solid State Structures 38 2.1 Introduction 38 2.2 Janus AT molecules; novel DNA-inspired Janus-AT heterocycles 39 2.3 Design of Janus AT heterocycles 1-3 40 V2.4 Janus AT 1 .44 2.4.1 Synthesis of Janus AT 1 44 2.4.2 Insight into the characterization and self-organization of Janus AT 1 45 2.4.2.1 1H-NMR studies of Janus AT 1 45 2.4.2.2 Variable concentration1H-NMR studies of Janus AT 1 46 2.4.2.3 Phase-sensitive 2D 15N-’H HMQC NMR analysis of Janus AT 1 47 2.4.2.4 Solid state analysis of Janus AT 1 by X-ray crystallographic diffraction 48 2.5 Janus AT 2 52 2.5.1 Synthesis of Janus AT 2 52 2.5.2 Insights into the characterization and self-organization of Janus AT 2 54 2.5.2.1‘5N-H HMQC Analysis of Janus AT 2b 54 2.5.2.2 Variable concentration ‘H-NMR studies of Janus AT 2b 56 2.5.2.3 Solid state analysis of JAr2 by X-ray crystallographic diffraction 57 2.5.2.4 Electron spray ionization mass spectrometry analysis on Janus AT 2a&b 60 2.6 Janus AT 3a and 3b 62 2.6.1 Synthesis of 11 62 2.6.2 Synthesis of 5-Amino-7-thioxo-1,7-dihydrothiopyrano[4,3-d]pyrimidine-2,4-dione analogs 12a & 12b .63 2.6.1.3 Synthesis of 5-Amino-7-thioxo-6,7-dihydro-1H-pyrido[4,3-d]pyrimidine-2,4-dione 13a &b 64 2.6.1.4 Synthesis of Janus AT 3 65 2.7 Insight into the characterization and self-organization of Janus AT 3a&b 67 2.7.1 ‘H-NMR of Janus AT 3a 67 2.7.2 Variable concentration1H-NMR of Janus AT 3a 68 2.7.3 Solid state analysis of Janus AT 3 by X-ray crystallographic diffraction 69 2.8 Bis-Janus AT heterocycles and investigation of their biological activity (abasic site targeting molecules) 75 2.9 Conclusion 78 2.10 Current and future trends 80 2.10.1 Current trends; Janus AT deoxynucleosides 80 2.10.1.1 Janus-DNA, phosphoramidite method 83 2.10.2 Self-organized hydrogen bonded sheets consists of two different sizes of rosettes 84 2.10.3 Peripheral crowding of Janus AT as origin of the exclusive formation of rosette 86 2.10.4 Janus AT heterocycles: potential small molecule DNA and RNA binders 87 2.11 Experimental 88 2.11.1 General materials and equipment 88 2.11.2 Synthesis of (Z)-2-cyano-3-ethoxyacrylethyl carbamate (5) 88 2.11.3 Synthesis of 1-butyl-1,2,3,4-tetrahydro-2,4-dioxopyrimidine-5-carbonitrile (6) 89 2.11.4 Synthesis of Janus AT 1 90 2.11.5 Synthesis of potassium 2-cyano-3-(ethoxycarbonylamino)-3-oxoprop-1-ene-1, 1-bis-thiolate (7)..91 2.11.6 Synthesis of 2-cyano-3,3-bis (methylthio)acrylethyl carbamate (8) 91 2.11.7 Synthesis of 1-butyl-1,2,3,4-tetrahydro-6-(methylthio)-2,4dioxopyrimidine-5-carbonitrile (9a) 92 2.11.8 Synthesis of 1-heptyl-6-(methylthio)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (9b) 93 2.11.9 Synthesis of Janus AT 2a 94 2.11.10 Synthesis of Janus AT 2b 95 2.11.11 Synthesis of (2-cyano-3-ethoxy-but-2-enoyl)carbamic acid ethyl ester (10) 96 2.11.12 Synthesis of 1-butyl-6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (ha) 97 vi 2.11.13 Synthesis of 1-hexyl-6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (lib) 98 2.11.14 Synthesis of 5-amino-1-butyl-7-thioxo-1,7-dihydro-thiopyrano[4,3-d]pyrimidine-2,4-dione (12a) 98 2.11.15 Synthesis of 5-amino-1-hexyl-7-thioxo-i,7-dihydro-thiopyrano{4,3-d]pyrimidine-2,4-dione (12b) 99 2.11.16 Synthesis of 5-amino-i-butyl-7-thioxo-6,7-clihydro-1H-pyrido[4,3-d]pyrimidine-2,4-dione (13a) 100 2.11.17 Synthesis of 5-amino-1-hexyl-7-thioxo-6,7-dihydro-1H-pyrido[4,3-dlpyrimidine-2,4-dione (13b) 101 2.11.18 Synthesis of 5-amino-i-butyl-7-methylsulfanyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (14a) 101 2.11.19 Synthesis of 5-amino-i-butyl-7-methytsulfanyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (14b) 102 2.11.20 Synthesis of 5-amino-1-butyl-7-methanesulfonyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (isa) 103 2.11.21 Synthesis of 5-amino-1-hexyl-7-methanesulfonyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (iSb) 104 2.11.22 Synthesis of 5,7-diamino-i-butyl-1H-pyrido[4,3-d]pyrimidirie-2,4-dione (Janus AT 3a) 104 2.11.23 Synthesis of 5,7-diamino-1-hexyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (Janus AT 3b) 105 2.11.24 1-a-2-deoxy-3, 5-di-O-p- toluoyl-D-ribosylchloride (28) 106 2.11.25 Synthesis of 2,4-dioxo-i,2,3,4-tetrahydro-pyrimicline-5-carbonitrile (31) 107 2.11.26 Synthesis of bis-cyanouracil (18) 108 2.11.27 Synthesis of bis-Janus AT (19) 109 2.11.28 Synthesis of 3,5-O-bis(4-methybenzoyI)-cyano-3-D-uracii (30) 110 2.11.29 Synthesis of Janus AT deoxynucleoside (22) lii 2.11.30 Synthesis of bis-(methylthio)-2,4dioxopyrimidine-5-carbonitrile (20) 112 2.11.31 Synthesis of bis-Janus AT 21 113 2.11.32 Synthesis of Janus AT 34 114 2.11.33 Synthesis of Janus AT 35 114 2.11.34 Synthesis of 6-Methylsulfanyl-2,4-dioxo-1-(2,4,6-tri methoxy-phenyl)-1,2,3,4-tetrahydro-pyrimidine-5- carbonitrile 115 2.11.35 Synthesis of Janus AT 36 116 3. Chapter 3: The GC Quartet- a DNA-Inspired Janus-GC Heterocycle: Synthesis, Structural Analysis and Self organization Studies 117 3.1 Introduction 117 3.2 The GC quartet - a novel DNA-inspired Janus-GC heterocycle 118 3.3 Design of Janus GC 1 119 3.4 Synthesis of Janus GC 1 122 3.4.1 Synthesis of 7-cyano-7-deazaguanine 4 122 3.4.2 Bromination of pyrrole core and the unsuccessful attempt to introduce the key nitrogen 122 3.4.2 Nitration of pyrrole core; successful introduction of key nitrogen 123 3.4.3 Catalytic hydrogenation of the nitro group of 6’ to afford 6-amino-5-cyano pyrrolo 7a 125 3.4.3 Finalizing and optimizing the multi-step synthesis of Janus GC 1 126 3.5 Preliminary insights into the self-organization of Janus GC 1 129 3.6 Confirmation of formation of Janus GC 1 quartet ensemble in the gas phase by electron spray ionization mass spectrometry 131 3.7 ‘H-NMR spectroscopy studies 132 3.7.1 Variable-temperature iH-NMR spectroscopy studies on Janus GC 1 134 3.7.2 Size determination of GC quartet using diffusion-ordered NMR spectroscopy (DOSY) 136 3.7.2.1 Concept of translational self-diffusion 137 3.7.2.2 Measuring diffusion with pulse field-gradient (PFG) NMR spectroscopy 139 3.7.2.3 Characterization data obtained for size determination of Janus GC 1 142 VII 3.7.2.3.1 Size determination calculations based on the observed diffusion coefficients 144 3.7.2.3.2 2D-Diffusion ordered spectroscopy DOSY 147 3.8 Conclusion 149 3.9 Current and future trends 150 3.9.1 Formation of GC quartet nanotubes 150 3.9.2 Introduction of Janus GC 1 into the deoxyribose backbone 152 3.9 Experimental 153 3.9.1 General materials and equipment 153 3.9.1.1 Synthesis of 2-amino-6-butylamino-3H-pyrimidin-4-one (3): 154 3.9.1.2 Synthesis of 2-amino-7-butyl-4-oxo-4, 7-dihydro-3H-pyrrolo [2, 3-d] pyrimidine-5-carbonitrile (4) 155 3.9.1.13 Synthesis of N-(7-butyl-5-cyano-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yI)- isobutyramide (5) 156 3.9.1.4 Synthesis of N-(7-butyl-5-cyano-6-nitro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2- yI)isobutyramide (6) 157 3.9.1.5 Synthesis of 2-amino-7-butyl-6-nitro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (6b) 158 3.9.1.6 Synthesis of N-(6-amino-7-butyl-5-cyano-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-dl pyrimidin-2-yI)- isobutyramide (7) 159 3.9.1.7 Synthesis of 2,6-diamino-7-butyl-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]pyrimidine-5-carbonitrile (7b) 160 3.9.1.8 Synthesis of N-[6-(3-benzoyl-ureido)-7-butyl-5-cyano-4-oxo-4,7-dihydro-3H-pyrrolo[2,3- d]pyrimidin-2-yl]isobutyramide (8) 161 3.9.1.9 Synthesis of Janus GC 1 162 3.9.2 DOSY and PFG NMR spectroscopic experiments 163 3.9.2.1 General 163 3.9.2.2 Experimental (Bruker) parameters for PFG NMR of Janus GC 1 164 References 165 Appendix 174 Co-Authorship 174 1H-NMR and‘3C-NMR spectra of compounds presented in Chapter 2 175 ‘HNMRof7 176 13CNMR0f7 176 ‘HNMRofS 177 13CNMRof8 177 ‘HNMRof9a 178 13CNMR0f9a 178 ‘HNMRof2a 179 ‘3CNMRof2a 179 VIII ‘HNMRof2b .180 ‘3CNMRof2b .180 ‘HNMRof6 181 ‘3C NMR of 6 181 ‘HNMRofl 182 ‘3CNMR0f1 182 1HNMR0f11 183 ‘3CNMR0f11 183 ‘HNMR0f12 184 ‘HNMR0f13 185 ‘3CNMR0f13 185 ‘H NMR0f 14 186 13CNMRof14 186 1HNMRof15 187 13CNMRof1S 187 ‘HNMRof3 188 ‘3CNMRof3 188 ‘H-NMRof18 189 ‘3C-NMR of 18 190 ‘H NMR of Janus AT 19 191 ‘3C NMR of Janus AT 19 192 ‘H NMR of bis-(methytthio)-2,4 dioxopyrimidine-5-carbonitrile (20) 193 ‘3c NMR of bis-(methylthio)-2,4 dioxopyrimdine-5-carbonitriIe (20) 194 ‘H NMR of bis-Janus AT 21 195 ‘3c NMR of bis-Janus AT 21 196 ‘3C NMR of bis-Janus AT 21 196 ‘H-NMR of Janus ATdeoxynucleoside 22 197 ‘3c-NMR of Janus AT deoxynucleoside 22 198 ‘H-NMRof28 199 ‘3c-NMR of 28 200 1H-NMR of 30 201 ‘3c-NMR of 30 202 ‘HNMRof27 202 Ix 1H-NMRof31 .203 ‘3C-NMRof31 .204 ‘H NMR of Janus AT 34 205 13C NMR of Janus AT 34 206 1H N MR of 6-Methylsu Ifanyl-2,4-dioxo-1-(2,4,6-trimethoxy-phenyl)-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile 207 ‘3C N MR of 6-Methylsu Ifany-2,4-dioxo-1-(2,4,6-trimethoxy-phenyI)-1,2,3,4-tetrahydro-pyrimidine-5-carbonitriIe 208 ‘H NMRofJanusAT3S 209 NMR Janus AT 35 210 ‘H NMR of Janus AT 36 211 ‘3c NMR of Janus AT 36 212 1H-NMR and 13C-NMR spectra of compounds presented in Chapter 3 213 1HNMRof3 214 ‘3CNMRof3 215 1H-NMRof4 215 ‘H-NMRof4 216 ‘3C-NMRof4 217 1H-NMRof6a 218 13C-NMR of Ga 219 ‘H-NMRof7a 220 ‘3c-NMR of 7a 221 ‘H-NMRofS 222 ‘3C-NMRofS 223 ‘H-NMRof6 224 13C-NMRof6 225 1H-NMRof7 226 ‘3C-NMRof7 227 ‘H-NMRof8 228 13C-NMRof8 229 ‘H-NMR of Janus GC 1 230 ‘3c-NMR of Janus GC 1 231 UVVIS spectrum of Janus GC 1 232 xList of tables Table 1.1. General characteristics of the three main types of hydrogen bonding.5° 5 Table 1.2. Hydrogen bonding distances (in A) in Watson—Crick base pairs in the crystalline state.’213 . 10 Table 3.1. Optimization of the nitration reaction of the pyrrolo core of 4 124 Table 3.2. Diffusion coefficients (D) of 1 and carbazole at 25 °C in DMSO-d6 143 xi List of figures Figure 1.1. Most common supramolecular structures adopted by DNA 1 Figure 1.2. A) Jorgensen’s secondary electrostatic modeL B) The first example of a (AAA-DDD) system reported by Zimmerman and its conversion to (ADA-DAD) system along with their association constants. 7 Figure 1.3. Canonical Watson-Crick base pairing modes 10 Figure 1.4. Major non-Watson-Crick hydrogen bonding modes 11 Figure 1.5. Different natural supramolecular structures of guanosine derivatives; (a) G-ribbon, (b) G quartet, (c) G-quadruplex 14 Figure 1.6. Self-association of the nucleosides guanine and isoguanine in the presence of cations to give G4-quartets or iso G5-pentamer cycles 16 Figure 1.7. Similar to guanine, pterins also can self-organize and create Hoogsteen bonded quartets. Pterin subunit is present in the skeleton of folic acid 17 Figure 1.8. Helical self-assembly of 8-oxogua nosine 18 Figure 1.9. i-Motif hydrogen bonding motif in natural systems occurring in C.CH paring 18 Figure 1.10. Supramolecular synthons based on two or three hydrogen bonds (favourable secondary interactions are shown as dashed lines, and unfavourable secondary interactions are shown as double headed arrows) 20 Figure 1.11. Hexagonal network (rosette) formed between cyanuric acid and melamine with a cavity diameter of approximately 4 A 22 Figure 1.12. Cyanuric or barbituric acid (ADA) and melamine (DAD) hydrogen bonding motif and the two competing rosette and ribbon supramolecular architectures 23 Figure 1.13. Cyclic rosette (ADA-DAD) aggregate templated with rigid and flexible spacers 24 Figure 1.14. An interesting example of using (ADA-DAD) hydrogen bonding motif, to create supramolecular nanotube 25 Figure 1.15. ORTEP view of the three dimensional hydrogen bonded network present in crystals inclusion tecton 1.242 27 Figurel.16. Lehn’s Janus AG molecule capable of forming a triad with an analogue of thymine and cytosine in CHCI3 28 Figure 1.17. Lehn’s Janus wedge concept: Janus wedges with two hydrogen bonding faces are designed to bind by insertion between base-pairs forming a triplet with the maximum number of Watson- Crick interactions. In contrast, common DNA binding molecules target hydrogen bonding sites of the intact base-pair, as illustrated by solid black (or slim black) arrows for the major (or minor) groove side of the AT base-pair 28 Figure 1.18. Trimesic acid self-assembles into a cyclic supramolecular aggregate 30 Figure 1.19. Zimmerman’s cyclotrimerization of self-complementary pyrido[4,3-g]quinoline 30 Figure 1.20. Lehn’s proposed self-assembly of the self-complementary GC base hybrids with (DDA-AAD) hydrogen bonding pattern into a hexagonal rosette ensemble 31 Figure 1.21. Mascal’s X-ray crystal structure of hexagonal rosette array of self-complementary GC base hybrid with (DDA-AAD) hydrogen bonding pattern.252 32 xli Figure 1.22. Fenniri’s helical self-assembly of GC hybrid base into a six-membered rosette and resulting nanotube based on GC base hybrid skeleton as demonstrated. The nanotube figure is adapted from Fenniri, H.; Deng, B. L.; Ribbe, A. E. i. Am. Chem. Soc. 2002, 124, 11064-11072 33 Figure 1.23. Kolotuchin-Zimmerman’s (DDA-ADD) hexameric rosette assembly 33 Figure 1.24. Self-assembly of Sessler’s guanosine-cytidine dinucleoside into cyclotrimer supramolecule. 34 Figure 2.1. Janus AT heterocycles 1, 2a, 2b, 3. Arrows indicate expected hydrogen bond donating or accepting functionalities 40 Figure 2.2. ‘H-NMR spectrum of Janus AT 1 in DMSO-d6 45 Figure 2.3. Variable concentration ‘H-NMR studies of Janus AT 1 in DMSO-d5show no change in chemical shifts upon dilution (25 °C, 400 MHz) 46 Figure 2.4. 2D-Phase-Sensitive‘5N-’H HMQC NMR of Janus AT 1 in DMSO-d6. 47 Figure 2.5. The ORTEP view of the X-ray crystal structure of Janus AT 1 48 Figure 2.6. A) Illustrated hydrogen bonded ribbon array of Janus AT 1, B) Single crystal X-ray structure of supramolecular ribbon of Janus AT 1 49 Figure 2.7. Sliced-away X-ray diffraction of the extended unit cell showing the lattice of 2 sets of parallel arrays 50 Figure 2.8. A) X-ray crystal structure viewed along the array planes (blue ellipses), clearly showing that the dihedral angle of two arrays is roughly 5 ° or less; B) the dihedral angle of the other two arrays is roughly 15 0; C) Propeller twist of a DAD-ADA hydrogen bonding interaction in one of the four arrays. .51 Figure 2.9. The ORTEP view of the X-ray crystal structure of 9a 53 Figure 2.10. Phase sensitive‘5N-1H HMQC NMR of Janus AT 2a in DMSO-d5 55 Figure 2.11 ‘H-NMR spectrum of Janus AT 2b in DMSO-d6 56 Figure 2.12. Variable concentration ‘H-NMR studies of Janus AT 2b in DMSO-d6,showing no change in chemical shifts upon dilution 57 Figure 2.13. The ORTEP view of the X-ray crystal structure of Janus AT 2b 58 Figure 2.14. A: Scheme of formate anion bridging the array, B: X-ray single crystal structure of supramolecular ribbon of Janus AT 2b, C: The extended structure with inter-lattice formate bridges. ... 59 Figure 2.15. ES! Multimeric Mass Spectrum of Janus AT 2a 60 Figure 2.16. ES! Multimeric Mass Spectrum of Janus AT 2b 61 Figure 2.17. The ORTEP view of the X-ray crystal structure of 12a 64 Figure 2.18. The ORTEP view of the X-ray crystal structure of 13b 65 Figure 2.19. ‘H-NMR spectrum of Janus AT 3a in DMSO-d5 68 Figure 2.20. Variable concentration ‘H-NMR studies of Janus AT 3a in DMSO-d6,show no change in chemical shifts upon dilution 69 Figure 2.21 The ORTEP view of the X-ray crystal structure of Janus AT 3a 70 Figure 2.22. A) Scheme of formate anion bridging the hydrogen bonded array of Janus AT 3a, B) X-ray single crystal structure of supramolecular crinkled ribbon of Janus AT 3a with formate bridging 71 Figure 2.23. The ORTEP view of the X-ray crystal structure of Janus AT 3b 72 Figure 2.24. A) Scheme of formate anion bridging the hydrogen bonded network of Janus AT 3b. B) X-ray single crystal structure of supramolecular crinkled ribbon of Janus AT 3b with formate bridging. C) it-it stacking of individual ribbons at the van der Waals contact distance (3.4-3.5 A) giving rise to infinite aggregates including sheets of ribbons 74 XIII Figure 2.25. Bis-Janus AT heterocycles 75 Figure 2.26. Inhibitory effect of Bis Janus AT 21 (0 jiM to 150 jiM) on the activity of DNAzyme 925-11 8 jiM 77 Figure 2.27. Janus AT deoxynucleosides and their potential for oligomerization and formation of the Janus-DNA 80 Figure 2.28. The ORTEP view of the X-ray crystal structure of a-anomer 29 82 Figure 2.29. Janus AT 30, a triply hydrogen bonding faced heterocycle 84 Figure 3.1. Janus GC 1 and the corresponding tetrameric rosette structure 119 Figure 3.2. The ORTEP view of the X-ray crystal structure of derivative 7a 126 Figure 3.3. ORTEP view of the X-ray crystal structure of Janus GC las a formate salt grown in the presence of dioxane 129 Figure 3.4. The formation of a thickened gel, upon dissolution of Janus GC 1 in DMF (3 mg/mL) 130 Figure 3.5. ESI-MS of a solution of 50 IIM Janus GC 1 in a DMSO/MeOH solution. The peaks seen are consistent with the presence of the quartet in the gas phase. Two major peaks for the monomer and dimer are observed along with a peak of lower intensity for the quartet consistent with the association of Janus GC 1 into a cyclic tetrameric species (Intensity of peaks from 1000 — 1350 m/z has been increased 3 times) 131 Figure 3.6.1H-NMR spectrum of Janus GC 1 in DMSO-d6 132 Figure 3.7. Portion of the 2D-NOESY spectrum of Janus GC 1(400 MHz, DMSO-d6) showing strong cross- coupling between the guanosine imino (NH) and the cytosine amino (NaH2)protons on the guanosine and cytosine-like faces as well as the guanosine amino (NLH2)and guanosine imino (NH) protons on the guanosine face 133 Figure 3.8. VT1H-NMR results suggest that the two amino groups in the GC base pair rotate via two different mechanisms. In a model GC base pair, one amino group rotates and the other does not 134 Figure 3.9 400-MHz VT ‘H-NMR spectra of Janus GC 1 in DMSO-d6/CDCI3(60/40%) featuring the four distinct resonances of the amino protons of the C and G faces at -65 °C that are putatively assigned above (* large peak at 7.6 ppm is corresponding to the CHCI3) 135 Figure 3.10 Chemical structures of Janus GC 1 and the internal standard carbazole 136 Figure 3.11 Schematic diagram of the pulsed-field gradient (PFG) method for measuring diffusion coefficients 139 Figure 3.12 The bi-polar longitudinal eddy current (BPLED) pulse sequence which consists of short gradients of contrary polarity, separated by a 180° pulse. In this sequence, each gradient pulse contains of two pulses of different polarity (6 and -G) separated by a 180 ° rf pulse with a duration of 6/2. The 6- l80-(-G) sequence causes the eddy currents to be cancelled out, while the diffusion gradients build up. 140 Figure 3.13. The schematic diagram of the signal decay for the period of diffusion. 6 (gradient pulse) is a variation in the magnetic field in one direction. As a result of diffusion during the delay time (s), the local magnetic field felt by the molecule A during the initial gradient pulse (molecule shown in red) is not exactly equivalent to that felt during the next gradient pulse (molecule shown in black). Therefore the signal of molecule A is decayed. Bigger signal decay is detected for the quicker diffusing molecule B due to the higher dissimilarity in local fields it feels for the period of the gradient pulses 141 Figure 3.14. A typical Stejskal-Tanner plot of experimental peak areas and normalized signal decay as a function of the b-value at 25 °C for ‘H PFG-BPLED NMR of Janus GC land carbazole in DMSO-d5.The xiv representative signals are NH2 of Janus GC land NH of the carbazole. The solid lines represent linear least-squares fits to the data 143 Figure 3.15 A) Normalized signal decay as a function of the g (gradient strength) at 25°C for NH2 (6.63- 6.66 ppm ) Janus GC in DMSO-d6/CDCI, B) Normalized signal decay as a function of the g (gradient strength) at 25 °C for NH 11.25-11.21 ppm) carbazole in DMSO-d6/CDCI3 144 Figure 3.16. 2D-diffusion ordered ‘H spectrum (DOSY) of the equimolar mixture of Janus GC land carbazole in DMSO-d5. The horizontal axis displays the conventional proton spectrum which is spreaded along the upright dimension by individual diffusion coefficients 148 Figure 3.17. The proposed GC quartet and a schematic illustration of the expected discotic liquid crystalline mesophase structure in its likely form 150 Figure 3.18. The proposed derivatizations of Janus GC’s tail 151 Figure 3.19. Janus GC deoxynucleosides and their potential for oligomerization and formation of the Janus-DNA 152 xv List of schemes Scheme 2.1. Retrosynthetic analysis of A) Janus AT 1, B) Janus AT 2 and C) Janus AT 3 43 Scheme 2.2. a) Triethylorthoformate, acetic anhydride, reflux, 45 mm, 95% b) Butyl amine, water, 85 °C, 15 mm, 93% c) Dicyandiamide, K0H, DM50, 100°C, 4 hours, 80% 44 Scheme 2.3. Synthesis of Janus AT 2. a) CS2,K2C03,DMF, RT, 95% b) Mel,H20:CH3CN (7:3), reflux, 85% c) Butyl or heptyl amine, EtOH, reflux, 87% d) Guanidinium hydrochloride, NaQEt, EtOH, reflux, 85% 52 Scheme 2.4. Synthesis of Janus AT 3a & 3b. a) Triethyl orthoacetate, acetic anhydride, reflux, 40 mm, 90% b) R-NH2,H20, 20 mm, 90 °C, 91% c) i. CS2, t-BuOK, dry THF, rt, 2h ii. acetic acid, H20, 89% d) Ammonium hydroxide, sealed tube, 100 °C, 12 h, 93% e) Mel, NaOH, H20, 90% f) (H20,formic acid, 4 h, RT) or (MCPBA, CHCI3 4 h, RT), 95% g) NH3, MeOH, sealed tube, 100 °C, 24 h, 92% 62 Scheme 2.5. Intended route for synthesis of Janus AT 3 63 Scheme 2.6. A nucleophilic aromatic (SNAr) amination reaction, utilized to complete the syntheses of Janus AT 3 a) Mel, NaOH, H20, 90% b) (H20,formic acid, 4 h, RT) or (MCPBA, CHCI3 4 h, RT), 95% c) NH3, MeOH, sealed tube, 100 °C, 24 h, 92% 66 Scheme 3.1. Retrosynthetic analysis of the target molecule Janus GC 1 121 Scheme 3.2. Synthesis of 7-cyano-7-deazaguarimne 4. a) N-butylamine, water, reflux, 5 hours, 90% b) 2- Chloro-3-oxopropionitrile, sodium acetate, water, 80°C, 75% 122 Scheme 3.3. Bromination of pyrrole core of 4 and unsuccessful amination attempts. i) NBS, CH2I RT, 90% ii.1) Sodium azide, DMF, RT, 48 h, NR. ii.2) Methylamine, sealed tube, H20, reflux, 24 h, NR. 11.3) Butylamine, sealed tube, H20, reflux, 24, NR 123 Scheme 3.4. Nitration of the pyrrole core of 7-cyano-7-deazaguanosine 4 124 Scheme 3.5. Catalytic hydrogenation of the nitro group of 6a 125 Scheme 3.6. Synthesis of Janus GC 1 a) N-butylamine, water, reflux, 5 hours, 90% b) 2-Chloro-3- oxopropionitrile, sodium acetate, water, 80°C, 75% c) Isobutyric anhydride, reflux, 2 hours, 90% d) Ammonium nitrate, TFAA, CH2I rt, 8 hours, 93% e) H2 latm, 10%-Pd/C, MeOH, rt, 2 hours, 97% f) Benzoyl isocyanate, pyridine, CH2I rt, 1 hour, 93% g) Sodium hydride, ethanol, toluene, reflux, 15 hours, 89% 127 xvi List of abbreviations AcOH Acetic acid AcOEt Ethyl acetate bs Broad singlet CD Circular dichroism CH21 Dichloromethane CHC13 Chloroform CS2 Carbon disulfide d Doublet dd Doublet of doublet DMT Dimethoxytriphenylmethyl chloride DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DOSY Diffusion ordered spectroscopy ESI Electro-spray ionization Et20 Diethyl ether HC1 Hydrochloric acid HMDS Hexamethyldisilane HSQC Heteronuclear single quantum coherence HRMS High resolution mass spectrometry m Multiplet MCPBA Meta-chloroperoxybenzoic acid Me Methyl MeOH Methanol xvii MS Mass spectrometry NMR Nuclear magnetic resonance NOE Nuclear overhauser enhancement NOESY Nuclear overhauser enhancement spectroscopy ppm Part per million q Quartet RNA Ribonucleic acid rt Room temperature s Singlet t Triplet t-Bu tert-Butyl TLC Thin layer chromatography TFA Trifluoroacetic acid TFAA Trifluoroacetic anhydride TOF Time of flight 6 Chemical shift XVIII Acknowledgments First and foremost I would like to thank my supervisor Professor David Perrin for his support, constant insight and availability. His enthusiasm and broad knowledge for science have been always inspiring and encouraging. I am truly indebted for the opportunities that he has given me throughout my graduate career to think independently, lucidly and professionally as a researcher. The years of my graduate studies have been an exhilarating experience. In particular, I would like to express my special appreciation to my kind wife Behnaz Aziz Souna for her understanding, patience and serenity. I would like to express my love to my son Yousef for his amusing presence and the blessing of his love. I thankfully acknowledge Professor Marco Ciufolini for his exceptionally helpful discussions about chemistry. His vision in chemistry has been always inspirational for me. I would like to thank Professor John Sherman for his very helpful discussion about DOSY experiment and for proof reading my dissertation. I would like to thank Dr. Mehran Nikan and Dr. Maria Ezhova for their very helpful technical assistance on DOSY experiment. I would like to express my ultimate appreciation to my parents. I have attained this accomplishment only because of their inspiration, guidance, and support. I would like to thank my father, Professor Bijan Asadi and my mother, Zohreh Bayanian for their unwavering love, compassion, faith and encouragement. My thanks go to all my colleagues in the Perrin lab. Their friendship and companionship will stay with me without end. In particular, I would like to thank Dr. Marcel Hollenstein for proofreading my dissertation. xix Dedication Dedicated to my eternally beloved Someone said “Love, pleasant folly...”, I say “Love, lunatic happiness...” True love doesn’t know how to speak! 11. Chapter 1: Background and Significance 1.1 Introduction Study of non-covalent intermolecular interactions such as hydrophobic, van der Waals, iu-t stacking, ion—dipole and hydrogen bonding is one of the most interesting topics in chemistry.’8 This study involves all areas of chemistry, predominantly the flourishing areas of structural biology,”2 supramolecular, and materials chemistry.38 The spontaneous association of definite classes of molecules into well-organized assemblies or structures, held together by non-covalent interactions, is also central to understanding the process of moving from simple molecules toward nature’s most complex functions.9 Among different non-covalent intramolecular interactions, hydrogen bonding is largely responsible for the specificity that is uniquely characteristic of life and plays an essential role in storage, replication, and transcription of genetic information. Indeed, correct hydrogen bonding ensures sequence specificity in the templated recognition and synthesis of DNA and RNA as well as in the self-association of single-stranded DNA leading to various regular supramolecular architectures including A-, B-, and Z-form double helices (duplex), hairpin, triplex (H-motif), G-quadruplexes (observed in G rich sequences) and i-motif structures formed by the anti-parallel intercalation of two C-rich parallel duplexes (Figure 1.1). ‘° Figure 1.1. Most common supramolecular structures adopted by DNA TTT 3, 5, \ / II TT A G---C T T c IC’T CjC C-i 1 ) 5’ 3’ 5’ A’ Duplex Hairpin Triplex (H-motif) G-quadruplex i-motif 2The complexity of natural systems always has been a stimulus to inspire chemists. Thus, it is not surprising that the biological importance and elegance of hydrogen bond driven base-pairing has triggered substantial research efforts to both understand this phenomenon and in so doing, to mimic it. For example, intensive research focused on questioning whether base pairing interactions could be exploited for applications other than those seen in naturally occurring biological systems. Such ideas are not new; in fact, researchers have used natural long chain DNA oligo-nucleotides as well as artificially modified DNA strands to create well-designed macroscopic assemblies with possible applications in nanotechnology and materials science.’ 1-13 Other research groups, encouraged by the hydrogen bonding seen in double-helical DNA, have prepared smaller synthetic ensembles based on the development of unconventional hydrogen bond driven recognition motifs. This class of research, known as non-covalent or supramolecular chemistry,’4”5intends to use chemical interactions beyond the molecular level and has become a very powerful strategy for the construction of well-defined nanostructures. This research deals with the molecular recognition properties of individual molecules and their self-organization to form well defined higher order structures and assemblies utilizing primarily hydrogen bonds. These aggregates have been found to form in solution or in the solid state.14’67 This dissertation illustrates the research that has been carried out with regard to the design and the synthesis of several new classes of DNA-inspired heterocycles. Specifically, the design, syntheses and self-organization studies described herein of a number of new one component self-complementary nucleobases, address a variety of specific issues, as will be discussed in this thesis. This chapter will provide an introduction to the major terms and definitions used in the context of self-organized base-pairing motifs by focusing on the background research that has been performed mainly on self-complementary and self-organized systems. That being noted a comprehensive discussion involving many other elegant hydrogen 3bond driven recognition moieties and classifications is not included. For a systematic discussion on this subject, the reader is referred to an in-depth review by Rheinhoudt,’4”8and various extensive and comprehensive reviews which encompass the entire subject.17’93 1.2 Major terms and definitions 1.2.1 Hydrogen bonding; a definition The powerful and directional character of hydrogen bonding interactions 32 explains their extensive presence in self-assembling systems: initially proposed by Bemal and Huggins in 1935, the term hydrogen bonding (abbreviated as H-bonding) is described as a non-covalent, attractive interaction between a proton donor D—H and a proton acceptor A group in the same or in a different molecule as shown below. Where: D = N, 0, F A = N, 0, F, Ct, Br According to conventional definitions, a hydrogen atom is bonded to electronegative atoms such as 0, N, and F. The acceptor A can be either an electronegative atom or functional group with t-electrons.6’344°However, in some instances the experimental results reveal that even C—H can be involved in hydrogen bonds and it-electrons can act as proton acceptors in the stabilization of a particularly weak hydrogen bonding interaction that may exist in many chemical and biological systems.37’4145 The it-hydrogen bond is an interaction between a partially positively charged hydrogen atom and the electrons of double andlor triple bonds or aromatic ic-bonds.46 In classical hydrogen bonding, there is a shortening of the distance between 4A and D when D—H is hydrogen bonded to A such that the distance between A-D is less than the sum of the van der Waals radii of the two atoms D and A separately. Hydrogen bonding interactions lead to an increase in the D—H bond distance. Formation of the D—H””A bond decreases the mean magnetic shielding of the proton involved in the hydrogen bonding thus leading to a downfield shift in chloroform. In the ‘H-NMR the downfield shift in hydrogen bonded complexes is of the order of a few ppm and the anisotropy of the proton magnetic shielding can be amplified by as much as 20 ppm.47’8 The strength of strong hydrogen bonding interactions is at least 1—2 kcal/mol in water, and 15 to 45 kcal/mol in the gas phase.36’49 For weak hydrogen bonds (i.e. CH--O and OH-it) and moderate (i.e. conventional OH--O and NH--O), the strengths vary from 0.5—4 in water to 4— 15 kcallmol in organic solvents, respectively. If either the donor or the acceptor is charged, the electronic attraction will be amplified, and consequently the implicated hydrogen bonds become much stronger (10-45 kcal/mol in organic solvents).36’495 The strength of hydrogen bonded interactions in diverse molecular systems has been classified (Table 1.1). Desiraju has proposed a comprehensive portrayal of the hydrogen bonding interactions in a range of systems and the concept of “hydrogen bridge”.49 To maximize the interaction, the geometrical requirement is that the D—H”A angle be near 180° (as in HCN H—F and F—HF) and that the hydrogen aligns itself approximately with an unshared pair of electrons of the acceptor atom A. The hydrogen bond length refers to the H--A distance. Evidence for a D—HA hydrogen bond is generally based on the observation of D-A distances that are less than the sum of the van der Waals radii of the individual atoms (for N-H”O hydrogen bonds, dDA= 3.07 A). Characteristic values for the H-A distance are 1.80 to 2.00 A for NH”0 hydrogen bonds and 1.60 to 1.80 A for OHO bonds. The three types of hydrogen bonding interactions which are 5most often discussed in the literature are weak, moderate, and strong. The properties of these three types are listed in Table 1.1. 50 H-bond parameters - Strong Moderate Weak Type of interaction Strongly covalent Mostly electrostatic Electrostatic/dispersed Bond lengths 1.2—l.5 1.5—2.2 2.2 (H-A [A]) Extension of 0.08—0.25 0.02—0.08 0.02 D-H (A) D-H Vs. H--A D-H approx H--A D-H < H--A D-H << H---A H-bond length 2.2—2.5 2.5—3.2 >3.2 (D-A [A]) H-bond angles (a) 170_1800 [ >1300 >90° H-bond strength 15—40 4—15 <4 (kcal/mol) Table 1.1. General characteristics of the three main types of hydrogen bonding.5° The hydrogen bond strength depends on its length and angle. However, small deviations from linearity in the bond angle (up to 20° degrees) appear to have marginal effects on hydrogen bond strength. The dependency of hydrogen bond strength on length is also very important and has been shown to decay exponentially with distance, on the order of (1/d2). The nature and polarity of the solvent also affects the strength of hydrogen bonding.52 The issue of “what is the 6essential nature of hydrogen bonding?” has been the subject of numerous studies in the literature.9’5355 In a classical sense, hydrogen bonding is highly electrostatic and partly covalent. From a precise theoretical perspective, hydrogen bonding is not a simple interaction. It has contributions from electrostatic interactions (acid/base), polarization (hard/soft) effects, van der Waals (dispersion/repulsion: intermolecular electron correlation) interactions and covalency (charge transfer). Whitesides,9”’53666 Wuest,677° Rebek,’9’775 Ghadiri,768° and others 81.84 have methodically categorized the structures of hydrogen bonding interactions and identified prototypes of hydrogen bonding taking place between specific functional groups. Etter, in particular, has proposed rules that describe the selectivity of different functional groups in forming hydrogen bonds.85 1.2.2 Jorgensen’s secondary interactions Hydrogen bond has contributions from electrostatic interactions, and therefore when several hydrogen bonds are present, secondary interactions need to be considered in addition to the primary attractive interactions.86’7These secondary interactions can be either attractive or repulsive, as demonstrated for the systems containing two and three hydrogen bonds illustrated in Figure 1 .2a. The free energy of a secondary interaction has been calculated to be approximately (+/-) 0.7 kcal/mol for a system containing three parallel hydrogen bonds.88 Stabilization derives from electrostatic attraction between positively and negatively polarized atoms in neighbouring hydrogen bonds, and similarly destabilization is the result of electrostatic repulsion between two positively or negatively polarized atoms. 7Figure 1.2. A) Jorgensen’s secondary electrostatic model. B) The first example of a (AAA DDD) system reported by Zimmerman and its conversion to (ADA-DAD) system along with their association constants. As a result of these secondary interactions, the DD-AA motif is expected to be more favourable than the DA-AD motif whereas for systems containing three hydrogen bonds, DDD AAA is more favourable than DDA-AAD, which in turn is more favourable than ADA-DAD. Based on an assessment of experimental binding data for 60 different synthetic hydrogen bonded a) ADA-DAD 4 repulsive secondary interactions - DAA-ADD O total repulsive & attractive secondary interactions AAA-DDD 4 total attractive secondary interactions Attractive secondary interactions Repulsive secondary interaction Primary H-Bonding G=A G=D 8complexes, Sartorius and Schneider proposed a simple empirical rule that can be used to predict the binding energy of a particular complex.89 They postulate that the free energy for hydrogen bond promoted dimerization consists only of two increments: a contribution of 1.88 kcal/mol for each hydrogen bond and (+1-) 0.7 kcal!mol for each attractive or repulsive secondary interaction in chloroform. To validate Jorgensen’s secondary electrostatic model, Murray and Zimmerman reported the first example of a (AAA-DDD) hydrogen bonding module that has the ability to switch to the tautomeric form (ADA-DAD) hydrogen bonding pattern (Figure 1 .2b). Comparing their corresponding association constants allowed for the careful assessment of structurally similar complexes and showed consistency with the Jorgensen proposal.9° 1.3 Hydrogen bonding in nature As mentioned previously, hydrogen bonding is a unique interaction which is significant in biologically important interactions. It is well recognized that the hydrogen bonding interaction is important for the structure and function of bio-molecules. Indeed hydrogen bonding in a- helical and B-sheet structures in proteins has been elucidated by Pauling et a!. ,91 cohesion of DNA base pairs by hydrogen bonds has been disclosed by Watson and Crick, 92 and the triple helical structure of collagen has been unravelled by Ramachandran and Kartha just to name a few. The hydrogen bonding interaction in nucleic acids plays a crucial role in the double helical structure of DNA and RNA along with stacking interactions facilitating molecular recognition via replication processes and protein synthesis. Extensive theoretical methodologies have been used to derive information about the hydrogen bonding in DNA base pairing and base stacking.94”8The hydrogen bonding pattern for the G-C and A-T DNA base pairs is shown in 9Figure 1.3. The ab initio calculation on the G-C and A-T base pairs provides the required information about the strength of hydrogen bonding in these systems and the respective binding energies are 20 and 17 kcal/mol in gas phase.’°° Hydrogen bonding is mainly accountable for DNA—ligand recognition, and studies on molecular recognition has guided the design new drug- like molecules.”9An attractive means of constructing supramolecular assemblies is through the use of synthetic molecules infused with recognition units known as supramolecular synthons, in precise spatial arrangements. Such encoded molecules can be mixed and matched with other molecules possessing complementary recognition units to form distinct supramolecular assemblies. 1.3.1 DNA/RNA base pairing Inferred from the influential structural studies of the DNA double-helix by James Watson and Francis Crick, two canonical base-pairing motifs were determined (See Figure 1.3).120 There are three kinds of hydrogen bonded dimers in nature that are formed by complementary nucleobases. These are the base pairs adenine-thymine (A-T), adenine-uracil (A-U), and guanine-cytosine (G-C) (Figure 1.3 and Table 1.2). The intrinsic interactions in these nucleic acid base pairs are commonly referred to as Watson-Crick type and they play in a key role in the storage and decoding of genetic information. 10 Figure 1.3. Canonical Watson-Crick base pairing modes. T---A N3-H Ni 2.835 04 H-N6 2.940 02 H-N2 2.86 C---G N3 H-Ni 2.95 N4-H 06 2.91 Table 1.2. Hydrogen bonding distances (in A) in Watson—Crick base pairs in the crystalline state. 121.123 It should be considered that Watson-Crick type base-pairing is not the only base-pairing motif available to nucleobases. In an alternative mode, hydrogen bonding may take place on the Hoogsteen edge as well. 124-127 A Hoogsteen base pair applies the N7 position of the purine base as hydrogen bond acceptor and the C6 amino group as a donor, which bind the Watson-Crick N3 face of the pyrimidine base (Figure 1.4). In fact, nucleobases in their unsubstituted forms can self-assemble using a variety of non-Watson-Crick, homo and hetero, base-pairs (See Figure 1.4). These non-Watson-Crick binding modes along with the other modes are responsible for the variety of tertiary structures formed by RNA and DNA. Within the framework of preparing synthetic self-assemblies, these modes are extremely important since they can be used to bring I;1 Watson-Crick (A-T) Watson-Crick (G-C) 11 together new ensembles with architectures that are not achievable by normal Watson-Crick base pairing. Figure 1.4. Major non-Watson-Crick hydrogen bonding modes. The Hoogsteen interaction, i.e., the one that utilizes the C6-N7 face of purine nucleobases, is arguably the most important of the non-Watson-Crick base pairing modes. This is because the Hoogsteen motif is the primary hydrogen-bonding interaction available to purine nucleic acids which are already involved in Watson-Crick hydrogen bonds. Watson-Crick driven duplex DNA also can interact with another oligonucleotide strand to form triple helices using Hoogsteen hydrogen bonds.’24’7 In addition, the Hoogsteen edge can also be used by proteins and small molecules to bind to double stranded DNA. Another example where Hoogsteen interactions play a critical role is in the stabilization of guanine rich oligonucleotide sequences R [;I .N. H 8 ‘4 Hoogsteen (AT) Seif-Dimerization (GG) G-quartetWobble (GT) Ribbon-like aggregation of guanine Reverse Watson-Crick (AT) i-motif’s C-CH base pair UAU base triplet (Watson-Crick and Hoogsteen 12 by the formation of quadruplex structure. These latter quadruplexes have been implicated in the maintenance of eukaryotic chromosomes. 136-138 Other non Watson-Crick base-pairing motifs include the wobble (or mismatched) base- pairs and various homo dimers (self-pairing). Variant base-pairing modes can also result from the conformation of the sugar residues with respect to what is normally seen in, e.g., Watson- Crick base-pair (i.e., cis vs. trans conformation with regard to the sugar on the complementary nucleobase). For instance, the ‘reverse’ base-pairing mode is characterized by a trans, or anti- parallel, conformation of the two respective sugar residues. An example of a reverse Watson- Crick hydrogen bonding mode is illustrated in Figure 1.4. Other, albeit less prevalent, dimerization modes are also possible as a result of tautomerization and ionization. In addition to nucleobase dimerization, individual bases can also form trimers and other higher order oligomers)21 These higher order species can be useful for researchers interested in the preparation of hydrogen bond driven polymeric materials. On the other hand, ill-defined oligomeric systems can produce various complications for synthetic chemists trying to prepare discrete, well defined ensembles. One method used to circumvent the formation of such oligomers involves the judicious monitoring of nucleobase concentrations, since oligomers are usually favored at higher solute concentrations. Inspired by the elegance of hydrogen bonding elements present in natural DNA/P.NA base-pairing motifs, as well as by the amenability of preparing hydrogen bonding donor/acceptor sites that act in a co-operative and directional fashion, many research groups have visualized the self-assembly of elegant supramolecular structures through hydrogen bonding driven molecular recognition. The endeavour of the current work is to create supramolecular architectures using DNA inspired hydrogen bonded driven base-pairing. In the current dissertation X-ray crystallography 13 has been utilized extensively to reveal the supramolecular architecture in the solid state and therefore part of this project has been focused on crystal engineering. 1.3.2 G-quartets in nature and self-assembly of guanosine derivatives Higher order complexes that are associated by hydrogen bonding interactions of one component nucleobases have the potential to be exploited in a number of applications. These applications range from the construction of self-organized ionophores to the generation of liquid crystalline mesophases.’28”9In the quest of such objectives, guanine and its hydrophilic and hydrophobic derivatives are of particular interest. For instance, supramolecular polymers of guanine are attractive for use in various electronic applications since guanine possesses a low oxidation potential.’30”’ The first study on the self-assembly of guanosine was carried out about 80 years before its potential biological implications were studied in vitro by Sen et.al.’32 It has long been known that guanosine nucleosides in oligo- and polynucleotides can readily self-associate, provided that a monovalent cation such as potassium or sodium is present to provide the needed additional stabilization by chelation.’33 This remarkable ability to support the formation of a number of architectures is largely due to the fact that guanine can employ both Watson-Crick and Hoogsteen base-pairing interactions. In addition, guanine derivatives can self-assemble into ribbons,’34 tetramers (G-quartets),’29 and higher order structures depending on the conditions.89”35 A prime example of this is the metal-templated self-assembly of guanine derivatives into G-quartets which involves a total of eight hydrogen bonds between the Watson— Crick face of one guanine base and the Hoogsteen major-groove face of another, in a similar manner to that observed in some G-G mismatched duplexes (Figure 1.5). 14 X-Ray diffraction patterns from fibers of poly(G) have been interpreted as arising from a novel four-stranded helix,’36”7with four guanine bases, one from each strand, being involved in a planar tetrad pattern with G—G base-pairing between them. This cyclic self-assembled structure, formed by the single-stranded overhang regions of telomeric DNA,’38 has implications in research since it might be triggering the discovery of cures against cancer as well insights into the process of aging.’39”4°Telomeric DNA is comprised of tandem repeats of short guanine tracts, sometimes including adenines/thymines, such as or (TTAGGG), Ra) G-ribbon N o u—NH H N NN 0 N N />N ‘I-IR H’° H R — Metal ion i- Metal ion b) G-quartet c) G-quadruplex Figure 1.5. Different natural supramolecular structures of guanosine derivatives; (a) G ribbon, (b) G-quartet, (c) G-quadruplex. 15 together with associated telomeric proteins.’4’The nature of the repeat is species-dependent, with (TTAGGG) being the most common repeat observed in mammals.’42’7The function of telomeres is to protect chromosomal ends from unwanted recombination and nuclease damage. It is thought that the formation of such guanine quadruplexes at the end of human chromosomes inhibits the activity of the enzyme telomerase which is upregulated in most cancer cell lines.’42 147 Molecules that bind to G-quadruplexes and or i-motif structures (see section 1.3.3) may inhibit cancer growth by two individual mechanisms. First, the over-expression of oncogenes like c-Myc, c-Kit, and K-Ras could be down regulated by small molecule stabilization of G quadruplex or i-motif structures in the promoter areas of these genes.’48 The second, and the most extensively studied mechanism, is the inhibition of telomerase, a ribo-nucleoprotein complex that catalyzes the 3’-extension of telomeric DNA, which is essential for the continued division of most immortalized cells ‘ as well as cancer cells.’50 Inspired by the beauty and biological significance of G-quadruplexes, supramolecular chemists have used lipophilic guanine derivatives to create self-assemblies that exhibit a variety of functions. The research groups of Gottarelli, Davis, and Spada have comprehensively studied the self-assembly and material properties of guanine derivatives; the interested reader is directed to a comprehensive review on the topic by Davis and Spada.” Davis and colleagues have demonstrated that isoguanine can form self-assembled cyclic structures and that the size of the resultant self-assembled supra-molecules can be tuned by altering the templating metal cation. For instance, iso-G (Figure 1.6) can form a pentameric assembly in the presence of cesium cations. When cesium cation is used in templation, iso-G self-assembles into sets of doubly stacked pentamers, which wrap around the central metal cation.’52 An illustration of such a pentamer is shown in Figure 1.6. 16 Figure 1.6. Self-association of the nucleosides guanine and isoguanine in the presence of cations to give G4-quartets or iso G5-pentamer cycles. Folic acid is an important, biologically active molecule bearing a characteristic pterine heterocycle. Similarly, analogs of folic acid and pterin, which resemble guanine, also form tetrameric macrocycles closely related to a G-quartet (Figure 1.7). Self-association of the folic acid salts and pterin in water to form cyclic tetrameric rosettes in solution has been examined by Gottarelli, Bonazzi, Sakai and Ciuchi.’53’6 Recently, the Kato and Matile groups have also reported a synthetic ion channel formed by the self-organization of a lipophilic folate derivatives.’56 M+. Na+ or K+ Guanine N. <I R Isoguanine 67’ angIe 17 Helical structures are found extensively in nature and are responsible for many of the essential biological functions, and therefore a number of research groups have focused their studies on the building of supramolecular helical In addition, Giorgi and Gottarelli have verified that 8-oxoguanosines also self-organize into supramolecular helical architectures (Figure 1.8) in solution and solid state, but were only able to form ribbon-like aggregates from the parent guanine under the same conditions. The helical assemblies formed by 8-oxoguanosine also have electrical properties, leading the authors to suggest that such compounds may be useful for the fabrication of optoelectronic devices. Circular dichroism, NMR, scanning tunnelling microscopy (STM) and X-ray diffraction reveal the presence of helical structures in solution and solid phase.’58 Figure 1.7. Similar to guanine, pterins also can self-organize and create Hoogsteen bonded quartets. Pterin subunit is present in the skeleton of folic acid. 18 Figure 1.8. Helical self-assembly of 8-oxoguanosine. 1.3.3 The i-Motif; self-assembly of cytosine in nature The DNA strand complementary to a guanine-rich double-stranded telomeric sequence is by definition a cytosine-rich strand. An NMR study on the cytosine-rich strand d[TCCCC] at acidic pH revealed that this sequence did not form a duplex, but instead a novel tetra-stranded quadruplex which was termed the i-motif.’76 (Figure 1.9) The basic unit is the C•C+ mismatch base pair, with one of the two cytosines protonated at the N3 position such that three hydrogen bonds are formed. The i-motif sequence is present in duplex telomeric DNA as the complement to the quadruplex-forming G-rich strand. Various crystal structures of such sequences have been reported and all show this structural i-motif.’77’9 A A 0 C9H19 D D Hydrocarbon solvents (e.g. Heptane) 0Y °r° C9H19 C9H19 i-motif Figure 1.9. i-Motif hydrogen bonding motif in natural systems occurring in C.CH paring. 19 1.4 Supramolecular chemistry According to Lehn, who first used the term, a supramolecule is described as an organized complex that is formed from the association of two or more chemical species held together by intermolecular 80 Beyond the perspective of molecular chemistry based on the covalent bonds, supramolecular structures are the result of not only additive but also co-operative, non- covalent intermolecular interactions (including hydrogen bonding, hydrophobic interactions and coordination) to develop highly complex chemical systems displaying properties which are different from individual interacting components.9”81’4Supramolecular chemistry has started to make a great impact in current scientific research.’85 Supramolecular chemistry has become a significant tool in a whole congregation of multidisciplinary research fields including the preparation of molecular machines,’86’8 sensing of biologically and environmentally relevant analytes,’89’93 elucidating and mimicking protein-protein and protein-small molecule and preparing novel polymeric materials.’95 1.4.1 Supramolecular synthons The term supramolecular synthon, analogous to the synthons of organic synthesis, was introduced by Desiraju to describe the “structural units within supermolecules which can be produced andlor assembled by known or plausible synthetic operations involving intermolecular rnteractions”.196 Such supramolecular synthons can involve two identical or different components (Fig. 1.10). 20 ONO 1 I :\Fl a) DD-AA b) DA-AD c) DAD-ADA ONN N’N’N XN HFyNyO N1O d) DDA-AAD e) AAA-DDD Attractive secondary interactions Repulsive secondary interaction . Primary H-Bonding Figure 1.10. SupramolecuJar synthons based on two or three hydrogen bonds (favourable secondary interactions are shown as dashed lines, and unfavourable secondary interactions are shown as double headed arrows). 1.4.2 Crystal engineering Crystal engineering 197 is the division of supramolecular chemistry focusing on the design and synthesis of one-, two- and three dimensional extended structures with predictable shape and function, mainly by the use of hydrogen bonding. In-depth analysis of the arrangement of molecules in the solid state is central to the science of organic crystal engineering, a growing technology that is producing new, well-designed materials.5”98 Crystal engineering involves controlling and utilizing the association of molecules in crystals, or more simply crystalline phase supramolecular chemistry.55”9920’One of the aims of crystal engineering is to construct 21 crystalline molecular or polymeric materials with highly ordered networks or infinite framework structures. Initially the term crystal engineering was used by Schmidt 30 years ago in a discussion of photo-dimerization reactions in crystalline cinnamic acids. However, it is only in the past 15 years that interest in the area has flourished and that the fundamental concepts extended as such that several books and reviews are covering the topic.49’5”839679202 Modern crystal engineering is an interdisciplinary topic, with contributions from and implications in organic, inorganic, organometallic, theoretical and materials chemistry, biology, crystallography and crystal growth. A useful modern definition is that provided by Desiraju who defined crystal engineering as “the understanding of intermolecular interactions in the context of crystal packing and the utilization of such understanding in the design of new solids with desired physical and chemical properties”.202 When crystallization is considered as a supramolecular reaction, sometimes the outcome depends on whether the system is able to select the path which will minimize the energy to the global minimum (thermodynamic crystallization) or the self- organized aggregate is one of the kinetic possibilities of crystallization (kinetic crystallization).202 1.5 One and two component self-complementary hydrogen bonding systems In supramolecular chemistry, strategies that utilize hydrogen bonding generally involve either one or two component systems. Thus, in the following discussion the molecular components that together program a hydrogen-bonded network have been introduced and categorized in these two classes. 22 1.5.1 Two component self-complementary hydrogen bonding systems One of the most studied motifs using the two-component complementary system is cyanuric or barbituric acid and melamine with the complementary ADA-DAD hydrogen bonding motif 57,59,6,M,65,90,203224 Whitesides and colleagues have comprehensively studied the two- component (1:1) hydrogen bonded complex between cyanuric acid and melamine which self- organize into a hexameric rosette1.This is a prime example of non-covalent synthesis and the resulting structure was unequivocally proven and illustrated by single crystal X-ray crystallography (Figure l.ll).225364225226 ‘Rosette is a circular array of parts, radiating out from the center and suggesting the petals of a rose or the windows of a gothic cathedal. I+I I+1 HfN% N-N..Y H1’H.. NH Figure 1.11. Hexagonal network (rosette) formed between cyanuric acid and melamine with a cavity diameter of approximately 4 A. 23 The DAD-ADA hydrogen bonding motif has two disadvantages; it is the weakest hydrogen bonding module among the triply hydrogen bonded arrays and its intrinsic symmetry causes competition between formation of a ribbon versus a cyclic aggregate (Figure l.12).1458 60,208,222,227-229 To counter this problem two different strategies, namely peripheral crowding and covalent pre organization, have been used to ensure the selective construction of cyclic rosette assemblies. The application of these general startegies has been studied extensively by both the Whitesides et al. 16,56-62,64,65,222,223,230 and Reinhoudt et at 16,230-232 and a few selected examples are discussed. Figure 1.12. Cyanuric or barbituric acid (ADA) and melamine (DAD) hydrogen bonding motif and the two competing rosette and ribbon supramolecular architectures. 24 As shown in Figure 1.13 Whitesides and his colleagues designed a tris-melamine complex containing three subunits of melamine which are covalently attached to a central hub in order to assemble into rosette aggregate. 64,65,223,233-236 Figure 1.13. Cyclic rosette (ADA-DAD) aggregate templated with rigid and flexible spacers. Aggregates based on hub and spoke architectures have a greater tendency to organize the un-complexed tris-melamine such that its conformation in solution and in the absence of molecules of isocyanuric acid resembles that in the aggregate. An investigation of the formation of this complex revealed the importance of thermodynamic factors during supramolecular formation (Fig 1.13). Fixation of the melamine units is beneficial when forming the closed complex and in turn, the efficiency of complex formation was found to be dependent on the flexibility of the spacer chains. Use of a tris-melamine unit with a rigid spacer resulted in a more stable complex than that yielded by a tris-melamine unit with flexible spacers. 233-236 general, such a flexible supramolecular complex formation is enthalpically favorable but is sometimes entropically unfavorable. If the latter entropic disadvantage is suppressed, the complex formation becomes thermodynamically more favorable. The conformational freedom in the tris-melamine ‘—, —-: ‘ • c3 • • Jt FIe>bIe SDactr Pigid Spacer 25 unit with inflexible spacers is restricted. Therefore, the loss of entropy upon binding with cyanuric acids is not as important as for the tris-melamine unit with flexible spacers, leading to a more favorable complex formation. Aforementioned examples show the importance of templating and constructing the molecular units appropriately before supramolecular assembly in directing the supramolecular structure. Another interesting example of using ADA-DAD hydrogen bonding motif in a two component complementary system can be found in the supramolecular nanotube designed by Kimizuka and Kunitake (Figure 1.14). TEM evidence illustrates that a hydrogen bonded aggregate is present as strands with Ca. 80 A width.237’8 Figure 1.14. An interesting example of using (ADA-DAD) hydrogen bonding motif, to create supramolecular nanotube. 26 Hydrogen bond driven self-organization of two-component complementary recognition motifs has been exploited extensively to create a variety of supramolecular architectures.90’203 207,209,210,212-221,224 There are far fewer examples of using one-component self-complementary motifs, in which the proper hydrogen bonding pattern itself is sufficient to unambiguously govern the structural outcome of the self-organization. Since all of the molecules that are designed, synthesized and investigated in this dissertation belong to this family of hydrogen bonding motifs, the following section will focus on introducing the background research on one component self-complementary systems. 1.5.2 One component self-complementary hydrogen bonding systems 1.5.2.1 Tectons and molecular tectonics The term tecton 239 (from the Greek word tekton; builder) was invented by Wuest and colleagues 67,240 to represent a molecule whose interactions are dominated by particular associative forces that induce the self-assembly of an ordered network with particular structural features. These building units are structurally and energetically defined active molecular building blocks bearing inside their backbone an assembling program based on molecular recognition processes. The molecular encoding leading to the formation of molecular networks with predefined dimensionality and connectivity is ensured by the nature and localization of recognition sites within the structure of tectons. The tectons are self-complementary 241 and hence possess the potential to self-assemble into the desired one-, two- or three-dimensional structures. A selected example is the tecton 1 that programs a molecular tectonic via the AD-DA hydrogen bonding motif of the diaminotriazine (Figure 1.15)242 27 Figure 1.15. ORTEP view of the three dimensional hydrogen bonded network present in crystals inclusion tecton 1.2 1.5.2.2 Janus molecules The term Janus molecule (from the Roman god Janus, who possesses faces both on the front and at the back of his head) was first used by Lehn and colleagues 237,243-245 to illustrate a single molecule containing two different but often complementary hydrogen bonding faces. Lehn et al. prepared the first two-faced “Janus-type” heterocyclic molecule that could simultaneously recognize Ni -modified cytosine and thymine in solution to give triad hydrogen bonded motif which was proven based on ‘H-NMR studies (Figure 1.1 6).245 Based on the use of Janus-type heterocycle, Lehn proposed the “Janus wedge” strategy for recognition of nucleobase pairs by wedging or inserting the given heterocycle in between mismatched and canonical base pairs via forming a triplet with the maximum number of Watson- Crick interactions (Figure 1.17). This idea represents a fundamentally new approach to duplex H - NA\ NC ‘N—H \ N— A/ N_HD D Tecton 1 28 DNA targeting that is distincted from other approachs such as triplex and PNA-loop forming strategies. Figurel.16. Lehu’s Janus AG molecule capable of forming a triad with an analogue of thymme and cytosine in CHC13. Figure 1.17. Lehn’s Janus wedge concept: Janus wedges with two hydrogen bonding faces are designed to bind by insertion between base-pairs forming a triplet with the maximum number of Watson- Crick interactions. In contrast, common DNA binding molecules target hydrogen bonding sites of the intact base-pair, as ifiustrated by solid black (or slim black) arrows for the major (or minor) groove side of the AT base-pair. 29 By utilizing the aforementioned Janus wedge heterocycle on a PNA backbone, Chen et al. have elucidated several important characteristics of Janus-wedge triplexes capable of hydrogen bonding to the Watson-Crick faces of both strands of a target DNA duplex. Janus wedge triplex formation, therefore has a DNA2-(Janus wedge-PNA) stoichiometry, with no Hoogsteen face interactions.246 1.5.2.2.1 Janus type heterocycles; formation of rosette architectures Hydrogen bonding has also been broadly used to express the self-association of properly prearranged motifs into a variety of supramolecular architectures, including linear, cyclic or three dimensional arrays. In almost all cases, the formation of the cyclic array depends on additional factors such as metal ion binding, proper templation or steric effects. There are few examples in which the array of hydrogen bonding itself has been sufficient to exclusively dictate the result of the self-assembly. Due to their regularity and the suggestive appeal of nanotube formation, cyclic structures are of particular interest and a number of supramolecular cycles have been obtained with the tetrameric motif formed by four guanine nucleotides. A common strategy to create cyclic supramolecular architectures is the use of complementary hydrogen bonding motifs in which two complementary hydrogen bonding faces are fixed at 1200 angles in order to direct the system towards the formation of cyclic rosettes.58’247 Kolotuchin and Zimmerman showed a noticeable example in which the DA-AD interaction present in trimesic acid self-assembles into a supramolecular sheet in which the driving force was hydrogen bonding (see Figure 1.1 8).70248249 Another interesting report of the cyclic self-association of Janus type self-complementary heterocyclic component with a DA-AD hydrogen bonding motif, comprises the self-assembly of pyrido[4,3-g]quinoline into a trimeric 30 cyclic arrangement reported by Zimmerman (Figure 1 .19). The cyclic trimer is formed in a cooperative fashion through the formation of six hydrogen bonds.25° H H Hd acid Figure 1.18. Trimesic acid self-assembles into a cyclic supramolecular aggregate H’H6 pyrido[43-gjquinoline Figure 1.19. Zimmerman’s cyclotrimerization of self-complementary pyrido[4,3- g] quinohne. 31 1.5.2.2.2 Rosettes; Janus type heterocycles containing a (DAA-ADD) hydrogen bonding motif The groups of Lehn 243 and Mascal 251-253 both reported the self-organization of the self- complementary Janus-type DNA base analogue GC (Figure 1.20 and 1.21) which possesses complementary guanine and cytosine AAD and DDA hydrogen bonding arrays at an angle of 120° degrees into a hexameric rosette architecture. This system has been designed in such a way that it can only assemble into a cyclic hexameric motif (rosettes) maintained by 18 hydrogen bonds, which then self-associates into rosette nano-tubes defining a 1.1 nm channel. Assembly studies of GC hetercycle analogue in apolar solvents were held back by solubility problems, but ‘H-NMR spectroscopic and VPO measurements indicated that the desired hexamer is indeed formed in solution. Moreover, X-ray crystallographic studies confirmed the presence of the cyclic hexamer in the solid state (Figure 1.21). Figure 1.20. Lehn’s proposed self-assembly of the self-complementary GC base hybrids with (DDA-AAD) hydrogen bonding pattern into a hexagonal rosette ensemble. 32 Figure 1.21. Mascal’s X-ray crystal structure of hexagonal rosette array of self- complementary GC base hybrid with (DDA-AAD) hydrogen bonding pattern.252 Fermiri and co-workers have also synthesized and investigated several GC motifs, featuring a number of different hydrophilic and hydrophobic moieties, which self-organize into stable and architecturally complex one-dimensional helical nanotubes, representing an Un occluded central channel (1 rim) (selected example is illustrated in Figure 1.22). These structures have been characterized by transmission electron microscopy, circular dichroism (CD), dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and circular dichroism (CD).’67’254 256 As illustrated in Figure 1.23, Kolotuchin and Zimmerman also designed and synthesized a unique molecule with complementary AAD and DDA hydrogen bonding sites that self-organize into a hexameric rosette.27 33 Figure 1.22. Fenniri’s helical self-assembly of GC hybrid base into a six-membered rosette and resulting nanotube based on GC base hybrid skeleton as demonstrated. The nanotube figure is adapted from Fenniri, H.; Deng, B. L.; Ribbe, A. E. .L Am. Chem. Soc. 2002, 124, 11064-11072. HH HNALN HNN N 0 6X 0 Fenniris GAC hybrid Base r ji ‘ -JT b. tr L/ ., j__ a irirn, -4Y - - Ar. Ar -N O(Ar _J ii NN Iii .j CH3 N. °Ar ONNCH3 H3C I: CH N HN H C H3CNNQ ArN 9 - — N N- -. - - N. - W NH--.0 N /N. N 0 çT0Ar Figure 1.23. Kolotuchin-Zimmerman’s (DDA-ADD) hexameric rosette assembly. 34 Sessler and Jayawickramarajah synthesized a guanosine—cytidine Janus type molecule that self-associates into a cyclic trimer in organic solvents (Figure 1 .24).258259 They used the strong GC hydrogen bonding module to express a self-organized cyclic triad array. An ethylene bridge separates the guanosine and cytidine moieties and preorganizes the GC complementary faces in configuration of the macrocycle using 18 hydrogen bonding interactions. The cyclic trimer structure has been characterized by ESI mass spectrometry and gel permeation chromatography (OPC) to some extent. Figure 1.24. Self-assembly of Sessler’s guanosine-cytidine dinucleoside into cyclotrimer supramolecule. TBDI 35 1.5.2.2.3 New Janus type DNA-inspired heterocycles presented in this dissertation As mentioned above, the correct positioning of pyrimidine and purine heterocycles within a well-designed one-component self-complementary molecular skeleton can lead to the formation of purposeful higher order systems that can be exploited to create new nanostructured materials. This approach has been used in this thesis for the design, synthesis and self organization studies of several new molecules of this class and will be discussed in Chapters 2 and 3. The abovementioned hydrogen bonded systems demonstrate that elegant, higher order self-organized assemblies can be prepared using the natural base-pairing capabilities of guanine, cytosine, adenine and thymine derivatives. With regard to this issue, cyclic self-assemblies are especially promising since the cavities present in these systems can be used for molecular recognition and transport of various analytes. Inspired by these cyclic ensembles, an originally unique, cyclic quartet supramolecule via guanine and cytosine Watson-Crick base-pairing has been designed and prepared in this thesis. A detailed discussion of this work can be found in Chapter 3. Three different Janus-type heterocycles based on the diaminoadenine-thymine H bond pairing have been designed and prepared, and their unique hydrogen bonded supramolecular structures obtained in different solvents are illustrated. A detailed discussion of this work can be found in Chapter 2. 36 1.6 Objective of this dissertation In this dissertation the research work includes the design, synthetic preparation and structural analysis of self-organized supramolecular ensembles derived from Janus type one- component self-complementary nucleic acid base pairing. Specifically, two projects concerning new Janus-type DNA nucleobase derived self-organization will be explained. Each of these projects addresses the high yielding syntheses of heterocycles with defined hydrogen bond accepting and donating capabilities designed to self-assemble under the general purview of base pairing. Importantly, each of the research projects, even though associated intrinsically through the concept of base pairing, will address a new opportunity in the field of DNA based self organization as well as the potential for DNA recognition. The first project involves the preparation of new Janus-type DNA nucleobase and the derived supramolecular structures. Chapter 2 discusses the high yielding syntheses of three DNA-inspired self-complementary heterocycles with defined DAD-ADA hydrogen bond accepting and donating capabilities provides for the design of self-assembling structures and recognition of biological targets. These heterocycles, which have never before been reported or characterized, represent diaminopurine thymine hydrogen bonding codes. All three heterocycles crystallized to afford the first X-ray crystal structures of self-complementary heterocycles capable of ADA-DAD pairing. The potential use in DNA and RNA recognition is briefly discussed. This chapter also introduces the synthetic development to build the Janus AT deoxynucleosides, capable of being oligomerized. Inspired by the significance of G-quartet ensembles in nature, chapter 3 will disclose the synthesis and characterization of a self-complementary heterocycle capable of AAD-DAA hydrogen bond pairing capable of forming a quartet rosette. Whereas in the past such AAD DAA seif-complementarity has given rise to trimeric and hexameric rosettes, it is now 37 demonstrated that the same hydrogen bonding pattern, when properly arrayed, can be used to program a tetrameric rosette, that unlike a 0-quartet, requires no metal binding or peripheral components for pre-organization. ‘H-NMR,‘3C-NMR spectra can be found in the appendix. For research work presented in this dissertation, each of the faces of the prepared Janus molecules have the complementary hydrogen bonding codes capable of recognizing adenine and thymine (Janus AT see Chapter 2) or cytosine and guanine (Janus GC see Chapter 3) respectively. This family of compounds could be screened for their ability to recognize DNA and RNA strands containing nucleic bases available for hydrogen bonding interactions (i.e. double stranded DNA’s mismatch regions, D-loops 260 as well as RNA’s loops’), which means that they might be used as a tool with an intriguing potential for sequence specific DNA or RNA that would compete with self-association. Since AT-rich sequences are abundantly found at important regions of both ribozymes and DNA promoters, properly functionalized Janus AT that could recognize such sequences may be useful for studying the biological processes mediated by these sequences. ‘A D-loop (displacement loop) is a DNA structure where the two strands of a double-stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA. An unrelated use of the term D-Ioop is for the RNA’s loop that forms the end of the D-arm (the D arm is a feature in the tertiary structure of transfer RNA) of a transfer RNA molecule. 38 2. Chapter 2: Novel DNA-Inspired Janus-AT Heterocycles: Synthesis, Self-Assembly, and Solid State Structures 2.1 Introduction Using heteroaromatic molecules containing geometrically well-defined arrays of hydrogen bond donor (D) and acceptor (A) groups present on the edge of the molecule is a powerful strategy for creating self-organized supramolecular structures from smaller motifs. Several classes of heteroaromatic molecules, as supramolecular synthons, have been broadly synthesized and studied by different research groups.’4Flat shape and rigidity of heteroaromatic molecules ensures the formation of two dimensional self-organized assemblies that have potential applications in nanotechnology, molecular tectonics, and crystal engineering where single crystal X-ray diffraction provides visualization of hydrogen bonded arrangements.24’85085”4061265 Despite the fact that hydrogen bond mediated self-organization of two components with self-complementary recognition sites (e.g., melamine and cyanuric or barbituric acid) has been used extensively to create a variety of supramolecular architectures,5962’65902068°’1 217,219,221,223,224 there are far fewer antecedent examples in which one-component self- complementary motifs have been designed for self-organized assemblies.’67’245512 Nucleobases designed to be simultaneously complementary to two nucleobases by Watson-Crick base pairing (i.e. A and T, or G and C), known as Janus wedges,245 have an intriguing potential for sequence specific DNA recognition that would compete with self- association. The first example of such Janus nucleobases was a Janus-GA that that had been designed to simultaneously recognize cytosine and uridine.243245 Chen and McLaughlin recently 39 incorporated the same Janus base into an octameric PNA homopolymer third strand that afforded recognition of heteroduplex DNA containing C-T mismatches.246 Although there have been several reports on the design, synthesis and self-assembly of a number of one-component, self-complementary Janus GC nucleobases which self-associate via a DDA-AAD interaction 167,243,251-253,255,257 (see section 1.5.2.2.1.2 in Chapter 1), the synthesis of a Janus AT heterocycle has never been reported, either with the intention of DNA recognition, or in terms of an exploration of self-organization via potential DAD-ADA hydrogen bonding interactions. This work will focus on the syntheses and solid-state structures of three self- complementary DNA-inspired heterocycles which contain ADA-DAD hydrogen bond acceptor- donor patterns (Janus AT 1-3, in Figure 2.1). These novel heterocycles represent diaminopurine thymine hybrids that, in two of the three cases, relate to previously reported heterocyclic hybrids of guanidine and cytosine. All three heterocycles crystallized and afforded the first X-ray crystal structures of such heterocycles and revealed their extended hydrogen bonded arrays. This chapter also introduces the synthetic development to build the Janus AT deoxynucleosides, capable of being oligomerized, as the current trend of the project. 2.2 Janus AT molecules; novel DNA-inspired Janus-AT heterocycles 40 Inspired by the aforementioned findings, preparation of three different types of one component self-complementary Janus AT molecules containing DAD-ADA hydrogen bonding motifs has been investigated. In this chapter, the design, high yielding syntheses, and, to the best of our knowledge, the first X-ray crystal structures of solid state supramolecular arrays of all of the three Janus AT heterocycles capable of DAD-ADA hydrogen bonding interactions are disclosed. Although these nucleobase hybrids are in principle closer to a thymine-diaminopurine or a uracil-diaminopurine hybrid nucleobase, the term “Janus AT” has been used to describe this family of molecules. The Janus AT heterocycles are shown in Figure 2.1. Figure 2.1. Janus AT heterocycles 1, 2a, 2b, 3. Arrows indicate expected hydrogen bond donating or accepting functionalities. HN Janus AT I R= Butyl (I, 2a, 3a),R Heptyl (2b) Hexyl (3b) R H Janus AT 2 (X=N), Janus AT 3(X=CH) 2.3 Design of Janus AT heterocycles 1-3 41 Janus AT heterocycles 1-3 are imbued with two complementary recognition units (a diamino-adenine face and a thymine face). It was thought likely that these two units would self organize through the AT (donor-donor-acceptor to acceptor-acceptor-donor, DDA—AAD) pair. Regarding the design of Janus 1-3 a number of thoughts have been considered. A diaminopurine-like recognition unit, instead of an additional natural adenine (aminopurine) face, has been chosen in order to afford three hydrogen bonds instead of two with the complementary thymine T-face. Although the ADA-DAD hydrogen bonding array is not generally found in regular DNA base-pairs, it exists in the cyanophage S-2L viral genome that contains 2,6- diaminopurine. 2,6-Diaminopurine has been found to increase duplex stability and specificity in synthetic systems relative to natural adenine and thymine (AT) base pair.266269 In addition, previous reports on ADA-DAD associated networks involving melamine have been used as a pattern for our designs. From a synthetic point of view, Janus AT 1-3 with two exocyclic amines would be somewhat more accessible compared to analogues with only one exocyclic amine particularly for the synthesis of Janus AT 1. Janus AT 2 and Janus AT 3 are more rigid compared to Janus AT 1 in which there is a single bond between A and T heteroaromatic rings. Janus AT 2 and 3 were designed based on the Janus GC base reported by Lehn,243 Mascal 251-253 and Fenniri.’67’255 Furthermore, the diaminopurine-like face in Janus AT 3 was installed on a pyridinyl ring system (as opposed to a pyrimidinyl ring system in Janus AT 2) to afford the final possibility of regioselective attachment to a deoxyribose backbone on either the nitrogen of the uracil-like face or on the carbon of the diaminopurine-like face. The proposed preparation of Janus AT heterocycles (1-3) can be best illustrated using a retrosynthetic analysis. As illustrated in Scheme 2.1, the synthesis of target compounds Janus AT 1-3 might be accessible by starting with the key compound N-cyanoacetylurethane through a 42 Knoevenagel type condensation of its activated methylene carbon with proper orthoesters (Janus AT 1 and 3) or carbon disulfide (Janus AT 2). 43 H.N.H o N’N NH HNNH HN N1 ON + H R JanusATi R 0 0 Butyl-NH2 + + H 0” N-cyanoacetylurethane 0 H..NH 5N NHHN N HN I + H2NANH0 N,’N N O”N”sI I R H R JanusAT2 00 00 N S — + C II S S N-cyanoacetylurethane S QH..NH 0 HNfN HN jrS 0N N - H + NH3 I I R H R Janus AT3 JJ, 0 0 -N N N N + HR—NH2+ H 0_ 0) N-cyanoacetylurethane Scheme 2.1. Retrosynthetic analysis of A) Janus AT 1, B) Janus AT 2 and C) Janus AT 3. 44 2.4 Janus AT 1 2.4.1 Synthesis of Janus AT 1 The synthesis of Janus AT 1 (Scheme 2.2) began with the facile construction of 5- cyanouracil, 6, which was obtained from condensation of commercially available N cyanoacetylurethane 4, with triethylorthoformate in acetic anhydride at reflux to afford 527O Adduct 5 was then treated with butylamine in water to trigger the ring closure and afford 5- Cyanouracil 6 in high yield (94%). 5-cyanouracil 6 was then condensed with dicyandiamide 67,263 in DMSO in the presence of KOH at 100 °C to afford 1 in 80% yield. NH2 a b c HNNNH2 5 NH LNJanus AT I H2N dicyandiamide Scheme 2.2. a) Triethylorthoformate, acetic anhydride, reflux, 45 mm, 95% b) Butyl amine, water, 85 °C, 15 mm, 93% c) Dicyandiamide, KOH, DMSO, 100°C, 4 hours, 80%. 45 2.4.2 Insight into the characterization and self-organization of Janus AT 1 2.4.2.1 1H-NMR studies ofJanusATl Compound 1 was very soluble in formic acid, moderately soluble (-1O mg/mL) in polar aprotic solvents (DMSO, DMF, NMP) that are known to break up hydrogen bonds, and was generally insoluble in other common organic solvents. Investigation of the self-organization process by ‘H-NMR (i.e. DOSY, NOESY) could not be monitored, due to the very low solubility of Janus AT 1 in chloroform or other aprotic apolar solvents where hydrogen bonds would have formed appreciably. A typical NMR spectrum of Janus AT 1 recorded in DMSO-d6 at room temperature is illustrated in Figure 2.2. Figure 2.2. ‘H-NMR spectrum of Janus AT 1 in DMSO-d6. 46 The downfield resonance observed at 11.4 ppm (Figure 2.2) was attributed to the imino proton (i.e., NH) of the thymine like face, which is exchangeable with D20. The resonance at 6.6 ppm was assigned to the four amino protons of the diaminotriazine. 2.4.2.2 Variable concentration ‘H-NMR studies ofJanus A Ti As the only experiment to study the self-assembly of Janus AT 1 in solution, a variable concentration ‘H-NMR experiment was carried out at room temperature in DMSO-d6.Neither of the amino and imino ‘H-NMR signals of Janus AT 1 were seen to change with an increase of the concentration of Janus AT 1 (Figure 2.3). This observation is consistent with the two hydrogen bonding faces of the Janus AT 1 participating in strong hydrogen boding interaction with the solvent. 0.05mM__j___________ ____ ________ ____ 0.25 mM ____________ 0.5 mM __ __ _ __ __ _ __ _ __ __ __ __ __ __ 1 mM __ _ _ _ _ _ _ __ __ 2mM 3mM 5 mM_________ _ 7.5 mM_____________ _ _ 10 mM ____ _ Figure 2.3. Variable concentration ‘H-NMR studies of Janus AT 1 in DMSO-d6show no change in chemical shifts upon dilution (25 °C, 400 MHz). - I ——.‘- ..- —. — — -‘.——.. — — .. --‘-- .- — — -- -________ -. . ,.. . — — I - .—‘ .. . — . ..— .. ,.. . i’j — -_k____________ J L 12 11 10 9 8 7 6 5 4 3 2 1 ppm 47 2.423 Phase-sensitive 2D‘5N-’HHMQCNMR analysis ofJanus AT 1 In order to further confirm the suggested tautomeric states of the JAr-i’s faces (DAD ADA) in solution, a 2D-Phase-Sensitive ‘5N-’H HMQC NMR experiment was performed in DMSO-d6on 1 (Figure 2.4). This verified that two sets of two protons correlated with two nitrogens and not with three or four nitrogens as would have been the case for other tautomers. ppm 20 40 N’ 60 / H.Na.H 120 Nb 0 NN 140 H.NbI*NNa.H 160 0N H 12 8 7 6 ppm Figure 2.4. 21)-Phase-Sensitive‘5N-1HHMQC N1VIR of Janus AT 1 in DMSO-d6. 48 2.424 Solid state analysis ofJanusAT 1 by X-ray crystallographic diffraction Single-crystal X-ray diffraction was utilized to verify the supramolecular architecture of Janus AT 1 in the solid state. A single crystal of Janus AT 1, appropriate for X-ray crystallographic diffraction, was obtained by dissolving 1 in dry DMSO’ at high temperature. After several days at room temperature, colorless prismatic crystals formed. An ORTEP view of l’s X-ray crystal structure is shown in Figure 2.5. N27 C43 N24 08 N25 C35 C42 C44 C36 23 C’ N26 C37 C34 2 C38 C39 C40 C41 Figure 2.5. The ORTEP view of the X-ray crystal structure of Janus AT 1. 1 DM50 was incubated with the dry molecular sieves (type 4A, 14 x 30 mesh) in an air-tight bottle for 48 h (molecular sieves was activated by heating 12 hours at 300CC). 49 Crystals of 1 diffracted in a P-21/c space group in which the asymmetric cell comprised four monomers and no molecules of DMSO were apparent. The asymmetric cell was propagated using Mercury software version 1.4.1 to discover a repeating matrix containing four slightly different hydrogen bonded arrays of the type illustrated in Figure 2.6 and Figure 2.7. A/ ON H H. H 0 NNHIXH H H H B + 1%) Lf Figure 2.6. A) Illustrated hydrogen bonded ribbon array of Janus AT 1, B) Single crystal X-ray structure of supramolecular ribbon of Janus AT 1. 50 The crystal structure of the hydrogen bonded arrays shows two repeating parallel arrays that are interdigitated with another two parallel replicated arrays at an angle of approximately 600. Two of the four arrays demonstrate a dihedral angle flanked by the two aromatic rings of approximately 5° with relatively coplanar hydrogen bonds (Figure 2.8A) whereas the other two arrays demonstrate a surprisingly high dihedral angle of nearly 15 ° (Figure 2.8B-C). Figure 2.7. Sliced-away X-ray diffraction of the extended unit cell showing the lattice of 2 sets of parallel arrays. 51 Figure 2.8. A) X-ray crystal structure viewed along the array planes (blue ellipses), clearly showing that the dihedral angle of two arrays is roughly 5 or less; B) the dihedral angle of the other two arrays is roughly 15 0; C) Propeller twist of a DAD-ADA hydrogen bonding interaction in one of the four arrays. As illustrated in Figure 2.8-C, the three hydrogen bonds in one of the parallel hydrogen bonded arrays diverge considerably from co-planarity and an analogous twist angle is visible between the hydrogen bonded faces of Janus AT 1. Similar irregularities in hydrogen bonding has been seen for Watson-Crick bases pairs in duplex B-DNA and (described as roll and propeller twist).’° This structure properly shows the flexibility of this Janus AT heterocycles as well as the degree to which the DAD-ADA hydrogen bonding motif itself can be different in the same crystal structure. 52 2.5JanusAT2 2.5.1 Synthesis of Janus AT 2 The synthesis of Janus AT heterocycles 2a and 2b (Scheme 2.3) started with the condensation of N-cyanoacetylurethane 4 with carbon disulfide 271 in the presence of potassium carbonate in DMF yielding intermediate 7 in good yield (95%). The resulting ketene dithioacetal dipotassium salt 7 was reacted with two equivalents of methyl iodide in water-acetonitrile to give ketene dithioacetal 8. a b C JH R= Butyl (9a), d HNN Heptyl (9b) o N N NH2 R Janus AT 2 R= Butyl (2a), Heptyl (2b) Scheme 2.3. Synthesis of Janus AT 2. a) CS2,K2C03,DMF, RT, 95% b) Mel,H20:CH3CN (7:3), reflux, 85% c) Butyl or heptyl amine, EtOH, reflux, 87% d) Guanidinium hydrochloride, NaOEt, EtOH, reflux, 85%. Intermediates 9a and 9b were obtained via dropwise addition of the corresponding amine to an ethanolic solution of 8 at room temperature to afford a presumed monoamine adduct that was cyclized in situ at reflux to afford the 5-cyano-6-methylthiouracils 9a and 9b which were characterized by both ‘H/’3C-NMR and high resolution mass spectroscopy. As illustrated in 53 Figure 2.9, 1 -butyl-6-methylsulfanyl-2,4-dioxo- 1,2,3 ,4-tetrahydropyrimidine-5-carbonitrile 9a was crystallized and characterized by single crystal X-ray crystallography to verify the correct substitution at positions 5 and 6. Compounds 9a and 9b were reacted with guanidine (free base generated in situ) in ethanol at reflux to afford the corresponding bicyclic AT heterocycles 2a and 2b in very good yields. Figure 2.9. The ORTEP view of the X-ray crystal structure of 9a. 54 2.5.2 Insights into the characterization and self-organization of Janus AT 2 2.5.2.1‘5N-’H HMQCAnaIysis ofJanus AT 2b Janus AT heterocycles 2a and 2b were mostly insoluble in common polar and apolar solvents and manifested low solubility even in DMSO, DMF, and NMP (<2 mg/mL) but dissolved in 99% formic acid to a significant extent. Study of self-organization in the solution phase (i.e. DOSY, NOESY) could not be carried out due to the insolubility of the monomers in various proper NMR solvents such as CDC13 in which hydrogen bonding is usually easily detectable and strong. Due to the limited solubility of these compounds, we were unable to achieve suitably high concentrations in chloroform where hydrogen bonds would have formed appreciably to be monitored by NMR spectroscopy. A 2D-Phase-Sensitive‘5N-’H HSQC NMR experiment was performed in DMSO-d6on compound 2b in order to verify the suggested tautomeric states of the faces of Janus AT 2b (DAD-ADA) in solution (Figure 2.10). This experiment verified the exchangeable proton at 11.1 ppm correlated with a single nitrogen, two exchangeable protons at 6.9 ppm correlated with a single nitrogen and two exchangeable protons at 7.4 ppm and 8.1 ppm correlated with a single nitrogen. Indeed five exchangeable protons (with D20) in Janus AT 2b correlated with three nitrogens and not with two nitrogens, as would have been expected in the case for other pyrimidol tautomeric form of the thymine like face (Figure 2.11). 55 ppm 20- 40 Na N° 60- o 1::: Nb__ 120- H.NbJLN 140- ONNNcH2} 160- 180- 12 11 10 9 8 7 6 5 4 3 2 1 ppm Figure 2.10. Phase sensitive 15N-HHMQC NMR of Janus AT 2a in DMSO-d A representative1H-NMR spectrum of JAT-2b recorded at room temperature in DMSO-d6 is shown in Figure 2.11. Based on the‘5N-’H HMQC NMR experiment, the downfield resonance at 11.1 ppm was attributed to the imino protons (i.e. NbH) of the thymine like faces and was shown to be exchangeable with D20. The resonance at 8.1 ppm was assigned to the (NaHa) amino proton of the diaminopyrimidine face which is involved in the internal hydrogen bonding with the carbonyl group (C=O) of the thymine like face. The resonance at 7.4 ppm was assigned to the (NaHb) amino proton of the diaminopyrimidine face. The resonance at 6.9 ppm was assigned to the (N°H2)amino protons of the diaminopyrimidine face. 56 2.5.2.2 Variable concentration ‘H-NMR studies offanus AT2b As the only experiment to study the self-assembly of Janus AT 2b in solution, a variable concentration ‘H-NMR experiment was carried out at room temperature in DMSO-d6.Neither of the amino and imino ‘H-NMR signals of Janus AT 2b were seen to change as the concentration of Janus AT 2b was altered (Figure 2.12), an observation that is also consistent with the two Figure 2.11 ‘H-NMR spectrum of Janus AT 2b in DMSO-d6. 57 hydrogen bonding faces of the Janus AT 1 participating in strong hydrogen bonding interactions with the solvent. 0.05 mM 0.1 mM 0.25 mM 0.5 mM 1 mM 2 mM 13 12 11 10 9 8 7 6 5 4 3 2 1 0 rppm Figure 2.12. Variable concentration ‘H-NMR studies of Janus AT 2b in DMSO-d6,showing no change in chemical shifts upon dilution. 2.5.2.3 Solid state analysis OfJA-2by X-ray crystallographic diffraction The single-crystal X-ray diffraction technique was applied to verify the nature of Janus AT 2’s supramolecular architecture in the solid state. Attempts for acquiring single crystals of 2a and 2b for X-ray diffraction in DMSO, DMF and NMP were ineffective. Several unsuccessful attempts were made with a range of mixed solvents applied in combination with either vapor or solvent diffusion techniques (using diethyl ether or dioxane) or slow cooling. Finally, compound 2b crystallized as colorless plates of two-component twin crystals via slow evaporation of a dilute solution of 2b in 98% formic acid (2a also crystallized but provided twin crystals that did not cleanly diffract) (Figure 2.13). 58 Figure 2.13. The ORTEP view of the X-ray crystal structure of Janus AT 2b. X-ray diffraction identified a P-i space group with a unit cell that included one molecule of 2b and two molecules of formic acid. Extension of the unit cell indicated a formate-ion mediated co-crystal as illustrated in Figure 2.14. As can be seen in the X-ray crystal structure of 2b, formic acid mediates the self-assembly by forming three complementary hydrogen bonds within the polymeric hydrogen bonded array as it protonates the DAD face. The carboxylate functionality of the formate molecule is hydrogen bonded to a proton from one of the exocylic amines (i.e. NH2), and one imino proton (i.e. NH) of the thymine-like face. The formate salt essentially alters the DAD face’s hydrogen bonding motif and provides for a DDD-AD pairing interaction with the thymine-like face. 59 Figure 2.14. A: Scheme of formate anion bridging the array, B: X-ray single crystal structure of supramolecular ribbon of Janus AT 2b, C: The extended structure with inter lattice formate bridges. 60 This unique asymmetrical hydrogen bonding pattern between the pyrimidinium cation face and the thymine-like face mediated by formate anion along with an AA hydrogen bonding motif, gave rise to formation of an infinite hydrogen-bonded molecular ribbon lattice. In addition, a second molecule of formic acid serves to bridge one lattice with another. 232.4 Electron spray ionization mass spectrometry analysis on Janus AT 2a&b An ESI-MS (positive ion mode) experiment was carried out on Janus AT 2a and 2b in order to further reveal self-association in the gas-phase and in the absence of formic acid. As is illustrated in Figure 2.15, the analysis of the ESI-MS spectrum of Janus AT 2a suggested the presence of several multimeric sodiated species: (Ma+Na) where a = 1-6, as well as putative (Mb+2Na)2where b = 7, 9, and 11. This data is suggestive of the formation of hydrogen bonded aggregates of Janus AT 2a in the gas phase. Intensity eooo [M+Na] 523.2 273 IM2+Na1 6000 \s.i 523\ EM3+Na] [M4+Na] [M9+2Na] [M6+Na] 773\ 1023 1149 1524 [M5+Na] 4000 354 10237 1274 1524.2 1149.0 [M11+2Na] \ 1398 1274.0 2000 j 610.4 195.0 8990 _____ .Z ALLA, 200 400 600 800 1000 1200 1400 inlz Figure 2.15. ESI Multimeric Mass Spectrum of Janus AT 2a. 61 An ESI analysis of 2b revealed several multimeric protonated species (M0+H) where c = 4-6, as well as putative (Md+2H)2 where d = 8-11 which is suggestive of the formation of hydrogen bonded aggregates of Janus AT 2b in the gas-phase (Figure 2.16). Intensity [M +H] or 11 699 [M8+21112+ [M5+H] or [M9+2H] [Mio +21112+ LMii+2H]2 13 \4625 \608.4 49 OQ ixo 1Q 17CC m/z Figure 2.16. ESI Multimeric Mass Spectrum of Janus AT 2b. 62 2.6 Janus AT 3a and 3b 2.6.1 Synthesis of 11 The synthesis of Janus AT heterocycles 3 (Scheme 2.4) started with the condensation of N-cyanoacetylurethane 4 with triethyl orthoacetate in acetic anhydride at reflux yielding compound 10 in good yield (90%). Reaction of the resulting adduct 10 with the corresponding amine in water induced the annulation to provide the desired thymine face and afforded 6- methyl-5-cyanouracil 1 la and 1 lb in excellent yields. a b c ONH2 d R=Buty (ha) R=Butyl (12a) Hexyl (hib) Hexyl (12b) 0 NH 0 NH 0 NH2 0 NH2 HNJH e f 9 HNflj NH2 Janus AT3 R=Butyl (13a) R=Butyl (14a) R=Butyl (15a) Hexyl (13b) Hexyl (14b) Hexyl (15b) R=Butyl (3a) Hexyl (3b) Scheme 2.4. Synthesis of Janus AT 3a & 3b. a) Triethyl orthoacetate, acetic anhydride, reflux, 40 mm, 90% b) R-NH2,H20, 20 mm, 90 °C, 91% c) i. CS2, t-BuOK, dry THF, rt, 2h ii. acetic acid, H20, 89% d) Ammonium hydroxide, sealed tube, 100 °C, 12 h, 93% e) Mel, NaOH, H20, 90% 1) (11202, formic acid, 4 h, RT) or (MCPBA, CHC13 4 h, RT), 95% g) NH3,MeOH, sealed tube, 100 °C, 24 h, 92%. 63 2.6.2 Synthesis of 5-Amino-7-thioxo-1,7-dihydrothiopyrano[4,3-d]pyrimidine-2,4-dione analogs 12a & 12b In the retrosynthetic analysis, the synthesis of target heterocycle Janus AT 3 was hypothesized to be accessible using condensation of 11 with carbon disulfide and methyl iodide in order to build the ketene dithioacetal 17, which, upon reaction with the ammonium hydroxide would yield compound 3 (Scheme 2.5). Scheme 2.5. Intended route for synthesis of Janus AT 3. Based on the aforementioned retrosynthesis, 6-methyl-5-cyanouracil ha was condensed with carbon disulfide in dry THF in the presence of potassium t-butoxide at room temperature followed by acidic workup affording a product in 89% yield that did not react with methyl iodide rapidly. X-ray crystallography along with ‘H/13C-NMR and HRMS were used in order to characterize the product of the condensation reaction between ha and carbon disulfide. A single crystal of this product was grown in acetonitrile by slow evaporation and characterized by X-ray diffraction to verify the formation of the novel structure 12a that never have been reported before in literature (Figure 2.17). 64 9 N4 05 C4 C5 N3 Cs Q N5 C7 C6 C3 ClO C” C2 Si Ci S2 Figure 2.17. The ORTEP view of the X-ray crystal structure of 12a. 2.6.1.3 Synthesis of5-Amino-7-thioxo-6, 7-dihydro-1H-pyrido[4,3-d]pyrimidine-2,4-dione 13a &b Thiopyranopyrimidines 12a and 12b were treated with 30% ammonium hydroxide at reflux in a sealed tube to afford 5-amino-7-thioxo-6,7-dihydro- 1 H-pyrido [4,3 -d]pyrimidine-2,4- dione 13a and 13b in high yields (93%). As illustrated in Figure 2.18, the hexyl analogue of 13 was characterized by X-ray diffraction. After several attempts at crystallization, using different techniques, a single crystal of 13b was grown in formic acid 98% by using the slow evaporation. 65 H4a C2 C24 I IN1 04 C3 N4 C8 C9 C7 LI cii H4b C4 N3 ClO C13 H3Ø C6 C5 C 12 Figure 2.18. The ORTEP view of the X-ray crystal structure of 13b. 2.6.1.4 Synthesis ofJanusAT3 The reaction of 13a and 13b with methyl iodide gave the methyl sulfide 14a and 14b in very good yields. Attempts to affect direct displacement of the methyl sulfide group of 14a&b failed to give any recognizable product upon reaction with ammonia in methanol. On the other hand the corresponding sulfone prepared by oxidation of 14a and 14b with hydrogen peroxide or MCPBA, reacted satisfactory with ammonia in methanol giving the desired Janus AT 3a and 3b good yields (Scheme 2.6). 66 0 NH 0 NH 0 NH2 0 NH2 HN’jH e f HN-Y5o g HN)5 NHJ Janus AT3 R=Butyl (13a) RButyI (14a) R=Butyf (15a) Hexyl (13b) Hexy (14b) Hexyl (15b) R=Butyl (3a) Hexyl (3b Scheme 2.6. A nucleophilic aromatic (SNAr) amination reaction, utilized to complete the syntheses of Janus AT 3 a) Mel, NaOH, H20, 90% b) (H20, formic acid, 4 h, RT) or (MCPBA, CHC134 h, RT), 95% c) NH3,MeOH, sealed tube, 100 °C, 24 h, 92%. Methyl sulfides 14a and 14b were obtained via dropwise addition of methyl iodide to an aqueous solution of 13a and 13b, correspondingly, in the presence of NaOH at room temperature in high yields (90%). Oxidation of methyl sulfide l4aIb to methanesulfonyl iSa/b was achieved in high yields through two routes: i) by using MCPBA in chloroform at room temperature, or ii) by using hydrogen peroxide in formic acid at room temperature (95%). Compounds iSa and iSb were reacted with anunonia in methanol at 100° C in a sealed tube to afford the bicyclic Janus AT heterocycles 3a and 3b in good yields. 67 2.7 Insight into the characterization and self-organization of Janus AT 3a&b 2.7.1 1H..NMR of Janus AT 3a Janus AT heterocycles 3a and 3b were insoluble in conventional organic solvents and were soluble in DMSO, DMF, HMPA and NMP and 99% formic acid. Study of self organization in the solution phase could not be carried out due to the insolubility of the Janus AT 3a and 3b in various appropriate NMR solvents for such as CDC13 in which hydrogen bonding is significant and strong. ‘H NMR spectrums of 3a and 3b were carried out at room temperature in DMSO-d6.A representative NMR spectrum of Janus AT 3a recorded in such conditions is shown in Figure 2.19. As in the case of compound Janus AT 1 and Janus AT 2 and based on their 2D-Phase- Sensitive‘5N-’H HMQC NMR study, a downfield resonance at 11.1 ppm was observed. This signal was attributed to the imino proton (i.e. NbH) of the thymine like faces and was shown to be exchangeable with D20. The resonance at 8.1 ppm was assigned to the (NaHa) amino proton of the diaminopyridine face which is internally hydrogen bonded to the carbonyl group (C=O) of the thymine-like face. 68 H Hb0” NH HbjJ\/i..N -‘LNC. H / \} // NbH NaHa NaH1 ____ __ _ I _ 11 10 9 8 7 6 5 4 3 2 1 ppm o o C <N < <NC cc j — 0 — <N C (N <0 Figure 2.19.1H-NMR spectrum of Janus AT 3a in DMSO-d6 The resonance at 6.7 ppm was assigned to the (NaHb) amino proton of the diaminopyrimidine face. The resonance at 6.4 ppm was assigned to the (NcR2)amino protons of the diaminopyridine face. 2.7.2 Variable concentration1H-NMR of Janus AT 3a As the only experiment to study the self-assembly of Janus AT 3a in solution, a variable concentration ‘H-NMR experiment was performed in DMSO-d6at 25 °C. Similar to the other VC ‘H-NMR experiments on the two Janus AT heterocycles, none of the amino and imino ‘H- 69 NMR signals of Janus AT 3a were seen to change as the concentration of Janus AT 3a was altered (Figure 2.20), an observation that is also consistent with the two hydrogen bonding faces of the Janus AT 3 participating in strong hydrogen bonding interaction with solvent. Figure 2.20. Variable concentration ‘H-NMR studies of Janus AT 3a in DMSO-d6,show no change in chemical shifts upon dilution. 2.7.3 Solid state analysis of Janus AT 3 by X-ray crystallographic diffraction Janus AT 3a and 3b were practically insoluble in alcohols, ethers, and halogenated solvents but dissolved in dimethyl formamide, dimethyl sulfoxide, N-methylpyrrolidone and formic acid. Initially Janus AT 3a crystallized as colourless plate crystals by the use of slow evaporation of a dilute solution (2 mg/ml) of Janus AT 3a in 98% formic acid and in the presence of methylene chloride (An ORTEP view of the X-ray crystal structure of Janus AT 3a is shown in Figure 2.21). 4mM 2mMJLJ 1mM 0.5mM J. 0.2mM 0.05mM L L . :zE’i:___ 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm 70 As illustrated in Figure 2.22, formate mediated the pairing interaction with the thymine like face of 3a due to the protonation of the nitrogen in diaminopyridine ring. The protonation, in this array, allowed for alignment of the DDD-D hydrogen bonding motif of the diaminopurine face with an ADA hydrogen bonding pattern of the thymine-face intervened through an (AA) hydrogen bonding motif. In close resemblance to the extended hydrogen bonding network that has been observed for Janus AT 2b, this unique asymmetrical hydrogen bonding pattern between the pyridinium cation face and the thymine-like face, mediated by formate anion, enabled the formation of a zigzagged hydrogen-bonded molecular ribbon association. Extension of the unit cell, using Mercury 2.1, indicated a formate-ion mediated co-crystal as shown in Figure 2.22b. Figure 2.21 The ORTEP view of the X-ray crystal structure of Janus AT 3a. 71 Figure 2.22. A) Scheme of formate anion bridging the hydrogen bonded array of Janus AT 3a, B) X-ray single crystal structure of supramolecular crinkled ribbon of Janus AT 3a with formate bridging. Finally, after several different attempts, X-ray quality crystals of Janus AT 3b were obtained from a dilute solution of 3b at slightly elevated temperatures in DMF, followed by slow H H N—H cooling of the solution. 72 Figure 2.23. The ORTEP view of the X-ray crystal structure of Janus AT 3b. In spite of the noticeable quality of the crystals, initial attempts to characterize Janus AT 3b by X-ray diffraction were ineffective, and it was only when a more powerful technique, synchrotron X-ray crystallography (U.C. Berkeley synchrotron) was applied that the crystals yielded to analysis (An ORTEP view of the X-ray crystal structure of Janus AT 3b is shown in Figure 2.23). As shown in Figure 2.24, the self-organization of Janus AT 3b in the solid state did not give rise to formation of a hexameric hydrogen bonded rosette, but instead a triply hydrogen bonded molecular ribbon via (ADA-DAD) hydrogen bonding motif was detected. The crystal plane was identified by the ir-t stacking of individual ribbons at the van der Waals contact 73 distance (3.4-3.5 A) giving rise to infinite aggregates including sheets of ribbons. This work displays the programmability of hydrogen bond triplex systems (ADA-DAD) and its potential for engineering molecular solids designed for proliferation in set dimensions. The self-organized molecular ribbon is placed in a new context as part of a two-dimensional assembly by the programmed (ADA-DAD) hydrogen bonding motif. 74 Figure 2.24. A) Scheme of formate anion bridging the hydrogen bonded network of Janus AT 3b. B) X-ray single crystal structure of supramolecular crinkled ribbon of Janus AT 3b with formate bridging. C) ir-ir stacking of individual ribbons at the van der Waals contact distance (3.4-3.5 A) giving rise to infinite aggregates including sheets of ribbons. - I yN- N I + ± — ç I _c I :4: C Y —-— — S ;: -Q 1 75 2.8 Bis-Janus AT heterocycles and investigation of their biological activity (abasic site targeting molecules) Janus AT heterocycles can potentially act as a small molecule DNA and RNA binders 272 due to their inherent DNA!RNA recognition capability via hydrogen bonding. Consequently screening for biological activity of the compounds presented in this work is under investigation. It is hypothesized that the synthesis of bis analogues of Janus AT would help increase the potency of these derivatives as DNA and RNA binders (abasic site targeting, D-loop and R-loop binders) (Figure 2.25). All of the proposed bis-Janus AT heterocycles contain a positively charged ammonium ion, which in physiological conditions would increase the binding affinity by ionic interaction with the negatively charged phosphate backbone of DNA and RNA. Bis Janus AT 19 and 21 have been synthesized using similar procedures described earlier in this chapter (Scheme 2.6 and 2.7). O NH2 ONH2 (CH2), NH (CH2) NH (CH2) oNçNH O NH2 X= N or CH n=2 or 3 NI-f2 o NN HN NNH2 ON (CH2), (CH2) NH (CH2) N HNy_LyN1NH2 ONN n=2 or 3 N 19 21 Figure 2.25. Bis-Janus AT heterocycles. 76 NH2 NNH a b : C H H2Nç’ 0NN NH2 Scheme 2.6. Synthesis of bis-Janus AT 19 a) Triethylorthoformate, acetic anhydride, reflux, 45 mm, 90% b) 3,3’-Diamino-N-methyldipropylamine, water, 85°C, 15 mm, 93% c) Dicyandiamide, KOH, DMSO, 100 °C, 4 hours, 70%. 0 NH2 HN HNN 0 C N N NN H a b 21 2 SNQ H2NXC C NH2O Scheme 2.7. Synthesis of bis-Janus AT 21 a) 3,3’-Diamino-N-methyldipropylamine, CH2J, rt, 48 hours, 80% c) Guanidinium hydrochloride, NaOEt, EtOH, reflux, 24 hours, 80%. 77 As a preliminary study, inhibitory effect of a solution of bis-Janus AT 21 on the activity of DNAzyme 925-1 i has been investigated by Dr. Marcel Hollenstein (Figure 2.26). 0 1 .tM 10 M 50 M 150 tM 4 4 Cleaved — -m * —. — — ‘• — — . — — substrate Uncleaved k substrate Time Time Time Time Time Figure 2.26. Inhibitory effect of Bis Janus AT 21 (0 p.M to 150 jiM) on the activity of DNAzyme 925-11 8 p.M. Varying concentrations of bis-Janus AT 21 (0 p.M to 150 jiM) were added to a solution of DNAzyme 925-11 273 (8 p.M final concentration). Substrate cleavage of the ribophosphodiester bond was initiated by the addition of trace amount of radio-labeled substrate. The figure 2.26 shows that increasing amounts of bis-Janus AT impeded DNAzyme 925-11 from cleaving the substrate. Indeed, the band corresponding to cleavage decreases with an increase of bis-Janus AT 21’s concentration. However, relatively high concentrations (>50 jiM) of bis-Janus AT 21 are required for an appreciable inhibitory effect to become apparent. The potency of the bis-Janus AT 21 as an inhibitor of DNAzyme is modest compared to strong bis-intercalators such as ethidium bromide dimer. 78 2.9 Conclusion This work is the first report of syntheses and high resolution X-ray structural data for supramolecular solids derived from three self-complementary monomers capable of DAD-ADA hydrogen bonding recognition. In the case of compound 1, four different H-bonded networks co-crystallize to demonstrate the microscopic diversity of such DAD-ADA hydrogen bonds that assume different dihedral angles of considerable variance within the same crystal lattice. The observation of such irregularity in the crystal lattice may have implications for the design of cyanuric/melamine-barbituric acid based structures that are supposed to be rigid and planar but may indeed be more polymorphic or flexible than thought. In the case of Janus AT 2, no crystals could be grown that would have suggested direct self-association via ADA-DAD and the only lattices that could be formed were obtained in the presence of formate. In the case of Janus AT 2b, a formate-bridged lattice was observed. This completely flattened network was revealed not to be a DAD-ADA H-bonded lattice but rather a DDD motif instigated through the DA-AD interaction of formate anion. Moreover, the alkyl tails of 2b were shown to align on the same side of the formed linear ribbon. Janus AT 3b displayed a formate-bridged network in which the alkyl tails of Janus AT 3b were on the opposite sides of the zigzagged ribbon. In this case we observed a DDD-D hydrogen bonding motif of diaminopurine face aligned with an ADA hydrogen bonding pattern of thymine face mediated by AA hydrogen bonding motif of formate anion. In the case of Janus AT 3b, a triply hydrogen bonded molecular ribbon via (ADA-DAD) hydrogen bonding motif was ultimately identified. This structure resulted from the crystallization of Janus AT 3b in DMF in which the t-t stacking of individual ribbons gave rise to infinite 79 aggregates. In no case did we observe rosette-like structures for any of the heterocycles (mentioned before). Not only are these the first crystal structures of compounds with both ADA and DAD faces, but these structures featured herein are as well as proof of the constitutional connectivity of two unique Janus heterocycle compositions which have not been previously reported. ESI MS in the case of Janus AT 2a, 2b provided further evidence for the formation of hydrogen bonded aggregates in the gas phase. The potential of using Janus heterocycles to recognize Watson Crick bases pairs and DNA sequences is an attractive thought that has yet to be fully investigated. However self- association of Janus heterocycles might interfere with the independent association with other DNA bases and recognition process might be hampered as a result. Difficulties in prepareation of soluble oligomers of Janus heterocycles that will have the appropriate length and resulting energetics to permit stable recognition of nucleic acid sequences might be another problem for such investigation. The DNA recognition properties of the heterocycles reported in this work wait for full investigation. Based on the synthetic routes reported in this work, a varied functionalization of Janus AT 1-3 for utilization in bioconjugation and oligomerization would be possible. For example, via using properly protected lysine (e.g. NBoc,COOtBu) instead of an alkylamine, oligomerization of Janus AT heterocycles can be available easily. Since AT-rich sequences are abundant at critical regions of both ribozymes and DNA sequences, properly functionalized Janus AT analogous that recognizes such sequences should be useful for studying the biological processes (e.g. replication, transcription and translation) governed by these sequences. 80 2.10 Current and future trends 2.10.1 Current trends; Janus AT deoxynucleosides An important application of Janus ATs and DNA derived homopolymers of them will be in the sequence specific recognition of AT-rich sequences in RNA and DNA. Such work is currently under investigation. In this context, an interesting and significant modification is the introduction of Janus AT 1-3 skeleton on the 2-deoxyribose backbone (Figure 2.27), since this would allow for the oligomerization of 24 and 25 and the formation of functionalized DNA. Figure 2.27. Janus AT deoxynucleosides and their potential for oligomerization and formation of the Janus-DNA. H H2 22 23 Ph 0 HN0 H N N DMTO N N NH1c:J 0P 25 N 81 Herein the synthetic development for making Janus AT deoxynucleosides, using Vorbruggen-type49coupling reaction, is briefly introduced (Scheme 2.9 and 2.10). Such work is currently under investigation. XX HO 0 Cba OH 26 0 27 0 28 H 0 O N<NH2 H O<NN f HO / N-+LJ0 NH H H NH2OH 0 /4 0 JanusATdeoxynucleoside22 N 29 30 Alpha anomer Beta Anomer -Si HN e IN H d ON -O N >5 31 32 Scheme 2.8. Synthesis of Janus AT deoxynucleoside 22. a) CH3O , acetyl chloride then p Toluoyl chloride, pyridine, 85 % b) HC1, Et20, AcOH, 0 °C, 70% c) Compound 32, anisole, 50 °C, S hours then workup 90 % f3:c ratio of 70:30 d) Ammonium hydroxide, 50 °C, 30 mm, 98% e) Hexamethyldisilane (HMDS), (NH42S0 reflux 1) Sodium methoxide, ethanol, rt then dicyandiamide 100 °C, 18h. 82 3 ,5-Di-toluoyl- 1 -a-chloro-2-deoxy-D-ribose 28 was first prepared by treating 2-deoxy- D-ribose 26 with catalytic HC1 in methanol for 2 h and then protecting the 3,5-hydroxyl groups with p-toluoyl chloride followed by reaction with anhydrous HC1 gas in diethyl ether. Using a similar procedure for making 6, compound 5 was transferred to cyanopyrimidine 31 via treatment with ammonium hydroxide. Persilylation of the cyanopyrimidine 31 was achieved in situ with hexamethyldisilazane (HMDS) and ammonium sulphate. As expected, the N glycosylation reaction via N-glycoside bond formation between the persilylated aglycon base 32 and the protected cL-sugar 28 (glycosyl donor), in anisole or chloroform, led to formation of both alpha and beta anomers (with a 13:a ratio of 70:30). Therefore fractional crystallization in ethanol was applied and this led to selective separation of the anomers. As a result of this crystallization, X-ray quality crystals of the cL-anomer 29 were obtained and diffracted (Figure 2.28). ClO 03 H2n22 C19 Figure 2.28. The ORTEP view of the X-ray crystal structure of cL-anomer 29. 83 Synthesis of Janus AT deoxynucloside 22 was achieved through the following steps (Scheme 2.7). The p-toluoyl protecting group removed trough reaction of compound 30 with two equivalents of NaOMe in EtOH at room temperature. Treatment of the formed product with dicyandiamide at 100 °C gave rise to formation of 22 (as expected from the synthesis of Janus AT 1, section 2.4, Chapter 2). The synthesis of Janus AT nucleoside 23 (Scheme 2.9) is envisioned via introduction of iodine at the 6-position of cynanouracil 30 using LDA/12.Treatment of the supposed product 33 with NaOMe in MeOH at room temperature and then guanidine at reflux in ethanol would give rise to formation of Janus AT nucleoside 23 (as expected from the synthesis of JAT-2). 2.10.1.1 Janus-DNA, phosphoramidite method Th phosphoramidite method 50,51 for oligomerization of Janus AT deoxynucleosides and making the first Janus-DNA is envisioned via utilization of the air-sensitive reagent 2-cyanoethyl tetraisopropyiphosphorodiamidite {[(CH3)2CHJN}POCH2CHN or 2-cyanoethyl AN diisopropylchlorophosphoramidite(iPr)NP(Cl)OCHCHNfor activation of nucleoside donor (Figure 2.27, compounds 24 and 25). This intermediate can be acquired by treatment of PCi3 Scheme 2.9. Janus AT deoxynucleoside 23; proposed steps for finalizing the syntheses. a) LDA, iodine, -78°C, Tifi? b) Sodium methoxide, methanol, rt iii. Guanidine, EtOH, reflux. 84 with 2 equivalents of diisopropylamine, and 1 equivalent of cyanoethanol. The general phosphoramidite approach begins with a nucleoside earlier-protected at the 5-OH position with 4,4-dimethoxytrityl group (DMT), also attached to a silica support. The trityl group is then removed from the 5-OH position and allowed to react with a nucleoside donor protected at position 5-OH with DMT group and activated at position 3 with 2-cyanoethyl diisopropylphophoroamidite. The coupling reaction being the important step is catalyzed by tetrazole, and the process is repeated for the installation of subsequent nucleoside units. Once the oligonucleotide chain is formed, the phosphoramidite group is transformed to phosphate with 12- H2O and released from the resin with ammonia. 2.10.2 Self-organized hydrogen bonded sheets consists of two different sizes of rosettes Janus AT 34 is an attractive heterocycle since it contains the three hydrogen bonding faces (ADA-DAD-ADAD) with a 1200 spatial orientation to each other (Figure 2.29). D% A A Aft All 0 D Figure 2.29. Janus AT 30, a triply hydrogen bonding faced heterocycle. 85 It is hypothesized that self-organization of such triply Hydrogen bondingfaced heterocycle should give rise to the formation of infinite hydrogen bonded sheets of supramolecular aggregates including hexameric rosettes in the network (Scheme 2.10). Analysis of such a hydrogen bonded array would be possible using either X-ray crystallography or atomic force microscope (AFM)/ transmission electron microscope (TEM). Scheme 2.10. The triply faced AT 34 and the possible network of two different hydrogen bonded rosettes (as shown in blue and red colors) H 86 2.10.3 Peripheral crowding of Janus AT as origin of the exclusive formation of rosette As mentioned in Chapter 1, a wide variety of hexameric rosettes have been obtained from two-component self-complementary hydrogen bonding motifs (e.g. cyanuric acid and melamine). The formation of the cyclic arrays in most of the examples depends on additional factors such as peripheral crowding or covalent pre-organization. The strategy of peripheral crowding might be utilized for Janus AT hetrocycles presented in this work, by introducing the Janus ATs on a protected ribose backbone, to induce the preferred formation of cyclic rosette ensemble (Figure 2.30). Figure 2.30. Janus ribo-ATs and the proposed cyclic hexameric rosette structure. 87 2.10.4 Janus AT heterocycles: potential small molecule DNA and RNA binders As a result of intrinsic DNA!RNA recognition capability of Janus AT heterocycles via hydrogen bonding , their potential application as a small molecule DNA and RNA binders 272 is an interesting issue. In addition to the aforementioned study at section 2.8, screening for biological activity of some their analogues (Figure 2.31) is under investigation. Figure 2.31. Janus AT heterocycles: potential small molecule DNA and RNA binders 88 2.11 Experimental 2.11.1 General materials and equipment N-cyanoacetylurethane, anhydrous dimethylformamide (DMF), N-butylamine, N-hexylamine, methyl iodide and carbon disulfide were purchased from Aldrich. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. All purchased chemicals were used without further purification. ‘H NMR spectra were recorded on either a Bruker AV-300, AV 400 or AV-600 spectrometer and calibrated to the residual protonated solvent at ö 2.49 for deuterated DMSO and 7.24 for deuterated chloroform CDC13. Electron ionization (El) mass spectra were obtained at the UBC Mass Spectrometry facility. Single crystal X-ray diffraction measurement for Janus AT 3b was run at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Single crystal X-ray diffraction measurements for the rest of the compounds were made on a Bruker X8 APEX diffractometer with graphite monochromated Mo-Ka radiation. 2.11.2 Synthesis of (Z)-2-cyano-3-ethoxyacrylethyl carbamate (5) 00 A stirring mixture of N-cyanoacetylurethane 4 (1.56 g, 10 mmol), triethyl orthoformate (1.64 mL, 10 mmol) and acetic anhydride (4 mL) was refluxed at 110 °C for 45 minutes. The reaction mixture was then allowed to cool to room temperature. The colorless crystals of product were filtered and washed with light petroleum ether (20 mL) and diethyl ether (5 mL), (yield 95 %). ‘H NMR (300 MHz, CDC13 25 °C) c5=1.24 (t, J= 7.14 Hz, 3H, CR3), 1.38 (t, J 7.14 Hz, 89 3H, CH3), 4.17 (q, J= 7.14 Hz, 2H, CH2), 4.36 (q, J= 7.14 Hz, 2H, CH2), 7.91 (br s, 1H, NH), 8.17 (s, 1H, CH). ‘3C NMR (400 MHz, CDC13) 5 62.5, 74.7, 81.5, 87.0, 104.7, 113.3, 150.1, 159.2, 174.2. ESI-MS (m/z) 235 (M+Na). HRMS (ESf) calcd forC9H,2NO4a 235.0695, found 235.0697. 2.11.3 Synthesis of 1-butyl- 1,2,3,4-tetrahydro-2,4-dioxopyrimidine-5-carbonitrile (6) 0 To a stirring solution of 5 (2.12 g, 10 mmol) in water (10 mL), butylamine (0.99 mL, 10 mmol) was added and the reaction mixture was heated at 90 °C for 25 minutes. The reaction mixture was then allowed to cool down to room temperature and acidified by SN HC1. The colorless crystals were filtered and washed with cold water (10 mL). (Colorless crystals recrystallized from water, yield 93%). ‘H NMR (300 MHz, DMSO-d625 °C) 50.87 (t, J 7.36 Hz, 3H, CH3), 1.22-1.30 (m, 2H, CH2), 1.55-1.60 (m, 2H, CH2), 3.70 (t, J 7.32 Hz, 2H, CH2), 8.69 (s, 1H, CH), 11.94 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6, 25°C) &nlS.S, 19.0, 30.3, 48.6, 87.3, 114.4, 149.6, 154.7, 160.6. ESI-MS (m/z) 216 (M+Na). HRMS (ESI) calcd for C9H11N3O2a216.0749, found 216.0751. 90 2.11.4 Synthesis of Janus AT 1 HN.H 0 NN H N N’NN - H 0N H A stirring mixture of cyanouracil 6 (0.97 g, 5 mmol), dicyandiamide (0.46 g, 5.5 mmol) and powdered potassium hydroxide (0.61 g, 11 mmol) in dry DMSO (5 mL) was heated at 100 °C for 5 hours. The reaction mixture was then allowed to cool down to room temperature, and water (20 mL) was added. The formed solution was acidified by 5N HC1 and the suspended solid product was filtered and washed with water (25 mL) and diethylether (50 mL) to afford pure product 1 (white powder, yield, 80%). ‘H NMR (300 MHz, DMSO-d6)ö0.89 (t, J = 7.41 Hz, 3H, CH3), 1.24-1.32 (m, 2H, CH2), 1.55-1.60 (m, 2H, CH2), 3.73 (t, J= 7.3 Hz, 2H, N-CH2), 6.69 (br s, 4H, NH2), 8.15 (s, 1H, CH), 11.98 (br s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6 25°C,) ö13.6, 19.1, 30.7, 47.6, 139.7, 147.3, 150.4, 160.6, 166.9, 168.3. HRMS (ESI) calcd for C11H6N702278.1365, found 278.1366. 91 2.11.5 Synthesis of potassium 2 -cyano-3- (ethoxycarbonylamino)-3-oxoprop-1-ene-1, 1-bis- thiolate (7) A solution of N-cyanoacetylurethane 4 (1.56 g, 10 mmol) and powdered potassium carbonate (1.38 g, 10 mmol) in dry DMF (25 mL) was vigorously stirred at room temperature for 2 hours. Carbon disulfide (1.3 mL, 20 mmol) was added all at once to the suspension and the stirring was continued for 4 hours. Absolute ethanol (100 mL) was added to the mixture and the precipitate that formed was filtered off, washed with diethyl ether (150 mL), and dried under reduced pressure (Pale yellow powder, yield 95%). ‘H NMR (400 MHz, DMSO-d6 25 °C, TMS): ö=14.9 (s, 1H, NH), 1.1 (t, J=13 Hz, 3H, CH3), 4 (q, J=7 Hz, 2H, CH2). ‘3C NMR (400 MHz, DMSO-d6 25 °C, TMS): ô= 14.5, 59.5, 97.5, 125.6, 152.4, 164.9, 222.1. ESI-MS (m/z) 308 (M). HRMS (ESI) m/z calcd forC7HN203SK308.9172 found 308.9171. 2.11.6 Synthesis of 2-cyano-3,3-bis (methylthio)acrylethyl carbamate (8) 00 ii H Ii s—s-- A solution of 7 (3.08 g, 10 mmol) in a water-acetonitrile (40 mL, 7:3) mixture was stirred at room temperature. Methyl iodide (1.3 mL, 22 mmol) in 10 mL acetonitrile was added dropwise to the solution and the mixture was stirred for 0.5 hour at room temperature. The flask was fitted with a reflux condenser and the reaction mixture was heated under reflux at 95 °C for 92 3 hours. The reaction mixture was then allowed to cool down to room temperature and then was concentrated to 25 mL under reduced pressure (with a rotary evaporator). The product was extracted with ethyl acetate (3x50 mL). The combined organic phases were washed with brine (100 mL), dried over sodium sulfate and concentrated on a rotary evaporator to give a viscous oil bale yellow), which solidified on standing at room temperature under reduced pressure (yield 85%). TLC Rf 0.87 (CH2J/MeOH, 95/5). 1H NMR (300 MHz, CDC13 25 °C, TMS) c=1.27 (t, J 7.1 Hz, 3H, CR3), 2.62 (s, 3H, S-CR3), 2.8 (s, 3H, S-CH3), 4.24 (q, J= 7.08 Hz, 2H, CH2), 7.95 (br s, 1H, NH). ‘3C NMR (400 MHz, CDC1325 °C, TMS) ö=14.40, 19.6, 21.1, 62.8, 98.8, 116.9, 150.6, 159.2, 183. El-MS (m/z) 260 (M). HRMS (El) m/z calcd forC9H12N203S 260.02894 found 260.02905. 2.11.7 Synthesis of 1-butyl- 1,2,3,4-tetrahydro-6-(methylthio)-2,4dioxopyrimidine-5- carbonitrile (9a) 0 A solution of 8 (1.3 g, 5 mmol) in absolute ethanol (30 mL) was stirred at room temperature. Butylamine (0.49 mL, 5 mmol) in 10 mL absolute ethanol was added dropwise over a period of 30 mm. The flask was fitted with a reflux condenser and the reaction mixture was heated under reflux at 100 °C for 16 hours. The reaction mixture was then allowed to cool down to room temperature and the solvent evaporated under reduced pressure (with a rotary evaporator). The resulting white powder was recrystallized in ethanol-water (1:1) (yield 83%). ‘H NMR (300 MHz, DMSO-d6)&0.95 (t, J 7.3 Hz, 3H, CR3), 1.33-1.4 (m, 2H, CR2), 1.58- 93 1.66 (m, 2H, CH2), 2.92 (s, 3H, S-CH3), 4.13 (t, J 7.8 Hz, 2H, N-CH2), 8.6 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6)ô=13.5, 19.2, 19.3, 30.3, 46.3, 92.0, 114.6, 149.1, 159.4, 166.3. ESI-MS (m/z) 262 (M+Na). HRMS (ES11) mlz calcd forC10H3N3O2aS 262.0626 found 262.0625. 2.11.8 Synthesis of 1-heptyl-6- (methylthio)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5- carbonitrile (9b). 0 A solution of 8 (1.3 g, 5 mmol) in absolute ethanol (30 mL) was stirred at room temperature. Heptylamine (0.74 mL, 5 mmol) in 10 mL absolute ethanol was added dropwise over 30 mm. The flask was fitted with a reflux condenser and the reaction mixture was heated under reflux at 100 °C for 16 hours. The reaction mixture was then allowed to cool dwon to room temperature and the solvent evaporated under reduced pressure (with a rotary evaporator). The resulting white powder was recrystallized in methanol-water (1:1) (yield 8 7%). Rf 0.53 (CH2C1/MeOH, 95/5).’H NMR (300 MHz, DMSO-d6)5=0.85 (t, J= 7.3 Hz, 3H, CH3), 1.1-1.4 (m, 8H, CH2), 1.6-1.8 (m, 2H, CH2), 2.8 (s, 3H, S-CH3), 4 (t, J 7.8 Hz, 2H, N-CH2), 8.5 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6)c514.1, 18.1, 19.26, 22.9, 25.0, 27.9, 31.8, 45.7, 92.0, 114.4, 148.5, 159.9, 167.5. ESI-MS (m/z) 281 (M). HRMS (ESIt) m/z calcd forC13H9N302S 281.1198 found 281.1202. 94 2.11.9 Synthesis of Janus AT 2a 0 NH2 NH2 A mixture of guanidinium hydrochloride (0.52 g, 5.5 mmol) and sodium ethoxide (0.37 g, 5.5 mmol) in 5 mL absolute ethanol was heated at 45 °C for 5 minutes and a precipitate formed (NaC1) from which the solvent and free guanidine base was filtered off. The filtrate was added to a solution of 9a (1.2 g, 5 mmol) in absolute ethanol (20 mL). The reaction mixture was heated under reflux at 100 °C for 16 hours. The reaction mixture was then allowed to cool down to room temperature and a white precipitate formed, which was filtered off and washed with water (2x25 mL). The product was then crystallized from formic acid (Yield 87%). 1H NMR (400 MHz, DMSO-d6). ô0.88 (t, J 7.5 Hz, 3H, CH3), 1.22-1.31(m, 2H, CH2), 1.49-1.56 (m, 2H, CH2), 3.94 (t, J 7.28 Hz, 2H, N-CH2), 6.86 (br s, 2H, NH2), 7.40 (br s, 1H, NH), 8.01 (br s, 1H, NH), 11.06 (br s, 1H, NH). 13C NMR (600 MHz, DMSO-do) ô13.8, 19.5, 29.6, 48.6, 82.9, 150.4, 159.4, 162.3, 163.6, 163.7. ESI-MS (m/z) 251 (M+H). HRMS (El) m/z calcd for C10H5N602251.1256 found 251.1258. 95 2.11.10 Synthesis of Janus AT 2b 0 NH2 H N N 0 NN NH2 A mixture of guanidinium hydrochloride (0.52 g, 5.5 mmol) and sodium ethoxide (0.37 g, 5.5 mmol) in 5 mL absolute ethanol was heated at 45°C for 5 minutes and a precipitate formed (NaC1) which was filtered off. The filtrate was added to a solution of 9b (1.4 g, 5 mmol) in absolute ethanol (20 mL). The reaction mixture was heated under reflux at 100 °C for 16 hours. The reaction mixture was then allowed to cool to room temperature and a white precipitate formed, which was filtered and washed with water (2x25 mL). The product was then crystallized from formic acid (yield 85%). 1H NMR (300 MHz, DMSO-d625°C, TMS): 1 1.1 (s, 1H; NH), 8.1(s, 1H, NH), 7.4 (s, 1H, NH),6.9 (s, 2H, NH2), 3.9 (t, J= 7.28 Hz, 2H, CR2), 1.5-1.6 (m, 2H, CH2), 1.2-1.4(m, 8H, CH2), 0.8 (t, J = 6.8 Hz, 3H, CH3). ‘3C NMR (600 MHz, DMSO-d6) ô14.3, 22.2, 26.5, 27.6, 28.9, 31.6, 83.1, 151.8, 159.8, 162.8, 163.4, 164.2. El-MS (m/z) 293 (M+H). HRMS (El) m/z calcd forC13H21N60293.1647 found 293.1656. 96 2.11 11 Synthesis of (2-cyano-3-ethoxy-but-2 -enoyl)carbamic acid ethyl ester (10) A stirring mixture of N-cyanoacetylurethane 4 (1.56 g, 10 mmol), triethyl orthoacetate (1.82 mL, 10 mmol) and acetic anhydride (4 mL) was refluxed at 110 °C for 40 minutes. The reaction mixture was then allowed to cool down to room temperature. The colorless crystals of product were filtered off and washed with light petroleum ether (20 mL) and cold diethyl ether (5 mL), (yield 90%). ‘H NMR (300 MHz, CDC13 25 °C) L5=1.27 (t, J= 7.3 Hz, 3H, CH3), 1.53 (t, J 7.3 Hz, 3H, CH3),2.49 (s, 3H, CH3), 4.2 (q, J= 7.3 Hz, 2H, CH2), 4.39 (q, J 7.3 Hz, 2H, CH2), 9.14 (br s, 1H, NH). 13C NMR (400 MHz, CDC13) ô 15.2, 15.6, 19.9, 62.5, 69.9, 93.5, 118.0, 152.3, 160.1, 178.2. ESI-MS (m/z) 235 (M+Na). HRMS (ESI) calcd forC9H12N2O4a 235.0695, found 235.0697. 97 2.11.12 Synthesis of 1-butyl-6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (ha) 0 To a stirred solution of 10 (2.26 g, lOmmol) in water (10 mL), butyl amine (0.99 mL, lOmmol) was added and the reaction mixture was heated at 90 °C for 20 minutes. The reaction mixture was then allowed to cool down to room temperature and acidified to pH 5 by the addition of 5N HC1. The colorless crystals were filtered off and washed with cold water (10 mL) and cold diethyl ether (10 mL). Colorless crystals recrystallized from water-methanol (1.87 g, yield 91%). ‘H NMR (400 MHz, DMSO-d625 °C) c5=O.99 (t, J= 7.6 Hz, 3H, CH3), 1.36-1.45 (m, 2H, CH2), 1.61-1.70 (m, 2H, CH2), 2.61 (s, 3H, CH3), 3.9 (t, J 7.6 Hz, 2H, CH2), 9.92 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6 25 °C) 513.8, 19.6, 20.1, 30.7, 46.1, 90.6, 113.9, 149.6, 159.8, 163.5. ESI-MS (m/z) 208 (M+H). ERMS (ESI) calcd forC10H,4N302208.1086, found 208.1088. 98 2.11.13 Synthesis of 1-hexyl-6-methyl-24-dioxo- 1,2,3,4-tetrahydro-pyrimidine-5- carbonitrile (lib) 0 Using the procedure for synthesis of ha, compound 10 (2.26 g, 10 mmol) was converted into 211mg (90%) of hib as a white powder.’H NMR (300 MHz, DMSO-d625°C) ô1 (t, J 7.6 Hz, 314, CR3), 1.38-1.48 (m, 6H, CH2), 1.61-1.72 (m, 2H, CH2), 2.60 (s, 3H, CH3), 4 (t, J 7.6 Hz, 2H, CH2), 9.9 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d625°C) 313.8, 19.6, 20.1, 26.5, 27.6, 30, 46, 91, 113.9, 149.6, 160, 163.5. ESI-MS (m/z) 236 (M+H). HRMS (ESI) calcd forC12H8N302236.1399, found 236.1400. 2.11.14 Synthesis of 5-amino- 1-butyl-7-thioxo- 1,7-dihydro-thiopyrano[4,3-d]pyrimidine 2,4-dione (12a) 0 NH2 HN 0NS To a stirred solution of ha (2.07 g, 10 mmol) in dry THF (100 mL) 35 mmol of potassium t-butoxide were added. The reaction mixture was stirred for 10 mm at room temperature followed by dropwise addition of carbon disulfide (25 mmol, 1.6 mL) in THF (10 mL) over period of 10 mm. The reaction mixture was stirred vigorously at room temperature until full conversion of starting material (3 h). Upon completion, the solvent was evaporated under reduced pressure. The crude product was dissolved in water (30 mL) and the solution was 99 acidified to pH 5 using glacial acetic acid. The orange precipitate was filtered off and washed with petroleum ether (10 mL) and cold diethyl ether (10 mL). Further purification was obtained by re-precipitating in methanol (2.52 g, yield 89 %). Single orange colored crystals for x-ray analysis were obtained from acetonitrile. Rf= 0.66 (CH2C1/MeOH, 95/5). ‘H NMR (300 MHz, DMSO-d6,25 °C) c50.94 (t, J 7.5 Hz, 3H, CR3), 1.3-1.38 (m, 2H, CR2), 1.48-1.56 (m, 2H, CH2), 3.94 (t, J 7.5 Hz, 2H, CR2), 6.73 (s, 1H, CR), 9.84 (br s, 1H, NH), 10.57 (br s, 1H, NH), 11.65 (br s, 1H, NH). 13C NMR (400 MHz, DMSO-d6 25 °C) ö=15.1, 20.8, 30.3, 44.5, 92.9, 111.3, 151.0, 151.3, 163.9, 173.5, 188.7. ESI-MS (m/z) 282 (M). HRMS (ESF) calcd for C1,H,2N30S282.0371, found 282.374. 2.11.15 Synthesis of 5-amino- 1-hexyl-7-thioxo-1,7-dihydro-thiopyrano[4,3 -d]pyrimidine 2,4-dione (12b) o NH2 H N S ONS Using the procedure for synthesis of 12a, compound lib (2.36 g, lOmmol) was converted into 283mg (91%) of 12b as an orange colored powder. ‘H NMR (400 M}{z, DMSO d6, 25°C) 50.9 (t, J= 7.5 Hz, 3H, CH3), 1.32-1.40 (m, 6H, CR2), 1.49-1.56 (m, 2H, CR2), 3.94 (t, J= 7.5 Hz, 2H, CR2), 6.7 (s, 1H, CR), 9.9 (br s, 1R, NH), 10.7 (br s, 1H, NH), 11.7 (br s, 1R, NH). ‘3C NMR (400 MHz, DMSO-d625 °C) ö=15.1, 20.8, 26.5, 27.6, 30.3, 44.5, 92.9, 111.3, 151.0, 151.3, 163.9, 173.5, 188.7. ESI-MS (m/z) 310 (M). HRMS (ESF) calcd forC,3H16N02S 310.0689, found 310.0691. 100 2.11.16 Synthesis of 5-amino-1-butyl-7-thioxo-6,7-dihydro-1H-pyrido[4,3-d]pyrimidine-2,4- dione (13a) 0 NH2 HN NH ONLS 5-amino-1-butyl-7-thioxo 12 (0.89 g, 3 mmol) and 30% ammonium hydroxide (30 mL) were placed in a 150 mL Teflon-sealed thick-walled glass tube equipped with a magnetic stirrer and was heated while stirring to 100 °C (12 h) (Caution! use blast shield). The mixture was cooled to room temperature and the excess ammonia was evaporated under reduced pressure. The formed tan coloured precipitate was filtered off and washed with water (10 ml) and cold diethyl ether (20 mL) (0.74 g, yield 93%, tan powder). Single crystal for X-ray crystallography analysis was obtained by using 99% formic acid as crystallization solvent. Rf = 0.35 (CH2C1/MeOH, 95/5). ‘H NMR (300 MHz, DMSO-d625 °C) ó=0.9 (t, J= 7.26 Hz, 3H, CH3), 1.25-1.33 (m, 2H, CH2), 1.43-1.61 (m, 2H, CH2), 3.82 (t, J= 7.32 Hz, 2H, CH2), 6.23 (s, 1H, CH), 7.1 (br s, 1H, NH), 8.35 (hr s, 1H, NH), 11.33 (br s, 1H, NH), 12 (hr s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d625 °C) 3=14.0, 15.12, 20.8, 30.3, 43.3, 91.4, 94.5, 150.1, 151.5, 160.5, 164.7, 167.1. ESI-MS (m/z) 267 (M+H). HRMS (ESI) calcd forC11H5N402S267.0916, found 267.0914. 101 2.11.17 Synthesis of 5-amino-1-hexyl-7-thioxo-6,7-dihydro-1H-pyrido[4,3-d]pyrimidine-2,4- dione (13b) 0 NH HN N H ONLS Using the procedure for synthesis of 13a, compound 12b (0.93 g, 3mmol) was converted into 810mg (92%) of 12b as a tan colored powder. ‘H NMR (400 MHz, DMSO-d6 25 °C) ô0.92 (t, J 7.26 Hz, 3H, CH3), 1.26-1.37 (m, 6H, CH2), 1.45-1.65 (m., 2H, CH2), 3.82 (t, J= 7.32 Hz, 2H, CH2), 6.23 (s, 1H, CH), 7.1 (br s, IH, NH), 8.35 (br s, 1H, NH), 11.33 (hr s, 1H, NH), 12 (hr s, 1H, NH). ‘3C NMR (300 MHz, DMSO-d625 °C) 14.0, 15.12, 20.8, 26, 27.2, 30.3, 43.3, 91.4, 94.5, 150.1, 151.5, 160.5, 164.7, 167.1. ESI-MS (m/z) 312 (M±H). HRMS (ESI) calcd forC,3H8N02S312.0840, found 312.0843. 2.11.18 Synthesis of 5-amino-1-butyl-7-methylsulfanyl-1H-pyrido[4,3-d]pyrimidine-2,4- dione (14a) 0 NH2 Compound 13a (2.66 g, 10 mmol) was added to an aqueous solution of NaOH (2.4 %, 50 mL). The reaction mixture was stirred for 5 mm at room temperature followed by dropwise addition of methyl iodide (10 mmol, 0.62 mL) in 3 mm. The reaction mixture was stirred at room 102 temperature (1.5 h) and upon completion of the reaction the mixture was acidified to pH 6 by dilute HC1 and the formed white precipitate was filtered off and washed with water (20 mL). Further purification was obtained by re-precipitating in methanol (2.65 g, yield 90 %), white powder. ‘H NMR (400 MHz, CDC13 25 °C) Y=1.01 (t, J= 7.6 Hz, 3H, CR3), 1.39-1.48 (m, 2H, CH2), 1.62-1.70 (m, 2H, CH2), 2.54 (s, 3H, CH3), 3.93 (t, J= 7.6 Hz, 2H, CR2), 5.52 (br s, 1H, NH), 6.17 (s, 1H, CH), 8.35 (br s, 1H, NH), 8.54 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6 25 °C) ô=14.O, 15.1, 20.8, 30.2, 43.3, 91.4, 94.5, 150.1, 151.5, 160.5, 164.7, 167.1. ESI-MS (m/z) 281 (M+H). HRMS (ESI) calcd forC12H,7N402S281.1072, found 281.1071. 2.11.19 Synthesis of 5-amino-1-butyl-7-methylsulfanyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (14b) 0 NH2 HNAj5 Using the procedure for synthesis of 14a, compound 13b (3.11 g, 1 Ommol) was converted into 2.92 g (95%) of 14b as a white powder. ‘H NMR (400 MHz, CDC13 25 °C) ö=1 (t, J= 7.6 Hz, 3H, CR3), 1.42-1.50 (m, 6H, CH2), 1.6-1.72 (m, 2H, CH2), 2.5 (s, 3H, CH3), 3.96 (t, J 7.6 Hz, 2R, CR2), 5.5 (br s, 1H, NH), 6.3 (s, 1H, CR), 8.4 (br s, 1H, NH), 8.6 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6 25°C) ô=14.0, 15.1, 20.8, 26.3, 27.4, 30.2, 43.3, 91.4, 94.5, 150.1, 151.5, 160.5, 164.7, 167.1. ESI-MS (m/z) 309 (M+R). RRMS (ESI) calcd for C,4H21N0S309.4071, found 309.4075. 103 2.11.20 Synthesis of 5-amino-1-butyl-7-methanesulfonyl-1H-pyrido[4,3-d]pyrimidine-2,4- dione (iSa) 0 NH2 HN NI 0 0N Method A. 5-Amino-1-butyl-7-methylsulfanyl 14a (5 mmol, 1.4 g) was dissolved in formic acid 88% (10 mL) and hydrogen peroxide 30% (15 mmol, 1.7 mL) was added dropwise to the solution at 0 °C over 5 mm. The reaction mixture was stirred for 5 h at room temperature. Upon completion, the solvent was evaporated under reduced pressure. The crude product was washed with water (10 mL) and diethyl ether (5 mL) to afford 1.48 g (95 %) of a pale yellow solid. Method B. 5-Aniino-1-butyl-7-methylsulfanyl 14a (5 mmol, 1.4 g) was dissolved in chloroform (50 mL) along with 3-chloroperoxybenzoic acid 77% (15 mmol, 2.5 g).The reaction mixture was stirred for 4 h at room temperature. Upon completion, the solvent was evaporated under reduced pressure. The crude product was washed with diethyl ether (50 ml) to afford 1 .2g (81 %) of a pale yellow solid. Rf= 0.44 (CH2C1/MeOH, 95/5). ‘H NMR (400 MHz, DMSO-d6 25 °C) ô=0.92 (t, J 7.6 Hz, 3H, CH3), 1.3-1.39 (m, 2H, CH2), 1.52-1.59 (m, 2H, CH2), 3.24 (s, 3H, CH3), 3.97 (t, J 7.6 Hz, 2H, CH2), 6.9 (s, 1H, CH), 7.97 (br s, 1H,NH), 8.41 (br s, 1H, NH), 1 1.85 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6 25 °C) 5=15.0, 20.7, 30.3, 40.7, 95.3, 96.7, 151.2, 152.4, 161.3, 162.2, 164.5. El-MS (m/z) 312 (M). HRMS (El) calcd for C,2H16N4053 12.08923, found 3 12.08917. 104 2.11.21 Synthesis of 5-amino-1-hexyl-7-methanesulfonyl-1H-pyrido[4,3-d]pyrimidine-2,4- dione (15b) 0 NH2 :xx5 Using the procedure for synthesis of 15a, compound 14b (1.54 g, 5mmol) was converted into 1.51 g (90%) of 15b as a white powder. ‘H NMR (400 MHz, DMSO-d625 °C) (5=0.99 (t, J = 7.6 Hz, 3H, CR3), 1.34-1.43 (m, 6H, CH2), 1.5-1.59 (m, 2H, CH2), 3.3 (s, 3H, CH3), 3.99 (t, J= 7.6 Hz, 2H, CR2), 6.92 (s, 1H, CR), 7.9 (br s, 1H, NH), 8.4 (br s, 1H, NH), 11.85 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6 25 °C) (5=15.0, 20.7,26.1, 27.2, 30.3, 40.7, 95.3, 96.7, 151.2, 152.4, 161.3, 162.2, 164.5. El-MS (m/z) 341 (M). HRMS (El) calcd forC,4H21N0S341.1284, found 341.1280. 2.11.22 Synthesis of 5,7-diamino-1-butyl-1H-pyrido [4.3-d]pyrimidine-2,4-dione (Janus AT 3a) 0 NH2 5-Amino-1-butyl-7-methanesulfonyl 15a (2 mmol , 0.62 g) was suspended in ammonia dissolved in methanol (7 N, 10 mL) and the mixture was placed in a 150 mL Teflon-sealed thick walled glass tube equipped with a magnetic stirrer and heated to 100 °C while stirring (24 h) 105 (Caution! use blast shield). The mixture was cooled down to room temperature and the excess ammonia was evaporated under reduced pressure. The precipitate was washed with water, methanol and diethyl ether to afford 0.45 g (92 %) of a white solid. ‘H NMR (400 MHz, DMSO-d6,25 °C) c50.93 (t, J= 7.6 Hz, 3H, CH3), 1.29-1.38 (m, 2H, CH2), 1.48-1.58 (m, 2H, CH2), 3.75 (t, J 7.6 Hz, 3H, CH3), 5.47 (s, 1H, CH), 6.45 (hr s, 2H, NH2), 6.72 (hr s, 1H, NH), 8.09 (hr s, 1H, NH), 10.79 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d625 °C) ô=13.7, 19.5, 28.6, 41.7, 79.9, 84.8, 149.2, 150.3, 160.7, 162.5, 162.6. ESI-MS (m/z) 248 (M). HRMS (ESI) calcd forC,1H4N502248.1147, found 248.1149. 2.11.23 Synthesis of 5,7-diamino- 1-hexyl-1H-pyrido[4,3-d]pyrimidine-2,4-dione (Janus AT 3b) 0 NH2 Using the procedure for synthesis of Janus AT 3a, compound 15b (0.68 g, 2mmol) was converted into 0.5rng (9 1%) of Janus AT 3b as a tan colored powder. ‘H NMR (400 MHz, DMSO d6, 25 °C) ö=0.9 (t, J 7.6 Hz, 3H, CH3), 1.32-1.39 (m, 6H, CH2), 1.5-1.59 (m, 2H, CH2), 3.75 (t, J= 7.6 Hz, 3H, CH3), 5.47 (s, 1H, CH), 6.5 (hr s, 2F1, NH2), 6.74 (hr s, 1H, NH), 8.1 (br s, 1H, NH), 10.8 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d625 °C) ö13.7, 19.5, 26.1,27.5, 28.6, 106 41.7, 79.9, 84.8, 149.2, 150.3, 160.7, 162.5, 162.6. ESI-MS (m/z) 278 (M). HRMS (ESI) calcd forC13H20N50278.1617, found 278.1621. 2.11.24 1-a-2 -deoxy-3, 5-di-O-p- toluoyl-D-ribosylchloride (28) Under an argon atmosphere, acetyl chloride (0.2 mL, 2.95 mmol) in MeOH (10 rnL) was stirred for 1.5 h at room temperature. The solution was added to 2-deoxy-D-ribose (10.0 g, 74.6 mmol) and stirred for 2.5 h at room temperature. And then AgCO3 was added to the reaction mixture and stirred for 30 mm at room temperature. This solution was filtered to remove the resulting precipitate of AgCO3,and the filtrate was evaporated to close to dryness in vacuo for 1 h and further co-evaporated with pyridine (15 mL) gave honey-like oil. To a solution this oil in pyridine (100 mL) was added DMAP (1.2 g, 9.7 mmol) and p-toluoylchloride (20.7 mL, 156 mmol) and stirred at 0 °C for 12 h. The slurry solution was poured into water/dichloromethane (2:3, 300 mL), the organic phase washed with sat. NaHCO3solution (2 x 20 mL), 3N H2S04(2 x 20 mL), water (2 x 20 mL), NaCl (50 ml), dried with MgSO4 and the solvents evaporated in vacuo. After evaporated of the solution residue was co-evapolated with toluene (30 mL, three times). The residue is (28.6 g, 74.4 mmol, 99%) as orange oil. Rf (hexane/ethyl acetate = 2/1) 0.47. To the solution of Methyl 2- deoxy-3,5-di-O-p-a-D-ribose (28.6 g, 74.4 mmol) in diethyl ether (20 mL) was introduced dry 107 HC1 gas and continued during the crystallization of the product for 45 mm at 0 °C. The crystals were filtered and washed with cold diethyl ether (60 mL, two times). Drying for 18 h in vacuo gave the product (20.5 g, 52.8 mmol, 72%) as white solid. Rf= 0.2 (hexane/ethyl acetate = 2/1). ‘H-NMR (300 MHz, CDC13 25°C) ö=8-7.8 (m, 4H, arom. H), 7.3—7.4 (m, 4H, arom. H), 6.4 (d, 1H, J = 5.1, H-C1), 5.5 (ddd, 1H, J 7.2, 4.5, 2.7, H-C3), 4.8 (m, 1H, H-C4), 4.66 (dd, 1H, J = 12.1, 9.0, H-C5), 4.57 (dd, 1H, J = 12.1, 7.8, H-C5), 2.85 (m, 1H, H-C2), 2.73 (d, 1H, J = 15.0, H- C2), 2.40 (s, 3H, CH3), 2.39 (s, 3H, CH3), 13C NMR (300 MHz, CDC13 25°C) c544.6, 53.6, 84.4, 84.8, 95.2, 95.5, 126.5, 129.2, 129.3, 12.4, 129.8, 129.9, 130.1, 144.2, 144.4, 166.2, 166.5. (Synthesis in analogy to Wang, Z.-X.; Duan, W.; Wiebe, L. I.; Baizarini, J.; de Clercq, E.; Knaus, E. E. Nucleosides Nucleotides 2001, 20, 11-40.) 2.11.25 Synthesis of 2,4-dioxo- 1,23,4-tetrahydro-pyrimidine-5-carbonitrile (31) 0 To a stirring solution of 5 (2.12 g, 10 mmol), ammonium hydroxide (40 mL, 30 %) was added and the reaction mixture was heated at 90 °C for 60 minutes. The reaction mixture was then allowed to cool down to room temperature and acidified by 5N HC1. The colorless crystals were filtered and washed with cold water (10 mL). (Colorless crystals recrystallized from water, yield 89%). ‘H NMR (300 MHz, DMSO-d625 °C) ô=8.3 (s, 1H, CH), 11 (br s, 2H, NH). ‘3C 108 NMR (400 MHz, DMSO-d6, 25°C) 87.2, 115.5, 151.3, 153.5, 161.9. ESI-MS (m/z) 137 (M+Na). 2.11.26 Synthesis of bis-cyanouracil (18) N N III I oyL91 HNy N N Ny N H To a stirring solution of 5 (4.24 g, 20 mmol) in water (15 mL), 3,3’-Diamino-N- methyldipropylamine (1.45 g, 10 mmol) was added and the reaction mixture was heated at 90 °C for 30 minutes. The reaction mixture was then allowed to cool down to room temperature and acidified by SN HC1. The colorless crystals were filtered and washed with cold water (10 mL). (Colorless crystals recrystallized from water, yield 90%). ‘H NMR (300 MHz, DMSO-d625 °C) ö=1.7 (rn, 4H, 2xCH), 2.1 (s, 3H, CH3), 2.3 (t, J 7.36 Hz, 4H, 2xCH), 3.7 (t, J 7.32 Hz, 4H, 2xCH), 8.6 (s, 2H, 2xCH), 11.9 (br s, 2H, 2xNH). ‘3C NMR (400 MHz, DMSO-d6, 25°C) ö23.5, 46.7, 52.5, 88.1, 115.1, 150.3, 155.2, 161.2. ESI-MS (m/z) 386 (M+H). HRMS (ESI) calcd forC17H20N704385.1498, found 385.1510. 109 2.11.27 Synthesis of bis-Janus AT (19) H2NyNyNH H2NNrNH2 N-N NN HNyN N Ny N H A stirring mixture of bis-cyanouracil 18 (3.85 g, 10 mmol), dicyandiamide (2.1 g, 25 mmol) and powdered potassium hydroxide (1.4 g, 25 mmol) in dry DMSO (15 mL) was heated at 100 °C for 6 hours. The reaction mixture was then allowed to cool down to room temperature, and water (20 mL) was added. The formed solution was acidified by 5N HCI and the suspended solid product was filtered and washed with water (25 mL) and diethylether (100 rnL) and recrystalized from DMF to afford pure product bis-Janus AT 19 (70%). ‘H NMR (300 MHz, DMSO-d6,25 °C) 2 (m, 4H, 2xCH), 2.8 (s, 3H, CH3), 3.2 (t, J= 7.36 Hz, 4H, 2xCH), 3.8 (t, J 7.32 Hz, 4H, 2xCH), 6.6 (br s, 8H, 4xNH2), 8.7 (s, 2H, 2xCH), 12 (br s, 2H, 2xNH). ‘3C NMR (600 MHz, DMSO-d625°C,) ö22.5, 45.6, 51.6, 87.1, 113.9, 118.1, 149.3, 154.2, 160.1, 162.4. ESI-MS (rn/z) 554 (M+H)’. 110 2.11.28 Synthesis of 3,5-O-bis(4-methybenzoyl) -cyano--D-uraci1 (30) 0N iN0 2,4-Dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile 31 (1.37 g, 10 mmol) and ammonium sulphate (0.02 g, 0,1 mmol) in HMDS (7 g, 40 mmol) were heated at 125 °C for 6 h. The low-boiling point reagents were removed by evaporation under reduced pressure. Toluene (2x10 mL) was then added and removed by evaporation under reduced pressure. The residual colorless liquid is dissolved in 5 mL anisole. To the reaction mixture l-a-2-deoxy-3,5-di-O-p- toluoyl-D-ribosylchloride 28 (1.94 g, 5 mmol) was added at 50 °C and the reaction continued at 50 °C for 5 h. After cooling, EtOH (5 mL) was added. The resulting suspension was filtered and washed with EtOH (10 mL) (a single crystal of a anomer was obtained from the ethanolic solution as the minor product of the reaction). The crude product crystallized using hot EtOH and the obtained wet powder was dried in vacuo (40 °C, 5 h) to afford j3 anomer 30 as a colorless solid (70%). Rf= 0.43 (EtOAc/Hexane, 50/50). ‘H-NMR (300 MHz, CDC13 25°C) ö=8.8 (br s, 1H, NH), 8.3 (s, 1H, CH), 7.8-8 (m, 4H, arom. H), 7.2—7.4 (m, 4H, arom. H), 6.25 (dd, 1H, J = 12, 9 Hz, C1H), 5.6 (dd, 1H, 3 = 6 Hz, CH), 4.8 (ddd, 1H, 3 = 7.2, 4.5, 2.7, CH), 4.15 (m, 1H, CH), 3.5 (s, 3H, CH3), 2.9 (m, 1H, CH), 2.2 (m, 1H, CH). ‘3C NMR (300 MHz, CDC13 25°C) =23.7, 38.4, 63.6, 75.2, 82.3, 85.2, 88.2, 89.2, 112, 127.1, 127.2, 129.7, 129.9, 130, 144.5, 149.5, 150.2, 161, 165.4, 166. 111 2.11.29 Synthesis of Janus AT deoxynucleoside (22) HON2 To a solution of 3,5-O-Bis(4-methybenzoyl)-cyano-f3-D-uracil 30 (2.45 g, 5 mmol) in EtOH (50 mL) was added 28% NaOMe in MeOH (4.9 g, 20 mmol) at 4 °C, and 5 h later, dicyandiamide (1.05 g, 12.5 mmol) was added at 100 °C and the reaction continued at 100 °C for 18 h. After cooling the reaction mixture was neutralized with 10% w/w AcOH in MeOH at 4 °C. The resulting suspension was filtered. The residual solids were dissolved in DMF (10 mL), and the precipitates were filtered off. The filtrate was concentrated in vacuo, and to the residue was added AcOBu (50 mL). The resulting suspension was filtered and washed with AcOBu (50 mL) and diethyl ether (50 ml). The obtained wet powder was dried in vacuo (40 °C, 4 h) to afford Janus AT deoxynucleoside 22 (40%) as a cream colored solid. ‘H-NMR (300 MHz, CDC13 25°C) ö=12 (hr s, 1H, NH), 8.8 (s, 1H, CH), 6.7 (hr s, 4H, 2xNH), 6.1 (t, 1H, J = 12 Hz, C1H), 5 (dt, 2H, J 7.2, 12 Hz, 2xCH), 4.25 (m, 1H, CH), 3.8 (m, 1H, CH), 3.5-3.7 (m, 3H, 3xCH), 3.3 (s, 2H, OH), 2.2 (t, 2H, 12 Hz, CH2). ‘3C NMR (300 MHz, CDC1325°C) ö61.2, 70, 86.5, 88.5, 88.7, 115, 119, 149, 151, 161, 164. ESI-MS (m/z) 338 (M+H). 112 2.11.30 Synthesis of bis-(methylthio)-2,4dioxopyrimidine-5-carbonitrile (20) I H N N ..— N—- A solution of 8 (1.04 g, 4 mmol) in dichioromethane (50 mL) was stirred at room temperature. 3,3’-Diamino-N-methyldipropylamine (0.29 g, 2 mmol) in 30 mL dichioromethane was added dropwise over a period of 5 hours. The reaction mixture was then allowed to stir at room temperature for 48 hours and a light yellow precipitate formed, which was filtered off and washed with dichioromethane (10 mL) and methanol (10 mL) (80%). Rf 0.66 (CH2C1/MeOH, 95/5). ‘H NMR (300 MHz, DMSO-d625 °C) ö=l.7 (m, 4H, 2xCH), 2.1 (s, 3H, CH3), 2.3 (t, J 7.36 Hz, 4H, 2xCH), 2.8 (s, 6H, 2xCH3),4.1 (t, J 7.32 Hz, 411, 2xCH), 12 (hr s, 2H, 2xNH). ‘3C NMR (400 MHz, DMSO-d6, 25°C) ö=19.7, 26.1, 41.1, 45.8, 54.7, 93.1, 115.2, 149.7, 160.1, 166.9. ESI-MS (m/z) 478 (M+H). 113 2.11.3 1 Synthesis of bis-Janus AT 21 H2N1NyNH2 oyN j NH N(O H2N N NH2 A mixture of guanidinium hydrochloride (0.52 g, 5.5 mrnol) and sodium ethoxide (0.37 g, 5.5 mmol) in 5 mL absolute ethanol was heated at 45 °C for 5 minutes and a precipitate formed (NaC1) from which the solvent and free guanidine base was filtered off. The filtrate was added to a solution of 20 (1.19 g, 2.5 mmol) in absolute ethanol (70 mL) and DMF (5 mL). The reaction mixture was heated under reflux at 100 °C for 24 hours. The reaction mixture was then allowed to cool down to room temperature and a white precipitate formed, which was filtered off and washed with water (2x25 mL) and methanol (2x10 mL) (yield 85%). ‘H NMR (400 MHz, DMSO-d6)5l.7 (m, 4H, 2xCH), 2.1 (br s, 3H, CH3), 2.3 (t, J 7.36 Hz, 4H, 2xCH), 3.9 (t, J = 7.32 Hz, 4H, 2xCH), 6.8 (br s, 4H, 2xNH), 7.4 (br s, 2H, 2xNH), 8.l(br s, 2H, 2xNH), 11.1 (hr s, 2H, 2xNH). ‘3C NMR (400 MHz, DMSO-d6, 25°C) &r19.8, 26.1, 41.1, 45.8, 54.6, 92.7, 115.3, 149.9, 158.9, 160.3, 166.8. ESI-MS (m/z) 500 (M+H). 114 2.11.32 Synthesis of Janus AT 34 0 NH2 NH2 A mixture of guanidinium carbonate (1.83 g, 10 mmol) and 6-Methylsulfanyl-2,4-dioxo- 1,2,3 ,4-tetrahydro-pyrimidine-5 -carbonitrile’ (1.83 g, 10 mmol) in diphenyl ether (10 g) was was heated at 220 °C for 18 hours. The reaction mixture was then allowed to cool down to room temperature by adding 20 mL of methanol. A white precipitate formed, which was filtered off and washed with hot methanol (2x20 mL) and diethyl ether (2x20 mL). The resulting white powder was recrystallized in hot DMF to afford 1.5 g of Janus AT 34 (yield 80%). ‘H NMR (300 MHz, DMSO-d625 °C) ô= 6.7 (br s, 2H, NH2), 7.4 (br s, 1H, NH), 7.9 (br s, 1H, NH), 10.85 (br s, 1H, NH), 11.1 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6, 25°C) ö 151.1, 160.5, 163.8, 164.1, 164.5, 168.2. ESI-MS (m/z) 195 (M+H). 2.11.33 Synthesis of Janus AT 35 H.NH o NN H NrN N - H 0N H A stirring mixture of cyanouracil 31 (1.37 g, 10 mmol), dicyandiamide (1 g, 11 mmol) and powdered potassium hydroxide (1.2 g, 22 mmol) in dry DMSO (20 mL) was heated at 100 °C for 10 hours. The reaction mixture was then allowed to cool down to room temperature, and 1 Tominaga, Y.; Ohno, S.; Kohra, S.; Fujito, H.; Mazume, H. Journal of Heterocyclic Chemistry 1991, 28, 10394042. 115 water (20 mL) was added. The formed solution was acidified by 5N HC1 and the suspended solid product was filtered and washed with water (25 mL) and diethylether (50 mL) to afford crude product 35. Further purification was obtained by re-precipitating in DMF (white powder, yield, 70%). ‘H NMR (300 MHz, DMSO-d6)(5=6.6 (br s, 4H, NH2), 8.1 (s, 1H, CH), 11.9 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d625°C,) (5=115.2, 119.1, 150.1, 152.7, 161.7, 163.9. ESI MS (m/z) 222 (M+H). 2.11.34 Synthesis of 6-Methylsulfanyl-2,4-dioxo-1-(2,4,6-trimethoxy-phenyl) - 1,2,3,4- tetrahydro-pyrimidine-5-carbonitrile 0 0NXS A solution of 8 (1.3 g, 5 mmol) in absolute ethanol (20 mL) was stirred at room temperature. 2,4,6-Trimethoxy-phenylamine (2.02 g, 5 mmol) in 10 mL absolute ethanol was added dropwise over 50 mm. The flask was fitted with a reflux condenser and the reaction mixture was heated under reflux at 100 °C for 16 hours. The reaction mixture was then allowed to cool dwon to room temperature and the resulting white powder was filtered off and recrystallized in methanol-water (1:1) (yield 85%). ‘H NMR (300 MHz, DMSO-d6)ô2.7 (s, 3H, CH3), 3.65 (s, 3H, CH3), 3.8 (s, 6H, 2xCH3), 6.5 (s, 2H, 2xCH), 12.15 (br s, 1H, NH). ‘C NMR (400 MHz, DMSO-d6)(5=19.9, 50.1, 56.6, 60.6, 104.5, 115.2, 132.3, 137.4, 150.2, 153.6, 160.1, 167.4. ESI-MS (m/z) 349 (M). 116 2.11.3 5 Synthesis of Janus AT 36 0 NH HN N 0N NNH2 A mixture of guanidinium hydrochloride (0.52 g, 5.5 mmol) and sodium ethoxide (0.37 g, 5.5 mmol) in 5 mL absolute ethanol was heated at 45 °C for 5 minutes and a precipitate formed (NaC1) from which the solvent and free guanidine base was filtered off. The filtrate was added to a solution of 6-Methylsulfanyl-2,4-dioxo- 1 -(2,4,6-trimethoxy-phenyl)- 1,2,3 ,4-tetrahydro- pyrimidine-5-carbonitrile (1.75 g, 5 mmol) in absolute ethanol (20 mL). The reaction mixture was heated under reflux at 100 °C for 18 hours. The reaction mixture was then allowed to cool down to room temperature and a white precipitate formed, which was filtered off and washed with hot water (2x25 mL) (yield 87%). ‘H NMR (300 MHz, DMSO-d6)ô3.65 (s, 3H, CH3), 3.75 (s, 6H, 2xCH3), 5.1 (s, 2H, 2xCH), 6.7 (br s, 2H, NH2), 7.50 (br s, 1H, NH), 8.05 (br s, 1H, NH), 11.2 (br s, 1H, NH). ‘3C NMR (300 MHz, DMSO-d6)&56.5, 60.5, 83.5, 106.3, 133.8, 137.3, 151.2, 153.2, 159.9, 162.9, 164, 164.4. ESI-MS (m/z) 361 (M+H). 117 3. Chapter 3: The GC Quartet - a DNA-Inspired Janus-GC Heterocycle: Synthesis, Structural Analysis and Self-Organization Studies 3.1 Introduction Hydrogen bonding has been widely used to express the self-organization of properly encoded, one or two-component self-complementary molecules into a series of supramolecular architectures, including linear structures (ribbon, crinkled tape), cyclic rosettes or three- dimensional arrays.’°”4’30”85 Among different supramolecular architectures, cyclic rosette structures are of special significance. The majority of the work carried out on nucleobase derived cyclic architectures focused on the generation of G-quartets that are the most important cyclic hydrogen bonding pattern that can be found in nature. In G-quartets the tetrameric unit is formed by four guanine nucleotides that self-associate through Hoogsteen hydrogen bonding interactions. The cavity formed by this self-assembly process is capable of binding. cations with + + + + •+ 129 151 198 .a selectivity of K > Na > Rb >> Cs , Li . ‘ Similarly, analogues of fohc acid and pterine, which resemble guanine, also form a tetrameric macrocycle of analogous structure to the G-quartets.150156,13,8 A wide variety of hexameric cyclic rosettes have been obtained from two- component self-complementary hydrogen bonding motifs (e.g. cyanuric or barbituric acid and melamine based components).59’602226303 36 However, the formation of the cyclic arrays in most of the above mentioned examples depends on additional factors such as metal ion binding, peripheral crowding or covalent pre-organization. There are far fewer examples of one component self-complementary motifs where the proper hydrogen bonding pattern itself drives the system to an unambiguous and determined self-organized structure (Janus type 244,245,274 molecules and Tectons 2O2325) This strategy involves the use of the relatively strong self complementary hydrogen bonding interaction of guanine and cytosine (ADD-DAA, Ka1O4M1 118 in CDC13)and leads to the formation of different cyclic supramolecules including trimeric and hexameric rosettes. 90,167,243,251-255 The self-organization of cyclic rosette ensembles, assembled through hydrogen bonding, is one of the more captivating subfields within the common purview of supramolecular chemistry. Indeed, quite a number of elegant architectures, such as two dimensional arrays, self- assembling dendrimers and helical rosette nanotubes, have been prepared by means of such cyclic systems.6° Several studies have exploited the strong self-complementary hydrogen bonding interaction of guanine and cytosine, to afford different cyclic architectures including trimeric 90,168,244,252,253,255,256 and hexameric rosettes (see section 1.5.2.2.1.2 in chapter 1). In particular, Mascal and Fenniri have pioneered the synthesis of GC heterocycles that form well- ordered hexameric rosettes.90’67243255 Inspired by their reports, this chapter focuses on the synthesis, structural analysis and self-organization studies of the Janus type DNA base hybrid GC 1 that forms a well-ordered tetrameric rosette (Figure 3.1). Similar to both Mascal and Fenniri GC bases and now GC quartet 1, and unlike guanine/pterin systems, no cation induces self-association because the lone pair on the oxygen is doubly hydrogen bonded, which leaves no site accessible for metal chelation. 3.2 The GC quartet - a novel DNA-inspired Janus-GC heterocycle 119 Inspired by the above findings, the preparation of a novel hydrogen bonded cyclic structure based on nucleic acid base-pairing has been prepared and investigated. Indeed, the Watson-Crick complementary hydrogen bonding paradigm of guanine and cytosine was incorporated in the design of Janus GC heterocycle 1 to program novel quartet architecture. The current chapter will discuss the design, synthesis, and self-organization studies of this novel Janus GC heterocycle 1, which was found to self-assemble through Watson-Crick hydrogen bonds to form a tetrameric rosette (see Ensemble, Figure 3.1). Figure 3.1. Janus GC 1 and the corresponding tetrameric rosette structure. 3.3 Design of Janus GC 1 120 Janus GC 1 displays the self-complementary hydrogen bonding faces of both guanine (ADD) and cytosine (DAA), and in terms of spatial orientation a 900 angle is expected between the two Hydrogen bondingfaces of the molecule. Therefore, these two complementary faces are hypothesized to promote regular association through the GC (DDA—AAD) Hydrogen bonding motif. The guanine-cytosine pair is an attractive building block because it associates predominantly in one well-defined manner and is significantly more stable than the two-point adenine-thymine recognition unit. In addition, literature precedence indicated that the recognition features found in the GC structural unit (i.e., other DDA—AAD DNA-inspired heterocycles can facilitate the formation of cyclic rosette ensembles. Based on the architecture of the molecule, the self-organization of four molecules of Janus GC 1 is thought to give rise to a quartet macrocycle through the formation of 12 strong hydrogen bonds (Figure 3.1). To the best of our knowledge, this represents a new approach for the exclusive formation of quartet cyclic architecture via self-complementary hydrogen bonding interactions of a molecular assembly capable of self-organization. As is apparent from inspection of Figure 3.1, the guanine-like and cytosine-like faces of Janus GC 1 are fused through a pyrrole core, such that the faces are placed in a 90° orientation with respect to each other. It was rationalized that the introduction of the alkyl chain would enhance the solubility of Janus GC 1. The proposed preparation of Janus GC 1 can be best illustrated using the following retrosynthetic analysis (Scheme 3.1). 121 Scheme 3.1. Retrosynthetic analysis of the target molecule Janus GC 1. As illustrated in Scheme 3.1, the synthesis of Janus GC 1 was hypothesized to be accessible starting from commercially available 2-amino-6-chloro-3H-pyrimidin-4-one 2 which already bears the G-face hydrogen bonding motif. Different alkyl chains can be introduced into the skeleton of GC 1 by means of reacting the appropriate amines with the 2-amino-6-chloro-3H- pyrimidin-4-one 2. The pyrrole core of the Janus GC 1 can be introduced by a Bischler-Möhlau type reaction of pyrimidine 3 with 2-chloro-3-oxopropionitrile.75’6 The important amino functionality at the 2-position of the pyrrole core can be introduced using an electrophilic aromatic substitution (i.e. nitration). Construction of the cytosine-face of Janus GC 1 can be achieved via annulation reaction using proper isocyanate functionality or urea. H oHNH H /N H \ NNINH2NN Janus GCI H N _____ H H 7a ÷ 4 0cPh or H2NANH Benzoyl isocyanate Urea 9 HN H2N N ‘NH 3L 9 HN H2N N CI N. + I OH 2-Chloro-3-oxo-propionitrile + Butyl amine 2 122 3.4 Synthesis of Janus GC 1 3.4.1 Synthesis of 7-cyano-7-deazaguanine 4 As mentioned in the retrosynthetic pathway (Scheme 3.1), the pyrrole core of the Janus GC 1 can be introduced during the synthesis of 7-cyano-7-deazaguanine 4 (Scheme 3.2). 2- Amino-6-chloro-4-hydroxypyrimidine 2 was refluxed in an aqueous solution of N-butylamine to give the diaminopyrimidine 3 in 90% yield. Condensation of the diaminopyrimidine 3 with 2- chloro-3-oxopropionitrile in an aqueous solution, in the presence of sodium acetate, provided 7- cyano-7-deazaguanine 4 in 75% yield. 3.4.2 Bromination of pyrrole core and the unsuccessful attempt to introduce the key nitrogen In order to introduce the important amino functionality at the 2-position of pyrimidine 4’s pyrrole core, electrophilic aromatic bromination followed by amination has initially been attempted. Bromination of pyrimidine 4 was carried out in dichioromethane at room temperature using N-bromosuccinimide in 90% yield. All of the attempts for amination of the pyrrole core of Scheme 3.2. Synthesis of 7-cyano-7-deazaguanine 4. a) N-butylamine, water, reflux, 5 hours, 90% b) 2-Chloro-3-oxopropionitrile, sodium acetate, water, 80°C, 75%. 123 9 by substitution of the bromine using different reagents and conditions including sodium azide (DMF, room temperature, 48 hours), animonium hydroxide, methylamine and butylamine (sealed tube, 110 °C, 24 hours) were unsuccessful, and unreacted starting material was recovered (Scheme 3.3). Scheme 3.3. Bromination of pyrrole core of 4 and unsuccessful amination attempts. i) NBS, CH2I,RT, 90% ii.1) Sodium azide, DMF, RT, 48 h, NR. ii.2) Methylamine, sealed tube, H20, reflux, 24 h, NR. ii.3) Butylamine, sealed tube, H20, reflux, 24, NR. 3.4.2 Nitration of pyrrole core; successful introduction of key nitrogen In view of these unsuccessful attempts, the electrophilic aromatic nitration seemed to be a bottleneck in the synthesis of Janus GC 1. After testing several different conditions (Table 3.1) for optimizing the nitration of the pyrrolo ring, the nitro derivative 6a was obtained in excellent yields using ammonium nitrate and trifluoroacetic anhydride 277 in dichioromethane at room temperature (Scheme 3.4). 0 HN Br N N 9 cX H2N N N No products were detected X= N, NHCH3NHC4H9,NH2 124 Scheme 3.4. Nitration of the pyrrole core of 7-cyano-7-deazaguanosine 4. Nitrating reagent Yield nitric acid, acetic anhydride 297 10O C, 24 h 25% silver nitrate, triphenylphosphine297R.T., 2 h 70% ammonium nitrate, trifluoroacetic anhydride 95% 277 , R.T., overnight HN H2N N Ammonium nitrate 0 TFAA, 95 % HN H2N N’ 6a Table 3.1. Optimization of the nitration reaction of the pyrroio core of 4. 125 3.4.3 Catalytic hydrogenation of the nitro group of 6’ to afford 6-amino-5-cyano pyrrolo 7a The reduction of the nitro group of 6a using catalytic hydrogenation over palladium on activated charcoal in methanol occurred in near quantitative yield to afford 6-amino-5-cyano pyrrolo 7a (Scheme 3.5). The single crystal of the 7a was grown in DMF in order to verify the regiochemical orientation of the —NH2 and —CN groups (Figure 3.2). Attempts for annulation reaction between urea and 7a using different conditions were ineffective (Scheme 3.5). N lI2 latm, N¶ 1O%-PdIC, 0 Ii 4 hoursHNP MeOH, HN Neat Urea, Melt rt, 2 hours, 97 % NH2 or s H2N N N C H2N N 61J 7a1 H2N ______ I’ — Urea, EtOH, NaOEt H2N N N Reflux, 72 hours H Janus GC I No product was detected Scheme 3.5. Catalytic hydrogenation of the nitro group of 6a. 126 3.4.3 Finalizing and optimizing the multi-step synthesis of Janus GC 1 The synthesis of Janus GC 1 finally was achieved through seven successive high yielding steps as shown in Scheme 3.6. As mentioned in the retrosynthetic pathway (Scheme 3.1), Janus GC 1 was hypothesized to be accessible through the reaction of 7 reacting with a suitable isocyanate derivative. To prevent the undesirable side reaction between the amino group of the N5 01 112 1165 1145 Figure 3.2. The ORTEP view of the X-ray crystal structure of derivative 7a. 127 G-face and the isocyanate functionality, the two amino groups of 7 have to be differentiated by adequate protection. In order to protect the amino group of the G-face, 7-cyano-7-deazaguanine 4 was refluxed in isobutyric anhydride and then in ethanol to obtain compound 5 in 90%. N N o 0 0 /1/ 0 Ii H N a H N b H N C 0 H N H2N N CI 2NNNH2NNN NN N 2 HJ N N 0 Ii 0 Ii 0 HNNHd OHN N I H H2N 00 0 __ HN o 2NNNN N N H81 Scheme 3.6. Synthesis of Janus GC 1 a) N-butylamine, water, reflux, S hours, 90% b) 2- Chloro-3-oxopropionitrile, sodium acetate, water, 80°C, 75% c) Isobutyric anhydride, reflux, 2 hours, 90% d) Ammonium nitrate, TFAA, C112rt, 8 hours, 93% e) 112 latm, 10%-Pd/C, MeOH, rt, 2 hours, 97% 1) Benzoyl isocyanate, pyridine, CH21 rt, 1 hour, 93% g) Sodium hydride, ethanol, toluene, reflux, 15 hours, 89%. 128 As mentioned before, the nitro derivative 6 was obtained in excellent yield using the optimized conditions for the nitration of the pyrroio ring (ammonium nitrate and TFAA). The reduction of the nitro group of 6 by catalytic hydrogenation over palladium on activated charcoal in methanol occurred in near quantitative yield to afford 6-amino-5-cyano pyrrolo 7. Ultimately, treatment of N-(6-amino-7-butyl-5-cyano-4-oxo-4,7-dihydro-3H-pyrrolo [2,3 - d]pyrimidin-2-yl)-isobutyramide 7 with benzoyl isocyanate 278,279 in the presence of pyridine in CH21 exclusively provided the desired benzoyl urea 8 in excellent yields. Treatment of benzoyl urea 8 with sodium hydride in a refluxing mixture of toluene and ethanol cleanly removed the benzoyl and isobutyryl groups and simultaneously induced the annulation to provide the desired cytosine face and afforded the Janus GC 1 in excellent yields. In order to verify the formation of the supramolecular, tetrameric rosette architecture of Janus GC 1 in the solid state, several different crystallization methods and a variety of solvents mixtures have been exploited. Attempts to obtain a single crystal suitable for X-ray diffraction of Janus GC 1, in terms of size and quality, in other polar solvents (DMF, DMSO, NMP, acetic acid) using vapor diffusion and solvent diffusion (with dioxane or diethyl ether) were unsuccessful. The single crystal of Janus GC 1 was obtained as a formate salt by diffusing dioxane into a solution of 1 in formic acid (99%) (An ORTEP view of the X-ray crystal structure of Janus GC 1 is illustrated in Figure 3.3). 129 Oo C21 01 H2n H4a C) H5a H5b N4 H4b C14 C13 08 C16 Cl 5N7 do C9 02 HGoH7n 0 C12\C11 05 906 U Figure 3.3. ORTEP view of the X-ray crystal structure of Janus GC 1 as a formate salt grown in the presence of dioxane. 3.5 Preliminary insights into the self-organization of Janus GC 1 Gel-phase materials are constructed via the hierarchical self-assembly of molecular building blocks. Over the past decade, there have been many reports on the self-assembly of small organic molecules into supramolecular gels (known as organo-gelators) in a range of solvents due to their distinctive and dynamic properties.280283 Intermolecular interactions, such 130 as t-n stacking, van der Waals interactions, solvophobicity and mainly hydrogen bonding are the driving force of the self-assembly of organogelators and can induce the formation of molecular gel-phase materials. Guanosine derivatives are also well-known to form gels in aqueous solvents via formation of hydrogen bonded linear tapes, known as G-ribbons 128,284,285 or through formation of a G-G dimer.286 G-quartets can also self-assemble into colunmar gels, primarily driven by hydrophobic it-t stacking and hydrogen bonding.’29’15 Based on the aforementioned idea, initial qualitative observations from solubility properties of Janus GC 1 suggested that an ordered supramolecule is formed in the solution state. While Janus GC 1 was only soluble in DMSO, DMF, NMP, acetic acid and formic acid, the gelation process as a result of hydrogen bonding was observable after dissolving Janus GC 1 in DMF and NMP. As is illustrated in Figure 3.4, an early evidence that self-organization was taking place came from the formation of a thick gel upon dissolving of Janus GC 1 in DMF (‘ 3 mg/mL). Figure 3.4. The formation of a thickened gel, upon dissolution of Janus GC 1 in DMF (‘-3 mglmL). 131 3.6 Confirmation of formation of Janus GC 1 quartet ensemble in the gas phase by electron spray ionization mass spectrometry ESI-MS analysis of Janus GC 1 showed two major peaks corresponding to the monomer and dimer (Figure 3.5) and a peak with lower intensity attributed to the quartet stemming from the association of Janus GC 1 into a cyclic tetrameric species. There was no detectable peak corresponding to trimer or any other higher ordered aggregates in the ESI-MS spectrum of Janus Gd. in Intensity % 200.2 [1+Hf 290 [12+11] [14+Na1 1179.6 1179 12.2 1180.7 254.0 02 1177.1 1181 7 0 ]jr1 100 200 300 400 500 600 700 600 900 1000 110’] 1200 1300 1400 1500 Figure 3.5. ESI-MS of a solution of 50 iM Janus GC 1 in a DMSO/MeOH solution. The peaks seen are consistent with the presence of the quartet in the gas phase. Two major peaks for the monomer and dimer are observed along with a peak of lower intensity for the quartet consistent with the association of Janus GC 1 into a cyclic tetrameric species (Intensity of peaks from 1000 — 1350 m/z has been increased 3 times). 132 3.71H-NMR spectroscopy studies In an effort to elucidate the nature of the species formed by the presumed self organization of Janus GC 1, ‘H-NMR spectroscopic studies were undertaken. Initially, ‘H-NMR experiments were carried out at room temperature in DMSO-d6.A typical NMR spectrum of Janus GC 1 recorded under such conditions is illustrated in Figure 3.6. As is noticeable by inspection of this Figure 3.7, two downfield resonances at 10.9 and 10.6 ppm are observed. These resonances, assigned to the guanosine and cytosine-like imino protons (i.e., NH), are exchangeable with D20. None of the amino and imino signals was seen to change as the concentration of Janus GC 1 was varied, an observation that is also consistent with the guanosine subunit participating in rather strong hydrogen bonding interaction with solvent. It seems noteworthy that no intramolecular seven-membered ring hydrogen bond interactions can be detected by1H-NMR at room temperature in DMSO-d6. H-ja JanusGCl NaH ..z, Hi Figure 3.6. ‘H-NMR spectrum of Janus GC 1 in DMSO-d6. 133 2D-NOESY experiments were undertaken to investigate whether any through space interactions are present, as would be expected given the proposed close proximity between the NH protons on the respective guanine-like and cytosine-like faces (Figure 3.7). This experiment revealed the presence of strong cross-peaks between all of the amino and imino protons of the G’”C 1 faces, including putative guanosine imino (N H) and the cytidine amino (NaH,) protons. This evidence is fully consistent with the suggestion that these protons (guanosine imino (N H) and the cytidine amino (NaH2) protons) lie in close spatial proximity. Moreover, these results support the notion that self-assembly takes place through Watson-Crick like interactions between two faces of Janus GC 1 (Figure 3.8). Figure 3.7. Portion of the 2D-NOESY spectrum of Janus GC 1 (400 Mhz, DMSO-d6) showing strong cross-coupling between the guanosine imino (NH) and the cytosine amino (NaH2) protons on the guanosine and cytosine-like faces as well as the guanosine amino bH2)and guanosine imino (NH) protons on the guanosine face. 134 3.7.1 Variable-temperature 1H-NMR spectroscopy studies on Janus GC 1 Williams used variable-temperature ‘H NMR experiment 287 to show that within a GC base pair the amino group of the guanine freely rotates whereas the amino group of cytosine does not. Only at very low temperature is the rotation slowed down significantly such that the exocyclic-NH2protons of a GC base pair exhibit four distinct ‘H-resonances (Figure 3.8) in an NMR spectrum. No rotation H N R’iN /) R Rotation Figure 3.8. VT ‘H-NMR results suggest that the two amino groups in the GC base pair rotate via two different mechanisms. In a model GC base pair, one amino group rotates and the other does not. Similarly a variable-temperature1H-NMR experiment was carried out by changing the temperature from 25 °C to -70 °C on solution of 1 in DMSO-d6/CDC13in order to verify the base pairing between the two faces of the Janus GC 1 (Figure 3.9). The observation of new peaks at very low temperature is consistent with a G-C pairing scheme and hints at formation of a quartet rosette in the solution phase. As illustrated in Figure 3.9, the NaH2protons of the G-face rotate 135 rapidly on the NMR time scale at higher temperatures and both protons appear as a single broad coalesced resonance at 6.3 ppm. At -65 °C they resolve as two distinct resonances at 5.75 and 7.3 ppm. In contrast, the amino protons of N”H2 of the C-face appear as a very broad, almost undetectable resonance (6.75-7.65 ppm), which at -10 °C splits into two well-resolved resonances at approximately 6.8 and 7.5 ppm. At -65 °C the NH2 protons are present as four distinct resonances. Figure 3.9 400-MHz VT ‘H-NMR spectra of Janus GC 1 in DMSO-d6/CDC13(60/40%) featuring the four distinct resonances of the amino protons of the C and G faces at -65 °C that are putatively assigned above (* large peak at 7.6 ppm is corresponding to the CHC13). H H-Na NH.J\ 14 NNN N”H2 25°C 11.0 10.5 10.0 9.5 9.0 8.5 8:0 7.5 7.0 6.5 6.0 5.5 ChemcaI Shift (ppm) 136 3.7.2 Size determination of GC quartet using diffusion-ordered NMR spectroscopy (DOSY) Diffusion ordered spectroscopy (DOSY) has recently been proven to be a significant and very useful tool in the field of supramolecular chemistry.288’9 Diffusion NMR spectroscopy effectively has been utilized to record intermolecular interactions of a variety of self-organized assemblies and multi-component systems in order to verifi their sizes.290293 In the following sections the fundamental features and basic description of this useful method is briefly provided, followed by the DOSY characterization data obtained for Janus GC 1. The acquired data is consistent with the formation of a quartet rosette ensemble in DMSO-d6. For precise measurements it is essential to use an internal standard with a similar structure to the analyte. Consequently, carbazole was chosen as an internal standard for the diffusion study of Janus GC 1 because it is believed to show no interaction with itself or with the Janus GC 1, and has similar shape and comparable molecular weight (Figure 3.10). Figure 3.10 Chemical structures of Janus GC 1 and the internal standard carbazole. 137 3. Z2.1 Concept of translational self-diffusion Translational self-diffusion is the arbitrary movement of molecules or ions driven by their internal thermal energy. This motion is the most important type of transport in chemical and biological systems. It is well known that diffusion is closely related to the molecular size of the diffusing species and is governed by the Einstein—Smoluchowski equation (1) or the Stokes— Einstein equation (3) which is a combination of equation (1) and the Stokes equation (2): 288,289 kb.TEquation 1 D = f Equation 2 f=6.irii.R Equation3D= Kb’T 6• . . R Where D is defined as the diffusion coefficient (cm2sec’), R is the hydrodynamic radius (cm) often called the Stokes’ radius and r’ is the viscosity in Poise (g.cm’sec’). Kb, T, f are the Boltzmann constant (gcm2secK’), the absolute temperature and the so-called hydrodynamic frictional coefficient, respectively. The Stokes radius (Ri) of a spherical molecule is correlated to its partial specific volume (v) and its molecular weight (M) by equation 4 which is derived from the hydrodynamic volume of the molecule (Equation 5). Where M is the molecular weight, V is the hydrodynamic volume, NA is Avogadro’s number, and (v) is the partial specific volume. 3 • 4•,r•R M•vEquation 4 V = S = 3 NA • 3•M•vEquation5R =3JS \I4iz.NA 138 The substitution of Equation (5) into Equation (3) allows for the description of the reciprocal cubic root dependency of the diffusion coefficient on the molecular weight. This means that, under the same conditions for two molecules with diffusion coefficient (Di and D2) and with molecular weights of M1 and M2, the following relation holds (Equation 6): D [iT Equation6 —‘-3I--—- D2 ‘JM1 Equation (3) illustrates also the reciprocal dependency of the diffusion coefficient on the Stokes radius (R3). This means that, under the same conditions for two molecules with diffusion coefficients (Di and D2) and with Stokes radii of R1 and R2, the following relation holds (Equation 7): D1 R2Equation 7 = R1 Since there is a correlation between R and the molecular size, molecular weight and the shape of the molecular species, it is apparent that diffusion coefficients give an insight into the structural properties and aggregation modes of these species. As a consequence, the diffusion coefficient can be utilized to investigate binding phenomena and intermolecular interactions and to provide information on the shape and size of the diffusing molecule.288 139 3.7.2.2 Measuring diffusion with pulsefield-gradient (PFG) NMR spectroscopy Because of its totally nondestructive nature and simplicity of use, nuclear magnetic resonance (NMR) spectroscopy is a distinctive tool for investigating molecular dynamics in chemical and biological systems and measuring their diffusion coefficients. In pulse field gradient (PFG) NMR spectroscopy, the attenuation of a spin-echo signal resulting from the dephasing of the nuclear spins due to the combination of the translational motion of the spins and the imposition of spatially well-defined gradient pulses is used to determine the motion of molecules and their diffusion coefficients (Figure 3.11 )•289 Stej skal and Tanner294’5 introduced a convenient method for measuring diffusion coefficients by NMR acquisition, combining a pair of pulse magnetic field gradients with a spin echo sequence. Currently the bi-polar longitudal eddy current (BPLED) pulse method is the sequence of choice for many PFG NMR experiments including the diffusion ordered spectroscopy (DOSY) (Figure 3.1 2).289296 This pulse sequence reduces the eddy currents to a minimum and also increases the effective gradient output at the same time. Relationship between phase iuation change and displacement in ____.[sio the presence of magnetic field Figure 3.11 Schematic diagram of the pulsed-field gradient (PFG) method for measuring diffusion coefficients. As illustrated in Figure 3.12, the absence of gradient pulses will permit a refocusing of the chemical shift progression in a manner that the detected signal is decayed just by transverse relaxation. Utilization of a field gradient causes the field to be dependent on the physical 140 location. As a result the refocusing state happens only if the spin remains in the same physical location when the pulse field gradients are utilized.288’9 Figure 3.12. The bi-polar longitudinal eddy current (BPLED) pulse sequence which consists of short gradients of contrary polarity, separated by a 180 O pulse. In this sequence, each gradient pulse contains of two pulses of different polarity (G and -G) separated by a 180 ° rf pulse with a duration of 6/2. The G-180-(-G) sequence causes the eddy currents to be cancelled out, while the diffusion gradients build up. If the molecule were to move away from its primary spot during the diffusion delay time (A), then the local field experienced throughout the subsequently pulse field gradient would not be precisely the same as that of the first, and only incomplete refocusing of the signal would take place (Figure 3.13). Therfore how far the molecule moved during the phase of (A) (specified by it irJ2 ‘t/2 ii t/2T RFpulse Jj I irJ2 it itf2 itJ2 Gradient (Gz) 8/2 Echo and Acquisition 8/2 4-, A delay 141 molecular diffusion coefficient) would affect the amount of the detected signal’s reduction. To find out diffusion coefficient, it is possible to progressively change the delay time (A), the duration of the gradient pulses or the intensity of them and to examine the signal decay.49D0 ‘II Figure 3.13. The schematic diagram of the signal decay for the period of diffusion. G (gradient pulse) is a variation in the magnetic field in one direction. As a result of diffusion during the delay time (A), the local magnetic field felt by the molecule A during the initial gradient pulse (molecule shown in red) is not exactly equivalent to that felt during the next gradient pulse (molecule shown in black). Therefore the signal of molecule A is decayed. Bigger signal decay is detected for the quicker diffusing molecule B due to the higher dissimilarity in local fields it feels for the period of the gradient pulses. The signal attenuation is correlated to the diffusion coefficient by equation 8 (Stejskal and Tanner equation 8).294295 (8): 1n =_D.(2..y.g.6)2.[A_=_D.bvajuve Gz 142 The bvaiue is the diffusion weighting factor and is defined as [b = (2it.y.g.6) (A-6/3) s/rn2)] in which y is the gyromagnetic ratio, 6 is the gradient pulse duration, and the g and A are the gradients strength and the time between the start of the first and the second gradient pulses respectively. As shown by equation (8), the diffusion coefficients (D) can be determined by plotting the peak areas to the Stejskal-Tanner equation via plotting the natural logarithm of the signal intensity versus diffusion weighting factor. The slope of the Stej skal-Tanner equation is(-D). 3.7.2.3 Characterization data obtainedfor size determination ofJanus GC 1 A mixture of Janus GC 1 and carbazole in DMSO-d6 was prepared and subjected to diffusion analysis. Diffusion measurements of Janus GC 1 were carried out in a coaxial NMR tube under identical conditions of concentration and temperature (to diminish the effect of convection) in the presence of equimolar concentrations of carbazole, which was chosen as a standard because of its analogous geometry with Janus GC 1. The experiments were carried out at 25 °C using a bipolar longitudinal eddy current delay (BPLED) gradient pulse sequence to suppress convection artifacts in DOSY spectra. The diffusion coefficients (D) of Janus GC 1 and carbazole were determined by fitting the peak areas to the Stejskal-Tanner equation by plotting the natural logarithm of the signal intensity versus the b-value (Figure 3.14). 143 Diffusion Constant G”C Quartet 1 Carbazole D [x10’° m2/s] 0.702 ± 0.02 1.43± 0.01 Table 3.2. Diffusion coefficients (D) of 1 and carbazole at 25 °C in DMSO-d6. bvaiue f 2 s/m1010 0 I I 5 1 1.5 2 2.5 -0.5 —1 11 I ____ in — carbazoe ‘o -2 —.— GC -2.5 -3 -3.5. -4 Figure 3.14. A typical Stejskal-Tanner plot of experimental peak areas and normalized signal decay as a function of the b-value at 25 °C for 1H PFG-BPLED NMR of Janus GC 1 and carbazole in DMSO-d6.The representative signals are NH2 of Janus GC 1 and NH of the carbazole. The solid lines represent linear least-squares fits to the data. No interaction was observed between Janus GC 1 and carbazole (No change was observed in the appearance of ‘H-NMR spectrum of the Janus GC 1 after addition of carbazole in DMSO-d6).The results are summarized in Table 3.2 and Figure 3.14. Figure 3.15 shows the exponential decay of the signals which are used for the calculation of the diffusion coefficients 144 as a function of the gradient strength (g). As it would be explained in the next section (section 3.7.2.3.1), the results of diffusion coefficient analysis are in a very good agreement with the results obtained from the ES I-MS and validate the presence of a tetrameric rosette in DMSO-d6. -1Difusjon Variable Gradient i=I[dlmgp(_D*SQR(2*P2*gaa.oa*Gi*LD)*(BD_LD/3)*1e4) Region 2 9qm 6.669 to 6.635 ppm Diff.Con.7.1—l1m2/S II - __________________________ 0 5 10 15 20 25 30 [GIcm] Figure 3.15. A) Normalized signal decay as a function of the g (gradient strength) at 25°C for NH2 (6.63-6.66 ppm ) Janus GC in DMSO-ddCDC13B) Normalized signal decay as a function of the g (gradient strength) at 25 °C for NH 11.25-11.2 1 ppm) carbazole in DMSO ddCDC13. 3.7.2.3.1 Size determination calculations based on the observed diffusion coefficients As mentioned above, Janus GC 1 was analyzed by PFG NMR spectroscopy in order to verify the presence of quartet assembly in the solution. Before interpreting the results obtained from the PFG analysis, it is necessary to clarify the relationship between the diffusion coefficients (D) of the monomer (carbazole as the internal standard) and of quartet. As —Djfusion Variable Gradient r I=i\exp(_D.sQR(2*pr.gammaoGi*w)*(ED_LD/3)*1e4) Region ]N6rom 11.254 to 11.216 ppm C —Dif6.Con.=l.437E—10m2/S I I C C - C ‘N B) 0 5 10 15 20 25 30 £GIcm] 145 mentioned in section 3.7.2.1, Equation 7 illustrates the reciprocal dependency of the diffusion coefficient on the Stokes radius (R) and Equation 6 shows the reciprocal cubic root dependency of the diffusion coefficient on the molecular weight: D TT Equation6 —-=3J—-- D2 jM1 D1 R2Equation 7 — = D2 R31 Thus the relationship between the diffusion coefficient of a monomer (carbazole as the internal standard) and a quartet will be given by Equation 8 and 9: Dquartet Rmonomer Equation 8 =Dmonomer Rquartet Dquartet JYlmonomer Equation 9 =3( Dmonomer V .ZV[quartet The hydrodynamic radii (R) of carbazole and GC quartet are related to their molecular weights and partial specific volumes and can be calculated from their correlated hydrodynamic volumes via Equation 4 (Assuming that carbazole and GC quartet are spherical molecules): 4 M•v Equation 4 V = — R =3 NA Where V is the hydrodynamic volume, v is the partial specific volume, NA is the Avogadro’s number and M is the molecular weight. Considering Equation 4 for monomer and quartet gives: 146 /3 Vmonomer Rmonomer = 3 I and V 4,r 13(4T”monomer) — j— I3VmonomerRquarret= — V 4•2z V 4•.ir By replacing the above equations into the Equation 8: 13 Vmonomer 31 Dquarter — 4 . — 1 —o 62 — 3/3._Vmonomer V 4r The theoretical ratio of 0.62 between the diffusion coefficients of the quartet and monomer is obtained ( Dquarzet/D,nonamer ). According to the Table 1, the experimental ratio of diffusion coefficients (DQuarlcI/Dmonomer0.5) is indeed in good agreement with the theoretical value obtained for the spherical molecule. Furthermore using Equation 9 for molecular weights of GC quartet (MW4x 289) and carbazole (MW=1 67) gives more precise value for the theoretical ratio of diffusion coefficients (Dquartet/Dmonomer): Dquarret .IY[monomer 1 67 =3f =I =0.52 Dmonomer II/Iquarte’ V 4 x 289 Thus, considering the reciprocal cubic root dependency of the diffusion coefficient on the molecular weight, the theoretical diffusion coefficient of the GC quartet is approximately 52 % 147 of that of a monomer (carbazole). Comparing to experimental data (Table 3.2), this number matches almost exactly with the experimental ratio of diffusion coefficients (50%) and is indeed in excellent agreement with the theoretical value obtained based on the molecular weights of the GC quartet and carbazole. 3.7.2.3.2 2D-Diffusion ordered spectroscopy DOSY One of the most significant characteristics of 2D-DOSY is its capability to separate signals of compounds in a mixture, derived from their diffusion coefficients which, actually, reflect their size and shape, hence offering a way for “virtual separation”.288 Once diffusion coefficients have been calculated from proper data fitting measurements, often it became possible to utilize these data to produce a diffusion dimension in a 2D spectrum. In a 2D-DOSY experiment, the diffusion coefficient is obtained by plotting the signal attenuation as a function of a diffusion increase and utilizing an inverse Laplace transform. Because each resonance of a molecule should be associated with the same diffusion coefficient, the resonances may disperse along the diffusion dimension of a 2D-DOSY plot. As depicted in Figure 3.16, the 2D-diffusion- ordered ‘H spectrum (DOSY) of the equimolar mixture of Janus GC 1 and carbazole in DMSO d6 shows the presence of a GC quartet ensemble, carbazole, DMSO-d6and water molecules in solution with different diffusion coefficients. 148 D ([m2/s] x 106) __AJ GC Quartet 4 4 -;.. H20 % DMSOCarbazole ppm Figure 3.16. 2D-diffusion ordered 111 spectrum (DOSY) of the equimolar mixture of Janus GC 1 and carbazole in DMSO-d6. The horizontal axis displays the conventional proton spectrum which is spreaded along the upright dimension by individual diffusion coefficients. 149 3.8 Conclusion The results of the diffusion coefficient analysis are in very good agreement with the results obtained from the ESI-MS and validate the presence of a tetrameric rosette in DMSO-d6. Furthermore, this work highlights the utility of DOSY-NIvIR for characterizing the stoeichiometry and the size of non-covalently associated supramolecules. Mascal and Fenniri have pioneered the synthesis of GC heterocycles that form well- ordered hexameric rosettes. Inspired by their reports, the synthesis, structural analysis and self organization of a tetrameric Janus type GC 1 is reported. As with both their Janus GC heterocycles and now ours, and unlike guanine/pterin systems, no cation induces association because the lone pair on the oxygen is doubly hydrogen bonded, which leaves no site accessible for metal chelation. In conclusion, the most important points of this work are the following: The synthesis of a heretofore unknown self-complementary Janus GC heterocycle has been fully disclosed for the first time. The central pyrrole ring arrays the self complementary DDA-AAD faces at precisely 90 degrees which programs self-assembly into hydrogen bonded tetrameric structures. These tetrameric structures are inferred from gas-phase data as well as VT and DOSY NMR experiments that provide conclusive evidence of this interaction in solution. The self-organization of Janus GC 1 and its potential for formation of functional higher order systems such as organic nanotubes and discotic liquid crystalline mesophases via t stacking of quartets are briefly discussed as a current trend which is under investigation. A noteworthy application of Janus GC 1, and homopolymers thereof based on DNA and PNA, will be in the sequence specific recognition of GC-rich sequences in RNA and DNA. Such work is currently under investigation. 150 3.9 Current and future trends 3.9.1 Formation of GC quartet nanotubes The preceding section detailed the design and self-organization of a novel cyclic quartet formed via Watson-Crick hydrogen bonding motifs of Janus GC 1. This latter molecule contains a rigid core (the heterocyclic unit) and flexible tails (the alkyl tethers). On the basis of previous literature precedence it is thought that further self-assembly of Janus GC 1 via n-stacking of quartets into tubular columns would result in the formation of discotic liquid crystalline mesophases (see Figure 3.17). “ Figure 3.17. The proposed GC quartet and a schematic illustration of the expected discotic liquid crystalline mesophase structure in its likely form. 151 The self-organization study of Janus GC 1 in the solid state using transmission electron microscopy (TEM) and its potential for the formation of functional higher order systems such as organic nanotubes and discotic liquid crystalline mesophases are currently under investigation. It is envisaged that suitable modifications to the design of Janus GC 1 would also facilitate the formation of quartet cyclic systems that could assemble into liquid crystalline mesophases and nanotubes in conventional organic solvents and water. For instance, replacing the alkyl moieties for longer alkyl chains (e.g. octadecyl, compound 11) or simple derivatization on the periphery of Janus GC 1 (e.g. introduction of lysine, as in the proposed compound 10) should increase the solubility of Janus GC 1 in usual organic solvents or water, respectively (Figure 3.18). Introducing an easily derivatizable functional group (e.g. aldehyde, primary alcohol or primary bromide as in the proposed compound 12, 13) in the Janus GC l’s tail allows the introduction and self-organization of functional species in the quartet periphery (Figure 3.18). Applications might be found wherever quartet states of self-aggregation impart unique properties or function, whether in solution or the solid state. The examples portrayed in this section are designed to underscore the fact that many additional novel self-assembled structures can potentially be formed as the result of making appropriate chemical modifications to Janus GC 1. H0 N Octadecyl 11 Octadecyl tail can be introduced in Janus GC for solubilizing purposes 10 Proposed water soluble version of Janus CC x, 13 X= Br, OH Proposed Janus CC heterocycles with functional tails Figure 3.18. The proposed derivatizations of Janus GC’s tail. 152 It is thought that these new systems will make fundamental contributions to supramolecular chemistry and may also demonstrate potential for the invention of materials with dynamic properties. 3.92 Introduction of Janus GC 1 into the deoxyribose backbone A noteworthy application of Janus GC 1 and DNA or PNA derived homopolymer of Janus GC 1, will be in the sequence specific recognition of GC-rich sequences in RNA and DNA. Such work is currently under investigation. In this context, an interesting and important modification is the introduction of Janus GC 1 skeleton on the deoxyribose backbone (Figure 3.19), since this would allow for the oligomerization of Janus GC deoxynucleoside 15 and the formation of functionalized DNA. It is predicted that introduction of deoxyribose backbone, due to its intrinsic chirality would facilitate the investigation of the self-organization process of Janus GC deoxynucleoside 14 by circular dichroism (CD) spectroscopy as well. OHN 14 15 Figure 3.19. Janus GC deoxynucleosides and their potential for oligomerization and formation of the Janus-DNA. 153 3.9 Experimental 3.9.1 General materials and equipment 2-Amino-6-chloro-3H-pyrimidin-4-one, anhydrous dimethylformamide (DMF), N-butylamine, and ammonium nitrate and TFAA were purchased from Aldrich. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. All purchased chemicals were used without further purification.’H NMR spectra were recorded on either a Bruker AV-300 , AV 400 or AV- 600 spectrometer and calibrated to the residual protonated solvent at 8 2.49 for deuterated DMSO and 6 7.24 for deuterated chloroform CDC13. Electron ionization (El) mass spectra were obtained at the UBC Mass Spectrometry facility. Single crystal X-ray diffraction measurements were made on a Bruker X8 APEX diffractometer with graphite monochromated Mo-Kc radiation. 154 3.9.1.1 Synthesis of2-amino-6-butylamino-3H-pyrimidin-4-one (3): 0 H N2 H A solution of N-butylamine (40 mmol, 2.92 g) in 2 mL of ethanol is added all at once to a stirred mixture of 2-amino-6-chloro-3H-pyrimidin-4-one (2) (20 mmol, 2.9 g) in 50 ml of deionised water. The mixture is heated gently to reflux for 4 hours. The reaction mixture is cooled down to room temperature and is acidified by a solution of (5 N) hydrochloric acid (pH6). The resulting creamy colored precipitate is filtered off and washed with 40 mL of water and 15 mL of diethyl ether respectively (Yield 90%). Rf 0.33 (CH2C1/MeOH, 95/5). ‘H NMR (300 MHz, DMSO-d625 °C) ö= 0.87 (t, J= 7.2 Hz, 3H, CH3), 1.2 (m, 2H, CH2), 1.4 (m, 2H, CH2), 3 (m, 2H, CH2), 4.4 (s, 1H, CH), 6.15 (br s, 2H, NH2), 6.35 (br s, 1H, NH), 9.88 (hr s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6)& 13.2, 21.1, 100.2, 156.1, 161.7, 164.1, 165.1, 166.7. ESI-MS (m/z) 281 (M). HRMS (ESI) m/z calcd forC8H15N40183.1246 found 183.134. 155 3.9.1.2 Synthesis of2-amino-7-butyl-4-oxo-4, 7-dihydro-3H-pyrro!o [2, 3-dJpyrimidine-5- carbonitrile (4) N0 9/ HNr$ H2N N N To a stirred solution of 2-amino-6-butylamino-3H-pyrimidin-4-one 3 (10 mmol, 1.8 g) in water/THF (5OmL/lOmL) sodium acetate trihydrate (20 mmol, 2.7 g) was added and the temperature was maintained at 75° C. 2-Chloro-3-oxo-propionitrile (15 mmol, 1.5 g) in 5 mL of THF was added dropwise to the above solution over a period of 30 mm. The deep blue colored mixture was then heated for 6 hours at 75° C. After cooling, the resulting mixture was filtered off and the brown formed powder was washed with 30 mL of cold water. The dried precipitate was dissolved in a mixture of 15 ml of aqueous 2N KOH and 0.5 g carbon charcoal was added to the mixture. After stirring for 5 mm the mixture was filtered over Celite. The filtrate was then acidified by acetic acid while cooling. The resulting creamy powder was filtered off and dried under reduced pressure and used for the next step without further purification (Yield 75%). Rf= 0.85 (CH2C1/MeOH, 95/5). ‘H NMR (400 MHz, DMSO-d625 °C) = 0.86 (t, J 7.2 Hz, 3H, CH3), 1.19 (m, 2H, CH2), 1.66 (m, 2H, Cl2), 3.92 (t, J = 7.2 Hz, 2H, CH2), 6.49 (br s, 2H, NH2), 7.68 (br s, 1H, NH). 13C NMR (400 MHz, DMSO-d6)& 14.8, 20.6, 32.8, 45.6, 86.1, 100.2, 117.1, 131.7, 152.1, 155.2, 158.9. ESI-MS (m/z) 235 (M + Na). HRMS (ESIj calcd for C,1H3N5ONa 254.1018, found 254.1024. 156 3.9.1.13 Synthesis ofN-(7-butyl-5-cyano-4-oxo-4, 7-dihydro-3H-pyrrolof2,3-djpyrimidin-2-y!)- isobutyramide (5) N H 2-Amino-7-butyl-4-oxo-4, 7-dihydro-3H-pyrrolo [2, 3 -d] pyrimidine-5-carbonitrile 4 (5 mmol, 1.16 g) is added to 5 mL of isobutyric anhydride under constant stirring. The mixture was then heated to 110 °C for 3.5 hours. After cooling, the resulting precipitate was filtered off and re-crystallized in hot ethanol (Yield 85 %). Rf = 0.79 (CH2C1/MeOH, 95/5). ‘H NMR (400 MHz, DMSO-d625 °C) & 0.87 (t, J= 7.3 Hz, 3H, CH3), 1.1 (d, J 7.5 Hz, 6H, 2xCH3), 1.23 (m, 2H, CH2), 1.72 (m, 2H, CH2), 2.78 (m, 1H, CH), 4.05 (t, J = 7.3 Hz, 2H, CH2), 8 (s, 1H, CH), 11.6 (hr s, 2H, NH2), 12 (hr s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6)& 13.4, 18.9, 19.2, 31.5, 34.6, 34.7, 44.8, 85.3, 103.1, 114.9, 147.6, 148.7, 155.3, 180.3. ESI-MS (m/z) 235 (M + Na). HRMS (ESI) calcd forC,5H19NO2a324.1436, found 324.1426. 157 3.91.4 Synthesis ofN-(7-butyl-5-cyano-6-nitro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-dJpyrimidin- 2-yl)isobutyramide (6) N 0 9/ 0 9 i- I NN N 0 N-(7-butyl-5-cyano-4-oxo-4, 7-dihydro-3H-pyrrolo[2,3-dj pyrimidin-2-yl)isobutyramide (4 mmol, 1.38 g), ammonium nitrate (4 nunol, 0.32 g) and trifluoroacetic anhydride (12 mmol, 1.7 mL) are charged along with 40 mL of dry dichioromethane into an oven dried, round bottomed flask equipped with a magnetic stirrer, reflux condenser and drying tube, under a nitrogen atmosphere. The mixture is stirred at room temperature (25 °C) for 4 hours (disappearance of solid ammonium nitrate could be used as an indication of reaction completion. However for more accuracy the reaction progress was monitored by TLC). The solvent was removed by evaporation under reduced pressure. Toluene (3x10 mL) was then added and removed by rotary evaporation under reduced pressure. The residual was dissolved in dichloromethane and was washed with saturated sodium carbonate (2x25 mL) and then brine (20 mL) and dried over sodium sulfate. The solvent was then removed by rotary evaporation and the deep yellow powder was suspended in 20 mL diethyl ether, filtered, dried under reduced pressure and used for the next step without further purification (yield 93%). Rf = 0.83 (CH2C1/MeOH, 95/5). ‘H NMR (400 MHz, CDC13 25 °C) 8 0.92 (t, J= 7.2 Hz, 3H, CR3), 1.31 (d, J= 12 Hz, 6H, 2xCH3), 1.35 (m, 2H, CH2), 1.7 (m, 2H, CH2), 4.57 (t, J = 7.2 Hz, 2H, CH2), 8.25 (br s, 1H, NH), 12.05 (br s, 1H, NH). ‘3C NMR (400 MHz, DMSO-d6)8= 13.7, 19.1, 20.1, 32.3, 37.2, 46.1, 77.4, 91.9, 105.2, 110.9, 117.7, 150.6, 155.2, 173.3. ESI-MS (m/z) 235 (M + Na). HRMS (ESI) calcd forC,5H18N6O4a369.1287, found 369.1297. 158 3.9.1.5 Synthesis of2-amino-7-butyl-6-nitro-4-oxo-4, 7-dihydro-3H-pyrrolo[2,3-dJpyrimidine-5- carbonitrile (6b) N 2-Amino-7-butyl-4-oxo-4, 7-dihydro-3H-pyrrolo [2,3 -dj pyrimidine-5-carbonitrile (4 mmol, 1.38 g), ammonium nitrate (4 mmol, 0.32 g) and trifluoroacetic anhydride (12 mmol, 1.7 mL) are charged with 40 mL of dry dichioromethane into an oven dried, round bottomed flask equipped with a magnetic stirrer, reflux condenser and drying tube, under a nitrogen atmosphere. The mixture is stirred at room temperature (25 °C) for 5 hours (disappearance of solid ammonium nitrate could be used as an indication of reaction completion. However the reaction’s progress was monitored by TLC). The solvent was removed by evaporation under reduced pressure. Toluene (3x10 mL) was then added and removed by evaporation under reduced pressure. The residual is dissolved in dichioromethane and was washed with saturated sodium carbonate (2x25 mL) and then brine (20 mL) and dried over sodium sulfate. The solvent was then removed by rotary evaporation and the deep yellow powder was suspended in 20 mL diethyl ether, filtered, dried under reduced pressure and used for the next step without further purification (yield 95%). Rf= 0.16(Et3N/MeOH/CH2C1,2/4/94). ‘H NMR (600 MHz, DMSO d6, 25 °C) 6 0.89 (t, J= 7.3 Hz, 3H, CH3), 1.31 (m, 2H, CH2), 1.68 (m, 2H, CH2), 4.38 (t, J = 7.3 Hz, 2H, CH2), 7.2 (br s, 2H, NH2), 11.3 (br s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6)ô= 13.5, 19.3, 31.1, 44.5, 90.9, 101.27, 112.1, 137.9, 151.2, 155.9, 157.1. ESI-MS (m/z) 235 (M + Na). HRMS (ESI) calcd forC11H2N6O3a299.0869, found 299.0865. 159 3.9.1.6 Synthesis ofN-(6-amino- 7-butyl-5-cyano-4-oxo-4, 7-dihydro-3H-pyrrolo[2,3-d] pyrimidin-2-yI)-isobutyramide (7) N o qi o HN A solution of N-(7-butyl-5-cyano-6-nitro-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]pyrimidin- 2-yl)-isobutyramide 6 (3 mmol, 1.04 g) in 20 mL of degassed methanol was treated with palladium on carbon (10 wt. % loading (dry basis), matrix activated carbon, wet support, Degussa type ElOl ) (50 mg, 5 % w/w). The mixture was placed inside a flask equipped with a plastic septum, a bubbler syringe and a rubber balloon containing hydrogen gas and was stirred. The bubbling of hydrogen gas inside of the solution was continued for 15 mm at atmospheric pressure (1 atm) and room temperature to ensure the saturation of solution. The reaction mixture was kept under 1 atm pressure of hydrogen gas at room temperature for another hour and the mixture was then filtered over a Celite plug. The colorless filtrate was evaporated under reduced pressure and the obtained product was re-crystallized in methanol-diethyl ether to obtain 94 mg of a creamy colour powder which was used without further purification for the next step (yield 98%).’H NMR (300 MHz, DMSO-d625 °C) & 0.87 (t, J= 7.3 Hz, 3H, CH3), 1.08 (d, J= 7.5 Hz 6H, 2xCH3), 1.25 (m, 2H, CH2), 1.57 (m, 2H, CH2), 2.74 (m, 1H, CH), 3.89 (t, J = 7.3 Hz, 2H, CH2), 6.8 (br s, 2H, NH2), 11.41 (br s, 1H, NH), 11.9 (br s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6)5= 13.7, 18.9, 19.3, 30.5, 34.5, 40.5, 60.7, 100.5, 116.7, 144.3, 146.9, 150.2, 154.2, 179.6. ESI-MS (m/z) 235 (M + Na). HRMS (ESI) calcd forC15H20N6Oa 339.1545, found 339.1542. 160 3.9.1.7 Synthesis of2,6-diamino-7-butyl-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-dJpyrimidine-5- carbonitrile (7b) N H2N A solution of 2-amino-7-butyl-6-nitro-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-djpyrimidine- 5-carbonitrile 6b (3 mmol, 1.04 g) in 20 mL of degassed methanol was treated with palladium on carbon (10 wt. % loading (dry basis), matrix activated carbon, wet support, Degussa type ElOl) (50 mg, 5 % w/w). The mixture was placed inside a flask equipped with a plastic septum, a bubbler syringe and a rubber balloon containing hydrogen gas and was stirred. The bubbling of hydrogen gas inside solution was continued for 15 mm at atmospheric pressure (1 atm) and room temperature to ensure the saturation of the solution. The reaction mixture was kept under 1 atm pressure of hydrogen gas for another hour at room temperature and the mixture was then filtered off over a Celite plug. The colorless filtrate was evaporated under rotary evaporation and the obtained product re-crystallized in methanol-diethyl ether to obtain 90 mg of a creamy color powder which was used without further purification for the next step (yield 98%). ‘H NMR (600 MHz, DMSO-d625 °C) 6 0.89 (t, J= 7.3 Hz, 3H, CH3), 1.25 (m, 2H, CH2), 1.56 (m, 2H, CH2), 3.83 (t, J = 7.3 Hz, 2H, CH2), 6.23 (br s, 2H, NH2), 6.4 (br s, 2H, NH2), 10.5 (br s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6)L5= 13.6, 19.4, 30.5, 59.8, 95.1, 119.2, 147.5, 149, 152.4, 156.4. ESI-MS (m/z) 235 (M + Na). HRMS (ESI) calcd for C,,H14N6ONa269.1127, found 269.1131. 161 3.9.1.8 Synthesis ofN-[6-(3-benzoyl-ureido)-7-butyl-5-cyano-4-oxo-4, 7-dihydro-3H-pyrrolo[2,3- djpyrimidin-2-yl]isobutyramide (8) NN N Benzoyl isocyanate (3.5 mmol, 0.53 g) was added to a solution of N-(6-amino-7-butyl-5- cyano-4-oxo-4,7-dihydro-3H-pyrrolo[2,3 -djpyrimidin-2-yl)isobutyramide (0.74 g, 2.4mmol) in CH21 (15 mL) and pyridine (1 mL, 10 mmol). The solution was stirred for 2h at room temperature. The solvent was then removed under reduced pressure by rotary evaporation and the obtained white powder was suspended in 20 mL of warm water 500 C under constant stirring in order to remove the formed benzamide. The precipitate was filtered and the precipitate re crystallized from methanol to afford 1.05 g white crystals (Yield 93%). Rf = 0.68 (CH2C1/MeOH, 95/5). ‘H NMR (300 MHz, DMSO-d625 °C) & 0.88 (t, J= 7.2 Hz, 3H, CH3), 1.19 (d, J= 7.2 Hz, 6H, 2xCH3), 1.22 (m, 2H, CH2), 1.66 (m, 2H, CH2), 2.78 (m, 1H, CH), 4 (t, J = 7.2 Hz, 2H, CH2), 7.5 1-7.66 (m, 3H, aromatic CR), 8.04 (d, J = 8.3 Hz, 2H, aromatic CH), 10.65 (br s, 1H, NH), 11.4 (hr s, 1H, NH), 11.65 (hr s, 1H, NH), 12.1 (hr s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6)& 13.4, 18.8, 19.1, 30.9, 34.7, 42.1, 82.5, 101.6, 113.8, 128.4, 128.9, 131.8, 133.4, 135.4, 145.5, 148.3, 151.9, 154.9, 168.5, 180.3. ESI-MS (m/z) 235 (M + Na). HRMS (ESr) calcd forC23H5N7O4a486.1866, found 486.1859. 162 3.9.1.9 Synthesis ofjanus GC 1 ‘-I H-N0 H2N N N Sodium hydride (0.38 g, 10 mmol) was added to a solution of N-{6-(3-benzoyl-ureido)-7- butyl-5-cyano-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]pyrimidin-2-yl]isobutyramide 8 (2 mmol, 0.93 g) in a mixture of anhydrous ethyl alcohol (5 mL) and toluene (2 mL). The mixture was refluxed for 18 hours. After cooling the solvent was removed by evaporation under reduced pressure. 20 mL of deionized water was then added to dissolve the formed precipitate and the solution was acidified with acetic acid. The resulting precipitate was washed with 20 mL of hot water, 10 mL of hot methanol and 10 mL of diethyl ether, respectively. Crystals of the final compound were grown from formic acid,fdioxane solution (1/5 w/w). ‘H NMR (300 MHz, DMSO-d6,25 °C) 8 0.88 (t, J= 7.2 Hz, 3H, CH3), 1.25 (m, 2H, CR2), 1.66 (m, 2H, CR2), 3.89 (t, J = 7.2 Hz, 2H, CH2), 6.6 (br s, 2H, NH2), 7.2 (br s, 2H, NH2), 10.7 (br s, 1H, NH), 10.9 (br s, 1H, NH). ‘3C NMR (600 MHz, DMSO-d6)& 13.6, 19.5, 30.5, 90.7, 154.1, 157.8. & ESI-MS (m/z) 235 (M + H). HRMS (ESI) calcd forC12H6N702290.1365, found 290.1375. 163 3.9.2 DOSY and PFG NMR spectroscopic experiments 3.9.21 General All diffiusion NMR experiments were carried out on a Bruker AV-400inv spectrometer equipped with a 5mm BBI Z-gradient probe (inverse broadband probe with z-gradient coil). All measurments were performed using a BPLED gradient pulse sequence (ledbpgp2s). The length of the diffusion gradient was optimized for each diffuision time to obtain at least 94 % signal attenuation due to diffusion. Eddy current (te) was set at 5 ms. All spectra acquired bu using coaxial tubes (Wilmad-Lab Glass 1002 Harding Highway, Buena, NJ) to minimize the effects of convection. All measurments were performed at 25 °C on a 1.5 x103M solution of Janus GC 1 and carbazole. A total of 300 jiL of solution was used for each analysis. The Spectra were processed and plotted by topSpin 1.3 and TopSpin Plot Editor 1.3 respectively. All data are means of at least five measurments. 3.9.2.2 Experimental (Bruker) parametersfor PFG NMR ofJanus GC 1 NS [scans]: 32 D20 []:0.2sec D21 [delay]: 5ms P19: 1.25 ms P30 [6/2]: 3000 J.tsec GPZ8: -13.17% GPZ7: -17.13 % TD (Fl): 16 TD (F2): 8K DS: 8 gpz6[G]: 100% Gradients were standardized from 2% up to 95% in 16 linear steps (TD 1). 164 165 References 1 Guo, H.; Salahub, D. R. Angew. Chem. mt. Ed. 1998, 37, 2985-2990. 2 Somoza, A.; Chelliserrykattil, J.; Kool, E. T. Angew. Chem. mt. Ed. 2006, 45, 4994-4997. 3 Haiduc, I.; Edelmann, F. T. 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Dr. Marcel Hollenstein performed the inhibition tests of bis-Janus AT on the activity of DNAzyme 925-11. ‘H-NMR and13C-NMR spectra of compounds presented in Chapter 2 175 176 1HNMRof7 15 14 13 12 11 10 9 C 0 0 -1 1 ppm w 13C NMR of 7 — j. *is 220 200 180 160 140 120 100 80 60 40 20 0 ppm 177 1HNMROf8 8 7 6 5 4 3 2 1 ppm I 13C NMR of 8 180 160 140 120 100 80 60 40 20 ppm 1H NMR of 9a 13C NMR of 9a I ç1 11 10 19.8 19.6 19.4 19.2 ppm I —_.- 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 178 0 IN 0N’S 9 8 kL ; e I ppm 1H NMRof2a — 0. (‘4 0. to 1 LO to — (N to to UD to <0 (0 — 0 0 GD to <‘4<’ to to to 04 to CD CD (0 UD (‘4 to L<’ (‘4 (‘4 040401 04 0< (0(004 (0 (‘4 (‘4 (‘4 — — !1 4 — 0 179 11 10 9 8 7 6 5 4 3 2 1 ppm 13C NMR of 2a 164 162 160 158 156 154 152 ppm 0 0 NH2 NH2 oto UD 04 toOL 04 0- 00 to to Vii IA j 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 180 1H NMR of 2b o NH2 HNN ONNNH2 L) 13C NMR of 2b 36 34 32 30 28 26 24 22 20 18 16 14 12 ppm 114 162 160 158 156 154 152 ppm 010ppm 181 1HNMRof6 0 N ON 11 10 9 8 7 6 5 4 3 2 1 ppm c 13C NMR of 6 IfrW I 180 160 140 120 100 80 60 40 20 I .á IA I.,. . ii .Ii . - LAd ppm iidd09090800!00!00!09!001 IJOUIINi wdd!0!11 NO HNyN__NH TJOUNNHi 1H NMR of 11 183 10 9 8 0 0 0 7 6 5 4 3 2 1 ppm 0 0 w 13C NMR of 11 to 0 0 C) N to3c too —— — 34 32 30 28 26 24 — 000 r— 0 0 c—rto -iii 22 20 18 16 14 ppm 0 to t0 0 to Nto 0 (- to o 0 m I I VI 0 H N )CN 0N .1 I. 90 80170 160 150 140 130 120 110 100 70 60 50 40 30 20 ppm 1H NMR of 12 184 AA 12 11 10 a a a a a 0 NH2 L 9 8 7 6 5 4 a a a a 3 2 1 a ø a a - a o a a ppm 13C NMR of 12 154 152 150 148 .1 .1 ppm I umiir. $*Iwu ø. I.-, 180 160 140 120 100 80 60 40 20 ppm 1H NMR of 13 12 11 C — — C C — o NH2 10 9 cc C C I 8 7 6 cc C C C C ,-1 .-4 5 4 C C 3 2 1 C C C CC C c’ (‘ 13C NMR of 13 ijit ... •l.ii.h iL 1 .1. . I—I..I.V 1111 _________________________ I I I I I 180 160 140 120 100 80 60 40 20 ppm 185 ppm .1, .1.. .1 1 i. 186 1H NMR of 14 7 0 NH2 I 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 56 x, 4 0 0 0 34 (N C C (N 13C NMR of 14 ppm 187 1H NMR of 15 0 NH2 HNYLJ 0_I H 12 11 C C 10 9 8 7 6 5 4 3 2 1 LD (N - ‘ C C C 13C NMR of 15 m c’- C CC (N mo ppm j U 41.5 41.0 40.5 ppm ill.. ii I I I I I I - 160 140 120 100 80 60 40 20 ppm 188 1HNMROf3 0 NH H N N N N H 11O9 13C NMR of 3 ONO (NON 0 0 ‘0 (N — 0 ONON .- N UD ‘0 ON ON (0 (0 (0 N (0 — (0 N (0 (0 (N N — 0 ON ON N 0 N ON ON N - ON N 0 (0 0 ON ON ON (0 NONO 00 . . to to to U, ON ,-1 0 ON ON ON ON ON ON ON ON ON ii 162 161 ppm - . . -r. -- 0r .. -_(y• Ut -uo I., . . ‘ .J - I I I I I I I 160 140 120 100 80 60 40 20 ppm O O (N f lC O Z J O Z E E — ( > = o (N O N 0 0 0 0 2 0 N O (N (N (2 0 N N N \/ / \ I/ 12 11 10 9 8 7 6 5 0) z 0 3. 75 3. 70 3. 65 pp m J L 2 .3 2 .2 2 .1 2 .0 1 .9 1 .8 1 .7 pp m 4 3 2 1 pp m 00 cD , - ‘ ‘ ‘ C — (fl , fl t , O ‘ 0 • fl t . 0 ‘ 0 ‘ fl ‘ 0 n r ’1 0 0 0 ; B R U K E R 0 ‘ 0 0 ; 0 ; z o \ ‘ t 0 II 17 0 16 5 16 0 15 5 15 0 14 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 10 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 op m 0 a to 0 a a pp m z C Cl) a a a to to to 0 to to a (N 12 11 10 9 8 7 6 5 4 3 2 1 Co 0 ‘ 0 Co cm 2 0 C o C oC o - , Co r C • 0 Co MØ t # s i- L I I Co ‘ 0 C o ’ 0 C o ’ 0 Co C . 0 C Co Co ‘ 0 ‘ 0 • — ‘ 0 ‘ 0 C o C o C o C o Co C o r Co ‘ tC o C o C o C o C o C o C o C o r 17 0 16 5 16 0 15 5 15 0 14 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 10 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 25 10 pm 2.07M t)J I : H — 4.00 w 2.497 __________ —2.491 —2.485 ____ 2.355 2.138 E’)-Z 4.04N NNNH HNyN 0 HN rj (0z)Vz-(oqjAqiow)-sqJoUNNHi E61 F fl 0% 0% 0% 0% 0% 0% 0% ‘ 0 0% 0 iW T T i I. 17 0 16 5 16 0 15 5 15 0 i4 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 10 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 pm cD z 0 z 0 -3 11 10 0 p p m z 1) 9 8 7 6 5 4 3 2 1 L - ø Q c (N (N u -I 13C NMR of bis-Janus AT 21 9’C61 OO[9 O8Z6 gcc6 9Fg6f fr(T Ut 1L9Ot ooct cc9.frc 196 4 NH2 NH2 H2N I (N (N (fl ‘0 (0 • (N —‘N • ‘0 -‘0 O9ZSf I 6?6tI f16 “sd E5r091 LS99l z3 .5 pp m 5. 35 5. 30 5. 25 5. 20 5. 15 5. 10 5. 05 7 0 I) 0 0 6. 0 pp m 12 11 10 2. 2 pp m o 0 0 0 CN C IC lC ) c 0 0 0 0 0 0 0 0 0 , - c I 9 8 7 6 5 4 3 2 1 pp m pp m w ç% j C CD 0 CD 0 CD 88 87 pp m 16 0 14 0 12 0 10 0 80 60 40 20 cD 00 z C 6. 55 6. 50 6. 45 pp m 8 .1 8 .0 7 .9 pp m 4 .9 4 .8 4 .7 4 .6 pp m F 2 .4 pp m 8 .5 8 .0 7 .5 7 .0 6 .5 6. 0 5 .5 5 .0 4 .5 4 .0 3 .5 3 .0 2 .5 2 .0 1 .5 pp m pp m II f , r ‘ fl” r’ ’T jT ’ “ T ’ I r r r T T T T rw rI ” t’ rr r’ w cm 0 1’. ) I IJ - ‘ , o ’ d ’ V V V \/ V 13 C BB EX PT te f to cD CI 3 a t 7?. 23 p p m T V 144. 5 pp m 16 7 ii L .J 0 17 5 17 0 16 5 16 0 15 5 15 0 14 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 10 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 o p, n L . II 0 0 1H o b se rv e r e f. to C D C 13 a t 7 .2 7 z 0 w d 0 z - Q -\ K 00f l D c 1-z L pp m 2. 2 6 .2 5 pp m L — . I 9. 5 9. 0 8. 5 8. 0 7 .5 7. 0 6. 5 6. 0 5. 5 5. 0 4 .5 4. 0 3. 5 3. 0 2 .5 pp m * C C C C co C 0 C C C 0 0 0 C c’ 1 C - 1 - 1 0 I- . 13C-NMR of 30 hi I i i ‘.iW( / )191i) —, fi4Iit i1i/(I (!tIit — JhssIl 202 U. 0 if I U I J_f( i —P—,— Lfl - I In Q I 1. I - LII it tI -‘ 1•Yi I wirjiI — 1’?J JI —‘—.___ flY,)f Ii r4 ‘H-NMR of 31 888 a a - - - - - L() - 203 0 oj - Io. (N w z 0 C ’ ii LI I, IIL IL L J ii II Lb k I L , IL IL B R U K E R L .> K J r z z . 1 IL iJI LI III, IIfl IL LL LIL L II I I I I LI IL J L I UI IA II IJ Jk li f f ‘ 41 ’ 1 17 0 16 5 16 0 15 5 15 0 14 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 10 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 ‘ pr n 1’ ) 0 z -3 z 5 4 3 2 pp m 0 -C w w z I’) 16 9 161 1 16 7 16 6 16 16 1 1 6 16 2 16 1 16 0 15 9 1 1 75 7 15 6 15 5 15 4 I5 h ’ jjm - . I-. -.— .— I-. --- .— .-I --— .-. --- I-. — .— .— . 17 0 16 5 16 0 15 5 15 0 14 5 14 0 13 5 13 0 12 5 12 0 11 5 11 0 20 5 10 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 1’ ) 0 —tO I— 0• ‘0 a’ —3. 2.195 c-fl. 6.006 3.045 CA3 —z- 3.000 —Th •0- t S a.nuoqsu3-s-aupwsAd-ospAqe.nai -Vc’fl-(Auaqd-Axo1pawai-9’)-JJO}.lliINHT C Loz -________________ — —167408 —-—160.094 U! -J53,652 -—150.226 137.370 -132.314 U. J — ——115.191 - 104.521 SXNO NI -0 —60.588 _. — 50.055 .-.j—40.947 —40,391 —401fl ‘—39.835 U.\“—39.557 39.230 IEEE— /,-40.669 —19.904 -OU!pIwJAd-o.ipAqtuiai -Jqlow-9JOHNNi 8oz z 0 1 2 .0 1 1 .5 1 1 .0 1 0. 5 1 0 .0 9 .5 9 .0 8 .5 8 .0 7 .5 7 .0 6 .5 6. 0 5 .5 5 .0 4 .5 4 .0 3 .5 3 .0 2 .5 2 .0 1 .5 1 .0 0 .5 PP m 0 cD z - C,3 C” - K R 17 0 i6 16 0 1 11 J 1 4 (4 0 14 5 H O 1 2 12 0 lf 11 11 lO 1(1( 1 ) JO 5 0 /5 70 05 4; tI 45 40 011 25 10 15 10 pm r’- ) 0 12 .0 11 .1 11 .0 10 .5 10 .0 9. 5 9. 0 8 .5 8. 0 7 .5 7. 0 6 .5 6 .6 . 5 9 .1 4 .5 4. 0 3 . 3 .0 2 .5 2 .0 1. 5 1. 0 0 .5 p 7 0 0’ r’ J z C (j.j - C3 \I/I II 0- / 17 0 16 5 /6 0 15 5 15 0 14 5 i4 0 /3 5 J3 0 12 5 /2 0 1/ 5 /1 0 10 5 1(W ) 95 90 & 6/) 75 70 65 60 55 50 45 30 35 30 25 20 15 l0 pp m r\) 1H-NMR and 13C-NMR spectra of compounds presented in Chapter 3 213 C u rr en t D at a P a ra m e te rs NA M E x a b u ty l p re EX PN O 1 PR O CN O 1 F2 — A c q u is it io n P a ra m e te rs Z D at e 2 00 80 61 7 Z z T im e 1 6 .2 8 >— Z IN ST R U M s p e c t o z ) ) = o PR O BH D 5 m m QN P 1H /1 — PU LP R O G z g3 O TD 16 46 6 z z SO LV EN T D M SO N S 69 D S 2 SW H 3 7 4 2 .5 1 5 H z FI D R E S 0 .2 2 7 2 8 7 H z A Q 2 .1 9 9 9 0 7 5 s e c RG 1 6 1 .3 DW 1 3 3 .6 0 0 u s e c DE 6 .0 0 u s e c TE 2 9 8 .2 K D l 1 .0 0 0 0 0 0 0 0 s e c M CR ES T 0 .0 0 0 0 0 0 0 0 s e c M CW RK 0 .0 1 5 0 0 0 0 0 s e c C H A N N EL fl N U C 1 1H P1 1 0 .7 5 u s e c PL 1 0 .0 0 dB SF 01 3 0 0 .1 3 1 8 0 0 8 M H z F2 — P ro c e ss in g p a ra m e te rs L L S I 32 76 8 SF 3 0 0 .1 3 0 0 0 6 9 M H z W DW EM SS B 0 LB 0 .5 0 H z GB 0 PC 1 .0 0 1D NM R p lo t p a ra m e te rs 10 9 8 7 6 5 4 3 2 1 p p m C X 2 0 .0 0 c m CY 1 2 .0 0 c m F1 P 1 2 .2 1 2 pp m I I I m I O F2 P — 0. 25 8 pp m F l 3 6 6 5 .1 1 H z lI IQ JIQ I 10 1 IcD I Io jo oI F2 — 77 .4 1 H z H H H HH H PP M CM 0 .6 2 3 4 8 pp m /c m H ZC M 1 8 7 .1 2 5 7 5 H z/ cm N ) z 13 C S O S C r - C ) z C u rr en t D at a P ar am et er s NA J1 E g cp re cl 3 EX PN O 1 Z PR O CN O 1 — F2 — A c q u is it io n P ar am et er s D at e 20 08 06 02 T im e 6 1 7 IN ST RI JM s p e c t PR OB HD 5 m m BB O B B -1 H PU LP RO G z gp g3 o PD 32 76 8 SO LV EN T 14 4S 0 40 0 DS 4 SW H 2 51 25 .6 29 H r F lI R tS 0 .7 66 77 3 H z AQ 0 .6 52 13 32 s e c RG 4 59 7. 6 tIN 1 9. 90 0 5 5 cC DE 1 0 .0 0 u s e c TN 2 9 8 .2 K D l 1 .0 0 0 0 0 0 0 0 s e c d li 0 .0 3 0 0 0 0 0 0 s e c D EL TA 0 .8 99 99 99 8 s e c M CR ES T 0 .0 0 0 0 0 0 0 0 s e c HC W RI < 0 .0 1 50 00 00 n e c CH A N N EL fl N O d 13 C 91 7 .8 8 u s e r P1 .1 — 3. 00 SB SF 01 1 0 0 .6 37 91 88 M H z CH A N N EL f2 CP D PR G 2 w a lt z i6 NT JC 2 1H PC PD 2 9 0 .0 0 o s e c PL 2 0 .0 0 dB P1 .1 2 1 6 .0 0 dB PL 13 2 0 .0 0 SB SF 02 4 0 0 .1 91 60 08 M H z F2 — P ro c e ss in g p ar am et er s SI 32 76 8 SF 1 0 0 .6 27 75 78 M H z W OW EM H z 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 90 80 70 60 50 40 30 20 pp m 1. ) 1. -s U , 1H NM R d6 -D M SO 25 C / R .T . C ya no G b u ty l z C u rr en t D at a P a ra m e te rs Z z NA M E a a C N U bu ty l 0 EX PN O 1 1 / z \Q PR O CN O 1 D at e 20 08 04 08 F2 — A c q u is it io n P a ra m e te rs Z T im e 1 2 .3 5 IN ST R tJ M s p e c t PR O BH D 5 m m BB O B B -1 H PU LP R O G z g3 O TD 32 76 8 SO LV EN T DM SO N S 32 D S 2 SW H 6 4 1 0 .2 5 6 H z FI D R E S 0 .1 9 5 6 2 5 H z n I I I OW 78 00 0 u s e c _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ AQ 2 .5 5 5 9 5 4 0 s e c RG 12 8 3 .5 3 .0 2 .5 2 .0 1 .5 1 .0 pp m DE 6. 00 u s e c TE 2 98 .1 K D l 1. 00 00 00 00 s e c M CR ES T 0 .0 0 0 0 0 0 0 0 s e c M CW RK 0 .0 1 5 0 0 0 0 0 s e c CH AN NE L fi N U C1 lB - P ro c e ss in g p a ra m e te rs P1 1 4 .0 0 u s e c PL 1 1 .0 0 dB SF 01 4 0 0 .1 9 2 6 0 1 2 M H z S I 32 76 8 40 0. 19 00 11 6 M Hz 11 10 9 8 7 6 5 4 3 2 1 pp M DW EM SS B 0 LB 0 .5 0 H z Li U U UL L) GB 0 Lo l Ir I I I I I PC 1 .0 0 Io Io P II H H H H HH r. J C u rr en t D at a P ar am et er s NA M E a a C N U bu ty lC l3 EX PN O 1 PR O CN O 1 F2 — A c q u is it io n P a ra m e te rs D at e_ 20 08 04 08 T im e 1 2 .5 2 IN ST R U M s p e c t PR O BH D 5 m m BB O B B -1 H PU LP R O G z gp g3 0 TD 32 76 8 SO LV EN T DM SO N S 25 8 DS 4 SW H 2 5 1 2 5 .6 2 9 H z FI D R E S 0 .7 66 77 3 H z AQ 0 .6 52 13 32 s e c RG 64 5. 1 DW 1 9 .9 0 0 u s e c DE 1 0 .0 0 u s e c TE 2 9 8 .3 K D l 1 .0 0 0 0 0 0 0 0 s e c d li 0 .0 3 0 0 0 0 0 0 s e c D EL TA 0 .8 99 99 99 8 s e c M CR ES T 0 .0 0 0 0 0 0 0 0 s e c M CW RK 0 .0 1 50 00 00 s e c CH AN NE L fi 1 3 C 7 .8 8 — 3. 00 10 0. 63 79 18 8 CH AN NE L f2 CP D PR G 2 w a lt zl 6 N UC 2 1H PC PD 2 90 .0 0 u s e c PL 2 0 .0 0 dE PL 12 1 6 .0 0 dB PL 13 2 0 .0 0 dB SF 02 4 0 0 .1 91 60 08 M H z F2 — P ro c e ss in g p a ra m e te rs pp m S I 32 76 8 SF 1 0 0 .6 27 75 78 M H z W DW EM SS B 0 LB 1 .0 0 H z GB 0 PC 1 .0 0 r O C co c o 0 c ’i N o ) (N c o 0 (N m H H 0 (5 c c N c o L I) . - N . . . . . . c o N c O c o c O Q ( N N 0 Is ) L I) ) - l 0 c o LI - . - . - C ) C ) 0 C ) (N 0 C ) C ) (N H \H I’ e 13 C d6 -D M SO 13 C NM R C ya no G b u ty l 25 C / z z = o / - N U C1 P1 PL 1 SF 01 u s e c dB M H z I I I I I 16 0 14 0 12 0 10 0 80 60 40 20 r’ J JO B N O :1 17 42 A u A sa d i a a 11 74 2 1 1 1H c r y o p ro b e r e f. G CN O 2 to DM SO a t 2 .5 pp m F2 — A c q u is it io n P ar am et er s D at e_ 20 08 05 02 T im e 1 1 .5 7 IN ST RU M a v 60 0c p PR O BH D 5 m m C PT C I 1W - PU LP RO G z g3 0 TD 32 76 8 SO LV EN T DM SO N S 32 DS 2 SW H 90 57 .9 71 H z FI D R E S 0. 27 64 27 H z AQ 1 .8 08 89 88 s e c RG 18 DW 55 .2 00 u s e c DE 6 .0 0 u s e c TE 2 9 8 .0 K D l 0. 50 00 00 00 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH A N N EL fi 1H 9 .2 0 3 .5 0 60 0. 15 33 33 0 P ro c e ss in g p a ra m e te rs 32 76 8 60 0. 15 00 07 7 M H z EM 0 0 .3 0 H z 0 1 .0 0 C u rr en t NA M E EX PN O PR O CN O D at a P ar am et er s a a 1 1 7 4 2 1 1 C 0’ 4 .8 4 .6 4 .4 4 .2 pp m “ 3 N U C1 P1 PL 1 SF 01 JL F 2SI SF WDW LB I ’ I I I T 12 11 10 9 8 - 1 u s e c dB M H z ] p p m C” ) C” ) c c c’ C” ) C” ) C” “ .3 00 I-i . em JO B N O :1 17 42 A ll A sa d i G CN O 2 a a 1 1 7 4 2 2 1 13 C c r y o p ro b e r e f. to D M SO a t 3 9 .5 1 pp m P ar am et er s a a 11 74 2 2 F2 — A c q u is it io n P ar am et er s D at e_ 20 08 05 02 T im e 5. 07 IN ST Rt JM a v 60 0c p PR OB HD 5 m m C PT C I iN PU LP RO G c a r b o n sp in e c h o sp TD 65 53 6 SO LV EN T DM SO N S 39 63 OS 4 SW H 37 59 3. 98 4 H z FI D R ES 0. 57 36 39 H z AQ 0. 87 16 92 1 s e c RG 26 00 8 DW 13 .3 00 u s e c DE 33 .2 5 u s e c TE 2 98 .0 K D l 1. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c D 20 0. 00 00 00 00 s e c d2 1 0. 00 00 15 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fi 1 3 C 1 5. 00 u s e c 20 00 .0 0 u s e c — 1. 90 dB 15 0. 92 28 38 0 M Hz 2 .7 4 dB C rp 6O co m p. 4 0. 00 H z CH AN NE L f2 CP D PR G 2 w a lt zl 6 N U C2 1H PC PD 2 10 0. 00 u s e c PL 2 3. 50 dB PL 12 2 4 .2 2 dB PL 13 1 2 0. 00 dB SF 02 60 0. 15 30 00 0 M Hz F2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 07 6 M Hz W DW EM SS B 0 LB 2 .0 0 H z GB 0 17 0 16 0 15 0 PC N 10 N N H U ) 04 5 ) H 01 40 0 1 0 )1 0 N 1 0 0 5 1 0 . . . . . . O s N 1 0 - 1 N 04 - 1 . L I) L I) 11 ) 10 - 4 0 0 0 U ) 05 U ) 0 5 0 5 0 5 0 5 - i 1 . - - 1 - 4 U ) 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 \/ 0 ) (1 ) 0 ) LI ) 05 0 ) L - 4 1 I 0 0’ C u rr en t D at a NA M E EX PN O PR OC NO I Z z = o N UC 1 P1 P8 PL 1 SF 01 SP 13 SP N A N 13 SP O FF 13 14 0 13 0 12 0 11 0 10 0 90 80 70 60 50 40 30 20 10 pp m F’ J ‘ . 0 1 .0 0 JO B N O :1 17 41 a a 11 74 1 1 1 A u A sa d i 1H c r y o p ro b e G CN H 2 r e f. to DM SO a t 2 .5 pp m F2 - A c q u is it io n P a ra m e te rs D at e 20 08 05 02 T im e 1 0 .0 3 IN ST RU M a v 60 0c p PR O BH D 5 m m C PT C I 1H - PU LP R 0G z g3 O TO 32 76 8 SO LV EN T D M S0 NS 32 OS 2 SW H 90 57 .9 71 H z FI D R E S 0 .2 7 64 27 H z AQ 1 .8 0 8 8 98 8 s e c RG 18 OW 55 .2 00 u s e c DE 6 .0 0 u s e c TE 2 9 8 .0 K D l 0. 50 00 00 00 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH A N N EL fl 1H 9 .2 0 u s e c 3 .5 0 dB 60 0. 15 33 33 0 M H z P ro c e ss in g p ar am et er s 32 76 8 60 0. 15 00 07 7 M H z EM 0 0 .3 0 H z 0 1 .0 0 0 C u rr en t NA M E EX PN O PR O CN O I’ ) z = o O at a P a ra m e te rs a a 1 1 7 4 1 1 1 6. 8 6. 6 6. 4 6. 2 pp m 3. 9 3. 8 3. 7 3. 6 pp m NU C 1 P1 PL 1 SF 01 F2 — S I SF W OW SS B LB GB PC 10 9 8 7 6 5 4 3 2 1 0 pp m c’ .) c ID C C 1’. ) 0 JO B N O :1 17 41 a a 1 1 7 4 1 2 1 A u A sa d i 13 C c r y o p ro b e G CN H 2 r e f. to D M SO a t 3 9 .5 1 pp m F2 - A cq u is it io n P ar am et er s D at e_ 20 08 05 02 T im e 3. 35 IN ST RU M a v 60 0c p PR OB HD 5 m m C PT C I iN — PU LP RO G c a r b o n sp in e c h o sp TO 65 53 6 SO LV EN T M eO D NS 32 33 OS 4 SW H 37 59 3. 98 4 H z FI D R ES 0. 57 36 39 H i AQ 0. 87 16 92 1 s e c RG 23 17 0. 5 OW 13 .3 00 u s e c DE 33 .2 5 u s e c TE 2 98 .0 K D l 1. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c D 20 0. 00 00 00 00 s e c d2 i 0. 00 00 15 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c F2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 07 6 M Hz WO W EM SS B 0 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ LB 2 .0 0 H z ----. GB 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 90 80 70 60 50 40 30 20 10 p p m Pc 1 .4 0 (9 (N OC CO In (N OC N CD In 1 0 1 0 — — (N (N (N — CC N (N (N (9 1 0 (N .0 0 O CC 0 (N N (9 0 tO t- 1 0- (9 0 L I) (N N (N CO O N CX ) CX ) 0 1 0 0 )0 - ‘ 0 tO (9 (N O . 0 O r - 01 t— i N CD (0 0 0 0 0 0 0 0 0 0 ) 0 0 0 0 0 0 0 0 ( 9 ( 9 C O CD (9 (9 )9 )9 (9 (9 )9 (9 0 1 0 1 N (N (N N 1 (N z C C u rr en t D at a P ar am et er s N PN E a a 11 74 1 EX PN O 2 PR OC NO 1 : - z I’) NU C1 P1 PB PL 1 SF 01 S P1 3 SP N A N 13 S PG FF 13 CP D PR G 2 H UC 2 PC PD 2 PL 2 PL 1 2 PL 1 3 SF 02 CH AN NE L fi 1 3 C 15 .0 0 u s e c 20 00 .0 0 u s e c — 1. 90 dB 15 0. 92 28 38 0 M Hz 2. 74 dB C rp 6o co m p. 4 0. 00 H z CH AN NE L f2 w a lt zi 6 iN 10 0. 00 u s e c 3. 50 dB 2 4 .2 2 dB 12 0. 00 dB 60 0. 15 30 00 0 M Hz C u rr en t D at a P ar am et er s NA )4E a a G is o p ro p y lp ro te ct ed EX PN O 1 PR OC HO 1 F2 — A cq u is it io n P ar am et er s D at e_ 20 06 04 22 T im e 7. 01 IN ST RU M s p ec t PR OB HD 5 m m BB O B B -1 H PU LP RO G z g3 O TO 32 76 8 SO LV EN T D M 50 M S 32 DS 2 OW N 64 10 .2 56 H z FI D R ES 0. 19 56 25 H z AQ 2. 55 59 54 0 s e c KG 12 8 OW 78 .0 00 u s e c DE 6. 00 u s e c TE 29 8. 1 K D l 1. 00 00 00 00 s e c H CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fi NU C1 11 1 P1 14 .0 0 u s e c PL 1 1. 00 dB SF 01 40 0. 19 26 01 2 M H z F2 - P ro ce ss in g p ar am et er s SI 32 76 8 SF 40 0. 19 00 10 7 M Hz WO W EM SS B 0 LB 0. 50 H z GB 0 PC 1. 00 gfl ’ , - : c’ j c ’ to r 1H N M R P ro te c te d C ya no de az aG B u ty l R .T . 25 C D 6- D M SO 0 (0 ( . - c co 0 0 (0 z z z C C;’ r— 0) (N - ( r- 0 () (0 (N c o o , - (N (0 N 0) 0 () (0 N (I) () ) 0 . ) 0 N (0 n 0) 0) U ’ (0 (‘ (( N O (0 N U ) (‘7 (N 0 Cc - 1 0 0 0 (‘) (0 N N N N N N N N (0 (N (N (N (N (N - . - 1 0 ) (N (N (N (N (N (N (N (N . , 1 , 1 , ( U , 1 . 4 . - ( H 4 H \W 1 .4 1 .2 2 .9 2 .8 2 .7 pp m pp m 12 II 10 9 8 7 6 5 4 3 2 lp p m C u rr en t D at a P ar am et er s NA M E a s a d il l7 2 7 EX PN O 1 PR OC NO 1 P2 — A cq u is it io n P ar am et er s D at e 20 08 04 23 T im e 7. 12 IN ST RU M a v 60 0c p PR OB HD 5 su e C PT C I iN PU LP RO G c a rb on _s pi ne ch o_ sp TD 65 53 6 SO LV EN T DM SO NS 60 7 DS 4 SW H 37 59 3. 98 4 H z FI D R ES 0. 57 36 39 H z AQ 0. 87 16 92 1 s e c KG 26 00 8 OW 13 .3 00 u s e c DE 33 .2 5 u s e c TE 29 8. 0 K 01 1. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c 02 0 0. 00 00 00 00 s e c d2 1 0. 00 00 15 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fl NO d 13 C 91 15 .0 0 u s e c P8 20 00 .0 0 u s e c PL 1 — 1. 90 dB SF 01 15 0. 92 30 11 5 M Hz SF 13 2. 74 dB SP N A M I3 C rp 6o co m p. 4 SP O FF S3 0. 00 H z CH AN NE L 12 CP O PR G 2 w a lt zl 6 NU C2 1H PC PD 2 10 0. 00 u s e c PL 2 3. 50 dB PL 12 24 .2 2 dB PL 13 12 0. 00 dB SF 02 60 0. 15 30 00 0 M Hz F2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 09 0 14 Hz WO W EM SI B 0 LB 2. 00 H z GS 0 PC 1. 40 I S am pl e N o: 11 72 7 A u A sa d i / ZD a s a d il l7 2 7 1 1 13 C SE / DM SO U BC B ru k er 60 0M H z T C I o r o b er - T = 2 K 22 D A o rl 20 08 Z (9 (‘1 C’ ) ‘ D aD Co C ) U ) C’ ) (0 (‘1 Co (0 . - ( C’ - C’ ) 0 ) . — 4 0 ) C’ - Co (0 C o . . . . . . . C’ ) (0 0 Co C- . ‘ 0 U ) (9 C’ .) C ) C- ’ U ’) Co — (0 Co 0 (0 (0 r- C’ ) C o (9 CO (0 Co Co C- ) . — ) 0 (0 Co 0 Co Co Co Co Co Co Co Co C o Co CO C” ) — , - 1 , - 4 , ‘ 1 , (0 Co Co (9 C’ ) (9 (9 (9 (9 (9 (9 (9 (9 ‘ I , “ ) , - 4 H)11 19 18 17 14 4 14 2 pp m 16 pp m I I I I I 22 0 20 0 18 0 16 0 14 0 12 0 10 0 80 60 40 20 0 pp m 1’ .) 1’ ) w C” 0 C” . - I 0 O c O C O c. m B R U K E R C u rr en t D at a P ar am et er s NA M E a a n it ro p ro tc b u ty l EX PN O 1 PR O CN O 1 F2 — A c q u is it io n P ar am et er s D at e 20 08 04 28 T im e 1 5. 13 IN ST RU M s p e c t PR O BH D 5 m m QN P 1H /1 PU LP RO G z g3 O TO 16 46 6 SO LV EN T C D C 13 N S 67 OS 2 SW H 37 42 .5 15 H z FI D R E S 0. 22 72 87 H z AQ 2 .1 99 90 75 s e c RG 51 2 OW 1 33 .6 00 u s e c DE 6. 00 u s e c TE 2 98 .2 K D l 1 .0 00 00 00 0 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH A N N EL fi M Ud iN P1 1 0. 75 u s e c PL 1 0 .0 0 dB SF 01 30 0. 13 18 00 8 M H z P ro c e ss in g p ar am et er s 32 76 8 30 0. 13 00 06 9 M H z EM 0 0 .5 0 H z 0 1 .0 0 1H o b se rv e r e f. to C D C 13 a t 7 .2 7 pp m z a a’ 2 .9 2 .8 2 .7 2 .6 pp m 4 .8 4 .6 4 .4 4 .2 pp m A A 1 .4 1 .3 1 .2 1 .1 pp m 0 0 0 F2 - SI SF W OW SS B LB GB PC . — 1 12 11 10 9 8 7 6 5 4 3 2 1 pp m C’ ) 0 ‘-4 C u rr en t D at a P ar am et er s N A M E a s a d il l7 3 3 EX PN O 1 PR OC NO 1 F2 — A cq u is it io n P ar am et er s D at e 20 08 04 29 T im e 9. 25 IN ST RU M s p ec t PR OB HD 5 ss s BB O B B -1 H PU LP RO G z gp g3 0 TO 32 76 8 SO LV EN T CD C1 3 NS 53 93 DS 4 SW H 25 12 5. 62 9 H z FI D R ES 0. 76 67 73 H z AQ 0. 65 21 33 2 s e c HG 64 5. 1 DW 19 .9 00 u s e c DE 10 .0 0 u s e c TE 29 8. 1 K D l 1. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fi NU C1 13 C P1 7 .8 8 u s e c PL 1 — 3. 00 dO SF 01 10 0. 63 79 18 8 M Hz CH AN NE L f2 CP D PR G 2 w a lt zl 6 HU C2 11 1 PC PD 2 90 .0 0 u s e c PL 2 0. 00 dB PL 12 16 .0 0 dB PL 13 20 .0 0 dB SF 02 40 0. 19 16 00 8 M Hz P2 - P ro ce ss in g p ar am et er s SI 32 76 8 SF 10 0. 62 78 35 5 M Hz MD W EM SS B 0 LB 1 .0 0 H z GB 0 PC 1. 40 80 60 S am pl e N o: 11 73 3 A u A sa d i N it ro p ro te c te d G / ZD a s a d il l7 3 3 1 1 13 C / C D C 13 U BC B ru ke a, 40 0M H z B B O c a io be T =2 98 K 2 9 A ir il 20 08 CO Co C - C - C ’ (f l — l (‘ F) CC F ) C ) C - Z . . . . . O ) “ C) 0, 0 C) C. ) C) , - . ) C— C o C ,) O r— C ) F) . . . . . . . C- - F) F) a ’ — 0 , - C- C- - C— ) C o C - C. ) C) 0) F) I ‘ITT Ti T Il L /i :Q 7 7 .0 7 6. 5 pp m 77 . 5 ..,,, , , , , . “ I W W. W ø, . I, , 18 0 16 0 14 0 12 0 10 0 40 20 pp m B R U K E R C u rr en t D at e P ar am et er s NA M E a a N H 2G C is op ro pl EX PN O 1 PR O CN O 1 F2 — A c q u is it io n P ar am et er s D at e_ 20 08 04 22 T im e 1 1 .4 7 IN ST RU M s p e c t PR O BH D 5 m m QN P 1H /1 PU LP R O G z g3 O TD 16 46 6 SO LV EN T DM SO N S 12 8 US 2 SW H 3 7 4 2 .5 15 H z FI B R E S 0. 22 72 87 H z AQ 2 .1 99 90 75 s e c RG 91 2. 3 DW 1 3 3 .6 00 u s e c BE 6. 00 u s e c TE 2 98 .2 K D l 1 .0 00 00 00 0 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH A N N EL fl 1H 1 0. 75 u s e c 0 .0 0 dB 30 0. 13 18 00 8 M Hz P ro c e ss in g p ar am et er s 32 76 8 30 0. 13 00 06 9 M Hz EM 0 0 .5 0 H z 0 1 .0 0 12 11 10 9 8 7 6 5 o L o o 0 C c 0 4 3 2 1 p p m z z 2. 95 2. 90 2. 85 2. 80 2. 75 2. 70 z 0 2. 60 2. 55 pp m Li 1. 4 1. 3 1. 2 1. 1 1. 0 0. 9 II pp m NU C 1 P1 PL 1 SF 01 F2 - SI SF W DW SS B LB GB PC N) C u rr en t D at a P ar am et er s NA S4 E a s a d il l7 2 3 EX PI OO 1 PR O CH O 1 P2 - A cq u is it io n P ar am et er s D at e 20 08 04 23 T im e 3. 39 IN ST RU M a v 60 0c p PR OB HD 5 m m C PT C I 10 - PU LP RO G c a rb o n _ sp in ec h o _ sp TD 65 53 6 SO LV EN T DM SO NS 10 17 OS 4 SW H 37 59 3. 98 4 H z FI D R ES 0. 57 36 39 H z AG 0. 87 16 92 1 s e c KG 26 00 8 DW 13 .3 00 u s e c DE 33 .2 5 u s e c TE 29 8. 0 K D l 3. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c D 20 0. 00 00 00 00 s e c d2 1 0. 00 00 15 00 s e c D EL TA 2. 90 00 00 10 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH A N N EL 01 NU C1 13 C P1 15 .0 0 u s e c P8 20 00 .0 0 u s e c PL 1 — 1. 90 dB SF 01 15 0. 92 30 11 5 M Hz SF 13 2. 74 dB SP SL ON 13 C rp 60 cc m p. 4 SP O FF 13 0. 00 Hz CH A N N EL 52 CP D PR G 2 w a lt zl 6 NU C2 10 PC PD 2 10 0. 00 u s e c PL 2 3. 50 dB PL 12 24 .2 2 dB PL S3 12 0. 00 dB SF 02 60 0. 15 30 00 0 M Hz P2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 09 3 M Hz WO W EM SO B 0 LB 2. 00 H z GB 0 PC 1. 40 22 0 20 0 18 0 16 0 14 0 12 0 10 0 0 pp m I.. ) S am pl e N o: 11 72 3 A li A sa di G C — N H 2- pr t / ZD a s a d il l7 2 3 1 1 13 C SE / DM SO UB C B ru k er 60 0M H z T C I o r o e m c T = 2 9 8 K 22 A o ri a 20 08 CO (N C’ ) C ) çz ) (C’ C ) (0 07 0 fl C C) CO C (C) (0 • . . r— U ) CD 0 ) C - ‘ 0 (1) C ) C’ ) C ) (C ) (C) ti 0 C 0 ) CD SO (0 CD r- (C ’ ((3 — 4 CD CD CD CD 0 0 )0 ) (0 0 )0 ) (3) C ) 0 ) CC ) C ) ‘ ) . 4 . . 4 SO C ) 07 CO (‘ 7( 00 7 0 7 0 7 (0 r4 . 4 ii i I I / C z 0 z 3: p3 21 20 19 18 17 16 pp m (.1 80 60 40 20 B R U K E R C .> K ) C u rr en t D at a P ar am et er s NA M E a a is o cy n ad ct b u t EX PN O 1 PR O CN O 1 F2 — A c q u is it io n P ar am et er s D at e_ 20 08 04 11 T im e 1 2 .2 0 IN ST RU M s p e c t PR O BH D 5 m m QN P lB /i PU LP RO G z g3 O TD 19 78 2 SO LV EN T DM SO N S 57 DS 2 SW H 4 4 96 .4 03 H z FI D R E S 0. 22 72 98 H z AQ 2 .1 99 80 84 s e c RG 4 06 .4 DW 11 1. 20 0 u s e c DE 6. 00 u s e c TE 2 98 .2 K D l 1 .0 00 00 00 0 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fl 1H 10 .7 5 u s e c 0. 00 dB 30 0. 13 19 50 8 M Hz P ro c e ss in g p ar am et er s 32 76 8 30 0. 13 00 06 9 M Hz EM 0 0. 50 H z 0 1. 00 z C / / NU C 1 P1 PL 1 SF 01 F2 - SI SF W DW SS B LB GB PC ± I I 12 11 10 9 8 7 0 0 H LO C\ j C C H C j c’ i m I ’ I 6 5 4 3 2 1 pp m r’ J 00 C u rr en t D at a P ar am et er s BA NE a 1i 11 71 5 EX PN O 1 PR OC NO 1 F2 — A cq u is it io n P ar am et er s D at e_ 20 08 04 17 T im e 6. 29 IN ST RU M a v 60 0c p PR OB IID 5 m m C PT C I 111 — PU LP RO G c a r b o n sp in ec h o sp TD 65 53 6 SO LV EN T DM SO NS 68 OS 4 SW N 37 59 3. 98 4 Hz FI D R ES 0. 57 36 39 H z AQ 0. 87 16 92 1 s e c RG 26 00 8 DW 13 .3 00 u s e c DE 33 .2 5 u s e c TE 29 8. 0 K D l 1. 00 00 00 00 s e c d li 0. 03 00 00 00 s e c 02 0 0. 00 00 00 00 s e c d2 1 0. 00 00 15 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fi NU C1 13 C P1 15 .0 0 u s e c 98 20 00 .0 0 u s e c PL 1 — 1. 90 dB SF 01 15 0. 92 28 38 0 M Hz SP 13 2. 74 dB SP N PN 13 C rp 60 co m p. 4 SP O FF 13 0. 00 H z CH A N N EL f2 CP DP RG 2 w a lt zl 6 NU C2 iN PC PD 2 10 0. 00 u s e c PL 2 3. 50 dB PL 12 24 .2 2 dB PL 13 12 0. 00 dB SF 02 60 0. 15 30 00 0 M Hz F2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 08 5 M Hz WO W EM SS B 0 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ LB 2. 00 Hz GB 80 60 40 20 0 pp m PC (N (N 0 — 4 (0 0 ’ r— 0 (0 4 0 40 (‘4 40 0 ’ 0 ’ C’ ) 40 C’ ) C’ ) 0 ) (0 (0 (0 40 r— C’ ) (5 ) 0 ) — 4 C’ - C’ ) 0 ) (N 0 4 0 ) (0 0 ) 0 (0 , - 4 (0 (0 (C ) C’ ) - 4 (0 CC C (4 ) H . (5 ) (0 (0 (0 C’ ) C’ ) C’ ) (N (N - 4 0 (N (N (3 )0 )0 ) (3 )0 )0 ) 0) 0 0 )0 4 C’ ) H , - 1 H , - 4 - 4 - 4 - 4 (0 (0 1 (H (H 4- 1 04 (4 ) () (4 ) C’ ) C’ ) CV ) C’ ) C’ ) C’ ) - 4 (0 (0 z C I I I I I 22 0 20 0 18 0 16 0 14 0 12 0 10 0 r’ rw r r i ’ r 0 1. 40 F’- , ‘ . 0 B R U K E R 0 C u rr en t D at a P ar am et er s NA M E x a G C q u rt et b u ty l EX PN O 1 PR OC NO 1 Cl) F2 — A c q u is it io n P ar am et er s D at e_ 20 08 03 25 T im e 16 .4 1 IN ST RU M s p ec t z i PR O BE D 5 m m QN P 1H /1 PU LP RO G z g3 O TD 19 78 2 // N S 12 8 SW H 44 96 .4 03 H z DS 2 SO LV EN T DM SO FI D R ES 0. 22 72 98 H z AQ 2. 19 98 08 4 s e c RG 81 2. 7 , OW 11 1. 20 0 u s e c DE 6. 00 u s e c TE 29 8. 2 K D l 1. 00 00 00 00 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L fi N U C1 1H P1 10 .7 5 u s e c PL 1 0. 00 dB SF 01 30 0. 13 19 50 8 M Hz L F2 — P ro ce ss in g p ar am et er s SI 32 76 8 SF 30 0. 13 00 06 9 M Hz W DW EM SS B 0 LB 0. 50 H z GB 0 PC 1. 00 I 11 10 9 8 7 6 5 4 3 2 1 pp m H rH oH o o II o I a l 10 1 10 1 Io II cj H jI Ir Jr (\J (‘41 ( ‘ 4H C ’ 4l l C C u rr en t D at a P ar am et er s NA M E a 1i 11 73 9 EX PN O 1 PR OC NO 1 P2 - A cq u is it io n P ar am et er s D at e 20 08 05 07 T im e 2. 51 IN ST R O M a v 60 0c p PR OB HD 5 m m C PT C I iN PI JL PR O G c a r b o n _ sp in ec h o sp TD 65 53 6 SO LV EN T D M 50 NS 61 44 DS 4 SW H 37 59 3. 98 4 H z FI D R ES 0. 57 36 39 H z AQ 0. 87 16 92 1 s e c RG 13 31 2 DW 13 .3 00 u s e c 05 30 .0 0 u s e c TE 29 8. 0 K D l 1. 00 00 00 00 s e c dl ]. 0. 03 00 00 00 s e c D 20 0. 00 00 00 00 s e c d2 1 0. 00 00 15 00 s e c D EL TA 0. 89 99 99 98 s e c M CR ES T 0. 00 00 00 00 s e c M CW RK 0. 01 50 00 00 s e c CH AN NE L 61 NU C1 13 C 81 . 15 .0 0 u s e c 88 20 00 .0 0 05 cc PL S — 1. 90 dB SF 01 15 0. 92 30 11 5 M Hz SF 13 2. 74 dB 5P N5 51 13 C rp 6S co m p. 4 SP O FF 13 0. 00 H z CH AN NE L 62 CP D PR G 2 w a lt zl 6 NU C2 11 1 FC PD 2 10 0. 00 u s e c PL 2 3. 50 dB PL S2 24 .2 2 dB PL 13 12 0. 00 dB SF 02 60 0. 15 30 00 0 M Hz F2 — P ro ce ss in g p ar am et er s SI 13 10 72 SF 15 0. 90 79 09 4 M H z HD H EM SO B 0 LB 2. 00 H z GB 0 PC 1. 40 CO C - a a C Cl) S am pl e N o :1 1 7 39 A ll A sa d i GC Q ua rt et / ZD a 1 i1 1 7 3 9 1 1 13 C SE / D M SO — d6 U BC B ru k er 60 0M H z T C I o re 1a e T = 29 8K 7 M ay 20 08 CO C - CO U -, 07 i. ijk Ii. s iu i Ii I, :. , l,I iII .i, i d0I L h II i 0 ii L / f ‘ 1 (‘.3 (0 07 - 3 N a z i (‘4 (0 a a , — CO (‘1 r’ c a ‘. o c l CO a a a a a a a a a a CO C’ ) C’ ) 07 (‘1 (0 1’ ) (0 (0 - 3 / ) ,) ii ik L I L j,[ .sI i1 L i’ ,’ i’ ’r IIl rq çI 31 vI j ,I p’ i II ),F II Pr 22 0 20 0 18 0 16 0 14 0 12 0 10 0 80 inr i 60 40 20 0 pp m 0.6 0,4 0.2 II II II U I k ) U I (- i,) 4 U I 00 e- o o ‘ • 1 - i o o I I - - N N ri m D U C - L 1 r J 1.2 1 0.8 N O O r Q L ó t.n N N Un fl L (3 L fl rn m I - lu , m c Un r.. J w 1’ .)

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