Citation
From Sugars to Nucleic Acids: Synthesis of an N-Linked Inositol Dimer and Advances toward a Fully Functional Expanded Genetic Information System

Material Information

Title:
From Sugars to Nucleic Acids: Synthesis of an N-Linked Inositol Dimer and Advances toward a Fully Functional Expanded Genetic Information System
Creator:
MARTINOT, THEODORE A. ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Acetates ( jstor )
Dimers ( jstor )
DNA ( jstor )
Epoxy compounds ( jstor )
Indoles ( jstor )
Inositols ( jstor )
Nucleosides ( jstor )
Reagents ( jstor )
Solvents ( jstor )
Tetrahedrons ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Theodore A. Martinot. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2009
Resource Identifier:
439074716 ( OCLC )

Downloads

This item is only available as the following downloads:


Full Text

PAGE 1

FROM SUGARS TO NUCLEIC ACIDS: SYNTHESIS OF AN N -LINKED INOSITOL DIMER AND ADVANCES TOWARD A FULLY FUNCTIONAL EXPANDED GENETIC INFORMATION SYSTEM By THEODORE A. MARTINOT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Theodore A. Martinot

PAGE 3

“What have you lost, Mulla?” “My key,” said Nasrudin. “Where did you drop it?” “At home.” “Then why, for heaven’s sake, are you looking for it here?” “There is more light here.” a Sufi Parable This work is dedicated to my family and friends.

PAGE 4

iv ACKNOWLEDGMENTS I would like to begin by thanking Profs. Tomas Hudlicky and Steven Benner for giving me the opportunity to do re search in their lab. I am particularly indebted to Prof. Hudlicky because of the many things I learne d from him and his group; the teaching I received from the time I was an undergraduate has allowed me to grow into the chemist that I am today. For serving on my committee, I would like to thank Profs. Merle Battiste, William Dolbier, Jon Stewart, and Linda Bloom. Pr of. Battiste has been a positive influence for me ever since I took his undergraduate or ganic class many years ago, and I am very thankful to have him see me through this point. Dr. David Gon zalez, my undergraduate mentor in the Hudlicky research group, ha s taught me much of the chemistry and techniques that I use today. Without his help and advice through the years of working with him, I certainly would not be here now . Prof. Dennis Wright, because of the close relationship his group and Prof . Hudlicky’s had, has also helped solve many problems along the way, as well as offer useful advice throughout my stay here. I would like to thank Lori Clark and Prof. James Deyrup for their counsel and advice when I needed it most. Also, Dr. Ion Ghiviriga has been inst rumental in the proper NMR assignment of many of the compounds synthesi zed in this dissertation. I would like to thank Bernhard Paul and Jerremey Willis, both of whom worked alongside me on the aminoinositol dimer project . Vu Bui and Yngve Stenstrøm, because they have both provided the group with the chir al starting material (diol) and taught me

PAGE 5

v how to perform the biotransformation, were a great resource in the group. Likewise, Daniel Hutter, Harch Ooi, Cynthia Hendric kson, and Alonso Ricardo have dedicated innumerable hours of their time to either he lping me solve problems or simply explaining concepts of nucleic acid chemistry to me. Nick Pasteur and Sonia Reback, both talented undergraduate assistants, have provided a great deal of help in the synthesis of nucleic acid precursors described in this dissertation. For their friendship and advice throughout the years, I would like to acknowledge David Gonzalez, Ben Novak, Dean Frey, Luke Koroniak, Bernhard Paul, Josef Zezula, Uwe Rinner, Heshan Illangkoon, Alonso Ricard o, Mike Sismour, Matt Carrigan, Harch Ooi, and the rest of the members of the H udlicky and Benner groups, as well as my many colleagues throughout the years. Without Ro maine Hughes, who in addition to keeping everything organized for the boss (from taxes to treats), has been a great source of laughter, I doubt that I would have ma de it through in only five years. I would like to thank Alons o Ricardo, Frances Chang, A. J. Kamath, and Heshan Illangkoon for help in editing this document. Last, but certainly not least, I would like to thank my family. In spite of not necessarily understanding the t opics of my dissertation, they have always been the ones who expressed the most interest in my da y-to-day work and in the progress of my research. My wife, Mandy, has brought me joy beyond words, and I am exceptionally fortunate to have her by my side.

PAGE 6

vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii LIST OF SCHEMES.........................................................................................................xii LIST OF ABBREVIATIONS.........................................................................................xvii ABSTRACT....................................................................................................................xxi i CHAPTER 1 INTRODUCTION........................................................................................................1 2 INOSITOL DERIVATIVES........................................................................................3 Background...................................................................................................................3 The Inositols..........................................................................................................3 Cis -inositol (1)................................................................................................4 Epi -inositol (2)...............................................................................................6 Allo -inositol (3)..............................................................................................7 Myo -inositol (4)............................................................................................10 Muco -inositol (5)..........................................................................................17 Neo -inositol (6)............................................................................................19 Scyllo -inositol (7).........................................................................................22 LChiro -inositol (8) and DChiro -inositol (9).............................................24 Nonspecific or branched syntheses of inositols...........................................29 Inositol Oligomers and Related Compounds.......................................................37 Introduction..................................................................................................37 Synthesis and molecular properties..............................................................38 Glycosidase Inhibition.........................................................................................46 Diaminoinositols..................................................................................................49 Inositol Signaling Pathways................................................................................50 Discussion...................................................................................................................52 Synthesis..............................................................................................................53 Glycosidase Inhibition.........................................................................................62 Calcium-binding affinity.....................................................................................63

PAGE 7

vii 3 7-DEAZA-ISOGUANINE AND DERIVATIVES....................................................66 Background.................................................................................................................66 iso Guanine and iso Guanosine.............................................................................67 Natural occurrence and synthesis.................................................................68 Tautomerism.................................................................................................75 2-Hydroxyadenine and DNA damage..........................................................81 Base-pairing of 2-hydroxyadenine and duplex structure.............................87 Why is iso guanine not part of the genetic code?..........................................92 Applications in biotechnology.....................................................................93 7-Deazaiso Guanine and 7-Deazaiso Guanosine................................................94 Synthesis.......................................................................................................96 Behavior.......................................................................................................99 Synthesis of Indoles...........................................................................................100 The Bartoli indole synthesis (1989)...........................................................100 The Bischler-Möhlau indole synthesis (1881)...........................................101 The Borsche-Drechsel cyclization (1858)..................................................102 The Fischer indole synthesis (1883)...........................................................102 The Gassman indole synthesis (1974)........................................................103 The Hegedus indole synthesis (1976)........................................................104 The Larock indole synthesis (1991)...........................................................106 The Madelung indole synthesis (1912)......................................................107 The Nenitzescu indole synthesis (1929).....................................................109 Discussion.................................................................................................................109 Tautomerism of 2’-deoxy-7-deazaiso Guanosine.............................................110 Synthesis.....................................................................................................110 pKa measurements......................................................................................114 Tautomerism measurements.......................................................................116 Synthesis of Derivatives....................................................................................121 Synthesis of a versatile precursor...............................................................123 Purine synthesis..........................................................................................124 Synthesis of an internal alkyne fo r the generation of functionalized 7-deazaiso guanine...............................................................................125 Heteroannulation and optimization............................................................125 4 CONCLUSIONS......................................................................................................128 APPENDIX EXPERIMENTAL SECTION.........................................................................................129 LIST OF REFERENCES.................................................................................................162 BIOGRAPHICAL SKETCH...........................................................................................192

PAGE 8

viii LIST OF FIGURES Figure page 1. The nine possible inositols a nd their respective IUPAC names.................................3 2. Hexaric acids............................................................................................................13 3. Land Dchiro -inositols and their correspondi ng methylated derivatives, quebrachitol and pinitol............................................................................................24 4. Some naturally-occurring glycopyranosyl cyclitols and possible insulin mimics and modulators.........................................................................................................37 5. Potential glycosidase inhibitors................................................................................38 6. Thermal ellipsoids drawing of the ex tended secondary helical structure of 184 shown (a) along the b -axis and (b) down the b -axis................................................40 7. Molecular modeling structures of (a) 181 , (b) 195 , (c) 196 , (d) an Lchiro inositol octamer, and (e) an L-proline octamer........................................................43 8. X-ray diffraction of naphthalene dimer 208 . (a) Three dimensional structure showing both hydrophilic and lipophilic regi ons. (b) Molecular drawing shown with 50% ellipsoids..................................................................................................45 9. Catalytic mechanism for configuration-retaining glycosidases according to Koshland182...............................................................................................................47 10. Classic inhibitors of glycosidases............................................................................48 11. Unnatural diaminoinositol derivatives.....................................................................49 12. The key step of the inositol signaling pathway........................................................50 13. The synthesis and role of PIP2 in intracellular signaling.........................................51 14. A general structure of phorbol esters.......................................................................52 15. A previously synthesized aminoinositol dimer ( 184 ) and three related targets.......53 16. Molecular modeling of dimers (a) 184 , (b) 227 , (c) 228 , and (d) 229 .....................53

PAGE 9

ix 17. The general structure of epoxy resins.......................................................................54 18. Generating high molecular weight poly mers using epoxides and aziridines...........54 19. Retrosynthetic analysis of dimer 229 .......................................................................57 20. Normalized inhibition of commerc ially-available glycosidases by 229 ..................62 21. Partial 1H NMR assignment of dimer 229 ...............................................................63 22. Calcium interaction study of dimer 184 ...................................................................64 23. Calcium interaction study of dimer 229 ...................................................................65 24. Graphical representation of the chemi cal shift changes associated with the binding of dimer 229 to calcium..............................................................................65 25. The twelve possible Watson-Crick type base-pairing interact ions containing three H-bonds...........................................................................................................67 26. Naturally-occurring iso guanine derivatives.............................................................68 27. The tautomerism of iso guanosine, as determined by Sepiol et al. ...........................76 28. Nucleosides synthesized by Seela et al. to investigate the tautomerism of 2’deoxyiso guanosine ( 291 ) and their respective pKa values......................................78 29. Crystal structure of the comple x of Hoechst 33342 and isoG-DODE.....................79 30. Possible configurations of the isoG:T base pair with varying tautomeric forms of isoG. The chemical structure of Hoechst 33342.....................................................80 31. The standard A:T Watson-Crick base pair and the two possible isoG:T and isoG:isoCM base pairs.............................................................................................83 32. The structure of tirapazamine...................................................................................86 33. Two possible Watson-Crick ge ometries are possible for iso guanine.......................89 34. Schematic representation of antiparalle land parallel-oriented Watson-Crick base pairs..................................................................................................................91 35. DNA quartets (left) and qui ntets (right), as formed by isoG, are anomalous structures that may affect the beha vior of the oligomer as a whole.........................92 36. The bDNA assay, such as the Versant® HIV-1 RNA assay (marketed by Bayer), benefits from the use of AEGIS to produ ce a five-fold increase in S/N over the assay without AEGIS...............................................................................................94

PAGE 10

x 37. The two iso guanine tautomers each have probl ems not found in the native guanine heterocycle ( 291 )........................................................................................94 38. Aromatic and heteroaromatic compounds and their relative aromaticity (% aromaticity based on benzene) as well as possibly less problematic derivatives of iso guanine............................................................................................................95 39. Perspective view of 7-deaza-2'-deoxy iso guanosine showing the atomic numbering scheme. Displacement ellipso ids of non-H atoms are drawn at 50% probability. H atoms are shown as spheres of an arbitrary size..............................99 40. Four possible tautomeric forms of 2'-deoxy-7-deazaiso guanosine and the fixed derivatives used to study the ta utomerism of the free nucleoside..........................110 41. Multiwavelength analysis of dC7isoG at varying pH (box)..................................115 42. pKa determination of dC7isoG...............................................................................115 43. UV spectra of compounds 311 , 310 , and 383 in water..........................................116 44. The linear regressions obtained with high R2 values indicate that the only species of dC7isoG present in solution are the N1-H keto and the enol forms......118 45. UV curves of dC7isoG with varying wa ter and dioxane concentrations (legend refers to % (v/v) water)..........................................................................................119 46. UV curves of disoG with varying wate r and dioxane concentrations (legend refers to % (v/v) water)..........................................................................................119 47. Comparison of the tautomerism of 2'-deoxyiso guanosine and 2'-deoxy-7-deaza iso guanosine. (A¹ = Absorption at ¹; A² = Absorption at ²)..............................120 48. Plot of log([ enol ]/[ keto ]) versus ET(30) for dC7isoG ( 311 ) and disoG ( 291 ) in mixtures of dioxane (ET(30) = 36.0) and water (ET(30) = 63.1)............................121 49. HPLC of 4-Amino-3-hydro-7-(2-d eoxy-ß-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidin-2-one ( 311 ).........................................................................................158 50. HPLC of 2-Methoxy-7-(3,5-diO -toluoyl-2-deoxy-D-ribofuranosyl)-pyrrolo [2,3-d]pyrimidin-4-ylamine ( 384 )..........................................................................159 51. HPLC of 2-Methoxy-7-(2-deoxy-D-ribofuranosyl)-pyrro lo[2,3-d]pyrimidin-4 -ylamine ( 310 )........................................................................................................159 52. HPLC of 4-Amino-3-hydro-7-(3,5-diO -toluoyl-2-deoxy-ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one ( 385 ).....................................................................160 53. HPLC of 4-( N , N -Diisobutyl-formamidine )-3-hydro-7-(3,5-diO -toluoyl-2deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 386, top isomer )......160

PAGE 11

xi 54. HPLC of 4-( N , N -Diisobutyl-formamidine )-3-hydro-7-(3,5-diO -toluoyl-2-deoxy -ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 386, bottom isomer )..........161 55. HPLC of 4-Amino-3-methyl-7-(2-d eoxy-ß-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidin-2-one ( 383 ).........................................................................................161

PAGE 12

xii LIST OF SCHEMES Scheme page 1. Angyal and Hickman’s synthesis of cis -inositol........................................................5 2. Chung and Kwon’s synthesis of cis -inositol..............................................................6 3. Posternak’s synthesis of epi -inositol..........................................................................6 4. Synthesis of epi -inositol by Pistarà et al....................................................................7 5. Dangschat and Fisher’s synthesis of allo -inositol......................................................7 6. Angyal and Gilham’synthesis of allo -inositol...........................................................8 7. Hudlicky’s synthesis of allo -inositol..........................................................................9 8. Mehta and Lakshminath’s synthesis of allo -inositol..................................................9 9. Maquenne’s proof for the structure of inositols.......................................................11 10. Dangschat’s proof for the structure of myo -inositol.................................................15 11. Posternak’s synthesis of myo -inositol......................................................................15 12. Chiara and Martìn-Lomas’s synthesis of myo -inositol............................................16 13. Danschat and Fisher’s synthesis of muco -inositol...................................................17 14. Angyal’s synthesis of muco -inositol........................................................................17 15. Synthesis of muco -inositol by Suami et al. ..............................................................18 16. Angyal and Odier’s synthesis of muco -inositol.......................................................19 17. Angyal and Matheson’s synthesis of neo -inositol....................................................20 18. Synthesis of neo -inositol by Riley et al. ..................................................................21 19. Synthesis of neo -inositol by Hudlicky et al. ............................................................21 20. Kohne and Praefcke’s synthesis of scyllo -inositol...................................................22

PAGE 13

xiii 21. Sarmah and Shashidhar’s synthesis of scyllo -inositol..............................................23 22. Chiara and Valle’s synthesis of Lchiro -inositol hexaacetate..................................25 23. Synthesis of Dchiro -inositol by Kim et al. ..............................................................26 24. Synthesis of Dchiro -inositol by Takahashi et al. ....................................................27 25. Synthesis of Dchiro -inositol by Catelani et al. .......................................................28 26. Synthesis of inositols by Nakajima et al. .................................................................29 27. Kowarski and Sarel’s synthesis of myo -, allo -, neo -, and epi -inositols...................30 28. Mandel and Hudlicky’s approach to Dchiro -, neo -, and allo -inositols...................31 29. Synthesis of muco, (±)chiro -, allo, and epi -inositols by Carless et al. ................32 30. Brammer and Hudlicky’s synthesis of Lchiro and muco -inositols........................33 31. Chung and Kwon’s synthesis of inositols................................................................34 32. Approach to inositols by Takahashi et al. ................................................................35 33. Synthesis of neo and Dchiro -inositols by Heo et al. ..............................................36 34. Hudlicky and Thorpe's synthesis of a Lchiro -inositolgala -quercitol conjugate....39 35. Synthesis of aminoinositol dimer 184 by Hudlicky et al. ........................................40 36. Synthesis of a conduritol-aminoinositol conjugate by Hudlicky et al. ....................41 37. Synthesis of aminoinositol dimer 191 by Hudlicky et al. ........................................41 38. Synthesis of Lchiro -inositol trimer and tetramers by Hudlicky et al. .....................42 39. Synthesis of an O -linked pseudosugar conjugate by Hudlicky et al. .......................44 40. Synthesis of a tetrahydrona phthalene dimer by Desjardins et al. ............................45 41. Synthesis of an amino-bridged te trahydronaphthalene dimer by Lallemand et al. ..46 42. Synthesis of chiral epoxides and aziridines..............................................................55 43. Model study for aziridine openings..........................................................................56 44. Aziridine opening with ammonia.............................................................................57 45. Synthesis of dimer 244 .............................................................................................58

PAGE 14

xiv 46. Protection and debromination of dimer 244 .............................................................59 47. Dihydroxylation and protection of dimer 247 ..........................................................60 48. Final deprotections to afford dimer 229 as its hydrochloride..................................61 49. Overview of the synthesis of dimer 229 ...................................................................61 50. The conversion of iso guanine to xanthine................................................................68 51. Synthesis of iso guanine by Andrews et al. ..............................................................69 52. Shaw's synthesis of iso guanine................................................................................70 53. Synthesis of iso guanine by Taylor et al. ..................................................................71 54. Synthesis of iso guanine by Yamazaki et al. .............................................................71 55. Synthesis of iso guanosine by Yamazaki et al. .........................................................72 56. Synthesis of iso guanine by Leonard et al. ................................................................72 57. Second synthesis of an iso Guanosine derivative by Yamazaki et al. ......................73 58. The different effects of vari ed base strength on imidazole 276 ...............................74 59. Oxidation of adenine to iso guanine by Pratt and Kraus...........................................75 60. Synthesis of iso guanine by Itaya et al. .....................................................................75 61. The oxidation of adenine with hydrogen peroxide...................................................82 62. Synthesis of 7-deazaiso guanine..............................................................................96 63. First synthesis of 2’-deoxy-7-deazaiso guanosine by Seela et al. ...........................97 64. Syntheses of 7-deazaiso guanosine by Seela et al. ..................................................98 65. The Bartoli indole synthesis...................................................................................100 66. Use of the Bartoli methodology by Fonseca et al. for the construction of complex indole structures.......................................................................................101 67. The Bischler-Möhlau indole synthesis...................................................................101 68. Synthesis of an indole derivative by Golob et al. ..................................................101 69. The Borsche-Drechsel cyclization.........................................................................102

PAGE 15

xv 70. Jiricek and Blechert's synthesis of ()-gilbertine using a Borsche-Drechsel cyclization..............................................................................................................102 71. The Fischer indole synthesis..................................................................................103 72. Synthesis of 3-substitute d indoles toward the genera tion of fused polycycles by Campo et al. ...........................................................................................................103 73. The Gassman indole synthesis...............................................................................104 74. Application of the Gassman indole synthe sis toward the tota l synthesis of (+)paspalicine and (+)-paspalinine by Smith et al. .....................................................104 75. The Hegedus indole synthesis................................................................................105 76. Pd(II)-mediated synthesis of an indole derivative from an alkynyl aniline...........105 77. Cu(I)-mediated synthesis of an indole derivative from an alkynyl aniline............105 78. TBAF-mediated synthesis of an indole derivative from an alkynyl aniline..........106 79. Base-mediated synthesis of an indol e derivative from an alkynyl aniline.............106 80. The Larock indole synthesis...................................................................................107 81. Use of the Larock heteroannulation in their synthesis of tryptophan analogues by Ma et al. .............................................................................................................107 82. The Madelung indole synthesis..............................................................................108 83. Use of a modified Madelung methodology to ward the synthesis of (-)-penitrem D by Smith et al. ....................................................................................................108 84. The Nenitzescu indole synthesis............................................................................109 85. The synthesis of an indomethacin analogue for the evaluation as MRP modulators using the Nenitzescu indole synthesis.................................................109 86. Improved synthesis of 309 .....................................................................................111 87. Synthesis of the nucleosides of C7isoG and the O2-methyl-C7isoG.....................112 88. Synthesis of 2'-deoxy-N1-methyl-7-deazaiso guanosine.......................................113 89. Synthesis of dC7isoG and derivatives....................................................................114 90. The iodination of 307 and 384 gives only the diiodinated derivatives..................122 91. Retrosynthetic analysis of 7-deazaiso guanine derivatives....................................123

PAGE 16

xvi 92. Synthesis of pyrimidines 392 and 394 ...................................................................124 93. Heteroannulation of pyrimidine 394 and 1-phenyl-2-trimethylsilylacetylene.......125 94. Synthesis of enyne 399 ...........................................................................................125 95. Heteroannulation of pyrimidine 394 and enyne 399 and assignments of the proton and carbon NMR spectra............................................................................126 96. Attempts to react BTMSA and TMS-acetylene with pyrimidine 394 ...................127

PAGE 17

xvii LIST OF ABBREVIATIONS °C degrees Celsius A adenine or adenosine Ac2O acetic anhydride AEGIS Artificially Expanded Genetic Information System AIBN 2,2’-azobisisobutyronitrile AICA 4-amino-5-imidazolecarboxamide AMP adenosine monophosphate AMV RT avian myeloblastosis virus reverse transcriptase aq. aqueous ATP adenosine triphosphate BDA butane-2,3-diacetal Bn benzyl BnBr benzyl bromide BTMSA bis(trimethylsilyl)acetylene Bz benzoyl BzCl benzoyl chloride C cytosine or cytidine C7isoG 7-deazaiso guanine CBM carboxymethyl CDP-DG diacylglycerol cytidine diphosphate

PAGE 18

xviii CH3CN acetonitrile CSA (±)-10-camphorsulphonic acid dA 2’-deoxyadenosine dAMP 2’-deoxyadenosine monophosphate dATP 2’-deoxyadenosine triphosphate DBH 1,3-dibromo-5,5dimethylhydantoin DBU 1,8-diazabicyc lo[5.4.0]undec-7-ene dC 2’-deoxycytidine dC7isoG 2’-deoxy-7-deazaiso guanosine DEP diethyl pyrocarbonate dG 2’-deoxyguanosine DG diacylglycerol disoG 2’-deoxy iso guanosine DMA dimethylacetamide DMAP N , N -dimethyl-4-aminopyridine DMF N , N -dimethylformamide DMP 2,2-dimethoxypropane DNA deoxyribonucleic acid dNTP 2’-deoxynucleotide triphosphate EDTA ethylenediaminetetraacetate ER endoplasmic reticulum EtOH ethanol Fe-NTA ferric nitrilotriacetate

PAGE 19

xix G guanine or guanosine h hour(s) HIV human immunodeficiency virus HOAc acetic acid iG-DODE iG-containing DNA dode camer d(CGC[iG]AATTTGCG) IP3 inositol 1,4,5-trisphosphate Ipc isopinocamphenyl isoCM 5-methylisocytosine isoG iso guanine or iso guanosine KOBz potassium benzoate L liter M molar m CPBA meta -chloroperbenzoic acid Me methyl MeOH methanol mg milligram min. minute(s) mL milliliter NaOMe sodium methoxide NIS N -iodosuccinimide NMMO N -methylmorpholineN -oxide NMP N -methylpyrrolidinone N -PSP N -(phenylseleno)phthalimide

PAGE 20

xx NsCl p -nitrobenzenesulphonyl chloride PCR polymerase chain reaction Ph phenyl PI phosphatidylinositol PIP2 phosphatidylinositol 4,5-bisphosphate PKC protein kinase C PLC phospholipase C ppm parts per million PS phosphatidylserine p TsCl p -toluenesulfonyl chloride p TsOH p -toluenesulfonic acid pu purine Py pyridine py pyrimidine RNA ribonucleic acid RT room temperature SARS severe acute respiratory syndrome T thymine or thymidine TBAF tetran -butylammonium fluoride TBDPSCl t -butyldiphenylsilyl chloride Tf2O trifluromethanesulfonic anhydride TFA trifluoroacetic acid TFAA trifluoroacetic anhydride

PAGE 21

xxi THF tetrahydrofuran TMS trimethylsilyl or tetramethylsilane Tol p -toluoyl TPAP Tetran -propyl ammonium perruthenate Ts p -toluenesulfonate TTMSS tris(trimethylsilyl)silane U uracil or uridine UV ultraviolet VIS visible µL microliter µM micromolar

PAGE 22

xxii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FROM SUGARS TO NUCLEIC ACIDS: SYNTHESIS OF AN N -LINKED INOSITOL DIMER AND ADVANCES TOWARD A FULLY FUNCTIONAL EXPANDED GENETIC INFORMATION SYSTEM By Theodore A. Martinot August 2004 Chair: Steven A. Benner Major Department: Chemistry This dissertation deals with the synthe sis of biologically-important products. Though none of the structures s ynthesized are naturally-occu rring, their importance either as potential drugs or as bi otechnology tools is discusse d. In the first section, a diaminoinositol dimer is synthesized as a poten tial glycosidase inhibitor. The synthesis of the novel carbohydrate derivative is describe d, as well as the enzyme inhibition and metal-chelating studies. In the seco nd section, the problem with the use of iso guanosine in biotechnology is explained, a nd a solution is provided. The synthesis of derivatives of 7-deaza-isoguanine, as well as the tautomer ism studies on the nucleoside, are described. The research performed for this dissertation integrated the skills of synthetic organic chemistry with physical organic and biochemistry to provide advances in all of these fields.

PAGE 23

1 CHAPTER 1 INTRODUCTION The purpose of a scientific dissertation is to purvey adequately to the reader that the experiments performed and the conclusions draw n are valid. This work was performed at the University of Florida unde r the direction of Professors Tomas Hudlicky and Steven Benner. After many years of graduate study, it becomes challenging to characterize correctly the dissertation as a tool to disseminate information rather than as a means to obtain a degree. Nevertheless, it is my intention to desc ribe unambiguously the work performed during my stay here. As has already been alluded to, this docum ent contains two parts in its main body. The first part describes work performed in the Hudlicky laboratory, whereas the second part describes work performed in the Benner laboratory. The research performed in the Hudlicky gr oup was concerned with the synthesis of inositol derivatives, in particular ami noinositol dimers. The synthesis of these compounds will be described in detail, and th eir intrinsic molecular properties such as biological activity and metal-binding affinity will be discussed. The research performed in the Benner group involved the general concept of expanding the genetic alphabet. The problem at hand was two part: First, the tautomeric ambiguity of iso Guanine (and iso Guanosine) had to be resolved in order for that base to be used practically in PCR as well as in vitro evolution studies. S econd, it was important to devise the synthesis of a nucleobase th at could be “picked out” of solution through

PAGE 24

2 affinity chromatography. Both of these issu es were, to some extent, resolved, and the experimental details will be highlighted. These two projects, though very different on the surface, are intrinsically very similar in that they try to br idge the gap between chemistry and biology; that is, synthetic targets were used to investigate biological pr oblems. Furthermore, rather than having only performed the synthesis of these targets, much of the biology was also studied in the same laboratories. So as many of todayÂ’ s questions become interdisciplinary, the scientists must learn either to work t ogether or to perform additional skills.

PAGE 25

3 CHAPTER 2 INOSITOL DERIVATIVES Background The Inositols The inositols (or hexahydrocyclohexanes, cyclitols) are a class of organic molecules that have interested chemists and biologists for many years. Though initially spurred because cyclitols are isomeric with su gars, the interest of scientists soon focused on the physical properties of inositols as well as their st ereochemical relationships. OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH cis -Inositol 1 epi -Inositol 2 allo -Inositol 3 neo -Inositol 6 myo -Inositol ( meso -Inositol) 4 muco -Inositol 5 scyllo -Inositol (Scyllitol) 7Lchiro -Inositol ( levo -Inositol) 8Dchiro -Inositol ( dextro -Inositol) 9 Figure 1. The nine possible i nositols and their respective IUPAC names. In parentheses are the older names.

PAGE 26

4 With six potentially asymmetric cente rs, the maximum possible number of structures for hexahydrocyclohexane is 26, or 64. However, a closer look at the resulting structures and their relative axes and planes of symmetry indicates th at there are actually only nine inositol isomers, two of which are mirror images.1 Structures of the nine inositols ( 1 9 ) are provided in Figure 1. The following nine sections will focus on some of the hist orical aspects of inositols, with an emphasis on their total synthe ses. They are by no means intended to be comprehensive. Rather, because of the la rge number of publications on the subject,2-5 these sections are used to illustrate the comp lexity associated with what would otherwise be a seemingly innocuous carbohydrate analog. Furthermore, these sections will be limited to inositols, as many articles and manuscripts have been published on the synthesis of inositol derivativ es, including inositol phosphates6-8 and conduritols;9 the reader is referred to these and othe r publications for further information. Cis -inositol (1) In addition to the challenges a ssociated with its synthesis, cis -inositol has attracted considerable interest because of its particular affinity to borate10, 11 and other ions.12, 13 With its six hydroxyl groups bearing a cis relationship, the two equivalent chair conformers each have three syn -axial hydroxyl groups. The first practical synthesis of cis -inositol was published by Angyal and Hickman in 197114 (other publications15, 16 afforded cis -inositol as a mixture of other cyclitols, and presented mixtures that were diff icult to analyze), starting from epi -inositol ( 2 ). Condensation of epi -inositol with cyclohexanone gave the triO -cyclohexylidene derivative of epi -inositol, which was then hydrolyzed to the diacetal ( 10 ) in good yield. Partial benzoylation of acetal 10 gave predominantly the 5-be nzoate. The authors justify

PAGE 27

5 the selectivity of the reaction to the assistance of the acetal oxygens in the delivery of the benzoyl group to the intramolecularly hydrogen-bonded hydroxyl group.17, 18 Then, oxidation of the remaining hydroxyl afforded ketone 11 , which was reduced to 12 , and finally deprotected to give cis -inositol ( 1 ). OH OH OH OH HO HO 2 Reagents and Conditions: a. cyclohexanone, benzene, p TsOH b. light petroleum, benzene, p TsOH c. BzCl, Py d. DMSO, Ac2O, benzene, 80 ° C e. NaBH4, MeOH f. NaOMe, MeOH g. HOAc, H2O O O O O OH OH a, b 10 O O O O O OBz c, d 11 O O O O OH OBz e 12 f, g OH OH OH OH HO HO 1 Scheme 1. Angyal and Hickman’s synthesis of cis -inositol14 In 1995, Angyal et al. published a synthesis of cis -inositol, whereby the key elements are a hydrogenation of tetr ahydroxyquinone and an ion-exchange chromatography separation of the products (the authors take advant age of the reported characteristic binding of cis -inositol to cations to isolate the product).19 Though the isolated yield of cis -inositol increased from 4% (in previous syntheses15) to 25-39%, the preparation is not original or interesting in a synthetic viewpoint, and so will not be discussed further. In 1999, Chung and Kwon synthesized the hexabenzoate of cis -inositol20 in five steps from 2O -benzoylmyo -inositol orthoformate ( 13 ).21, 22 The target cyclitol was generated by sequential benzylation, hydr olysis, benzoylation, hydrogenation, and

PAGE 28

6 triflation to obtain triflate 17 in good overall yield. Lastly, the triflates were displaced by benzoate to generate the targ eted hexabenzoate ester of 1 ( 18 ). R O O O OBz R OBz OBz R OBz R BzO OBz OBz OBz OBz BzO BzO 18 R = OH, 13 R = OBn, 14 R = OBn, 15 R = OH, 16 R = OTf, 17 a b, c d e f Reagents and Conditions: a. BnBr, NaH, 4 Ã… mol. sieve, DMF b. p TsOH, MeOH, reflux c. BzCl, Py d. Pd(OH)2/C, MeOH, H2 (50 psi) e. Tf2O, CH2Cl2, Py f. KOBz, DMSO Scheme 2. Chung and KwonÂ’s synthesis of cis -inositol20 Epi -inositol (2) The first synthesis of epi -inositol was published in 1946 by Posternak.23 The author generates the unnatural cyclitol by first oxidizing myo -inositol to two inososes ( 19 and 20 ), a transformation that can be effected ei ther with nitric acid or by biochemical means with Acetobacter suboxydans,24, 25 then reducing the inosos es (Scheme 3). This methodology was further developed in a patented preparation.26 OH OH OH OH HO HO 4 a OH O OH OH HO HO b OH OH OH OH HO HO 2 OH OH OH OH HO O 20 Reagents and Conditions: a. HNO3 or Acetobacter suboxydans b. H2, Pt or Na, Hg 19 Scheme 3. PosternakÂ’s synthesis of epi -inositol23

PAGE 29

7 Likewise, several epi -inositol derivatives were prepared by Gigg and Gigg from the ketone obtained by oxidation of 3,4,5-triO -benzyl-1,2O -isopropylidenemyo -inositol.27 In 1992, the Tschamber group has also synthesized epi -inositol (as well as other cyclitols) by catalytic osmylation of cyclohexadiene diols.28 In 2000, Pistarà et al. synthesized epi -inositol from D-galactose.29 -Dgalactopyranoside ( 21 ) was converted to 22 using procedures described by Barili et al. ,30 which was then treated with DB U to generate, after workup, 2L-2,4-diO -benzyl(2,3,5,6/4)-pentahydroxycyclohexanone ( 23 ) in good yields. Inosose 23 was then sequentially reduced and debenz ylated to give exclusively epi -inositol (Scheme 4). OOMe OH OH HO HO 21 OH H OCH2OBn OH H CHO H BnO 22 a O OBn OH OBn HO HO 23 b, c OH OH OH OH HO HO 2 Reagents and Conditions: a. DBU, toluene b. H2, Raney-Ni or NaBH4c. H2, Pd(C), MeOH Scheme 4. Synthesis of epi -inositol by Pistarà et al.29 Allo -inositol (3) OH OH OH OH Conduritol A 24 Reagents and Conditions: a. Acetone, HCl b. Ac2O, Py c. KMnO4, MgSO4, EtOH, H2O a, b O O OAc OAc 25 c O O OAc OAc HO HO (+ Isomer) 26 d, e OH OH OH OH HO HO 3 d. NaOMe, MeOH e. 50 % HOAc, H2O, 100 °C Scheme 5. Dangschat and Fisher’s synthesis of allo -inositol31, 32

PAGE 30

8 The work of Dangschat and Fisher31, 32 is noteworthy because it effectively determined the configuration of conduritol A (S cheme 5). It was also the first synthesis of allo -inositol (completed in 1939). O O O O NsO NsO OH OH OH OH HO HO 2 f, b 29 c, d OH OH OH OH 30 OH OH OH OH HO HO 3 e O O O O NsO NsO 27 OH OH OH OH HO HO 8 c, d OH OH OH OH 28 e a, b Reagents and Conditions: a. Acetone, ZnCl2, HOAc b. NsCl, Py c. NaI, Acetone, 100 °C d. H+e. AgClO3, OsO4f. Acetone, ZnCl2Conduritol D Conduritol E Scheme 6. Angyal and Gilham’synthesis of allo -inositol33 Another synthesis of allo -inositol was offered by Angyal and Gilham in 1958.33 Though their initial atte mpt was to synthesize cis -inositol, hydroxyla tion of conduritol D ( 30 ), formed by elimination of bisO -isopropylidene 29 ,34 only took place from the less hindered face to yield, after deprotection, allo -inositol 3 . In the same publication, the authors also synthesized 3 from Lchiro -inositol ( 8 ) in the same manner. In 1997, the Hudlicky group published a practical synthesis of allo -inositol on a multigram scale from bromobenzene.35 The synthesis uses three different cis dihydroxylations, the first of which is th e enzymatic biooxidation of bromobenzene ( 31 )36, 37 (whole cell fermentation using E. coli JM109 (pDTG601)) to furnish the

PAGE 31

9 corresponding bromodienediol ( 32 ). After osmylation, the vinyl bromide is removed, and the bond is again dihydroxylated to afford, after deprotection, allo -inositol (Scheme 7). Br Br OR OR R = H, 32 R = C(Me)2, 33 31 HO OH X O O X = Br, 34 X = H, 35 O O O O OH OH OH OH HO HO 3 36 a b c d b e, f Reagents and Conditions: a. E. coli JM109 (pDTG601) b. DMP, p TsOH, acetone c. OsO4, NMMO, acetone, H2O d. Bu3SnH, AIBN, THF, reflux e. RuCl3, NaIO4, CH3CN, H2O, 0 °C f. HCl, EtOH Scheme 7. Hudlicky’s synthesis of allo -inositol35 The latest synthesis of allo -inositol has been published by Mehta and Lakshminath (Scheme 8).38 OMe MeO OH 37 Reagents and Conditions: a. p TsCl, Py, DMAP, CH2Cl2b. OsO4, NMMO, acetone, H2O c. Amberlyst-15, aq. acetone d. NaOMe, MeOH e. LiAlH4, THF, 0 ° C f. NaI, acetone, reflux a-d OOMe O O 38 b-c e, a OTs O O 39 O O g. KO t Bu, t BuOH, reflux h. O3, NaHCO3, CH2Cl2, -78 ° C, Me2S i. NaBH4, MeOH j. MsCl, Et3N, CH2Cl2, -10 ° C k. 5 % HCl, H2O:EtOH (4:1) f-h O O O 40 O O i-j, g O O 41 O O b, k HO HO OH OH OH OH 3 Scheme 8. Mehta and Lakshminath’s synthesis of allo -inositol38

PAGE 32

10 Bicyclic alcohol 37 , which is readily available39-42 from 5,5-dimethoxy-1,2,3,4tetrachlorocyclopentadiene and vinyl acetate, was sequentially tosylated, dihydroxylated with osmium tetroxide, protected, and finally fragmented with s odium methoxide to afford cyclohexene methyl ester 38 .43 Ester 38 is then dihydroxyl ated, protected, reduced, and tosylated to give tosyl ester 39 . After elimination and ozonolysis of the exocyclic olefin, ketone 40 is reduced, mesylated, and elim inated to give cyclohexene derivative 41 , which is again dihydroxylated a nd deprotected to finally afford allo inositol. Though more recent in terms of publication date, Mehta’s synthesis by no means rivals Hudlicky’s (Scheme 7), which is not only amenable to sc aleup, but is also a very short approach to a stereo chemically complex molecule. Myo -inositol (4) Myo -inositol, because of its prevalence in na ture, has been the most investigated of all inositols. This cyclitol has been studied so extensively that it is often simply referred to in the literature as “inositol.” It was first described in 1850 by Scherer, as a meat extract, and given the name inositol.44, 45 However, the general structure of this compound would not be determined un til 1887, with the work of Maquenne.46 Taking inositol, Maquenne treated it with hydr oiodic acid, which generated benzene ( 44 ), phenol ( 45 ), and triiodophenol ( 46 ). The latter was unambiguously characterized by generating the potassium picrate salt ( 47 ). Also, when inositol was treated with concentrated nitric acid, both tetrahydroxyp -benzoquinone ( 43 ) and rhodizonic acid ( 42 ) were generated. Interestingly, these quinones ( 42 and 43 ) produce distinctive colors as alkaline earth salts, a behavior that has long been used as a ge neral qualitative test for the presence of inositols (a test that is often referred to as Scherer’s test ).45, 47 This work in effect

PAGE 33

11 confirmed not only the cyclic nature of inosito l, but also the secondary characteristic of its six alcohol substituents. a Reagents and Conditions: a. HI b. HNO3c. K2CO3OH OH Inositol O O O O O OOH OH HO HO b b, c O NO2O2N NO2K++ + 4243 45 44 4647 HO HO I3 Scheme 9. Maquenne’s proof fo r the structure of inositols46 The first synthesis of natural inositol, or myo -inositol, was produced by Wieland and Wishart in 1914.48 Though little experimental expl anation is provided, the authors simply reduced hexahydrobenzene with a pa lladium catalyst at room temperature and atmospheric pressure to afford the natural produ ct. With this synthesis, the authors then reaffirmed the discoveries of Maquenne, t hough they provide little explanation for the selectivity in the hydrogenation other th an acknowledging that it had a fortunate outcome. It is important to note that severa l attempts were made to reproduce the work of Wieland and Wishart, most to no avail. Neither Stetten and Stetten49 nor Anderson and Wallis16 could generate myo -inositol from hexahydroben zene using the procedures listed. However, using Raney nickel, A nderson and Wallis did manage to reduce hexahydrobenzene to scyllitol, myo -inositol, as well as anot her inositol unknown at the time. Nevertheless, Kuhn et al. ,50 in 1949, did manage to generate a mixture of cyclitols, 13% of which was myo -inositol, from hydrogenation (at 20 °C and 55 °C) of both

PAGE 34

12 hexahydrobenzene and tetrahydrobenzoquinone (the latter of which is more stable and readily available, and which is reduced in situ to hexahydrobenzene). The most difficult aspect in the characterization of myo -inositol was the assignment of the relative geometry of each of the al cohols around the ring. Despite having made the discovery of the configuration of myo -inositol second, the work of Posternak, because of his greater contribution, w ill be described first. Being aware of the nine po ssible inositol structures,1 Posternak was able to dismiss both 8 and 9 because of their optical activity ( myo -inositol, containing a plane of symmetry, is not optically active). From myo -inositol hexaphosphoric acid, and using phosphatase to hydrolyze the molecule, Posternak and Posternak51, 52 were able to isolate optically-active diand tetraphos phate esters of the molecule. This discovery could then eliminate configuration 1 , since all possible diand tetraphosphates of 1 contain a plane of symmetry. Further action of the phosphatase then pr oduced a monophophate that was inactive. This then eliminated configuration 3 , since a resolvable monophosphate derivative of biochemical origin is unlik ely. When a mixture of the monoand diphosphate esters of myo -inositol was oxidized with ni tric acid, then hydrolyzed, a mixture of mesoand racemic tartaric acids was obt ained. Given that no inversion of stereochemistry (Walden inversion53, 54) takes place, structures 1 and 7 can then be ruled out. When Posternak oxidized myo -inositol with alkaline po tassium permanganate, the product was the hexaric acid (the term hexari c acid refers to the dicarboxylic acids of aldohexoses55) known as allomucic acid ( 49 , Figure 2), which had previously been reported by Fischer.56 In principle, since this conf iguration places four hydroxyl groups

PAGE 35

13 in a cis cis cis relationship, all of the remaining inositol configurations except 2 ( 1 and 3 have already been eliminated) are inappl icable. Additionally, Posternak isolated D,Lglucosaccharic acid from th e mother liquor of the oxidati on; this finding only showed that 6 was not a possible form for myo -inositol. CO2H OH H H HO H HO OH H CO2H Mucic acid 48 CO2H H HO H HO H HO H HO CO2H Allomucic acid 49 CO2H H HO OH H OH H OH H CO2H CO2H OH H H HO H HO H HO CO2HD,L-Talomucic acid 50 CO2H OH H H HO OH H OH H CO2HD,L-Glucosaccharic acid 51 CO2H HOH HOH HOH HOH CO2H CO2H OH H H HO OH H H HO CO2HD,L-Idosaccharic acid 52 CO2H HOH HOH HOH HOH CO2H Figure 2. Hexaric acids Fortunately, Posternak later revealed a flaw in this proof of configuration,57 and thus re-opened the debate. Essentially, Poster nak showed that Fisher had been mistaken in the configuration of his “allomucic aci d” (a problem that Fisher himself had suspected58). In Fisher’s work, mucic acid ( 48 ) was treated with pyridine at high temperature to afford a product that he expect ed to have inverted configuration at both carbons two and five. However, when Post ernak, using an unequivocal method, prepared D,L-talomucic acid ( 50 ), he found this product identical to that which he had obtained from myo -inositol.

PAGE 36

14 This discovery proved not only that Fisc her was wrong in his assumption, but also that the configuration of myo -inositol was once again in doubt. Of the remaining possible forms not excluded from previous evidence ( 2 , 4 , 5 ), only 2 and 4 may lead to D,Ltalomucic acid on oxidation. Further consideration of structures 2 and 4 indicate that oxidative cleavage of 2 would give D,L-talomucic ( 50 ), D,L-glucosaccharic ( 51 ), and allomucic acids ( 49 ), whereas oxidative cleavage of 4 would generate D,L-talomucic ( 50 ), D,L-glucosaccharic ( 51 ), and D,L-idosaccharic acid ( 52 ). Therefore, the isolation of either allomucic ( 49 ) or D,L-idosaccharic acid ( 52 ) would answer the problem. This question, however, would remain unanswered for another six years (from 1935 to 1941). In 1941, Posternak oxidized scyllo meso -inosose with permanganate to give a hexaric acid, which he believed to be D,L-glucosaccharic acid.59 Since, of configurations 2 and 4 , only 2 can, upon oxidative cleavage, generate in situ an optically inactive inosose that would then lead to D,L-glucosaccharic acid, the evidence seemed to settle the configuration of myo -inositol. However, in 1942, Dangschat announced the isolation of D,L-idosaccharic acid from the oxidation of a derivative of myo -inositol.60 This work was followed several months later by Posternak with a proof that th e hexaric acid he isol ated was, in fact, D,Lidosaccharic acid and not D,L-glucosaccharic acid.61 Thus, by two separate endeavors, the structure of myo -inositol was finally settled to 4 . The structure proof of Dangschat invol ved generating the cyclic ketal of 4 . As opposed to their acyclic anal ogs—the sugar alcohols—the r eactivity of cyclitols with aldehydes and ketones is markedly diminished . So much is that the case that many previous attempts to generate cyclic acetals of myo -inositol and quercitol had failed.62-64

PAGE 37

15 However, using a mixture of zinc chloride a nd acetic acid (a mixture Meerwein described as an “ansolvo acid”65), Dangschat was able to isolate the 5,6-isopropylidenemyo inositol ( 53 ) and the 4,5-isopropylidenemyo -inositol ( 54 ) in a 1:1 mixture. Acetylation, then hydrolysis of the cyclic acetal, followed by oxidation, and finally esterification with diazoethane furnished a compound which wa s identical with diethyl tetraacetyl-D,Lidosaccharate ( 56 , Scheme 10). myo -inositol 4 OH OH OH OH O O O O HO OH OH OH 53 54 Reagents and Conditions: a. Acetone, ZnCl2, HOAc b. Acetylation, then H+c. Pb(OAc)4d. CH3CO3H e. CH3CHN2b a OAc OAc OAc OAc 55 HO HO c-e CO2Et H AcO OAc H H AcO CO2Et OAc H 56 Scheme 10. Dangschat’s proof for the structure of myo -inositol60 O O O HO HO OH 57 a, b, c Reagents and Conditions: a. Pb(OAc)2, C6H6b. NaOEt, CH3NO2c. 0.1 M H2SO4CHO OH H H HO OH H CH2NO2OH H 58 d, e NH2OH OH OH HO HO 59 f OH OH OH OH HO HO 4 d. Ba(OH)2e. H2, PtO2f. HNO2 Scheme 11. Posternak’s synthesis of myo -inositol66 In 1950, Posternak synthesized myo -inositol from D-glucose.66 Using the procedure of Grosheintz an d Fischer, 6-nitro-6-deoxy-D-glucose ( 58 ) is first synthesized,67 then cyclized68 with barium hydroxide to generate nitrodeoxyinositol,

PAGE 38

16 which is reduced to the aminodeoxyinositol 59 , and finally transformed to myo -inositol, 4 (Scheme 11). A more recent synthesis of myo -inositol was provided by Schubert et al. in 1983, where the conduritol is generated from di anhydroinositol deriva tives obtained from benzene.69 Perhaps the latest synthesis of a myo -inositol derivative was published in 1994 by Chiara and Martìn-Lomas.70 Using a known procedure,71 D-mannitol is converted, in five steps, to diepoxide 61 . Opening of the epoxides by benzyl alcohol, followed by debenzylation and a Swern oxidation afforded dialdehyde 63 , which was cyclized using samarium diiodide to generate, as a major product, the diol with myo -inositol configuration. Subsequent protection of the cis -diol followed by protodesylation gave the bis-acetonide derivative of myo -inositol, 66 (Scheme 12). CH2OH HO HO OH CH2OH OHD-mannitol 60 a-e O O 61 O O Reagents and Conditions: a. Acetone, H2SO4b. HOAc, H2O, 40 °C c. BzCl, Py, CH2Cl2, -78 °C d. p TsCl, Et3N, DMAP, CH2Cl2O O 62 TBDPSO OTBDPS BnO OBn f, g i. (COCl)2, DMSO, Et3N, THF j. SmI2, t BuOH, THF k. DMP, p TsOH l. TBAF, THF CHO O O CHO 63 TBDPSO OTBDPS h, i O O OTBDPS OTBDPS O O j OH OH OTBDPS OTBDPS O O (+ minor diastereomer) 64 j OTBDPS OTBDPS O O 65 j OH OH O O 66 O O O O e. MeOH, K2CO3f. BnOH, NaH, DMF g. TBDPSCl, imidazole, DMAP, DMF h. H2, Pd(C), i PrOH, EtOAc Scheme 12. Chiara and Martìn-Lomas’s synthesis of myo -inositol70

PAGE 39

17 Muco -inositol (5) In addition to their work on allo -inositol ( vide supra ), Dangschat and Fischer also provided the first synthesis muco -inositol. This time, they took advantage of the tetraacetate of conduritol A ( 67 ), which offered a different steric bias from 25 (Scheme 5), favoring the other oxidation product ( 68 , Scheme 13). OH OH OH OH Conduritol A 24 Reagents and Conditions: a. Ac2O, Py b. KMnO4, MgSO4, EtOH, H2O c. NaOMe, MeOH a OAc OAc 67 b, a OAc OAc AcO AcO (+ Isomer) 68 c OH OH OH OH HO HO 5 OAc OAc OAc OAc Scheme 13. Danschat and FisherÂ’s synthesis of muco -inositol31, 32 Another synthesis of muco -inositol was presented by Angyal et al.72. In this synthesis, the authors propose treatment of 1O -toluenep -sulphonyl-myoinositol ( 70 , synthesized from 1,4,5,6-tetraO -acetylmyoinositol, 6918, 73) with mild base to afford epoxide 71 , which did not isomerise to the more st able epoxide. Instead, acid hydrolysis then afforded muco -inositol (Scheme 14). A more in-d epth account of epoxide openings was later published by the same authors.74 OTs OH OH OH HO HO a OH OH OH HO c OH OH OH OH HO HO 5 O 71 70 Reagents and Conditions: a. p -TsCl, Py; then HCl b. Deacidite FF, CO2 (s), MeOH, H2O c. HCl OAc OH OH OAc AcO AcO b 69 Scheme 14. AngyalÂ’s synthesis of muco -inositol72

PAGE 40

18 In a similar approach, Suami et al.75 used 1,4,5,6-tetraO -acetyl-3O methanesulfonylmyo -inositol ( 7276) to generate, in two steps, muco -inositol. The procedure calls for the displacement of a mesylate by acetate anion followed by acetylation of the remaining free alcohol, after which the hexaacetate can then be saponified to the title compound (Scheme 15). OAc OAc OAc OAc AcO AcO a OH OH OH OH HO HO 5 73 Reagents and Conditions: a. NaOAc, then Ac2O b. HCl (conc.), EtOH OAc OH OMs OAc AcO AcO b 72 Scheme 15. Synthesis of muco -inositol by Suami et al.75 In a study of the hexaacetates of Lchiro -, myo -, and muco -inositol, Paulsen and Höhne77 showed that when any of these compo unds were treated with HF (liquid), the isomerization product had the muco -configuration. The author s claim that the dication formed with the muco -configuration can generate a boatform of cyclohexane wherein all of the substituents are “quasi-equatorial.” These findings were corroborated in a nearly identical study by Hedgley and Fletcher.78 A more recent synthesis by Angyal and Odier79 provide a facile synthesis of muco inositol from quebrachitol. The authors firs t oxidize the pentabenzoate of quebrachitol ( 74 ),80 followed by hydrolysis of the formyl gr oup using methanolic HCl. The free hydroxyl is then tosylated, solvolyzed, and the benzoates were saponified to afford 5 (Scheme 16).

PAGE 41

19 b 5 Reagents and Conditions: a. CrO3, HOAc b. HCl, MeOH c. TsCl, Py OBz OR OBz OBz BzO BzO d OBz OR OBz OBz BzO BzO R = Me 74 R = CHO 75 a R = H 76 R = Ts 77 c OBz OH OBz OBz BzO BzO OH OH OH OH HO HO e 78 d. H2O, DMF e. NaOMe, MeOH Scheme 16. Angyal and Odier’s synthesis of muco -inositol79 Neo -inositol (6) In 1955, Angyal and Matheson synthesized an inositol that had not yet been reported.81 For that reason, they suggested the name “ neo -inositol.” Though no proof of structure is given to this new cyclitol, the authors distinguish it fr om others by its high melting point (315 °C), low solubi lity (0.1 g in 100 mL of cold water), different synthetic route, and the fact that it was not identical with any of the six (at the time) inositols known. Starting from the known 1,2,5,6-diO -isopropylidene-(-)-inositol ( 79 ),34, 82 ditosylate 80 is formed, and then solvolyzed in sodium methoxide to give epoxide 81 . Though this method provided an easy venue into epoxide 81 , and though the formation of epoxides from ditosyl compounds had been reported elsewhere,83-87 neither the mechanism of action, nor the direction of the ring formation have been discussed by the authors. In this case, though, because of the symmetry element provided by this compound, only one epoxide can form. The correct assignment of epoxide 81 was also demonstrated by an alternate synthesis (n amely, monotosylation followed by acetylation, then hydrolysis of the acetate, and displacement of the tosyl). When epoxide 81 was

PAGE 42

20 heated in dilute sulfuric acid, the new inositol ( 6 ) crystallized out of solution (Scheme 17). O O O O OH OH 79 a O O O O OTs OTs 80 b O O O O 81 c O OH OH OH OH HO HO 6 Reagents and Conditions: a. p TsCl, Py b. NaOMe, MeOH, reflux c. 0.1 M H2SO4 Scheme 17. Angyal and Ma thesonÂ’s synthesis of neo -inositol81 Since its discovery by Angyal and Matheson, neo -inositol has also been extracted from Croton celtifidolius88 as well as mammalian tissues.89 Also, a crystal study has been performed to explain the low solubility of the cyclitol.90 As it turns out, Angyal and Craig found that the crystal structure had unus ual stability and that its lattice energy was exceptionally large, exceeding that of the so lvated molecules in water and the difference in entropy between the solid and dissolved states. In 1998, Riley et al. synthesized neo -inositol from myo -inositol in a preparation that could be amenable to scaleup b ecause it did not require chromatography.91 From myo -inositol, the authors firs t selectively protect the trans -diols with the butane-2,3diacetal (BDA) group92, 93 to generate 82 . Inversion of stereochemistry at C-5 was done by first selectively generating the triflate at that position, then displacing that with dimethylacetamide (DMA) to give the acetat e, which was subsequently hydrolyzed to give diol 83 . Finally, hydrolysis of the protec tive groups gave, after crystallization, neo inositol in 60% yield from protected myo -inositol 82 (Scheme 18).

PAGE 43

21 OH OH OH OH HO HO 4 a O O O O OH OH MeO MeO OMe OMe 82 b-d O O O O OH OH MeO MeO OMe OMe 83 e OH OH OH OH HO HO 6 Reagents and Conditions: a. butanedione, MeOH, CH(OMe)3, CSA, reflux b. Tf2O, Py, CH2Cl2c. DMA, H2O d. NaOMe, MeOH, reflux e. HOAc, H2O, reflux Scheme 18. Synthesis of neo -inositol by Riley et al.91 The most recent synthesis of neo -inositol was published by Hudlicky et al. in 2000.94 This approach also allowed for a multi-gram scale synthesis of this cyclitol. Starting from known diol 32 , the compound is first protected as the acetonide, then converted to bromohydrin 84 using DBH in acetone/water (Scheme 19). Br OH OH a, b Br O O HO Br c Br O O O Br O O HO OH d O O HO OH e O O HO OH f HO OH OH HO HO OH OH OH 87 6 32 84 85 86 88 Reagents and Conditions: a. DMP, pTsOH b. DBH, H2O, acetone c. 10 % aq. KOH, DME, RT then reflux d. Bu3SnH, AIBN, PhH, reflux e. OsO4, NMMO, tBuOH, acetone, H2O f. HCl, MeOH Scheme 19. Synthesis of neo -inositol by Hudlicky et al.94 Treatment of 84 with aqueous KOH afforded the epoxide ( 85 ) in situ , which was hydrolyzed to give trans -diol 86 . Radical debromination followed by osmylation gave

PAGE 44

22 tetrol 88 , which was then deprotected in methanolic HCl, affording neo -inositol in 77% yield. The overall yield from diol 32 was 17%. Scyllo -inositol (7) Scyllitol, another natura lly-occurring cyclitol, wa s first reported in 1858 by Staedeler and Frerichs in the orga ns of various plagiostomous fish.95 In 1887, Vincent and Delachanal isolated a carbohydrate from acorns that they named quercitinol,96 while Müller, in 1907, repor ted the presence of a similar compound from the leaves of Cocos plumose and Cocos nuciferia , the extract of which he named cocositol.97 Further investigation by Müller on inositols98 and these extracts99 led him to conclude that all of these compounds were, in fact, identical, and ha d the structure for th e cyclitol that we now refer to as scyllo -inositol ( 7 ). Since then, others ha ve found the compound in the muscle100 and liver101 of the spur dogfish ( Acantia vulgaris ), as well as in flowers and bracts of the dogwood tree ( Cornus florida ).102 The configuration of scyllo -inositol was determined by Posternak in the course of his work on myo -inositol ( vide supra ).24, 59, 61 It was also obtained through the hydrogenati on of hexahydrobenzene by Anderson and Wallis.16 OH OH OH OH HO HO 4 a, b O OAc OAc OAc AcO AcO c, d OH OH OH OH HO HO 7 Reagents and Conditions: a. Acetobacter suboxydansb. Ac2O, H2SO489 c. NaBH4, MeOH d.2MHCl Scheme 20. Kohne and Praefcke’s synthesis of scyllo -inositol103

PAGE 45

23 The first synthesis of scyllo -inositol was provided by Kohne and Praefcke in 1985.103 Myo -inositol was first oxidized by Acetobacter suboxydans24, 25, 104 to the inosose, then acetylated. The pentaacetate was reduced105 and finally deprotected to afford scyllo -inositol ( 7 , Scheme 20). More recently, a preparation of scyllo -inositol by equilibration of myo -inositol using Raney-nickel in water followed by ort hoacetate derivatizati on and deprotection has been reported as a viable approach.106 The latest synthesis of scyllo -inositol has been published in 2003 by Sarmah and Shashidhar.107 From the orthoformate of myo -inositol ( 90 ),108 the equatorial benzoate is selectively formed, after which th e remaining hydroxyl groups are tosylated, and the benzoate ester is cleaved. The free hydroxyl group is oxidized, then reduced, and eventu ally deprotected to afford the desired cyclitol (Scheme 21). The interest in scyllo -inositol has grown considerably since it was found that some of its derivatives will form liquid crystals.109 Also, this cyclitol has been detected by magnetic resonance in the brain of healthy hum an subjects, a discovery that suggests that the scyllo -inositol metabolism may be independently regulated from that of myo inositol.110 HO OO O HO OH 90 TsO OO O OTs 91 O HO OO O OH 92 HO a-d Reagents and Conditions: a. NaH, BzCl, DMF b. p TsCl, Py c. i BuNH2, MeOH d. (COCl)2, DMSO, CH2Cl2, Et3N e-g, c h OH OH OH OH HO HO 7 e. NaBH4, MeOH, THF f. NaOMe, MeOH, reflux g. Ac2O, Py h. TFA, H2O Scheme 21. Sarmah and ShashidharÂ’s synthesis of scyllo -inositol107

PAGE 46

24 LChiro -inositol (8) and DChiro -inositol (9) DChiro -inositol occurs naturally in various plants as its monomethyl ether (pinitol), and was first mentioned by Berthelot in 1855111 as an extract of Pinus lambertiana . LChiro -inositol is also found as a m onomethyl ether and was first discovered by Tanret in the bark of the quebracho tree,112 giving the new compound the name quebrachitol. The latex of the rubber tree, Hevea brasiliensis , has been found to be one of the most convenient sources of quebrachitol.113-116 The sweetness of quebrachitol, which is less than that of gluc ose or sucrose, has led to its investigation as a sweetener for diabetics. However, the negative side effects (colic and severe diarrhea) associated with the ingestion of considerable amounts (two to three times as much quebrachitol as natural cane sugar is required) of que brachitol made the potential sweetener unsuitable for this purpose.117 The physical properties of pinitol and quebrachitol indicate that the two compounds are not enantiomorphic (therefore, the methyl groups are not in the same position). Treatment of either pinitol or quebrachitol with hot concentrated hydroiodic acid will generate the corresponding cycl itol in nearly qu antitative yields. OH OH OH OH HO HOLchiro -Inositol 8 OH HO HO OH OH OHDchiro -Inositol 9 OH OH OH OH O HO Quebrachitol 93 OH HO HO O OH OH (+)-Pinitol 94 Me Me Figure 3. Land Dchiro -inositols and their correspondi ng methylated derivatives, quebrachitol and pinitol The absolute assignment of stereochemistry of Dand Lchiro -inositol was done by Posternak in 1936.116 Before that, a racemic mixture of inositols had previously been described by Maquenne and Tanret in 1890,118 and again in 1907.119 In 1948, Fletcher

PAGE 47

25 and Findlay prepared racemic chiro -inositol,120 and with this work were able to conclude that the compounds synthesized were identical to those described by Maquenne and Tanret. The ready source of both enantiomers of th ese cyclitols has limited the number of syntheses of these compounds. The first such synthesis came as a m eans to establish the absolute configuration of synthetic in termediates and was published by Berlin et al. in 1991.121 CH2OH OH HO OH CH2OH OHD-sorbitol 95 Reagents and Conditions: a. acetone, H2SO4b. HOAc, H2O c. BzCl, Py, CH2Cl2d. TBDPSCl, imidazole, DMAP, DMF e. DIBALH, toluene f. HCl, H2O g. (COCl)2, DMSO, i Pr2NEt, THF h. SmI2, t BuOH, THF i. PPTS, CH2Cl2, MeOH j. TBAF, THF k. Ac2O, Py, DMAP a-c CH2OBz OH O O CH2OBz OH 96 d-g CHO OTBDPS O O CHO OTBDPS 97 O O OTBDPS OTBDPS O O OH OH OTBDPS OTBDPS O O h 98 major product OAc OH OAc OAc AcO AcO i-k 99 Scheme 22. Chiara and ValleÂ’s synthesis of Lchiro -inositol hexaacetate122 In a similar synthesis to their work on myo -inositol, Chiara and Valle synthesized Lchiro -inositol from D-sorbitol in 1995.122 Using a known procedure,71 D-sorbitol ( 95 ) is converted to dibenzoate 96 . After silation of the remaining hydroxyl groups followed by saponification of the benzoate esters, the terminal hydroxyls were oxidized to afford

PAGE 48

26 dialdehyde 97 , which was then subsequently treated with samarium diiodide to effect the intramolecular pinacol coupling, giving diol 98 as the major product. Subsequent acidcatalyzed hydrolysis of the acetonide follo wed by protodesylation a nd acetylation of the six hydroxyls gave the pentaacetate of Lchiro -inositol, 99 (Scheme 22). A tedious approach to Dchiro -inositol was published in 1998 by Kim et al. .123 Oxyselenylation of cyclohexene ( 100 ) with N -(phenylseleno)phthalimide ( N -PSP) and ( S,S )-hydrobenzoin to afford two diastereom ers in ca. 1:1 ratio (Scheme 23). 100 SePh O Ph OH Ph 101 O Ph OH Ph 102 Reagents and Conditions: a. ( S,S )-hydrobenzoin, N -PSP, BF3-OEt2, CH2Cl2b. NaIO4, NaHCO3, MeOH, H2O c. PhSeOTf, CH2Cl2d. m CPBA, NaHCO3, CH2Cl2e. PhSeSePh, NaBH4, EtOH or n BuOH (+ diastereomer) 103 O O SePh Ph Ph a b c 104 O O Ph Ph b, d O 105 O O Ph Ph e OH PhSe f, g 106 O OPh Ph OTBDPS 107 O OPh Ph d, e OTBDPS PhSe HO b, h 108 O OPh Ph OH HO 9 OH OH i, j OH HO OH HO f. 30 % H2O2, THF, EtOH g. TBDPSCl, imidazole, DMF h. TBAF, THF i. K2OsO4, NMMO, acetone, H2O j. H2 (15 psi), Pd(OH)2, HCl, EtOH Scheme 23. Synthesis of Dchiro -inositol by Kim et al.123 Oxyselenide 101 was converted into olefin 102 by oxidation of the selenoxide followed by elimination. Intramol ecular oxyselenylat ion of olefin 102 generated selenide

PAGE 49

27 103 , which was subsequently eliminated and oxidized to give epoxide 104 . Trans -diaxial opening of epoxide 104 with NaSePh (generated in situ from PhSeSePH and NaBH4) generated exclusively hydroxyselenide 105 , which was then eliminated and protected to give silyl ether 106 . After another series of epoxi dation, diaxial opening with NaSePh, and elimination, alkene 108 was obtained, and finally dihydroxylated to give, after deprotection, Dchiro -inositol ( 9 , Scheme 23). In spite of th e lengthy approach to this naturally-occurring cyclitol, the chemistry is interesting because it essentially takes a innocuous cyclohexene and effectively functionaliz es it selectively to generate a chiral molecule. Another synthesis of Dchiro -inositol was published in 2001 by Takahashi et al. .124 The authors converted myo -inositol to its 2 ,3-(+)-camphor acetal, then introduced selectively the leaving group at C-1 by treating the resulting acetal with trifluoromethanesulfic anhydride (Scheme 24). OH OH OH OH HO HO 4 O O OTf OH OH OH Reagents and Conditions: a. (1 R )-(+)-camphor dimethyl acetal, H2SO4, DMSO b. NaOMe, MeOH c. p TsOH, CHCl3, MeOH, H2O a-d 109 O O OBz OAc OAc OAc e, f 110 HO HO OH OH OH OH b, g 9 d. Tf2O, Py, CH2Cl2e. Ac2O, Py, CH2Cl2f. LiOBz, DMF g. HOAc, H2O Scheme 24. Synthesis of Dchiro -inositol by Takahashi et al.124 The authors attribute the selectivity at the 1-position to the intramolecular hydrogen-bonding that may take place with the cis -vicinal oxygen at C-2. A similar selectivity was observed by Suami et al. .125 The remaining free hydroxyls were then

PAGE 50

28 protected as acetates, and the triflate was displaced by a benzoate to afford 110 . Finally, the protective groups were cleaved, affording cyclitol 9 . The latest synthesis of Dchiro -inositol was published in 2002 by Catelani et al. .126 The starting material for this synt hesis was the 1,5-bis-methyl glycoside 111 ,30 a masked forme of 2,6-diO -benzyl-Larabino -hexos-5-ulose that Pistarà et al.29 used in their preparation of epi -inositol. The preparat ion of hexos-5-ulose 113 was achieved by acid hydrolysis of bis-glycoside 112 , which itself was obtaine d through a sequence of oxidation and stereoselective reduction of the 4O -benzyl ether of 111 .127 Treatment of crude 113 with a catalytic amount of DBU gave -hydroxy-inosose 114 with high diastereoselectivity. The aut hors then took advantage of th e free hydroxyl group to effect an intramolecular hydride delivery128 that would set the correct stereochemistry of Dchiro -inositol. Finally, deprotection of the obtained structure affo rded the title cyclitol in high yield (Scheme 25). OOMe HO OH OBn BnO MeO OOMe HO OH OBn BnO MeO O HO OH OBn BnO O OH HO OH OBn O OBn OH HO OH OH HO OH 9 113 112 111 a b cd, e Reagents and Conditions: a. see Barili et al. 1998 b. CF3CO2H, H2O, CH3CN c. DBU, PhMe, CH2Cl2d. NaBH(OAc)3, HOAc, CH3CN e. H2, Pd(C), MeOH 114 Scheme 25. Synthesis of Dchiro -inositol by Catelani et al.126

PAGE 51

29 Nonspecific or branched syntheses of inositols Because they are often a more efficient approach to these biologically-interesting cyclitols, branched syntheses (or synthese s that have a common intermediate) will be looked at independently. Thes e few examples are provided either because of their simplicity, their historical importance, or their synthetic ingenuity. In 1959, Nakajima and coworkers devised a synthesis for all i nositols except for cis -inositol based on the hydroxyl ation of the various conduritol isomers (Scheme 26).129 OH OH OH OH cis or transhydroxylation OH OH OH OH HO HO 2 9 115 Scheme 26. Synthesis of inositols by Nakajima et al.129 In 1973, Kowarski and Sarel provided a synthesis of myo, allo -, neo -, and epi inositols from the two Diels-Alder adducts ( 116 and 117 ) of furan and vinylene carbonate (Scheme 27).130 The endo and exo adducts were epoxidized, and, upon basic hydrolysis, afforded 1,4-anhydro-Dallo ( 119 ) and cis -inositols ( 121 ), respectively. These were then converted to allo ( 3 ) and myo -inositols ( 4 ). The same adducts were osmylated, and after hydrolysis, yielded neo -inositol ( 6 , endo -adduct) and epi -inositol ( 2 , exo -adduct).

PAGE 52

30 O O O O O O O O Reagents and Conditions: a. HOAc, H2O2, H2SO4, 40 °C b. (i) OsO4, EtOAc, Py, 24 hrs.; (ii) Na2SO3, reflux; (iii) NaOH, 1 hr. c. Ac2O, Py d. 2 M NaOH, EtOH e. HOAc (80 %), H2O (19 %), H2SO4 (1 %) O OAc OAc AcO AcO OH OH OH OH HO HO 6 O O O O O OH OH OH OH HO HO 4 OH OH OH OH HO HO 3 + d, e e b, c a O AcO AcO OH OH OH OH HO HO 2 O O OH OH OH OH HO HO 4 OH OH OH OH HO HO 3 + d, e e b, c a O O O OAc OAc 116 117 118 119 120 121 Scheme 27. Kowarski and Sarel’s synthesis of myo -, allo -, neo -, and epi -inositols130 An elegant approach by Mandel and Hudlicky131-133 has used the oxidation of aromatic hydrocarbons through whole cell fermentation to generate a common intermediate that was then further transformed into three different inositols selectively. In effect, by controlling the reactivity of a key epoxide ( 124 ) and its dechlorinated derivative ( 125 ), the authors were able to synthesize dextro ( 9 ), neo( 6 ), and alloinositol ( 3 ) with good yields, Scheme 28.

PAGE 53

31 Cl Cl OH OH Reagents and Conditions: a. P. putida 39 D b. DMP, p TsOH, acetone c. KMnO4, MgSO4, H2O, acetone d. TTMSS, AIBN, toluene, 110 °C e. H2O, Amberlite IR 118, 110 °C OR H2O, 100 °C a b, c Cl O O OH HO O 122 123 124 d O O OH HO O OH OH OH OH HO HO 3 OH OH OH OH HO HO 6 + OH OH OH OH HO HO 9 125 ef g, h f. Amberlyst A21 + Amberlite IRA 904 (1:1), 100 °C OR NaOBz, H2O g. H2O, Al2O3h. H2, RaNi, MeOH OH OH OH OH HO HO 9 Scheme 28. Mandel and Hudlicky’s approach to Dchiro -, neo -, and allo -inositols131-133 Another divergent approach that uses bi otransformation was de veloped by Carless et al.134 in their syntheses of muco, (±)chiro -, allo, and epi -inositol. Microbial oxidation of benzene by P. putida , followed by photo-oxidation and reduction of the endoperoxides gave conduritol A ( 24 ) and conduritol D ( 30 ). After dihydroxylation or epoxidation followed by hydrolysis of these c onduritols, the five inositols mentioned are obtained selectively depending on th e steps taken (Scheme 29).

PAGE 54

32 a OH OH 126 44 OH OH OH OH OH OH OH OH Conduritol A 24 Conduritol D 30 b, c OH OH OH OH O OH OH OH OH O 127 128 OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO OH OH OH OH HO HO 2 3 5 9 8 + d e, f g e j h, i, f Reagents and Conditions: a. P. putidab. 1O2c. thiourea, MeOH d. MeCO3H, MeCO2H e. H3O+, 80 °C f. K2CO3, MeOH g. mCPBA, CH2Cl2h. Ac2O, Py i. OsO4, NMMO Scheme 29. Synthesis of muco, (±)chiro -, allo, and epi -inositols by Carless et al.134 In 1998, Brammer and Hudlicky proposed a synthesis of both Lchiro and muco inositols from a common intermediate.135 Diol 32 was obtained in high yield from bromobenzene through whol e-cell fermentation using E. coli JM109 (pDTG601),136 and was protected and epoxidized to afford 129 .137 Epoxide 129 was thus either opened with benzyl alcohol or with potassium hydroxide toward the syntheses of Lchiro and muco inositols, respectively. When trans -diol 131 was treated with m CPBA, a mixture of epoxides 133a and 133b in a 1:1.8 ratio was obtained. Th e authors explain this ratio by

PAGE 55

33 pointing out the apparent competition between the syn -directing effect of the free allylic alcohol and the hindering effect of the a cetonide moiety; when the free alcohol was replaced by a benzyl ethe r, the ratio of epoxides shifted to favor the -epoxide (Scheme 30). Br Br O O O 31 O O O O 129 130 131 HO OH OH BnO OH OH 132 OH BnO OH HO OH OH 8 OH HO OH HO 133a OH HO OH OH 5 OH HO OH HO O O O 133b OH HO O O O (+ ca. 5 % myo -inositol) a-c Reagents and Conditions: a. E. coli JM109 (pDTG601) b. DMP, acetone, p TsOH c. m CPBA, CH2Cl2d. 10 % aq. KOH, H2O, DME e. BnOH, BF3-OEt2e, f d, f g, h c j i f. n Bu3SnH, AIBN, THF g OsO4, acetone, H2O, NMMO h. HCl, EtOH i. 10 % Pd(C), H2, H2O j. 10 % aq. H2SO4 Scheme 30. Brammer and HudlickyÂ’s synthesis of Lchiro and muco -inositols135 In 1999, Chung and Kwon published a synthesis of all inositol stereoisomers from myo -inositol.20 Myo -inositol diols 134 ,138 141 ,139 and 145140 were first synthesized,141-143 then converted to conduritols C ( 132 ), B, and F ( 143 ) derivatives, respectively (Scheme 31).

PAGE 56

34 O O OBz OH OH BzO O O O O OH OH OH OH OBz OBz BzO BzO 134 141 145 Reagents and Conditions: a. PPh3, imidazole, I2, toluene, reflux b. BnBr, NaH, DMF c. HOAc, H2O d. BzCl, Py e. Pd(OH)2/C, H2 (50 psi), EtOAc, EtOH OBn BnO BnO OBn OBn OBn BnO BnO 135 142 146 a, c, g, h a, g, h, i b-e, a g, h OBn BnOOH OH 136 f OBn BnOOH OH 137 neo epi OBn BnO OBn BnO BnO OBn 147 f OH OH chiro OBn BnO OBn BnO OBn BnO 138 OBn BnO OBn BnO O OBn BnOOH OH 139 j OBn BnOOH OH 140 allo myo OBn BnO OBn BnO i O OH BnOOBn OBn 143 j OH BnOOBn OBn 144 chiro scyllo OH BnO OH BnO 148 149 j 150 muco myo i OBn BnO BnO OBn OBn BnO BnO OBn OBn BnOBnO OBn O OH OH OH OH f. OsO4, NMMO, H2O, acetone g. NaOMe, MeOH h. BnBr, NaH, DMF i. I2, Ag2O, aq. dioxane j. CF3CO2H, THF Scheme 31. Chung and KwonÂ’s synthesis of inositols20

PAGE 57

35 Through a series of functional group interconversions and subsequent transformations, several inositols were prep ared. The procedures used, however, were tedious and certainly not, despite claims by th e authors, practical, as most routes yielded more than one inositol derivative, and would therefore necessitate fu rther purification to generate the individual cyclitol. More recently, a synthesis of all inositol diastereomers from 6O -acetyl-5enopyranosides ( 151 ) has been published.144 These enopyranosides were converted into the corresponding substituted cyclohexanones ( 152 ) through a Ferrier-II rearrangement, then stereoselectively reduced to give these cyclitols ( 1 9 ) in good to excellent yield, and with high stereoselectivity (o ften as high as 99:1 in favor of one diastereomer, Scheme 32). O AcO RO OR OR OR 151 Glucose-, Galactose-, or Mannose-type enopyranosides a O AcO HO OR OR OR b OH HO HO OH OH OH 1 9 Reagents and Conditions: a. Pd(II)-mediated Ferrier-II Reaction b. StereoselectiveReduction 152 Scheme 32. Approach to inositols by Takahashi et al.144 In 2003, Heo et al. synthesized Dchiro and neo -inositols (as well as conduritols B-D and F) using a common-intermediate strategy.145 Brown allylboration146 of aldehyde 153 using either enantiomers of 157147 gave selectively both diastereomers 154a and 154b . The rest of the synthesis was carried out nearly identically for both diastereomers. Dienes 154a and 154b were first treated with GrubbsÂ’s second generation catalyst to

PAGE 58

36 effect a Ring Closing Metathesis, then the cyclohexene generated was dihydroxylated to afford 155 and 156 , respectively. A Fleming-Tamao oxidation of hydroxysilanes 155 and 156 generated, after deprotection, Dchiro ( 9 ) and neo -inositols ( 6 ), respectively (Scheme 33). OBn OBn H O 157 = Ipc B Ipc PhMe2Si Ipc = OR Reagents and Conditions: a. 157 from (-)-IpcBOMe ( 154a ) or from (+)-IpcBOMe ( 154b ) b. Grubbs' catalyst (2nd. generation) c. OsO4, NMMO, acetone d. TBSOTf e. K2OsO4, H2O, K3Fe(CN)6, K2SO4, MeSO2NH2, quinuclidine f. HF, Py g. Hg(OAc)2, AcOOH, AcOH h. H2, Pd(C), EtOH OBn OBn OH SiMe2Ph OBn OBn OH SiMe2Ph 154a 154b a b, c b, d, e SiMe2Ph OH OBn OBn HO HO 153 155 SiMe2Ph OTBS OBn OBn HO HO 156 g, h f-h OH OH OH OH HO HO 9 OH OH OH OH HO HO 6 Scheme 33. Synthesis of neo and Dchiro -inositols by Heo et al.145 Most recently, Podeschwa et al. published a stereoselective synthesis of myo -, neo -, Lchiro -, Dchiro -, allo -, scyllo -, and epi -inositols. These cyclitols were prepared from p benzoquinone via enzymatic resolution of derived conduritol B key intermediate. The products of reactions were purified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPIC-IPAD).148

PAGE 59

37 Inositol Oligomers an d Related Compounds Introduction Over the years, a great deal of natural product containing inosito ls linked to a sugar (saccharide unit such as glucose such as 158 or 159 ) have been isolated from a number of sources.149, 150 Considerable interest, both in the synthetic community and elsewhere, has been devoted to the study and prepara tion of glycosyl phosphatidyl inositol derivatives;151, 152 this interest has in part been s purred by the discovery, in 1986, that rat hepatocytes contained insulin mimetic phosphooligosaccharides.153 In addition to these findings, phosphorylated inosityl glycans we re found to act as substitutes for insulin in vitro ,154 and it was therefore suggested that glyc opyranosyl cyclitols co uld act as insulin mimics. For this reason, targets such as 160155-157 and 161158 have attracted considerable synthetic interest. In 1994, Billington et al.159 also synthesized a number of conduritolsrelated compounds that acted as insulin modulators ( 162 , 163 , 164 , Figure 4). OO HO HO OH OH OH OH OH OH HO OO HO HO OH OH OH OH OH OH HO OO HO HO OH NH2OPO3H2OH OH OH HO OO HO HO OH NH2OPO3H2OH OH OH HO 158 159 160 161 HO HO OH HO HO OH HO HO OH OH O OH OH OH OH OH OH 162163164 Figure 4. Some naturally-occurring glycopyranos yl cyclitols and possible insulin mimics and modulators155-159

PAGE 60

38 Since then, a number of syntheses of th ese potential insulin mimics have been published. For example, the Balci group has prepared bis -homo-conduritols D and F ( 172 and 173 )160, 161 and the Mehta group has synthe sized polyhydroxylated hindrindanes ( 167 , 168 , and 169 ),162, 163 decalin and diquinane polyols ( 165 and 166 ),164 and bicyclitols ( 174 177 ),165, 166 all as potential insulin mimics or compounds of related biological activity (Figure 5). All of these cyclitol derivatives were tested for their glycosidase inhibition, and were all found inactive except for 174 , which showed moderate inhibition of –glucosidase with a Ki of 87 µM. OH HO HO OH 172 OH HO HO OH 173 OH OH OH HO HO HO OH 167 OH OH R OH HO HO HO Kara et. al. 1994 ; Balci 1997 Mehta et al. 1999 HO HO HO OHOH OH OH OH 165 HO HO HOOH OH OH 166 HO OH HO OHOH OH OH OH 170 HO OH HO OHOH OH OH OH 171 OH HO HO OH OH OH OH 174 Mehta et al. 2000 OH HO HO OH OH OH OH 176 OH HO HO OH OH OH OH 177 OH HO HO OH OH OH OH 175 MehtaandRamesh 2001 168 , R = OH, down 169 , R = OH, up Figure 5. Potential glycosidase inhibitors160-166 Synthesis and molecular properties The first synthesis of a cylitol dime r was published in 1994 by Hudlicky and Thorpe.167 In this approach, epoxide 129 (obtained as described in Scheme 30) was first

PAGE 61

39 debrominated, then reacted w ith known secondary alcohol 179 .168, 169 The coupling was performed using boron trifluoride diethyl et herate as a Lewis acid and afforded dimer 180 in 75% yield. Subsequent dihydroxyl ation followed by deprotection gave Lchiro inositolgala -quercitol conjugate 181 (Scheme 34). O O Br O O OH 179 O O O O a b c O O O OH O O 180 178 129 O OH 181 HO OH OH OH OH OH HO HO d, e Reagents and Conditions: a. n Bu3SnH, AIBN, benzene b. LiAlH4c. 179 , BF3-OEt2, CH2Cl2d. OsO4, NMMO, H2O, acetone, t BuOH e. Amberlyst resin, MeOH, H2O Scheme 34. Hudlicky and Thorpe's synthesis of a Lchiro -inositolgala -quercitol conjugate167 Using a similar strategy, Hudlicky et al. synthesized 1,2-Lchiro -inositol conjugates and oligomers.170, 171 This time, an aziridine (inst ead of an epoxide) served as an electrophile and was opened with secondary alcohol 179 . The resulting dimer was then dihydroxylated, protected, and af ter all of the protective groups were removed, aminoinositol dimer 184 was isolated as its hydrochloride (Scheme 35). The metal-chelating ability of aminoinositol dimer 184 was tested by doping an aqueous solution of the amine hydrochloride with an equimolar aqueous solution of calcium chloride.172 The solvent was allowed to evapor ate slowly at room temperature, generating crystals that were analyzed by single crystal Xray diffraction. The crystal structure obtained exhibited striking exte nded secondary helical structure (Figure 6),

PAGE 62

40 whereby an ordered array of calcium ions br idged two amino residues. In short, the crystal structure possessed a 3dimensional H-bonding network. O O Br O O TsN a, b c O O O NHTs O O 183 182 33 O NH3184 HO OH OH OH OH OH HO HO d-g Reagents and Conditions: a. PhINTs, Cu(acac)2b. n Bu3SnH, AIBN, THF c. 179 , BF3-OEt2, CH2Cl2Cl d. OsO4, NMMO, H2O, acetone, t BuOH e. DMP, acetone, p TsOH f. Na, NH3 (l) g. HCl, MeOH Scheme 35. Synthesis of aminoinositol dimer 184 by Hudlicky et al.170, 171 Figure 6. Thermal ellipsoids drawing of the extended secondary helical structure of 184 shown (a) along the b -axis and (b) down the b -axis170, 171 Conduritol-aminoinositol dimer 187 was synthesized using again the same strategy. When bromoepoxide 129 was reacted with secondary alcohol 179 , monobrominated dimer 185 was obtained in high yields. The free olefin was epoxidized, and the epoxide was opened with sodium azi de to give azido-diol 186 . Reduction of the vinyl bromide under radical conditions not only afforded the debrominated olefin, but also reduced the azide to the corresponding amino-diol, whic h gave, after hydrolysis of the protective groups, dimer 187 as the hydrochloride (Scheme 36).

PAGE 63

41 O O Br O a O O O OH O O 185 129 O OH 187 OH OH NH3OH HO HO b, c Reagents and Conditions: a. 179 , BF3-OEt2, CH2Cl2b. m CPBA, CH2Cl2Br O OH 186 N3OH d, e O O O O Br Cl c. NaN3, NH4Cl, DME, H2O, EtOH d. n Bu3SnH, AIBN, THF e. HCl, MeOH Scheme 36. Synthesis of a conduritolaminoinositol conjugate by Hudlicky et al.171 The fully hydroxylated analog of 187 was also synthesized using the same approach. Vinyl epoxide 178 was reacted with alcohol 188 to give dimer 189 . Dihydroxylation of the free vinyl group afforde d, after protection of the free diols, and debromination, alcohol 190 . Then, the remaining olefin was treated with m CPBA, and the resulting epoxide was again opened with sodium azide to give , after reduction and cleavage of the protective groups, aminoinositol dimer 191 as the corresponding hydrochloride (Scheme 37). O O O a O O O OH O O 189 178 O OH 191 HO OH OH OH NH3OH HO HO b-d Reagents and Conditions: a. BF3-OEt2, CH2Cl2b. OsO4, NMMO, acetone, H2O, t BuOH c. DMP, acetone, p TsOH d. n Bu3SnH, AIBN, THF O OH 190 e-h O O O O O O Br OH 188 Br O O Cl e. urea hydroperoxide, TFAA, Na2HPO4, CH2Cl2f. NaN3, NH4Cl, DME, H2O, EtOH g. LiAlH4, THF h. HCl, MeOH Scheme 37. Synthesis of aminoinositol dimer 191 by Hudlicky et al.171

PAGE 64

42 The same methodology was again employed to synthesize Lchiro -inositol oligomers (trimers, tetramers). Bromoepoxide 129 is opened with benzyl alcohol using CSA as a catalyst for the transformation. This generates a new nucleophile within the dimer that can then be used to open another molecule of epoxide 129 , affording trimer 194 . If the procedure is repeated once more , the corresponding tetramer is generated. Subsequent debromination and dihydroxylation, followed by cl eavage of the benzyl ether and of the acetonides generated Lchiro -inositol trimer 195 and tetramer 196 (Scheme 38). This approach has also been us ed to generate higher order oligomers.173 O O O a 129 Reagents and Conditions: a. Benzyl alcohol, CSA, CH2Cl2b. 129 , BF3-OEt2, CH2Cl2c. n Bu3SnH, AIBN, THF d. OsO4, NMMO, acetone, H2O, t BuOH e. DMP, acetone, p TsOH f. H2, Pd(C), MeOH g. HCl, MeOH Br O O 192 Br OH BnO b O O 193 Br OH O OBn O O Br O O OH OH OH OH OH OH HO HO HO HO OH OH OH OH O O OH OH OH OH OH HO HO HO HO OH O OH OH OH OH OH HO OH OH O O OBn OH O O O O O O b c-g 195 196 194 Br Br Br b-g Scheme 38. Synthesis of Lchiro -inositol trimer and tetramers by Hudlicky et al.171

PAGE 65

43 The three-dimensional structures of these oligomers has been studied theoretically, and it was found that thes e kinds of molecules are prone to forming helices.171 Molecular mechanics calculations were performed on dimer 181 (Scheme 34), trimer 192 (Scheme 38), and tetramer 196 (Scheme 38), as well as the hypothetical octamers of Lchiro inositol and of L-proline. The results (Figure 7) indicate that even the smaller fragments are prone to the formation of -turns. Furthermore, though the amino-acid oligomer is stabilized by hydrogen bonds, the similarity between the two octamer structures is striking. Figure 7. Molecular modeling structures of (a) 181 , (b) 195 , (c) 196 , (d) an Lchiro inositol octamer, and (e) an L-proline octamer170 In the same publication, the synthesis of O -linked pseudosugar conjugate 202 is reported. Alkene 198 was prepared from the corresponding diol ( 197 ) following standard

PAGE 66

44 procedure. Methoxycarbonylation of this alkene afforded ester 199 , which was reduced twice to generate primary alcohol 200 . The remaining steps involve using the synthesized alcohol to open bromoepoxide 129 , and the dimer generated is sequentially debrominated, dihydroxylated , and deprotected to affo rd pseudosugar conjugate 202 (Scheme 39). I O O O O I OH OH CO2Me O O O O O O O O HO Br OH O O O O O O O OH O HO HO HO OH OH OH OH OH a, b, a Reagents and Conditions: a. DMP, acetone, p TsOH b. OsO4, NMMO, acetone, H2O, t BuOH c. t BuLi, Et2O, CO2d. MeI, K2CO3, acetone c, d e, f g h, b, i e. H2 (50 psi), Pd(C), EtOH, EtOAc f. Dibal-H, toluene g. 129 , BF3-OEt2, CH2Cl2h. n Bu3SnH, AIBN, THF i. MeOH, HCl 197198 199 200 201 202 Scheme 39. Synthesis of an O -linked pseudosugar conjugate by Hudlicky et al.171 Polyhydroxylated tetrahydronaphthalene dimers were also synthesized by Desjardins et al. (Scheme 40).174, 175 Naphthalene diol 204 , obtained by whole cell fermentation of naphthalene ( 203 ), was first protected, then epoxidized ( 205 ), and finally opened with sodium methoxide to give secondary alcohol 206 (the epoxide was also opened with potassium hydroxide to afford the trans -diol). When alcohol 206 was reacted with another mol ecule of epoxide, dimer 208 was obtained after cleavage of the acetonides (likewise, when the trans -diol obtained from opening the epoxide with

PAGE 67

45 potassium hydroxide was treated with anothe r equivalent of epoxide, two dimers were obtained). OH OH O O O O O OH OMe O O O OMe O O HO O OMe HO 203 204 205 206 207 208 Reagents and Conditions: a. E. coli JM109 (pDTG601) b. DMP, acetone, p TsOH c. m CPBA, CH2Cl2d. NaOMe, MeOH e. 205 , BF3-OEt2, CH2Cl2f. THF, TFA, H2O a b, c d e f OH OH OH OH Scheme 40. Synthesis of a tetrahyd ronaphthalene dimer by Desjardins et al.174, 175 Figure 8. X-ray diffraction of naphthalene dimer 208 . (a) Three dimensional structure showing both hydrophilic and lipophilic regions. (b) Molecular drawing shown with 50% ellipsoids174 The stereochemistry of dimer 208 was confirmed by single crystal X-ray diffraction analysis (Figure 8, b), which also clearly indicated an aggregate with strong hydrogen

PAGE 68

46 bonding (Figure 8, a). The three-dimensional st ructure shows sheets in the ab plane. Among those sheets, a hydrophilic region (containing water molecules linked by hydrogen bonding) and two lipophilic regions (com posed of the aromatic portions of the molecules) are discernable. This suggests th at chelation of metals can occur via either hydroxyl groups or –chelation in the lipophilic region. A similar N -linked dimer was synthesized in 1997 by Lallemand et al. .176 This time, epoxide 205 was opened with sodium azide, and the azido group was reduced using triphenylphosphine. The aminoalcohol was then used as the nucleophile to open another molecule of epoxide, which afforded, after deprotection, dimer 211 . O O O O O OH NH2205 209 Reagents and Conditions: a. NaN3, DME, H2O b. PPh3c. 202 , t BuOH, 120 ° C, sealed tube d. THF, TFA, H2O a, b NH O O OH O O HO NH OH HO OH OH OH HO c d 210 211 Scheme 41. Synthesis of an amino-bri dged tetrahydronaphthalene dimer by Lallemand et al.176 Glycosidase Inhibition The role of carbohydrates and their corres ponding enzymes in biochemical systems has continued to be an area of interest for ma ny researchers. Of pa rticular interest are glycosidases, or enzymes that hydrolyt ically cleave glycosidic bonds, and the fundamental roles that these molecules play in biochemistry and metabolism.177 Biochemical research focused on glycomimetic s has shown especially promising results

PAGE 69

47 pertaining to diabetes control178, 179 and treatment of HIV.180 In particular, new techniques and synthetic pr otocols have spurred deve lopments in this field.181 Many researchers have therefore been trying to de velop sugar mimetics capable of glycosidase inhibition.182 There exists two types of glycosidases: exoglycosidases and endoglycosidases. The former cleave oligosaccharides one sugar at a time; these enzymes are responsible for the breakdown of starch and glycogen, fo r the biosynthesis and modification of glycosphingolipids, and for the catabolism of peptidoglycans and other glycoconjugates. On the other hand, endoglycosidases can cleave glycosidic bonds within an oligosaccharide; they are respons ible for alteration of cell wall s in bacteria and plants, as well as for the hydrolysis of highly insoluble polysaccharides such as chitin and cellulose. The enzymatic cleavage of a glycosidic bond will liberate a sugar hemiacetal that will either have the same configuration as the substrate (retention) or, less commonly, will have the opposite configuration (inversion). In 1953, Koshland proposed the catalytic mechanism for configur ation-retaining glycosidases.183 Despite few elaborations, this mechanism has remained unchallenged (Figure 9). O OH OR OH HO HO O O OO H B-HA O OH HO HO HO O O OO B A-H O H O OH HO HO HO O HO OO B-HA OH Figure 9. Catalytic mechanism for configura tion-retaining glycosid ases according to Koshland183

PAGE 70

48 In this mechanism, the aglycon is activat ed as a leaving grou p by coordinating the exocyclic oxygen with an enzymic acidic func tion (HA), which partially protonates the aglycon. A carboxylate (B-) then can attack in a more or less synchronous fashion, leading to a glycosyl ester intermediate. The degree of concertedness of the reaction (bond breaking vs. bond making) is unclear, though both extremes have been ruled unlikely. The aglycon then diffuses from th e active site and is replaced by a water molecule. The conjugate ba se of the catalytic acid (A-) then partially deprotonates this water, which then attacks the anomeric ca rbon and cleaves the newly formed glycosyl ester bond. Retention of stereochemistry is achieved therefore th rough double inversion. NH OH OH HO HO OR Nojirimycin 212 H N HO OH HOOH 1,4-Dideoxy-1,4-imino-D-mannitol (DIM) 213 N OH OH OH Swainsonine 214 N OH OH Swainsonine 215 N H O O N Kifunensine 216 HO HO HO HO HO OH Figure 10. Classic inhib itors of glycosidases Some of the typical glycosid ase inhibitors are shown in Figure 10. The activity of these and other potential inhibitors are t ypically monitored through UV assays. The assay is based on the hydrolysis of commercially-available p -nitrophenyl glycosides using the corresponding enzymes; the rate of hydrolysis is compared to the various inhibitor concentrations. The assay can be performed as an endpoint assay, whereby the enzymatic reaction is stopped by addition of a strongly basic buffer; at the same time, the

PAGE 71

49 p -nitrophenol is converted to its anion, which ha s a characteristic yellow color. A more advanced assay instead m onitors the generation of p -nitrophenol/ p -nitrophenolate as the reaction proceeds at 400 nm with a UV/Vis sp ectrophotometer. A detailed procedure for this assay is presented in the Appendix. Diaminoinositols Non-natural cyclitol derivatives have at tracted synthetic interest for many years because of their potential as glycosidase inhib itors or as probes for the investigation of the inositol phosphate cycle. Furthermore, many of these compounds have been found to exhibit antibiotic activity. NH2H2N OH OH OH NH2H2N OH OH OH HO O O NH2NH2NH2OH NHCH3HOO R H 219 , R = H 220 , R = CH3217218 NHAc NHAc OAc OAc AcO AcO OH OH NH2NH2HO HO 221 222 Figure 11. Unnatural diam inoinositol derivatives184-186 Typical structures of these aminoglycosid e antibiotics include 2-deoxystreptamine ( 217 ) and streptamine ( 218 ) as well as fortimicins ( 219 and 220 ). Guédat et al. have prepared 1,2-dideoxy-1,2-diaminomyo -inositol ( 221 ),184 while Kresze et al. synthesized 222,185 both of which were suggested as possi ble streptamine analogs. More recently,

PAGE 72

50 Arcelli et al. synthesized several 2,3-diamino c onduritols that showed good glycosidase inhibition (Figure 11).186 Inositol Signaling Pathways It is clear, from the number of manuscr ipts and publi cations on the subject, that inositols play an important role in cell signaling pathways.8, 187, 188 In the 1980Â’s, it was demonstrated that inositol phospholipids pl ay a part as secondary messenger in the phosphoinositide cascade.188, 189 Second messengers are in tracellular mediators of externally received ho rmonal messages, and are charac terized by Berridge as follows: Stimulation by hormones or neurotransmitters will result in a net increase in concentration or turnover of the second messenger. Once the extracellular agonist has been removed, a mechan ism must exist to turn off the internal signal. Internal signals responsible for altering cellula r activity must be activated by the second messenger. HO PO3 -2PO3 -2OH OH O P O O O OR2R1O 223 PIP2HO PO3 -2PO3 -2OH OH-2O3P OH OR2R1O 225 IP3224 DG PLC H2O GTP GDP Pi Figure 12. The key step of the inositol signaling pathway In spite of not identifying the system at play, the early work of Hokin and Hokin in 1952190 discovered such a system, and determined that acetylcholine stimulates the turnover of phosphatidylinositol in exocrine pancreas. In the process, th e turnover of phosphatidate also increased, yet the turnove r of the major membrane phospholipids did not. The key step in this mechanism involve s the hydrolysis (when an agonist binds to the receptor) of phosphatidylinositol 4,5-bisphosphate (PIP2, 223 ) to produce

PAGE 73

51 diacylglycerol (DG, 224 ) and inositol 1,4,5-trisphosphate (IP3, 225 ), both of which can then act as second messengers in the cell (F igure 12). This reaction is catalyzed by phospholipase C (PLC), a G protein. Figure 13. The synthesis and role of PIP2 in intracellular signali ng. Image property of John Wiley & Sons, Inc. PIP2 itself is synthesized by sequential phosphorylation (first catalyzed by PI kinase, then PIP kinase) of phosphatidylinositol (PI). PI is formed in the reaction of myo inositol and diacylglycerol cy tidine diphosphate (CDP-DG), a transformation catalyzed by PI synthase. Figure 13 illustrates how second messengers can induce a cellular response. After PIP2 is hydrolyzed by PLC, IP3 will diffuse through the cytoplasm to the endoplasmic reticulum (ER), where it will stimul ate the release of intr acellular calcium. The lipophilic DG will remain in the plasma membrane. If phosphatidylserine (PS) and calcium ions are present, DG will activate protein kinase C (PKC). Once IP3 and DG have completed their messenger functions, they get converted to myo -inositol and CDP-

PAGE 74

52 DG, which will then recombine to form PI. From this point, the cycle can then start again. Protein kinase C has a wide distribution in animal cells, and in particular, brain cells. The purpose of this pr otein is to activate various pr oteins by phosphorylating their serine and threonine residues. Interestingly, it was found that PKC can also be activated by phorbol esters ( 226 ). The activity of these esters stems from their three dimensional structure similarity with DG. However, phorbo l esters have a much stronger affinity for PKC than does DG; the KD of DG to PKC is ~ 20-100 µM, whereas that for phorbol esters ranges from 1-10 nM. Because of this activity, tumor-promoting phorbol esters have been the subject of many research endeavors. O HO HO OH OCOR OCOR 226 Figure 14. A general structure of phorbol esters Discussion Based on the work previously done on inositol dimers and oligomers, as well as the potential applications of th ese kinds of compounds (e.g., as metal-chelating agents, scaffolds in parallel or combinatorial synthe sis, heparin analogs, in sulin mediators, or glycosidase inhibitors), new targets could easily be devised. Using dimer 184 as a template, three new compounds beca me of interest (Figure 15).

PAGE 75

53 O HO HO OH OH NH3OH OH OH HO 184 O HO HO OH OH NH3OH OH OH HO HO Cl NH2HO HO OH OH OH OH OH OH HO HO 228 NH2HO HO OH OH NH3OH OH OH HO HO 229 Cl ClCl Cl 227 Figure 15. A previously synt hesized aminoinositol dimer ( 184 ) and three related targets The targets were modeled using molecular mechanics calculations, and were compared to known dimer 184 (Figure 16). As such, we set out to synthesize these compounds. Compounds 227191 and 228192 were the subject of two dissertations. Diaminoinositol dimer 229 is therefore the remaining dimer in this family.193, 194 Figure 16. Molecular modeling of dimers (a) 184 , (b) 227 , (c) 228 , and (d) 229 . Calculations were performed using SPARTAN 5.1.1 SGI version on a Silicon Graphics O2. Synthesis The general approach to the synthesis of these dimers can be related to epoxy resins (Figure 17). In these resins, a hardener containing amino groups is used to crosslink with an epoxide-containing linker. The electrophilic and energetically-str ained oxirane reacts with the nucleophilic amine to generate an aminoalcohol.

PAGE 76

54 O Linker O NH2Hardener H2N N N H a r d e n e r OH OH OH OH Linker Linker n Figure 17. The general st ructure of epoxy resins In other words, when an aziridine or epoxide ( 230 ) is treated with a nucleophile ( 231 ), a new compound ( 232 ) bearing a new nucleophile, XH, is generated. This nucleophile can then react w ith another el ectrophile ( 230 ); this process can, much like epoxy resins do, generate high molecu lar weight polymers (Figure 18). R R X X = O, NH, NR' Y = O, NH, NR' R'YH YR' R XH R 230 231 232 YR' R X R 230 233 R X R H n Figure 18. Generating high mol ecular weight polymers using epoxides and aziridines Therefore, we set out to investigate wh ether this process could take place using chiral epoxides (such as 129 and 178 ) and aziridines ( 234 and 235 ). The synthesis and relative stereochemistry of both of these compounds is well known. Bromobenzene ( 31 ) is oxidized via whole-cell fermentation of E. coli JM109 (pDTG601) to give cyclohexadienediol 32 with greater than 99% ee . Diol 32 is then protected as the acetonide ( 33 ), and the free olefin is oxidized using m CPBA or PhINTs/Cu(acac)2 to give epoxide 129 or aziridine 234 , respectively. Each can then be debrominated using tributyltin hydride and AIBN in THF. The sy nthesis of aziridines with other protective

PAGE 77

55 groups (such as CBM) is performed through a hetero-Diels-Alder reaction using diene 33 , followed by reductive cleavage of the N-O bond, and final cyclization using Mitsunobu protocol.195 CBM Br 31 Br OH OH 32 O O Br 33 O O Br O O Br O TsN 129 234 Reagents and Conditions: a. E. coli JM109 (pDTG601) b. DMP, acetone, pTsOH c. mCPBA d. PhINTs, Cu(acac)2e. nBu3SnH, AIBN, THF, reflux f. MeOCONHOH, NaIO4, H2O, MeOH g. Al(Hg), THF, H2O h. PPh3, DEAD, THF ab c d e e O O O O O TsN 178 235 Br O O N O 236 g, h O O N 237 CBM f Scheme 42. Synthesis of chiral epoxides and aziridines An early study of the aziri dine opening with a nucleophile was performed using debrominated tosyl aziridine 235 as well as debrominated carbomethoxyl aziridine 237 . First, aziridine 237 was treated with benzylamine in TH F with a variety of Lewis acids. Ytterbium triflate, trimethylsilyl triflate, tin tetrachloride, and bor on trifluoride diethyl etherate were used to catalyze the reaction. Only ytterbium trifla te and trimethylsilyl triflate generated diamine 238 in appreciable amount. In the other cases, only product decomposition (witnessed as greater than fi ve products could be seen by TLC) was

PAGE 78

56 observed. Because ytterbium triflate is much easier to handle, it was chosen as the catalyst for these reactions. When tosyl aziridine 235 was treated under the same conditions, diamine 239 was isolated in 75% yield. O O N Ts O O N CBM 237 235 Reagents and Conditions: a. Catalyst , BnNH2, THF, reflux b. Yb(OTf)3, BnNH2, THF, reflux a b O O HN BnHN CBM Catal y st Yb(OTf)3TMSOTf SnCl4BF3-OEt2Outcome product obtained product obtained decomposition decomposition O O HN BnHN Ts 238 239 Scheme 43. Model study for aziridine openings In order to generate the N -linked dimer ( 229 ), however, it would be necessary to debenzylate compounds 238 or 239 . Nevertheless, if a more simple nucleophile were used, such as azide or even ammonia, little further transformation would be necessary to have a diamino compound containing an accessible nucleophile. Therefore, the ytterbium(III)-catal yzed opening of bromoaziridine 234 was attempted using ammonia as the nucleophile. The reaction did indeed work, but the diamine generated ( 240 ) was too unstable to isolate; instead, in order to adequately characterize the new compound obtained, the crude reaction mixture was treated with acetic anhydride to generate both the monoacetamide ( 242 ) and the diacetamide ( 241 ) derivatives.

PAGE 79

57 O O TsN 234 Reagents and Conditions: a. NH3, Yb(OTf)3, THF b. Ac2O, Py, DMAP a O O NHTs H2N 240 Br Br b O O TsN N H Br O O NHTs N H Br Ac Ac Ac 241 242 Scheme 44. Aziridine opening with ammonia The reaction, which is quantit ative, and which was later optimized to require no solvent other than the ammonia itself (in a seal ed tube or using a cold finger condenser), was then applied to the synthesis of the target dimer ( 229 ). A retrosynthetic analysis of this compound therefore would establish that diamine 240 should open epoxide 129 , after which a series of oxidations and deprot ections would offer the desired dimer ( 229 , Figure 19) NH2HO HO OH OH NH3OH OH OH HO HO 229 Cl Cl NH OH OH NH2OH HO HO 243 Br Br O O NHTs H2N 240 Br O O O 129 Br Br 31 Figure 19. Retrosyntheti c analysis of dimer 229 With this strategy in mind, it became clear wh at course would be necessary for this synthesis. Furthermore, building on the obs ervations made on the stability of diamine 240 , it became obvious that a one-pot process, wherein the intermediate nucleophile

PAGE 80

58 would not be isolated, would be th e only viable approach to dimer 243 . Also, due to observations made in the laboratory that epoxide 129 , when mixed with Yb(OTf)3 in THF, would form a viscous polymeric material, it was also necessary to vary the solvent for the coupling reaction of diamine 240 and epoxide 129 . The reaction was first attempted in methylene chloride and benzene, and neither was successful. Because of its structural and intrinsic (e.g., polarity) similarities with te trahydrofuran, 1,4-dioxane was the next logical choice. The reaction was carried out as follows: In a thick walled tube equipped with a screw thread, ammonia (l) was condensed in such a way to occupy ca. one third of the tube volume, after which aziridine 234 and ytterbium(III) triflate as well as methylene chloride (as co-solvent to di ssolve the aziridine) were a dded at once, and the tube was sealed. The mixture was heated to 50 °C for 30 minutes, at which point no more starting material could be detected. The tube was ope ned to the atmosphere and the solvents were removed under reduced pressure. The dry sa mple was then redissolved in dioxane, and epoxide 129 was added. This second transforma tion was run at 60 °C for 3 hours, and afforded dimer 244 in > 75% yield. O O TsN 234 Reagents and Conditions: a. NH3, Yb(OTf)3, CH2Cl2b. 129 , dioxane a O O NHTs H2N 240 Br Br b 244 O O NHTs Br HN Br HO O O Scheme 45. Synthesis of dimer 244 The protocol used in the s ynthesis of this dimer was th erefore extremely successful not only because of the high yi eld of the reaction and because it did not allow for the

PAGE 81

59 intermediate diamine to decom pose, but also because it was more economical in terms of the catalyst used since one load wa s necessary for the entire process. In order to obtain dimer 229 , it would therefore be n ecessary to debrominate and osmylate the two double bonds of 244 . However, it is well know that amines are very susceptible to oxidation. It would then be n ecessary to protect the secondary amine prior to effecting the dihydroxylati on of the two double bonds. As it turned out, the steric bulk around this central nitrogen more than accounted for its expected increased nucleophilicity. Therefore, when 244 was treated with acetic anhydride in pyridine, only the O -acetate ( 245 ) was obtained. The central nitrogen would have to be protected using trifluoacetic anhydride in refluxing methylene ch loride/pyridine. Last, the two bromines were removed using tributyltinhydride in THF, affording diene 247 , and setting up the system for the following dihydroxylations (Scheme 46). Reagents and Conditions: a. Ac2O, Py, CH2Cl2, DMAP, RT b. TFAA, Py, CH2Cl2, DMAP, reflux c. nBu3SnH, AIBN, THF a b 244 O O NHTs Br HN Br HO O O 245 O O NHTs Br HN Br AcO O O 246 O O NHTs Br N Br AcO O O TFA c 247 O O NHTs N AcO O O TFA Scheme 46. Protection and debromination of dimer 244

PAGE 82

60 This iteration (i.e., first protection, then debromination) was preferable to first debromination, then protection, as it afforded a higher overall yield for the three step process (67% over three step s vs. 43% over three steps). The dihydroxylation was performed usi ng osmium(VIII) tetroxide and NMMO in acetone. The reaction was carried overnight , after which the crude bisdihydroxylated product was treated with DMP to protect the diols for the subseque nt reactions (Scheme 47). Reagents and Conditions: a. OsO4, NMMO, acetone b. DMP, CH2Cl2, pTsOH a, b 247 O O NHTs N AcO O O 248 O O NHTs N AcO O O O O O O TFA TFA Scheme 47. Dihydroxylation and protection of dimer 247 In the final steps, the acetate and acetam ides were first saponified (to give aminoalcohol 249 ). Then the tosyl group was reducti vely cleaved in sodium/ammonia, the acetonides were hydrolyzed in methanolic HCl, and the resulting hydrochloride was precipitated out of solution by addition of Et2O. The ratio of HCl to dimer 229 was determined by combustion analysis to be 1.3 (Scheme 48). In summary, the synthesis of dimer 229 , as its hydrochloride sa lt, was effected in 8 steps (10 operations), with an overall 7% yi eld from bromoaziridine 234 . The overview of the synthesis is provided in Scheme 49.

PAGE 83

61 Reagents and Conditions: a. NaOMe, MeOH b. Na, NH3c. HCl, MeOH, then Et2O a 248 O O NHTs N AcO O O O O O O b, c 249 O O NHTs HN HO O O O O O O NH HO HO OH OH NH2OH OH OH HO HO 229 1.3 HCl TFA Scheme 48. Final deprotections to afford dimer 229 as its hydrochloride Reagents and Conditions: a. DMP, acetone, p TsOH b. m CPBA c. PhINTs, Cu(acac)2d. NH3, Yb(OTf)3, CH2Cl2e. 129 , dioxane f. Ac2O, Py, CH2Cl2, DMAP 248 O O NHTs N AcO O O O O O O k-m NH HO HO OH OH NH2OH OH OH HO HO 229 1.3 HCl TFA 244 O O NHTs Br HN Br HO O O i, j 247 O O NHTs N AcO O O TFA Br OH OH 32 O O Br O O Br O TsN 129 234 a, c a, b d, e f-h g. TFAA, Py, CH2Cl2, DMAP, reflux h. n Bu3SnH, AIBN, THF, reflux i. OsO4, NMMO, acetone j. DMP, CH2Cl2, p TsOH k. NaOMe, MeOH l. Na, NH3m. HCl, MeOH, then Et2O Scheme 49. Overview of the synthesis of dimer 229

PAGE 84

62 Glycosidase Inhibition The protocol for the glycosidase inhibiti on studies was established by Bernhard Paul.192 Though the fine aspects of this experime nt are worth noting, the fact that they were discussed in that disserta tion precludes its inclusion in this section; only the results of the study will be discussed herein. Diaminoinositol dimer 229 was tested for its inhibition of six commerciallyavailable glycosidases: and -galactosidase, and -glucosidase, and and mannosidase. The inhibition curves as a func tion of inhibitor concentration are provided in Figure 20. On the whole, this particular dimer showed little inhibi tion of any of these six enzymes (IC50 > 1 mM in all cases). It only tu rned out to be a mild inhibitor of galactosidase and -mannosidase. 50 60 70 80 90 100 110 120 00.20.40.60.811.21.41.61.82Concentration of Inhibitor (mmol/L)Enzyme Activity (%) -galactosidase -galactosidase -Mannosidase -Mannosidase -Glucosidase -Glucosidase Figure 20. Normalized inhibition of co mmercially-available glycosidases by 229

PAGE 85

63 Calcium-binding affinity In order to understand the behavior of this compound as calcium is added to a solution, it was first necessary to perform a partial assignment of the structure in D2O. This assignment was done us ing long and short-distance 1H-13C correlations. The chemical shifts of the non-exchangeable protons are provided in Figure 21. NH OH OH NH2HO HO HO HO OH OH OH Formal C2 axis for top ring4.02 4.06 4.10 3.98 3.28 3.39 3.05 3.78 4.04 4.04 3.92 3.71 Figure 21. Partial 1H NMR assignment of dimer 229 . The formal C2 axis indicates that the chemical shift assignments of the t op ring may be inverted about that axis A solution containing calcium acetate (f ormed by mixing calcium chloride and acetic acid) was used to dope a NMR tube containing a solution of dimer 229 . The acetate in the sample was simply used to have a reference of the relative ratio of calcium to dimer throughout the experiment . If a coordination of dimer 229 with the calcium ions in solution were to happen, then a variation in chemical shifts of the dimer should be observed. However, this vari ation should not be constant fo r all of the signals; rather, because it is expected that as the dimer ch anges shapes to chelate to the metal ion, only some signals would vary noticea bly, whereas others would not. The known solid state coordination of dimer 184 with calcium ( vide supra ) prompted us to study the solution interact ions of this compound. When a NMR tube containing 184 was doped with the standard calcium acetate solution, the chemical shifts

PAGE 86

64 of the compound all shifted equivalently, indi cating that at the c oncentration used, the inositol derivative possessed no calcium-seque stering ability (Figur e 22). A plot of chemical shifts vs. concentration showed a sm all variation of the ch emical shifts (< 0.03 ppm for a calcium-to-dimer ratio of 5) for this dimer. The change in the chemical shifts during titration of CH’s 1-6 in these co mpounds also paralleled the temperature coefficients of the corresponding hydroxyl grou ps. Therefore, they simply reflect the change in ionic stre ngth of the solution. Figure 22. Calcium interaction study of dimer 184 . The top spectrum is in the absence of calcium, whereas the bottom spectrum is with excess calcium When the same experiment was perfor med on the newly synthesized dimer ( 229 ), a considerable difference was observed (Figure 23) from the previous experiment on dimer 184 . This time, a variable change in chemical shifts indicated that this compound was, in fact, a reasonable ligand of calcium. In this case, a net change in chemical shift (0.1 to 0.2 ppm) is observed for what would be the pr otons nearest the nitrogens in the molecule, whereas the protons assigned to the “periphery” of the dimer barely shifted at all. This is clearly an indication of the mo lecule binding with calcium. A graphical representation of

PAGE 87

65 this behavior is provided in Figure 24. However, a dissociation constant (KD) has not been established for this complexation. Nevertheless, Figure 24 shows a plateau (contrasted by a linear relationship at low Ca2+:dimer ratios), which qualitatively indicates a saturation of the dimer with calcium. Figure 23. Calcium interaction study of dimer 229 . The top spectrum is in the absence of calcium, whereas the bottom spectrum is with excess calcium -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 00.511.522.53 mol Calcium/mol dimer (ppm) 4.10 4.06 4.04 4.03 3.98 3.92 3.78 3.72 3.39 3.28 3.06 Figure 24. Graphical representa tion of the chemical shift changes associated with the binding of dimer 229 to calcium

PAGE 88

66 CHAPTER 3 7-DEAZA-ISOGUANINE AND DERIVATIVES Background The nucleobase pair inter actions in DNA, as desc ribed by Watson and Crick,196-198 follow two rules of complementarity: size comp lementarity (large purines (pu) pair with small pyrimidines (py)) and hydrogen-bondi ng complementarity (hydrogen-bond donors from one partner complement hydrogen-bond accep tors from another partner). A decade ago, Benner et al. noted that,199-201 following the Watson-Crick base-pairing rules, 12 nucleobases (6 base pairs) joined by mutu ally exclusive hydroge n-bonding patterns are possible (Figure 25). The use of an Artificially Expanded Genetic Information System (AEGIS) continues to shed light on th e idiosyncrasies of DNA (such as “Why are there only four letters?”)202 as well as prove their use in medici ne. By providing on demand molecular recognition similar to nucleic acids, but with a coding system that is orthogonal to the systems in DNA and RNA, AEGIS has enabled c linical assays such as Bayer’s branched DNA diagnostics tool that monitors the viral load in patients infected with HIV and hepatitis C viruses.203 Furthermore, AEGIS components were responsible for the enabling of an early detecti on assay of the SARS virus.204 With the emergence of the first six-letter PCR,205 AEGIS now supports the development of a synthetic biology where higher order processes of living systems, including reproduction and Darwinian evolu tion, are duplicated by designed artificial systems.

PAGE 89

67 N N N R O N N N N R N O H H H H H N N O R O N N N N R N N H H H H H N N O R N NN N N R O N H H H H H N N R N H H N H H NN N N R O O H H N N R O N H H N N N N R N O H H H N N R N H H O N N N N R O N H H HH donor acceptor acceptor acceptor donor acceptor acceptor acceptor donor acceptor donor donor donor acceptor donor donor donor acceptor donor acceptor donor donor donor acceptor acceptor donor donor acceptor donor acceptor acceptor acceptor donor donor acceptor acceptor py D AA py A D A py AA D pu A DD pu D A D pu DD A py D A D py DD A py A DD pu A D A pu AA D pu D AA Cytosine Guanine Thymine 2-amino-Adenine Figure 25. The twelve possibl e Watson-Crick type base-pai ring interactions containing three H-bonds However, while synthetic chemistry could overcome NatureÂ’s inability to generate 12 bases, some problems including epimerizat ion, oxidizability, and tautomerism of the non-standard nucleosides, remain.206-208 iso Guanine and iso Guanosine This first section on the native heterocycle and its derivatives will be used to highlight a variety of sy ntheses of the purine as well as to showcase interesting biological aspects and biotechnological applications of this compound. Because the synthesis of iso Guanine (isoG) is fairly representative of th e syntheses of purines in general, a few of these will be first considered for their historical as well as synthetic importance.

PAGE 90

68 Natural occurrence and synthesis Naturally occurring iso guanine (2-hydroxy-6-aminopur ine; 2-hydroxyadenine) was first isolated in 1932 by Cherbuliez and Bernhard209, 210 from the croton bean ( Croton tiglium L. ) as the D-riboside;211 because of its natural source, this glycoside was first named “crotonoside.” Iso guanine, however, was known as early as 1897, when Fischer noted that, unlike guanine,212, 213 it could not be converted to xanthine by treatment with nitrous acid.214 Iso guanine has also been isolated from the butterfly wings of Prioneris thestylis , where it was found to exhi bit antineoplastic activity.215 Furthermore, iso guanosine, its N1-methyl(doridosine, 252 ) and O2-methyl-derivative (spongosine, 253 ) have been found to be widespread am ong marine organisms, and have been extensively studied for thei r pharmacological properties.216, 217 Also, iso guanine has been found to be present among the other oxidized bases in DNA of normal as well as cancerous human tissues.218, 219 HN N N N R NH2O 250 , iso guanine (R = H) 251 , iso guanosine (R = D-ribose) N N N N R NH2O 252 , doridosine (R = D-ribose) H3C N N N N R NH2O 253 , spongosine (R = D-ribose) H3C Figure 26. Naturally-occurring iso guanine derivatives HN N N H N O NH2iso Guanine 250 HN N H N H N O O Xanthine 254 HCl/H2O Scheme 50. The conversion of iso guanine to xanthine220

PAGE 91

69 In 1939, Spies reported220 that it was possible to effect the transformation of iso guanine to xanthine us ing hydrochloric acid.221 This conversion has been used as proof of structure for iso guanine (Scheme 50). In 1949, Andrews et al.222 built on previous work by Holland et al.223 to effect the synthesis of iso guanine as well as its 9-D-glucopyranoside. In the synthesis of the heterocycle, 2-methylthioadenine (prepared according to Holland et al. ) was oxidized to generate the sulfone, which was s ubsequently hydrolyzed to afford iso guanine, 250 (Scheme 51). N N N H N NH2S N N N H N NH2MeS Reagents and Conditions: a. Cl2, H2O, then NH3 (aq.)b. NaOH, H2O, reflux a HN N N H N NH2O b 255 256 250 Me O O Scheme 51. Synthesis of iso guanine by Andrews et al.222 A year later, Shaw synthesized iso guanine from malononitrile in four steps (in the publication, preparations of xanthine, hypoxanthine, and aden ine are also described).224 The preparation calls for generating malonamidine dihydrochloride ( 258 ) from malononitrile ( 257 ), and then reacting it with benzen ediazonium chloride to afford phenylazomalonamidine dihydrochloride ( 259 ). The azo compound was then reduced with zinc dust in formic acid to genera te a mixture of formamidomalonamidine ( 260 ) and imidazole ( 261 ). Rather than isolat ing the two products, the remaining formamide was thermally converted to the imidazole . In the last step, imidazole 261 was treated with urea in a melt reaction to afford iso guanine ( 250 , Scheme 52).

PAGE 92

70 N N 257 Reagents and Conditions: a. EtOH, HCl, dioxane, then NH3, MeOH b. PhN2Cl c. HCO2H, Zn d. urea, melt NH2NH HN NH2258 * 2 HCl a b NH2NH HN NH2259 * 2 HCl N N Ph c NH2NH HN NH2260 * 2 HCl NH H NH2NH N NH2NH 261 d NNH N HN NH2O * 2 HCl 250 O Scheme 52. Shaw's synthesis of iso guanine224 Taylor et al. synthesized iso guanine in 1959 from isonitrosomalononitrile ( 262 ) and urea by way of nitrosopyrimidines 263 and 264 .225 The silver salt of isonitrosomalononitrile226 was first converted to the potassi um salt, then reacted with urea to give 2-hydroxy-4,6-diamino-5-nitrosopyrimidine ( 263 ) in 60% yield. Heating this pyrimidine with a mixture of formamide, formic acid, and sodium hydrosulfite227 effected the cyclization to generate 2-hydroxyadenine, 250 . In an effort to improve the yield of this synthesis, methyl isothiourea hydroiodide and the silver salt of isonitrosomalononitrile were mixed in metha nol (forming the methyl isothiourea salt of isonitrosomalononitrile), then heated in pyr idine to give 2-methylthio-4,6-diamino-5nitrosopyrimidine ( 264 ), which was directly converted to 2-mercaptoadenine ( 255 ) with the procedure described above. Treatment of 255 with hydrogen peroxide in ethanol

PAGE 93

71 gave iso guanine ( 250 ) in one step, which is a clear improvement over the two-step conversion used by Andrews et al. (Scheme 51). N N 262 Reagents and Conditions: a. KI b. urea c. formamide, formic acid, Na2S2O4d. methyl isothiourea hydroiodide, MeOH, then Py, heat e. H2O2, EtOH a, b N O Ag+N N NH2NO NH2HO 263 c HN N NH2O 250 N H N c N N NH2NO NH2MeS 264 N N NH2MeS 255 N H N d e Scheme 53. Synthesis of iso guanine by Taylor et al.225 In 1968, Yamazaki et al. synthesized iso guanine and its ribos ide as well as a variety of other nucleosides.228 4-Amino-5-imidazolecarboxamide (AICA, 265 ) was treated with phosphorous pentasulfide229 to afford 266 , which was fused with urea to give thioxanthine derivative 267 .224 Methylation of 267 with methyl iodide in alkaline solution gave 6-methylthio-2-hydroxypurine ( 268 ), which was aminated with ammonia in an autoclave at 130 °C to yield iso guanine, 250 (Scheme 54). N H N H2N O H2N 265 N H N H2N S H2N 266 N H N HN S N H 267 O N H N N SMe N 268 HO N H N HN NH2N 250 O Reagents and Conditions: a. P2S5b. urea c. CH3I d. NH3a b c d Scheme 54. Synthesis of iso guanine by Yamazaki et al.228

PAGE 94

72 In the same publications, the authors used similar conditions and treated 5-amino4-cyano-1-(2,3O -isopropylidene-D-ribofuranosyl)-imidazole ( 269 , prepared by dehydration of 5-amino-4-carbamoyl-1-(2’,3’O -isopropylidene-D-ribofuranosyl)imidazole with p -toluenesulfonyl chloride) with hydr ogen sulfide to afford 5-amino-4thiocarbamoyl-1-(2’,3’O -isopropylidene-D-ribofuranosyl)-imidazole ( 270 ). The acetonides in 270 were then hydrolyzed, and the resu lting nucleoside was treated with diethyl carbonate to give thioxanthosine derivative 271 . Finally, methylation followed by amination of the nucleoside gave iso guanosine, 251 (Scheme 55). 269 Reagents and Conditions: a. H2S b. H+c. EtOCO2Et, NaOEt d. CH3I e. NH3H2N NC N N O HO O O 270 H2N N N O HO O O H2N S 271 N H N N O HO OH OH HN S O 251 N N N O HO OH OH HN NH2O a b, c d, e Scheme 55. Synthesis of iso guanosine by Yamazaki et al.228 N N N H N NH2Reagents and Conditions: a. DEP, HCl, H2O b. NH3, H2O, 120 °C N HN N H N NH2EtO O EtOO HN N N H N NH2272 273 250 O Scheme 56. Synthesis of iso guanine by Leonard et al.230

PAGE 95

73 In their studies of the inte raction of diethyl pyrocarbonate (DEP) w ith nucleic acid components, Leonard et al. also synthesized iso guanine from adenine ( 272 ).230 Treatment of adenine with DEP generated 5(4)-carbethoxyaminoimidazole-4(5)N ’carbethoxy-carboxamidine ( 273 ) in 57% yield. When 273 was treated with concentrated aq . ammonia in a sealed flask at 120 °C, iso guanine was isolated in 71% yield (Scheme 56). In 1976, Yamazaki et al. published an updated synthesis of iso guanine and iso guanosine.231 For the synthesis of the nucleoside, the isopropylidene riboside of AICA ( 274 ) was treated with benzoyl isothiocyanate then methyl ated to give imidazole derivative 276 . 274 Reagents and Conditions: a. PhCONCS b. CH3I c. 0.1 M NaOH H2N N N O HO O O 275 HN N N H2N O 276 HN N N H2N O 277 N N N O HO O O HN NH2O a b c H2N O R HNS O Ph R NSMe PhO R = 2',3'O -isopropylidene--D-ribofuranose Scheme 57. Second synthesis of an iso Guanosine derivative by Yamazaki et al.231 The specific strength of the base used for converting 276 to iso guanosine derivative 277 was critical in that should more concentr ated sodium hydroxide be used instead, the guanosine derivative would be obtained. The authors propose a mechanism for this transformation which is shown in Scheme 58. The authors de scribe this process as an initial cyclization by the carboxamide oxygen of 278 to give the intermediate oxazine

PAGE 96

74 ( 279 ). Subsequent ring opening would give rise to imidazole 280 , which could tautomerize, then cyclize again to give the closed structure 277 . On the other hand, when stronger base is used, formation of aminonitrile 283 would be immediately followed by cyclization to give guanine derivative 284 . 276 HN N N H2N O R NSMe PhO 278 N N N O NH2R 282 HN N N H2N O R NSMe H OH N O N N N R NH2HN N C N N R N O H2N H N H C N N R N O H2N N H N N H2N O R N N N HN N R O H2N HN N N N R NH2O 279 280 281 277 283 284 0.1 M NaOH 6 M NaOH Scheme 58. The different effects of varied base strength on imidazole 276231 In 1979, Saxena and Bhakuni synthesized crotonoside by two methods, one of which was from iso guanine.232 Two years later, Pratt a nd Kraus reacted adenine with phenyl chloroformate at pH 4.5230, 233 to give a white precipitate that was characterized as iso guanine. A proposed mechanism for the tr ansformation is provided in Scheme 59; some of its aspects are similar to the Dimroth rearrangement of 6-aminopurines.234 More recently, Chern et al. synthesized iso guanosine in a one-pot process from 5amino-1-( -D-ribofuranosyl)imidazole-4 -carboxamide in 68% yield.235 The methodology

PAGE 97

75 employed, however, is extremely similar to that of Yamazaki et al. ,231 and only makes the distinction that DCC instead of NaOH is used . Nevertheless, the authors claim that this approach, because of its ease and high yield, is a novel entry into iso guanosine. N N N H N NH2PhOCOCl N N N H N NH2CO2Ph H2O N N N H N NH2CO2Ph H HO N HN N H N NH2CO2Ph H O HN N H N H N NCHO O HN N N H N NH2O 272 285 286 287 288 250 H2O Scheme 59. Oxidation of adenine to iso guanine by Pratt and Kraus230, 233 The latest approach to 2-hydroxya denine was published by Itaya et al. .236 The synthesis started with the N3-oxide of adenine237, 238 ( 289 ), which was methylated to give 3-methoxyadenine ( 290 ). This methoxy derivative wa s then hydrolyzed to give iso guanine, 250 . N N N H N NH2O 289 a N N N N NH2OMe 290 b HN N N H N NH2O 250 Reagents and Conditions: a. MeI, AcNMe2b. NaOH, H2O Scheme 60. Synthesis of iso guanine by Itaya et al.236 Tautomerism One of the first studies on the tautomerism of iso guanine was published in 1942 by Stimson.239 However, this work only investigated the ultraviolet absorption properties of

PAGE 98

76 the heterocycle at varying pH and compared them to those of adenine and 2-hydroxy-6,8diaminopurine. The author does not, how ever, draw clear conclusions on the tautomerism of 2-hydroxyadenine. Nearly twen ty years later, the advent of nuclear magnetic resonance allowed Jardetzky and Jard etzky to investigate the structures of purines, including iso guanine,240 but their studies did not lo ok at the tautomerism of the heterocycle. An answer on the tautomerism of 2-hydroxya denosine would not be provided until 1976, when Sepiol et al. investigated th e solvent-induced keto enol tautomerism of various 9-substituted heterocycles and nucleosides.241 Using a series of model compounds in solvents of varying ET(30) values,242 the authors found that the predominant form of iso guanosine in water is the N(1)H, 2-keto-6-amino form ( 291a ). Furthermore, the paper establis hes the tautomeric constant, KT, to be equal to 10 in water; this would therefore lead to a content of the enol form ( 291b ) of about 10% (Figure 27). HN N N N NH2O R N N N N NH2HO R R = 2'-deoxyribose kT = 10 291b 291a H2O Figure 27. The tautomerism of iso guanosine, as determined by Sepiol et al.241 This work was based on the fact that for a series of heterocyclic compounds, it had previously been shown243 that the logarithm of the tautomeric constant ( keto enol ) exhibited a linear dependen ce on the solvent parameter Z , which was introduced by Kosower.244-246 The polarity parameter ET(30) was later introduced, and was demonstrated to be a linear function of the parameter Z for various solvents. Because the

PAGE 99

77 parameter ET(30)242, 247 was determined for a broader vari ety of solvents, it is often more convenient to use by researchers. More recently, a number of computer-g enerated calculations on both the tautomerism248-250 and the properties251-255 of iso guanine and iso guanosine have been published, but few have experimental evid ence to back their claims. Among these publications, Jiang et al.252 suggest that the iso guanosine: iso cytidine base pair would be even more stable than the guanosine:cytidine base pair. At hear t in the tautomerism problem of isoG is the dilemma between th e assertion that nucleic acid bases exist predominantly in their respective keto -amino forms256-259 and fundamental rules of aromaticity (i.e., molecules will be aromatic if they can) coupled with the particular environment that the molecule is in (i.e., aqueous or gas phase). In 1995, Seela et al. investigated the substituent reactivity of iso guanosine and related nucleosides260 and applied the findings to draw conclusions on the tautomerism of these compounds. One of the driving forces for this publication wa s the observation that the synthesis of oligonucleotides containing iso guanine using phosphoramidite chemistry with the 2O -unprotected base was inefficient.261 Whereas protecting groups, such as the diphenylcarbamoyl262-266 or 4-nitrophenylethyl267 residues, had been used to improve the solid-phase synthesis using phosphoramidites. And whereas phosphonate chemistry268, 269 had also been used successfully to synthesize oligonucleot ides with the 2O unprotected iso guanine building blocks, the many problems associated with the incorporation of iso guanine residues warranted, in th e authorsÂ’s opinion, a study of the reactivity and tautomeris m of these nucleosides.

PAGE 100

78 N N N O HO OH HN NH2O N N N O HO OH N NH2O H3C N N N O HO OH N NH2O H3C N N N O HO OH HN N O OH H3CCH3N N N O HO OH HN NH2O OH NMe2N N N O OH N NH2O 291 pKa 3.45, 9.80 292 pKa 3.45 293 pKa 3.20 294 pKa 3.55, 9.70 295 pKa 1.35, 3.80, 9.65 296 pKa 3.95 OH Figure 28. Nucleosides synthesized by Seela et al. to investigate the tautomerism of 2Â’deoxyiso guanosine ( 291 ) and their respective pKa values. The lower pKa value corresponds to the pr otonation of the base a nd the higher one to the deprotonation.260 First, a series of acylations (with a vari ety of electrophiles and catalysts) and silylations indicated that the 2-oxo functiona lity was the most r eactive whereas the 6amino group of iso guanine nucleosides was found to be comparably electron-deficient. For the tautomerism study, which may be cons idered an extension of the previouslydiscussed Sepiol et al. publication,241 a series of derivatives having the H-atoms replaced by alkyl groups (thereby fixing the tautomeric states of the base moiety) were prepared (Figure 28). The conclusion drawn from bot h NMR and UV studies was that, apart from the 2O -substituted and the 3N -substituted derivatives, the iso guanine nucleosides exist in neutral aqueous phase predominantly as the 1 H /6-amino/2-oxo tautomers. Lastly, the authors corroborate the findings of Sepiol et al. that the tautomerism of iso guanine

PAGE 101

79 nucleosides is greatly influenced by the pol arity of the solvent, but fail to expound a value for KT. More recently, Robinson et al. analyzed, both by X-ray crystallography and NMR, the three-dimensional structure of an iso guanine-containing DNA dodecamer, d(CGC[isoG]AATTTGCG) (denot ed isoG-DODE), wherein iso guanine is base-paired with T in the duplex.270 Figure 29. Crystal structure of the comple x of Hoechst 33342 and isoG-DODE. (Top Left) The van der Waals drawing. DNA is shown in green and light orange, except for the two isoG:T base pairs, which are shown in red, with the Hoechst 33342 (yellow) covering the cen tral isoGAATT base pairs. (Top Right) Stereoscopic skeletal drawin g. The hydrogen bonds between the NH groups of the aromatic benzimidazoles of Hoechst 33342 and the A:T base pairs are shown as white dotted lines. (Bottom) 2 Fo-Fc electron density maps of the A5:T20, isoG4:T21, and isoG16:T9 base pairs.270

PAGE 102

80 The refined crystal structur e of the isoG-DODE comple xed with the minor groove binder Hoechst 33342 revealed that the two independent isoG:T base pairs in the dodecamer duplexes adopt different conforma tions (one Watson-Crick and one wobble, Figure 29, Figure 30). Although the isoG:T ba se pair was “forced” in this study, the results do offer considerable insight on the tautomerism of isoG in DNA, which may explain the facile incorporat ion of T across isoG during in vitro DNA replication.199, 267 The base-pairing properties of isoG will be discussed in greater detail in a later section. N N RO H O H H3C O N N N H H N N R Wobble (iG in the N1-H keto form) T iG N N H3C R O O H H N H O N H N N N R iG T Watson-Crick (iG in the O2-H enol form) N N H3C R O O H H N O N H N N N R iG T Watson-Crick (iG in the N3-H keto form) H N H N N H N N N H3C H O 297 Hoechst33342 Figure 30. Possible configurations of the isoG:T base pair with varying tautomeric forms of isoG. The chemical structure of Hoechst 33342.270 Most recently, Blas et al. have studied the tautomeric properties of iso guanine in the gas phase, in different pure solvents, and in the DNA e nvironment using theoretical methods.271 Though no experimental evidence is pr ovided to corroborate the theoretical results (which really only reaffirmed previous findings), the publication provides an interesting review of the properties of bot h the heterocycle and the nucleoside, and investigates the various ta utomeric forms possible for iso guanine (up to 23 tautomers are

PAGE 103

81 considered). Clearly, iso guanine and its nucleoside are uniqu e in that their behavior will vary so broadly depending on their environmen t. This attribute and its consequences have attracted considerable interest to modulate recogniti on, stability, and enzymatic susceptibility, among others. 2-Hydroxyadenine and DNA damage In the 1950Â’s, when the role of iso guanine was still unclear, researchers were trying to understand what purpose it played as a biomolecule. The work of Friedman and Gots in 1951 established that E. coli strain B could transform iso guanine to xanthine under aerobic conditions.272 Though this provided little info rmation on the purpose of this molecule, it offered at least insight into its metabolism (in 1961, Goodwin and Passorn tried to deaminate iso guanine with Rhodospirillum rubrum ,273 but could not conclusively determine whether they were succ essful). Also in 1951, Elion et al. , in searching for antagonists for nucleic acid c onstituents, found no activity for iso guanine in the assay.274 In 1968, 2-hydroxyadenine was es tablished for the first ti me as being one of the products of oxidative damage of adenine.275 When adenine was treated with hydrogen peroxide at concentrations less than 0.1 M, adenineN 7-oxide was obtained. At higher concentrations, two new products, namel y, 8-hydroxyand 2-hydroxyadenine were generated. Furthermore, when adenineN 7-oxide was irradiated under UV light, both adenine and 8-hydroxyadenine were obtained (Scheme 61). Interes tingly, however, when the three oxidation products were tested for th eir ability to induce poi nt mutations in the phage T4, none were found to be mutagenic base analogs. Nevertheless, the assay only employed the free bases; therefore, the induc tion of mutation when the damage is done within DNA remained, at that time, still under investigation.

PAGE 104

82 N N N H N NH2272 < 0.1 M H2O2N N N H N NH2298 O > 0.1 M H2O2N N N H N NH2299 N N N H N NH2250 OH HO h Scheme 61. The oxidation of adenine with hydrogen peroxide275 Though much work was performed on the subject of oxidative damage of DNA during the 70Â’s and 80Â’s, most of the work done on the oxidation of A and its implication on mutation has been done in recent years. In 1995, Kamiya and Kasai established that oxygen radical treatment (Fe2+ EDTA) of dA, dATP, and both singleand doublestranded DNA produced hydroxylation at the C-2 position of adenine.276-278 Before that, much emphasis had been placed on the oxida tive damage of DNA to generate purines oxidized at C-8 such as 8-hydr oxyguanine (one of the most commonly used markers of DNA damage)279, 280 and the G T mutation that it created in cells.281, 282 In this later work, not only is the oxidized product of ad enine residues in and out of DNA described, but so is the successful incorporation of 2-hydroxy-dAMP into DNA opposite C and T using polymerase and opposite T using the Klenow fragment of E. coli DNA polymerase I. Furthermore, the exonuclease-deficient Klenow fragment, as well as DNA polymerases and incorporated dCMP and dAMP in addition to dTMP opposite iso guanine in oxidatively-damage d DNA fragments (the mutant Klenow fragment also incorporated dGMP opposite the lesion). Further wo rk by the same group also investigated the effect of sequence on the misincorporat ion of nucleotides opposite 2-

PAGE 105

83 hydroxyadenine and found that alteration of the 5Â’and/or 3Â’flanki ng base(s) affected the incorporation of nucleotides.283 All of these results s uggest that formation of 2hydroxyadenine in DNA from A will induce A T, A G, and A C transversions in cells. N N N N N O R N N N N N O R iCM, pyAADT, pyADA H H H N O O H H H N O N H H N R CH3N R H 291b , enol 291a , keto R = 2'-deoxyribose Major Groove Major Groove Minor Groove Minor Groove CH3donor donor donor donor donor donor acceptor acceptor acceptor acceptor acceptor acceptorN N N N N R T, pyADA H H N O O H N R CH3A, puDA_ Major Groove Minor Groovedonor donor acceptor acceptor acceptor Figure 31. The standard A:T Watson-Crick ba se pair and the two possible isoG:T and isoG:isoCM base pairs. Because isoCM is not a natural nucleobase, Bukowska and Ku mierek assert that oxidation of A to isoG will not lead to mutations in DNA.284 In 1996, Bukowska and Ku mierek studied the miscoding properties of iso guanine in an AMV RT in vitro system.284 The authors performed competitive studies using the four standard nucleotides and found that iso guanine can pair with thymine as well as the non-standard base, 5-methylisocytosine (isoCM). They established th ese parings to have a ratio of 1:10, respectively. Interestingly, this ratio also corresponds to the tautomeric ratio of enol : keto established by Sepiol et al. ( vide supra ). The authors address the clear

PAGE 106

84 difference in their results from those of Kamiya et al. by first pointing out a fundamental difference in experiment: th e results obtained by Kamiya et al were performed under non-competitive conditions (i.e., only one dNTP could be tested at a time), and the polymerases used possessed no correcting abil ity. In spite of ac knowledging that more work needs to be done to study the behavior of iso guanine in DNA, Bukowska and Ku mierek assert that because T is the only na tural base that can pa ir with isoG in a Watson-Crick fashion (albeit in its minor ta utomeric form), formation of isoG in DNA should not lead to mutation by the simple mispairing mechanism (Figure 31). A year later, Kamiya and Kasai again studied the mutation frequency and the mutation spectrum of iso guanine when it is site-specifica lly incorporated into a unique restriction enzyme site in singleand doubl e-stranded vectors, a nd those vectors are transfected into E. coli .285 The authors observed that lit tle or no replication block was induced by the iso guanine residue. Further, they determined that this residue was indeed mutagenic (inducing substitution and deletion mu tations), and that the mutation spectra were affected by the sequence context and the strands upon which the base was located. That same year, the research was repeated in mammalian cells, and the same end results (with slightly different mutati on frequencies) were obtained.286 The authors therefore concluded that 2-hydroxyadenine is mutagenic in both eukaryotic and prokaryotic cells. More recently, the author s published a more thorough publication describing the formation of iso guanine with Fenton-type reagents in vitro and the consequences of this DNA damage on mutation.287 The repair of these mutations was st udied the following year by Tsurudome et al. .288 Recognizing that 8-hydroxyg uanine may be one of the major forms of oxidative

PAGE 107

85 DNA damage,279 that it is often considered a useful marker of DNA damage, and that its mechanism of repair has been reported by several researchers,289-295 they chose to compare the repair of 2-hydroxya denine to that of 8-hydroxyguanine in 7 week-old male Sprague-Dawley rat organs. The repair ac tivities were measur ed by an endonuclease nicking assay using 22-mer 32P-end-labeled double-strand ed DNA substrates, which contained either 8-hydroxyguanine (opposite C) or 2-hydroxyadenine (opposite T or C). In all cases, no nicking of the 2-hydr oxyadenine was detected, while the 8hydroxyguanine was clearly detected under the same conditions. Furthermore, ferric nitrilotriacetate (Fe-NTA) tr eatment, which is known to increase 8-hydroxyguanine repair activity,296, 297 failed to induce 2-hydroxyadenine nick ing activity. Although the repair of 2-hydroxyadenine may be done through another mechanism, these results suggest that iso G residues are not repaired by the glycosylase type mechanism in mammalian cells. In 2000, Kamiya and Kasai tested the E. coli MutY and MutM proteins for their ability to act as a 2-hyd roxyadenine glycosylase.298 They found that, irrespective of the base in the complementary strand, iso guanine was a very poor substrate of MutY. Moreover, a strand containing A or G opposite isoG was rarely cleaved with MutY. Last, the cleavage of oligonucleot ides containing isoG by MutM was not observed. The authors conclude that in E. coli , neither MutM nor MutY play an important role in the removal of isoG from DNA (however, a mo re recent publication ha s characterized an E. coli MutY protein that possesses 2-h ydroxyadenine DNA glycosylase activity299). In stark contrast to this work, Ohtsubo et al. identified, in 2000, a human MutY homolog (hMYH) that possessed both ad enine and 2-hydroxyadenine glycosylase activities.300 Previously, Fujikawa et al. reported that 2-hydroxydATP is efficiently

PAGE 108

86 hydrolyzed to 2-hydroxy-dAMP by hMTH1 (which had previously been identified as 8oxo-dGTPase); this indicated that some orga nisms come equipped with the ability to eliminate isoG from their nucleotide pools.301 However, since isoG forms a stable base pair with T in DNA, it was speculated that this pair may escape repair.270 Nevertheless, Jaruga and Dizdaroglu reported, in 1996, that isoG detected in DNA of human cells after hydrogen peroxide exposure is repaired to background level w ithin 4 h, indicating that human cells possess repair activ ity for 2-hydroxyadenine in DNA.302 This work by Ohtsubo et al. identifies at least one of the possible enzymes responsible for this repair. Among the most recent publications discus sing oxidative damage of DNA, Giray and Hincal have established a relationship between iodine defi ciency (such as is found in goitrous children) and the increase in the number of mutations found due to oxidative stress.303 Also, Limoli et al. determined that chronic oxidativ e stress derived in part from dysfunctional mitochondria—and not elevated le vels of reactive oxygen species (which would correlate directly with the levels of oxidatively-damaged DNA)—was responsible for the decreased stability of cells.304 N N N O O NH2300 Figure 32. The structure of tirapazamine303 Last, tirapazamine ( 300 ), a bioreductively activate d DNA-damaging agent that selectively kills the hypoxic cells found in solid tumors, has been found to generate, among other oxidized species of DNA, iso guanine.305

PAGE 109

87 Finally, Moser and Prudent have recently reported that the excision repair machinery of a thermophilic bacterium ha s been shown to recognize and repair mismatches found between non-standard base pairs.306 Thermus aquaticus DNA polymerase will remove mismatched natural bases opposite isoG and replace them with non-natural isoCM to form an expanded ba se pair. Furthermore, DNA ligase will covalently attach the strands when a nick formed by polymerase between two nonstandard bases is found. This work indi cates that cellular replication and repair machinery of an organism containing AEGIS could recognize and pr operly repair a site containing unnatural bases. Base-pairing of 2-hydroxyadenine and duplex structure The first enzymatic incorporation of a nucleotide with a novel hydrogen-bonding pattern was published in 1989 by Switzer et al. .199 This ground-break ing work, in which both DNA (Klenow enzyme) and RNA (T7) pol ymerases were used to direct the incorporation of iso guanosine into an oligonucleotide opposite iso cytidine in a DNA template, demonstrated for the first time that the general concept of an Artificially Expanded Genetic Information System could be applied in vitro . A year later, the concept was expanded even further to other base pairs,200 and later to more complex processes, such as the incorporation of a non-standard amino-acid into a peptide through AEGIS.307 Four years later, Tor and Dervan dem onstrated the site-specific enzymatic incorporation of N6-(6-aminohexyl) iso guanine opposite isoCM into RNA.308 This work was particularly significant because the authors discovered that T7 RNA polymerase neither incorporate isoG de rivatives opposite T when ATP was present, nor did they

PAGE 110

88 incorporate A opposite isoCM when isoGTP was present. This work falls well with the previously-discussed resu lts of Bukowska and Ku mierek. That same year, Switzer et al. studied the enzymatic recognition of the isoC:isoG base pair.267 This time, several different polymerases were used and thei r behavior noted. When isoC was in the template, T7 RNA polymerase, AMV reverse transcriptase, and the Klenow fragment of DNA polymerase all co rrectly incorporated isoG opposite isoC, whereas T4 DNA polymerase did not, and seve ral other polymerases also incorporated isoG opposite T. On the other hand, both is oC and T were incorporated opposite isoG when Klenow fragment was used, and only U was incorpor ated opposite isoG when T7 RNA polymerase was employed. These results o ffer valuable information on some of the idiosyncrasies of polymerases such as the bi as that they may generate toward a certain tautomer of a nucleobase present in the template. In 1995, two different accounts validated the stability of the isoG:isoC (or isoG:isoCM) base pair. First, Horn and co workers studied the hybrid ization properties of the isoG:isoCM base pair in synthetic oligodeoxynucleotides.309 The authors determined that the base pair is isoenergetic with the na tive G:C base pair (a re sult slightly different from the theoretical data252) and that each base can effectively discriminate mismatches. Second, Roberts et al. , compared the melting temperatur es of three DNA/RNA duplexes, each containing a central isoG:isoC, G:C, or A:U base pair, and found that the stability (expressed as a function of melting temperat ure) of the DNA/RNA duplex containing the isoG:isoC base pair was the same as that of the duplex containing the G:C base pair.310 Two years later, Roberts et al. studied the theoretical and experimental implications of an isoG:isoC base pair.311 Once again, both theoretical and experimental data showed

PAGE 111

89 that the isoG:isoC and G:C base pairs are is oenergetic. However, the authors found that the next most stable unnatural pair is that formed between C and isoG, and that this pair also happens to be as stable as a U:A base pair. Furthermore, ab initio studies showed that isoG may form an unprecedented imino oxo tautomer to favor that particular base pair (Figure 33). The im plications of these results are discussed in terms of the viability of a six-letter genetic system containing isoC and isoG. In short, the authors see no problem with such a system other than the possible problems associated with proofreading the C:isoG mismatch because it adopts a Watson-Crick geometry as opposed to the G:U (or G:T) mismatch, which is a Wobble. OH N N N H H R H O N N N H N N R iC iG N N O N R H O N N N H N N R iG H H NN OHN N N N N O R OH R U G MATCH Watson-Crick geometry OH N O N R HN N N H N N R MISMATCH Watson-Crick geometry MISMATCH Wobble geometry MATCH Watson-Crick geometry H H H C U A Figure 33. Two possible Watson-Cric k geometries are possible for iso guanine. The G:U mismatch, unlike the isoG:C mism atch, adopts a wobble geometry.311 In 2001, Kawakami et al. investigated the thermodynamic stabilit y of base pairs between iso guanine and incoming nucleotides depending on the location of isoG base within the sequence (i.e., in the middle or at the end).312 The results obtained indicated that when isoG was located at the end, the stabilities of the duplexes (indicated by

PAGE 112

90 melting temperatures) were dependent on the se quence. In this study, the authors only looked at the selectiv ity of misincorporation opposite is oG. When the sequence was 5Â’G[isoG]C-3Â’, the order of stability of the is oG:N base pair was T > G > C >> A. When the sequence was 5Â’-T[isoG]A-3Â’, the order of stability for the same base pair was T > A > C > G. Furthermore, the T/G/C and T/A in corporation opposite isoG in the respective sequences above agree with the tendency of mutagenic misincorporation of the nucleotides opposite isoG in vitro . In 2003, Maciejewska et al. looked at the effect of temperature as well as neighboring bases on the base-pairing of iso guanine.313 The authors assume that the efficiency of incorporation of isoCM and T op posite isoG is a measure of the tautomeric ratio of keto and enol species, respectively, within the Thermus aquaticus DNA polymerase active center ( c.f. the work of Bukowska and Ku mierek284). On the basis of experiments with four sequences at various temperatures, the au thors found that 3Â’neighbors decrease the fraction of the keto tautomer (and therefor e increase the number of isoG:T mismatches) in the order C > G T, whereas temperature apparently did not influence the tautomeric equilibrium of isoG. Iso guanine-containing oligonucleotides have been known to generate interesting structures. Of those is the parallel-stranded structure of DNA containing isoCM (or isoC) and isoG (Figure 34).264, 314-316 It has been reported that strong base pairs such as the parallel isoG:C and isoC:G will help favor pa rallel DNA, especially when the oligomer is rich in A:T base pairs.314, 317

PAGE 113

91 A T C H N N H N O H N N N N O H N N N O H N N N N NH O N N NH N N N N O H O H N H H H H iG iC G O O PO O O OPO O O O O PO O O O O PO O O O O P O O O O P O O O O O P O O O O O P O O O 5' 3' 5' 3' ANTIPARALLEL A T C H N NHN O H NN N N O H N NN O H N N N N NH O NN NH N N N N O H O H N H H H H O O P O O O O P O O O O O P O O O O O P O O O O O P O O O O P O O O O O P O O O O O P O O O PARALLEL 5' 3' 5' 3' iG GiC Figure 34. Schematic representation of antip aralleland parallel-oriented Watson-Crick base pairs.264, 314-316

PAGE 114

92 Another interesting aspect of iso guanine is its propensity to form quartets265, 315, 317319 and quintets265, 320-323 under certain conditions (Figure 35). In a practical sense, this means that DNA oligonucleotides containing shor t runs of isoG may have a propensity to aggregate, and may therefore affect the physi cal behavior of the oligomer, such as its migrating time on ion-exchange HPLC.317, 318, 324 Furthermore, it wa s found that certain isoG tetrads are more stable with regard s to their thermal denaturation and their resistance against enzymatic phosphodiester hy drolysis than tetrads formed from G.318 N N N N O N HH R N N N N O N H H R N N N N O N H H R N N N N O N H H R N N N N O N H H R H H H H H M+NN N N O N H H R N N N N O N H H R H H N N N N O N H H R N N N N O NH H R H H M+ Figure 35. DNA quartets (left) and quintets (right), as formed by isoG, are anomalous structures that may affect the behavior of the oligomer as a whole. The metal ion (M+) can be sodium, cesium, potassium, etc. 265, 315, 317-323 Why is iso guanine not part of the genetic code? The problems associated with evolution and information transfer have been discussed for many years, particularly as they refer to the four canonical DNA bases.202, 325, 326 In general, there are four constraints to a successful incorporation of a new base pair: 1. Chemical stability. The base should not decompose readily. 2. Thermodynamic stability. The nucleic aci d structure should not be negatively affected by the new base.

PAGE 115

93 3. Enzymatic processability. Polymerases shoul d be able to accept the base pair as substrate, recognize the pattern, and carry ou t the process of addition to the primer. 4. Kinetic selectivity. The hydrogen-bonding pattern should be orthogonal to that of other bases. In the case of iso guanine, the first two requirements seem to be met. However, the last two, which may be referred to as re plicability of the code, would become problematic. Though some enzymes have be en found to adequately incorporate the isoG:isoC base pair, that be havior is not universal ( vide supra ). Furthermore, it is mostly in the selectivity field that the isoG:isoC ba se pair clearly lacks. And the problematic recognition is primarily attributed to the tautomerism of these bases, particularly iso guanine. Even if, at best, a correct inco rporation of isoC oppos ite isoG (and vice versa) occurs 90% of the time, this interaction is not sufficiently specific in the sense that it is observed for the rest of the DNA bases. Inasmuch as a genetic system whereby only few mutations over a certain time (e.g., 1 in 105 or more replicati ons) are desired, the existence of a base pair that would generate considerably more changes, might have, over time, been selected against. Applications in biotechnology One of the most significant applications of AEGIS in biot echnology is in the sandwich assay (or branched DNA, bDNA, assay) for the detection of DNA.203 Whereas in the original bDNA assay, na tural interference from adve ntitious DNA barely afforded a signal-to-noise ratio (S/N) that could be analyzed, the use of AEGIS in the selfassembly pieces of the machinery increased the S/N by five or ders of magnitude. Such an assay enables real-time monitoring of viru s loads in patients infected with HIV and hepatitis.

PAGE 116

94 Analyte DNA Capture Strand Branched DNA Signal Molecules NSBs here improve the signal-to-noise ratio Solid Support NSB-containing duplex Figure 36. The bDNA assay, such as the Versant® HIV-1 RNA assay (marketed by Bayer), benefits from the use of AEGIS to produce a five-fold increase in S/N over the assay without AEGIS.203 7-Deazaiso Guanine and 7-Deazaiso Guanosine N N N N R N HH O N N N N R N HH O 291a , keto 291b , enol Problem: cross-conjugated imidazole ring Problem: less favored enol form N N N N N H H O H H H 301 R R = 2'-deoxy-D-ribose Figure 37. The two iso guanine tautomers each have probl ems not found in the native guanine heterocycle ( 291 ). Some of the intrinsic characteristics of iso guanine (such as its tautomerism problem) may warrant a closer look, particularly in terms of how it may be improved upon as a nucleobase. As it has already been discussed, perhaps one of the clearest

PAGE 117

95 problems in iso guanine (and its nucleoside) is the competition between having a fully aromatic imidazole ring and a keto form of the tautomer (Figure 37). Therefore, it may be possible to favor the keto tautomer by destabilizing the enol tautomer. In other words, if a 5-membered heterocyclic ring that was less aromatic than imidazole were to replac e that same ring in the iso guanine system, the equilibrium might shift toward the keto form. Given that pyrrole is onl y 59% as aromatic as benzene, whereas imidazole is 64% as aromatic as benzene (Figure 38),327 the first suggestion for a replacement of iso guanine might be its 7an d/or 9-deaza-derivatives ( 302 and 303 , respectively). S H NO >>> 100665943 N 86 N N 73 N H N 64 > >> N N N N HH H O R N N H N N HH H O R 7-deazaiso guanine 302 (R = H) 9-deazaiso guanine 303 (R = H) Figure 38. Aromatic and heteroaromatic co mpounds and their relative aromaticity (% aromaticity based on benzene)327 as well as possibly less problematic derivatives of iso guanine. However, 7-deazaiso guanine (C7isoG) might be a more versatile derivative because it possesses a N -glycosylic linkage (which is more easily synthesized). Also, the absence of the nitrogen at the 7-position would allow for func tionalization at that carbon, which may be useful to append functional grou ps or labels (such as biotin) for possible molecular biology using that nucleotide. Furthermore, the 7-deaza-derivative would be

PAGE 118

96 less susceptible to non-Watson-Cr ick interactions (such as H oogsteen base-pairing) of the nucleosides. Synthesis The synthesis of 7-deazaiso guanine was first published by Davoll in 1960.328 Bromoacetaldehyde diethyl acetal ( 304 ) is reacted with ethyl cyanoacetate ( 305 ) under basic conditions to disp lace the bromine. This affords cyanoester 306 , which is transaminated and dehydrated to afford dinitrile 307 (Scheme 62). Dinitrile 307 is then reacted with O -methylisourea hydrogensulfate, which is neutralized stoi chiometrically using sodium ethoxide, to afford pyrimidine 308 . Acetal 308 can then be hydrolyzed to heterocycle 309 , which is finally taken to iso G ( 302 ) using refluxing concentrated hydrochloric acid (Scheme 62). O O Br + N O O N O O N N EtO OEt EtO OEt N N ONH2O O NH2N N O NH2N H HN N O NH2N H Reagents and Conditions: a. K2CO3, NaI b. NH3, MeOH c. P2O5, Et3N, PhH a b, c d e f d. O -methylisourea sulfate, NaOEt, EtOH, reflux e. 1 M HCl, RT f. conc. HCl, reflux 304 305 306 307 308 309 302 Scheme 62. Synthesis of 7-deazaiso guanine328

PAGE 119

97 Seela et al. have done considerable work on the nucleoside of 7-deazaiso guanine. It seems apparent that coup ling of the free heterocycle ( 302 ) using typical methodology may not be practical, as there is no report of th is direct reaction (thi s has been confirmed, as will be described shortly). The first attempt was made using the O -methyl protected nucleoside.329 Nucleoside 310 was treated with 7 M HCl in refluxing dioxane. The reaction affords the desired nucleoside ( 311 ) in marginal yield (59% reported on a 300 mg scale), but the authors warn against the use of aqueous acid, as in this particular case, only isomerization was observed (Scheme 63). N N O NH2N OOH HOHN N O NH2N OOH HO 311N N O NH2NOH HO OH isomerization 310 Reagents and Conditions: a. HCl, water b. HCl (conc.), dioxane, reflux a b 312 Scheme 63. First synthe sis of 2Â’-deoxy-7-deazaiso guanosine by Seela et al.329 SeelaÂ’s other two syntheses of 2Â’-deoxy-7-deazaiso guanosine follow similar methodologies. They also employ the simple coupling of a heterocycle with halogenose 320 . In both cases, the syntheses start with 7deazaguanine. After a series of steps, the desired nucleoside ( 311 ) is obtained (Scheme 64). In a first attempt, 7-deazaguanine ( 313 ) is converted to 2-amino-4-ch loro-7H-pyrrolo[2,3-d]pyrimidine ( 314 ) by treatment with benzyltriethylammonium chloride.330, 331 Antimony(III) chloride is then used to obtain the dichloropurine ( 315 ).332 Purine 315 is coupled with halogenose 320 to afford nucleoside 316 , which undergoes aminolysis to gi ve 6-amino-2-chloro nucleoside 317 .333

PAGE 120

98 This last nucleoside is finally photolyz ed in water to give 2’-deoxy-7-deazaiso guanosine, 311 .334 The overall yield of the prepar ation (from 7-deaza guanine) is 6%. A better synthesis of 2’-deoxy-7-deazaiso guanosine still goes through 2-amino-4chloro-7H-pyrrolo[2,3-d]pyrimidine ( 314 ). This time, the authors chose to first form the nucleoside ( 318 ), which then undergoes aminolysis (giving 319 ), and finally selective deamination with NaNO2 in aq. acetic acid.319 The overall yield of the preparation (from 7-deazaguanine) is 27%. N N N H H2N OH N N N H H2N Cl 313 7-deazaguanine Sigma D1035 250 mg -$ 209.30 aN N ClNH2N OOH HON N ClClN OOTol TolO eHN N O NH2N OOH HO 311 dN N ClClN HcN N H2NClN OOTol TolO fN N H2NNH2N OOH HO gh bOOTol TolO 320 Cl Reagents and Conditions: a. R4NCl, CH3CN, reflux b. SbCl3, t BuNO c. Bu4NHSO4, then 320 d. NH4OH, 60 °C e. h, water f. NaH, DMF, then 320 g. NH3, dioxane, 120 °C, 3 days h. NaNO2, aq. HOAc 314 315 316 317 318 319 Scheme 64. Syntheses of 7-deazaiso guanosine by Seela et al.319, 334

PAGE 121

99 It seems apparent that the authors could not solve the problem of deprotecting the methyl ether in nucleoside 310 , and so had to turn to more complex alternatives for the synthesis of a deceivingly simple molecule. Behavior Though the tautomeric behavior of 7-deazaiso guanine has not been determined, the Seela laboratory, among others, has studied this nucleoside extensively. In 1997, Seela and Wei established that oligonuc leotides containing consecutive 7-deazaiso guanine residues form self-assembled quartets, indicating that the purine N7 of isoG is not participating in the hydrogen bonding patterns.319 Also, in 1999, Seela et al. established that the 2Â’-deoxynucleoside of C7isoG was in the N1-H2-keto-6-amino tautomeric form in the solid state (Figure 39).335 And more recentl y, the base-pairing properties of C7isoG have also been studied.264 Figure 39. Perspective view of 7-deaza-2'-deoxy iso guanosine showing the atomic numbering scheme. Displacement ellip soids of non-H atoms are drawn at 50% probability. H atoms are shown as spheres of an arbitrary size.335

PAGE 122

100 Synthesis of Indoles Since 7-deazaiso guanine may be either regard ed as a deazapurine or as a diazaindole, the synthesis of indol es may be considered to be at the heart of this problem. A brief review of some of the classical a nd more modern approaches to indoles will therefore be considered. The Bartoli indole synthesis (1989) The Bartoli indole synthesi s reacts 2-nitroarenes ( 321 ) with vinyl Grignard to afford the corresponding 7-substituted indole ( 322 ).336 The reaction proceeds first via a nucleophilic attack of the nitroarene by th e vinyl Grignard, followed by a series of sigmatropic rearrangements and nucleophilic displacements. The reaction uses a total of three equivalents of vinyl Grignard for every molecule of indole generated (Scheme 65). R N 321 R = Me, F, Cl, Br, OTMS, etc. O O a Reagents and Conditions: a. CH2CHMgBr, THF, -78 °C H N R 322 Scheme 65. The Bartoli indole synthesis336 More recently, Fonseca et al. have used this methodology for the synthesis of indole 324 in an efficient (70% yiel d) one step conversion from ortho -bromonitro derivative 323 (Scheme 66).337 Indole 324 was found to inhibit bo th varicella-zoster virus (VZV) and cytomegalovirus (CMV) replic ation at a concentra tion ca.5to 10-fold lower than the cytotoxic concentration (MCC or CC50), when tested in human embryonic lung (HEL) cells.

PAGE 123

101 Br NO2CO2CH3a Reagents and Conditions: a. CH2CHMgBr, THF, -78 °C Br CO2CH3H N 323 324 Scheme 66. Use of the Bartoli methodology by Fonseca et al. for the construction of complex indole structures337 The Bischler-Möhlau indole synthesis (1881) This classical approach to indoles uses aniline ( 326 ) and bromoacetophenone ( 325 ) to afford the 2-substitued indole ( 327 ).338 The reaction proceeds first through the formation of a Schiff base, followed by ther mal nucleophilic displacem ent of the bromide by the “aniline” aromatic ring, and final el imination and tautomerism (Scheme 67). Br O 325 NH2+H N 326 327 Scheme 67. The Bischle r-Möhlau indole synthesis338 In 2002, Golob et al. used this methodology to synthesize precursors of 2phenylindole-based inhibitors of steroid sulfatase (Scheme 68).339 Br O 328 NH2+ a H N 329 330 Me MeO OMe O MeO O OMe Me Reagents and Conditions: a. PhNMe2, Xylenes, Scheme 68. Synthesis of an indole derivative by Golob et al.339

PAGE 124

102 The Borsche-Drechsel cyclization (1858) Though it may be one of the oldest synthe ses of indole derivatives, the BorscheDrechsel cyclization340 is often simply looked at as a Fischer indole synthesis because of their resemblance. N H N Reagents and Conditions: a. HCl, a N H 331 332 Scheme 69. The Borsche-Drechsel cyclization340 Jiricek and Blechert, in 2004, have use this methodology in their enantioselective synthesis of (-)-gilbertine ( 335 ), a member of the Aspidosperma alkaloids, in a 17-step sequence with a 5.5% overall yield (Scheme 70).341 N N H N Reagents and Conditions: a. p TsOH, toluene, reflux a N H 333 334 O OTPS O OTPS (+cis) N H O 335 Scheme 70. Jiricek and Blechert' s synthesis of (-)-gilbertine via a Borsche-Drechsel cyclization The Fischer indole synthesis (1883) The Fischer indole synthesis342 may be regarded as one of the more classical approaches to indoles. In it s simplest form, phenylhydrazine ( 336 ) is reacted with an

PAGE 125

103 aldehyde or ketone ( 337 ), generating the hydrazone, which is rearranged into indole 339 by treating it with acid (lewis or otherwise, Scheme 71). N H NH2Reagents and Conditions: a. ZnCl2a N H R2R1336 339 N H N R2R1338 O R2R1337 + Scheme 71. The Fischer indole synthesis342 Campo et al. has used this methodology for the s ynthesis of iodina ted aryl indole 342 , which was then used to generate fused polycycles (Scheme 72).343 N H NH2Reagents and Conditions: a. MeSO3H a N H 340 342 341 + I I H O Scheme 72. Synthesis of 3-substituted indoles toward the generation of fused polycycles by Campo et al.343 The Gassman indole synthesis (1974) In the prototypical Gassman synthesis,344 aniline is sequentially reacted with t butylhypochlorite (to give the N -chloroaniline 343 ), methylthio-2-propanone (generating the sulfonium ion 344 through nucleophilic attack of the sulfur atom onto the N chloroaniline), and finally triethylamine (e ffecting the [2,3]-sigm atropic rearrangement and deprotonation/eliminations) to finally generate indole 345 . The latter can then be desulfurized using Raney-Ni to give 2-methylindole, 346 (Scheme 73).

PAGE 126

104 NH2Reagents and Conditions: a. t BuOCl b. methylthio-2-propanone a N H 326 345 NH b 343 Cl S N H 346 N H 344 S O Cl c d c. Et3N d. Raney-Ni Scheme 73. The Gassman indole synthesis344 In their work on (+)-paspalicine and (+)-paspalinine, Smith et al. used a modified version of the Gassman synthesis to generate the indole core (rings A and B) of their natural products targets (Scheme 74).345 Reagents and Conditions: a. tBuOCl, PhNH2, Et3N b. RaNi, EtOH c. pTsOH, PhH, reflux O MeS O O N H a-c 347 348 N H O O O R 349 , R = H, (+)-paspalicine 350 , R = OH, (+)-paspalinine O O Scheme 74. Application of the Gassman indol e synthesis toward th e total synthesis of (+)-paspalicine and (+)-paspalinine by Smith et al.345 The Hegedus indole synthesis (1976) This methodology calls for the stoichiometric Pd(II)-mediated oxidative cyclization of alkenyl anilines to indoles.346, 347 The mechanism itself is remiscent of that of the Wacker oxidation348 because of the coordination of th e palladium to the olefin in the early part of the mechanism. Vinylaniline 351 is reacted with a Pd (II) species to form the insoluble complex 352 , which is then treated with triethylamine to effect the transformation to indole 353 (Scheme 75).

PAGE 127

105 NH2MeO O a Reagents and Conditions: a. PdCl2(CH3CN)2, THF b. Et3N NH2MeO O Pd Cl Cl 352 351 b N H MeO O 353 Scheme 75. The Hegedus indole synthesis346, 347 The methodology has not been used consid erably in the synthesis of indole alkaloids and other na tural products. However, a va riant of the above methodology has used alkynyl anilines to generate indoles. Th e reagent used will ofte n vary from the more classical Pd(II) (Scheme 76)349 to Cu(I) (Scheme 77),350-353 TBAF (Scheme 78),354 and even base (Scheme 79).355-360 NH2NCO2Me Boc Boc NCO2Me Boc Boc N H 354 355 Reagents and Conditions: a. PdCl2(MeCN)2, MeCN a Scheme 76. Pd(II)-mediated synthesis of an indole derivative from an alkynyl aniline349 NH2N N OEt OEt a N H N N OEt EtO 356 357 Reagents and Conditions: a. CuI, DMF, Ethylene glycol Scheme 77. Cu(I)-mediated synthesis of an i ndole derivative from an alkynyl aniline353

PAGE 128

106 R1NH R2N a R1R2R1 = Ph, Bu, H, CH2CH2CO2Et, Hex, TMS R2 = CO2Et, CHO, Ac, Ms, Boc N R1H + 358 359 360 Conditions and Reagents: a. TBAF, THF Scheme 78. TBAF-mediated synthesis of an indole derivative from an alkynyl aniline354 NH2Ph a N H Ph Base NaH NaOEt KOtBu KH CsOH*H2O CsOtBu Temp/Time 60 °C/8 h 80 °C/15 h 25 °C/4 h 25 °C/5h 90 °C/5 h 25°C / 5h Yield (%) <5 66 79 72 68 71 361 362 Conditions and Reagents: a. Base (1-2 eq.), NMP Scheme 79. Base-mediated synthesis of an indole derivative from an alkynyl aniline359, 360 The Larock indole synthesis (1991) Larock has recently developed a methodology for the palladium-mediated synthesis of indoles from o -iodoanilines ( 363 ) and internal alkyne ( 364 ) derivatives.361, 362 This heteroannulation, as the author calls it, involves first an oxidative addition of the palladium species into the aryl-iodide bond, followed by complexation and addition into the alkyne, affording, after reductive elimina tion of the palladium, indole derivative 365 . If the alkyne used is not symmetric, the transformation will occur with some steric bias, such that the larger group (such as TMS) will be preferentially put in the 2-position of the indole (Scheme 80).

PAGE 129

107 I NH2TMS + Reagents and Conditions: a. Pd(OAc)2, NaOAc, nBu4NCl, DMF N H TMS 363 364 365 a Scheme 80. The Larock indole synthesis361, 362 Recently, this methodology has been used extensively, such as by Ma et al. in their approach to biologically im portant tryptophan analogues like 369 (Scheme 81).353 I NH2TES N N OEt EtO + Reagents and Conditions: a. Pd(OAc)2, Na2CO3, LiCl, DMF N H TES N N OEt EtO 367 366 a 368 N H NH2O HO 369 H Scheme 81. Use of the Larock heteroannulati on in the synthesis of tryptophan analogues by Ma et al.353 The Madelung indole synthesis (1912) Indoles can also be synthesized from 2-( acylamino)-toluene using very strong bases in a process call the Madelung indole synthesis.363 The starting material ( 370 ) is deprotonated twi ce (either with n BuLi at RT or with NaNH2 at 250 °C), generating a methyl anion, which performs a nucleophilic attack onto the carbony l to generate the five-membered ring (Scheme 82).

PAGE 130

108 N H R O a or b N H R 370 371 Reagents and Conditions: a. nBuLi, RT b. NaNH2, 250 ° C Scheme 82. The Madelung indole synthesis363 Because the reagents used in the Made lung synthesis are so harsh, it may be difficult to incorporate this methodology into a synthesis with sensitive functional groups. Nevertheless, the Smith group, using a modifi ed protocol, has used this procedure extensively, such as in their tota l synthesis of (-)-penitrem D ( 375 , Scheme 83).364 NH2OTMS TIPSO H H 372 O O O OTES H OH H 373 TIPSO H H 374 N H OTMS O OH OH OTES H H Reagents and Conditions: a. 1.1 eq. nBuLi, THF, -78 °C -> RT b. 1.1 eq. TMSCl, 0 °C c. 2.1 eq. sBuLi, 0 °C d. 0.1 eq. 373 , THF, Et2O, 0 ° C e. Silica gel, CHCl3+ a-e N H O O OH H H OH H H (-)-Penitrem D 375 Scheme 83. Use of a modified Madelung methodology toward the synthesis of (-)penitrem D by Smith et al.364

PAGE 131

109 The Nenitzescu indole synthesis (1929) From substituted p -quinones ( 376 ), aminoacrylates ( 377 ) will add in a conjugate fashion to generate 5-hydroxyindoles such as 378 in what is known as the Nenitzescu indole synthesis (Scheme 84).365 O O R1H OR3O HN R2+ N O OR3R1HO R2376377 378 Scheme 84. The Nenitzescu indole synthesis365 In 2001, Maguire et al. used this methodology in the synthesis of compounds that were selected to allow systematic investigati on of the influence of each of the structural components of indomethacin on the enhancem ent of cytotoxicity of doxorubicin and other anticancer agents in combinati on toxicity assays (compounds such as 382 , Scheme 85).366 O O HN O O + 379 380 HO N O O 381 O N O 382 NH2(HO)2OP Scheme 85. The synthesis of an indome thacin analogue for the evaluation as MRP modulators using the Nenitzescu indole synthesis366 Discussion Given the previously-dis cussed properties of iso guanine and its nucleoside, as well as the opportunity to establish whether the “a romaticity formalism” used to reason that 7deazaiso guanine would not suffer from the tautomeric ambiguity of iso guanine, the first

PAGE 132

110 goal was to establish the extent to which the enol tautomer of 7-deazaiso guanosine existed in solution. The second goal was to develop a synthesis of a functionalized derivative of 7-deazaiso guanine that would enable the molecular biologist to use the C7isoG:isoC base pair to, among other things, probe polymerases and select for particular properties using in vitro evolution experiments. Tautomerism of 2Â’-deoxy-7-deazaiso Guanosine Synthesis Because the tautomerism of C7isoG (a nd dC7isoG) would be measured using standard spectroscopic methodology as Sepiol et al. did for isoG, a few derivatives whereby the tautomeric form would be fixed either as a methyl ether ( 310 ) or as a methylamine ( 383 ), would need to be synthesized (Figure 40). N N N NH2HO R 311, enolHN N N NH2O R 311, N1-H ketoN N N NH2O R Me KTN N N NH2O R Me HN N N NH HO R 311, iminoN N H N NH2O R 311, N3-H ketoR = 2'-deoxyribose 310383 Figure 40. Four possible tautomer ic forms of 2'-deoxy-7-deazaiso guanosine and the fixed derivatives used to study the tautomerism of the free nucleoside Naturally, O -methyl derivative 310 has already been descri bed, both as the free heterocycle, and as the 2Â’-deoxy-D-riboside. An improved protocol for the synthesis of

PAGE 133

111 heterocycle 309 was devised based on the procedures of Davoll (Scheme 62). In a three step, two-pot reaction, malononitrile is condens ed with bromoacetaldehyde diethyl acetal in acetonitrile using KOH as the base. The reaction is simply worked up the following day by filtration of the inorganic KBr formed. Afterwards , one more equivalent of KOH is added, followed by methylisourea hydrogensul fate. Upon complete formation of the intermediate pyrimidine, the reaction is quenched with HCl, and the product is recrystallized from ethanol/water mixtur es. Although the prev ious methodology did afford the desired product, the yield was low and the preparation was tedious, as two vacuum distillations were necessary to purify al l of the intermediates. With this approach (Scheme 86), no intermediate purification is needed, and product is available after two days of work (instead of two weeks). N N N H O NH2a-c CN NC Reagents and Conditions: a. KOH, CH3CN then bromoacetaldehyde diethyl acetal, reflux b. KOH then O-methylisourea hydrogensulfate, reflux c. HCl (aq.) to pH 3 257 309 Scheme 86. Improved synthesis of 309 A first attempt at synthesizi ng the nucleoside of 7-deazaiso guanine was made by reacting the heterocycle ( 302 ) with halogenose 320 (Scheme 87). However, the yield for this direct transformation was dismal. It is suspected that, in sp ite of not finding any reference to this in the literature, this same result was obtained by, among others, Seela et al. , as indicated by the lengthy syntheses of th is otherwise simple nucleoside (Scheme 63,

PAGE 134

112 Scheme 64). On the other hand, the O -methyl nucleoside ( 384 ) was synthesized in the same manner with high yields (Scheme 87). HN N N H O NH2N N N H O NH2HN N O NH2N O OTol TolO 320 , Tol = p-MePhCOO OTol TolO Cl N N O NH2N O OTol TolO a b Reagents and Conditions: a. NaH, then 320 ; yield < 5 % b. NaH, then 320 ; yield 60 80 % c. PhSH, ethylene glycol, 130 °C d. 1 M NaOMe, MeOH e. HCl (conc.), dioxane, reflux cf. Seela et al., 1986 f. CH2N2, EtOH, Et2O 302 309 385 384 c HN N O NH2N O OTol TolO 385 N N O NH2N O OH HO 310 e HN N O NH2N O OH HO 311 d d f Scheme 87. Synthesis of the nucleosides of C7isoG and the O2-methyl-C7isoG Naturally, in order to genera te the free nucleoside from 384 , the O -methyl ether would need to be removed. Seela’s proce dure (conc. HCl, dioxane, reflux, Scheme 63) for this deprotection was repeated, and although it was successful, it gave low yields in our hands. Instead, thiophenol in ethylene glycol367 was found to give excellent yields of the demethylated compound ( 385 ). Although problematic at first, the excess ethylene glycol from the transformation was rem oved upon workup by complexation with sodium

PAGE 135

113 borate in the form of a slurry , which has a tendency to st ick, thus allowing the organic phase in the workup to be easily decante d from the undesired material. After saponification of the two tol uoyl esters, 2Â’-deoxy-7-deazaiso guanosine (dC7isoG, 311 ) was found to have identical spectroscopic proper ties to those reported by Seela et al. . The structure of this synthetic dC7isoG was further confirmed by treatment with diazomethane to afford 310 , which was identical in all as pects to the parent compound obtained through methanolysis of 384 . The N1-methyl derivative was synthesized by first protecting the exocyclic amino group using diisobutylformamidine,368, 369 which produced a mixture of diastereomers ( 386 ). Subsequently, 386 was treated with NaH and CH3I, after which, upon workup, all protecting groups were hydrol yzed using methanolic NH3/NaOMe to give 383 . Methylation at the N1-position was c onfirmed using both long range (HMBC370) and short range (HMQC371) 1H-13C NMR correlations (Scheme 88). HN N O NH2N O OTol TolO N( i Bu)2OMe MeO HN N O N N O OTol TolO N( i Bu)2H mixture of isomers N N O N N O OTol TolO N( i Bu)2H One product only Not isolated N N O NH2N O OH HO Reagents and Conditions: a. 388 , RT b. NaH, MeI, CH3CN c. NaOMe, NH3, MeOH 388 a b c 385 386 387 383 Scheme 88. Synthesis of 2'-deoxy-N1-methyl-7-deazaiso guanosine

PAGE 136

114 In summary, three compounds, two corresponding to individual “fixed” tautomers, and a third the native dC7iso G, were made to study the tautomerism of this last nucleoside. The overall synthe sis of each of these compounds is summarized in Scheme 89. N N O NH2N O OTol TolO HN N O NH2N O OTol TolO HN N O N N O OTol TolO H N N O NH2N O OH HO N N O NH2N O OH HO HN N O NH2N O OH HO Reagents and Conditions: a. NaOMe, MeOH b. HCl, dioxane, reflux c. PhSH, ethylene glycol, 130 °C N(iBu)2c a b d a e f, g d. CH2N2, MeOH, Et2O e. 388 f. NaH, MeI, CH3CN g. NaOMe, NH3, MeOH 384 310 311 385 386 383 Scheme 89. Synthesis of dC7isoG and derivatives pKa measurements In order to establish whether dC7isoG ( 311 ) would behave adequately at physiologic pH (i.e., would not exist as the anion or cation at neutral pH), the pKas of this species would need to be establishe d. Using spectrophoto metric titration372 from pH 2.8 to 13.7, at wavelengths between 240 and 350 nm, values for pK1 and pK2 were obtained as 4.3 (± 0.1) and 9.9 (± 0.2), respectively (Figure 41, Figure 42). These are comparable

PAGE 137

115 to the pKa values of natural nucleobases, and ensure that the C7isoG heterocycle is largely uncharged in neutral water. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 240260280300320340 Wavelength (nm) Absorbance 2.8 2.9 3.0 3.1 3.3 3.5 3.7 4.0 4.6 5.0 5.1 5.2 5.3 5.5 5.8 6.0 6.2 6.4 6.6 6.8 6.9 7.0 7.2 7.3 7.5 7.6 7.8 8.0 8.2 8.3 8.6 8.9 9.1 9.2 9.4 9.6 9.8 10.0 10.3 10.5 10.7 10.8 11.0 11.3 11.5 11.6 11.9 12.1 12.3 12.5 12.7 12.9 13.1 13.4 Figure 41. Multiwavelength analysis of dC7isoG at varying pH (box) 0.5 1 1.5 2 2.5 2468101214pHAbsorbance Ratio at 258/307 Figure 42. pKa determination of dC7isoG

PAGE 138

116 Tautomerism measurements Given the four likely tautomeric forms of dC7isoG (Figure 40), as well as literature precedent241, 373-375 on this kind of tautomerism, the imino form was considered the least likely. This was further confirmed by 1H NMR studies of dC7isoG: the –NH2 group was easily identified by integration and D2O exchange. At first glance, the UV spectrum of dC7isoG ( 311 ) in water resembles closely that of the N -methylated derivative ( 383 ), and not at all that of the O -methylated nucleoside ( 310 , Figure 43). Following the same logic as Sepiol et al. for isoG, this qualitative analysis suggests—but does not prove—that the keto tautomer predominates in aqueous solutions. Furthermore, the only slight deviation from the spectrum of the fixed N methyl derivative in 99% dioxane also suggests the strong preponderance of the keto tautomer, even in nonpolar solvents. 0.00E+00 1.00E+03 2.00E+03 3.00E+03 4.00E+03 5.00E+03 6.00E+03 7.00E+03 8.00E+03 9.00E+03 1.00E+04 240260280300320340 wavelength (nm) HN N N NH2O R N N N NH2O R Me N N N NH2O R MeR =2'-deoxyribose Figure 43. UV spectra of compounds 311 , 310 , and 383 in water. Inset: UV spectra of the same compounds, in 99:1—1,4-dioxane:water.

PAGE 139

117 In order to confirm that only the enol and N1-H keto tautomers of 311 were present in the solution at all times, the multiwavelength analysis described by Dewar and Urch376 was used. The method allows for an easy anal ysis of mixtures based on BeerÂ’s law. Given an unknown solution containing substances , , , Â… i at known concentrations , , , Â… i g per L, and given that solutions of , , , Â… i , at known concentrations c, c, c, Â… ci g per L, give rise to absorptions of A, A, A, Â… Ai at wavelength , if R is the observed value of the unknown solution, and if BeerÂ’s law is obeyed, then: A A c A A c A A c c A R gives A by throughout Dividing A c A c A c A c Ri i i i i i : Therefore, in a two-component system, if the values of A R are plotted against the corresponding values of A A, a straight line of slope c with intercept c will be obtained. Any deviation from a straight line indicates the presence of other components. Once again, the two derivatives of 311 , 310 and 383 , were used to represent the keto and enol tautomers, respectively. In our case, the wavelengths between 280 and 300 nm were used. The linear correlation observed le d us to conclude deci sively that only the enol and the N1-H keto tautomers exist in solutions of water and dioxane (Figure 44).

PAGE 140

118 R2 = 1 R2 = 1 R2 = 1 R2 = 1 R2 = 1 R2 = 0.9975 R2 = 0.9999 0 1 2 3 4 5 6 7 8 9 00.511.522.533.54Aketo/AenolR/Aenol 100 % water 85 % water 65 % water 50 % water 35 % water 15 % water 1 % water Figure 44. The linear regressi ons obtained with high R2 values indicate that the only species of dC7isoG present in solution are the N1-H keto and the enol forms. In order to measure the tautomeric ratios of the keto and enol forms of 311 , the UV curves were measured with a gradient of wa ter and 1,4-dioxane. To validate our data, we also measured the curves of disoG, 291 (Figure 45, Figure 46). The procedure of Voegel et al.377 was then used to estimate a value of the keto:enol tautomers in pure water for dC7isoG. This procedure exploits the fact that in nonpolar solvents, the UV spectra for both disoG and dC7isoG shift from their fo rm in pure water (which resembles the Nmethylated derivatives) to a form th at resembles the UV spectra of the O-methylated derivatives. This is interpreted as evidence that the tautomeric equilibrium shifts from one favoring the keto tautomer in pure water to one favoring the en ol tautomer in pure dioxane.

PAGE 141

119 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 240250260270280290300310320 wavelength (nm) Absorbance (AU) 100 % 95 % 90 % 85 % 80 % 75 % 70 % 65 % 60 % 55 % 50 % 45 % 40 % 35 % 30 % 25 % 20 % 15 % 10 % 9 % 8 % 7 % 6 % 5 % 4 % 3 % 2 % 1 % Figure 45. UV curves of dC7isoG with va rying water and dioxane concentrations (legend refers to % (v/v) water) 0 0.1 0.2 0.3 0.4 0.5 0.6 240250260270280290300310320 wavelength (nm) Absorbance (AU) 100 % 95 % 90 % 85 % 80 % 75 % 70 % 65 % 60 % 55 % 50 % 45 % 40 % 35 % 30 % 25 % 20 % 15 % 10 % 9 % 8 % 7 % 6 % 5 % 4 % 3 % 2 % 1 % Figure 46. UV curves of disoG with varying water and dioxane concentrations (legend refers to % (v/v) water) To obtain quantitative data, the ratio of the extinction coefficients at two wavelengths ( = 296 and 255 nm for disoG and = 305 and 254 nm for dC7isoG; Figure 47), chosen to maximize the difference between the keto and enol forms, was selected as

PAGE 142

120 a metric for the tautomeric equilibrium cons tant in dioxane:water mixtures in varying proportions. This was plotted ag ainst the corresponding Dimroth ET(30) value, which provides a measure of the local dielectric constant.242-244, 378 -0.4 -0.3 -0.2 -0.1 0 0.1 4146515661ET(30) (kcal/mol)log(A¹/A²) disoG ( ¹= 296 nm; ² = 255 nm) dC7isoG ( ¹ = 305 nm; ² = 254 nm) Figure 47. Comparison of the tautomerism of 2'-deoxy-isoguanosine and 2'-deoxy-7deaza-isoguanosine. (A¹ = Absorption at ¹; A² = Absorption at ²) Even qualitatively, disoG and dC7isoG behave differently in these experiments. The UV spectrum of disoG changes well before the water is completely removed. In contrast, the UV spectrum of dC7isoG is not identical to that of the O-methylated derivative even at the highest concentrations of dioxane tested. Further, the UV spectrum of disoG continues to change even in mixt ures approaching pure water. This suggests that the conversion of the enol tautomer to the keto tautomer of disoG is not complete even in pure water (the conclusion of Sepiol et al.). With dC7isoG, however, the UV spectrum ceases to be solvent dependent when the fraction of water is greater than 50% (v/v). These results suggest that our hypothe sis at outset had manipulative value: the

PAGE 143

121 keto tautomer of dC7isoG is mo re stable relative to its enol tautomer than the keto tautomer of disoG is relative to its enol tautomer. We then estimated the KT (= [keto]/[enol]) for dC7isoG using the method of Voegel et al.. Here, the fraction of enol form was estimated over a range of ET(30) values where the KT 1, regions where an estimate coul d be made with some reliability. This is the region where ET(30) 40-50. The log fraction of tautomers was plotted against ET(30) (Figure 48), and the line was extrapolated to the ET(30) of pure water (63.1).247 This generated a value of KT 103 for dC7isoG. Recalculating the KT for disoG gave a value of 10, consistent with that previously reported. These values can be compared with the value for the natural guanosine nucleoside (KT 104 – 105).219 y = -0.29x + 14.83 R2 = 0.98 y = -0.11x + 5.59 R2 = 0.99-0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 4042444648505254ET(30)log([enol]/[keto]) HN N O NH2N N O OH HO HN N O NH2N O OH HO Figure 48. Plot of log([enol]/[keto]) versus ET(30) for dC7isoG ( 311 ) and disoG ( 291 ) in mixtures of dioxane (ET(30) = 36.0) and water (ET(30) = 63.1). Synthesis of Derivatives The initial strategy used in the synthesis of C7isoG derivatives functionalized at C7 was to first iodinate the heterocycle (or the nucleoside) at C7, then functionalize using

PAGE 144

122 Heck or Sonogashira methodology. However, when nucleoside 384 or heterocycle 309 was treated with N-iodosuccinimide (NIS), the only product obtained was the diiodinated derivative, no matter whether excess or less than 1 equivalent of NIS was used (Scheme 90). The reaction was attempted with variou s solvents and iodinating agents, but the result was invariably the same. Likewise , no selectivity was obs erved when diiodides 389 and 390 were treated under Pd-mediated coupling conditions (such as Heck, Sonogashira or Suzuki couplings). Furtherm ore, no isolable product could be obtained when 3Â’,5Â’-di-O-toluoyl-2Â’-deoxy-7-deaza-isoguanosine ( 385 ) was used as the starting material. N N O NH2N HN N O NH2N OOTol TolO N N O NH2N HN N O NH2N OOTol TolO I I I I a a Reagents and Conditions: a. NIS, 1,2-dichloroethane 384 309 390 389 Scheme 90. The iodination of 307 and 384 gives only the diiodinated derivatives Direct functionalization was also attempted by reacting 384 directly with electrophiles such as 3-ethoxyacrylonitrile379, 380 and POCl3/DMF (Vilsmeyer reagent) as well as through palladium-mediated transformations.381-383 However, none of the conditions tried generated any product that co uld be characterized. At this point, it

PAGE 145

123 became extremely clear that a different strate gy would be necessary to construct these structures effectively (Scheme 91). Because the approach provided in path A (Scheme 91) had already been considered, paths B, C, and D (with D bei ng the most unlikely disconnection) appeared as viab le solutions to this problem. N N N H R2R1NH2RO N N NH2NH2RO O R2R1N N NH2NH2RO N N NH N H RO I R2R1O R2X R1N R2R1N NH NH2RO A B C D R3R3 Scheme 91. Retrosynthetic analysis of 7-deaza-isoguanine derivatives Synthesis of a versatile precursor Both paths B and C contain a diaminopyrim idine moiety that should be assembled fairly easily. The condens ation of malononitrile ( 257 ) with O-methylisourea hydrogensulfate ( 391 ) afforded the aromatic product ( 392 ) as well as a byproduct ( 393 ) that stemmed from the malononitrile acting as a nucleophile onto the urea derivative (Scheme 92). The two compounds are very simi lar in terms of their polarity and weight, and so chromatography to isolate the desire d product proved to be tedious. However, because 392 is aromatic, it is po ssible to selectively crys tallize one co mpound over the

PAGE 146

124 other selectively. Nevertheless, by selecti ng a mild base that would not deprotonate malononitrile quite as easily, it was also possible to minimize the amount of undesired byproduct that would form. Furthermore, in generating a product for the Larock heteroannulation path (Path C), it was also possible to synthesize iodinated pyrimidine 394 in a two-operation, one-pot process, wher eby the product is simply isolated using acid/base extraction followed by a simple recrystallization. CN NC NH NH2O 1/2 H2SO4+ NCCN NH2H2N 257 391 N N NH2NH2O + 392 393 Reagents and Conditions: a. NaOMe, MeOH, reflux b. I2N N NH2NH2O 394 I a b Scheme 92. Synthesis of pyrimidines 392 and 394 Purine synthesis All attempts to react simple pyrimidine 392 with -haloketones, -aldehydes, or derivatives thereof (such as sugars) resulted in the simple decomposition of the carbonyl species, while leaving diamine 392 usually intact. However, iodopyrimidine reacted quite readily under the La rock conditions with 1-Phenyl-2 -trimethylsilylacetylene to give a mixture of the purine ( 395 ) and the deiodinated pyrimidine ( 392 ). The regioselectivity of the reaction was conclusively establis hed as previously reported, with the trimethylsilyl at C8 and the phenyl at C7 (S cheme 93). Also, protodesylation with TBAF in THF proceeded smoothly to yield purine 396 .

PAGE 147

125 N N NH2NH2I O N N N H NH2Ph TMS O aReagents and Conditions:a. Pd(OAc)2, PPh3, iPr2NEt, Et4NCl, DMF, 1-Phenyl-2-trimethylsilylacetylene, 100 °C b. TBAF, THF, RT394 395N N N H NH2Ph O b396 Scheme 93. Heteroannulation of pyrimidine 394 and 1-phenyl-2-trimethylsilylacetylene Synthesis of an internal alkyne for th e generation of functionalized 7-deazaiso guanine Because of steric requireme nts on behalf of the polymerases, it has been suggested that an E-allylamino functionality w ould be suitable to append functional groups onto the nucleoside (such as biotin, etc.). Therefore, E-1,3-dichloropropene was treated under basic conditions with trifluoroacetamide384, 385 to yield allylamido compound 398 . Subsequently, a Sonogashira coupling386, 387 was used to couple TMS-acetylene with the vinyl chloride 398 to give conjugated enyne 399 (Scheme 94). H N H CF3O Cl H NCF3O 80-90 % H NCF3O TMS 399 50-60 % Reagents and Conditions: a. NaH, THF, then E -1,3-dichloropropene b. PdCl2(PPh3)2, CuI, piperidine, TMS-acetylene a b 397 398 Scheme 94. Synthesis of enyne 399 Heteroannulation and optimization The cyclization to give purine 400 was first attempted using a known procedure.388 The yield of the reaction, however, was quite low, and it was clear that the reaction

PAGE 148

126 would need to be optimized (Scheme 95). The selectivity of the reaction was as predicted and confirmed using 1H-13C NMR correlation spectroscopy. N N NH2NH2O I H NCF3O TMS + Reagents and Conditions: a. Pd(OAc)2, PPh3, Et4NCl, Huenig's Base, DMF, 120 °C; yield: 12 % b. PdCl2(PPh3)2, i Pr2NEt, CH3CN, 80 °C; yield: 60-80 % N N N H TMS NHCOCF3NH2O a or b 394 399 400 N N N H SiMe3NH NH2O CF3O1 6 2 . 5 1 5 4 . 9 5 3 . 9 1 3 0 . 6 9 8 . 1 1 2 1 . 5 1 5 8 . 5 1 2 7 . 7 1 2 7 . 5 4 1 . 7 1 5 7 . 6 ( q , J = 1 5 0 H z ) 1 1 5 . 8 ( q , J = 1 1 4 0 H z ) 0 . 9 N N N H SiMe3NH NH2O CF3O3.87 5.79 9.09 0.27 6.83 5.81 4.11 8.23 Scheme 95. Heteroannulation of pyrimidine 394 and enyne 399 and assignments of the proton and carbon NMR spectra. By varying the catalyst, the ligand, th e base, and the solvent as well as the temperature of the reaction, the yield was optimized from 12% to 60-80% on repeated attempts. As it is, the only side-product of the reaction is the de iodinated pyrimidine, 392 , which conclusively shows that palladiu m does insert, but that the alkyne may be sterically challeng ing, and could prove problematic should it be any larger. In an attempt to better understand this r eaction as well as to provide yet another possible synthesis of heterocycle 309 , iodopyrimidine 394 was first reacted under the optimized conditions with bis(trimethylsilyl) acetylene (BTMSA). Unfortunately, neither these nor other conditions afforded any isolable product other than deiodinated pyrimidine 392 . Furthermore, when TMS-acetylene was used instead, surprisingly, only

PAGE 149

127 the Sonogashira product ( 403 ) could be isolated (copper-free Sonogashira couplings have been reported, but are still rare389), albeit in very low yield. The yield of this last transformation was greatly improved unde r more common conditions (Scheme 96). N N I NH2NH2O TMS TMS + a N N H NH2NH2O N N I NH2NH2O H TMS + N N NH2NH2O b or c TMS Reagents and Conditions: a. Various Pd, base, solvent, ligand conditions b. Pd(OAc)2, LiCl, PPh3, i Pr2NEt; yield: < 5 % c. Pd(PPh3)2Cl2, CuI, i Pr2NEt; yield: 60 % 401 402 394 392 394 403 Scheme 96. Attempts to react BTMS A and TMS-acetylene with pyrimidine 394 Having alkynylpyrimidine 403 in hand, the subsequent Hegedus-type cyclization was also attempted. However, none of th e conditions tried (base, TBAF, Pd, Cu) afforded anything other than decomposed starting material. It is possible that molecular modeling of this compound and its products and precursors might shed light on the interactions present in this as well as the heteroannulation r eactions. Nevertheless, it is clear that sterics play a very impor tant role in these transformations.

PAGE 150

128 CHAPTER 4 CONCLUSIONS In the first part of this dissertation, the synthesis of a novel aminoinositol dimer was described. The key step in the synt hesis was the sequential aziridine/epoxide opening to generate the dimeric structure. The synthesized dimer was tested for its ability to inhibit six commercially-available glycosidases, but showed no impressive behavior in that regard. However, the dime r did show a surprising affinity for calcium ions, as indicated by the selective shift of certain signals as observed using NMR spectroscopy. In the second part of this diss ertation, a case is made against isoguanosine as a good nucleobase due, in part, to its undesired propensity to tautomerize. Using an aromaticity “formalism,” it wa s suggested that the 7-deaza-isoguanosine would not behave nearly as poorly. A series of derivatives of 2’-deoxy-7-deaza-isoguanosine were synthesized to study the tautomerism of this unnatural nucleobase. The conclusion reached is that the first original analysis had manipulative value, as 2’-deoxy-7-deaza-isoguanosine was shown to exhibit a tautomeric ratio two orders of magnitude more favorable than 2’-deoxy-isoguanosine. These results, combined with a facile synthesis of functionalized derivatives of 7-deaza-isoguanine, have solved the last problem identified in AEGIS components exploi ting the full range of hydrog en-bonding complementarity. Thus, with this application, we now have th e ingredients for a fu lly-functiona l genetic alphabet that meets the specificati ons met by natural nucleic acids.

PAGE 151

129 APPENDIX EXPERIMENTAL SECTION General All nonaqueous reactions were performe d using standard techniques for the exclusion of moisture and air. Methylen e chloride, pyridine, and acetonitrile were distilled from calcium hydrid e under nitrogen. Tetrahydr ofuran was distilled from sodium/potassium/benzophenone under nitrogen. 1,4-Dioxane (anhydrous) was purchased from ACROS or distilled fr om sodium/potassium/benzophenone under nitrogen. Ethylene glycol (anhydrous) wa s purchased from Aldrich. Water was deionized using a Millipore Mi lli-Q purification system. Th in-layer chromatography was performed on Whatman 60 Å plates with fl uorescent indicator and flash chromatography was performed using Whatman 230-400 Mesh sili ca gel. Melting points were recorded either on a Hoover Unimelt apparatus or on a Mel-Temp electrotherm al apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FT-IR spectrometer or on a Perkin-Elmer Spectrum One FT-IR spectrome ter. Preparative HPLC was performed using a Waters PrepLC4000 system with a Wa ters 486 Tunable Absorbance detector and a Waters PrepLC 25 mm module equipped with a Waters NovaPak HR C18 6 µ m 60 Å cartridge and guard inset. Analytical HP LC was performed using a Waters Alliance 2690 Separation Module with a Waters 996 Photodiode Array detector and a Waters NovaPak C18 (3.9 x 150 mm) column. UV spectra were measured using a Vari an Cary 1 Bio UVVisible spectrophotometer. 1H and 13C NMR spectra were recorded on a Varian Gemini (300 MHz), a Varian VXR (300 MHz), a Varian Mercury (300 MHz), or a Varian Inova

PAGE 152

130 500 (500 MHz) instrument and are referenced to an internal TMS standard. Combustion analyses were performed by the University of Florida or by Atlantic Microlab, Norcross, GA. Mass spectrometry was performed by the an alytical division of the University of Florida, Gainesville, FL. General Procedure for the Inhibition Assays All measurements were performed on a Hewlett Packard 8452A diode array spectrophotometer with a multi-cell attachment. The following buffers were used: Sodium phosphate buffer (25 mM) pH = 7.3 for -galactosidase Sodium phosphate buffer (25 mM) pH = 6.8 for -glucosidase, -glucosidase Sodium phosphate buffer (25 mM) pH = 6.0 for -mannosidase, -galactosidase Sodium phosphate buffer (25 mM) pH = 5.5 for -mannosidase The inhibitor concentration was kept around 1 mg/mL. All tests were performed at 37 °C, except for the -mannosidase assay, which was performed at 25 °C. The substrate concentration was 5 mM except for the -mannosidase and -mannosidase, where it was 2.5 mM. The enzyme concentration was ad justed to produce a slope of approx 0.020.025 except for the -mannosidase assay, where it was ad justed to give a slope of 0.0050.01. The final volume in each cuvette was 1.000 mL for all cases. To a solution of varying amounts of i nhibitor (0-0.400 mL), in the corresponding buffer solution, was added 0.100 mL of the enzyme solution, except for one cuvette, which was used as a blank to correct for the autohydrolysis of the substrate. All samples were pre-incubated for 30 minutes, after wh ich the reaction was started by adding the substrate (0.400 mL). The absorption of each sample was continually measured over a

PAGE 153

131 period of 20 minutes. The slopes obtained for each individual sample was then plotted against the inhibitor concentra tion to obtain the inhi bition curves described in the text. Structure Data N -(5-Benzylamino-2,2-dimethyl-3a,4,5,7a-te trahydro-benzo[1,3]dioxol-4-yl)-4methyl-benzenesulfonamide Aziridine 235 (1.00 g, 3.11 mmol) and Yb(OTf)3 (482 mg, 0.78 mmol) were dissolved in THF (30 mL) at RT and benzylamine (1.7 mL, 15.55 mmol) was added using a syringe. The reaction mixture was heated to reflux for 18 hours, then allowed to cool to room temperature and concentrated in vacuo. The residue was dissolved in CH2Cl2 and extracted with water (1 x 25 mL) and brine (1 x 25 mL). The organic layer was dried (MgSO4) and evaporated on a rotary evaporator, yielding a light pink oil. The latter was further purified by flashchromatography (hexanes : ethyl acetate 1:1) to give a slightly yellow solid (1.171 g). Subsequent recrystallization fr om hexanes / ethyl acetate (1 0:1) gave the title compound as white crystals (992 mg, 75%). Rf 0.2 (hexanes:ethyl acetate / 1:1); mp= 116-118°C; [ ]D 26= -10.7 (c 1.24, CH3CN); 1H NMR (CDCl3, 300 MHz) 7.79 (d, 1H), 7.18 – 7.32 (m, 7H), 5.90 (dd, J = 2.3, 10.0 Hz, 1H), 5.77 (ddd, J = 2.0, 3.2, 10.2 Hz, 1H ), 5.33 (bs, 1H), 4.51 (dd, J = 4.6, 4.4 Hz, 1H), 4.07 (dd, J = 6.1, 7.6 Hz, 1H), 3.69 (dd, J = 13.2 Hz, 2H), 3.50 (t, J = 7.3 Hz, 1H), 2.96 (d, J = 6.8 Hz, 1H), 2.48 (s, 3H), 2.15 (bs, 1H), 1.25 (s, 3H), 1.16 (s, 3H) ppm; 13C NMR (CDCl3, 75 MHz) 143.2, 139.8, 137.5, 131.9, 129.4, 128.3, 128.0, 127.3, 126.8, 127.5, 110.0, 76.4, 71.6, 56.1, 54.3, 50.1, 27.4, 25.8, 21.4; MS (FAB) m/z 429 N H HN O O SO O 239

PAGE 154

132 ([M+1]+); HRMS calcÂ’d. For C23H28N2O4S: 429.1846; Found: 429.1812; Anal calcÂ’d.: C 64.46, H 6.59; Found : C 64.54, H 6.61. (5-Benzylamino-2,2-dimethyl-3a,4,5,7a-tetr ahydro-benzo[1,3]dioxol-4-yl)-carbamic acid methyl ester To a solution of Yb(OTf)3 (220 mg, 0.36 mmol) in THF (15 mL) was added aziridine 237 (500 mg, 1.8 mmol) in portions at room temperature. Benzylamine (0.22 mL, 1.96 mmol) was then added dropwise, and the resulting solution was heated to reflux overnight. The next day, brine (5 mL) and me thylene chloride (20 mL) were added to the solution, and the aqueous layer was extracted (3 x 20 mL) with methylene chloride. The organic layers were combined, dried with anhydrous magnesium sulfate, and concentrated in vacuo. The resulting oil was purified with column chromatography using a gradient of hexanes and ethyl acetate to afford the title compound (267 mg, 45%). Rf 0.2 (hexanes:ethyl acetate / 6: 4); IR (KBr) 3325, 2985, 2935, 1704, 1667, 1604, 1538, 1454, 1381, 1287, 1245, 1164, 1055, 968, 899, 867, 752, 701, 666 cm-1; 1H NMR (CDCl3, 300 MHz) 7.28 (m, 5), 6.02 (dd, J = 10.0, 1.5 Hz, 1), 5.87 (dq, J = 10.3, 2.4 Hz, 1), 4.94 (d, J = 9.4 Hz, 1), 4.55 (t, J = 4.96 Hz, 1), 4.09 (dd, J = 9.4, 6.2 Hz, 1), 3.81 (m, 3), 3.67 (s, 3), 3.15 (d, J = 8.8 Hz, 1), 2.16 (s, 1), 1.51 (s, 3), 1.36 (s, 3) ppm; 13C NMR (CDCl3, 75 MHz) 140.1, 134.0, 128.5, 128.2, 128.1, 126.9, 123.7, 110.0, 76.6, 76.2, 72.9, 56.2, 53.1, 52.1, 49.7, 28.1, 26.1; MS (CI, methane) m/z 333, 301, 176, 91; HRMS calcÂ’d. for C18H25N2O4: 333.1814; Found: 333.1810. N H HN O O 238 O O

PAGE 155

133 N-(5S-amino-7-bromo-2,2-dimethyl-3Ra,4S ,5S,7a-tetraydro-benzo[1S,3R]dioxol-4Syl)-4-metyl-benzenesulfonamide To a solution of aziridine 234 (1000 mg, 2.5 mmol) in THF (50 mL) at –78 ºC, was added Yb(OTf)3 (310 mg, 0.5 mmol). Using a dry ice/acetone cold finger, ammonia gas was bubbled through the solution with a bent needle, and allowed to reflux on the condenser. After 15 minutes of mild bubbli ng, the ammonia was turned off, and the reaction vessel was allowed to warm to room temperature slow ly, while still keeping the cold finger at 78 ºC. When no more starting material remained by TLC, the cold finger was removed, and the reaction was concentrated in vac uo. Product was used as is in further transformations. Crude yiel d: 1.46 g, brown solid. Rf 0.2 (methylene chloride:methanol / 85:15 with a drop of ammonium hydroxide), stains very brightly orange with a ninhydrin solution. 1H NMR (CDCl3, 300 MHz) 7.77 (d, J = 8.1 Hz, 2), 7.26 (d, J = 6.2 Hz, 2), 6.21 (s, 1), 4.55 ( d, J = 5.8 Hz, 1), 4.25 (bs, 2+1), 4.15 (t, J = 6.3 Hz, 1) , 3.41 (bs, 1), 3.30 (m, 1), 2.40 (s , 3), 1.24 (s, 3), 1.11 (s, 3) ppm; 13C NMR (CDCl3, 75 MHz) 143.6 (up), 137.3 (up), 129.5 (down), 127.3 (down), 110.6 (up), 76.4 (down), 27.2 (down), 25.7 (dow n), 21.5 (down) ppm; MS (FAB) m/z 419 ([81BrM+H]+), 417 ([79BrM+H]+), 361, 359, 344, 342, 263, 204, 190, 188; HRMS calcd. for C16H22O4N2SBr: 417.0483; Found: 417.0474. N-(4S-(acetyl-(toluene-4-sulfonyl)-a mino-7-bromo-2,2-dimethyl-3Ra,4S,5S,7atetrahydro-benzo[1S,3R]dioxol-5S-yl)-acetamide To crude diamine (835 mg) in pyridine (6 mL), was added Ac2O (3 mL) and a spatula tip of DMAP. The solution was allowe d to stir for 3 hours at RT until no more starting material could be observed by TLC. The reaction was dissolved in ethyl acetate Br H2N NHTs O O 240

PAGE 156

134 (20 mL), and transferred to a se paratory funnel. The resulting mixture was washed 5% CuSO4 (3 x 5 mL) and with brine (2 x 5 mL). The organic phase was dr ied with anhydrous magnesium sulfate and concentrated to afford a crude brown solid. Two products from the reaction were isolated and purified using flash column chromatography with a 1:1 mixture of hexanes-ethyl acetate. Yield: 250 mg, 35% fr om the aziridine. Rf 0.4 (hexanes:ethyl acetate / 1:1); mp 128-130 ºC; [ ]D 25 -39.3 (c = 1.01, CHCl3); IR (KBr) 3293, 3064, 2986, 1702, 1654, 1596 , 1541, 1438, 1352, 1242, 1218, 1165, 1090, 1028, 949, 902, 868, 816, 795, 798, 662, 590, 547 cm-1; 1H NMR (CDCl3, 300 MHz) 8.08 (d, J = 8.2 Hz, 2), 7.36 (d, J = 8.2 Hz , 2), 6.21 (d, J = 2.1 Hz, 1), 5.79 (d, J = 10.0 Hz, 1), 5.45 (t, J = 10.0 Hz, 1), 5.33 (dd, J = 9.6, 6.3 Hz, 1), 4.80 (dd, J = 6.3, 1.1 Hz, 1), 4.47 (t, J = 9.6 Hz, 1), 2.46 (s, 3), 2.11 (s, 3), 2.06 (s, 3), 1.60 (s, 3), 1.44 (s, 3); C NMR (CDCl , 75 MHz) 171.3 (up), 170.9 (up), 168.8 (up), 145.0 (up), 144.5 (up), 137.0 (up), 135.9 (up), 135.3 (down), 129.9 (down), 129.3 (down), 128.2 (down), 127.7 (down), 118.0 (up), 110.9 (up), 77.7 (down) , 73.0 (down), 62.2 (down), 47.4 (down), 27.7 (down), 25.9 (down), 23.1 (down), 21.5 (down); MS (FAB) m/z 501 (81BrM+H), 499 (79BrM+H), 403, 401, 365, 343, 291, 289, 232, 230, 189, 155, 139, 123, 109; HRMS calc’d. for C20H26O6N2SBr: 501.0695; Found: 501.0666. The sample gave a positive Beilstein test for the presence of halogen. N-[7-bromo-2,2-dimethyl-4S-(toluene-4 -sulfonylamino)-3Ra,4S,5S,7a-tetrahydrobenzo[1S,3R]dioxol-5S-yl]-ac Yield: 450 mg, 68% from aziridine. Rf 0.3 (1:1 hexanes-ethyl acetate); mp 189-190 ºC; [ ]D 28 + 6.47 (c = 1.72, CHCl3); IR (KBr) 3335, 3289, 3052, 2989, 2921, 2867, 1654, Br N H N O O 241 Ac Ts Ac

PAGE 157

135 1539, 1437, 1328, 1257, 1383, 1328, 1257, 1214, 1160, 1071, 1023, 962, 866, 810, 705, 671, 606, 573, 550, 509, 488 cm-1; 1H NMR (CDCl3, 300 MHz) 7.78 (d, J = 8.3 Hz, 2), 7.28 (d, J = 8.3 Hz, 2), 6.75 (d, J = 8.0 Hz , 1, exchanges with D2O), 6.59 (d, J = 8.6 Hz, 1), 6.16 (d, J = 2.3 Hz , 1), 4.65 (d, J = 5.7 Hz, 1) , 4.52 (dd, J = 8.0, 6.3 Hz, 1), 4.25 (dd, J = 8.0, 6.3 Hz, 1), 3.41 (q, J = 8.4 Hz, 1), 2.40 (s, 3), 2.01 (s, 3), 1.27 (s, 3), 1.12 (s, 3); 13C NMR (CDCl3, 75 MHz) 170.9 (up), 143.1 (up), 137.9 (up), 133.3 (down), 129.3 (down), 127.1 (down), 120.1 ( up), 110.3 (up), 76.6 (down), 76.0 (down), 55.2 (down), 50.3 (down), 27.2 (down), 25.7 ( down), 23.1 (down), 21.4 (down); MS (FAB) m/z 461 (81BrM+H), 459 (79BrM+H), 403, 401, 334, 281, 221, 150, 90; HRMS calcd. for C18H24O5N2SBr: 459.0589; Found: 459.0586; An al. Calc’d.: C 47.06, H 5.05, N 6.10; Found: C 47.09, H 5.17, N 5.85. The sample gave a positive Beilstein test for the presence of halogen. N-[7-Bromo-5S-(7-bromo-4S-hydroxy-2,2dimethyl-3Ra,4S,5S,7a-tetrahydrobenzo[1S,3R]dioxol-5S-ylamino)-2,2dimethyl-3Ra,4S,5S,7a-tetrahydrobenzo[1S,3R]dioxol-4S-yl]-4methyl-benzenesulfonamide In a flame-dried 30 cm long (5 cm diameter) sealed tube was condensed ammonia gas (appr oximately 20 mL) at –78ºC. To the liquid ammonia solution was added aziridine 234 (3.1 g, 7.75 mmol) and Yb(OTf)3 (830 mg, 1.34 mmol) as solids to the reaction mixture. Methylene ch loride (10 mL) was then added, and the tube was sealed and heated to 50 ºC. After 30 minutes, the solids disappeared, and the reaction was checked by TLC. At this time, no more aziridine remained, so the solvents were removed under reduced pressure . To the dry mixture was added freshly Br N H HN O O 242 Ac Ts Br HN NHTs O O 244 HO O O Br

PAGE 158

136 distilled dioxane (25 mL) and epoxide 240 (2.19 g, 8.85 mmol). The tube was again sealed and heated to 60-70 ºC with an o il bath for 3 hours. The dioxane was then removed under reduced pressure, and the cr ude mixture was purified by flash column chromatography using a gradient of ethyl acetate and hexanes. Yield: 4.072 g (6.1 mmol), 78%. Rf 0.5 (1:1 / hexanes:ethyl acetate); mp 158-158.5 ºC; [ ]D 26 + 41.7 (c = 0.36, CHCl3); IR (KBr) 3905, 3822, 3752, 3677, 3651, 3589, 3504, 2346, 1846, 1794, 1752, 1735, 1710, 1702, 1654, 1638, 1560, 1508, 1458, 1382, 1330, 1220, 1161, 1074, 866, 814, 791, 669, 575, 544 cm-1; 1H NMR (CDCl3, 300 MHz) 7.79 (d, J = 8.0 Hz, 2); 7.36 (d, J = 8.0 Hz, 2), 6.22 (d, J = 3.9 Hz, 1), 5.93 (s, 1) ; 4.86 (m, 1, exchanges with D2O), 4.64 (d, J = 6.6 Hz, 2), 4.37 (d, J = 5.3 Hz, 1), 4.26 (t, J = 5.8 Hz, 1), 4.05 (m, 1), 3.62 (m, 1), 3.34 (t, J = 8.8 Hz, 1), 3.03 (s, 1), 2.92 ( d, J = 8.8 Hz, 1),2.70 (s, 1), 2.46 (s, 3), 1.55 (s, 3), 1.42 (s, 3), 1.32 (s, 3), 1.26 (s, 3) ppm; 1H NMR (DMSO, 300 MHz) 8.01 (d, J = 7.5 Hz, 1), 7.71 (d, J = 8.5 Hz, 2), 7.37 (d, J = 8.5 Hz, 2), 6.31 (d, J = 3.5 Hz, 1), 5.77 (s, 1), 5.27 (d, J = 5.5 Hz, 1), 4.62 (d, J = 5.7 Hz, 1), 4.57 (d, J = 6.2 Hz, 1), 4.03 (m, 2), 3.28 (q, J = 6.4 Hz, 1), 3.01 (m, 3), 2.39 (s, 3), 2.19 (dd, J = 9.9, 3.3 Hz, 1), 1.40 (s, 3), 1.30 (s, 3), 1.27 (s, 3), 1.22 (s, 3) ppm; 13C NMR (CDCl3, 75 MHz, APT) 144.2 (up), 136.9, (up), 133.4 (down), 131.3 (down), 130.0 (dow n), 127.3 (down), 122.8 (up), 118.8 (up), 111.4 (up), 110.7 (up), 78.2 (down), 76.7 (d own), 75.7 (down), 72.3 (down), 57.8 (down), 56.7 (down), 54.0 (down), 28.1 (down), 27.4 (dow n), 26.0 (down), 25.8 (down), 21.7 (down); MS (FAB) m/z 665 (M + H) , 607, 419, 329, 249, 176; HRMS calc’d. for C25H33O7N2Br2S: 663.0375; Found: 663.0372. Anal. Calc’d.: C 45.19, H 4.85, N 4.22; Found: C 45.49, H 5.04, N 3.94.

PAGE 159

137 Acetic acid 7-bromo-5S-[7-bromo-2,2-di methyl-4S-(toluene-4-sulfonylamino)3Ra,4S,5S,7a-tetrahydro-benzo[1S,3R ]dioxol-5S-ylamino]-2,2-dimethyl3Ra,4S,5S,7a-tetrahydro-benzo [1S,3R]dioxol-4S-yl ester To a solution of dimer 244 (2.025 g, 3.05 mmol) in methylene chloride (10 mL), was added a spatula tip of DMAP. The solution was sti rred, and pyridine (2.5 mL, 30.5 mmol) was added, followed by Ac2O (0.3 mL, 3.35 mmol). The solution was stirred at room temperature for 5 hours. The excess acetic anhydride was then quenched w ith methanol (15 mL), and the solution was concentrated under reduced pressure. Th e resulting solution in pyridine was then dissolved in Et2O (200 mL), and washed with 1% (v/v ) hydrochloric acid (4 x 15 mL), and brine (2 x 20 mL). The solution was dr ied with anhydrous magnesium sulfate, and concentrated to afford a yellow solid, which was purified using flash column chromatography with a gradient of methylene ch loride and ethyl acetat e. Yield: 1.996 g (93%). Rf 0.7 (1:1 / hexanes:ethyl acetate); mp 105.5-107 ºC; [ ]D 28.5 + 31.8 (c = 0.335, CHCl3); IR (Neat) 3530, 3261, 2988, 2935, 1742, 1644, 1598, 1454, 1373, 1334, 1223, 1162, 1076, 1040, 989, 911, 860, 814 , 793, 732, 6666, 574, 546 cm-1; 1H NMR (CDCl3, 300 MHz) 7.81 (d, J = 8.1 Hz, 2), 7.35 (d, J = 8.4 Hz, 2), 6.25 (d, J = 3.9 Hz, 1), 5.99 (d, J = 3.6 Hz, 1), 5.20 (d, J = 8.1 Hz, 1), 5.00 (t, J = 6.0 Hz, 1), 4.56 (dd, J = 8.8, 5.5 Hz, 2), 4.21 (m, 1), 3.05 (bs, 2), 2.45 (s, 3), 2.01 (s, 3), 1.49 (s, 3), 1.38 (s, 3), 1.33 (s, 3), 1.30 (s, 3) ppm; 13C NMR (CDCl3, 75 MHz) 170.3, 145.2, 144.0, 137.1, 132.4, 131.9, 129.9, 129.7, 128.3, 127.3, 122.3, 120.6, 111.2, 76.2, 75.5, 75.4, 71.1, 58.4, 57.5, 54.6, 31.8, 29.0, 27.8, 27.5, 26.3, 16.1, 23.5, 22.7, 21.7, 21.6, 21.1, 14.1 ppm; MS (FAB) m/z 709 Br HN NHTs O O 245 AcO O O Br

PAGE 160

138 (81Br81BrM+H), 707 (81Br79BrM+H), 705 (79Br79BrM+H),627, 502, 401; HRMS calc’d. for C27H35O8N2SBr2: 705.0481; Found: 705.0454. Anal. Calc’d.: C 45.91, H 4.85, N 3.97; Found: C 46.33, H 4.80, N 4.14. Acetic acid 7-bromo-5S-[[7-bromo-2,2-di methyl-4S-(toluene-4-sulfonylamino)3Ra,4S,5S,7a-tetrahydro-benzo[1S,3R]dioxol -5S-yl]-(2,2,2-trifluor o-acetyl)-amino]2,2-dimethyl-3Ra,4S,5S,7a-tetrahydro-b enzo[1S,3R]dioxol-4S-yl ester To a crude solution of acetylated dimer 245 (1.996 g, 2.83 mmol) in methylene chloride (12 mL), was added a spatula tip of DMAP. The so lution was stirred, and pyridine (2.3 mL, 28.3 mmol) was added, followed by trifluoroacetic anhydride (0.6 mL, 4.25 mmol). The solution was heated to reflux, and stirred for 3 hours. The excess trifluoracetic anhydrid e was quenched with methanol (15 mL), and the solution was c oncentrated under reduced pressure. The resulting crude in pyridine was then dissolved in diethyl ether (200 mL), and washed four times with 1% (v/v) hydrochlo ric acid (4 x 15 mL), and brine (2 x 20 mL). The solution was dried with anhydrous magnesium sulfate, and concentrated to afford a yellow solid, which was purified using flash column chro matography with a gradient of methylene chloride and ethyl acetate. Yield: 2.092 g (92%). Rf 0.7 (1:1 hexanes-ethyl acetate), co-spots with starting material; mp 108-110 ºC; [ ]D 28.5 + 99.6 (c = 0.105, CHCl3); IR (Neat) 3522, 3275, 2989, 2926, 2855, 1747, 1705, 1455, 1376, 1337, 1217, 1161, 1079, 989, 869, 795, 729, 665, 575.4 cm-1; 1H NMR (CDCl3, 300 MHz) 7.76 (d, J = 8.2 Hz, 2), 7.25 (d, J = 8.2 Hz, 2), 6.54 (d, J = 3.5 Hz, 1), 6.01 (s, 1), 5.94 (d, J = 2.7 Hz, 1), 5.17 (d, J = 8.3 Hz, 1), 4.67 (s, 1), 4.58 (m, 2), 4.44 (dd, J = 5.1, 4.0 Hz, 1), 4.05 (m, 3), 3.85 (dd, J = 9.1, 6.4 Hz, 1), Br N NHTs O O 246 AcO O O Br TFA

PAGE 161

139 2.38 (s, 3), 2.16 (s, 3), 1.49 (s, 3), 1.38 (s, 3), 1.33 (s, 3), 1.30 (s, 3); 13C NMR (CDCl3, 75 MHz) 169.4, 147.4, 143.6, 137.1, 134.2, 129.5, 128.3, 126.5, 117.7, 111.6, 110.8, 76.81, 74.24, 74.0, 68.2, 52.2, 29.9, 28.1, 16.7, 26.6, 25.5, 21.7, 21.0; 19F NMR (CDCl3, 282 MHz) -69.4; MS (FAB) m/z 747 (81Br81BrM+H-CH3COCH3), 745 (81Br79BrM+HCH3COCH3), 743 (79Br79BrM+H-CH3COCH3), 735, 707, 705, 703; HRMS calcd. for C26H28O8N2SF3Br2: 742.9885; Found: 742.9869; HRMS calcd. for C26H28O8N2SFBr2: 704.9917; Found: 704.9870. Acetic acid 5S-[[2,2-dimethyl-4S-(t oluene-4-sulfonylamino)-3Ra,4S,5S,7atetrahydro-benzo[1S,3R]dioxol-5S-yl]-(2,2,2 -trifluoro-acetyl)-am ino]-2,2-dimethyl3Ra,4S,5S,7a-tetrahydro-benzo [1S,3R]dioxol-4-yl ester To a flame-dried 100 mL flask, was added the dibromide ( 246 , 1.874 g, 2.3 mmol) and THF (40 mL). The solution was stirred and degassed with argon for 30 minutes. Tri-nbutyltinhydride (1.4 mL, 5.1 mmol) was then added to the solution, and it was heated to re flux. Upon reflux, a spatula tip of AIBN was added, and the solution was stirre d at reflux for 2 hours. A change in color was observed (solution went from dark yello w to a light straw color). The resulting solution was concentrated under reduced pr essure, and purified using flash column chromatography and a gradient of methylene ch loride and ethyl acetate . Yield: 823 mg (56%); on repeated trials, the yield ranged from 50 to 80%. Rf 0.3 (1:1 hexanes-ethyl acetate) ; mp 219-220 ºC (decomposes); [ ]D 29 + 29.9 (c = 0.98, CHCl3); IR (KBr) 3270, 3049, 2988, 2935 , 2873, 1748, 1702, 1599, 1455, 1374, 1338, 1217, 1161, 1065, 971, 915, 868, 735, 664, 573, 549 cm-1; 1H NMR (CDCl3, 300 MHz) 7.78 (d, J = 8.2 Hz, 2), 7.24 (d, J = 8.2 Hz, 2), 6.04 (dt, J = 10.3, 2.6 Hz, 1), 5.84 N NHTs O O 247 AcO O O TFA

PAGE 162

140 (dt, J = 9.9, 2.9 Hz, 1), 5.75 (m, 2), 5.03 (d, J = 8.7 Hz, 1), 4.67 (s, 1), 4.56 (m, 2), 4.32 (t, J = 5.3 Hz, 1), 4.08 (dd, J = 9.6, 9.1 Hz, 1), 3.83 (m , 2), 2.37 (s, 3), 2.17 (s, 3), 1.44 (s, 3), 1.41 (s, 3), 1.18 (s, 3), 0.84 (s, 3); 13C NMR (CDCl3, 75 MHz) 170.1, 158.2, 157.7, 142.9, 137.4, 133.5, 130.3, 129.1, 128.1, 126.3, 123.9, 111.5, 117.8, 115.0, 114.0, 110.8, 110.0, 78.0, 73.7, 72.4, 70.3, 68.7, 57.3, 52.6, 31.9, 29.7, 29.0, 26.6, 26.5, 25.2, 22.7, 21.4, 21.2; MS (FAB) m/z 667 (M+Na), 491, 145, 276, 264, 211, 155; HRMS calcÂ’d. for C29H35O9N2F3SNa: 667.1913; Found: 667.1909. An al. CalcÂ’d.: C 54.03, H 5.47, N 4.35; Found: C 53.82, H 5.53, N 4.06. Acetic acid 2,2,7-trimethyl-5-[[2,2,7,7-tetr amethyl-5S-(toluene-4-sulfonylamino)hexahydro-benzo[1,2-d;3,4-d']bis[1S,3R]d ioxol-4S-yl]-(2,2,2-trifluoro-acetyl)amino]-hexahydro-benzo[1,2-d;3,4-d' ]bis[1S,3R]dioxol-4S-yl ester To a solution of the diene ( 247 , 2.996 g, 4.6 mmol) in acetone (35 mL) was added N-methylmorpholine-N-oxide-1hydrate (1.57 g, 11.6 mmol) and th e solution was stirred at room temperature. A small cr ystal of osmium(VIII) tetroxide was then added, and the solu tion was stirred under argon overnight. The following day, the solution, now black, was quenched with excess saturated sodium bisulf ite, and the pH was adjusted to 2 with concentrated sulfuric acid. The aqueous la yer was extracted with ethyl acetate (10 x 150 mL), and the organic fractions were dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting dark oil was then redissolved in methylene chloride (25 mL) and 2,2-dimethoxypr opane (25 mL), to which a crystal of ptoluenesulfonic acid was added. The solution was heated to reflux for three hours, after which aqueous concentrated sodium bicarbona te (25 mL) was added, and the aqueous N O O 248 AcO O O TFA O O O O NHTs

PAGE 163

141 layer was extracted with diethy l ether (6 x 100 mL). The organic layers were combined and washed with saturated sodium bicarbonate (2 x 25 mL), then with saturated sodium carbonate (2 x 25 mL). The organic fracti ons were finally dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to afford a crude oil, 2.523 g. The desired material was purif ied using flash column chromatography with gradients of methylene chloride and hexane s. Yield: 0.939 g (26%), amorphous solids (in other runs, the yields will vary between 20-50%). Rf 0.5 (50:50 / hexane s:ethyl acetate); [ ]D 29 –34.3 (c = 0.285, CHCl3); IR (KBr) 3545, 3284, 2988, 2938, 1744, 1697, 1457, 1383, 1373, 1342, 1217, 1162, 1057, 858, 815, 666 cm-1; 1H NMR (CDCl3, 300 MHz) 7.76 (d, J = 8.0 Hz, 2), 7.23 (d, J = 8.0 Hz, 2), 5.96 (s, 2), 5.52 (t, J = 4.9 Hz, 1), 5.16 (d, J = 9.7 Hz, 1) , 4.94 (t, J = 7.0 Hz, 1), 4.64 (d, J = 3.9 Hz, 1), 4.58 (d, J = 5.4 Hz, 1), 4.28 (m, 3), 4.08 (t, J = 6.8 Hz, 1), 3.24 (dd, J = 11.8, 7.5 Hz, 1), 2.39 (s, 3), 2.19 (s , 3), 1.59 (s, 3), 1.49 (s, 3), 1.46 (s, 3), 1.42 (s, 3), 1.30 (s, 3), 1.26 (s, 3), 1.16 (s, 3), 1.06 (s, 3); 13C NMR (CDCl3, 75 MHz) 143.0 (up), 129.2 (down), 128.3 (down), 128.0 (down), 111.3 ( up), 109.9 (up), 79.2 (down), 77.0 (down), 74.1 (down), 73.7 (down), 70.7 (down), 69.5 (down), 60.0 (down), 29.9 (up), 28.2 (down), 27.8 (down), 27.2 (down), 26.7 (down), 25.5 (dow n), 24.9 (down), 21.6 (down), 21.5 (down); MS (FAB) m/z 793 (M+H), 777, 735, 661, 647, 530, 441, 280, 219; HRMS calc’d. for C35H48O13N2F3S: 793.2829; Found: 793.2843.

PAGE 164

142 N-[5-(5-Hydroxy-2,7,7-trimethyl-hexahydro -benzo[1,2-d;3,4-d']bis[1,3]dioxol-4ylamino)-2,2,7,7-tetramethyl-hexahydrobenzo[1,2-d;3,4-d']bis [1,3]dioxol-4-yl]-4methyl-benzenesulfonamide Fully hydroxylated dimer ( 248 , 1.106 g, 1.39 mmol) was dissolved in of 0.2 M NaOMe (25 mL) and the solution was stirred at room temperature overnight. The reaction was checked by TLC for consumption of starting material, then concentrated under reduced pressure to afford the crude material (1.083 g). Flash column chroma tography with gradients of hexanes and ethyl acetate afforded the desired aminoalcohol as a pale yellow powder. Yield: 518 mg (57%); Rf 0.2 (50:50-hexanes:ethyl acetate); mp 103-105 ºC; [ ]D 29.5 –19.6 (c = 0.37, CHCl3); IR (KBr) 3453, 3313, 3251, 2988, 2937, 2254, 1456, 1383, 1331, 1246, 1217, 1161, 1063, 913, 861, 814, 733, 667, 549, 513 cm-1; 1H NMR (CDCl3, 300 MHz) 7.82 (d, J = 8.2 Hz, 2), 7.28 (d, J = 8.2 Hz, 2), 5.85 (dt, J = 10.1, 2.6 Hz, 1), 5.76 (d, J = 10.1 Hz, 1), 5.54 (d, J = 5.0 Hz, 1), 4.61 (m, 1), 4.25 (m, 2), 4.16 (m, 2), 4.05 (m, 1), 3.33 (m, 2), 3.16 (m, 2), 2.71 (dd, J = 9.4, 6.6 Hz, 1), 2.43 (s, 3), 1.61 (bs, 3), 1.52 (s, 3), 1.49 (s, 3), 1.39 (s, 3), 1.32 (s, 3) , 1.26 (s, 6), 1.15 (s, 3); 13C NMR (CDCl3, 75 MHz) 143.5, 137.9, 129.7, 129.4, 127.9, 127.6, 109.8, 109.3, 1 09.2, 80.8, 80.3, 79.0, 76.3, 75.8, 75.7, 71.3, 63.5, 59.9, 56.5, 28.0, 27.9, 27.8, 27.3, 25.4, 25.3, 25.2, 21.7; MS (FAB) m/z 581 (M+H–C3H6O2), 428, 387, 386, 281, 221, 149; HRMS calc’d. for C28H41O9N2S: 581.2533; Found: 581.2557. HN O O 249 HO O O O O O O NHTs

PAGE 165

143 6-(2-Amino-3,4,5,6-tetrahydroxy-cycloh exylamino)-cyclohexane-1,2,3,4,5-pentaol hydrochloride Using a dry ice/acetone cold finger, amm onia (25 mL) was condensed in a 100 mL 2-neck round-bottomed flask. Sodium metal (172 mg, 7.5 mmol) was then added to the liquid ammonia, and a blue color immediately developed. Tosyldiaminoalcohol 249 (460 mg, 0.58 mmol) in THF (20 mL) was then added to the dissolving metal solution and allowed to stir for 1 min, after which the reaction was quenche d with excess 2-propanol. The crude solution was then concentrated under reduced pressure, and finally redissolved in deionized water (25 mL) and diethyl ether (150 mL). The layers were separated, and the aqueous la yer was then further extracted with diethyl ether (8 x 125 mL). The ethereal layer wa s dried (magnesium sulfate) and filtered, followed by concentration under reduced pres sure, which afforded 222 mg of light yellow solids, which were used as -is in the next transformation. The newly-formed diaminoalcohol was then dissolved in methanol (7.5 mL) at room temperature, and concentrated hydrochl oric acid (1 mL) was added. The solution was stirred for 5 minutes, then stirring was interrupted, allowing the solution to stand overnight. The following day, diethyl ether (30 mL) was added to the solution, which resulted in precipitation of the hydrochloride, which was filtered and washed with diethyl ether, affording the pure hydrochloride as off-white crystals. Yield: 136 mg of hydrochloride (47% over two steps). mp > 250 ºC; [ ]D 28.5 –51.5 (c = 0.325, CH3OH); IR (neat) 3341 (very broad), 2931, 1624, 1501, 1401, 1321, 1068, 664 cm-1; 1H NMR (CD3OD, 300 MHz) 4.22 (dd, 229 NH HO HO OH OH NH2OH OH OH HO HO 1.3 HCl

PAGE 166

144 J = 11.1, 2.4 Hz, 1), 4.11 (dd, J = 9.4, 2.8 Hz, 1), 4.05-3.85 (m, 6), 3.73 (dd, J = 9.4, 3.5 Hz, 1), 3.70-3.58 (m, 2), 3.41 (t, J = 10.6 Hz, 1); 1H NMR (D2O, 500 MHz) 4.09 (t, J = 3.5 Hz, 1), 4.04 (t, J = 3.7 Hz, 1), 4.01 (m, 2), 3.99 (dd, J = 10.8, 3.1 Hz, 1), 3.86 (dd, J = 10.3, 3.2 Hz, 1), 3.76 (dd, J = 10.1, 2.7 Hz, 1), 3.74 (dd, J = 10.6, 2.7 Hz , 1), 3.56 (t, J = 9.9 Hz, 1), 3.16 (t, J = 11.0 Hz, 1), 2.97 (t, J = 10.5 Hz, 1), 2.78 (t, J = 10.1 Hz, 1) Note: Chemical shifts will change with concentration; 13C NMR (CD3OD, 75 MHz) 73.33, 73.29, 73.15, 72.65, 72.30, 71.78, 69.86, 69.35, 69.23, 63 .49, 62.42, 53.40; MS (FAB) m/z 341 (M+), 291, 234, 186, 93; HRMS calc’d. for C12H25O9N2: 341.1560; Found: 341.1561. Anal. Calc’d. for C12H24N2O9•1.3 HCl: C 37.18, H 6.58, N 7.23; Found: C 36.82, H 6.94, N 6.87. N-[5-(4-Hydroxy-2,2-dimethyl-3a,4,5,7a-tetr ahydro-benzo[1,3]dioxol-5-ylamino)2,2-dimethyl-3a,4,5,7a-tetrahydro-ben zo[1,3]dioxol-4-yl]-4-methylbenzenesulfonamide To a flame-dried 100 mL flask, was added dibrominated dimer 244 (4.072 g, 6.13 mmol) and THF (40 mL). The solution was stirred and degassed with argon/sonication for 1.5 hours. The solution was then treated with AIBN (197 mg, 1.2 mmol) and heated to reflux. When the reaction began refluxing, tri-n-butyltinhydride was added (4 mL, 14.7 mmol) and the reaction was stirred and refluxed overnight. The following day, the solution was concentrated in vacuo and the resulting dimer was purified using flash column chromatography and a gradient of ethyl acetate and hexanes. Yield: 1.807 g (58%). Rf 0.12 (1:1 hexanes-ethyl acetate); mp 181-183 ºC (decomposes); [ ]D 28 + 1.3 (c 0.89, CHCl3); IR (KBr) 3479, 3259, 3038, 2987 , 2933, 1737, 1599, 1455, 1380, 1327, HN NHTs O O HO O O

PAGE 167

145 1248, 1216, 1159, 1061, 973, 896, 869, 815, 734, 666, 548 cm-1; 1H NMR (CDCl3, 300 MHz) 7.80 (d, J = 7.9 Hz, 2), 7.29 (d, J = 7.9 Hz, 2), 5.82 (m, 4), 5.12 (d, J = 7.74 Hz, 1), 4.6 (dd, J = 6.5, 2.1 Hz; 1), 4.52 (dd, J = 5.2, 3.2 Hz, 1), 4.07 (m, 2), 3.22 (m, 2), 3.05 (d, J = 7.0 Hz, 1), 3.01 (s, 1), 2.89 (d, J = 9.4 Hz, 1), 2.42 (s, 3) , 1.98 (bs, 2), 1.52 (s, 3), 1.39 (s, 3), 1.25 (s, 3), 1.11 (s, 3); 13C NMR (CDCl3, 75 MHz) 143.6, 137.2, 132.4, 131.6, 129.6, 127.6, 124.3, 124.2, 110.3, 110.1, 78.2, 76.1, 73.2, 72.4, 71.6, 56.7, 55.8, 55.2, 28.1, 27.3, 25.8, 25.6, 32.5; MS (FAB) m/ z 507 (M + H), 461, 383, 335, 327, 281, 207, 147; HRMS calcd. for C25H35O7N2S: 507.216498951; Found: 507.2151. Acetic acid 5-[2,2-dime thyl-4-(toluene-4-sulfon ylamino)-3a,4,5,7a-tetrahydrobenzo[1,3]dioxol-5-ylamino]2,2-dimethyl-3a,4,5,7a-tetrahyd ro-benzo[1,3]dioxol-4-yl ester To a solution of N-[5-(4-Hydroxy-2,2-dimethyl3a,4,5,7a-tetrahydro-benzo[1,3] dioxol-5-ylamino)-2,2dimethyl-3a,4,5,7a-tetrahydr o-benzo[1,3]dioxol-4-yl]-4methyl-benzenesulfonamide (1.81 g, 3.57 mmol) in pyridine (2.9 mL, 35.7 mmol) was added a spatula tip of DMAP followed by acetic anhydride (0.51 mL, 5.36 mmol). The reaction was stirre d at room temperature overnight and checked by TLC for consumption of starting ma terial. When no more starting material remained, methanol (5 mL) was added to th e mixture, and the solution was evaporated under reduced pressure. The excess pyridine was removed by forming the azeotrope with toluene. The product was purified using ei ther flash column chromatography or preparatory TLC. Yield: 1.84 g (94%). Rf 0.3 (1:1 hexanes-ethyl acetate); mp 169-170.5 ºC; [ ]D 27 + 5.9 (c = 1.2, CHCl3); IR (KBr) 3329, 3263, 3036, 2985, 2926, 2856 , 1743, 1598, 1456, 1380, 1330, 1244, HN NHTs O O AcO O O

PAGE 168

146 1218, 1160, 1063, 973, 916, 890, 867, 815, 733, 665, 547 cm-1; 1H NMR (CDCl3, 300 MHz) 7.79 (d, J = 8.1 Hz, 2), 7.29 (d, J = 7.8 Hz, 2), 5.79 (m, 4), 4.96 (t, J = 8.5 Hz, 1), 4.82 (d, J = 7.5 Hz, 1), 4.58 (d, J = 5.3 Hz, 1), 4.51 (s, 1), 4.09 (m, 2), 3.29 (q, J = 7.2 Hz, 1), 3.19 (d, J = 8.1 Hz, 1), 3.03 (d, J = 6.6 Hz, 1) , 2.42 (s, 3), 2.11 (s, 3), 1.71 (bs, 2), 1.50 (s, 3), 1.37 (s, 3), 1.24 (s, 3), 1.14 (s, 3); 13C NMR (CDCl3, 75 MHz) 170.7, 143.5, 137.5, 133.3, 132.4, 129.5, 127.6, 124.1, 123.5, 119.5, 115.0, 110.5, 110.3, 76.1, 76.0, 73.1, 72.4, 71.6, 56.1, 55.9, 55.5, 29.7, 27.9, 27.4, 26.3, 25.7, 21.5, 21.4; MS (FAB) m/z 549 (M + H), 530, 491, 441, 393, 295, 219, 154, 136, 91; HRMS calcd. for C27H37O8N2S: 549.227063612; Found: 549.2257. 4-Amino-3-hydro-7-(2-deoxy-ß-D-ribofuranosyl)-pyrrolo [2,3-d]pyrimidin-2-one 4-Amino-3-hydro-7-(3,5-di-O-to luoyl-2-deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d] pyrimidin-2-one ( 385 , 399 mg, 0.79 mmol) is dissolved in a methanolic solution of sodium methoxide (1 M, 10 mL) and the solution is stirred at room temperature for 2 hours. After checking for consumption of starting material (TLC), the mixture is concentrated in vacuo and purified using Prep HPLC with an aqueous 5 mM Et3N and acetonitrile gradient. Yield: 137 mg (65%) as a white powder. Rf 0.05 (80:20 / chloroform:methanol); mp 188-192 °C (ethanol); Lit. 229-231 °C (water); max (H2O) 225 ( 1.66 x 105), 254 ( 7.0 x 104) and 299 ( 6.5 x 104); 1H NMR (300 MHz, DMSO-d6) 6.95 (m, 1H), 6.22 (m, 1H), 6.18 (m, 1H), 4.4-4.5 (m, 1 H), 4.04.2 (m, 1H), 3.6-3.8 (m, 1 H), 2.4-2.8 (m, 1 H), 2.2-2.4 (m, 1 H) ppm; 13C NMR (75 MHz, DMSO-d6) 157.0, 152.2, 121.3, 101.1, 93.6, 86.8, 83.7, 71.5, 62.0, 38.9 ppm; FTIR (KBr) 3346, 3158, 2925, 1793, 1772, 1665, 1623, 1561, 1514, 1499, 1433, 1405, HN N O NH2N O OH HO 311

PAGE 169

147 1092, 1059, 703, 670 cm-1; HRMS (FAB, POS) Calc’d for C11H15N4O4 267.1093 m/z, Found 267.1102 m/z. 2-Methoxy-7-(3,5-diO -toluoyl-2-deoxy-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin4-ylamine 2-Methoxy-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine (1.043 g, 6.35 mmol) is stirred as a suspension in dry acetonitrile (50 mL) at room temperature. Sodium hydr ide (60% weight dispersion in mineral oil; 0.331 g, 8.27 mmol) is then added under argon, and the reaction mixture is stirred at r oom temperature for 15 minutes. 1( )-chloro-3,5-di-O-(p-toluoyl)-2-deoxy-D-ribose (2.787 g, 7.17 mmol) is added in two portions, and the resulting solution is stir red at room temperature overnight. Consumption of starting material is m onitored by TLC. The reaction mixture is concentrated on a rotary evaporator to afford a crude oil, which is purified using flash column chromatography with a gradient of methylene chloride and methanol (100% methylene chloride (500 mL) 50:1 – methylene chloride:methanol (500 mL) 25:1 methylene chloride:methanol (500 mL)). Yield: 2.457 g (75%) of 384 as a white powder. Repeating this procedure will afford a yield between 70-90%. Unstable compound. Rf 0.2 (50:50 / hexanes:ethyl acetate); mp 86-88 °C (hexanes/methylene chloride); 1H NMR (300 MHz, CDCl3) 7.95 (dd, J = 12.9, 8.2 Hz, 4 H), 7.26 (dd, J = 12.9, 8.2 Hz, 4 H), 6.96 (d, J = 3.7 Hz, 1H), 6.69 (dd, J = 8.4, 6.0 Hz, 1 H), 6.28 ( d, J = 3.7 Hz, 1 H), 5.75 (m, 1H), 5.08 (bs, 2 H), 4.45-4.74 (m, 3 H), 3.98 (s, 3 H), 2.80-2.88 (m, 1 H), 2.67 (ddd, J = 14.3, 5.9, 2.4 Hz, 1 H), 2. 44 (s, 3 H), 2.41 (s, 3 H) ppm; 13C NMR (75 MHz, N N O NH2N H3C O OTol TolO 384

PAGE 170

148 CDCl3) 166.4, 166.2, 162.6, 158.1, 152.9, 144.5, 144.2, 130.0, 129.8, 127.0, 126.8, 119.8, 99.8, 99.4, 84.0, 81.9, 75.3, 64.6, 54.6, 37.8, 21.9, 21.8 ppm; FTIR (neat) 3456, 3365, 1717, 1633, 1596, 1369, 1272, 1104 cm-1; HRMS (FAB, POS) Calc’d for C28H29N4O6 517.2087 m/z, Found 517.2092 m/z; Anal. Calc’d for C28H28N4O6: C, 65.11; H, 5.46; N, 10.85. Found: C, 66.75; H, 5.69; N, 9.24. 2-Methoxy-7-(2-deoxy-D-ribofuranosyl)-pyrrolo[ 2,3-d]pyrimidin-4-ylamine 2-Methoxy-7-(3,5-di-O-toluoyl-2-deoxy-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-ylamine ( 384 , 98 mg, 0.19 mmol) is dissolved in a methanolic solution of sodium methoxide (1 M, 2 mL) and the solution is stirred at room temperature for 2 hours. After checking for consumption of starting material (TLC), the mixture is concentrated in vacuo and purified using fl ash column chromatography with 25% (w/w) deactivated silica gel and elut ed with gradients of methylene chloride, methanol and aqueous concentrated ammonium hydroxide (95:5:0 – methylene chloride:methanol:ammonium hydroxide 90:10:0 – methylene chloride:methanol:ammonium hydroxide 90:10:1 – methylene chloride:methanol:ammonium hydroxide). Yiel d: 23 mg (43%) as a white crystalline material. Rf 0.7 (80:20 / chloroform:methanol); mp 182-183 °C (ethyl acetate); max (H2O) 261 ( 1.0 x 105) and 273 ( 1.0 x 105); 1H NMR (300 MHz, DMSO-d6) 7.12 (d, J = 3.8 Hz, 1 H), 7.03 (bs, 2 H), 6.49 (d, J = 3.8 Hz, 1 H), 6.38 (dd, J = 7.8, 6.2 Hz, 1 H), 5.25 (d, J = 4.1 Hz, 1 H), 4.95 (t, J = 5.8 Hz, 1 H), 4.33 (bs, 1H), 3.79 (s, 3 H), 3.40-3.60 (m, 2 H), 2.48 (m, 2 H), 2.12 (m, 1 H) ppm; 13C NMR (75 MHz, DMSO-d6) 162.0, 158.6, N N O NH2N H3C O OH HO 310

PAGE 171

149 151.6, 119.8, 100.1, 98.6, 87.1, 82.8, 71.2, 62.2, 53.5 ppm; FTIR (KBr) 3402, 2913, 1735, 1717, 1700, 1653, 1604, 1578, 1511, 1490, 1386, 1291, 1222, 1099, 1084, 1010, 935 cm-1; HRMS (FAB, POS) Calc’d for C12H17N4O4 281.1250 m/z, Found 281.1249 m/z; 4-Amino-3-hydro-7-(3,5-diO -toluoyl-2-deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidin-2-one 2-Methoxy-7-(3,5-di-O-toluoyl-2-deoxy-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-ylamine ( 384 , 3.135 g, 6.06 mmol) is suspended in dry ethylene glycol (20 mL) at room temperature. Benzenethiol (3 mL, 29.2 mmol) is then added, and the mixture is heated at 100-120 °C (but not above 130 °C). The reaction is monitored for consumption of starting mate rial by TLC (1-3 days). When no more starting nucleoside remains, the mixture is cool ed to room temperatur e, and ethyl acetate (150 mL) is added. The resulting solution is transferred to an Erlenmeyer flask and sodium tetraborate decahydrate (37.0 g, 97 mmol) is added as a powde r. The mixture is stirred for 5 min., after which the supernatan t is decanted and the solid mixture washed with ethyl acetate (3 x 50 mL ). The organic fractions are combined, concentrated in vacuo and chromatographed using gradient s of methylene chlo ride, methanol and ammonium hydroxide (conc. aq ueous) (50:1:0.2 – methylene chloride:methanol:ammonium hydroxide 50:3.5:0.2 – methylene chloride:methanol:ammonium hydroxide). Yiel d: 2.630 g (86%) of 5 as an off-white semisolid that can be recrystallized from methylene chloride/hexanes. Unstable compound. HN N O NH2N O OTol TolO 385

PAGE 172

150 Rf 0.13 (90:10 – methylene chloride:m ethanol); mp 70-75 °C (methylene chloride/hexanes); 1H NMR (300 MHz, DMSO-d6) 10.6-11.0 (bs, 2H), 10.66 (s, 1H), 7.91 (m, 4 H), 7.36 (m, 4H), 6.93 (m, 1H), 6.43 (d, J = 4.3 Hz, 1H), 6.41 (m, 1H), 5.61 (m, 1H), 4.61 (m, 1H), 4.53 (m, 2 H), 2.90 (m, 1H), 2.55 (m, 1H), 2.39 (s, 3 H), 2.38 (s, 3 H) ppm; 13C NMR (75 MHz, CDCl3) 170.6, 166.2, 166.0, 157.8, 157.5, 144.2, 144.0, 130.0, 129.6, 129.2, 128.9, 127.9, 126.7, 126.5, 93.7, 83.6, 83.2, 81.7, 74.9, 63.9, 37.7, 21.6 ppm; FTIR (KBr) 3340, 3119, 2926, 2861, 1720, 1659, 1613, 1499, 1443, 1389, 1273, 1178, 1103 cm-1; HRMS (LSIMS, POS) Calc’d for C27H27N4O6 503.1931 m/z, Found 503.1935 m/z; HRMS (FAB, POS) Calc’d for C27H27N4O6 503.1931 m/z, Found 503.1902 m/z. 4-( N , N -Diisobutyl-formamidine)-3-hydro-7-(3,5-diO -toluoyl-2-deoxy-ß-Dribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one 4-Amino-3-hydro-7-(3,5-di-O-toluoyl-2-deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 385 , 1014 mg, 2.02 mmol) is dissolved in methylene chloride (10 mL), and dimethoxymethyldiisobutylamine ( 2.5 mL of a 70% w/w solution in diisobutylformamide) is added dropwise. The solution is allowed to stir at room temperature overnight , after which a TLC of the reaction showed complete conversion of the starting material to two products. The mixture is concentrated to small bulk (yellow oil) and chromatographed using gradients of methylene chloride and methanol to afford a mixture of the two isomers. Yield: 945 mg (73%) as light yellow semisolids. The mixt ure was then repurified to resolve the two isomers. HN N O N N O OTol TolO N H 386

PAGE 173

151 Data for the first isomer (less polar): Rf 0.7 (90:10 – methylene chloride:m ethanol); mp 112-115 °C (methylene chloride/hexanes); 1H NMR (300 MHz, CDCl3) 8.60 (s, 1 H), 7.97 (d, J = 8.1 Hz, 2 H), 7.96 (d, J = 8.1 Hz, 2 H), 7.26 (d, J = 8.0 Hz, 2 H), 7.25 (d, J = 8.0 Hz, 2 H), 6.85 (d, J = 3.9 Hz, 1 H), 6.76 (dd, J = 8.8, 5.5 Hz, 1 H), 6.30 ( d, J = 3.9 Hz, 1 H), 5.66 (d, J = 6.1 Hz, 1 H), 4.45-4.70 (m, 3 H), 3.42 (d, J = 7.6 Hz, 2 H), 3.28 (d, J = 7.6 Hz, 2 H), 2.5-2.7 (m, 2 H), 2.43 (s, 3 H), 2.41 (s, 3 H), 2.14 (m, 1 H), 2.02 (m, 1 H), 0.95 (d, J = 6.7 Hz, 6 H), 0.94 (d, J = 6.5 Hz, 6 H) ppm; 13C NMR (75 MHz, CDCl3) 166.2, 166.1, 158.4, 144.2, 144.0, 129.8, 129.7, 129.6, 129.2, 129.1, 126.9, 126.5, 119.9, 102.2, 83.0, 81.2, 75.3, 64.6, 60.2, 53.0, 37.9, 27.2, 26.5, 21.7, 21.6, 20.2, 19.8 ppm; FTIR (KBr) 3422, 2961, 2931, 2872, 1722, 1611, 1553, 1402, 1271, 1178, 1104, 754 cm-1; HRMS (LSIMS, POS) Calc’d for C36H44N5O6 642.3292 m/z, Found 642.3279 m/z.; Data for the second isomer (more polar): Rf 0.6 (90:10 – methylene chloride:metha nol); mp 94-97 °C (methylene chloride/hexanes); 1H NMR (300 MHz, CDCl3) 8.57 (s, 1 H), 7.95 (d, J = 8.2 Hz, 2 H), 7.84 (d, J = 8.2 Hz, 2 H), 7.25 (d, J = 8.2 Hz, 2 H), 7.21 (d, J = 8.2 Hz, 2 H), 7.06 (d, J = 4.0 Hz, 1 H), 6.73 (dd, J = 7.4, 2.4 Hz, 1 H), 6.33 ( d, J = 4.0 Hz, 1 H), 5.61 (d, J = 7.3 Hz, 1 H), 4.81 (m, 1 H), 4.54 (m, 2 H), 3.44 (d, J = 7.6 Hz, 2 H), 3.27 (d, J = 7.3 Hz, 2 H), 3.02 (m, 1 H), 2.67 (m, 1 H), 2.42 (s, 3 H), 2.40 (s , 3 H), 2.25 (m, 1 H), 2.05 (m, 1 H), 0.97 (d, J = 6.6 Hz, 6 H), 0.95 (d, J = 6.5 Hz, 6 H) ppm; 13C NMR (75 MHz, CDCl3) 166.3, 166.0, 158.3, 144.2, 144.0, 129.7, 129.7, 129.2, 129.2, 126.8, 126.6, 121.1, 101.3, 84.5, 83.6, 75.5, 64.5, 60.3, 53.1, 38.5, 29.7, 27.3, 26.5, 22.6, 21.7, 20.2, 19.8 ppm; FTIR

PAGE 174

152 (KBr) 3434, 2960, 2921, 2872, 2851, 1722, 1612, 1551, 1445, 1387, 1271, 1101, 754 cm-1; HRMS (LSIMS, POS) Calc’d for C36H44N5O6 642.3292 m/z, Found 642.3316 m/z. 4-Amino-3-methyl-7-(2-deoxyß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one 4-(N,N-Diisobutyl-formamidine )-3-hydro-7-(3,5-di-O-toluoyl-2deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 386 , 250, 0.39 mmol) is dissolved in acetonitrile (5 mL) and sodium hydride (60% w/w dispersion in mineral o il, 43 mg, 1.1 mmol) is added. The solution is stirred at room temperature fo r 10 min., after which iodomethane (1 mL, 16 mmol) is added. The mixture is allowed to react at room temperature overnight. Consumption of starting material is confir med by TLC, after which methanolic ammonia (5 mL) and methanolic sodium methoxide (1 M, 2 mL) are added. The resulting mixture is stirred for 3 hours at 50 °C, after which the solids are removed by filtration. The filtrate is then concentrated to small bulk and triturated with absolute ethanol (2 x 50 mL). The product is purifie d using flash column chroma tography with 25% (w/w) deactivated silica gel and gr adients of methylene chlori de, methanol and ammonium hydroxide (conc. aq.). Yield: 25 mg (23%) as a white powder. The reaction has not been optimized. Rf 0.4 (80:20 – chloroform:methanol); mp 115-119 °C (methanol); 1H NMR (300 MHz, DMSO-d6) 7.79 (bs, 2 H), 6.82 (d, J = 3.9 Hz, 1 H), 6.46 (d, J = 3.9 Hz, 1 H), 6.24 (dd, J = 7.8, 6.1 Hz, 1 H), 5.30 (d, J = 4.1 Hz, 1 H), 4.22 (m, 1 H), 3.83 (m, 1 H), 3.46 (m, 2 H), 3.27 (s, 3 H), 2.0-2.25 (m, 2 H) ppm; 1H NMR (300 MHz, D2O) 7.01 (d, J = 3.8 Hz, 1 H), 6.51 (d, J = 3.9 Hz, 1 H), 6.40 (t, J = 7.1 Hz, 1 H), 4.52 (dt, J = 6.3, 3.6 Hz, 1 H), 4.11 (dt, J = 6.1, 3.8 Hz, 1 H), 3.62 (dt, J = 11.0, 6.3 Hz, 2 H), 3.39 (s, 3 H), N N O NH2N O OH HO 383

PAGE 175

153 2.60 (dt, J = 14.4, 6.8 Hz, 1 H), 2.41 (J = 14.4, 6.7, 3.9 Hz, 1 H) ppm; 13C NMR (75 MHz, DMSO-d6) 154.0, 153.1, 152.4, 117.7, 100.8, 91.2, 84.5, 81.5, 73.0, 58.5, 29.6 cm-1; HMBC (500 MHz, D2O), relevant correlations ( numbering corresponds to the purine numbering): 156.3 (C6), 3.33 (N-M e); 152.6 (C2), 3.33 (N-Me); 151.5 (C4), 6.85 (C7-H); 151.5 (C4), 6.35 (C8-H) ; 102.5 (C5), 6.85 (C7-H); 9 4.8 (C7), 6.35 (C8-H); FTIR (KBr) 3392, 3213, 2966, 2861, 1684, 1673, 1649, 1636, 1559, 1457, 1262, 1095, 1026, 804 cm-1; MS (FAB, POS) m/z 281 ([M+H]+, 21%), 267, 251, 221, 207, 191, 177, 165, 147; MS (HPLC/MS; (+)ESI-MS) m/z 281([M+H]+), 165 ([Heterocycle+H]+); HRMS (FT-ICR, POS) CalcÂ’d for C12H17N4O4 281.1250 m/z; Found 281.1175 m/z. 5-Iodo-2-methoxy-pyrimidine-4,6-diamine O-Methylisourea hemisulfate ( 14.35 g, 117 mmol) is dissolved in methanolic sodium methoxide (1 M, 117 mL, 117 mmol) and malononitrile (7.73 g, 117 mmol) is a dded at once. The mixture is heated to reflux for 3 hours and mon itored by TLC. Analytical samples of 392 are obtained by purificatio n (flash column chromatography using gradients of methylene chloride and methanol). After cooling the reaction mixture, I2 (22.33 g, 88 mmol) is added, and the mixture is allowe d to stir at room temperatur e for an additional 2 hours. Upon complete consumption of the intermedia te pyrimidine, ethyl ether (500 mL) is added and the solution is transferred to a se paratory funnel, after which aq. HCl (1 M, 300 mL) is added, and the layers are separated. The aqueous layer is kept and washed with ethyl ether (2 x 300 mL), after which it is neutralized with NaOH (pellets) to a pH ~ 8, and extracted with ethyl acetate (3 x 300 mL). The organic pha se is dried (MgSO4) N N NH2NH2O I 394

PAGE 176

154 and concentrated under reduced pressure. Yi eld: 11.27 g (36%) as a light yellow solid that can be recrystallized from toluene or hexanes. Rf 0.7 (90:10 – CH2Cl2:MeOH); mp 171-172 °C (hexanes); 1H NMR (300 MHz, CDCl3) 5.20 (bs, 4 H), 3.84 (s, 3 H) ppm; 13C NMR (75 MHz, CDCl3) 165.8, 163.8, 54.6, 47.7 ppm; FTIR (KBr) 3446, 3400, 3337, 3134, 1 646, 1608, 1578, 1542, 1452, 1433, 1354, 1141, 998, 778 cm-1; HRMS (EI, POS) Calc’d for C5H7IN4O ([M]+) 265.9665 m/z, Found 265.9666 m/z; Anal. Calc’d for C5H7IN4O: C, 22.57; H, 2.65; N, 21.06. Found: C, 22.97; H, 2.64; N, 20.66. 2-Methoxy-pyrimidine-4,6-diamine Synthesized as an interm ediate to 5-Iodo-2-methoxypyrimidine-4,6-diamine. Rf 0.3 (90:10 – CH2Cl2:MeOH); mp 138-139 °C (CH2Cl2/Hexanes); 1H NMR (300 MHz, CD3OD) 5.72 (bs, NH2), 5.24 (s, 1 H), 3.79 (s, 3 H) ppm; 13C NMR (75 MHz, CD3OD) 166.8, 166.6, 78.5, 54.2 ppm; FTIR (NaCl) 3448, 3339, 3147, 1630, 1585, 1448, 1359, 1244, 1196, 1145, 1096, 1067, 1032, 979, 799 cm-1; HRMS (EI, POS) Calc’d for C5H8N4O ([M]+) 140.0698 m/z, Found 140.0697 m/z; Anal. Calc’d for C5H8N4O: C, 42.85; H, 5.75; N, 39.98. Found: C, 43.27; H, 5.79; N, 39.55. 2-Diaminomethylene-malononitrile Byproduct of the synthesis of 2-Methoxy-pyrimidine-4,6-diamine. Rf 0.2 (90:10 – CHCl3:MeOH); mp 237-239 °C; 1H NMR (300 MHz, N N NH2NH2O 392 NCCN NH2H2N 393

PAGE 177

155 DMSO-d6) 6.923 (bs, NH2) ppm; 13C NMR (75 MHz, DMSO-d6) 165.1, 118.2, 31.5 ppm; FTIR (NaCl) 3438, 3327, 3226, 2210, 217 6, 1655, 1637, 1549, 1506 cm-1; HRMS (EI, POS) CalcÂ’d for C4H4N4 ([M]+) 108.0436 m/z, Found 108.0433 m/z; N-(3-Chloro-allyl)-2,2, 2-trifluoro-acetamide Trifluoroacetamide (10.44 g, 92.4 mmol) was dissolved in THF (30 mL), and sodium hydride (60% weight dispersion in mineral oil, 3.1 g, 77 mmol) was added in small portions at room temperature while regulating the temperature of the reaction with a water bath. The mi xture was stirred at room temperature for 5 min., after whic h E-1,3-dichloropropene (4.5 mL, 49.4 mmol) was added at once. After 2 min., the mixture thickened, and the reacti on had to be heated to reflux to melt the slurry. Reflux was ma intained for 45 min., after which the reaction was cooled to RT, and saturated aq. amm onium chloride (20 mL) and water (40 mL) were added. The mixture was transferred to a separatory funnel and extracted with ether (2 x 400 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure to obtained a colorless oil that was used as-is for the next transformation. Rf 0.45 (80:20 hexanes:ethyl acetate); 1H NMR (300 MHz, CDCl3) 7.33 (bs, 1H), 6.27 (d, J = 13.4 Hz, 1 H), 5.94 (dt, J = 13.3, 6.6 Hz, 1 H), 3.96 (d, J = 6.5 Hz, 2 H) ppm; 13C NMR (75 MHz, CDCl3) 157.5, 157.0, 127.0, 123.1, 121.3, 117.6, 113.7, 109.9, 39.7, 36.5 ppm; 19F NMR (282 MHz, CDCl3) -76.5 ppm. 2,2,2-Trifluoro-N-(5-trimethylsilany l-pent-2-en-4-yn yl)-acetamide 398 O H NCF3Cl

PAGE 178

156 N-(3-Chloro-allyl)-2,2,2trifluoro-acetamide (863 mg, 4.62 mmol) was dissolved in piperidine (3 mL), and dichlorobis(triphenylphosphi ne)palladium (131 mg, 0.19 mmol ), and copper(I) iodide (60 mg, 0.32 mmol) were added under n itrogen. After 2 min., the so lution turned light green, and TMS-acetylene (1 mL) was then added. Th e solution was allowed to stir overnight at RT under nitrogen, after which mixture was found to be dark red in color. The reaction was concentrated to small bulk under reduced pressure, loaded onto silica gel, and purified using flash column ch romatography with methylene chloride as the solvent. Yield: 617 mg (54%) as a colorless oil. Rf 0.5 (80:20—Hexanes:Ethyl acetate); 1H NMR (300 MHz, CDCl3) 6.93 (bs, 1H), 6.12 (dt, J = 15.9, 6.3 Hz, 1 H), 5.70 (dt, J = 15.9, 1.7 Hz, 1 H), 4.00 (dt, J = 6.0, 1.2 Hz, 2 H), 0.18 (s, 9 H) ppm; 13C NMR (75 MHz, CDCl3) 157.7, 157.2, 137.2, 117.8, 114.0, 113.8, 102.1, 96.7, 41.4, -0.1 ppm; 19F NMR (282 MHz, CDCl3) -76.3 ppm. N-[3-(4-Amino-2-methoxy-6-trimethylsil anyl-7H-pyrrolo[2,3d]pyrimidin-5-yl)allyl]-2,2,2-trifluoro-acetamide 5-Iodo-2-methoxy-pyrimidine-4,6-diamine (3.267 g, 12.28 mmol) is dissolved in acetonitrile (50 mL) and 2,2,2-trifluoro-N-(5-trimethyl silanyl-pent-2-en-4-ynyl)acetamide (3.209 g, 12.87 mmol), dichlorobis(triphenylphosphi ne)palladium (0.328 g, 0.467 mmol), and Huenigs base (12 mL, 69 mmol) are added. The mixture is heat ed to reflux for 3 hours, after which TLC shows complete consumption of the starting pyrimidine. The mixture is concentrated N N N H NHCOCF3TMS NH2O 400 399 O H NCF3TMS

PAGE 179

157 under reduced pressure and purified using flas h column chromatography. Yield: 3.580 g (75%) as an off-white solid. Rf 0.1 (95:5 – CH2Cl2:MeOH); mp 196-197 °C (Hexanes); 1H NMR (300 MHz, CDCl3) 8.90 (s, 1 H), 7.92 (m, 1 H), 6.84 (dd, J = 15.9, 1.5 Hz, 1 H), 5.81 (dt, J = 15.9, 6.1 Hz, 1 H), 5.69 (bs, 2 H), 4.12 (t, J = 5.8 Hz , 2 H), 3.89 (s, 3 H), 0.28 (s, 9 H) ppm; 13C NMR (75 MHz, CDCl3) 162.6, 158.5, 157.6, 154.9, 130.7, 127.8, 127.3, 121.5, 115.8, 98.1, 54.0, 41.8 ppm; 19F NMR (282 MHz, CDCl3) -76.1 ppm; FTIR (KBr) 3362, 3205, 2957, 2923, 2205, 1718, 1636, 1578, 1467, 1359, 1205, 1178, 1146, 841 cm-1; HRMS (FAB, POS) Calc’d for C15H21F3N5O2Si ([M+H]+) 388.1417 m/z, Found 388.1414 m/z; Anal. Calc’d for C15H21F3N5O2Si: C, 46.50; H, 5.20; N, 18.08; Found: C, 41.63; H, 5.35; N, 27.36. 2-Methoxy-5-trimethylsilanylethynyl-pyrimidine-4,6-diamine 5-Iodo-2-methoxy-pyrimidine-4,6-diamine (2.573 g, 9.67 mmol) was combined with dichlorobis(triphenylphosphine)palladium (138 mg, 0.20 mmol), copper(I) iodide (49 mg, 0.26 mmol), and diisopropylethylamine (10 mL) in dimethylformamide (1 mL) under nitrogen at RT. After 5 min., TMS-acetylene (2.7 mL, 11.91 mmol) was added, and the mixture was st irred at RT overnight. Upon complete consumption of the starting material (monito r by TLC), ethyl acetate (200 mL) and water (250 mL) were added, and the layers were separated. The aqueous layer was washed again with ethyl acetate (2 x 200 mL), and the combined organic phases were dried (MgSO4), filtered, and concentrated under reduced pressure. The product was purified N N NH2NH2O TMS 403

PAGE 180

158 using flash column chromatogr aphy using gradients of methyl ene chloride and methanol. Yield: 1.328 g (58%) as a light yellow powder. Rf 0.6 (95:5—CH2Cl2:MeOH); mp 155-155 °C; 1H NMR (300 MHz, CDCl3) 5.47 (bs, 4 H), 3.81 (s, 3 H), 0.22 (s, 9 H) ppm; 13C NMR (75 MHz, CDCl3) 165.2, 163.9, 105.3, 96.6, 75.3, 54.1, 0.1 ppm; FTIR (KBr) 3483, 3328, 3298, 2958, 2134, 1629, 1561, 1459, 1384, 1247, 1153, 1079 cm-1; HRMS (FAB, POS) Calc’d for C10H17N4O1Si ([M+H]+) 237.1172 m/z, Found 237.1159 m/z; Anal. Calc’d for C10H16N4O1Si: C, 50.82; H, 6.82; N, 23.71; Found: C, 51.21; H, 6.77; N, 23.94. HPLC Traces Figure 49. HPLC of 4-Am ino-3-hydro-7-(2-deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidin-2-one ( 311 ). Isocratic with 90% 5 mM Et3N (aq.) and 10% acetonitrile. Flow is 1 mL /min. Wavelength = 259 nm.

PAGE 181

159 Figure 50. HPLC of 2-Methoxy-7-(3,5-di-O-toluoyl-2-deoxy-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-ylamine ( 384 ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL/min. Wavelength = 254 nm. Figure 51. HPLC of 2-Methoxy-7-(2-deoxy-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin4-ylamine ( 310 ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL/min. Wavelength = 254 nm.

PAGE 182

160 Figure 52. HPLC of 4Amino-3-hydro-7-(3,5-di-O-toluoyl-2-deoxy-ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-2-one ( 385 ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL/min. Wavelength = 300 nm. Figure 53. HPLC of 4-(N,N-Diisobutyl-formamidine )-3-hydro-7-(3,5-di-O-toluoyl-2deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 386, top isomer ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL/min. Wavelength = 390 nm.

PAGE 183

161 Figure 54. HPLC of 4-(N,N-Diisobutyl-formamidine )-3-hydro-7-(3,5-di-O-toluoyl-2deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3-d]pyrimidin-2-one ( 386, bottom isomer ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL/min. Wavelength = 390 nm. Figure 55. HPLC of 4-Am ino-3-methyl-7-(2-deoxy-ß-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidin-2-one ( 383 ). Solvent A is 5 mM Et3N (aq.). Solvent B is acetonitrile. Flow is 1 mL /min. Wavelength = 300 nm.

PAGE 184

162 LIST OF REFERENCES 1. Bouveault, L., De l'isomérie optique dans les corps à chaînes fermées. Bull. Soc. Chim. Paris 1894 , 11, 144-147. 2. Fletcher, H. G., Jr., The chemistry a nd configuration of the cyclitols. In Advances in Carbohydrate Chemistry, Pigman, W. W.; Wolfrom, M. L., Eds. Academic Press: New York, 1948; Vol. 3, pp 45-77. 3. Angyal, S. J.; Anderson, L., The cyclitols. In Advances in Carbohydrate Chemistry, Wolfrom, M. L.; Tipson, R. S., Eds. A cademic Press: New York, 1959; Vol. 14, pp 135-212. 4. Hudlicky, T.; Cebulak, M., Cyclitols and their derivatives: A handhook of physical, spectral, and synthetic data. VCH: New York, 1993. 5. Posternak, T., Les cyclitols: chimie, biochimie, biologie. Hermann: Paris, 1962. 6. Billington, D. C., Recent devel opments in the synthesis of myo-inositol phosphates. Chem. Soc. Rev. 1989 , 18, 32-122. 7. Cosgrove, D. J., Studies in organic chemistry 4: Inositol phosphates; their chemistry, biochemistry and physiology. Elsevier: New York, 1980. 8. Reitz, A. B., Inositol phosphates and derivatives. Synthesis, biochemistry, and therapeutic potential. American Chemical Society: Washington, DC, 1991; Vol. 463. 9. Balci, M.; Sütbeyaz, Y.; Seçen, H., Conduritols and related compounds. Tetrahedron 1990 , 46, (11), 3715-3742. 10. Angyal, S. J.; McHugh, D. J., Cyclitols. V. Paper ionophoresis, complex formation with borate, and the rate of periodic acid oxidations. J. Chem. Soc. 1957 , 1423-1431. 11. Angyal, S. J.; Greeves, D.; Pickles, V. A., Stereochemistry of complex-formation of polyols with borate and periodate anions, and with metal cations. Carbohydr. Res. 1974 , 35, (1-2), 165-173. 12. Hancock, R. D.; Hegetschweiler, K ., A molecular mechanics study of the complexation of metal-ions by inositols. J. Chem. Soc. Dalton 1993 , (14), 21372140.

PAGE 185

163 13. Angyal, S. J., Complexes of metal ca tions with carbohydrates in solution. In Adv. Carbohyd. Chem. Bi., Tipson, R. S.; Horton, D., Eds. Academic Press: San Diego, 1989; Vol. 47, pp 1-43. 14. Angyal, S. J.; Hickman, R. J., Cyclit ols XXXIII. A practical synthesis of cisinositol. Carbohydr. Res. 1971 , 20, 97-104. 15. Angyal, S. J.; Mchugh, D. J., Cy clitols. VI. The hydrogenation of hexahydroxybenzene. J. Chem. Soc. 1957 , 3682-3691. 16. Anderson, R. C.; Wallis, E. S., The catalytic hydrogenation of polyhydridic phenols. I. The synthesis of meso-Inositol, scyllitol and a new isomeric cyclitol. J. Am. Chem. Soc. 1948 , 70, 2931-2935. 17. Angyal, S. J.; Hoskinson, R. M., Cyc litols XIII. The conformation of cyclic diketals: The skew conformation. J. Chem. Soc. 1962 , 2991-2995. 18. Angyal, S. J.; Tate, M. E.; Gero, S. D., Cyclitols. 9. Cyclohexylidene derivatives of myo-inositol. J. Chem. Soc. 1961 , 4116-4122. 19. Angyal, S. J.; Odier, L.; Tate , M. E., A simple synthesis of cis-inositol. Carbohydr. Res. 1995 , 266, (1), 143-146. 20. Chung, S.-K.; Kwon, Y.-U., Practical synthesi s of all inositol stereoisomers from myo-inositol. Bioorg. Med. Chem. Lett. 1999 , 9, (15), 2135-2140. 21. Ozaki, S.; Koga, Y.; Ling, L.; Watanabe, Y.; Kimura, Y.; Hirata, M., Synthesis of 2-substituted myo-inositol 1,3,4,5-tetrakis(phosphate) and 1,3,4,5,6pentakis(phosphate) analogs. Bull. Chem. Soc. Jpn. 1994 , 67, (4), 1058-1063. 22. Chung, S.-K.; Chang, Y.-T., Synthesis of all possible regioisomers of myo-inositol pentakisphosphate. Bioorg. Med. Chem. Lett. 1996 , 6, (17), 2039-2042. 23. Posternak, T., Recherches dans la série des cyclites. VIII. Sur la configuration du d,l-épi-ms-inosose et de sa forme lé vogryre obtenue par voie biochimique. Helv. Chim. Acta 1946 , 29, 1991-1998. 24. Posternak, T., Recherches dans la série de s cyclites. V. Sur un inosose préparé par voie biochimique. Helv. Chim. Acta 1941 , 24, 1045-1058. 25. Kluyver, A. J.; Boezaardt, A. G. J., Note on the biochemical preparation of inosose. Rec. Trav. Chim. Pays-Bas 1939 , 38, 956-958. 26. Atsushi, T.; Tsuyoshi, T.; Kenji, K.; Yuichi, K.; Tetsuya, M.; Tomio, T. Novel process for producing L-epi-2-inosose and novel process for producing epi-Inositol. EP1197562; WO0075355, 2002.

PAGE 186

164 27. Gigg, J.; Gigg, R., Reactions of th e ketone derived from (±)-3,4,5-tri-O-benzyl-1,2-O-isopropylidene-myo-inositol: preparation of racemic derivatives of epi-inositol and of 4-C-methyl-epi-[(±)-iso-laminitol] and 4-C-methyl-myo-inositol [(±)laminitol]. Carbohydr. Res. 1997 , 299, 77-83. 28. Tschamber, T.; Backenstrass, F.; Fritz, H.; Streith, J., Cataly tic one-pot osmylation of cyclohexadienes: Stereochemical and conformational studies of the resulting polyols. Helv. Chim. Acta 1992 , 75, 1052-1060. 29. Pistarà, V.; Barili, P. L.; Catelani, G. ; Corsaro, A.; D'Andrea, F.; Fisichella, S., Rare and complex saccharides from D-galactose and other milk-derived carbohydrates. Part 12. A new highly diastere oselective synthesi s of epi-inositol from D-galactose. Tetrahedron Lett. 2000 , 41, (17), 3253-3256. 30. Barili, P. L.; Berti, G.; Catelani, G.; Dandrea, F., New synthetic pathways to 5-Calkoxypyranosides and to hexos-5-ulose derivatives. Gazz. Chim. Ital. 1992 , 122, (4), 135-142. 31. Dangschat, G.; Fischer, H. O. L., Üb er die Konstitution des Condurits und über einige Cyclohexantetrole. Naturwissenschaften 1939 , 27, 756-757. 32. Dangschat, G.; Fischer, H. O. L., The c onfiguration of conduritol and the synthesis of muco-inositol and allo-inositol. Carbohydr. Res. 1987 , 164, 343-355. 33. Angyal, S. J.; Gilham, P. T., Cyclitols . VIII. Elimination of vicinal sulfonyloxy groups by iodide ion. J. Chem. Soc., Abstracts 1958 , 375-379. 34. Angyal, S. J.; MacDonald, C. G., Cyclitols. Part I. isoPropylidene derivatives of inositols and quercitols. The stru cture of pinitol and quebrachitol. J. Chem. Soc. 1952 , 686-695. 35. Desjardins, M.; Brammer Jr., L. E.; Hudlicky, T., A practical multigram-scale synthesis of allo-inositol. Carbohydr. Res. 1997 , 304, 39-42. 36. Gibson, D. T.; Hensley, M.; Yoshioka, H.; Mabry, T. J., Formation of (+)-cis-2,3dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry 1970 , 9, (7), 1626-1630. 37. Hudlicky, T.; Stabile, M. R.; Gibson, D. T.; Whited, G. M., 1-Chloro-(2S,3S)dihydroxycyclohexa-4,6-diene. Org. Synth. 1999 , 76, 77-85. 38. Mehta, G.; Lakshminath, S., A norbornyl ro ute to cyclohexitols: stereoselective synthesis of conduritol-E, allo-inositol, MK 7607 and gabosines. Tetrahedron Lett. 2000 , 41, 3509-3512. 39. Mehta, G.; Mohal, N., Norbornyl r oute to cyclopentitols: Synthesis of trehazolamine analogues and the pur ported structure of salpantiol. Tetrahedron Lett. 1999 , 40, (31), 5795-5798.

PAGE 187

165 40. Mehta, G.; Mohal, N., A norbornyl route to cyclopentitols via novel regiospecific fragmentation of a 2,7-disubstituted norbornane. Tetrahedron Lett. 1999 , 40, (31), 5791-5794. 41. Mehta, G.; Mohal, N., Norbornyl route to polyoxygenated cycl ohexanes. A facile entry into carbasugars and shikimic acid. Tetrahedron Lett. 1998 , 39, (20), 32853286. 42. Mehta, G.; Mohal, N., Norbornyl r oute to polyoxygenated cyclohexanes. An approach to pancratistatin a nd narciclasine alkaloids. Tetrahedron Lett. 1998 , 39, (20), 3281-3284. 43. Mehta, G.; Mohal, N.; Lakshminath, S ., A norbornyl route to cyclohexitols: structural diversity in fr agmentation through functiona l group switching. Synthesis of and -galactose, -talose, and -fucopyranose carbasugars. Tetrahedron Lett. 2000 , 41, 3505-3508. 44. Scherer, J., Ueber eine neue, aus de m Muskelfleische gewonnene Zuckerart. Annalen 1850 , 73, 322-328. 45. Scherer, J., Ueber der Inosit. Annalen 1852 , 81, 375-375. 46. Maquenne, M., Chimie organique. Sur les proprétés de l'inosite. C. R. Hebd. Acad. Sci. 1887 , 104, 297-299. 47. Salkowski, E., Kleinere Mitteilungen. Z. physiol. Chem. 1910 , 69, 466-481. 48. Wieland, H.; Wishart, R. S., Die Synthese des natürlichen Inosits. Chem. Ber. 1914 , 47, 2082-2085. 49. Stetten, M. R.; Stetten, D ., Jr., Biological conversion of inositol into glucose. J. Biol. Chem. 1946 , 164, 85-91. 50. Kuhn, R.; Qadbeck, G.; Röhm, E., Zur Synthese des meso-Inosits. Annalen 1949 , 565, 1-6. 51. Posternak, S.; Posternak, T., Ch imie biologique. Sur un éther inositoltétraphosphorique naturel optiquement actif. C. R. Hebd. Acad. Sci. 1928 , 186, 261-263. 52. Posternak, S.; Posternak, T., Sur la configuration de l'inosite inactive. Helv. Chim. Acta 1929 , 12, 1165-1171. 53. Walden, P., Weiters über optisch active Halogenverbindungen. Berichte 1895 , 28, 2766-2773. 54. Walden, P., Ueber optisch active Halogenverbindungen. Berichte 1895 , 28, 12871297.

PAGE 188

166 55. Peirce, G., The configuration of some of the higher monosaccharides. J. Biol. Chem. 1915 , 23, 327-337. 56. Fischer, E., Ueber ein neues Isomer s des Schleimsäure und die sogennante Paraschleimsäure. Berichte 1891 , 24, 2136-2143. 57. Posternak, T., Sur l'acide allomucique er sur un nouvel acide tétraoxy-adipique. Helv. Chim. Acta 1935 , 18, 1283-1287. 58. Fischer, E., Synthesen in der Zuckergruppe II. Berichte 1894 , 27, 3189-3232. 59. Posternak, T., Sur l'inosine de Kluyver et su r la configuration de la méso-inosite et de la scyllite. Compt. Rend. Soc. Phys. Hist. Nat. Genève 1941 , 58, 44-47. 60. Dangschat, D., Acetonier ung und Konfiguration des meso-Inosits. Naturwissenschaften 1942 , 30, 146-147. 61. Posternak, T., Recherche dans la série des cy clites. VI. Sur la configuration de la méso-inosite, de la scyllite, et d'un inos ose obtenu par voie biochimique (scyllo-msinosose). Helv. Chim. Acta 1942 , 25, 746-753. 62. Böeseken, J.; Julius, A., La configura tion de l'inosite et de la quericte. Rec. Trav. Chim. Pays-Bas 1926 , 45, 489-490. 63. Karrer, P., Die Konfiguration des d-Quercits. Helv. Chim. Acta 1926 , 9, 116-117. 64. Micheel, F.; Ruhkopf, H.; Suckfüll, F., Zur Kenntnis der Umwandlung von Hexosen in Inosite. Berichte 1935 , 68, 1523-1527. 65. Meerwein, H., Über die Vergrößeru ng der Ionisationsfähigkeit schwacher Elektrolyte durch Komplexbildung und ihre Bedeutung für katalytische Prozesse. Annalen 1927 , 455, 227-253. 66. Posternak, T., 209. Recherches dans la série des cyclitols XIV. Désamination nitreuse d'amino-cyclitols. Synthèse du d,l-viburnitol et nouvelle synthèse complète du méso-inositol. Helv. Chim. Acta 1950 , 33, 1597-1605. 67. Grosheintz, J. M.; Fischer, H. O. L., Synthesis of 6-nitro-6-deoxy-D-glucose and 6nitro-6-desoxy-L-idose. J. Am. Chem. Soc. 1948 , 70, 1476-1479. 68. Grosheintz, J. M.; Fischer, H. O. L., Cyclization of 6-nitrodesoxyaldohexoses to nitrodesoxyinositols. J. Am. Chem. Soc. 1948 , 70, 1479-1484. 69. Schubert, J.; Keller, R.; Schwesinger, R.; Prinzbach, H., Polyfunktionalisierte Cylohexane aus Dianhydroinositen cis-1,3(1,4)-Inosadiamine aus Benzol. Chem. Ber. 1983 , 116, 2524-2545.

PAGE 189

167 70. Chiara, J. L.; Martín-Lomas, M., A ster eoselective route to enantiomerically pure myo-inositol derivatives starting from D-mannitol. Tetrahedron Lett. 1994 , 35, (18), 2969-2972. 71. Le Merrer, Y.; Duréault, A.; Greck, C. ; Micas-Languin, D.; Gravier, C.; Depezay, J.-C., Synthesis of diepoxides and diaziridi nes, precursors of enantiomerically pure -hydroxy and -amino aldehydes or acids, from D-mannitol. Heterocycles 1987 , 25, 541-548. 72. Angyal, S. J.; Bender, V.; Curtin, J. H ., Cyclitols. Part XIII. Suppression of epoxide migration. Synthesis of muco-inositol. J. Chem. Soc. C 1966 , 798-800. 73. Angyal, S. J.; Gilham, P. T.; Melrose, G. J. H., Cyclitols.16. Toluene-p-sulphonyl derivatives of myo-inositol . Acetyl migration in anhydrous pyridine solution. J. Chem. Soc. 1965 , 5252-5255. 74. Angyal, S. J.; Stewart, T. S., XXVII. The reaction of tosylinositols with sodium benzoate in dimethylformamide. Anomalous opening of epoxides. Aust. J. Chem. 1967 , 20, 2117-2136. 75. Suami, T.; Lichtenthaler, F. W.; Ogawa, S., Aminocyclitols. XI. Studies on the synthesis of muco-inosadiamines and inosatetraamine. Bull. Chem. Soc. Jpn. 1967 , 40, 1488-1495. 76. Suami, T.; Ogawa, S., Aminocyclitol s .4. The new synthesis of inosamine. Bull. Chem. Soc. Jpn. 1964 , 37, (8), 1238-1239. 77. Paulsen, H.; Höhne, H., Acyloxonium-Unlagerung von myo-Inosit zu einem Dikation des muco-Inosits. Chem. Ber. 1972 , 105, 3445-3455. 78. Hedgley, E. J.; Fletcher, H. G., Behavior of esters in liquid hydrogen fluoride facile inversions in cyclitol series. J. Am. Chem. Soc. 1962 , 84, (19), 3726-3731. 79. Angyal, S. J.; Odier, L., An easy synthesis of muco-inositol. Carbohydr. Res. 1980 , 80, 203-206. 80. Angyal, S. J.; James, K., Oxidative demet hylation with chromium trioxide in acetic acid. Carbohydr. Res. 1970 , 12, 147-149. 81. Angyal, S. J.; Matheson, N. K., Cyclitols. III. Some tosyl esters of inositols. Synthesis of a new inositol. J. Am. Chem. Soc. 1955 , 77, (16), 4343-4346. 82. Ballou, C. E.; Fischer, H. O. L., Derivatives of D-mannohexodialdose (6-aldo-Dmannose). J. Am. Chem. Soc. 1953 , 75, (15), 3673-3675. 83. Richtmyer, N. K.; Hudson, C. S., Crystalline -methyl-D-altroside and some new derivatives of D-altrose. J. Am. Chem. Soc. 1941 , 63, (6), 1727-1731.

PAGE 190

168 84. Ansell, E. G.; Honeyman, J., Sugar nitr ates. 1. Removal of nitrate and toluene-para-sulphonyl groups from esters of subs tituted methyl-deuterium-glucosides. J. Chem. Soc. 1952 , 2778-2789. 85. Wiggins, L. F., The conversion of galactose into derivatives of D-idose. J. Chem. Soc. 1944 , 522-526. 86. Robertson, G. J.; Whitehead, W., Walde n inversions in the altrose series. J. Chem. Soc. 1940 , 319-323. 87. Robertson, G. J.; Griffith, C. F., The c onversion of derivatives of glucose into derivatives of altrose by simple optical inversions. J. Chem. Soc. 1935 , 1193-1201. 88. Mukherjee, R.; Axt, E. M., Cyclitols from Croton celtidifolius. Phytochemistry 1984 , 23, (11), 2682-2684. 89. Sherman, W. R.; Goodwin, S. L.; Gunnell, K. D., neo-Inositol in mammalian tissues. Identification, measurement, and enzymic synthesis from mannose 6phosphate. Biochemistry 1971 , 10, (19), 3491-3499. 90. Angyal, S. J.; Craig, D. C., The unus ually stable crystal structure of neo-inositol. Carbohydr. Res. 1994 , 263, 149-154. 91. Riley, A. M.; Jenkins, D. J.; Potter, B. V. L., A concise synthesis of neo-inositol. Carbohydr. Res. 1998 , 314, (3-4), 277-281. 92. Montchamp, J.-L.; Tian, F.; Hart, M. E.; Frost, J. W., Butane 2,3-bisacetal protection of vicinal diequatorial diols. J. Org. Chem. 1996 , 61, (11), 3897-3899. 93. Hense, A.; Ley, S. V.; Osborn, H. M. I.; Owen, D. R.; Poisson, J.-F.; Warriner, S. L.; Wesson, K. E., Direct preparation of diacetals from 1,2-diketones and their use as 1,2-diol protecting groups. J. Chem. Soc., Perkin Trans. 1 1997 , (14), 20232032. 94. Hudlicky, T.; Restrepo-Sanchez, N.; Kary, P. D.; Jaramillo-Gomez, L. M., A short, stereoselective synthesis of neo-inositol. Carbohydr. Res. 2000 , 324, (3), 200-203. 95. Staedeler, G.; Frerichs, F. T., Ueber di e allotropischen Zustände des Schewefels. J. Prakt. Chem. 1858 , 73, 48-49. 96. Vincent, C.; Delachanal, S., Sur un hydrat e de carbone contenu dans le gland du chêne. C. R. Hebd. Acad. Sci. 1887 , 104, 1855-1858. 97. Muller, H., Cocositol (cocosite) , a constituent of the leaves of Cocos nucifera and Cocos plumosa. J. Chem. Soc. 1907 , 91, 1767-1780. 98. Muller, H., Inos itol (inosite). J. Chem. Soc. 1907 , 91, 1780-1793.

PAGE 191

169 99. Muller, H., Inositol and some of its isomerides. J. Chem. Soc. 1912 , 101, 23832410. 100. Mohr, M., Nitrogen-containing constituent of shark muscle (Acanthias vulgaris). Z. Biol. 1937 , 98, 276-280. 101. Ackermann, D.; Mohr, M., N itrogen-containing constituent of the liver of the shark (Acanthias vulgaris). Z. Biol. 1937 , 98, 37-42. 102. Sando, C. E.; Markley, K. S.; Matlack, M. B., Some chemical constituents of flowering dogwood (Cornus florida). J. Biol. Chem. 1936 , 114, (1), 39-45. 103. Kohne, B.; Praefcke, K., Notiz zur Darstellung von scyllo-Inosit. Liebigs Ann. Chem. 1985 , (4), 866-868. 104. Posternak, T., Myo-epi-Inose-2 and Dl-epi-Inosose-2. Biochem. Prep. 1952 , 2, 5764. 105. Reymond, D., Recherches dans la serie des cyclitols .23. Sur la reduction de deux inososes par le borohydrure de sodium. Helv. Chim. Acta 1957 , 40, (2), 492-494. 106. Husson, C.; Odier, L.; Vottéro, P. J. A., New conditions for the synthesis of scylloinositol starting from myo-inositol. Carbohydr. Res. 1998 , 307, 163-165. 107. Sarmah, M. P.; Shashidhar, M. S., Sulf onate protecting groups. Improved synthesis of scyllo-inositol and its ort hoformate from myo-inositol. Carbohydr. Res. 2003 , 338, (9), 999-1001. 108. Praveen, T.; Shashidhar, M. S., C onvenient synthesis of 4,6-di-O-benzyl-myoinositol and myo-inositol 1,3,5-orthoesters. Carbohydr. Res. 2001 , 330, (3), 409411. 109. Kohne, B.; Praefcke, K., Hexa-O-alkanoyl-scyllo-inositols, the first alicyclic, saturated, discotic liquid crystals. Angew. Chem. Int. Ed. 1984 , 23, (1), 82-83. 110. Seaquist, E. R.; Gruetter, R., Iden tification of a high concentration of scyllo-inositol in the brain of a healt hy human subject using H1and C13-NMR. Magnet. Reson. Med. 1998 , 39, (2), 313-316. 111. Berthelot, M., Sur quelques matières sucrées. C. R. Hebd. Acad. Sci. 1855 , 41, 392396. 112. Tanret, C., Sur deux sucres nouveaux retirés du québracho. C. R. Hebd. Acad. Sci. 1889 , 109, 908-910. 113. DeJong, A. W. K., La présence de québrachite dans le latex de Hevea brasiliensis. Rec. Trav. Chim. Pays-Bas 1906 , 25, 48-49.

PAGE 192

170 114. Contardi, A., Derivati forforici dell'inosite dalla Hevea brasiliensis. Ann. Chim. Applicata 1924 , 14, 281-289. 115. Levi, T. G., Quebrachitol from the serum of Hevea latex. Rubber Chem. Tech. 1930 , 3, 1-2. 116. Posternak, T., Recherches dans les série des cyclites. III. Sur la configuration des inosites actives. Helv. Chim. Acta 1936 , 19, 1007-1010. 117. McCance, R. A.; Lawrence, R. D., An i nvestigation of quebrachitol as a sweetening agent for diabetics. Biochem. J. 1933 , 27, 986-989. 118. Maquenne, M.; Tanret, C., Sur une i nosite nouvelle, la racémo-inosite. C. R. Hebd. Acad. Sci. 1890 , 110, 86-88. 119. Tanret, G., Sur les inosites du Gui. C. R. Hebd. Acad. Sci. 1907 , 145, 1196-1198. 120. Fletcher, H. G.; Findlay, G. R., The identity of Müller's iso-inositol with racemic inositol. J. Am. Chem. Soc. 1948 , 70, (12), 4050-4052. 121. Berlin, W. K.; Zhang, W.-S.; Shen, T. Y., Glycosyl inositol derivatives. III. Synthesis of hexosamine-inositol-phosphates related to putative insulin mediators. Tetrahedron 1991 , 47, (1), 1-20. 122. Chiara, J. L.; Valle, N., Synthesis of L-chiro-inositol and (-)-conduritol F from sorbitol by a highly stereoselective intr amolecular pinacol coupling promoted by samarium diiodide. Tetrahedron: Asymmetry 1995 , 6, (8), 1895-1898. 123. Kim, K. S.; Park, J. I.; Moon, H. K.; Yi, H., Transformation of cyclohexene to enantiopure cyclitols mediated by sequential oxyselene nylation with (S,S)hydrobenzoin: synthesis of D-chiro-inositol and muco-quercitol. J. Chem. Soc., Chem. Commun. 1998 , (18), 1945-1946. 124. Takahashi, Y.; Nakayama, H.; Katagiri , K.; Ichikawa, K.; Ito, N.; Takita, T.; Takeuchi, T.; Miyake, T., Facile and pr actical synthesis of optically pure 1D-chiroinositol from myo-inositol. Tetrahedron Lett. 2001 , 42, (6), 1053-1056. 125. Suami, T.; Ogawa, S.; Tanaka, T.; Otake, T., para-Toluenesulfonylation of 1,2-Ocyclohexylidene-myo-inositol. Bull. Chem. Soc. Jpn. 1971 , 44, (3), 835-841. 126. Catelani, G.; Corsaro, A.; D'Andrea, F.; Mariani, M.; Pistarà, V., Intramolecular aldol cyclization of L-lyxo-hexos-5-ulose derivatives: A new diastereoselective synthesis of D-chiro-Inositol. Bioorg. Med. Chem. Lett. 2002 , 12, (22), 3313-3315. 127. Barili, P. L.; Berti, G.; Catelani, G.; D ’Andrea, F.; de Rensis, F.; Goracci, G. J., Preparation of L-lyxo-hexos-5-ulose through C-3 epimerization of bisglycopyranosides of L-arabino-hexos-5-ulose. J. Carbohydr. Chem. 1998 , 17, (8), 1167-1180.

PAGE 193

171 128. Evans, D. A.; Chapman, K. T.; Carreira, E. M., Directed reduction of beta-hydroxy ketones employing tetramethyla mmonium triacetoxyborohydride. J. Am. Chem. Soc. 1988 , 110, (11), 3560-3578. 129. Nakajima, M.; Tomida, I.; Kurihara, N.; Ta kei, S., Zur Chemie des Benzolglykols. V. Eine neue Synthese der Inosite. Chem. Ber. 1959 , 173-178. 130. Kowarski, C. R.; Sarel, S., To tal stereoselective synthesis of myo-, allo-, neo-, and epi-inositols. J. Org. Chem. 1973 , 38, (1), 117-119. 131. Mandel, M.; Hudlicky, T., Corrigendum. J. Chem. Soc., Perkin Trans. 1 1993 , (13), 1537-1537. 132. Mandel, M.; Hudlicky, T.; Kwart, L. D. ; Whited, G. M., Unusual oxidation of 1halo-1,3-dienes with permanganate. Expedient syntheses of (+)-D-chiro-3-inosose and (+)-D-chiro-inositol from chlorobenzene. J. Org. Chem. 1993 , 58, (8), 23312333. 133. Mandel, M.; Hudlicky, T., General synt hesis of inositols by hydrolysis of conduritol epoxides obtained biocatalyt ically from halogenobenzenes: (+)-D-chiroinositol, allo-inositol muco-inositol and neo-inositol. J. Chem. Soc., Perkin Trans. 1 1993 , (7), 741-743. 134. Carless, H. A. J.; Busia, K.; Oak, O. Z ., Microbial oxidation of benzene as a route to inositol stereoisom ers and (±)-quebrachitol. Synlett 1993 , (9), 672-674. 135. Brammer Jr., L. E.; Hudlicky, T., Inosito l synthesis: concise preparation of L-chiroinositol and muco-inositol from a common intermediate. Tetrahedron: Asymmetry 1998 , 9, (12), 2011-2014. 136. Zylstra, G. J.; Gibson, D. T., Toluene degradation by Pseudomonas putida F1 nucleotide-sequence of the Todc1c2b ade genes and their expression in Escherichia coli. J. Biol. Chem. 1989 , 264, (25), 14940-14946. 137. Hudlicky, T.; Tian, X. R.; Konigsberger , K.; Rouden, J., Oxabicyclo[4.1.0]heptene and azabicyclo[4.1.0]heptene as new synthons for C-disaccharide and alkaloid synthesis reactiv ity trends with carbon nucleophiles. J. Org. Chem. 1994 , 59, (15), 4037-4039. 138. Chung, S.-K.; Chang, Y.-T., Synthesis of all possible regioisomers of myo-inositol tetrakisphosphates. J. Chem. Soc., Chem. Commun. 1995 , (1), 11-12. 139. Garegg, P. J.; Lindberg, B.; Kvarnstrom, I. ; Svensson, S. C. T., Synthesis of some benzyl and methyl ethers of myo-inositol. Carbohydr. Res. 1988 , 173, (2), 205-216. 140. Chung, S.-K.; Ryu, Y., Regioselective functionalizations and conformational studies of di-O-isopropylidene-myo-inositol derivatives. Carbohydr. Res. 1994 , 258, 145-167.

PAGE 194

172 141. Gigg, J.; Gigg, R.; Payne, S.; Conant, R., (±)-1,2-4,5-di-O-isopropylidene-myoInositol. Carbohydr. Res. 1985 , 142, (1), 132-134. 142. Gigg, J.; Gigg, R.; Payne, S.; Conant, R., The allyl group for protection in carbohydrate-chemistry. 21. (±)-1,2-5.6-di-O-isopropylidene-myo-Inositol and (±)-1,2-3,4-di-O-isopropylidene-myo-inositol the unusual behavior of crystals of (±)-3,4-di-O-acetyl-1,2,5,6-tetra-O-benzyl-myo-inositol on heati ng and cooling a thermosalient solid. J. Chem. Soc., Perkin Trans. 1 1987 , (11), 2411-2414. 143. Desai, T.; Fernandez-Mayoralas, A.; Gigg, J.; Gigg, R.; Payne, S., The allyl group for protection in carbohydrate-chemistry. 22. The synthesis a nd resolution of (±)1,5,6-tri-O-benzyl-myo-inositol. Carbohydr. Res. 1990 , 205, 105-123. 144. Takahashi, H.; Kittaka, H.; Ikegami, S., Novel synthesis of enantiomerically pure natural inositols and th eir diastereoisomers. J. Org. Chem. 2001 , 66, (8), 27052716. 145. Heo, J.-N.; Holson, E. B.; Roush, W. R., Common-intermediate strategy for synthesis of conduritol s and inositols via -hydroxy cyclohexenylsilanes. Org. Lett. 2003 , 5, (10), 1697-1700. 146. Racherla, U. S.; Brown, H. C., Chiral synthesis via organoboranes. 27. Remarkably rapid and exceptionally enantioselective (approaching 100% ee) allylboration of representative aldehydes at -100 °C under new, salt-free conditions. J. Org. Chem. 1991 , 56, (1), 401-404. 147. Roush, W. R.; Pinchuk, A. N.; Micalizio, G. C., [(E)(Dimethylphenylsilyl)allyl]diisopinocamphe ylborane: a highly enantioselective reagent for the synthesis of anti-hydroxyallylsilanes. Tetrahedron Lett. 2000 , 41, (49), 9413-9417. 148. Podeschwa, M.; Plettenburg, O.; vom Broc ke, J.; Block, O.; Adelt, S.; Altenbach, H.-J., Stereoselec tive synthesis of myo-, neo-, L-chiro, D-chiro, allo-, scyllo-, and epi-inositol systems via c onduritols prepared from p-benzoquinone. Eur. J. Org. Chem. 2003 , (10), 1958-1972. 149. Ogawa, K.; Watanabe, T.; Ikeda, Y.; Kondo, S., A new glycoside, 1D-2-O-Dgalactopyranosyl-chiro-inositol from jojoba beans. Carbohydr. Res. 1997 , 302, 219-221. 150. Obendorf, R. L.; Steadman, K. J.; Fuller, D. J.; Horbowicz, M.; Lewis, B. A., Molecular structure of fagopyritol A1 (O-D-galactopyranosyl-(1 3)-D-chiroinositol) by NMR. Carbohydr. Res. 2000 , 328, 623-627. 151. Murakata, C.; Ogawa, T., Synthetic studies on cell-surface glycan s. 75. Synthetic study on glycophosphatidyl inos itol (Gpi) anchor of Trypanosoma brucei glycoheptaosyl core. Tetrahedron Lett. 1990 , 31, (17), 2439-2442.

PAGE 195

173 152. Murakata, C.; Ogawa, T., Synthetic studi es on cell-surface gly cans. 78. A total synthesis of Gpi anchor of Trypanosoma Brucei. Tetrahedron Lett. 1991 , 32, (5), 671-674. 153. Saltiel, A. R.; Cuatrecasas, P., Insulin stimulates the generation from hepatic plasma membranes of modulators deri ved from an inositol glycolipid. Proc. Nat. Acad. Sci. USA 1986 , 83, 5793-5797. 154. Czech, M. P.; Klarlund, J. K.; Yagaloff, K. A.; Bradford, A. P.; Lewis, R. E., Insulin receptor signaling. Activation of multiple serine kinases. J. Biol. Chem. 1988 , 263, (23), 11017-11020. 155. Berlin, W. K.; Wang, S. N.; Shen, T. Y ., Glycosyl inositol derivatives. 2. Synthesis of 2-amino-2-deoxy-D-galactosyl-1,3-D-chiro-inositol. Tetrahedron Lett. 1990 , 31, (8), 1109-1112. 156. Plourde, R.; Dalarcao, M., Synthesi s of a potentially insulin-mimetic phosphodisaccharide. Tetrahedron Lett. 1990 , 31, (19), 2693-2696. 157. Ley, S. V.; Yeung, L. L., Microbial oxida tion in synthesis: preparation of a potential insulin mimic from benzene. Synlett 1992 , 997-998. 158. Reddy, K. K.; Falck, J. R.; Capdevila, J., Insulin second messengers synthesis of 6-O-(2-Amino-2-deoxy-D-glucopyranosyl)-D-chiro-inositol-1-phosphate. Tetrahedron Lett. 1993 , 34, (49), 7869-7872. 159. Billington, D. C.; Perronsierra, F.; Picard, I. ; Beaubras, S.; Duhault, J.; Espinal, J.; Challal, S., Total synthesis of novel conduritol related-compounds capable of modulating insulin release. Bioorg. Med. Chem. Lett. 1994 , 4, (19), 2307-2312. 160. Kara, Y.; Balci, M.; Bourne, S. A.; Watson, W. H., Synthe sis of new conduritol analogs derived from bicyclooctatriene bis-homo-conduritol-D and bis-homoconduritol-F. Tetrahedron Lett. 1994 , 35, (20), 3349-3352. 161. Balci, M., Synthesis of c onduritols and related compounds. Pure Appl. Chem. 1997 , 69, (1), 97-104. 162. Mehta, G.; Reddy, D. S., Polycylitols: Stereoselective synthesis of enantiopure polyhydroxylated hydrindanes (annulated carbasugars). Tetrahedron Lett. 1999 , 40, 9137-9140. 163. Mehta, G.; Reddy, D. S., Enzy matic resolution of dioxygenated dicyclopentadienes: enan tiopure hydrindanes, hydrois oquinolones, diquinanes and application to a synthesi s of (+)-coronafacic acid. Tetrahedron Lett. 1999 , 40, 991994.

PAGE 196

174 164. Mehta, G.; Reddy, D. S.; Ramesh, S. S.; Ta tu, U., Polycyclitols: stereoselective synthesis of decalin and diquinane ba sed polyols as potential glycomimics. Tetrahedron Lett. 1999 , 40, 9141-9144. 165. Mehta, G.; Ramesh, S. S., Polycylitols . Novel conduritol and carbasugar hybrids as a new class of potent glycosidase inhibitors. Chem. Commun. 2000 , 2429-2430. 166. Mehta, G.; Ramesh, S. S., Polycyclitol s: synthesis of novel carbasugars and conduritol analogues as potenti al glycosidase inhibitors. Tetrahedron Lett. 2001 , 42, 1987-1990. 167. Hudlicky, T.; Thorpe, A. J., Glycoconjuga te coupling strategy: Synthesis of a L-chiro-inositol-gala-quercitol conjugate and the synthesis of (+)-proto-quercitol. Synlett 1994 , 899-901. 168. Hudlicky, T.; Luna, H.; Olivo, H. F.; A ndersen, C.; Nugent, T.; Price, J. D., Biocatalysis as the strategy of choice in the exhaustive enantiomerically controlled synthesis of conduritols. J. Chem. Soc., Perkin Trans. 1 1991 , (12), 2907-2917. 169. Hudlicky, T.; Mandel, M.; Rouden, J.; Lee, R. S.; Bachmann, B.; Dudding, T.; Yost, K. J.; Merola, J. S., Microbial oxi dation of aromatics in enantiocontrolled synthesis. Part 1. Expedient and genera l asymmetric synthesi s of inositols and carbohydrates via an unusual oxidation of polarized diene with potassium permanganate. J. Chem. Soc., Perkin Trans. 1 1994 , 1553-1567. 170. Hudlicky, T.; Abboud, K. A.; Bolonick, J.; Maurya, R.; Stanton, M. L.; Thorpe, A. J., Concise syntheses of 1,2-L-chiro-inositol conjugates and oligomers-a novel class of saccharide mimics with promising molecular properties. Chem. Commun. 1996 , 1717-1718. 171. Hudlicky, T.; Abboud, K. A.; Entwistle, D. A.; Fan, R.; Maurya, R.; Thorpe, A. J.; Bolonick, J.; Myers, B., Toluene-dioxygenase-mediated cis-dihydroxylation of aromatics in enantioselective synthesis. Iterative glycoconjuga te coupling strategy and combinatorial design for the synthe sis of oligomers of nor-saccharides, inositols and pseudosugars with in teresting molecular properties. Synthesis 1996 , 897-911. 172. Cook, W. J.; Bugg, C. E., Metal-ligand interactions in organic chemistry and biochemistry. D. Reidel Publishi ng Company: Boston, 1977. 173. Restrepo-Sanchez, N. Ph.D. Dissertation, Universidad del Valle, Cali, Colombia, 2001. 174. Desjardins, M.; Lallemand, M.-C.; H udlicky, T.; Abboud, K. A., Polyhydroxylated tetrahydronaphthalene ethers: Synt hesis and molecular properties. Synlett 1997 , 728-730.

PAGE 197

175 175. Desjardins, M.; Lallemand, M.-C.; Freeman, S.; Hudlicky, T.; Abboud, K. A., Synthesis and biological evaluation of conduritol and conduramine analogs. J. Chem. Soc., Perkin Trans. 1 1999 , 621-628. 176. Lallemand, M.-C.; Desjardins, M.; Fr eeman, S.; Hudlicky, T., Synthesis of conduritol, conduramine, and validoxylamin e analogs from tetrahydronaphthalene-cis-diol. Tetrahedron Lett. 1997 , 38, (44), 7693-7696. 177. Legler, G., Glycoside hydrolases mech anistic information from studies with reversible and irreversible inhibitors. Adv. Carbohyd. Chem. Bi. 1990 , 48, 319-384. 178. Anzeveno, P. B.; Creemer, L. J.; Daniel, J. K.; King, C.-H. R.; Liu, P. S., A facile, practical synthesis of 2,6-dideoxy-2,6-imino-7-O-D-glucopyranosyl-D-glycero-L-gulo-heptitol (MDL 25,637). J. Org. Chem. 1989 , 54, (11), 2539-2542. 179. Elbein, A. D., Inhibitors of th e biosynthesis and processing of N-linked oligosaccharide chains. Ann. Rev. Biochem. 1987 , 56, 497-534. 180. Fleet, G. W. J.; Karpas, A.; Dwek, R. A.; Fellows, L. E.; tyms, A. S.; Petursson, S.; Mangoong, S. K.; Ramsden, N. G.; Smith, P. W.; Son, J. C.; Wilson, F.; Witty, D. R.; Jacob, G. S.; Rademacher, T. W., Inhi bition of HIV replication by amino-sugar derivatives. FEBS Lett. 1988 , 237, (1,2), 128-132. 181. Alper, J., Searching for medicine's sweet spot. Science 2001 , 291, (5512), 23382343. 182. Heightman, T. D.; Vasella, A. T., Recent insights into inhibition, structure, and mechanism of configurati on-retaining glycosidases. Angew. Chem. Int. Ed. 1999 , 38, 750-770. 183. Koshland, D. E., Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. 1953 , 28, (4), 416-436. 184. Guédat, P.; Spiess, B.; Schlewer, G. , Synthesis of (±)1,2-dideoxy-1,2-diamino-myo-inositol. Tetrahedron Lett. 1994 , 35, (40), 7375-7378. 185. Kresze, G.; Weiss, M. M.; Dittel, W., Polyhydroxyamino compounds via diene synthesis with nitroso-compounds. 9. Trans-6-azido-1,3-cycl ohexadien-5-ol as educt for the synthesis of condurodiamine and streptamine isomers. Liebigs Ann. Chem. 1984 , (2), 203-212. 186. Arcelli, A.; Cerè, V.; Peri, F.; Pollici no, S.; Ricci, A., Synthesis and glycosidase inhibition of new enantiopure 2,3-diamino conduritols. Tetrahedron 2001 , 57, 3439-3444. 187. Michell, R. H.; Drummond, A. H.; Downes, C. P., Inositol lipids in cell signalling. Academic Press: San Diego, 1989.

PAGE 198

176 188. Berridge, M. J., Inositol trisphosphate and diacylglycer ol: Two interacting second messengers. Ann. Rev. Biochem. 1987 , 56, 159-193. 189. Berridge, M. J.; Irvine, R. F., Inositol phosphates and cell signaling. Nature 1989 , 341, (6239), 197-205. 190. Hokin, M. R.; Hokin, L. E., Enzyme secretion and the in corporation of P32 into phospholidides of pancreas slides. J. Biol. Chem. 1953 , 203, 967-977. 191. Willis, J. Synthesis of O-linked amino-conduritol and amino-inositol dimers and their biological properties. Ph.D. Disserta tion, University of Florida, Gainesville, 2002. 192. Paul, B. J. Synthesis, st ructural and biological evalua tion of inositol oligomers and diaminoinositols. Ph.D. Dissertation, Un iversity of Florida, Gainesville, 2002. 193. Paul, B. J.; Martinot, T. A. ; Willis, J.; Hudlicky, T., Novel Oand N-linked inositol oligomers: A new class of unnatural saccharide mimics. Synthesis 2001 , (6), 952956. 194. Paul, B. J.; Willis, J.; Martinot, T. A. ; Ghiviriga, I.; Abboud, K. A.; Hudlicky, T., Synthesis, structure, and biological evaluation of novel Nand O-linked diinositols. J. Am. Chem. Soc. 2002 , 124, (35), 10416-10426. 195. Olivo, H. F.; Hemenway, M. S.; Hartwig, A. C.; Chan, R., New preparation of activated 2-vinylaziridines from 1,4-aminoalcohols. Synlett 1998 , (3), 247-248. 196. Watson, J. D.; Crick, F. H. C., Molecular st ructure of nucleic ac ids a structure for deoxyribose nucleic acid. Nature 1953 , 171, (4356), 737-738. 197. Watson, J. D.; Crick, F. H. C., The structure of DNA. Cold Spring Harb. Sym. 1953 , 18, 123-131. 198. Watson, J. D.; Crick, F. H. C., Genetic al implications of the structure of deoxyribonucleic acid. Nature 1953 , 171, (4361), 964-967. 199. Switzer, C.; Moroney, S. E.; Benner, S. A ., Enzymatic incorporation of a new base pair into DNA and RNA. J. Am. Chem. Soc. 1989 , 111, 8322-8323. 200. Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A., Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 1990 , 343, (6253), 33-37. 201. Geyer, C. R.; Battersby, T. R.; Benner, S. A., Nucleobase pairing in Watson-Cricklike genetic expanded information systems. Structure 2003 , 11, (12), 1485-1498. 202. Szathmary, E., Why are there four letters in the genetic alphabet? Nature Rev. Genetics 2003 , 4, (12), 995-1001.

PAGE 199

177 203. Collins, M. L.; Irvine, B.; Tyner, D.; Fine, E.; Zayati, C.; Chang, C. A.; Horn, T.; Ahle, D.; Detmer, J.; Shen, L. P.; Kolberg, J.; Bushnell, S.; Urdea, M. S.; Ho, D. D., A branched DNA signal amplification a ssay for quantification of nucleic acid targets below 100 molecules/mL. Nucl. Acids Res. 1997 , 25, (15), 2979-2984. 204. EraGen: SARS Diagnostic. http://www.eragen.com/diagnostics/sars.html (June 9, 2004) 205. Sismour, A. M.; Lutz, S.; Park, J.-H.; Lu tz, M. J.; Boyer, P. L.; Hughes, S. H.; Benner, S. A., PCR amplification of DN A containing non-standard base pairs by mutants of reverse transcriptase fr om Human Immunodeficienty Virus-1. Nucl. Acids Res. 2004 , 32, 728-735. 206. Voegel, J. J.; Altorfer, M. M.; Benner , S. A., The donor-acceptor-acceptor purine analog: Transformation of 5-aza-7-deaza1H-isoguanine (= 4-aminoimidazo-[1,2a]-1,3,5-trazin-1(1H)-one) to 2'-deoxy-5 -aza-7-deaza-isoguanosine using purine nucleoside phosphorylase. Helv. Chim. Acta 1993 , 76, 2061-2069. 207. Voegel, J. J.; Benner, S. A., Synthe sis and characterization of non-standard nucleosides and nucleotides bearing the acceptor-donor-donor pyrimidine analog 6amino-3-methylpyrazin-2-one. Helv. Chim. Acta 1996 , 79, 1863-1880. 208. Hutter, D.; Benner, S. A., Expanding the genetic alphabet : Non-epimerizing nucleoside with the pyDDA hydrogen-bonding pattern. J. Org. Chem. 2003 , 68, (25), 9839-9842. 209. Cherbuliez, E.; Bernhard, K., Recherches sur la graine de croton. I. Sur le crotonoside (2-oxy-6-amino-purine-D-riboside). Helv. Chim. Acta 1932 , 15, 464471. 210. Cherbuliez, E.; Bernhard, K., Remarques sur le crotonoside. Helv. Chim. Acta 1932 , 15, 978-980. 211. Spies, J. R.; Drake, N. L., D-Ribose from the croton bean. J. Am. Chem. Soc. 1935 , 57, (4), 774-774. 212. Strecker, Untersuchunger über die chem ischen Beziehungen zwischen Guanin, Xanthin, Theobromin, Caffeïn und Kreatinin. Annalen 1861 , 118, 151-177. 213. Fischer, E., Ueber Caffeïn, Theobromin, Xanthin und Guanin. Annalen 1882 , 215, 253-320. 214. Fischer, E., Synthese des Hypoxanthi ns, Xanthins, Adenins und Guanins. Berichte 1897 , 30, 2226-2254. 215. Pettit, G. R.; Ode, R. H.; Coomes, R. M. ; Ode, S. L., Antineoplactic agents. 42. The butterfly, Prioneris thestylis. Lloydia 1976 , 39, (5), 363-367.

PAGE 200

178 216. Fuhrman, F. A.; Fuhrman, G. J.; Nachma n, R. J.; Mosher, H. S., Isoguanosine isolation from an animal. Science 1981 , 212, (4494), 557-558. 217. Bergmann, W.; Burke, D. C., Contributions to the study of marine products. 40. The nucleosides of sponges. 4. Spongosine. J. Org. Chem. 1956 , 21, (2), 226-228. 218. Olinski, R.; Zastawny, T.; Budzbon, J.; Skokowski, J.; Zegarski, W.; Dizdaroglu, M., DNA-base modifications in chro matin of human cancerous tissues. FEBS Lett. 1992 , 309, (2), 193-198. 219. Topal, M. D.; Fresco, J. R., Complement ary base-pairing and origin of substitution mutations. Nature 1976 , 263, (5575), 285-289. 220. Spies, J. R., Isoguanine from the croton bean. J. Am. Chem. Soc. 1939 , 61, 350351. 221. Fischer, E., Verwandlung des Guan ins in Xanthin durch Salzsaüre. Berichte 1910 , 43, 805-806. 222. Andrews, K. J. M.; Anand, N.; Todd, A. R.; Topham, A., Synthesis of purine nucleosides. XXVI. 9-D-Glucopyranosidoisoguanine. J. Chem. Soc., Abstracts 1949 , 2490-2497. 223. Holland, A.; Lythgoe, B.; Todd, A. R., Experiments on the synthesis of purine nucleosides. 18. A synthesis of 9-D-glucopyranosidoadenine. J. Chem. Soc. 1948 , 965-967. 224. Shaw, E., New synthesis of the purin es adenine, hypoxanthine, xanthine, and isoguanine. J. Biol. Chem. 1950 , 185, 439-447. 225. Taylor, E. C.; Vogl, O.; Cheng, C. C., St udies in purine chemistry. II. A facile synthesis of 2-substituted adenines. J. Am. Chem. Soc. 1959 , 81, 2443-2448. 226. Longo, G., Dioximes. LXXVIII. Gazz. Chim. Ital. 1931 , 61, 575-583. 227. Bredereck, H.; Edenhofer, A., Synthesen in der Purinreihe. 6. Mitteil Synthesen mit 4und 5-amino-uracil. Chem. Ber.-Recl. 1955 , 88, (8), 1306-1312. 228. Yamazaki, A.; Kumashiro, I.; Takenishi, T.; Ikehara, M., Synthesis of thio-AICA, 6-thioxanthine, isoguanine an alogs, and their ribosides. Chem. Pharm. Bull. 1968 , 16, (11), 2172-2181. 229. Hitchings, G. H.; Elion, G. B. Nove l process for the manufacture of 6mercaptopurine from aminoglyoxaline5-thiocarboxamide. 2,756,228, July 24, 1956, 1956.

PAGE 201

179 230. Leonard, N. J.; McDonald, J. J.; Henderson, R. E. L.; Reichmann, M. E., Reaction of diethyl pyrocarbonat e with nucleic acid co mponents. Adenosine. Biochemistry 1971 , 10, (18), 3335-3342. 231. Yamazaki, A.; Okutsu, M.; Yamada, Y., S ynthesis of guanosine and its derivatives from 5-amino-1-D-ribofuranosyl-4-imidazolecarboxamide. IV. A new route to guanosine via cyanamide derivative. Nucl. Acids Res. 1976 , 3, (1), 251-259. 232. Saxena, N. K.; Bhakuni, D. S., New syntheses of crotonoside. Indian Journal of Chemistry, Section B: Organic Chemis try Including Medi cinal Chemistry 1979 , 18B, (4), 344-347. 233. Leonard, N. J.; McDonald, J. J.; Reichmann, M. E., Reaction of diethyl pyrocarbonate with nucleic ac id components .1. Adenine. Proc. Nat. Acad. Sci. USA 1970 , 67, 93-98. 234. Lunt, E., In Comprehensive Organic Chemistry, Sammes, P. G., Eds. Pergamon Press: Oxford, 1979; Vol. 4, pp 522. 235. Chern, J. W.; Lee, H. Y.; Huang, M.; Shish, F. J., Novel and efficient synthesis of isoguanosine. Tetrahedron Lett. 1987 , 28, (19), 2151-2154. 236. Itaya, T.; Kanai, T.; Takada, Y.; Kaneko, M.; Yasuhara, K.; Fujii, T., Purines. LXXIX. Synthesis and hydr olysis of 3-methoxyadenine and its N6-benzyl derivative leading to the co rresponding 2-hydroxyadenines. Chem. Pharm. Bull. 1999 , 47, (4), 554-556. 237. Scheinfeld, I.; Parham, J. C.; Murphy, S.; Brown, G. B., Purine N-oxides. 25. 3-Noxides of adenine and hypoxanthine. J. Org. Chem. 1969 , 34, (7), 2153-2156. 238. Giner-Sorolla, A., Purine N-oxides: XXXVII. Derivati ves from 6-chloropurine 3oxide. J. Heterocycl. Chem. 1971 , 8, 651-655. 239. Stimson, M. M., Ultraviolet absorption spectra of nitrogenous heterocycles. IV. Effect of pH and irradiation on the spectra of isoguanine and 2-hydroxy-6,8diaminopurine. J. Am. Chem. Soc. 1942 , 64, 1604-1605. 240. Jardetzky, C. D.; Jardetzky, O., Inves tigation of the structure of purines, pyrimidines, ribose nucleosides and nucleo tides by proton magnetic resonance. II. J. Am. Chem. Soc. 1960 , 82, 222-229. 241. Sepiol, J.; Kazimierczuk, Z.; Shugar, D ., Tautomerism of isoguanosine and solventinduced keto-enol equilibrium. Z. Naturforsch. C 1976 , 31, (7-8), 361-370. 242. Dimroth, K.; Reichardt, C.; Siepman n, T.; Bohlmann, F., Ueber Pyridinium-Nphenol-betaine und ihre Verwendung zur Charakterisierung der Polaritaet von Loesungsmitteln. Liebigs Ann. Chem. 1963 , 661, 1-37.

PAGE 202

180 243. Gordon, A.; Katritzky, A. R., A quantita tive relation between heteroaromatic tautomeric equilibrium constants and solvent polarity. Tetrahedron Lett. 1968 , 23, 2767-2770. 244. Kosower, E. M., The effect of solvent on spectra. I. A new empirical measure of solvent polarity: Z-values. J. Am. Chem. Soc. 1958 , 80, 3253-3260. 245. Kosower, E. M., The effect of solvent on spectra. II. Co rrelation of spectral absorption data with Z-values. J. Am. Chem. Soc. 1958 , 80, 3261-3267. 246. Kosower, E. M., The effect of solvent on spectra. III. The use of Z-values in connection with kinetic data. J. Am. Chem. Soc. 1958 , 80, 3267-3270. 247. Reichardt, C.; Harbusch-Goernert, E., Erweiterun, Korrektur un Neudefinition der ET-Loesungsmittelpolaritaetsskala mit Hilf e eines lipophilen penta-tert-butylsubstituierten Pyridinium-N -phenolat-Betainfarbstoffes. Liebigs Ann. Chem. 1983 , 721-743. 248. Kwiatkowski, J. S.; Leszczynski, J ., Temperature-dependent thermodynamic contributions to the relative tautomeric stabilities of nucleic acid bases. Chemical Physics Letters 1993 , 204, (5-6), 430-434. 249. Les, A.; Kukawska-Tarnawska, B., Ta utomerism of biologically important pyrimidines and purines. Part I. Theoretical predictions. Theochem 1986 , 33, (1-2), 45-60. 250. Shcherbakova, I.; Elguero, J.; Katritzky, A. R., Tautomerism of heterocycles: Condensed five-six, five-five, and six-si x ring systems with heteroatoms in both rings. Adv. Het. Chem. 2000 , 77, 51-113. 251. Cysewski, P.; Madjar, C. V.; Noinville, V. ; Olinski, R., Structur e and properties of 2-hydroxy-adenine and 2-hydroxy-2'-deoxy-adenosine. Bulletin de la Societe Chimique de France 1995 , 132, (4), 453-460. 252. Jiang, S. P.; Raghunathan, G.; Ting, K. L.; Xuan, J. C.; Jernigan, R. L., Geometries, charges, dipole moments and interaction energies of normal, tautomeric and novel bases. Journal of Biomolecular Structure & Dynamics 1994 , 12, (2), 367-382. 253. Parkanyi, C.; Boniface, C.; Aaron, J.-J .; Bulaceanu-MacNair, M.; Dakkouri, M., Theoretical and experimental dipole moments of purines. Collect. Czech. Chem. Comm. 2002 , 67, (8), 1109-1124. 254. Rogstad, K. N.; Jang, Y. H.; Sowers, L. C.; Goddard, W. A., First principles calculations of the pKa values and tautomers of isoguanine and xanthine. Chem. Res. Toxicol. 2003 , 16, (11), 1455-1462.

PAGE 203

181 255. Cysewski, P.; Jeziorek, D.; Olinski, R.; Woznicki, W.-l., Ab initio studies on the structure and properties of the hydroxyl-radi cal-modified adenin e derivatives in different tautomeric forms. J. Phys. Chem. 1995 , 99, (24), 9702-9708. 256. Kwiatkowski, J. S.; Bartlett, R. J.; Pe rson, W. B., Contributions from electron correlation to the relative stabilities of the tautomers of nucleic-acid bases. J. Am. Chem. Soc. 1988 , 110, (8), 2353-2358. 257. Leszczynski, J., Tautomerism of uracil the final chapter 4th-order electron correlation contributions to the re lative energies of tautomers. J. Phys. Chem. 1992 , 96, (4), 1649-1653. 258. Les, A.; Adamowicz, L., Theoretical ab initio study of the protomeric tautomerism of 2-hydroxypyrimidine, 4-hydroxypyrimidine, and their derivatives. J. Phys. Chem. 1990 , 94, (18), 7021-7032. 259. Les, A.; Adamowicz, L., Oxo hydroxy taut omerism of uracil and 5-fluorouracil. J. Phys. Chem. 1989 , 93, (20), 7078-7081. 260. Seela, F.; Wei, C.; Kazimierczuk, Z., Substituent reactivity and tautomerism of isoguanosine and related nucleosides. Helv. Chim. Acta 1995 , 78, 1843-1854. 261. Tuschl, T.; Ng, M. M. P.; Pieken, W.; Benseler, F.; Eckstein, F., Importance of exocyclic base functional groups of cent ral core guanosines for hammerhead ribozyme activity. Biochemistry 1993 , 32, 11658-11668. 262. Seela, F.; Chen, Y. M.; Melenewski, A.; Rosemeyer, H.; Wei, C. F., Synthesis and application of novel nucle oside phosphonates and phosphoram idites modified at the base moiety. Acta Biochim. Pol. 1996 , 43, (1), 45-52. 263. Seela, F.; Wei, C. F., Oligonucle otides containing consecutive 2'deoxyisoguanosine residues: Synthesis, dupl exes with parallel chain orientation, and aggregation. Helv. Chim. Acta 1997 , 80, (1), 73-85. 264. Seela, F.; Wei, C., The base-pairing properties of 7-deaza-2'-deoxyisoguanosine and 2'-deoxyisoguanosine in oligonucleotide duplexes with parallel and antiparallel chain orientation. Helv. Chim. Acta 1999 , 83, 726-745. 265. Seela, F.; Kroschel, R., Quadrupl ex and pentaplex self-assemblies of oligonucleotides containing short runs of 8-aza-7-deaza-2 '-deoxyisoguanosine or 2 '-deoxyisoguanosine. Bioconjugate Chem. 2001 , 12, (6), 1043-1050. 266. Seela, F.; Debelak, H.; Andrews, L. ; Beigelman, L., Synthesis and enzymic hydrolysis of oligoribonucleotides incorporating 3-deazaguanosine: The importance of the nitrogen-3 atom of single conserved guano sine residues on the catalytic activity of the hammerhead ribozyme. Helv. Chim. Acta 2003 , 86, (8), 2726-2740.

PAGE 204

182 267. Switzer, C. Y.; Moroney, S. E.; Benner, S. A., Enzymic recognition of the base pair between isocytidine and isoguanosine. Biochemistry 1993 , 32, (39), 10489-10496. 268. Seela, F.; Fröhlich, T., Synthesis of o ligoribonucleotides containing isoguanosine. Helv. Chim. Acta 1994 , 77, (1), 399-408. 269. Seela, F.; Mertens, R.; Kazimierczuk, Z., Synthesis of phosphonates and oligodeoxyribonucleotides derived from 2'-deoxyisoguanosine and 2'-deoxy-2haloadenosines. Helv. Chim. Acta 1992 , 75, (7), 2298-2306. 270. Robinson, H.; Gao, Y.-G.; Bauer, C.; Robe rts, C.; Switzer, C.; Wang, A. H.-J., 2'Deoxyisoguanosine adopts more than one tautomer to form base pairs with thymidine observed by high-resolutio n crystal structure analysis. Biochemistry 1998 , 37, 10897-10905. 271. Blas, J. R.; Luque, F. J.; Orozco, M., Uni que tautomeric properties of isoguanine. J. Am. Chem. Soc. 2004 , 126, (1), 154-164. 272. Friedman, S.; Gots, J. S., Deamination of isoguanine by Escherichia coli. Archives of Biochemistry and Biophysics 1951 , 32, 227-229. 273. Goodwin, T. W.; Passorn, P. N. A., Purine and pyrimidine metabolism in Rhodospirillum rubrum. Nature (London, United Kingdom) 1961 , 191, 276-277. 274. Elion, G. B.; Hitchings, G. H.; Vande r-Werff, H., Antagonists of nucleic acid derivatives. VI. Purines. J. Biol. Chem. 1951 , 192, 505-518. 275. Rhaese, H. J., Chemical analysis of DNA alterations. III. Isolation and characterization of adenine oxidation products obtained from oligoand monodeoxyadenylic acids treate d with hydroxyl radicals. Biochimica et Biophysica Acta 1968 , 166, (2), 311-326. 276. Kamiya, H.; Kasai, H., 2-Hydroxyadenine (isoguanine) as oxidative DNA damage: its formation and mutation inducibility. Nucleic Acids Symposium Series 1995 , 34, (Twentysecond Symposium on Nuclei c Acids Chemistry, 1995), 233-234. 277. Kamiya, H.; Ueda, T.; Ohgi, T.; Matsuka ge, A.; Kasai, H., Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine. Nucl. Acids Res. 1995 , 23, (5), 761-766. 278. Kamiya, H.; Kasai, H., Formation of 2-hydroxydeoxyadenosin e triphosphate, an oxidatively damaged nucleotide, and its incorporation by DNA polymerases. Steady-state kinetics of the incorporation. J. Biol. Chem. 1995 , 270, (33), 1944619450. 279. Kasai, H.; Nishimura, S., Hydroxylation of deoxyguanosine at the C-8 position by ascorbic-acid and other reducing agents. Nucl. Acids Res. 1984 , 12, (4), 2137-2145.

PAGE 205

183 280. Whiteman, M.; Hong, H. S.; Jenner, A. ; Halliwell, B., Loss of oxidized and chlorinated bases in DNA treated with re active oxygen species: implications for assessment of oxidative damage in vivo. Biochemical and Biophysical Research Communications 2002 , 296, (4), 883-889. 281. Kamiya, H.; Miura, H.; Kato, H.; Nishim ura, S.; Ohtsuka, E., Induction of mutation of a synthetic C-Ha-Ras gene containing hypoxanthine. Cancer Research 1992 , 52, (7), 1836-1839. 282. Kamiya, H.; Muratakamiya, N.; Koizume, S.; Inoue, H.; Nishimura, S.; Ohtsuka, E., 8-Hydroxyguanine (7,8-di hydro-8-oxoguanine) in hot-spots of the C-Ha-Ras gene effects of sequence c ontexts on mutation spectra. Carcinogenesis 1995 , 16, (4), 883-889. 283. Kamiya, H.; Kasai, H., Effects of se quence contexts on misincorporation of nucleotides opposite 2-hydroxyadenine. FEBS Lett. 1996 , 391, (1,2), 113-116. 284. Bukowska, A. M.; Kusmierek, J. T., Miscoding properties of isoguanine (2oxoadenine) studied in an AMV revers e transcriptase in vitro system. Acta Biochim. Pol. 1996 , 43, (1), 247-254. 285. Kamiya, H.; Kasai, H., Substitution and deletion mutations induced by 2hydroxyadenine in Escherichia coli: effects of sequence c ontexts in leading and lagging strands. Nucl. Acids Res. 1997 , 25, (2), 304-311. 286. Kamiya, H.; Kasai, H., Mutations indu ced by 2-hydroxyadenine on a shuttle vector during leading and lagging strand syntheses in mammalian cells. Biochemistry 1997 , 36, (37), 11125-11130. 287. Kamiya, H.; Kasai, H., 2-Hydroxyadeni ne formation by reactive oxygen species and mutagenic effects. Recent Research Developments in Biochemistry 2000 , 2, 4150. 288. Tsurudome, Y.; Hirano, T.; Kamiya, H.; Yama guchi, R.; Asami, S.; Itoh, H.; Kasai, H., 2-Hydroxyadenine, a mutagenic form of oxidative DNA damage, is not repaired by a glycosylase type mechanism in rat organs. Mutation Research 1998 , 408, (2), 121-127. 289. Bessho, T.; Roy, R.; Yamamoto, K.; Kasai, H.; Nishimura, S.; Tano, K.; Mitra, S., Repair of 8-hydroxyguanine in DNA by mammalian N-methylpurine-DNA glycosylase. Proc. Nat. Acad. Sci. USA 1993 , 90, (19), 8901-8904. 290. Bessho, T.; Tano, K.; Kasai, H.; Ohtsuka, E.; Nishimura, S., Evidence for 2 DNArepair enzymes for 8-hydroxyguanine (7,8dihydro-8-oxoguanine) in human cells. J. Biol. Chem. 1993 , 268, (26), 19416-19421.

PAGE 206

184 291. Yamamoto, F.; Kasai, H.; Bessho, T.; Chung, M. H.; Inoue, H.; Ohtsuka, E.; Hori, T.; Nishimura, S., Ubiquitous presence in mammalian-cells of enzymatic-activity specifically cleaving 8-h ydroxyguanine-containing DNA. Jpn. J. Cancer Res. 1992 , 83, (4), 351-357. 292. Chung, M. H.; Kim, H. S.; Ohtsuka, E.; Kasai, H.; Yamamoto, F.; Nishimura, S., An endonuclease activity in human polymor phonuclear neutrophils that removes 8hydroxyguanine residues from DNA. Biochemical and Biophysical Research Communications 1991 , 178, (3), 1472-1478. 293. Chung, M. H.; Kasai, H.; Jones, D. S.; Inoue, H.; Ishikawa, H.; Ohtsuka, E.; Nishimura, S., An endonuclease activity of Escherichia coli that specifically removes 8-hydroxyguanine residues from DNA. Mutation Research 1991 , 254, (1), 1-12. 294. Tchou, J.; Kasai, H.; Shibutani, S.; Chung, M. H.; Laval, J.; Grollman, A. P.; Nishimura, S., 8-Oxoguanine (8-hyd roxyguanine) DNA glycosylase and Its substrate-specificity. Proc. Nat. Acad. Sci. USA 1991 , 88, (11), 4690-4694. 295. Michaels, M. L.; Tchou, J.; Grollman, A. P.; Miller, J. H., A repair system for 8oxo-7,8-dihydrodeoxyguanine. Biochemistry 1992 , 31, (45), 10964-10968. 296. Yamaguchi, R.; Hirano, T.; Asami, S.; Chung, M. H.; Sugita, A.; Kasai, H., Increased 8-hydroxyguanine levels in DNA and its repair acti vity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate. Carcinogenesis 1996 , 17, (11), 2419-2422. 297. Yamaguchi, R.; Hirano, T.; Asami, S.; Sugita, A.; Kasai, H., Increase in the 8hydroxyguanine repair activity in the rat kidney after the administration of a renal carcinogen, ferric nitrilotriacetate. Environ. Health Persp. 1996 , 104, 651-653. 298. Kamiya, H.; Kasai, H., 2-Hydroxyadenine in DNA is a very poor substrate of the Escherichia coli MutY protein. Journal of Radiation Research 2000 , 41, (4), 349354. 299. Hashiguchi, K.; Zhang, Q. M.; Sugiyama, H. ; Ikeda, S.; Yonei, S., Characterization of 2-hydroxyadenine DNA gl ycosylase activity of Escherichia coli MutY protein. International Journal of Radiation Biology 2002 , 78, (7), 585-592. 300. Ohtsubo, T.; Nishioka, K.; Imaiso, Y.; Iwai, S.; Shimokawa, H.; Oda, H.; Fujiwara, T.; Nakabeppu, Y., Identification of huma n MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria. Nucl. Acids Res. 2000 , 28, (6), 1355-1364. 301. Fujikawa, K.; Kamiya, H.; Yakushiji, H.; Fujii, Y.; Nakabeppu, Y.; Kasai, H., The oxidized forms of dATP are substrat es for the human MutT homologue, the hMTH1 protein. J. Biol. Chem. 1999 , 274, (26), 18201-18205.

PAGE 207

185 302. Jaruga, P.; Dizdaroglu, M., Repair of pr oducts of oxidative DNA base damage in human cells. Nucl. Acids Res. 1996 , 24, (8), 1389-1394. 303. Giray, B.; Hincal, F., Oxidative DNA base damage, antioxidant enzyme activities and selenium status in highly io dine-deficient goitrous children. Free Radical Research 2002 , 36, (1), 55-62. 304. Limoli, C. L.; Giedzinski, E.; Morgan, W. F.; Swarts, S. G.; Jones, G. D. D.; Hyun, W., Persistent oxidative stress in chromosomally unstable cells. Cancer Research 2003 , 63, (12), 3107-3111. 305. Birincioglu, M.; Jaruga, P. ; Chowdhury, G.; Rodriguez, H.; Dizdaroglu, M.; Gates, K. S., DNA base damage by the antitu mor agent 3-amino-1,2,4-benzotriazine 1,4dioxide (tirapazamine). J. Am. Chem. Soc. 2003 , 125, (38), 11607-11615. 306. Moser, M. J.; Prudent, J. R., Enzymatic repair of an expanded genetic information system. Nucl. Acids Res. 2003 , 31, (17), 5048-5053. 307. Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A., Ribosome-nediated incorporation of a nonstandard amino-acid into a peptide through expansion of the genetic code. Nature 1992 , 356, (6369), 537-539. 308. Tor, Y.; Dervan, P. B., Site-specific en zymatic incorporation of an unnatural base, N6-(6-aminohexyl)isoguanosine, into RNA. J. Am. Chem. Soc. 1993 , 115, (11), 4461-4467. 309. Horn, T.; Chang, C.-A.; Collins, M. L., Hybridization properties of the 5-methylisocytidine/isoguanosine base pair in synthetic oligodeoxynucleotides. Tetrahedron Lett. 1995 , 36, 2033-2036. 310. Roberts, C.; Bandaru, R.; Switzer, C., S ynthesis of oligonucleotides bearing the non-standard bases iso-C and iso-G. Co mparison of iso-C-iso-G, C-G and U-A base-pair stabilities in RNA/DNA duplexes. Tetrahedron Lett. 1995 , 36, (21), 3601-3604. 311. Roberts, C.; Bandaru, R.; Switzer, C., Theoretical and expe rimental study of isoguanine and isocytosine: Base pair ing in an expanded genetic system. J. Am. Chem. Soc. 1997 , 119, (20), 4640-4649. 312. Kawakami, J.; Kamiya, H.; Yasuda, K.; Fujiki, H.; Kasai, H.; Sugimoto, N., Thermodynamic stability of base pair s between 2-hydroxyadenine and incoming nucleotides as a determinant of nucle otide incorporation specificity during replication. Nucl. Acids Res. 2001 , 29, (16), 3289-3296. 313. Maciejewska, A. M.; Lichota, K. D.; Kusmierek, J. T., Neighbouring bases in template influence base-pairing of isoguanine. Biochem. J. 2003 , 369, (3), 611-618.

PAGE 208

186 314. Sugiyama, H.; Ikeda, S.; Saito, I., Remarkably stable parallel-stranded oligonucleotides containing 5-me thylisocytosine and isoguanine. J. Am. Chem. Soc. 1996 , 118, 9994-9995. 315. Yang, X.-L.; Sugiyama, H.; Ikeda, S.; Sait o, I.; Wang, A. H. J., Structural studies of a stable parallel-stranded DNA duplex incorporating isoguanine:cytosine and isocytosine:guanine base pairs by nucl ear magnetic res onance spectroscopy. Biophysical Journal 1998 , 75, (3), 1163-1171. 316. Seela, F.; Wei, C.; Becher, G.; Zulauf, M.; Leonard, P., The influence of modified purine bases on the stability of parallel DNA. Bioorg. Med. Chem. Lett. 2000 , 10, 289-292. 317. Seela, F.; Wei, C.; Melenewski, A.; Le onard, P., Parallel-stranded duplexes and quartet assemblies formed by oligon ucleotides containing isoguanine. Nucleic Acids Symposium Series 1997 , 37, (Symposium on Nucleic Acids Chemistry, 1997), 149-150. 318. Seela, F.; Wei, C.; Melenewski, A ., Four-stranded DNA formed by isoguanine quartets: complex stoichiometry, ther mal stability and resistance against exonucleases. Origins of life and evolution of th e biosphere : journal of the International Society for the Study of the Origin of Life 1997 , 27, (5-6), 597-608. 319. Seela, F.; Wei, C., 7-Deazaisoguanine quartets: self-assembled oligonucleotides lacking the Hoogsteen motif. Chem. Commun. 1997 , 1869-1870. 320. Chaput, J. C.; Switzer, C., A DNA pentap lex incorporating nu cleobase quintets. Proc. Nat. Acad. Sci. USA 1999 , 96, (19), 10614-10619. 321. Gu, J.; Leszczynski, J., Isoguanine complexes: quintet versus tetrad. Journal of Physical Chemistry B 2003 , 107, (27), 6609-6613. 322. Switzer, C.; Chaput, J. C., Probing struct ure and function with alternative nucleic acids bearing 2',5'-linked, zwitterionic, and isocytosine-isoguanine components. Methods (San Diego, Calif.) 2001 , 23, (2), 141-148. 323. Meyer, M.; Suehnel, J., Self-association of isoguanine nucleobases and molecular recognition of alkaline ions: te trad vs pentad structures. Journal of Physical Chemistry A 2003 , 107, (7), 1025-1031. 324. Demushkin, V. P.; Plyashkevich, Y. G., Analysis of nucleic acid bases by highperformance liquid ion chromatography. Analytical Biochemistry 1978 , 84, (1), 1218. 325. Rich, A., On the problems of evolution and biochemical information transfer. In Horizons in Biochemistry, Kasha, M.; Pullman, B., Eds. Academic Press: New York, 1962; Vol. pp 103-126.

PAGE 209

187 326. Jaworski, A.; Kwiatkowski, J. S.; Les yng, B., Why isoguanine and isocytosine are not the components of the genetic code. International Journal of Quantum Chemistry, Quantum Biology Symposium 1986 , 12, 209-216. 327. Katritzky, A. R.; Pozharskii, A. F., Handbook of heterocyclic chemistry. Pergamon Press: Oxford, 2000. 328. Davoll, J., Pyrrolo[2,3-d]pyrimidines. J. Chem. Soc. 1960 , 131-138. 329. Seela, F.; Menkhoff, S.; Behrendt, S., Furanoside-pyranoside isomerization of tubercidin and its 2'-deoxy de rivatives: Influence of nuc leobase and sugar structure on the proton-catalyzed reaction. J. Chem. Soc. Perkin Trans. II 1986 , 525-530. 330. Seela, F.; Winkeler, H. D., 2-Amino-7-beta-D-arabinofuranosyl-4-methoxy-7hpyrrolo[2,3-d]pyrimidine a facile pr eparation and anomerization of a 7deazapurine nucleoside. Carbohydr. Res. 1983 , 118, (JUL), 29-35. 331. Seela, F.; Kehne, A.; Winkeler, H., Synthesis of acyclo-7-deazaguanosine by regiospecific phase-transfer alkylati on of 2-amino-4-methoxy-7h-pyrrolo[2,3d]pyrimidine. Liebigs Ann. Chem. 1983 , (1), 137-146. 332. Seela, F.; Driller, H., 7-(Beta-D-arabinofuranosyl)-2,4-di chloro-7h-pyrrolo[2,3d]pyrimidine synthesis, selective disp lacement of halogen, and influence of glyconic protecting groups on the reactivity of the aglycone. Liebigs Ann. Chem. 1984 , (4), 722-733. 333. Seela, F.; Steker, H.; Driller, H.; Bindig, U., 2-Amino-2'-desoxytubercidin und verwandte Pyrrolo[2,3-d]pyrimidi nyl-2'-desoxyribofuranoside. Liebigs Ann. Chem. 1987 , 15-19. 334. Kazimierczuk, Z.; Mertens, R.; Kawczynski, W.; Seela, F., 168. 2'Deoxyisoguanosine and base-modified anal ogues: chemical and photochemical synthesis. Helv. Chim. Acta 1991 , 74, 1742-1748. 335. Seela, F.; Wei, C.; Reuter, H.; Kast ner, G., 7-Deaza-2'-deoxyisoguanosine adopts the form of an N1-H,2-keto tautomer. Acta Cryst. 1999 , C55, 1335-1337. 336. Bartoli, G.; Palmieri, G.; Bosco, M.; Dal pozzo, R., The reaction of vinyl Grignard reagents with 2-substituted nitroarenes: a new approach to the synthesis of 7substituted indoles. Tetrahedron Lett. 1989 , 30, (16), 2129-2132. 337. Fonseca, T.; Gigante, B.; Marques, M. M.; Gilchrist, T. L.; De Clercq, E., Synthesis and antiviral evaluation of be nzimidazoles, quinoxalines and indoles from dehydroabietic acid. Bioorganic & Medicinal Chemistry 2004 , 12, (1), 103112. 338. Möhlau, R., Ueber die Einwirkung prim ärer aromatischer Aminbasen auf Acetophenonbromid. Berichte 1881 , 14, 171-175.

PAGE 210

188 339. Golob, T.; Liebl, R.; von Angerer, E. , Sulfamoyloxy-substituted 2-phenylindoles. Antiestrogen-based inhibitors of the steroid sulfatase in human breast cancer cells. Bioorganic & Medicinal Chemistry 2002 , 10, (12), 3941-3953. 340. Drechsel, E., J. Prakt. Chem. 1858 , 38, 69-. 341. Jiricek, J.; Blechert, S., Enantioselectiv e synthesis of (-)-gilbertine via a cationic cascade cyclization. J. Am. Chem. Soc. 2004 , 126, (11), 3534-3538. 342. Fischer, E.; Jourdan, F., Ueber di e Hydrazine der Benztraubensaüre. Berichte 1883 , 16, 2241-2245. 343. Campo, M. A.; Huang, Q.; Yao, T.; Tian, Q.; Larock, R. C., 1,4-Palladium migration via C-H activation, followed by aryl ation: synthesis of fused polycycles. J. Am. Chem. Soc. 2003 , 125, (38), 11506-11507. 344. Gassman, P. G.; van Bergen, T. J.; Gilbert, D. P.; Cue, B. W., General method for the synthesis of indoles. J. Am. Chem. Soc. 1974 , 96, (17), 5495-5508. 345. Smith, A. B.; Kingery-Wood, J.; Leenay, T. L.; Nolen, E. G.; Sunazuka, T., Indole diterpene synthetic studie s. 8. The total synthesis of (+)-paspalicine and (+)paspalinine. J. Am. Chem. Soc. 1992 , 114, (4), 1438-1449. 346. Hegedus, L. S.; Allen, G. F.; Waterman, E. L., Palladium assisted intramolecular amination of olefins new synthesis of indoles. J. Am. Chem. Soc. 1976 , 98, (9), 2674-2676. 347. Hegedus, L. S.; Allen, G. F.; Bozell, J. J.; Waterman, E. L., Palladium-assisted intra-molecular amination of olefins synthesis of nitrogen-heterocycles. J. Am. Chem. Soc. 1978 , 100, (18), 5800-5807. 348. Tsuji, J., Synthetic applications of the palladium-catalyzed oxid ation of olefins to ketones. Synthesis 1984 , (5), 369-384. 349. van Esseveldt, B. C. J.; van Delft, F. L.; de Gelder, R.; Rutjes, F. P. J. T., Palladium-catalyzed synthesis of novel optically active tryp tophan analogues. Org. Lett. 2003 , 5, (10), 1717-1720. 350. Kumar, V.; Dority, J. A.; Bacon, E. R. ; Singh, B.; Lesher, G. Y., Synthesis of 7azaindole and 7-azaoxindole derivatives through a palladium-catalyzed crosscoupling reaction. J. Org. Chem. 1992 , 57, (25), 6995-6998. 351. Fujiwara, J.; Fukutani, Y.; Sano, H.; Maruoka, K.; Yamamoto, H., Nucleophilic aromatic-substitution by organo-aluminum reagents application to the synthesis of indoles. J. Am. Chem. Soc. 1983 , 105, (24), 7177-7179.

PAGE 211

189 352. Ezquerra, J.; Pedregal, C.; Lamas, C.; Barl uenga, J.; Perez, M.; GarciaMartin, M. A.; Gonzalez, J. M., Efficient reagents for the synthesis of 5-, 7-, and 5,7substituted indoles star ting from aromatic amines: Scope and limitations. J. Org. Chem. 1996 , 61, (17), 5804-5812. 353. Ma, C.; Liu, X.; Li, X.; Flippen-Anderson, J.; Yu, S.; Cook, J. M., Efficient asymmetric synthesis of biologically important tryptophan analogues via a palladium-mediated heteroannulation reaction. J. Org. Chem. 2001 , 66, (13), 45254542. 354. Yasuhara, A.; Kanamori, Y.; Kaneko, M.; Numata, A.; Kondo, Y.; Sakamoto, T., Convenient synthesis of 2substituted indoles from 2-ethynylanilines with tetrabutylammonium fluoride. J. Chem. Soc., Perkin Trans. 1 1999 , (4), 529-534. 355. Kondo, Y.; Kojima, S.; Sakamoto, T., Genera l and facile synthesi s of indoles with oxygen-bearing substituents at the benzene moiety. J. Org. Chem. 1997 , 62, (19), 6507-6511. 356. Dai, W. M.; Guo, D. S.; Sun, L. P ., Chemistry of aminophenols. Part 1: Remarkable additive effect on Sonogash ira cross-coupling of 2-carboxamidoaryl triflates and application to novel synthesis of indoles. Tetrahedron Lett. 2001 , 42, (31), 5275-5278. 357. Dai, W. M.; Sun, L. P.; Guo, D. S., Chem istry of aminophenols. Part 2: A general and efficient synthesis of indoles possessi ng a nitrogen substituent at the C4, C5, C6, and C7 positions. Tetrahedron Lett. 2002 , 43, (43), 7699-7702. 358. Belley, M.; Scheigetz, J.; Dube , P.; Dolman, S., Synthesis of N-aminoindole ureas from ethyl 1-amino-6-(trifluor omethyl)-1H-indole-3-carboxylate. Synlett 2001 , (2), 222-225. 359. Rodriguez, A. L.; Koradin, C.; Dohle, W. ; Knochel, P., Versatile indole synthesis by a 5-endo-dig cyclization mediated by potassium or cesium bases. Angew. Chem. Int. Ed. 2000 , 39, (14), 2488-2490. 360. Koradin, C.; Dohle, W.; Rodriguez, A. L. ; Schmid, B.; Knochel, P., Synthesis of polyfunctional indoles and rela ted heterocycles mediated by cesium and potassium bases. Tetrahedron 2003 , 59, (9), 1571-1587. 361. Larock, R. C.; Yum, E. K., Synthesi s of indoles via palladium-catalyzed heteroannulation of internal alkynes. J. Am. Chem. Soc. 1991 , 113, 6689-6690. 362. Larock, R. C.; Yum, E. K.; Refvik, M. D., Synthesis of 2,3-disubstituted indoles via palladium-catalyzed annul ation of internal alkynes. J. Org. Chem. 1998 , 63, 7652-7662. 363. Madelung, W., Über eine neue Darstell ungsweise für substituierte Indole. I. Berichte 1912 , 45, 1128-1134.

PAGE 212

190 364. Smith, A. B.; Kanoh, N.; Ishiyama, H.; Minakawa, N.; Rainier, J. D.; Hartz, R. A.; Cho, Y. S.; Cui, H.; Moser, W. H., Tr emorgenic indole alkaloids. The total synthesis of (-)-penitrem D. J. Am. Chem. Soc. 2003 , 125, (27), 8228-8237. 365. Nenitzescu, C. D., Derivativ es of 2-methyl-5-hydroxyindole. Bull. Soc. Chim. Romania 1929 , 11, 37-43. 366. Maguire, A. R.; Plunkett, S. J.; Papot, S.; Clynes, M.; O'Connor, R.; Touhey, S., Synthesis of indomethacin analogues for evaluation as modulators of MRP activity. Bioorganic & Medicinal Chemistry 2001 , 9, (3), 745-762. 367. Wildes, J. W.; Martin, N. H.; Pitt, C. G.; Wall, M. E., The synthesis of (-)9-transtetrahydrocannabinol. J. Org. Chem. 1971 , 36, (5), 721-723. 368. Zemlicka, J.; Holy, A., Preparation of N-dimethylaminomethylene derivatives a new method of a selective substitu tion of nucleoside amino groups. Collect. Czech. Chem. Comm. 1967 , 32, (9), 3159-3168. 369. Froehler, B. C.; Matteucci, M. D., Dial kylformamidines depurination resistant N6-protecting group for deoxyadenosine. Nucl. Acids Res. 1983 , 11, (22), 80318036. 370. Bax, A.; Summers, M. F., H-1 and C-13 A ssignments from sensitivity-enhanced detection of heteronuclear multiple-b ond connectivity by 2-D multiple quantum NMR. J. Am. Chem. Soc. 1986 , 108, (8), 2093-2094. 371. Hurd, R. E.; John, B. K., Gradient-e nhanced proton-detected heteronuclear multiple-quantum coherence spectroscopy. J. Magn. Reson. 1991 , 91, (3), 648-653. 372. Kazimierczuk, Z.; Shugar, D., Preparation of some N-methyl isoguanines via 6methyl-thio-2-oxopurines, and of 8-methylisoguanine. Acta Biochim. Pol. 1974 , 21, (4), 455-463. 373. Katritzky, A. R.; Lagowski, J. M., Prot otropic tautomerism of heteroaromatic compounds. 2. 6-Membered rings. Adv. Het. Chem. 1963 , 1, 339-437. 374. Cox, R. H.; Bothner-By, A. A., Proton nuclear magnetic resonance of diand trisubstituted pyrazines and their cations. J. Phys. Chem. 1968 , 72, (5), 1642-1645. 375. Cox, R. H.; Bothner-By, A. A., Proton nuclear magnetic resonance spectra of monosubstituted pyrazines. J. Phys. Chem. 1968 , 72, (5), 1646-1649. 376. Dewar, M. J. S.; Urch, D. S., Electrophilic substitution. Part VIII. The nitration of dibenzofuran and a new method of ultrav iolet spectrophotometric analysis of mixtures. J. Chem. Soc. 1957 , 345-347.

PAGE 213

191 377. Voegel, J. J.; von Krosigk, U.; Benner, S. A., Synthesis and tautomeric equilibrium of 6-amino-5-benzyl-3-methylpyrazin-2one. An acceptor-donor-donor nucleoside base analog. J. Org. Chem. 1993 , 58, 7542-7547. 378. von Jouanne, J.; Palmer, D. A.; Kelm, H ., The use of solvent ET-values in high pressure kinetics. Bull. Chem. Soc. Jpn. 1978 , 51, (2), 463-465. 379. Kamlet, M. J., Methyl 2-nitro-3ethoxyacrylate and related compounds. J. Org. Chem. 1959 , 24, (5), 714-715. 380. Horwell, D. C.; Nichols, P. D.; Ratc liffe, G. S.; Roberts, E., Synthesis of conformationally constrained tryptophan derivatives. J. Org. Chem. 1994 , 59, (16), 4418-4423. 381. Lee, K. L.; Goh, J. B.; Martin, S. F., Novel entry to the Ergot alkaloids via ring closing metathesis. Tetrahedron Lett. 2001 , 42, (9), 1635-1638. 382. Yokoyama, Y.; Kondo, K.; Mitsuhashi, M. ; Murakami, Y., Total synthesis of optically active chanoclavine-I. Tetrahedron Lett. 1996 , 37, (52), 9309-9312. 383. Yokoyama, Y.; Matsumoto, T.; Murakami, Y., Synthetic studies on indoles and related compounds .36. optically-active to tal synthesis of clavicipitic acid. J. Org. Chem. 1995 , 60, (6), 1486-1487. 384. Harland, P. A.; Hodge, P.; Maughan, W. ; Wildsmith, E., Synthesis of primary amines via alkylation of the sodium-salt of trifluoroacetamide an alternative to the gabriel synthesis. Synthesis 1984 , (11), 941-943. 385. Landini, D.; Penso, M., A convenient synthesis of N-alkyl and N,Ndialkyltrifluoroacetamides in a solid-liqui d 2-phase system in the presence of phase-transfer catalysts. Synthetic Commun 1988 , 18, (8), 791-800. 386. Sonogashira, K.; Tohda, Y.; Hagihara, N., Convenient synthesis of acetylenes catalytic substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes, and bromopyridines. Tetrahedron Lett. 1975 , (50), 4467-4470. 387. Alami, M.; Crousse, B.; Ferri, F., Weakly ligated palladium complexes PdCl2(RCN)2 in piperidine: versatile catalysts for Sonogashira reaction of vinyl chlorides at room temperature. J Organomet Chem 2001 , 624, (1-2), 114-123. 388. Gathergood, N.; Scammells, P. J., Prepara tion of the 4-hydroxytryptamine scaffold via palladium-catalyzed cylization: A pract ical and versatile s ynthesis of psilocin. Org. Lett. 2003 , 5, (6), 921-923. 389. Leadbeater, N. E.; Tominack, B. J., Rapid, easy copper-free Sonogashira couplings using aryl iodides and ac tivated aryl bromides. Tetrahedron Lett. 2003 , 44, (48), 8653-8656.

PAGE 214

192 BIOGRAPHICAL SKETCH Theodore Martinot was born in Paris, Fran ce. Shortly after his birth, his family moved to Canada, where he spent the first part of his life in and around Montreal. When he was twelve, his family returned to France, this time to Toulouse, where he first attended College Le Caousou, then College Pier re de Fermat, until he was fifteen, when they again relocated, this time to Florida. He attended Lake Brantley High School for the last three years, then move d to Gainesville, where he ma jored in chemistry (highest honors) and French (high honors) at the Univ ersity of Florida. Early on as an undergraduate, he joined the Hudlicky group, an d acquired such experience that he chose to continue on to graduate school to study or ganic chemistry. He stayed in Gainesville and spent the first part of his time there wo rking with Prof. Tomas Hudlicky, after which he joined the laboratory of Prof. Steven Benner.