SEEKING mRNA METHYLATION INHIBITORS AS ANTIVIRAL AGENTS Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. ________________________ Weikuan Li Certificate of Approval: ________________________ Peter Livant Associate Professor Chemistry and Biochemistry ________________________ Douglas C. Goodwin Associate Professor Chemistry and Biochemistry ________________________ Stewart W. Schneller, Chair Professor Chemistry and Biochemistry ________________________ Anne Gorden Assistant Professor Chemistry and Biochemistry ________________________ George T. Flowers Interim Dean Graduate School SEEKING mRNA METHYLATION INHIBITORS AS ANTIVIRAL AGENTS Weikuan Li A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 9, 2008 iii SEEKING mRNA METHYLATION INHIBITORS AS ANTIVIRAL AGENTS Weikuan Li Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________ Signature of Author ______________________ Date of Graduation iv VITA Weikuan Li, son of Chengmao Li and Enzhen Zhang, was born in Longchuan County, Yunnan Province, China, on June 16, 1979. In September 1997, he began his study in Lanzhou University, and received a Bachelor degree in chemistry in July 2001. He attended Nanjing University in September 2001 for three years and obtained a Master degree in analytical chemistry. He entered Graduate School, Auburn University, in August, 2004, under the direction of Dr. Stewart W. Schneller. v DISSERTATION ABSTRACT SEEKING mRNA METHYLATION INHIBITORS AS ANTIVIRAL AGENTS Weikuan Li Doctor of Philosophy, August 9, 2008 (M. S., Nanjing University, Nanjing, P. R. China, 2004) (B. S., Lanzhou University, Lanzhou, P. R. China, 2001) 159 Typed Pages Directed by Stewart W. Schneller Aristeromycin and neplanocin A are two examples of potent S-adenosylhomocysteine hydrolase inhibitors. As a consequence, they show significant broad-spectrum antiviral activity, however, their clinical potential is limited by toxicity, which is associated with phosphorylation at their 5?-hydroxyl groups. 5?-Noraristeromycin has been found to exhibit wide-spectrum antiviral activity with reduced toxicity due to its inability to form the corresponding nucleotide. To explore new antiviral agents retaining aristeromycin- based activity while reducing undesired toxicity, 5?-fluoro-5?-deoxyaristeromycin (1), 4?- fluoro-4?-deoxynoraristeromycin (2), 3,7-dideazaaristeromycin (3) and 3,7- dideazanoraristeromycin (4) were synthesized. Compounds 1 and 2 showed moderate activity against measles but were inactive in other antiviral assays. Compounds 3 and 4 exhibited no significant activity against all viruses tested. vi Another member of the adenosine (Ado) set is sinefungin (5), which is a naturally occurring analog of S-adenosyl-L-homocysteine (AdoHcy) or S-adenosylmethionine (AdoMet). The wide-spectrum biological activities (anti-fungal, anti-virus, anti-bacteria and anti-malarial) are associated with its inhibition of AdoMet dependent methyltransferases. The application of sinefungin is limited by its toxicity. A convenient synthesis of sinefungin and related compounds (6, 7 and 8) was developed. vii ACKNOWLEDGMENTS This dissertation would not have been possible without the help of many people. First of all, the author would express great appreciation to his research advisor Dr. Stewart W. Schneller for insightful direction, untiring guidance and encouragement throughout this endeavor. He is also indebted to his committee members, Drs. Douglas C. Goodwin, Peter Livant, Anne Gorden and Dr. Jack DeRuiter for their intellectual assistance and constructive suggestions. The great help from Dr. Xueqiang Yin is greatly appreciated. The constructive suggestion from Drs. Atanu Roy, Yan Zhang, Wei Ye and Haisheng Wang are sincerely acknowledged. The cooperation of Drs. Mark Prichard, Eric De Clercq, Lieve Naesens, Michael Murray, Donald Smee, Brent Korba and Robert Sidwell in providing the antiviral data is especially appreciated. In addition, the author extends his thanks to Department of Chemistry and Biochemistry at Auburn University and National Institute of Health for their financial support. viii Style manual or journal used: Computer software used: Journal of Organic Chemistry Microsoft Office 2003 for PC CS Chemoffice 2005 for PC KiNG Viewer 2.13 for Windows ix TABLE OF CONTENTS LIST OF FIGURES ?????????????...?????????????xi LIST OF SCHEMES??????????????????????????.xii LIST OF TABLES??????????????????????????...xiii INTRODUCTION?????????????????????????1 Life cycle of virus?????????????????????????1 Viral Disease and Bioterrorism????????????????????2 Prevention and Antivirus Strategies??????????????????..3 Nucleoside Analogs as Antiviral Agents????????????????5 AdoHcy Hydrolase as the Antiviral Therapy Target???????????..7 Mechanism of AdoHcy Hydrolase??????????????????..10 Inhibitors of AdoHcy hydrolase ???????????????????13 Methyl Transferase Inhibitors: Sinefungin and Its Analogs????????..19 5?-DEOXY-5?-FLUORO ARISTEROMYCIN???????????????22 4?-DEOXY-4?-FLUORO NORARISTEROMYCIN????????????..29 3,7-DIDEAZA ARISTEROMYCIN AND NORARITSTEROMYCIN?????33 DEVELOPING AN ALTERNATIVE SYNTHESIS OF SINEFUNGIN AND RELATED COMPOUNDS????????????????????????????.39 x SYNTHESIS OF OXA-ADOHCY AND RELATED COMPOUNDS?????52 BIOLOGICAL RESULTS??????????????????????.57 CONCLUSIONS???????????????????????????66 EXPERIMENTAL?????????????????????????69 REFERENCES??????????????????????????.127 LICENSE FROM ELSEVIER??????????????????????.145 xi LIST OF FIGURES Figure 1. Naturally Occurring Nucleosides?????????????????..5 Figure 2. FDA Approved NRTI?s and NtRTI?????????????????.6 Figure 3. Feedback Inhibition of Methyltransferases by AdoHcy?????????.8 Figure 4. Capping of mRNA and Structure of Capped 5?End of mRNA??????.9 Figure 5. Mechanism of AdoHcy Hydrolase?????????????????11 Figure 6. Ribbon Structure of AdoHcy Hydrolase ??????????????.12 Figure 7. Examples of Potent AdoHcy Hydrolase Inhibitors??????????.14 Figure 8. Phosphorylation of Ari and NpcA by Cellular Kinases?????????15 Figure 9. Design of Ari or NpcA Analogs by 5? Position Modifications??????16 Figure 10. Truncated Analog of Ari and NpcA????????????????16 Figure 11. Chain-length Modified and Sterically Encumbered Analogs of Ari???..17 Figure 12. Targets 1 and 2????????????????????????17 Figure 13. Natural and Synthesized 7-Deazapurine Nucleosides?????????..18 Figure 14. Targets 3 and 4????????????????????????18 Figure 15. Sinefungin????????????????????????.20, 39 Figure 16. Target 5: Sinefungin and Related Compounds????????????20 Figure 17. Target 6: Oxa-AdoHcy?????????????????????.21 xii Figure 18. Targets 7 and 8: Ari and NpcA Version of Oxa-AdoHcy???????..21 Figure 19. NOESY and HMBC Correlationship in DMSO-d6??????????32 Figure 20. Stereoselectivity of Sch?llkopf Auxiliary??????????????40 Figure 21. Stereoselectivity of Brown Allylation???????????????41 Figure 22. Compounds for Antiviral Evaluation???????????????..57 LIST OF SCHMEMES Scheme 1. Initial Retrosynthetic Analysis to 1????????????????23 Scheme 2. Optimization of Enone Synthesis?????????????????24 Scheme 3. Synthesis of Key Intermediate 16?????????????????25 Scheme 4. Initial Attempt to 1??????????????????????26 Scheme 5. Revised Retrosynthetic Analysis towards Synthesis of 1???????.27 Scheme 6. Synthesis of Target 1?????????????????????..28 Scheme 7. Retrosynthetic Analysis of 2???????????????????29 Scheme 8. Synthesis of 2????????????????????????31 Scheme 9. Retrosynthetic Analysis to 3 and 4????????????????34 Scheme 10. Synthesis of 6-Chloro-3,7-dideazapurine?????????????35 Scheme 11. Synthesis of 3,7-Dideaza Ari?????????????????..37 Scheme 12. Synthesis of 3,7-Dideaza Norari?????????????????38 Scheme 13. Retrosynthetic Analysis of Sinefungin??????????????41 Scheme 14. Protection of Anomeric Hydroxyl as Methyl Ether?????????42 xiii Scheme 15. Synthesis of Compound 68??????????????????.43 Scheme 16. Proposed Side Reactions in Double Bond Cleavage by OsO 4 /NaIO 4 ............44 Scheme 17. Attempted Synthesis of Compound 79??????????????.45 Scheme 18. Synthesis of Coumpound 79??????????????????46 Scheme 19. Unexpected Result of PMB Ether Cleavage????????????..47 Scheme 20. Attempt to Introducing Base at Early Stage????????????48 Scheme 21. Attempted Synthesis of Compound 93?????????????......49 Scheme 22. Synthesis of Compound 97??????????????????.50 Scheme 23. Synthesis of Azidosinefungin???????..??????????51 Scheme 24. Retrosynthetic Analysis towards Compound 6, 7 and 8???????..53 Scheme 25. Synthesis of Compound 6???????????????????..54 Scheme 26. Synthesis of Ari-oxa-AdoHcy?????.????????????55 Scheme 27. Attempted Synthesis of Compound 8??..????????????56 LIST OF TABLES Table 1. Viruses Used for Bioassay????????????????????.58 Table 2. Antiviral Activity towards HCV??????????????????58 Table 3. Antiviral Activity towards Influenza in MDCK Cell Cultures??????.59 Table 4. Anti-Feline Corona Virus (FIPV) Activity and Cytotoxicity in CRFK Cell Cultures???????????????????????????????.59 xiv Table 5. Activity and Cytotoxicity towards Vesicular Stomatitis Virus, Coxsackie Virus B4 and Respiratory Syncytial Virus in HeLa Cell Cultures???????????60 Table 6. Activity and Cytotoxicity towards Para-influenza 3 Virus, Reovirus-1, Sindbis Virus, Coxsackie Virus B4 and Punta Toro Virus in Vero Cell Cultures?????..60 Table 7. Activity and Cytotoxicity towards Herpes Simplex Virus-1 (KOS), Herpes Simplex Virus-2 (G), Vaccina Virus, Vesicular Stomatitis Virus, Herpes Simplex Virus-1 TK - KOS ACV + in IL Cell Cultures????????????????????61 Table 8. Activity and Cytotoxicity towards Cowpox and Vaccinia Viruses?????61 Table 9. Activity and Cytotoxicity towards EBV???????????????..61 Table 10. Activity and Cytotoxicity towards West Nile Virus??????????62 Table 11. Activity and Cytotoxicity towards Parainfluenza Virus????????..62 Table 12. Activity and Cytotoxicity towards Adenovirus???????????..62 Table 13. Activity and Cytotoxicity towards Measles?????????????63 Table 14. Activity and Cytotoxicity towards Respiratory Syncytial A Virus????.63 Table 15. Activity and Cytotoxicity towards Rhinovirus????????????63 Table 16. Activity and Cytotoxicity towards VZV??????????????.64 Table 17. Activity and Cytotoxicity towards HCMV?????????????.64 Table 18. Activity and Cytotoxicity towards HSV-1 and HSV-2 in HFF Cells???.64 Table 19. Activity and Cytotoxicity towards Human Corona (SARS) Virus?????64 Table 20. Activity and Cytotoxicity towards HBV??????????????65 1 INTRODUCTION Viruses are small, infectious particles whose growth, reproduction and propagation depend on hosts. The components of a viral particle, or virion, include genetic material (DNA or RNA) and a protective shell called a capsid. Many viral sizes and shapes have evolved. The shapes vary from simple helical to more complex structures with tails or envelopes. 1 Life Cycle of a Virus The life cycle of a virus can be separated into several basic stages, i.e. attachment, entry, replication, assembly, and release, although the details may differ significantly between species. The attachment of viruses to host cells is achieved by a specific binding between viral capsid proteins and specific receptors on the host cellular surface, which determines the site and infectious scope of a virus. 2-5 The attached virus then enters the host cell by receptor mediated endocytosis or membrane fusion. The viral capsid is removed and degraded by viral enzymes or host enzymes to release the viral genomic nucleic acid. 6-9 Replication follows with the synthesis of viral messenger RNA (mRNA, except for positive sense RNA viruses, where the viral nucleic acid core serves as an 2 mRNA) leading to viral protein synthesis and viral genome replication. 1, 10-11 The necessary viral building blocks synthesized in the host cell, including protein shells,structure units, capsids, segmented genomes are assembled into a progeny virions. Maturation of the viral proteins often occurs after the assembly of the virus particles. In some viruses, this maturation occurs after release of virions. 12-14 Viral Disease and Bioterrorism Virus-induced common human diseases (these diseases are usually self-limiting, but antiviral therapy is necessary for immune suppressed patients) include the common cold, 15 the flu, 16 and chickenpox. 17 Very serious diseases, like Ebola, 18 acquired immune deficiency syndrome (AIDS), 19 avian influenza 20 and severe acute respiratory syndrome (SARS) 21 are also caused by viruses. In addition, viruses can induce life-long or chronic infections where the viruses continue replicating at a certain rate in the body despite the adaptive or innate immune response. This is usually seen with people who are infected with hepatitis B virus (HBV) 22 and hepatitis C virus (HCV) 23 viruses. These types of viruses can be transmitted through high-risk intimate interaction between infected and healthy people such as unprotected body contact and blood transfusion. An important point to be considered is the lethal threat that might arise from utilizing viruses as biological weapons. 24-28 The necessity for anti-biological terrorism plans was evidenced by the anthrax attacks in 2001 in the United States. Although some viruses, such as smallpox, were eradicated, the possible use of ?eradicated? viruses in bioterrorism must be taken into account. 29 Other viruses, such as those that cause 3 hemorrhagic fevers, which can be generated in large amounts in cell culture, and then made transmissible in aerosol form, may become weapons of terrorism. 30-33 This threat is compounded by the fact that currently there are no vaccines or antivirals available for such viruses. 30 Prevention and Antivirus Strategies Vaccination is a tactic towards combating illness or the spread of virus by intentional introduction of antigenic material (antigen) to develop immunity to a disease. The antigen administered can be either live, or weakened forms of viruses, killed or inactivated forms of viruses, or purified material such as proteins. 1 Once a proper vaccine is developed and administered, it is a very good means to prevent viral diseases, however, for people already infected, the benefit of vaccination is limited. Developing a vaccine for fast mutating viruses, such as human immunodeficiency virus (HIV) and influeanza, poses major challenges, due to the high genetic variable nature of these viruses. 34-38 Therapeutics agents directly towards viral infections can be broadly categorized as agents that assist and fortify host immune defenses, or that attack the virus and its replicative cycle directly. 30 Stimulation or protection of the immune system is a strategy to render host immunity as the viral defense, instead of attacking the viruses directly. Usually, these sorts of agents stimulate the immune system to fight against a range of pathogens. Interferon is one example of this class. The well-known "interferon alpha" is established as a treatment for hepatitis B and C, 22, 23 and other interferons are also being investigated as treatments for various diseases. 39 Some viruses, like vaccinia, influenza 4 virus, Ebola, and Marburg viruses produce interferon antagonists, 40-47 which allow them to evade the assault of host immune system. Modifying the innate immune response to fight against specific virus infections is calling forth a need for a deeper understanding of the interaction between virus and immune response. 30 In principle, any stage of viral replication cycle can be selected for antiviral target success. To enter the host cell, a virus must go through a sequence of steps. The initial step is binding to a specific "receptor" on the surface of the host cells. In this regard, the cell receptors can be selected as antiviral targets that would block that site. 48-54 Other strategies include inhibiting uncoating and envelope fusion. 53 During viral replication in a host cell, a number of viral or cellular functional proteins can be targeted. 55-59 For example, HIV reverse transcriptase has been a widely investigated viral specific enzyme. Inhibition of this enzyme suppresses the transition of single-stranded viral RNA into double-stranded proviral DNA (to be integrated into host chromosome), which, in turn, reduces the HIV replication and the human immunity is preserved. 60 Another example is blocking the RNA dependent RNA polymerase of HCV. 61 The last step of the virus life cycle is release of progeny virion from the host cell. Preventing their release is a strategy that has attracted many researchers. One example of an antiviral agent of this type is oseltamivir (Tamiflu), which has been successfully introduced to treat influenza. The antiviral mechanism of Tamiflu is through the inhibition of neuraminidase, which prevents the release of virus from host cell. 56 Nucleoside Analogs as Antiviral Agents Nucleosides (Figure 1) are naturally occurring biomolecules, which serve as fundamental building blocks for DNA and RNA. O HO OH N HO N N N NH 2 Adenosine O HO OH N HO N N N NH 2 NH 2 Guanosine O HO OH N HO N NH 2 O Cytidine O HO OH N HO NH O O Uridine O HO N HO NH O O Thymidine Figure 1. Naturally Occurring Nucleosides The distinguishing structural characteristic of natural nucleosides shows a heterocyclic base moiety bonded to a ribofuranose in the beta configuration. The major purine base components in these nucleosides are adenine and guanine. The major pyrimidine base residues are cytosine, uracil (in RNA), and thymine (in DNA). Nucleosides are involved in a number of important biological metabolisms. 62 As a consequence, nucleoside analogs show a variety of biological activities, including medicinal effects of anti-viral, 63 anti-cancer, 64-67 anti-fungal 68, 69 and anti-malarial. 70, 71 Particularly relevant to this research are the antiviral activities of nucleoside and 5 nuleotide analogs with great clinical potential. 72-81 Among more than thirty FDA approved antiviral agents (not including interferons and immunoglobulins), many are nucleoside/nucleotide analogs. For example, FDA-approved HIV reverse transcriptase inhibitors zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, and tenofovir disoproxil (Figure 2) are among them. 56 These nucleosides are inhibitors of reverse transcriptase after conversion into the corresponding 5? triphosphates by cellular or viral kinases. Subsequently, they are incorporated into the viral DNA chain, leading to viral DNA chain termination by their lacking a 3?-hydroxyl and inhibition of reverse transcriptase. 56 O N HN O O CH 3 HO N 3 Zidovudine O HO N N N NH O Didanosine O N N O NH 2 HO Zalcitabine O N HN O O CH 3 HO Stavudine S O N N O NH 2 OH Lamivudine HO N N N N NH 2 NH HO 2 C CO 2 H Abacavir succinate S O N N O NH 2 OH F Emtricitabine N N N N NH 2 O P O O O O O O O O O CO 2 H HO 2 C Tenofovir disoproxil fumarate Figure 2. FDA-approved NRTI?s and NtRTI 6 7 AdoHcy Hydrolase as an Antiviral Therapy Target The symbiotic relationships between viral pathogens and hosts have led to viral adaptation to host cellular mechanisms for entry, replication, assembly and progeny releasing. In addition to the viral specifically encoded components, the host cell also persents possible synthetic targets for developing of antiviral agents. The advantage of targeting a host process opens the door to development of broad spectrum antiviral agents. Once a common host pathway is blocked, all pathogens that rely on this pathway will be inhibited. This is especially useful for fighting against emerging or gene-engineered bio-pathogens, since valid vaccines or specific antivirals are not already available. A further advantage of this approach is less likelihood of drug resistance, since there are a limited number of alternative cellular pathways. 30 S-Adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1), which is a cellular enzyme, fits this promise and has been regarded as a target for wide spectrum antiviral agent development. 57, 82-93 AdoHcy hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The reaction is favored in the synthetic (reverse) direction. However, the fast removal of Ado and Hcy by metabolism (Ado is removed by Ado deaminase and Ado kinase, and Hcy follows to the cysteine synthesis and methionine regeneration) moves the reaction forward. The intracellular AdoHcy level will increase if AdoHcy hydrolase is inhibited. The accumulation of AdoHcy, in turn, inhibits essential S-adenosylmethionine (AdoMet)-dependent methylation reactions, where the AdoHcy is both the product and a potent feedback inhibitor (Figure 3). AdoMet dependent methylation is required for final maturation of the capped mRNA (Figure 4) from cellular and viral sources. Therefore, by inhibition of AdoHcy hydrolase, the processing and maturation of viral mRNAs may be inhibited. As a consequence, requisite proteins and enzymes for progeny virion assembly are not generated. O N N N N NH 2 S CH 3 HO OH H 2 N CO 2 H O N N N N NH 2 S HO OH H 2 N CO 2 H AdoMet AdoHcy mRNA methylated mRNA methyl transferases feedback inhibition O N N N N NH 2 SH HO OH H 2 N CO 2 H HO + AdoHcy hydrolase inosine, AMP/ADP/ATP Ado Hcy Figure 3. Feedback Inhibition of Methyltransferases by AdoHcy 8 O O OH Base P O O O O O OH Base P O O O P O P O P O O O O O O O O N HO OH N N NH NH 2 O O O O Base P O O O O O O Base P O O O P O P O P O O O O O O O O N HO OH N N NH NH 2 O H 3 C CH 3 CH 3 AdoMet AdoHcy 5' end of capped mRNA methylated 5' end of mRNA (cap 2) methyl transferases Figure 4. Methylation of Capped mRNA Since the AdoHcy hydrolase inhibitors exert their antiviral activities through suppressing the methylation of mRNA, it is not surprising that some viruses, like poliovirus, have polypeptide capped 5? end of mRNA, which was not methylated, 94-96 are intrinsically insensitive to this type of compounds. 90 On the other hand, because AdoHcy hydrolase is a cellular enzyme, it might be expected that its inhibition would lead to general suppression of cellular protein synthesis and subsequent host toxicity. However, examples exist where this is not a major drawback. For example, the successful treatment of filovirus by an AdoHcy hydrolase inhibitor without significant toxicity is noteworthy, and this may be the result of two mechanisms. 97 First, the fast replicative nature of the virus leads to increased demand for protein synthesis in the virally infected cells compared to uninfected cells. This would result in a greater demand for viral mRNA 9 10 methylation, which would render the methyltransferase more sensitive to inhibition by the bio-feedback partner, AdoHcy. Therefore, the elevated AdoHcy levels would significantly inhibit the activity of methyltransferases. Second, the virally encoded methyltransferase may have a different binding constant with AdoHcy compared to cellular methyl transferase. As a consequence, the preferential inhibition of viral methyltransferase would occur, producing significant antiviral activity with tolerable, or no toxicity. Undoubtedly, long term inhibition of AdoHcy hydrolase will suppress general cellular protein synthesis, leading to severe toxicity; Wolfe and Borchardt have suggested that ?a temporary and partial inhibition, while not seriously altering cell function, may allow phosphatases and ribonucleases to destroy the foreign (that is, viral) mRNAs. After removal of the AdoHcy hydrolase inhibitor, favorable cellular mRNA cap methylation could resume and full protein synthesis would ensue?. 82 Mechanism of AdoHcy Hydrolase The mechanism by which AdoHcy hydrolase acts is shown in Figure 5. 99-108 The 3? hydroxyl group of AdoHcy (I, forward direction) or Ado (VII, reverse or synthetic direction) are first converted to 3?-keto derivatives (II or VI) via the oxidation by enzyme-bound NAD + . This increases the acidity of 4?-proton and causes it to be abstracted by an enzyme base. The resulting carbanion species (III or V) facilitates an elimination of the 5?-substituents, Hcy (forward) or water (reverse), to give the central intermediate 3?-keto-4?,5?-dehydroadenosine (IV). A Michael type addition of water (forward) or Hcy (reverse) to this central intermediate, followed by reduction of corresponding 3?-keto intermediates (II, VI) by the NADH, completes the AdoHcy hydrolase catalyzed ?hydrolysis? (forward) or synthesis (reverse) of AdoHcy, resulting in the formation of the final product, Ado (VII) or AdoHcy (I). O N N N N NH 2 S H 2 N HO 2 C HO OH O N N N N NH 2 S H 2 N HO 2 C OHO H H O N N N N NH 2 S H 2 N HO 2 C OHO O N N N N NH 2 OHO O N N N N NH 2 OHO HO O N N N N NH 2 OH HO HO NAD + EB 1 NADH EB 1 H O N N N N NH 2 OHO HO Hcy H 2 O EB 2 EB 2 H I II III IV VVI VII Figure 5. Mechanism of AdoHcy Hydrolase AdoHcy hydrolase contains four identical subunits (denoted as A, B, C, and D). 106, 107, 109-121 In each subunit, there exists three domains: a large N-terminal domain for substrate-binding, a large cofactor binding domain, and a smaller C-terminal domain. The small C-terminal domain of A extends to the adjacent subunit B and forms part of the cofactor-binding site in that subunit. Also, the small C-terminal of B subunit inserts into A in the same manner and serves as part of cofactor binding domain. The same reciprocal penetration between A and B subunits also appears in C and D. However, the similar 11 linkage is not present between AB and CD. Therefore, the AdoHcy hydrolase can be regarded as dimer of dimers. A typical crystal structure of AdoHcy hydrolase is shown in Figure 6, where the substrate binding domain is bound to neplanocin A in its 3?-keto form ( PDB code: 1LI4). 107 Figure 6. Ribbon Structure of AdoHcy Hydrolase Up to now, ten three dimensional structures of AdoHcy hydrolase from different sources are available. 106, 107, 113, 114, 116, 118-121 The protein data bank codes are: 1LI4, 1B3R, 1A7A, 1D4F, 1KY4, 1KY5, 1V8B, 1XWF, 1K0U, and 2H5L. The crystal structures show that in the absence of substrate, the NAD + binding domain and substrate binding domain 12 13 are quite far from each other. The enzyme is in an ?open? state. 114 Upon binding of substrate, the enzyme shifts to a ?closed? state, where the cofactor binding domain and substrate binding form come close. 107 Based on the crystal structure, a detailed catalytic mechanism is proposed for this enzyme from rat liver, which bears 431 amino acid residues in each identical subunit. 114, 119 In this case, Glu155 abstracts the O 3? -H proton, while the C 3? -H proton is removed by NAD + . General acid-base catalysis is performed by His54 or Asp130. The oxidation state of the bound NAD + is regulated by Cys194. Inhibitors of AdoHcy hydrolase Since inhibition of AdoHcy hydrolase has been targeted for antiviral drugs design, a number of potent inhibitors have been synthesized and evaluated. 84 Several examples of this class are shown in Figure 7, i.e., aristeromycin (Ari), noraristeromycin (Norari), neplanocin A (NpcA), 3-deazaaristeromycin, 3-deazaneplanocin A, and 2-fluoro and 6?- methyl derivatives of neplanocin A. Poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus) are very sensitive to these compounds. 84 HO OH N N N N NH 2 HO aristeromycin HO OH N N N N NH 2 HO noraristeromycin HO OH N N N N NH 2 HO neplanocin A HO OH N N N NH 2 HO 3-deazaneplanocin A HO OH N N N N NH 2 HO F HO OH N N N N NH 2 HO 2-fluoroneplanocin A 6'-methyneplanocin A HO OH N N N NH 2 HO 3-deazaaristeromycin Figure 7. Examples of Potent AdoHcy Hydrolase Inhibitors Ari and NpcA were two naturally occurring nucleosides. Ari was separated from the metabolites of Steptomyces citricolor in 1968, 122 and NpcA was isolated from Ampullariella regularis in 1981. 123, 124 Ari showed antiviral activity by inhibition of AdoHcy hydrolase. 82 Two mechanisms of NpcA?s antiviral activity have been proposed 125, 126 : (1) inhibition of AdoHcy hydrolase and (2) transformation into S-neplanocylmethionine, which is an AdoMet analog and acts by inhibiting the methyltransferase directly. In the first case, NpcA anchors to the active site of AdoHcy hydrolase with a high affinity and is oxidized by enzyme bound NAD + into its 3?-keto form. This causes the enzyme to enter the closed state and lose catalytic capability. 107 The cytotoxicity, which was believed to be a result of cellular C5? phosphorylation, has limited the further application of Ari and NpcA as broad spectrum antiviral agents. This phosphorylation by the sequence of adenosine kinase, adenylate kinase and nucleotide diphosphate kinase convert Ari or NpcA into the 5?-mono/di/triphosphates respectively (Figure 8). 127-131 The monophosphate of Ari can also be converted into the 14 inosine analog by adenosine monophosphate deaminase, and further to the carbocyclic guanosine monophosphate derivative. Instead of deamination of the monophosphate, NpcA is directly converted to the inactive inosine form by adenosine deaminase. The triphosphates of Ari and NpcA, resemble ATP in structure, leading to deleterious effects. 132-134 N HO OH HO N N N NH 2 Ari N HO OH O N N N NH 2 P O O O Carbocyclic AMP N HO OH O N N N NH 2 P O O O 2 N HO OH O N N N NH 2 P O O O 3 Carbocyclic ADP Carbocyclic ATP N HO OH HO N N N NH 2 NpcA N HO OH O N N N NH 2 P O O O NpcA monophosphate N HO OH O N N N NH 2 P O O O 2 N HO OH O N N N NH 2 P O O O 3 NpcA diphosphate NpcA triphosphate Figure 8. Phosphorylation of Ari and NpcA by Cellular Kinases The further design of Ari and NpcA analogs, with the expectation of retaining their high activities while decreasing toxicity, focused on introducing structure modifications, which prevent or decrease the tendency of C5? phosphorylation. Not surprisingly, much effort has been devoted in this undertaking. Since the 5?-hydroxyl group is the site at which phosphorylation occurs, a number of Ari analogs with a modified 5? position were designed and synthesized. This strategy includes (but is not limited to) (1) removal of the 5? hydroxyl group; (2) increasing the steric hindrance at the 5? position; and (3) a change in chain length at the 5? position (Figure 9). 15 HO OH N N N N NH 2 HO Ari or NpcA R=groups without OH group HO OH N N N N NH 2 R HO OH N N N N NH 2 HO R HO OH N N N N NH 2 HO n n=0,1,2, ... Figure 9. Design of Ari or NpcA Analogs by 5? Position Modifications For example, Borchardt?s group synthesized truncated analogs of Ari and NpcA, as well as their 3-deaza counterparts (Figure 10). 135-138 These compounds showed potent antiviral activity, while the associated toxicity was greatly decreased. HO OH N N X N NH 2 X=N, DHCaA X=C, c 3 -DHCaA HO OH N N X N NH 2 X=N, DHCeA X=C, c 3 -DHCeA Figure 10. Truncated Analog of Ari and NpcA The Schneller group developed chain-length modified analogs of Ari, as well as a sterically encumbered analog of Ari (Figure 11). 139-142 5?-Noraristeromycin (Norari) showed significant antiviral activity against human cytomegalovirus (HCMV), hepatitis B virus (HBV), measles, influenza, and vaccinia virus. This compound is much less toxic than Ari. 139-140 Homoaristeromycin showed potent activity towards vaccinia, cowpox, and monkeypox viruses, with little associated toxicity. 141 Subsequently, targets 1 and 2 (Figure 12), the derivatives of Ari and Norari, where the 5? and 4? hydroxyl groups were replaced by fluoride, were designed and synthesized for this dissertation project, since the fluoride is incapable of phosphorylation. 16 HO OH N N N N NH 2 HO HO OH N N N N NH 2 HO HO OH N N N N NH 2 HO Noraristeromycin Homoaristeromycin 5'-methylaristeromycin Figure 11. Chain-length Modified and Sterically Encumbered Analogs of Ari HO OH N N N N NH 2 F HO OH N N N N NH 2 F 5'-Fluoro-5'-deoxyaristeromycin (1) 4'-Fluoro-4'-deoxynoraristeromycin (2) Figure 12. Targets 1 and 2 A second method to reduce the possibility of phosphorylation was to modify the base moiety, since it was well known the 3-deazaadenosine is not phosphorylated. The 3-deaza version of Ari and NpcA were synthesized (Figure 7). As expected, these analogs show potent antiviral activities and decreased toxicity. 143, 144 Examples of other modified base analogs include 2-halopurine, 8-methylpurine, 1-deazapurine, and 7-deazapurine. 7-Deazapurine nucleosides, either naturally occurring or synthesized, exhibit antibacterial, antifungal, antiviral and anticancer activity (Figure 13). 145 While, of course, replacement of a nitrogen atom in the purine ring with a CH does not change the shape of the base, other properties, such as basicity of the 6-amino group and hydrogen bonding formation capability in various positions (that is 1, 3, 7), 17 will be different from the purine base. Furthermore, deazapurines make it possible to attach substituents at the 1, 3 and 7 positions, which is not possible for the parent purine ring. With this in mind, it is interesting to consider pursuing 3,7-dideazapurine nucleoside analogs. Therefore, targets 3 and 4 (Figure 14) for this dissertation research (the dideaza counterparts of Ari and Norari analogs) were sought. O HO OH N N N NH 2 HO R R: H, CN, CONH 2 , NH 2 OH, I, etc. O HO OH N N NH HO R R: H, COOH, etc. NH 2 O 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Figure 13. Natural and Synthesized 7-Deazapurine Nucleosides HO OH N N NH 2 HO HO OH N N NH 2 HO 3,7-dideazaaristeromycin (3) 3,7-dideazanoraristeromycin (4) 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Figure 14. Targets 3 and 4 18 19 Methyl Transferase Inhibitors: Sinefungin and Its Analogs As mentioned above, the Ado derivatives exert their antiviral activity by inhibition of AdoHcy hydrolase. Meanwhile, some Ado analogs (like NpcA) are metabolized into AdoHcy analogs, 125, 126 which may inhibit cellular methyltransferases as a result of their effects on AdoHcy hydrolase, or selectively inhibit virally coded methyltransferases. Another member of the Ado set is sinefungin (SF, or A9145), which is a naturally occurring analog of AdoHcy or AdoMet (Figure 15). It was isolated from Streptomyces griseolus (NRRL 3739) 146, 147 and showed potent antimalarial, 148-152 antibacterial, 153 antifungal 154-156 and antiviral 157-160 activities. The bioactivity of sinefungin is associated with its inhibition of AdoMet dependent methyltransferases. 154, 161-174 However, sinefungin was limited in application by its toxicity. 146-148 Thus, the synthesis of sinefungin, 175-185 sinefungin analogs with modification on the side chain or the base, 186-194 AdoHcy analogs, 195-198 carbocyclic sinefungin (analogs), 199, 200 and biosynthesis (effect) of sinefungin 201,202 , have attracted a lot of researchers. An efficient, practical and diverse synthesis of sinefungin and related compounds has been developed in the Schneller group. This method has been successfully applied to the total synthesis of carbocyclic sinefungin. 199 For this dissertation research, an enantioselective, efficient and high yielding route to sinefungin that is adaptable to various modified compounds including modification at C6? position (Figure 16) was sought. O HO OH N NH 2 H 2 N O OH N N N NH 2 5 Figure 15. Sinefungin O HO OH N H 2 N O OH N N N NH 2 X X = H, OH, NH 2 , N 3 , F and OMe (R or S) Figure 16. Target 5: Sinefungin and Related Compounds Since AdoHcy is a potent feedback inhibitor of AdoMet-dependent methyltransferases, it is interesting to design and synthesize analogs of AdoHcy itself that highly resemble AdoHcy and can serve as a competing inhibitor of methyltransferases. The AdoHcy analog may also acts as an alternative substrate for AdoHcy hydrolase, leading to elevated levels of parent AdoHcy and inhibition of methylation. With these guidelines, oxa-AdoHcy, an AdoHcy analog, where the side chain sulfur was replaced by an oxygen, was designed and synthesized (Figure 17) in this project. Simultaneously, the Ari and NpcA version of oxa-AdoHcy analogs were also investigated (Figure 18). 20 O HO OH N O H 2 N O OH N N N NH 2 6 Figure 17. Target 6: Oxa-AdoHcy HO OH N O H 2 N O OH N N N NH 2 HO OH N O H 2 N O OH N N N NH 2 7 8 Figure 18. Targets 7 and 8: Ari and NpcA Version of Oxa-AdoHcy 21 22 5?-DEOXY-5?-FLUORO ARISTEROMYCIN Experimental Design and Synthesis Adenosine, the natural product of AdoHcy ?hydrolysis?, is in the D-configuration. The naturally occurring Ado analogs, Ari and NpcA are D-like analogs. It is not surprising that most of the high ranking AdoHcy hydrolase inhibitors are of D-like configuration. 84 For example, D-like Noari is much more potent than its L-like counterpart. 139 As a consequence, in the search for new hydrolase inhibitors, the D-like Ado analogs are designed and synthesized as a priority. As mentioned previously, the toxicity of Ari is associated with the 5?-phosphorylation. Thus, Ado analogs incapable of C-5? phosphorylation (or with less tendency to do so), are worthy targets. In that direction, the D-like 5?-fluoro-5?-deoxyaristeromycin (1) was selected as a synthetic target for this dissertation research. The initial retrosynthetic plan for this goal is shown in Scheme 1. HO OH N N N N NH 2 F 1 O O N N N N Cl HO O O OH O O O O HO OH HO OH D-Ribose 14 16 19 Scheme 1. Initial Retrosynthetic Analysis to 1 To ensure the D-like configuration, the synthesis was envisioned as starting from commercially available D-ribose, which has the predefined requisite stereochemistry. The key intermediate, the D-like cyclopentenone 14, is a common beginning point for various carbocyclic adenosine analogs. Thus, the large scale synthesis of 14 was sought. In the Schneller group, a facile synthesis of (4R, 5R)-4,5-O-isopropylidene-2-cyclopentenone has been developed. 203 Optimization of this pathway to avoid the volatility of the intermediate aldehyde (2R, 3R)-2,3-O-isopropylidene-pent-4-enal has been achieved in this dissertation research by shifting the glycol protecting group from isopropylidene to cyclopentylidene (Scheme 2). 23 Scheme 2. Optimization of Enone Synthesis OO O OO O 11 OO O OO O 14 O HO OH HO OH D-Ribose Volatile intermediate 49 As shown in Scheme 3, protection of D-ribose with cyclopentanone and methanol in the presence of trimethyl orthoformate and a catalytic amount of sulfuric acid gave 9. The primary alcohol moiety of 9 was substituted with iodine using the combination of triphenylphosphine (TPP), imidazole, and iodine chips to give derivative 10. 204 Reductive elimination of 10 with activated zinc powder in hot methanol yielded aldehyde 11. 205 Treatment of 11 with vinyl magnesium bromide gave diene 12 as mixture of two diastereoisomers. Ring closure metathesis of olefin in the presence of Grubbs catalyst (1 st generation) gave 13, 206 which was not isolated. Instead, compound 13 was further oxidized with a modified Swern protocol 207 to provide the key intermediate 14. With 14 in hand, a Michael addition of vinyl magnesium bromide under the catalysis of copper (I) salt and chlorotrimethylsilane (TMSCl) afforded 15, 208 which was not isolated but transformed into 16 by a Luche reduction. 209 24 Scheme 3. Synthesis of Key Intermediate 16 O HO OH HO OH D-Ribose O HO OMe OO O I OMe OO OO O OO OH OO OH OO O 9 10 111213 14 Cyclopentanone HC(OMe) 3 , MeOH H 2 SO 4 (cat.),79% Ph 3 P, I 2 imidazole,89% n-BuLi -78 o C "105%" VinylMgBr, 84% Grubbs cat. 1st generation DMSO, SO 3 -pyridine complex DIPEA, 77% for two steps OO O 15 OO OH 16 VinylMgBr CuBr-SMe 2 TMSCl NaBH 4 , CeCl 3 -7H 2 O 63% Introduction of the base moiety was next considered (Scheme 4). The 6-chloropurine was used as adenine building block. Thus, 6-chloropurine was installed by a Mitsunobu coupling 210, 211 with 16 to give 17 as a mixture with diisopropyl hydrazine-1,2-dicarboxylate, which was used for next step withouth further purification. Oxidative cleavage of the double bond of 17 using sodium metaperiodate with a catalytic amount of osmium tetroxide 212, 213 resulted in 18. This was not isolated, but reduced with sodium borohydride to yield 19. The desired fluoro compound 20 could not be achieved 25 by various methods of fluorination (for example, combination of N,N-diethylaminosulfur trifluoride (DAST) and pyridine; conversion of 19 into corresponding ?mesylate/tosylate? and treatment with sodium fluoride). Careful examination of NMR spectrum of the fluorination products mixture indicated that the 6-chloropurine moiety may interfere with the transformation of the hydroxyl group into fluoride, since the hydroxyl group was missing, while the expected fluoromethyl group was not present. Scheme 4. Initial Attempt to 1 OO OH 16 OO N 17 N N N Cl OO NO 18 N N N Cl OO NHO N N N Cl 19 OO NF N N N Cl 20 Ph 3 P, DIAD 6-chloropurine 58% NaIO 4 , OsO 4 NaBH 4 CeCl 3 ?7H 2 O 41% (from 17) DAST Realizing the difficulty of introducing fluoride in the presence of 6-chloropurine moiety, it was decided to introduce the purine base at a later stage, while introducing the fluorine at an earlier stage. A revised retrosynthetic plan is shown in Scheme 5. 26 Scheme 5. Revised Retrosynthetic Analysis towards Synthesis of 1 HO OH N N N N NH 2 F 1 O O OH F O O HO OPMB O O OPMB O O OH 16 21 2224 The execution of this plan is shown in Scheme 6. Protection of the secondary hydroxyl group of 16 with the para-methoxybenzyl (PMB) group gave 21. Oxidative cleavage of the double bond of 21 followed by Luche reduction produced 22. Fluorination of 22 with DAST 214 successfully yielded the desired 23. Removal of the PMB protecting group of 23 with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) formed 24 in good yield. Subjecting 24 to a Mitsunobu coupling with 9H-6-di(butoxylcarbonyl)aminopurine (Ad(Boc) 2 ) provided 25. Acid deprotection of 25 successfully gave 1. 27 Scheme 6. Synthesis of Target 1 HO OH N N N N NH 2 F 1 O O X F O O HO OPMB O O OPMB O O OH O O N N N N N(Boc) 2 F 23, X=OPMB 24, X=OH 16 21 22 25 PMBCl, NaH 81% 1)NaIO 4 , OsO 4 2)NaBH 4 , CeCl 3 ?7H 2 O 53% DAST 76% Ph 3 P, DIAD Ad(Boc) 2 DDQ, 60% 3N HCl/MeOH 50 o C, 58% 28 4?-DEOXY-4?-FLUORONORARISTEROMYCIN Experimental Design and Synthesis D-Noraristeromycin, designed and synthesized by the Schneller group, shows potent antiviral activity with little associated toxicity. 139 Encouraged by this result, similar compounds were sought. Out of this, 4?-fluoro-4?-deoxynoraristeromycin (2) was sought, since replacing the 4?-hydroxyl group with fluoride would abolish the potential capability of undesired 5?-noraristeromycin phosphorylation by a cellular kinase. A convergent synthetic strategy was planned. The chosen retrosynthetic analysis is shown in Scheme 7. The basic idea was to build the desired carbocyclic moiety 37, then couple it with a purine base under Mitsunobu conditions to give desired compound. Scheme 7. Retrosynthetic Analysis of 2 O O F N N N N NH 2 2 O O F OH O O OH OPMB HO OAc 37 35 26 29 30 The synthesis began with (1R,4S)-4-hydroxycyclopent-2-enyl acetate 26, which was prepared in large scale by a routine method used in the Schneller group. 203 Protection of the secondary hydroxyl group with the tert-butyldimethylsilyl (TBS) group gave 27. Dihydroxylation 215 of 27, followed by glycol protection, yielded 28. Removal of the acetyl group of 28 with ammonia in methanol gave 29. Pyridinium chlorochromate (PCC) oxidation of 29 yielded 30. Luche reduction of 30 afforded 31. The secondary hydroxyl group of 31 was protected as a PMB ether furnishing 32. Removal of the silyl group of 32 with tetra-n-butylammonium fluoride (TBAF) formed 33, which was subjected to a PCC oxidation and the Luche reduction to avail 35. Fluorination of 35 with DAST yielded 36. The PMB protecting group of 36 was removed by DDQ, producing the key intermediate 37. Mitsunobu coupling of 37 with 6-chloropurine afforded 38. Subsequent amination of 38 led to 39, which was deprotected to give 2. Scheme 8. Synthesis of 2 HO OAc TBSCl imidazole 80% TBSO OAc TBSO X OO 28, X=OAc 29, X=OH TBSO OO O Y X OO 31, X=OH, Y=OTBS 32, X=OPMB, Y=OTBS 33, X=OPMB, Y=OH OPMB OO O OPMB OO HO X OO F 36, X=OPMB 37, X=OH N OO F N N N Cl N OO F N N N NH 2 N HO OH F N N N NH 2 2 26 27 30 3435 38 39 1)NMO, OsO 4 2)Me 2 CO, Me 2 C(OMe) 2 pTSA, 85% on 29 PCC, 85% NH 3 /MeOH 90% NaBH 4 CeCl 3 ?7H 2 O 97% PMBCl NaH, 84% TBAF, 90% on 33 PCC, 81% DAST 69% 6-chloropurine DIAD Ph 3 P 30% NH 3 /MeOH 83% 1N HCl/MeOH 93% NaBH 4 CeCl 3 ?7H 2 O 81% DDQ, 86% To elucidate the relative configuration of 2, ge-NOESY, ge-COSY and ge-HMBC were conducted on Bruker 400 NMR spectrometer. The following correlations were observed (Figure 19): NOESY: H1??H5? beta (weak), H5? alpha (strong), H2?, H4?, 2?OH, 3?OH H2??H5? beta (strong), H5? alpha (very weak), H3? (strong), 2?OH, 3?OH H3??H5? beta (moderate), H2?, H4?, 3?OH, 2?OH 31 H4??H1?, H5? alpha (strong), H5? beta (moderate), H3? (moderate), H2? (very weak), 2?OH (moderate), 3?OH (moderate) H8?H1? (strong), H2? strong), H3? (very weak), H5? beta (strong), 2?OH (very weak), 3?OH (very weak) HMBC: H1??C4 (150.2), C8 (140.2), C2? (73.7), C5? (33.3) H2?C4 (150.2), C6 (156.0) H8?C4 (150.2), C5 (119.4) The dependence of NOE on the inverse of the distance to the sixth power allows for an estimation of the internuclear distance. According to this principle, a NOE between H1? and H4? indicates that they are in syn configuration, since the distance between H1? and H4? is shorter when they are in syn configuration, compared to anti configuration. Similarly, the NOE correlations of H8?H2? and H8?H5? (beta) supports the conclusion that adenine is connected to cyclopentane ring in the desired beta configuration. Normal N 9- product was confirmed by three-bond coupling between H1? and C4 (150.2), C8 (140.2). (S) (S) (R) (S) HO OH F N H N N N NH 2 H H 150.2 140.2 119.4 156.0 152.2 73.7 33.3 HMBC correlationship (R) N N N N NH 2 H H H H H H OH OH F H H NOESY correlationship Figure 19. NOESY and HMBC Correlationship in DMSO-d 6 32 33 3,7-DIDEAZA ARISTEROMYCIN AND NORARISTEROMYCIN Experimental Design and Synthesis 3-Deaza purine nucleosides show potent antiviral activity. For example, 3-deaza Ari and 3-deaza NpcA display significant activity, while their associated toxicity (relative to Ari and NpcA) was greatly reduced. 84 Similarly, 7-deazapurine nucleosides demonstrate a wide range of biological effects. 145 Combining these two structural features to create new modified base analogs of Ari or NpcA with decrease in related toxicity, leads to investigating 3,7-dideaza adenine as a novel modified purine base. The synthesis was planned in a convergent strategy (Scheme 9), where the proper carbocyclic sugar moiety was seen as coupling with 3,7-dideaza-6-chloropurine to give the desired compounds. The carbocyclic sugar was to be derived from the common intermediate 26. The modified purine base was foreseen from commercially available pyrrole-2-carboxaldehyde, by modifying a literature process. 216 Scheme 9. Retrosynthetic Analysis of 3 and 4 HO OH HO N N NH 2 n 3, n=1 3,7-dideaza Ari 4, n=0, 3,7-dideaza Norari O O PGO LG n PG=protecting group LG=leaving group + N N Cl H 6-Cl-3,7-dideazapurine HO OAc H N O H 26 1H-pyrrole-2-carbaldehyde Synthesis of 6-Chloro-3,7-dideazapurine Synthesis of 6-chloro-3,7-deazapurine started from pyrrole-2-carboxaldehyde (Scheme 10). Protecting the ring nitrogen of pyrrole-2-carboxaldehyde gave 40. Aldol condensation of 40 with malonic acid resulted in 41, which spontaneously decarboxylated to 42. Transformation of the carboxylic acid of 42 into mixed anhydride afforded 43, which was further treated with sodium azide to provide 44. A thermally induced Curtius rearrangement 217 of 44, followed by a subsequent Friedel-Crafts like ?acylation? provided 46. Removal of the benzyl protecting group of 46 with sodium metal in liquid ammonia furnished 47. Chlorination of 47 gave 48. 34 Scheme 10. Synthesis of 6-Chloro-3,7-dideazapurine N H O H N O H Bn BnBr NaOH 82% pyrrole-2-carboxaldehyde 40 PhNH 2 malonic acid N H Bn COOH COOH N H Bn COOH 41 42 N H Bn 43 O O O OEt N H Bn O N 3 N H Bn N C O N N OH Bn N N OH H N N Cl H 444546 47 48 ClCO 2 Et TEA heatheat Na, NH 3 (liquid) 70% POCl 3 60% heat NaN 3 96%40% Synthesis of 3,7-Dideaza Aristeromycin The synthesis of 3,7-dideaza Ari began with 26 (Scheme11), which was transformed into 49 in moderate scale (up to 20 g) using a methodology developed in Schneller group. 203 A Michael type 1,4-addition of the lithium salt of tert-butyl methyl ether catalyzed by copper (I) provided 50. Luche reduction of 50 gave 51. Since Mitsunobu coupling of 48 with various carbocyclic pseudosugars failed to yield desired compounds, a classical S N 2 reaction approach was taken into account. To minimize the possible accompanying elimination, triflate was selected as the S N leaving group. Thus, treatment 35 36 of 51 with trifluoromethanesulfonic anhydride in the presence of pyridine in dichloromethane yielded 52. It must be mentioned that the triflate 52 was not stable for long storage at room temperature (it became black). Coupling of triflate 52 with the sodium salt of dideaza base 48 gave 53 in moderate yield. Removal of the isopropylidene protecting group and tert-butyl group of 53 in strong acid conditions afforded 54. Direct amination of 54 with ammonia in methanol failed to yield desired product. Therefore, a two-step, one pot procedure was applied. The chloride in 54 was replaced by hydrazine at high temperature to produce 55. Reduction of 55 with Raney nickel gave 3 as its hydrochloride salt. Scheme 11. Synthesis of 3,7-Dideaza Ari HO OAc 26 OO O 49 OO O O 50 OO O OH 51 OO O OTf 52 OO O N 53 N Cl HO OH HO N N Cl 54 HO OH HO N N NHNH 2 HO OH HO N N NH 2 55 3 Ref.203 t-BuOCH 2 Li CuBr-SMe 2 77% NaBH 4 CeCl 3 ?7H 2 O 83% Tf 2 O Pyr NaH, 48 43% TFA/H 2 O 83% Raney Ni 41% N 2 H 4 ?H 2 O N H N Cl 48: Synthesis of 3,7-Dideaza Noraristeromycin The synthesis of 4 started from 31, as shown in Scheme 12. Transformation of 31 into the corresponding triflate gave 56, which was coupled with the sodium salt of 6-chloro-3,7-dideazapurine to produce 57. Compound 58 was obtained by removal of the 37 38 protecting groups of 57. Using the aforementioned two-step, one pot procedure, 58 was converted to the hydrazine derivative 59, which was, in turn, reduced with Raney nickel to produce 4 as its hydrochloride salt. Scheme 12. Synthesis of 3,7-Dideaza Norari TBSO OO OH 31 TBSO OO OTf TBSO OO N N Cl HO HO OH N N Cl HO HO OH N N NHNH 2 HO HO OH N N NH 2 56 57 5859 4 Tf 2 O, Pyr NaH, 48 46% 1N HCl/MeOH 92% N 2 H 4 ?H 2 O Raney Ni 60% DEVELOPING AN ALTERNATIVE SYNTHESIS OF SINEFUNGIN AND RELATED COMPOUNDS Experimental Design and Synthesis Despite of a number of total syntheses of sinefungin (Figure 15) and related compounds, 175-200 a high yielding and versatile synthetic method remains to be decribed. To explore the preparation and activities of sinefungin and its derivatives, a high-efficiency synthetic pathway was sought in this dissertation research. For this purpose, the structure of sinefungin was separated into several building blocks: the terminal amino acid residue, the 6?-amino moiety, the furanose sugar and the adenine base. A practical and versatile synthesis of sinefungin will require bringing together these building blocks in the proper order. O HO OH N NH 2 H 2 N O OH N N N NH 2 5 1' 2'3' 4' 5' 6' 7' 8' 9' Figure 15. Sinefungin A detailed screening of the literature showed that the amino acid residue can be introduced by means of a Sch?llkopf chiral auxiliary (Figure 20), 218-220 which is an 39 40 O-alkyl ether of a cyclic dipeptide. The stereoselectivity in using this auxiliary comes from the directing effect of the isopropyl group via masking one surface of the intermediate (that is the Sch?llkopf carbanion). N N RO OR R=Me or Et N N RO OR X LG LG X favored disfavored n BuLi X=other functional groups LG=leaving group N N RO OR X H 2 N O OH X Figure 20. Stereoselectivity of Sch?llkopf Auxiliary Since azide is a good synthon for an amino group, 221 it was designed to be the source of the 6?-amino group, which, in turn, can be derived from an alcohol substituent. To build the correct stereochemistry for the 6? position, a stereoselective addition of carbanionic species to an aldehyde was considered. Previous reports from the literature indicated that a Brown asymmetric allylation of an aldehyde would meet this criterion. 222-225 The stereoselectivity of the Brown allylation is achieved by a cyclic six-membered ring transition state mechanism, in which one face of aldehyde is shielded by chiral auxiliary (Figure 21). 225 The sugar moiety of sinefungin can be derived from D-ribose, since it has the desired stereochemistry. The adenine base segment follows from 6-chloropurine. Me H H B Me H H O H R B *L *L + R O H Re face is shielded R OH Figure 21. Stereoselectivity of Brown Allylation With these ideas in mind, a retrosynthetic analysis arose (Scheme 13). In this plan, the furanose sugar segment serves as the scaffold upon which to build the structure. All of the desired functional groups, i.e., the 6?-amino group, the amino acid residue and the purine are connected to this scaffold by the methods just described. Scheme 13. Retrosynthetic Analysis of Sinefungin O HO OH H 2 N NH 2 HO 2 C N N N N NH 2 5 O RO OR X 1 X 2 X 3 X 1 =masked amino acid X 2 =amino synthon X 3 =base moiety R=protecting group FGI O RO OR P 3 O OP 2 OP 1 P 1-3 =protecting groups R=glycol protecting groups O RO OR O OP P=protecting groups R=glycol protecting groups H O OO HO OPMB O HO OH HO OH D-ribose I II III FGI = Functional Group Interconversion 61 41 In previous investigations in the Schneller group (Scheme 14), a methyl ether was selected as the anomeric hydroxyl protecting group, due to its ease of introduction. However, eventual cleavage of the methyl ether under strong acid conditions gave intractable products (Scheme 14). Thus, the anomeric hydroxyl group must be protected by a group that can be removed under mild conditions. To meet this criterion, PMB ether was considered as a protecting group, since its removal could be achieved conveniently by DDQ. Scheme 14. Protection of Anomeric Hydroxyl as Methyl Ether D-ribose O OO OMe N N O O N 3 strong acid intractable products The synthetic pathway towards sinefungin started from D-ribose (Scheme 15). The vicinal hydroxyl groups were protected in the isopropylidene framework to give 60. The anomeric hydroxyl of 60 was protected with PMB group to produce 61. Dimethyl sulfoxide (DMSO) oxidation of 61 formed 62, which was converted to 63 via a Wittig reaction. Hydroboration and oxidation of 63 led to 64, which was further treated with PCC to yield 65. Brown allylation of 65 gave 66. The stereochemistry of 66 was tentatively assigned according to methodology established in the literature: 222-225 when (+)-B-methoxydiisopinocamphenylborane ((+)-Ipc 2 BOMe) is used as chiral auxiliary, the Re face of the aldehyde is masked (the nucleophile attacks the aldehyde from the Si face). Mesylation of 66, followed by treatment with sodium azide, provided 67. Double bond cleavage of 67 was performed by a combination of osmium tetroxide and sodium 42 metaperiodate. However, this method resulted in a low yield of the desired compound 68, together with 69, 70 and intractable materials. Scheme 15. Synthesis of Compound 68 O OO OH HO O HO OH OH HO D-ribose 60 O OO OPMB HO 61 O OO OPMB O 62 O OO OPMB 63 O OO OPMB 64 HO O OO OPMB H O O OO OPMB OH O OO OPMB N 3 O OO OPMB HO N 3 6566 67 68 O OO OPMB O N 3 HO O OO OPMB HO N 3 HO 69 70 + + NaH, PMBCl 66% 1)9-BBNPCC, 93% 1)(+)Ipc 2 BOMe AllylMgBr 1)NaIO 4 , OsO 4 Acetone pTSA, 69% DMSO SO 3 ?Pyr DIPEA Ph 3 PMeBr, t BuOK,60% 2)NaOH, H 2 O 2 88% 2)NaOH, H 2 O 2 74% 1)TsCl, TEA, DABCO (cat.) 2)NaN 3 , 89% 2)NaBH 4 or 1)NMO, OsO 4 2)NaIO 4 3)NaBH 4 , 89% A possible pathway to this complication was analyzed via Scheme 16. Compound 67 was converted to 71 by osmium tetroxide. Cleavage of the vicinal hydroxyls of 71 with sodium metaperiodate gave intermediate 72, which was reduced by NaBH 4 to produce desired compound 68. The intermediate 72 also tautomerizes to 73. Since 73 bears a double bond, this can be oxidized to give 74. Compound 74 underwent a dehydration 43 reaction to yield 69. Also, 74 can be cleaved by sodium metaperiodate to give 75, which can tautomerize to 76 and undergo further reactions, leading to intractable products, making the isolation of 68 difficult. Similar results have been reported in the literature. 226 To circumvent this complication, a three step procedure was used to prepare the desired 68. Hence, compound 67 was converted to 71 by a combination of osmium tetroxide and 4-methylmorpholine N-oxide (NMO). The application of the Sharpless dihydroxylation protocol (AD-mix-alpha or AD-mix-beta) also gave 71. Treatment of 71 with sodium metaperiodate gave 72, which was reduced with sodium borohydride to provide 68. Scheme 16. Proposed Side Reactions in Double Bond Cleavage by OsO 4 /NaIO 4 O OO OPMB N 3 67 OsO 4 O OO OPMB N 3 OH HO O OO OPMB O N 3 O OO OP MB HO N 3 68 O OO OPMB HO N 3 O OO OPMB HO N 3 HO OH O OO OPMB O N 3 HO 69 71 72 7374 O OO OPMB HO N 3 70 O OO OPMB N 3 O O OO OPMB N 3 OH 75 76 intractable products NaIO 4 NaBH 4 OsO 4 NaBH 4 OsO 4 NaIO 4 HO To install the Sch?llkopf chiral auxiliary, the hydroxyl group of 68 was transformed into a good leaving group (Scheme 17). Thus, treatment of 68 with imidazole, iodine and 44 triphenylphosphine (TPP) gave 77 in moderate yield (30%-60%). The low yield may be due to involvement of the azido group in an undesired side mechanism, since it is well known that azido group can be reduced to an amino group by a phosphine species at room temperature. 221 Consequently, another leaving group was sought. Previous reports 220 indicated that the tosylate would serve this purpose. Converting the hydroxyl of 68 into the tosylate gave 78 in high yield (80%-95%). Compound 77 and 78 were subjected to coupling with the Sch?llkopf chiral auxiliary. However, the desired compound 79 was not obtained. Careful NMR analysis of the products showed that the azido group and the leaving groups (iodo, tosyl) disappeared, while the desired dihydropyrazine moiety was not present. The starting materials were not recovered. This observation indicated that azido group was not compatible with organolithium species in this case. Scheme 17. Attempted Synthesis of Compound 79 O OO OPMB HO N 3 68 O OO OPMB X N 3 X=I, 77 X=OTs, 78 O OO OPMB N N O O N 3 79 Ph 3 P, I 2 , imidazole,60% (77) Schollkopf auxiliary n-BuLi or TsCl, TEA DABCO (cat.) 79% (78) N N O O Schollkopf auxiliary: To avoid this, introduction of the azido functionality was considered after installation of the Sch?llkopf auxiliary (Scheme 18). For this reason, the secondary hydroxyl in 66 45 was protected as a silyl ether to provide 80. Conversion of the double bond of 80 into a hydroxyl yielded 81. Compound 81 was transformed into tosylate 82, which was coupled with the Sch?llkopf auxiliary to give 83. Removal of the silyl group of 83 by TBAF led to 84, which was further converted to 85 by another tosylation. Introduction of the azido group by sodium azide gave 79 in high yield. Scheme 18. Synthesis of Compound 79 O OO OPMB OH 66 O OO OPMB OTBS 80 O OO OPMB OTBS X X=OH, 81 X=OTs, 82 O OO OPMB X N N O O X=OTBS, 83 X=OH, 84 X=OTs, 85 O OO OPMB N 3 N N O O 79 TBSCl, imidazole 93% 1)AD-mix-beta 2)NaIO 4 3)NaBH 4 68% TsCl, TEA DABCO(cat.) 90% Schollkopf auxiliary n-BuLi, 84% TsCl, TEA DABCO(cat.) 93% TBAF, 89% NaN 3, 78% Schollkopf auxiliary: N N O O Upon initial effort, removal of PMB ether was performed using DDQ oxidation (Scheme 19), however, the desired compound 86 was obtained only in low yield (usually 10-20%). In addition, purification of 86 was complicated with unknown side products. Other methods, such as oxidation with cerium ammonium nitrate (CAN), catalytic hydrogenation or strong acid hydrolysis did not give the expected results. Subjecting the 46 Sch?llkopf auxiliary to DDQ or CAN also gave intractable products. These facts pointed to the necessity of removal of the PMB group prior to introduction of the Sch?llkopf auxiliary. Scheme 19. Unexpected Result of PMB Ether Cleavage O OO OPMB N N O O N 3 79 O OO OH N N O O N 3 86 DDQ, 10% yield CAN Pd/C H 2 N N O O (R)-3,6-diethoxy-2-isopropyl-2,5-dihydropyrazine DDQ or CAN intractable products A new synthetic pathway, in which the PMB was removed before the Sch?llkopf auxiliary installation, was sought (Scheme 20). In this scheme, the base moiety was added at an earlier stage. Thus, the removal of PMB from 80 produced 87. 6-Chloropurine was introduced by a two-step one-pot procedure (converting the anomeric hydroxyl of 87 into corresponding chloride followed by an S N 2 reaction with sodium salt of 6-chloropurine) to provide 88. Double bond cleavage followed by Luche reduction converted 88 into 89, however, transformation of 89 into 90 was not successful. Instead of giving the desired compound 90, the reaction formed water soluble products. An attempt to couple this with the Sch?llkopf auxiliary failed to form expected compound. 47 48 This indicated that 6-chloropurine moiety is incompatible with a good leaving group within the same molecule. Accordingly, introducing of 6-chloropurine as base moiety must be conducted after installation of Sch?llkopf auxiliary. Scheme 20. Attempted Introduction of Base at Early Stage O OO OPMB OTBS 80 O OO X OTBS X=OH, 87 X=6-Cl-purin-9-yl, 88 O OO N OTBS OH N N N Cl 89 O OO N OTBS TsO N N N Cl 90 DDQ, 76% 1)AD-mix-beta TsCl, TEA DABCO (cat.) 1)HMPT,CCl4 2)NaH 6-Chloropurine 60% 2)NaIO 4 3)NaBH 4 , 87% Based on the results described above, the best order of introducing functional groups incorporation was concluded to be (1) the Sch?llkopf auxiliary, (2) the azido group and (3) the purine base moiety. For this reason, a new synthetic cascade was designed (Scheme 21). Scheme 21 Attempted Synthesis of Compound 93 O OO OPMB OTBS TsO 82 O OO OH OTBS TsO 91 O OO OPiv OTBS TsO 92 O OO OPiv OTBS N N O O DDQ, 75% PivCl, TEA DABCO (cat.) 70% 93 Schollkopf auxiliary n-BuLi Schollkopf auxiliary: N N O O Removal of the PMB group of 82 produced 91. The anomeric hydroxyl group was protected as its pivalate ester to give 92. Installation of the Sch?llkopf auxiliary on 92 failed to provide 93, indicating that the pivalate is not a suitable protective group for this purpose. An alternative pathway to circumvent this complication was planned (Scheme 22). Compound 81 was converted into 94 by desilylation and tosylation. The PMB ether of 94 was removed and the anomeric hydroxyl was reprotected with tert-butyldimethylsilyl (TBS) group to afford 95. Introduction of the Sch?llkopf auxiliary onto 95 gave 96, which was further transformed into 86 by installation of azido group and desilylation. Compound 86 was converted to 97 by installation of base moiety. 49 Scheme 22. Synthesis of Compound 97 O OO OPMB OTBS HO 81 O OO OPMB OTs TsO 94 O OO OTBS OTs TsO O OO OTBS OTs N N O O O OO OH N N O O N 3 95 96 86 1)TBAF 2)TsCl, TEA DABCO (cat.),70% 1)DDQ 2)TBSCl imidazole 56% 1)NaN 3 2)TBAF 83% O OO N N 3 N N O O N N N Cl 97 N N O O Schollkopf auxiliary n-BuLi, 62% 1)HMPT, CCl 4 2)NaH, 6-Chloropurine 60% Schollkopf auxiliary: Transformation of the 6-chloropurine moiety of 97 into the adenine moiety failed to give desired product. Thus, Ad(Boc) 2 was introduced as base building block on 87 (Scheme 23) to provide 98. An attempt to convert the azido group of 98 under catalytic 50 51 hydrogenation conditions failed to give desired compound 100. Consequently, removal of protecting groups at earlier stage was considered. Liberation of the amino acid residue and removal of the isopropylidene of 98 gave 99. However, hydrogenation of 99 did not afford desired compound. Scheme 23. Synthesis of Azidosinefungin O OO OH N 3 N N O O 87 O OO N N 3 N N O O N N N N(Boc) 2 98 O OO N NH 2 N N O O N N N N(Boc) 2 100 O HO OH N N 3 H 2 N HO O N N N NH 2 O HO OH N NH 2 H 2 N HO O N N N NH 2 99 5 1)HMPT, CCl 4 2)NaH, Ad(Boc) 2 60% Pd(OH) 2 /C H 2 (50 psi) 1)TFA/H 2 O 2)K 2 CO 3 , MeOH/H 2 O 78% Pd(OH) 2 /C cyclohexene 52 SYNTHESIS OF OXA-ADOHCY AND RELATED COMPOUNDS Experimental Design and Synthesis Since AdoHcy is a potent bio-feedback inhibitor of AdoMet dependent methyltransferases, it is interesting to synthesize compounds resembling AdoHcy to mimic AdoHcy, which may serve as methyltranferases inhibitors. For this purpose, oxa-AdoHcy in which the side chain sulfur was replaced by an oxygen was investigated. Two closely related compounds, the Ari and NpcA versions of oxa-AdoHcy (Ari-oxa-AdoHcy and NpcA-oxa-AdoHcy) were also in the scope of study. Based on chemistry developed in synthesis of sinefungin, a retrosynthetic analysis was established (Scheme 24). Scheme 24. Retrosynthetic Analysis of Compounds 6, 7 and 8 X HO OH O H 2 N O EtO N N N N NH 2 X OO OH O N N O O X OO OPG O LG LG=Leaving Group PG=Protective Group X OO OPG O X OO OPG HO X=O, 6 X=CH 2 , 7 X=CH-, 8 O OO OPMB HO OO OTBS HO OO OTBS HO 61 OO OO OH TBSO 118 111 119 49 O Synthesis of Oxa-AdoHcy The synthesis of oxa-AdoHcy started from compound 61 (Scheme 25). Allylation of primary hydroxyl group of 61 afforded 101. Double bond cleavage followed by reduction converted 101 into 102. Compound 102 was tosylated to give 103. Removal of the PMB group of 103 led to 104. The anomeric hydroxyl of 104 was protected as a silyl ether to yield 105, which was coupled with the Sch?llkopf auxiliary to provide 106. Desilylation of 106 afforded 107. Installation of the 6-chloropurine was achieved by a 53 two-step one pot procedure which converted 107 into 108. However, amination of 108 at elevated temperature gave intractable products. Thus, Ad(Boc) 2 was introduced as base moiety on 107 to give 109. Global deprotection of 109 finished the synthesis of oxa-AdoHcy 6. Scheme 25. Synthesis of Compound 6 O OO OPMB HO 61 O OO OPMB O 101 O OO OY O X X=OH, Y=PMB, 102 X=OTs, Y=PMB, 103 X=OTs, Y=H, 104 X=OTs, Y=TBS, 105 O OO X O N N O O X=OTBS, 106 X=OH, 107 O OO N O N N O O N N N Cl O HO OH N O H 2 N HO O N N N NH 2 108 6 NaH, AllylBr 1)NMO, OsO 4 79% 2)NaIO 4 3)NaBH 4 , 75% TsCl, TEA DABCO (cat.), 82% DDQ, 77% TBSCl, imidazole 66% TBAF, 60% 1)HMPT, CCl 4 2)NaH 6-chloropurine 28% O OO N O N N O O N N N N(Boc) 2 1)HMPT, CCl 4 2)NaH,Ad(Boc) 2 25% 109 1)TFA/H 2 O 2)K 2 CO 3 , 61% Schollkopf auxiliary n-BuLi 68% Synthesis of Ari-oxa-AdoHcy The synthesis begins with 49. After a Michael addition, a subsequent Luche reduction followed by protecting the hydroxyl as a silyl ether gave 110. Oxidative cleavage of the double bond of 110 followed by reduction afforded 111, which was allylated at the primary hydroxyl to provide 112. Compound 112 was converted into 113, which was transformed into tosylate 114. Coupling of 114 with the Sch?llkopf auxiliary gave 115. Removal of the silyl protecting group of 115 afforded 116. Installation of the base moiety 54 55 was achieved by a Mitsunobu reaction on 116 to yield 117. Global deprotection of 117 gave 7. Scheme 26. Synthesis of Ari-oxa-AdoHcy OO OO OTBS OO OTBS HO OO OTBS O OO OTBS O X X=OH, 113 X=OTs, 114 OO X O N N O O X=OTBS, 115 X=OH, 116 OO O N N O O N N N N N(Boc) 2 HO OH O H 2 N N N N N NH 2 O HO 49 110 111 112 1177 1)VinylMgBr CuBr-SMe 2 TMSCl 2)NaBH 4 CeCl 3 -7H 2 O 3)TBSCl imidazole, 43% 1)NMO, OsO 4 2)NaIO 4 3)NaBH 4 CeCl 3 -7H 2 O 84% NaH AllyBr 83% Schollkopf auxiliary n-BuLi 89% Ph 3 P, DIAD Ad(Boc) 2 57% 1)TFA/H 2 O 2)K 2 CO 3 MeOH/H 2 O 46% O 1)NMO, OsO 4 2)NaIO 4 3)NaBH 4 CeCl 3 -7H 2 O 86% TsCl, TEA DABCO (cat.) 83% TBAF, 43% Attempted Synthesis of NpcA-oxa-AdoHcy Starting from 60, compound 118 was prepared according to the literature (Scheme 27). 227 Protection of the secondary hydroxyl as a TBS ether and selectively removal of the primary silyl protecting group afforded 119. Allylation of 119 provided 120, which was converted to a primary alcohol 121. Tosylation of 121 gave 122. Scheme 27. Attempted Synthesis of Compound 8 O OO OH HO OO TBSO OH OO HO OTBS OO O OTBS 60 119 120 OO O OTBS X X=OH,121 X=OTs, 122 OO O OX N N O O X=TBS, 123 X=H, 124 OO O N N O O N N N N N(Boc) 2 HO OH O H 2 N N N N N NH 2 HO O 8 Ref. 227 118 125 1)TBSCl, imidazole 2)TBAF, -40 o C 63% NaH AllylBr 85% Schollkopf auxiliary n-BuLi DIAD, Ph 3 P Ad(Boc) 2 1)AD-mix-beta 2)NaIO 4 3)NaBH 4 96% TsCl DABCO (cat.) TEA 95% TBAF 1)TFA/H 2 O 2)K 2 CO 3 , MeOH/H 2 O 56 BIOLOGICAL RESULTS The synthesized compounds 1-4 were evaluated against a wide variety of viruses (Table 1) to determine their antiviral activity. The detailed results were shown in Tables 2-19. N HO OH F N N N NH 2 1 N HO OH N N N NH 2 2 F N HO OH HO N NH 2 3 HO N HO OH N NH 2 4 Figure 22. Compounds for Antiviral Evaluation 57 58 Table 1. Viruses Used for Bioassay Virus Family Individual Virus Adenoviridae Adenovirus Arenaviridae Pichinde Virus Bunyaviridae Punta Toro Virus Coronaviridae Human Coronavirus, Severe Acute Respiratory Syndrome (SARS) virus Filoviridae Ebola Virus Flaviviridae Hepatitis C Virus (HCV), West Nile Virus, Yellow Fever Virus Hepadnaviridae Hepatitis B Virus (HBV) Herpesviridae Epstein-Barr Virus (EBV), Human Cytomegalovirus (HCMV), Varicella-Zoster Virus (VZV), Herpes Simplex Virus (HSV) Orthomyxoviridae Influenza A Virus, Influenza B Virus Paramyxoviridae Parainfluenza Virus, Measles Virus, Respiratory Syncytial Virus (RSV) Piconoviridae Rhinovirus Poxviridae Cowpox Virus, Vaccinia Virus Reoviridae Reovirus Rhabdoviridae Vesicular Stomatitis Virus Togaviridae Venezuelan Equine Encephalitis Virus (VEE), Sindbis Virus Table 2. Antiviral Activity Towards HCV a Compound Activity (% Inh Virus Control ) Cytotoxicity (% Cell Control) SI 1 0.0 104.3 <1 2 0.0 109.8 <1 3 N.D. N.D. 4 N.D. N.D. a Assay: HCV RNA replicon; Cell type: Huh7ET; Assay type: Single dose (primary); High test concentration: 20 ?M. N.D.: not determined 59 Table 3. Antiviral Activity Towards Influenza in MDCK Cell Cultures EC 50 b Influenza A H1N1 subtype Influenza A H3N2 subtype Influenza B Compound Minimum cytotoxic concentration a Visual CPE score MTS Visual CPE score MTS Visual CPE score MTS 1 100 ?M. N.A. N.A. N.A. N.A. N.A. N.A. 2 100 ?M. N.A. N.A. N.A. N.A. N.A. N.A. 3 N.D. N.A. N.A. N.A. N.A. N.A. N.A. 4 N.D. N.A. N.A. N.A. N.A. N.A. N.A. a Minimum compound concentration that causes a microscopically detectable alteration of cell morphology b 50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay. MDCK cells: Madin Darby canine kidney cells N.A.: not active at the highest concentration tested, or at subtoxic concentrations. Table 4. Anti-Feline Corona Virus (FIPV) Activity and Cytotoxicity in CRFK Cell Cultures Compound CC 50 a (?M.) EC 50 b (?M.) 1 >100 >100 2 >100 >100 3 >100 >100 4 >100 >100 a 50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay b 50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by 60 measuring the cell viability with the colorimetric formazan-based MTS assay. CRFK cells: Crandell-Rees Feline Kidney cells Table 5. Activity and Cytotoxicity Towards Vesicular Stomatitis Virus, Coxsackie Virus B4 and Respiratory Syncytial Virus in HeLa Cell Cultures EC 50 (?M) Compound Minimum cytotoxic concentration (?M) Vesicular stomatitis virus Coxsackie virus B4 Respiratory syncytial virus 1 >100 100 >100 N.D. 2 >100 >100 >100 N.D. 3 >200 200 >200 >200 4 >200 >200 120 >200 Table 6. Activity and Cytotoxicity Towards Para-influenza 3 Virus, Reovirus-1, Sindbis Virus, Coxsackie Virus B4 and Punta Toro Virus in Vero Cell Cultures EC 50 (?M) Compou nd Minimum cytotoxic concentratio n (?M) Para-influenza 3 Virus Reovirus -1 Sindbis virus Coxsackie virus B4 Punta toro virus 1 >100 >100 >100 >100 >100 >100 2 >100 >100 >100 >100 >100 >100 3 >200 >200 >200 >200 >200 >200 4 >200 >200 >200 >200 >200 >200 61 Table 7. Activity and Cytotoxicity Towards Herpes Simplex Virus-1 (KOS), Herpes Simplex Virus-2 (G), Vaccina Virus, Vesicular Stomatitis Virus, Herpes Simplex Virus-1 TK - KOS ACV + in IL Cell Cultures EC 50 (?M) compound Minimum cytotoxic concentration (?M) Herpes simplex virus-1 (KOS) Herpes simplex virus-2 (G) Vaccina virus Vesicular stomatitis virus Herpes simplex virus-1 TK - KOS ACV + 1 >100 >100 >100 >100 >100 >100 2 >100 >100 >100 >100 >100 >100 3 >200 >200 >200 >200 >200 >200 4 >200 >200 >200 >200 >200 >200 Table 8. Activity and Cytotoxicity Towards Cowpox and Vaccinia Viruses a compound virus EC 50 EC 90 CC 50 SI CDV EC 50 CDV EC 90 Cowpox >300 >300 >300 0 7.3 58 1 Vaccinia >300 >300 >300 0 7.6 12 Cowpox >300 >300 >300 0 7.3 58 2 Vaccinia >300 >300 >300 0 7.6 12.1 Cowpox >300 >300 >300 0 N.D. N.D. 3 Vaccinia >300 >300 >300 0 N.D. N.D. Cowpox >300 >300 >300 0 N.D. N.D. 4 Vaccinia >300 >300 >300 0 N.D. N.D. a Assay: CPE; Cell Line: HFF Cells; Drug Unit: ?M Table 9. Activity and Cytotoxicity Towards EBV a Compound EC 50 EC 90 CC 50 SI ACV EC 50 1 3.3 14.4 24.1 6.5 2.4 2 17.5 >20 51.8 3 2.4 3 N.D. N.D. N.D. 4 N.D. N.D. N.D. a Assay: DNA Hybridation; Cell Line: Akata Cells; Drug Unit: ?M 62 Table 10. Activity and Cytotoxicity Towards West Nile Virus a Compound Assay EC 50 IC 50 SI Neutral Red >100 >100 0 1 Visual >100 >100 0 Neutral Red >100 >100 0 2 Visual >100 >100 0 Neutral Red N.D. N.D. 3 Visual N.D. N.D. Neutral Red N.D. N.D. 4 Visual N.D. N.D. a Vehicle: DMSO; Cell Line: Vero 76; Drug Unit: ?g/mL; Virus Strain: New York Isolate Table 11. Activity and Cytotoxicity Towards Parainfluenza Virus a Compound Assay EC 50 IC 50 SI Neutral Red >100 >100 0 1 Visual >81 81 0 Neutral Red >100 >100 0 2 Visual >100 >100 0 Neutral Red >100 >100 0 3 Visual >100 >100 0 Neutral Red >100 >100 0 4 Visual >100 >100 0 a Vehicle: DMSO; Cell Line: MA-104; Drug Unit: ?g/mL; Virus Strain: 14702 Table 12. Activity and Cytotoxicity Towards Adenovirus a Compound Assay EC 50 IC 50 SI Neutral Red >58 58 0 1 Visual >100 >100 0 Neutral Red >100 >100 0 2 Visual >100 >100 0 Neutral Red N.D. N.D. 3 Visual N.D. N.D. Neutral Red N.D. N.D. 4 Visual N.D. N.D. a Vehicle: DMSO; Cell Line: A-549; Drug Unit: ?g/mL; Virus Strain: 65089/Chicago 63 Table 13. Activity and Cytotoxicity Towards Measles a Compound Assay EC 50 IC 50 SI Neutral Red 2.8 >100 >35 1 Visual 13 >100 >7.8 Neutral Red 1.2 21 18 2 Visual 14 36 2.5 Neutral Red N.D. N.D. 3 Visual N.D. N.D. Neutral Red N.D. N.D. 4 Visual N.D. N.D. a Vehicle: DMSO; Cell Line: CV-1; Drug Unit: ?g/mL; Virus Strain: MO6 Table 14. Activity and Cytotoxicity Towards Respiratory Syncytial A Virus a Compound Assay EC 50 IC 50 SI Neutral Red >100 >100 0 1 Visual >100 >100 0 Neutral Red >100 >100 0 2 Visual >100 >100 0 Neutral Red >100 >100 0 3 Visual >100 >100 0 Neutral Red >100 >100 0 4 Visual >100 >100 0 a Vehicle: DMSO; Cell Line: MA-104; Drug Unit: ?g/mL; Virus Strain: A2 Table 15. Activity and Cytotoxicity Towards Rhinovirus a Compound Assay EC 50 IC 50 SI Neutral Red >100 >100 0 1 Visual >100 >100 0 Neutral Red >100 >100 0 2 Visual >100 >100 0 Neutral Red N.D. N.D. 3 Visual N.D. N.D. Neutral Red N.D. N.D. 4 Visual N.D. N.D. a Vehicle: DMSO; Cell Line: Hela Ohio-1; Drug Unit: ?g/mL; Virus Strain: HGP 64 Table 16. Activity and Cytotoxicity Towards VZV a Compound EC 50 EC 90 CC 50 SI 1 N.D. N.D. N.D. 2 N.D. N.D. N.D. 3 >60 >60 214 <3.6 4 >300 >300 >300 0 a Assay: CPE; Cell Line: HFF Cells; Drug Unit: ?M Table 17. Activity and Cytotoxicity Towards HCMV a Compound EC 50 EC 90 CC 50 SI 1 N.D. N.D. N.D. 2 N.D. N.D. N.D. 3 >60 >60 202 <3.4 4 >60 >60 269 <4.5 a Assay: CPE; Cell Line: HFF Cells; Drug Unit: ?M Table 18. Activity and Cytotoxicity Towards HSV-1 and HSV-2 in HFF Cells a EC 50 EC 90 CC 50 SI Compou nd HSV-1 HSV-2 HSV-1 HSV-2 HSV-1 HSV-2 HSV-1 HSV-2 1 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 2 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 3 >60 >60 >60 >60 227 227 <3.8 <3.8 4 >300 >300 >300 >300 >300 >300 0 0 a Assay: CPE; Cell Line: HFF Cells; Drug Unit: ?M Table 19. Activity and Cytotoxicity Towards Human Corona (SARS) Virus Compound EC 50 (?M) CC 50 (?M) 1 N.D. N.D. 2 N.D. N.D. 3 >100 >100 4 >100 >100 65 Table 20. Activity and Cytotoxicity Towards HBV a Compound EC 50 EC 90 CC 50 1 N.D. N.D. N.D. 2 N.D. N.D. N.D. 3 >10 >10 >300 4 >10 >10 >300 a Assay: VIR; Drug Unit: ?M Compound 1 and 2 show moderate activity against measles virus. Against most of tested viruses, compounds 1-4 did not show any activity. The bioassay data for 5, 6, 7 and 8 will be forthcoming as part of future study in the Schneller group. 66 CONCLUSIONS S-Adenosylmethionine (AdoMet)-dependent methylation reactions are essential for maturation of mRNA (including host and viral mRNA). These reactions are regulated by S-adenosylhomocysteine (AdoHcy), the product of AdoMet-dependent methylation, via a biofeedback inhibition mechanism. AdoHcy is ?hydrolyzed? by AdoHcy hydrolase into adenosine and homocysteine. By inhibiting the AdoHcy hydrolase, the cellular AdoHcy level is elevated, which, in turn, inhibits the biomethylation, including 5?-capping of mRNA. Aristeromycin is a naturally occurring potent AdoHcy hydrolase inhibitor and shows significant antiviral activity. However, its clinical potential is limited by its toxicity, which is associated with phosphorylation at its 5?-hydroxyl. To avoid or decrease the tendency of phosphorylation at the 5?-hydroxyl center while retaining the aristeromycin-based antiviral activity, two strategies were sought: (1) replacing the 5?-hydroxyl of aristreomycin (and the 4?-hydroxyl of noraristeromycin) with a substituent incapable of phosphorylation and (2) using a modified purine (3,7-dideazapurine) as the base moiety since some deazapurine nucleosides are not phosphorylated by the cellular kinases. Compounds 1 and 2 were designed according to the first strategy. Their synthesis was achieved by a convergent approach in which the desired carbocyclic pseudo-sugar moieties containing fluoride were coupled with the 67 purine precursors (6-chloropurine and 6-di-(tert-butoxylcarbonyl)aminopurine) under the Mitsunobu conditions. The synthetic design for compounds 3 and 4 occurred by the second strategy. The key steps in these synthesis were S N 2 reactions between the sodium salt of 3,7-dideaza-6-chloropurine in N,N-dimethylformamide (DMF) and the triflates (prepared in situ) of corresponding carbocyclic units. Compounds 1 and 2 were evaluated against a variety of viruses. They showed moderate activity against measles, but lacked antiviral activity against the other viruses tested. No cytotoxicity was observed. This indicated that the replacement of the 5?-hydroxyl of aristeromycin and 4?-hydroxyl of 5'-noraristeromycin with fluoride abolish the undesired phosphorylation (reduced cytotoxicity) at the expense of losing antiviral activity. Thus, 5?-hydroxyl of aristeromycin and the 4?-hydroxyl of 5'-noraristeromycin are necessary for activity and, likely, the inhibition of AdoHcy hydrolase. Compounds 3 and 4 exhibited no significant activity against all viruses tested. They also showed no cytotoxicty to host cells. This suggested that the 3,7-dideazapurine carbocyclic nucleosides were not inhibiting AdoHcy hydrolase. An alternative strategy to aristeromycin analogs for the inhibition of biomethylations is to design AdoHcy analogs, since AdoHcy is a natural methyltransferases inhibitor. Sinefungin is a naturally occurring AdoHcy/AdoMet analog that shows significant biological activity, including antiviral, antimalarial, antifungal, antibacterial, and antitumor effects. However, the potential of sinefungin is limited by its toxicity. To explore structural variations and corresponding activity of AdoHcy/AdoMet analogs based on sinefungin, chemistry towards compounds 5, 6, 7 and 8 was investigated. A convenient, diverse and high yield strategy to these analogs was explored. In this 68 direction, (1) the stereochemistry at the 6? position of sinefungin (5) was built by a Brown allylation procedure, (2) the terminal amino acid moieties of 5-8 were introduced through the Sch?llkopf auxiliary and (3) the 6-di-(tert-butoxylcarbonyl)aminopurine served as the purine precursor for the synthesis of theses analogs. EXPERIMENTAL Materials and Methods Melting points were recorded on a Meltemp II melting point apparatus and are uncorrected. 1 H and 13 C NMR were performed on Bruker Avance 250 MHz or 400 MHz spectrometers. Two dimensional NMR experiments were conducted on the Bruker Avance 400 MHz spectrometer. NMR spectra were reported in ? relative to internal standard (TMS, 0.00) or solvent (DMSO-d 6, 2.50; D 2 O, 4.87). The spin multiplicities are shown by the symbols s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Elemental analyses were performed by the Atlantic Microlabs, Atlanta, GA. Reactions were monitored by silica thin layer chromatography (TLC, Whatman ? TLC K6F plates). Flash columns were performed on silica (Silicycle ? Siliaflash ? F60) columns. 1-Methoxy-2,3-(cyclopentylidenedioxy)-4-hydroxymethyl Tetrahydrofuran (9). D-ribose (100g, 0.667 mol), cyclopentanone (200 mL), MeOH (300 mL) and trimethylorthoformate (200 mL) were added to a 1 L flask. H 2 SO 4 (3.0 mL) was also added. The mixture was stirred at room temperature for 2 days. Ammonia hydroxide (29.6%) was added to neutralize the mixture. The solvent was removed under reduced pressure. The residue was dissolved in EtOAc. The organic layer was washed with brine, dried over Na 2 SO 4 , and concentrated to give 9 as yellow oil (122 g, 79.7%). 1 H NMR (250 MHz, CDCl 3 ), ? 4.98 (s, 1H), 4.77 (d, J=6.0 Hz, 1H), 4.53 (d, J=6.0 Hz, 1H), 69 4.44 (t, J=2.8 Hz, 1H), 3.67 (m, 2H), 3.44 (s, 3H), 3.29 (dd, J1=9.7 Hz, J2=3.5 Hz, 1H), 1.93 (m, 2H), 1.66 (m, 6H). 13 C NMR (62 MHz, CDCl 3 ), ? 121.8, 109.8, 88.2, 85.6, 81.5, 64.1, 55.6, 35.8, 35.7, 23.8, 23.3. Anal. Calcd for C 11 H 18 O 5 : C, 57.38; H, 7.88; Found: C, 57.17; H, 7.99. 1-Methoxy-2,3-(cyclopentylidenedioxy)-4-iodomethyl Tetrahydrofuran (10). 9 (122 g, 0.529 mol) was dissolved in MeCN/toluene (1/1, 500 mL). Imidazole (60.0 g, 0.999 mol), triphenylphosphine (TPP) (188 g, 0.701 mol) was added. I 2 was added in portions until the solution turned black. The solution was stirred at room temperature for 2 hours. H 2 O (300 mL), sodium thiosulfate (10 g) were added. The organic layer was separated, dried over sodium sulfate, concentrated, and purified with column chromatography (Hex/EtOAc=5/1). The product 10 was isolated as colorless oil (161 g, 89.6%). 1 H NMR (250 MHz, CDCl 3 ), ? 5.06 (s, 1H), 4.72 (d, J=0.7Hz, 1H), 4.69 (d, J=0.7 Hz, 1H), 4.44 (m, 1H), 3.37 (s, 3H), 3.28 (m, 1H), 3.16 (m, 1H), 1.90 (m, 2H), 1.67 (m, 6H). 13 C NMR (62 MHz, CDCl 3 ), ? 122.1, 109.4, 87.0, 85.1, 82.7, 55.2, 35.8, 35.7, 23.6, 23.2, 6.7. Anal. Calcd for C 11 H 17 IO 4 : C, 38.84; H, 5.04; Found: C, 39.07; H, 5.05. 2,3-(Cyclopentylidenedioxy)-pent-4-enal (11). 10 (24.8 g, 72.9 mmol) was dissolved in ether. n-BuLi (38.0 mL, 2.5M in hexanes, 95.0 mmol) was added in portions over 15 minutes at ?78 o C. The solution was stirred at this temperature for 2 hours. NH 4 Cl (10 g) was added after the reaction mixture was warmed to ?40 o C. H 2 O (100 mL) was added. The mixture was extracted with ether (3?100 mL). The organic layer was dried over Na 2 SO 4 , concentrated under reduced pressure to give crude 11 with solvent (14.0 g, ?105%?), which was used immediately without further purification. 1 H NMR (250 MHz, CDCl 3 ), ? 9.54 (d, J=3.2 Hz, 1H), 5.7 (m, 1H), 5.3-5.5 (m, 2H), 4.75 (m, 1H), 70 71 4.34 (m, 1H), 2.09 (m, 2H), 1.75 (m, 6H). 13 C NMR (62 MHz, CDCl 3 ), ? 200.9, 131.3, 121.2, 120.0, 81.9, 79.4, 36.9, 36.8, 24.1, 23.3. 2,3-(Cyclopentylidenedioxy)-hepta-1,6-dien-3-ol (12). 11 (14.0 g, crude product) was dissolved in DCM. At ?78 o C, vinylmagnesium bromide (120 mL, 1M in THF, 120 mmol) was added. The mixture was warmed to 0 o C. Saturated NH 4 Cl (40 mL) was added. The organic layer was separated, dried over sodium sulfate, and concentrated using a rotavapor (bath temperature 10 o C). The residue was purified with silica gel column (Hex/EtOAc=5/1) to give 12 as colorless oil (mixture of two diastereomers, 12.9 g, 84.8% from 10). 1 H NMR (250 MHz, CDCl 3 ), ? 6.13 (m, 1H), 5.79 (m, 1H), 5.20-5.59 (m, 4H), 4.48-4.62 (m, 1H), 4.2 (m, 1H), 4.02 (m, 1H), 2.01 (m, 2H), 1.65 (m, 6H). 13 C NMR (62 MHz, CDCl 3 ), ? 137.7, 136.9, 134.0, 133.9, 119.8, 119.0, 118.7, 117.2, 116.6, 80.7, 80.6, 79.0, 78.7, 71.3, 70.9, 37.1, 37.0, 36.9, 36.6, 24.2, 24.1, 23.4, 23.3. 2,3-(Cyclopentylidenedioxy)-cyclopent-2-enone (14). 12 (1.4 g, 6.6 mmol) was dissolved in dry DCM (100 mL). N 2 was bubbled to remove O 2 for 10 minutes. Grubbs catalyst (5.3 mg, 0.0060 mmol) was added. The solution was stirred at room temperature for 12 hours. Dimethyl sulfoxide (DMSO) (5.0 mL) was added. Diisopropylethylamine (DIPEA) (2.3 mL, 13.3 mmol) was added. The solution was cooled to 0 o C. SO 3 -pyrridine complex (2.2 g, 13 mmol) was added portionwise. The mixture was warmed to room temperature, stirred 2h. Water (20.0 mL) was added. The organic layer was separated, dried over Na 2 SO 4 , concentrated, and purified with column chromatography (Hexanes/EtOAc=2/1) to give 14 as white needle-like solid (0.92 g, 77%), m.p.: 54-55 o C. 1 H NMR (400 MHz, CDCl 3 ), ? 7.63 (dd, J1= 4.8 Hz, J2= 2.4 Hz, 1H), 6.28 (d, J= 6.0 Hz, 1H), 5.23 (dd, J1= 5.2 Hz, J2= 2.0 Hz, 1H), 4.40 (d, J= 5.2 Hz, 72 1H), 1.86 (m, 2H), 1.66 (m, 6H). 13 C NMR (100MHz, CDCl 3 ), ? 204.0, 159.9, 135.5, 124.3, 78.2, 76.2, 37.9, 37.4, 24.1, 23.3. Anal. Calcd for C 10 H 12 O 3 : C, 66.65; H, 6.71; Found: C, 66.99; H, 7.08. 2,3-(Cyclopentylidenedioxy)-4-vinyl-cyclopentanol (16). To a suspension of CuBr?SMe 2 (1.59 g, 7.73 mmol) in THF was added vinylmagnium bromide (155.0 mL, 0.155 mol) at ?78 o C. A solution of 14 (14.23 g, 78.97 mmol), trimethylsilyl chloride (TMSCl) (19.6 mL, 0.155 mol), hexamethylphosphoramide (HMPA) (13.0 mL, 77.7 mmol) in THF was added dropwise at ?78 o C. The mixture was stirred at ?78 o C for 2 hours, and then warmed slowly to room temperature. The reaction was quenched with saturated NH 4 Cl solution (100 mL). After the removal of solvent, the mixture was extracted with EtOAc (3?200 mL). The combined organic layer was dried over sodium sulfate, concentrated to give brown oil. THF (100 mL) was added. At 0 o C, LiAlH 4 (4.60 g, 121 mmol) was added slowly. The mixture was stirred at room temperature overnight. The reaction was quenched with water. The mixture was filtered through celite, and then extracted with EtOAc. The organic layer was dried over sodium sulfate, concentrated, and purified by silica column chromatography (Hexanes:EtOAc=10:1 to 2:1) to isolate 16 as a colorless oil (10.60 g, 63.84%). 1 H NMR (400 MHz, CDCl3), ? 5.72 (m, 1H), 5.04-5.10 (m, 2H), 4.39 (m, 2H), 4.07-4.13 (m, 1H), 2.76 (m, 1H), 2.41 (d, J=7.6 Hz, 1H), 1.89-1.96 (m, 4H), 1.7 (m, 6H). 13 C NMR (100 MHz, CDCl 3 ), ?138.2, 121.6, 115.4, 84.4, 79.0, 71.3, 44.3, 36.3, 35.7, 35.5, 24.2, 23.0. Calcd HRMS for C 12 H 18 O 3 : 210.1253; Found: 210.1256. 6-Chloro-9-(2?,3?-(cyclopentylidenedioxy)-4?-vinyl-cyclopentyl)-purine (17). 16 (10.9 g, 51.8 mmol) was dissolved in THF (100 mL). 6-Chloropurine (10.0 g, 64.7 mmol), 73 (Ph 3 )P (17.0g, 64.8 mmol) was added. The solution was cooled to ?40 o C. Diisopropyl azodicarboxylate (DIAD) (12.5 mL, 64.5 mmol) was added dropwise. The mixture was warmed to room temperature, then heated to 60 o C for 24 hours. The solvent was removed under reduced pressure. The residue was purified by silica column (Hex:EtOAc=5:1 to 2:1) to give 17 as organce oil, contaminated with diisopropyl hydrazine-1,2-dicarboxylate (10.4 g, 57.9%). The mixture was used in next step without further purification. 6-Chloro-9-(2?,3?-(cyclopentylidenedioxy)-4?-hydroxymethyl-cyclopentyl)-purine (19). 17 (0.73 g, 2.1 mmol) was dissolved in MeOH (50 mL). Water (0.5 mL) was added. NaIO 4 (0.99 g, 4.6 mmol) was added. The mixture was cooled to 0 o C. OsO 4 (10 mg, 0.040 mmol, 2% mol) was added. The mixture was stirred at 0 o C for 2 hours. The mixture was filtered. MeOH was removed by reduced pressure. The residue was extracted with DCM (3?50 mL). The organic layer was washed with brine, dried over sodium sulfate, concentrated. The residue was dissolved in methanol (20 mL). NaBH 4 (0.10 g, 2.6 mmol) was added portionwise at 0 o C. The mixture as stirred at 0 o C for 1 hour. Saturated NH 4 Cl solution (20 mL) was added. The mixture was filtered through Celite. The solvent was removed with reduced pressure. The residue was extracted with EtOAc (3?20 mL). The combined organic layer was dried over sodium sulfate, concentrated. The residue was purified by silica column (Hex:EtOAc=1:1 to 1:10) to provide 19 as a colorless oil (0.30 g, 41%). 1 H NMR (400 MHz, CDCl 3 ), ? 8.74 (s, 1H), 8.28 (s, 1H), 4.99 (m, 1H), 4.91 (m, 1H), 4.66 (m, 1H), 3.87 (m, 2H), 2.93 (t, J=4.8 Hz, 1H), 2.44-2.64 (m, 3H), 2.03 (m, 2H), 1.70 (m, 6H). 13 C NMR (100 MHz, CDCl 3 ), ? 151.8, 151.7, 151.4, 144.9, 132.4, 123.4, 83.8, 82.0, 63.6, 62.7, 45.0, 36.8, 36.6, 33.3, 74 23.8, 23.2. Calcd HRMS for C 16 H 19 ClN 4 O 3 (-cyclopentanone):284.0677, Foud: 284.0667. 2?,3?-(Cyclopentylidenedioxy)-4?-vinylcyclopentyl 4-Methoxybenzyl Ether (21). 16 (0.70 g, 3.32 mmol) was dissolved in dry DMF (50 mL). The solution was cooled to 0 o C. NaH (147 mg, 60% in mineral oil, 3.67 mmol) was added in one portion. The solution was stirred for 30 minutes. para-Methoxybenzyl chloride (PMBCl) (0.54 mL, 4.0 mmol) was added at 0 o C in one portion. The solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. Saturated NH 4 Cl solution (10 mL) was added. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was dried over sodium sulfate, concentrated, and purified by silica gel column (Hex:EtOAc=20:1) to provide 21 as a colorless oil (0.90g, 81%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.30 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 5.63-5.72 (m, 1H), 4.97-5.01 (m, 2H), 4.51-4.63 (m, 2H), 4.43-4.46 (m, 2H), 4.31 (d, J=5.6 Hz, 1H), 3.80 (s, 3H), 3.76-3.79 (m, 1H) 2.66 (m, 1H), 2.07-2.10 (m, 3H), 1.90-1.93 (m, 1H), 1.78-1.81 (m, 1H), 1.70 (m, 4H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.2, 138.6, 130.5, 129.5, 120.7, 114.8, 113.7, 83.7, 78.2, 77.6, 71.4, 55.2, 43.9, 35.5, 35.3, 31.8, 24.0, 23.0. Anal. Calcd for C 20 H 26 O 4 : C, 72.70; H, 7.93; Found: C, 72.37; H, 7.87. 2?,3?-(Cyclopentylidenedioxy)-4?-hydroxymethylcyclopentyl 4-Methoxybenzyl Ether (22). 21 (0.90 g, 2.7 mmol) was dissolved in MeOH (50 mL), and cooled to 0 o C. Water (5 mL) was added. NaIO 4 (1.45 g, 6.81 mmol) was added. OsO 4 (20 mg) was added. The solution was stirred at 0 o C for 2 hours. The mixture was filtered through Celite. The solvent was removed under reduced pressure. The residue was extracted with DCM (3?50 mL). The combined organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH (50 mL), cooled to 0 o C. 75 NaBH 4 (0.20 g, 5.4 mmol) was added in portionwise. The mixture was stirred for 2 hours. Saturated NH 4 Cl solution (30 mL) was added. MeOH was removed under reduced pressure. The residue was extracted with EtOAc (3?50 mL). The combined organic layer was dried over sodium sulfate, concentrated and purified by silica gel column (Hex:EtOAc=3:1) to give 22 as colorless oil (0.49 g, 53%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.30 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.52-4.63 (m, 2H), 4.43-4.45 (m, 1H), 4.36 (m, 1H), 3.86 (m, 1H), 3.80 (s, 3H), 3.49-3.52 (m, 1H), 3.41-3.45 (m, 1H), 2.17 (m, 1H), 2.10 (m, 2H), 2.04 (m, 1H), 1.70 (m, 7H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.2, 130.5, 129.4, 120.9, 113.7, 81.6, 79.0, 77.9, 71.4, 64.3, 55.2, 44.2, 35.7, 35.5, 30.7, 24.01, 23.0. Calcd HRMS for C 19 H 26 O 5 : 334.1780, Found: 334.1777. 2?,3?-(Cyclopentylidenedioxy)-4?-fluoromethyl-cyclopentyl 4-Methoxybenzyl Ether (23). 22 (0.35 g, 1.0 mmol) was dissolved in dry DCM (20 mL), and cooled to ?78 o C. Pyridine (0.17 mL, 2.1 mmol), (Diethylamino)sulfur trifluoride (DAST) (0.20 mL, 1.5 mmol) was added. The solution was warmed to room temperature, and then refluxed under protection of N 2 for 12 h. the reaction was quenched with saturated Na 2 CO 3 solution (20 mL). The organic layer was separated, washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column (Hex:EtOAc=10:1) to provide 23 as a colorless oil (0.27 g, 76%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.30 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.51-4.63 (m, 2H), 4.42-4.46 (m, 1.5H), 4.39 (m, 1H), 4.30-4.34 (m, 1H), 4.19-4.22 (m, 0.5H), 3.80-3.86 (m, 1H), 3.80 (s, 3H), 2.15 (dm, J=10 Hz, 1H), 2.05-2.20 (m, 2H), 1.93 (m, 1H), 1.74-1.76 (m, 1H), 1.70 (m, 6H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.2, 130.4, 129.5, 121.1, 113.7, 85.5 (d, J=168Hz), 81.2 (d, J=6Hz), 79.1, 78.0, 79.0, 71.5, 55.2, 42.6 (d, J=19 Hz), 35.6 (d, J=15 Hz), 30.8, 24.0, 23.0. Calcd 76 HRMS for C 19 H 25 FO 4 : 336.1744; Found: 336.1737. 2,3-(Cyclopentylidenedioxy)-4-fluoromethyl-cyclopentanol (24). 23 (0.78 g, 2.3 mmol) was dissolved in 19:1 DCM/H 2 O (50 mL). DDQ (0.63 g, 2.8 mmol) was added in one portion. The mixture was stirred at room temperature for 2 hours. Saturated Na 2 CO 3 solution (30 mL) was added. The organic layer was separated, washed with saturated Na 2 CO 3 solution (30 mL), brine (30 mL), dried over sodium sulfate, concentrated and purified by silica gel column (Hex:EtOAc=5:1) to provide 24 as a colorless oil (0.31 g, 60%). 1 H NMR (250 MHz, CDCl 3 ), ? 4.11-4.69 (m, 5H), 2.43-2.46 (m, 2H), 2.30-2.40 (m, 1H), 1.85-2.02 (m, 3H), 1.65-1.75 (m, 6H). 13 C NMR (62 MHz, CDCl 3 ) ? 121.9, 84.9 (d, J=160 Hz), 81.9 (d, J=4.8 Hz), 79.6, 71.3 (d, J=1.8 Hz), 42.6 (d, J=17.4 Hz), 35.5 (d, J=10.8 Hz), 34.8, 34.7, 23.9, 22.8. Anal. Calcd for C 11 H 17 FO 3 : C, 61.10; H, 7.92; Found: C, 60.94; H, 8.00. 5?-Fluoro-5?-deoxy aristeromycin (1). 24 (0.14 g, 0.65 mmol) was dissolved in dry THF (50 mL), TPP (0.34 g, 1.3 mmol), 6-di(tert-butoxylcarbonyl)aminopurine (Ad(Boc) 2 )(0.26 g, 0.78 mmol) was added. The solution was cooled to 0 o C, DIAD (0.20 mL, 0.98 mmol) was added in one portion. The solution was warmed to room temperature and stirred 5 hours. The solvent was removed under reduced pressure, the residue was purified by silica gel column (Hex:EtOAc=2:1) to give intermediate 25 as an orange oil, contaminated with diisopropyl hydrazine-1,2-dicarboxylate (from DIAD). Without further purification, 25 was dissolved in 3N HCl MeOH solution, stirred at 50 o C overnight. NaHCO 3 was added to neutralize the solution until it no longer bubbled. The mixture was filtered. The solvent was removed under reduced pressure, and the residue was purified by silica gel column (EtOAc:MeOH:NH 3 ?H 2 O=4:1:0.3) to provide 1 as a 77 white solid (0.10 g, 58%), mp=168-169 o C. 1 H NMR (400 MHz, DMSO), ? 8.19 (s, 1H), 8.12 (s, 1H), 7.20 (s, 2H), 5.06 (d, J=6.0 Hz, 1H), 4.91 (d, J=4.0 Hz, 1H), 4.70 (m, 1H), 4.58 (m, 1H), 4.45 (m, 1H),4.35 (m, 1H), 3.89 (m, 1H), 2.27 (m, 2H), 1.80 (m, 1H). 13 C NMR (100 MHz, DMSO) ? 156.0, 152.1, 149.6, 140.1, 119.3, 84.55(d, J=165 Hz), 74.2, 70.7 (d, J=5.0 Hz), 59.1, 43.4 (d, J=18.0 Hz), 27.8 (d, J=6.0 Hz). Anal. Calcd for C 11 H 14 FN 5 O 2 (+0.2H 2 O): C, 48.77; H, 5.35; N, 25.85; Found: C, 48.71; H, 5.47; N, 25.96. Calcd mass for C 11 H 14 FN 5 O 2 : 267.1132; Found: 267.1131 (1R,4S)-4-(tert-Butyldimethylsilyloxy)cyclopent-2-enyl Acetate (27). 26 (5.0 g, 0.035 mol) was dissolved in dry DCM (60 mL). 4-(Dimethylamino)pyridine (DMAP) (20 mg) was added. The solution was treated with imidazole (5.9 g, 0.087 mol), and tert-butyldimethylsilyl chloride (TBSCl) (6.4 g, 0.042 mol) at 0 o C. The solution was then warmed to room temperature and stirred for 2 hours. Water (20 mL) was added to quench the reaction. The organic layer was separated, washed with brine, dried over sodium sulfate, concentrated under reduced pressure. The residue was purified by silica column (Hexanes/EtOAc=15:1) to give 27 as a colorless oil (7.3 g, 80%). The NMR spectra are consistent with the literature. 228 (3aS,4R,6S,6aS)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydro-3aH-cyc lopenta[d][1,3]dioxol-4-yl Acetate (28). 27 (7.0 g, 27 mmol) was dissolved in THF (50 mL). 4-Methylmorpholine N-oxide (NMO) (8.8 mL, 50% water solution, 42 mmol) was added. The solution was cooled to 0 o C. OsO 4 (20 mg, 0.078 mmol, 0.28% moles) was added. The mixture was warmed to room temperature and stirred overnight. Sodium thiosulfate (2.2 g, 14 mmol) was added and stirred 30 minutes. The solvent was removed under reduced pressure. The residue was dissolved in EtOAc, dried over sodium sulfate, 78 and filtered through a short silica column (5 cm). The filtrate was concentrated. The resulting oil was dissolved in dry acetone (100 mL). 2,2-dimethoxypropane (30 mL) was added. p-Toluenesulfonic acid monohydrate (100 mg) was added. The solution was stirred at room temperature overnight. Ammonium hydroxide (29.6%, 1 mL) was added. The solution was dried over sodium sulfate and concentrated. The residue was purified by silica column (hexanes/EtOAc=10:1) to provide 28 as a colorless oil (8.3 g, 85%). The NMR spectra are consistent with literature. 229 (3aS,4R,6S,6aS)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydro-3aH-cyc lopenta[d][1,3]dioxol-4-ol (29). 28 (8.3 g, 25 mmol) was dissolved in methanol (200 mL) in a high pressure reaction vessel. The solution was cooled to 0 o C and saturated with ammonia. The reaction vessel was sealed and warmed to room temperature overnight. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=5:1) to give 29 as a colorless oil (5.0 g, 90%). The NMR spectra were consistent with literature. 229 (3aR,6S,6aS)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-dihydro-3aH-cyclopent a[d][1,3]dioxol-4(5H)-one (30). 29 (1.0 g, 3.5mmol) was dissolved in dry DCM (20 mL). PCC (1.6 g, 6.9 mmol) was added. The solution was stirred at room temperature for 3 hours. The solution was filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=7:1) to give 30 as a colorless oil (0.85 g, 85%). The NMR spectra were consistent with literature. 229 (1S,2S,3S,4S)- 4-O-(tert-Butyldimethylsilyl)-2,3-O-isopropylidenecyclopentane-1,2,3,4-tetrol (31). To a solution of 30 (0.35 g, 1.2 mmol) in MeOH (10 mL) was added CeCl 3 ?7H 2 O (0.45 g, 79 1.2 mmol). The mixture was cooled to 0 o C. NaBH 4 (60.0 mg, 1.58 mmol) was added portion-wise. The mixture was stirred for 30 minutes at 0 o C, then warmed to room temperature, stirred for 1 hour. The reaction was quenched with saturated NH 4 Cl solution (5 mL). The solvent was removed under reduced pressure. The residue was poured to water and extracted with EtOAc (3?10 mL). The combined organic layer was dried over sodium sulfate, and concentrated. The residue was purified by silica gel column (EtOAc/Hexanes=3:1) to produce 31 as a colorless oil (0.34 g, 97%). 1 H NMR (400 MHz, CDCl 3 ) ?4 .56 (t, J= 5.4 Hz,1H), 4.34-4.26 (m, 2H), 4.01 (dd, J1=3.5 Hz, J2=0.4 Hz, 1H), 2.25 (d, J=10.4 Hz, 1H), 1.89-1.93 (m, 1H), 1.68-1.75(m, 1H), 1.46 (s, 3H), 1.34 (s, 3H), 0.86 (s, 9H), 0.05 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) ?111.3, 85.8, 78.6, 73.4, 72.0, 39.3, 26.1, 25.9, 24.4, 18.1, -4.6, -4.7. Anal. Calcd for C 14 H 28 O 4 Si: C, 58.29; H, 9.78; Found: C, 58.34; H, 9.85. tert-Butyl((3aS,4S,6S,6aS)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydro-3a H-cyclopenta[d][1,3]dioxol-4-yloxy)dimethylsilane (32). 31 (7.56 g, 26.2 mmol) was dissolved in dry DMF (100 mL). The solution was cooled to 0 o C, NaH (1.26 g, 60.0% in mineral oil, 31.4 mmol) was added portionwise. The solution was stirred at 0 o C for 30 minutes. PMBCl (4.30 mL, 31.4 mmol) was added in one portion. The solution was warmed to room temperature, stirred for 3 hours. The solvent was removed under reduced pressure. The residue was quenched with water, and extracted by EtOAc. The organic layer was dried over sodium sulfate, concentrated, and purified with a silica column (Hex: EtOAc=10:1) to provide 32 as a colorless oil (9.00 g, 84.1%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.29 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 4.61 (m, 1H), 4.45-4.58 (m, 2H), 4.25 (dd, J1=5.6 Hz, J2=1.6 Hz, 1H), 4.02-4.05 (m, 1H), 3.94 (d, J=4.0 Hz, 1H), 80 3.79 (s, 3H), 1.90-1.94 (m, 1H), 1.75-1.76 (m, 1H), 1.48 (s, 3H), 1.30 (s, 3H), 0.83 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.2, 130.4, 129.7, 113.7, 110.9, 85.7, 77.7, 77.5, 73.4, 71.5, 55.2, 35.7, 26.1, 25.7, 24.0, 17.9, -4.8. Anal. Calcd for C 22 H 36 O 5 Si: C, 64.67; H, 8.88; Found: C, 64.99; H, 8.64. (3aR,4S,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydro-3aH-cyclope nta[d][1,3]dioxol-4-ol (33). 32 (9.00 g, 22.0 mmol) was dissolve in THF (100mL). Tetrabutylammonium fluoride (TBAF) (33.0 mL, 1.0M in THF, 33.0 mmol) was added. The mixture was stirred at room temperature for one hour. The mixture was quenched with water, extracted with EtOAc (3?300 mL). Combined organic layer was dried over sodium sulfate, concentrated, and purified with a silica column (Hex:EtOAc=5:1 to 1:1) to give 33 as a colorless oil (5.80 g, 89.6%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.31 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.52-4.66 (m, 3H), 4.32 (dd, J1=5.6 Hz, J2=1.2 Hz, 1H), 4.04-4.09 (m, 2H), 3.80 (s, 3H), 2.00-2.07(m, 1H), 1.83-1.88 (m, 1H), 1.58 (s, 3H), 1.32 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 171.4, 159.4, 130.5, 129.7, 113.9, 111.3, 85.5, 77.9, 73.4, 71.7, 55.4, 35.8, 26.3, 24.2. Anal. Calcd for C 16 H 22 O 5 : C, 65.29; H, 7.53; Found: C, 65.03; H, 7.62. (3aS,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-dihydro-3aH-cyclopenta[d][ 1,3]dioxol-4(5H)-one (34). 33 (5.80 g, 19.7 mmol) was dissolved in dry DCM (200 mL). DMSO (10 mL), N,N-Diisopropylethylamine (DIPEA) (6.95 mL, 39.4 mmol) was added. The solution was cooled to 0 o C. SO 3 ?Py complex (6.25 g, 39.4 mmol) was added portionwise. The solution was stirred at 0 o C for 1 hour. The mixture was quenched with ice cold water (200 mL). The organic layer was separated, washed with saturated NaHCO 3 and brine, and concentrated. the residue was purified by silica gel column (Hex: 81 EtOAc=5:1 to 1:1) to give 34 as a colorless oil (4.71 g, 81.8%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.31 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 4.80 (t, J=4.2 Hz, 1H), 4.60-4.68 (m, 2H), 4.18 (dt, J1=4.8 Hz, J2=1.2 Hz, 1H), 4.05-4.11 (m, 1H), 3.81(s, 3H), 2.68-2.76 (m, 1H), 2.47-2.53 (m, 1H), 1.48 (s, 3H), 1.38 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 211.0, 159.6, 129.7, 129.2, 113.9, 113.5, 80.5, 77.6, 71.4, 70.0, 55.3, 39.8, 26.9, 25.2. Anal. Calcd for C 16 H 20 O 5 : C, 65.74; H, 6.90. Found: C, 65.60; H, 6.91. (3aR,4R,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydro-3aH-cyclope nta[d][1,3]dioxol-4-ol (35). 34 (0.27 g, 0.92 mmol) was dissolved in dry THF (20 mL). LiAlH 4 (52.3 mg, 1.38 mmol) was added portionwise at 0 o C. The mixture was stirred at 0 o C for 3 hours. The mixture was quenched with water, and filtered through celite. The filtrate was extracted with EtOAc (3?50 mL). The combined organic layer was dried over sodium sulfate and concentrated to give 35 as colorless oil (0.22 g, 81%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.28 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.52-4.59 (m, 3H), 4.40 (t, J=5.6 Hz, 1H), 3.80 (s, 3H), 3.69-3.78 (m, 1H), 3.45-3.51 (m, 1H), 2.41 (d, J=10.8 Hz, 1H), 2.10-2.15(m, 1H), 1.72-1.80 (m, 1H), 1.56 (s, 3H), 1.37 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.5, 130.1, 129.7, 114.0, 111.5, 78.4, 78.0, 73.7, 71.3, 68.5, 55.4, 34.7, 25.9, 24.4. Anal. Calcd for C 16 H 22 O 5 : C, 65.29; H, 7.53; Found: C, 65.13; H, 7.53. (3aS,4S,6S,6aS)-4-Fluoro-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydro-3aH -cyclopenta[d][1,3]dioxole (36). 35 (4.70 g, 15.9 mmol) was dissolved in dry DCM (100 mL). Pyridine (2.5 mL, 31 mmol) was added at 0 o C. DAST (4.1 mL, 31 mmol) was added through a syringe at 0 o C. The mixture was warmed to room temperature, then refluxed 2 days under protection of nitrogen. The mixture was quenched with saturated NaHCO 3 solution (100 mL). The organic layer was separated, dried over sodium sulfate, 82 concentrated, and purified with a silica column (Hex: EtOAc=5:1) to give 36 as a colorless oil (3.27 g, 69.4%). 1 H NMR (400 MHz, CDCl 3 ), ? 7.3 (d, J=8.8 Hz, 2H), 6.88 (d, J=8.8 Hz, 2H), 4.47-4.81 (m, 5H), 4.00-4.05 (m, 1H), 3.81 (s, 3H), 2.04-2.14 (m, 2H), 1.48 (s, 3H), 1.32 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 159.6, 130.2, 129.8, 114.1, 111.8, 94.4 (d, J=174.5 Hz), 82.82(d, J=33.4 Hz), 77.8, 77.1, 71.9, 55.5, 33.7 (d, J=20.1 Hz), 26.3, 24.2. Anal. Calcd for C 16 H 21 FO 4 : C, 64.85; H, 7.14. Found: C, 64.72; H, 7.19. (3aS,4S,6S,6aS)-6-Fluoro-2,2-dimethyl-tetrahydro-3aH-cyclopenta[d][1,3]dioxol -4-ol (37). 36 (0.98 g, 3.3 mmol) was dissolved in DCM/H 2 O mixture (100 mL DCM, 5 mL H 2 O). DDQ (0.90 g, 4.0 mmol) was added. The mixture was stirred at room temperature for 1 hour. Saturated NaHCO 3 (20 mL) was added to quench the reaction. The organic layer was separated, washed with brine, dried over sodium sulfate, concentrated, and purified by silica chromatography (Hex:EtOAc=5:1) to give 37 as white solid (0.49 g, 86%). Mp=51~52 o C. 1 H NMR (400 MHz, CDCl 3 /D 2 O), ? 4.68 (dd, J1=46.0 Hz, J2=3.6 Hz, 1H), 4.56-4.60 (m, 2H), 4.26-4.32 (m, 1H), 2.26-2.32 (td, J1=15.2 Hz, J2= 5.6Hz, 1H), 1.83 (dddd, J1=44.4 Hz, J2=14.4 Hz, J3=10.8 Hz, J4=3.6 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ), ? 111.7, 93.8 (d, J=172.3 Hz), 82.4 (d, J=33.2 Hz), 78.2, 71.4, 36.8 (d, J=20.8 Hz), 25.9, 24.1. 6-Chloro-9-((3aS,4R,6S,6aS)-6-fluoro-2,2-dimethyl-tetrahydro-3aH-cyclopenta[ d]-[1,3]dioxol-4-yl)-9H-purine (38). 37 (0.73 g, 4.1 mmol) was dissolved in dry THF (50 mL). TPP (1.30 g, 4.96 mmol), 6-chloropurine (0.76 g, 4.9 mmol) were added. The mixture was cooled to 0 o C. DIAD (0.95 mL, 4.9 mmol) was added. The mixture was warmed to room temperature, then heated and stirred at 50 o C overnight. The solvent was removed under reduced pressure. The residue was purified by silica column to give 38 83 with diisopropyl hydrazine-1,2-dicarboxylate (0.40 g, 30%). This product was used directly without further purification. 9-((3aS,4R,6S,6aS)-6-Fluoro-2,2-dimethyl-tetrahydro-3aH-cyclopenta[d][1,3]dio xol-4-yl)-9H-purin-6-amine (39). In a higher pressure reaction vessel, 38 (0.50 g, 1.6 mmol) was dissolved in dry MeOH (100 mL). The solution was cooled to 0 o C, and saturated with NH 3 . The solution was sealed, and heated to 120 o C overnight. The solvent was removed under reduced pressure, and the residue was purified by silica column to give 39 as white solid (0.37 g, 83%).mp=158-160 o C. 1 H NMR (250 MHz, DMSO), ? 8.16 (s, 1H), 8.10 (s, 1H), 7.25(br, 2H), 4.8-5.2 (m, 4H), 2.55-2.80 (m, 2H), 1.45 (s, 3H), 1.28 (s, 3H). 13 C NMR (62.8 MHz, DMSO), ? 156.0, 152.4, 149.4, 139.1 (d, J=6.5 Hz), 118.8, 111.4, 96.9 (d, J=177.6 Hz), 83.5 (d, J=23.1 Hz), 83.2, 58.6, 35.1(d, J=20.3 Hz), 26.2, 24.1. Anal. Calcd for C 13 H 16 N 5 FO 2 : C, 53.24; H, 5.50; N, 23.88; Found: C, 53.05; H, 5.47; N, 23.74. (1S,2S,3R,5S)-3-(6-Amino-9H-purin-9-yl)-5-fluorocyclopentane-1,2-diol (2). 39 (0.40 g, 1.4 mmol) was dissolved in 0.5 M HCl MeOH solution (100 mL). The mixture was stirred at room temperature overnight. The mixture was neutralized with Amberlite ? IRA-67 (commercially available from Aldrich) ion exchange resin. The mixture was filtered, concentrated and purified by silica chromatography ( EtOAc:MeOH:NH 4 OH (29.6%)=3:1:0.2) to give 2 as a white solid (0.32 g, 93%). Mp=218-220 o C ?sample recrystalized from MeOH/H 2 O?. 1 H NMR (250 MHz, DMSO), ? 8.16 (s, 1H), 8.12 (s, 1H), 7.22 (s, 2H), 5.36 (d, J=4.2 Hz, 1H), 5.25 (d, J=6.5 Hz, 1H), 4.59-4.97 (m, 2H), 4.58-4.63 (m, 1H), 4.04 (dm, J=12.0 Hz, 1H), 2.68-2.76 (m, 1H), 2.20-2.34 (m, 1H). 13 C NMR (62 MHz, DMSO), ? 156.0, 152.2, 149.6, 140.2, 119.4, 95.1 (d, J=177.7 Hz), 73.7 84 (d, J=10.5 Hz), 73.4, 57.8 (d, J=3.1 Hz), 33.3 (d, J=22.6 Hz). Anal. Calcd for C 10 H 12 FN 5 O 2 : C, 47.43; H, 4.78; N, 27.66; Found: C, 47.28; H, 4.86; N, 27.36. 1-Benzyl-1H-pyrrole-2-carbaldehyde (40). Pyrrole-2-carboxaldehyde (25 g, 0.27 mol), benzene (125 mL), NaOH solution (50%, 125 mL), tetrabutylammonium iodide (TBAI) (1.0 g) were mixed and refluxed over 24 hours. The mixture was cooled to room temperature. The organic layer was separated, washed with water (3?100mL), dried over sodium sulfate, and concentrated to give crude 40 as a yellow oil (43 g, 82%), which was used without further purification. 3-(1-Benzyl-1H-pyrrol-2-yl)acrylic Acid (42). 40 (43 g, 0.23 mol) was dissolved in dry ethanol (200 mL). Malonic acid (26 g, 0.25 mol), aniline (23 g, 0.25 mol) were added. The mixture was refluxed until white solid formed. The flask was cooled with ice-water. The precipitate was filtered, and washed with benzene. The solid was dried under vacuum to give 42 as white solid (37 g, 72%). The NMR spectra are consistent with literature. 216 3-(1-Benzyl-1H-pyrrol-2-yl)acryloyl Azide (44). 42 (10.0 g, 44.0 mmol) was dissolved in dry acetone (200 mL). Triethylamine (TEA) (5.8 g, 57 mmol) was added. Chloroethyl formate (6.2 mL, 66 mmol) was added dropwise via a syringe at 0 o C. The solution was stirred at the same temperature for 1 hour. Sodiam azide (4.4 g, 66 mmol) in minimum water was added dropwise. The mixture was warmed to room temperature and stirred 1 hour. The solvent was removed under reduced pressure. Water was added. The mixture was extracted with EtOAc (200 mL). The organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated to give 44 as yellow oil (11 g, 96%). The NMR spectra were consistent with literature. 216 85 1-Benzyl-1H-pyrrolo[3,2-c]pyridin-4-ol (46). 44 (10.6 g, 42.0 mmol) in diphenyl ether (20 mL, 60 o C) was added dropwise to a solution of n-Bu 3 N (2.0 mL) in diphenyl ether (30 mL) at 220 o C (maintaining this temperature during addition of 44). The solution was stirred at same temperature for 15 minutes. The solvent was removed under reduced pressure. The residue was purified with silica column (EtOAc/MeOH=5:1) to give 46 as a yellow solid (3.80 g, 40.4%). The NMR spectra are consistent with literature. 216 1H-Pyrrolo[3,2-c]pyridin-4-ol (47). 46 (7.6 g, 34 mmol) was dissolved in liquid ammonia at -78 o C (about 100 mL). Sodium metal (in small pieces) was added until the solution remained blue for 5 minutes. Ammonium chloride (15 g) was added to quench the reaction. The mixture was warmed to room temperature. The mixture was purified by silica column (EtOAc/MeOH=3:1) to give 47 as white solid (3.1 g, 70%). The NMR spectra are consistent with literature. 216 4-Chloro-1H-pyrrolo[3,2-c]pyridine (48). 47 (3.1 g, 25 mmol) was dissolved in POCl 3 (20 mL) in high pressure reaction vessel. The mixture was heated to 170 o C overnight. The POCl 3 was removed under reduced pressure. The residue was poured to ice-water. The resulting mixture was extracted with EtOAc (4?100 mL). The combined organic layer was washed with brine (3?20 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (EtOAc) to give 48 as white solid (2.3 g, 60%). The NMR spectra are consistent with literature. 216 (3aR,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-dihydro-3aH-cyclopenta[d]-[1, 3]dioxol-4(5H)-one (50). tert-BuOMe (10 mL), tert-BuOK (2.0 g, 17 mmol) was dissolved in dry THF (30 mL) and cooled to -78 o C. sec-BuLi (16 mL, 1.4 M in hexane, 86 22 mmol) was added dropwise. The mixture was stirred at -78 o C for 2 hours. LiBr (2.9 g, 34 mmol) in THF (10 mL) was added over 10 min at -70 o C. The mixture was warmed to -30 o C, stirred 30 minutes. The mixture was recooled to -78 o C. CuBr?SMe 2 (1.8 g, 8.7 mmol), SMe 2 (10 mL) in THF (10 mL) was added dropwise over 5 minutes. 49 (0.70 g, 4.5 mmol) in THF (5 mL) was added dropwise at the same temperature. The mixture was warmed to -30 o C and stirred 30 minutes. MeOH (15 mL), saturated NH 4 Cl solution (15 mL) were added. The mixture was extracted with diethyl ether (3?100 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified by silica column (hexanes/EtOAc=10:1) to give 50 as a colorless oil (0.84 g, 77%). The NMR spectra were consistent with literature. 230 (3aS,4S,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-tetrahydro-3aH-cyclopenta [d]-[1,3]dioxol-4-ol (51). 50 (0.30 g, 1.2 mmol) was dissolved in MeOH (10mL) at 0 o C. CeCl 3 ?7H 2 O (0.46 g, 1.2 mmol) was added. NaBH 4 (0.12 g, 2.4 mmol) was added portionwise, and stirred 30 minutes. Saturated NH 4 Cl solution (3 mL) was added. The solvent was removed under reduced pressure. The residue was extracted with EtOAc (3?30 mL). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate and concentrated. The residue was purified with silica column (hexanes/EtOAc=5:1) to give 51 as colorless oil (0.25 g, 83%). The NMR spectra were consistent with the literature. 230 1-((3aS,4R,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-tetrahydro-3aH-cyclope nta[d]-[1,3]dioxol-4-yl)-4-chloro-1H-pyrrolo[3,2-c]pyridine (53). To a chilled (-20 o C) solution of 51 (0.9 0g, 3.7 mmol) and pyridine (0.59 mL, 7.4 mmol) in dry CH 2 Cl 2 (30 mL) was added dropwise trifluoromethansulfonic anhydride (0.93 mL, 5.5 mmol). The 87 mixture was placed in an ice/water bath, and stirred for 1 hour. Ice cold water was added to quench the reaction. The organic layer was washed with ice cold water (3?10 mL). The organic layer was separated and dried by Na 2 SO 4 . The solvent was removed at reduced pressure to give an orange oil (1.2 g, 84%). The resulting oil was dissolved in anhydrous DMF (20 mL) and cooled to ?20 o C. The resulting solution was added to pre-prepared sodium salt of 48 in DMF (by addting NaH (0.22 g, 60% in mineral oil, 5.5 mmol) to a solution of 48 (0.84 g, 5.5 mmol) in DMF at romm temperature) at ?40 o C. The mixture was stirred at room temperature for 2 days. The solvent was removed under reduced pressure. The residue was dissolved in water, extract with EtOAc (3?10 mL). The combined organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by silica gel column (Hexanes/EtOAc=5:1) to give 53 as a colorless oil (0.60 g, 43%). 1 H NMR (400 MHz, CDCl 3 ) ? 8.09 (d, J=9.4 Hz, 1H), 7.46 (d, J=9.6 Hz, 1H), 7.31 (d, J=5.6 Hz, 1H), 6.68 (d, J=5.4 Hz, 1H), ?4.74 (m, 1H), 4.60 (m, 2H), 3.54 (m, 2H), 2.51 (m, 2H), 2.33 (m, 1H), 1.64 (s, 3H), 1.33 (s, 3H), 1.25 (s, 9H). 13 C NMR (100 MHz, CDCl 3 ) ? 144.1, 141.1, 139.9, 126.1, 123.9, 113.3, 105.6, 101.5, 85.5, 81.3, 77.2, 72.9, 63.2, 61.6, 43.9, 33.2, 27.6, 27.5, 27.0, 25.1. Anal. Calcd for C 20 H 27 ClN 2 O 3 : C, 63.40; H, 7.18; Cl, 9.36; N, 7.39; Found: C, 63.63; H, 7.27; Cl, 9.06; N, 7.14. 3-(4-Chloro-pyrrolo[3,2-c]pyridin-1-yl)-5-hydroxymethyl-cyclopentane-1,2-diol (54). 53 (0.40 g, 1.06 mmol) was dissolved in CF 3 COOH/H 2 O=2:1 (30 mL). The mixture was refluxed under N 2 overnight. The solvent was removed at reduced pressure, and the residue was neutralized with saturated NaHCO 3 solution. The solvent was removed under reduced pressure. The residue was purified with a silica gel column ( CH 2 Cl 2 /MeOH=8:1) to give 54 as a yellow solid (0.25 g, 83%), mp=194-196 o C. 1 H NMR (400 MHz, MeOD) 88 ? 7.96 (d, J=6.0 Hz, 1H), 7.66 (d, J=3.6 Hz, 1H), 7.60 (d, J=6.0 Hz, 1H), 6.71 (d, J=3.6 Hz, 1H), 4.8 (m, 1H), 4.21 (m, 1H), 3.98 (m, 1H), 3.68 (m, 2H), 2.40 (m, 1H), 2.24 (m, 1H), 1.80 (m, 1H). 13 C NMR (100 MHz, MeOD) ? 144.1, 143.3, 139.6, 128.7, 124.9, 107.2, 102.5, 77.9, 73.8, 64.3, 62.6, 46.7, 30.2. Anal. Calcd for C 13 H 15 ClN 2 O 3 ?0.5H 2 O: C, 53.52; H, 5.52; Cl, 12.15; N, 9.60; Found: C, 53.75; H, 5.44; Cl, 12.26; N, 9.39. 3-(4-Amino-pyrrolo[3,2-c]pyridin-1-yl)-5-hydroxymethyl-cyclopentane-1,2-diol Hydrochloride Salt Monohydrate (3). 54 (1.0 g, 3.7 mmol) was dissolved in 2-methoxy ethanol (30 mL). The solution was degassed with N 2 for 10 minutes. Hydrazine monohydrate (20 mL) was added in one portion. The mixture was heated to reflux under N 2 overnight. The solvent was removed under reduced pressure, co-evaporated with ethanol three times (20 mL each). Water (30 mL) was added. The solution was degassed with N 2 for 10 minutes. Raney ? 2800 Nickel (3.0 g in water suspension) was added. The mixture was refluxed under N 2 overnight. The mixture was filtered, concentrated. The residue was purified by silica gel column (EtOAc/MeOH/NH 4 OH (29.6%)=3:1:0.1) to give 3 as gel. Recrystallization from MeOH/EtOAc give 3 as white solid (as hydrochloric acid salt, 0.40 g, 41%). m.p.: decomposed above 200 o C. 1 H NMR (400 MHz, MeOD) ? 7.61 (d, J=3.2 Hz, 1H), 7.46 (d, J=7.2 Hz, 1H), 7.22 (d, J= 7.2 Hz, 1H), ?7.00 (d, J=3.2 Hz, 1H), 4.81 (m, 1H), 4.18 (m, 1H), 3.98 (m, 1H), 3.68 (m, 2H), 2.38 (m, 1H), 2.24 (m, 1H), 1.80 (m, 1H). 13 C NMR (100 MHz, MeOD) ?151.1, 142.1, 127.4, 127.1, 111.1, 104.5, 100.8, 78.1, 73.8, 64.3, 62.6, 46.7, 30.4. Anal. Calcd for C 13 H 18 ClN 3 O 3 (+H 2 O): C, 49.14; H, 6.34; Cl, 11.16; N, 13.22; Found: C, 49.26; H, 6.28; Cl, 11.17; N, 13.09. 1-[6-(tert-Butyl-dimethyl-silanyloxy)-2,2-dimethyl-tetrahydro-cyclopenta[1,3]dio xol-4-yl]-4-chloro-1H-pyrrolo[3,2-c]pyridine (57). To a chilled (-20 o C) solution of 31 89 (2.6 g, 9.0 mmol) and pyridine (0.76 mL, 9.1 mmol) in dry CH 2 Cl 2 (30 mL) was added dropwise trifluoromethansulfonic anhydride (1.5 mL, 9.1 mmol). The mixture was placed in an ice/water bath, and stirred for 1 hour. Ice chilled water (5 mL) was added to quench the reaction. The organic layer was washed with ice cold water (3?10 mL). The organic layer was separated and dried by Na 2 SO 4 . The solvent was removed at reduced pressure to give 56 as orange oil. The resulting 56 was dissolved in anhydrous DMF (20 mL) and cooled to ?20 o C. The resulting solution was added to sodium salt of 48 in DMF solution (prepared by addition of NaH (0.54 g, 60% dispersion in mineral oil, 13 mmol) to a solution of 48 (2.1 g, 13 mmol) in DMF) at ?40 o C. The mixture was stirred at room temperature for 2 days. The solvent was removed under reduced pressure. The residue was dissolved in water, extracted with EtOAc (3?50 mL). The combined organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by silica gel column (Hexanes/EtOAc=2:1) to give 57 as yellow oil (1.4 g, 46%). 1 H NMR (400 MHz, CDCl 3 ) ? 8.08 (d, J=10.6 Hz, 1H), 7.55 (d, J=3.3 Hz, 1H), 7.32 (d, J=5.8 Hz, 1H), 6.60 (d, J=3.3 Hz, 1H), 4.81 (d, J=8.3 Hz, 1H), 4.71(d, J=5.7 Hz, 1H), 4.55 (d, J=5.8 Hz, 1H), 4.43 (m, 1H), 2.75 (m, 1H), 2.18 (d, J=2.5 Hz, 1H), 1.54 (s, 3H), 1.26 (s, 3H), 0.91 (s, 9H), 0.14 (s, 3H), 0.09 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 114.1, 140.0, 128.1, 111.84, 105.2, 101.0, 87.6, 86.6, 63.1, 37.9, 32.4, 27.0, 26.0, 24.5, 23.0, 18.2, 14.5, -4.6, -4.7. Anal. Calcd for C 21 H 31 ClN 2 O 3 Si: C, 59.62; H, 7.39; Cl, 8.38; N, 6.62; Found: C, 59.69; H, 7.38; Cl, 8.08; N, 6.33. 4-(4-Chloro-pyrrolo[3,2-c]pyridin-1-yl)-cyclopentane-1,2,3-triol (58). 57 (1.2 g, 2.8 mmol) was dissolved in 0.5 N HCl methanol solution (20 mL). The mixture was stirred at room temperature for 24 hours. The mixture was neutralized with ammonium 90 hydroxide (29.6%). The solvent was removed under reduced pressure. The residue was purified by silica gel column (EtOAc/MeOH=10:1) to give 58 as white solid (0.70 g, 92%). m.p.: 173-175 o C. 1 H NMR (400 MHz, DMSO) ? 7.97 (d, J=5.8 Hz, 1H), 7.68-7.66 (m, 2H), 6.62 (d, J=3.1 Hz, 1H), 5.27 (s, 1H), 4.98 (s, 2H), 4.78-4.72 (m, 1H), 4.32-4.29 (m, 1H), 3.96 (d, J=5.9 Hz, 1H), 3.75 (d, J=3.6 Hz, 1H), 2.72-2.64 (m, 1H), 1.75-1.69(m, 1H). 13 C NMR (100 MHz, DMSO) ? 142.3, 140.7, 139.0, 128.9, 122.9, 106.3, 100.2, 76.8, 76.7, 73.5, 60.7, 36.5. Anal. Calcd for C 12 H 13 ClN 2 O 3 : C, 53.64; H, 4.88; Cl, 13.19; N, 10.43; Found: C, 53.58; H, 4.79; Cl, 13.39; N, 10.21. 4-(4-Amino-pyrrolo[3,2-c]pyridin-1-yl)-cyclopentane-1,2,3-triol Hydrochloride Salt (4). 58 (0.40g, 1.5 mmol) was dissolved in 2-methoxyethanol (20 mL). The solution was degassed by N 2 for 10 minutes. Hydrazine monohydrate (20 mL) was added in one portion. The mixture was heated to reflux under N 2 overnight. The solvent was removed under reduced pressure, co-evaporated with ethanol three times (30 mL each). Water (30 mL) was added. The solution was degassed with N 2 for 10 minutes. Raney 2800 Nickel (2.0 g in water suspension) was added. The mixture was refluxed under N 2 overnight. The mixture was filtered, and concentrated. The residue was purified by silica gel column (EtOAc/MeOH/NH 4 OH (29.6%)=3:1:0.1) to give 4 as gel. Recrystallization from MeOH/EtOAc give 4 as white solid (as hydrochloride salt, 0.20 g, 53%). Mp=218-219 o C. 1 H NMR (400 MHz, MeOD) ? 7.60 (d, J=3.4 Hz, 1H), 7.46 (d, J=7.2 Hz, 1H), 7.26 (d, J=6.7 Hz, 1H), 7.01 (d, J=3.6 Hz, 1H), 4.84 (m, 1H), 4.44 (m, 1H), 4.13 (m, 1H), 3.93 (m, 1H), 2.81-2.87 (m, 1H), 1.84-1.87 (m, 1H). 13 C NMR (100 MHz, DMSO) ? 149.4, 139.5, 126.9, 126.6, 109.3, 103.5, 99.2, 77.3, 76.6, 73.5, 60.7, 36.7; Anal. Calcd for C 12 H 16 ClN 3 O 3 : C, 50.44; H, 5.64; Cl, 12.41; N, 14.71; Found: C, 50.35; H, 5.67; Cl, 91 12.44; N, 14.44. (3aR,4R,6R,6aR)-6-(Hydroxymethyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dio xol-4-ol (60). D-Ribose (123 g, 0.823 mol), dry acetone (500 mL) and sulfuric acid (1 mL) were mixed and stirred at room temperature overnight. Ammonium hydroxide (29.6% water solution) was added to neutralize the solution. The solution was dried over Na 2 SO 4 and directly filtered through a silica column. The column was rinsed with acetone/THF mixture. The combined filtrate was concentrated, and coevaporated with dry THF (3?50 mL) to give 60 as gel-like foam (103 g, 69.3%). The NMR spectra are consistent with literature. 231 ((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d]-[ 1,3]dioxol-4-yl)methanol (61). 60 (2.98 g, 16.6mmol) was dissolved in DMF. The solution was cooled to 0 o C. NaH (0.68 g, 60% in mineral oil, 17 mmol) was added in portionwise. The mixture was stirred at same temperature for 1 hour. PMBCl (2.76 mL, 20.4 mmol) was added. The solution was warmed to room temperature, stirred overnight. The solvent was removed under reduced pressure. The residue was partitioned between water and EtOAc. The aqueous phase was extracted with EtOAc (3?30 mL). The combined organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanse/EtOAc=2:1) to give 61 as colorless oil contaminated with para-methoxybenzyl alcohol (PMBOH) (3.38 g, 65.6%). 1 H NMR (400 MHz, CDCl 3 ) ? 7.26 (m, 2H), 6.89 (m, 2H), 5.16 (s, 1H), 4.83 (d, J=6.0 Hz, 1H), 4.69 (d, J=11.2 Hz, 1H), 4.62 (m, 2H), 4.50 (d, J=11.2 Hz, 1H), 4.43 (m, 1H), 3.80 (s, 3H), 3.68 (m, 2H), 1.47 (s, 3H), 1.30 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.7, 130.0, 128.6, 114.1, 112.1, 107.7, 88.4, 86.0, 81.5, 69.9, 55.3, 26.4, 24.7. Calcd 92 HRMS for C 16 H 22 O 6 : 310.1416; Found: 310.1409. (3aR,4R,6R,6aR)-4-(4-Methoxybenzyloxy)-2,2-dimethyl-6-vinyl-tetrahydrofuro[ 3,4-d]-[1,3]dioxole (63). 61 (50 g, 0.16 mol) was dissolved in DCM (300 mL). DIPEA (33 mL, 0.19 mol), SO 3 ?Py (31 g, 0.19 mol) were added in portion. The mixture was stirred at the same temperature for 2 hours. Ice chilled water (50 mL) was added. The organic layer was separated, washed with ice water (3?50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated. The residue was filtered through a short silica column. The filtrate was concentrated to give intermediate 62, which was directly used in next step. (Ph) 3 PMeBr (70 g, 0.20 mol) was suspended in THF (300 mL). tert-BuOK (23 g, 0.20 mol) was added portionwise at 0 o C. The mixture was warmed to room temperature and stirred 3 hours. The solution was recooled to -78 o C. 62 in THF (100 mL) was added dropwise. The mixture was warmed to room temperature and stirred overnight. Water (50 mL) was added to quech the reaction. The solvent was removed under reduced pressure. The residue was extracted with EtOAc (3?200 mL). The combined organic layer was washed with water (100 mL), brine (100 mL), dried over sodium sulfate, and concentrated. The residue was purified by silica column (hexanse/EtOAc=10:1) to give 63 as a colorless oil (30 g, 60%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.29 (d, J=8.8 Hz, 2H), 6.93 (d, J=1.8 Hz, 2H), 6.03 (m, 1H), 5.36 (d, J=17.6 Hz, 1H), 5.23 (d, J=17.6 Hz, 1H), 5.20 (s, 1H), 4.18 (m, 2H), 4.50 (s, 2H), 4.46 (d, J=12.0 Hz, 1H), 3.84 (s, 3H), 1.53 (3H), 1.35 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.4, 137.2, 129.8, 129.3, 117.5, 130.4, 112.3, 106.9, 88.7, 85.7, 84.6, 68.5, 55.3, 26.5, 24.9. 2-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d] -[1,3]dioxol-4-yl)ethanol (64). To a solution of 63 (4.6 g, 15 mmol) in dry THF (20 mL) 93 at 0 ?C was added 9-borabicyclo[3.3.1]nonane (9-BBN) (36 mL, 0.5M in THF, 18 mmol) dropwise. The reaction mixture was stirred at room temperature for overnight and MeOH (10 mL), NaOH (3N aq, 20 mL) and H 2 O 2 (20 mL, 30% aq. solution) were added dropwise sequentially. The resulting mixture was evaporated after 4 hours at room temperature and extracted with EtOAc (3?50 mL). The combined organic layers were dried (Na 2 SO 4 ), filtered, concentrated, and purified by column chromatography. Elution with hexane-EtOAc (1:l) gave 64 (4.3 g, 88%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.28 (d, J=8.8 Hz, 2H), 6.86 (d, J=8.8 Hz, 2H), 5.17 (s, 1H), 4.72-4.64 (m, 3H), 4.54 (t, J=7.6 Hz, 2H), 3.85 (m, 2H), 3.83 (s, 3H), 2.07 (br, 1H), 1.98 (m, 1H), 1.87 (m, 1H), 1.53 (s, 3H), 1.32 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 129.7, 129.3, 113.9, 112.4, 107.5, 85.6, 85.4, 84.5, 69.2, 60.4, 55.3, 37.4, 26.5, 25.0; Anal. Cald for C 17 H 24 O 6 : C, 62.95; H, 7.46; Found: C, 63.13; H, 7.52. 2-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d] -[1,3]dioxol-4-yl)acetaldehyde (65). To a solution of 64 (3.8 g, 12 mmol), DMSO (20 mL) and DIPEA (5 mL) in CH 2 Cl 2 (100 mL) was added dropwise a solution of SO 3 ?Py (3.7 g, 23 mmol) in DMSO (20 mL) at 0 ?C. The reaction was then warmed to room temperature and stirred for 2 hours. The reaction mixture was then diluted with CH 2 Cl 2 (200 mL), washed with water (20 mL), saturated NaHCO 3 (30 mL) and brine (30 mL). The mixture was dried (Na 2 SO 4 ), filtered, concentrated, and purified by column chromatography. Elution with hexane-EtOAc (5:l) gave 65 (3.4 g, 93%) as a white solid. MP=87-88 ?C. 1 H NMR (CDCl 3 , 400 MHz) ? 9.83 (dd, J=2.4, 1.2 Hz, 1H), 7.29 (d, J=8.8 Hz, 2H), 6.92 (d, J=8.8 Hz, 2H), 5.17 (s, 1H), 4.80 (dd. J=8.8, 6.4 Hz, 1H), 4.74 (d, J=6.0 Hz, 1H), 4.64 (dd, J=8.0, 6.0 Hz, 2H), 4.41 (d, J=11.2 Hz, 1H), 3.83 (s, 3H), 2.94 94 (ddd, J=16.8, 8.8, 2.4 Hz, 1H), 2.76 (ddd, 16.8, 6.4, 1.2 Hz, 1H), 1.51 (s, 3H), 1.33 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 199.9, 159.4, 129.9, 128.9, 113.9, 112.7, 107.5, 85.6, 84.1, 81.7, 69.1, 55.3, 48.9, 26.5, 25.0; Anal. Cald for C 17 H 22 O 6 : C, 63.34; H, 6.88; Found: C, 63.55; H, 7.01. (R)-1-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3, 4-d]-[1,3]dioxol-4-yl)pent-4-en-2-ol (66). Allyl magnesiumbromide (18 mL, 1M in diethyl ether, 18 mmol) was added dropwise to solution of (+)-B-methoxy diisopinocampheylborane ((+)-Ipc 2 BOMe) (5.7 g, 18 mmol) in diethyl ether (100 mL) at 0 ?C. The reaction mixture stirred at room temperature for 1 hour to give a white suspension. This suspension was cooled to -78 ?C and 65 (3.4 g, 11 mmol) in THF (30 mL) was then added dropwise. The new reaction mixture was then stirred at the same temperature for 3 hours and allowed to warm to room temperature in 2 hours. MeOH (10 mL), followed by NaOH aqueous solution (3M, 25 mL) and H 2 O 2 (25 mL) was added into reaction mixture. The suspension was then stirred at room temperature for another 4 hours and concentrated. The residue was diluted with EtOAc (200 mL), washed with water (50 mL) and brine (50 ml), dried over Na 2 SO 4 , filtered, concentrated in vacuo, and purified by column chromatography. Elution with hexane-EtOAc (10:l to 5:1) gave 66 (3.2 g, 74 %) as colorless oil. 1 H NMR (CDCl 3 , 400 MHz) ? 7.30 (d, J=8.8 Hz, 2H), 6.91 (d, J=8.8 Hz, 2H), 5.87 (m, 1H), 5.17 (m, 3H), 4.74 (d, J=10.8, 1H), 4.69 (d, J=6.0 Hz, 1H), 4.62 (d, J=6.0 Hz, 1H), 4.46 (m, 2H), 3.93 (s, 1H), 3.83 (s, 3H), 2.76 (s, 1H), 2.33 (m, 2H), 1.91-1.76 (m, 2H), 1.51 (s, 3H), 1.33 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 134.5, 129.9, 129.2, 118.1, 113.9, 112.5, 107.6, 86.6, 85.5, 84.4, 69.7, 69.4, 55.3, 41.8, 41.1, 26.5, 25.0; Anal. Calcd for C 20 H 28 O 6 : C, 65.91; H, 7.74; Found: C, 65.48; H, 95 7.72. (3aR,4R,6R,6aR)-4-((S)-2-Azidopent-4-enyl)-6-(4-methoxybenzyloxy)-2,2-dimeth yl-tetrahydrofuro[3,4-d][1,3]dioxole (67). To a solution of 66 (2.7 g, 7.4 mmol) and triethylamine (3 mL) in anhydrous CH 2 Cl 2 (20 mL) was added MsCl (0.84 mL, 15 mmol) at 0?C. The reaction mixture became a yellow solution after stirring for 1 hour at room temperature. It was then diluted with CH 2 Cl 2 (20 mL) and washed with icy water (20 mL), dried (Na 2 SO 4 ) and filtered. Evaporation of filtrate provided a sticky yellow oil which was directly used for the next step. A suspension of above oil in dry DMF (20 mL) was added NaN 3 (2.0 g, 30.8 mmol). The mixture was stirred at 100 ?C overnight. The solvents was evaporated, quenched with water (10 mL), extracted with EtOAc (3?20 mL) and dried (Na 2 SO 4 ). The organic layers was filtered and concentrated to give yellow oil. The residue was purified by column chromatography with hexances-EtOAc (5:1) to give 67 (2.0 g, 89 %) as colorless oil%), 1 H NMR (CDCl 3 , 400 MHz) ? 7.30 (d, J=8.8 Hz, 2H), 6.92 (d, J=8.8 Hz, 2H), 5.82 (m, 1H), 5.18 (m, 3H), 4.70 (d, J=6.4 Hz, 1H) 4.66 (d, J=11.6 Hz, 1H), 4.61 (d, J=6.0 Hz, 1H), 4.49 (m, 2H), 3.83 (s, 3H), 3.67 (m, 1H), 2.38 (m, 2H), 1.87(m, 1H),1.61 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 133.4, 129.4, 129.2, 118.7, 113.9, 112.4, 107.9, 85.7, 84.4, 84.0, 69.5, 69.4, 59.4, 55.3, 39.4, 26.5, 25.0; Anal. Calcd for C 20 H 27 N 3 O 5 : C, 61.68; H, 6.99; N, 10.79; Found: C, 61.91; 7.02; N, 10.65. (S)-3-Azido-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydr ofuro[3,4-d][1,3]dioxol-4-yl)butan-1-ol (68). Method A: 67 (2.3 g, 6.0 mmol) was dissolved in MeOH/H 2 O (2:1) (50 mL) at 0 o C. NaIO 4 (3.2 g, 15 mmol) was added. OsO 4 (20 mg, 0.078 mmol) was added. The mixture was stirred at 0 o C for 2 hours. The mixture 96 was filtered. The filtrate was concentrated under reduced pressure and extracted with DCM (3?100 mL). The combined organic layer was washed with water (50 mL), brine (30 mL), dried over Na 2 SO 4 and concentrated. The residue was dissolved in MeOH (30 mL) at 0 o C. NaBH 4 (0.45 g, 12 mmol) was added portionwise. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 mL) was added. The mixture was filtered through a celite pad. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 68 (oil, 0.50 g, 20%), 69 (oil, 0.20 g, 10%), 70 (oil, 0.10 g, 5%) and a lot of intractable products. 68: 1 H NMR (CDCl 3 , 400 MHz) ? 7.28 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 5.18 (s, 1H), 4.72 (d, J=6.0 Hz, 1H), 4.67 (d, J=11.6Hz, 1H), 4.61 (d, J=6.0Hz, 1H), 4.48 (m, 2H), 3.84 (m, 1H), 3.83 (s, 3H), 3.76 (q, J=6.0 Hz, 2H), 2.02 (s, 1H), 1.88(ddd, J=14.0, 11.2, 3.2 Hz, 1H), 1.77 (m, 2H), 1.68 (ddd, J=14.0, 11.2, 3.6 Hz, 1H), 1.61 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 129.5, 129.3, 113.9, 112.4, 107.9, 85.6, 84.4, 84.0, 69.5, 59.2, 57.2, 55.3, 40.0, 37.5, 26.5, 25.0; Anal. Calcd for C 19 H 27 N 3 O 6 : C, 58.00; H, 6.92; N, 10.68; Found: C, 58.27; H, 7.14; N, 10.45; 69: 1 H NMR (CDCl 3 , 400 MHz) ? 9.55 (d, J=8.0 Hz, 1H), 7.23 (m, 2H), 6.89 (m, 2H), 6.23 (m, 1H), 5.12 (s, 1H), 4.72 (d, J=6.0 Hz, 1H), 4.60-4.65 (m, 2H), 4.38-4.46 (m, 2H), 3.82 (s, 3H), 2.76 (m, 1H), 2.65 (m, 1H), 1.50 (s, 3H), 1.33 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 193.7, 159.5, 153.6, 134.8, 129.6, 128.9, 113.9, 112.6, 107.5, 85.5, 85.4, 83.7, 69.3, 55.3, 38.4, 26.4, 24.9; 70: 1 H NMR (CDCl 3 , 400 MHz) ? 7.23 (m, 2H), 6.89 (m, 2H), 5.72-5.74 (m, 2H), 5.13 (s, 1H), 4.64-4.69 (m, 2H), 4.58 (m, 1H), 4.40 (m, 1H), 4.25 (m, 1H), 4.12 (m, 2H), 3.80 (s, 3H), 2.50 (m, 1H), 2.35 (m, 1H). 1.47 (s, 3H), 1.30 (s, 3H). 13 C NMR ?159.4, 132.2, 129.7, 129.3, 128.2, 113.9, 112.3, 86.8, 85.7, 83.5, 68.9, 63.6, 97 55.3, 37.9, 26.5, 24.9. Method B: 67 (1.9 g, 5.2 mmol) was dissolved in THF (50 mL). NMO (2.4 mL, 50% in water, 10 mmol) was added. OsO 4 (5 mg, 0.020 mmol) was added. The mixture was stirred at room temperature overnight. Sodium thiosulfate (5 g) was added to quench the reaction. The mixture was stirred for another 2 hours. The mixture was filtered through a short silica column (5 cm). The column was rinsed with EtOAc. The combined organic liquid was concentrated. The residue was dissolved in DCM/H 2 O (1:1, 30 mL). NaIO 4 (3.2 g, 15 mmol) was added at room temperature. The mixture was stirred 3 hours. The organic layer was diluted with DCM (100 mL), separated, washed with water (20 mL), dried over sodium sulfate and concentrated. The residue was dissolved in MeOH (30 mL) at 0 o C. NaBH 4 (450 mg, 12 mmol) was added portionwise. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 mL) was added. The mixture was filtered through a celite pad. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 68 exclusively as colorless oil (1.7 g, 89%) Method C: 67 (1.0 g, 2.6 mmol) was dissolved in tert-BuOH/H 2 O (1:1 30 mL). AD-mix-beta (3.6 g) was added at room temperature. The mixture was stirred at room temperature overnight. Sodium thiosulfate (5 g) was added. The mixture was stirred for another 2 hours. The mixture was filtered through celite pad. The pad was rinsed with tert-BuOH. The organic layer was sperated. NaIO 4 (0.80 g, 7.6 mmol) in water (10 mL) was added at room temperature. The mixture was stirred 3 hours. The mixture was filtered to remove precipitate. NaBH 4 (0.10 mg, 2.7 mmol) was added portionwise. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 98 mL) was added. The mixture was filtered through a celite pad. The solvent was removed under reduced pressure. The residue was extracted with EtOAc (3?50 mL). The combined organic layer was washed with water (10 mL), brine (50 mL), dried over sodium sulfate. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 68 exclusively as a colorless oil (0.72 g, 72%) (3aR,4R,6R,6aR)-4-((R)-2-Azido-4-iodobutyl)-6-(4-methoxybenzyloxy)-2,2-dimet hyl-tetrahydrofuro[3,4-d][1,3]dioxole (77). 68 (1.4 g, 3.5 mmol) was dissolved in PhMe/MeCN (1:1, 50 mL). Ph 3 P (1.0 g, 3.8 mmol), imidazole (1.2 g, 18 mmol) were added. I 2 chip was added until the solution turned purple. The mixture was stirred at room temperature for 2 hours. Water (10 mL), sodium thiosulfate (1 g) was added. The organic layer was separated. The aqueous phase was extracted with EtOAc (50 mL). The combined organic layer was dried over sodium sulfate and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 77 as white solid (1.1 g, 60%). MP= 99-100 ?C; 1 H NMR (CDCl 3 , 400 MHz) ? 7.29 (d, J=8.4 Hz, 2H), 6.93 (d, J=8.4 Hz, 2H), 5.20 (s, 1H), 4.74 (m, 2H), 4.69 (d, J=6.0 Hz, 1H), 4.61 (d, J=11.2 Hz, 1H), 4.48 (dd, J=11.2, 2.8 Hz, 1H), 3.84 (s, 3H), 3.73 (m, 1H), 3.28 (m, 2H), 1.97 (m, 2H), 1.87(dt, J=14.0, 2.7Hz, 1H), 1.70 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 129.5, 129.2, 114.0, 112.5, 107.9, 85.6, 84.4, 83.8, 69.6, 60.4, 55.3, 39.9, 39.0, 26.5, 25.0, 1.63; Anal. Cald for C 19 H 26 IN 3 O 5 ?0.6EtOAc: C, 46.17; H, 5.41; N, 7.55; Found: C, 46.12; H, 5.19; N, 7.42. (S)-3-Azido-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydr ofuro[3,4-d][1,3]dioxol-4-yl)butyl 4-Methylbenzenesulfonate (78). 68 (0.25 g, 0.64 mmol) was dissolved in DCM (10 mL). Triethylamine (TEA) (0.5 mL), p-toluenesulfonyl 99 chloride (TsCl) (0.24 g, 1.2 mmol), and 1,4-diazabicyclo[2.2.2]octane (DABCO) (20 mg) were added at room temperature. The mixture was stirred at room temperature for 30 minutes. Water (10 mL) was added. The mixture was stirred for 30 minutes. The organic layer was separated, washed with brine (5 mL), dried over sodium sulfate and concentrated. The residue was purified by silica column (hexanse/EtOAc=5:1) to give 78 as yellow oil (0.28 g, 79%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.78 (m, 2H), 7.32 (m, 2H), 7.21 (m, 2H), 6.89 (m, 2H), 5.15 (s, 1H), 4.67 (d, J=6.0 Hz, 1H), 4.57 (d, J=11.6 Hz, 1H), 4.53 (d, J=6.0 Hz, 1H), 4.38 (d, J=11.6 Hz, 1H), 4.37 (dd, J=11.2, 3.6 Hz, 1H), 4.12 (m, 2H), 3.80 (s, 3H), 3.71 (m, 1H), 2.43 (s, 3H), 1.65-1.84 (m, 4H), 1.48 (s, 3H), 1.30 (s, 3H). 13 C NMR (CDCl 3 , 100 MHz) ? 159.6, 145.3, 132.9, 130.2, 129.7, 128.1, 114.2, 112.7, 108.1, 85.8, 84.6, 83.9, 69.8, 66.8, 56.6, 55.5, 52.3, 40.5, 34.6, 26.6, 25.1, 21.8. tert-Butyl((R)-1-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahy drofuro[3,4-d][1,3]dioxol-4-yl)pent-4-en-2-yloxy)dimethylsilane (80). 66 (2.9 g, 8.1 mmol) was dissolved in DCM (50 mL). The solution was treated with imidazole (1.0 g, 15 mmol), TBSCl (1.8 g, 12 mmol) and DMAP (20 mg). The mixture was stirred at room temperature overnight. Water (10 mL) was added. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=20:1) to give 80 as colorless oil contaminated with tert-butyldimethylsilanol (TBSOH) (3.6 g, 93%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.20 (m, 2H), 6.82 (m, 2H), 5.78 (m, 1H), 5.04 (m, 3H), 4.6 (m, 2H), 4.56 (m, 1H), 4.35 (m, 2H), 3.85 (m, 1H), 3.76 (s, 3H), 2.27 (m, 2H), 1.82 (m, 1H), 1.73 (m, 1H), 1.42 (s, 3H), 1.25 (s, 3H), 0.87 (s, 9H), 0.06 (s, 6H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.5, 134.8, 129.9, 117.5, 114.0, 112.3, 107.2, 85.9, 84.5, 69.4, 68.9, 66.1, 55.5, 41.9, 100 41.7, 26.7, 26.2, , 25.8, 25.2, 18.2, 18.1, 15.5, -3.4, -3.5. (R)-3-(tert-Butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2 ,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butan-1-ol (81). 80 (1.1 g, 2.3 mmol) was dissolved in THF (50 mL). NMO (2.4 mL, 50% in water, 10.4 mmol) was added. OsO 4 (5 mg) was added. The mixture was stirred at room temperature overnight. Sodium thiosulfate (5 g) was added. The mixture was stirred for another 2 hours. The mixture was filtered through a short silica column (5 cm). The column was rinsed with EtOAc. The combined organic liquid was concentrated. The residue was dissolved in DCM/H 2 O (1:1, 30 mL). NaIO 4 (1.0 g, 4.7 mmol) was added at room temperature. The mixture was stirred vigorously 3 hours. The organic layer was diluted with DCM (100 mL), separated, washed with water (20 mL), dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH (30 mL) at 0 o C. NaBH 4 (0.24 mg, 6.5 mmol) was added portionwise. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 mL) was added. The mixture was filtered through celite. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 81 exclusively as a colorless oil (0.75 g, 68%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.25 (m, 2H), 6.87 (m, 2H), 5.09 (s, 1H), 4.64-4.67 (m, 2H), 4.58-4.60 (m, 1H), 4.42 (d, J=11.6 Hz, 1H), 4.3 (m, 1H), 4.12 (m, 2H), 3.80 (s, 3H), 3.75 (m, 1H), 2.22 (m, 1H), 1.83-1.95 (m, 2H), 1.45-1.79 (m, 2H), 1.45 (s, 3H), 1.29 (s, 3H), 0.87 (s, 9H), 0.09 (s, 3H), 0.06 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 129.5, 129.3, 113.9, 112.3, 107.3, 85.7, 84.5, 84.2, 69.1, 68.9, 59.9, 55.3, 41.9, 37.7, 26.5, 25.8, 25.0, 17.9, -4.4, -4.6. (R)-3-(tert-Butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2 101 ,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl 4-Methylbenzenesulfonate (82). 81 (2.0 g, 4.2 mmol) was dissolved in DCM (50 mL). TEA (2 mL), TsCl (1.2 g, 6.3 mmol), DABCO (20 mg) were added. The mixture was stirred at room temperature 30 minutes. Water (10 mL) was added. The mixture was stirred at room temperature 1 hour. The organic layer was separated, washed with brine (10 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=5:1) to give 82 as orange oil (2.4 g, 90%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.78 (m, 2H), 7.33 (m, 2H), 7.21 (m, 2H), 6.88 (m, 2H), 5.07 (s, 1H), 4.53-4.63 (m, 3H), 4.39 (d, J=11.6 Hz, 1H), 4.29 (m, 1H), 4.13 (m, 2H), 3.95 (m, 1H), 3.80 (s, 3H), 2.43 (s, 3H), 1.81-1.91 (m, 3H), 1.66-1.71 (m, 1H), 1.44 (s, 3H), 1.28 (s, 3H), 0.81 (s, 9H), 0.01 (s, 3H), -0.01 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.4, 144.7, 133.1, 129.8, 129.6, 129.3, 127.9, 113.9, 112.3, 107.2, 85.6, 84.5, 83.7, 69.0, 67.4, 66.1, 65.8, 55.3, 42.1, 35.6, 26.5, 25.7, 25.0, 21.6, 17.9, 15.3, -4.3, -4.8. (2S,5R)-2-((R)-3-(tert-Butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4-methoxyb enzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl)-3,6-diethoxy-5-i sopropyl-2,5-dihydropyrazine (83). (R)-3,6-Diethoxy-2-isopropyl-2,5-dihydropyrazine (1.2 mL, 5.7 mmol) was dissolved in THF (2 mL). The solution was cooled to -78 o C. n-BuLi (2.4 mL, 2.5 M in hexanes, 6.0 mmol) was added dropwise. The mixture was stirred at -78 o C 1 hour. 82 (2.4 g, 3.8 mmol) was dissolved in THF (5 mL) was added dropwise via syringe. The mixture was slowly warmed up to -30 o C and kept 2 hours, and then warmed to room temperature, stirred overnight. Water (10 mL) was added to quench the reaction. The mixture was extracted with ethyl ether (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and 102 concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 83 as colorless oil (2.2 g, 84%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.25 (m, 2H), 6.86 (m, 2H), 5.08 (m, 1H), 4.60-4.66 (m, 3H), 4.38-4.40 (m, 2H), 4.09-4.13 (m, 6H), 3.98 (m, 1H), 3.89 (m, 1H), 3.82 (m, 1H), 3.79 (s, 3H), 2.25 (m, 1H), 1.83-1.88 (m, 2H), 1.72-1.78 (m, 2H), 1.45 (s, 3H), 1.23-1.28 (m, 9H), 1.04 (d, J=6.8 Hz, 3H), 0.88 (s, 9H), 0.71 (d, J=6.8 Hz, 3H), 0.03 (s, 3H), 0.01 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.16, 163.14, 159.32, 141.6, 129.7, 129.4, 113.9, 112.1, 106.9, 85.8, 84.4, 84.3, 69.6, 68.7, 60.53, 60.49, 60.40, 55.3, 55.2, 41.8, 31.9, 31.5, 29.6, 26.5, 26.0, 25.9, 25.0, 19.1, 18.1, 16.6, 14.4, 14.2, -4.4, -4.5. (R)-4-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin-2-yl)-1-((3aR,4R,6R ,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d]-[1,3]dioxol-4-yl)b utan-2-ol (84). 83 (1.5 g, 2.2 mmol) was dissolved in THF (5 mL). TBAF (5 mL, 1.0M in THF, 5% water content) was added. The mixture was heated to 60 o C for 1 hour. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=5:1) to give 84 as colorless oil (1.1 g, 89%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.26 (m, 2H), 6.86 (m, 2H), 5.14 (s, 1H), 4.66-4.70 (m, 2H), 4.58-4.61 (m, 1H), 4.37-4.40 (m, 2H), 4.05-4.20 (m, 5H), 3.88-3.97 (m, 4H), 3.79 (s, 3H), 2.26 (m, 1H), 2.08 (m, 1H), 1.89 (m, 1H), 1.71 (m, 2H), 1.47 (s, 3H), 1.28 (m, 9H), 1.03 (d, J=6.8 Hz, 3H), 0.73 (d, J=6.8 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.8, 163.0, 159.4, 129.0, 129.3, 113.9, 112.4, 107.4, 86.2, 85.6, 84.4, 69.4, 69.2, 65.9, 61.0, 60.98, 55.3, 55.2, 41.8, 33.1, 32.1, 29.9, 26.5, 24.9, 19.1, 16.8, 14.4, 14.3; (R)-4-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin-2-yl)-1-((3aR,4R,6R ,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d]-[1,3]dioxol-4-yl)b 103 utan-2-yl 4-Methylbenzenesulfonate (85). 84 (0.19 g, 0.36 mmol) was dissolved in DCM (50 mL). TEA (2.0 mL), TsCl (0.12 g, 0.63 mmol), DABCO (20 mg) were added. The mixture was stirred at room temperature 30 minutes. Water (10 mL) was added to quench the reaction. The mixture was stirred at room temperature 1 hour. The organic layer was separated, washed with brine (10 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=5:1) to give 85 as orange oil (0.24 g, 93%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.78 (m, 2H), 7.24-7.28 (m, 4H), 6.85 (m, 2H), 5.06 (s, 1H), 4.83 (m, 1H), 4.58-4.62 (m, 2H), 4.51 (m, 1H), 4.35 (d, J=11.6 Hz, 1H), 4.05-4.20 (m, 7H), 3.90 (m, 1H), 3.86 (m, 1H), 3.79 (s, 3H), 2.40 (s, 3H), 2.23 (m, 1H), 2.13 (m, 1H), 1.95 (m, 1H), 1.72-1.85 (m, 2H), 1.42 (s, 3H), 1.27 (m, 9H), 0.88 (d, J=6.8 Hz, 3H), 0.69 (d, J=6.8 Hz, 3H). 13 C NMR (CDCl 3 , 100 MHz) ? 163.5, 162.7, 159.4, 144.4, 134.5, 129.8, 129.7, 129.3, 127.7, 113.9, 112.3, 107.2, 85.5, 84.4, 83.6, 81.4, 69.0, 65.9, 60.9, 60.5, 55.3, 54.9, 39.3, 31.9, 28.9, 28.7, 26.4, 24.9, 21.62, 19.1, 16.7, 15.3, 14.4; (2S,5R)-2-((S)-3-Azido-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2-dimethy l-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl)-3,6-diethoxy-5-isopropyl-2,5-dihydrop yrazine (79). 85 (6.0 g, 8.4 mmol) was dissolved in DMF. NaN 3 (1.3 g, 20 mmol) was added. The mixture was heated to 80 o C overnight. The solvent was removed under reduced pressure. Water (20 mL) was added to the residue. The mixture was extracted with EtOAc (3?100 mL). The combined organic layer was washed with water (50 mL), brine (50 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 79 as white solid (3.9 g, 78%). MP=65-67 o C. 1 H NMR (CDCl 3 , 400 MHz) ? 7.22 (m, 2H), 6.87 (m, 2H), 5.13 (m, 1H), 104 4.67 (d, J=6.0 Hz, 1H), 4.55-4.57 (m, 2H), 4.43-4.47 (m, 2H), 4.05-4.21 (m, 4H), 3.92-3.97 (m, 1H), 3.88-3.91 (m, 1H), 3.79 (s, 3H), 3.51-3.58 (m, 1H), 2.23-2.28 (m, 1H), 1.76-1.95 (m, 3H), 1.52-1.65 (m, 3H), 1.47 (s, 3H), 1.25 (m, 9H), 1.03 (d, J=6.8 Hz, 3H), 0.71 (d, J=6.8 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.4, 162.7, 159.4, 129.5, 129.2, 113.9, 112.4, 107.7, 85.6, 84.4, 84.2, 69.4, 60.9, 60.7, 60.6, 59.9, 55.2, 54.9, 39.9, 32.0, 30.5, 30.0, 26.5, 24.9, 19.1, 16.7, 14.4, 14.3; (3aR,4R,6R,6aR)-6-((R)-2-(tert-Butyldimethylsilyloxy)pent-4-enyl)-2,2-dimethyl- tetrahydrofuro[3,4-d][1,3]dioxol-4-ol (87). 80 (1.2 g, 2.5 mmol) was dissolved in DCM/H 2 O (20:1, 30 mL). 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) (0.63 g, 2.7 mmol) was added. The mixture was stirred vigorously at room temperature 3 hours. The precipitate was removed by filtration. The filtrate was diluted with DCM (100 mL) and washed with saturated NaHCO 3 solution (3?30 mL). The organic layer was dried over sodium sulfate and concentrated. The residue was purified by a very careful silica column (hexanes/EtOAc=20:1) to give 87 as colorless oil (mixture of ? and ? isomers, ?:?=6:1, 0.68 g, 76%). 1 H NMR (CDCl 3 , 400 MHz) ? 5.81 (m, 1H), 5.41 (d, J=2.4 Hz, 1H), 5.08 (d, J=6.0 Hz, 1H), 5.04 (m, 1H), 4.61 (m, 2H), 4.33 (t, J=8.4 Hz, 1H), 3.90 (m, 1H), 2.76 (d, J=2.4 Hz, 1H), 2.31-2.34 (m, 1H), 2.25-2.26 (m, 1H), 1.85 (m, 1H), 1.75 (m, 1H), 1.47 (s, 3H), 1.31 (s, 3H), 0.88 (s, 9H), 0.07 (s, 6H); 13 C NMR (CDCl 3 , 100 MHz) ? 134.7, 117.3, 112.3, 130.2, 86.2, 84.8, 84.4, 69.3, 42.0, 41.2, 26.5, 25.8, 24.9, 18.0, -4.4, -4.5. 9-((3aR,4R,6R,6aR)-6-((R)-2-(tert-Butyldimethylsilyloxy)pent-4-enyl)-2,2-dimeth yl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-6-chloro-9H-purine (88). 87 (0.20 g, 0.56 mmol) was dissolve in THF (10 mL). CCl 4 (0.06 mL, 0.7 mmol) was added. At -78 o C, 105 hexamethylphosphorous triamide (HMPT) (0.1 mL, 0.7 mmol) was added dropwise. The mixture was stirred at same temperature for 1 hour then warmed to 0 o C, stirred 1 hour. The solution was recooled to -78 o C. Sodium salt of 6-chloropurine in DMF (10 mL) (prepared by addition of 40 mg 60% NaH in mineral oil to 0.15 g 6-chloropurine in 10 mL DMF) was added dropwise. The mixture was warmed to room temperature. Water (20 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 88 as yellow oil (0.16 g, 60%). 1 H NMR (CDCl 3 , 400 MHz) ? 8.80 (s, 1H), 8.26 (s, 1H), 6.14 (d, J=2.4 Hz, 1H), 5.72-8.85 (m, 1H), 5.50 (m, 1H), 5.03-5.15 (m, 2H), 4.94 (m, 1H), 4.45 (m, 1H), 3.82 (m, 1H), 2.28 (m, 2H), 1.86 (m, 2H), 1.65 (s, 3H), 1.42 (s, 3H), 0.88 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 152.1, 151.6, 150.9, 144.5, 134.3, 117.5, 114.8, 112.2, 103.3, 90.7, 84.7, 84.4, 68.91, 41.2, 40.4, 27.1, 25.9, 25.8, 25.4, 24.9, 18.1, -4.4, -4.8. (R)-3-(tert-Butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(6-chloro-9H-purin-9-yl )-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butan-1-ol (89). 88 (0.90 g, 1.9 mmol) was dissolved in tert-BuOH/H 2 O (1:1, 20 mL). AD-mix-beta (2.8 g) was added. The mixture was stirred at room temperature overnight. Sodium thiosulfate (2 g) was added and stirred 1 hour. The mixture was extracted with EtOAc (3?30 mL). The organic layer was passed through a short silica pad (5 cm), concentrated, and dissolved in MeOH/H 2 O (1:1, 50 mL). NaIO 4 (089 g, 4.2 mmol) was added. The mixture was stirred vigorously at room temperature for 1 hour. The precipitate was removed by filtration. The solvent was removed at reduce pressure. The residue was extracted with EtOAc (3?30 106 mL). The combined organic layer was concentrated, and dissolved in MeOH. NaBH 4 (0.15 g, 4.1 mmol) was added portionwise at 0 o C. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (10 mL) was added. The mixture was filtered through celite. The solvent was removed at reduced pressure. The residue as extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 89 as yellow oil (0.78 g, 87%). 1 H NMR (CDCl 3 , 400 MHz) ? 8.88 (s, 1H), 8.31 (s, 1H), 6.18 (d, J=2.4 Hz, 1H), 5.54 (dd, J=6.4 Hz, 2.4 Hz, 1H), 5.02 (dd, J=6.4 Hz, 2.4 Hz, 1H), 4.42 (m, 1H), 4.07 (m, 1H), 3.82 (m, 2H), 2.03-2.14 (m, 3H), 1.90 (m, 1H), 1.80 (m, 1H), 1.71 (s, 3H), 1.48 (s, 3H), 0.93 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 152.2, 151.6, 150.9, 144.5, 132.5, 115.0, 90.7, 84.6, 84.4, 83.9, 68.3, 59.8, 40.9, 37.7, 27.2, 25.7, 25.4, 17.9, -4.8, -4.9. (R)-3-(tert-Butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-hydroxy-2,2-dimethyl-t etrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl 4-Methylbenzenesulfonate (91). 82 (0.20 g, 0.31 mmol) was dissolved in DCM/H 2 O (20:1, 30 mL). DDQ (0.11 g, 0.48 mmol) was added. The mixture was stirred vigorously at room temperature 3 hour. The precipitate was removed by filtration. The filtrate was diluted with DCM (100 mL) and washed with saturated NaHCO 3 solution (3?30 mL). The organic layer was dried over sodium sulfate and concentrated. The residue was purified by a silica column (hexanes/EtOAc=3:1) to give 91 as organge oil (mixture of ? and ? isomers, ?:?=5:1, 0.12 g, 75%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.80 (m, 2H), 7.35 (m, 2H), 5.42 (s, 1H), 4.53-4.63 (m, 2H), 4.25 (m, 1H), 4.15 (m, 2H), 3.95 (m, 1H), 2.68 (d, J=2.8 Hz, 1H), 107 2.45 (s, 3H), 1.85-1.95 (m, 2H), 1.75-1.82 (m, 1H), 1.62-1.67 (m, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 0.82 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 144.7, 133.1, 129.8, 127.9, 112.4, 103.2, 86.1, 84.9, 83.7, 67.6, 66.1, 42.4, 35.3, 26.5, 25.7, 24.9, 21.6, 17.9, -4.4, -4.8. (3aR,4S,6R,6aR)-6-((R)-2-(tert-Butyldimethylsilyloxy)-4-(tosyloxy)butyl)-2,2-dim ethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl Pivalate (92). 91 (1.5 g, 2.9 mmol) was dissolved in DCM (50 mL). Pivaloyl chloride (PivCl) (0.54 mL, 4.4 mmol), DABCO (0.50 g, 4.5 mmol) were added. The mixture was stirred at room temperature overnight. Water (20 mL) was added and stirred 1 hour to quench the reaction. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 92 as orange oil (1.2 g, 69%). 1 H NMR (CDCl 3 , 250 MHz) ? 7.80 (m, 2H), 7.35 (m, 2H), 6.07 (d, J=4.8 Hz, 1H), 4.80 (m, 1H), 4.40 (m, 2H), 4.21 (m, 1H), 4.10 (m, 1H), 3.95 (m, 1H), 2.45 (s, 3H), 1.85 (m, 2H), 1.73 (m, 2H), 1.53 (s, 3H), 1.34 (s, 3H), 1.25 (s, 9H), 0.82 (s, 9H), 0.03 (s, 3H), -0.01 (s, 3H); 13 C NMR (CDCl 3 , 60 MHz) ? 177.3, 144.7, 133.0, 129.8, 127.9, 116.6, 95.6, 83.8, 80.3, 79.8, 67.4, 65.7, 40.2, 38.9, 35.8, 27.1, 26.4, 25.7, 25.3, 21.6, 17.9, -4.4, -4.5; (R)-4-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3, 4-d][1,3]dioxol-4-yl)butane-1,3-diyl bis(4-Methylbenzenesulfonate) (94). A solution of 81 (5.0 g, 10 mmmol) was treated with TBAF (13 mL, 1M in THF, 5% water content, 13 mmol) at room temperature, stirred 1 hour. Water (10 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (2?20 mL), dried over sodium sulfate and concentrated. The 108 residue was filtered through a short silica column (10 cm, elute with hexanes/EtOAc=2:1). The fluorescent fraction was collected, concentrated and dissolved in DCM (50 mL). TEA (10 mL), TsCl (6.0 g, 32 mmol), DABCO (50 mg) were added. The mixture was stirred at room temperature 1 hour and quenched with water (10 mL). The organic layer was separated, washed with brine (10 mL), dried over sodium sulfate and concentrated. The residue was purified by silica column (hexanse/EtOAc=5:1) to give 94 as orange gel (4.7 g, 70%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.75 (m, 4H), 7.33 (m, 6H), 6.88 (m, 2H), 5.05 (s, 1H), 4.86 (m, 1H), 4.61 (m, 2H), 4.46 (m, 1H), 4.37 (m, 1H), 4.12 (m, 2H), 3.95 (m, 1H), 3.80 (s, 3H), 2.44 (s, 3H), 2.42 (s, 3H), 2.05 (m, 3H), 1.86 (m, 1H), 1.42 (s, 3H), 1.27 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 171.1, 159.4, 144.9, 133.7, 132.9, 130.0, 129.9, 129.7, 129.2, 127.9, 127.8, 113.9, 112.4, 107.4, 85.4, 84.3, 83.1, 69.2, 65.9, 55.3, 52.2, 45.9, 39.5, 33.1, 26.5, 24.9, 21.7. (R)-4-((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydrofu ro[3,4-d]-[1,3]dioxol-4-yl)butane-1,3-diyl bis(4-Methylbenzenesulfonate) (95). 94 (2.0 g, 3.2 mmol) was dissolved in DCM/H 2 O (20:1, 30 mL). DDQ (1.1 g, 4.8 mmol) was added. The mixture was stirred vigorously at room temperature 3 hour. The precipitate was removed by filtration. The filtrate was diluted with DCM (100 mL) and washed with saturated NaHCO 3 solution (3?30 mL). The organic layer was dried over sodium sulfate and concentrated. The residue was purified by a silica column (hexanes/EtOAc=3:1) to give the intermediate as orange oil. This intermediate was dissolved in dry DCM (50 mL) and treated with imidazole (0.42 g, 6.4 mmol), DMAP (20 mg) and TBSCl (0.72 g, 4.8 mmol) at room temperature. The mixture was stirred overnight. Water (10 mL) was added to quench the reaction. The organic layer was 109 separated, washed with brine (20 mL), dried over sodium sulfate, and concentrated. The residue was purified by silica column (hexanes/EtOAc=10:1) to give 95 as an orange oil (1.2 g, 56%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.74 (m, 4H), 7.33 (m, 4H), 5.29 (s, 1H), 4.81 (m, 1H), 4.47 (m, 2H), 4.10 (m, 1H), 4.05 (m, 1H), 3.94 (m, 1H), 2.45 (s, 3H), 2.44 (s, 3H), 1.97-2.04 (m, 4H), 1.43 (s, 3H), 1.29 (s, 3H), 0.87 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 145.1, 145.0, 133.8, 132.9, 130.1, 130.0, 128.0, 127.9, 112.6, 103.6, 87.5, 84.5, 82.7, 77.1, 66.2, 39.6, 33.6, 26.7, 25.8, 25.2, 21.8, 18.1, -4.2, -5.3. (R)-1-((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydrofu ro-[3,4-d][1,3]-dioxol-4-yl)-4-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihydropyrazin-2 -yl)-butan-2-yl 4-Methylbenzenesulfonate (96). (R)-3,6-diethoxy-2-isopropyl-2,5-dihydropyrazine (0.96 mL, 4.5 mmol) was dissolved in THF (2 mL). The solution was cooled to -78 o C. n-BuLi (2.0 mL, 2.5 M in hexanes, 6.0 mmol) was added in dropwise. The mixture was stirred at -78 o C 1 hour. 95 (2.4 g, 3.8 mmol) was dissolved in THF (5 mL) was added dropwise via syringe. The mixture was slowly warmed up to -30 o C and kept 2 hours, and then warmed to room temperature, stirred overnight. Water (10 mL) was added. The mixture was extracted with ethyl ether (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 96 as orange oil (2.0 g, 62%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.80 (m, 2H), 7.30 (m, 2H), 5.30 (s, 1H), 4.75 (m, 1H), 4.55 (m, 1H), 4.45 (m, 1H), 4.05-4.18 (m, 5H), 3.85-3.95 (m, 2H), 2.42 (s, 3H), 2.24 (m, 1H), 1.95-2.05 (m, 2H), 1.65-1.72 (m, 4H), 1.45 (s, 3H), 1.25 (m, 9H), 1.03 (d, J=6.8 Hz, 3H), 0.88 (s, 9H), 0.68 110 (d, J=6.8 Hz, 3H), 0.11 (s, 3H), 0.09 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.4, 162.7, 144.3, 134.5, 129.7, 127.7, 112.3, 103.4, 87.5, 84.3, 83.1, 81.2, 60.8, 60.6, 60.5, 60.3, 54.7, 69.5, 31.9, 26.5, 25.7, 25.0, 21.6, 21.0, 19.0, 17.9, 16.7, 14.4, 14.3, -4.25, -5.3. (3aR,4R,6R,6aR)-6-((S)-2-Azido-4-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihydro pyrazin-2-yl)butyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-ol (86). 96 (1.2 g, 1.7 mmol) was dissolved in DMF (10 mL). NaN 3 (0.50 g, 7.7 mmol) was added. The mixture was stirred at 80 o C overnight. The solvent was removed at reduced pressure. TBAF (5.0 mL, 1.0M in THF, 5% water content, 5.0 mmol) was added. The mixture was heated to 60 o C for 2 hour. The solvent was removed under reduced pressure. The residue was purified with silica column (hexanes/EtOAc=5:1) to give 86 as colorless oil (mixture of two isomers, beta:alpha=3:1, 0.66 g, 83%). 1 H NMR (mixture of two isomers, CDCl 3 , 250 MHz) ? 5.44 (m, 1H), 4.57-4.65 (m, 2H), 4.33-4.38 (m, 2H), 4.05-4.20 (m, 5H), 3.92 (m, 2H), 2.45 (m, 1H), 2.25 (m, 1H), 1.87 (m, 2H), 1.6 (m, 3H), 1.48 (s, 3H), 1.3 (m, 9H), 1.02 (d, J=6.8 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H); 13 C NMR (mixture of two isomers, CDCl 3 , 62 MHz) ? 163.7, 163.5, 162.9, 162.8, 114.9, 112.4, 103.3, 95.6, 86.1, 85.0, 84.7, 84.5, 84.3, 84.0, 79.3, 77.2, 68.5, 67.7, 60.9, 60.8, 60.76, 60.70, 60.4, 59.4, 54.8, 54.6, 53.7, 39.8, 37.6, 32.0, 31.9, 30.4, 30.2, 30.0, 27.8, 26.5, 25.0, 24.9, 23.9, 22.2, 20.8, 19.1, 16.8. 9-((3aR,4R,6R,6aR)-6-((S)-2-Azido-4-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihy dropyrazin-2-yl)butyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-6-chloro- 9H-purine (97). 86 (0.17 g, 0.36 mmol) was dissolve in THF (10 mL). CCl 4 (0.04 mL, 0.4 mmol) was added. At -78 o C, HMPT (0.08 mL, 0.4 mmol) was added dropwise. The mixture was stirred at same temperature for 1 hour then warmed to 0 o C, stirred 1 hour. The solution was recooled to -78 o C. Sodium salt of 6-chloropurine in DMF (10 mL) 111 (prepared by addition of 40 mg 60% NaH in mineral oil to 0.15 g 6-chloropurine in 10 mL DMF) was added dropwise. The mixture was warmed up to room temperature. Water (20 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 97 as yellow oil (0.13 g, 60%). 1 H NMR (CDCl 3 , 250 MHz) ? 8.89 (s, 1H), 8.63 (s, 1H), 6.61 (d, J=4.3 Hz, 1H), 5.05 (m, 1H), 4.73 (m, 2H), 4.05-4.25 (m, 4H), 3.92 (m, 1H), 3.62 (m, 1H), 2.26 (m, 2H), 1.91 (m, 2H), 1.64-1.73 (m, 4H), 1.26-1.33 (m, 12H), 1.03 (d, J=6.8 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H). 13 C NMR (CDCl 3 , 62 MHz) ? 163.8, 163.8, 152.5, 147.9, 142.5, 121.9, 114.7, 87.4, 84.6, 81.1, 80.5, 61.1, 60.9, 60.8, 60.6, 59.2, 54.8, 37.2, 32.2, 30.5, 30.0, 25.7, 24.6, 19.2, 16.9, 14.5, 14.4. 9-((3aR,4R,6R,6aR)-6-((S)-2-Azido-4-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihy dropyrazin-2-yl)butyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin -6-bis(tert-butoxylcarbobyl)amine (98). 86 (0.80 g, 1.8 mmol) was dissolve in THF (10 mL). CCl 4 (0.21 mL, 2.2 mmol) was added. At -78 o C, HMPT (0.40 mL, 2.3 mmol) was added dropwise. The mixture was stirred at same temperature for 1 hour then warmed to 0 o C, stirred 1 hour. The solution was recooled to -78 o C. Sodium salt of Ad(Boc) 2 in DMF (10 mL) (prepared by addition of 200 mg 60% NaH in mineral oil to 1.5 g Ad(Boc) 2 in 10 mL DMF) was added dropwise. The mixture was warmed to room temperature. Water (20 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 98 as yellow oil (1.3 g, 60%). 1 H NMR (CDCl 3 , 112 250 MHz) ? 8.86 (s, 1H), 8.14 (s, 1H), 6.08 (d, J=2.5 Hz, 1H), 5.46 (m, 1H), 4.92 (m, 1H), 4.38-4.42 (m, 1H), 4.05-4.15 (m, 4H), 3.92 (m, 1H), 3.35 (m, 1H), 2.25 (m, 2H), 1.75-1.90 (m, 6H), 1.47 (s, 21H), 1.39 (s, 3H), 1.25 (m, 6H), 1.03 (d, J=6.8 Hz, 3H), 0.69 (d, J=6.8 Hz, 3H). 13 C NMR (CDCl 3 , 62 MHz) ? 163.6, 162.9, 152.5, 152.4, 150.9, 150.6, 144.3, 129.8, 115.2, 90.7, 84.4, 84.1, 84.0, 83.9, 60.8, 60.7, 60.6, 59.5, 54.9, 38.4, 32.2, 30.4, 30.1, 27.9, 27.4, 25.6, 19.2, 16.9, 14.5, 14.4. (2S,5S)-2-Amino-6-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxy-tet rahydrofuran-2-yl)-5-azidohexanoic Acid (99). 98 (0.20 g, 0.34 mmol) was dissolved in TFA/H 2 O (2:1, 2 mL) at -20 o C. The solution was stirred at 0 o C 6h. IRA-67 ion exchange resin was added until pH=7. The solvent was removed at reduce pressure. The residue was dissolved in MeOH/H 2 O (2:1, 2 mL). K 2 CO 3 (0.10 g, 0.72 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed at reduce pressure. The residue was purified with silica column (EtOAc/MeOH/NH 4 OH (29.6%) =2:1:0.5) to give 99 as white solid (0.10 g, 78%). Mp>200 o C (decomposed). 1 H NMR (D 2 O, 400 MHz) ? 8.32 (s, 1H), 8.27 (s, 1H), 6.07 (d, J=5.2 Hz, 1H), 4.85 (m, 1H), 4.33 (m, 1H), 4.15 (m, 1H), 3.80 (m, 1H), 3.65 (m, 1H), 2.00-2.10 (m, 6H). 13 C NMR (D 2 O, 250 MHz) ? 174.4, 155.2, 152.2, 149.0, 140.8, 119.2, 88.2, 81.5, 73.7, 73.6, 69.8, 54.7, 37.5, 29.4, 27.3. (3aR,4R,6R,6aR)-4-(Allyloxymethyl)-6-(4-methoxybenzyloxy)-2,2-dimethyl-tetra hydrofuro[3,4-d][1,3]dioxole (101). 61 (10.02 g, 32.32 mmol) was dissolved in DMF (50 mL). NaH (1.54 g, 38.5 mmol, 60% in mineral oil) was added in portions. AllylBr (5.50 mL, 64.1 mmol) was added dropwise via a syringe. The mixture was stirred at room temperature 12 hours. Water (10 mL) was added to quench the reaction. The mixture was 113 extracted with ethyl ether (3?100 mL). The combined organic layer was washed with water (3?20 mL), brine (50 mL), dried over sodium sulfate, and concentrated. The residue was purified by silica column (hexanes/EtOAc=20:1) to give 101 as colorless oil (8.97 g, 79.5%). 1 H NMR (CDCl 3 , 250 MHz) ? 7.22 (m, 2H), 6.89 (m, 2H), 5.85-5.92 (m, 1H), 5.13-5.31 (m, 3H), 4.61-4.68 (m, 3H), 4.37-4.46 (m, 2H), 4.02 (m, 2H), 3.82 (s, 3H), 3.50 (m, 2H), 1.48 (s, 3H), 1.30 (s, 3H); 13 C NMR (CDCl 3 , 62 MHz) ? 159.4, 134.6, 129.8, 129.2, 117.3, 113.8, 112.3, 106.9, 85.3, 85.2, 82.2, 72.2, 71.0, 68.8, 55.3, 26.4, 24.9; Calcd HRMS for C 19 H 26 O 6 (M-CH 3 ): 335.1499; Found: 335.1445. 2-(((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d ]-[1,3]dioxol-4-yl)methoxy)ethanol (102). 101 (7.11 g, 20.31 mmol) was dissolved in THF (50 mL). NMO (7.20 mL, 50% in water, 31.2 mmol) was added. OsO 4 (20 mg, 0.078 mmol) was added. The mixture was stirred at room temperature overnight. Sodium thiosulfate (10 g) was added. The mixture was stirred for another 2 hours. The mixture was filtered through a short silica column (5 cm). The column was rinsed with EtOAc. The combined organic liquid was concentrated. The residue was dissolved in DCM/H 2 O (1:1, 30 mL). NaIO 4 (6.40 g, 30.0 mmol) was added at room temperature. The mixture was stirred 3 hours. The organic layer was diluted with DCM (100 mL), separated, washed with water (20 mL), dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH (30 mL) at 0 o C. NaBH 4 (1.80 g, 48.6 mmol) was added portionwise. The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 mL) was added. The mixture was filtered through celite. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 102 exclusively as a colorless oil (5.41 g, 75.2%). 1 H 114 NMR (CDCl 3 , 400 MHz) ? 7.25 (m, 2H), 6.88 (m, 2H), 5.14 (s, 1H), 4.69 (d, J=6.0 Hz, 1H), 4.62-4.65 (m, 2H), 4.36-4.40 (m, 2H), 3.80 (s, 3H), 3.72 (m, 2H), 3.54-3.60 (m, 4H), 2.38 (m, 1H), 1.47 (s, 3H), 1.31 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.6, 129.9, 129.3, 114.1, 112.6, 107.5, 85.7, 85.5, 82.3, 72.6, 72.4, 69.2, 61.9, 55.5, 26.7, 25.2; Calcd HRMS for C 18 H 26 O 7 (M-CH 3 ): 339.1444; Found: 339.1444. 2-(((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyl-tetrahydrofuro[3,4-d ]-[1,3]dioxol-4-yl)methoxy)ethyl 4-Methylbenzenesulfonate (103). 102 (6.05 g, 17.0 mmol), TEA (20 mL), TsCl (3.90 g, 20.5 mmol), DABCO (50 mg) were mixed in DCM (100 mL) and stirred at room temperature 30 minutes. Water (10 mL) was added. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate and concentrated. The residue was purified by silica column (hexanse/EtOAc=5:1) to give 103 as organe oil (7.15 g, 82.3%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.80 (m, 2H), 7.31 (m, 2H), 7.22 (m, 2H), 6.87 (m, 2H), 5.10 (s, 1H), 4.55-4.60 (m, 3H), 4.37 (d, J=11.6 Hz, 1H), 4.24 (m, 1H), 4.09-4.16 (m, 2H), 3.80 (s, 3H), 3.67 (m, 2H), 3.43 (m, 2H), 2.43 (s, 3H), 1.47 (s, 3H), 1.30 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 159.6, 145.0, 133.2, 130.0, 129.9, 129.4, 128.2, 114.1, 112.6, 107.2, 85.5, 85.1, 82.3, 72.4, 69.3, 69.0, 68.9, 55.5, 26.6, 25.1, 21.8; Calcd HRMS for C 25 H 32 O 9 S: 508.1767; Found: 508.1774. 2-(((3aR,4R,6R,6aR)-6-Hydroxy-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4 -yl)methoxy)ethyl 4-Methylbenzenesulfonate (104). 103 (4.87 g, 9.59 mmol) was dissolved in DCM/H 2 O (20:1, 30 mL). DDQ (1.10 g, 4.85 mmol) was added. The mixture was stirred vigorously at room temperature 3 hours. The precipitate was removed by filtration. The filtrate was diluted with DCM (100 mL) and washed with saturated NaHCO 3 solution (3?30 mL). The organic layer was dried over sodium sulfate and 115 concentrated. The residue was purified by a silica column (hexanes/EtOAc=3:1) to give 104 as orange oil (?:?=6:1, 2.88 g, 77.4%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.82 (m, 2H), 7.37 (m, 2H), 5.27 (d, J=10.8 Hz, 1H), 4.71 (m, 1H), 4.45 (d, J=5.6 Hz, 1H), 4.32 (m, 1H), 4.11-4.20 (m, 3H), 3.72-3.78 (m, 2H), 3.55-3.65 (m, 2H), 2.45 (s, 3H), 1.48 (s, 3H), 1.31 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 145.2, 132.6, 129.9, 128.0, 112.1, 103.9, 87.2, 85.3, 81.8, 72.6, 69.1, 68.2, 26.4, 24.8, 21.7; Calcd HRMS for C 17 H 24 O 8 S (M+NH 4 ): 406.1537; Found: 406.1536. 2-(((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydrofuro- [3,4-d][1,3]dioxol-4-yl)methoxy)ethyl 4-Methylbenzenesulfonate (105). 104 (1.54 g, 3.97 mmol) was dissolved in dry DCM (60 mL). DMAP (20 mg) was added. The solution was treated with imidazole (0.68 g, 10.4 mmol), and TBSCl (1.21 g, 8.07 mmol) at 0 o C. The solution was then warmed to room temperature and stirred for 2 hours. Water (20 mL) was added to quench the reaction. The organic layer was separated, washed with brine, dried over sodium sulfate, concentrated under reduce pressure. The residue was purified by silica column (hexanes/EtOAc=5:1) to give 105 as a colorless oil (1.32 g, 66.2%). 1 H NMR (CDCl 3 , 400 MHz) ? 7.79 (m, 2H), 7.35 (m, 2H), 5.34 (s, 1H), 4.65 (dd, J=6.0, 0.4 Hz, 1H), 4.49 (d, J=6.0 Hz, 1H), 4.13 (m, 3H), 3.66 (m, 2H), 3.43 (m, 2H), 2.45 (s, 3H), 1.47 (s, 3H), 1.32 (s, 3H), 0.87 (s, 9H), 0.10 (s, 3H), 0.06 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 144.8, 132.9, 129.9, 128.0, 112.3, 103.3, 87.2, 84.9, 82.5, 72.6, 69.1, 68.7, 26.5, 25.7, 25.1, 21.7, 17.9, -4.3, -5.4; Calcd HRMS for C 23 H 38 O 8 SSi (M+NH 4 ): 520.2406; Found: 520.2400. (2S,5R)-2-(2-(((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetra hydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)ethyl)-3,6-diethoxy-5-isopropyl-2,5-dihydr 116 opyrazine (106). (R)-3,6-Diethoxy-2-isopropyl-2,5-dihydropyrazine (1.50 mL, 7.04 mmol) was dissolved in THF (3 mL). The solution was cooled to -78 o C. n-BuLi (3.00 mL, 2.5 M in hexanes, 7.50 mmol) was added in dropwise. The mixture was stirred at -78 o C for 1 hour. 105 (2.76 g, 5.49 mmol) was dissolved in THF (5 mL) was added dropwise via syringe. The mixture was slowly warmed up to -30 o C and kept 2 hours, and then warmed to room temperature, stirred overnight. Water (10 mL) was added to quench the reaction. The mixture was extracted with ethyl ether (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=10:1) to give 106 as orange oil, which contaminated with (R)-3,6-diethoxy-2-isopropyl-2,5-dihydropyrazine (2.03 g, ?68.4%?). This product was used directly in next step without further purification. 1 H NMR (CDCl 3 , 400 MHz) ? 5.35 (s, 1H), 4.72 (m, 1H), 4.51 (m, 1H), 3.85-4.25 (m, 6H), 3.42-3.65 (m, 4H), 2.25 (m, 2H), 2.15 (m, 1H), 1.85 (m, 1H), 1.47 (s, 3H), 1.25 (m, 9H), 1.03 (d, J= 6.8 Hz, 3H), 0.89 (s, 9H), 0.77 (d, J= 6.8 Hz, 3H), 0.11 (s, 3H), 0.09 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 164.2, 163.4, 163.1, 161.8, 112.1, 103.3, 87.3, 85.1, 82.7, 72.0, 67.7, 61.0, 60.7, 60.6, 60.5, 52.6, 46.7, 34.1, 32.6, 31.9, 26.5, 25.6, 25.0, 19.1, 19.0, 17.8, 17.0, 16.7, 14.3, -4.3, -5.5. Calcd HRMS for C 27 H 50 N 2 O 7 Si: 542.3387; Found: 542.3379. (3aR,4R,6R,6aR)-6-((2-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin-2- yl)ethoxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (107). 106 (1.72 g mixture, ?3.17? mmol) was dissolved in THF (5 mL). TBAF (10.0 mL, 1M in THF) was added. The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The residue was purified by silica column 117 (hexanes/EtOAc=3:1) to give 107 as colorless oil (0.81 g, 59.7%). 1 H NMR (CDCl 3 , 250 MHz) ? 5.28 (d, J=11.0 Hz, 1H), 4.95 (d, J=11.0 Hz, 1H), 4.75 (d, J=6.0 Hz, 1H), 4.37 (m, 1H), 4.00-4.22 (m, 5H), 3.85-3.95 (m, 1H), 3.55-3.72 (m, 4H), 2.35-2.45 (m, 1H), 2.15-2.30 (m, 2H), 1.78-1.90 (m, 1H), 1.48 (s, 3H), 1.28 (m, 9H), 1.03 (d, J=6.8 Hz, 3H), 0.71 (d, J=6.8 Hz, 3H); 13 C NMR (CDCl 3 , 60 MHz) ? 163.6, 162.9, 111.9, 103.8, 87.5, 85.6, 81.9, 71.9, 68.8, 60.8, 53.9, 52.3, 33.6, 29.2, 26.4, 24.8, 20.8, 19.1, 16.8, 14.4, 14.3; Calcd HRMS for C 21 H 36 N 2 O 7 : 428.2523; Found: 425.2518. 6-Chloro-9-((3aR,4R,6R,6aR)-6-((2-((2S,5R)-3,6-diethoxy-5-isopropyl-2,5-dihydr opyrazin-2-yl)ethoxy)methyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9 H-purine (108). 107 (0.18 g, 0.42 mmol) was dissolve in THF (2 mL). CCl 4 (0.15 mL, 1.56 mmol) was added. At -78 o C, HMPT (0.09 mL, 0.50 mmol) was added dropwise. The mixture was stirred at same temperature for 1 hour then warmed to 0 o C, stirred 1 hour. The solution was recooled to -78 o C. Sodium salt of 6-chloropurine in DMF (10 mL) (prepared by addition of 260 mg 60% NaH in mineral oil to 1.00 g 6-chloropurine in 10 mL DMF) was added dropwise. The mixture was warmed to room temperature. Water (20 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (3?30 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=3:1) to give 108 as yellow oil (0.07 g, 28%). 1 H NMR (CDCl 3 , 400 MHz) ? 8.72 (s, 1H), 8.41 (s, 1H), 6.64 (m, 1H), 4.91 (m, 1H), 4.51 (m, 1H), 4.05-4.21 (m, 6H), 3.90 (m, 1H), 3.60-3.72 (m, 4H), 2.18-2.25 (m, 2H), 1.90 (m, 1H), 1.43 (s, 3H), 1.28 (m, 9H), 1.03 (d, J=7.2 Hz, 3H), 0.71 (d, J=7.2 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.4, 163.0, 151.9, 151.1, 150.7, 145.1, 131.5, 113.4, 86.5, 82.5, 82.2, 79.8, 118 72.7, 68.8, 60.8, 52.7, 34.1, 31.9, 25.7, 24.0, 19.1, 16.7, 14.4, 14.3, 14.2, 14.1; Calcd HRMS for C 26 H 37 ClN 6 O 6 : 564.2463; Found: 564.2448. 9-((3aR,4R,6R,6aR)-6-((2-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin- 2-yl)ethoxy)methyl)-2,2-dimethyl-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-6-bis(ter t-butoxylcarboxyl)aminopurine (109). 107 (0.61 g, 1.43 mmol) was dissolve in THF (10 mL). CCl 4 (0.20 mL, 2.0 mmol) was added. At -78 o C, HMPT (0.35 mL, 2.0 mmol) was added dropwise. The mixture was stirred at same temperature for 1 hour then warmed to 0 o C, stirred 1 hour. The solution was recooled to -78 o C. Sodium salt of Ad(Boc) 2 in DMF (10 mL) (prepared by addition of 260 mg 60% NaH in mineral oil to 1.80 g Ad(Boc) 2 in 10 mL DMF) was added dropwise. The mixture was warmed to room temperature and stirred overnight. Water (20 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (3?30 mL), dried over sodium sulfate and concentrated. The residue was purified with silica column (hexanes/EtOAc=3:1) to give 109 as yellow oil (0.23 g, 25%). 1 H NMR (CDCl 3 , 400 MHz) ? 8.88 (s, 1H), 8.38 (s, 1H), 6.28 (d, J=2.8 Hz, 1H), 5.23 (m, 1H), 4.95 (m, 1H), 4.52 (m, 1H), 3.95-4.23 (m, 6H), 3.55-3.65 (m, 4H), 2.25 (m, 1H), 2.28 (m, 1H), 1.82 (m, 1H), 1.65 (s, 3H), 1.38-1.48 (m, 27H), 1.02 (d, J=7.2 Hz, 3H), 0.70 (d, J=7.2 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.3, 163.0, 152.9, 152.3, 150.4, 150.3, 143.4, 133.6, 137.0, 129.3, 114.2, 91.4, 85.8, 85.1, 83.7, 81.8, 70.9, 68.4, 60.75, 60.71, 60.62, 60.56, 52.6, 33.8, 31.9, 27.8, 27.3, 25.3, 19.1, 14.4, 14.3; Calcd HRMS for C 36 H 55 N 7 O 10 : 745.4010; Found: 740.3993. (S)-2-Amino-4-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxy-tetrah ydrofuran-2-yl)methoxy)butanoic Acid (6). 109 (0.10 g, 0.92 mmol) was dissolved in 119 TFA/H 2 O (2:1, 2 mL) at -20 o C. The solution was stirred at 0 o C for 6 hours. IRA-67 ion exchange resin was added to neutralize the mixture. The resin was removed by filtration. The solvent was removed at reduce pressure. The residue was dissolved in MeOH/H 2 O (2:1, 2 mL). K 2 CO 3 (0.20 g, 1.4 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed under reduced pressure. The residue was purified with silica column (EtOAc/MeOH/NH 4 OH (29.6%) =1:1:1) to give 6 as white foam (0.030 g, 61%). m.p.>210 o C (decomposed). 1 H NMR (D 2 O, 250 MHz) ? 8.35 (s, 1H), 8.22 (s, 1H), 6.07 (d, J=5.0 Hz, 1H), 4.78 (m, 2H), 4.43 (m, 1H), 4.32 (m, 1H), 3.65-3.85 (m, 4H), 2.15-2.25 (m, 2H). 13 C NMR (D 2 O, 62 MHz) ? 175.3, 155.8, 152.8, 140.7, 88.3, 83.7, 74.2, 71.0, 70.8, 68.8, 54.0, 30.6. Calcd HRMS for C 14 H 20 N 6 O 6 (M+H): 369.1522; Found: 369.1525. tert-Butyl((3aR,4S,6R,6aR)-2,2-dimethyl-6-vinyl-tetrahydro-3aH-cyclopenta-[d]- [1,3]dioxol-4-yloxy)dimethylsilane (110). VinylMgBr (25 mL, 1.0 M in THF, 25 mmol) was added to a suspension of CuBr?SMe 2 (0.41 g, 2.0 mmol) in THF (30 mL) at -78 o C. The mixture was stirred at this temperature for 1 hour. Compound 49 (3.0 g, 19 mmol), HMPA (10 mL), TMSCl (3.2 mL, 25 mmol) in THF (30 mL) was added dropwise at -78 o C. The mixture was warmed to room temperature and stirred overnight. Saturated NH 4 Cl solution (30 mL) was added to quench the reaction. The mixture was exctracted with ethyl ether (3?100 mL). The combined organic layer was washed with bine (100 mL), dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH (100 mL). CeCl 3 ?7H 2 O (7.4 g, 20 mmol) was added at 0 o C. NaBH 4 (0.74 g, 20 mmol) was added portionwise. The mixture was stirred at this temperature for 1 hour. Saturated NH 4 Cl solution (20 mL) was added to quench the reaction. The mixture was filtered 120 through celite. The filtrated was concentrated. The residue was extrated with ethyl ether (3?100 mL). The combined organic layer was dried over sodium sulfate, and filtered through a short silica column (10 cm). The filtrate was concentrated. The residue was dissolved in DCM (100 mL). TBSCl (3.0 g, 20 mmol), imidazole (2.0 g, 31 mmol) were added at room temperature. The mixture was stirred 3 hours. Water (10 mL) was added to quench the reaction. The organic layer was sperated, washed with water (20 mL), dried over sodium sulfate, and concentrated. The residu was purified by silica column (Hexanes/EtOAc=20:1) to give 110 as a colorless oil (2.5 g, 43%). 1 H NMR (CDCl 3 , 400 MHz) ? 5.76 (m, 1H), 5.02-5.08 (m, 2H), 4.37 (m, 2H), 4.07 (m, 1H), 2.66 (m, 1H), 2.03 (m, 1H), 1.73 (m, 1H), 1.49 (s, 3H), 1.31 (s, 3H), 0.91 (s, 9H), 0.09 (s, 6H); 13 C NMR (CDCl 3 , 100 MHz) ? 139.1, 114.7, 111.3, 84.1, 80.1, 72.5, 44.3, 35.6, 26.3, 25.7, 24.7 18.4, -4.4, -4.7. Cacld HRMS for C 16 H 30 O 3 Si (M+H): 299.2042; Found: 299.2043. ((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydro-3aH-cy clopenta[d][1,3]dioxol-4-yl)methanol (111). 110 (2.5 g, 8.4 mmol) was dissolved in THF (50 mL). NMO (3.6 mL, 50% in water, 16 mmol) was added. OsO 4 (20 mg, 0.078 mmol) was added. The mixture was stirred at room temperature overnight. Sodium thiosulfate (5 g) was added. The mixture was stirred for another 2 hours. The mixture was filtered through a short silica column (5 cm). The column was rinsed with EtOAc. The combined organic liquid was concentrated. The residue was dissolved in DCM/H 2 O (1:1, 30 mL). NaIO 4 (2.1 g, 9.9 mmol) was added at room temperature. The mixture was stirred 3 hours. The organic layer was diluted with DCM (90 mL), separated, washed with water (20 mL), dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH (30 mL) at 0 o C. NaBH 4 (0.30 g, 6.4 mmol) was added portionwise. 121 The mixture was stirred at same temperature for 30 minutes. Saturated NH 4 Cl solution (30 mL) was added. The mixture was filtered through celite. The solvent was removed under reduced pressure. The residue was purified by silica column (hexanes/EtOAc=3:1 to 1:1) to give 111 exclusively as a colorless oil (2.1 g, 84%). The NMR spectra are consistent with the literature. 232 ((3aR,4S,6R,6aR)-6-(Allyloxymethyl)-2,2-dimethyl-tetrahydro-3aH-cyclopenta-- [d][1,3]dioxol-4-yloxy)(tert-butyl)dimethylsilane (112). Compound 111 (1.6 g, 5.3 mmol) was dissolved in DMF (50 mL). NaH (0.25 g, 6.3 mmol, 60% in mineral oil) was added in portions. AllylBr (1.1 mL, 12 mmol) was added dropwise via a syringe. The mixture was stirred at room temperature 12 hours. Water (10 mL) was added to quench the reaction. The mixture was extracted with ethyl ether (3?100 mL). The combined organic layer was washed with water (3?20 mL), brine (50 mL), dried over sodium sulfate, and concentrated. The residue was purified by silica column (hexanes/EtOAc=20:1) to give 112 as a colorless oil (1.6 g, 83%). 1 H NMR (CDCl 3 , 400 MHz) ? 5.89 (m, 1H), 5.14-5.26 (m, 2H), 4.38 (m, 2H), 4.19 (m, 1H), 3.94 (m, 2H), 3.37 (m, 1H), 3.30 (m, 1H), 2.24 (m, 1H), 2.05 (m, 1H), 1.68 (m, 1H), 1.48 (s, 3H), 1.31 (s, 3H), 0.91 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 134.8, 116.7, 111.3, 82.5, 80.9, 77.3, 72.9, 72.0, 42.3, 34.8, 26.6, 26.1, 24.9, 18.5, -4.4, -4.7. Calcd HRMS for C 18 H 34 O 4 Si (M+H): 343.2305; Found: 343.2312. 2-(((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydro-3aH -cyclopenta[d][1,3]dioxol-4-yl)methoxy)ethanol (113). Compound 113 was prepared from 112 by the same procedure used in synthesis of 111. 1 H NMR (CDCl 3 , 400 MHz) ? 4.38 (m, 2H), 4.14 (m, 1H), 3.72 (m, 2H), 3.55 (m, 2H), 3.42 (m, 1H), 3.35 (m, 1H), 2.29 122 (br, 1H), 2.03 (m, 2H), 1.62 (m, 1H), 1.49 (s, 3H), 1.31 (s, 3H), 0.91 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 111.6, 82.3, 80.9, 73.0, 72.9, 72.2, 61.8, 42.4, 34.6, 26.5, 26.0, 24.9, 18.5, -4.4, -4.8. Calcd HRMS for C 17 H 34 O 5 Si (M+H): 347.2254; Found: 347.2249. 2-(((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetrahydro-3aH -cyclopenta[d][1,3]dioxol-4-yl)methoxy)ethyl 4-Methylbenzenesulfonate (114). Compound 114 was prepared from 113 by the same procedure used in synthesis of 103. 1 H NMR (CDCl 3 , 400 MHz) ? 7.80 (m, 2H), 7.33 (m, 2H), 4.31 (m, 2H), 4.13 (m, 3H), 3.62 (m, 2H), 3.37 (m, 1H), 3.28 (m, 1H), 2.45 (s, 3H), 2.18 (m, 1H), 2.05 (m, 1H), 1.53 (m, 1H), 1.47 (s, 3H), 1.30 (s, 3H), 0.90 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 144.9, 133.0, 129.9, 127.9, 111.4, 82.2, 80.9, 77.2, 73.2, 72.9, 69.1, 68.5, 68.0, 42.2, 34.6, 26.6, 25.6, 24.9, 21.7, 18.5, -4.4, -4.7. Calcd HRMS for C 24 H 40 O 7 SSi (M+H): 501.2342; Found: 501.2338. (2S,5R)-2-(2-(((3aR,4R,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-tetra hydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methoxy)ethyl)-3,6-diethoxy-5-isopropyl-2, 5-dihydropyrazine (115). Compound 115 was prepared from 114 by the same procedure used in synthesis of 106. 1 H NMR (CDCl 3 , 400 MHz) ? 4.28-4.38 (m, 3H), 4.15 (m, 2H), 4.10 (m, 2H), 4.05 (m, 1H), 3.87 (m, 1H), 3.55 (m, 1H), 3.45 (m, 1H), 3.25-3.35 (m, 2H), 2.25 (m, 1H), 2.17 (m, 2H), 2.05 (m, 1H), 1.77 (m, 1H), 1.62 (m, 1H), 1.48 (s, 3H), 1.25-1.32 (m, 9H), 1.03 (d, J=6.8 Hz, 3H), 0.91 (s, 9H), 0.72 (d, J=6.8 Hz, 3H), 0.09 (s, 3H), 0.08 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.4, 163.0, 111.1, 82.5, 81.1, 73.1, 72.7, 67.7, 61.9, 61.8, 60.6, 60.5, 52.6, 42.3, 31.9, 29.2, 26.6, 26.1, 24.8, 19.1, 18.5, 16.7, 14.7, 14.4, -4.4, -4.7. Calcd HRMS for C 28 H 52 N 2 O 6 Si: 540.3595; Found: 540.3584. 123 (3aS,4S,6R,6aR)-6-((2-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin-2-y l)ethoxy)methyl)-2,2-dimethyl-tetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (116). Compound 116 was prepared from 115 by the same procedure used in synthesis of 107. 1 H NMR (CDCl 3 , 400 MHz) ? 4.49 (m, 1H), 4.45 (m, 1H), 4.08-4.21 (m, 5H), 3.96 (m, 1H), 3.87 (m, 1H), 3.53 (m, 1H), 3.45 (m, 1H), 3.35 (m, 1H), 3.25 (m, 1H), 2.40 (d, J=8.4 Hz, 1H), 2.25 (m, 2H), 2.12 (m, 1H), 1.82 (m, 3H), 1.49 (s, 3H), 1.34 (s, 3H), 1.25-1.28 (m, 6H), 1.03 (d, J=6.8 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.2, 163.1, 111.2, 83.1, 79.6, 76.3, 72.2, 67.8, 60.69, 60.65, 60.57, 60.51, 52.7, 42.0, 35.5, 34.2, 31.9, 26.2, 26.1, 14.4, 14.3. Calcd HRMS for C 22 H 38 N 2 O 6 : 426.2730; Found: 426.2733. 9-((3aS,4R,6R,6aR)-6-((2-((2S,5R)-3,6-Diethoxy-5-isopropyl-2,5-dihydropyrazin- 2-yl)ethoxy)methyl)-2,2-dimethyl-tetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-9H- purin-6-di(tert-butoxylcarbonyl)amine (117). Comound 116 (0.14 g, 0.33 mmol) was dissolved in THF. Ph 3 P (0.17 g, 0.66 mmol), Ad(Boc) 2 (0.22 g, 0.66 mmol) were added. DIAD (0.13 mL, 0.66 mmol) was added portionwise via a syringe at 0 o C. The mixture was warmed to room temperature and stirred overnight. The solvent was removed under reduced pressure. The residu was purified with a silica column to give 117 as an organge oil (0.14 g, 57%). 1 H NMR (CDCl 3 , 400 MHz) ? 8.85 (s, 1H), 8.21 (s, 1H), 5.09 (m, 1H), 4.95 (m, 1H), 4.85 (m, 1H), 4.63 (m, 1H), 4.05-4.21 (m, 6H), 3.85 (m, 1H), 3.55 (m, 3H), 2.45 (m, 2H), 2.25 (m, 1H), 2.15 (m, 1H), 1.90 (m, 1H), 1.55 (s, 3H), 1.47 (s, 18H), 1.35 (s, 3H), 1.27 (m, 6H), 1.03 (d, J=6.8 Hz, 3H), 0.70 (d, J=6.8 Hz, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 163.4, 163.2, 153.5, 151.8, 150.6, 150.4, 143.9, 129.4, 113.7, 83.7, 71.8, 67.9, 61.9, 60.6, 60.5, 59.6, 52.7, 43.8, 33.9, 33.7, 31.9, 27.9, 27.8, 27.7, 25.1, 21.9, 21.8, 124 19.2, 16.8, 14.4, 14.2. Calcd HRMS for C 37 H 57 N 7 O 9 : 743.4218; Found: 743.4208. (S)-2-Amino-4-(((1R,2R,3S,4R)-4-(6-amino-9H-purin-9-yl)-2,3-dihydroxycyclope ntyl)methoxy)butanoic Acid (7). Compound 7 was prepared from 117 by the same procedure used in synthesis of 6. 1 H NMR (D 2 O/MeOD, 400 MHz) ? 8.24 (s, 1H), 8.17 (s, 1H), 4.75 (m, 1H), 4.50 (m, 1H), 4.09 (m, 2H), 3.85 (m, 1H), 3.65 (m, 2H), 3.55 (m, 2H), 2.40 (m, 2H), 2.25 (m, 1H), 2.15 (m, 1H); 13 C NMR (D 2 O/MeOD, 100 MHz) ? 173.4, 155.4, 152.0, 149.1, 140.5, 118.7, 74.7, 72.5, 72.2, 67.9, 59.6, 53.7, 42.8, 29.9, 28.9. Calcd HRMS for C 15 H 22 N 6 O 5 (M+H): 367.1730; Found: 367.1740. ((3aR,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-6,6a-dihydro-3aH-cyc lopenta[d][1,3]dioxol-4-yl)methanol (119). Compound 118 (0.85 g, 2.8 mmol) was dissolve in DCM (30 mL). TBSCl (0.85 g, 5.6 mmol), imidazole (0.36 g, 5.6 mmol) were added at room temperature. The mixture was stirred at room temperature for 5 hours. Water (10 mL) was added to quench the reaction. The organic layer was separated, dried over sodium sulfate, and filtered through a short silica column. The filtrate was concentrated under reduce pressure (water bath temperature: 80 o C). The resulting colorless oil was dissolved in THF and cooled to -78 o C. TBAF (2.8 mL, 1.0 M in THF, 2.8 mmol) was added. The solution was slowly warmed to room temperature. Saturated NH 4 Cl solution (10 mL) was added. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (3?20 mL), dried over sodium sulfate, and concentrated. The residue was purified with silica column (hexanes/EtOAc=2:1) to give 119 as a colorless oil (0.54 g, 63%). 1 H NMR (CDCl 3 , 400 MHz) ? 5.67 (m, 1H), 4.89 (m, 1H), 4.64-4.67 (m, 2H), 4.30-4.38 (m, 2H), 2.05 (br, 1H), 1.42 (s, 3H), 1.37 (s, 3H), 0.92 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 143.7, 130.5, 125 112.4, 83.2, 79.1, 74.5, 60.2, 27.4, 26.7, 25.9, 18.5, -4.4, -4.7. ((3aR,4S,6aR)-6-(Allyloxymethyl)-2,2-dimethyl-4,6a-dihydro-3aH-cyclopenta[d][ 1,3]dioxol-4-yloxy)(tert-butyl)dimethylsilane (120). Compound 120 was prepared from 119 by the same procedure used in synthesis of 101. 1 H NMR (CDCl 3 , 400 MHz) ? 5.85-5.95 (m, 1H), 5.70 (m, 1H), 5.26 (dm, J=17.2 Hz, 1H), 5.19 (dm, J= 10.4 Hz, 1H), 4.88 (d, J=4.8 Hz, 1H), 4.65 (m, 2H), 4.13 (m, 2H), 4.03 (m, 2H), 1.39 (s, 3H), 1.37 (s, 3H), 0.91 (s, 9H), 0.12 (s, 6H); 13 C NMR (CDCl 3 , 100 MHz) ? 141.8, 134.6, 131.6, 117.2, 112.2, 82.8, 78.9, 74.6, 71.9, 66.5, 27.5, 26.8, 25.9, 18.5, -4.4, -4.7. 2-(((3aR,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-6,6a-dihydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)methoxy)ethanol (121). Compound 120 (0.35 g, 1.0 mmol) was dissolved in tert-BuOH/H 2 O (1:1, 10 mL). AD-mix-beta (1.4 g) was added. The mixture was stirred at room temperature for 24 hours. Sodium thiosulfate (2.0 g) was added to quench the reaction. The mixture was extracted with EtOAc (3?50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was dissolved in MeOH/H 2 O (1:1, 10 mL). NaIO 4 (0.26 g, 1.2 mmol) was added. The mixture was stirred at room temperature for 3 hours. NaBH 4 (0.24 g, 5.1 mmol) was added portionwise. The mixture was stirred at room temperature for 30 minutes. Saturated NH 4 Cl solution (10 mL) was added to quench the reaction. The mixture was extracted with EtOAc (3?100 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was purified by a silica column (Hexanes/EtOAc=2:1) to produce 121 as colorless oil (0.34 g, 96%). 1 H NMR (CDCl 3 , 400 MHz) ? 5.69 (m, 1H), 4.89 (d, J=5.2 Hz, 1H), 4.65 (m, 2H), 4.19 (m, 2H), 3.75 (m, 2H), 3.59 (m, 2H), 1.41 (s, 3H), 1.37 (s, 3H), 0.92 (s, 9H), 0.14 (s, 126 3H), 0.13 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 141.6, 131.9, 112.3, 82.9, 78.9, 74.5, 71.9, 67.5, 61.8, 27.4, 26.7, 25.9, 18.5, -4.4, -4.7. 2-(((3aR,6S,6aR)-6-(tert-Butyldimethylsilyloxy)-2,2-dimethyl-6,6a-dihydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)methoxy)ethyl 4-Methylbenzenesulfonate (122). Compound 122 was prepared from 121 by the same procedure used in synthesis of 103. 1 H NMR (CDCl 3 , 400 MHz) ? 7.80 (dm, J=8.4 Hz, 2H), 7.34 (dm, J=8.4 Hz, 2H), 5.64 (d, J=1.2 Hz, 1H), 4.80 (d, J=4.0 Hz, 1H), 4.63 (m, 2H), 4.08-4.18 (m, 4H), 3.64-3.67 (m, 2H), 2.44 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 0.92 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H); 13 C NMR (CDCl 3 , 100 MHz) ? 144.8, 141.2, 133.0, 132.0, 129.9, 128.0, 112.2, 82.7, 78.9, 74.5, 69.1, 68.2, 67.6, 27.4, 26.8, 25.9, 21.7, 18.2, -4.4, -4.7. 127 REFERENCES (1) Flint, S. J.; Enquist, L. W.; Krug, R. M.; Racaniello, V. R.; Skalka, A. M. Principles of virology: molecular biology, pathogenesis, and control. 2000, ASM Press?ISBN 1-55581-127-2. (2) Weiss, R. A.; Tailor, C. S. Cell 1995, 82, 531-533. (3) Norkin, L. C. Clin. Microbiol. Rev. 1995, 293-315. (4) Harrison, S. C. Curr. Opin. Struct. Biol. 1995, 5, 157-164. (5) Evans, D. J.; Almond, J. W. Trends Microbiol. 1998, 6, 198-202. (6) Marsh, M.; Helenius, A. 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