SYNTHETIC EXPLORATIONS TO SINEFUNGIN ANALOGS Except where references are made to the work of others, the research is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. ____________________________ Guoxia Zhao Certificate of Approval: ____________________________ ____________________________ Peter Livant Stewart W. Schneller, Chair Associate Professor Professor Chemistry and Biochemistry Chemistry and Biochemistry. ____________________________ ____________________________ Edward Parish Susanne Striegler Professor Associate Professor Chemistry and Biochemistry Chemistry and Biochemistry ____________________________ George. T. Flowers Dean Graduate School SYNTHETIC EXPLORATIONS TO SINEFUNGIN ANALOGS Guoxia Zhao 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 May 9, 2009 iii SYNTHETIC EXPLORATIONS TO SINEFUNGIN ANALOGS Guoxia Zhao 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 Guoxia Zhao, daughter of Yijun Zhao and Hailian Zhang, was born in Shenxian County, Liaocheng City, Shandong Province, China, on April 6, 1978. After graduating from high school, she began her college studies first at Liaocheng University in September 1997 and then entered to Qufu Normal University at September 1999 as the fifth in Shandong Province University Entrance Examination (SPUEE) and received a Bachelor Degree in Chemistry in July 2001. Then, she attended national graduate-student entrance examination (NGEE) in December 2000. She entered the University of Science and Technology of China (USTC) to begin her graduate study at September 2001 and obtained a Master Degree in Organic Chemistry in July 2004 under the Professors Guan- wu Wang and Li-feng Yan. After she graduated from USTC in 2004, she got a research position (Assistant Professor) in a famous Institute of Chinese Academy in Beijing. However, she chose to study abroad to Auburn University. She began her Ph.D studies in Organic Chemistry under the direction of Prof. S. W. Schneller at Auburn University in August 2004. She is married to Hai, Li, son of Zhiheng Li and Yiqing Liu. v DISSERTATION ABSTRACT SYNTHETIC EXPLORATIONS TO SINEFUNGIN ANALOGS Guoxia Zhao Doctor of Philosophy, May 9, 2009 (M. S., University of Science and Technology of China, USTC, July 3, 2004) (B. S., Qufu Normal University, July 4, 2001) 173 Typed Pages Directed by Stewart W. Schneller Sinefungin, a natural complex nucleoside, has been isolated from the cultures of Streptomyces griseolous, and from that of Streptomyces incarnatus. It is a structural analog of S-adenosylmethionine (AdoMet) and since its discovery, its biological activities have been thoroughly studied, including antifungal, antitumor, antiparasitic, antiviral and amoebicidal activities. The nature of these biological activities results from its inhibition of a variety of AdoMet-dependent methyltranferase enzymes. These methylation reactions are crucial in many biochemical processes, including virus-encoded mRNA cap methyltransferases, of which sinefungin is a selective inhibitor. As a vi consequence of the latter observation, sinefungin and related compounds have attracted the attention of synthetic chemists in the design of antiviral chemotherapeutic agents. However, the clinical usefulness of sinefungin has been precluded by its toxicity in in vivo assays. To seek a novel approach to toxic-free sinefungin-based compounds, the main body of this dissertation research considers the design and possible synthetic routes to the analog of carbocyclic sinefungin (target I) and related intermediates (47, 53, 55 and 79, 81, 86) for other sinefungin-based compounds. In this research, the key synthetic steps that have been investigated are the installment of the C-6' and C-9' stereocenters by efficient asymmetric synthetic methods. In the C-6' case, a highly diastereoselective allylborating agent, (+) and/or (-)-B- allydiisopinocampheylborane (Ipc 2 BCH 2 CH=CH 2 ) has been called forth. The requisite C- 9' amino acid stereochemistry has been established by alkylation reactions with the Sch?llkopf reagent. Finally, coupling of cyclopentyl or ribosyl units with the heterocyclic base (adenine) has been evaluated by standard S N 2 substituted reactions. These synthetic approaches are adaptable to a number of structural investigations relevant to analogs of carbocyclic sinefungin and the parent sinefungin. vii ACKNOWLEDGMENTS Many people have contributed to this dissertation and have given me endless support. I am extremely grateful to each of them. First and foremost, I would like to express my sincere gratitude to my research advisor, Professor Stewart W. Schneller, for his continuous support and encouragement during my graduate career. Without his dedication much of the research discussed in this dissertation would not have been possible. My gratitude is extended to the distinguished members of Committee including Drs. Peter Livant, Edward J. Parish, Susanne Striegler and Jack DeRuiter as my outside reader for their intellectual assistance and constructive suggestions. I am grateful to all Schneller group members for their kind help, useful discussions, and friendship. In particular, I would like to thank these lab mates, Drs. Xue- qiang Yin, Weikuan Li, Mingming Yang, Atanu Roy, Yan Zhang, Wei Ye, and Mingzhu He. Additionally, I extend my gratitude to Department of Chemistry and Biochemistry and National Institutes of Health for their financial support. Finally, I wish to thank my family and friends in China for their infinite love and support which made this dissertation possible. Especially, I am deeply indebted to my husband, Hai Li for his patience, support, and never-ending encouragement. viii Style manual or jounal used: American Chemical Society Style-The Journal Organic Chemistry ___________________________________________________________________ Computer software used: Microsoft Word 2003, ISIS Draw 2.0, EndNote X1 ____________________________________________________________________ ? 2009 Guoxia Zhao All Right Reserved ix TABLE OF CONTENTS LIST OF SCHEMES............................................................................................................x LIST OF FIGURES .......................................................................................................... xii CHAPTER 1.INTRODUCTION .........................................................................................1 CHAPTER 2. SYNTHESIS OF THE IMPORTANT INTERMEDIATES FOR TARGET I AND CARBOCYCLIC SINEFUNGIN ..........................................................................31 CHAPTER 3. SYNTHESIS OF TARGET I......................................................................38 CHAPTER 4. SYNTHESIS OF CARBOCYCLIC SINEFUNGIN BUILDING BLOCKS (53 and 55) .........................................................................................................................59 CHAPTER 5. SYNTHESIS OF TARGET II ....................................................................65 CONCLUSION ..................................................................................................................87 EXPERIMENTAL SECTION ...........................................................................................89 REFERENCES ................................................................................................................144 x LIST OF SCHEMES Scheme 1. AdoMet-dependent methyltransferases and AdoHcy metabolism ...................11 Scheme 2. The principle of aristeromycin as inhibitor in AdoMet methylation reaction ..17 Scheme 3. Metabolic pathway of Ari ................................................................................19 Scheme 4. The metabolism of NpcA .................................................................................20 Scheme 5. Synthesis the important precursor 6 .................................................................32 Scheme 6. Synthesis 11 from monoacetate 6 .....................................................................33 Scheme 7. Another approach to form 11 ...........................................................................34 Scheme 8. Synthesis of the important precursor 21 from D-ribose ...................................35 Scheme 9. Dissociative mechanism of olefin metathesis ..................................................37 Scheme 10. Retrosynthetic analysis of target I ..................................................................39 Scheme 11. First approach to building block A .................................................................41 Scheme 12. The second approach for 26 ...........................................................................42 Scheme 13. Allylation reaction of 26 ................................................................................45 Scheme 14. Optimized approach for target I .....................................................................46 Scheme 15. Synthesis of Sch?llkopf?s reagent (35, building block C) .............................48 Scheme 16. Synthesis the important intermediates 42 (building block B) ........................50 xi Scheme 17. Synthesis of iodide 44 ....................................................................................54 Scheme 18. Optimized approach for the synthesis of tosylate 45 .....................................56 Scheme 19. Synthesis of 47, a close precursor to target I ..................................................58 Scheme 20. Retrosynthetic analysis of carbocyclic sinefungin .........................................60 Scheme 21. Synthesis of 53 ...............................................................................................61 Scheme 22. Synthesis of 55 ...............................................................................................62 Scheme 23. Retrosynthetic analysis of target II .................................................................66 Scheme 24. Synthesis of important intermediate 59 ..........................................................67 Scheme 25. Synthesis of 63 ...............................................................................................68 Scheme 26. Synthesis of 64 ...............................................................................................69 Scheme 27. Optimized approach for target II ....................................................................70 Scheme 28. Synthesis of 65 ...............................................................................................71 Scheme 29. Synthesis of 70 ...............................................................................................72 Scheme 30. Another method for 70 ...................................................................................73 Scheme 31. Synthesis of iodide 78 ....................................................................................75 Scheme 32. Optimization approach for 77 ........................................................................78 Scheme 33.Synthesis of 79 ................................................................................................82 Scheme 34. Synthesis of 80 ...............................................................................................83 Scheme 35. Synthesis of 81 ...............................................................................................83 Scheme 36. Synthesis of 86 ...............................................................................................85 xii LIST OF FIGURES Figure 1. Monomeric components of DNA and RNA-nucleosides .....................................2 Figure 2. Structures of approved nucleoside drugs ..............................................................4 Figure 3. In vivo metabolic deactivation ..............................................................................5 Figure 4. Carbocyclic nucleosides, Ari and NpcA ..............................................................6 Figure 5. 5'-methylated and capped structure of eukaryotic mRNA ...................................8 Figure 6. Structural analogs of AdoHcy based on the amino acid modification ...............13 Figure 7. Base-modified AdoHcy analogs .........................................................................14 Figure 8. Sugar modification of analogs of AdoHcy .........................................................15 Figure 9. The lead inhibitors of AdoHcy hydrolase ..........................................................22 Figure 10. Structural analogs of AdoMet ..........................................................................24 Figure 11. Sinefungin and its C-6' epimers ........................................................................26 Figure 12. Some nucleoside derivatives of sinefungin: modification on the side ?chain ..27 Figure 13. Base modified derivatives of sinefungin ..........................................................28 Figure 14. Initial targets of dissertation research ...............................................................29 Figure 15. Initial target of dissertation research ................................................................30 Figure 16. Important precursor 2 for target II ....................................................................30 Figure 17. Important intermediates for the targets .............................................................31 Figure 18. Grubbs? catalysts ..............................................................................................36 Figure 19. Proposed intermediates for target I ...................................................................39 Figure 20. Boron-based allylating reagents .......................................................................43 xiii Figure 21. Proposed mechanism of allylation of aldehydes with Brown?s reagent ...........44 Figure 22. Configurations of amino acids..........................................................................47 Figure 23. COSY spectrum of compound 41 in deuteriochloroform: (a) the small one chemical shift ? (1.0-7.5) and (b) the magnified one ? (1.0-2.5) .......................................53 Figure 24. Transition state for the alkylation reaction .......................................................57 Figure 25. Horeau's method ...............................................................................................64 Figure 26. COSY spectrum of compound 75 in deuteriochloroform: (a) the small one chemical shift ? (1.0-7.8) and (b).the magnified one ? (1.0-6.1) .......................................77 Figure 27. COSY spectrum of compound 79 in deuteriochloroform: (a), the small one chemical shift ? (-0.30-5.5) and (b).the magnified one ? (-0.30-2.5) ................................81 1 CHAPTER 1 INTRODUCTION Nucleosides are glycosylated nitrogen heterocyclic molecules and function biologically in their monomeric state or as units in the large molecules of nucleic acids: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). In their latter role, DNA is a polymer of three components: phosphate, heterocyclic base, and 2-deoxy-D-ribose. The four bases in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). The structurally similar RNA is also composed of three components: phosphate, heterocyclic base, and D-ribose. In ribonucleosides, the bases are adenine (A), guanine (G), uracil (U) and cytosine (C). The structural difference between the deoxy-ribose of DNA and ribose of RNA is the presence of a hydroxyl group at the 2' position of the later ribose sugar. In both DNA and RNA, the ribose is in its furanose form. The nitrogen heterocyclic bases of nucleosides attach themselves to the C-1' sugar unit (Figure 1). N N N H N NH 2 NH N N H N O NH 2 N N H NH 2 O NH N H O O Me adenine guanine cytosine thymine NH N H O O uracil R=H, for DNA B= A, G, C, T R=OH, for RNA B =A, G, U, C Figure 1. Monomeric components of DNA and RNA-nucleosides. O HO HO R B 1' The nucleic acid DNA carries the inherited genetic code that cells use to direct their replication and behavior. DNA is transcribed into mRNA, which is then translated into protein sequence at the ribosomes. Monomeric nucleosides play important roles in the fundamental biological processes. For example, S-adenosylmethionine (AdoMet) serves directly as the methyl donor for numerous methyltransferases, 1a-b such as small molecular weight neurotransmitters (histamine, norepinephrine) and macromolecules (proteins, lipids, nucleic acid) and as an important substrate for enzymatically catalyzed group transfer reactions [AdoMet is also decarboxylated by AdoMet decarboxylase to dcAdoMet. dcAdoMet serves as an aminopropyl donor toward the synthesis of polyamines and in the process is converted to 5'-methylthioadenosine (MTA)], 1c-d and as substrate for the enzyme 1-aminocyclopropanecarboxylic acid synthase. 1e-f Because of their biological pervasiveness nucleosides have found a prominent place in drug discovery (for example, Severe Acute Respiratory Syndrome or SARS; Human Immunodeficiency Virus or HIV; and, Epstein-Barr virus). 2 It is reported that of 2 3 the thirty compounds currently marketed in the United States for treatment of viral infections, fifteen are nucleosides analogs. 3 This demonstrates the wide uses of the nucleosides as a source of antiviral drugs. To discover new therapeutic nucleosides, two approaches exist: (1) molecular modification of the sugar moiety and (2) variation of the heterocyclic base. 3 In the former case, Figure 2 presents nucleosides clinically used for treating herpes simplex virus, HSV (acyclovir 4 and ganciclovir 5 ) and HIV (AZT 6 , ddC 7 , ddI 8 , d4T 9 , (-)-3TC 10 , avacavir 11 ). Variation of the heterocyclic base has also led to effective anti-HSV drugs (5-iodo-2'- deoxyuridine, IDU, Idoxudine; and, 5-trifluoromethyl-2'-deoxyuridine, Trifluridine). 12 O NH N N N O NH 2 HO O NH N N N O NH 2 HO HO NH N N N O NH 2 HO HO acyclovir gancyclovir pencyclovir O HO NH N O N 3 O H 3 C O HO NH N NH 2 O ddC AZT O HO NH N N N O ddI O HO NH N O O H 3 C d4T O OH N N O NH 2 (-)-3TC Figure 2. Structures of approved nucleoside drugs. O OH HO N HN O O I 5-Iodo-2'-deoxyuridine (IDU) O OH HO N HN O O CF 3 5-trifluoromethyl-2'-deoxyuridine 4 However, the clinical application for these nucleoside drugs has been restricted by their inherent undesirable properties, such as toxicity, drug resistance and stability. 13 Among them, IDU and 5-trifluromethyl-2'-deoxyuridine show very little selectivity against virus-infected cells and are highly toxic to host cells and mutagenic. 14 Another major problem with 5-substituted 2'-deoxyuridine derivatives is that they are easily cleaved by pyrimidine phosphorylases at the glycosidic bond to their respective uracil bases (Figure 3). 15 Because of these limitations, the search for nucleoside derived antiviral agents continues in many laboratories. O OH HO N HN O O R O OH HO N H HN O O R OH 5-Substituted uracil 2'-Deoxyribose Figure 3. In vivo metabolic deactivation. phosphorylase R =I, CF 3 , CH 2 CH 3 ,CH 2 CH 2 Cl, CH=CHBr + In that regard, among the sugar moiety modified nucleosides, the carbocyclic nucleoside or carbanucleoside is an important type of nucleoside. In this series of compounds, a methylene group replaces the oxygen atom of the furanose ring of the traditional nucleosides. Carbanucleosides have important biological properties, including: (i) metabolic stability and (ii) recognition by the enzymes and acceptors common to the standard nucleosides. 16 In the first case, their half-life in vivo is prolonged since they are not affected by the phosphorylases and hydrolases, which cleave the glycosidic bonds of natural nucleosides. 17 Their second relevant property arises from the similarity between the cyclopentane ring of carbocyclic nucleosides and the tetrahydrofuran ring of natural nucleosides. This renders carbocyclic nucleosides recognizable as substrates for numerous cellular enzymes. Moreover, the higher lipophilicity of carbocyclic nucleosides is beneficial for increasing their oral availability and cellular membrane penetration. 18 5 Two of the most important carbocyclic nucleosides, aristeromycin 17d,19 (Ari) and neplanocin A 20 (NpcA), are naturally occurring nucleosides and have been enantioselectively synthesized and found to exhibit various antiviral activities. As shown in Figure 4, both Ari and NpcA are carbocyclic analogs of adenosine with the only difference being the presence of a double bond between C-4' and C-6' of the carbocyclic ring of NpcA. Aristeromycin was first isolated from Streptomyces citricolor by Kusaka et al. 19a and synthesized by Shealy and Clayton in 1966 in racemic form. 17d Its adenosine-like biological properties and the chemical stability have made it a lead compound in the development of new drugs. Neplanocin A, the olefinic analog of Ari, first isolated from the culture of Ampullariella regularis A11079 20f and then synthesized by some groups, 20g-i possesses apparent in vivo antileukemic activity. 20a X HO HO OH N N N N NH 2 HO HO OH N N N N NH 2 X=O, Adenosine X=CH 2 , Aristeromycin Neplanocin A Figure 4. Carbocyclic nucleosides, Ari and Npc A. Ari and NpcA have been subjected to various biological assays and both exhibit broad-spectrum antiviral activities. The antiviral activity of Ari has been correlated with its inhibition of the enzyme S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase or 6 7 SAH hydrolase) and, subsequently, to inhibit viral mRNA methylation. Furthermore, only its (-) enantiomer shows biological activity in inhibiting tumor cell growth and viral replication. 21 Neplanocin A has activity against a broad spectrum of viruses, 22 such as DNA viruses (vaccinia virus), (-) RNA viruses (such as parainfluenza, measles, and vesicular stomatitis), and double-stranded RNA viruses (such as reo) in vitro in cell culture. Its in vivo activity has been established against the vaccinia virus in mouse L-929 cells with little or no toxicity to the host cells. 22b Neplanocin A also possesses significant antitumor activity in vivo against murine leukemias. 23 Based on its structural similarity to adenosine and aristeromycin, the pharmacological activity of NpcA has been investigated. This compound is a potent inhibitor of AdoHcy hydrolase both in vitro and in vivo as mentioned. Because of the common inhibitory mechanism of antiviral effect of Ari and Npc A, AdoHcy hydrolase has been recognized as an important target for broad-spectrum antiviral agents. 24 It is known that S-adenosyl-L-homocysteine hydrolase plays an important role in regulating biological methylation reactions, for example, small molecules (triptamine and histamine) and macromolecules (nucleic acids, proteins and phospholipids). 25 The latter methylation reactions are catalyzed by specific AdoMet- dependent methyltransferases, which themselves are attracting the attention of medicinal chemists as potential targets for the design and synthesis of antitumor and antiviral agents. It has been found that eukaryotic mRNA, but not prokaryotic mRNA, possesses a methylated 5'-capped structure linked to the 5' end of the transcript by a 5'-5' triphosphate bridge (Figure 5). This is essential to stabilize the mRNA against phosphatase and ribonuclease and is crucial for the proper binding of the mRNA to the ribosome for translation to proteins. 25b,26,27 Shuman and co-workers 30 found that the 5'-end of eukaryotic mRNAs is capped with a 7'-methyl-GTP (guanosine triphosphate) residue, which protects the RNA from digestion by the aforementioned enzymes, and is linked to a polyadenylated tail of the code mRNA transcript (Figure 5). 31 Those adenosines also carry a 2' ? methyl group. The capping is essential for successful translation into protein at the ribosomes. OH OH O O P O O_ O P O_ O OP O O O PO O CH 3 Base O O O_ HN N O H 2 N N N CH 3 + guanosine N-7 methyl cap 2'-O methyl cap Figure 5. 5'-methylated and capped structure of eukaryotic mRNA. O O O P_O O O CH 3 Base 2'-O methyl cap O_ CH 2 2' 3' 5' 5' 2'3' 5' mRNAm 7 GpppN 1 pN 2 mRNA The relevant capping process of mRNA involves three enzymatic reactions. 28 The first is the hydrolysis of the 5'-triphosphate end of the pre-mRNA to a diphosphate by RNA 5'-triphosphatase. The second reaction involves a RNA guanylyltransferase, which adds guanosine monophosphate (GMP) to the just formed diphosphate RNA end. The final step is the methylation of the guanyl capped mRNA by an RNA (guanine N-7) 8 methyltransferase, with a methyl group from S-adenosylmethionine (AdoMet) serving as the methyl source. This occurs at the N-7 position of the GpppN of the mRNA to produce a m 7 GpppN RNA and S-adenosylhomocysteine (AdoHcy). This important biochemical process is conserved in all eukaryotic organisms and many eukaryotic viruses. 29 The latter observation produces the basis for drug development of inhibition of the cap methylation reaction as an anti-infective drug strategy, including antiviral agent design. pppN(pN) n ppN(pN) n + P i ppN(pN) n + pppG (i) G(5')pppN(pN) n + PP i (ii) (iii) G(5')pppN(pN) n + AdoMet m 7 G(5')pppN(pN) n + AdoHcy Inhibition of AdoMet methylation as a mechanism for antiviral drug design and discovery Since the first discovery of S-adenosyl-L-methionine (AdoMet), 32 a large number of AdoMet-dependent methyltransferases have been extensively studied. 33 In the early 1980s, Borchardt 1 investigated AdoMet-dependent methyltransferases and found they were inhibited by AdoHcy, the AdoMet product (Scheme 1). Attention then turned to the AdoHcy catabolic enzyme, AdoHcy hydrolase. As seen in Scheme 1, AdoHcy hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine in mammals, and thus controls the intracellular levels of the essential metabolites AdoHcy, adenosine and homocysteine. Cellular inhibition of the hydrolase leads to increased levels 9 10 of AdoHcy. This increased level of AdoHcy causes bio-feedback inhibition (product inhibition) of the essential AdoMet bio-methylation reactions. 35,42 Therefore, increasing the cellular concentration of AdoHcy by inhibition of AdoHcy hydrolase, results in limiting metabolic processes requiring methylations (for example, mRNA maturation) and blocking the chain of the viral replication. 22a As a consequence, inhibitors of AdoHcy hydrolase can function indirectly on the viral mRNA methylation reactions by focusing on the steps of the mechanism by which AdoHcy hydrolase proceeds. Several approaches have lent themselves to inhibitor design as a means to potential antiviral chemtherapeutic agents. 43,44 O HO OH N N N N NH 2 S + H 2 N CO 2 H H 3 C AdoMet O HO OH N N N N NH 2 S NH 2 HO 2 C AdoHcy Nu: Nu-CH 3 methyl transferase biofeedback inhibition O HO OH N N N N NH 2 HO H CCO 2 HHSCH 2 CH 2 H 2 N + AdoHcy hydrolase Scheme 1. AdoMet-dependent methyltransferases and AdoHcy metabolism. ATP + methionine adenosyl transferase adenosine (mammals) adenine + S-ribohomocysteine AdoHcy nucleosidase (bacteria) In that direction, and referring to Scheme 1, inhibitor categories have been designated: direct and indirect. 1 Direct inhibitors, such as analogs of AdoMet and AdoHcy, function on a particular methyltransferase. They exert their effects by binding to the active site (AdoMet analogs) of the methyltransferase 34 or the AdoHcy inhibitory binding locale site of the transferases. The indirect inhibitors block the biosynthesis of AdoMet (that is via adenosyltransferase) or AdoHcy metabolism (that is, AdoHcy hydrolase, AdoHcy nucleosidase). In the first category, Borchardt and co-workers 36,37 have made many important contributions with AdoHcy analogs and their effects in viral (Newcastle and vaccinia) 11 12 AdoMet-dependent mRNA (guanine-7)-methyltransferase. Their analogs focused on modifications in the amino acid, base and sugar moiety areas of AdoHcy. Among of the amino acid modified analogs (Figure 6), it was found that there are important requirements for manifesting into inhibitors of virion mRNA (guanine-7)- methyltransferase: (i) with modifications of the sulfur atom (AdoHcy sulfone and AdoHcy sulfoxide) showed reduction (but less) of AdoHcy inhibitory properties; (ii) the S chirality of the amino acid (that is C-9') is necessary for inhibition; 38 (iii) the amino acid amine and carboxyl groups are necessary for binding to the methyltransferase; and, (iv) the two carbon atom distance between the sulfur atom and ?-carbon of the amino acid is crucial for successful interaction with the methyltransferase. From these results, it is noteworthy that the virion mRNA (guanine-7)-methyltransferase appears capable of accommodating changes at the sulfur atom of AdoHcy. 36a O HO OH N N N N NH 2 S + H 2 N CO 2 H H 3 C AdoMet O HO OH N N N N NH 2 S H 2 N CO 2 H AdoHcy O HO OH N N N N NH 2 S H 2 N CO 2 H O HO OH N N N N NH 2 S H 2 N CO 2 H O HO OH N N N N NH 2 S H 2 N CO 2 H D-AdoHcy O AdoHcy sulfoxide O O AdoHcy sulfone O HO OH N N N N NH 2 S AcHN CO 2 H Ado-N-AcHcy Figure 6. Structural analogs of AdoHcy based on amino acid modifications. The base-modified analogs (Figure 7) of AdoHcy indicated that only N 6 -methyl- AdoHcy and 3-deaza-AdoHcy show significant inhibitory activity toward the vaccinia (guanine-7)-methyltransferase. The other base-modified analogs displayed little or no inhibitory activity toward this enzyme. 36b For the Newcastle disease viral transferase, 7- deaza-AdoHcy (TubHcy), N 6 -methyl-AdoHcy, and 8-aza-AdoHcy were potent inhibitors. It was also found that replacement of the heterocyclic base (adenine) of AdoHcy with a 13 pyrimidine unit (cytosine or uracil) or a guanine base, resulted in complete loss of inhibitory activities towards the mRNA (guanine-7) methyltransferase. 36a O HO OH N N N N NH 2 S H 2 N CO 2 H AdoHcy O HO OH N N N NH 2 S H 2 N CO 2 H O HO OH N N N N N NH 2 S H 2 N CO 2 H 8-Aza-AdoHcy O HO OH N N N N NHCH 3 S H 2 N CO 2 H N 6 -Methyl-AdoHcy O HO OH N N N NH 2 S H 2 N CO 2 H 3-Deaza-AdoHcy Figure 7. Base-modified AdoHcy analogs. 7-Deaza-AdoHcy (TubHcy) In the sugar-modified analog group (Figure 8), only the carbocylic AdoHcy (AriHcy) had significant inhibitory ability on the vaccinia (guanine-7)- methyltransferase. 36b Other sugar-modified AdoHcy analogs (for example, 2'-deoxy- AdoHcy or 3'-deoxy-AdoHcy) are substantially less active or lose their inhibitory activity toward the vaccinia (guanine-7)-methyltransferase. 36b,37b Thus, the roles of the 2'- and 3'- hydroxyl groups of the AdoHcy are very significant for the binding AdoHcy derivatives to the active site of the vaccinia methyltransferase. 14 On the other hand, for the Newcastle disease (guanine-7)-methyltransferase, 38a both 2'-deoxy-AdoHcy and 3'-deoxy-AdoHcy exhibit some inhibitory activity (K i = 26 ?8 pM and K i = 78?3 pM, respectively, where K i represents the inhibition constants for the methyltransferase). This observation shows that the roles of these AdoHcy functional groups are not stringent in the NDV methyltransferase as they are with the vaccinia system. Johnson and co-workers 39 later reported the synthesis of 4'-methyl-AdoHcy. However, its biological activity remains unreported. O HO OH N N N N NH 2 S H 2 N CO 2 H AdoHcy HO OH N N N N NH 2 S H 2 N CO 2 H AriHcy O HO N N N N NH 2 S H 2 N CO 2 H 2'-deoxy-AdoHcy O OH N N N N NH 2 S H 2 N CO 2 H 3'-deoxy-AdoHcy Figure 8. Sugar modification analogs of AdoHcy. O HO OH N N N N NH 2 S H 2 N CO 2 H 4'-methyl-AdoHcy H 3 C Among the indirect inhibitors (Scheme 1), two major designs exist: (i) blocking the biosynthesis of AdoMet by inhibiting the adenosyltranferase; and, (ii) preventing the catabolism of AdoHcy by inhibiting AdoHcy hydrolase or AdoHcy nucleosidase. Of 15 16 these, most research has concentrated on designing and synthesizing inhibitors of the catabolism of AdoHcy. To be sure, the pervasiveness of AdoHcy hydrolase in yeasts, plants, birds, and mammals, 40 has stimulated studies in this regard. On the other hand, the enzyme AdoHcy nucleosidase, has only been found in bacteria. 40b, 41 From these investigations, De Clercq and co-workers 45 have shown that a close correlation exists between the antiviral potency of Ado-derived analogs and their inhibitory effects on AdoHcy hydrolase. Logically, Borchardt and co-workers 46 also have found that a close correlation exists between the antiviral potency of the nucleosides and their ability to increase the cellular levels of AdoHcy. As a result, inhibitors of AdoHcy hydrolase have exhibited broad-spectrum antiviral activities, 45a,47 by disrupting the essential viral mRNA methylation processes. 42e-f. Among the AdoHcy hydrolase inhibitors, the adenosine analogs, (-)-Ari 17d,19 and (-)-NpcA 20 have exhibited broad and significant antiviral activities, 44 that correlate with their inhibitory effects of AdoHcy hydrolase. 45a,48 Reviewing Scheme 2 clarifies the inhibiting mechanism of Ari (I). A similar mechanism exits for NpcA and other analogs of adenosine. 42c Aristeromycin?s effect results from its conversion to carbocyclic AriHcy (C-AriHcy, II), which functions as a product inhibitor (just like AdoHcy) of AdoMet- dependent methylations and is responsible for its antiviral characteristics. O HO OH N N N N NH 2 S + H 2 N CO 2 H H 3 C AdoMet X HO OH N N N N NH 2 S NH 2 HO 2 C X=O AdoHcy (II) X=CH 2 C-AriHcy Nu: Nu-CH 3 methyl transferase biofeedback inhibition X HO OH N N N N NH 2 HO H CCO 2 HHSCH 2 CH 2 NH 2 + AdoHcy hydrolase X=CH 2 carba-ATP (III) carba-AdoMet (IV) X=O adenosine (I) X=CH 2 aristeromycin Scheme 2. The principle of aristeromycin as inhibitor in AdoMet methylation reaction. Scheme 2 demonstates another relevant metabolic pathway for Ari: that is, it can be phosphorylated to carbocyclic ATP (III) by adenosine kinase. Subsequently, the latter compound is converted to carbocyclic AriMet (IV) under the catalysis of AdoMet synthetase. 42c,49 Then, carbocyclic AriMet (IV), the counterpart of AdoMet, can bind to the active site of the methyltransferase and inhibit the enzyme. At the same time, these metabolites possess undesirable side effects for drug discovery, such as toxicity, both in vitro and in vivo testing, which preclude their clinical applications. Consequently, studies concerning the source of toxicity of carbocyclic 17 18 analogs of adenosine become important to the development of this series as antiviral agents. The possible cause for this toxicity may exist in its metabolites: carba-ATP (III) (Scheme 2) and/or carba-GMP (V) (Scheme 3). 50 Carba-ATP (III), may replace ATP in uninfected cellular processes requiring the latter nucleotide for growth and/or it may be incorporated into the host DNA cells, leading to mutations. In the case of carba-GMP (V), 42c,51 that is formed from the monophosphate of Ari catalyed by AMP deaminase and other enzymes, inhibition of cellular hypoxanthine-(guanine) phosphoribosyltransferase can occur. 52 This enzyme is part of the purine salvage pathway and its inhibition can lead to complete blockade of the availability of hypoxanthine and guanine essential for disease free cellular metabolism. 52,53 HO N N N N NH 2 HO HO OH H 2 O 3 PO NH N N N O NH 2 HO OH N N N N NH 2 H 2 O 3 PO carba-ATP (III) OH adenosine kinase Scheme 3. Metabolic pathway of Ari. carba-GMP (V) Ari For NpcA, Marquez and co-workers 54 and, Glazer and co-workers 55 found that NpcMet, the AdoMet-like metabolite of NpcA, inhibits cellular mRNA methylation (HT- 29 cells), producing toxic effects (Scheme 4). Until now, there is no direct evidence for the effect of NpcMet on virus mRNA methyltransferase. On the other hand, there are no studies for NpcTP, as a source of NpcA toxicity. Glazer?s laboratory 55 also found that NpcTP, the counterpart of ATP, forms NpcMet under the catalysis of AdoMet synthetase and is minimally incorporated into mRNA. 19 NpcMet (AdoMet) NpcHcy (AdoHcy) AdoHcy hydrolase AdoMet synthetase Adenosine kinase (Adenosine) + Homocysteine HO OH Adenine HOH 2 C Npc A (Adenosine) NpcD (Inosine) Adenosine deaminase RXH RXCH 3 Methyl transferase NpcTP (ATP) Scheme 4. The metabolism of Npc A. Met Adohcy hydrolase inhibition From the metabolic studies above, adenosine kinase plays a major role in the toxicity of Ari and NpcA by promoting formation of the 5'-nucleotides. Thus, in order to develop potencial inhibitors of AdoHcy hydrolase based on Ari and NpcA, their susceptibility to adenosine kinase must be eliminated/reduced. Guided by the conclusion that elimination/reduction of the effects by adenosine kinase (due to the toxicity caused by this adenosine kinase), inhibitor design has fallen into three categories: (i) analogs of AdoMet/AdoHcy; (ii) variation of the heterocyclic base 56 of Ari and NpcA; and, (iii) various cyclopentyl modifications (cyclic and acyclic). 57 Many research groups have made important contributions to this area (Figure 9). For example, Montgomery and co-workers 58 synthesized the 3-deaza Ari. Marquez and his colleagues 59 designed and synthesized 3-deaza NpcA. Borchardt?s laboratory 60 synthesized DHCeA (dihydroxycyclopentenyladenine) and its saturated counterpart, DHCaA. The Schneller group designed and synthesized 5'-noraristeromycin and related 20 21 coumpounds. 61 Finally, acyclic nucleosides, such as Acycliovir 57g and Penciclovir, 57h-i were synthesized and reported as potent antiviral agents. In all of these examples, the aim is to increase the selectivities toward AdoHcy hydrolase (it means that these modified compounds lack substrate activity toward these two enzymes: adenosine kinase and adenosine deaminase) and reduce the nucleotide-related toxcities. This goal was accomplished by the secondary generation AdoHcy hydrolase inhibitors (compared with their corresponding parent compounds). HO N N N NH 2 HO OH HO N N N N NH 2 OH HO N N N N NH 2 OH HO HO N N N N NH 2 OH HO N N N NH 2 OH 3-deazaaristeromycin (3-deaza Ari) HO 3-deazaneplanocin A (3-deaza NpcA) DHCeA HO N N N N NH 2 OH DHCaA HO 5'-noraristeromycin 8-Aza-7-deazanoraristeromycin Figure 9. The lead inhibitors of AdoHcy hydrolase. NH N N N O NH 2 NH N N N O NH 2 O HO Acyclovir HO HO Penciclovir Very little effort has been devoted to the aforementioned category i, analogs of AdoMet/AdoHcy. This dissertation research addresses this situation by the design and synthesis of analogs of AdoMet/AdoHcy. 22 23 Direct inhibitors targets: analogs of sinefungin It is known that inhibition of cap methylation of virus mRNA is the basis for two anti-infective strategies: (i) designing and developing inhibitors of AdoHcy hydrolase, which indirectly block the virus replication by the intracellular accumulation of AdoHcy; and, (ii) direct AdoMet methyltransferase inhibition by analogs of AdoMet/AdoHcy. This latter category can be addressed by screening structural analogues of AdoMet, such as sinefungin, AdoazaMet, MeAzaAdoMet, and carbocyclic AdoazaMet (Figure 10). Within this group, the neutral AdoazaMet derivative was first designed and synthesized by Minnick and co-workers. 62 Latter, Blackburn?s group accomplished the total synthesis of AdoazaMet and MeAzaAdoMet. 63 It was found that AdoazaMet can act as a substrate of the AdeMet-dependent methylation reactions and can serve as a competitive inhibitor for the methyltransferases. 64 Recently, the Schneller research group described the total synthesis of the carbocyclic AdoazaMet. 65 The biological activities of these compounds have been studied by Shuman and co-workers. 28c They found that, based on the cellular cap-methylating enzyme (Ecm1, the smallest known cap methyltransferase), the inhibitory effects of sinefungin were the IC 50 = 1.5?M (IC 50 is the half maximal inhibitory concentration of a substrate), while AdoazaMet and carbocyclic AdoAzaMet were relatively weak inhibitors of this Ecm1 (100 and 35?M, respectively). O HO OH N N N N NH 2 S + H 2 N CO 2 H H 3 C AdoMet Figure 10. Structural analogs of AdoMet. O HO OH N N N N NH 2 N H 2 N CO 2 H H 3 C AdoazaMet HO OH N N N N NH 2 N H 2 N CO 2 H H 3 C carbocyclic AdoazaMet O HO OH N N N N NH 2 N + H 2 N CO 2 H H 3 C MeAzaAdoMet H 3 C O HO OH N N N N NH 2 H 2 N CO 2 H sinefungin H 2 N Among these related derivatives, sinefungin has attracted the most interest in the past few decades. It is known that sinefungin is an efficient inhibitor of cap methylation in vitro and can inhibit the growth of diverse viruses, fungi and protozoa. 66 Furthermore, recently, sinefungin was found to exhibit selectivity in inhibiting the yeast cap methyltransferase versus that of the human enzyme in vivo. 67 24 25 Sinefungin, a natural complex nucleoside, 68 was first isolated from the cultures of Streptomyces griseolus, 69 and from that of Streptomyces incarnatus. 70 The structure of this nucleoside is very close to that of AdoMet (Figure 10). Since the discovery of sinefungin, its biological activities have been thoroughly studied, including antifungal, 71,72 antitumor, antiparasitic and antiviral activities. 36b,73 The nature of these biological activities results from the inhibition of a variety of AdoMet- dependent methyltransferase enzymes. 74 Moreover, it was reported that sinefungin is a selective inhibitor of virus-encoded cap methyltransferases. 74c Unfortunately, the clinical use of sinefungin has been severely precluded by its toxicity in in vivo assays. 75 To seek ways to eliminate the undesired side effects, the past decade has seen the total synthesis of sinefungin 76 and the preparation of structurally modified derivatives (side chain, sugar and base unit) 77 conducted for in-depth toxicity mechanistic studies. To pursue sinefungin, Moffatt and co-workers were the first to complete a total synthesis, but they could not separate the C-6' epimers (Figure 11). 76a In 1991, Barton and co-workers published a convenient synthesis of natural sinefungin and its C-6' epimer by radical coupling reactions. 76f Later, Fourrey?s group succeeded in synthesizing sinefungin and separating its C-6' epimer by a practical method different from that of Moffatt's attempts. 76b Rapoport and co-workers adopted another stereoselective appoach for synthesizing sinefungin and confirmed its C-6' chiral center as the S configuration by X-ray crystallographic analysis method. 76e O HO OH N N N N NH 2 H 2 N CO 2 H sinefungin H 2 N O HO OH N N N N NH 2 H 2 N CO 2 H C-6' epimer sinefungin H 2 N Figure 11. Sinefungin and its C-6' epimers. Later, Rapoport?s group reported the total synthesis of two analogs of sinefungin: (S)-6'-methyl-6'-deaminosinefungin and 6'-deaminosinefungin (Figure 12). 77f Almost concurrently, Secrist and co-workers reported the synthesis of a series of analogs of sinefungin, 77b for example, C-5' chain-extended adenosine derivatives related to sinefungin. Robert-Gero and colleagues synthesized a series of side-chain analogues of sinefungin and found that all completely lost activities. 77d These studies indicated that the terminal amino and carboxyl groups, connecting at C-9' in (S) configuration, are necessary for maintaining the biological activities of the parent compound. In 1990, Fourrey?s group 77c succeeded in preparing three analogues of sinefungin, and recently reported the total synthesis of higher homologs of sinefungin by a radical- based strategy. 77j These latter studies lack any biological data. 26 O HO OH N N N N NH 2 H 2 N CO 2 H (S)-6'-methyl-6'-deaminosinefungin H 3 C O HO OH N N N N NH 2 H 2 N CO 2 H 6'-deaminosinefungin O HO OH N N N N NH 2 HO 2 C H 2 N O HO OH N N N N NH 2 H 2 N O HO OH N N N N NH 2 H 2 N CO 2 H H 2 N Homosinefungin Figure 12. Some nucleoside dervatives of sinefungin: modification on the side-chain. O HO OH N N N N NH 2 H 2 N R decarboxylated sinefungin analogs R=NH 2 , CH 2 NH 2 9'-deaminosinefungin vinylsinefungin analog O HO OH N N N N NH 2 HO R R=NH 2 , CONH 2 O HO OH N N N N NH 2 H 2 N H 2 N 9'-decarboxylsinefungin The Ghosh laboratories were the first to use efficient asymmetric synthetic methods to construct the two stereogenic centers (C-6' and C-9') of sinefungin. 76f-g 27 Almost concurrently, the Fourrey group reported the first sugar modified analog of sinefungin: carbocyclic sinefungin. 77i This grew out of the known bio-properties of carbocyclic nucleosides, whether naturally occurring or chemically synthesized. 78 In addition to seeking the side-chain modified derivatives of sinefungin, changing the heterocyclic base is relevant to guiding this dissertation project. In that regard, Barton and co-workers reported the total synthesis of uracil derivatives of sinefungin (Figure 13). 77e When compared to sinefungin, biological activities of these derivatives were dramatically reduced, as were their affinities for the methyltransferases. These observations point to the significance of the adenine portion of sinefungin for its biological properties. O HO OHH 2 N CO 2 H H 2 N NH N O O O HO OHH 2 N CO 2 H H 2 N NH N O O Figure 13. Base modified derivatives of sinefungin. uracil analog of sinefungin dihydrouracil analog of sinefungin Based on the various biological activities of sinefungin and its undesirable side effects (in vitro and in vivo), and its unique mechanism of action at viral mRNA methyltransferase level, the structurally related derivative target I (Figure 14), where the 6'-amino of carbocyclic sinefungin is replaced by the isoelectronic hydroxyl group, 28 become a focus for this dissertation. Also, preliminary work on a new method towards carbocyclic sinefungin is decribed. O HO OH N N N N NH 2 H 2 N CO 2 H sinefungin H 2 N HO OH N N N N NH 2 H 2 N CO 2 H carbocyclic sinefungin H 2 N HO OH N N N N NH 2 H 2 N CO 2 H HO Figure 14 Initial targets of dissertation research. target I1 For comparative purposes, target II (Figure 15) was seen as a possible goal. For this purpose, the synthesis of its sugar segment became a feature of this dissertation research. 29 O HO OH N N N N NH 2 H 2 N CO 2 H target II HO O HO OH N N N N NH 2 H 2 N CO 2 H sinefungin H 2 N Figure 15. Initial target of dissertation research. In the meantime, due to the difference methods for coupling the heterocyclic base with the carbocyclic pseudo sugar and natural sugar, there was a challenge for target II to couple the heterocyclic base. Therefore, in the second project of this dissertation, the important precursor 2 toward target II was finished here (Figure 16). O HO OH N N N N NH 2 H 2 N CO 2 H target II HO O O O OPMB TBSO N N MeO OMe 2 Figure 16. Important precursor 2 for target II. 30 CHAPTER 2 SYNTHESIS OF THE IMPORTANT INTERMEDIATES FOR TARGET I AND CARBOCYCLIC SINEFUNGIN In order to pursue target I and carbocyclic sinefungin, three important intermediates (Figure 17) were needed in large scale. OAc HO O O O 6 11 O O O 21 Figure 17. Important intermediates for the targets. Compound 6, (+)-(1R,4S)-4-hydroxy-2-cyclopentyl acetate, has served as a general building block for the synthesis of carbocyclic nucleosides in our laboratories and other groups. 61b,79 It can be obtained from the enzymatic-catalyzed chemical reaction of diacetate 5 (Scheme 5) with PCL (Pseudomonas cepacia lipase). The use of various enzymes in chemical reactions is now a common technique for the enantioselective synthesis of nucleosides. 80 This procedure has been developed more extensively in the Schneller laborotary. 61a,82 31 O OAc AcO OAc HO 3 4 5 6 heat, 170 0 C CH 3 CO 3 H, Na 2 CO 3 , CH 2 Cl 2 , -10-0 0 C Pd(PPh 3 ) 4 , (CH 3 CO) 2 O THF, N 2 , -20 0 C-rt Buffer solution, pH=7.0-7.4 (HPO 4 -2 and H 2 PO 4 -1 ), PCL Scheme 5. Synthesis the important precursor 6. The requisite diacetate 5 was synthesized following reported procedures 81 : epoxidation of freshly prepared cyclopentadiene 3 gave epoxide 4, that was, in turn, treated with tetrakis(triphenylphosphine)palladium(0) followed by subjecting the resultant palladio zwitterion to acetic anhydride. Compound 6 permitted access to precursor 11 again using a procedure developed in our group and other groups (Scheme 6). 83 Thus, treatment of monoacetate 6 with diethylchlorophosphate provided 7, whose the double bond was oxidized to the diol 8 under catalytic osmium tetraoxide and N-methylmorpholine N-oxide (NMO) conditions. After protecting the diol 8 with an isopropylidene group to compound 9, lithium hydroxide hydrolysis formed 10. Finally, oxidative-elimination by pyridinium chlorochromate (PCC) on 10 afforded compound 11. 83b-c The advantage of this method is that 11 can be obtained in large scale (more than 20 grams). 32 O O OAc (EtO) 2 OPO O O OH (EtO) 2 OPO O O O HO OH OAc (EtO) 2 OPO OAc (EtO) 2 OPO OAc HO 6 7 8 9 10 11 Py, CH 2 Cl 2 , (EtO) 2 POCl 0 0 C-rt, 4 h 95% OsO 4, NMO, acetone/H 2 O, rt, 12 h 55% 2, 2-dimethoxypropane, acetone, cat. H 2 SO 4 , 1.5 h 85% LiOH?H 2 O, THF/H 2 O rt, 12 h 93% PCC, celite, CH 2 Cl 2 , rt, 36 h 40% Scheme 6. Synthesis of 11 from monoacetate 6. In addition to the above method, other groups developed alternative approaches to compound 11. 84 However, some of these methods use extreme reaction conditions and the yields are not reproducible. Based on the previous procedures for the synthesis of 11, the Schneller group developed another more efficient and practical method to 11 (Scheme 7). 65 33 O OHHO HO OH O O O OCH 3 HO O O O OCH 3 I O O O O O OH O O OH O O O PDC, 4? MS acetic acid, rt, 12 h 12 13 14 15 11 acetone, CH 3 OH, 2, 2-dimethoxypropane cat. H 2 SO 4 , rt, 12 h D-ribose 98% PPh 3 , imidazole toluene/acetonitrile, reflux, I 2 55% n-BuLi, THF, -78 0 C, 2 h (115%) vinylmagnesium bromide, THF, -40-0 0 C 26% Grubbs' catalyst 1 st generation CH 2 Cl 2, 40 0 C, 12 h Scheme 7. Another approach to form 11. 40% This optimized method began with the commercially available D-ribose. Its protection using 2, 2-dimethoxypropane and methanol catalyzed by sulfuric acid afforded 12, which was converted into the iodide 13. 85 To secure the desired compound 14, reductive elimination (Boord elimination) of 13 was used with n-butyllithium at low temperature. 86 Exposure of 14 to vinylmagnesium bromide yielded 15. Ring-closing metathesis (RCM) of 15 catalyzed by Grubbs? 1 st generation catalyst, 87 followed by oxidation of the intermediate allylic alcohol, provided 11. The overall yield of 11 from D- ribose was very low, possibly due to instability and volatility of 14. In order to overcome this disadvantage in the synthesis of 11, the cyclopentylidiene protected derivative (21) was proposed (Scheme 8). 88 In contrast to Scheme 7, the first step in Scheme 8 replaced 2, 2-dimethoxypropane with 34 cyclopentanone. In addition, aldehyde 18, which was more stable than 14, was obtained by reductively cleaving 17 with activated zinc in hot methanol. 89 This eliminated the difficulty of scale-up associated with the n-butyl lithium step of Scheme 7. In this synthetic methodology, all the reaction conditions are mild and give good yields of 21, up to 15 grams. The overall yield of this method was 40% for 5 steps. O OHHO HO OH O O O OCH 3 HO O O O OCH 3 I O O O O O OH O O OH O O O 16 17 19 20 21 CH 3 OH,cyclopentanone, trimethyl orthoformate, PTSA, reflux TPP, imidazole toluene/acetonitrile, reflux, I 2 70% CH 3 OH, Zinc, reflux 94% 18 vinylmagnesium bromide, Et 2 O, 0 0 C 75% Grubbs' catalyst 1 st , CH 2 Cl 2, 40 0 C, 12 h DMSO, DIPEA, SO 3 ?Py CH 2 Cl 2 , rt, 4 h 81% Scheme 8. Synthesis of the important precursor 21 from D-ribose. D-ribose quantitative 35 The key step for achieving the important intermediates (11 and 21) was the ring closing metathesis using the Grubbs? catalysts (Figure 18). In the past two decades, olefin metathesis has become the most important method for the formation of carbon-carbon single bonds. 90 The Grubbs? catalysts are the most prominent for this purpose. 91 Some of the ruthenium-based catalysts are commercially available, such as those designated as Grubbs? 1 st and 2 nd generation. These agents are highly efficient catalysts, can tolerate various functional groups, and are stable to air, water, and acids. 92 These advantages make this class of catalysts attractive for practical applications in organic chemistry and related areas. Ru PCy 3 Cl Cl N N Cy=Cyclohexyl 2nd generation Figure 18. Grubbs' catalysts. Ru PCy 3 PCy 3 Ph Cl Cl 1st generation Ph The mechanism of olefin metathesis catalysis has been proposed via two routes: associative and dissociative. 87b The latter one is considered the dominant mechanism (Scheme 9). During the catalytic cycle, one ligand (the phosphine) first dissociates from the ruthenium center. Then, ruthenium binds the olefin, forming a 16, 18, or 14-electron coordinate complex (Scheme 9, a 14-electron coordinate complex). This complex undergoes the metathesis to form the four-member ring at the ruthenium center, which follows with disconnection of one olefin from the system. The newly formed ruthenium center coordinates another part of the olefin and forms a new 16, 18, or 14-electron coordinate complex. Finally, after the metathesis (forming the four-member ring product), 36 the desired olefin is obtained and the catalyst can be regenerated by recombining the first phosphine ligand, and enters the next catalytic cycle. (PR 3 ) 2 X 2 Ru=CHR' (PR 3 )X 2 Ru=CHR' + PR 3 (R 3 P)X 2 Ru R' CH 2 =CHR' + RuX 2 (PR 3 ) (PR 3 ) 2 X 2 Ru=CH 2catalytic cycles Scheme 9. Dissociative mechanism of olefin metathesis. RuX 2 (PR 3 ) PR 3 37 38 CHAPTER 3 SYNTHESIS OF TARGET I Experiment Design and Approaches In order to design the synthesis of target I, a retrosynthetic analysis was considered (Scheme 10). The key steps in this approach are installation of the C-6' and C- 9' stereo centers by efficient asymmetric synthetic methods. The C-6' hydroxyl chiral center arises from building block A that can be constructed by calling on a highly diastereoselective allylborating agent. 93 The amino acid at C-9' was seen available in three steps: (i) cleaving the double bond of the allylic alcohol of A and oxidizing the resultant primary alcohol (D) (Figure 19) to the corresponding aldehyde; (ii) using a Wittig-Horner reaction, 94 with the phosphonate B, which can be generated from Boc- glycine ethyl ester, 95 to deliver the amino acid moiety (E), while extending the side chain needed for target I; and, (iii) asymmetric hydrogenation of the newly formed double bond of E to give the desired configuration at C-9', (S). HO OH N N N N NH 2 H 2 N CO 2 H ( target I ) HO 1 O O N N N N NH 2 HO O O N N N H N NH 2 PO(OEt) 2 BocHN EtO 2 C OH EtO 2 C + OAc HO O O O 6 11 Scheme 10. Retrosynthetic analysis of target I. A B C + 6' 9' OO N N N N NH 2 PGO D HO OO N N N N NH 2 PGO BocHN CO 2 Et E Figure 19. Proposed intermediates for target I. PG = protecting group 39 40 It was seen from Scheme 11 that building block C (24) was achieved by two steps: (i) a regioselective and stereoselective 1,4 Michael addition with ethyl tri(methylsilyl) acetate to 11 followed by hydrolysis to remove the TMS group to furnish 23 (Scheme 11), in 55% yield for two steps ; and, (ii) reduction of 23 with sodium borohydride/cerium chloride heptahydrate (Luche reduction) stereoselectively to afford 24 (also C of Scheme 11). Then, compound 24 was subjected to standard Mitsunobu conditions (diisopropyl azodicarboxylate and triphenylphosphine, TPP) to provide 25 in 40% yield. Finally, reduction the ester of 25 with diisobutylaluminum hydride (DIBAH) at low temperature yielded a mixture of 26 and 27 (27 is a byproduct confirmed by TLC and H NMR). The mixture was difficult to separate by standard silica gel column chromatography. 96 97 98 1 99 O O O O O O TMS EtO 2 C O O O EtO 2 C O O EtO 2 C OH OO EtO 2 C N N N N Cl O O N N N N Cl O OO N N N N Cl HO + 1. DIPA, n-Buli, -78 0 C 2. Ethyl tri(methylsilyl)acetate, HMPA/THF KF/EtOH/H 2 O or NH 4 Cl 55% NaBH 4 , MeOH CeCl 3 ?7H 2 O 86% TPP, DIAD, 6-chloropurine, THF 11 22 23 24 25 26 27 Scheme 11. First approach to building block A. 40% 82% DIBAH, CH 2 Cl 2 , -78 0 C 41 To resolve this problem, an alternative approach (Scheme 12) was explored. In this method, introduction of a vinyl group to 11 by a cuprate-catalyzed Michael addition selectively afforded 28 in 80% yield. Adding trimethylsilyl chloride (TMSCl) and hexamethylphosphoramide (HMPA) accelerates the selectivity for the 1,4-addition. 65 Reduction of 28 using lithium aluminum hydride selectively provided 29 in high yield, 92%. Employing Mitsunobu reaction conditions, compound 30 was successfully obtained. Hydroboration-oxidation was conducted with 30 to produce 27. Unfortunately, the yield in this step was very low, 25%. A possible explanation may be the steric hindrance of the heterocyclic base, affecting the attack of the bulky hydroborating agent on the double bond from the desirable syn face. To move forward with this project, subjecting compound 27 to a modified Swern oxidation reaction 100 produced aldehyde 26. Having authentic compound 27 was used to confirm its presence in the mixture with 26 in Scheme 11. O O O O O O O O OH LiAlH 4 , THF OO O O N N N N Cl HO OO N NN N Cl O N N N N Cl CuBr?Me 2 S , vinylmagnesium bromide TMSCl, HMPA, THF, -78 0 C 80% 92% TPP, DIAD, THF 6-chloropurine, 60% 1. 9-BBN, THF 2. MeOH, NaOH, H 2 O 2 25% DMSO, DIPEA CH 2 Cl 2 , SO 3 ?Py 70% 11 28 29 30 27 26 Scheme 12. The second approach for 26. The aldehyde 26 was then employed to set up the C-6' stereocenter and to extend the side-chain by two carbon atoms. Asymmetric allylation was pursued for this purpose. 101 In this direction, there are many asymmetric allylation methods and related chiral auxiliaries developed for this purpose. 93,102,103 The most common are boron-based 42 agents as shown in Figure 20: (i) the tartrate-based allylboron compound (Roush?s reagent); (ii) Corey?s allylating reagent; and, (iii) Brown?s reagents. From the view of the target preparation, focus was on the highly efficient and easy synthesized chiral allylborane auxiliary: (+) and (-)-B-allyldiisopinocampheylborane (Ipc 2 BCH 2 CH=CH 2 ), which can be achieved in situ from the commercially available precursor (B- methoxydiisopinocampheylborane) upon reaction with allylmagnesium bromide. 93a 2 (-)-B-allyldiisocampheylborane B 2 B (+)-B-allyldiisocampheylborane Figure 20. Boron-based allylating reagents. O B O O O i Pr O O i Pr N B N Ph Ph Ts Ts Ts = -SO 2 C 6 H 4 CH 3 Roush's reagent Corey's reagent Brown's reagents A comment about the mechanism of allylation of aldehydes with Brown?s reagent (Ipc 2 BAll) is appropriate at this point (Figure 21). 93f It has been shown that the allylboration reagent (Ipc 2 BAll) can form a chair-like six-membered ring transition state with the aldehyde. Due to the face selectivity of the aldehyde, there are two possible transition states: Si face and Re face (the allylborane attacking the aldehyde from two different sides). The nature of face selectivity results from the minimization of the steric 43 interactions in space between the bulky axial Ipc ligand and the aldehyde group. In this allylation reaction, the chirality of the boron ligands induces the chirality of the new asymmetric center (asymmetric allylic alcohol). 101a B H H 3 C H 3 C H H 3 C H CH 3 CH 3 H 3 C H H H H H O H R Figure 21. Proposed mechanism of allylation of aldehydes with Brown's reagent. Si Face 2 (-)-B-allyldiisocampheylborane B Unfortunately, allylation reaction of 26 with Ipc 2 BAll (synthesized in situ following the literature 93a ) led to a complex mixture (by TLC; not the desired 31) (Scheme 13). It was concluded that this observation was due to the instability of the newly formed Ipc 2 BAll to air or moisture, even though the filtration was handled under dry nitrogen. Finally, a one-pot synthetic method (without removal the magnesium salt) was developed to successfully carry out this allylation reaction to give the desired enantiomeric pure product. 44 OO N N N N Cl O OO N N N N Cl HO 26 31 Scheme 13. Allylation reaction of 26. 1. (-)-B-Ipc 2 OMe, ethyl ether, allylmagnesium bromide, -78 0 C 2. NaOH, MeOH, H 2 O 2 At this point attention returned to the limitations to the routes being pursued. In that regard, while Scheme 12 represents a successful process to 26, it is limited by several disadvantages. First, the yield in the hydroboration-oxidation step (i.e., 30 to 27) is low. Also, the pathway from compound 11 to 27 is very time consuming and the yield of coupling the 6-chloropurine with 29 to 30 was poor. Additionally, there was some uncertainty whether the 6-chloropurine of 26 could withstand further requisite side chain conversions. Based on the previous studies in our group, another concern was whether the asymmetric hydrogenation of the Wittig-Horner product E (Figure 19) would be straightforward. Other difficulties were foreseen (such as low yields and numerous protection/deprotection steps) for installing the side chain of target I, from 26. Therefore, a more suitable strategy to target I was sought. The alternative approach (Scheme 14) was to replace 6-chloropurine with adenine as the coupling heterocyclic base with the building block A near the end of the synthesis and to replace the isopropylidene protected 11 with the cyclopentanone as protected product 21 (see earlier in dissertation). The amino acid unit in this approach was to be 45 introduced via Sch?llkopf?s bis-lactim ether chiral auxiliary (building block C, Scheme 14). 104 Building block A was to be achieved from precursor B, and B to be obtained from the allylation reaction under Brown?s reagent from the corresponding aldehyde, which was expected from 21. HO OH N N N N NH 2 H 2 N CO 2 H (target I ) HO 1 N N N H N NH 2 + Scheme 14. Optimized approach for target I. OGP PGO O O N N MeO OMe OGP PGO O O + N NMeO OMe O O O D-ribose A B C 21 PG= Protecting Group 6' 9' The selection of Schollkopf?s bis-lactim ether chiral auxiliary for the purpose of this project arose from the following consideration: it is known that ?-amino acids exist 46 in either the D or L form (Figure 22), with the majority of naturally occurring amino acids being L-amino acids. For target I, the terminal amino acid is L (by the Cahn- Ingold-Prelog designation, it is S configuration). Often in the laborotary, L-amino acids are obtained by biological methods, such as enzymatic syntheses and resolution techniques. 105 Alternatively, asymmetric synthesis methodology using chiral reagents or auxiliaries or synthons, is a powerful tool for preparing the optically active ?-amino acid derivatives. 106 Among the synthetic methods, various asymmetric derivatives of glycine or corresponding glycine equivalents, provide convenient precursors for direct formation of the new stereogenic amino acid center. 107 The first efficient derivative of a glycine equivalent was developed by Sch?llkopf and co-workers, 104d which later was called Sch?llkopf reagent (building block C, Scheme 14), and was proved to be a highly efficient enantioselective reagent for preparing ?-amino acids. 108 Although building block C is commercially available, it is very expensive. Therefore, a modified literature procedure 109 was designed for C as shown in Scheme 15. CO 2 HH 2 N RH (S)-L-amino acid CO 2 HH 2 N RH (R)-D-amino acid Figure 22. Configurations of amino acids. 47 H 2 N BocHN 32 BocHN O N H 33 N H H N O O 34 N N O OH O OH O OMe OMe MeO 35 Boc 2 O, NaHCO 3 THF-H 2 O D-valine 1. ClCO 2 i-Bu, Et 3 N 2. CH 3 O 2 CCH 2 NH 2 -HCl CH 2 Cl 2 Dichlorobenzene Heat, 175 0 C (CH 3 ) 3 O BF 4 CH 2 Cl 2 , pH=8-9 Scheme 15. Synthesis of the Sch?llkopf reagent (35, building block C). 90% 82% 65% 78% Protection of commercially available D-valine with di-tert-butyl dicarbonate led to 32 in quantitative yield. Treatment of 32 with isobutyl chloroformate under basic conditions followed by glycine methyl ester hydrochloride, provided the Boc-protected dipeptide 33. Then, cyclization of 33 in 1, 2-dichlorobenzene gave 34 in excellent yield. Finally, methylation of 34 with the commercially available trimethyloxonium tetrafluoroborate in dichloromethane at room temperature resulted in (2R)-2, 5-dihydro-3, 6-dimethoxy-2-isopropylpyrazine, 35 (also C in Scheme 14) in good yield, after the high vacuum distillation. The NMR ( 1 H and 13 C) data of 35 was in agreement with the literature. 109c After building block C (compound 35) was in hand, attention was shifted to constructing additional components of Scheme 14, shown in Scheme 16. With compound 48 49 21 as the starting material, the first two steps were adopted from Scheme 12. Thus, a Michael addition reaction employing vinylmagnesium bromide and the enone 21 provided compound 36 as the single isomer. Reduction of 36 using lithium aluminum hydride selectively afforded the secondary alcohol 37 in 90% yield. Scheme 14 shows that a modification of the side chain of 37 was necessary for coupling of the heterocyclic base (adenine). Therefore, protection of 37 with p-methoxybenzyl chloride under strongly basic conditions, provided 38. The hydroboration-oxidation reaction was then conducted on 38, resulting in the primary alcohol 39 in good yield. The oxidation of 39 using modified Swern oxidation conditions, yielded aldehyde 40. 1. CuBr?Me 2 S vinylmagnesium bromide 2. TMSCl, HMPA/THF, -78 0 C 75% LiAlH 4 , THF 90% O O O 21 O O O 36 O O OH O O OPMBNaH, DMF, PMBCl 90% O O OPMB O O OPMB 1. 9-BBN,THF 2. MeOH, NaOH, H 2 O 2 HO DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 O 70% 90% O O OPMB 1. (-)-B-Ipc 2 OMe, allylmagnesium Bromide, -78 0 C 2. MeOH, NaOH, H 2 O 2 90% HO O O OPMB MOMO MOMCl, DIPEA, CH 2 Cl 2 37 38 39 40 41 42 72% Scheme 16. Synthesis of the important intermediate 42 (building block B). 1' 4' 5'6' 8' 9' 5 The aforementioned Brown stereospecific hydroboration was next performed on the aldehyde 40, using freshly prepared (-)-B-allyldiisopinocampheylborane (in situ), to afford the desired enantiomerically pure homoallylic alcohol 41 in 90% yield for two steps. This product was confirmed by two-dimensional NMR Spectroscopy ( 1 H, 1 H- COSY) illustrated in Figure 23. The ( 1 H, 1 H-COSY) 2D spectrum of compound 41 in CDCl 3 (Scheme 16) shows that. the diagonal peak labeled 8'-H on the left-hand corner 50 51 has the symmetric cross-peaks with 9'-H (2H) around the diagonal. When proceeding along the diagonal line, the symmetric cross-peaks are the CH 2 in PMB group. Then further moving along the diagonal line, 2'-H has a symmetric cross-peak with 3'-H and it has another upper cross-peak in the vertical line, which is deduced for 1'-H. From the diagonal peak of 1'-H, the cross-peaks of 5-H (2H) are confirmed. Besides the symmetric cross-peaks of 5-H (2H) around the diagonal line, both of them has another symmetric cross-peaks, which is deduced as 4'-H. Continuing moving along the diagonal line, the diagonal peak of 6'-H has a weaker cross-peak, which is identified as 7'-H (2H) and also has two cross-peaks. The much stronger intensity is identified as 5'-H (trans with the H atom of C-6') and the other is considered as the cis 5'-H. The remaining diagonal peaks are not accompanied by any cross-peak. 52 Figure 23. COSY spectrum of compound 41 in deuteriochloroform: (a) the small one chemical shift ? (1.0-7.5) and (b) the magnified one ? (1.0-2.5). With 41 available, its C-6' hydroxyl group was protected as the methoxymethyl ether to provide 42 (also building block B in Scheme 14). 110 Combining 42 and 35 was next addressed. Based on the literature, 104,108 the use of the Sch?llkopf?s auxiliary, 35 (building block C), is best conducted using its lithiated 53 (2R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine derivative together with haloderivatives as co-reactant. The most common halo derivative is the bromo species. However, in this case, the iodo derivative 44 was chosen for its ease of preparation and efficiency as a good leaving group (Scheme 17). O O OPMB MOMO 42 O O OPMB HO MOMO 43 1. NaIO 4 , OsO 4 , MeOH/H 2 O 2. NaBH 4 , CeCl 3 ?7H 2 O, MeOH 50% O O OPMB I MOMO 44 TPP,imidazole, I 2 , toluene/acetonitrile, reflux 40% Scheme 17. Synthesis of iodide 44. To achieve 44, compound 42 was subjected to oxidative-cleavage conditions using sodium (meta) periodate (under osmium tetroxide catalysis). This was followed by reduction of the resulting aldehyde to afford 43 in moderate yield. Then, compound 43 was converted to iodide 44 according to standard iodination conditions (triphenylphosphine, imidazole, iodine), but resulted in very low yield. 85b This low yield was believed to be due to the formation of unwanted intermediates in the conversion of 42 to 43. 54 55 To move past this problem, Scheme 18 shows an optimized approach to afford the tosylate 45, which we hope to use in place of the iodide 44. In this method, oxidation of the double bond of 42 under osmium tetraoxide/N-methylmorpholine N-oxide (NMO) conditions provided the diol 42-1'. This was followed by cleaving 42-1' under sodium periodate standard conditions to 42-1'' in quantitative yield. 111 Compound 42-1' and 42- 1'' were proved by 1 H NMR Spectroscopy data (also TLC analysis). In turn, Luche reduction of aldehyde 42-1'' gave 43 in excellent yield (three steps, 75%). Treatment of 43 with p-toluenesulfonyl chloride under basic conditions using triethylamine and catalytic amount of 4-dimethylaminopyridine (DMAP) afforded the tosylate 45 in good yield. 112 O O OPMB MOMO 42 O O OPMB MOMO HO HO O O OPMB O MOMO 42-1'' O O OPMB HO MOMO 43 OsO 4 , NMO, THF, 0 0 C NaIO 4 , CH 2 Cl 2 /H 2 O, 15-crown-5 NaBH 4 , CeCl 3 ?7H 2 O MeOH 42-1' O O OPMB TsO MOMO 45 TsCl, Et 3 N, DMAP, 0 0 C 75% 90% Scheme 18. Optimized approach for the synthesis of tosylate 45. Compound 45 was then used in the next stage of the synthesis (Scheme 19), that is, to construct the terminal amino acid unit. Thus, treating the tosylate 45 with the lithio derivative of 35 (building block C, Scheme 14) in anhydrous THF at low temperature gave compound 46. The stereospecificity of the transformation from tosylate 45 to 46 was attributed to steric interaction imposed by the bulky moiety of 45 with the chiral dihydropyrazine to the less hindered face of lithio (2R)-2,5-dihydro-3,6-dimethoxy-2- isopropylpyrazine masked amino acid (Figure 24) to afford diastereoselectively the trans 56 product 46 in 70% yield. Then compound 46 was subjected to 2,3-dichloro-5,6-dicyano- 1,4-benzoquinone (DDQ) to provide the product 47. 119 From compound 47 to 49 (target I), these two steps could not be finished in the time available. N N OMe H H 3 C CH 3 H H R' C X H H Li Figure 24. Transition state for the alkylation reaction. MeO 57 O O OPMB TsO MOMO 45 O O OPMB MOMO N N OMe MeO 46 O O OH MOMO N N OMe MeO 47 O O MOMO N N OMe MeO 48 N N N N NH 2 HO OH HO N N N N NH 2 H 2 N CO 2 H 49 (target I) Scheme 19. Synthesis of 47, a close precursor to target I. 1. n-BuLi, 35, THF, -78 0 C, 5 h; -20 0 C, 12 h 2. EtOH or NH 4 Cl 70% DDQ, CH 2 Cl 2 /H 2 O r.t., 30 min 65% 58 59 CHAPTER 4 SYNTHESIS OF CARBOCYCLIC SINEFUNGIN BUILDING BLOCKS (53 and 55) Experimental Design and Approaches Carbocylic sinefungin is a target of this dissertation research. Scheme 20 represents a retrosynthetic analysis to this compound. Installation of the C-6' and C-9' stereocenters was seen by adapting the efficient asymmetric synthetic methods of Scheme 14. In that direction, the C-6' (S) amine group of building block A was foreseen to be constructed in three steps: (i) employing a highly diastereoselective allylborating agent to introduce a C-6' (R) hydroxyl group; (ii) a S N 2 substitution reaction by sodium azide under the basic conditions; and, (iii) hydrogenation of the azide species to the desired primary amine. The amino acid at C-9' was to be achieved using Sch?llkopf?s auxiliary alkylation reaction whereby the absolute configuration of the newly introduced C-9' stereo center (as S) was to be set based on the findings in Sch?llkopf?s asymmetric amino acid synthesis. 104e HO OH N N N N NH 2 H 2 N CO 2 H Carbocyclic sinefungin H 2 N N N N H N NH 2 + Scheme 20. Retrosynthetic analysis of carbocyclic sinefungin. OPG PGO O O N N MeO OMe OPG PGO O O + N NMeO OMe A B C PG = Protecting Group O O O 11 HO OAc 6 Working collaboratively with Dr. Xue-Qiang, Yin (a postdoctor fellow in our group) on this investigation, building block B was established for this dissertation project. Thus, beginning with enone, compound 11 (see chapter 2), a conjugate addition (Michael reaction) was conducted with vinylmagnesium bromide in the presence of a copper bromide dimethylsulfide complex to afford 28 in 80% yield (Scheme 21). Reduction of 28 with lithium aluminum hydride selectively provided 29 in 92% yield. At this stage, modification of the side chain of 29 was necessary. Thus, protection of the secondary alcohol of 29 was carried out with p-methoxybenzyl chloride under strong basic 60 conditions to yield 50. Hydroboration of compound 50 with 9-borabicyclo[3.3.1]nonane (9-BBN) was followed by oxidation under hydrogen peroxide conditions to achieve the primary alcohol 51 in good yield. Finally, oxidation of 51 by modified Swern oxidation conditions provided intermediate 52. O O O 11 O O O 28 O O OH O O OPMB O O OPMB HO O O OPMB O 29 50 51 52 CuBr?Me 2 S, vinylmagnesium bromide TMSCl, HMPA, THF, -78 0 C 80% LiAlH 4 , THF 92% DMF, NaH, p-methoxybenzyl chloride 60% 1. THF, 9-BBN 2. MeOH, NaOH, H 2 O 2 92% DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 87% O O OPMB 53 HO 1. (+)-B-Ipc 2 OMe, allylmagnesium bromide, Et 2 O, THF, -78 0 C 2. MeOH, NaOH, H 2 O 2 93% Scheme 21. Synthesis of 53. At this point, attention was drawn to establishing the C-6' (S) chiral center by an allylation reaction with the aldehyde 52. For this purpose, the aforementioned Brown?s stereospecific hydroborating reagent, freshly prepared (+)-B- allydiisopinocampheylborane in situ was used to accomplish the allylation reaction with 61 aldehyde 52. This afforded the desired enantiomerically pure homoallylic alcohol 53 in two steps (93%). 93 Compound 53 was provided to Dr. Yin to finish the latter total synthesis of carbocyclic sinefungin, which has subsequently been published. 113 To investigate the allylation procedure more extensively, in this instance the aldehyde 52 was subjected to the freshly prepared (-)-B-allydiisopinocampheylborane (in situ) to provide the enantiomerically pure homoallylic alcohol 54 in two steps in excellent yield (Scheme 22). Then, protection of the homoallylic alcohol with benzyl bromide under the basic conditions afforded compound 55 in good yield (85%). 117 O O OPMB O 52 O O OPMB 54 HO 1. (-)-B-Ipc 2 OMe, allylmagnesium bromide, Et 2 O,THF, -78 0 C 2. MeOH, NaOH, H 2 O 2 90% Scheme 22. Synthesis of 55. O O OPMB 55 BnO benzyl bromide, NaH, DMF, 0 0 C 85% Compound 55 was initially considered as an important intermediate for accomplishing of target I (Chapter 3, Scheme 14). But later, based on that fact that 62 63 compound 21 was developed in our group as a good building block for carbocyclic nucleoside derivatives, 88 replacing 11 with 21 (Chapter 2, Figure 17) became acceptable for target I (Scheme 14). The stereochemistry of the homoallylic alcohol center was established by the Horeau method. There are several methods available to determine the absolute configuration of secondary alcohols in chiral molecules, such as X-ray diffraction method, 114a chemical interrelation method, 114b Horeau?s method 115 and Nuclear Magnetic Resonance method. 114c-d Among the methods, Horeau?s method was chosen here as an empirically correlative approach (based on the kinetic resolution of 2-phenylbutyric anhydride), and its determination of the absolute configuration of secondary alcohols has been successfully applied to natural products. 116 In the method, the excess anhydride is hydrolyzed and the net rotation of the resulting acid is measured. It has been found experimentally that there is a relationship between the sign of optical rotation of the recovered 2-phenylbutyric acid and the absolute configuration of the stereo-alcohol involved. 115 Thus, if the resulting rotation of 2-phenylbutyric acid recovered is levorotatory, the secondary alcohol will be configured in the (S) form that is shown in Figure 25 (L and M refer to the large and medium substituents.). On the contrary, if the rotation of 2-phenylbutyric acid recovered is dextrorotatory, the secondary alcohol will be in the (R) configuration. CO 2 H C 6 H 5 H C 2 H 5 (R)-(-)-2-phenylbutanoic acid recovered H OH M L Figure 25. Horeau's method. (S) correlated with Based on Horeau?s method, a modified approach using racemic 2-phenylbutyryl chloride to replace the anhydride as the resolving reagent has been shown to be more effective for the secondary alcohols with bulky groups. 114d Therefore, experimentally, the excess racemic 2-phenylbutyryl chloride reacted with compound 53 (Scheme 21) under the basic conditions, followed by the hydrolysis, the rotation of the resulting 2- phenylbutyric acid was measured as levorotatory. So, the secondary alcohol of 53 was deduced to be in (S) configuration. At the same time, this result is consistent with the Si face selectivity with the Brown allyboration conditions employed in Figure 21. 93f 64 65 CHAPTER 5 SYNTHESIS OF TARGET II Experiment Design and Approaches A retrosynthetic design to the 6'-hydroxyl analog of sinefungin (target II) is shown in Scheme 23. As before the key steps in this synthesis are establishment of the C- 6' and C-9' stereo centers by efficient asymmetric synthetic methods, as employed to target I. The C-6' hydroxyl chiral center of building block A (Scheme 23) should be available by the highly diastereoselective allylborating conditions. 93 Likewise, the C-9' amino acid was to be introduced via now well established Schollkopf?s bis-lactim ether chiral auxiliary, (building block C) of Scheme 14. 104 Building block A was envisioned from B, and B, in turn, was obtainable from commercially available D-ribose in several steps. O HO OH N N N N NH 2 H 2 N CO 2 H target II HO N N N H N NH 2 + Scheme 23. Retrosynthetic analysis of target II. O PGO O O N N MeO OMe O PGO O O + N NMeO OMe A B C PG = Protecting Group OMe OMe O HO HO OH OH D-ribose Realization of precursor B began with commercially available D-ribose (Scheme 24). Its protection using 2, 2-dimethoxypropane and methanol catalyzed by surfuric acid afforded 12, which was then subjected to oxidation under modified Swern oxidation conditions to provide 56 in moderate yield. Compound 56 underwent a Wittig reaction with methyl triphenylphosphonium bromide under the strong basic conditions to furnish 57. 84a It was noted that the transformation from 56 to 57 was accompanied epimerization at C-4, due to the strong basic conditions, so the yield for the desired product 57 was moderate. Hydroboration-oxidation conducted on 57, resulted in the primary alcohol 58 in good yield. Oxidation of 58 using a modified Swern oxidation afforded aldehyde 59. 66 O HO OH HO OH O O O HO OMe O O O OMe O O O OMe O H acetone, MeOH, CH 3 C(OMe) 2 CH 3 , cat. H 2 SO 4 quantitative DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 70% P(Ph) 3 MeBr, t-BuOK, Et 2 O 75% O O O OMe HO 1.9-BBN, THF 2.MeOH, NaOH, H 2 O 2 92% DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 87% O O O OMe O 12 56 57 58 59 Scheme 24. Synthesis of important intermediate 59. D-ribose 4 With aldehyde 59 available, focus turned to the allylation reaction with the aforementioned Brown?s reagent to be followed by side chain modification (Scheme 25). 93 Thus, freshly prepared (-)-B-allyldiisopinocampheylborane (in situ) was reacted with the aldehyde 59 at low temperature to provide the desired enantiomerically pure 60 in a yield of 95% in two steps. The C-6' hydroxyl group of compound 60 was protected by benzyl bromide under the strong basic conditions to furnish 61 in 70% yield. 117 67 O O O OMe O 59 O O O OMe 60 HO 1.(-)-B-Ipc 2 OMe, allylmagnesium bromide, -78 0 C, 2.MeOH, NaOH, H 2 O 2 95% DMF, NaH, benzyl bromide 70% O O O OMe 61 BnO O O O OMe 62 BnO HO 1. NaIO 4 , MeOH/H 2 O, OsO 4 , 0 0 C 2. NaBH 4 , MeOH 45% O O O OMe 63 BnO I Ph 3 P, imidazole, toluene/acetonitrile, I 2 , reflux Scheme 25. Synthesis of 63. 77% With compound building block B (61) and building block C (35) obtained, the alkylation coupling reaction to the requisite amino acid was considered. As mentioned in Chapter 3, the asymmetric synthesis of terminal ?-amino acids via Sch?llkopf?s chiral auxiliary is best conducted using lithiated (2R)-2,5-dihydro-3,6-dimethoxy-2- isopropylpyrazine and halogen derivatives of the co-reactant. 104 The most common haloderivative is the bromospecies. However, iododerivative 63 was chosen for its ease of preparation and efficiency as a good leaving group. Therefore, compound 61 was subjected to a two-step process: (i) the oxidative-cleavage with osmium tetroxide and sodium periodate followed by (ii) reduction with sodium borohydride to yield the primary 68 alcohol in the side chain of 62 with the moderate yield in two steps. Then, the primary alcohol in 62 was converted to iodide 63 in moderate yield, according to standard iodination conditions. 85b Finally, the alkylation reaction (Scheme 26) of iodide 63 with lithiated (2R)-2, 5-dihydro-3, 6-dimethoxy-2-isopropylpyrazine (in situ) was performed at low temperature with copper cyanide catalysis. Unfortunately, this alkylation reaction did not provide the desired product 64, giving only recovered reactant 63 and a very strong UV absorbing material (TLC). The explanation for this failure may lie in two areas: (i) the pyrazine unit may decompose with n-butyl lithium at the reaction temperature (> - 78 0 C) to give the UV product, and/or (ii) the reaction intermediate was extremely susceptible to moisture. O O O OMe 63 BnO I O O O OMe BnO N N MeO OMe 64 1. 35, THF, n-BuLi -78 0 C, 2. CuCN, THF, 0 0 C 3.NH 4 Cl, NH 4 OH Scheme 26. Synthesis of 64. Another possible concern arose when thinking further about using Sch?llkopf?s chiral auxiliary: that is, after installation of the pyrazine part on iodide 63, in the removal of the C-1 methoxyl group step under the usual strong acid conditions for coupling with the heterocyclic base may affect the pyrazine center. Thus, removal of the methoxyl group before setting in place the pyrazine unit was reviewed as a more suitable approach 69 for target II, where p-methoxybenzyl protecting seemed viable, building block B of Scheme 27. O HO OH N N N N NH 2 H 2 N CO 2 H target II HO N N N H N NH 2 + Scheme 27. Optimized approach for target II. O PGO O O N N MeO OMe O PGO O O + N NMeO OMe A B C PG = Protecting Group OPMB OPMB O HO HO OH OH D-ribose To explore conditions to B, a simple method was investigated (Scheme 28). Thus, protection of D-ribose using anhydrous acetone in the presence of excess of p- methoxybenzyl alcohol afforded the desired 65. However, 65 and unreacted p- methoxybenzyl alcohol showed the same polarity (TLC), and could not be separated by routine silica gel column chromatography. 70 O O O HO OPMB O HO OH HO OH acetone, p-methoxybenzyl alcohol cat. H 2 SO 4 65 Scheme 28. Synthesis of 65. D-ribose Thus, another strategy was evaluated (Scheme 29) to construct the building block B. In that regard, compound 57 (Scheme 29) was treated with 4 M hydrochloric acid in dioxane to afford the intermediate-1,2,3-triols. That was followed by 2,3-diol protection with anhydrous acetone and sulfuric acid catalysis to give 66 in 60% yield in two steps. Protection of 66 with p-methoxybenzyl chloride under strongly basic conditions furnished the desired 67 as a major product and 68, as a minor product. At this stage, compound 67 and 68 could be distinguished by 1 H NMR. After separation by silica gel column chromatography, 67 was subjected to hydroboration-oxidation to result in the primary alcohol 69 in moderate yield. Oxidation of 69 using modified Swern oxidation conditions afforded the aldehyde 70 in 87% yield. 71 O O O OH O O O OPMB O O O OPMB + O O O OMe 57 1. 4 M HCl, 1, 4-dioxane, 72 h, r.t. pH=7.0 2. acetone, cat. H 2 SO 4 , r.t. 2 h 60% DMF, NaH, p-methoxybenzyl chloride , 0 0 C, 4 h 1. 9-BBN, THF 2. MeOH, NaOH, H 2 O 2 75% O O O OPMB HO 66 67 major 68 minor 69 O O O OPMB O 70 DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 87% 60% Scheme 29. Synthesis of 70. 15% Although the important intermediate 70 could be obtained by the approach shown in Scheme 29, the transformation from 57 to 66 was time-consuming and led to only a moderate yield. Furthermore, this route encountered the by-product 68 when producing 67. To overcome these disadvantages, a more efficient approach for constructing 70 was envisioned and shown in Scheme 30. 72 O HO OH HO OH O O O HO acetone, cat. H 2 SO 4 celite 71 OH DMF, NaH, p-methoxybenzyl chloride, 0 0 C, 12 h O O O HO OPMB O O O HO OPMB + DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 85% O O O H OPMB O O O O OPMB O O O OPMB HO O O O OPMB O 72, major, 75% 73, minor, 5% 74 67 69 70 D-ribose Scheme 30. Another approach for 70. 95% P(Ph) 3 MeBr, t-BuOK, Et 2 O 80% 1. 9-BBN, THF 2. MeOH, NaOH, H 2 O 2 75% DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 87% 80% In this method, protection of D-ribose by acetone under anhydrous conditions provided 71. Then, compound 71 was placed in the vacuum drying oven for 12 h before it was subjected to the next modification. Using p-methoxybenzyl chloride to protect the primary alcohol of 71 under the basic conditions afforded 72 as the major product in 75% yield and, disappointingly, 73 as a by-product in 5% yield. Following separation of these 73 74 two products by silica gel column chromatography, oxidation of 72 using modified Swern oxidation conditions furnished aldehyde 74 in 85% yield. Subjecting 74 to a Wittig reaction with methyl triphenylphosphonium bromide under the strong basic conditions formed 67 in good yield. 84a The transformation of 67 to 70 was the same that in Scheme 29. With aldehyde 70 in hand, further modifications toward building block B as outlined were pursued in Scheme 31. Subjecting aldehyde 70 to the Brown stereospecific hydroboration reagent of freshly prepared (-)-B-allyldiisopinocampheylborane (in situ), furnished the desired enantiomerically pure 75 in 95% yield in two steps, whose structure was confirmed by two-dimensional NMR spectroscopy ( 1 H, 1 H-COSY) shown in Figure 26. The ( 1 H, 1 H-COSY) 2D NMR spectrum of compound 75 in CDCl 3 (Scheme 31) was explained here. The diagonal peak labeled 8'-H has two cross-peaks: one is the coupled with 9'-H (2H) and is placed symmetrically around the diagonal line; and, the other is coupled with 7'-H (2H). From the latter cross-peak, the position of 6'-H is deduced. The diagonal peak of 6'-H has two cross-peaks: one results from the protons of the neighboring 7'-H (2H) group and the other belongs to the proton of a trans 5'-H. Beginning with the diagonal peak of trans 5'-H, a symmetrical cross-peaks of cis 5'-H is confirmed. From the cross-peaks of trans 5'-H and cis 5'-H, the diagonal peak of 4'-H is identified. When moving from the diagonal line, the diagonal peaks (2'-H and 3'-H) and their symmetric cross-peaks are proved. The remaining diagonal peaks are not accompanied by any cross-peak. O O O OPMB O O O O OPMB HO 76 O O O OPMB TBSO O O O OPMB TBSO I 78 1. (-)-B-Ipc 2 OMe, allylmagnesium bromide, -78 0 C, 2. MeOH, NaOH, H 2 O 2 95% 1. NaIO 4 , MeOH/H 2 O, OsO 4 , 0 0 C 2. NaBH 4 , MeOH 45% TBSCl. imidazole, DMAP, CH 2 Cl 2 , 0 0 C, 24 h 96% O O O OPMB HO TBSO 77 70 75 Ph 3 P, imidazole, toluene/acetonitrile, I 2 , reflux 90% Scheme 31. Synthesis of iodide 78. 1'4' 5'6' 9' 75 76 Figure 26. COSY spectrum of compound 75 in deuteriochloroform: (a) the small one chemical shift ? (1.0-7.8) and (b).the magnified one ? (1.0-6.1). Protection of the newly formed secondary alcohol of 75 as a tert- butyldimethylsilyl ether with tert-butyldimethylsilyl chloride and imidazole catalyzed by DMAP provided 76 in 96% yield. 118 Under oxidative-cleavage and reduction conditions with osmium tetraoxide/sodium periodate, followed by sodium borohydride, the C-8 77 primary alcohol of 77 was achieved in moderate yield. Finally, iodination of 77 under the standard reaction conditions afforded iodide 78 in 90% yield. Considering the poor yield in the oxidative-cleavage/reduction series (from 76 to 77), an optimization method was sought (Scheme 32) that was based on the approach in Scheme 18. Thus, double bond of 76 was oxidized under osmium tetraoxide/N- methylmorpholine N-oxide (NMO) conditions to provide the diol 76-1'. After isolating 76-1', it was subjected to cleavage with sodium periodate to furnish aldehyde 76-1''. The intermediates 76-1' and 76-1'' could be identified by 1 H NMR spectroscopy and TLC. Luche reduction of the aldehyde 76-1'' achieved 77 in excellent yield (three steps, 80%). O O O OPMB TBSO HO HO 76 O O O OPMB TBSO O O O OPMB HO TBSO 77 O O O OPMB TBSO O 76-1' 76-1'' OsO 4 , NMO, THF/H 2 O, 0 0 C, 2 h NaIO 4 , CH 2 Cl 2 /H 2 O 15-crown-5, r.t., 12 h NaBH 4, MeOH r.t., 12 h 80% Scheme 32. Optimization approach for 77. 78 79 Compound 78 was then used for the Sch?llkopf coupling reaction (Scheme 33), for the purpose of constructing the terminal amino acid unit. Thus, 78 was alkylated with the lithio derivative of 35 (Scheme 15) in anhydrous THF at low temperature to afford compound 79, whose structure was proved by spectral data: NMRs ( 1 H and 13 C) and two- dimentional NMR ( 1 H, 1 H, COSY) shown in Figure 27. The ( 1 H, 1 H-COSY) 2D NMR spectrum of compound 79 in CDCl 3 (Scheme 33) was addressed here. The diagonal peak of 2'-H has one symmetric cross-peak, belonging to the proton of 3'-H. When proceeding on the diagonal peak of 4'-H, two cross-peaks are confirmed: a trans 5'-H and a cis 5'-H. From the diagonal peak of 6'-H, the trans 5'-H is identified by their cross-peak. When proceeding along the diagonal line, the diagonal peak of pyrazine (2H) has two cross- peaks, one is the the proton of CH group of isopropyl in pyrazine; and the other belongs to the proton of 9'-H (1H). From the cross-peak of the latter, 8'-H (2H) are proved. Finally, the remaining diagonal peaks are not accompanied by any cross-peak. 80 Figure 27. COSY spectrum of compound 79 in deuteriochloroform: (a), the small one chemical shift ? (-0.30-5.5) and (b).the magnified one ? (-0.30-2.5). As before, the stereospecificity transformation (78 to 79) arises from the steric interactions imposed by bulky moiety of 78 with chiral dihydropyrazine anion (lithio (2R)-2, 5-dihydro-3, 6-dimethoxy-2-isopropylpyrazine) to the less hindered face of the masked amino acid to afford diastereoselectively the trans product 79 in 85% yield. 104,108 81 O O O OPMB TBSO I 78 O O O OPMB TBSO N N MeO OMe 1. 35, THF, n-Buli, -78-90 0 C, 5 h 2. EtOH, sat. NH 4 Cl 85% 79 Scheme 33. Synthesis of 79. 1' 2'3' 4' 5'6' 9' To this point, compound 79 (building block A) was successfully obtained from D- ribose in 13 steps. Attention then shifted to coupling the heterocyclic base (adenine) with building block A (79). Therefore, deprotection of the PMB group became very important. In this direction, treatment of 79 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was expected to follow oxidative cleavage to compound 80 (Scheme 34). 119 However, this transformation (79 to 80) resulted in a complex mixture (from 1 H NMR analysis). Possible reasons for this result are: (i) decomposition (by 1 H NMR spectra and TLC analysis) of the masked amino acid unit (pyrazine) during the oxidation-reduction process of removal of the PMB group; and/or, (ii) the anomeric center of 79 could not withstand the oxidative-reduction cleavage conditions. 82 O O O OPMB TBSO N N MeO OMe 79 O O O TBSO N N MeO OMe 80 OH Scheme 34. Synthesis of 80. DDQ, CH 2 Cl 2 /H 2 O, r.t., 2 h Because of the failure of the 79 to 80 transformation (Scheme 34), another strategy was explored (Scheme 35) with compound 78 that lacked the pyrazine unit. This proved successful by producing the desired compound 81. DDQ, CH 2 Cl 2 /H 2 O, r.t., 30 min O O O OPMB TBSO I 78 O O O TBSO I 81 OH Scheme 35. Synthesis of 81. 78% During these explorations, the minor product 73 was accumulated for the modifications in Scheme 36. Oxidation of compound 73 under standard Swern oxidation conditions provided 82 in 80% yield. Compound 82 was subjected to Wittig reaction conditions to give 68 in moderate yield (compare to 68 in Scheme 29). Hydroboration- oxidation of compound 68 was carried out to afford the primary alcohol 83 in 75% yield. Oxidation of 83 again using modified Swern oxidation conditions afforded aldehyde 84 83 84 in good yield (85%). Calling again the Brown stereospecific hydroboration reagent freshly prepared (-)-B-allyldiisopinocampheylborane (in situ) on aldehyde 84, furnished the desired enantiomerically pure 85 in 90% yield for two steps. Finally, protection of the newly formed side chain secondary alcohol of 85 as a tert-butyldimethylsilyl ether with tert-butyldimethylsilyl chloride and imidazole catalyzed by DMAP provided 86 in 92% yield. O O O HO OPMB 73 O O O H OPMB O O O O OPMB O O O OPMB HO O O O OPMB O O O O OPMB HO O O O OPMB TBSO 68 82 83 84 85 86 DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 80% P(Ph) 3 MeBr, t-BuOK, Et 2 O 70% 1. 9-BBN, THF 2. MeOH, NaOH, H 2 O 2 75% DMSO, DIPEA, SO 3 ?Py, CH 2 Cl 2 85% 1. (-)-B-Ipc 2 OMe, allylmagnesium bromide, -78 0 C, 2. MeOH, NaOH, H 2 O 2 90% TBSCl. imidazole, DMAP, CH 2 Cl 2 , 0 0 C, 24 h 92% Scheme 36. Synthesis of 86. Compound 86, just like intermediate compound 76, was also an important building block with further modifications to synthesize target II. Due to difficulties in dealing with deprotection of the PMB group and possible challenge in the installation of the heterocyclic base (adenine) on the 5-member ring, this dissertation research finished the important intermediates: 79, 81 and 86. Compound 79 85 86 was the most direct precursor of target II and it needs more research process to target II from here. 87 CONCLUSION Sinefungin, a naturally occurring complex nucleoside, has many important biological activities, including antifungal, antitumor, antiparasitic, antiviral and amoebicidal activities. The nature of these biological activities results from its inhibition of a variety of AdoMet-dependent methyltranferase enzymes. In one instance, the resulting inhibition of AdoMet methyltransferase leads to the disruption of important biomethylations, such as the viral mRNA 5'-capping process, which leads to blocking viral replication. However, the clinical usefulness of sinefungin has been precluded by its toxicity in in vivo assays. To circumvent the detrimental side-effects of sinefungin, structural analogs became the focus of this dissertation. The synthesis of a carbocyclic analog of sinefungin (target I) in a stereocontrolled fashion from D-ribose and important intermediates (47 for target I and 53, 55 for carbocyclic sinefungin) has been investigated. Variation on this approach to sinefungin-based analog using 79, 81 and 86 (to target II) is described. The key strategies for the construction of C-6' and C-9' stereocenters of targets I and II brought forth efficient catalytic asymmetric syntheses. The C-6' hydroxyl chiral center was found accessible by the highly diastereoselective allylborating agent: (+) 88 and/or (-)-B-allydiisopinocampheylborane (Ipc 2 BCH 2 CH=CH 2 ). This asymmetric homoallylic alcohol reagent provided access to either diastereomeric possibility at C-6'. The C-9' amino acid stereochemistry of targets I and II was established by a diastereoselective alkylation reaction with the Sch?llkopf reagent. Finally, coupling of the heterocyclic base (adenine) with these 5-membered rings employed standard S N 2 substitution reactions. The synthetic processes analyzed provide adaptable possibilities to a number of structural sinefungin analogs for biological evaluation. 89 EXPERIMENTAL SECTION Materials and Methods: Melting points were recorded on a Meltemp II melting point apparatus and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Bruker AV 250 Spectrometer (operated at 250 or 62.9 MHz, respectively) or AV 400 Spectrometer (operated at 400 or 100 MHz, respectively). The assignments of various NMR spectra were assisted by homonuclear ( 1 H/ 1 H) correlation spectroscopy (COSY) on the AV 400 Spectrometer. All 1 H chemical shifts are reported in ? relative to the internal standard tetramethylsilane (TMS, ? 0.00). 13 C chemical shifts are reported in ? relative to CDCl 3 (center of triplet, ? 77.23) or relative to DMSO-d 6 (center of septet, ? 39.51). The spin multiplicities are indicated 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, Georgia. Reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm E. Merck silica gel 60-F254 precoated silica gel plates with visualization by the irradiation with Mineralight UVGL-25 lamp, or exposure to iodine vapor, or exposure to the basic solution of KMnO 4 . Column chromatography was performed on Whatman silica gel (average particle size 2-25 ?m, 60 ?) and elution with the indicated solvent system. Yields refer to chromatographically and spectroscopically ( 1 H and 13 C NMR) homogeneous materials. 90 (1R,3S)-Cyclopent-4-ene-1,3-diyldiacetate (5). The freshly synthesized cyclopentadiene (189 g, 2.85 mol) was immediately dissolved in a 5 L three-neck round bottom flask containing 2.2 L methylene chloride, and sodium carbonate (724 g, 6.83 mol) was added to the above reaction mixture. The mixture of sodium acetate (14.5 g, 0.177 mol), peracetic acid (363 mL, 5.45 mol) and methylene chloride (150 mL) were added dropwise to the stirring cyclopentadiene solution, which was pre-cooled to -20 0 C. During the adding process, the temperature between -10 0 C and 0 0 C, was maintained until all the mixture was completely added. The whole reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was evaporated to give pale yellow liquid product, which was crude monoepoxide 4 and could be directly used to the next step. The monoepoxide 4 dissolved in anhydrous THF (145 mL) was added dropwise to a stirred solution of tetrakis(triphenylphosphine)palladium(0) (4.98 g, 1.72 mmol) and acetic anhydride (255 mL, 2.70 mol) in THF (350 mL). The reaction mixture was maintained from -5 0 C to 0 0 C and under nitrogen condition stirred at room temperature overnight. After evaporation of THF at room temperature under reduced pressure, the resulting solution was passed through a pad of silica gel and anhydrous magnesium sulfate using ether as eluant. The solvent was removed and the residue was distilled using high vacuum at the temperature 90-95 0 C to afford a pale yellow liquid (139 g, 36 % for two steps), which was diacetate 5 in agreement with the literatures. 61b,79 (1R,4S)-4-Hydroxycyclopent-2-enyl acetate (6). Diacetate 5 (139 g, 752 mmol) was added to a three-neck 5 L round flask, which contained the 0.1 M phosphate buffer (1.00 91 L) and the pH was maintained at 7.00-7.40 by the addition of 6 M NaOH. The enzyme, pseudomonas cepacia lipase (5.18 g), was added to the above flask and the pH of the reaction mixture was kept constant (7.00-7.40) during the whole hydrolysis process by adding dropwise the 1 M NaOH solution (753 mL). After the reaction was finished (it was seen from the pH meter that the pH was constant within 30 min), the solution was filtered through celite and extracted with EtOAc. The combined organic layer was dried with anhydrous MgSO 4 and the solvent was removed under reduced pressure to afford the crude yellow liquid. Finally, the yellow liquid was distilled at the reduced pressure (at the temperature 88-93 0 C) to get the pure monoacetate 6 (86.7 g, 82 %) as a light yellow solid. The 1 H NMR data agreed with the literature. 83a (1R,4S)-4-(Diethoxyphosphoryloxy)cyclopent-2-enyl acetate (7). Diethyl chlorophosphate (9.32 g, 52.7 mmol) was added dropwise to the solution of monoacetate 6 (5.75 g, 40.5 mmol) dissolved in CH 2 Cl 2 and pyridine (6.00 mL, 72.9 mmol), which was cooled to 0-5 0 C. The reaction mixture was stirred at room temperature for 5 h and 5% aqueous HCl (30.0 mL, cooled with ice-water) was added to the above reaction mixture. After dilution of the reaction mixture with CH 2 Cl 2 (50.0 mL) and washing with saturated equeous NaHCO 3 and brine, the organic phase was obtained. After drying over anhydrous MgSO 4 and removing the solvent under reduced pressure, 7 (11.0 g, 98 %) was obtained as a pale yellow liquid and could be used in the further steps without purification. (1R, 2R, 3S, 4S)-4-(Diethoxyphosphoryloxy)-2,3-dihydroxycyclopentyl acetate (8). 50 % aqueous NMO (N-methylmorpholine N-oxide, 27.0 mL, 54.0 mmol) was added to a solution of 7 (16.8 g, 60.3 mmol) in 120 mL acetone at 0 0 C. H 2 O (50.0 mL) was added 92 until the solution became clear. The oxidant, OsO 4 (119 mg, 0.468 mmol) was slowly added to the above clear solution. The whole reaction mixture was stirred at room temperature overnight. After evaporating the solvents under reduced pressure, the residue was purified by silica gel column chromatography with EtOAc as eluent to afford the colorless liquid 8 (9.24 g, 50 %). (1R,2R,3S,4S)-4-(Diethoxyphosphoryloxy)-2,3-(isopropylidenedioxy)-cyclopentyl acetate (9). A solution of 8 (9.24 g, 29.6 mmol) in anhydrous acetone (85.0 mL, 1.16 mol) and 2,2-dimethoxypropane (22.0 mL, 178 mmol) was treated with p-toluenesulfonic acid monohydrate (288 mg, 5.0 % mmol). The reaction was stirred at r.t. overnight. Solvents were evaporated under vacuum, the residue was dissolved in EtOAc and washed with Na 2 CO 3 (20.0 mL) and brine. The organic layer was dried over MgSO 4 , filtered and solvents removed under reduced pressure to provide 9 (9.90 g, 95 %) as pale yellow liquid, which could be used in the next step without further purification. (1R,2R,3S,4S)-4-Hydroxy-2,3-(isopropylidenedioxy)cyclopentyl-1-yl diethyl phosphate (10). Compound 9 (9.90 g, 28.1 mmol) was dissolved in THF (25.0 mL) and added to a solution of LiOH?H 2 O (1.83 g, 43.6 mmol) in H 2 O (40.0 mL). The reaction was stirred at room temperature overnight. After removal of THF and most of H 2 O under vacuum, the residue was extracted by CH 2 Cl 2 (3?50 mL). The organic layer was dried over anhydrous MgSO 4 and evaporated the solvent to give 10 (8.72 g, 82 %). The 1 H NMR data was agreed with the literature. 83a (-)-(4R,5R)-4,5-O-isopropylidene-2-cyclopentenone (11)-based on the monoacetate 6. Pyridinium chlorochromate (15.1 g, 70.1 mmol) and celite (10.0 g) were added to a solution of compound 10 (8.72 g, 28.1 mmol) in anhydrous CH 2 Cl 2 (105 mL). The 93 reaction mixture was stirred at room temperature for 36 h. The reaction mixture was diluted with CH 2 Cl 2 and filtrated to remove the celite. The filtrate was evaporated under reduced pressure to give the residue, which was purified by column chromatography (1/1, Hexane/EtOAc) to afford pure 11 (1.95 g, 50 %) as the yellow liquid: 1 H NMR (250 MHz, CDCl 3 ) ? 7.62 (dd, J=2.25, 6.0, 1H), 6.23 (d, J=6.0, 1H), 5.28 (dd, J= 2.25, 5.50, 1H), 4.48 (d, J=5.50, 1H), 1.42 (s, 6H). The NMR spectral data agreed with the literature. 84a Methyl-2,3-O-isopropylidene-?-D-ribofuranoside (12). 2,2-dimethoxypropane (76.0 mL, 1.06 mol) was added to a solution of D-ribose (32.0 g, 213 mmol) in acetone (300 mL) and methanol (100 mL). Sulfuric acid (4.00 mL, 5 % mmol) was added to the above solution and the reaction mixture was stirred at room temperature overnight. After neutralized with ammonium hydroxide, H 2 O (50.0 mL) and EtOAc (200 mL) were added to the reaction mixture. The aqueous layer was extracted with EtOAc, and the combined organic phase was concentrated under reduced pressure to afford 12 (58.4 g, 98 %) as colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 5.0 (s, 1H), 4.87 (d, J=5.90, 1H), 4.62 (d, J=5.90, 1H), 4.46 (t, J=2.80, 1H), 3.75-3.60 (m, 2H), 3.46 (s, 3H), 3.28 (dd, J=10.50, 2.50, 1H), 1.51 (s, 3H), 1.34 (s, 3H). Methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-?-D-ribofuranoside (13). The furanoside 12 (58.4 g, 286 mmol) was dissolved in toluene (600 mL) and acetonitrile (150 mL). After the addition of imidazole (29.2 g, 429 mmol) and triphenylphosphine (90.0 g, 343 mmol), the iodine was introduced in approximately 100 mg portions to the above reaction mixture until the reaction mixture remained dark-brown in color. The dark-brown solution was heated to reflux for 30 min and cooled to room temperature. 94 Diethyl ether (200 mL) was added to dilute the solution and then the solution was washed with 10% sodium thiosulfate solution and brine. The organic phase was dried over anhydrous MgSO 4 and concentrated in vacuo to obtain a residue, which was purified through a short plug of silica gel (elution with 20:1 Hexanes/EtOAc) to provide 13 (47.1 g, 60 %) as a colorless oil. The 1 H NMR data was in agreement with the literature. 85 4,5-Dideoxy-2,3-O-isopropylidene-D-erythro-4-pentenose (14). The iodide 13 (47.1 g, 150 mmol) was dissolved in anhydrous THF (300 mL) in a flame-dried flask under a nitrogen atmosphere. The above solution was cooled to -78 0 C and n-BuLi (89.7 mL, 224 mmol, 10. 0 M solution in THF) was added dropwise over a period of 30 min by syringe. The reaction mixture was stirred at -78 0 C for 2 h and quenched with solid NH 4 Cl (15.0 g). The mixture was allowed to warm to -40 ?C and H 2 O (50.0 mL) was slowly added and the aqueous phase was extracted with diethyl ether (3 ? 50.0 mL). The combined organic solution was dried with anhydrous MgSO 4 and concentrated in vacuo (since the aldehyde 4 is somewhat volatile, the temperature of the rotorvapor was kept below 30 0 C). The aldehyde 14 (36.2 g, 115 %) was obtained as a colorless oil, which was used in the next reaction without further purification. (4S,5R)-4,5-O-isopropylidenehepta-1,6-diene-3,4,5-triols (15). The aldehyde 14 (36.2 g, 232 mmol) was dissolved in anhydrous CH 2 Cl 2 (100 mL) under a nitrogen atmosphere and the above solution was cooled to -40 0 C. Vinylmagnesium bromide (350 mL, 1.0 M in THF) was added dropwise via a cannula to the above stirred solution over 1 h. The reaction mixture was kept at the same temperature for 2 h and allowed to warm to room temperature. Saturated NH 4 Cl (30.0 mL) was added to quench the reaction and EtOAc (3?150 mL) was used to extracted the organic phase. The organic phase was 95 washed with brine and dried over anhydrous MgSO 4 . The solvent was removed under reduced pressure and the residue was purified on silica gel column chromatography (7/1, Hexane/EtOAc) to afford the diene 15 (15.6 g, 30 %): 1 H NMR (400 MHz, CDCl 3 ) ? 6.10 (m, 1H), 5.55 (m, 1H), 5.23 (m, 4H), 4.54 (t, J=6.0, 1H), 4.16 (dd, J=6.40, 2H), 3.23 (dd, J=6.50, 1H), 1.54 (s, 3H), 1.39 (s, 3H). (-)-(4R,5R)-4,5-O-isopropylidene-2-cyclopentenone (11)-based on D-ribose. The diene 15 (15.6 g, 84.2 mmol) was dissolved in anhydrous CH 2 Cl 2 (300 mL) and the above solution was flushed with N 2 for 30 min at room temperature. Grubbs? catalyst 1 st generation (1 % mmol, 693 mg) was added to the diene solution. After stirred at 40 0 C for 48 h, pyridinium dichromate(63.4 g, 168 mmol), 4 ? molecular sieves (15.0 g) and acetic acid (0.24 mL, 5 mmol %) were added to the above reaction mixture. The reaction mixture was stirred at room temperature for 12 h and filtrated over celite pad with EtOAc. After removal of the solvent, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 11 (2.90 g, 22 %): 1 H NMR (250 MHz, CDCl 3 ) ? 7.62 (dd, J=2.25, 6.0, 1H), 6.23 (d, J=6.0, 1H), 5.28 (dd, J= 2.25, 5.50, 1H), 4.48 (d, J=5.50, 1H), 1.42 (s, 6H). Methyl-2,3-O-cyclopentanyl-?-D-ribofuranoside (16). To a solution of D-ribose (20.0 g, 133 mmol) in anhydrous methanol (200 mL) under a nitrogen atmosphere at room temperature, were added cyclopentanone (23.7 mL, 267 mmol), trimethyl orthoformate (66.5 mL, 400 mmol) and catalyzed amount p-toluenesulfonic acid monohydrate (1.27 g, 6.66 mmol, 5 % mmol). The reaction mixture was refluxed for 30 min. After removed the excess cyclopentanone at high vacuum pump, the residue was diluted with EtOAc and brine. The organic phase was extracted with EtOAc, dried with 96 anhydrous MgSO 4 and evaporated. The residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc, confirmed by preparative TLC) to afford pale yellow liquid 16 (35.9 g, quantitative), which was the major epimer. Methyl 5-deoxy-5-iodo-2,3-O-cyclopentanyl-?-D-ribofuranoside (17). The furanoside 16 (35.9 g, 156 mmol) was dissolved in toluene (400 mL) and acetonitrile (100 mL). After the addition of imidazole (21.2 g, 311 mmol) and triphenylphosphane (49.0 g, 187 mmol), the iodine was introduced in approximately 100 mg portions to the above reaction mixture until the mixture remained dark-brown in color. The dark-brown solution was heated to reflux for 55 minutes and cooled to room temperature. The solution was diluted with diethyl ether (400 mL) and washed with 10 % sodium thiosulfate solution and brine. The organic phase was dried over anhydrous MgSO 4 and concentrated in vacuo to obtain a residue, which was purified through a short plug of silica gel column chromatography (5/1, Hexanes/EtOAc) to provide 17 (37.2 g, 70 %) as a colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 5.06 (s, 1H, 1'-H), 4.72 (d, J=6.0, 1H, 2'-H), 4.59 (d, J=6.00, 1H, 3'-H), 4.45 (m, 1H, 4'-H), 3.38 (s, 3H, OCH 3 ), 3.35-3.13 (m, 2H, 5'- Ha, 5'-Hb), 1.95-1.65 (br, 8H); 13 C NMR (62.90 MHz, CDCl 3 ) ? 122.4, 109.7, 87.3, 85.4, 83.0, 55.5, 36.1, 36.0, 23.8, 23.4, 6.9. 4,5-Dideoxy-2,3-O-cyclopentanyl-D-erythro-4-pentenose (18). To a solution of the iodide 17 (37.2 g, 111 mmol) in anhydrous methanol (300 mL) in a flame-dried flask under a nitrogen atmosphere, was added zinc power (7.86 g, 120 mmol) activated with acetic acid. The above solution was refluxed for 1 h and cooed to room temperature. After filtrating the precipitate with a celite pad, the filtrate was concentrated in vacuum at 35 0 C and the residue was loaded to a short silica gel column chromatography (4/1, 97 Hexane/EtOAc) to yield the colorless oil 18 (18.4 g, 94%): 1 H NMR (250 MHz, CDCl 3 ) ? 9.55 (d, J=3.0, 1H, 1'-H), 5.83-5.70 (m, 1H), 5.51 (d, J=17.3, 1H), 5.36 (d, J=10.3, 1H), 4.75 (t, J=7.50, 1H, 3'-H), 4.36 (dd, J=3.0, J=7.50, 1H, 2'-H), 2.06 (m, 2H), 1.76 (br, 6H). (4S,5R)-4,5-O-cyclopentanyl-1,-6-diene-3,4,5-triols (19). The aldehyde 18 (18.4 g, 102 mmol) was dissolved in anhydrous diethyl ether (100 mL) under a nitrogen atmosphere and the above solution was cooled to -20-0 0 C. Vinylmagnesium bromide (113 mL, 113 mmol, 1.0 M in THF) was added dropwise to the above stirred solution over 1 h via a cannula. The reaction mixture was kept at the same temperature for 2 h and allowed to warm to room temperature. Saturated NH 4 Cl (30.0 mL) was added to quench the reaction and diethyl ether (3?150 mL) was used to extracted the organic phase. The combined organic phases were washed with brine and dried over anhydrous MgSO 4 . The solvent was removed under reduced pressure and the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford the diene 19 (12.2 g, 60 %) as clear yellow oil: 1 H NMR (250.1 MHz, CDCl 3 ) ? 6.05 (m, 4H), 5.35 (m, 8H), 4.59 (t, J=4.0, 1H), 4.51 (d, J=4.0, 1H), 4.18-4.11 (m, 2H), 4.00-3.93 (m, 2H), 2.04-1.91 (m, 4H), 1.78-1.66 (m, 14H). (-)-(4R,5R)-4,5-O-cyclopentanylidene-2-cyclopentenone (21). The diene 19 (12.2 g, 58.6 mmol) was dissolved in anhydrous CH 2 Cl 2 (300 mL) and the above solution was flushed with dry nitrogen for 45 min at room temperature. Grubbs? catalyst 1 st generation (1 % mmol, 482 mg) was slowly added to the diene solution. After stirred at 40 0 C for 48 h, the formed 20 intermediate was oxidized by a modified Swern oxidation method. Dimethyl sulphoxide (62.4 mL, 879 mmol), diisopropylethylamine (30.6 mL, 176 mmol) and SO 3 ?Py complex (14.0 g, 87.9 mmol) were added to the above solution of 20 at -10-0 98 0 C. The reaction mixture was kept 0 0 C for 2 h and continued stirring for 4 h at room temperature. After the reaction completed, the solution was diluted with CH 2 Cl 2 (200 mL) and H 2 O (20.0 mL). The organic phase was extracted with CH 2 Cl 2 , washed with H 2 O, saturated NaHCO 3 and brine, and dried over anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 21 (8.45 g, 81 %) as yellow liquid: 1 H NMR (250 MHz, CDCl 3 ) ? 7.67 (dd, J=2.50, J=6.0, 1H), 6.29 (d, J=6.0, 1H), 5.26 (dd, J=5.5, J=2.50, 1H), 4.42 (d, J=5.50, 1H), 1.92-1.83 (m, 2H), 1.73-1.60 (br, 6H). Ethyl 2-((3aR,4R,6aR)-2,2-dimethyl-6-oxotetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)acetate (23). n-BuLi (3.0 mL, 7.50 mmol, 2.50 M in hexane) was added dropwise to a three-neck flask at -10-0 0 C by syringe, which contained anhydrous THF (20.0 mL) and DIPA (diisopropylamine, 1.00 mL). The above solution was cooled further to -40 0 C. Ethyl (trimethylsilyl) acetate (1.00 mL, 5.47 mmol) was added dropwise over 10 min to the above solution by syringe and the reaction mixture was stirred at the same temperature for 40 min. HMPA (hexamethylphosphoramide) / THF(v/v, 1/1, 6.0 mL) was added to the above mixture and the above solution was further cooled to -78 0 C. Compound 11 (0.770 g, 5.00 mmol) in anhydrous THF (3.0 mL) was added dropwise via a cannula to the above reaction mixture at -78 0 C and the mixture was kept stirring at the same temperature for 1.5 h to provide the intermediate 22. After the temperature of the reaction mixture warmed to -40 0 C, saturated NH 4 Cl (10.0 mL) was used to quench the reaction. When the amount of 11 was increased, the mixture of KF/EtOH/H 2 O (20/1/1, v/v/v, saturated KF solution) was introduced to quench the above reaction mixture. The aqueous phase was extracted with 99 CH 2 Cl 2 (3?50.0 mL) and the organic layer was dried over anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was separated on silica gel column chromatography (5/1, Hexane/EtOAc) to afford 23 (0.490 g, 55 % for two steps): 1 H NMR (250.1 MHz, CDCl 3 ) ? 4.61 (d, J=5.50, 1H), 4.39 (d, J=5.50, 1H), 4.18 (q, 2H), 2.88 (m, 1H), 2.81 (m, 2H), 2.50 (m, 1H), 2.16 (m, 1H), 1.44 (s, 3H), 1.34 (s, 3H), 1.29 (t, 3H). Ethyl 2-((3aR,4R,6S,6aS)-6-hydroxy-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)acetate (24). Sodium borohydride (115 mg, 3.04 mmol) and cerium chloride heptahydrate, CeCl 3 ?7H 2 O (0.640 g, 1.72 mmol) were added to the solution of 23 (0.490 g, 2.03 mmol) in methanol (12.0 mL) at -10-0 0 C. The reaction mixture was kept 0 0 C for 1.5 h and continuing stirring at room temperature for 2 h. Saturated NH 4 Cl (4.00 mL) was used to quench the above reaction. Extracted with CH 2 Cl 2 (3?20.0 mL) and evaporated the solvents under reduced pressure, the residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to yield 24 (0.420 g, 86%): 1 H NMR (250 MHz, CDCl 3 ) ? 4.53 (d, J=5.50, 1H), 4.42 (d, J=5.50, 1H), 4.19 (q, 2H), 2.55 (br, 1H), 2.43 (d, J=7.85, 1H), 2.29 (m, 2H), 1.98 (m, 1H), 1.76 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H), 1.29 (t, 3H). Ethyl 2-((3aR,4R,6R,6aS)-6-(6-chloro-9H-purin-9-yl)-2,2-dimethyltetrahydro- 3aH-cyclopenta[d][1,3]dioxol-4-yl)acetate (25). DIAD (diisopropyl azodicarboxylate, 0.450 mL, 2.32 mmol) was added by syringe to a solution of TPP (triphenyl phosphine, 0.480 g, 1.83 mmol) in anhydrous THF (10.0 mL) at -10 0 C under a nitrogen atmosphere. The above solution was stirred at the same temperature for 20 min. 6-chloropurine (292 mg, 1.89 mmol) and a solution of 24 (0.420 g, 1.72 mmol) in anhydrous THF (5.00 mL) 100 were added to the above reaction mixture at room temperature. The whole reaction mixture was stirred at room temperature for 1 h and then was heated to 50 0 C for 36 h. After the reaction completed, the solvent was removed under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 25 (0.250 g, 40 %): 1 H NMR (250 MHz, CDCl 3 ) ? 8.74(s, 1H), 8.17(s, 1H), 5.12(t, J=5.80, 1H), 4.84(m, 1H), 4.64(m, 1H), 4.21(q, 2H), 2.78-2.40(m, 5H), 1.58(s, 3H), 1.31(s, 3H), 1.27(t, 3H). 2-((3aR,4R,6R,6aS)-6-(6-Chloro-9H-purin-9-yl)-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)acetaldehyde (26). Diisobutylaluminum (DIBAH, 0.220 mL, 1.32 mmol, 1.5 M in toluene) was added dropwise by syringe to a solution of 25 (0.250 g, 0.660 mmol) in CH 2 Cl 2 (10.0 mL) at -78 0 C. The reaction mixture was stirred at the same temperature for 3 h and quenched by methanol (2.0 mL) and H 2 O (10.0 mL) at - 78 0 C. The reaction mixture was warmed to room temperature and extracted with CH 2 Cl 2 (3?20.0 mL). After the solvent was removed under reduced pressure, the residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford 26 (800 mg, 82 %): 1 H NMR (400 MHz, CDCl 3 ) ? 9.84 (t, J=1.04, 1H), 8.84 (s, 1H), 8.17 (s, 1H), 5,12 (q, 1H), 4.85 (m, 1H), 4.62 (q, 1H), 2.92 (m, 1H), 2.74-2.64 (m, 3H), 2.41 (m, 1H), 1.58 (s, 6H). It was noted that if the above reduction reaction was quenched at -50 0 C or room temperature, from TLC analysis, two products (26 and 27) were obtained, which are inseparated by standard silica gel column chromatography method. (2R,3R,4R)-4-Vinyl-2,3-O-isopropylidene-1-cyclopentenone (28). Vinylmagnesium bromide (16.5 mL, 16.5 mmol, 1.0 M in THF) was added dropwise by syringe to a 101 suspension of CuBr?Me 2 S (0.230 g, 1.12 mmol) in anhydrous THF (40.0 mL) at -90-78 0 C. The reaction mixture was stirred at the same temperature for 30 min before a solution of 11 (2.00 g, 13.0 mmol) and TMSCl (3.40 mL, 26.5 mmol) and HMPA (5.80 mL, 33.1 mmol) in THF (20.0 mL) were added dropwise via a cannula to the above reaction mixture. The reaction mixture was kept stirring at -78 0 C for 5 h and warmed to room temperature. Saturated NH 4 Cl (15.0 mL) and tert-n-butylammonium fluoride (TBAF, 3.0 mL) were added to quench the reaction and the reaction mixture was stirred for 30 min. The reaction mixture was diluted with EtOAc (100 mL) and extracted with EtOAc (3?50.0 mL). The combined organic phases were washed with brine, dried with anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 28 (1.89 g, 80 %): 1 H NMR (250 MHz, CDCl 3 ) ? 5.81 (m, 1H), 5.19-5.07 (m, 2H), 4.66 (d, J=5.50, 1H), 4.22 (d, J=5.50, 1H), 3.11 (m, 1H), 2.90 (m, 1H), 2.33 (d, 18.0, 1H), 1.45 (s, 3H), 1.36 (s, 3H). (1S,2S,3R,4R)-4-Vinyl-2,3-O-isopropylidenecyclopentan-1-ol (29). Sodium borohydride (405 mg, 10.7 mmol) and cerium chloride heptahydride, CeCl 3 ?7H 2 O (2.22 g, 5.95 mmol) were added to a solution of 28 (1.30 g, 7.14 mmol) in anhydrous methanol (50.0 mL) at -10-0 0 C. The reaction mixture was kept at 0 0 C for 1.5 h and continued stirring at room temperature for 2 h. Saturated NH 4 Cl (9.00 mL) was used to quench the reaction. The reaction mixture was diluted with EtOAc (50.0 mL) and extracted with EtOAc (3?30.0 mL). The combined organic phases were washed with brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to provide 29 (1.21 g, 92 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 5.59 (m, 1H), 5.11 (m, 2H), 4.49 (d, J=3.30, 102 2H), 4.10 (br s, 1H), 2.76 (m, 1H), 2.44 (d, J=8.0, 1H), 1.92-1.86 (m, 2H), 1.52 (s, 3H), 1.36 (s, 3H). 6-Chloro-9-((3aS,4R,6R,6aR)-2,2-dimethyl-6-vinyltetrahydro-3aH cyclopenta[d][1,3]dioxol-4-yl)-9H-purine (30). A solution of DIAD (diisopropyl azodicarboxylate, 2.13 mL, 11.0 mmol) was added by a syringe to a solution of TPP (triphenyl phosphine, 2.88 g, 11.0 mmol) in anhydrous THF (50.0 mL) at -10 0 C under a nitrogen atmosphere. The above solution was stirred at the same temperature for 30 min. 6-chloropurine (1.27 g, 8.24 mmol) and a solution of 29 (1.01 g, 5.49 mmol) in THF (10.0 mL) were slowly added to the above reaction mixture. The whole reaction mixture was stirred at room temperature for 12 h and then was heated to 50 0 C for 8 h. The solvent was removed under reduced pressure, and the residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to afford 30 (0.990 g, 60 %) contaminated with the azadicarboxylate byproduct. 2-((3aR,4R,6R,6aS)-6-(6-Chloro-9H-purin-9-yl)-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)acetaldehyde (26)?the Second Method. Dimethyl sulphoxide (1.15 mL, 12.2 mmol), diisopropyl ethylamine (0.420 mL, 2.43 mmol) and SO 3 ?Py complex (0.260 g, 1.62 mmol) were added to the solution of compound 27 (0.260 g, 0.81 mmol) in CH 2 Cl 2 (10.0 mL) at 0 0 C. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. After the reaction completed, the solution was diluted with CH 2 Cl 2 (50.0 mL) and H 2 O (10.0 mL). Extracted with CH 2 Cl 2 , the organic phase was washed with H 2 O, NaHCO 3 and brine and dried over MgSO 4 . After removing the solvent under the reduced pressure, the residue 103 was purified by column chromatography (3/1, Hexane/EtOAc) to afford 26 (0.170 g, 66 %). The 1 H NMR data was the same as that of the first method. (S)-1-((3aR,4R,6R,6aS)-6-(6-Chloro-9H-purin-9-yl)-2,2-dimethyltetrahydro-3aH- cyclopental[d][1,3]dioxol-4-yl)pent-4-en-2-ol (31). Allylmagnesium bromide (1.10 mL, 1.10 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B- methoxydiisocampheylboron (348 mg, 1.10 mmol) in anhydrous diethyl ether (22.0 mL) at 0 0 C at room temperature under a nitrogen atmosphere. The above solution was well stirred at -78 0 C for 15 min and allowed to warm to room temperature for 3 h. The formation of Ipc 2 BAll was indicated by precipitation of the magnesium salts. The reaction mixture was filtered under nitrogen to remove the solid residue (magnesium salts) and washed the solid residue with pentane. Evaporation the solvents at reduced pressure, the resulting solution was added dropwise to a solution of 26 (0.380 g, 1.00 mmol), previously cooled to at -78 0 C, in a mixture of anhydrous diethyl ether (20.0 mL) and methylene chloride (5.00 mL). The reaction mixture was stirred at -78 0 C for 3 h, quenched with methanol (1.50 mL) and stirred at room temperature for 1 h. Saturated sodium bicarbonate (3.00 mL) and H 2 O 2 (3.00 mL, 32 wt % in H 2 O) were carefully added. The resulting mixture was stirred at room temperature for 14 h. The solvent was removed under reduced pressure and the residue was extracted with EtOAc. The combined organic layer was dried over anhydrous MgSO 4 and evaporated under reduced pressure. TLC analysis showed that it formed a complex and no desired product (31) was found from NMR. The reactant 26 was recovered. 104 It was noted that the allylation reaction with Brown?s reagent using this procedure failed. Later, with the help of Dr. Yin, the one-pot allylation approach was developed and succeeded in getting the desired product, such as 41, 53. N-(tert-butoxycarbonyl)-D-valine (32). Solid NaHCO 3 (21.5 g, 256 mmol) was added to a solution of D-valine (15.0 g, 128 mmol) in H 2 O (100 mL) and then a solution of di- tert-butyl dicarbonate (28.0 g, 128 mmol) in THF (100 mL) was added to the above reaction mixture. The reaction mixture was stirred and heated under reflux for 16 h and then concentrated under vacuum to remove THF. EtOAc (150 mL) was added to the above residue and the mixture was cooled to 10 ?C and then adjusted the solution to pH=3.0 with 1.0 M sulfuric acid solution. The aqueous layer was extracted with EtOAc (3?200 mL) and the combined organic layers were washed with water (200 mL) and brine (200 mL), dried over anhydrous MgSO 4 , and concentrated under vacuum to give 32 (24.3 g, 87 %) as colorless oil, or a whiteless foam: 1 H NMR (400 MHz, CDCl 3 ) ? 10.89 (br s, 1H), 6.50 (d, J=7.60, 1H), 5.24 (d, J=9.20, 1H), 2.20 (m, 1H), 1.45 (s, 9H), 1.00 (d, J=6.80, 3H), 0.94 (d, J=7.20, 3H). Methyl N-(tert-butoxycarbonyl)-D-valyl glycinate (33). Isobutyl chloroformate (14.6 mL, 112 mmol) was added to a stirred mixture of 32 (24.3 g, 112 mmol) and triethylamine (15.6 mL, 112 mmol) in CH 2 Cl 2 (200 mL) at 0-5?C. When the addition was complete, the mixture was stirred at 0-5 ?C for 30 min. In a separate flask, a mixture of glycine methyl ester hydrochloride (14.0 g, 112 mmol), triethylamine (15.6 mL, 112 mmol) and CH 2 Cl 2 (200 mL) was stirred for 30 min and this mixture was then added to the first flask containing 32 over 1 h. After the addition was complete, the mixture was stirred at room temperature for 16 h and then washed with water (3?50.0 mL) and brine 105 (100 mL), dried with anhydrous MgSO 4 and concentrated under vacuum to give 33 (27.2 g, 89 %) as yellow solid: 1 H NMR (400 MHz, CDCl 3 ) ? 6.52 (br s, 1H), 5.06 (br s, 1H), 3.98-4.02 (m, 1H), 3.76 (s, 3H), 2.20 (m, 1H), 1.45 (s, 9H), 0.99 (d, J=6.80, 3H), 0.94 (dd, J=7.20, 4.00, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 172.1, 170.3, 52.6, 46.0, 41.3, 31.1, 31.0, 28.5 (3C), 19.5, 19.1, 8.9. (2R)-Isopropylpiperazine-3,6-dione (34) A solution of 33. (27.2 g, 99.1 mmol) dissolved in 1,2-dichlorobenzene (150 mL) was heated at 175-180 ?C for 18 h and during this process, formed methanol was removed by distillation. After removing 1,2- dichlorobenzene with high-vacuum pump, the residue was cooled to 50 ?C. Tert- butyl methyl ether (200 mL) was slowly added to the above residue. The reaction mixture was further cooled to room temperature and filtered. The resulting solid was washed with tert- butyl methyl ether (200 mL) and ethyl ether (100 mL), dried under vacuum at 100 ?C to give 34 (8.83 g, 60 %) as white needle solid: 1 H NMR (400 MHz, DMSO-d 6 ) ? 8.19 (br s, 1H), 8.01 (br s, 1H), 3.84 (AB, 1H), 3.64 and 3.60 (AB, 1H), 3.52 (t, J= 3.20, 1H), 2.11 (m, 1H), 0.93 (d, J=6.80, 3H), 0.86 (d, J=6.80, 3H). (2R)-2,5-Dihydro-3,6-dimethoxy-2-isopropylpyrazine (35). Trimethyloxonium tetrafluoroborate (10.0 g, 67.6 mmol) was slowly added to a suspension of 34 (3.52 g, 22.5 mmol) in anhydrous CH 2 Cl 2 (50.0 mL) at room temperature. The mixture was stirred at room temperature for 84 h. The resulting solid was added in portions under a nitrogen atmosphere to a vigorously stirred mixture of saturated aqueous NaHCO 3 (150 mL) and CH 2 Cl 2 (100 mL) at 0-4 ?C, while maintaining the pH between 8.0 and 9.0 by the simultaneous addition of 3 M aqueous NaOH as required. The mixture was separated, and the aqueous phase was extracted with CH 2 Cl 2 (3?200 mL). The combined organic 106 phases were washed with brine (50.0 mL), dried with anhydrous MgSO 4 and concentrated under vacuum. The pale yellow residual oil was purified by reduced- pressure distillation to give 35 (2.91 g, 70 %, > 95 % ee from 1 H NMR) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 3.98-4.02 (m, 3H), 3.72 (s, 3H), 3.68 (s, 3H), 2.26 (m, 1H), 1.04 (d, J=6.80, 3H), 0.76 (d, J=6.8, 3H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 165.1, 162.5, 61.3, 52.7, 52.9, 46.8, 32.7, 19.2, 17.2. (2R,3R,4R)-4-Vinyl-2,3-O-cyclopentanyl-1-cyclopentenone (36). Vinylmagnesium bromide (89.2 mL, 89.2 mmol, 1.0 M in THF) was added dropwise to a suspension of CuBr?Me 2 S (0.780 g, 3.79 mmol) in THF (100.0 mL) at -90-78 0 C. The reaction mixture was stirred at the same temperature for 30 min before a solution of 21 (8.04 g, 44.6 mmol) and TMSCl (12.6 mL, 98.1 mmol) and HMPA (19.6 mL, 112 mmol) in anhydrous THF (30.0 mL) were added dropwise via a cannula to the above reaction mixture over 50 min. The reaction mixture was kept stirring at the same temperature for 5 h and warmed to room temperature. Saturated NH 4 Cl (15.0 mL) and tetra-butylammonium fluoride (3.00 mL) were added to quench the reaction and the reaction mixture was stirred for 30 min. EtOAc (200 mL) was added to dilute the reaction mixture and the organic phase was extracted with EtOAc (3?100 mL). The combined organic layer was washed with brine and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 36 (6.78 g, 73 %) as a clear yellow oil: 1 H NMR (400 MHz, CDCl 3 ) ? 5.84 (m, 1H), 5.17-5.08 (m, 2H), 4.56 (d, J=5.60, 1H), 4.18 (d, J= 5.20, 1H), 3,12 (m, 1H), 2.85 (dd, J=8.40, 1H), 2.30 (dm, J=18.0, 1H), 1.94-1.65 (m, 8H); 13 C NMR (62.9 MHz, CDCl 3 ) 107 ? 213.2, 137.3, 122.3, 116.5, 81.6, 77.7, 40.0, 38.8, 36.3, 36.2, 23.9, 23.1. Anal. Calcd for C 12 H 16 O 3 : C, 69.21; H, 7.74. Found: C, 67.36; H, 7.79. (1S,2S,3R,4R)-4-Vinyl-2,3-O-cyclopentanyl-cyclopentan-1-ol (37). A solution of 36 (6.87 g, 32.6 mmol) in anhydrous THF (30.0 mL) was added dropwise to a suspension of lithium aluminum hydride (3.18 g, 81.4 mmol) in anhydrous THF (50.0 mL) at -10-0 ?C. The reaction mixture was stirred at room temperature overnight. H 2 O (2.0 mL) and NaOH (5.0 mL, 6 M in H 2 O) were added to quench the above reaction. The resulting solid was removed by filtration and the solvent was evaporated under vacuum to afford 37 (6.06 g, 89 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 5.75 (m, 1H), 5.11- 5.04 (m, 2H), 4.39 (m, 2H), 4.08 (m, 1H), 2.78 (m, 1H), 2.45 (d, J=8.0, 1H), 1.99-1.84 (m, 2H), 1.71 (m, 8H); 13 C NMR (100.6 MHz, CDCl 3 ) ? 138.3, 121.7, 115.5, 84.5, 79.1, 71.4, 44.4, 36.4, 35.8, 35.6, 24.2, 23.1. Anal. Calcd for C 12 H 18 O 3 : C, 68.54; H, 8.63. Found: C, 67.31; H, 8.64. (3aS,4S,6R,6aR)-4-(4-Methoxybenzyloxy)-6-vinyltetrahydro-3aH- spiro[cyclopenta[d][1,3]dioxole-2,1'-cyclopentane] (38). Sodium hydride (1.15 g, 28.8 mmol) was added in portions to a solution of 37 (6.06 g, 28.8 mmol) in anhydrous N, N- dimethylformamide (DMF, 30.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature for 30 min and then p-methoxybenzyl chloride (4.70 mL, 34.6 mmol) was added dropwise by a syringe to the above reaction mixture. The reaction was kept at 0 0 C for 1 h and at room temperature for 4 h. After removed DMF by high pressure vacuum, the residue was quenched by H 2 O (3.0 mL) and extracted with EtOAc (3?100 mL). The combined organic layer was washed with brine and dried with anhydrous MgSO 4 . After removal of the solvents under vacuum, the residue was purified on silica 108 gel column chromatography (4/1, Hexane/EtOAc) to afford 38 (8.63 g, 90 %) as a shallow yellow oil: 1 H NMR (250 MHz, CDCl 3 ) ? 7.32 (dd, J=8.50, 2.00, 2H), 6.89 (dd, J=8.75, 2.00, 2H), 5.68 (m, 1H), 5.03 (m, 1H), 4.97 (m, 1H), 4.65 (d, J=11.76, 1H), 4.55 (d, J=11.51, 1H), 4.43 (t, J=6.50, 5.25, 1H), 4.30 (d, J=5.75, 1H), 3.81 (m, 4H), 2.66 (t, J= 7.25, 6.75, 1H), 2.16-2.03 (m, 2H), 1.94-1.70 (m, 8H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.5, 138.9, 130.8, 129.7, 121.0, 115.0, 114.0, 84.0, 78.5, 77.9, 71.7, 55.5, 44.1, 35.8, 35.6, 32.1, 24.3, 23.3. Anal. Calcd for C 20 H 36 O 4 : C, 72.70; H, 7.93. Found: C, 72.55; H, 8.06. 2-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro-3aH- spiro[cyclopenta[d][1,3]dioxole-2,1'-cyclopentane]-6-yl)ethanol (39). 9-Borabycyclo [3.3.1] nonane (9-BBN, 78.4 mL, 39.2 mmol, 0.5 M in THF) was added dropwise by syringe to a solution of 38 (8.63 g, 26.1 mmol) in anhydrous THF (50.0 mL) at -10-0 ?C. The reaction mixture was stirred at room temperature overnight. Methanol (10.0 mL), NaOH (6 M, 12.0 mL) and H 2 O 2 (15.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 0 C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (50.0 mL), the organic phase was washed with saturated NaHCO 3 and brine and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford 39 (6.70 g, 66 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (dd, J=8.80, 2.00, 2H), 6.89 (dd, J=8.80, 2.00, 2H), 4.65 (d, J=11.61, 1H), 4.54 (d, J=12.01, 1H), 4.45 (t, J=5.20, 5.60, 1H), 4.21 (d, J= 6.00, 1H), 3.83-3.78 (m, 4H), 3.72-3.62 (m, 2H), 2.11-2.04 (m, 2H), 1.92 (m, 1H), 1.71-1.56 (m, 9H), 1.49-1.41 (m, 2H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.5, 130.8, 129. 7, 121.5, 109 114.0, 84.8, 79.0, 77.8, 71.7, 61.7, 55.5, 38.4, 36.0, 35. 8, 33.7, 24.1, 23.3, 14.4. Anal. Calcd for C 20 H 28 O 5 : C, 68.94; H, 8.10. Found: C, 68.05; H, 8.18. 2-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro-3aH- spiro[cyclopenta[d][1,3]dioxole-2,1'-cyclopentane]-6-yl)acetaldehyde (40). Dimethyl sulphoxide (20.4 mL, 287 mmol) and diisopropyl ethylamine (8.33 mL, 47.8 mmol) were added to the solution of 39 in CH 2 Cl 2 (50.0 mL) at -10-0 ?C. After the reaction was stirred at 0 0 C for 30 min, SO 3 ?Py complex (4.56 g, 28.7 mmol) was added to the above solution. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. The reaction mixture was diluted with CH 2 Cl 2 (100 mL) and H 2 O (10.0 mL) and extracted with CH 2 Cl 2 (3?100 mL). The combined organic phases were washed with saturated NaHCO 3 , H 2 O and brine, dried over MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford the aldehyde 40 (6.41 g, 97 %) as the colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 9.70 (t, J=2.00, 1.75, 1H), 7.31 (dd, J= 8.50, 2.00, 2H), 6.89 (dd, J=8.75, 2.25, 2H), 4.64 (d, J=11.76, 1H), 4.55 (d, J=11.76, 1H), 4.44 (t, J=5.25, 5.00, 1H), 4.16 (d, J=5.75, 1H), 3.82-3.74 (m, 4H), 2.54 (m, 1H), 2.36-2.04 (m, 3H), 1.95 (m, 1H), 1.68-1.54 (m, 8H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 200.9, 159.5, 130.5, 129.7, 121.5, 114.0, 84.0, 78.7, 77. 6, 71.7, 55.5, 46.9, 35.8, 35.6, 35.4, 33.0, 24.3, 23.3. Anal. Calcd for C 20 H 26 O 5 : C, 69.34; H, 7.56. Found: C, 69.00; H, 7.50. (S)-1-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro-3aH-spiro- [cyclopenta[d][1,3]dioxole-2-1'-cyclopentane]-6-yl)pent-4-en-2-ol (41). Allylmagnesium bromide (34.2 mL, 34.2 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B-methoxydiisocampheylboron (10.8 g, 34.2 mmol) in 110 anhydrous diethyl ether (80.0 mL) at -10-0 ?C under a nitrogen atmosphere. The reaction was stirred at room temperature for 2 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 40 (6.41 g, 18.5 mmol) dissolved in anhydrous THF (25.0 mL) was added dropwise via a cannula over 50 min to the above reaction mixture. The newly reaction mixture was kept at -78 ?C for 6 h and allowed to warm to room temperature for another 4 h. Finally, methanol (10.0 mL), NaOH (6 M, 13.0 mL) and H 2 O 2 (25.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (150 mL) and extracted with EtOAc (3?150 mL), the combined organic phase was washed with H 2 O and brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 41 (6.43 g, 90 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (dd, J=8.80, 2.00, 2H), 6.89 (dd, J=8.80, 2.00, 2H), 5.85 (m, 1H), 5.14-5.10 (m, 2H), 4.64 (d, J=11.61, 1H), 4.53 (d, J=12.01, 1H), 4.45 (t, J=5.60, 5.20, 1H), 4.28 (d, J=7.60, 1H), 3.81 (m, 4H), 3.72-3.64 (m, 1H), 2.26 (m, 1H), 2.18-2.05 (m, 4H), 1.91 (m, 1H), 1.70-1.56 (m, 8H), 1.39-1.29 (m, 2H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.4, 134.8, 130.8, 129.7, 121.5, 118.5, 114.0, 85.1, 79.1, 77.6, 71.7, 69.9, 55.5, 42.4, 39.9, 38.9, 36.1,35.8, 34.0, 24.2, 23.3. Anal. Calcd for C 23 H 32 O 5 : C, 71.11; H, 8.30. Found: C, 71.26; H. 8.43. (3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)-6-((S)-2-(methoxymethoxy)pent-4- enyl)tetrahydro-3aH-spiro-[cyclopenta[d][1,3]dioxole-2-1'-cyclopentane] (42). To a solution of 41 (6.43 g, 16.6 mmol) in anhydrous CH 2 Cl 2 (25.0 mL) was added diisopropyethylamine (DIPEA, 10.1 mL, 57.9 mmol) at -10-0 ?C under a nitrogen 111 atmosphere. After the mixture was stirred for 30 min and kept at -10 ?C, chloromethyl methyl ether (2.52 mL, 16.6 mmol) was slowly added by a syringe. The whole reaction mixture was then stirred for 1 h, warmed to room temperature and transferred to stir for 24 h at 40 ?C in oil bath. After the reaction mixture was diluted with EtOAc and saturated NaHCO 3 at -10-0 ?C. The organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (3?150 mL). The combined organic phases were washed with water and brine, dried with MgSO 4 , and then evaporated in vacuo to afford the crude product, which was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to furnish 42 (5.07 g, 72%) as a yellow oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (dd, J=8.75, 2.00, 2H), 6.86 (dd, J=8.50, 2.00, 2H), 5.75 (m, 1H), 5.08-5.03 (m, 9'-H, cis and trans, 2H), 4.68 (d, mom-H, J=7.20, 1H), 4.62 (d, pmb-H, J=12.0, 1H), 4.59 (d, mom-H, J=7.20, 1H), 4.54 (d, pmb-H,J=12.0, 1H), 4.42 (t, J=5.25, 2'-H, 1H), 4.21 (d, 3'-H, J=5.60, 1H), 3.80 (s, PMB, -OCH 3 , 3H), 3.77-3.72 (m, multi, 5, 6'-H, 1H), 3.65-3.59 (m, 1'-H, 1H), 3.35 (s, MOM, -OCH 3 , 3H), 2.30-2.26 (m, 7'-H, 2H), 2.16-2.01 (m, 5'-H, 2H; 5-H, 1H), 1.92-1.83 (m, 4'-H, 1H; 5-H, 1H), 1.68-1.61 (m, cyclopentanone, 8H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.4, 134.5 (8'-C), 130.6, 129.6 (2C), 120.9 (2C), 117.6 (cyclopentanone, 1-C), 113.9 (9'-C), 95.6 (MOM, CH 2 ), 85.0 (2'-C), 78.4 (3'-C), 77.6 (1'- C), 75.4 (6'-C), 71.5 (PMB, -OCH 2 ), 55.8 (PMB, OCH 3 ), 55.4 (MOM, OCH 3 ), 39.1, 37.7 (CYCLO), 37.2 (7'-C), 35.7 (5'-C), 35.5 (5-C), 32.6 (4'-C), 24.2, 23.2 (CYCLO). Anal. Calcd for C 25 H 36 O 6 : C, 69.42; H, 8.39. Found: C, 69.48; H, 8.49. (R)-4-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro-3aH-spiro- [cyclopenta[d][1,3]dioxole-2-1'-cyclopentane]-6yl)-3-(methoxymethoxy)butan-1-ol (43). Sodium periodate (4.95 g, 23.4 mmol) was added to a solution of 42 (5.07 g, 11.7 112 mmol) in MeOH /H 2 O (160 mL, v/v=3/1) at -10-0 ?C. Catalytic amount of osmium tetroxide (488 mg) was slowly added to the above reaction mixture. The reaction mixture was stirred at 0 ?C for 1 h and kept for 2 h at room temperature. The mixture was filtrated with a little pad of celite, diluted with CH 2 Cl 2 and the aqueous layer was extracted with CH 2 Cl 2 . The combined organic phases was dried over anhydrous MgSO 4 and then evaporated under reduced pressure to give a yellow syrup, which was used to the next reaction without further purification. To a stirred solution of the yellow synrup in anhydrous MeOH (50.0 mL) at -10- 0 ?C was added sodium borohydride (532 mg, 14.1 mmol) and the reaction mixture was stirred at 0 ?C for 1 h and kept for 2 h at room temperature. Saturated NH 4 Cl solution (5.0 mL) was added to quench the reaction mixture. The residue was diluted with EtOAc (120 mL), dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to furnish 43 (2.54 g, 50 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (dd, J=8.50, 2.00, PMB, 2H), 6.88 (dd, J=8.50, 2.00, PMB, 2H), 4.65-4.60 (m, 3H, PMB, 2H; MOM, 1H),4.53 (d, 1H, J=12.00, MOM, 1H), 4.43 (t, 2'-H, J=5.25 1H), 4.19 (d, 3'-H, J= 5.75, 1H), 3.80 (s, PMB, OCH 3 , 3H), 3.78-3.74 (m, 3H; 6'-H, 1H; 8'-H, 2H), 3.69-3.62 (m, 1H; 1'-1H), 3.38 (s, 3H, MOM),2.52 (br, 1H, OH), 2.12-2.02 (m, 3H; 4'-H, 1H; 7'-H, 2H), 1.91-1.88 (m, 1H; 5'-H, 1H), 1.83-1.77 (m, 1H; cyclopentane), 1.71-1.64 (m, 7H; cyclopentane), 1.63-1.60 (m, 1H; 5'-H), 1.56-1.49 (m, 1H; 5-H, 1H), 1.29-1.22 (m, 1H; 5- H, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.4, 130.6, 129.6 (2C), 121.1 (1C, cyclopentane), 113.9 (2C), , 96.3 (1C, MOM, CH 2 ), 84.9 (2'-C), 78.6 (3'-C), 77.7 (1'-C), 75.0 (6'-C), 71.5 (1C, PMB, CH 2 ), 59.5 (1C, 8'-C,OH), 56.0 (PMB, OCH 3 ), 55.4 (MOM, 113 OCH 3 ), 38.0, 37.7, (2C, cyclopentane, 2 & 5 position), 37.1 (7'-C), 35.8 (5'-C), 35.5 (5-C), 33.3 (4'-C), 24.1, 23.2 (2C, cyclopentane, 3 & 4 position). Anal. Calcd for C 24 H 36 O 7 : C, 66.03; H, 8.31. Found: C, 65.58; H, 8.33. (R)-4-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro-3aH-spiro- [cyclopenta[d][1,3]dioxole-2-1'-cyclopentane]-6yl)-3-(methoxymethoxy)butan-1-ol (43)-the Optimized Method. To a solution of 42 (7.22 g, 16.7 mmol) in THF (80.0 mL) was added a catalytic amount of osmium tetroxide (100 mg) and 50 % aqueous N- methylmorpholine N-oxide (NMO, 20.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature for 2 h and warmed to room temperature and kept stirring for 12 h. After evaporated the solvent (THF) under reduced pressure, the residue was diluted and extracted with EtOAc. The organic phase was concentrated again to give a yellow liquid, which was purified on silica gel column chromatography (EtOAc) to furnish the diol 42-1' (6.33 g, 83 %). 42-1' was not necessary to be conducted with NMR and further used to the next reaction after separation. To a stirred solution of 42-1' in a mixture of CH 2 Cl 2 /H 2 O (200 mL, 3/1, v/v) at - 10-0 ?C was slowly added sodium periodate (7.44 g, 34.0 mmol). A catalytic amount of 15-crown-5 (1.00 mL) was added to the above reaction mixture. The reaction mixture was stirred at the same temperature for 2 h and warmed to room temperature and kept stirring for 12 h. The solution was diluted with CH 2 Cl 2 (150 mL), and the aqueous layer was extracted with CH 2 Cl 2 (3?150 mL). The combined organic phases were washed with H 2 O and brine, dried over anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to 114 afford the aldehyde 42-1'' (5.59 g, 95 %) as a colorless oil and the aldehyde 42-1'' was used to the next reaction without NMR data. The aldehyde 42-1'' (5.59 g, 12.9 mmol) thus obtained was dissolved in anhydrous MeOH (80.0 mL) at -10-0 ?C. To this solution were added sodium borohydride (771 mg, 32.2 mmol) and CeCl 3 ?7H 2 O (5.75 g, 15.4 mmol). The reaction mixture was stirred at -10-0 ?C for 1 h and kept for 2 h at room temperature. Saturated NH 4 Cl solution (5.0 mL) was added to quench the reaction mixture. The residue was diluted with EtOAc (2?60.0 mL), dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (2/1, Hexane/EtOAc) to furnish 43 (4.32 g, 77%) as a colorless oil in 80% yield in three steps from 43. The NMRs ( 1 H, 13 C) of 43 were the same as that of 43, which was achieved from the first oxidative-cleavage method. (3aR,4S,6S,6aS)-4-((R)-4-Iodo-2-(methoxymethoxy)butyl)-6-(4 methyoxybenzyloxy)tetrahydro-3aH-spiro-[cyclopenta[d][1,3]dioxole-2-1'- cyclopentane] (44). A solution of 43 (6.42 g, 14.7 mmol) in toluene (50.0 mL) and acetonitrile (10.0 mL) was treated with imidazole (2.51 g, 36.8 mmol) and triphenylphosphane (9.65 g, 36.8 mmol). Then, iodine was introduced in approximately 10.0 mg portions to the above reaction mixture until the reaction mixture remained dark- brown in color. The dark-brown solution was heated to reflux for 10 minutes and cooled to room temperature. The reaction mixture was diluted with Et 2 O (50.0 mL) and washed with 10 % sodium thiosulfate solution and brine, dried over anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified through a short plug of silica gel column chromatography (1/1, Hexanes/EtOAc) to provide 44 (3.22 g, 40%) as a 115 yellow oil: 1 H NMR (400 MHz, CDCl 3 ) 7.31 (dd, J=8.40, 2.00), 6.88 (dd, J=8.80, 2.00), 4.65-4.59 (m, 3H), 4.55 (d, J=12.00, 1H), 4.43 (t, J=5.60, 5.20, 1H), 4.18 (d, J=5.60, 1H), 3.79 (s, 3H), 3.77-3.74 (m, 1H), 3.68-3.59 (m, 1H), 3.36 (s, 3H), 3.24-3.13 (m, 2H), 2.15- 1.88 (m, 5H), 1.68-1.62 (m, 8H), 1.47 (m, 1H), 1.22 (m, 1H); 13 C NMR (100 MHz, CDCl 3 ) 159.3, 130.5, 129.5, 121.0, 113.8, 96.0, 84.8, 78.4, 77.4, 76.6, 71.4, 55.8, 55.3, 38.8, 37.5, 37.1, 35.7, 35.4, 32.9, 24.1, 23.1, 1.8. (R)-4-((3aS,4S,6S,6aR)-4-(4-Methoxybenzyloxy)tetrahydro--3aH-spiro- [cyclopenta[d][1,3]dioxole-2-1'-cyclopentane]-6-yl)-3-(methoxymethoxy)butyl 4- methylbenzenesulfonate (45). To a solution of 43 (2.79 g, 6.39 mmol) in anhydrous CH 2 Cl 2 (30.0 mL) were added triethylamine (22.3 mL, 160 mmol) and a catalytic amount of DMAP (500 mg) at room temperature under a nitrogen atmosphere. After the reaction mixture was stirred for 30 min and then cooled to -10-0 ?C, p-toluenesulfonyl chloride (3.05 g, 16.0 mmol) was slowly added. The whole reaction mixture was stirred at the same temperature for 2 h, warmed to room temperature and stirred overnight. After the reaction mixture was diluted with ice-water (10.0 mL), the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (3?80.0 mL). The combined organic phases were washed with 1 % HCl and brine, dried with anhydrous MgSO 4 and concentrated in vacuuo to give the crude product, which was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to furnish 45 (3.40 g, 90%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.79 (dd, J=8.40, 1.60, 2H, Tos 2H), 7.35 (dd, J=9.20, 2.00, Tos 2H), 7.31 (dd, J=8.40, 2.00, PMB, 2H), 6.89 (dd, J=8.40, 2.00, PMB 2H), 4.63 (d, J=12.00, PMB, CH 2 , 1H), 4.55-4.49 (m, PMB, CH 2 , 1H; MOM, CH 2 , 2H), 4.42 (t, J=5.20, 2'-H, 1H), 4.14 (m, 3'-H, 1H; 8'-H, 2H), 3.80 (s, PMB, OCH 3 , 3H), 3.77-3.72 (m, 116 1'-H, 1H), 3.66-3.58 (m, 6'-H, 1H), 3.27 (s, MOM, OCH 3 , 3H), 2.44 (s, TOS, CH 3 , 3H), 2.08-2.00 (m, 5-H, 1H; cyclopentane, 2H), 1.92-1.77 (m, 7'-H, 2H; 4'-H, 1H), 1.72-1.63 (m, cyclopentane, 6H), 1.59-1.55 (m, 5-H, 1H), 1.46-1.38 (m, 5'-H, 1H), 1.23-1.18 (m, 5'- H, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.4, 145.0 (Tos, 1C), 133.3 (Tos, 1C), 130.7 (Tos, 1C), 130.0 (Tos, 2C), 129.7 (Tos, 2C), 128.1 (2C), 121.1 (cyclopentane, tert-C), 113.9 (PMB, 2C), 96.3 (MOM, CH 2 , 1C), 84.9 (2'-C), 78.6 (3'-C), 77.7 (1'-C), 73.4 (6'-C), 71.6 (Tos, CH 2 , 1C), 67.3 (8'-C), 55.9 (PMB, OCH 3 ), 55.5 (MOM, OCH 3 ), 37.9 and 37.6 (cyclopentane, 2 and 5 position), 35.8 (7'-C), 35.6 (5'-C), 34.2 (5-C), 33.2 (4'-C), 24.2 and 21.8 (cyclopentane, 3 and 4 position), 23.2 (tosyl, CH 3 ). (2R,5S)-2-Isopropyl-3,6-dimethoxy-5-((S)-4-((3aS,4S,6S,6aR)-4-(4- methoxybenzyloxy)tetrahydro-3aH-spiro[cyclopenta[d][1,3]dioxole-2-1'- cyclopentane]-6-yl)-3-(methoxymethoxy)butyl)-2,5-dihydropyrazine (46). To a solution of (2R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine 35 (0.450 mL, 2.71 mmol) in anhydrous THF (10.0 mL) under a nitrogen atmosphere was added n-BuLi (1.1 mL, 2.71 mmol, 2.5 M in THF) dropwise at -90-78 ?C by a syringe. This solution was stirred at -90-78 ?C for 30 min. Then, to this solution was slowly added a solution of 45 (0.800 g, 1.35 mmol) in anhydrous THF (10.0 mL) via a cannula over 40 min at -90- 78 ?C. The reaction mixture was stirred at -90-78 ?C for 5 h, kept at -20 ?C for 12 h and allowed to slowly warm to room temperature. The solution was then quenched with the addition of saturated NH 4 Cl (3.0 mL) and ammonium hydroxide (3.0 mL) and ethanol (3.0 mL). The THF was removed under reduced pressure, and the residue was diluted with EtOAc. The organic phase was separated and the aqueous layer was extracted with EtOAc (3?50.0 mL). The combined organic phases were washed with brine and dried 117 over anhydrous MgSO 4 . After removal of solvent under reduced pressure, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 46 (1.96 g, 85 %) as the yellow oil, the only diastereoisomer: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (d, J=8.80, 2H), 6.89 (d, J=8.80, 2H), 4.64-4.49 (m, PMB, CH 2 ; MOM, CH 2 , 4H), 4.43 (t, J=5.60, 2'-H, 1H), 4.13 (t, J=6.0, 3'-H, 1H), 4.05-3.91 (AB, pyrazine, 2H), 3.80 (s, 3H), 3.78-3.73 (br s, 6'-H, 1H), 3.69 (s, 3H), 3.66 (s, 3H), 3.65-3.60 (m, 1'-H, 1H), 3.27 (s, MOM, OCH3, 3H), 2.28-2.22 (m, pyrazine, 1H), 2.08-2.01 (m, 5-H, cyclopentane, 3H), 1.90-1.81(m, 4'-H, 7'-H, 3H), 1.70-1.65(m, cyclopentane, 6H), 1.62-1.58 (m, 5-H, 1H), 1.48-1.43 (m, 5'-H, 8'-H, 3H), 1.27-1.24 (m, 5'-H, 1H), 1.05 (d, J=6.80, 3H), 0.70 (d, J=6.80, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 161.1 (pyrazine), 159.1, 155.3 (pyrazine), 138.1, 130.7 (2C), 121.0, 113.9 (2C), 93.8 (PMB, CH 3 OCH 2 -), 84.9 (2'-C), 78.4 (3'-C), 77.8 (1'-C), 76.1 (6'-C), 71.6 (8'-C), 56.2, 55.8 (PMB, OCH 3 ), 55.5 (MOM, OCH 3 ), 54.0 (pyrazine, OCH 3 ), 53.9 (pyrazine, OCH 3 ), 38.6 and 37.5 (cyclopentane, 2, 5 positions), 38.51(pyrazine, 9'-C), 35.8 (7'-C), 35.6 (5'-C), 32.8 (5-C), 28.5 (pyrazine, CH(CH 3 ) 2 ), 28.3 (4'-C), 24.2 and 21.3 (cyclopentane, 3, 4 positions), 21.2 and 20.7 (pyrazine, CH(CH 3 ) 2 ). ((3aS,4S,6R,6aR)-6-((S)-4-((2S,5R)-Isopropyl-3,6-diemethoxy-2,5-dihydropyrazin- 2-yl)-2-(methoxymethoxy)butyl)tetrahydro-3aH-spiro[cyclopenta[d][1,3]dioxole-2- 1'-cyclopentan]-4-ol (47). To a solution of 46 (70.0 mg, 0.12 mmol) in CH 2 Cl 2 (10.0 mL) at -10-0 ?C were added H 2 O (0.50 mL) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (39.6 mg, 1.17 mmol). The reaction mixture was stirred at -10-0 ?C for 30 min and allowed to slowly warm to room temperature. After removal of solvent, the residue was purified by column chromatography (3/1, Hexane/EtOAc) to afford the desired product 118 47 (32.0 mg) as a yellow oil: 1 H NMR (250 MHz, CDCl 3 ) ? 4.64 (m, MOM, CH 2 , 2H), 4.49 (t, J=5.75, 1H), 4.35(dd, J=2.0, 6.25, 1H), 4.09 (AB, pyrazine, 2H), 3.95 (t, J=3.25, 1H), 3.70 (s, 3H), 3.69 (s, 3H),3.62 (m, C-6', 1H) 3.39 (s, OCH 3 , 3H), 2.43 (br s, C-1, 1H), 2.31 (br s, CH(CH 3 ) 2 , 1H), 1.97-1.86 (m, 2H), 1.73-1.66 (m, 4H), 1.63-1.50 (m, cylcopentanone, 8H), 1.42-1.40 (m, 3H), 1.07 (d, J=7.0, 3H), 0.72 (d, J=7.0, 3H). (3aS,4S,6R,6aR)-4-(4-Methoxybenzyloxy)-2,2-dimethyl-6-vinyltetrahydro-3aH- cyclopenta[d][1,3]dioxle (50). Sodium hydride (1.83 g, 49.2 mmol) was added in portions to a solution of 29 (7.53 g, 41.0 mmol) in anhydrous N, N-dimethylformamide (DMF, 70.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature for 30 min and then p-methoxybenzyl chloride (6.52 mL, 49.2 mmol) was added dropwise by syringe to the above reaction mixture. The reaction was kept at 0 ?C for 1 h and at room temperature for 4 h. After removed DMF by high pressure vacuum, the residue was quenched by H 2 O (5.0 mL) and extracted with EtOAc (3?150 mL). The combined organic layers were washed with brine and dried with anhydrous MgSO 4 . After removal the solvents under reduced pressure, the residue was purified on silica gel column chromatography (10/1, Hexane/EtOAc) to afford 50 (6.90 g, 60 %) as a yellow oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.32 (d, J=8.50, 2H), 6.89 (d, J=8.50, 2H), 5.72 (m, 1H), 5.02-4.97 (m, 2H), 4.63 (t, J=4.0, 8.0, 2H), 4.57-4.46 (m, 2H), 4.41 (d, J=6.0, 1H), 3.82 (s, 3H), 3.81-3.76 (m, 3H), 1.52 (s, 3H), 1.28 (s, 3H). 2-((3aR,4S,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxl-4-yl)ethanol (51). 9-Borabycyclo [3.3.1] nonane (9-BBN, 68.1 mL, 34.0 mmol, 0.5 M in THF) was added dropwise to a solution of 50 (6.90 g, 22.7 mmol) in anhydrous THF (50.0 mL) at -10-0 ?C. The reaction mixture was stirred at 119 room temperature overnight. Methanol (15.0 mL), NaOH (6 M, 15.0 mL) and H 2 O 2 (20.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (100 mL), the organic phase was washed with saturated NaHCO 3 and brine and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford 51 (6.73 g, 92 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.32 (dd, J=9.60, 2.00, 2H), 6.89 (dd, J=9.60, 2.00, 2H), 4.65-4.62 (d, J=12.0, 2H), 4.57-4.50 (m, 3H), 4.30 (d, J=5.60, 2H), 3.81 (s, 3H), 3.80-3.77 (m, 1H), 3.70-3.65 (m, 3H), 2.10-2.08 (m, 2H), 1.52 (s, 3H), 1.33 (s, 3H). 2-((3aR,4R,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxl-4-yl)acetaldehyde (52). Dimethyl sulphoxide (65.0 mL, 914 mmol) and diisopropyl ethylamine (14.7 mL, 84.4 mmol) were added to the solution of 51 (6.73 g, 20.9 mmol) in anhydrous CH 2 Cl 2 (50.0 mL) at -10-0 ?C. After the reaction was stirred at 0 ?C for 30 min, SO 3 ?Py complex (6.80 g, 33.6 mmol) was added to the above solution. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. The reaction mixture was diluted with CH 2 Cl 2 (150 mL) and H 2 O (10.0 mL) and extracted with CH 2 Cl 2 (3?100 mL). The combined organic phases were washed with saturated NaHCO 3 , H 2 O and brine, dried over anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford the aldehyde 52 (5.82 g, 87 %) as the colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 9.71 (t, J=1.75, 1H), 7.31 (d, J=8.50, 2H), 6.90 (d, J=8.50, 2H), 4.64-4.50 (m, 3H), 4.25 (d, J=5.50, 1H), 3.81 120 (s, 3H), 3.79-3.75 (m, 1H), 2.56-2.13 (m, 5H), 1.53 (s, 3H), 1.32 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 200.6, 157.6, 130.2, 129.5 (2C), 113.8 (2C), 111.5, 84.0, 78.5, 77.3, 71.4, 55.3, 46.6, 35.5, 32.7, 26.3, 24.4. (R)-1-((3aR,4S,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-diemthyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)pent-4-en-2-ol (53). Allylmagnesium bromide (17.6 mL, 17.6 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (+)-B- methoxydiisocampheylboron (5.60 g, 17.6 mmol) in anhydrous diethyl ether (30.0 mL) at -10-0 ?C under dry nitrogen. The reaction was stirred at room temperature for 2 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 52 (3.75 g, 11.7 mmol) dissolved in THF (15.0 mL) was added dropwise for 50 min to the above reaction mixture by a cannula. The newly reaction mixture was kept at -78 ?C for 5 h and allowed to warm to room temperature for another 4 h. Finally, methanol (9.00 mL), NaOH (6 M, 7.50 mL) and H 2 O 2 (15.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (150 mL), the organic phase was washed with H 2 O and brine and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 51 (3.95 g, 93%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (d, J=8.40, 2H), 6.88 (d, J=8.40, 2H), 5.80-5.73 (m, 1H), 5.12-5.04 (m, 2H), 4.64- 4.48 (m, 3H), 4.27 (d, J=5.60, 1H), 3.83-3.80 (m, 1H), 3.78 (s, 3H), 3.68-3.60 (br, s, 1H), 2.29-1.88 (m, 6H), 1.73-1.57 (m, 2H), 1.52 (s, 3H), 1.32 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.1, 134.6, 130.5, 129.4 (2C), 118.0, 113.7 (2C), 111.2, 84.3, 78.7, 77.8, 71.3, 68.8, 55.2, 42.0, 39.7, 37.9, 33.9, 26.4, 24.5. 121 (S)-1-((3aR,4S,6S,6aS)-6-(4-Methoxybenzyloxy)-2,2-diemthyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-yl)pent-4-en-2-ol (54). Allylmagnesium bromide (20.8 mL, 20.8 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B- methoxydiisocampheylboron (6.60 g, 20.8 mmol) in anhydrous diethyl ether (30.0 mL) at -10-0 ?C under a nitrogen atmosphere. The reaction was stirred at room temperature for 2 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 52 (4.43 g, 13.8 mmol) in THF (10.0 mL) was added dropwise via a cannula over 50 min to the above reaction mixture. The reaction mixture was kept at -78 ?C for 6 h and allowed to warm to room temperature for another 4 h. Finally, methanol (8.0 mL), NaOH (3.0 M, 15.0 mL) and H 2 O 2 (15.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 ?C, respectively. After being stirred at room temperature for 4 h, the reaction mixture was diluted with EtOAc (100 mL) and washed with H 2 O and brine, dried with anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 54 (4.51 g, 90%) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.31 (d, J=8.40, 2H), 6.89 (d, J=8.40, 2H), 5.83 (m, 1H), 5.15 (m, 2H), 4.65-4.49 (m, 3H), 4.36 (dd, J=5.20, 3'-H, 1H), 3.80 (s, 3H), 3.79-3.75 (m, 1'-H, 1H), 3.70 (br s, 6'-H, 1H), 2.30-2.03 (m, 6H), 1.87-1.77 (m, 1H), 1.52 (s, 3H), 1.31 (s, 3H). (3aR,4S,6S,6aS)-4-((S)-2-(Benzyloxy)pent-4-enyl)-6-(4-methoxybenzyloxy)-2,2- diemthyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (55). To a solution of 54 (3.96 g, 10.9 mmol) in anhydrous N, N-dimethylformamide (DMF, 50.0 mL) at -10-0 ?C, was added sodium hydride (524 mg, 13.1 mmol). The reaction mixture was stirred at 0 ?C for 30 min before benzyl chloride (1.55 mL, 13.1 mmol) was added dropwise by syringe. 122 The reaction mixture was kept at 0 ?C for 2 h and continued stirring for 6 h at room temperature. After removal of DMF by high pressure vacuum, the residue was quenched by H 2 O (5.0 mL) and extracted with EtOAc (3?80.0 mL). The combined organic layers were washed with brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. Purification of the residue on silica gel column chromatography (4/1, Hexane/EtOAc) afforded 55 (4.20 g, 85 %): 1 H NMR (400 MHz, CDCl 3 ) ? 7.33-2.27 (m, 7H), 6.87 (d, J=8.0, 2H), 5.83 (m, 1H), 5.09-5.05 (m, 2H), 4.60-4.33 (m, benzyl, CH 2 ; PMB, CH 2 ; 2'-H; 3'-H; 6H), 3.79 (s, 3H), 3.72 (m, 1'-H, 1H), 3.45 (br s, 6'-H, 1H), 2.33- 1.90 (m, 7'-H, 2H; 4'-H; 5-H, 2H; 5H), 1.52 (s, 3H), 1.42-1.38 (m, 5'-H, 1H), 1.30 (s, 3H), 1.23 (m, 5'-H, 1H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.2, 138.6, 134.4 (8'-C), 130.6, 129.5 (2C, PMB), 128.4 (2C, benzyl), 128.0 (2C, benzyl), 127.6, 117.5 (9'-C), 113.8 (2C, PMB), 111.1 (C(CH 3 ) 2 ), 84.9 (2'-C), 78.6 (3'-C),78.2 (1'-C), 77.6 (6'-C), 71.3 (CH 2 , PMB), 71.2 (CH 2 , benzyl), 55.3 (OCH 3 ), 38.2 (7'-C), 37.4 (5'-C), 37.0 (5-C), 32.7 (4'-C), 26.4, 24.5. (3aR,4S,6R,6aR)-6-Methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4- caraldehyde (56). Dimethyl sulphoxide (53.0 mL, 560 mmol), diisopropyl ethylamine (27.0 mL, 155 mmol) and SO 3 ?Py complex (25.5 g, 155 mmol) were added to a solution of 55 (12.7 g, 61.8 mmol) in CH 2 Cl 2 (100 mL) at 0 ?C. The reaction mixture was kept at 0 ?C for 2 h and continued stirring for 4 h at room temperature. After the reaction completed, the solution was diluted with CH 2 Cl 2 (100 mL) and H 2 O (10.0 mL). Extracted with CH 2 Cl 2 (3?100 mL), the organic phase was washed with H 2 O, saturated NaHCO 3 and brine, and dried over anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (4/1, 123 Hexane/EtOAc) to afford the aldehyde 56 (8.82 g, 70 %). 1 H NMR (400 MHz, CDCl 3 ) ? 9.58 (t, J=9.25, 4.50, 1H), 5.10 (s, 1H), 5.03-4.96 (m, 1H), 4.52-4.48 (m, 2H), 3.44 (s, 3H), 1.52 (s, 3H), 1.36 (s, 3H). (3aR,4R,6R,6aR)-4-Methoxy-2,2-dimethyl-6-vinyltetrahydrofuro[3,4- d][1,3]dioxole (57). To a suspension of methyl triphenylphosphonium bromide (6.57 g, 18.4 mmol) in anhydrous diethyl ether (100 mL) at -10-0 ?C was added potassium tert- butoxide (2.10 g, 18.4 mmol) portionwise. The yellow mixture was stirred at 0 ?C for 1 h and kept at room temperature for 1.5 h. The reaction mixture was re-cooled to -10-0 ?C, and a solution of the aldehyde 56 (2.50 g, 12.3 mmol) in Et 2 O (10.0 mL) was added dropwise. The mixture was stirred at room temperature overnight. H 2 O (10.0 mL) was used to quench the reaction. The organic layer was separated and the aqueous layer was extracted with Et 2 O (3?30.0 mL). The combined organic phases were dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (10/1, Hexane/EtOAc) to afford 57 (1.75 g, 75 %). 1 H NMR (400 MHz, CDCl 3 ) ? 5.88 (m, 1H), 5.31-5.13 (m, 2H), 4.99 (s, 1H), 4.63 (m, 3H), 3.34 (s, 3H), 1.50 (s, 3H), 1.32 (s, 3H). 2-((3aR,4R,6R,6aR)-6-Methoxy-2,2-dimethyltetrahydro[3,4-d][1,3]dioxol-4- yl)ethanol (58). 9-Borabycyclo [3.3.1] nonane (9-BBN, 119 mL, 59.3 mmol, 0.5 M in THF) was added dropwise to a solution of 57 (7.99 g, 39.6 mmol) in anhydrous THF (50.0 mL) at 0 ?C. The reaction mixture was stirred at room temperature for 12 h. Methanol (15.0 mL), NaOH (6 M, 11.0 mL) and H 2 O 2 (20.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at 0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (100 mL), the organic 124 phase was washed with saturated NaHCO 3 and brine and dried with MgSO 4 . After removal of the solvents, the residue was purified by column chromatography (1/1, Hexane/EtOAc) to afford 58 (8.00g, 92%). Compound 58 was then directly used to the next step without NMR data. 2-((3aR,4R,6R,6aR)-6-Methoxy-2,2-dimethyltetrahydro[3,4-d][1,3]dioxol-4- yl)acetaldehyde (59). Dimethyl sulphoxide (79.5 mL, 840 mmol), diisopropyl ethylamine (36.0 mL, 207 mmol) and SO 3 ?Py complex (15.8 g, 99.4 mmol) were added to a solution of 58 (18.2 g, 82.8 mmol) in anhydrous CH 2 Cl 2 (150 mL) at 0 ?C. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 6 h at room temperature. After the reaction completed, the solution was diluted with CH 2 Cl 2 (150 mL) and H 2 O (15.0 mL). Extracted with CH 2 Cl 2 (3?150 mL), the organic phase was washed with H 2 O, saturated NaHCO 3 and brine, and dried over anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (5/1, Hexane/EtOAc) to afford the aldehyde 59 (15.7 g, 87%). 1 H NMR (400 MHz, CDCl 3 ) ? 9.81 (t, 1H), 4.98 (s, 1H), 4.75-4.71 (m, 1H), 4.65-4.58 (m, 2H), 3.33 (s, 3H), 2.86-2.79 (qd, J=2.30, 1H), 2.72-2.66 (qd, J=1.20, 1H), 1.50 (s, 3H), 1.33 (s, 3H). (S)-1-((3aR,4R,6R,6aR)-6-Methoxy-2,2-dimethyltetrahydro[3,4-d][1,3]dioxol-4- yl)pent-4-en-2-ol (60). Allylmagnesium bromide (37.3 mL, 37.3 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B-methoxydiisocampheylboron (11.8 g, 37.3 mmol) in anhydrous diethyl ether (50.0 mL) at -10-0 ?C under a nitrogen atmosphere. The reaction was stirred at room temperature for 2 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 59 (5.37 g, 125 24.8 mmol) dissolved in anhydrous THF (25.0 mL) was added dropwise over 50 min to the above reaction mixture by a cannula. The newly reaction mixture was kept at -78 ?C for 6 h and allowed to warm to room temperature for another 4 h. Finally, methanol (10.0 mL), NaOH (6 M, 12.0 mL) and H 2 O 2 (22.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at -10-0 ?C, respectively. The reaction was stirred for 4 h at room temperature. The reaction mixture was diluted with EtOAc (150 mL) and extracted with EtOAc (3?150 mL). The combined organic phases were washed with H 2 O and brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 60 (6.09 g, 95 %) as a colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 5.91-5.75 (m, 1H), 5.18-5.09 (m, 2H), 4.98 (s, 1'-H, 1H), 4.61-4.56 (m, 2H), 4.41 (t, J=7.75, 1H), 3.89 (br s, 6'-H, 1H), 3.36 (s, 3H), 2.66 (d, J=2.50, 1H), 2.33-2.22 (m, 2H), 1.76-1.70 (m, 2H), 1.49 (s, 3H), 1.32 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 134.5, 117.5, 112.2, 109.7, 85.6, 85.3, 84.1, 68.9, 54.9, 41.6, 41.1, 26.4, 24.9. (3aR,4R,6R,6aR)-4-((S)-2-(Benzyloxy)pent-4-enyl)-6-methoxy-2,2- dimethyltetrahydro[3,4-d][1,3]dioxole (61). To a solution of 60 (2.09 g, 8.10 mmol) in anhydrous N, N-dimethylformamide (DMF, 20.0 mL) at -10-0 ?C, was added sodium hydride (363 mg, 9.72 mmol). The reaction mixture was stirred at -10-0 ?C for 20 min before benzyl chloride (1.15 mL, 9.72 mmol) was added by a syringe. The reaction mixture was kept at 0 ?C for 2 h and continued stirring for 6 h at room temperature. After removal of DMF by high pressure vacuum, the residue was quenched by H 2 O (3.0 mL) and extracted with EtOAc (3?50.0 mL). The combined organic layers were washed with brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. Purification 126 of the residue on silica gel column chromatography (4/1, Hexane/EtOAc) afforded 61 (1.97 g, 70 %): 1 H NMR (250 MHz, CDCl 3 ) ? 7.36-7.30 (m, 5H), 5.80 (m, 1H), 5.14 (m, 2H), 4.94 (s, 1H), 4.56-4.63 (m, Bn, CH 2 , 4'-H, 2'-H, 4H), 4.44-4.51 (m, 3'-H, 1H), 3.65- 3.76 (m, 6'-H, 1H), 3.32 ( s, 3H), 2.25-2.45 (m, 7'-H, 2H), 1.66-1.82 (m, 5'-H, 2H), 1.49 (s,3H), 1.30 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 138.2, 134.3, 128.3 (2C), 127.8, 127.5 (2C), 117.5, 112.1, 109.9, 85.6, 84.6, 84.3, 76.2, 71.8, 55.1, 40.1, 38.8, 26.5, 25.0. (S)-3-(Benzyloxy)-4-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)butan-1-ol (62). Sodium periodate (2.72 g, 12.7 mmol) was added to a solution of 60 (2.21 g, 6.34 mmol) in MeOH and H 2 O (80 mL, v/v=3/1) at 0 ?C. Catalytic amount of osmium tetroxide (100 mg) was slowly added to the above reaction mixture. The reaction mixture was stirred at 0 ?C for 1 h and kept for 2 h at room temperature. The mixture was filtrated with a little pad of celite, diluted with CH 2 Cl 2 and the aqueous layer was extracted with CH 2 Cl 2 . The combined organic phases were dried over anhydrous MgSO 4 and then evaporated under reduced pressure to give a yellow syrup, which was used to the next reaction without further purification. To a stirred solution of the yellow synrup in anhydrous MeOH (40.0 mL) at -10- 0 ?C was added sodium borohydride (254 mg, 6.34 mmol) and the reaction mixture was stirred at 0 ?C for 1 h and kept for 2 h at room temperature. Saturated NH 4 Cl solution (5.0 mL) was used to quench the reaction mixture. The residue was diluted with EtOAc (2?60.0 mL), dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to furnish 62 (0.940 g, 44 %): 1 H NMR (250 MHz, CDCl 3 ) ? 7.38-7.29 (m, 5H), 4.95 (s, 1H), 4.580-4.610 (m, 4H), 4.41-4.47 (m, 1H), 3.88-3.3.86 (m, 6'-H, 1H), 3.78-3.67 (m, 8'- 127 H, 2H), 3.30 (s, 3H), 2.80-2.50 (br, OH, 1H), 1.88-1.71 (m, 7'-H, 5'-H, 4H), 1.49 (s, 3H), 1.31 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 138.2, 128.4 (2C), 127.9 (2C),127.8, 112.2, 109.9, 85.5, 84.6, 84.2, 75.3, 72.2, 59.8, 55.1, 40.3, 36.6, 26.5, 25.0. (3aR,4R,6R,6aR)-4-((R)-2-(Benzyloxy)-4-iodobutyl)-6-methoxy-2,2- dimethyltetrahydrofuro[3,4-d][1, 3]dioxole (63). A solution of 62 (940 mg, 2.67 mmol) in toluene (20.0 mL) and acetonitrile (10.0 mL) was treated with imidazole (273 mg, 4.0 mmol) and triphenylphosphane (841 mg, 3.20 mmol). Then, iodine was introduced in approximately 10.0 mg portions to the above reaction mixture until the reaction mixture remained dark-brown in color. The dark-brown solution was heated to reflux for 10 min and cooled to room temperature. Diethyl ether (20.0 mL) was added to dilute the solution and then the solution was washed with 10% sodium thiosulfate solution and brine. The combined organic phases were dried over anhydrous MgSO 4 and concentrated in vacuo to obtain a residue, which was purified through a short plug of silica gel column chromatography (6/1, Hexanes/EtOAc) to provide 63 (0.940 g, 77 %): 1 H NMR (250 MHz, CDCl 3 ) ? 7.37-7.29 (m, 5H), 4.98 (s, 1H), 4.65-4.56 (m, 4H), 4.49-4.42 (m, 1H), 3.82-3.72 (m, 6'-H, 1H), 3.39 (s, 3H), 3.31-3.18 (m, 8'-H, 2H), 2.13-2.02 (m, 7'-H, 2H), 1.79-1.70 (m, 5'-H, 2H),1.49 (s, 3H), 1.31 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 138.4, 128.5 (2C), 127.9 (2C), 127.8, 112.3, 110.1, 85.6, 84.6, 84.0, 76.5, 72.8, 55.5, 40.3, 39.5, 26.5, 25.0, 1.9 (-CH 2 I). (2R,5R)-2-((S)-3-(Benzyloxy)-4-((3aR,4R,6R,6aR)-6-methoxy-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl-5-isopropyl-3,6-dimethoxy-2,5- dihydropyrazine (64). To a solution of (2R)-2,5-dihydro-3,6-dimethoxy-2- isopropylpyrazine 35 (0.380 mL, 2.08 mmol) in anhydrous THF (5.0 mL) under a 128 nitrogen atmosphere was added n-BuLi (0.830 mL, 2.08 mmol) dropwise at -90-78 ?C. This solution was stirred at -78 ?C for 10 min. Then, this solution was transferred via a cannula over 10 min to a suspension of CuCN (93.6 mg, 1.04 mmol) in THF (10.0 mL) at -10-0 0 C. The reaction mixture was stirred at -10-0 ?C for 5 min then it was cooled to -90- 78 ?C. A solution of 63 (0.480 g, 1.04 mmol) in anhydrous THF (10.0 mL) was added dropwise via a cannula over 20 min to the above reaction mixture. The reaction mixture was stirred at -90-78 ?C for 5 h and kept at -20 ?C for 12 h. The solution was quenched with the addition of saturated NH 4 Cl (5.0 mL) and ammonium hydroxide (3.0 mL). From TLC analysis, it formed a complex, including a very strong UV activity point. Finally, THF was removed at reduced pressure, and the residue was separated to recover the reactant 63. The desired product 64 was not obtained, due to the possible decomposition of the pyrazine unit. ((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)-methanol (65). To a solution of D-ribose (5.00 g, 33.3 mmol) in anhydrous acetone (100 mL), p-methoxybenzyl alcohol (6.20 mL, 50.0 mmol) and catalytic amount of sulfuric acid (0.5 mL) were added. The reaction mixture was stirred at room temperature overnight. After neutralized with ammonium hydroxide, H 2 O (10.0 mL) and EtOAc (100 mL) were added to the solution. The aqueous layer was extracted with EtOAc, and the combined organic phases were concentrated under reduced pressure to afford the crude product 65 and the excess unreacted p-methoxybenzyl alcohol, which were very difficult to be separated by the general silica gel column chromatography (the same polarity from TLC analysis after several developments in Hexane/EtOAc, 2/1). 129 (3aR,6R,6aR)-2,2-Dimethyl-6-vinyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (66). To a solution of 57 (7.68 g, 38.4 mmol) in 1,4-dioxane (60.0 mL), 4 M hydrochloric acid (30.0 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 72 h. 6 M NaOH was used to neutralize the excess acid until the solution was pH = 7.0. After removal of the solvents in vacuo, the residue (triols) was obtained, diluted with MeOH, and dried with anhydrous MgSO 4 . Evaporating the solvent under reduced pressure, the residue was used in the next step without purification. Then, the residue was dissolved in anhydrous acetone (30.0 mL) and catalytic amount of sulfuric acid (1.0 mL) was added to the above solution. The reaction mixture was stirred at room temperature for 2 h. After removal of the solvent under reduced pressure, the residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to furnish 66 (3.16 g, 60 %): 1 H NMR (250 MHz, CDCl 3 ) ? 6.05 (m, 1H), 5.48 (d, J=3.0, 1H), 5.32-5.13 (m, 2H), 4.69-4.59 (m, 3H), 4.21 (d, J=3.0, 1H), 1.50 (s, 3H), 1.32 (s, 3H). (3aR,4R,6R,6aR)-4-(4-Methoxybenzyloxy)-2,2-dimethyl-6- vinyltetrahydrofuro[3,4-d][1,3]dioxole (67 & 68). Sodium hydride (489 mg, 20.4 mmol, 60 % in material oil) was added in portions to a solution of 66 (3.16 g, 17.0 mmol) in anhydrous N, N-dimethylformamide (DMF, 30.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature for 30 min and then p-methoxybenzyl chloride (2.80 mL, 20.4 mmol) was added dropwise to the above reaction mixture. The reaction was kept at 0 ?C for 2 h and at room temperature for 4 h. After removal of DMF by high pressure vacuum, the residue was quenched by H 2 O (3.0 mL) and extracted with EtOAc (3?50.0 mL). The combined organic layera were washed with H 2 O and brine, and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the 130 residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to privide 67 (3.16 g, 60 %) as a shallow yellow oil, a major product and a by-product 68 (0.620 g, 15 %). For 67: 1 H NMR (250 MHz, CDCl 3 ) ? 7.26 (dd, J=6.75, 2.25, 2H), 6.89 (dd, J=6.75, 2.25, 2H), 5.96 (m, 1H), 5.33-5.19 (m, 2H), 5.15 (s, 1H), 4.70-4.56 (m, 4H), 4.42-4.34 (m, 1H), 3.79 (s, 3H), 1.49 (s, 3H), 1.31 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) 159.4, 132.6, 130.0, 129.6 (2C), 119.4, 114.3, 114.0 (2C), 105.2, 85.6, 81.5, 71.6, 68.8, 55.5, 26.2, 25.0. For 68: 1 H NMR (250 MHz, CDCl 3 ) 7.34 (d, J=6.75, 2H), 6.91 (d, J=6.75, 2H), 5.84 (m, 1H), 5.80-5.20 (m, 2H), 5.06 (d, J=4.25, 1H), 4.84-4.45 (m, PMB, CH 2 ; 2'-H; 3'-H; 4'-H; 5H), 3.80 (OCH 3 , 3H), 1.56 (s, 3H), 1.35 (s, 3H). 2-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)ethanol (69). 9-Borabycyclo [3.3.1] nonane (9-BBN, 29.5 mL, 14.8 mmol, 0.5 M in THF) was added dropwise to a solution of 67 (3.16 g, 9.83 mmol) in anhydrous THF (30.0 mL) at -10-0 ?C. The reaction mixture was stirred at room temperature for 12 h. Methanol (13.0 mL), NaOH (6 M, 8.0 mL) and H 2 O 2 (15.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at -10-0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (50.0 mL), the organic phase was washed with saturated NaHCO 3 and brine, and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to afford 69 (2.51 g, 75 %): 1 H NMR (400 MHz, CDCl 3 ) ? 7.25 (dd, J=8.80, 2.80, 2H), 6.89 (dd, J=8.80, 2.80, 2H), 5.14 (s, 1H), 4.69-4.61(m, 3H), 4.41-4.38 (m, 2H), 3.82-3.79 (m, 2H), 3.78 (s, 3H), 2.51 (br s, 1H), 1.97-1.85 (m, 1H), 1.83-1.77 (m, 1H), 1.47 (s, 3H), 1.30 (s, 131 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.5, 129.8 (2C), 129.5, 114.0 (2C), 112.4, 107.5, 85.7, 85.1, 84.5, 69.2, 60.1, 55.4, 37.6, 26.6, 25.3. 2-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)acetaldehyde (70). Dimethyl sulphoxide (80.4 mL, 1.13 mol), diisopropyl ethylamine (28.2 mL, 162 mmol) and SO 3 ?Py complex (7.72 g, 48.5 mmol) were added to a solution of 69 (10.5 g, 32.3 mmol) in CH 2 Cl 2 (80.0 mL) at -10-0 ?C. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. The solution was diluted with CH 2 Cl 2 (100 mL) and H 2 O (10.0 mL) and extracted with CH 2 Cl 2 (3?100 mL). The combined organic phases were washed with saturated NaHCO 3 , H 2 O and brine, dried over anhydrous MgSO 4 and evaporated at reduced pressure. The residue was purified on silica gel column chromatography (5/1, Hexane/EtOAc) to afford the aldehyde 70 (9.07 g, 87 %): 1 H NMR (250 MHz, CDCl 3 ) ? 9.80 (dd, J=2.50, 1.25, 1H), 7.27 (dd, J=8.80, 2.80, 2H), 6.89 (dd, J=8.80, 2.80, 2H), 5.14 (s, 1H), 4.79-4.68 (m, PMB, CH 2 , 2H), 4.62 (d, J=2.50, 2'-H, 1H), 4.58 (d, J=2.50, 3'-H, 1H), 4.38 (d, J=11.25, 4'-H, 1H), 3.79 (s, 3H), 2.93-2.83 (qd, J=2.50, 5'-H, 1H), 2.75-2.65 (qd, J=1.25, 5'-H, 1H), 1.49 (s, 3H), 1.30 (s, 3H). (3aR,6R,6aR)-6-(Hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- ol (71). A catalytic amount sulfuric acid (1.0 mL) and celite (5.0 g) were slowly added to a suspension of D-ribose (22.3 g, 149 mmol) in anhydrous acetone (200 mL). The reaction mixture was stirred at the same temperature for 2 h. Solid NaHCO 3 was used to neutralize the reaction mixture to pH = 7.0. The solution was filtrated and the residue was washed with EtOAc. The filtrate was dried over anhydrous MgSO 4 and evaporated under reduced pressure to give a viscous liquid, which was purified on silica gel column 132 chromatography (3/1, Hexane/EtOAc) to afford 71 (26.8 g, 95 %): 1 H NMR (250 MHz, CDCl 3 ) ? 5.44 (d, J=6.25, 1H), 4.86 (d, J=5.75, 1H), 4.61 (d, J=6.00, 1H), 4.46-4.41 (m, 2H), 3.76-3.72 (m, 2H), 3.38 (br s, 1H), 1.49 (s, 3H), 1.33 (s, 3H). ((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)methanol (72 & 73). Sodium hydride (6.43 g, 161 mmol, 60 % in material oil) was added in portions to a solution of 71 (30.6 g, 161 mmol) in anhydrous N, N-dimethylformamide (DMF, 90.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature for 50 min and then p-methoxybenzyl chloride (21.8 mL, 161 mmol) was added dropwise by a syringe over 40 min to the above solution. The reaction mixture was kept at -10-0 ?C for 2 h and at room temperature for 12 h. After removal of DMF under high pressure vacuum, the residue was quenched by H 2 O (12.0 mL) and extracted with EtOAc (3?150 mL). The combined organic layers were washed with H 2 O and brine, dried with anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to privide a major product 72 (37.4 g, 75 %) as and a possible by-product 73 (< 10%). For 72: 1 H NMR (250 MHz, CDCl 3 ) ? 7.24 (d, J=8.40, 2H), 6,87 (d, J=8.40, 2H), 5.15 (s, 1H), 4.84 (d, J=6.0, 1H), 4.71-4.58 (m, 3H), 4.46 (d, J=15.2, 1H), 4.41 (t, J=4.0, 1H), 3.79 (s, 3H), 3.72-3.64 (m, 2H), 1.47(s, 3H), 1.30 (s, 3H). (S)-1-((3aR,4R,6R,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)pent-4-en-2-ol (75). Allylmagnesium bromide (40.2 mL, 40.2 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B- methoxydiisocampheylboron (12.7 g, 40.2 mmol) in anhydrous diethyl ether (50.0 mL) at -10-0 ?C under a nitrogen atmosphere. The reaction was stirred at room temperature for 2 133 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 70 (8.64 g, 26.8 mmol) dissolved in anhydrous THF (25.0 mL) was added dropwise over 50 min to the above reaction mixture by a cannula. The newly reaction mixture was kept at -78 ?C for 6 h and allowed to warm to room temperature for another 4 h. Finally, methanol (10.0 mL), NaOH (6 M, 10.0 mL) and H 2 O 2 (25.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at -10-0 ?C, respectively. The reaction was stirred for 4 h at room temperature. The reaction mixture was diluted with EtOAc (150 mL) and extracted with EtOAc (3?150 mL). The combined organic phases were washed with H 2 O and brine, dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 75 (9.28 g, 95 %) as a white solid: 1 H NMR (400 MHz, CDCl 3 ) ? 7.24 (dd. J=8.50, 2.00, 2H), 6.89 (dd, J=8.75, 2.00, 2H), 5.74-5.85 (m, 8'-H, 1H), 5.13- 5.15 (m, CH2=CH, CH 2 , 2H), 5.10 (s, 1'-H, 1H), 4.66-4.69 (m, PMB, CH 2 , 2H), 4.64 (d, J=11.25, 2'-H, 1H), 4.48-4.51 (dd, J=4.80, 4'-H, 1H), 4.43 (d, J=11.25, 1H), 3.91 (br s, 6'- H, 1H), 3.80 (s, 3H), 2.15-2.28 (m, 7'-H, 2H), 2.09 (br s, -OH, 1H), 1.79-1.86 (qd, J=2.80, 5'-H, trans with 6'-H, 1H), 1.59-1.67 (qd, J=4.8, 5'-H, cis with 6'-H, 1H), 1.48(s, 3H), 1.31(s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.5, 134.6, 129.7 (2C), 129.5, 118.5 (CH 2 =CH), 114.0 (2C), 112.4 (isopropyl C), 107.8 (C-1'), 85.8 (5'-H), 84.8 (C-2'), 84.7 (PMB, CH 2 ), 69.3 (C-4'), 68.1 (C-6'), 55.4 (OCH 3 ), 42.7 (C-7'), 41.8 (C-5'), 26.7, 25.2. HRMS: calcd for C 20 H 28 O 6 [(M - CH 3 ) + ] 349.1651; found 349.1653. Tert-butyl((S)-1-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pent-4-en-2-yloxy)dimethylsilane (76). To a solution of 75 (1.08 g, 2.96 mmol) in anhydrous CH 2 Cl 2 (25.0 mL) were added 134 imidazole (504 mg, 7.40 mmol) and a catalytic amount of DMAP (2 % mmol, 7.23 mg) at -10-0 ?C under a nitrogen atmosphere. After the mixture was stirred for 30 min at room temperature and then re-cooled -10 ?C, tert-butyldimethylsilyl chloride (670 mg, 4.45 mmol) was slowly added. The whole reaction mixture was then stirred at the same temperature for 1 h, warmed to room temperature and stirred for 24 h. After the reaction mixture was diluted with ice-water, the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (3?50.0 mL). The combined organic phases were washed with H 2 O and brine, dried with anhydrous MgSO 4 , and then evaporated in vacuo to afford the crude product, which was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to furnish 76 (1.36 g, 96 %) as a colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 7.09 (d, J=8.00, 2H), 6.72 (d, J=8.00, 2H), 5.58 (m, 1H), 4.96 (s, 1H), 4.90 (irregular t, J=8.75, 9.25, 2H), 4.52 (m, 3H), 4.26 (d, J=11.50, 2H), 3.80 (br s, 1H), 3.61 (s, 3H), 2.08 (m, 2H), 1.57-1.66 (m, 1H), 1.39-1.47 (m, 1H), 1.32 (s, 3H), 1.14 (s, 3H), 0.78 (s, 9H), 0.00 (s, 3H), -0.02 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.4, 134.5, 129.5 (2C), 127.6, 117.4, 113.9, 112.1, 107.6, 85.9, 84.9, 84.3, 69.12, 69.0, 55.2, 43.1, 42.1, 26.6, 26.0 (3C), 25.8, 25.2, 18.2, -4.2, -4.6. Anal. Calcd for C 26 H 42 O 6 Si: C, 65.24; H, 8.84; Found: C, 65.38; H, 8.91. (S)-3-(Tert-butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)- 2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butan-1-ol (77)-the First Method. Sodium periodate (4.65 g, 21.7 mmol) was slowly added to a solution of 76 (5.20 g, 10.9 mmol) in a mixture of MeOH /H 2 O (120 mL, v/v=3/1) at -10-0 ?C. Catalytic amount of osmium tetraoxide (200 mg) was slowly added to the above reaction mixture. The reaction mixture was stirred at 0 ?C for 1 h and kept for 2 h at room temperature. The 135 mixture was filtrated with a little pad of celite, diluted with CH 2 Cl 2 and the aqueous layer was extracted with CH 2 Cl 2 . The combined organic phases were dried over anhydrous MgSO 4 and then evaporated under reduced pressure to give a yellow syrup, which was used to the next reaction without further purification. To a stirred solution of the yellow synrup in anhydrous MeOH (50.0 mL) at -10- 0 ?C was added sodium borohydride (495 mg, 13.1 mmol). The reaction mixture was stirred at -10-0 ?C for 1 h and kept for 2 h at room temperature. Saturated NH 4 Cl solution (5.0 mL) was used to quench the reaction mixture. The residue was diluted with EtOAc (2?60.0 mL), dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to furnish 77 (2.36 g, 45 %) as a colorless oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.12 (dd, J=8.50, J=2.0, 2H), 6.75 (dd, J=8.75, J=2.0, 2H), 4.98 (s, 1H), 4.54 (d, J=6.0, 1H), 4.44- 4.48 (m, 2H), 4.29 (d, J=11.6, 1H), 4.20 (t, J=11.6, 1H), 3.99 (m, 1H), 3.65 (s, 3H), 3.51- 3.66 (m, 2H), 2.13 (br s, 1H), 1.68-1.77 (m, 1H), 1.61-1.64 (m, 2H), 1.43-1.50 (m, 1H), 1.33 (s, 3H), 1.16 (s, 3H), 0.77 (s, 9H), -0.02 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.5, 129.6 (2C), 129.6, 114.1 (2C), 112.4, 107.7, 85.9, 84.9, 84.1, 69.2, 68.5, 59.8, 55.5, 42.1, 39.3, 26.7, 26.1 (3C), 25.3, 18.2, -4.3, -4.5. Anal. Calcd for C 25 H 42 O 7 Si: C, 62.21; H, 8.77. Found: C, 61.99; H, 8.84. (S)-3-(Tert-butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)- 2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butan-1-ol (77)- the Optimized Method. To a solution of 76 (10.3 g, 21.4 mmol) in THF (100 mL) was added a catalytic amount of osmium tetroxide (200 mg) and 50 % aqueous N-methylmorpholine N-oxide (NMO, 30.0 mL) at -10-0 ?C. The reaction mixture was stirred at the same temperature 136 for 2 h and warmed to room temperature and kept stirring for 12 h. After evaporated the solvents under reduced pressure, the residue was diluted and extracted with EtOAc. The organic phase was concentrated again to give a yellow liquid, which was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to furnish the diol 76-1' (8.92 g, 85 %). 76-1' was not necessary to be conducted with NMR and further used to the next reaction. To a stirred solution of 76-1' in a mixture of CH 2 Cl 2 /H 2 O (160 mL, 3/1, v/v) at - 10-0 ?C was slowly added sodium periodate (7.44 g, 34.8 mmol). A catalytic amount of 15-crown-5 (1.0 mL) was added to the above reaction mixture. The reaction mixture was stirred at the same temperature for 2 h and warmed to room temperature and kept stirring for 12 h. The solution was diluted with CH 2 Cl 2 (150 mL), and the aqueous layer was extracted with CH 2 Cl 2 (3?150 mL). The combined organic phases were washed with H 2 O and brine, dried over anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified on silica gel column chromatography (1/1, Hexane/EtOAc) to afford the aldehyde 76-1'' (7.74 g, 93 %) as a colorless oil and the aldehyde 76-1'' was used to the next reaction. The aldehyde 76-1'' (7.74 g, 16.1 mmol) thus obtained was dissolved in anhydrous MeOH (80.0 mL) at -10-0 ?C. To this solution were added sodium borohydride (772 mg, 32.2 mmol) and CeCl 3 ?7H 2 O (6.00 g, 16.1 mmol). The reaction mixture was stirred at -10-0 ?C for 1 h and kept for 2 h at room temperature. Saturated NH 4 Cl solution (5.0 mL) was used to quench the reaction mixture. The residue was diluted with EtOAc (2?60.0 mL), dried with anhydrous MgSO 4 and evaporated under reduced pressure. The residue was purified on silica gel column chromatography (2/1, 137 Hexane/EtOAc) to furnish 77 (6.27 g, 80 %) as a colorless oil in three steps from 76. The NMRs ( 1 H, 13 C) of 77 were the same as that of 77, which was achieved from the first oxidative-cleavage method. Tert-butyl(R)-4-iodo-1-((3aR,4R,6R,6aR)-6-(4-methoxybenzyloxy)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butan-2-yloxy)dimethylsilane (78). A solution of 77 (5.17 mg, 483 mmol) in toluene (100 mL) and acetonitrile (20.0 mL) was treated with imidazole (1.10 g, 16.1 mmol) and triphenylphosphane (4.20 g, 16.1 mmol). Then, iodine was introduced in approximately 10.0 mg portions to the above reaction mixture until the reaction mixture remained dark-brown in color. The dark-brown solution was heated to reflux for 30 min and cooled to room temperature. The reaction mixture was diluted with Et 2 O (50.0 mL) and washed with a 10% sodium thiosulfate solution and brine, dried over anhydrous MgSO 4 and concentrated under reduced pressure. The residue was purified through a short plug of silica gel column chromatography (6/1, Hexanes/EtOAc) to provide 78 (5.92 g, 90 %) as a yellow oil: 1 H NMR (400 MHz, CDCl 3 ) ? 7.25 (dd, J=8.40, 2.0, 2H), 6.89 (d, J=8.80, 2.0, 2H), 5.12 (s, 1H), 4.55-4.67 (m, 2H), 4.62-4.65 (d, J=11.60, 1H), 4.43-4.46 (d, J=11.60, 1H), 4.36 (dd, J=4.80, 1H), 3.91-3.94 (m, 1H), 3.81 (s, 3H), 3.07-3.17 (m, 2H), 1.98-2.05 (m, 2H), 1.64- 1.76 (m, 2H), 1.47 (s, 3H), 1.30 (s, 3H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 159.6, 129,7 (2C), 129.6, 114.1 (2C), 112.5, 107.6, 86.0, 85.0, 84.0, 69.9, 69.4, 55.5, 42.7, 42.4, 26.8, 26.1 (3C), 25.4, 18.3, -4.0. -4.3. (2R,5R)-2-((S)-3-(Tert-butyldimethylsilyloxy)-4-((3aR,4R,6R,6aR)-6-(4- methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)butyl)-5- isopropyl-3,6-dimethoxy-2, 5-dihydropyrazine (79). To a solution of (2R)-2,5-dihydro- 138 3,6-dimethoxy-2-isopropylpyrazine 35 (1.20 mL, 7.20 mmol) in anhydrous THF (20.0 mL) under a nitrogen atmosphere was added n-BuLi (2.90 mL, 7.20 mmol) dropwise at - 90-78 ?C by a syringe. This solution was stirred at -78 ?C for 30 min. Then, to this solution was slowly added a solution of 78 (2.10 g, 3.60 mmol) in anhydrous THF (20.0 mL) via a cannula over 40 min at -90-78 ?C. The reaction mixture was stirred at -90- 78 ?C for 5 h, kept at -20 ?C for 12 h and allowed to slowly warm to room temperature. The solution was then quenched with the addition of saturated NH 4 Cl (5.0 mL) and ammonium hydroxide (3.0 mL). The THF was removed under the reduced pressure, and the residue was diluted with EtOAc. The organic phase was separated and the aqueous layer was extracted with EtOAc (3?100 mL). The combined organic phases were washed with brine and dried over anhydrous MgSO 4 . After removal of solvent under reduced pressure, the residue was purified on silica gel column chromatography (3/1, Hexane/EtOAc) to afford 79 (1.96 g, 85 %) as the yellow oil, the only diastereoisomer, which was confirmed by NMRs and ( 1 H, 1 H, COSY). 1 H NMR (400 MHz, CDCl 3 ) 7.12 (d, J=8.40, 2H), 6.76 (d, J=8.80, 2H), 4.99 (s, 1H), 4.55 (d, J=6.00, 2'-H, 1H), 4.50 (m, PMB, CH2, 2H), 4.29 (d, J=11.20,3-H', 1H), 4.24 (m, 4-H', 1H), 3.89 (m, 6-H', 1H), 3.82 (m, 2H), 3.68 (s, 3H), 3.55 (s, 3H), 3.54 (s, 3H), 2.15 (m, 1H), 1.77-1.71 (m,7-H', 1H),1.65-1.47 (m, 5-H' down, 5-H' up, 8-H', 4H), 1.35 (s, 3H), 1.28 (m, 7-H', 1H), 1.18 (s, 3H), 0.94 (d, J=6.80, 3H), 0.80 (s, 9H), 0.59 (d, J=6.80, 3H), 0.00 (s, 3H), -0.02 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) 163.8, 163.8, 159.5, 129.8 (2C), 129.6, 114.0 (2C), 112.2, 107.6, 85.9, 84.9, 84.5, 69.5, 69.2, 60.9, 55.4, 52.5, 52.5, 42.3, 33.1 (7-C'), 31.9 (5-C'), 29.3, 26.7, 26.1 (3C), 26.1 (8-C'), 25.3, 19.3, 18.3, 16.8, -4.2, -4.5. 139 ((3aR,6R,6aR)-6-((S)-2-(Tert-butyldimethylsilyloxy)-4-((2R,5R)-5-isopropyl-3,6- dimethoxy-2,5-dihydropyrazin-2-yl)butyl)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-ol (80). To an ice-cooled solution of 79 (220 mg, 3.39?10 -4 mmol) in CH 2 Cl 2 (20.0 mL) were added H 2 O (2.0 mL) and 2,3-dichloro-5,6-dicyano-1,4- benzoquinone (115 mg, 0.510 mmol). The reaction mixture was stirred at -10-0 ?C for 2 h and allowed to slowly warm to room temperature. The reaction mixture was diluted with CH 2 Cl 2 , filtered with a pad of celite, and washed with saturated NaHCO 3 , H 2 O, and brine, and dried with anhydrous MgSO 4 . After removal of solvent under reduced pressure, the residue was purified on silica gel column chromatography (6/1-1/1, Hexane/EtOAc). However, no the desired product 80 was obtained from the separated several points (from 1 H NMR data). (3aR,6R,6aR)-6-((R)-2-(Tert-butyldimethylsilyloxy)-4-iodobutyl)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (81). To a solution of 78 (850 mg, 1.43 mmol) in CH 2 Cl 2 (20.0 mL) at -10-0 ?C were added H 2 O (1.0 mL) and 2,3-dichloro-5,6- dicyano-1,4-benzoquinone (391 mg, 1.72 mmol). The reaction mixture was stirred at -10- 0 ?C for 2 h and allowed to slowly warm to room temperature. The reaction mixture was diluted with CH 2 Cl 2 , filtrated with a pad of celite, and washed with saturated NaHCO 3 , H 2 O, and brine, and dried with MgSO 4 . After removal of solvent, the residue was purified by column chromatography (2/1, Hexane/EtOAc) to afford the desired product 81 (0.530 g, 78 %) as a yellow oil: 1 H NMR (400 MHz, CDCl 3 ) ? 5.13 (m, 1H), 4.64-4.61 (d, J=11.60, 2'-H, 1H), 4.47-4.42 (d, J=11.60, 3'-H, 1H), 4.32 (dd, J=4.80, 4'-H, 1H), 3.94- 3.92 (br s, 6'-H, 1H), 3.17-3.00 (m, 2H), 2.08-1.98 (m, 2H), 1.78-1.64 (m, 2H), 1.49 (s, 140 3H), 1.31 (s, 3H), 0.92 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) ? 112.5, 107.6, 85.9, 85.1, 69.8, 69.3, 42.6, 42.3, 26.8, 26.2(3C), 25.4, 18.3, 1.7, -4.0. -4.3. (3aR,4S,6S,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxole-4-carbaldehyde (82). Dimethyl sulphoxide (20.6 mL, 290 mmol), diisopropyl ethylamine (12.6 mL, 72.5 mmol) and SO 3 ?Py complex (3.46 g, 48.5 mmol) were added to a solution of 73 (4.50 g, 14.5 mmol) in CH 2 Cl 2 (50.0 mL) at 0 ?C. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. The solution was diluted with CH 2 Cl 2 (50.0 mL) and H 2 O (8.0 mL) and extracted with CH 2 Cl 2 (3?80.0 mL). The combined organic phases were washed with saturated NaHCO 3 , H 2 O and brine, dried over anhydrous MgSO 4 and evaporated at reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford the aldehyde 82 (3.58 g, 80%). From TLC analysis, 82 was confirmed compared with the reactant 73 was further used to the next step. (3aR,4S,6S,6aR)-4-(4-Methoxybenzyloxy)-2,2-dimethyl-6-vinyltetrahydrofuro[3,4- d][1,3]dioxole (68)-the Second Approach from 73. To a suspension of methyltriphenylphosphonium bromide (6.21 g, 17.4 mmol) in anhydrous diethyl ether (50.0 mL) at -10-0 ?C was added potassium tert-butoxide (1.99 g, 17.4 mmol) portionwise. The yellow mixture was stirred at 0 ?C for 1 h and kept at room temperature for 1.5 h. The reaction mixture was re-cooled to -10-0 ?C, and a solution of the aldehyde 82 (3.58 g, 11.6 mmol) in Et 2 O (15.0 mL) was added dropwise. The mixture was stirred at room temperature overnight. H 2 O (5.0 mL) was used to quench the reaction. The organic layer was separated and the aqueous layer was extracted with Et 2 O (3?30.0 mL). The combined organic phase was dried with anhydrous MgSO 4 and evaporated under 141 reduced pressure. The residue was purified on silica gel column chromatography (5/1, Hexane/EtOAc) to afford 68 (2.49 g, 70 %). The NMR data of 68 was the same with that of the first method obtained. 2-((3aR,4R,6S,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)ethanol (83). 9-Borabycyclo [3.3.1] nonane (9-BBN, 41.0 mL, 20.3 mmol, 0.5 M in THF) was added dropwise to a solution of 68 (4.35 g, 13.5 mmol) in anhydrous THF (30.0 mL) at -10-0 ?C. The reaction mixture was stirred at room temperature for 12 h. Methanol (12.0 mL), NaOH (6 M, 8.0 mL) and H 2 O 2 (12.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at -10-0 ?C, respectively. The reaction was stirred for 4 h at room temperature. After diluted the reaction with EtOAc (50.0 mL), the organic phase was washed with saturated NaHCO 3 and brine and dried with anhydrous MgSO 4 . After removal of the solvents under reduced pressure, the residue was purified on silica gel column chromatography (2/1, Hexane/EtOAc) to afford 83 (3.45 g, 75%). From TLC analysis, 83 was confirmed and used to the next step. 2-((3aR,4R,6S,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)acetaldehyde (84). Dimethyl sulphoxide (10.3 mL, 145 mmol), diisopropyl ethylamine (6.30 mL, 36.3mmol) and SO 3 ?Py complex (776 mg, 10.9 mmol) were added to the solution of 83 (2.46 g, 7.25 mmol) in CH 2 Cl 2 (30.0 mL) at -10-0 ?C. The reaction mixture was kept at the same temperature for 2 h and continued stirring for 4 h at room temperature. The solution was diluted with CH 2 Cl 2 (30.0 mL) and H 2 O (8.0 mL) and extracted with CH 2 Cl 2 (3?80.0 mL). The combined organic phases were washed with saturated NaHCO 3 , H 2 O and brine, dried over anhydrous MgSO 4 and evaporated at reduced pressure. The residue was purified on silica gel column chromatography (4/1, 142 Hexane/EtOAc) to afford the aldehyde 84 (2.08 g, 85 %): 1 H NMR (400 MHz, CDCl 3 ) 9.74 (t, J=2.80, 1H), 7.31 (dd, J=8.40, 1.60, 2H), 6.91 (dd, J=8.40, 1.60, 2H), 5.03 (d, J=4.40, 1H), 4.77-4.45 (m, PMB, CH 2 ; 2'-H; 3'-H; 4'-H; 5H), 3.82 (s, 3H), 2.69-2.36 (m, 5'-H, 2H), 1,56 (s, 3H), 1.37 (s, 3H). (S)-1-((3aR,4R,6S,6aR)-6-(4-Methoxybenzyloxy)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)pent-4-en-2-ol (85). Allylmagnesium bromide (5.0 mL, 4.65 mmol, 1.0 M in diethyl ether) was added dropwise to a solution of (-)-B- methoxydiisocampheylboron (1.47 g, 4.65 mmol) in anhydrous diethyl ether (30.0 mL) at -10-0 ?C under a nitrogen atmosphere. The reaction was stirred at room temperature for 2 h to give a white suspension. Then, the reaction flask was cooled to -90-78 ?C and the aldehyde 84 (1.44 g, 3.10 mmol) dissolved in anhydrous THF (15.0 mL) was added dropwise over 30 min to the above reaction mixture by a cannula. The newly reaction mixture was kept at -78 ?C for 6 h and allowed to warm to room temperature for another 4 h. Finally, methanol (8.0 mL), NaOH (6 M, 8.0 mL) and H 2 O 2 (15.0 mL, 32 wt % in H 2 O) were added dropwise to the above solution at -10-0 ?C, respectively. The reaction was stirred for 4 h at room temperature. The reaction mixture was diluted with EtOAc (100 mL) and extracted with EtOAc (3?100 mL). The combined organic phases were washed with H 2 O and brine, dried with anhydrous MgSO 4 and evaporated at reduced pressure. The residue was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to afford 85 (1.47 g, 90 %): 1 H NMR (250 MHz, CDCl 3 ) ? 7.33 (dd, J=8.75, 2.50, 2H), 6.91 (dd, J=8.75, 2.50, 2H), 5.91-5.76 (m, 1H), 5.74-5.5.72 (d, J=3.75, 1'-H, 1H), 5.16-5.01 (m, 2H), 4.77-4.43 (m, PMB, CH 2 ; 2'-H; OH; 4H), 4.32-4.12 (m, 3'- H; 4'-H; 2H), 3.81 (s, OCH 3 ), 3.52-3.47 (m, 6'-H, 1H), 2.28-2.20 (m, 7'-H, 2H), 1.80-1.71 143 (m, 5'-H, 2H), 1.60 (s, 3H), 1.36 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.5, 134.6, 129.6 (2C), 129.5, 118.4, 114.0 (2C), 112.4, 107.7, 85.8, 84.8, 84.6, 69.3, 68.1, 55.4, 42.6, 41.7, 26.6, 25.1. Tert-butyl((S)-1-((3aR,4R,6S,6aR)-6-(4-methoxybenzyloxy)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pent-4-en-2-yloxy)dimethylsilane (86). To a solution of 85 (1.50 g, 4.10 mmol) in anhydrous CH 2 Cl 2 (20.0 mL) were added imidazole (697 mg, 10.3 mmol) and a catalytic amount of DMAP (5 % mmol, 25 mg) at - 10-0 ?C under a nitrogen atmosphere. After the mixture was stirred for 30 min at the room temperature and then re-cooled -10 ?C, tert-butyldimethylsilyl chloride (1.60 mg, 10.3 mmol) was slowly added. The whole reaction mixture was then stirred at the same temperature for 1 h, warmed to room temperature and stirred for 24 h. After the reaction mixture was diluted with ice-water, the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (3?50.0 mL). The combined organic phases was washed with water and brine, dried with anhydrous MgSO 4 , and then evaporated in vacuo to afford the crude product, which was purified on silica gel column chromatography (4/1, Hexane/EtOAc) to furnish 86 (1.81 g, 92 %) as a colorless oil: 1 H NMR (250 MHz, CDCl 3 ) ? 7.15 (dd, J=11.50, 2.50, 2H), 6.79 (dd, J=11.50, 2.50, 2H), 5.73-5.58 (m, 1H), 4.99-4.87 (m, 9'-H; 1'-H; 3H), 4.56-4.47 (m. PMB, CH2; 2'-H; 3H), 4.32-4.23 (m, 3'-H; 4'-H; 2H), 3.88-3.77 (br s, 6'-H, 1H), 3.69 (s, OCH 3 , 3H), 2.21-2.09 (m, 7'-H, 2H), 1.80- 1.47 (m, 5'-H, 2H), 1.36 (s, 3H), 1.19 (s, 3H), 0.80 (s, C(CH 3 ) 3 , 9H), 0.02 (s, 3H), -0.01 (s, 3H); 13 C NMR (62.9 MHz, CDCl 3 ) ? 159.5, 134.7, 129.7 (2C), 129.7, 117.5, 114.1 (2C), 112.3, 107.7, 86.0, 85.0, 84.4, 69.2, 55.5, 43.1, 42.2, 29.9, 26.7, 26.1 (3C), 25.3, 18.3, - 4.1, -4.5. 144 REFERENCES 1. (a) Borchardt, R. T. J. Med. Chem. 1980, 23, 347. (b) Biological Methylation and Drug Design; Borchardt, R. T.; Creveling, C. R.; Ueland, P. M.; Eds.; Humana Press: Clifton, NJ 1986. (c) Tabor, C. W.; Tabor, W. Annu. Rev. Biochem. 1976, 45, 286. (d) Russell, D. H.; Durie, B. G. M. Progr. Cancer Res. Ther. 1978, 8, 1. (e) Miura, G. A.; Chiang, P. K. Anal. Biochem. 1985, 147, 217. (f) Smith, Jr., D. D.; Norton, S. J. Biochem. Biophys. Res. Commun. 1980, 94, 1458. (g) Icekson, I.; Apelbaum. A. Biochem. Biophys. Res. Commun. 1983, 113, 586. 2. 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