A Convergent Synthetic Route to the Tunicamycin Antiobiotics. Synthesis of (+)-Tunicamycin V Citation Gin, David Y. (1994) A Convergent Synthetic Route to the Tunicamycin Antiobiotics. Synthesis of (+)-Tunicamycin V. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/p6qx-ng34. https://resolver.caltech.edu/CaltechTHESIS:05072013-095813413 Abstract A concise synthetic route to the tunicamycin antibiotics is described, illustrated by the preparation of (+)-tunicamycin-V (1-V). Key features of the synthesis include: (1) the development and application of a silicon-mediated reductive coupling of aldehydes and allylic alcohols to construct the undecose core of the natural product; and (2) the development of an efficient procedure for the synthesis of the trehalose glycosidic bond within the antibiotic. These innovations allow for the coupling of a uridine-derived aldehyde fragment with a preformed trehalose-linked disaccharide allylic alcohol to form the carbohydrate core (1) of the natural product in a highly convergent manner. The resultant amino polyol is a versatile intermediate for the synthesis of any of the homologous tunicamycin antibiotics. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: Chemistry Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Myers, Andrew G. Thesis Committee: Unknown, Unknown Defense Date: 10 December 1993 Record Number: CaltechTHESIS:05072013-095813413 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:05072013-095813413 DOI: 10.7907/p6qx-ng34 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 7667 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 07 May 2013 17:35 Last Modified: 09 Nov 2022 19:19 Thesis Files Preview PDF - Final Version See Usage Policy. 43MB Repository Staff Only: item control page

A Convergent Synthetic Route to the Tunicamycin Antibiotics. Synthesis of (+)-Tunicamycin V. Thesis by David Y_ Gin In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 1994 (Submitted December 10, 1993) 11 © 1994 David Y. Gin All Rights Reserved lll To my mother and father, for their undying love and support. Acknowledgments I would like to thank my research advisor, Professor Andy Myers, for his guidance throughout my graduate studies. During this time, I have found myself in a highly motivating environment in which he has offered me continuous support and encouragement. It is an honor to have been associated with him and his research group. I would also like to express my gratitude to Dr. Dan Rogers, who was directly involved with the work presented in this thesis. This project certainly would not have been completed as quickly without his many contributions. My thanks goes out to all of the past and present members of the Myers' research group (Katherine, Kevin, Elaine, Pete, Nat, Mark, Scott, Susan, Steve, Bryant, Tommy, Erik, Cindy, Marlys, Norma, Phil, Paivi, Byoung, Jia Ning, Dan, and Brad) with whom I have had the pleasure of being associated. Without exception, you all have been extremely helpful and have provided a pleasant atmosphere in which to work. In particular, I consider myself extremely lucky to have worked with Mark Fraley, my lab-mate for the past four years. He is an outstanding chemist and, more importantly, an exceptional friend. My thanks also goes to Professor Erick Carreira for the many helpful suggestions and discussions. I am indebted to Professor Thomas Money, who first sparked my interest in synthetic organic chemistry. His support throughout the years has been invaluable. Finally, I am grateful to my family and to Mary for their encouragement and unwavering confidence in my abilities to see my research project to the end. A four-year pre-doctoral fellowship from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. v Abstract A concise synthetic route to the tunicamycin antibiotics is described, illustrated by the preparation of (+)-tunicamycin-V (1- V). Key features of the synthesis include: ( 1) the development and application of a silicon-mediated reductive coupling of aldehydes and allylic alcohols to construct the undecose core of the natural product; and (2) the development of an efficient procedure for the synthesis of the trehalose glycosidic bond within the antibiotic. These innovations allow for the coupling of a uridine-derived aldehyde fragment with a preformed trehalose-linked disaccharide allylic alcohol to form the carbohydrate core (1) of the natural product in a highly convergent manner. The resultant amino polyol is a versatile intermediate for the synthesis of any of the homologous tunicamycin antibiotics. Vl Table of Contents Acknowledgments .............................................................................................................. .iv Abstract ................................................................................................................................ v Table of Contents.................................................................................................................vi List of Schemes..................................................................................................................viii List of Figures and Tables.................................................................................................... x List of Abbreviations ............................................................................................................ xi Chapter 1: Introduction The Tunicamycin Antibiotics .................................................................................... 2 Synthetic Plan ............................... ............... ............................................................. 6 Chapter 2: Synthesis of Tunicaminyluracil Synthesis of Tunicaminyluracil (2)......................................................................... 13 Chapter 3: Synthesis of Tunicamycin-V Synthesis of the ~. a- Trehalose Linkage .................................................................25 Other Glycosylation Attempts ................................................................................ .35 Carbon-Carbon Bond Formation - Construction of the Trisaccharide Core...................................................................................... 39 Final Stages ............................................................................................................ 50 Preparation of C5'-epi-Tunicamycin-V ................................................................... 54 Vl1 Summary ........................................................................................................................ 56 Experimental Section ....................................................................................................57 References and Notes..................................................................................................... 204 Appendix: Catalog of Spectra .............................. ....................................................... 209 viii List of Schemes Scheme I. Tunicamycin Inhibition of the Lipid Cycle in Peptidoglycan Biosynthesis ......................................................... .4 Scheme II. Tunicamycin Inhibition of Asparagine-Linked Glycoprotein Biosynthesis ............................................................... 5 Scheme ill. Previous Synthetic Studies: Suami et al.. .........................................7 Scheme IV. Previous Synthetic Studies: Danishefsky et al .... ............. ........ 8 Scheme V. Reductive Coupling of Hydrocinnamaldehyde and Allyl Alcohol. ...........................................................................9 Scheme VI. Proposed Synthesis of the U ndecose Core ........................... . 10 Scheme VII. Synthesis of Galactosyl Allylic Alcohol 15 .. ......................... 14 Schemevm. Attempted Reductive Coupling of 15 and 17............................. 16 Scheme IX. Synthesis of Uridine-5'-Aldehyde 22 ......................................... 18 Scheme X. Reductive Coupling of 15 and 22. Formation of the Undecose Core.......................................................................... 19 Scheme XI. Synthesis of Tunicaminyluracil and Structure Correlation .......... 21 Scheme XII. Synthesis of Galactosyl Hemiacetal 38 ................................ 30 Scheme XIII. Synthesis of Glucosyl Trichloroacetimidate 44 ..................... .32 Scheme XIV. Synthesis of I3,a-Trehalose-Linked Allylic Alcohol Disaccharide 62 ..............................................................................40 Scheme XV. Reductive Coupling of Disaccharide 62 and Uridine-5'Aldehyde 64....................................................................................43 Scheme XVI. Synthesis of Uridine-5'-Aldehyde 73 .................... ...... ...... .47 ix SchemeXVll. Reductive Coupling of Adduct 75. Formation of the Trisaccharide Framework of Tunicamycin ................................... .49 SchemeXVIll. Synthesis of Fatty Acid 82 .......... ................... ......................... 51 Scheme XIX. Synthesis of Tunicamycin-V .........................................................53 Scheme XX. Synthesis of C5'-epi-Tunicamycin-V ............................................. 55 X List of Figures and Tables Figure 1. The Tunicamycins, Corynetoxins, Streptovirudins, and Tunicaminyluracil. ..................................................................... .3 Figure 2. Reductive Coupling of 0-Silylhemiselenoacetal Adduct 23. Proposed Transition Structures ..................................22 Figure 3. Glycosylation Coupling Partners: Survey of Periphery Functionality .................................................. 27 Figure 4. Reductive Coupling of 0-Silylhemiselenoacetal Adduct 65. Proposed Transition Structures ................................ .45 Table I. Reductive Coupling of 0-Silylhemiselenoacetal Adduct 23. Solvent and Temperature Effects................ ........ 20 Table II. Screening of 3-D-Protecting Groups for 32 ................ ......... 29 X1 List of Abbreviations [a]o25 optical rotation (589 nm, 25 "C) A angstrom Ac acetyl AIBN 2,2'-azobis(isobutyronitrile) Aoc allyloxy carbonate arom. aromatic Boc tert-butyl carbamate BOM benzyloxy methyl Bu butyl Bz benzoyl c grams per 100 mL of solution oc degrees Celsius CI chemical ionization CAN eerie ammonium nitrate cat catalyst Cbz benzyl carbamate cm-1 reciprocal centimeters CSA camphorsulfonic acid 8 chemical shift DBU 1,8-diazobicylo[5.4.0]undec-7 -ene DCC 1,3-dicyclohexylcarbodiimide DMAP 4-(N,N-dimethylamino)pyridine DME dimethoxyethane X11 DMF N,N-dimethylformamide DMS dimethyl sulfide DMSO dimethyl sulfoxide DMT para-dimethoxytri ty I Dol dolichol EI electron impact equiv equivalent Et ethyl FAB fast atom bombardment FT Fourier transform g gram GDP guanosine diphosphate Glc glucose GlcNAc N-acetylglucosamine h hour Hal- halide HPLC high performance liquid chromatography HRMS high resolution mass spectroscopy Hz Hertz i iso IR infrared J coupling constant L liter LDA lithium diisopropylamide m meta Xlll M molar M+ molecular ion m-CPBA meta-chloroperoxybenzoic acid Man mannose Me methyl MEM methoxyethoxy methyl MeOH methyl alcohol mg milligram MHz megahertz min minute mL milliliter mM millimolar mmol millimole mol mole mol. sieves molecular sieves MOM methoxy methyl mp melting point MS mass spectroscopy MurNAc N-acetylmuramic acid llL microliter n normal N normal (concentration) NBS N-bromosuccinimide nm nanometer NMR nuclear magnetic resonance xiv 0 ortho p para p phosphate p protecting group pep peptide Ph phenyl PMB para-methoxybenzyl ppm parts per million Pr propyl PhthN phthalimide Py pyridine R,Re rectus Rt retention factor S, Si sinister SEM trimethylsilylethoxymethyl t tertiary TBAF tetrabutylammonium fluoride TBS tert-butyldimethylsilyl Tf trifluoromethanesulfony1 THF tetrah ydrofuran 1LC thin layer chromatography TMS trimethylsilyl UDP uridine diphosphate UMP uridine monophosphate vlv volume-to-volume ratio XV w Watt w/w weight-to-weight ratio 1 Chapter 1 Introduction 2 The Tunicamycin Antibiotics The tunicamycins, corynetoxins, and streptovirudins make up a unique class of microbial metabolites that inhibit various enzymatic processes involving the formation of phospholipid-linked intermediates. As a consequence, they elicit a range of biological responses, to include potent antimicrobial, antiviral, and antitumor activities. I Structurally, the more than thirty members of the class may be categorized as long-chain N-acyl derivatives of the core substructure 1 or, in the case of certain streptovirudins, its dihydrouracil analog. TheN-acyl appendages vary in length and in degree of unsaturation, branching, and hydroxylation. Representative examples are depicted below (Figure 1). Consideration of these antibiotic structures has led to the proposal that they function as bisubstrate analogs for the enzymes they inhibit.2 In prokaryotic systems, for example, the tunicamycins block the exchange of uridine diphosphate N-acetylmuramic acidpentapeptide (UDP-MurNAc-peps) with a phospholipid carrier (Lipid-P, Scheme I), thus inhibiting cell wall biosynthesis} In eukaryotic systems, they block the transfer of Nacetylglucosamine-1-phosphate from its UDP-activated precursor uridine diphosphate Nacetylglucosamine (UDP-GlcNAc) to the phospholipid dolichol phosphate (Dol-P, Scheme ll), thereby inhibiting oligosaccharide biosynthesis.4 Given the substantial structural differences between the substrates of these enzymatic transformations, it is reasonable to propose that these processes might respond differently to variations in antibiotic structure, and that the relative inhibitory activities of different tunicamycins might differ as a consequence. An ambiguity in most biological studies of the tunicamycins conducted thus far is that complex and varying mixtures of tunicamycins, as obtained by fermentation, are typically assayed . Separation of these mixtures is tedious, requiring the use of reverse-phase HPLC; thus, in practical terms, only 3 Figure 1 RNH 0 ~ cNH HO ~"" ~0 0 ~~· 1' H ,. H 0 HO e· 1 R=H 0 Tunicamycin-v (1-V) R = H \V (CH,),CH(CH,J, H 0 Corynetoxin-H 19a* R = ~Jl CH,CH(OH)(CH, ),CH(CH,)CH,CH, Streptovirudin-A1 R= \v 0 H (CH,),CH(CH,), H * Stereochemistry of acyl appendage undetermined. HO 0-i Tunicaminyluracil (2) 4 Schemel Tunicamycin Inhibition of the Lipid Cycle in Peptidoglycan Biosynthesis UDP-MurNAc-peps l Lipid-P p UMP UDP-GicNAc UDP *) .. \_ Lipid-PP-MurNAc-pep 5 translocase ~ transferase polymerase Lipid-PP ~·---7--=-\:::---- Lipid-PP-MurNAc(GicNAc)-pep5 peptidoglycan acceptor * = site of tunicamycin inhibition Lipid-Phosphate (Lipid-P) 0 O~#cNH I 00 HO HO 0II ~H - H 0 NH ~ o-~..,.O-p..-0~0 CH3 H02C-(O.Aia)z-L-Lys-D-Gin--L-Aia-NH C O 0 0I_ H HO H OH Uridine DiphosphateN-Acetylmuramic Acid-Pentapeptide (UDP-MurNAc-pep 5 ) 5 SchemeD Tunicamycin Inhibition of Asparagine-Linked Glycoprotein Biosynthesis UDP-GicNAc UMP Doi-P \.._*.)• UDP-GicNAc UDP Doi-PP-GicNAc transferase L \... Doi-PP-(GicNAc)2 transferase ManMan I Man(Man)2 I Doi-P P-(GicNAc)2Man I (Man)3(Gic)3 peptide 5GDP 7 Doi-P "core" glycoprotein 5 GOP-Man 4 Doi-P-Man 3 Doi-P-Gic * = site of tunicamycin inhibition 0 II P, o'l o- o Dolichol Phosphate (Doi-P) Uridine Diphosphate N-Acetylglucosamine (UDP-GicNAc) 6 milligram quantities of any given pure tunicamycin are available.s For this reason, and in light of the potent biological activity of the tunicamycins, we have developed an efficient synthetic route to the tunicamycin antibiotics, 6 described in full herein. Synthetic Plan Retrosynthetic analysis of the tunicamycins as a class suggests that one highly versatile strategy for their preparation would involve the selective N-acylation of the precursor 1 as the final synthetic step. Two challenges emerge upon consideration of the simplified precursor 1 as a synthetic target: (1) the construction of the undecose fragment, tunicaminyluracil (2), from readily available precursors; and (2) the efficient formation of the trehalose disaccharide linkage with proper stereochemistry. Given the complexity of the tunicaminyluracil substructure (2) in terms of stereochemistry and functionality, it is not surprising that previous efforts to synthesize the tunicamycins have focused initially on the preparation of 2, deferring formation of the trehalose disaccharide linkage to a later stage_7,8 These same studies have shown, however, that the latter problem is perhaps as difficult as the former. In the single reported synthesis of a natural tunicamycin prior to studies described herein, Suami et al. 7 described the successful late-stage formation of the trehalose linkage via a modified Koenigs-Knorr carbohydrate construction, albeit proceeding in poor yield (18%, Scheme ill). In a detailed study, Danishefsky and co-workers8 noted that a similar coupling reaction involving nearly identical precursors did not proceed according to precedent (Scheme IV) ; these authors point out that the earlier successful coupling had been achieved with retrosyntheticallyderived material and was conducted on relatively large scale. These observations proved invaluable in the development of our synthetic plan, where it was determined to conduct the trehalose bond-forming step at an early stage in the synthesis. 7 Scheme ill Previous Synthetic Studies: Suami et al. 1' O:zN~o.., / AcO 51% H~Q AcO 10 steps t O+CH3 CH3 AcO CbzNH .. 4 steps 1-V OAc 1' H A cO 18% (+15% C1 "!)--C11'~) OAc 8 Scheme IV Previous Synthetic Studies: Danishefsky et al. 0 0~(NP:B o H 0 ~~,HCN.-C 1) TMSCH 2 CH=CH 2 , BF3o0Et2 , 67% O H H 2) MOMCI, (i-PrbNEt, 96% 3) Os04 ; Pb(OAc)4 , 85% o A o CH3 CH3 0 H ~steps A cO [plus (7'-S, 8'-R)diastereomer] CbzNH /TBgo~'" CH3--r--o::::r-- c) ,...-------, No desired coupled products CH3 AgCI04, Ag co 2 3 9 The undecose substructure within 1 may be viewed as the product of the coupling of uridine and galactosamine residues through carbons C5' and C6, respectively. Suami et al. employed a related coupling reaction in their synthesis of tunicaminyluracil (2) (Scheme III),7c while Danishefsky et al. established this bond by an organometallic addition reaction with subsequent development of the galactosamine fragment by de novo construction (Scheme IV).8 Our retrosynthetic analysis of 1 also targeted the C5'-C6' bond for disconnection; however, we planned to employ a new method for this bond formation that allowed for the use of simple precursors derived from uridine and galactosamine.6b Exploratory studies had shown (Scheme V) that when a solution of dihydrocinnamaldehyde (3, 1 equiv) in pyridine was treated sequentially with benzeneselenol9 (1.0 equiv, 23 °C, 15 min), excess dichlorodimethylsilane (14 equiv, 23 OC, Scheme V 0 Ph~H 3 CH3 ~. PhSeH {1 equiv), Py, 23 oc , CH 3 ~H'c1 15 min; Me2 SiCI2 (14 equiv), 23 oc, 16 h Ph CHz=CHCH 20H {1 equiv), 23 oc, SePh l 1 h, >90% 5 (62%) , Sl Bu3SnH {2.2 equiv), AIBN {0.06 equiv), PhCH3 , 60 °C, 6 h 4 10 16 h, excess reagent removed in vacuo), and allyl alcohol (1.0 equiv, 23 OC, 1 h), the 0silylhemiselenoacetals 4 were formed in high yield (>90% ).10 Subsequent exposure of 0silylhemiselenoacetals 4 to tributyltin hydride (2.2 equiv) at 60 OC in toluene in the presence of the radical initiator 2,2'-azobis(isobutyronitrile) (AIBN, 0.06 equiv) led to a 7-end.o-trig ring closure to form the siloxane 5 (62%) together with a small amount of non-cyclized reduction product 6 (11%). No product arising from 6-exo-trig cyclization was observed, perhaps a consequence of the length of the Si-0 bonds. II This procedure allowed for the mild and efficient coupling of aldehydes and allylic alcohols and generated a siloxaneprotected 1,4-diol functionality, a retron that maps onto the C5'-C8' substructure within 1 (Scheme VI). A substrate such as the 0-silylhemiselenoacetal 7, prepared from an Scheme VI N ,. H 8 OP 0 ~ CH3 sL )lNP u__ ~ *:o::f »e· H"r-?'~ CH31,. S e Ph 0 0 o/ N -H~O~CH2 P = various protecting groups PO OP OP 7 appropriate uridine 5'-aldehyde derivative and a galactosamine-derived allylic alcohol, was thus envisioned to undergo a similar silicon-mediated reductive coupling to form the siloxane 8. For the proposed retrosynthetic disconnection to be valid, it was critical that the newly-formed stereogenic centers at C5' and C7' be established with correct 11 stereochemistry. The stereochemical outcome at C7' was predicted to be the desired R configuration for the following reasons: (1) glycosyl radicals are known to react to form axial bonds preferentially, 12 and (2) equatorial C-H bond formation would result in a prohibitively strained ring system. The stereochemistry of C5', on the other hand, was less easily predicted (vide infra). To investigate the feasibility of the proposed bond formation and its potential application in a synthesis 1, the reductive coupling procedure was first examined in the context of a synthesis of tunicarninyluracil (2). 12 Chapter 2 Synthesis of Tunicaminyluracil 13 Synthesis of (+)- Tunicaminyluracil (2) To apply the hemiselenoacetal reductive coupling methodology described above in a synthesis of tunicaminyluracil (2), the allylic alcohol 15 was synthesized, employing commercially available N-acetylgalactosamine (9) as the starting material (Scheme Vll). Heating a solution of 9 and hydrochloric acid in methyl alcohol at reflux for 1.5 h provided the corresponding a.-methylgalactopyranoside which, without purification, was treated with benzaldehyde (11 equiv) and zinc chloride (1.6 equiv) at 23 "C for 25 h to provide the 4,60-benzylidene-a.-methylgalactopyranoside 1013 as a single anomer in 55% yield (mp 165.0-166.3 "C, ethyl alcohol). In initial investigations, alcohol 10 was protected as the corresponding 3-0-t-butyldimethylsilyl ether derivative; however, the silyl protecting group was later shown to be inappropriate due its propensity to migrate upon deprotection of the C4-hydroxyl functionality. methoxyethoxymethyl Consequently, (MEM) ether14 11 10 was protected as the 3-0(MEM chloride (5 .0 equiv), diisopropylethylamine (10 equiv), tetrahydrofuran (THF), 60 "C, 2 h, 75%). Generation of the C6-exo-methylene functional group from 11 was initiated by oxidative cleavage of the 4,6-0-benzylidene acetal. Thus, exposure of 11 to N-bromosuccinimide (NBS, 1.3 equiv) and barium carbonate (1.6 equiv) in refluxing carbon tetrachloride for 2 h15 efficiently formed the bromide 12 (87 %). It was necessary to employ the anomeric methyl galactopyranoside in the latter transformation because the corresponding benzyl galactopyranoside underwent competitive oxidation of the benzyl group. In efforts to generate the C6-exo-methylene functionality from 12 by the direct elimination of hydrogen bromide (e.g., silver fluoride, pyridine;I 6 triethylamine, benzene, reflux; silver carbonate, isooctane, reflux), 12 was found to be unreactive, presumably a consequence of steric shielding of the C5-hydrogen by the axial C1-methoxyl substituent. Elimination was therefore induced in a two-step procedure involving the initial treatment of the bromide 12 14 Scheme VII #o P\_,,,H 2% HCI: MeOH, reflux,1.5h; o""" ------------~~ AcNH PhCHO (11 equiv), ZnCI2 (1.6 ~iv), 23 °C, 25 h, 55% OCH3 10 9 ~ MO MEMCI (5 equiv), (i-Pr) 2 NEt~ (10 equiv), THF, 60 °C, 2 h, 75% OCOPh AcNH H O OCHa H NBS (1.3 equiv), BaC0 3 CH B r - - - - - - - - - - - - - - - 2 ;w o)(~ Ph H MMO AcNH 0 (1.6 equiv),CCI4 , reflux, 2 h, 87% OCH3 H 11 12 ~ ~ PhSeH (3 equiv), Et3 N (6 equiv), DME, reflux, 18 h,96% OCOPh MO m.CPBA (1.5 equiv), CC14 , AcNH 0 OCHa H CH2SePh _ 14 oc, 1 h; OMS (15 equiv); reflux, 5 h, 99% MO ~ AcNH OCOPh O CH2 OCH3 14 13 +I K2 C03 (3 equiv), MeOH, 23 °C, 3 h, 92% MO ~ AcNH OH O OCHa 15 CH2 15 with benzeneselenol (3.0 equiv) in the presence of triethylamine (6.0 equiv) in refluxing dimethoxyethane (DME) for 18 h to generate the phenylselenide 13 (96%). Oxidation of the phenylselenide 13 with m-chloroperoxybenzoic acid (m-CPBA, 1.5 equiv) in carbon tetrachloride at -14 OC for 1 h, followed by thermal elimination of the resulting selenoxide (carbon tetrachloride, reflux, 5 h),17 afforded the allylic acetate 14 in 99% yield. Removal of the C4-benzoyl ester group of 14 was accomplished by transesterification with potassium carbonate (3.0 equiv) in methyl alcohol at 23 OC for 3 h to yield the allylic alcohol15 (92% ). The uridine 5'-aldehyde derivative 17 was prepared as the initial coupling partner for the allylic alcohol15 and was synthesized from commercially available 2',3'-isopropylidene uridine (16). Oxidation of 16 following the procedure of Corey and Samuelsson (chromium trioxide (4.0 equiv), pyridine (8 .0 equiv), acetic anhydride (4.0 equiv), dichloromethane, N,N-dimethylformamide (DMF), 23 °C, 20 min)18 afforded 17 in 55% yield. The aldehyde 17, like many others synthesized during the course of our investigations, underwent ready hydration and was unstable to purification by conventional chromatography on silica gel. Aldehyde 17 was partially purified in its hydrated form by flash chromatography on silica gel at -14 °C. Regeneration of the aldehyde was readily accomplished by the azeotropic removal (toluene) of water from the hydrate. {NH HO~~o 0 H Y,. o H H{NH Cr03 (4 equiv), Py (8 equiv), A~O (4 equiv), DMF, CH 2CI2, 23 oc, 7 min, 46% ~~o 0 H Y,. o CH3 ~CH3 CH3 ~CH3 16 17 H 16 Initial attempts to construct the O-silylhemiselenoacetal18 (Scheme VITI) from 17 and the allylic alcohol 15 employed the procedure developed for the coupling of hydrocinnamaldehyde and allyl alcohol (see above). Thus, treatment of the aldehyde 17 (2.0 equiv) with benzeneselenol (2.0 equiv, 8 h) in pyridine at 23 T for 1 h, addition of dichlorodimethylsilane (20 equiv), removal of excess dichlorodimethylsilane in vacuo, and addition of a solution of allylic alcohol 15 (1 equiv) in pyridine at 23 OC produced the Schemevm PhSeH (2 equiv), Py, 23 oc, 1 h; ~12 {20 equiv); 15 {1 equiv) ~H3 CH3' :. 17 (-2 equiv, crude) 0 MEMO / 0 CNH I SePh 0 AcNH~ ~o~cH2 H H o.........,-o 1·':. OCH3 CH3 CH3 18 (-60%, crude) Bu3 SnH (2.5 equiv), AIBN {0.05 equiv), PhCH 3 , reflux, 10 h ; KF•2H 20 (15 equiv), MeOH,23 oc MO ~ AcNH H +HO~O{~H OH NH 0 O OCH3 15 CH2 0~0 CH3 CH3 I St-0~~ N 0 16 17 ad ducts 18 in modest yield as a 1: 1 mixture of diastereomers at C5'. Purification of the diastereomers 18 proved to be difficult due to their instability to column chromatography; as a result, the crude adducts (approximately 60% pure) were subjected directly to conditions conducive to free radical cyclization. Addition of a solution of tributyltin hydride (2.5 equiv, 10 mM) and 2,2'-azobis(isobutyronitrile) (0.05 equiv) in toluene over a period of 10 h to a solution of diastereomers 18 in refluxing toluene (1 mM), followed by treatment of the crude product mixture with potassium fluoride hydrate in methyl alcohol, afforded only the reduction product 16 and recovered allylic alcohol 15, indicating that trapping of the C5'radical with tributyltin hydride was faster than cyclization. This unfavorable result in the initial cyclization attempt prompted the preparation of a second 0-silylhemiselenoacetal derivative (23, Scheme X). This derivative incorporated a protecting group for the imido functionality and, with the greater steric bulk of the 2'-0- and 3'-0-silyl ethers, was felt to be better disposed toward intramolecular cyclization through shielding of the C5'-radical intermediate from bimolecular trapping. Preparation of 23 began with the treatment of uridine (19) with dimethoxytrityl chloride (1.0 equiv) in pyridine at 23 "C for 12 h (Scheme IX).19 The resultant dimethoxytrityl ether was combined with excess t-butyldimethylsilyl chloride (6.0 equiv) and imidazole (12 equiv) in DMF at 23 "C for 13 h to afford the bis(t-butyldimethylsilyl) ether 20 in 93% yield from uridine. Exposure of 20 to p-methoxybenzyl chloride (2.0 equiv) and sodium hydride (1.5 equiv) in DMF at 0 "C for 4.5 h afforded the corresponding N-(p-methoxybenzyl) derivative. Subsequent removal of the dimethoxytrityl protecting group with a solution of benzenesulfonic acid in chloroform (2% (w/w)) at 0 "C for 5 min20 provided the alcohol21 in 82% yield for the two steps. The use of the p-methoxybenzyl protecting group for the imido functionality of uracil is precedented in the work of Danishefsky and co-workers in their synthesis of tunicaminyluracil.8 Efforts to oxidize 21 to the corresponding aldehyde 18 Scheme IX DMTa (1oq""), Py, ""'0 23 °C,1h; TBSCI (6 equiv), imidazole (12 equiv), DMF, 23 °C, 13 h, 93% 19 {;:: ~O~N H " r - - ( 'H TBSO OTBS 20 PMBCI (2 equiv), NaH (1.5 equiv), DMF, o ac, 4.5 h; 2% PhS03 H : CHCI3, 0 oc, 5 min, 82% H { T~SO l ~ NPMB o~~~t~o HI)---{' OT~S 22 (crude) ° HO NPMB ll~o (COCih(6.2equiv), DMSO ~o~ (9.4 equiv), Et3N (15 equiv), CH 2CI2 , -78 °C, 45 min H " r - - ( 'H TBSO OTBS 21 (22) employing a number of standard reagents (pyridinium dichromate, chromium trioxide in pyridine, or 1,3-dicyclohexylcarbodiimide and dimethyl sulfoxide) were complicated by the formation of by-products that could not be readily separated from 22. Only the Swern oxidation21 (oxalyl chloride (6.2 equiv), dimethylsulfoxide (9.4 equiv), triethylamine (15 equiv), dichloromethane, -78 °C, 45 min) afforded product 22 of sufficient purity (-90%) to carry on with the reductive cyclization procedure. Subjection of the crude aldehyde 22 (2.0 equiv) to the coupling conditions described above (benzeneselenol (2.0 equiv), pyridine, 23 "C; dichlorodimethylsilane (20 equiv); 15 (1 equiv)) afforded the 0-silylhemiselenoacetals 23 in 92% yield as a 1:1 mixture of C5'-diastereomers after purification by flash column chromatography (Scheme X). The stability of 0-silylhemiselenoacetals 23 to silica gel is notable in light of the lability of the hemiselenoacetals 13 previously encountered. 19 Scheme X ~~0{;::' /)--?\ TBSO OTBS 22 (-2 equiv, crude) PhSeH (2 equiv), Py, 23 °C, 1 h; Me2 SiCI2 (20 equiv); 15 (1 equiv), 92% {0 CH3 CH 31 NPMB f SePh I~ ''sl-o~ / M~MO O O AcNH O H CH2 TBSO 0 H OTBS OCH3 23 Bu3 SnH (2 equiv), Et3 B (0.25 equiv), CH3 CN, -8 °C, 30 min; KFo2H 20 (excess), MeOH, 23 oc, 5 h 24 (18%) + 25 (62%) A series of experiments was performed to evaluate the feasibility of carbon-carbon bond formation within the hemiselenoacetals 23 (Table 1). Treatment of a solution of the hemiselenoacetals 23 with tributyltin hydride in the presence of a free radical initiator led to efficient intramolecular cyclization to form a mixture of epimeric adducts whose diastereomeric ratio varied markedly with the choice of solvent. Direct treatment of the 20 Table I Reductive Coupling of 23: Solvent and Temperature Effectsa Solvent Temperatureb ("C) PhCH3 PhCH3 PhCH3 PhCH3 THF 10% H20: THF 10% DMF: THF CH3CN 110 60 50 -78 --7 23 60 60 60 23 0 -5 -14 --7 0 CH3CN CH3CN CH3CN (CH3)2CHOH (CH3)2CHOH 0 -78 --7 23 CH3CH20H CH3CH20H 65 0 CH30H 0.06% H20 : CH3CN 0.06% H20 : CH3CN 20% CH30H : CH3CN 20% CH30H : CH3CN 0 0 -14 --7 23 0 -20--7 23 Product Ratioc 25(desired) : 24(undesired) 1.0: 3.0 1.0: 3.2 1.0:4.2 1.0: 5.3 1.0: 2.2 1.0: 2.0 1.0: 3.0 1.9: 1.0 2.4: 1.0 2.9: 1.0 3.1 : 1.0 1.9: 1.0 1.9: 1.0 1.0 : 1.0 1.7 : 1.0 3.7: 1.0 2.4 : 1.0 2.3: 1.0 2.9: 1.0 2.1: 1.0 a Reactions were performed employing 3-5 mg 23 (1 equiv, 1 mM) and 10 equiv Bu3SnH . b Reactions performed at or below 23 T were initiated with Et3B and oxygen. Reactions conducted at temperatures >23 T were initiated by the slow addition of a solution of Bu3SnH (10 equiv) and AIBN (0.05 equiv) at 23 "C. c Yields were generally >70%, except for those reactions carried out in protic solvents, which afforded yields of -40%. The major by-product in all cases was that of hydrogen-atom addition to the radical site. 21 crude product mixture with potassium fluoride hydrate in methyl alcohol produced the diastereomeric diols 24 and 25 in 40-80% yield from 23. These diastereomers could be separated by preparative thin-layer chromatography or radial chromatography to afford each C5'-diastereomer in pure form. To establish the stereochemistry of the cyclization products, each of the diols 24 and 25 was separately deprotected (eerie ammonium nitrate, acetonitrile, Scheme XI CAN (5.0 equiv), H2 0, CH3 CN, 60 oc, 3 h; 3 N HCI, reflux, 3 h C5'-epi-2 (crude) Ac2 0 (38 equiv), DMAP (45 equiv), CH 2CI2 , 0 °C, 2 h, Prep. TLC, 31% AcNH CAN (5.4 equiv), H 20, CH3CN, 60 oc, 3 h; 25 3 N HCI, reflux, 3 h Ac20 (38 equiv), DMAP (45 equiv), CH2 CI2 , 0 oc, 2 (crude) 2 h, Prep. TLC, 43% AcNH AcO Zl 2 Authentic Tunicamycins 3N HCI, reflux, 3 h 1d (crude) + GlcNAc A~O (42 equiv), DMAP (50 equiv), CH2CI2 , 0 oc, 2 h, Prep. TLC, 30% OAc 22 water, 60 ·c, 3 h;22 3 N hydrochloric acid, reflux, 3 h) and was peracetylated (acetic anhydride, 4-(N,N-dimethylamino)pyridine (DMAP), dichloromethane, 0 "C, 2 h) to give aheptaacetyl-5'-epi-tunicaminyluracil (26) from 24 and a-heptaacetyltunicaminyluracil (27) from 25 after preparative thin layer chromatography (Scheme XI). These products were compared with an authentic sample of a-heptaacetyltunicaminyluracil (27), prepared from a mixture of tunicamycins according to the procedure of Tamura et al. Id In general, the use of nonpolar solvents (toluene, TI-IF) in the free-radical cyclization was found to favor the formation of the undesired epimer (24) from 23, whereas polar solvents (acetonitrile, methyl alcohol) favored the formation of the desired isomer 25. Hypothetical transition structures 28 and 29 leading to these isomers, respectively, are depicted in Figure 2. In both structures, attack of the C5'-radical is invoked to occur opposite the bulky t-butyldimethylsilyl ether substituents. The manner in which the solvent polarity apparently influences the stereochemical outcome of the cyclization reaction is not at all evident, and the validity of transition structures 28 and 29 is certainly open to question. Figure 2 MEgMOO '· AcNH,, CH,O''.. 0 I CO Si-o I NPMB C.::1f ~0 .::r), H 0 .. M H / ) - - ( ' H TBSO 28 o-;l~ lNPMB OTBS MEMO:e~lo,J~H ~0 • CH3 •. AcNH'' CH , 0 3 0 0 H TBSO 29 H OTBS 23 Nevertheless, the role of the solvent in the reaction provides a useful device for practical syntheses of either stereoisomer. The optimal protocol for the preparation of the desired stereoisomer (25) involved the addition of tributyltin hydride (2.0 equiv) and triethylborane23 (0.25 equiv) to a solution of 0-silylhemiselenoacetals 23 (1 mM, 1 equiv) in acetonitrile at -8 °C. Treatment of the crude product mixture with potassium fluoride hydrate in methyl alcohol and purification of the resulting diol mixture by radial chromatography afforded the pure diol 25 in 62% yield and the pure diol 24, isolated in separate fractions, in 18% yield. Although reactions conducted in methyl alcohol produced an increased proportion of 25 relative to reactions conducted in acetonitrile, the increased formation of the reduction product 21 in methyl alcohol led to a lower absolute yield. Deprotection of the synthetic diol 25 (eerie ammonium nitrate (5.4 equiv), acetonitrile-water, 60 °C, 3 h; 3 N hydrochloric acid, reflux, 3 h) furnished synthetic tunicaminyluracil (2) in crude form. Peracetylation of synthetic 2 with excess acetic anhydride and DMAP in dichloromethane at 0 °C afforded, after preparative thin-layer chromatography, a-heptaacetyltunicaminyluracil (27, 43%), which was shown to be identical in all respects (lH NMR, 13C NMR, mp, FTIR, MS, HRMS, TLC, HPLC, optical rotation) with an authentic sample. 24 Chapter 3 Synthesis of Tunicamycin-V 25 Synthesis of (+)-Tunicamycin-V (1-V) The establishment of an efficient method for the formation of the C5'-C6' carboncarbon bond of tunicaminyluracil has provided the basis for a highly convergent synthesis of the tunicamycin antibiotics. In addition to the preparation of the undecose core, the latter endeavor required the development of a method for the construction of the l3,a-trehalose linkage within 1, a crucial problem that previously had been met with only modest success. In contrast to prior work, in which the trehalose bond was formed as a late-stage synthetic operation,7.8 the approach described herein focused on glycosidic bond formation early in the synthesis, as the initial convergent step. Construction of the C5'-C6' bond of the tunicaminyluracil core was deferred to a later stage in the synthesis, where the mild conditions of the silicon-mediated reductive coupling methodology projected to form this bond (see above) were anticipated to be compatible with the trehalose linkage. An advantage of this strategy was that it permitted the use of relatively simple carbohydrate precursors for the synthesis of the trehalose-linked disaccharide. Synthesis of the l3,a- Trehalose Linkage Although the stereocontrolled synthesis of any glycosidic bond is inherently challenging, the preparation of disaccharides containing an ether linkage between anomeric carbons (trehalose linkage) is particularly so. Any such linkage is potentially disconnected retrosynthetically in two ways; in both disconnections the glycosyl acceptor (nucleophilic component) contains an anomeric hydroxyl group as the nucleophile. This compounds the difficulty of glycosidic bond formation by virtue of the poorer nucleophilicity of the anomeric hydroxyl group as compared with other alcohols, and because the orientation of the anomeric hydroxyl group is ambiguous. saccharides are seldom prepared efficiently. As a consequence, trehalose-linked 26 The traditional method of glycosidic bond formation, originating with Koenigs and Knorr in 1901,24 suffers from several disadvantages, to include:25 (1) the difficulty of stereocontrolled synthesis of the glycosyl halide coupling partners; (2) the thermal and hydrolytic instability of these halides; (3) the use of toxic or potentially explosive heavymetal salts in the coupling reaction; and (4) the frequently poor efficiency of the coupling reactions, particularly with hydroxyl groups of low nucleophilicity. For these reasons, and given the documented poor performance of the KoenigsKnorr methodology in the context of synthesis of the tunicamycin trehalose linkage,7,8 our studies focused on the methodology of Schmidt et al. for glycosidic bond formation. Also known as the trichloroacetimidate method,26 the Schmidt protocol entails the coupling of an anomeric trichloroacetimidate (glycosyl donor) with the nucleophilic hydroxyl group of a glycosyl acceptor. Advantages of this method include: (1) the ease of synthesis of trichloroacetimidates of either a- or !)-configuration; (2) the thermal and hydrolytic stability of the glycosyl trichloroacetimidates; (3) mild conditions for glycosidic bond formation, typically catalyzed by Lewis acids; and (4) the efficiency and stereoselectivity of these coupling reactions. The trichloroacetimidate methodology has seen limited use in the synthesis of disaccharides containing the trehalose linkage. 27 In retrosynthetic analysis of the tunicamycin trehalose-linked disaccharide, primary consideration must be given to the assignment of the roles of electrophile and nucleophile to the galactosamine and glucosamine components (Figure 3). In addition, careful consideration must be given to the choice of protective groups for each C2-amino functionality and to the potential role of that protective group in the glycosylation reaction. Similar considerations apply to the hydroxyl groups of each sugar. In addition, the C6substituent ("Z") of the galactosamine residue must function as a precursor to an exo methylene group (C5-C6). 27 Figure 3 N-functionality: • differentiable • neighboring-9roup participation effects N ro:Yf Z-functionality: • alkene precursor X Electrophile or Nucleophile? glucosyl fragment galactosyl fragment Protecting groups (P): • stable during glycosytation and reductive coupling procedures • easily removable in the presence of trehalose linkage After extensive experimentation, the coupling partners 38 and 44 were found to serve as optimal substrates in an acid-promoted trichloroacetimidate glycosylation reaction to form the desired ~ , a-trehalose linkage (see below)_ This approach employed the galactosamine derivative 38 as the nucleophilic component in the reaction. This component was synthesized from tri-0-acetyl-D-galactal (30)28 in a sequence initiated by the azido nitration procedure of Lemieux et aL (eerie ammonium nitrate (3.0 equiv), sodium azide (1.5 equiv), acetonitrile, -20 "C, 10 h).2 9 Hydrolysis of the product anomeric nitrate esters with sodium nitrite (3.0 equiv) in a mixture of water and 1,4-dioxane at 80 "C for 12 h afforded the corresponding azido sugar as a mixture of anomers. The azido nitration procedure proved to be extremely valuable in the synthesis as it allowed for the facile incorporation of a masked amino group at C2 of galactose from a readily available carbohydrate precursor. Protection of the anomeric hydroxyl group of the intermediate azido sugar with t- 28 AcO:~H~ ~c?(" 'oAc H H CAN (3 equiv), NaN3 (1 .5 equiv), CH3CN, -20 oc, 10 h ; NaN02 (3 equiv), 1.4-dioxane, so oc. 12 h; HP. TBSCI (2 equiv), imidazole (4 equiv), CH2 Cl2 , 23 oc, 5 h, 33% 31 K2C03 (0.3 equiv), MeOH, 23°C, 1h; PhCH(O·i-Pr)2 (1 .2 equiv), CSA (0.01 equiv), CH 3CN, 23 OC, 30 min, 69% l P~,H 0 Na TBS H H 32 butyldimethylsilyl chloride (2.0 equiv) and imidazole (4.0 equiv) in dichloromethane provided the t-butyldimethylsilyl ether 31 as a single anomer(!)) in 33% yield from tri-0acetyl-D-galactal (30). Methanolysis of triester 31 with potassium carbonate (0.3 equiv) in methyl alcohol (23 °C, 1 h) and selective protection of the C4- and C6-hydroxyl groups of the resulting triol with diisopropoxybenzyl acetal (1.2 equiv) and camphorsulfonic acid (0.01 equiv) in acetonitrile (23 °C, 30 min) furnished the galactopyranoside 323° in 69% yield. The 4,6-0-benzylidene acetal not only served to protect the C4- and C6-hydroxyl groups, but also functioned as a precursor to the allylic alcohol functionality necessary for the reductive coupling that would form the undecose core of the tunicamycins (see Scheme Vll). Because of concerns that the benzylidene acetal would not be stable under the acidic conditions of the glycosidic coupling procedure, the oxidative cleavage of this group was performed prior to the glycosidic coupling. Initially, it was deemed prudent to protect the C3-hydroxyl group before oxidative cleavage of the benzylidene acetal; however, after 29 Table II. Screening of 3-D-Protective Groups (R) for 32a Px,H :\,H HO 0 N3 0 N3 TBS TBS H H H H 32 R Reagent Yield(%) Benzyl Benzyl bromide 84 Removal during NBS-cleavage of benzylidene SEM SEMCl 95 Removal during acid-catalyzed gl ycosy lation TBS TBSCl 92 Migration upon Incompatibility deprotection of C4- OH Acetyl acetic anhydride quantitative Migration to form C2-acetamide on azide reduction a SEM =trimethylsilylethoxymethyl, TBS =t-butyldimethylsilyl, NBS =N-bromosuccinimide. serious difficulties were encountered with four different types of protective groups (Table II), the direct oxidative cleavage of 32, with a free C3-hydroxyl group, was examined. Irradiation of a solution of 32 (0.077 M) in bromotrichloromethane with a 250-W sunlamp at 0 OC for 2.5 hours3I afforded the bromide 33 in 87% yield (Scheme XII). The C3- 30 SchemeXD 0.077 M in BrCCI3 , 250 W N3~h sunlamp, o oc, 2.5 h, 87% TBS0--1- o - r - - Br H ! 33 32 BOMCI (5 equiv), (i-PrhNEt (5.5 equ1v), CH2CI2 , 55 oc, 15h,89% H~~OPh TBSO --r- o..r-H PhSeH (3 equiv), Et:JN Br H 60 oc, 2.5 h, 98% N3~0Ph TBS0--1- o - r - - Br H H 35 34 PhthN~OPh TBS0--1- H H o..r-H BOMO PhSeH (3.2 equiv), Et3N Br . (12 eqUIV), DME, 90 °C, OCOPh PhthN~ 0 TBS0--1- -r--sePh H 10h, 95% H 36 I Et3 N•3HF (8.7 equiv), 23 °C, 6 h, 97% ' BOMO OCOPh H0--1- - r - - SePh PhthN~ 0 H H 38 31 hydroxyl group of 33 was then protected as the corresponding benzyloxymethyl (BOM) ether32 (34, BOM chloride (5.0 equiv), diisopropylethylamine (5.5 equiv), dichloromethane, 55 °C, 15 h, 98%). The BOM ether 34 was found to be unreactive toward a variety of standard reagents developed for the reduction of azides,33 presumably a consequence of steric shielding of the azido group by the adjacent t-butyldimethylsilyloxy group. Only the treatment of 34 with benzeneselenol (3.0 equiv) in triethylamine at 60 OC for 2.5 h34 efficiently formed the corresponding amine (35, 98%). Protection of the amine 35 with phthaloyl dichloride (2.0 equiv) in a mixture of toluene and 1,8-diazabicylo[5.4.0]undec-7ene (DBU) at 100 OC for 1.5 h afforded the phthalimide 36 in 86% yield. Displacement of the primary bromide within 36 using benzeneselenol (3.2 equiv) and triethylamine (12 equiv) in refluxing dimethoxyethane for 10 h efficiently furnished the phenylselenide 37 (95% ). It was also possible to both reduce the azido group and displace the bromide by the treatment of 34 with benzeneselenol and triethylamine; however, the two-step procedure outlined above proceeded in higher yield. The phenylseleno group served as a masked form of the exa-methylene functionality necessary for the planned reductive coupling protocol. Cleavage of the anomeric t-butyldimethylsilyl ether 37 with triethylamine trihydrofluoride (8.7 equiv) in acetonitrile (23 OC, 6 h, 97%) produced the hemiacetal 38 as a 10:1 (l3:a) mixture of anomers. The C2-phthalimido substituent is believed to favor the equatorial or 13-orientation of the anomeric hydroxyl group, by virtue of a non-bonding steric interaction between a phthalimido carbonyl group and the anomeric hydroxyl group within the axial or a-anomer.35 The 13-orientation is required to prepare the trehalose linkage within the tunicamycins. The preparation of the coupling partner 44 was initiated in a manner similar to that of 38, by the azido nitration29 of commercially available triacetoxy-D-glucal (39, Scheme Xlll) with eerie ammonium nitrate (3.0 equiv) and sodium azide (2.6 equiv) in acetonitrile at 32 SchemeXlll CAN (3 equiv), NaN 3 (2.6 equiv), CH3 CN, -20 oc, 10 h; N H NaN02 (1 eq.Jiv), 1 ,4-doxane, ~o. 10 oc, 12 h; AcO Aco-::!:JTBSCI (2 equiv), imidazole AcO (3 equiv), CH 2CI2 , 23 oc, H Ac:&~ AcO AcO . -,-..Z.+_ OTBS 0 ....._ / O H 13 h, 37% 40 NaOH (0.08 equiv ), MeOH, 23 °C, 45 min; 1:5 (v/v) 1 2,2-dmethoxypropane : acetone, r:r TSA (0.03 eq.Jiv), 23 °C, 1.5 h, 76% N3 H T B S # OTBS TBSCI (2 equiv), o imidazole (3 equiv), l 0 ~--------------CHa CH2CI2 , 23 oc, 1.5h, 95% J CHa 41 42 KF-2H 20 (5.5 equiv) , MeOH, 23 °C, 6.5 h, quant. l Na OH 5:1 (v/v) CCI3CN: #H TBS TBS~ CH 2CI 2 , K2 C03 0 0 O --------. )----C~ CHa- L CH -l- 0 3 1 (0.9 equrv , 23 o CHa 24 h, 64% (+12% rec. 43) 43 , )lNH 0 1CHa 44 CCb 33 -20 ·c for 10 h. The resulting 1-nitrato-2-azidosugar was readily hydrolyzed with sodium nitrite (1 equiv) in aqueous 1,4-dioxane at 70 ·c for 12 h. Protection of the resulting anomeric hydroxyl group with t-butyldimethylsilyl chloride (2.0 equiv) and imidazole (3.0 equiv) in dichloromethane at 23 ·c for 13 h produced the f)-oriented silyl ether 40 in 37% yield from 39. Methanolysis of the acetoxy substituents with a catalytic amount of sodium hydroxide (0.08 equiv) in methyl alcohol at 23 ·c for 45 min, followed by selective protection of the C4- and C6-hydroxyl groups with excess 2,2-dimethoxypropane and ptoluenesulfonic acid (0.03 equiv) in acetone at 23 T for 1.5 h afforded the acetonide 41 in 76% yield. Treatment of 41 with t-butyldimethylsilyl chloride (2.0 equiv) and imidazole (3.0 equiv) in dichloromethane at 23 ·c for 1.5 h produced the bis(silyl) ether 42 in 95% yield.36 Quantitative and selective cleavage of the anomeric silyl ether was accomplished by the treatment of 42 with potassium fluoride hydrate (5.5 equiv) in methyl alcohol at 23 ·c for 6.5 h. Exposure of the hemiacetal 43 to a suspension of potassium carbonate (0.9 equiv) in trichloroacetonitrile and dichloromethane at 23 ·c for 24 h provided the J3trichloroacetimidate 44 (64%) as well as recovered starting material (12%), after flash chromatography using triethylamine-treated silica gel. Literature procedures for the Schmidt coupling of anomeric trichloroacetimidates with alcohols typically involve the use of the Lewis acids boron trifluoride etherate or trimethylsilyltrifluoromethane sulfonate (TMS0Tf).26 Treatment of the coupling partners 38 and 44 with boron trifluoride etherate under a variety of conditions led only to the decomposition of 44. The use of TMSOTf as catalyst did produce the coupled products 46 and 47 in low yield (<30% combined yield); the competitive rearrangement of 44 to the amide 45 accompanied this transformation. The latter coupling reaction appeared to exhibit induction periods of varying length, suggesting that trifluoromethanesulfonic acid (TfOH) might function as the actual catalyst in the reaction.3 7 Indeed, coupling reactions employing 34 CCI, HN~O TMSOTf 3 TBS#N .. 0 H CH,--(-0 O CH, 45 44 TfOH as the catalyst were found to be both rapid and efficient In the optimum procedure, slow addition of a solution of TfOH (5% in toluene (v/v), 0.36 equiv total) to a solution of hemiacetal 38 (1 equiv) and trichloroacetimidate 44 (2.0 equiv) in dry toluene at 4-h intervals over a 24-h period at -20 OC produced the !),a.-linked trehalose 46 in 77% yield after flash column chromatography. The a.,a.-diastereomer (47) was also isolated as a minor product (11%) in separate fractions. This procedure was found to be equally efficient on the milligram to 10-gram scale and represents a highly practical solution to the problem of stereocontrolled formation of the trehalose linkage within 1. BOMO OCOPh PhlhN~ 0 H0__,- -,----- SePh H 38 (1 equiv) 44 (2 equiv) fI 46(77%) H TfOH (0.36 equiv), PhCH3 , -20 oc, 24 h, 4 A mol. sieves 47(11%) 35 Other Glycosylation Attempts Prior to the development of the glycosylation procedure described above, early investigations into trehalose construction centered on a reversal of the roles of the galactosamine- and glucosamine-derived coupling partners. Initial efforts employed theNacetylglucosamine derivative S08 as the nucleophilic component in the glycosylation reaction. This was a logical strategy to follow given that the tunicamycins contain anNCH3 a=< NH L ffi~ L = leaving group acetylglucosamine residue. The incompatibility of the C2-acetamido group with a leaving group at Cl (oxazoline formation) mandated that the N-acetylglucosamine residue serve as the nucleophilic component in the coupling reaction, if it were to be used at all. Protection of the known benzylgalactopyranoside 4838 with t-butyldimethylsilyl chloride (1.5 equiv) TBSCI (1.5 equiv), imidazole (2.9 ecpJiV), DMF, 23 °C, 12 h, 98% 2 "#1-cNH OCH Ph TB~O H CH3-(-0 O CH3 49 48 U (2 equiv), NH3 (1) • ~ -78 °C, 5 min, 72% AcNH TBSO~ CH3-fo!::T O CH3 a-! 36 and imidazole (2.9 equiv) in DMF at 23 "C for 12 h furnished the silyl ether 49 in 98% yield. Reduction of 49 with lithium (2.0 equiv) in liquid ammonia at -78 OC produced the N-acetylglucosamine derivative 50 as a 2:1 (a:~) mixture of anomers in 72% yield. The galactose-derived trichloroacetimidate 53, with a C2-azido substituent, was synthesized for initial coupling studies with 50. The azido alcohol 32, described above, was protected as its benzyl ether (51) using sodium hydride (1.2 equiv) and benzyl bromide (1.3 equiv) in THF at 23 "C for 8 h. Cleavage of the anomeric silyl ether with potassium fluoride hydrate in methyl alcohol at 23 OC furnished the anomeric alcohols 52 (1.5:1, a:~) in 75% yield. Treatment of the anomers 52 with excess trichloroacetonitrile and a catalytic quantity of DBU (0.05 equiv) in dichloromethane at 23 OC for 10 min afforded the ~trichloroacetimidate 53, which was used in the coupling reaction without purification. PhCH2 Br ( 1.3 equiv), NaH (1 .2 equiv), THF, 23 °C, 8 h, 85% 51 32 K~2H 2 0 (5 equiv), ~ MeOH, 23 oc, 7h, 75% Ph 5:1 (vlv) CH 2 CI 2 : CCI3 CN, DBU (0.05 equiv), 23 ac, 10 min H p~)(~ roY N3· __ HO H 53 (crude) Exposure of a mixture of anomers 50 (3.0 equiv) and the trichloroacetimidate 53 (1 equiv) to TMSOTf (0.2 equiv) in dry dichloromethane at -20 OC for 6 h provided only trace 37 Ph H P~hC~ O~~ Na 0 ~H NH CCia TBSO~OH 0 H 53 (1 equiv, crude) CHa-fo-!::/ O CHa 50 (3 equiv) quantities of coupling products; the anomeric alcohols 50 and 52 were the primary components of the reaction mixture. The disaccharide 54, containing the undesired a.~trehalose configuration, was isolated in -5% yield. It should be noted that this product was formed with the incorrect stereochemistry at both anomeric positions. Although the configuration of the anomeric nucleophile (50) could not be controlled in any obvious way, there was ample precedent for the use of a C2-phthalimido group within the electrophilic component to direct nucleophilic attack in the desired (~) sense.35 Toward this end, the glycosyl donor 57 was prepared, initiated by the reduction of the azido group of 51 with hydrogen sulfide in a mixture of pyridine and triethylamine (3.5: 1 (v/v), 23 OC, 20 h) to furnish the amine 55 in 98% yield. Introduction of the phthalimido protecting group was accomplished by the treatment of 55 with phthaloyl dichloride (3 equiv) and DBU (6.4 equiv) in toluene at 100 "C for 3 h to provide the phthalimide 56 in 94% yield. Cleavage of the anomeric silyl ether of 56 with potassium flu oride hydrate (20 equiv) in methyl alcohol 38 [c90~CH,OPl PhthN~CH;PP ~Lev.<•aOd PO OP ~H CCI3 PO OP - ~ H /) O H NH ROH P= various protecting groups ~ PO OP PhthN~ oo~OTCH20P H Ph H H P~hC20 °)<~ N3 TBSO 23 °C, 20 h, 98% O H H 55 51 o-C6 H4 (COCil2. (3 equiv), DBU (6.4 equiv), PhCH 3 , 100 °C, 3 h, 94% Ph H Ph Ph~H20 ~~ KFo2H 20 (20 equiv), MeOH, 23 °C, 8 h; 1H H 5:1 (vlv) CH2 CI2 : CCI3 CN, DBU (0.3 equiv), 23 oc , PhthN CCI3 O NH 51 l 5min, 52% H P~hC:P oX~ PhthN TBSO H H 56 at 23 OC for 8 h and activation of the resultant hemiacetal with excess trichloroacetonitrile in methylene chloride in the presence of a catalytic amount of DBU (0.3 equiv) at 23 OC for 5 min afforded the 13-trichloroacetimidate 57 in 52% yield from 56. Treatment of a solution of the coupling partners 50 (1.7 equiv) and 57 (1 equiv) in dry dichloromethane with TMSOTf (0.9 equiv) at -20 OC for 12 h produced the desired j3,a-trehalose 58 as the major 39 Ph H ·~~ PhthN~oy ~H NH H CCI3 SO (1.7 equiv) 57 (1 equiv) I TMSOTI (0.9 equiv), CH CI , T-20 oc, 4 Amol. sieves, 122 h2 59 (17%) product (24%) along with a significant amount of the l3,l3-linked diastereomer 59 (17% ). Although the problem of stereochemical control at the anomeric center of the galactosamine residue appears to have been solved with the introduction of the phthalimido group, the anomeric center of the nucleophilic N-acetylglucosamine residue was poorly controlled. In addition, the coupling yield was unacceptable for preparative purposes. For these reasons, this glycosylation approach was abandoned in favor of one in which the roles of electrophile and nucleophile in the coupling reaction were interchanged-an approach that evolved into the optimized glycosylation procedure with the substrates 44 and 38 described above. Carbon-Carbon Bond Formation-Construction of the Trisaccharide Core. With the establishment of an efficient procedure for the synthesis of the l3,atrehalose-linked disaccharide 46, efforts turned toward the development of a procedure for its transformation to the allylic alcohol62, required for reductive coupling with a uridine-derived 40 5'-aldehyde. Because of the lability of the phthalimido substituent, we first elected to replace this protective group with the more stable benzyl carbamate group (Scheme XIV). Treatment of 46 with a mixture ofhydrazine hydrate and ethyl alcohol (1:8 (v/v)) at 100 ·c in a sealed tube for 12 h led to cleavage of both the phthalimido and benzoyl substituents to afford the Scheme XIV O ~ PhthN 0 TBSO 0 O H SePh H H 46 CH3--rCH3 OCOPh ~ 1) 8:1 (vlv) EtOH: H2NNH2 , 100•C,12h,87% 2) PhCH20COCI (8.9 equiv), Py, 0 oc, 30 min, 91% #.0 0 B~Oi CbzNH TBSO 0 H sePh H H 60 CH3--rCH3 _j OAc ~ o CbzNH O 0 TBSO 0 CH 3- - r CH3 H H 1) PhSeH (14eq.Jiv) , Et3 N, 55 °C, 12h,91% 2)2:1(vlv)Py:A~O. 60 °C, 2.5 h, 91% SePh H 61 ~ 1) m-CPBA (3.5 equiv), CH 2CI2, 0 oc, 30 min ; OMS (12 equiv), Et3 N (3 eq.Jiv), 65 °C, 10 h, 88% 2) K2C03 (0.07 equiv), MeOH, 23 oc, 2 h, 92% BOMOi ~ ~CbzNH 0 TBSO 0 CH3-r H H CH2 41 corresponding amino alcohol in 87% yield. Selective protection of the amino group as the benzyl carbamate was accomplished by the treatment of the amino alcohol with benzyl ch1oroformate (8.9 equiv) in pyridine at 0 OC for 30 min, furnishing the disaccharide 60 in 91% yield. The hindered azido group of 60 was smoothly reduced with benzeneselenol34 (14 equiv) in triethylamine at 55 "C for 12 h (91 %), and the resultant amino alcohol was directly acetylated with acetic anhydride and pyridine (60 "C, 2.5 h, 91% ), providing the diacetyl derivative 61. Transformation of 61 to the allylic alcohol62 proceeded efficiently in a two-step procedure involving the initial oxidation of 61 to the selenoxide (m-CPBA (3.5 equiv), carbon tetrachloride, 0 "C, 30 min) followed by thermolysis of the selenoxide at 65 "C for 10 h. Exposure of the resulting allylic acetate to potassium carbonate (0.07 equiv) in methyl alcohol at 23 "C for 2 h produced the allylic alcohol 62 in 81 % yield from 61. The silicon-mediated reductive coupling of the allylic alcohol 62 with a undinederived 5'-aldehyde coupling partner represented the final convergent step in the construction of the core structure (1) of the tunicamycins. The aldehyde 64 was cho~n as the initial substrate for coupling. Unlike the aldehyde 22, used in the synthesis of tunicaminyluracil (2), 64 does not incorporate the p-methoxybenzyl group for protection of the uracil imide. Although the latter protective group functioned adequately in two previous syntheses of tunicaminyluracil, the rathe r harsh oxidative conditions necessary for its removaJ22 were believed to be incompatible with the trehalose linkage of 1. The t-butyl carbamate protective group was chosen as an alternative that was anticipated to undergo facile deprotection under mildly acidic conditions. Treatment of uridine derivative 20 with di-t-butyl dicarbonate (2.0 equiv) and DMAP (0.06 equiv) in pyridine at 23 OC for 12 h, and exposure of the resulting t-butyl carbamate to trichloroace tic acid (4.6 equiv) in dichloromethane at 0 "C for 15 min furnished the alcohol 63 in 53% yield from 20. Efficient oxidation of 63 to the corresponding aldehyde (64) was accomplished, as before, 42 0 0 ~0~0 TBSO OTBS ~0 (2 eq.Jiv), DMAP (0.06 equiv), Py, 23 °C, 12 h; CCI3 C02 H (4.6 equiv), CH2 2 , oc, 15 min, 53% a o ~~: TBSO OTBS 63 (COCI)2 (3 equiv), DMSO (5 equiv), CH 2CI2 , -78 oc, 15 min; E!:JN (10 equiv), -40 oc, 25 min ~ 0 ~~ct.;:: HI)----{'H TBSO OTBS 64 (crude) employing the Swem oxidation protocol (oxalyl chloride (3.0 equiv), dimethyl sulfoxide (5.0 equiv), triethylamine (10 equiv), dichloromethane, -40 "C, 25 min). Like uridine-5'aldehyde derivatives previously prepared, 64 was found to be unstable toward chromatography on silica gel and was therefore used in crude form for the formation of the 0-silylhemiselenoacetal in the next step. The procedure for hemiselenoacetal adduct formation (Scheme XV) was similar to that used for the preparation of 23 (see Scheme X). Thus, a deoxygenated solution of the aldehyde 64 (2.5 equiv) in toluene was treated with benzeneselenol (3.8 equiv) and pyridine (4.1 equiv) at 23 "C for 15 min, followed by a solution of dichlorodimethylsilane (10 equiv) in pyridine at 23 "C for 6 h; excess dichlorodimethylsilane and solvents were removed in vacuo and a solution of the allylic alcohol 62 (I equiv) in pyridine was added at 23 "C to form, within 5 min, the adduct 65 as a 5:1 mixture of C5'-epimers (50%). Free-radical cyclization 43 Scheme XV H 0 ():oc ~OT~S o 3o~~~), ~ 64 (2.5 equiv, crude) PhCH 3, 2 Py (4.1 MezSiCI2 (10 ' min; 62 (1 equiv) equlv) 23 oc, 6 h; ~ CH:l:__ CbzNH~ 8<>'0 o~s oP~ o-r 0~ T~SO TBS~AcNH 0 CH3-(-0 CH3 e~lv) ' PhSeH(3B TBSO 0 H CH2 {):" 0 0 H OTBS H 65 (50%) e~lv), . Bu3SnH (2 5 Et3B (0.2 e. PhCH ~IV), 3• 0 oc, 15 min l BOMO CbzNH 1' H OTBS 66(80%) 44 of 65 was induced by the dropwise addition of a solution of triethylborane in THF (1 M , 0.2 equiv) over a 15-min period to a solution of 65 and tributyltin hydride (2.5 equiv) in toluene ( 1 mM) at 23 "C. The cyclic siloxane 66 was isolated in 80% yield after chromatography on silica gel. Although carbon-carbon bond formation was highly efficient, the reaction produced exclusively the S-configuration at C5'-the configuration opposite to that of the desired product. This stereochemical assignment was determined by degradation of 66 with aqueous hydrochloric acid (3 N, reflux, 3 h) followed by peracetylation of the resulting amino polyol with acetic anhydride (40 equiv) and DMAP (51 equiv) in dichloromethane at 0 "C for 2 h, and comparison of the product with authentic samples of peracetyl a-tunicaminyluracil (27) and peracetyl C5'-epi-a-tunicaminyluracil (26). The observed preference for the formation of the undesired C5'-S diastereomer in this transformation paralleled our earlier OAc 3 N HCI, reflux, 3 h; AcNH 8) ~0 (40 equiv), DMAP 1' (51 equiv), CH 2CI2 , o "C, 2 h, Prep. TLC, 25% H 26 A cO OAc results in studies leading to a synthesis of tunicaminyluracil. Unfortunately, the use of polar solvents in the cyclization of substrate 65, a procedure which led to a reversal of selectivity in the previous study, failed to produce the desired diastereomer; cyclizations of 65 conducted in acetonitrile, methyl alcohol, and aqueous methyl alcohol all produced 66 exclusively. The high stereoselectivity of this free-radical cyclization reaction was rationalized by invoking the two hypothetical transition structures 67 and 68 (Figure 4). These structures, similar to structures 28 and 29 previously invoked (Figure 2), depict the olefin approaching the carbon-centered radical from the side opposite to that of the bulky t-butyldimethylsilyl 45 Figure 4 BOMO ?:y~O ~ O~~~OH{~' CbzNH,g. O . I H CH , , •. 3 f--o"' 0 CH3 -Si N ,,,,o .• CH3 H H 'NHAc T BSO OTBS o o OTBS H 0! Hypothetical tra .. leading to und n~ltlon structure (Observed). eslred product. H 68 ypothetical tran .. leading to d . Sltlon structure es1red od ( Not observed). pr uct. 69 46 ethers on the furanose ring. Structure 68, which leads to the formation of the desired C5'-R configuration in the product, is believed to possess a destabilizing steric interaction between the disaccharide and the 3'-0-t-butyldimethylsilyl ether. This destabilizing interaction is diminished in structure 67. The proposed steric interaction is believed to be exacerbated in 68 relative to 29 by the additional steric shielding of the N-acetylglucosamine residue. Further consideration of structures 67 and 68 suggested that the replacement of the bulky silyloxy groups of the uridine-derived fragment with hydroxyl groups would not only eliminate the disfavorable steric interaction, but might also induce an associative interaction that draws the N-acetylglucosamine residue closer to the furanose ring by virtue of an intramolecular hydrogen bond between the acetamide carbonyl group and the C3'-hydroxyl group. The proposed transition structure (69) should favor formation of the desired C5'-R stereochemistry in the cyclized product. In order to test this hypothesis, it was necessary to devise a synthesis of the diol 0silylhemiselenoacetal 75 (Scheme XVII). This required a protective group for the 2'- and 3'hydroxyl groups of the uridine moiety that could be removed in the presence of the sensitive 0 -silylhemiselenoacetal functional group. Toward this end, the aldehyde 73, incorporating allyloxy carbonate (Aoc) protective groups39 on the C2'- and C3'-hydroxyl groups, was prepared in a five-step sequence from 5'-0-dimethoxytrityl uridine (70) (Scheme XVI). Transient protection of the C2'- and C3'-hydroxyl groups within 70 (trimethylsilyl chloride (2.5 equiv), triethylamine (5.0 equiv), DMAP (0.02 equiv), dichloromethane, 23 °C, 2 h), followed by the sequential treatment of the resulting bis(trimethylsilyl) ether with di-t-butyl dicarbonate (1.4 equiv) and DMAP (0.02 equiv) in pyridine at 23 OC for 12 h and then potassium fluoride hydrate (2.4 equiv) in methyl alcohol at 23 OC for 3 h, afforded the diol 71 in 79% yield. Exposure of the diol 71 to allylchloroformate (1 0 equiv) in pyridine (-20 OC ~ 0 "C) produced the diallyloxycarbonate derivative (87% ). Subjection of the latter to 47 Scheme XVI c 0 0 C NH L DMTO};:f~O 0 H HO H a; TMSCI (2.5 equiv), Et3 N (5 equiv), DMAP (0.02 equiv), CH2CI2, 23 oc, 2 h; I NBoc l DMTO};:f~O 9oc.?O (1.4 equiv), DMAP, o (0.02 equiv), Py, 23 °C, 12 h; KF•2H 20 (2.4 equiv), MeOH, 23 °C, 3 h, 79% H H0 H a; 71 70 1) (allyi)OCOCI (10 equiv), Py (20 equiv), -20 °C->0 °C, 25min, 87% 2) PhS03H (1.4 equiv), CHCI3, 23 °C, 2 min, 76% 0 H CNBo< o~ ~o2'r''lo Dess-Martin periodinane (3 equiv), H"r--?'H AocO OAoc 73 (crude) AocO OAoc 72 benzenesulfonic acid (1.4 equiv) in chloroform at 23 "C for 2 min afforded the alcohol 72 in 76% yield. Swern oxidation of 72, as previously conducted, produced multiple products, presumably a consequence of the greater lability of the aldehyde 73 due to the electronwithdrawing character of the C2'- and C3'-substituents. Oxidation of 72 with the DessMartin periodinane44 (3.0 equiv) in dichloromethane at 23 OC for 20 min, by contrast, was found to furnish the desired aldehyde (73), isolated as a mixture with its hydrated form. Regeneration of the aldehyde from its hydrated form was readily accomplished prior to siloxane adduct formation by azeotropic drying with toluene. The coupling of the aldehyde 73 with the allylic alcohol 62 proceeded according to procedures described above, involving (1) the treatment of the aldehyde 73 (2.0 equiv) with 48 benzeneselenol (3.0 equiv) and pyridine (3.0 equiv) in dry toluene at 23 "C for 15 min, (2) exposure of the resulting solution to dichlorodimethylsilane (20 equiv) at 23 OC for 4.5 h, (3) removal of excess dichlorodimethylsilane and solvents in vacuo, and (4) treatment of the residue with a solution of the allylic alcohol62 (1 equiv) in pyridine at 23 "C for 5 min. The 0 0 C··" ~~ H H H AocO OAoc 0 PhSeH (3 equiv), Py (3 equiv), PhCH3 , 23 oc, 15 min; Me2SiCI2 (20 equiv), 23 °C, 4.5 h; ~9CJUIV) 0 73 (2 equiv, crude) CH3 cH 3 , BOMO ' CNBoc SePh I~ ··si-o~ / 0 0 O CbzNHO~ A~cO OA~c )--t- ~CH2 # 0 TB H H CH3-r 74 (81%) CH3 siloxane adducts 74 were isolated as an inseparable mixture of C5'-diastereomers (2: 1, stereochemistry not determined) in 81% combined yield. Selective removal of the allyloxy carbonate protective groups (Scheme XVII) proceeded efficiently employing a catalytic quantity of dichlorobis(triphenylphosphine) palladium (0.01 equiv) and tributyltin hydride (3.0 equiv) in moist dichloromethane at 23 OC for 6 min to afford the diols 75 in 85% yield.39 Free-radical cyclization of the diols 75 was initiated by the addition of aliquots of a solution oftriethylborane (1M, hexanes, 0.1 equiv each) at 15-min intervals to a solution of the diols 75 (1 mM) and tributyltin hydride (2.0 equiv) in toluene at 0 OC over 2 h. Subsequent treatment of the crude cyclization products with potassium fluoride hydrate (25 49 Scheme XVII 74 R=Aoc (Ph Bu3SnH 3 P)2PdCl2 (0.01 . (3 . equiV), 23 oc . equtv), CH Cl ' 6 mtn, 85% 2 2• [ ~~SnH(2 equiv), Et 8 ~ equtv), PhCH 0 C,2 h; ! 75 R = H 3 3• KF•2H 20 (25 equiv) MeOH '23 oc,2 h ' BOMO OH CbzNH 1' H OH (60 76 %, + -8% 5'-S ep1mer) · 50 equiv) in methyl alcohol to remove the siloxane tether produced a mixture of tetraols epimeric at C5' in a ratio of 7.5: 1. The major product was determined to possess the desired C5'-R configuration by its transformation to peracetyl a-tunicaminyluracil (27) and comparison with an authentic sample as above. The diastereomer 76 could be separated by careful column chromatography on silica gel eluting with benzene:acetonitrile:isopropyl alcohol (12:4: 1); the desired 5'-R diastereomer 76 was isolated in pure form in 60% yield. This observed reversal of stereoselectivity compared to that of the cyclization of 65 supports the proposed transition structure 69. In further support of this hypothesis, it was found that the cyclization of 7 5 in a pro tic solvent (methyl alcohol) led to an erosion in the stereoselectivity in the carbon-carbon bond forming process (1.6:1, C5'-R : C5'-S), presumably due to disruption of the proposed intramolecular hydrogen bond. Given the complexity of the system, however, it is certainly possible that other factors may be involved. Final Stages Having constructed the carbon framework of the tunicamycin antibiotics, there remained the deprotection steps and the attachment of the lipophilic N-acyl substituent to complete the synthesis. Tunicamycin-V, a major constituent of most fermentation broths of the natural tunicamycins, was selected for preparation, thus necessitating that the (E)-14methyl-2-tetradecenoic acid side chain be synthesized (Scheme XVIII). Ozonolysis of a commercial sample of cyclododecene (77) in a mixture of dichloromethane and methyl alcohol in the presence of sodium bicarbonate (0.6 equiv) at -78 "C for 3 hand treatment of the crude product mixture with triethylamine (2.8 equiv) and acetic anhydride (5.6 equiv) in dichloromethane at 23 OC for 6 h afforded the aldehyde 78 in 94% yield. 40 Olefination of 78 with isopropylidene triphenylphosphorane afforded the corresponding trisubstituted 51 SchemeXVill 0 3 , NaHC03 (0.6 equiv), 10:3 (vlv) CH2CI2 : MeOH, -78 oc, 3h; ~0 (5.6 equiv). Et3N (2.8 equiv), CH2 CI2, 23 oc, 5 h, 94% 78 77 (CHa)2C~Ph 31 (1.5 equiv), NaN(TMS) (1.3 equiv), THF, 2 -78 °C~23 °C, 30 min, 82% j 10% Pd-carbon (cat.), H2 (1 atm), PhCH 3 , 60 °C, 12 h, 96% 79 l LOA (1.2 equiv), (PhSe)2 (2 equiv), THF, -78 oc~ 23 oc, 5.5 h; m.CPBA, (1.2 eq.Jiv), OMS (4.9 equiv), Et3N (1 equiv), 23 oc, 6 h, 55% 1:1 (vlv) 1M NaOH(aql : t-BuOH, 60 oc, 1.5 h, 91% 81 82 52 olefin 79 (82%), which, upon hydrogenation under one atmosphere of hydrogen with 10% palladium on carbon as catalyst in toluene at 60 ·c for 12 h, furnished the methyl ester 80 in 96% yield. Introduction of a,~-unsaturation in the acyl chain was accomplished by the formation of the a-phenylselenide (lithium diisopropylamide (1.2 equiv), THF, -78 ·c, 25 ·c - > 23 ·c, 5.5 h) and oxidation of the crude selenide with m-CPBA (1.2 equiv) in dichloromethane at - 78 ·c for 2 h. Treatment of the oxidation mixture with dimethyl sulfide (4.9 equiv) and Et3N (1.0 equiv) at 23 ·c for 6 h min~ diphenyldiselenide (2 equiv), -78 induced elimination of the selenoxide to form the (E)-a,~-unsaturated ester 81 in 55% yield. Saponification of 81 with aqueous sodium hydroxide in t-butyl alcohol (60 ·c. 1.5 h) produced the crystalline fatty acid 82 (91 %). In the final stages of the synthesis, deprotection of the tetraol 76 (Scheme XIX) by catalytic transfer hydrogenolysis of both the benzyl carbamate and the benzyloxymethyl ether groups was performed with 10% formic acid in methyl alcohol in the presence of a catalytic amount of palladium black at 23 ·c for 1.5 h. Subsequent treatment of the crude amino pentaol with 13% formic acid in methyl alcohol at 40 "C for 5 h led efficiently to hydrolysis of the isopropylidene ketal and the t-butyl carbamate groups. Further treatment of the crude product from the latter reaction with excess hydrofluoric acid in a mixture of acetonitrile and methyl alcohol (1: 1) at 23 ·c for 2 h furnished the amino polyol 1. Purification of 1 was achieved by chromatography with RP-18 reverse-phase silica gel eluting with pyridine: methyl alcohol:water (1: 1: 1.5) ~ 1 was obtained in greater than 90% yield over the entire deprotection sequence. N-Acylation of 1 was accomplished under conditions similar to those described by Suami et az.7 The fatty acid (6.0 equiv) was activated by stirring 82 with 1,3- dicyclohexylcarbodiimide (9.0 equiv) in dichloromethane at 23 "C for 30 min. Aliquots of the latter solution were added (1.0 equiv each) to a solution of 1 in methyl alcohol at 8-h 53 Scheme XIX TBSO H o~c.-r­ CH3-{-o 76 CH3 10% (vlv) HC02 H : MeOH, Pd-black, 23 oc, 1.5 h; 13% (v!v) HC02 H: MeOH, 40 oc. 5 h; HF, 1:1 (vlv) CH3 CN : MeOH, 23 oc , 2 h l JlNH ~ l~o 0 H 1 (crude, >90%) 82 (6 equiv), DCC (9 equiv), CH 2CI2 , MeOH, 23 oc, 2 d fI )lNH ~ ~~0 0 HO---r-~'T'-... HO~.-?- HO Tunicamycin-V (83% from 76) 54 intervals over 2 days to afford, after flash column chromatography through RP-18 reversephase silica gel eluting with methyl alcohol:pyridine:water (1: 1:1) and trituration with chloroform, pure tunicamycin-V. It should be noted that the judicious selection of the protecting groups on the trisaccharide core (76) allowed for a highly efficient deprotection sequence. Thus, the entire deprotection procedure was performed without purification of any intermediate and provided, after fatty acid coupling, purified tunicamycin-V (88 mg) in 83% yield from the tetraol 76. Synthetic 1-V was shown to be identical in all respects (lH NMR, 13C NMR, melting point, mixed melting point, FTIR, HPLC, MS, HRMS, optical rotation) to that of a purified authentic sample. The route described for the synthesis of 1-V is potentially applicable to the preparation of any of the homologous tunicamycin antibiotics by the attachment of the appropriate fatty acid side chain in the final step. Preparation of C5'-epi- Tunicamycin-V Using the synthetic route described for the preparation of tunicamycin-V (1- V), the C5'-S diastereomer 66 was transformed efficiently into the nonnatural tunicamycin isomer C5'-epi-tunicamycin-V (84), outlined in Scheme XX. Thus, using the chemistry described, this stereoisomeric series of tunicamycins is also available for biological evaluation . 55 Scheme XX TBSO 10% (vlv) HC02H: MeOH, Pd-black, 23 °C, 30 min; 13% (vlv) HC02 H: MeOH, 40 °C, 4 h; HF, 1:1 (vlv) CH3 CN : MeOH, 23 °C, 2 h H OTBS l 83 (crude, >90%) 82 (9 equiv), DCC (13.5 equiv), CH 2CI2 , MeOH, 23 oc, 3d fI CH3 OH 0 CH3~ ~----~ 9 N H __..... HOHO#.H H HO CS'-epi-Tunicamycin-V (84) (74% from 66) 56 Summary A convergent, stereoselective synthesis of tunicamycin-V (1-V) and its C5'-epimer is described. Within this synthetic route, an efficient method for carbon-carbon bond formation was developed, involving the silicon-mediated reductive coupling of aldehydes and allylic alcohols. This protocol forms the basis for the stereoselective preparation of tunicaminyluracil (2) and its C5'-epimer, employing the uridine derivative 22 and the galactosamine derivative 15 as the coupling partners. An attractive feature of this reductive coupling procedure is its compatibility with the sensitive trehalose glycosidic linkage within the tunicamycins. This allowed for the synthesis of the carbohydrate core (1) by carboncarbon bond formation between a uridine 5'-aldehyde derivative and a trehalose-linked disaccharide allylic alcohol. Implementation of this synthetic plan led to the development of an efficient procedure for the previously problematic preparation of the ~.a-trehalose linkage within the natural product, using the glycosidic coupling partners 38 and 44 in a variation of the trichloroacetimidate glycosylation method. Subsequent reductive coupling of the trehalose-linked disaccharide allylic alcohol62 with uridine 5'-aldehyde derivatives 73 or 64 allowed for the highly convergent and selective preparation of 1 or its C5'-epimer (83), respectively. The synthesis of the amino polyol intermediates 1 and 83 should allow for the preparation of any of the homologous tunicamycin antibiotics in pure form, as well as of related structures of potential utility as biochemical probes. 57 Experimental Section General Procedures. All reactions were performed in flame-dried round bottom or modified Schlenk (Kjeldahl shape) flasks fitted with rubber septa under a positive pressure of argon, unless otherwise noted. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Where necessary (so noted), solutions were deoxygenated by alternate evacuation/argon flush cycles (greater than three iterations). Organic solutions were concentrated by rotary evaporation below 30 OC at ca. 25 Torr (water aspirator). Flash column chromatography was performed as described by Still et al. employing 230-400 mesh silica ge1. 41 Thin-layer chromatography (analytical and preparative) was performed using glass plates pre-coated to a depth of 0.25 mm with 230400 mesh silica gel impregnated with a fluorescent indicator (254 nm). Materials. Commercial reagents and solvents were used as received with the following exceptions. Tetrahydrofuran, ethyl ether, and dimethoxyethane were distilled from sodium benzophenone ketyl. Dichloromethane, N,N-diisopropylethylamine, diisopropylamine, triethylamine, pyridine, toluene, and acetonitrile were distilled from calcium hydride at 760 Torr. Dimethyl sulfoxide was distilled from calcium sulfate at 40 Torr and was stored over 4A molecular sieves. Carbon tetrachloride was distilled from phosphorous pentoxide at 760 Torr. Oxalyl chloride was distilled at 760 Torr immediately prior to use. The molarity of n-butyllithium solutions was determined by titration using diphenyacetic acid as an indicator (average of three determinations).42 Instrumentation. Infrared (IR) spectra were obtained using a Perkin-Elmer 1600 Ff-IR spectrophotometer referenced to a polystyrene standard. Data are presented as follows: frequency of absorption (cm-1), and intensity of absorption (s = strong, m= 58 medium, w =weak). Proton and carbon-13 nuclear magnetic resonance (lH NMR or 13C NMR) spectra were recorded with a JEOL JX-400 (400 MHz) or aGE QE-300-Plus (300 MHz) NMR spectrometer; chemical shifts are expressed in parts per million (8 scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent (CHCl3: 8 7.26, C6HDs: 8 7.20, CD3COCD2H: 8 2.04, CD2HOD: 8 3.30). Data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t =quartet, m = multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz), and assignment. High performance liquid chromatography (HPLC) was conducted with a Waters 501 HPLC equipped with a Beckman ODS C18 standard reverse phase column and an Isco y4 Absorbance detector set at 255 nm. Optical rotations were determined with a JASCO-DIP-181 polarimeter equipped with a sodium lamp source. High resolution mass spectra were obtained from the University of California, Riverside Mass Spectrometry Facility. Melting points were recorded with a Btichi SMP-20 melting point apparatus and are uncorrected. 59 9 10 a-Methyl Galactopyranoside 10. N-Acetylgalactosamine (9) (5.0 g, 22.6 mmol, 1 equiv) was dissolved in methanolic hydrochloric acid (2% (w/w), 250 mL), and the resulting solution was heated at reflux for 1.5 h. The reaction mixture was allowed to cool to 23 "C, whereupon solid silver carbonate (15.0 g, 54.4 mmol, 2.4 equiv) was added slowly to neutralize the acid catalyst. The solids were removed by filtration, and the filtrate was concentrated in vacuo. The residue, benzaldehyde (25.0 mL, 245.9 mmol, 10.9 equiv), and fused zinc chloride (5.0 g, 36.7 mmol, 1.6 equiv) were combined, and the resulting suspension was stirred vigorously at 23 "C for 25 h. The reaction mixture was shaken with a mixture of water and pentane ( 1:1 (v/v), 160 mL) to afford a white precipitate that was isolated by filtration. The crude, solid product was washed well with ice-cold pentane (100 mL), then was recrystallized from 95% ethyl alcohol to afford pure 10 as white crystals (4.0 g, 55%) (mp 165.0-166.3 "C). lH NMR (400 MHz, acetone-d6) 8: 7.53 (m, 2 H, arom) 7.34 (m, 3 H, arom) 6.95 (s, 1 H, NH), 5.63 (s, 1 H, benzylidene acetal) 4.75 (d, 1 H, J = 3.5 Hz, H 1), 4.31 (ddd, 1 H, J = 3.5, 8.8, 11.4 Hz, H 2), 4.26 (d, 1 H, J = 3.5 Hz, H 4), 4.13 (m, 2 H, H 6), 3.84 (m, 1 H, H 3), 3.73 (d, 1 60 H, J =9.4 Hz, OH), 3.70 (d, 1 H, J = 1.2 Hz, H 5), 3.33 (s, 3 H, OCH3), 1.89 (s, 3 H, Ac). FTIR (neat fllm) cm-1: 3320 (w, br), 2908 (w), 1614 (s), 1557 (m), 1455 (m), 1097 (s), 1045 (s), 994 (m), 773 (m), 700 (s). MS (FAB) m/z: 324 (MH)+, 292 (M+- CH30), 186 (M+- PhCHO, CH30). HRMS (FAB) mlz: Calcd for C16H22N06 (MH)+: 324.1447. Found: 324.1460. 1LC RJ (33% PhH in acetone): 0.42 61 =~,H ~-? AcNH~~ OCH3 p'··''H MEMCI (5 equiv), (i-PrhNEt 0~ M~MOo AcNH (10 equiv), THF, 60 °C, 2 h, 75% H 10 e 1 OCH3 H 11 Methoxyethoxymethyl Ether 11. Methoxyethoxymethyl chloride (4.4 mL, 30.7 mmol, 5.0 equiv) was added to a solution of 10 (2.5 g, 7.7 mmol, 1 equiv) and diisopropylethylamine (13.5 mL, 77.3 mmol, 10.0 equiv) in tetrahydrofuran (20 mL), and the resulting solution was heated at 60 QC for 2 h. The reaction mixture was partitioned between water (600 mL) and ethyl acetate (4 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. The residue was purified by flash column chromatography (20% acetone in ethyl acetate) to give the methoxyethoxymethyl ether 11 (2.37 g, 75%) as a white solid (mp 53.5- 54.0 QC). lH NMR (400 MHz, acetone-d6) 8: 7.51 (m, 2 H, arom), 7.34 (m, 3 H, arom), 6.98 (s, 1 H, NH), 5.65 (s, 1 H, benzylidene acetal), 4.79 (d, 1 H, J = 7.0 Hz, MEM CH2), 4.74 (d, 1 H, J = 3.5 Hz, H 1), 4.67 (d, 1 H, J =7.0 Hz, MEM CH2), 4.51 (d, 1 H, J = 3.2 Hz, H 4), 4.48 (m, 1 H, H 2), 4.12 (m, 2 H, H 6), 3.99 (dd, 1 H, J = 3.5, 11.4 Hz, H 3), 3. 7 5 (m, 1 H, H 5), 3.69 (m, 2 H, MEM CH2), 3.54 (m, 2 H, MEM CH2), 3.35 (s, 62 3 H, CH30), 3.34 (s, 3 H, CH30), 1.85 (s, 3 H, Ac). FI1R (neat ftlrn) crn-1: 3300 (w, br), 2898 (rn), 1659 (s), 1548 (rn), 1453 (w), 1370 (w), 1199 (w), 1134 (rn), 1112 (rn), 1050 (s), 984 (rn), 792 (w), 748 (w), 760 (rn). MS (FAB) mlz: HRMS (FAB) mlz: Calcd forC2oH3oNOg (MH)+: 412.1971. Found: 412.1970. TLC Rt (25% acetone in EtOAc): 0.32 63 Ph H -~)(~ AcNH~O~ OCHa 11 H MEMO NBS (1 .3 equiv), BaC03 (1.6 equiv), CCI4 , reflux, 2 h, 87% OCOPh AcNH~ H 0 1 OCHa CH:!Br e H 12 Bromobenzoate 12. Solid barium carbonate (1.80 g, 9.1 mmol, 1.6 equiv) and N-bromosuccinimide (1.32 g, 7.4 mmol, 1.3 equiv) were added sequentially to a solution of the methoxyethoxymethyl ether 11 (2.34 g, 5.7 mmol, 1 equiv) in carbon tetrachloride (47 mL). The resulting suspension was deoxygenated and was heated at reflux for 2 h, during which time the reaction mixture turned orange and then yellow. After allowing the reaction mixture to cool to 23 T , the solvent was removed in vacuo, and the residue was diluted with dichloromethane (500 mL). Solids were removed by filtration, and the filtrate was washed sequentially with 5% aqueous sodium bisulfate solution (500 mL) and saturated aqueous sodium chloride solution (300 mL). The organic layer was dried (magnesium sulfate) and concentrated, and the residue was purified by flash column chromatography (100% ethyl acetate) to afford 12 (2.42 g, 87 %) as a white solid (mp 57.0-57.5 ·c). lH NMR (400 MHz, acetone-d6) 8: 8.06 (m, 2 H, arom), 7.67 (m, 1 H, arom), 7.55 (m, 2 H, arom), 7.10 (d, 1 H, J = 9.4 Hz, NH), 5.82 (d, 1 H, J = 2.0 Hz, H 4), 4.83 (d, 1 H, J = 3.5 Hz, H 1), 4.81 (d, 1 H, 7.3 Hz, MEM CH2), 4.47 (d, 1 H, J = 7.3 Hz, MEM CH2), 4.47 (m, 1 H, H 2), 64 4.25 (m, 2 H, H 3, 5), 3.81-3.50 (m, 6 H, H 6, MEM CH2). 3.44 (s, 3 H, CH30), 3.37 (s, 3 H, CH30), 1.87 (s, 3 H, Ac). FTIR (neat film) cm-1: 3314 (w), 2924 (w), 1723 (s), 1670 (m), 1543 (m), 1451 (w), 1370 (w), 1268 (s), 1116 (s), 1038 (s), 981 (w), 942 (w), 846 (w), 711 (m). MS (FAB) mlz: 490 (MH)+, 414 (M+- CH30CH2CH20), 307 (M+ -MEMO, C6Hs). HRMS (FAB) mlz: Calcd for C2oH29BrNOg (MH)+: 490.1077. Found: 490.1091. TLC RJ(50% acetone in EtOAc): 0.50 65 ~Ph AcNH ~ OTCH;!Sr OCH3 H 12 MEMO PhSeH (3 equiv), Et3 N OCOPh AcNH~ 0 (6 equiv), DME, reflux, 18 h, 96% ' OCH3 H CH2SePh 6 13 Phenyl Selenide 13. Benzeneselenol (1.81 mL, 16.5 mmol, 3.0 equiv) was added to a solution of 12 (2.70 g, 5.5 mmol, 1 equiv) and triethylamine (4.60 mL) , 33.0 mmol, 6.0 equiv) in dimethoxyethane (40 mL), and the resulting mixture was heated at reflux for 18 h. The reaction solution was partitioned between saturated aqueous sodium bicarbonate solution (300 mL) and ethyl acetate (4 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. The residue was purified by flash column chromatography (100% ethyl acetate) to give 13 (2.98 g, 96%) as a white solid (mp 44.0 "C). lH NMR (400 MHz, acetone-d6) 8: 8.07 (m, 2 H, Bz arom), 7.67 (m, 1 H, Bz arom), 7.55 (m, 4 H, Bz arom, PhSe arom), 7.25 (m, 3 H, PhSe arom), 7.06 (s, 1 H, NH), 5.80 (d, 1 H, J = 2.0 Hz, H 4), 4.80 (m, 2 H, H 1, MEM CH2), 4.47 (m, 1 H, H 2), 4.46 (d, 1 H, J = 7.3 Hz, MEM CH2), 4.21 (m, 2 H, H 3, 5), 3.78 (m, 1 H, MEM CH2), 3.55 (m, 3 H, MEM CH2), 3.38 (s, 3 H, CH30), 3.35 (s, 3 H, CH30), 3.18 (dd, 1 H, J = 66 8.8, 12.6 Hz, H 6), 3.04 (dd, 1 H, J =5.0, 12.6 Hz, H 6), 1.86 (s, 3 H, Ac ). FTIR (neat film) cm-1: 3316 (w), 2934 (w), 2896 (w), 1722 (s), 1674 (m), 1539 (m), 1452 (w), 1371 (w), 1269 (s), 1116 (s), 1040 (s), 981 (w), 941 (w), 711 (m). MS (FAB) m/z: 568 (MH)+, 536 (M+ - CH30), 492 (M+ CH30CH2CH20). HRMS (FAB) mlz: Calcd for C26H34NOgSe (MH)+: 568.1450. Found: 568.1437. TLC Rt(lOO% EtOAc): 0.24 67 ~ MO AcNH OCOPh 0 OCH3 mCPBA (1.5 equiv), CCI4 , M~MO OCOPh AcNH 0 CH 2SePh H 13 -14 "C, 1 h; OMS (15 equiv); reflux, 5 h, 99% ' CH2 OCH3 14 Alkene 14. Solid m-chloroperoxybenzoic acid (ca. 60% (w/w), 2.22 g, 7.7 mmol, 1.5 equiv) was added to a solution of the selenide 13 (2.91 g, 5.14 mmol, 1 equiv) in carbon tetrachloride (40 mL) at -14 "C, and the resulting suspension was stirred at this temperature for 1 h. Excess oxidant was quenched by the addition of dimethyl sulfide (5.66 mL, 77.1 mmol, 15.0 equiv) and triethylamine (1.51 mL, 10.3 mmol, 2.0 equiv), and the mixture was heated at reflux for 5 h. The resulting yellow solution was partitioned between saturated aqueous sodium bicarbonate solution (400 mL) and ethyl acetate (3 x 200 mL); and the combined organic layers were dried (magnesium sulfate) and concentrated. The crude product was purified by flash column chromatography (100% ethyl acetate) to give 14 (2.07 g, 99%) as a white solid (mp 44.0-44.5 OC). lH NMR (400 MHz, acetone-d6) 8: 8.04 (m, 2 H, arom), 7.65 (m, 1 H, arom), 7.54 (m, 2 H, arom), 7.19 (d, 1 H, J = 8.1 Hz, NH), 6.02 (d, 1 H, J = 3.4 Hz, H 4), 4.91 (d, 1 H, J = 3.4 Hz, H 1), 4.83 (s, 1 H, H 6), 4.8 1 (d, 1 H, J = 7.3 Hz, MEM CH2), 4.78 (s, 1 H, H 6), 4.70 (m, 1 H, H 2), 4.57 (d, 1 H, J =7.3 Hz, MEM CH2), 4.26 (dd, 1 H, J = 3.4, 11.2 Hz, H 3), 3.75 (m, 1 H, MEM 68 CH2), 3.55 (m, 3 H, MEM CH2), 3.43 (s, 3 H, CH30), 3.34 (s, 3 H, CH30), 1.89 (s, 3 H, Ac). FfiR (neat film) cm-1: 3287 (w), 2933 (w), 17196 (s), 1663 (s), 1543 (m), 1451 (w), 1369 (w), 1266 (s), 1197 (w), 1132 (m), 1111 (s), 1025 (s), 950 (m), 884 (w), 713 (m). MS (FAB) mlz: 410 (MH)+, 378 (M+- CH30), 334 (M+CH3CH2CH20), 105 (MEMO)+. HRMS (FAB) mlz: Calcd for C2oH2gNOg (MH)+: 410. 1815. Found: 410.181. TLCRtOOO% EtOAc): 0.20 69 MO ~ AcNH AcNH 0 OCH3 ~ MO OCOPh CH2 23 °C, 3 h, 92% 14 Oi O 1 CH2 OCH3 15 Allylic Alcohol 15. Potassium carbonate (2.0 g, 14.5 mmol, 3.0 equiv) was added to a solution of alkene 14 (2.00 g, 4.9 mmol, 1 equiv) in methyl alcohol (35 mL), and the resulting suspension was stirred at 23 "C for 3 h. The reaction mixture was partitioned between saturated aqueous sodium chloride solution (200 mL) and dichloromethane (4 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. Flash column chromatography of the residue afforded 15 (1.37 g, 92%) as a white solid (mp 102.5 "C). 5.97 (d, I H, J = 8.6 Hz, NH), 5.12 (ddd, 1 H, J = 3.6, 8.6, 10.0 Hz, H 2), 4.95 (d, 1 H, J = 3.6 Hz, H 1), 4.77 (s, 1 H, H 6), 4.63 (s, 1 H, H 6), 4.49 (d, 1 H, J =7.7 Hz, MEM CH2), 4.47 (d, 1 H, J = 3.8 Hz, H 4), 4.42 (d, 1 H, J =7.7 Hz, MEM CH2), 4.08 (dd, 1 H, J = 3.8, 10.0 Hz, H 3), 3.48 (m, 1 H, MEM CH2), 3.39 (d, 1 H , J = 4.1 Hz, OH), 3.34 (m, 1 H, MEM CH2), 3.12 (s, 3 H, CH30), 3.11 (m, 2 H, MEM CH2), 3.03 (s, 3 H, CH30), 1.68 (s, 3 H, Ac). 70 FilR (neat film) cm-1: 3589-3096 (m), 2930 (m), 1660 (s), 1649 (s), 1556 (m), 1373 (w), 1249 (w), 1196 (w), 1138 (m), 1104 (s), 1038 (s), 973 (w), 944 (w). MS (FAB) mlz: 306 (MH)+, 289 (M+- CH3), 274 (M+- CH30), 230 (M+ - CH30CH2CH20). HRMS (FAB) mlz: Calcd for C13H24N07 (MH)+: 306.1553. Found: 306.1550. TLC RJ(50% acetone in PhH): 0.20 71 16 17 Uridine-5'-Aldehyde 17. Pyridine (645 !1-L, 8.0 mmol, 8.0 equiv) was added dropwise to a solution of chromium trioxide (400 mg, 4.0 mmol, 4.0 equiv) in a mixture of dichloromethane and N,Ndimethylformamide (4:1 (v/v), 5 mL) at 23 ·c. and the resulting solution was stirred at 23 °C for 15 min. To this solution were added sequentially a solution of 16 (284 mg, 1.0 mmol, 1 equiv) in a mixture of dichloromethane and N,N-dimethylformamide (4: 1 (v/v), 4 mL), and acetic anhydride (378 11L, 4 .0 mmol, 4.0 equiv). After stirring the reaction mixture at 23 ·c for 7 min, excess oxidant was quenched by the addition of anhydrous ethyl alcohol (0.50 mL). The product solution was diluted with ethyl acetate (250 mL) and was filtered through a short column of silica gel topped with sodium sulfate to remove chromium salts. The filtrate was concentrated, and the residue was passed through a column of silica gel at - 14 ·c (5% hexanes in ethyl acetate) to afford crude 17 (130 mg) as a white solid, which was used without further purification in the coupling attempt with allylic alcohol15. 72 0 0 ~~0 1-0 OH DMTO S_)lNH sll~o DMTCI (1 equiv), py, 23 °C, 1 h; ~ o~,. TBSCI (6 equiv), imidazole (12 equiv), DMF, 23 °C, 13 h, 93% TBSO 19 H ' ) - - { 'H OTBS 20 Hydroxy-protected Uridine 20. Uridine (19) (1.00 g, 4.1 mmol, 1 equiv), dimethoxytrityl chloride (1.40 g, 4.10 mmol, 1 equiv), and pyridine (7 mL) were combined, and the resulting solution was stirred at 23 ·c for 12 h. The orange mixture was poured into vigorously stirred ice-water (100 mL), and the resulting yellow precipitate was isolated by filtration . The solid was dried by azeotropic removal of residual water (toluene, 3 x 3 mL) and was dissolved in N ,Ndimethylformamide (3 mL). Imidazole (3.33 g, 49.0 mmol, 12.0 equiv) and t- butyldimethylsilyl chloride (3.70 g, 24.5 mmol, 6.0 equiv) were added sequentially, and the resulting viscous solution was stirred at 23 "C for 13.5 h at which point excess silyl chloride was quenched by the slow addition of methyl alcohol (10 mL). The product mixture was partitioned between water (500 mL) and ethyl acetate (3 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. The residue was purified by flash chromatography (33% ethyl acetate in hexanes) to afford 20 (2.95 g, 93%) as a pale yellow solid (mp 118.0-122.0 "C). IH NMR (400 MHz, CDCl3) 8: 8.47 (s, 1 H, NH), 8.19 (d, 1 H, J = 8.2 Hz, H 6), 7.4-7.2 (m, 9 H, arom), 6.85 (m, 4 H, arom), 5.84 (d, 1 H, J = 1.8 Hz, H 1'), 5.29 (dd, 1 H, J = 2.3, 73 8.2 Hz, H 5), 4.17 (m, 3 H, H 2', 3', 4'), 3.79 (s, 6 H, CH30), 3.71 (dd, 1 H, J = 1-2,9.4 Hz, H 5'), 3.34 (dd, 1 H, J = 1-2, 10.6 Hz, H 5'), 0.90 (s, 9 H, t-butyl), 0.77 (s, 9 H, t-butyl), 0.18 (s, 3 H, SiCH3), 0.10 (s, 3 H, SiCH3), 0.03 (s, 3 H, SiCH3), -0.06 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3184 (m), 3058 (m), 2930 (s), 2856 (s), 1684 (s), 1608 (m), 1509 (s), 1463 (s), 1253 (s), 1175 (m), 836 (s). MS (FAB) m/z: 775 (MH)+, 718 (MH+- t-butyl), 303 (DMT)+. HRMS (FAB) m/z: Calcd for C42H59N20gSi2 (MH)+: 775.3810. Found: 775.3774. TLC RJ (50% EtOAc in hexanes): 0.45 74 PMBCI (2 equiv), NaH (1.5 equiv), DMF, 0 oc, 4.5 h: 2"/o w/w PhS03 H : CHCI3 , o oc, 5 min, 82"/o TBSO OTBS 20 ~ ·l"p"" Jl~o ~~ TBSO OTBS 21 Uridine-5'-Alcohol 21. A solution of the imide 20 (2.90 g, 3.7 mmol, 1 equiv) in N,N-dimethylformamide (5 mL) and neat p-methoxybenzyl chloride (1.01 mL, 7.5 mmol, 2.0 equiv) were added sequentially to a suspension of sodium hydride (135 mg, 5 .6 mmol, 1.5 equiv) in N,Ndimethylformamide (4 mL) at 0 "C. The reaction mixture was stirred at 0 °C for 4.5 h, whereupon excess base was neutralized by the slow addition of methyl alcohol (5 mL). The resulting solution was partitioned between water (500 mL) and ethyl acetate (3 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. The residue was dissolved in a solution of benzenesulfonic acid in chloroform (2% (w/w), 20 mL) at 0 "C, and the resulting orange solution was stirred at 0 °C for 5 min. The product solution was partitioned between saturated aqueous sodium bicarbonate solution (500 mL) and ethyl acetate (3 x 200 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. Flash column chromatography of the residue (50% ethyl acetate in hexanes) afforded 21 contaminated with residual p-methoxybenzyl alcohol. The product was purified by flash column chromatography (gradient elution: dichloromethane --7 50% ethyl acetate in dichloromethane) to give pure 21 (1.82 g, 82%) as a white solid (mp 84.0 "C). 75 lH NMR (400 MHz, CDCl3) 8: 7.43 (d, 2 H, J = 8.8 Hz, arom), 7.39 (d, 1 H, J = 8.1 Hz, H 6), 6.80 (d, 2 H, J = 8.8 Hz, arom), 5.77 (d, 1 H, J = 8.1 Hz, H 5), 5.40 (d, 1 H, J = 6.3 Hz, H 1'), 5.07 (d, 1 H, J = 13.4 Hz, PMB CH2), 4.99 (d, 1 H, J = 13.4 Hz, PMB CH2), 4.62 (dd, 1 H, J = 4.6, 6.3 Hz, H 2'), 4.15 (dd, 1 H, J = 2.7, 4.6 Hz, H 3'), 4.06 (m, 1 H, H 4'), 3.91 (m, 1 H, H 5'), 3.77 (s, 3 H, CH30), 3.68 (m, 1 H, H 5'), 3.34 (dd, 1 H, J = unres., 5.9 Hz, OH), 0.90 (s, 9 H, t-butyl), 0.80 (s, 9 H, t-butyl), 0.08 (s, 3 H, SiCH3), 0.07 (s, 3 H, SiCH3), -0.03 (s, 3 H, SiCH3), -0.19 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3456 (w, br), 2930 (m), 2857 (m), 1710 (m), 1667 (s), 1513 (m), 1462 (m), 1250 (s), 1162 (w), 1097 (w), 836 (m), 776 (m). MS (FAB) mlz: 593 (MH)+, 535 (M+- t-butyl), 121 (PMB)+. HRMS (FAB) mlz: Calcd for C29H49N207Si2 (MH)+: 593.3078. Found: 593.3088. TLC Rt (50% EtOAc in hexanes): 0.43 76 (COCI)2 (6.2 equiv), DMSO (9.4 eq..~iv), E!:JN (15 eq..~iv), TBSO OTBS 21 TBSO OTBS 22 (crude) Uridine-5'-Aldehyde 22. Dimethyl sulfoxide (204 J.!L, 2.9 mmol, 4.7 equiv) was added dropwise to a solution of oxalyl chloride (167 J.!L, 1.9 mmol, 3.1 equiv) in dichloromethane (4 mL) at -78 °C, and the resulting solution was stirred at -7 8 °C for 5 min. To this solution was added dropwise via cannula a solution of 21 (365 mg, 0.62 mmol, 1 equiv) in dichloromethane (4 mL), and the mixture was stirred at -78 "C for 15 min. Triethylamine (669 J.!L, 4.8 mmol, 7.5 equiv) was added at -78 OC and, after 30 min, the cold reaction mixture was poured into saturated aqueous sodium bicarbonate solution (150 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated to afford the uridine 5'-aldehyde derivative 22 (375 mg) as a pale yellow solid. Due to the lability of the product and its susceptibility to hydration, the uridine 5'aldehyde derivative 22 was used in its crude form in the following experiment. 77 I PhSeH (2 equiv), py,23 °C,1h; 22 (2 equiv, crude) _ _ _ _...,.. Me2SiCI2 (20 equiv); 15 (1 equiv), 92% CH3 s(NPMB CH31 f SePh ~ ''SI o~ / M~MO 0 • s· 0 o ,. H AcNH 0 11 H , CH TBSO OCH3 "' 2 OTBS 23 0-Silylhemiselenoacetal Adduct 23. Freshly distilled benzeneselenol (68 )lL, 0.62 mmol, 2.0 equiv) was added to a solution of the aldehyde 22 (375 mg, ca. 0.62 mmol, ca. 2 equiv, azeotropically dried with two 3-mL portions of toluene) in pyridine (6 mL), and the resulting solution was deoxygenated. After stirring at 23 OC for 1 h, the reaction mixture was transferred via cannula to a solution of dichlorodimethylsilane (752 )lL, 6.2 mmol, 20 equiv) in pyridine (6 mL). The resulting solution was deoxygenated and was to stirred at 23 oc for 10 h. The cloudy yellow suspension was concentrated in vacuo at 23 T, and the residue was suspended in toluene (3 mL). Volatiles were removed in vacuo, and the residue was diluted with a mixture of toluene and pyridine (10: 1 (v/v), 6.6 mL). To the suspension was added via cannula a solution of the allylic alcohollS (94 mg, 0.31 mmol, 1 equiv) in pyridine (2 mL), and the resulting reaction mixture was stirred at 23 T for 10 min. The product was partitioned between a mixture of ethyl acetate and pentane (1: 1 (v/v), 200 mL) and water (100 mL). The organic layer was washed with water (100 mL), then was dried (magnesium sulfate) and concentrated. Flash column chromatography (ethyl acetate) of the residue afforded a 1:1 mixture of the C5'-diastereomers 23 (314 mg, 92%). For analytical 78 purposes, the diastereomers could be separated by preparative thin layer chromatography (1:1 ethyl acetate in hexanes). lJa;. 1H NMR (400 MHz, C6D6) 8: 8.04 (d, 1 H, J = 8.2 Hz, H 6), 7.76 (d, 2 H, J = 8.5 Hz, PMB arom.), 7.72 (d, 2 H, J = 7.0 Hz, PhSe arom), 7.10 (t, 2 H, J = 7.0 Hz, PhSe arom), 7.04 (d, 1 H, J =7.3 Hz, PhSe arom), 6.78 (d, 2 H, J = 8.5 Hz, PMB arom), 6.71 (d, 1 H, J = 8.4 Hz, NH), 6.61 (d, 1 H, J = 6.2 Hz, H 1'), 5.90 (d, 1 H, J = 7.9 Hz, H 5), 5.90 (d, 1 H, J = 2.3 Hz, H 5'), 5.28 (d, 1 H, J = 13.2 Hz, PMB CH2), 5.11 (d, 1 H, J = 13.2 Hz, PMB CH2), 5.11 (m, 1 H, H 10'), 5.09 (d, 1 H, J =3.5 Hz, H 11'), 4.67 (t, 1 H, J = 2.4 Hz, H 4'), 4.59 (s, 1 H, H 6'), 4.56 (d, 1 H, J =7.0 Hz, MEM CH2), 4.46 (m, 1 H, H 2'), 4.41 (d, 1 H, J =7.0 Hz, MEM CH2), 4.41 (d, 1 H, J = 2.9 Hz, H 8'), 4.35 (m, 1 H, H 3'), 4.31 (s, 1 H, H 6'), 4.25 (dd, 1 H, J = 2.9, 10.8 Hz, H 9'), 3.79 (m, 1 H, MEM CH2), 3.29 (s, 3 H, CH30), 3.17 (m, 2 H, MEM CH2), 3.14 (s, 3 H, CH30), 3.03 (m, 1 H, MEM CH2), 3.00 (s, 3 H, CH30), 1.84 (s, 3 H, Ac), 1.02 (s, 9 H, t-butyl), 0.99 (s, 9 H, t-butyl), 0.33 (s, 3 H, SiCH3), 0.24 (s, 3 H, 79 SiCH3), 0.23 (s, 6 H, SiCH3), 0.20 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3). FI1R (neat film) cm-1: 3332 (w), 2929 (m), 2856 (w), 1713 (m), 1670 (s), 1513 (m), 1455 (m), 1390 (w), 1251 (m), 1108 (m), 1038 (m), 839 (m), 777 (w), 742 (w). MS (FAB) m/z: 1111 (MH)+, 121 (PMB)+. HRMS (FAB) mlz: Calcd for CsoHsoN3014SeSi3 (MH)+: 1110.4113. Found: 1110.4136. mp: 57.0-59.0 oc. TLC Rt (100% EtOAc): 0.38 ~ 1H NMR (400 MHz, C6D6) 8: 7.75 (m, 5 H, H 6, PMB arom, PhSe arom), 7.12 (m, 2 H, PhSe arom), 7.07 (m, 1 H, PhSe arom), 6.78 (d, 2 H J = 8.5 Hz, PMB arom), 6.68 (d. 1 H, J = 7.3 Hz, H 1'), 6.59 (d, 1 H, J = 8.5 Hz, NH), 6.11 (d, 1 H, J = 8.2 Hz, H 5), 6.00 (d, 1 H, J = 4.7 Hz, H 5'), 5.30 (d, 1 H, J = 13.2 Hz, PMB CH2), 5.15 (m, 1 H, H 10'), 5.12 (d, 1 H, J = 13.2 Hz, PMB CH2), 5.01 (d, 1 H, J = 3.2 Hz, H 11 '), 80 4.60 (m, 4 H, H 2', 3', 6', MEM CH2). 4.52 (d, 1 H, J = 2.9 Hz, H 8'), 4.49 (m, 1 H, H 4'), 4.42 (d, 1 H, J =7.0 Hz, MEM CH2), 4.36 (s, 1 H, H 6'), 4.26 (dd, 1 H, J =2.9, 11.4 Hz, H 9'), 3.72 (m, 1 H, MEM CH2), 3.29 (s, 3 H, CH30), 3.20 (m, 2 H, MEM CH2), 3.11 (s, 3 H, CH30), 3.07 (m, 1 H, MEM CH2), 3.02 (s, 3 H, CH30), 1.79 (s, 3 H, Ac), 1.04 (s, 9 H, t-butyl), 0.97 (s, 9 H, t-butyl), 0.39 (s, 3 H, SiCH3), 0.28 (s, 3 H, SiCH3), 0.27 (s, 3 H, SiCH3), 0.24 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3), 0.06 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3335 (w), 2830 (m), 2856 (w), 1713 (m), 1668 (s), 1513 (w), 1455 (w), 1390 (w), 1251 (m), 1108 (m), 1039 (m), 837 (m), 775 (w), 743 (w). MS (FAB) m/z: 1111 (MH)+, 121 (PMB)+. HRMS (FAB) mlz: Calcd for CsoHsoN3014SeSi3 (MH)+: 1110.4113. Found: 1110.4111. mp: 59.5-61.0 oc. 1LC Rt (100% EtOAc): 0.35 81 0 23 5 Bu3 SnH (5 equiv), E!:JB (0.20 equiv), PhCH3 , -78 oc -> 23 oc, 4 h; • AcNH CNPMB ~0 ,. KF•2H 20 (excess), MeOH, 23 oc, 5 h H OTBS 24 (59%) Diol24. A solution of triethylborane (30 j..!L, 1.0 M in hexanes, 0.03 mmol, 0.2 equiv) was added to a deoxygenated solution of the siloxanes 23 (153 mg, 0.14 mmol, 1 equiv) and tributyltin hydride (185 j..!L, 0.69 mmol, 5.0 equiv) in toluene at -78 T, and the resulting solution was allowed to warm to 23 T over 4 h. Volatiles were removed in vacuo, and the residue was diluted with methyl alcohol (25 mL). Potassium fluoride hydrate (300 mg, 3.2 mmol, 16.0 equiv) was added, and the resulting suspension was stirred at 23 OC for 9 h. Mter dilution with dichloromethane (50 mL), the suspension was filtered, and the filtrate was concentrated. Flash column chromatography of the residue (25% acetone in ethyl acetate) afforded a 5:1 mixture of the diastereomers 24 and 25, respectively (88 mg combined, 71 %) as a white solid. ~ 1H NMR (400 MHz, CDCl3) 8: 8.02 (d, 1 H, J = 8.8 Hz, H 6), 7.44 (d, 2 H, J = 9.1 Hz, arom), 6.80 (d, 2 H, J = 9.1 Hz, arom), 6.21 (d, 1 H, J = 9.4 Hz, NH), 5.79 (d, 1 H, J = 4.5 Hz, H 1'), 5.75 (d, 1 H, J = 8.8 Hz, H 5), 5.03 (s, 2 H, PMB CH2), 4.80 (d, 1 H, J = 7.9 Hz, 82 MEM CH2), 4.79 (d, 1 H, J = 3.8 Hz, H 11 '), 4.67 (d, 1 H, J = 7.9 Hz, MEM CH2), 4.50 (m, 1 H, H 10'), 4.20 (t, 1 H, J =4.4 Hz, H 2'), 4.12 (t, 1 H, J =4.4 Hz, H 3'), 3.95 (m, 3 H, H 5', 7', 8'), 3.89 (m, 2 H, H 4', 9'), 3.84 (m, 1 H, MEM CH2), 3.75 (s, 3 H, CH30), 3.72 (m, 1 H, MEMCH2), 3.57 (m, 3 H, OH, MEM CH2), 3.41 (s, 3 H, CH30), 3.37 (s, 4 H, OH CH30), 2.26 (m, 1 H, H 6'), 1.98 (s, 3 H, Ac), 1.79 (m, 1 H, H 6'), 0.87 (s, 9 H, t-butyl), 0.83 (s, 9 H, t-butyl), 0.07 (s, 3 H, SiCH3), 0.06 (s, 3 H. SiCH3), 0.02 (s, 3 H, SiCH3), -0.02 (s, 3 H, SiCH3). FTIR (neat film) em- I: 3600-3213 (w, br), 2930 (m), 2857 (m), 1712 (m), 1668 (s), 1514 (m), 1455 (m), 1392 (w), 1249 (m), 1109 (m), 1052 (m), 837 (m), 778 (m). MS (FAB) mlz: 898 (MH)+, 866 (M+- CH30), 840 (M+- t-butyl), 822 (M+- CH30CH2CH20), 121 (PMB)+. HRMS (FAB) m/z: Calcd for C42H72N3014Si2 (MH)+: 898.4553. Found: 898.4551 . mp: 100.5-102.0 oc. 83 84 CAN (5.0 equiv), Hp, CH3 CN, 60 oc, 3 h; 3 N HCI, reflux, 3 h; AcNH Acp (38 equiv), DMAP (45 equiv), CH2 CI2 , 0 °C, 2 h, Prep. TLC, 31% (overall) AcO OAc 26 C5'-evi-a-Heptaacetyl Tunicaminyluracil 26. Ceric ammonium nitrate (60 mg, 0.11 mmol, 5.0 equiv) was added to a solution of the diol24 (20 mg, 0.022 mmol, 1 equiv) in a mixture of acetonitrile and water (1 0:1 (v/v), 3.3 mL), and the resulting solution was heated at 60 ·c for 3 h. The reaction mixture was partitioned between saturated aqueous sodium chloride solution (50 mL) and ethyl acetate (3 x 50 mL), and the combined organic layers were dried (sodium sulfate) and concentrated. The residue was diluted with aqueous hydrochloric acid (3 N, 2 mL), and the resulting suspension was heated at reflux for 3 h, at which time volatiles were removed in vacuo at 23 ·c. The solid residue of 5'-epi-tunicaminyluracil was diluted with dichloromethane (5 mL), and to the resulting solution was cooled to 0 ac and treated sequentially with DMAP (120 mg, 1.0 mmol, 45 equiv) and acetic anhydride (80 J.LL, 0.84 mmol, 38 equiv). The reaction mixture was stirred at 0 ac for 2 h, then was diluted with ethyl acetate (50 mL). The product solution was washed sequentially with 0.5 N aqueous hydrochloric acid solution (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and saturated aqueous sodium chloride solution (40 mL). The organic layer dried (sodium sulfate), and concentrated, and the residue was purified by preparative thin layer chromatography (7% methyl alcohol in dichloromethane) to afford 26 as the major product (6.5 mg, 31 %). 85 lH NMR (400 MHz, CDCl3) o: 8.42 (s, 1 H, imide NH), 7.53 (d, 1 H, J = 8.6 Hz, H6),6.17 (d, 1 H,l=3.7 Hz,H 11'),6.13 (d, 1 H, J = 5.6 Hz, H 1'), 5.85 (d, 1 H, J = 8.6 Hz, H 5), 5.44 (d, 1 H, J = 9.9 Hz, amide NH), 5.28 (d, 1 H, J = 2.9 Hz, H 8'), 5.26 (m, 1 H, H 5'), 5.22 (t, 1 H, J = 5.6 Hz, H 2'), 5.19 (dd, 1 H, J = 2.9, 12.0 Hz, H 9'), 5.11 (dd, 1 H, J = 4.0, 5.6 Hz, H 3'), 4.69 (ddd, 1 H, J = 3.7, 9.9, 12.0 Hz, H 10'), 4.22 (d, 1 H, J = 8.3 Hz, H 7'), 4.10 (d, 1 H, J = 4.0 Hz, H 4'), 2.19 (s, 6 H, Ac), 2.11 (s, 3 H, Ac), 2.11 (m, 1 H, H 6'), 2.08 (s, 3 H, Ac), 2.07 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 1.94 (s, 3 H, Ac), 1.62 (m, 1 H, H 6'). FTIR (neat film) cm-1 : 3289 (w, br), 2925 (w), 1747 (s), 1693 (s) 1546 (w), 1458 (w), 1370 (m), 1223 (s), 1123 (w), 1041 (m), 929 (w). MS (FAB) m/z: 700 (MH)+, 640 (M+- AcO). HRMS (FAB) m/z: Calculated for C29H38N3017 (MH)+: 700.2201. Found: 700.2231 . TLC Rt (10% MeOH in CH2Cl2): 0.33 86 0 5 23 tNPMB aiJL~o 8u:JSnH (2 equiv), Et3B (0.25 equiv), MeCN, ~ oc, 30 min; KF•2H2 0 (excess), MeOH, 23 oc, 5 h 0 25 (62%) Diol 25. A solution oftriethylborane (15 J..LL, 1.0 Min hexanes, 0.015 mmol, 0.2 equiv) was added to a deoxygenated solution of the siloxane adducts 23 (110 mg, 0.099 mmol, 1 equiv) and tributyltin hydride (53 J..LL, 0.20 mmol, 2.0 equiv) in acetonitrile (110 mL) at -8 OC. Mter 10 min, a second aliquot of triethylborane solution (5 J..LL, 0.005 mmol, 0.05 equiv) was added. The reaction mixture was stirred at -8 OC for 20 min, and volatiles were removed in vacuo. The residue was diluted with methyl alcohol (30 mL), and potassium fluoride hydrate (1.0 g) was added to the resulting solution. After stirring at 23 OC for 5 h, the reaction solution was concentrated, and the residue was partitioned between saturated aqueous sodium chloride solution (70 mL) and ethyl acetate (6 x 50 mL). The combined organic layers were dried (sodium sulfate) and concentrated, and the products were isolated by radial chromatography (5% methyl alcohol in dichloromethane) to afford separate fractions of25 (55 mg, 62%) and 24 (16 mg. 18%) as white solids. ~ lH NMR (400 MHz, CDCl3) 8: 7.41 (d, 2 H, J =7.5 Hz, arom), 7.22 (d, 1 H, J = 8.3 Hz, H 6), 6.77 (d, 2 H, J =7.5 Hz, arom), 6.16 (d, 1 H, J = 8.8 Hz, NH), 5.79 (d, 1 H, J = 8.3 Hz, 87 H 5), 5.26 (d, 1 H, J =7.9 Hz, H 1'), 5.06 (d, 1 H, J = 13.9 Hz, PMB CH2), 4.95 (d, 1 H, J = 13.9 Hz, PMB CH2), 4.81 (m, 2 H, H 2', MEM CH2), 4.74 (d, 1 H, J = 3.8 Hz, H 11 '), 4.70 (s, 1 H, OH), 4.68 (d, 1 H, J = 7.3 Hz, MEM CH2), 4.49 (m, 1 H, H 10'), 4.13 (d, 1 H, J = 4.4 Hz, H 2'), 4.06 (m, 2 H, H 5', 7'), 3.98 (m, 1 H, H 4'), 3.90 (m, 2 H, H 8', 9'), 3.82 (m, 1 H, MEM CH2), 3.75 (s, 3 H, CH30), 3.74 (m, 1 H, MEM CH2), 3.58 (m, 2 H, MEM CH2). 3.40 (s, 3 H, CH30), 3.38 (s, 3 H, CH30), 3.10 (s, 1 H, OH), 2.04 (m, 1 H, H 6'), 1.98 (s, 3 H, Ac), 1.57 (m, 1 H, H 6'), 0.89 (s, 9 H, t-butyl), 0.72 (s, 9 H, t-butyl), 0.09 (s, 3 H, SiCH3), 0.08 (s, 3 H, SiCH3), -0.10 (s, 3 H, SiCH3), -0.31 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3624-3178 (w, br), 2930 (m), 2860 (w), 1713 (m), 1667 (s), 1514 (m), 1456 (m), 1390 (w), 1250 (m), 1108 (m), 1052 (s), 876 (w), 838 (m), 775 (m). MS (FAB) mlz: 898 (MH)+, 866 (M+- CH30), 840 (M+- t-butyl), 822 (M+- CH30CH2CH20), 121 (PMB)+. HRMS (FAB) m/z: Calcd for C42H72N3014Si2 (MH)+: 898.4553. Found: 898.4528. 88 mp: 100.5-101.5 T. 89 CAN (5.4 equiv), Hp, CH3 CN, 60 oc, 3 h; 3 N HCI, reflux, 3 h; 25 ~0 (38 equiv), OMAP (45 equiv), CH2CI2 , 0 °C, AcNH 2 h, Prep. TLC, 43% (overall) 27 Synthetically-Derived a-Heptaacetyl Tunicaminyluracil 27. Ceric ammonium nitrate (165 mg, 0.30 mmol, 5.4 equiv) was added to a solution of the diol25 (50 mg, 0.056 mmol, 1 equiv) in a mixture of acetonitrile and water (10: 1 (v/v), 5.5 mL), and the resulting solution was heated at 60 OC for 3 h. The reaction mixture was partitioned between saturated aqueous sodium chloride solution (50 mL) and ethyl acetate (3 x 50 mL), and the combined organic layers were dried (sodium sulfate) and concentrated. The residue was diluted with aqueous hydrochloric acid (3 N, 3 mL), and the resulting suspension was heated at reflux for 3 h, at which time volatiles were removed in vacuo at 23 "C. The solid residue of crude tunicaminyluracil was diluted with dichloromethane (5 mL), and the resulting solution was cooled to 0 °C and was treated sequentially with DMAP (300 mg, 2.5 mmol, 45 equiv) and acetic anhydride (200 IlL, 2.1 mmol, 38 equiv). The reaction mixture was stirred at 0 °C for 2 h, then was diluted with ethyl acetate (50 mL). The product solution was washed sequentially with 0.5 N aqueous hydrochloric acid solution (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and saturated aqueous sodium chloride solution (40 mL). The organic layer was dried (sodium sulfate), and concentrated, and the residue was purified by preparative thin layer chromatography (7% methyl alcohol in dichloromethane) to afford 27 as the major product (17 mg, 43 %) (mp 174.5 OC (decomp)). 90 lH NMR (400 MHz, CDCI3) 8: 8.41 (s. 1 H, imide NH), 7.2 1 (d, 1 H, J =8.4 Hz, H 6), 6.12 (d, 1 H, J = 3.5 Hz, H 11'), 5.90 (d, 1 H, J = 5.9 Hz, H 1'), 5.80 (d, 1 H, J = 8.4 Hz, H 5), 5.46 (d, 1 H, J = 9.8 Hz, amide NH), 5.35 (t, 1 H, J =5.9 Hz, H 3'), 5.27 (t, 1 H, J =5.9 Hz, H 2'), 5.24 (d, 1 H, J = 3.3 Hz, H 8'), 5.20 (dd, 1 H, J = 3.3, 11.6 Hz, H 9'), 5.10 (m, 1 H, H 5'), 4.71 (ddd, 1 H, J = 3.5, 9.8, 11.6 Hz, H 10'), 4.07 (m, 2 H, H 4', 7'), 2.19 (s, 3 H, Ac), 2.17 (s, 3 H, Ac), 2.12 (s, 3 H, Ac), 2.10 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 1.95 (s, 3 H, Ac), 1.95 (obscured, 1 H, H 6'), 1.55 (ddd, 1 H, J = 1.9, 8.3, 9.9 Hz, H 6'). 13C NMR (100 MHz, CDCI3) 8: 171.2, 170.6, 170.0, 170.0, 169.6, 169.3, 169.1 (6 x CH3C02, CH3CONH), 162.2 (C 4), 149.9 (C 2), 139.7 (C 6), 103.5 (C 5), 91.1, 88.0, 82.6 (C 1', 4', 11'), 72.4, 69.6, 69.6, 69.4, 68.2, 67.7 (C 2', 3', 5', 8', 9', 10'), 46.8, 32.6 (C 6', 7'), 23.2 (CH3CONH), 20.9, 20.9, 20.8, 20.7, 20.5, 20.4 (6 X CH3C02). 91 FI1R (neat film) cm-1 : 3295 (w, br), 3013 (w), 1746 (s), 1694 (s), 1543 (w), 1455 (w), 1431 (w), 1373 (m), 1222 (s), 1046 (m), 933 (w), 756 (w). MS (FAB) mlz: 700 (MH)+, 640 (M+- AcO). HRMS (FAB) mlz: Calcd for C29H3sN3017 (MH)+: 700.2201. Found: 700.2177. [a]n25: +65.9° (c = 1.67, CHC13). HPLC See Appendix. 92 OAc Authentic Tunicamycin 3 N HCI, reflux, 3 h; AcNH ,. A~O (42 equiv), DMAP H (50 equiv), CH2 CI2 , 0 °C, 2 h, Prep. TLC, 30% (overall) OAc 27 Authentically-Derived a-Heptaacetyl Tunicaminyluracil27. A solution of commercial tunicamycin (20 mg, 0.024 mmol, 1 equiv) in aqueous hydrochloric acid (3 N, 1.5 mL) was heated at reflux for 3 h, and volatiles were removed in vacuo at 23 OC. The residue was diluted with dichloromethane (3 mL), and to this solution were added DMAP (150 mg, 1.2 mmol, 50 equiv) and acetic anhydride (100 J.!L, 1.0 mmol, 42 equiv) in sequence, at 0 °C. The resulting solution was stirred at 0 °C for 2 h, then was diluted with ethyl acetate (50 mL). The product solution was washed sequentially with 0.5 N aqueous hydrochloric acid solution (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and saturated aqueous sodium chloride solution (40 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by preparative thin layer chromatography (7% methyl alcohol in dichloromethane) to afford 27 as the major product (5 mg, 30%) (mp 175.0 T (decomp)). lH NMR (400 MHz, CDCl3) 8: 8.34 (s. 1 H, imide NH), 7.21 (d, 1 H, J = 8.4 Hz, H 6), 6.12 (d, 1 H, J = 3.4 Hz, H 11'), 5.90 (d, 1 H, J =5.9 Hz, H 1'), 5.80 (d, 1 H, J = 8.4 Hz, H 5), 5.47 (d, 1 H, J = 10.0 Hz, amide NH), 5.35 (t, 1 H, J = 5.9 Hz, H 3'), 5.27 (t, 1 H, J = 5.9 Hz, H 93 2'), 5.24 (d, 1 H, J = 3.3 Hz, H 8'), 5.20 (dd, 1 H, J =3.3, 11.4 Hz, H 9'), 5.10 (m, 1 H, H 5'), 4. 71 (ddd, 1 H, J =3.4, 10.0, 11.4 Hz, H 10'), 4.07 (m, 2 H, H 4', 7'), 2.19 (s, 3 H, Ac), 2.17 (s, 3 H, Ac), 2.12 (s, 3 H, Ac), 2.10 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 1.95 (s, 3 H, Ac), 1.95 (obscured, 1 H, H 6'), 1.55 (ddd, 1 H, J = 2.0, 8.2, 9.9 Hz, H 6'). 13C NMR (100 MHz, CDCI3) ~: 171.2, 170.6, 170.0, 170.0, 169.6, 169.3, 169.1 (6 x CH3C02, CH3CONH), 162.1 (C 4), 149.8 (C 2), 139.7 (C 6), 103.5 (C 5), 91.1, 88.0, 82.6 (C 1', 4', 11'), 72.4, 69.7, 69.6, 69.4, 68.2, 67.7 (C 2', 3', 5', 8', 9', 10'), 46.8, 32.6 (C 6', 7'), 23.2 (CH3CONH), 20.9, 20.9, 20. 8, 20.7, 20.5, 20.4 (6 X CH3C02). FTIR (neat film) cm-1 : 3307 (w, br), 3023 (w), 1747 (s), 1694 (s), 1538 (w), 1455 (w), 1431 (w), 1373 (m), 1223 (s), 1046 (m), 933 (w), 756 (w). MS (FAB) mlz: 700 (MH)+, 640 (M+- AcO). HRMS (FAB) mlz: Calcd for C29H38N3017 (MH)+: 700.2201. Found: 700.2229. 94 [a]o25: +63.4. (c = 1.33, CHCl3). HPLC See Appendix. 95 CAN (3 equiv), NaN3 (1.5 equiv), CH3CN, -20 oc, 10 h; OAc NaN02 (3 equiv), 1,4-doxane, ~cO OAc AcO~ H _ 0 ~OAc H H20, 80 °C, 12 h; TBSCI (2 equiv), imidazole (4 equiv), CH2CI2 , 23 °C, 5 h, 33% (overall) 30 N3 TBSO 0 6 1 H OA c H 31 2-Deoxy-2-azidogalactopyranoside 31. A solution of triacetoxy-D-galactal (30) (5.0 g, 18.2 mmol, 1 equiv) in acetonitrile (100 mL) at -40 OC was added via cannula to a powdered mixture of eerie ammonium nitrate (30 g, 54.7 mmol, 3.0 equiv) and sodium azide (1.78 g, 27.4 mmol, 1.5 equiv) at -20 OC over a 20-min period. The resulting orange slurry was stirred at -20 OC for 8 h, at which time the reaction mixture was diluted with water (400 mL). The product solution was extracted with ethyl ether (2 x 250 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated in vacuo. The residue was dissolved in a mixture of 1,4-dioxane (100 mL) and water (15 mL), and sodium nitrite (1.60 g, 23.0 mmol, 1.3 equiv) was added. After heating at 80 OC for 12 h, the reaction mixture was partitioned between water (250 mL) and ethyl ether (250 mL), and the organic layer was washed sequentially with water (200 mL) and saturated aqueous sodium chloride solution (200 mL). The ethereal solution was dried (sodium sulfate) and concentrated, and the residue was subjected to flash column chromatography (50% ethyl acetate in hexanes) to afford the azido sugar intermediate. The anomeric mixture of galactopyranosides was dissolved in dichloromethane (50 mL), and imidazole (3.46 g, 50.8 mmol, 2.8 equiv) and t-butyldimethylsilyl chloride (3.83 g, 25.4 mmol, 1.4 equiv) were added to the resulting solution in sequence. After stirring at 23 OC for 5.5 h, excess silyl chloride was quenched by the slow addition of methyl alcohol (10 96 mL). The product mixture was partitioned between water (200 mL) and dichloromethane (2 x 150 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. Purification of the residue by flash column chromatography (25% ethyl acetate in hexanes) afforded the pure ~-0-t-butyldimethylsilyl ether 31 as a viscous oil (2.7 g) in 33% overall yield from 30. lH NMR (300 MHz, CDCl3) 8: 5.3 1 (dd, 1 H, J = 1.12, 3.4 Hz, H 4), 4.75 (dd, 1 H, J = 3.4, 11.0 Hz, H 3), 4.58 (d, 1 H, J = 7.6 Hz, H 1), 4.15 (dd, 1 H, J = 7.2, 11.3 Hz, H 6), 4.07 (dd, 1 H, J =5.9, 11.3 Hz, H 6), 3.84 (m, 1 H, H 5), 3.60 (dd, 1 H, J = 7.6, 11.0 Hz, H 2), 2.15 (s, 3 H, Ac), 2.04 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 0.94 (s, 9 H, t-butyl), 0.17 (s, 6 H, 2 x SiCH3). FI1R (neat film) cm-1: 2932 (w), 2559 (w), 2114 (s), 1750 (s), 1368 (m), 1236 (s), 1179 (w), 1158 (w), 1115 (w), 1078 (m), 1046 (m), 840 (m), 784 (w). MS (CI (NH3)) mlz: 463 (MNH4)+, 388 (M+- t-butyl), 314 (M+OTBS). HRMS (CI (NH3)) mlz: Calcd for C1&H3sN40gSi (MNR4)+: 463.2224 Found: 463.2210. 97 lLC Rt (20% EtOAc in hexanes): 0.21 98 N3~Ac TBSO~O~OAc H H K2C03 (0.3 equiv), CH 30H, 23 °C, 1h; PhCH(O-f.Pr)2 (1.5 equiv), CSA (0.05 equiv), CH 3 CN, 23 oc, 30 min, 69% 31 32 4.6-0- Benzylidene-ealactopyranoside 32. Solid potassium carbonate (0.5 g, 3.62 mmol, 0.3 equiv) was added to a solution of 31 (5.2 g, 11.7 mmol, 1 equiv) in methyl alcohol (50 mL), and the resulting solution was stirred at 23 OC for 1 h, whereupon strongly acidic Amberlite IR-120 resin (1 g) was added to neutralize the base catalyst. The suspension was filtered, and the filtrate was concentrated in vacuo. The residue was passed through a short column of silica gel eluting with 5% methyl alcohol in ethyl acetate to afford the crude trio! intermediate. Acetonitrile (150 mL), diisopropoxybenzyl acetal (2.98 g, 13.7 mmol, 1.2 equiv), the crude trio! intermediate, and crushed 4A molecular sieves (9 g) were combined, and the resulting suspension was stirred at 23 OC for 1 h. (±)-Camphorsulfonic acid (20 mg, 0.10 mmol, 0.01 eq) was added to the reaction mixture, and the whole was stirred at 23 OC for 30 min. After neutralization of acid catalyst by the addition of excess triethylamine (3 mL), the reaction mixture was diluted with ethyl ether (300 mL), and the solids were removed by filtration. The ethereal solution was washed with water (300 mL), dried (magnesium sulfate), and concentrated, and the residue was purified by flash column chromatography (2.5% ethyl acetate in dichloromethane) providing 32 (3.24 g, 69%) as a viscous oil. 99 7.51 (m, 2 H, arom), 7.40 (m, 3 H, arom), 5.56 (s, 1 H, benzylidene acetal), 4.56 (d, 1 H, J =7.0 Hz, H 1), 4.29 (dd, 1 H, J = 1.4, 12.4 Hz, H 6), 4.16 (dd, 1 H, J = 3.2, 0.6 Hz, H 4), 4.06 (dd, 1 H, J = 12.4, 1.8 Hz, H 6), 3.51 (m, 2 H, H 2,3), 3.42 (d, 1 H, J = 1.4 Hz, H 5), 2.51 (d, 1 H, J = 8.9 Hz, OH), 0.96 (s, 9 H, t-butyl), 0.20 (s, 3 H, SiCH3), 0.18 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3401 (w, br), 2929 (w), 2858 (w), 2112 (s), 1396 (w), 1365 (w), 1252 (m), 1171 (m), 1088 (m), 1055 (m), 995 (m), 837 (m), 820 (w), 783 (w), 733 (w), 698 (w). MS (FAB) m/z: 408 (MH)+, 365 (M+- N3), 350 (M+- t-butyl), 330 (M+- C6Hs). HRMS (FAB) m/z: Calcd for Ct9H3oN30sSi (MH)+: 408.1955. Found: 408.1977. TLC Rt (EtOAc in hexanes): 0.50 100 o.on M in BrCCI3 , 250 w sunlamp, o oc, 2.5 h, 87% * o_,.-;- N3~0Ph TBSO H 32 Br H 33 Bromide 33. A solution of galactopyranoside 32 (4.70 g, 11.6 mmol, 1 equiv) in bromotrichloromethane (150 mL) was divided equally among 10 sealed Pyrex tubes (10 x 1.5 em) and the tubes were irradiated with a 275-watt sunlamp at 0 OC for 2.5 h. The reaction mixtures were combined and concentrated, and the residue was purified by flash column chromatography (25% ethyl acetate in hexanes) to give the bromide 33 (4.88 g, 87%) as a colorless oil. lH NMR (400 MHz, CDC13) 8: 8.11 (m, 2 H, arom), 7.63 (m, 1 H, arom), 7.50 (m, 2 H, arom), 5.63 (dd, 1 H, J = 3.4, 1.0 Hz, H 4), 4.63 (d, 1 H, J = 7.5 Hz, H 1), 3.87 (ddd, 1 H, J = 7.6, 5.9, 1.0 Hz, H 5), 3.71 (m, 1 H, H 3), 3.57 (dd, 1 H, J = 10.3, 7.5 Hz, H 2), 3.41 (m, 2 H, H 6), 2.45 (s(br), 1 H, OH), 0.98 (s, 9 H, t-butyl), 0.24, 0.22 (2 X s, 2 X 3H, Si(CH3h). Be NMR (100 MHz, CDCl3) 8: 166.4, 133.5-128.4 (arom), 97.4, 74.0, 70.5, 70.4, 66.0, 29.1' 25.6, 25.6, 25.5, 17 .8, -4.2, -5.2. 101 FITR (neat film) em- I: 3624-3248 (m, br), 2930 (m), 2858 (m), 2115 (s), 1725 (s), 1452 (w), 1362 (w), 1273 (s), 1115 (s), 1071 (s), 836 (s), 708 (s). MS (FAB) mlz: 486 (MH)+, 354 (M+- OTBS), 105 (C6lfsCO)+. HRMS (FAB) mlz: Calcd for C19H29N30sBrSi (MH)+: 486.1077. Found: 486.1060. TLC Rt (33% EtOAc in hexanes): 0.40 102 HO N OCOPh OCOPh ~ TB~O O H BOMCI (5equiv), (i-PrbNEt Br H 33 ~o . N3 (5.5 equtv), CH2 CI2 , 55 °C, 89"/o TBSO 0 , 0 H Br H 34 C3-0-Benzyloxymethyl Ether 34. Benzyloxymethyl chloride (26.0 mL, 188 mmol, 5.0 equiv) was added to a solution of the bromide 33 (18.3 g, 37.6 mmol, I equiv) and diisopropylethylamine (36.0 mL, 207 mmol, 5.5 equiv) in dichloromethane (150 mL), and the resulting solution was heated at 55 DC for 15 h. The reaction mixture was diluted with dichloromethane (200 mL), and the resulting solution was washed sequentially with water (50 mL) and saturated aqueous sodium chloride solution (50 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was filtered through a short column of silica gel (17% ethyl acetate hexanes) providing the corresponding benzyloxymethyl ether 34 (20.2 g, 89%) as a viscous oil. lH NMR (400 MHz, CDCl3) b: 8.12 (m, 2 H, arom), 7.62 (m, 1 H, arom), 7.50 (m, 2 H, arom), 7.41-7.28 (m, 5 H, arom), 5.71 (dd, 1 H, J = 3.2, 1.0 Hz, H 4), 4.93 (d, 1 H, J = 7.3 Hz, OCH20), 4.75 (d, 1 H, J = 6.6 Hz, Ph0CH20), 4.74 (d, 1 H, J = 7.3 Hz, OCH20), 4.64 (d. 1 H, J =7.6 Hz, H 1), 4.60 (d, 1 H, J =6.6 Hz, PhCH20), 3.83 (m, 1 H, H 5), 3.78 (dd, 1 H, J = 10.5, 3.2 Hz, H 3), 3.66 (dd, 1 H, 10.5, 7.6 Hz, H 103 2), 3.40 (m, 2 H, H 6), 1.00 (s, 9 H, t-butyl), 0.25 (s, 3 H, SiCH3), 0.24 (s, 3 H, SiCH3)~ FfiR (neat film) cm-1: 2930 (m), 2858 (m), 2113 (s), 1728 (s), 1602 (w), 1454 (w), 1267 (s), 1113 (s), 1027 (s), 836 (s), 784 (m), 709 (s). MS (FAB) mlz : 604 (M+- H), 526 (M+- Br). HRMS (FAB) mlz: Calcd for C27H35N306BrSi (M+- H): 604.1511. Found: 604.1479. TLC RJ (33% EtOAc in hexanes): 0.60 104 H2N~OPh N3~0Ph 0 TBS0_,_ - , - - B r H H 34 60 "C 2.5 h, 98% TBSO--t- O~ Br H H 35 Amine35. A solution of the benzyloxymethyl ether 34 (20.2 g, 33.4 mmol, 1 equiv) in triethylamine (50 mL) was added dropwise to a solution of benzeneselenol (10.2 mL, 96 mmol, 2.9 equiv) in triethylamine (150 mL) at 0 "C. The resulting solution was stirred at 0 °C for 5 min, then at 23 °C for 5 min, and finally at 60 "C for 2.5 h. The reaction mixture was concentrated in vacuo, and the yellow residue was dissolved in ethyl acetate (250 mL). The latter solution was washed sequentially with water (2 x 100 mL) and saturated aqueous sodium chloride solution (50 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (gradient elution: 20--? 33% ethyl acetate in hexanes) to furnish the amine 35 (18.9 g, 98%) as a colorless oil. lH NMR (400 MHz, CDCl3) 8: 8.09 (m, 2 H, arom), 7.60 (m, 1 H, arom), 7.47 (m, 2 H, arom), 7.34-7.27 (m, 5 H, arom), 5.68 (d(br), 1 H, J = 2.6 Hz, H 4), 4.96 (d, 1 H, J = 7.1 Hz, OCH20), 4.74 (d, 1 H, J = 7. 1 Hz, OCH20), 4.66 (d, 1 H, J = 11.7 Hz, PhCH20), 4.58 (d, 1 H, J = 7.6 Hz, H 1), 4.56 (d, 1 H, J = 11.7 Hz, PhCH20), 3.87 (dd(br), 1 H, J = 7.8, 5.6 Hz, H 5), 3.81 (dd, 1 H, J = 10.3, 2.6 Hz, H 3), 3.43 (dd, 105 1 H, J = 10.7, 5.4 Hz, H 6), 3.38 (dd, 1 H, J = 10.7, 7.8 Hz, H 6), 3.21 (dd, 1 H, J = 10.3, 7.6 Hz, H 2), 1.79 (m, 2 H, NH2), 0.96 (s, 9 H, tbutyl), 0.22, 0.20 (2 x s, 2x 3 H, Si(CH3)2). 13C NMR (300 MHz, CDCl3) 8: 166.5, 137.3-127.8 (arom), 99.4, 93.1, 77.0, 74.3, 70.0, 67.6, 54.7, 29.7, 25.8, 17.0, -3.8, -5.1. FI1R (neat film) cm-1: 2928 (m), 2857 (m), 1722 (s), 1452 (w), 1271 (s), 1170 (m), 1109 (s), 1044 (s), 837 (s), 783 (m), 708 (m). MS (F AB) mlz: 580 (MH)+, 502 (M+- Br). HRMS (FAB) mlz: Calcd for C27H39N06BrSi (MH)+: 580.1739. Found: 580.1730. 1LC Rt (50% EtOAc in hexanes): 0.20 106 OCOPh ~ H2N TBSO 0 H o-C6 H4 (COCI)2 (2 equiv), Et3 N OCOPh ~ (4 equiv), CH2 CI2 , 0 oc , 10 min; PhlhN Br H 35 6:1 v/v PhCH3 :DBU, 100 °C, 1.5 h, 86% TBS0 O 1 H eBr H 36 Phthalimide 36, A solution of the amine 35 (21.0 g, 36.5 mmol, 1 equiv) and triethylamine (20.0 mL, 146 mmol, 4.0 equiv) in dichloromethane (240 mL) at 0 T was treated with phthaloyl dichloride (10.5 mL, 72.9 mmol, 2.0 equiv), then was stirred at 0°C for 10 min. The reaction solution was concentrated in vacuo, and the residue was diluted with a mixture of toluene and DBU (6: 1 (v/v ), 280 mL). The resulting green solution was heated at 100 OC for 1.5 h, then was cooled to 23 OC. Ethyl acetate (300 mL) was added, and the product solution was washed sequentially with water (2 x 100 mL) and saturated aqueous sodium chloride solution (100 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (33% ethyl acetate in hexanes) to afford phthalimide 36 (22.1 g, 86%) as a colorless oil. lH NMR (400 MHz, CDCl3) 8: 8.17 (m, 2 H, ArH), 7.84-6.98 (m, 12 H, ArH), 5.85 (d(br), 1 H, J =4.4 Hz, H 4), 5.52 (d, 1 H, J =8.0 Hz, H 1), 4.88 (dd, 1 H, J = 11.5, 4.4 Hz, H 3), 4.84 (d, 1 H, J = 7.3 Hz, OCH20), 4.56 (dd, 1 H, J = 11.5, 8.0 Hz, H 2), 4.53 (d, 1 H, J = 7.3 Hz, OCH20), 4.18 (s, 2 H, PhCH20), 4.08 (m, 107 1 H, H 5), 3.47 (m, 2 H, H 6), 0.72 (s, 9 H, tbutyl), 0.14, 0.01 (2 x s, 2 x 3 H, Si(CH3)2); 13C NMR (400 MHz, CDCI3) 8: 168.2, 167.5, 166.1, 137.2-123.1 (arom), 94.0, 94.0, 93.3, 74.4, 71.7, 69.7, 68.3, 54.9, 29.4, 25.4, 17.6, -4.0, -5.4. FI1R (neat film) cm-1: 2955 (m), 2858 (m), 1775 (m), 1715 (s), 1267 (s), 1175 (m), 1110 (s), 1039 (s), 837 (s), 783 (m), 721 (m). MS (FAB) m/z : 708 (M- H)+, 652 (M- t-butyl)+. HRMS (FAB) mlz: Calcd for C35H39BrNOgSi (M+- H): 708.1628. Found: 708.1595. 1LC RJ (50% EtOAc in hexanes): 0.45 108 BOMO OCOPh PhthN~ TBS0--1- O _ , _ Br H BOMO PhSeH (3.2 equiv), Et:JN, (12 eqJiv), DME, 90 °C, 10h, 95% H 36 OCOPh PhthN~ TBSO~o-r--e- SePh H H 37 Phenylselenide 37. Benzeneselenol (10.6 mL, 100 mmol, 3.2 equiv) was added to a deoxygenated solution of bromide 36 (22.0 g, 31.2 mmol, 1 equiv) and triethylamine (50.0 mL, 359 mmol, 11.5 equiv) in anhydrous dimethoxyethane (280 mL), and the resulting solution was heated at 90 OC for 10 h. The reaction mixture was cooled to 23 °C, then was diluted with ethyl ether (300 mL). The resulting solution was washed sequentially with water (100 mL) and saturated aqueous sodium chloride solution (100 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (25% ethyl acetate in hexanes) to afford the phenylselenide 37 (23.3 g, 95%) as a pale yellow oil. lH NMR (400 MHz, CDCl3) o: 8.17 (m, 2 H, arom), 7.84-6.98 (m, 17 H, arom), 5.78 (d(br), 1 H, J = 3.4 Hz, H 4), 5.74 (d, 1 H , J =8.1 Hz, H 1), 4.84 (dd, 1 H, J = 11.2, 3.4 Hz, H 3), 4.82 (d, 1 H, J = 7.6 Hz, OCH20), 4.57 (dd, 1 H, J = 11.2, 8.1 Hz, H 2), 4.5 1 (d, 1 H, J = 7.6 Hz, OCH20), 4.17 (s, 2 H, PhCH20), 3.98 (ddd, 1 H, J = 8.6, 5.1, 1.0 Hz, H 5), 3.21 (dd, 1 H, J = 12.9, 8.6 Hz, H 6), 3.01 (dd, 1 H, J = 12.9, 109 5.1 Hz, H 6), 0.71 (s, 9 H, t-butyl), 0.15, 0.02 (2 X S, 2 X 3 H, Si(CH3h). 13C NMR (300 MHz, CDCI3) 8: 168.6, 168.0, 166.2, 137.1-123.0 (arom), 93.9, 93.0, 73.6, 71.6, 69.6, 54.9, 28.1, 25.4, 17.5,3.9, -5.5; IR (neat film) 2928 (m), 2849 (m), 1775 (m), 1715 (s), 1469 (w), 1389 (s), 1266 (s), 1173 (m), 1113 (s), 1038 (s), 839 (s), 783 (m), 721 (m). MS (FAB) mlz: 786 (M+ - H), 730 (M+ - t-butyl). HRMS (FAB) m/z: Calcd for C41f44NOgSeSi (M+- H): 786.2001. Found: 786.2013. 1LC RJ (25% EtOAc in hexanes): 0.40 110 OCOPh ~ PhthN TBSO 0 H H 37 OCOPh . SePh Et3No3HF (8.7 eqUIV), 23 °C,6h,97% ~ PhthN H0 O e 1 H SePh H 38 Hemiacetals 38. Triethylamine trihydrofluoride (40.0 mL, 250 mmol, 8.7 equiv) was added to a solution of the phenylselenide 37 (22.5 g, 28.6 mmol, 1 equiv) in acetonitrile (120 mL) contained in a 300-mL polyethylene reaction vessel. The resulting solution was stirred at 23 OC for 6 h, then was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic phase was washed sequentially with water (100 mL) and saturated aqueous sodium chloride solution (100 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (gradient elution: 25 -7 50% ethyl acetate in hexanes) to afford 38 as a mixture of anomers (>10:1; f) :a, 18.6 g, 97%) as a colorless oil. The f)-anomer could be fractionally crystallized (ethyl acetate in hexanes) to give white needles (mp 69.0-71.0 T). lH NMR (400 MHz, CDCl3) 0: 8.14 (m, 2 H, arom), 7.83-6.97 (m, 17 H, arom), 5.88 (d(br), 1 H, J =2.9 Hz, H 4), 5.53 (t(br), 1 H, J- 8.1 Hz, H 1), 4.87 (dd, 1 H, J = 11.0, 3.2 Hz, H 3), 4.84 (d, 1 H, J = 7.3 Hz, OCH20), 4.56 (dd, 1 H, J = 11.2, 8.5 Hz, H 2), 4.52 (d, 1 H, J = 7.3 Hz, OCH20), 4.16 (s, 2 H, PhCH20), 4.02 (t(br), 1 H, J- 6.8 Hz, H 5), 4.74 (d(br), I H, J = 111 7.5 Hz, OH), 3.19 (dd, 1 H, J = 12.9, 7.3 Hz, H 6), 3.02 (dd, 1 H, J = 12.9 , 6.3 Hz, H 6). 169.0, 168.2, 166.3, 137.9-123.2 (arom), 93.9, 93.4, 74.3, 72.5, 69.8, 54.9, 28.6. FI1R (neat film) cm-1: 3600-3300 (s), 3062 (m), 2857 (m), 1773 (m), 1714 (s), 1602 (w), 1453 (w), 1392 (s), 1268 (s), 1176 (m), 1114 (s), 1025 (s), 720 (m). MS (FAB) mlz: 673 (M)+, 626 (M+- OH), 566 (M+- C@1 5CH20). HRMS (FAB) mlz: Calcd for C3sH31NOsSe (M)+: 673.1191. Found: 673.1215. TLC Rt (33% EtOAc in hexanes): 0.40 112 H Aca--}..___ AcO~- O H CAN (3 equiv), NaN 3 (2.6 equiv), CH3CN, -20 •c, 10 h; NaN02 (1 equiv), 1,4-<lioxane, Hp, 70 OC, 12 h; TBSCI (2 equiv), imidazole ~ H OAc (3 equiv), CH2 Cl2 , 23 •c, 13 h, 37% (overall) 39 40 2-Deoxy-2-azido&lucopyranoside 40. A solution of triacetoxy-D-glucal (39) (25.0 g, 91.9 mmol, 1 equiv) in acetonitrile (500 mL) at -40 ·c was added via cannula to a powdered mixture of eerie ammonium nitrate (150 g, 274 mmol, 3 equiv) and sodium azide (10 g, 154 mmol, 2.6 equiv) at -20 ·cover a 20-min period. The resulting orange slurry was stirred at -20 · c for 1.5 h, whereupon a second portion of sodium azide (4.7 g, 72.3 mmol, 0.8 equiv) was added to the reaction mixture. The resulting suspension was stirred for 3 h at -20 ·c, and a third portion of sodium azide (4.7 g, 72.3 mmol, 0.8 equiv) was added, and the whole was stirred for an additional 5 h. Ethyl ether (2 L) was added, and the product solution was washed sequentially with water (2 x 1 L) and saturated aqueous sodium chloride solution (1 L). The combined organic layers were dried (magnesium sulfate) and concentrated. The residue was dissolved in a solution of 1,4-dioxane in water (3.5 : 1 (v/v), 450 mL), and sodium nitrite (6.5 g, 94.2 mmol, 1.0 equiv) was added. The resulting solution was heated at 80 °C 12 h, then was concentrated in vacuo to a volume of 100 mL. The product was extracted with three 200-mL portions of ethyl ether, and the combined organic layers were dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (33% ethyl acetate in hexanes), and the purified anomeric alcohols were dissolved in dichloromethane (130 mL). The resulting solution was treated sequentially 113 with imidazole (12.3 g, 181 mmol, 2 equiv) and t-butyldimethylsilyl chloride (18.2 g, 121 mmol, 1.3 equiv). After stirring at 23 OC for 13 h, the reaction mixture was partitioned between water (500 mL) and dichloromethane (500 mL), and the combined organic layers were dried (magnesium sulfate) and concentrated. Purification of the residue by flash column chromatography (25% ethyl acetate in hexanes) afforded the pure ~-0-t­ butyldimethylsilyl ether 40 as a viscous oil (14.7 g) in 37% yield from 39. lH NMR (300 MHz, CDCl3) 0: 4.07 (m, 2 H, H 3,4), 4.64 (d, 1 H, J = 7.7 Hz, H 1), 4.20 (dd, 1 H, J = 12.1 , 6.0 Hz, H 6), 4.12 (dd, 1 H, J = 12.1 2.6 Hz, H 6), 3.68 (m, 1 H, H 5), 3.44 (dd, 1 H, J = 10.3, 7.7 Hz, H 2), 2.09 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 0.94 (s, 9 H, t-butyl), 0.17 (s, 6 H, 2 x SiCH3). FTIR (neat film) cm-1: 2932 (w), 2859 (w), 2113 (s), 1755 (s), 1366 (m), 1228 (s), 1051 (s), 841 (s), 785 (m). MS (FAB) m/z: 446 (MH)+, 430 (M+- CH3), 388 (M+- t-butyl), 314 (M+- OTBS). HRMS (FAB) m/z: Calcd for C18H32N30-8Si (MH)+: 446.1959. Found: 446.1970. TLC Rt (25% EtOAc in hexanes): 0.26 114 H NaOH (0.08 equiv), MeOH, 23 °C, 45 min; CH 3 2 ,2-dmethoxypropane, acetone, p-TSA (0.03 eq.Jiv), 23 oc, 1 .5 h, 76% (overall) 40 ~3 ~ OTBS 1 --(-0 e O CH 3 41 4.6-0-Isopropylidene Glucopyranoside 41. Sodium hydroxide (100 mg, 2.50 mmol, 0.08 equiv) was added to a solution of triacetate 40 (14.50 g, 32.6 mmol, 1 equiv) in methyl alcohol (100 mL), and the resulting solution was stirred at 23 OC for 45 min. The base catalyst was neutralized by the addition of Amberlyst IR-45 acidic ion-exchange resin (2.5 g); after stirring for 5 min, the solids were removed by filtration. The filtrate was concentrated, and the residue, together with crushed activated 5A molecular sieves (9.5 g), was diluted with a solution of acetone and 2,2-dimethoxypropane (5: 1 (v/v), 120 mL). The resulting suspension was stirred at 23 T for 40 min, then solid p-toluenesulfonic acid monohydrate (200 mg, 1.05 mmol, 0.03 equiv) was added, and the reaction mixture was stirred at 23 OC for 1.5 h. The acid catalyst was quenched by the addition of triethylamine (2.0 mL), and the solids were removed by filtration. The filtrate was concentrated, and the residue was purified by flash column chromatography (25% ethyl acetate in hexanes) to afford 41 (8.90 g, 76%) as a viscous oil. lH NMR (300 MHz, CDCl3) o: 4.58 (d, 1 H, J = 10.2 Hz, H 1), 3.86 (dd, 1 H, J = 7.0, 14.8 Hz, H 6), 3.76 (t, 1 H, J = 14.8 Hz, H 6), 3.57 (t, 1 H, J = 12.5 Hz, H 4), 3.45 (ddd, 1 H, J = 3.6, 10.2, 12.5 Hz, H 3), 3.26 (dd, 1 H, J = 10.2, 12.5 Hz, H 2), 3.23 (m, 1 H, H 5), 2.89 (d, 1 H, J 115 =3.6 Hz, OH), 1.50 (s, 3 H, CH3), 1.41 (s, 3 H, CH3), 0.90 (s, 9 H, t-butyl), 0.15 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3458 (m, br), 2931 (m), 2860 (m), 2112 (s), 1473 (w), 1382 (m), 1267 (m), 1198 (m), 1181 (m), 1096 (s), 1039 (m), 966 (m), 842 (s), 784 (m), 682 (w). MS (FAB) mlz: 360 (MH)+, 344 (M+ - CH3), 302 (M+ - t-butyl), 228 (M+ - OTBS). HRMS (FAB) mlz: Calcd for C15H3oN306Si (MH)+: 360.1955. Found: 360.1974. TLC RJ (50% EtOAc in hexanes): 0.59 116 3 CH3 H ~ --.l. 1 ° 0 OTBS TBSCI (2 equiv), imidazole (3 equiv), CH2 0 2 , 23 oc, CH3 1.sh, 97% CH3 3 H TBS~N O OTBS --..t- 0 1 a ° 1 CH3 41 42 BisCTBS) Glucopyranoside 42. Solid t-butyldimethylsilyl chloride (7.45 g, 49.4 mmol, 2.0 equiv) was added to a solution of the glucopyranoside 41 (8.87 g, 24.7 mmol, 1 equiv) and imidazole (5.04 g, 74.1 mmol, 3.0 equiv) in dichloromethane (100 mL) at 23 "C. The reaction mixture was stirred at 23 °C for 1.5 h, then was partitioned between dichloromethane (100 mL) and water (100 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (10% ethyl acetate in hexanes) to give 42 (11.10 g, 95%) as clear, colorless oil. lH NMR (300 MHz, CDCl3) 8: 4.52 (d, 1 H, J = 10.3 Hz, H 1), 3.85 (dd, 1 H, J = 7.1, 14.1 Hz, H 6), 3.75 (t, 1 H, J = 14.1 Hz, H 6), 3.48 (t, 1 H, J = 12.1 Hz, H 4), 3.38 (t, 1 H, J = 12.1 Hz, H 3), 3.16 (dd, 1 H, J = 10.3, 12.1 Hz, H 2), 3.16 (m, 1 H, H 5), 1.45 (s, 3 H, CH3), 1.39 (s, 3 H, CH3), 0.93 (s, 9 H, t-butyl), 0.90 (s, 9 H, t-butyl), 0.14 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3), 0.10 (s, 3 H, SiCH3), 0.07 (s, 3 H, SiCH3). 117 FI1R (neat film) cm-1: 2931 (m), 2859 (m), 2111 (s), 1472 (w), 1380 (w), 1258 (m), 1126 (s), 1094 (s), 968 (w), 836 (s), 781 (m). MS (FAB) mlz: 474 (MH)+, 472 (M+- H), 458 (M+- CH3), 416 (M+ - t-buty1). HRMS (FAB) mlz: Calcd for C21H42N30sSi2 (MH)+: 472.2663,. Found: 472.2675. TLC RJ (25% EtOAc in hexanes): 0.67 118 N3 KFo2H 20 (5.5 equiv), MeOH, 23 °C, 6.5 h, q.Janl ClH TBS~ 0 1 CH3-{- 0 0 O CH3 42 43 Glucosyl Hemiacetal 43. Solid potassium fluoride hydrate (12.0 g, 127.5 mmol, 5.5 equiv) was added to a solution of 42 (11.0 g, 23.3 mmol, 1 equiv) in methyl alcohol (100 mL), and the resulting solution was stirred at 23 OC for 6.5 h. The reaction mixture was partitioned between ethyl acetate (700 mL) and water (300 mL). The organic layer was washed with saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (25% ethyl acetate in hexanes) to afford a mixture of the hemiacetals 43 (a:~ 2:1, 8.49 g, quantitative) as a clear, colorless oil. lH NMR (300 MHz, CDCI3) 8: 5.30 (t, 1 H, 1 =3.6 Hz, H 1a), 5.11 (d, 1 H, 1- 1 Hz, OH~). 4.62 (dd, 1 H, 1 = 1, 8.0 Hz, H 1~), 4.19 (dd, 1 H, 1 = 4.0, 8.4 Hz, H 6~). 4.0- 3.6 (m, H 6a&~. OHa), 3.55 - 3.40 (m, H 3, 4a&~). 3.30- 3.10 (m, H 5, 2 a&~). 1.47 (s, 3 H, CH3), 1.46 (s, 3 H, CH3), 1.40 (s, 3 H, CH3), 1.38 (s, 3 H, CH3), 0.92 (s, 9 H, t-butyl), 0.90 (s, 9 H, t-butyl), 0.16 (s, 3 H, SiCH3), 0.12 (s, 3 H, 119 SiCH3), 0.11 (s, 3 H, SiCH3), 0.08 (s, 3 H, SiCH3). FI1R (neat film) cm-1: 3391 (w, br), 2930 (m), 2859 (m), 2111 (s), 1472 (w), 1383 (w), 1267 (m), 1124 (m), 1088 (s), 838 (s). MS (FAB) m/z: 360 (MH)+, 344 (M+- CH3), 302 (M+- t-butyl), 228 (M+ - OTBS). HRMS (FAB) mlz: Calcd for C15H3oN30sSi: (MH)+ 360.1955. Found: 360.1960. 1LC RJ (50% EtOAc in hexanes): 0.57 120 43 44 l}-Trichloroacetimidate 44. Potassium carbonate (3.0 g, 21.7 mmol, 0.9 equiv) was added to a solution of the hemiacetals 43 (8.40 g, 23.4 mmol, 1 equiv) in a mixture of dichloromethane and trichloroacetonitrile (5:1 (v/v), 120 mL), and the resulting solution was stirred at 23 T for 24 h. Precipitated solids were removed by filtration, and the filtrate was concentrated. The residue was purified by flash column chromatography (10% ethyl acetate in hexanes containing 2% triethylamine) to afford 44 (7.53 g, 64%). Unreacted starting material 43, (1.0 g, 12%) was recovered in separate fractions. lH NMR (300 MHz, CDCl3) 8: 8.73 (s, 1 H, NH), 5.67 (d, 1 H, J = 6.7 Hz, H 1), 3.97 (dd, 1 H, J =4.8, 12.1 Hz, H 6), 3.79 (t, 1 H, J = 12.1 Hz, H 6), 3.55 (m, 3 H, H 2, 3, 4), 3.35 (m, 1 H, H 5), 1.48 (s, 3 H, CH3), 1.38 (s, 3 H, CH3), 0.90 (s, 9 H, t-butyl), 0.14 (s, 3 H, SiCH3), 0.11 (s, 3 H , SiCH3). FI1R (neat film) cm-1: 3347 (w), 2930 (m), 2858 (m), 2113 (s), 1679 (s), 1472 (w), 1373 (m), 1267 (s), 1203 (m), 1173 (m), 121 1065 (s), 971 (m), 835 (s), 798 (s), 781 (s), 647 (m). TLC Rt (20% EtOAc in hexanes): 0.30 122 38 (1 equiv) + 44 (2 equiv) BOMO TfOH (0.06 equiv), PhCH3 , OCOPh PhlhN~ -20 oc, 24 h, 4 Amol. sieves 0 ~ ~sePh 0 TBS~ 0 CH3-r H H 47(11%) . 6 CH3 ~.a-Trehalose Disaccharide 46. Hemiacetal 38 (0.90 g, 1.34 mmol, 1 equiv), imidate 44 (1.33 g, 2.64 mmol, 2.0 equiv), crushed 4 A molecular sieves (2 g), and toluene (10 mL) were combined, and the mixture was stirred at 23 "C for 2 h. The suspension was cooled to -20 "C, and 6 aliquots of a solution of trifluoromethanesulfonic acid in toluene (5% (v/v), 300 11L-aliquots, 0.17 mmol each, 0.06 equiv each) were added dropwise at 4-hour intervals over a 24-hour period. The acid catalyst was neutralized by the addition of triethylamine (100 IlL), and the reaction mixture was diluted with ethyl acetate (50 mL). The product solution was filtered through a pad of Celite and was concentrated. The residue was purified by flash column chromatography (25% ethyl acetate in hexanes) to provide disaccharide 46 (1.04 g, 77%), as well as the a,a-trehalose coupling product 47 (149 mg, 11 %) in separate fractions, both as colorless oils. 123 ~ 1H NMR (400 MHz, C6D6) 8: 8.24 (m, 2 H, arom), 7.64 (m, 1 H, arom), 7.57 (m, 1 H, arom), 7.48 (m, 2 H, arom), 7.08-6.87 (m, 13 H, arom), 5.67 (d(br), 1 H, J = 3.2 Hz, H 8'), 5.64 (d, 1 H, J = 8.8 Hz, H 11'), 5.24 (dd, 1 H, J = 11.0, 8.8 Hz, H 10'), 4.94 (dd, 1 H, J = 11.0, 3.2 Hz, H 9'), 4.83 (d, 1 H, J = 3.7 Hz, H 1"), 4.81 (d, 1 H, J = 7.3 Hz, OCH20), 4.43 (d, 1 H, J = 7.3 Hz, OCH20-, 4.27 (d, 1 H, J = 12.2 Hz, PhCH20), 4.26 (m, 2 H, H 6"), 4.10 (d, 1 H, J = 12.2 Hz, PhCH20), 3.98 (t, 1 H, J = 9.0 Hz, H 3"), 3.65 (m, 2 H, H 5, H 5"), 3.29 (t, 1 H, J = 9.6, H 4"), 3.25 (dd, 1 H, J = 12.5, 9.0 Hz, H 6'), 2.88 (dd, 1 H, J = 12.5, 4.4 Hz, H 6'), 2.82 (dd, 1 H, J =9.5, 3.7 Hz, H 2"), 1.39, 1.24 (2 x s, 2 x 3 H, C(CH3)2), 0.97 (s, 9 H, t-butyl), 0.08, 0.06 (2 X s, 2 X 3 H, Si(CH3)2). 169.2, 167.7, 166.2, 137.8-123.0 (arom), 101.3, 101.1, 99.4, 93.3, 74.8, 74.5, 72.3, 71.2, 69.7, 69.4, 66.4, 65.2, 62.5, 53.2, 29.1, 26.1, 25.9, 19.0, 18.3, -4.2, -5.1. 124 FfiR (neat ftlm) cm-1: 2952 (s), 2856 (s), 2107 (s), 1778 (m), 1714 (s), 1580 (w), 1470 (m), 1392 (s), 1265 (s), 1125 (s), 1025 (s), 970 (m), 865 (s), 720 (s). MS (FAB) mlz: 957 (M+- t-butyl), 156 (PhSe)+. Elemental Analysis: Calcd for CsoHssN4012SeSi: C, 59.22; H, 5.77; N,5.52. Found: C, 59.13; H, 5.79; N, 5.71. TLC RJ (25% EtOAc in hexanes): 0.36 47: 1H NMR (400 MHz, C6D6) S: 8.33 (m, 2 H, arom), 7.67 (m, 1 H, arom), 7.57 (m, 2 H, arom), 7.48 (m, 1 H, arom), 7.17-6.86 (m, 13 H, arom), 6.09 (d, 1 H, J = 3.2 Hz, H 8'), 5.92 (dd, 1 H, J = 11.5, 3.2 Hz, H 9'), 5.69 (d, 1 H, J = 3.9 Hz, H 11 '), 5.48 (dd, 1 H, J = 11.5, 3.9 Hz, H 10'), 5.38 (d, 1 H, J = 3.4 Hz, H 1"), 4.87 (d, 1 H, J = 7.0 Hz, OCH20), 4.83 (d, 1 H, J = 7.0 Hz, OCH20), 4.82 (m, 1 H, H 6"), 4.46 (d, 1 H, J = 12.2 Hz, PhCH20), 4.38 (d, 1 H, J = 12.2 Hz, PhCH20), 4.27 (dd, 1 H, J = 9.5, 8.8 Hz, H 6"), 3.32 (dd, 1 H, J = 12.9, 9.5 Hz, H 3"), 3.26 (m, 2 H, H 6', 7'), 3.19 (t, 1 H, J =9.5 Hz, H 4"), 3.12 125 (m, 1 H, H 5"), 2.99 (dd, 1 H, J = 12.9, 3.4 Hz, H 2"), 2.96 (dd, 1 H, J = 9.8, 3.4 Hz, H 6'), 1.18 (s, 9 H, t-butyl), 1.08 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.57 (s, 3 H, SiCH3), 0.42 (s, 3 H, SiCH3). 168.7, 167.9, 166.1, 138.3-123.4 (arom), 99.0, 94.8, 94.1, 93.1, 74.7, 71.9, 71.7, 70.7, 70.4, 70.3, 65.2, 65.1, 62.0, 52.1, 29.0, 28.9, 26.1, 18.7, 18.6, -3.9, -4.6. FfiR (neat film) cm-1: 2950 (w), 2856 (w), 2108 (s), 1775 (w), 1722 (s), 1470 (w), 1386 (m), 1267 (s), 1135 (m), 1071 (m), 970 (m), 869 (m), 711 (m). MS (FAB) m/z: 656 (galactosyl hemiacetal37- OH)+, 156 (PhSe)+, 131 (OTBS)+. TLC RJ (25% EtOAc in hexanes): 0.22 126 TBSCI (1.5 equiv), imidazole (2.9 equiv), DMF, 23 •c, 12 h, 98% 48 49 N- Acetylglucopyranoside 49. Imidazole (3.90 g, 57.4 mmol, 2.9 equiv) and t-butyldimethylsilyl chloride (4.53 g, 29.9 mmol, 1.5 equiv) were added sequentially to a solution of N-acetylglucosamine derivative 48 (7.00 g, 19.9 mmol, 1 equiv) in N,N-dimethylformamide (50 mL), and the resulting solution was stirred at 23 OC for 12 h. The reaction mixture was partitioned between water (100 mL) and ethyl acetate (100 mL). The organic layer was washed with saturated aqueous sodium chloride solution (50 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (gradient elution: 33% -7 50% ethyl acetate in hexanes) to afford 49 (9.13 g, 98%) as a colorless oil. lH NMR (400 MHz, CDCl3) 8: 7.35 (m, 5 H, arom), 5.50 (d, 1 H, J =9.8 Hz, NH), 4.83 (d, 1 H, J = 3.9 Hz, H 1), 4.68 (d, 1 H, J = 12.0 Hz, PhCH2), 4.44 (d, 1 H, J = 12.0 Hz, PhCH2), 4.23 (dt, 1 H, J = 9.8, 3.9 Hz, H 2), 3.82 (dd, 1 H, J = 10.0, 4.9 Hz, H 6), 3.74 (t, 1 H, J = 10.0 Hz, H 6), 3.68 (t, 1 H, J = 9.8 Hz, H 3), 3.67 (m, 1 H, H 5), 3.57 (t, 1 H, J =9.8 Hz, H 4), 1.93 (s, 3 H, Ac), 1.47 (s, 3 H, CH3), 1.40 (s, 3 H, 127 CH3), 0.84 (s, 9 H, t-butyl), 0.04 (s, 3 H, SiCH3), 0.03 (s, 3 H, SiCH3). 13C NMR (100 MHz, CDCl3) 8: 169.3, 136.9, 128.4, 128.1, 128.0, 99.2, 97.6, 74.7' 71.0, 69.5, 63.8, 62.2, 53.8, 28.9, 25.5, 23.2, 18.8, 18.0, -4.2, -5.1. FTIR (neat film) cm-1: 3291 (m), 2928 (s), 2856 (m), 1653 (s), 1552 (m), 1374 (m), 1200 (m), 1131 (s), 1042 (s), 838 (s). MS (FAB) m/z : 466 (MH)+, 450 (M+- CH3), 408 (M+- t-butyl), 350 (M+ - PhCH20). HRMS (FAB) m/z: Calcd for C24Ii4oN06Si (MH)+ : 466.2625. Found: 466.2604. TLC RJ (33% EtOAc in hexanes): 0 .39 128 U (2 equiv), NH 3(I)• -78 oc, 5 min, 72% 49 50 N-Acetyl~lucosyl Hemiaceta150. A solution of 49 (0.98 g, 2.1 mmol, 1 equiv) in tetrahydrofuran ( 15 mL) was added dropwise via cannula to a solution of lithium metal (30 mg, 4.3 mmol, 2 equiv) in freshly distilled liquid ammonia (75 mL) at -78 °C. The resulting solution was stirred at -78 oc for 5 min, whereupon excess solid ammonium chloride (3 g) was added. The reaction mixture was allowed to wann slowly to 23 T and was stirred at this temperature for 2 h to allow the solvent ammonia to evaporate. The residue was further concentrated in vacuo, then was purified by flash column chromatography (100% ethyl acetate) to afford an anomeric mixture of hemiacetals 50 (2: 1 a:p, 0.60 g, 72%) as a viscous oil. lH NMR (400 MHz, CDCl3) 8: 5.72 (d, 1 H, J =9.5 Hz, NHa), 5.68 (d, 1 H, J = 5.9 Hz, NHp), 5.19 (d, 1 H, J = 3.7 Hz, H la), 4.62 (d, 1 H, J = 8.0 Hz, H 1p), 4.14 (m, 2 H, H 2a,2b), 3.94 (dd, 1 H, J = 11.0, 5.4 Hz, H 6p), 3.8-3.5 (m), 3.24 (m, 1 H, H 5p), 2.05 (s, 3 H, Acp), 2.00 (s, 3 H, Aca), 1.47 (s, 6 H, CH3a&p), 1.40 (s, 6 H, CH3a&p), 0.87 (s, 9 H, t-butylp), 0.85 (s, 9 H, t-butyla), 0.10 (s, 3 H, SiCH3p), 129 0.09 (s, 3 H, SiCH3~). 0.06 (s, 3 H, SiCH3a), 0.05 (s, 3 H, SiCH3a). FI1R (neat film) cm-1: 3300 (m, br), 2930 (m), 2856 (m), 1654 (s), 1534 (m), 1377 (s), 1200 (s), 1130 (s), 965 (w), 868 (s). MS (FAB) mlz: 376 (MH)+, 318 (M+- t-butyl), 268 (M+PhCH20), 244 (M+ - OTBS). HRMS (FAB) mlz: Calcd for CnH3sN06Si (MH)+: 376.2155. Found: 376.2144. TLC Rt (100% EtOAc): 0.40 130 PhCH2 Br (1.3 equiv), NaH (1 .2 equiv), THF, 23 oc, 8 h, 85% 32 51 2-Azido-2-deoxy~alactopyranoside 51. A solution of alcohol32 (1.66 g. 4.1 mmol, 1 equiv) in tetrahydrofuran (25 mL) was added via cannula to a suspension of sodium hydride (120 mg, 4.9 mmol, 1.2 equiv) in tetrahydrofuran (10 mL), and the resulting mixture was stirred at 23 OC for 15 min. Benzyl bromide (630 ~L. 5.3 mmol, 1.3 equiv) was added, and the heterogeneous reaction mixture was heated at 60 "C for 10 h. The suspension was diluted with ethyl ether (200 mL), and the resulting ethereal solution was filtered through a short column of Celite. The filtrate was concentrated, and the residue was purified by flash column chromatography (15% ethyl acetate in hexanes) to provide 51 (1.72 g, 85%) as a pale yellow viscous oil. 1H NMR (400 MHz, CDCl3) 8: 7.53 (m, 2 H, arom), 7.37 (m, 8 H, arom), 5.47 (s, 1 H, benzylidene acetal), 4.74 (s, 2 H, PhCH2), 4.52 (d, 1 H, J =7.8 Hz, H 1), 4.24 (dd, 1 H, J = 12.2, 1.2 Hz, H 6), 4.06 (d(br), 1 H, J = 3.7 Hz, H 4), 4.00 (dd, 1 H, J = 12.2, 1.7 Hz, H 6), 3.78 (dd, 1 H, J = 10.5, 7.8 HZ, H 2), 3.32 (dd, 1 H, J = 10.5, 3.7 Hz, H 3), 3.28 (s(br), 1 H, H 5), 0.97 (s, 131 9 H, t-butyl), 0.19 (s, 3 H, SiCH3), 0.17 (s, 3 H, SiCH3). FI1R (neat film) cm-1: 2929 (w), 2857 (w), 2112 (s), 1454 (w), 1402 (w), 1365 (w), 1284 (w), 1253 (w), 1174 (m), 1108 (s), 1059 (m), 997 (m), 837 (s), 783 (w), 697 (m). MS (FAB) mlz: 496 (M+ - H), 440 (M+ - t-butyl), 420 (M+ - C6Hs). HRMS (FAB) m/z: Calcd for C26H34.N30sSi (M+- H): 496.2268. Found: 496.2289. TLC Rt (25% EtOAc in hexanes): 0.44 132 KF•2H 20 (5 equiv), MeOH, 23 °C , 7 h, 75% PhC~~ p~~ N3 O ' e HO 51 H 52 Galactosyl Hemiacetal 52. Solid potassium fluoride hydrate (0.93 g, 9.9 mmol, 5.0 equiv) was added to a solution of 51 (0.98 g, 1.97 mmol, 1 equiv) in methyl alcohol (40 mL), and the resulting solution was stirred at 23 OC for 7 h. Ethyl acetate (100 mL) was added, and the resulting solution was washed sequentially with water (2 x 100 mL) and saturated aqueous sodium chloride solution (100 mL). The organic phase was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to afford an anomeric mixture of hemiacetals 52 (1.5:1 a :l3, 0.57 g, 7 5%) as a colorless oil. 7.8-7.2 (m, arom), 5.26 (s, 2 H, benzylidene acetal a&l3), 4.86 (t(br), 1 H, J = 3.3 Hz, H 1a), 4.54 (s, 2 H, PhCH2I3), 4.52 (s, 2 H, PhCH2 a), 4.16 (t, 1 H, J =9.2 Hz, H 113), 4.07 (d(br), 1 H, J = 11.8 Hz, H 3a), 4.05 (d(br), 1 H, J = 12.3 Hz, H 613). 3.89 (s,(br), 2 H, H 6a), 3.74 (s(br), 1 H, H 4a), 3.70 (dd, 1 H, J = 10.2, 9.2 Hz, H 213). 3.51 (d, 1 H, J = 3.0 Hz, H 413), 3.42 (d(br), 1 H, J = 11.8 133 Hz, H 2a), 3.32 (d(br), 1 H, J = 12.3 Hz, H 6J3), 3.28 (s(br), 1 H, H 5a), 3.01 (dd, 1 H, J = 10.2, 3.0 Hz, H 3J3), 2.59 (d, 1 H, J = 9.2 Hz, OHI3), 2.31 (s, 1 H, H 5J3), 2.00 (d, 1 H, J = 3.8 Hz, OHa). FI1R (neat ftlm) cm-1: 3420 (w, br), 3049 (w), 2867 (w), 2112 (s), 1454 (w), 1363 (w), 1249 (w), 1170 (w), 1099 (m), 1049 (m), 995 (m), 744 (m), 698 (m). MS(FAB) m/z: 384 (MH)+, 366 (M+- OH), 341 (M+- N3). HRMS (FAB) m/z: Calcd for C2oH22N30s (MH)+: 384. 1559. Found: 384.1548. TLC Rt (50% EtOAc in hexanes): 0.34 134 Ph P~hC~ O)(~ 5:1 v/v CH2CiiCCI3 CN, N3 DBU (0.05 ~iv), 23 •c, 10min 0 O s 1 ~H CCI3 52 H H NH 53 (crude) Galactosyl Trichloroacetimidate 53. DBU (10 ~L. 0.07 mmol, 0.05 equiv) was added to a solution of hemiacetals 52 (500 mg, 1.31 mmol, 1 equiv) in a mixture of trichloroacetonitrile and dichloromethane (1 :5 (v/v), 12 mL). The resulting solution was stirred at 23 ·c for 10 min, then volatiles were removed in vacuo. The residue was filtered through a short column of silica gel, eluting with 33% ethyl acetate in hexanes containing 2% triethylamine to afford the trichloroacetimidate 53, which was used in the following glycosylation reaction without further purification. 135 P~,H PhC~tJ 0 .• 0 50 (3 equiv) + N3 TMSOTf (0.2 equiv), CH2 CI2 , -20 oc, 4 A mol. sieves, 6 h 53 (1 equiv, crude) ~H"' TB~o CH3-t- j 0 H •· o •• CH3 54 (5%) a.6-Disaccharide 54. The crude trichloroacetimidate from the previous experiment (53, 137 mg, -0.26 mmol, -1 equiv), hemiacetals 50 (308 mg, 0.79 mmol, 3.0 equiv), dichloromethane (2 mL), and crushed, activated 4A molecular sieves (200 mg) were combined, and the resulting suspension was stirred at 23 ·c for 2 h. The suspension was cooled to -20 °C, and a solution of trimethylsilyltrifluormethane sulfonate (5% (v/v) in dichloromethane, 250 1.1L, 0.14 mmol, 0.2 equiv total) was added portion-wise at 1-hour intervals over a 5-h period. The suspension was diluted with ethyl ether (10 mL) and was allowed to warm to 23 OC. Solids were removed by filtration through a short column of Celite, and the filtrate was concentrated. The residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to provide disaccharide 54 (12 mg, 5%) and recovered 52 (76 mg, 76%). lH NMR (400 MHz, CDCl3) 8: 7.5-7.2 (m, 10 H, arom), 5.62 (d, 1 H, 1 = 7.8 Hz, NH), 5.44 (s, 1 H, benzylidene acetal), 5.16 (d, 1 H, 1 = 3.2 Hz, H 11 '), 5.15 (d, 1 H, 1 = 8.5 Hz, H 1"), 4.71 (s, 2 H, PhCH2), 4.17 M, 2 H, H 6', 8'), 4.14 (dd, 1 H, 1 = 10.3, 9.5 Hz, H 3"), 3.95 136 (m, 3 H, H 6', 9', 10'), 3.83 (m, 2 H, H 6", 7'), 3.69 (t, 1 H, J = 10.3 Hz, H 4"), 3.40 (t, 1 H, J = 10.4 Hz, H 6"), 3.33 (m, 1 H, H 5"), 3.22 (m, 1 H, H 2"), 1.98 (s, 3 H, Ac), 1.45 (s, 3 H, CH3), 1.39 (s, 3 H, CH3), 0.87 (s, 9 H, t-butyl), 0.06 (s, 3 H, SiCH3), 0.04 (s, 3 H, SiCH3). FTIR (neat film) cm-1: 3286 (w), 2928 (w), 2856 (w), 2113 (s), 1657 (s), 1556 (w), 1372 (m), 1249 (m), 1099 (s), 1040 (s), 860 (m), 697 (m). MS (FAB) mlz: 741 (MH)+, 683 (M+- t-butyl). HRMS (FAB) mlz: Calcd for C37Hs3N4010Si (MH)+: 741.3531. Found: 741.3550. 1LC Rt (50% EtOAc in hexanes): 0.28 137 Ph H ~s. 3.5:1 v/v py:E~N. 23 °C, 20 h, 98% PhwH~ oX~ H~ TBSO H 51 o O 1 H 55 Galactosamine 55. Hydrogen sulfide gas was bubbled through a solution of azido sugar 51 (500 mg, 1.01 mmol, 1 equiv) in a mixture if pyridine and triethylamine (3.5: 1 (v/v), 40 mL) at 23 OC for 20 h. Volatiles were removed in vacuo at 23 "C, and the residue was partitioned between water (100 mL) and ethyl ether (100 mL). The organic layer was washed with saturated aqueous sodium chloride solution (100 mL), then was dried (magnesium sulfate) and concentrated. The residue was purified by flash column chromatography (67% ethyl acetate in hexanes) to afford the amine 55 (465 mg, 98%) as a viscous oil. lH NMR (400 MHz, CDCl3) 8: 7.50 (m, 2 H, arom), 7.30 (m, 8 H, arom), 5.45 (s, 1 H, benzylidene acetal), 4.71 (d, 1 H, J = 12.0 Hz, PhCH2), 4.63 (d, 1 H, J = 12.0 Hz, PhCHz), 4.51 (d, 1 H, J =7.3 Hz, H 1), 4.25 (d(br), 1 H, J = 12.2 Hz, H 6), 4.08 (d, 1 H, J = 2.7 Hz, H 4), 4.03 (dd, 1 H, J = 12.2, 1.9 Hz, H 6), 3.43 (dd, 1 H, J = 10.3, 2.7 Hz, H 3), 3.35 (s(br), 1 H, H 5), 3.27 (dd, 1 H, J = 10.3, 7.3 Hz, H 2), 0.92 (s, 9 H, t 138 -butyl), 0.17 (s, 3 H, SiCH3), 0.14 (s, 3 H, SiCH3). FI1R (neat film) cm-1: 2927 (m), 2856 (m), 1454 (w), 1405 (w), 1366 (w), 1251 (w), 1172 (m), 1105 (s), 1059 (s), 1027 (m), 1002 (m), 880 (w), 836 (s), 782 (m). MS (FAB) m/z: 472 (MH)+, 414 (M+- t-butyl), 340 (M+- OTBS). HRMS (FAB) mlz: Calcd for C26H3gNOsSi (MH)+: 472.2519. Found: 472.2532. TLC Rt (50% EtOAc in hexanes): 0.13 139 P~hC~ p\: H~ TBSO O H P\:,H 0 O:C6 H4 (COCI)2 , (3 equiv), P~hC~0 DBU (6.4 equiv), PhCH3, PhthN 100 °C, 3 h, 94% H 55 TBSO 1 6 O H H 56 Phthalimide 56. DBU (2.64 mL, 17.7 mmol, 6.4 equiv) and phthaloyl dichloride (1.20 mL, 8.26 mmol, 3 equiv) were added sequentially to a solution of amine 55 in toluene (15 mL), and the mixture was heated at 100 OC for 3 h. The reaction mixture was allowed to cool to 23 T, then was diluted with ethyl ether (100 mL). The ethereal solution was washed sequentially with water (2 x 50 mL) and saturated aqueous sodium chloride solution (50 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (33% ethyl acetate in hexanes) to provide 56 ( 1.56 g, 94%) as a colorless oil. 1H NMR (400 MHz, CDCl3) 8: 7.86 (m, 1 H, arom), 7.71 (m, 3 H, arom), 7.60 (m, 2 H, arom), 7.39 (m, 3 H, arom), 7.10 (m, 5 H, arom), 5.51 (s, 1 H, benzylidene acetal), 5.38 (d, 1 H, J = 8.1 Hz, H 1), 4.64 (dd, 1 H, J = 11.2, 8.1 Hz, H 2), 4.62 (d, 1 H, J = 12.4 Hz, PhCH2), 4.49 (d, 1 H, J = 12.4 Hz, PhCH2), 4.46 (dd, 1 H, J = 11.2, 3.4 Hz, H 3), 4.3 (d, 1 H, J = 12.2 Hz, H 6), 4.20 (d, 1 H, J = 3.4 Hz, H 4), 4.08 (d, 1 H, J = 140 12.2 Hz, H 6), 3.50 (s(br), 1 H, H 5), 0.68 (s, 9 H, t-butyl), 0.09 (s, 3 H, SiCH3), 0.04 (s, 3 H, SiCH3). FTIR (neat ftlm) cm-1: 2929 (w), 2857 (w), 1775 (w), 1714 (s), 1470 (w), 1389 (m), 1251 (w), 1172 (w), 1087 (m), 838 (m), 720 (w), 700 (w). MS (FAB) mlz: 600 (M+ - H), 544 (M+ - t-butyl), 470 (M+ OTBS). HRMS (FAB) m/z: Calcd for C34H3gN07Si (M+- H): 600.2418. Found: 600.2422. lLC RJ (50% EtOAc in hexanes): 0.57 141 p\. Ph 0~ PhC~20o PhthN TBSO O H H 56 KF•2H 20 (20 equiv), MeOH, 23 °C, 8h; H 0 PhC~~ )(~ •'H PhthN 5:1 v/v CH 2 CI2 :C03CN, DBU (0.3 equiv), 23 •c, 5 min, 52% CCI3 t 6 ~ H H NH 57 l}-Trichloroacetimidate 57. Solid potassium fluoride hydrate (4.80 g, 52 mmol, 20 equiv) was added to a solution of phthalimide 56 (1.55 g, 2.58 mmol, 1 equiv) in methyl alcohol (90 mL), and the resulting solution was stirred at 23 ·c for 8 h. The reaction mixture was diluted with ethyl acetate (100 mL), and the resulting solution was washed sequentially with water (2 x 100 mL) and saturated aqueous sodium chloride solution (100 mL). The organic phase was dried (sodium sulfate) and concentrated, and the residue was dissolved in a mixture of dichloromethane and trichloroacetonitrile (5: 1 (v/v), 24 mL). DBU (100 ~L. 0.70 mmol, 0.3 equiv) was added, and the mixture was stirred at 23 ·c for 5 min. Volatiles were removed in vacuo, and the residue was purified by flash column chromatography (50% ethyl acetate in hexanes with 3% triethylamine) to afford trichloroacetimidate 57 (876 mg, 52% from 56) as a white solid (mp 154-157 "C). 7.70 (m, 2 H, arom), 7.49 (m, 1 H, arom), 7.40 (m, 1 H, arom), 7.19-6.75 (m, 11 H, arom, H 1), 5.61 (dd, 1 H, J = 11.0, 8.8 Hz, H 2), 5.28 (s, 1 H, benzylidene acetal), 4.76 (dd, 1 H, J = 11.0, 3.4 Hz, H 3), 4.57 (d, 1 H, J = 12.4 Hz, PhCH2), 4.42 142 (d, 1 H, J = 12.4 Hz, PhCH2), 4.15 (dd, 1 H, J = 12.4, 1.5 Hz, H 6), 3.79 (d(br), 1 H, J = 3.4 Hz, H 4), 3.37 (dd, 1 H, J = 12.4, 1.7 Hz, H 6), 2.89 (s(br), 1 H, H 5). FTIR (neat ftlm) em- I: 3336 (w), 2871 (w), 1777 (w), 1715 (s), 1677 (m), 1455 (w), 1389 (s), 1297 (m), 1060 (s), 795 (m), 721 (m). MS (FAB) mlz: 471 (M+- OC(NH)CCl3). 1LC RJ (50% EtOAc in hexanes): 0.24 143 Ph X' H p~_j- \? PhthN~~ TBS~ H 11 0 50 (1.7 equiv) + 57 (1 equiv) CHa-(TMSOTf (0.9 equiv), CH2 CI2 , CHa -20 oc, H •· H o· 58 (24%) 4Amol. sieves, 12 h :){H P~H20O AcNH H PhthN ---.-~ ··~-o 1. 0 6' O •. 11 H H 59 (17%) Trehalose Disaccharides 58 and 59. Hemiacetals 50 (110 mg, 0.28 mmol, 1.7 equiv), trichloroacetimidate 57 (104 mg, 0.16 mmol, 1 equiv), crushed, activated 4A molecular sieves (200 mg), and dichloromethane (1.5 mL) were combined, and the resulting suspension was stirred at 23 "C for 2 h. The reaction mixture was cooled to -20 °C, and trimethylsilyltrifluormethane sulfonate (50 j..tL, 0.26 mmol. 0.9 equiv total) was added portion-wise at 4-h intervals over a 12-h period. The suspension was diluted with ethyl ether (20 mL) and was allowed to warm to 23 OC. Solids were removed by filtration through a short column of Celite, and the filtrate was washed sequentially with water (10 mL) and saturated aqueous sodium chloride solution (10 mL). The etherea1layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (25% ethyl acetate in hexanes) providing the disaccharides 58 (34 mg, 24%) and 59 (23 mg, 17% ), as colorless oils. 144 lH NMR (400 MHz, CDCI3) 8: 7.83-7.03 (m, 14 H, arom), 5.54 (s, 1 H, benzylidene acetal), 5.25 (d, 1 H, J = 8.6 Hz, H 11'), 5.24 (d, 1 H, J = 10.5 Hz, NH), 4.75 (dd, 1 H, J = 11.2, 8.6 Hz, H 10'), 4.72 (d, 1 H, J = 3.9 Hz, H 1"), 4.63 (d, 1 H, J = 12.4 Hz, PhCH2), 4.51 (dd, 1 H, J = 11.2, 3.4 Hz, H 9'), 4.46 (d, 1 H, J = 12.4 Hz, PhCH2), 4.30 (d, 1 H, J = 12.0 Hz, H 6'), 4.24 (d, 1 H, J = 3.4 Hz, H 8'), 4.11 (d, 1 H, J = 12.0 Hz, H 6'), 4.04 (m, 2 H, H 2", 5"), 3.73-3.60 (m, 3 H, H 6", 3"), 3.58 (s(br), 1 H, H 7'), 3.47 (t, 1 H, J =9.3 Hz, H 4"), 1.43 (s, 3 H, Ac), 1.41 (s, 3 H, CH3), 1.35 (s, 3 H, CH3), 0.79 (s, 9 H, t-butyl), 0.03 (s, 6 H, SiCH3). FfiR (neat film) cm-1: 3446 (w), 2928 (w), 2856 (w), 1774 (w), 1715 (s), 1507 (w), 1387 (m), 1174 (w), 1072 (s), 1028 (s), 868 (m), 723 (m). MS (FAB) mlz: 845 (MH)+, 787 (M+ - t-butyl). HRMS (FAB) m/z: Calcd for C4sHs7N2012Si (MH)+: 845.3681. Found: 845.3671 . 1LC RJ (15% EtOAc in CH2Cl2): 0.11 145 ~ IH NMR (400 MHz, CDCI3) 8: 7.86-7.05 (m, 14 H, arom), 5.74 (d, 1 H, J =7.1 Hz, NH), 5.49 (s, 1 H, benzylidene acetal), 5.22 (d, 2 H, 1= 8.3 Hz, H 11', 1"), 4.62 (m, 2 H, H 10', PhCH2), 4.56 (dd, 1 H, J = 11.2, 3.4 Hz, H 9'), 4.51 (d, 1 H, J = 12.4 Hz, PhCH2), 4.40 (t, 1 H, J = 8.6 Hz, H 3"), 4.25 (d, 1 H, J = 12.2 Hz, H 6'), 4.18 (d, 1 H, J = 3.4 Hz, H 8'), 4.05 (d(br), 1 H, J = 12.2 Hz, H 6'), 3.50 (s(br), 1 H, H 7'), 3.31 (dd, 1 H, J = 10.2, 4.6 Hz, H 6"), 3.10 (m, 2 H, H 4", 5"), 2.82 (t, 1 H, J = 10.2 Hz, H 6"), 2.68 (m, 1 H, H 2"), 1.93 (s, 3 H, Ac), 1.28 (s, 3 H, CH3), 1.21 (s, 3 H, CH3), 0.80 (s, 9 H, t-butyl), -0.04 (s, 6 H, SiCH3). FTIR (neat fllm) cm-1: 3260 (w), 2926 (w), 2849 (w), 1772 (w), 1715 (s), 1652 (m), 1393 (m), 1167 (w), 1108 (m), 1073 (s), 857 (m), 714 (m). MS (FAB) mlz: 845 (MH)+, 787 (M+- t-butyl). HRMS (FAB) mlz: Calcd for C4sH57N2012Si (MH)+: 845.3681. Found: 845.3692. 146 46 8:1 vlv EtOH:H 2NNH2 , 100 °C, 12 h, 87% # TBSO 0 CH3-{-0 BOMO Q-l H2N~ 3 0 ~* ,. H H o-y-;:- SePh H e· CH3 C 10'-Amino-disaccharide. A 25-mL heavy-walled Pyrex tube containing a solution of disaccharide 46 (0.81 g, 0.79 mmol, 1 equiv) in a mixture of ethyl alcohol and hydrazine hydrate (8: 1 (v/v), 22.5 mL) was placed under static vacuum and was sealed, then was immersed in an oil bath at 100 T for 12 h. The reaction mixture was cooled to 23 °C, and the product was partitioned between ethyl acetate (75 mL) and water (50 mL). The organic layer was washed sequentially with water (50 mL) and saturated aqueous sodium chloride solution (50 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to afford the intermediate amino alcohol (0.54 g, 87%) as a colorless oil. 7.47 (m, 3 H, arom), 7.30 (m, 2 H, arom), 7.236.99 (m, 5 H, arom), 5.00 (d, 1 H, J = 3.7 Hz, H 1"), 4.54, 4.48 (2 x d, 2 x 1 H, J = 6.8 Hz, OCH20), 4.44 (m, 2 H, PhCH20), 4.33 (m, 2 H, H 5', H 6"), 4.21 (t, 1 H, J = 9.5 Hz, H 3"), 4.17 (d, 1 H, J =7.8 Hz, H 11'), 3.79 (d(br), 1 H, J = 2.9 Hz, H 8'), 3.70 (m, 1 H, H 6"), 3.43 (t, 1 H, J = 9.5 Hz, H 4"), 3.32 (m, 2 H, H 6'), 3.27 (dd, 1 147 H, J = 11.0, 7.8 Hz, H 10'), 3.22 (dd, 1 H, J = 11.0, 2.9 Hz, H 9'), 3.08 (m, 1 H, H 7'), 3.01 (dd, 1 H, J =9.5, 3.7 Hz, H 2"), 1.91 (m(br), 1 H, OH), 1.45, 1.32 (2 x s, 2 x 3 H, C(CH3)2), 1.11 (s, 9 H, t-butyl), 0.50 (m(br), 2 H, NH2), 0.28, 0.21 (2 X s, 2 X 3 H, Si(CH3)2). 138.0-127.0 (arom), 105.9, 100.6, 99.5, 94.0, 81.0, 75.2, 75.0, 71.2, 70.1, 67.2, 66.2, 64.7, 62.6, 52.6, 29.2, 28.4, 26.0, 19.0, 18.5, -3.9, -4.9. FTIR (neat film) cm-1: 3600-2900 (m, br), 2930 (s), 2857 (s), 2107 (s), 1580 (w), 1472 (w), 1383 (m), 1265 (m), 1128 (s), 1024 (s), 970 (m), 856 (s), 737 (s). MS (FAB) mlz: 781 (MH)+, 723 (M+- t-butyl), 624 (M+- SePh). HRMS (FAB) mlz: Calcd for C35H53N409SeSi (MH)+: 781.2747. Found: 781.2731. 1LC Rt (50% EtOAc in hexanes): 0.18 148 H;2N~OMO OH SePh TBS H 0 ~ CH3-(CH3 H H OH equiv)~ CbzNH~ TBSO 0 CH3-L,... H PhCH 20COCI (6.9 py,0°C, 30min,91% 0 ~o,.q- ~sePh T H H o· CH3 60 Benzylcarbamate 60. To a solution of the intermediate amino alcohol (1.35 g, 1.73 mmol, 1 equiv) in pyridine (25 mL) at 0 "C was added benzyl chloroformate (2.30 mL, 15.0 mmol, 8.9 equiv), and the resulting mixture was stirred at 0 °C for 30 min. The reaction mixture was diluted with ethyl ether (150 mL), and the resulting solution was washed sequentially with water (3 x 75 mL) and saturated aqueous sodium chloride solution (50 mL). The ethereal solution was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (33% ethyl acetate in hexanes) to provide 60 (1.35 g, 91 %) as white needles (mp 114.0-115.0 °C). 7.50-6.98 (m, 10 H, arom), 5.15 (d, 1 H, J = 12.2 Hz, PhCH20CO), 5.06 (m(br), 1 H, PhCH20CO), 5.05 (d, 1 H, J = 3.9 Hz, H 1"), 5.00 (d(br), 1 H, J = 7.8 Hz, H 11'), 4.95 (m, 1 H, NH), 4.60 (d, 1 H , J = 6.8 Hz, OCH20), 4.57 (d, 1 H, J = 6.8 Hz, 149 OCH20), 4.47 (d, 1 H, J = 12.1 Hz, PhCH20), 4.45 (d, 1 H, J = 12.1 Hz, PhCH20), 4.25 (m, 2 H, H 5', H 6"), 4.16 (dd, 1 H, J =9.5, 9.0 Hz, H 3"), 4.02 (m(br), 1 H, H 10'), 3.87 (m, 1 H, H 8'), 3.67 (m, 1 H, H 6"), 3.59 (m(br), 1 H, H 9'), 3.46 (dd(br), 1 H, J = 7.3, 6.5 Hz, H 7'), 3.45 (dd, 1 H, J = 9.8, 9.0 Hz, H 4"), 3.30 (dd, 1 H, J = 12.5, 7.3 Hz, H 6'), 3.10 (dd, 1 H, J = 12.5, 6.5 Hz, H 6'), 3.03 (dd, 1 H, J =9.8, 3.7 Hz, H 2''), 2.19 (m(br), 1 H, OH), 1.43, 1.33 (2 X S, 2 X 3 H, C(CH3)2), 1.08 (s, 9 H, t-buty1), 0.28, 0.22 (2 x s, 2 x 3 H, Si(CH3)2). 155.5, 141.9-127.0 (arom), 100.2, 99.6, 93.8, 75.3, 74.9, 70.8, 70.0, 69.6, 68.0, 67.0, 65.8, 65.0, 64.8, 62.7, 54.1, 29.2, 28.4, 26.0, 19.0, 18.5, -3.8, -4.9. FTIR (neat film) cm-1: 3650-3175 (m, br), 3309 (s), 2930 (s), 2857 (s), 2107 (s), 1694 (s), 1556 (s), 1455 (m), 1384 (m), 1248 (s), 1023 (s), 949 (m), 860 (s), 696 (s). 150 Elemental Analysis: Calcd for C43H57N40nSeSi: C, 56.52; H, 6.29; N,6.14. Found: C, 56.17; H, 6.29; N, 6.11. 1LC Rt (33% EtOAc in hexanes): 0.63 151 PhSeH (14 equiv), El:JN, 55 ac, 12 h, 91% C2"-Amino-Disaccharide. Benzeneselenol (2.50 mL, 24.0 mmol, 14.0 equiv) was added to a deoxygenated solution of benzylcarbamate 60 (1.52 g, 1.66 mmol, 1 equiv) in triethylamine (50 mL), and the resulting mixture was heated at 55 "C for 12 h. The product was partitioned between dichloromethane (150 mL) and water (150 mL), and the organic phase was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (33% ethyl acetate in hexanes) to afford the intermediate C2"-amino disaccharide (1.35 g, 91 %) as white needles (mp 172.0-174.0 "C). 7.49-6.99 (m, 10 H, arom), 5.17 (d, 1 H; 1= 12.3 Hz, PhCH20CO), 5.14 (d, 1 H, J = 3.7 Hz, H 1"), 5.11 (d, 1 H, J = 12.3 Hz, PhCH20CO), 4.96 (m(br), 1 H, H 11'), 4.61 (d, 1 H, J =7.1 Hz, OCH20), 4.59 (d, 1 H, J = 7.1 Hz, OCH20), 4.54 (d, 1 H, J = 12.1 Hz, PhOCH2), 4.47 (d, 1 H, J = 12.1 Hz, Ph0CH2), 4.45 (d(br), 1 H, J = 8.3 Hz, NH), 4.31 (m, 2 H, H 5", H 6"), 4.00 (q(br), 1 H, J- 10.5 Hz, H 10'), 3.88 (d(br), 1 H, J = 2.9 Hz, H 8'), 3.79 (t, 1 H, J = 9.0 Hz, H 3"), 3.74 (t, 152 1 H, J = 10.0 Hz, H 6"), 3.52 (d(br), 1 H (obscured), H 3), 3.50 (dd, 1 H, J =9.3, 8.7 Hz, H 4"), 3.43 (dd(br), 1 H, J = 7.3, 6.6 Hz, H 7'), 3.28 (dd, 1 H, J = 12.5, 6.6 Hz, H 6'), 3.13 (dd, 1 H, J = 12.5, 7.3 Hz, H 6'), 2.80 (dd, 1 H, J =9.0, 3.7 Hz, H 2"), 2.61 (m(br), 1 H, OH), 1.46, 1.36 (2 x s, 2 x 3 H, C(CH3)2), 1.02 (s, 9 H, t-butyl), 0.18, 0.15 (2 X S , 2 X 3 H, Si(CH3h). 157.0, 138.1-126.9 (arom), 102.8, 99.3, 93.7, 75.2, 75.2, 74.7, 70.1, 67.7, 67.1, 65.2, 64.8, 63.0, 58.7, 53.5, 29.4, 28.2, 26.3, 19.1, 18.6, -3.7, -4.4. FI1R (neat film) cm-1: 3516-3100 (m, br), 3308 (m), 2928 (s), 2858 (m), 1700 (s), 1544 (s), 1478 (w), 1382 (m), 1248 (s), 1097 (s), 1038 (s), 945 (m), 864 (m), 735 (m). MS (FAB) m/z: 889 (MH+), 556 (galactopyranoside)+, 316 (glucopyranoside)+. HRMS (FAB) m/z: Calcd for C43H61N20nSeSi: (MH)+: 889.3210. Found: 889.3210. TLC RJ (50% EtOAc in hexanes): 0.28 153 61 Diacetyl-Disaccharide 61. The intermediate C2"-amino disaccharide (1.35 g, 1.52 mmol, 1 equiv) was dissolved in a mixture of pyridine and acetic anhydride (2: 1 (v/v), 30 mL), and the resulting solution was heated at 60 "C for 2.5 h. The reaction mixture was diluted with ethyl ether (100 mL), and the resulting solution was washed sequentially with water (50 mL) and saturated aqueous sodium chloride solution (30 mL). The ethereal solution was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (33% ethyl acetate in hexanes) to afford 61 (1.35 g, 91 %) as a colorless oil. 7.45-6.95 (m, 10 H, arom), 6.00 (m(br), 1 H, NHAc), 5.30 (d, 1 H, J = 3.2 Hz, H 8'), 5.24 (d(br), 1 H, J = 12.5 Hz, PhCH20CO), 5.13 (m(br), 1 H, NHC02R), 5.08 (d, 1 H, J = 3.2 Hz, H 1"), 4.92 (d(br), 1 H, J = 12.5 Hz, PhCH20CO), 154 4.70 (d, 1 H, J = 7.3 Hz, OCH20), 4.55 (dt, 1 H, J = 9.3, 3.2 Hz, H 2"), 4.54 (d, 1 H, J = 12.5 Hz, PhCH20), 4.50 (d, 1 H, J = 7.3 Hz, OCH20), 4.32 (m, 2 H, H 5", H 6"), 4.31 (d, 1 H, J = 12.5 Hz, PhCH20), 4.13 (q(br), 1 H, J =obscured, H 10'), 3.90 (t, 1 H, J =9.3 Hz, H 3"), 3.74 (m, 1 H, H 6"), 3.58 (t, 1 H, J = 9.3 Hz, H 4"), 3.50 (dd, 1 H, J = 11.0, 3.2 Hz, H 9'), 3.37 (dd(br), 1 H, J = 8.1, 6.4 Hz, H 7'), 3.03 (dd, 1 H, J = 13.5, 8.1 Hz, H 6'), 2.79 (dd, 1 H, J = 13.5, 6.4 Hz, H 6'), 2.04 (s, 3 H, NHCOCH3), 1.57 (s, 3 H, OCOCH3). 1.39, 1.32 (2 X s, 2 X 3 H, C(CH3)2), 0.96 (s, 9 H, t-butyl), 0.08, 0.04 (2 x s, 2 x 3 H, Si(CH3)2). 170.1, 157.2, 138.1-127.3 (arom), 101.2, 99.5, 92.8, 75.3, 74.6, 73.3, 71.8, 69.9, 67.7, 67.1, 65.2, 62.8, 54.5, 54.2. 29.3, 28.2, 26.0, 23.6, 20.2, 19.2, 18.4, -3.9, -4.7. FI1R (neat film) cm-1: 3525-3100 (m, br), 2929 (s), 2856 (m), 1722 (s), 1667 (s), 1538 (s), 1373 (s), 1296 (w), 1231 (s), 1117 (s), 1068 (s), 1027 (s), 864 (m), 737 (m). MS (FAB) mlz: 973 (MH+), 915 (M+- t-butyl). 155 HRMS (FAB) m/z: Calcd for C47H6sN2013SeSi: (MH)+: 973.3437. Found: 973.3421. 1LC RJ (50% EtOAc in hexanes): 0.63 156 OAc m-CPBA (3.5 equiv), CH2 CI2 , o oc, 30 min; OMS (12 equiv), 61 Et:JN (3 equiv), 65 °C, 10 h, 88% TBSO 0-..;_..,--- CHa-(-0 s· CHa Allylic Acetate Disaccharide. Solid m-chloroperoxybenzoic acid (-60% (w/w), 1.38 g, 4.8 mmol, 3.5 equiv) was added to a solution of selenide 61 (1.35 g, 1.39 mmol, 1 equiv) in carbon tetrachloride (10 mL) at -15 ·c. The resulting suspension was stirred at -15 °C for 20 min, and then at 0 T for 30 min. Excess oxidant was quenched by the sequential addition of dimethyl sulfide (1.20 mL, 16.0 mmol, 12.0 equiv) and triethylamine (0.5 mL, 4.0 mmol, 3.0 equiv), and the resulting solution then was heated at 65 ·c for 10 h. The product was partitioned between ethyl acetate (100 mL) and saturated aqueous sodium bicarbonate solution (40 mL). The organic layer was washed sequentially with water (40 mL) and saturated aqueous sodium chloride solution (40 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (gradient elution: 33 ~ 50% ethyl acetate in hexanes) to provide the intermediate allylic acetate disaccharide (1.09 g, 88%) as a colorless oiL 7.26-7.06 (m, 10 H, arom), 6.25 (d(br), 1 H, J = 9.8 Hz, NHAc), 5.86 (d, 1 H, J = 3.2 Hz, H 8'), 5.18 (d, 1 H, 1= 12.2 Hz, PhCH20CO), 5.10 (d(br), 1 H, J = 7.0 Hz, NHC02R), 5.04 (d, 1 H, J =3.7 Hz, H 1"), 4.97 (d(br), 1 H, J = 12.2 Hz, 157 PhCH20CO), 4.69 (d, 1 H, J = 1.0 Hz, H 6'), 4.65 (ddd, 1 H, J = 9.8, 9.8, 3.7 Hz, H 2"), 4.62 (d, 1 H,J=7.1 Hz,OCH20),4.61 (d, 1 H,J=6.1 Hz, H 11'), 4.54 (d, 1 H, J = 7.1 Hz, OCH20), 4.53 (d, 1 H, J = 1.0 Hz, H 6'), 4.52 (d, 1 H, J = 12.3 Hz, PhCH20), 4.42 (ddd, 1 H, J = 10.5, 7.0, 6.1 Hz, H 10'), 4.41 (d, 1 H, J = 12.3 Hz, PhCH20), 4.24 (ddd, 1 H, J = 10.5, 10.5, 5.3 Hz, H 5"), 3.96 (dd, 1 H, J = 9.8, 9.7 Hz, H 3"), 3.94 (dd, 1 H, J = 10.5, 5.3 Hz, H 6"), 3.73 (t, 1 H, J = 10.5 Hz, H 6"), 3.60 (td, 1 H, J = 10.0, 9.7 Hz, H 4"), 3.59 (dd, 1 H, J = 10.5, 3.2 Hz, H 9'), 2.13 (s, 3 H, NHCOCH3), 1.77 (s, 3 H, OCOCH3), 1.43, 1.34 (s, 9 H, t-butyl), 0.17, 0.15 (2 x s, 2 x 3 H, Si(CH3)2). 169.8, 169.7, 157.2, 152.2, 137.9-127.6 (arom), 103.8, 101.2, 99.5, 93.7, 75.1, 73.9, 72.0, 70.0, 68.0, 67.2, 65.3, 62.5, 54.4, 54.2, 53.5, 29.3, 26.0, 23.5, 20.5, 19.1, 18.5, -3.9, -4.7. FTIR (neat fllm) cm-1: 3530-3125 (m, br), 2952 (s), 1745 (s), 1715 (s), 1666 (s), 1523 (s),, 1372 (s), 1230 (s), 1113 (s), 1023 (s), 864 (m), 698 (m). 158 MS (FAB) mlz: 815 (MH)+, 757 (M+- t-butyl). HRMS (FAB) mlz: Calcd for C4tHs9N2013Si (MH)+: 815.3820. Found: 815.3786. 1LC Rt (50% EtOAc in hexanes): 0.46 159 BOMO OAc CbzNH~ )__,_ ~CH2 0 TBS CH 3 -f CHa H ~ H K2C0 3 (0.07 eq..~iv), MeOH, ~rn CbzNH o ~cNH 11 23 °C, 2 h, 92% TBSO 0 CHa-L-o T H 0 1' cH 2 o· H •. CHa 62 Allylic Alcohol Disaccharide 62. Solid potassium carbonate (10 mg, 0.07 mmol, 0.07 equiv) was added to a solution of the intennediate allylic acetate disaccharide (0.81 g, 1.0 mmol, 1 equiv) in methyl alcohol (25 mL), and the resulting suspension was stirred at 23 OC for 2 h. The product was partitioned between ethyl acetate (100 mL) and water (40 mL). The organic layer was washed sequentially with water (40 mL) and saturated aqueous sodium chloride solution (40 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to afford allylic alcohol 62 (703 mg, 92%) as a colorless oil. 7.29-7.08 (m, 10 H, arom), 6.06 (d(br), 1 H, J8.6 Hz, NHAc), 5.17 (d, 1 H, J = 12.2 Hz, PhCH20CO), 5.12 (d, 1 H, J = 3.7 Hz, H 1"), 5.08 (d(br), 1 H, J- 8.3 Hz, NHC02CH2Ph), 5.04 (d(br), 1 H, J = 12.2 Hz, PhCH20CO), 4.71 (s, 1 160 H, H 6'), 4.70 (d, 1 H, J = 6.5 Hz, H 11'), 4.63 (dt, 1 H, J = 10.8, 3.7 Hz, H 2"), 4.57 (m, 2 H, OCH20), 4.49 (s, 2 H, PhCHzO), 4.45 (s, 1 H, H 6'), 4.33 (dt, 1 H, J = 8.6, 6.5 Hz, H 10'), 4.28 (dt, 1 H, J = 10.3, 5.4 Hz, H 5"), 4.23 (m, 1 H, H 8'), 4.02 (dd, 1 H, J = 10.3, 5.4 Hz, H 6"), 3.97 (dd, 1 H, J = 11.2, 10.8 Hz, H 3"), 3.75 (t, 1 H, J = 10.3 Hz, H 6"), 3.60 (dd, 1 H, J = 11.2, 10.3 Hz, H 4"), 3.58 (dd, 1 H, J = 8.6, 3.4 Hz, H 9'), 2.58 (m, 1 H, OH), 2.06 (s, 3 H, Ac), 1.47, 1.35 (2 x s, 2 x 3 H, C(CH3)2), 1.06 (s, 9 H, t-butyl), 0.21, 0.18 (2 X s, 2 X 3 H, Si(CH3h). 170.3, 157.1, 155.9, 138.0-127.8 (arom), 102.7, 100.6, 99.6, 98.3, 94.4, 77.4, 75.2, 71.9, 70.2, 67.7, 67.2, 65.1, 62.6, 54.8, 53.8, 29.3, 26.1, 23.4, 19.2, 18.5, -3.8, -4.6. FI1R (neat film) cm-1: 3650-3100 (s), 2928 (s), 2856 (m), 1709 (s), 1662 (s), 1534 (s), 1375 (s), 1247 (s), 1116 (s), 1026 (s), 861 (m), 780 (m), 698 (m). MS (FAB) m/z: 773 (MH)+, 398 (ga1actopyranoside)+, 358 (g1ucopyranoside)+. 161 HRMS (FAB) mlz: Calcd for C39H57N2012Si (MH)+: 773.3676. Found: 773.3681. Elemental Analysis: Calcd for C39H56N2012Si: C, 60.60; H, 7.30; N, 3.62. Found: C, 60.28; H, 7.13; N, 3.90. TLC RJ (50% EtOAc in hexanes): 0.44 162 0 ~0 (2 py, 23DMAP (0.06 °C, 12 h; equiv), eq.Jiv), CCI3C02H (4.6 equiv), CH2CI2, 0 °C, 15 min, 53% (overall) 20 HO~' "l. ~ :{ [Bo c • N 0 H TBSO 0 1 H OTBS 63 Uridine-5'-Alcohol 63. DMAP (50 mg, 0.4 mmol, 0.06 equiv) and di-t-butyl dicarbonate (2.90 g, 13 mmol, 2 equiv) were added sequentially to a solution of uridine derivative 20 (5. 1 g, 6.5 mmol, 1 equiv) in pyridine (50 mL) at 0 T, and the resulting solution was stirred at 23 OC for 12 h. Volatiles were removed in vacuo, and the residue was dissolved in dichloromethane (100 mL). A solution of trichloroacetic acid (5.0 g, 30 mmol, 4.6 equiv) in dichloromethane (45 mL) was added dropwise over a 5-min period at 0 °C, and the resulting orange solution was stirred at 0 "C for 15 min. The product was partitioned between dichloromethane (150 mL) and saturated aqueous sodium bicarbonate solution (80 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to afford the product 63 (2.01 g, 53%) as a colorless oil. lH NMR (400 MHz, CDCl3) 8: 7.50 (d, 1 H. J = 8.3 Hz, H 6), 5.64 (d, 1 H, J = 8.3 Hz, H 5), 5.42 (d, 1 H, J = 5.4 Hz, H 1'), 4.43 (dd, 1 H, J = 5.4, 4.4 Hz, H 2'), 4.05 (dd, 1 H, J = 4.4, 2.9 Hz, H 3'), 3.97 (m, 1 H, H 4'), 3.82 (m, 1 H, J = 12.2 Hz, H 5'), 3.60 (m, 1 H, J = 12.2 Hz, 163 H 5'), 2.93 (m, 1 H, OH), 1.49 (s, 9 H, t-Boc), 0.81 (s, 9 H, t -butyl), 0.77 (s, 9 H, t -butyl), -0.01, -0.02, -0.05, -0.08 (4 X S, 4 X 4 H, 4 X SiCH3) FI1R (neat fllm) cm-1: 3495 (w), 2930 (m), 2857 (m), 1788 (s), 1722 (s), 1678 (s), 1449 (m), 1372 (m), 1253 (s), 1151 (s), 837 (s), 778 (s). MS (FAB) m/z: 515 (M+- t-butyl), 471 (M+- t-Boc), 457 (M+TBS). HRMS (FAB) mlz: Calcd for C26H49N20gSi2 (MH)+: 573.3027. Found: 573.3032. TLC RJ (50% EtOAc in hexanes): 0.43 164 H>\_.-ot):"~ l)--('H TBSO OTBS 63 0 5 ~NBoc JL ..,. l (COa)2 (3 ~uiv), D~SO (5 equiv), H CH2CI:!, -78 C, 15 mm; O)Az~ 0 0 Et3N (10 equiv), -40 oc, 25 min •· 1' H H TBSO OTBS 64 (crude) Uridine-5'-Aldehyde 64. Dimethylsulfoxide (311 ~L. 4.3 mmol, 5 equiv) was added dropwise to a solution of oxalyl chloride (224 ~L, 2.6 mmol, 3 equiv) in dichloromethane (12 mL) at-78 "C, and the resulting solution was stirred at -78 °C for 5 min. A solution of uridine derivative 63 (500 mg, 0.87 mmol, 1 equiv) in dichloromethane (10 mL) was added via cannula, and the resulting solution was stirred at -78 "C for 15 min. Triethylamine (1.21 mL, 8.7 mmol, 10 equiv) was added, and the resulting suspension was stirred at -78 "C for an additional 25 min. The reaction mixture was poured into saturated aqueous sodium bicarbonate solution (150 mL), the aqueous layer was extracted with ethyl acetate (2 x 100 mL), and the combined organic layers were dried (sodium sulfate) and concentrated to provide crude (64, 513 mg) as a pale yellow solid. Due to the lability of the product and its susceptibility to hydration, the uridine 5'-aldehyde derivative 64 was used in its crude form in the following experiment. 165 64 (2.5 equiv, crude) Me2SiCI2 (10 equiv) 23 °C, 6 h; 62 (1 equiv) / ~ CbzNH TBS;#P-11~ 0 CH3-(- O s NBoc I~ s t - O~e 0 0 c 0 PhSeH (3.8 Eq.Jiv), ~ (4.1 equiv), PhCH3, 23 0 C, 15 m1n; CH3 CH3,,, Y e~H2 SePh s· 0 0 1' H H TBSO OTBS H e· CH3 65 (50%) 0-Silylhemiselenoacetal Adduct 65. Benzeneselenol (56 !lL, 0.51 mmol, 3.8 equiv) and pyridine (45 !lL, 0.55 mmol, 4.1 equiv) were added sequentially to a freshly prepared, deoxygenated solution of aldehyde 64 (190 mg, -0.34 mmol, -2.5 equiv, azeotropically dried with 1.5 mL of toluene), and the resulting solution was deoxygenated. After stirring at 23 T for 15 min, the reaction mixture was transferred via cannula to a solution of dichlorodimethylsilane (340 !lL, 3.4 mmol, 10 equiv) in pyridine (2 mL). The resulting suspension was deoxygenated and was stirred in the dark at 23 T for 6 h. The reaction mixture was concentrated in vacuo at 23 OC, and the residue was suspended in toluene (2 mL). Volatiles were removed at 23 T, and the residue was diluted with toluene (3 mL). To the mixture was added via cannula a solution of allylic alcohol 62 (104 mg, 0. 13 mmol, I equiv) in pyridine (2 mL), and the resulting suspension was stirred at 23 OC for 5 min. The product was partitioned between ethyl acetate (100 mL) and water (100 mL). The organic layer was dried (sodium sulfate) and concentrated, and the residue was purified by flash column chromatography (gradient elution: 33% --7 50% ethyl acetate in hexanes) to afford a 5:1 mixture of C5'-diastereomers 65 (106 mg, 50%) as 166 a colorless oil. Preparative thin layer chromatography (50% ethyl acetate in hexanes) provided analytical samples of each diastereomer. ~ IH NMR (400 MHz, C6D6) 8: 7.78 (d, 1 H, J = 8.6 Hz, H 6), 7.7-7.0 (m, 15 H, arom), 6.46 (d, 1 H, J = 9.8 Hz, NH), 6.21 (d, 1 H, J =5.9 Hz, H 1'), 5.94 (d, 1 H, J = 8.6 Hz, H 5), 5.78 (d, 1 H, J = 3.7 Hz, H 5'), 5.23 (d, 1 H, J = 12.2 Hz, PhCH20CO), 5.12 (d, 1 H, J = 3.9 Hz, H 1"), 5.08 (d, 1 H, J = 12.2 Hz, PhCH20CO), 4.8-4.5 (m), 4.45 (m, 2 H), 4.32 (s, 1 H, H 6'), 4.31 (m, 1 H), 4.25 (dd, 1 H, J =4.4, 2.9 Hz, H 3'), 4.22 (m, 1 H, H 2"), 3.98 (m, 2 H), 3.75 (t, 1 H, J = 10.2 Hz, H 3"), 5.90 (m, 2 H)2.16 (s, 3 H, Ac), 1.51 (s, 9 H, t-Boc), 1.46, 1.35 (2 x s, 2 x 3 H, 2 X CH3). 1.11, 1.03, 0.98 (3 X s, 3 X 9 H, 3 X tbutyl), 0.31, 0.28, 0.27, 0.25, 0.21, 0.16, 0.15, 0.10 (8 X s, 8 X 3 H, 8 X SiCH3). FTIR (neat film) cm-1: 3324 (w), 2929 (m), 2850 (m), 1789 (s), 1724 (s), 1680 (s), 1535 (w), 1448 (m), 1371 (m), 1257 (s), 1150 (s), 1063 (s), 972 (m), 838 (s). MS (FAB) mlz: 1549 (M+- t-butyl), 1425 (M+ - OTBS), 1400 (M+ - PhSe). 167 1LC Rt (50% EtOAc in hexanes): 0.36 ~ lH NMR (400 MHz, C6D6) 8: 7.81 (d, 1 H, J =7.6 Hz, H 6), 7.5-7.0 (m, 15 H, arom), 6.56 (d, 1 H, J = 7.6 Hz, NH), 6.40 (d, 1 H, J = 8,3 H z, H 5), 5.83 (d, 1 H, J = 2.4 Hz, H 1"), 5.32 (d, 1 H, J =4.15 Hz, H 5'), 5.21 (d(br), 2 H, NH), 4.67 (m, 1 H, H 2"), 4.65-4.15 (m), 4.00 (dd, 1 H, J = 10.7, 5.4 Hz, H 6"), 3.78 (t, 1 H, J = 10.7 Hz, 3.63 (dd, 1 H, J =9.8, 8.6 Hz, H 3"), 3.56 (m, 1 H), 2.23 (s, 3 H, Ac), 1.51 (s, 9 H, tBoc), 1.44, 1.36 (2 x s, 2 x 3 H, 2 x CH3), 1.16, 1.06, 1.05 (3 x s, 3 x 9 H, 3 x t-butyl), 0.40, 0.32, 0.31, 0.29, 0.28, 0.25, 0.17, 0.16 (8 X S, 8 X 3 H, 8 X SiCH3). FTIR (neat film) cm-1: 3320 (w), 2929 (m), 2857 (m), 1789 (m), 1724 (s), 1684 (s), 1527 (w), 1448 (w), 1372 (m), 1257 (s), 1114 (s), 1023 (s), 838 (s), 779 (m). MS (FAB) m/z: 1549 (M+- t-butyl), 1400 (M+- PhSe). 1LC Rt (50% EtOAc in hexanes): 0.32 168 0 8u3SnH (2.5 equiv), Et:JB (0.2 equiv), 66 PhCH 3 , o °C, 15 min :{):: CbzNH 0 1' TBSO H OTBS 66 (80%) Siloxane 66. A solution of triethylborane (5 ~L. 1.0 M solution in hexanes, 0.005 mmol, 0.2 equiv) was added to a deoxygenated solution of 0-silylhemiselenoacetals 65 (50 mg, 0.03 mmol, 1 equiv) and tributyltin hydride (20 ~L. 0.07 mmol, 2.5 equiv) in toluene at 0 OC, and the resulting solution was stirred at 0 °C for 15 min. The solvent was removed in vacuo at 0 "C, and flash column chromatography of the residue (gradient elution: 33% --7 100% ethyl acetate in hexanes) afforded siloxane 66 (36 mg, 80%) as a colorless film. 7.90 (d, 1 H, J = 8.3 Hz, H 6), 6.50 (d, 1 H, J = 6.8 Hz, H 1'), 5.86 (d, 1 H, J =9.8 Hz, NH), 5.64 (d, 1 H, J = 8.3 Hz, H 5), 5.29 (d, 1 H, J = 12.4 Hz, PhCH20CO), 5.17 (d, 1 H, J = 3. 7 Hz, H 1 "), 5.00 (d, 1 H, J = 12.4 Hz, PhCH20CO), 4.83 (d, 1 H, J = 8.6 Hz, NH), 4.70-4.44 (m, 7 H), 4.42 (dd, 1 H, J = 6.8, 4.4 Hz, H 2'), 4.28 (m, 1 H, J = 8.8 Hz, H 10'), 4.15 (m, 1 H, H 2"), 4.13 (m, 1 H, H5'), 4.08 (m, 1 H, H 4'), 3.93 (d. 1 H, J = H 8'), 169 3.88 (m, 2 H, H 4", 6"), 3.74 (t, 1 H, J = 10.5 Hz, H 3"), 3.57 (t, 1 H, J =9.8 Hz, H 6"), 3.49 (dd, 1 H, J = 8.8, 2.7 Hz, H 9'), 3.16 (m, 1 H, H 7'), 2.07 (s, 3 H, Ac), 1.90 (m, 2 H, H 6'), 1.51 (s, 9 H, t-Boc), 1.50, 1.34 (2 x s, 2 x 3 H, 2 x CH3), 1.07, 1.06, 0.99 (3 x s, 3 x 9 H, 3 x t-buty1), 0.24, 0.22, 0.17, 0.12, 0.12, 0.09, 0.06, 0.04 (8 X S, 8 X 3 H, 8 X SiCH3). FTIR (neat film) cm-1: 3329 (w), 2930 (m), 2858 (m), 1789 (m), 1726 (s), 1688 (s), 1532 (w), 1447 (m), 1372 (m), 1258 (s), 1151 (s), 1029 (s), 838 (s), 779 (m). MS (FAB) mlz: 1346 (M+- t-butyl). 1LC RJ (50% EtOAc in hexanes): 0.22 170 3 N HCI, reflux, 3 h; AcNH ACz(> (40 equiv), DMAP (51 equiv), CH2CI2 , 0 •c, 2 h, Prep. TLC, 25% (overall) 26 C5'-evj-a-Heptaacetyl Tunicaminyluracil 26. A solution of siloxane 66 (23 mg, 0.02 mmol, 1 equiv) in 3 N aqueous hydrochloric acid (1.5 mL) was heated at reflux for 3 h, at which time the solvent was removed in vacuo at 23 ·c. The residue then was diluted with dichloromethane (2 mL), and to this solution at 0 ·c was added sequentially 4-(N,N-dimethylamino)pyridine (50 mg, 0.4 mmol, 20 equiv) and acetic anhydride (33 I..LL, 0.3 mmol, 15 equiv). The resulting reaction mixture was stirred at this temperature for 2 h before it was diluted with ethyl acetate (50 mL). The product solution then was washed sequentially with 0.5 N aqueous hydrochloric acid solution (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and saturated aqueous sodium chloride solution (40 mL), and the organic layer was dried (sodium sulfate) and concentrated. The residue was purified by preparative thin layer chromatography (7% methanol in dichloromethane) to yield 26 as the major product (4 mg, 36%). lH NMR (400 MHz, CDCl3) 8: 8.42 (s, 1 H, imide NH), 7.53 (d, 1 H, J = 8.6 Hz, H 6), 6.17 (d, 1 H, J = 3.7 Hz, H 11'), 6.13 (d, 1 H, J =5.6 Hz, H 1'), 5.85 (d, 1 H, J = 8.6 Hz, H 5), 5.44 (d, 1 H, J =9.9 Hz, amide NH), 5.28 (d, 1 H, J = 2.9 Hz, H 8'), 5.26 (m, 1 H, H 5'), 5.22 (t, 171 1 H, J = 5.6 Hz, H 2'), 5.19 (dd, 1 H, J = 2.9, 12.0 Hz, H 9'), 5.11 (dd, 1 H, J =4.0, 5.6 Hz, H 3'), 4.69 (ddd, 1 H, J = 3.7, 9.9, 12.0 Hz, H 10'), 4.22 (d, 1 H, J = 8.3 Hz, H 7'), 4.10 (d, 1 H, J = 4.0 Hz, H 4'), 2.19 (s, 6 H, Ac), 2.11 (s, 3 H, Ac), 2.11 (m, 1 H, H 6'), 2.08 (s, 3 H, Ac), 2.07 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 1.94 (s, 3 H, Ac), 1.62 (m, 1 H, H 6'). FTIR (neat film) cm-1: 3289 (w, br), 2925 (w), 1747 (s), 1693 (s) 1546 (w), 1458 (w), 1370 (m), 1223 (s), 1123 (w), 1041 (m), 929 (w). MS (FAB) mlz: 700 (MH)+, 640 (M+- AcO). HRMS (FAB) m/z: Calculated for C29H3sN3017 (MH)+: 700.2201. Found: 700.2231. 172 JNH ~00~0 HO OH c 0 TMSCI (2.5 equiv), Et3 N (5 equiv), DMAP (0.02 equiv), CH2CI 2, 23 oc, 2 h; ~MAP, DMTO Boc:!O (1A equiv), (0.02 equrv), py, 23 C, 12 h; KF-2H20 (2.4 equiv), MeOH, 23 oc, 3 h, 79% (overall} 70 5 I ~NBoc e ~· 0 ,. H HO 0 H OH 71 Uridine Diol 71 . Chlorotrimethylsilane (3.47 mL, 27.40 mmol, 2.5 equiv) was added to a solution of 5'-0-dimethoxytrityl uridine (70) (5.98 g, 10.94 mmol, I equiv), DMAP (30 mg, 0.25 mmol, 0.02 equiv), and triethylamine (7.62 mL, 54.70 mmol, 5.0 equiv) in dichloromethane (20.0 mL). The resulting white slurry was stirred at 23 T for 2 h and then was filtered. The filtrate was concentrated, and the residual oil was passed through flash grade silica gel (33% ethyl acetate in hexanes) to yield a viscous, yellow oil (6.95 g). The intermediate bis(trimethylsilyl) ether was dissolved in pyridine (25.0 mL), and DMAP (30 mg, 0.25 mmol, 0.02 equiv) and di-t-butyl dicarbonate (3.47 mL, 15.10 mmol, 1.4 equiv) were added sequentially. The resulting mixture was stirred at 23 T for 12 h. Volatiles were removed in vacuo, and the residue was diluted with methyl alcohol (20 mL). Potassium fluoride hydrate (2.50 g, 26.56 mmol, 2.4 equiv) was added to the resulting solution, and the reaction mixture was stirred at 23 OC for 3 h. The product was partitioned between ethyl acetate (600 mL) and water (200 mL). The organic layer was washed with saturated aqueous sodium chloride solution (200 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (40% ethyl acetate in hexanes) to provide 71 (5.58 g, 79%) as a white solid (mp 93.0 "C). 173 lH NMR (400 MHz, Acetone-d6) 8: 7.97 (d, 1 H, J = 8.2 Hz, H 6), 7.47 (m, 2 H, arom), 7.33 (m, 6 H, arom), 7.26 (m, 1 H, arom), 6.90 (m, 4 H, arom), 5.89 (d, 1 H, J =4.1 Hz, H 1'), 5.33 (d, 1 H, J = 8.2 Hz, H 5), 4.84 (d, 1 H, J =5.5 Hz, OH), 4.49 (m, 1 H, H 3'), 4.37 (m, 2 H, H 4' and OH), 4.13 (m, 1 H, H 2'), 3.78 (s, 6 H, OCH3), 3.50 (dd, 1 H, J = 3.0, 12.1 Hz, H 5'), 3.43 (dd, 1 H, J = 2.7, 12.1 Hz, H 5'), 1.55 (s, 9 H, t-butyl). FTIR (neat film) cm-1: 3436 (w, br), 2932 (w), 1784 (s), 1716 (s), 1668 (s), 1608 (m), 1509 (m), 1446 (m), 1392 (m), 1252 (s), 1177 (w), 1147 (m). MS (FAB) mlz: 646 (M)+, 303 (DMT )+. HRMS (FAB) mlz: Calcd for C3sH3gN2010 (M)+: 646.2526. Found: 646.2498. TLC RJ (67% EtOAc in hexanes): 0.46 174 0 'l:NBoo (AIIyi)OCOCI {10 equiv), DMTO~e~ o PY (20 equiv), -20 °C->0 °C, 25 min, 87% H AocO OAoc 71 2'.3'-BisCAoc) Uridine. Allyl chloroformate (10.20 mL, 95.80 mmol, 10.0 equiv) was added dropwise over a 10-min interval to a solution of diol 71 (6.20 g, 9.58 mmol, 1 equiv) in pyridine (15.50 mL, 191.6 mmol, 20.0 equiv) at -20 °C. The resulting slurry was allowed to warm to 23 OC and was stirred at this temperature for 25 min. Volatiles were removed in vacuo, and the residue was dissolved in dichloromethane (30 mL). The product was partitioned between ethyl acetate (500 mL) and water (300 mL). The organic layer was washed with saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. The crude product was purified by flash column chromatography (33% ethyl acetate in hexanes) to yield 2',3'-0-di(allyloxycarbonyl)-5'-0-dimethoxytrityl-3-N-tbutylcarbamate uridine (6.78 g, 87%) as a white solid (mp 77.0- 78.0 "C). 7.57 (m, 2 H, arom), 7.40 (m, 4 H, arom), 7.36 (d, 1 H, 1 = 8.2 Hz, H 6), 7.20 (m, 2 H, arom), 7.07 (m, 1 H, arom), 6.80 (m, 4 H, arom), 6.26 (d, 1 H, 1 = 3.7 Hz, H 1'), 5.73 (m, 3 H, Aoc), 5.67 (m, 1 H, H 3'), 5.62 (m, 1 H, H 2'), 5.19 (d, 1 H, 1 = 8.2 Hz, H 5), 5.14 (m, 1 H, Aoc), 5.08 (m, 1 H, 175 Aoc), 4.98 (m, 1 H, Aoc), 4.96 (m, 1 H, Aoc), 4.43 (m, 1 H, Aoc), 4.40 (m, 1 H, Aoc), 4.31 (m, 1 H, Aoc), 4.10 (m, 1 H, H 4'), 3.44 (dd, 1 H, J = 2.9, 11.5 Hz, H 5'), 3.35 (dd, 1 H, J = 2.9, 11.5 Hz, H 5'), 3.33 (s, 6 H, OCH3), 1.45 (s, 9 H, t-butyl). FI1R (neat film) cm-1: 2935 (w), 1787 (s), 1759 (s), 1724 (s), 1682 (s), 1608 (w), 1509 (m), 1440 (m), 1372 (m), 1256 (s), 1148 (m), 1033 (w), 833 (w). MS (FAB) m/z: 814 (M)+, 303 (DMT)+. HRMS (FAB) mlz: Calcd for C43lf46N2014 (M)+: 814.2949. Found: 814.2900. 1LC RJ (50% EtOAc in hexanes): 0.53 176 ~o\_.-o{.J:: l)-----{'H AocO OAoc 0 PhS03 H (1.4 equiv), CHCI3 , ·C"~' HO~·~ 0" 0 23 •c, 2 min, 76% H H Aoco OAoc 72 Uridine-5'-Alcohol 72. A solution of benzenesulfonic acid (1.80 g, 11.38 mmol, 1.4 equiv) in chloroform (60.0 mL) was poured onto 2',3'-0-di(allyloxycarbonyl)-5'-0-dimethoxytrityl-3-N-tbutylcarbamate uridine (6.78 g, 8.33 mmol, 1 equiv), and the resulting orange solution was stirred at 23 ·c for 2 min. The product was partitioned between ethyl acetate (500 mL) and saturated aqueous sodium bicarbonate solution (300 mL). The organic layer was washed with saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (67% ethyl acetate in hexanes) to afford 72 (3.26 g, 76%) as a white solid (mp 62.0 ·c). 7.01 (d, 1 H, J = 10.1 Hz, H 6), 5.96 (d, 1 H, J = 6.7 Hz, H 1'), 5.73 (m, 1 H, Aoc), 5.72 (m, 1 H, H 3'), 5.68 (m, 1 H, Aoc), 5.62 (m, 1 H, Aoc), 5.48 (dd, 1 H, J = 5.3, 6.7 Hz, H 2'), 5.34 (d, 1 H, J = 10.1 Hz, H 5), 5.20-4.94 (m, 4 H, Aoc), 4.454.28 (m, 3 H, Aoc), 3.95 (m, 1 H, H 4'), 3.53 (m, 1 H, H 5'), 3.30 (m, 1 H, H 5'), 2.57 (t(br), 1 H, OH), 1.45 (s, 9 H, t-butyl). 177 FI1R (neat film) em- I: 3499 (w, br), 2986 (w), 1784 (s), 1756 (s), 1722 (s), 1682 (s), 1451 (m), 1372 (m), 1265 '(s), 1147 (m), 1100 (w), 951 (w), 786 (w). MS (FAB) m/z: 513 (MH)+, 413 (MH+- t-Boc). HRMS (FAB) m/z: Calcd for CnH29N2012 (MH)+: 513.1720. Found: 513.1739. TLC RJ (67% EtOAc in hexanes): 0.42 178 Dess-Martin periodinane, (3 equiv), CH2 Ci:!, 23 oc, 20 min AocO 72 OAoc AocO OAoc 73 (crude) Aldehyde 73. A solution of alcohol 72 (355 mg, 0_693 mmol, 1 equiv) in dichloromethane (4.0 mL) was added via cannula to a suspension of the Dess-Martin periodinane (882 mg, 2.08 mmol, 3.0 equiv) in dichloromethane (4.0 mL), and the resulting suspension was stirred at 23 ·c for 20 min. The product was partitioned between ethyl acetate (130 mL) and a mixture of saturated aqueous sodium bicarbonate solution and saturated aqueous sodium thiosulfate solution (4: 1 (v/v), 50 mL). The organic layer was washed with saturated aqueous sodium chloride solution (50 mL), then was dried (sodium sulfate) and concentrated. The residue was filtered through a short column of silica gel (67% ethyl acetate in hexanes) to afford the crude aldehyde 73 (250 mg). Due to the extreme lability of the product and its high susceptibility to hydration, the crude uridine 5'-aldehyde derivative 73 was immediately used without further purification in the following experiment. 179 c 0 73 (2 equiv, crude) PhSeH (3 equiv), py (3 equiv), PhCH3 , 23 oc, 15 min; NBoc CH 3 CH:~<,. f Me2SiCI2 (20 equiv), 23 °C, 4.5 h; 62 (1 equiv) 0 SePh TBS 0 CH3- f ,,. O l ,. CbzNH 0 I . Sl- O~e~ 0 / s· 0 ~ 0 5 CH H H AocO OAoc 2 e· H H e· CH3 74 (81 %) 0-Silylhemiselenoacetal 7 4. Benzeneselenol (8lj...LL, 0.74 mmol, 3.0 equiv) and pyridine (59j...LL, 0.74 mmol, 3.0 equiv) were added sequentially to a deoxygenated solution of freshly prepared aldehyde 73 from the previous experiment (250 mg, -0.5 mmol, -2 equiv, azeotropically dried with 1.5 mL of toluene), and the resulting solution was deoxygenated. After stirring at 23 OC for 15 min, the reaction mixture was transferred via cannula to a solution of dichlorodimethylsilane (594 j...LL, 4.9 mmol, 20 equiv) in pyridine (2 mL). The resulting suspension was deoxygenated and was stirred in the dark at 23 OC for 6 h. The reaction mixture was concentrated in vacuo at 23 °C, and the residue was suspended in toluene (2 mL). The volatiles were removed at 23 OC, and the residue was diluted with toluene (3 mL). To the mixture was added via cannula a solution of allylic alcohol62 (189 mg, 0.24 mmol, 1 equiv) in pyridine (2.5 mL), and the resulting suspension was stirred at 23 OC for 5 min. The product was partitioned between ethyl acetate (100 mL) and water (100 mL). The organic phase was dried (sodium sulfate) and concentrated, and the residue was purified by flash 180 column chromatography (50% ethyl acetate in hexanes) to afford an inseparable mixture of diastereomers 74 (1.5: 1) (296 mg, 81% total) as a white solid (mp 78.0-80.5 ·c). lH NMR (400 MHz, Y>D6, 50 ·c) o: 7.69 (m, arom), 7.60 (m, arom), 7.30 (m, arom), 7.14 (m, arom), 7.10-7.03 (m, arom), 6.36 (d, 1 H, J = 10.1 Hz, H 1'), 6.27 (m, 2 H, NH, H 1'), 5.97 (m), 5.91 (m, 1 H, NH), 5.85 (m, H 5'), 5.78-5.54 (m, Aoc, H 11 ', H 1', H 2', H 3'), 5.25-4.95 (m), 4.74-4.53 (m), 4.50-4.20 (m), 4.14 (t, 1 H, J = 9.75 Hz, H 6"), 4.07-3.95 (m), 3.77 (t, 1 H, J = 9.75 Hz, H 4"), 3.65-3.56 (m), 2.18 (s, 3 H, Ac), 2.13 (s, 3 H, Ac), 1.49 (s, t-Boc), 1.45 (s, 3 H, CH3), 1.36 (s, 3 H, CH3), 1.33 (s, 3 H, CH3), 1.12 (s, 9 H, t-butyl), 1.09 (s, 9 H, t-butyl), 0.31 (s, 3 H. SiCH3), 0.28 (s, 3 H, SiCH3), 0.24 (s, 3 H. SiCH3), 0.21 (s, SiCH3), 0.20 (s, SiCH3), 0.19 (s, SiCH3). FTIR (neat film) cm-1: 3331 (w, br), 2943 (w), 1784 (m), 1760 (m), 1723 (m), 1682 (s), 1527 (w), 1450 (w), 1373 (m), 1262 (s), 1147 (m), 1079 (m), 1023 (m), 842 (w). MS (FAB) mlz: Elemental Analysis: 1498(MH)+, 772 (allylic alcohol disaccharide)+. 181 N,3.74. Found: C, 55.70; H, 6.18; N, 3.82. TLC Rt (67% EtOAc in hexanes): 0.59 182 74 75 Diol75. Tributyltin hydride (503 J.!L, 1.9 mmol, 3.0 equiv) was added to a deoxygenated solution of 0-silylhemiselenoacetals 74 (931 mg, 0.62 mmol, 1 equiv) and bis(triphenylphosphine)palladium(II) chloride chloride (3 mg, 4 J.lmol, 0.007 equiv) in a mixture of water in dichloromethane (2% (v/v ), 6 mL), and the resulting brown solution was stirred at 23 OC for 6 min. The reaction mixture immediately was subjected to flash column chromatography (gradient elution: 50 --7 67% ethyl acetate in hexanes) to afford diol 75 (700 mg, 85%) as a white solid (mp 94.0-95.5 OC). lH NMR (400 MHz, C()l)6, 50 OC) 8: 9.70 (d, 1 H, J = 9.7 Hz, H 6), 7.68-7.60 (m, arom), 6.16 (d, 1 H, J = 3.33 Hz, H 1'), 6.11 (d, 1 H, J = 10.0 Hz, NH), 6.01 (d, 1 H, J = 5.67 Hz, H 11 '), 5.80 (m, 2 H, NH, H I"), 5.70 (d, I H, J = 2.0 Hz, H 5'), 5.53 (d, 1 H, J = 8.67 Hz, H 5), 5.51 (d, 1 H, J = 10.0 Hz, NH), 5.27-5.15 (m), 183 4.73-3.95 (m), 3.84-3.73 (m), 3.64-3.55 (m), 2.13 (s, 3 H, Ac), 2.07 (s, 3 H, Ac), 1.51 (s, 9 H, tBoc), 1.47 (s, 3 H, CH3), 1.35 (s, 3 H, CH3), 1.33 (s, 3 H, CH3), 1.09 (s, 9 H, t-butyl), 1.06 (s, 9 H, t-butyl), 0.28 (s, 3 H, SiCH3), 0.25 (s, 3 H, SiCH3), 0.23 (s, SiCH3), 0.22 (s, SiCH3), 0.16 (s, SiCH3), 0.15 (s, SiCH3), 0.05 (s, SiCH3), -0.17 (s, SiCH3). FI1R (neat film) cm-1: 3334 (w, br), 2931 (w), 1786 (m), 1719 (s), 1667 (s), 1540 (w), 1452 (w), 1374 (m), 1256 (s), 1120 (s), 1022 (s), 861 (m), 840 (m). MS (FAB) m/z: 1329 (MH)+, 358 (glucopyranoside)+. TLC RJ (67% EtOAc in hexanes): 0.44 184 0 Bu3 SnH (2 equiv), Et3 B (0.8 equiv), PhCH3 , 0 oc, 2 h; 75 OH 0 :{_;:-: H HO OH 76 (60%) Tetraol76. Aliquots of a solution of triethylborane (25 j..!.L each, 1.0 M in hexanes, 0.025 mmol, 0.1 equiv each) were added to a deoxygenated solution of diol 7 5 (340 mg, 0.26 mmol, 1 equiv) and tributyltin hydride (138 j..!.L, 0.51 mmol, 2.0 equiv) in toluene (300 mL) at 0 OC at 15-min intervals over a 2-hour period. The resulting solution was concentrated at 0 "C, and the residue was diluted with methyl alcohol (10 mL). To this solution was added potassium fluoride hydrate (600 mg, 6.4 mmol, 25 equiv), and the resulting mixture was stirred at 23 OC for 2 h. The product was partitioned between ethyl acetate (300 mL) and saturated aqueous sodium chloride solution (150 mL). The organic layer was separated, and the aqueous layer was extracted further with ethyl acetate (100 mL). The combined organic layers were dried (sodium sulfate) and concentrated, and the residue, containing a 7.5:1 mixture of C5' diastereomers, was purified by careful flash column chromatography (12:4: 1 benzene:acetonitrile:isopropanol) to afford pure 76 (172 mg, 60%) as a white solid (mp 223.0 "C). 185 lH NMR (400 MHz, CDCl3, 50 OC) o: 7.52 (d, 1 H, J = 8.1 Hz, H 6), 7.35-7.20 (m, 10 H, arom), 5.83 (d, 1 H, J = 8.6 Hz, NH), 5.74 (d, 1 H, J =8.1 Hz, H 5), 5.65 (d, 1 H, J =4.0 Hz, H 1'), 5.18 (d, 1 H, J = 12.1 Hz, PhCH20CO), 5.03 (m, 2 H, H 1", H 11'), 4.98 (d, 1 H, J = 12.1 Hz, PhCH20CO), 4.79 (d, 1 H, J = 6.3 Hz, OCH20), 4.73 (d, 1 H, J = 6.3 Hz, OCH20), 4.62 (d, 1 H, J =7.5 Hz, NH), 4.56 (d, 1 H, J = 11.8 Hz, PhCH2), 4.52 (d, 1 H, J = 11.8 Hz, PhCH2), 4.32 (m, 2 H), 4.20-4.05 (m, 2 H, 3.96 (t, 1 H, J = 3.5 Hz, H 3'), 3.94 (m, 1 H, H6"), 3.84 (m, 1 H, H 4'), 3.82-3.71 (m, 3 H), 3.66 (t, 1 H, J = 10.1 Hz, H 6"), 3.55 (t, 1 H, J = 9.2 Hz, H 4"), 3.43 (s-br, 1 H), 3.27 (s-br, 1 H), 2.62 (s-br, 1 H), 2.20 (m, 1 H, H 6'), 1.91 (s, 3 H, Ac), 1.74 (m, 1 H, H 6'), 1.58 (s, 9 H, t-Boc), 1.45 (s, 3 H, CH3), 1.37 (s, 3 H, CH3), 0.85 (s, 9 H, t-butyl), 0.08 (s, 6 H, SiCH3). FTIR (neat film) cm-1: 3354 (m, br), 2933 (m), 1784 (m), 1713 (s), 1667 (s), 1544 (m), 1449 (w), 1374 (m), 1253 (m), 1120 (s), 1079 (m), 1026 (s), 867 (w), 838 (w), 726 (m). MS (FAB) mlz: 908 (MH+- uracil(Boc)), 211 (uracil(Boc))+. 186 TLC Rt (10% MeOH in CH2Cl2): 0.34 187 0 3 , NaHC03 (0.6 equiv), 10:3 vlv CH2 Cl2 :MeOH, -78 oc, 3h; Ac-,P (5.6 equiv). Et3N (2.8 equiv), CH2 CI2 , 23 oc, 5 h, 94% 77 1 O~CO.,CH3 121 \ ~ 17 ... H 78 Aldehyde 78. Ozone was bubbled through a mixture of sodium bicarbonate (1.60 g, 19.0 mmol, 0.6 equiv) and cyclododecene (77) (5.00 g, 30.1 mmol, 1 equiv) in a mixture of dichloromethane and methyl alcohol (10:3 (v/v), 130 mL) at -78 OC for 3 h until the solution became deep blue. To remove excess ozone, nitrogen was bubbled through the solution at -78 "C for 15 min until the solution became colorless; the reaction mixture was then allowed to warm to 23 OC. After filtration of the suspension, benzene (80 mL) was added to the filtrate, and the resulting solution was concentrated to a volume of 40 mL. The concentrate was diluted with dichloromethane (160 mL), and triethylamine (12.0 mL, 86.1 mmol, 2.8 equiv) and acetic anhydride (16.0 mL, 169.6 mmol, 5.6 equiv) were added sequentially at 23 DC over a 10-min period. After stirring for 5 h, the reaction mixture was diluted further with dichloromethane ( 150 mL). The solution was washed sequentially with 0.1 N aqueous hydrochloric acid (300 mL), saturated aqueous sodium bicarbonate solution (300 mL), and saturated aqueous sodium chloride solution (300 mL). The organic layer was dried (sodium sulfate) and concentrated, and the pale yellow residue was purified by flash column chromatography (10% ethyl acetate in hexanes) to give the aldehyde 78 (6.10 g, 94%) as a clear, colorless oil. 188 IH NMR (300 MHz, CDCl3) 8: 9.75 (s, 1 H, H 12), 3.62 (s, 3 H, OCH3), 2.41 (dt, 1 H, J =2.1, 7.5 Hz, H 2), 2.30 (t, 1 H, J =7.5 Hz, H 2), 1.62 (m, 2 H, H 11), 1.29 (m, 16 H, CH2). FI1R (neat film) cm-1: 2926 (s), 2853 (s), 1739 (s), 1436 (w), 1172 (m). MS (EI) m/z: 227 (M+ - H), 213 (M+- CH3). HRMS (EI) m/z: Calcd for C13H2303 (M+- H): 227.1647, Found: 227.1641. lLC Rt (20% EtOAc in hexanes): 0.39 189 + - (CH3hCH-PPh31(1.5 equiv), NaN(TMS)2 (1.3 equiv), THF, -78 °C->23 °C, CH3 ~ 3 CH3 :::::..., 1 CO~H3 • 30min, 82% 78 79 Alkenyl Methyl Ester 79. A solution of sodium bis(trimethysilyl)amide (12.0 mL, 1.0 M in tetrahydrofuran, 12.0 mmol, 1.3 equiv) was added to a suspension of isopropyl-triphenyl-phosphonium iodide (6.00 g, 13.9 mmol, 1.5 equiv) in tetrahydrofuran (100 mL) at -78 "C. The resulting red suspension was stirred at -78 "C for 5 min, then at 23 OC for 25 min, and finally at 0 OC for 5 min. A solution of aldehyde 78 (2.00 g, 9.25 mmol, 1 equiv) in tetrahydrofuran (25 mL) was added via cannula to the ylide solution at 0 OC, and the resulting suspension was stirred at 23 OC for 1 h. The product was partitioned between ethyl ether (500 mL) and water (300 mL). The organic layer was washed with saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. The resulting oil was purified by flash column chromatography (gradient elution: 3 ~ 5% ethyl acetate in hexanes) to afford the methyl ester 79 (1.92 g, 82%) as a clear, colorless oil. lH NMR (400 MHz, CDCl3) 8: 5.11 (m, 1 H, H 12), 3.66 (s, 3 H, OCH3), 3.30 (d, 2 H, J =7.7 Hz, H 2), 1.93 (m, 1 H, H 11), 1.65 (s, 3 H, CH3), 1.60 (s, 3 H, CH3), 1.60 (m, 1 H, H 11), 1.27 (m, 16 H, CH2). 190 FTIR (neat film) em- I: 2925 (s), 2854 (s), 1743 (s), 1436 (m), 1376 (w), 1171 (m). MS (EI) m/z: 254 (M)+, 223 (M+- OCH3). HRMS (EI) m/z: Calcd for C16H3o02 (M)+: 254.2246, Found: 254.2246. TLC Rt (10% EtOAc in hexanes): 0.57 191 10% Pd-aubon (cat), PhCH3 , 60 oc, 12 h, 96% 80 79 Methyl Ester 8(). A solution of the methyl ester 79 (2.02 g, 7.87 mmol, 1 equiv) in toluene (20 mL) was heated at 60 "C in the presence of 10% palladium on activated carbon (300 mg) under a hydrogen atmosphere (1 atm) for 12 h. The reaction mixture was filtered through a pad of Celite, washing well with ethyl ether (150 mL). The filtrate was concentrated to afford pure 80 ( 1.94 g, 96%) as a clear, colorless oil. lH NMR (300 MHz, CDCl3) 8: 3.64 (s, 3 H, OCH3), 2.29 (t, 2 H, J = 7.5 Hz, H 2), 1.52 (m, 1 H, H 13), 1.61 - 1.16 (m, 20 H, CH2), 0.84 (d, 6 H, J = 8.3 Hz, CH3). FTIR (neat film) cm-1: 2925 (s), 2853 (s), 1743 (s), 1461 (w), 1436 (w), 1170 (m). MS (EI) mlz: 256 (M)+, 225 (M+- OCH3). HRMS (EI) mlz: Calcd for C16H3202 (M)+: 256.2402, Found: 256.2382. TLC RJ (10% EtOAc in hexanes): 0.57 192 LOA {12 equiv), (PhSe)2 {2 equiv), THF, -78 °C--> 23 °C, 5.5 h; m.CPBA, CH3 4._, '~ CO~H3 CHi'"'M.~ {12 equiv), DMS (4.9 equiv), Et3N {1 equiv), 23 oc, 6 h, 55% 80 1 1 • 81 (l.~-Unsaturated Methyl Ester 81. n-Butyllithium (8.15 mL, 1.3 Min hexanes, 10.6 mmol, 1.2 equiv) was added to a solution of diisopropylamine (1.86 mL, 13.3 mmol, 1.5 equiv) in tetrahydrofuran (40 mL) at -78 °C. The reaction flask was transferred briefly to an ice bath (<1 0 min), and then was recooled to -78 OC. A solution of 80 (2.26 g, 8.83 mmol, 1 equiv) in tetrahydrofuran (20 mL) was transferred by cannula to the cold solution of lithium diisopropylamide, and the resulting solution was stirred at -78 OC for 25 min. Solid diphenyldiselenide was added to the reaction mixture in one portion, and the resulting suspension was allowed to warm to 23 °C. The deep yellow solution was stirred at 23 °C for 5.5 h. The product was partitioned between ethyl ether (700 mL) and water (300 mL). The organic layer was washed sequentially with water (300 mL) and saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. Excess diphenyldiselenide was removed from the residue by flash column chromatography (gradient elution: 20 --7 25% dichloromethane in hexanes). Solid m-chloroperoxybenzoic acid (3.13 g, 60% (w/w), 10.9 mmo1, 1.2 equiv) was added to a solution of the crude selenide residue in dichloromethane (100 mL) at -78 OC, and the resulting suspension was stirred at -78 °C for 2 h. Excess oxidant was quenched by the addition of dimethyl sulfide (3.20 mL, 43.6 mmol, 4.9 equiv) and triethylamine (1.22 mL, 8.83 mmol, 1 equiv). The resulting solution was stirred at 23 OC for 6 h, and the product was partitioned between ethyl ether (500 mL) and water (300 193 mL). The organic layer was washed with saturated aqueous sodium chloride solution (300 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (5% dichloromethane in benzene) to afford methyl ester 81 (1.11 g, 55%) as a clear, colorless oil. lH NMR (300 MHz, CDCl3) b: 6.97 (dt, 1 H, J =6.9, 15.5 Hz, H 3), 5.81 (dt, 1 H, J = 1.4, 15.5 Hz, H 2), 3.73 (s, 3 H, OCH3), 2.20 (m, 2 H, H 4), 1.50 (m, 1 H, H 13), 1.451.17 (m, 16 H, CH2), 0.82 (d, 6 H, J = 6.9 Hz, CH3). FTIR (neat film) cm-1: 2926 (s), 2854 (s), 1729 (s), 1658 (m), 1436 (w), 1269 (m), 1173 (w), 1042 (w). MS (EI) m/z: 255 (MH)+, 223 (M+- OCH3). HRMS (EI) mlz: Calcd for C16H3102 (MH)+: 255.2113, Found: 255.2313. TLC Rt (5% EtOAc in hexanes): 0.28 194 1:1vlv1M NaOH(aq):t-BuOH, 60 °C, 1.5 h, 91% 81 82 a.j3-Unsaturated Fatty Acid 82. Methyl ester 81 (364 mg, 1.43 mmol, 1 equiv) was dissolved in a mixture of 1 M aqueous sodium hydroxide solution and t-butyl alcohol (1: 1 (v/v), 8 mL), and the resulting solution was heated at 60 OC for 1.5 h. The product was partitioned between ethyl acetate (100 mL) and 0.5 N aqueous hydrochloric acid solution (100 mL). The organic layer was washed with saturated aqueous sodium chloride solution (100 mL), then was dried (sodium sulfate) and concentrated. The residue was purified by flash column chromatography (50% ethyl acetate in hexanes) to afford 82 (312 mg, 91%) as a white solid (mp 41.5 °C). lH NMR (300 MHz, CDCl3) 8: 7.09 (dt, 1 H, J = 7.1, 15.3 Hz, H 3), 5.82 (dt, 1 H, J = 1.2, 15.3 Hz, H 2), 2.22 (m, 2 H, H 4), 1.50 (m, 1 H, H 13), 1.47- 1.12 (m, 16 H, CH2), 1.86 (d, 6 H, J =7.0 Hz, CH3). FTIR (neat film) cm-1: 3300- 2300 (w, br), 2922 (s), 2849 (s), 1691 (s), 1651 (m), 1668 (w), 1420 (w), 1284 (w). MS (EI) mlz: HRMS (El) mlz: 241 (MH)+, 223 (M+- OH). 195 Found: 241.2178. TLC Rt (25% EtOAc in hexanes): 0.35 196 10% vlv HC02H:MeOH, Pd-black,23 oc, 1.5 h; 13% vlv HC02H:MeOH, 40 °C, 5 h; HF, 1:1 CH 3 CN:MeOH, 23 °C, 2 h; 82 (6 equiv), DCC (9 equiv), CH2CI2, MeOH, 23 oc, 2 d CH3 0 OH CH3~N-~~-.. ~~··~ HO~cf··-H HO ,. H OH e· Tunicarnycin-V (1-V) (83% from 76) Tunicamycin V Cl-V). Palladium black (60 mg) was added to a solution of tetraol 76 (140 mg, 0.13 mmol, 1 equiv) in a mixture of formic acid in methyl alcohol (10% (v/v), 10 mL), and the resulting suspension was stirred at 23 OC for 1.5 h. After filtration of the reaction mixture and concentration of the filtrate, the residue was diluted with a mixture of of formic acid in methyl alcohol (13% (v/v), 10 mL), and the resulting solution was heated at 40 OC for 5 h. Following removal of volatiles in vacuo, the residue was dissolved in a mixture of methyl alcohol and acetonitrile ( 1:1 (v/v), 15 mL), and an aqueous solution of hydrofluoric acid (48% (w/w), 300 J..LL) was added. The resulting solution w as stirred at 23 "C for 2 h. Concentration of the reaction mixture at 23 OC and filtration of the residue through a short column of RP-18 reverse phase silica gel (1: 1:1.5 methyl alcohol :pyridine: water) afforded the corresponding crude amino heptaol 1 (81 mg), which was used without further purification. 197 Solutions of the activated fatty acid 82 were prepared as follows : dichloromethane (3 mL) was added to a solid mixture of fatty acid 82 (31 mg, 0.13 mmol, 1 equiv) and 1,3dicyclohexylcarbodiimide (40 mg, 0.19 mmol, 1.5 equiv), and the resulting suspension was stirred at 23 ·c for 30 min. Freshly prepared solutions of activated 82 (1 equiv each, 6 equiv total) were added to a solution of the crude amino heptaol 1 (81 mg) in methyl alcohol (4 mL) at 8-h intervals over a period of 2 days. The reaction mixture was concentrated at 23 ·c, and the residue was purified by flash column chromatography through RP-18 reverse phase silica gel (1: 1:1 methyl alcohol pyridine: water) followed by trituration of the residue with chloroform to afford pure 1-V (88 mg, 83% from tetraol 76) as a white solid (mp 235236 ·c (decomp)). lH NMR (400 MHz, CD30D) (): 7.91 (d, 1 H, 1 = 8.0 Hz, H 6), 6.81 (dt, 1 H, 1 = 6.9, 15.5 Hz, fatty acid H~.). 5.93 (d, 1 H, 1 = 15.5 Hz, fatty acid Ha), 5.92 (d, 1 H, 1 =5.6 Hz, H 1'), 5.74 (d, 1 H, 1 = 8.0 Hz, H 5), 4.92 (d, 1 H, 1 = 3.9 Hz, H 1"), 4.58 (d, 1 H, 1 = 8.8 Hz, H 11 '), 4.20 (m, 2 H, H 3', H 2'), 4.07 (m, 1 H, H 10'), 4.01 (m, 2 H, H 5', H 5"), 3.84 (m, 3 H, H 3", H 4', H 8'), 3.77 (m, 1 H. H 7'), 3.64 (m, 4 H, H 3", H 6", H 9'), 3.33 (t, 1 H, 1= 9.1 Hz, H 4"), 2.19 (m, 2 H, fatty acid Hy), 2.09 (m, 1 H, H 6'), 1.92 (s, 3 H, Ac), 1.57-1.40 (m, 3 H, H 6', i-Pr-CH, fatty acid HS), 1.28 (s-br, 12 H, CH2). 1.16 (m, 2 H, CH2), 0.87 (d, 1 H, 1 = 6.7 Hz, i-Pr-CH3). 198 Be NMR (100 MHz, CD30D) 3: 173.5 (acetamide C=O), 169.8 (fatty acid amide C=O), 166.1 (C4), 152.6 (C2), 146.5 (C6), 142.8 (fatty acid C~), 125.0 (fatty acid Ca), 103.1, 102.1 (C5), 100.3 (Cll'), 90.1 (C1"), 90.1, 89.6 (C1'), 75.5, 74.3, 73.3, 72.9, 72.7, 72.6, 72.1, 70.9, 68.4, 63.2, 55.0, 54.5, 40.2, 35.9, 33.0, 31.0, 30.7, 30.6, 30.5, 30.3, 29.5, 29.1, 28.5, 23.2, 23.0. FTIR (neat film) cm-1: 3329 (s, br), 2924 (s), 2849 (m), 1667 (s, br), 1468 (m), 1267 (w), 1096 (s), 1020 (s). MS (FAB) m/z: 831 (MH)+, 223 (fatty acid acyl fragment)+. HRMS (FAB) m/z: Calcd for C38H63N4016 (MH)+: 831.4239. Found: 831.4229. +60.5 ° (c = 0.515, pyridine). HPLC See Appendix. TLC RJ (2:1:1 n-BuOH:AcOH:H20): 0.60 199 Authentic 1-V. A sample of authentic tunicamycins (25 mg, Sigma) was dissolved in methyl alcohol (5 mL) at 40 °C. The solution of authentc 1-V, in ten separate 500-j.lL injections, was loaded onto a Beckman Ultrasphere ODS (Cis, 5 j.lm) rp-HPLC column (10 x 250 mm, as part of a Waters 501 HPLC system, flow= 2.00 mUmin), eluting with 85:15 (v/v) methyl alcohol:water. Fractions containing authentic 1-V were collected and pooled. The combined fractions were concentrated to afford authentic tunicamycin-V (4 mg) as a white solid. lH NMR (400 MHz, CD30D) 8: 7.91 (d, 1 H, J = 8.1 Hz, H 6), 6.81 (dt, 1 H, J = 6.9, 15.5 Hz, fatty acid H~,), 5.93 (d, 1 H, J = 15.5 Hz, fatty acid Ha), 5.92 (d, 1 H, J = 5.6 Hz, H 1'), 5.74 (d, 1 H, J = 8.1 Hz, H 5), 4.92 (d, 1 H, J = 3.9 Hz, H 1"), 4.58 (d, 1 H, J = 8.6 Hz, H 11 '), 4.20 (m, 2 H, H 3', H 2'), 4.07 (m, 1 H, H 10'), 4.01 (m, 2 H, H 5', H 5"), 3.84 (m, 3 H, H 3", H 4', H 8'), 3.77 (m, 1 H, H 7'), 3.64 (m, 4 H, H 3", H 6", H 9'), 3.33 (t, 1 H, J = 9.1 Hz, H 4"), 2.19 (m, 2 H, fatty acid Hy), 2.09 (m, 1 H, H 6'), 1.92 (s, 3 H, acetamide), 1.57-1.40 (m, 3 H, H 6', i-PrCH, fatty acid HS), 1.28 (s-br, 12 H, CH2), 1.16 (m, 2 H, CH2), 0.87 (d, 1 H, J = 6.7 Hz, i-PrCH3). 200 Be NMR (100 MHz, CD30D) 8 : 173.5 (acetamide C=O), 169.8 (fatty acid amide C=O), 166.1 (C4), 152.6 (C2), 146.5 (C6), 142.8 (fatty acid Cp), 125.0 (fatty acid Ca), 103.1, 102.1 (C5), 100.3 (Cll '), 90.1 (C1 "), 90.1, 89.6 (C1'), 75.5, 74.3, 73.3, 72.9, 72.7' 72.6, 72.1 , 70.9, 68.4, 63.2, 55.0, 54.5, 40.2, 35.9, 33.0, 31.0, 30.7, 30.6, 30.5, 30.3, 29.5, 29.1, 28.5, 23.2, 23.0. FTIR (neat film) cm-1 : 3328 (s, br), 2924 (s), 2849 (m), 1666 (s, br), 1465 (m), 1267 (w), 1096 (s), 1020 (s). MS (FAB) mlz: 831 (MH)+. HRMS (FAB)m/z: Calcd for C3gH63N4016 (MH)+: 831.4239. Found: 831.4250. +59.1 ° (c = 0.501, pyridine). mp: 233-235 OC (decamp). HPLC See Appendix. lLC Rt (2:1:1 n-BuOH:AcOH:H20): 0.60 201 10% v/v HC02H:MeOH, Pd-black,23 oc, 30 min; 13% vlv HC02 H:MeOH, 40 °C, 4 h; HF, 1:1 CH3 CN:Me0H, 23 °C, 2h; 82 (9 equiv), DCC (13.5 equiv), CH2CI2, MeOH, 23 °C, 3 d CS'-epi-Tunicamycin-V (84) (74% from 66) C5'-epj-Tunicamycin V (84). Palladium black (45 mg) was added to a solution of tetraol 66 (55 mg, 0.039 mmol, 1 equiv) in a mixture of formic acid in methyl alcohol (10% (v/v), 10 mL), and the resulting suspension was stirred at 23 "C for 30 min. After filtration of the reaction mixture and concentration of the filtrate, the residue was diluted with a mixture of of formic acid in methyl alcohol (13% (v/v), 7 mL), and the resulting solution was heated at 40 "C for 4 h. Following removal of volatiles in vacuo, the residue was dissolved in a mixture of methyl alcohol and acetonitrile (1: 1 (v/v), 10 mL), and an aqueous solution of hydrofluoric acid (48% (w/w), 200 j.!L) then was added. The resulting solution was stirred at 23 "C for 2 h. Concentration of the reaction mixture at 23 "C and filtration of the residue through a short column ofRP-18 reverse phase silica gel (1 :1:1.5 methyl alcohol :pyridine:water) afforded the corresponding crude amino heptaol 83 (22 mg), which was used without further purification. 202 Solutions of the activated fatty acid 82 were prepared in a manner similar to that described above: dichloromethane (1 mL) was added to a solid mixture of fatty acid 82 (14 mg, 0.06 mmol, 1.5 equiv) and 1,3-dicyclohexylcarbodiimide (20 mg, 0.10 mmol, 2.6 equiv), and the resulting suspension was stirred at 23 "C for 30 min. Freshly prepared solutions of activated 82 (1.5 equiv each, 9 equiv total) were added to a solution of the crude amino heptaol 83 (22 mg) in methyl alcohol (2 mL) at 12-hour intervals over a period of 3 days. The reaction mixture was concentrated at 23 °C, and the residue was purified by flash column chromatography through RP-18 reverse phase silica gel (1: 1:1 methyl alcohol :pyridine:water) followed by trituration of the residue with chloroform to afford pure 84 (24 mg, 74% from siloxane 66). lH NMR (400 MHz, CD30D) 8: 8.11 (d, 1 H, J = 8.1 Hz, H 6), 6.81 (dt, 1 H, J = 15.2, 7.0 Hz, fatty acid HI)), 5.95 (d, 1 H, J = 15.2 Hz, fatty acid Ha), 5.93 (d, 1 H, J = 5.0 Hz, H 1'), 5.72 (d, 1 H, J = 8.1 Hz, H 5), 4.96 (d, 1 H, J = 3.5 Hz, H 1"), 4.56 (d, 1 H, J = 8.4 Hz, H 11 '), 4.20 (m, 2 H, H 2', 3'), 4.05 (dd, 1 H, J = 10.7, 8.4 Hz, H 10'), 4.00-3.60 (m, 10 H), 3.42 (t, 1 H, J = 9.8 Hz), 2.20 (m, 2 H, fatty acid Hy), 2.09 (m, 1 H, H 6'), 1.91 (s, 3 H, Ac), 1.88 (m, 1 H, H 6'), 1.60-1.15 (m, 16 H), 0.88 (s, 3 H, CH3), 0.86 (s, 3 H, CH3). Be NMR (100 MHz, CD30D) 8: 173.4 (acetamideC=O), 169.7 (fatty acid amide C=O), 166.3 (C4), 152.6 (C2), 146.6 (C6), 142.9 203 (fatty acid C~), 124.9 (fatty acid Ca.), 102.8, 102.5 (C5), 100.4 (Cll'), 90.1 (C1"), 90.1, 87.8 (C1'), 75.6, 74.3, 74.1, 73.1, 72.8, 72.5, 71.8, 70.8, 68.8, 62.3, 55.0, 54.4, 40.3, 35.9, 33.1, 31.1, 30.8, 30.7, 30.6, 30.4, 29.5, 29.2, 28.6, 23.3, 23.1. FTIR (neat film) cm-1: 3399 (s, br), 2919 (m), 2849 (w), 1666 (s), 1631 (m), 1561 (w), 1461 (w), 1414 (m), 1349 (m), 1094 (m), 1023 (m). MS (FAB) mlz: 853 (MNa)+, 223 (fatty acid acyl fragment)+. HRMS (FAB) m/z: Calcd for C38H62N4016 (MNa)+: 853.4059. Found: 853.4036. lLC Rt (2:1:1 n-BuOH:AcOH:H20): 0.60 204 References and Notes (1) Isolation and structure elucidation of tunicamycin: (a) Tak:atsuki, A.; Arima, K.; Tamura, G . J. Antibiot. 1971, 24, 215. (b) Kenig, M.; Reading, C.; J. Antibiot. 1979, 32, 549. (c) Hamill, R. L.; U. S. Patents 4,273,225, 1980; 4,336,333, 1982. (d) Ito, T.; Kodama, Y.; Kawamura, K.; Suzuki, K.; Tak:atsuki, A.; Tamura, G. Agric. Bioi. Chem. 1977,41, 2303. (e) Tak:atsuki, A.; Kawamura, K.; Okina, M.; Kodama, Y.; Ito, T.; Tamura, G. Agric. Bioi. Chem. 1977,41,2307. (f) Tak:atsuki, A.; Kawamura, K.; Kodama, Y.; Ito, T.; Tamura, G. Agric. Bioi. Chem. 1979, 43, 761. (g) Ito, T. ; Tak:atsuki, A.; Kawamura, K. ; Sato, K.; Tamura, G. Agric. Bioi. Chem. 1980, 44, 695. Reviews of tunicamycin: (h) Elbein, A. Trends Biochem. 1981 , 219. (i) Tunicamycin; Tamura, G., Ed.; Japan Scientific Press: Tokyo, Japan, 1982. Corynetoxins and streptovirudins: (j) Eckardt, K. J. Nat. Prod. 1983, 46, 544. (k) Vogel, P.; Patterson, D. S.; Berry, P. H. ; Frahn, J. L.; Anderton, N.; Cockrum, P. A.; Edgar, J. A.; Jago, M. V.; Lanigan, G. W.; Payne, A. L.; Culvenor, C. C. J. Aust. J. Exp. Bioi. Med. Sci. 1981, 59, 455. (1) Edgar, J. A.; Frahn, J. L.; Cockrum, P. A.; Anderton, N.; Jago, M. V.; Culvenor, C. C. J.; Jones, A, J.; Murray, K. E.; Shaw, K. J. J. Chern. Soc., Chern. Commun.. 1982, 222. (m) Thrum, H.; Eckardt, K.; Bradler, G .; Fuegner, R.; Tonew, E.; Tonew, M. J. Antibiot. 1975,28, 514. (n) Eckardt, K.; Thrum, H.; Bradler, G .; Tonew, E.; Tonew, M. J. Antibiot. 1975, 28, 274. (o) Eckardt, K. ; Ihn, W .; Tresselt, D.; Krebs, D. J. Antibiot. 1981, 34, 1631. (2) (a) Ito, T.; Kodama, Y.; Kawamura, K.; Suzuki, S.; Takatsuki, A.; Tamura, G. Agric. Bioi. Chern. 1979,43, 1187. (b) see ref 1(e). (3) (a) Takatsuki, A.; Shimizu, K.; Tamura, G. J. Antibiot. 1972, 25, 75. (b) Tamura, G.; Sasaki, T.; Matsuhashi, M.; Tak:atsuki, A.; Yamasaki, M. Agric. Bioi. Chern. 1976, 40,447. (4) (a) Tak:atsuki, A.; Kohno, K.; Tamura, G . Agric. Bioi. Chem. 1975, 39, 2089. (b) Tkacz, J. S.; Lampen, J. 0 . Biochem. Biophys. Res. Commun. 1975, 65, 248. 205 (5) (a) Mahoney, W. C.; Duksin, D. J. Chromatogr. 1980, 198, 506. (b) see ref l(g). (6) Preliminary accounts of this work: (a) Myers, A. G.; Gin, D. Y.; Rogers, D. H. J. Am. Chern. Soc. 1993, 115, 2036. (b) Myers, A. G.; Gin, D. Y.; Widdowson, K. L. J. Am. Chern. Soc. 1991, 113, 9661. (7) (a) Suami, T.; Sasai, H.; Matsuno, K. ; Suzuki, N. Carbohydr. Res. 1985, 143, 85. (b) Suami, T .; Sasai, H.; Matsuno, K.; Suzuki, N.; Fukuda, Y.; Sakanaka, 0. Tetrahedron Lett. 1984,25, 4533. (c) Suami, T.; Sasai, H.; Matsuno, K. Chern. Lett. 1983, 819. (8) (a) Danishefsky, S. J.; DeNinno, S. L.; Chen, S.; Boisvert, L.; Barbachyn, M. J. Am. Chern. Soc. 1989,111,5810. (b) Danishefsky, S.; Barbachyn, M. J. Am. Chem. Soc. 1985, 107, 7761. (9) Foster, D . G. Organic Synthesis, Collective Vol. 3 Horning, E. C., Ed.; John Wiley and Sons: New York, 1955; pp. 771-773. (10) For existing methods for the formation of 0-trimethylsilyl hemithio- and hemiselenoacetals, see: (a) Chan, T. H.; Ong, B. S. Tetrahedron Lett. 1976, 319. (b) Dumont, W.; Krief, A. Angew. Chem., Int. Ed. Engl. 1977, 16, 540. (c) Glass, R. S. Synth. Commun. 1976, 6, 47. (d) Evans, D. A.; Truesdale, L. K.; Grimm, K. G.; Nesbitt, S. L. J. Am. Chem. Soc. 1977, 99, 5009. (e) Liotta, D.; Paty, P. B .; Johnston, J .; Zima, G . Tetrahedron Lett. 1978, 5091. (f) Sassaman, M. B.; Surya Prakash, G . K.; Olah, G. A. Synthesis 1990, 104. (11) The increased tendency for endo attack in ring systems incorporating silicon, as opposed to all-carbon systems, has been rationalized by trajectory analysis: Wilt, J. W.; Lusztyk, J.; Perran, M.; Ingold, K. U. J. Am Chern. Soc. 1988, 107,281. (12) (a) Giese, B.; Dupuis, J. Angew. Chern., Int. Ed. Engl. 1983, 22, 622. (b) Adlington, R. M.; Baldwin, J. E.; Basak, A.; Kozyrod, R. P. J. Chem. Soc., Chem. Commun. 1983, 944. (c) Baumberger, F.; Vasella, A. Helv. Chim. Acta 1983,66, 2210. (d) Dupuis, J.; Giese, B.; Ruegge, D.; Fischer, H.; Korth, H.-G.; Sustmann, R. Angew. Chem., Int. Ed. Engl. 206 1984,23, 896. (e) Korth, H.-G.; Sustmann, R. ; Dupuis, J.; Giese, B. J. Chern. Soc., Perkin Trans. 2 1986, 1453. (13) Stoffyn, P. J.; Jeanloz, R. W. J. Am Chern. Soc. 1954, 76, 561, 563. (14) Corey, E. J.; Gras, J. L.; Ulrich, P. Tetrahedron Lett. 1976, 809. (15) Hanessian, S. Carbohydr. Res. 1966,2, 86. (16) Horton, D.; Weckerle, W. Carbohydr. Res. 1975, 44, 227. (17) Reich, H. J.; Wollowitz, S.; Trend, J. E.; Chow, F.; Wendelborn, D. F. J. Org. Chern. 1978,43, 1679. (18) Corey, E. J.; Samuelsson, B. J. Org. Chern. 1984, 49, 4735. (19) Smith, M.; Rammler, D. H.; Goldberg, H.; Khorana, H. G. J. Am. Chern. So c. 1962, 84,430. (20) Stawinski, J. ; Hozumi, T.; Narang, S, A.; Bahl, C. P.; Wu, R. Nucleic Acid Res. 1977, 4, 353. (21) Mancuso, A. J.; Swern, D. Synthesis, 1981, 165. (22) Johansson, R. ; Samuelsson, B. J. Chern. Soc., Perkin Trans. 11984,2371. (23) Nozaki, K.; Koichiro, 0.; Utimoto, K. J. Am. Chern. Soc. 1987,109,2547. (24) Koenigs, W.; Knorr, E. Chern. Ber. 1901, 34,957. (25) Reviews of the Koenigs-Knorr method: (a) Paulsen, H. Angew. Chern. , Int. Ed. Engl. 1982,21, 155. (b) Paulsen, H. Chem. Soc. Rev. 1984, 13, 15. 207 (26) Reviews of the trichloroacetimidate method: (a) Schmidt, R. R.; Pure Appl. Chern. 1989,61, 1257. (b) Schmidt, R. R. angew. Chern., Int. Ed. Engl. 1986, 25, 212. (27) (a) Isheda, H.; lmai, Y.; Kiso, M.; Hasegawa, A.; Sakurai, T.; Azuma, I. Carbohydr. Res. 1989, 195, 59. (b) Paulsen, H.; Sumfleth, B. Chern. Ber. 1979, 112, 3203. (28) Readily prepared from D-galactose: Shafizadeh, F. Methods Carbohydr. Chern. 1963,2,409. (29) Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244. (30) Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826. (31) Chana, J.; Collins, P.M.; Farina, F.; Peacock, D. J. J. Chem. Soc., Chem. Commun. 1988,94. (32) Stork, G.; lsobe, M. J. Am. Chern. Soc. 1975, 97, 6260. (33) Scriven, E. F.; Turnbull, K. Chem. Rev. 1988, 88, 351. (34) In our preliminary publication of this work, we were unaware of, and therefore did not cite, the following precedent for this transformation: (a) Bartra, M .; Romea, P .; Uprf, F.; Vilarrasa, J. Tetrahedron Lett. 1990, 46, 587. (b) Bartra, M.; Felix, U.; Vilarrasa, J. Tetrahedron Lett. 1992, 46, 587. We thank Professor Vilarrasa for bringing this work to our attention. (35) Lemiuex, R. U.; Takeda, T.; Chung, B. Y. A.C.S. Symposium Series 1976, 39, 90. (36) K.inzy, W.; Schmidt, R. R. Liebigs Ann. Chern. 1985, 1537. (37) For a related observation, see: Evans, D. A.; Kaldor, S. W.; Jones, K. J.; Clardy, J.; Stout, T. J. J. Am. Chern. Soc. 1990, 112,7001. 208 (38) Hasegawa, A .; Kaneda, Y.; Amano, M.; Kiso, M .; Azuma, I. Agric. Bioi. Chem. 1978,42,2187. (39) Guibe, F.; Dangles, 0.; Balavoine, G. Tetrahedron Lett. 1986, 27, 2365. (40) Schreiber, S. L.; Claus, R. E.; Reagan, J. Tetrahedron Lett. 1982, 23, 3867. (41) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978,43, 2923. (42) Kofron, W. G.; Baclawski, L. M. J. Org. Chem. 1976,41, 1879. 209 Appendix Catalog of Spectra ,. · 210 :)(H ~NHW, OCH 3 H 10 _L_____l_Ll.~ - 6 89.72 :n 71 - ~IJ_---,----~~----r-----r-----,-----------,------------r--------~~, 4000 3500 3000 2500 2000 1500 1000 211 Ph a-'\ MD AcNH H ~ O H , 6 H OCH3 11 6 941 . .ol1 IT .oiOOO 3500 3000 2500 2000 1500 1000 CI0- 1 500 212 ~ MO OCOPh AcNH H 0 , OCH 3 CH2Br 6 H 12 6 I 83 . 18 IT 81.~7~--~----~r-----r-----~----~-----------r----------~--------~--rCll-1 !100 1!100 3!100 3000 2!100 2000 JOOO 213 AcNH~OP:H,SePh H~C>~ 6 C>CH3 H 13 110! . 27 ST • . ~J---~-----r-----r-----r-----r-----------r-----------r--------L-~ 3!100 2!500 2000 1!100 1000 Cll... !100 214 ~ MO AcNH H OCOPh O , OCH3 CH2 s 14 a.72 S1 73.42~---,.-----~----,------r-----.------------,------------,.-------~---r- 4000 3500 l500 soco 215 AcNH?JH H-r-O~CH2 OCH 3 s 15 6 8Sl . 30 ST n . ~~~---.----.-----.-----r----.,----------.----------,-------~--~ 1000 l!IOO 1000 ea""' 500 - 216 0 92 . 79 :n 73 . 03~---..-----~----------~-----.r------------.------------.-------~~~ 4000 3500 3000 2500 2000 1!500 1000 217 0 6 IU . IIII ST 81.~~--~----~--~-----r----~--------~----------~------~~ 4000 !BOO JOOO 21!100 1000 11100 1000 c•~ 1100 218 0 CH3 S1-0 0/ H 6 CH 11 ' OCH 3 O 1' 0 H N 0 5' M~ MO AcNH NPMB 5 2 H TBSO 6' H OTBS 23a 91 . 61 IT 4000 3!500 3000 2!100 2000 S!500 sooo 219 0 CH3 SCNPMB f SePh I I '81-0~6 N~O 0/ s· 0 cH 3 ,,, M~MO AcNH 0 ,. H CH 2 TBSO H , . OCH3 6 . H OTBS 23b 112.73 lrT ~--~------r-----~----~----~------------~----------~--------4000 3500 2000 l!IOO 1000 220 OH H OTBS 24 10.2!1 ST 4000 2000 UIOO 1000 221 0 ·CNH OAc 6 AcNH NAO ,. H 26 AcO OAc _A__Ji_ _____ • • 111 IT .1 . 85~---,-----,----~r-----r-----~----------,------------r------~---,teoo 1000 - 222 0 s,)l OH NPMB oHJl.. ~ 1 AcNH N H 0 ,. 0 25 811 .11'5 IT 2000 UIOO tOOO 223 0 OAc:(J: 0 OAc ,. 0 H AcO 27 Synthetic - Authentic • - 224 AcNH 27 Synthetic Authentic 225 OAc ,. H • .e n Synthetic n.•~---r-----r----~----~----~----------,-----------,-------~--~ UIOO lOOO ••• n Authentic ..... ~---r-----r----~----.-----.-----------,-----------,---------~~ SIIOO 1000 1000 sooo I AcO \ OAc 1' "'0 => ••'•i <INTEGR4TOR> Juld2~'Ql l.C: t<Lt-4 FILE N4ME Ml 0 . 00 --- 32. 0 2 • in S• oothin1 NORMAL ~t nt au • arta 18 3 •i n Slo pt Ori f t OFF 3 0 n He i aht Widt h OFF CO - l NJECT A-4C Packin1 aaterial 3. 9 aa i D• 30. Oca Mobi It ph•se 0.10 al '•in Pre,surl!' ·c In J e ct t o n voluae 2~. 0 10.00 12 . 02 •• n 90-'1 0 DC Md~ EOH PSI ul AU" III n AU' •• n AU •• n 0 001 0 . 001 0. 001 0 l 0. 01 SILICA GEL AIJ••• n po 1 nt' 7 Srop It TEST I 0.0 .s Co-InJection ~0 1200 . 0 '·' ~ .. ............................................................................... Saaple naae Co I uan F low rate Ttaperaturt Ft ow cell Ml nus ptak Paper speed Bitt II M co rrect Tiat ranae Jntu·,·al Tlu double Wavtltnath Waters Oau 994 27 ................................................................................ ~ ; I 'CI NH AcNH~ \ ...--.:/OAc s NAO o_ 1 .H OAc 0 ,; 0 ~ = ~ 1 0.0 ~ .2 • .. .. Synthetic 1. 2 1.4 I.' ~ r Authentic " ~~~~~~~~~~~~~~~~~~~~~~= = ... ; ~ 0.0 N N 01 227 H H 31 d j_ l l ==r==r==~=,::;:-:-c · =;:=:::::;:::=~===;:~~ :, ___L_ __ , T - ':;:,:-=---,===;::= . ==;r==:~--r::...::.:.::==r--'--r-: ·=--c._:;:-...-:_-=, 4 0 2 F'F'!/ 84 . 30 IT 81.-~---r----~----~----r---~r----------r----------~--------~~ 1000 1000 11100 1000 228 )<H N3~ ~ is TBS0-1H 0 H 32 ~J L~ =---, = - ,=---:::::::---, --, ---, I ---, 6 8 -,· --, - ~ - ----, --, -- -, ---r-=-r==--.- --,-=----r= 4 2 113.87 ST 4000 31100 3000 11500 2000 11100 1000 ca-t 1100 229 OCOPh H H 33 I 0 - tw.eo IT • .• ~--~----r----r----~--~----------~--------~--------~ 1000 230 OCOPh Br H H 34 111.98~I I ST ! ; I I .., I I 1 I 153.03 --.----.----..----· . -,--·---'L..r- - - - -·-,---·- -- - --,-------.1.....,.... ~0 3!100 3000 2!SOO 2000 Sl500 sooo 231 OCOPh Br H H 35 ei! . SO n 78.15·~---,-----,r---~r-----r-----~----------~----------~--------~--r- 11100 S!IOO sooo 232 OCOPh Br H • 36 H . 67 In" 1 . . .L.--.-~---- - ---.... 4000 2!100 2000 ··-----, 1!100 1000 233 OCOPh SePh H H 37 e &4 . 7!5-'---,-- 4000 0 - ~-- -- T- - --r-- B)O 1000 11100 - , - - - - - - - - r - - - - - """-T-- 1!000 l!IOO lOOO 234 OCOPh SePh H H 38 __ U~-~-0 811.82 XT •.a.~---,----~r-----.-----r-----~----------.-----------~-----------L~ 2000 S!IOO sooo - 235 AcO~ AcO~c( 1 AcO OTBS 6 40 _L_ ... 8 6 4 2 92 . 68 IT 4000 3500 3000 2500 2000 1500 1000 ca-t 500 236 9 8 6 2 0 PPM e7.111 IT U . e7~--~----~-----r-----r-----r------~---r-----------r-----------+1000 4000 237 I I 9 8 L I 6 1 0 PPM .,. 17.!18 • . 71~---T-----T-----T-----r-----r-----------r-----------r----------~ 1000 ttiOO sooo 238 8 6 4 2 0 PPM IU . "nl liT --~~----~----~r-----~-----r------------~------------~------11500 1000 2000 4000 239 _j_ ____ L_ ____L __- ----- u_L_ .~~·-......-.--,.~·-~~~~·-~~~~~~~~~~~~~~~-~ 9 b r, " 100.00 liT O.OOJ----r----~----~----r---~~---------r----------~----------~ 3!100 2100 2000 1!100 tOOO 240 OCOPh H 46 93 . 87~ IT n I'~ ~\ II 'I ~ I ' 12 . 49 --- -, - -----, 4000 3500 ---- , -3000 - - ·,-- - - , - - -- - - - - , - -- - · -2!!00 2000 i~O - T - - ··- - - -!.000 Ctll-1 500 241 OCOPh SePh H 47 - 9 ~ . !!9 IT 4000 2000 1500 1000 242 I 9!..70 lrT - ~-''---1L--- - ·v I ~ -~~------~~---r----~--~----------~------------------~ SISOO !.000 243 AcNH OH TBS 0 CHJf 6 CH3 50 I 110 . 711 I"T ~--~--~----r----,-----r----~--------~----------T-------~~ 11100 1000 244 :x,H P~CH20O N3 TBSO 1 O H 6 H 51 .,. 83.18 ~.Q~J----r----~--~r---~--~. .---------,----------,-------~~~ 11100 1000 245 :)(" P~hH200 N3 , 0 HO 6 H 52 6 4 2 0 • . 30 IT 2000 1000 PPM 246 1111 . 82 IT !11 . 1!1..1..---.---...---...------,.-----r------r-------r-----.i.-~ 2!100 2000 1!100 1000 247 :x-" Ph~H200 H2N TBSO O 1 H s H 55 .,. 117 .73 111.& ~ 723.8 ce-< n.~~------r------r------r------r-------------r-------------.-------L 2000 t!IOO 4000 3000 tOOO 248 )<H Ph~H200 PhthN TBSO O s 1 H H 56 93.711 ST ~.2.~---r----~--~r----r----,----------,----------,--------J-,ca-l !500 11500 1000 4000 31500 3000 2!500 2000 249 Ph H O~ Phf-FVH,O PhthN O I, 6 ~H CCI 3 .... -- H NH 57 _____ ---- "-- ..__...._ 8!1 . 3~ S'!' ~.~~--~-----.-----,r-----r-----r-----------,------------r----------~4000 3500 3000 2'500 2000 1500 1000 250 8 0 118. «! ST ~.R·~--~----~----~r-----r-----r-----------,------------r--------~-,2000 t!!OO sooo 251 ' i 11!5.33 liT 74. 13~--.----r---..----r--"'T"""------,.------r----..L...-~ 4000 3!!00 3000 21100 2000 t!IOO sooo cr !100 252 SePh 1111 . 42 ST 81.21~--~r-----r-----,------r-----r-----------~----------~r---------~-r- 2000 1!500 1000 253 OH CbzNH--.--.L- TBS~O 11~ 0 CH 3 --i.l SePh H H 6" 60 CH3 !10.!5!1 IT ~-"~---r-----r--·---r----,-----,-----------~----------~--------~r- 2000 1!100 1000 254 OH SePh 9 2 0 - 81 . ~~---T-----T-----r-----r----~----------~----------,---------~~ 3000 2000 1!500 1000 255 SePh - '--" -------··~-- 90.98 ST S1.R~--r----r-----r----.-----,-----------,-----------~--------~~ 3000 2000 1!500 1000 256 OAc BOMO CbzNH O 0 11' 1· H TBSO H 0 0 6 • CH3--£-o I 6" CH 3 - 6 90.36 IT ~.13~---r----,-----~----r---~-----------r----------r-------~~- 2000 ~~ 1000 257 • . .a ItT 3000 2000 11500 1000 ca-4 1500 258 0 I L_--"-t--"-'H......._,....._ _.I_ _•....__.__.J,Jl,ll...___, ,______, '---q ~-~~--~--~----~--~----,---------.----------r-------L-,1!100 sooo 259 0 sePh H O ·rNBoc Jl. A N 0 1' H OTBS 65a 6 5 0 - n . R~--~----r---~-----r----~---------T----------~------~~ 4000 3800 3000 2!100 2000 UIOO 1000 260 0 5CNBoc I CH3 CH3''•· 1 ·si- O SePh 0/ BOMO CbzNH H 0 0 CH3-rO • 6 N~O 0 ,. H O TBSO H 6 TBSO 6 OTBS . ,. H 65b CH3 - 8 112 . 8!1 ST R.~t.~---r----~--~r----r----,---------~----------~------~..,U!OO 1000 261 1' H OTBS 91.110 ST 81.001~---r----,-----~----r---~~---------r----------,---------L-~ 4000 2000 S!IOO sooo 262 0 I h I I , ,1 , .t. 11 ·I 10 - 90 . 00 lET n . •~--~--~----~--~----~--------~--------~------~~ UIOO sooo 263 0 8 6 5 4 3 0 2 PPM 80.711 ST • . 0!~---r----~---,-----r----,----------,----------,---------~4000 !1100 3000 11100 1000 11100 1000 264 HO ,fNBo' 6 ~N~O )vo--J,. HI)-\\ AocO OAoc 72 9 10 8 6 5 4 3 2 0 PPiol 95.02 IT ~9 . 18~----r-----.-----~-----r-----.,-----------~----------~,---------~-r1000 2!500 2000 1!100 3000 4000 ~0 265 0 SCNBoc SePh 1 H 6 N~O 0 H AocO OAoc 74 6 10 II 93.86 IT 78.~~---r-----r-----r-----r----.r----------,-----------,-------~~~ 3000 2!100 2000 1!100 1000 ca-t !100 266 0 scNBoc 1 SePh s H N~O 0 H OH 0 10 II 1111 . 110 ST 3000 2!100 2000 !!lOCI !000 267 0 '~'NBoc l .A OH 6 OH 0 N 0 H HO OH 76 0 9 57.02 ST 47.1e~---r----,-----~----r----.,----------r----------,-------~--r3000 2!100 2000 1!100 1000 ca-f !500 268 78 I 10 I I 8 6 I 4 I 0 2 lilt . !!!! IT 4000 31500 3000 2!100 2000 1!100 1000 PPM 269 79 _ __..______ _-"--!_ g 6 __._A..__u_ _____ \....)...__ - 99 . 41. ST 21..~~--~----~------~----r-----~----------~----------~----------~~ ca-t !500 1.!500 1.000 2000 4000 3000 270 80 1 -,- I I p I 10 I I b 4 2 0 P PM 71.91~--~-----r-----r-----r-----r----------,-----------,-------~~,_ 2000 1!100 1000 271 81 ,. I l 8 4 0 PPM 93 . 06 ST 4000 3500 3000 2500 2000 1500 woo COl-o 500 272 82 [j I ' ' I ' 11 10 I ' a I ' 8 ' ' L_l__ I ' 6 ' ' ' I ' ~ .. I ' 4 I ' I' 0 1 84 . 36 IT 4000 2000 1!100 1000 ' I , I -1 -2 PPM 274 0 CH3 0 CH3~N--~~9~HH0 H HO HO 5~NH OH OH 0 sl..A N 11' H H • 6 0 H OH Tunicamycin-V Synthetic g Authentic - 275 CH3 0 CH3~N H 9 OH 0 _ __._ H~~> HO 6 • Tunicamycin·V Synthetic Authentic 276 Tunicamycin-V Ill )II Synthetic :.-. ... 0 0 I0 lO · :~i;·, Ill I' I''' 10 iii 40 10 ••r !!~ Authentic ... H a> "' 0 0 ·"l ;(: ;·:_ ___.,_-!"----~-----=-~---.:.......--~-~-~--~----f ....,_:>--t·_, .i· _______~..'f'·t-+-....jl.'·i.~. : ;-----· •:: : lO 10 10 30 40 · ~····.·~···•... . ,..,.. 2n , . Co-Injection ..... <C ~ ,'' .~ :;: 0 ';': -{-----""''v----_,.....____...,}.n.\.""!.:!--------~----'-~~~-.....;__.-~ I~ 10 lO l~ 40 30 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Wu~n 994 c M U L T I • C It I 0 M A T 0 G I A M , Sto" at 49.6~ al• Dat~ tt . . rant~ Ckvl6;92 U: 18:21 1.00 ·-· 40. 00 •lw Sa•,l~ las~lln~ corr~~• I nt ~rrval na.e Colwan Fl . . rate FILE IIAMI:: TESTI OFF IIORMAL Pa~r !kal~ T-Y CO-IWJtCT Pa~klna aaterlal 7.0 AUTO ~ALr AUTO SCAlE Tea~rat.re 4.5aaiD• 0 . 40 25.0 Fl . . cell IO.Dt .,..ed Ml M2 ~.Oca aJ;ala •c: • Mobile "hue IECJ:MAN OOS 15"'1 '5 CM JOH;fl20 lnJectlow vet ... '50 Pre•••r~ 1500.0 ,, •• •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• . ..... .. . 277 Tunlcamycin-V 76.~ IT Synthetic 63 . 931~----r-----~-----r-----.------r------------r------------r-------~--~ 4000 3500 3000 2000 1!500 1000 !13. 16 IT Authentic 74.12~---,------r-----,------r-----,------------,---------------r-------~-----r- 4000 3000 2000 1!500 1000