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SYNTHESIS OF MODIFIED MANNOSE OLIGOSACCHARIDES AS POTENTIAL INHIBITORS OF MYCOBACTERIAL LIFO ARAB INOMANN AN BIOSYNTHESIS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Vinodhkumar Subramaniam, M.S.
*****
The Ohio State University 2000
Dissertation Committee: Approvedyby
Dr. Todd L. Lowary. Adviser
Dr. T V . RajanBabu Adviser Dr. Christopher M. Hadad Department of Chemistry UMI Number; 9994944
UMI
UMI Microform 9994944 Copyright 2001 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT
Infections by Mycobacterium tuberculosis, the causative agent of tuberculosis
(TB). have reemerged as a public health threat in recent years. The resurgence in TB has sparked renewed interest in identifying new antibiotics that can be used to treat this disease, which claims nearly three million lives worldwide each year. This disease has been difficult to treat and can be attributed in part to the unusual structure of the cell wall of the organism, which presents a formidable barrier to the passage of drugs into the organism. In order to identify new anti-tuberculosis agents, our research goals are of two-fold: The first objective is to synthesize oligosaccharides which are potential substrates for the enzymes (mannosyltransferases) involved in the biosynthesis of one of the polysaccharides (mannosylated lipoarabinomannan, Man-LAM) found in the cell wall complex that surrounds the organism. The second objective is to prepare mannose-based disaccharides (type A and B, Page 38) which are potential inhibitors of the LAM biosynthesis. Such compounds are believed to be excellent lead candidates for new anti tuberculosis agents. During the synthesis of oligosaccharide substrates, a key step is the stereoselective synthesis of octyl P-arabinofuranoside via a 5^2 type displacement reaction of octanol with a benzyl-substituted arabinofuranosyl chloride. Selective deprotection followed by sequential addition of thioglycosides, afforded the oligosaccharide fragments of Man-LAM. In the synthesis of the disaccharide inhibitors, our strategy was to proceed via a common intermediate leading to more than one target.
We tlrst developed a methodology for the synthesis of type A mannose disaccharides, which are modified at C-2' of the mannose ring. We then made use of the intermediates used in the preparation of type A disaccharides in the synthesis of type B disaccharides, which contain an additional modification at C-6' of the ring. These compounds will play a significant role in understanding the carbohydrate-enzyme interactions and in turn, can be useful as lead compounds for drug design. Additionally, we also synthesized potential suicide inhibitors, which could function by forming a covalent bond with the active site of the enzyme. We characterized the target oligosaccharides by 800 MHz NMR spectroscopy to ensure purity of such compounds for biological studies. Biological investigations on type A disaccharides were performed in collaboration, which suggested that 4 out of 5 compounds in type A class are substrates of the enzyme a(l-^6)-ManT, but none inhibited the enzyme. Biological studies on type B disaccharides are in progress, as are studies with a ( I—>2)-ManT.
Ill This dissertation is dedicated to my parents, Subramaniam and Lalitha, for upbringing me
with Godly values, my best friend Juanda, for her love and support, my late sister,
Vidhya, for her respect and care toward me, all my friends, extended family and the one
whom I agapee (love) the most.
IV ACKNOWLEDGMENT
I want to take this opportunity to begin by thanking Dr. Todd Lowary for his project ideas, guidance and financial support. My sincere thanks to Dr. RajanBabu and
Dr. Hart, for their encouragement and motivation. I also, want to thank ail the professors who taught me courses at OSU, I IT, and at Loyola. Especially, to Dr. S. Govindarajan,
Dr. V. Sreenivasan, Dr. D. Loganathan and Dr. M.N.S. Rao for their personal attention and input toward my academic as well as personal growth.
My heart-felt gratitude to Dr. Charles Cottrell, Dr. Karl Vermillion, Susan
Hatcher, Dr. Kari Green-Church and others for their help toward NMR and MS experiments. I also, want to thank Dr. G. Besra and coworkers (from UK) for conducting biological experiments on my final compounds. My appreciation to Dr. Robert Field and
Phil, for their helpful suggestions toward my research. I want to thank Dr. Hart’s Group,
Dr. Babu’s Group, Dr. Parquette s Group and Dr. Paquette’s Group for letting me borrow chemicals under unavoidable circumstances.
I take this opportunity to convey my best wishes and thanks to all the Lowary group members (present and the past) for their help, support and encouragement. Matt, I thank God for you and fc. our friendship, words cannot be expressed to describe your love toward me. My gratitude to Joe, Grace, Wallace, Oana, Charla, Lori and Haifeng for their comradeship, to Prakxiti for her sincerity in lab maintenance and in sending elemental analysis samples, to Lori, Oana, Chris and Justin for ordering chemicals, to
Doug for helping with NMR and with resume writing. Special thanks to Raj for his brotherly love and helping me with synthesis of building blocks.
1 am grateful to the department of chemistry for their financial support as a GTA and instrumentation facility. 1 appreciate Bobbie Cassity, Kathie, Martha and others in the chemistry office for their sincerity and compassionate service toward me. I want to thank all my friends, brothers and sisters in faith for being there for me and helped me to have a family type atmosphere.
The pursuit of ones Ph.D. can be a challenging event; you work hard, teach hard, and sleep little. When the experiments are going well, you feel as if you are on top of the world, and when they are not. you may feel and experience isolation. It was during both of these times that God taught me some invaluable lessons: “Truly there is one that sticks closer than a brother." During these difficult times I learned to persevere and to trust God to help me with those situations that were beyond my control, whether it was a compound or a conflict. Being so close to finishing and having to deal with my sister's death, I remember Him comforting me and saying "Just show up, and I will help you with your work." God also, taught me to be compassionate and helpful to my colleagues, especially when their work was not going well. I know now that the good times are victories of the difficult. To the reader I want to leave with you this message: your belief in God and
His love will take you far beyond your comprehension.
vi VITA
May, 28 1973 ...... Born - Chennai. India
1990 - 1993 ...... B.S., Loyola College, India
1993 - 1995 ...... M.S. Chemistry, Indian Institute of
Technology, Chennai, India
1995 - present ...... Graduate Teaching and Research
Associate, The Ohio State University
PUBLICATIONS
1. Subramaniam, V.; Lowary, T. L. "Synthesis of Oligosaccharide Fragments of Mannosylated Lipoarabinomannan from Mvcobacteriiim tuberculosis ", Tetrahedron. 1999, 55, 5965-76.
2. Kannan, T.; Subram aniam , V.: Loganathan, D. “Syn th esis of Glycosyl phosphoramidates: Novel Isosteric Analogs of Glycosyl Phosphates ”, Molecules, Third International Electronic Conference on synthetic organic chemistry, 1999, ECSOC-3.
3. Subramaniam, V.; Kannan, T.; Loganathan, D. “Synthesis of Glycosyl phosphoramidates: Novel Isosteric Analogs of Glycosyl Phosphates ”, Paper presented in the National Carbohydrate Conference, 1997, Kanpur, India.
4. Subramaniam, V.: Pathak, T.; Ganesh, K. N. “Synthesis of New Class of Acyclonucleosides Containing Tertiary Nitrogen Atom”, Project report submitted to Jawaharlal Nehru Center for Advanced Scientific Research, June 1994, National Chemical Laboratory, Pune, India.
Vll FIELD OF STUDY
Major Field: Chemistry
vii; TABLE OF CONTENTS
Ease
A bstract...... ii
D edication ...... iv
Acknowledgements ...... v
\/ita ...... vii
List of T a b le s ...... xiv
List of F ig u res ...... xv
Abbreviations ...... xx
Chapters:
1. Introduction ...... 1
1.1 General Introduction ...... I
1.2 Biological background ...... 2
1.2.1 General aspects of mycobacteria ...... 2
1.2.2 Pertinent aspects of mycobacteria ...... 3
1.2.3 Structure of mycobacterial cell w all ...... 4
1.2.4 Structure of lipoarabinomannan ...... 8
1.2.5 Role of LAM in disease progression ...... 12
1.3 B iological formation of oligosaccharides ...... 14
ix 1.3.1 Glycosidases and glycosy[transferases ...... 14
1.3.2 Suggested glycosyltransferase mechanisms ...... 16
1.4 Biosynthesis of lipoarabinomannan ...... 19
1.4.1 Mannan biosynthesis ...... 19
1.4.2 Arabinan biosynthesis ...... 22
1.4.3 Addition of capping species ...... 23
1.4.4 Evidence for the postulated biosynthetic pathways ...... 24
1.4.5 Mode of action of ethambutol ...... 24
1.5 Inhibitors of glucosyltransferases ...... 27
1.5.1 Common carbohydrate-protein interactions ...... 27
1.5.2 Information on the active sites of glycosyltransferases ...... 28
1.5.3 N-acetylgalactosaminyltransferase inhibitor ...... 32
1.6 Scope of the project ...... 34
1.6.1 Design of glycan fragments of Man-LAM ...... 34
1.6.2 Design of mannan core inhibitors ...... 37
1.6.3 Modifications probing hydrogen bonding ...... 37
1.6.4 Modifications probing steric interactions ...... 39
1.6.5 Design of suicide inhibitors ...... 39
1.7 Synthesis of oligosaccharides ...... 41
1.7.1 Glycoside synthesis ...... 41
1.7.2 Alkylation of glycosyl alkoxides ...... 42
1.7.3 Alkylation of aglycosidic alkoxides ...... 43
1.7.4 Trapping of glycosyl cations with alcohol ...... 43
X 1.7.5 1,2-Trans mannopyranosyl linkages ...... 44
1.7.6 Role of substituents and stereocontrol ...... 49
1.7.7 1,2-Cis glycosyl linkages ...... 50
1.7.8 1,2-Cis arabinofuranosyl linkages ...... 51
2. Synthesis of glycan fragments of Man-LAM ...... 56
2.1 Introduction ...... 56
2.2 Synthetic strategy ...... 56
2.3 Retrosynthetic analysis ...... 58
2.4 Synthesis of thioglycoside donors ...... 59
2.5 Synthesis of octyl 3-D-arabinofuranoside ...... 62
2.5.1 .Attempted Yb(OTf)] mediated glycosylation ...... 62
2.5.2 Attempted glycosylation with imidate ...... 64
2.5.3 Glycosylation with arabinofuranosyl chloride ...... 65
2.5.4 Synthesis of acceptor alcohol 53 ...... 67
2.6 Synthesis of octyl 3-D-arabinofuranoside 16 ...... 69
2.7 Synthesis of oligosaccharides 17-19 ...... 69
2.8 NMR analysis ...... 74
3. Synthesis of mono modified mannose disaccharides as potential inhibitors of
LAM biosynthesis ...... 75
3.1 Introduction ...... 75
3.2 Retrosynthetic analysis ...... 76
xi 3.3 Synthesis of acceptor alcohol 75 ...... 78
3.4 Synthesis of disaccharide 20-22 ...... 80
3.5 Synthesis of trichloroaceiimidate 76 ...... 81
3.6 Synthesis of disaccharide 23 ...... 88
3.7 Synthesis of trichloroacetimidate 76 ...... 89
3.8 Synthesis of disaccharide 24 ...... 91
3.9 NMR analysis ...... 92
3.9.1 Effect of substituents on '^C chemical shifts of C-2' ...... 92
3.10 Biological investigations ...... 92
4. Synthesis of di-modified mannose disaccharides as potential inhibitors of LAM
biosynthesis ...... 94
4.1 Introduction ...... 94
4.2 Retrosynthetic analysis for the targets 25-28 ...... 95
4.3 Retrosynthetic analysis for the targets 29-30 ...... 97
4.4 Selective fluorination at C-6 ...... 99
4.5 Synthesis of disaccharide 25 ...... 101
4.6 Synthesis of disaccharide 26 ...... 103
4.7 Synthesis of disaccharide 27 ...... 106
4.8 Synthesis of disaccharide 28 ...... 107
4.9 Synthesis of trichloroacetimidate 109 ...... 109
4.10 Synthesis of disaccharide 29 and 30 ...... I l l
4.11 Synthesis of disaccharide 29 ...... 113
xii 4.12 Unexpected anhydro sugar formation ...... 115
4.13 Synthesis of disaccharide 30 ...... 117
4.14 NMR analysis ...... 119
4.14.1 'T NMR and coupling constants ...... 118
4.14.2 Effect of substitution on ‘^C chemical shifts ...... 120
5. Synthesis of potential suicide inhibitors of mannosyltransferases in LAM
biosynthesis ...... 121
5.1 Introduction ...... 121
5.2 Retrosynthetic analysis ...... 122
5.3 Synthesis of acceptor alcohol 133 ...... 123
5.4 Synthesis of disaccharide 31 and 32 ...... 123
5.5 Summary ...... 126
6. Experimental section ...... 127
Bibliographic references ...... 199
Appendix A ...... 207
Appendix B ...... 215
Xlll LIST OF TABLES
Table Page
3.1 Comparison of '^C chemical shifts in compounds 20-24, 3land 32 ...... 92
4.1 '‘’F chemical shifts and 'H-'^'F coupling constants ...... 119
4.2 Comparison of '^C chemical shifts of compounds 20 and 25-30 ...... 120
A. 1 Mannoside acceptor assay ...... 211
XIV LIST OF FIGURES
Figure Pnge
1.1 Structures of common antimycobacterial drugs ...... 5
1.2 Schematic representation of mycobacterial cell wall ...... 6
1.3 Structure of Lipomannan in LAM ...... 8
1.4 Structure of LAM with terminal ends are mannose capped ...... 9
1.5 Structure of terminal hexasaccharide Ara^, uncapped and capped ...... 11
1.6 .A) An example of a glycosyltransferase reaction and
B) other glycosyl donors ...... 15
1.7 An example of a glycosidase reaction ...... 16
1.8 Proposed mechanism for the glycosyltransferase mediated transfer of a
carbohydate with inversion of configuration ...... 17
1.9 Proposed mechanism for the glycosyltransferase mediated transfer of a
carbohydate with retention of configuration ...... 18
1.10 Structure of Phosphotidylinositols PIMi and PIM, ...... 19
1.11 Structures of mannosyl phosphoprenols found in mycobacteria ...... 20
1.12 Postulated pathways for the biosynthesis of the PIM’s, LM and LAM ...... 21
1.13 Structure of P-D-arabonofuranosyl phosphodecaprenol ...... 22
1.14 Inhibition of EMB and truncated LAM. structural comparison with AG ...... 26
XV 1.15 Synthesis of Gal-P-(l-^)-GlcNAc-R catalyzed by P4Gal-TI ...... 29
1.16 A representative example for a fucosyl transfer catalyzed by FucT ...... 31
1.17 Synthesis of GalNAca-(l->3)[Fuc(l->2)Gaip-OR] catalyzed by GalNAcT 33
1.18 Glycan fragments of ManLAM ...... 35
1.19 Proposed schematic representation of the role of the substrate analog ...... 36
1.20 Structures of analogs of type A and type B ...... 38
1.21 Structures of suicide inhibitors ...... 40
1.22 Glycoside synthesis via S^2 reaction using a glycosyl alkoxide ...... 42
1.23 Glycoside synthesis via Ss2 reaction using a glycosyl halide ...... 43
1.24 Glycoside synthesis via trapping of a glycosyl cation by an alcohol ...... 44
1.25 Mechanism for orthoester 35 and the 1,2-trans mannoside 36 ...... 45
1.26 Mechanism of formation of glycosyl cations from a thioglycoside ...... 47
1.27 Mechanism of activation in case of trichloroacetimidates ...... 48
1.28 Effect of substituents on sterocontrol of glycosylation of 2-deoxy substituted
trichloroacetimidates ...... 50
1.29 Methanolysis of a-D-arabinofuranosyl chloride 42 ...... 51
1.30 General strategy of Intramolecular Aglycone Delivery ...... 52
1.31 Synthesis of P-D-arabinofuranoside 46 via I AD methodology ...... 53
1.32 Synthesis of hexasaccharide Ara^ ...... 55
2.1 A representative example of our synthetic strategy ...... 57
2.2 Retrosynthetic analysis ...... 58
2.3 Synthesis of thioglycoside 51 ...... 59
xvi 2.4 Synthesis of thioglycoside 52 ...... 61
2.5 Stereoselective synthesis of octyl P-D-ribofuranoside ...... 62
2.6 Pictorial representation of intemediate 61 complexed with [Yb] ...... 63
2.7 Attempted Yb(0Tf)3 glycosylation of 60 ...... 64
2.8 Attempted glycosylation of imidate 62...... 65
2.9 Synthesis of Octyl 2.3,5-tri-O-benzyl-P-D-arabinofuranoside 64 ...... 66
2.10 Synthesis of building block 53 ...... 68
2.11 Synthesis of Octyl p-D-arabinofuranoside 16 ...... 69
2.12 Synthesis of disaccharide 17 ...... 71
2.13 Synthesis of triisaccharide 18 ...... 72
2.13 Synthesis of tetrasaccharide 19 ...... 73
3.1 Type A disaccharides 20-24...... 76
3.2 Retrosynthetic analysis for the disaccharides 20-22 ...... 77
3.3 Retrosynthetic analysis for the disaccharides 23 and 24 ...... 78
3.4 Synthesis of acceptor alcohol 75 ...... 79
3.5 Synthesis of disaccharide 20 ...... 81
3.6 Synthesis of disaccharides 21 and 22 ...... 83
3.7 Fluorination of D-glucal 86 with selectfluor 87 ...... 87
3.8 Synthesis of trichloroacetimidate 76 ...... 87
3.9 Synthesis of disaccharide 23 ...... 88
3.10 Synthesis of trichloroacetimidate 77 ...... 90
3.11 Synthesis of disaccharide 24 ...... 91
xvii 4.1 Type A disaccharides 25-30 ...... 95
4.2 Retrosynthetic analysis for the disaccharides 25-28 ...... 96
4.3 Retrosynthetic analysis for the disaccharides 29 and 30 ...... 98
4.4 Mechanism of fluorination with DAST reagent ...... 99
4.5 Some reported examples of DAST induced fluorination reactions ...... 100
4.6 Synthesis of disaccharide 25 involving selective fluorination at C-6' ...... 103
4.7 Synthesis of disaccharide 26 ...... 105
4.8 Synthesis of disaccharide 27 involving selective fluorination at C-6' ...... 106
4.9 Synthesis of disaccharide 28 ...... 108
4.10 Synthesis of trichloroacetimidate 109 ...... 110
4.11 Synthesis of the intermediate 129 ...... 112
4.12 Synthesis of disaccharide 29 ...... 114
4.13 Unexpected anhydro sugar formation from the reaction of 129 with DAST 115
4.14 Proposed mechanism for the formation of anhyro sugar 131 ...... 116
4.15 Synthesis of disaccharide 30 ...... 117
5.1 Retrosynthetic analysis for the disaccharides 31 and 32 ...... 122
5.2 Synthesis of building block 133 ...... 123
5.3 Synthesis of disaccharides 31 and 32 ...... 125
xviu A. 1 Synthesis of 14 (Cjo-Man) from GDP-Man ...... 208
A.2 An illustrative example of a reaction of with '■*€,-labeled 14 (C;o-Man)with a
potential substrate 20 catalyzed by a (l->6)-M anT ...... 209
A.3 Disaccharide acceptors and their values ...... 214
XIX ABBREVIATIONS
GDP Guanosine diphosphate
UDF Uridine diphosphate
GlcNAc 2-Acetamido-D-glucopyranose
GalNAc 2-Acetamido-D-galactopyranose
CMP Cytidine monophosphate
NeuAc N-Acetylneuraminic acid
Xyl Xylose
Phe Phenylalanine
Arg Arginine
Lys Lysine
Asp Aspartic acid
GIu Glutamic acid
Lac N Ac 2-Acetamido-lactose
NIS N-Iodosuccinimide
TMSOTf Trimethylsilyl triflate
DDQ 2,3-Dichloro-5.6-dicyano-1,4-benzoquinone
HOAc Acetic acid
NMR Nuclear Magnetic Resonance
XX AIBN a-a'-Azobisisobutyronitrile
Im Imidazole
DMF Dimethy Iformamide
TBAF Tetrabutylammonium fluoride
Py Pyridine
THF Tetrahydrofuran
COSY 'H-'H Correlation spectroscopy
TOCS Y Totally correlated spectroscopy
HSQC ‘H-''C Heteronuclear Single Quantum transfer correlation
TBDPS Tetrabutyldiphenylsilyl
HRMS High Resolution Mass spectrometry
DMAF 4,4-Dimethylaminopyridine
DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
DIAD Diisopropyl azodicarboxylate
TLC Thin layer chromatography
TMS Trimethylsilane
MALDI Matrix assisted laser desorption/ionization
ATP Adenosine triphosphate
XXI CHAPTER 1
INTRODUCTION
1.1 GENERAL INTRODUCTION
Mycobacterium tuberculosis, the causative agent of tuberculosis, claims nearly three million lives each year and a third of the world's population has been estimated to be infected by this pathogen.' ' Other mycobacterial infections which are of serious human health threats are Mycobacterium leprae, the causative agents of leprosy claims one million lives annually^ and Mycobacterium avium infections are increasingly the cause of death in AIDS patients.'* These infections have attracted renewed attention.
This is due not only to their increasing incidence in both industrialized and developing world/ but also due to the emergence of strains of these organisms resistant to commonly used anti-mycobacterial drugs.* Historically, the treatment of these diseases has been difficult, requiring strict adherence to a regimen of three or more antibiotics that must be taken for several months.^ The difficulty in treating these diseases is directly related to the unusual structure of the cell wall of the organism, which presents a formidable barrier to the passage of antibiotics into the organism.* These drug regimens employ at least one
I antibiotic that inhibits the cell wall biosynthesis {e.g., ethambutol or isoniazid), in combination with others that have intracellular targets {e.g., rifampicin or streptomycin).’
Such combinations slowly destroy the integrity of the cell wall and allow other antibiotics to pass into the organism more easily.
There has been increasing interest in the development of new drugs that act by inhibiting the enzymes involved in the assembly of mycobacterial cell wall.'’"'’ A significant portion of the cell wall complex is carbohydrate as evident from its detailed structure. We were interested in the study of biosynthesis of LAM (Lipoarabinomannan), a major antigenic component of cell wall. Our research focuses on designing and synthesis of oligosaccharides which will interfere with the normal biosynthetic assembly of LAM making it weaker or more permeable to other antibiotics. Such compounds are of potential use as drugs in prevention and treatment of tuberculosis.
1.2 BIOLOGICAL BACKGROUND
1.2.1 GENERAL ASPECTS OF MYCOBACTERIA
Mycobacteria are a genus of prokaryotes most of which are soil saprophytes that are 0.2 to 0.6 x I to 10 p.m in size. The cell wall is extremely thick and rich in lipids.
This cell wall is responsible for many of the characteristic properties of the bacteria {e.g., slow growth, resistance to detergents, resistance to common antibacterial antibiotics, antigenicity).'^ The basic structure of the cell wall is typical of gram-positive bacteria: a cytoplasmic membrane overlayed with a thick peptidoglycan layer and with free lipids, carbohydrates and polypeptides.'* This hydrophobic barrier impedes the entry of nutrients into the cells, and necessitates the expenditure of large quantities of energy to synthesize lipids.'^
1.2.2 PERTINENT ASPECTS OF M. tuberculosis
Tuberculosis is the classic human mycobacterial disease and the causative agent
M. iiiberculosis was discovered by Robert Koch in 1982, when TB was referred to as the
“ The Great White Plague of Europe.'" The infection is acquired through the inhalation of aerosolized infectious particles, which then travel to the terminal airways. At this site the bacteria penetrate into unactivated alveolar macrophages and replicate freely.
Infected macrophages can also spread during the initial phase of the disease to the local lymph nodes, as well as into the bloodstream and other tissues {e.g.. bone marrow, spleen, kidneys, central nervous system).'* The TB infection can be identified by performing a tuberculin skin test using purified antigenic protein derivatives as the skin test reagent and the test can be used to measure a patient’s cellular immune response to infection.
The modem, standard “ short-course “ therapy for tuberculosis involves treatment of patients with a combination of four drugs (Figure l.l).'* including isoniazid
(isonicotinic acid hydrazide, INH, 1), rifampicin (2), ethambutol (EMB, 3) and pyrazinamide (4) for several months. Globally, INH and rifampicin together constitute the backbone of M. tuberculosis chemotheraphy. INH functions as a pro-drug which inhibits biosynthesis of the mycotic acid (lipid) portion of the mycobacterial cell wall,'* whereas rifampicin targets mycobacterial RNA polymerase.'" Recent studies suggest that
EMB primarily inhibits the arabinofuranosyltransferases which are responsible for the synthesis of arabinan components of the cell wall.'' ” Both EMB and INH lead to truncated structures, resulting in organisms with weakend cell walls, which in turn makes them more susceptible to the passage of other antibiotics across this barrier.
Pyrazinamide is an active semidormant bacilli, and displays strong synergy with INH and rifampicin, shortening the chemotherapeutic schedule from 12-18 months to 6 months.
These three drugs together form the basis of the current WHO DOTS (World Health
Organization, Directly Observed Therapy. Short course).'^ NHg
f] NHz V
Isoniazid, 1 Pyrazinamide, 4
H jO H
H CH2CH3 H3CH2C N CHaOH Rifampicin, 2
Ethambutol, 3
Figure 1.1 Structures of common antimycobacterial drugs
1.2.3 STRUCTURE OF MYCOBACTERIAL CELL WALL
The mycobacterial cell wall is an intricate structure of polysaccharides, proteins, lipids, and glycolipids.'^ A schematic drawing illustrating its general structural motifs is shown in Figure 1.2.'^ '“ The cell wall complex has five major components: Proteins ^
Phosphate ® QD QDD Mycolic Acids Q Glycolipids O
Arabinogalactan O n # /) # Lipoarabinomannan O r * r *â Peptidoglycan # lllllllllllilllllllllllllllllllllllllllllllllllllllllllllllllllllll
Figure 1.2 Schematic representation of mycobacterial cell wall 1 . the plasma membrane;
2 . peptidoglycan, a polymer common to many bacteria;
3. the mycolyl-arabinogalactan (AG) complex, a polymer of galactofuranosyl and arabinofuranosyl residues covalently linked to peptidoglycan and capped at its non reducing terminus by hydrophobic mycolic esters;
4. lipoarabinomannan (LAM) and its truncated form, lipomannan (LM), a polymer of
mannopyranosyl and arabinofuranosyl residues which are postulated to be linked to the
plasma membrane through a phosphotidylinositol (PI) anchor and;
5. lipids and glycolipids noncovalently bound to the mycolate esters. Finally, there are a
number of polysaccharides and proteins at the outermost periphery of the cell wall envelope which are often referred to as the capsule.
Although the structures of glycolipids and mycolic acids are specific to a
particular member of the genus, the polysaccharide components are very similar, if not
virtually identical in all mycobacteria. This has been proved important in studying these
organisms, because working with M. tuberculosis and M. leprae is difficult. Aside from
the obvious health threat, these two species are examples of “slow-growing”
mycobacteria which are difficult to culture in the laborator).'^ Consequently, many of
these structural and biosynthetic investigations have been conducted using “fast-
growing” mycobacteria such as M. sm egm atis. Furthermore, this species is non-
pathogenic to humans. 1.2.4 STRUCTURE OF LIPOARABINOMANNAN
Lipoarabinomannan (LAM) and its arabinan-devoid counterpart, lipomannan
(LM) are considered prokaryotic versions of a growing body of biologically important phosphotidylinositol (PI) "membrane anchors". Structural analysis of LAM suggest that in addition to arabinose and mannose residues, it contains glycerol, inositol, phosphate, palmitate and tuberculostearate. Both LAM and LM are believed to be non-covalently attached to the cytoplasmic membrane via the lipid portion of the PI moiety.^
Mana(l — 6)Mana( i-*6)Manoi(l— 6)Mana( 1 — 6)Mana( I— 2) a( a(l-> 2 )| I I ct( 1 -^ 2 ) I OUTER Man I M anl PI------MEMBRANE OF MYCOBACTERIAL L. ..J 10 CELL t w MANNAN CORE Mana(l —► 6)
Figure 1.3 Structure of Lipomannan in LAM
(Ara = Arabinofuranose polysaccharide) ï Ë -et' A
A
At-Ù)
A
A A>, A -^A A ^ 4 ^
nositol Palmitate M -+M -t>M 0-T uberculostearate
A = a-(1->5) Ara/ A = a-(1->3) Ara/ A = P-(1->2) Ara/ M = a-(1-»6) Manp M= a-(1-^2) M anp M = a-(1->5) Manp
Figure 1.4 Structure of LAM where the terminal ends are mannose capped It has been proposed that phosphotidylinositolmannoside (PIM,, 11, Page 19)
serves as the foundation upon which LAM and LM are built. The mannan core of both
polymers consists of a linear a ( l—> 6 ) linked mannan which extends from the mannose
residue linked to the 6 -position of PIM,."'’ The length of the glycan is dependent upon the
organism'^ and the polymer is further substituted by the attachment of a ( l—> 2 ) linked
mannopyranose residues as shown in Figure 1.3. In LAM. this mannan is further elaborated by the addition of arabinofuranose residues in a linear a(1^5) linked chain
with periodic branch points. At the distal ends of this polysaccharide is the
hexasaccharide motif. 5 (Page 11). The complete structure of LAM is shown in Figure
1.4.
In the case of LAM isolated from virulent strains of M. tuberculosis, both the
linear a (l—>5) Araf termini and the hexasaccharide Ara^, (5) termini are capped with
a(l->2) Manp residues and this polymer is termed as Mannosylated LAM (ManLAM,
6 ) . In another structural modification of LAM. discovered recently from strains of
M. smegmatis, this hexasaccharide is capped with inositol phosphates thus providing a
new type of LAM termed phosphotidylinositol-glyceroarabino-mannan (PI-GAM,
as shown in Figure 1.5.
10 OH
HO
HO
5 R = H HP
HO' 6 A R = H?
H P HP HP 6 C R = HO'
6 B R = HO'
.n/vv HO
.A/VV*
0 H_ HP 7 R = HO OH
Figure 1.5 Structure of terminal hexasaccharide Ara«„ uncapped and capped
11 1.2.5 ROLE OF LAM IN DISEASE PROGRESSION
It is believed that LAM is anchored in the plasma membrane by its “lipid anchor" and protrudes through the thickness of the cell wall so that its terminal arabinose or
mannose-capped or inositol-capped arabinose units are accessible outside. This would
result in the presentation of all or part of the polymer outside the cell envelope where it
would interact with the immune system of the host. ’'
LAM has been implicated in many immunomodulatory events occurring during
progression of the disease. These include inhibition of macrophage activation/' the
induction of cytokines.the neutralization of potentially cytotoxic oxygen free
radicals,'* the inhibition of kinase activities,'*'' the promotion of tyrosine
dephosphorylation’ and inducing the expression of collagenases that destroy the
extracellular matrix of the lung.'"^ A detailed understanding of the structural features of
LAM responsible for these events is not available; however, in some cases there are
reports suggesting that they arise from a particular structural domain. For example, the
ability of LAM to scavange oxygen free radicals is increased when the fatty acyl groups
of the PI portion are removed by basic hydrolysis, indicating that this property is
localized to the glycan portion of the polymer.^* In contrast, the ability of LAM to induce
cytokines was eliminated by removal of the lipid portion of the PI. Furthermore, small
phosphatidyl inositol mannoside structures also elicit cytokines and thus it appears that
the PI portion of LAM is critical for this purpose.^'^
12 Many of the biological activities associated with LAM appear to be localized at the periphery of the structure. LAM from M. smegmatis is capped with inositol phosphate residues (7),"‘ and this form of the polymer is a potent inducer of mmor necrosis /actor-a (TNF-a). LAM from M. tuberculosis, capped with mannopyranosyl
residues (6 ), have been suggested to be involved in the initial stages of infection by adhering to human cells through their interaction with mannose binding proteins, and this recognition event may be a critical first step in the phagocytosis of the organism by the host.'”’'“ '*' Recently, it was reported that polystyrene microspheres, when bound to LAM, are internalized by human macrophages,"'' and this confirms earlier reports on the uptake of mycobacteria by host cells via mannose receptors."*^ However, the mechanism by which LAM functions is not well understood and is currently an expanding area of research.
Finally, a major recent discovery is that T-cells recognize LAM via major histocompatibility complex (MHC)-independent antigen presentation pathways."'"'"'^
Through a sequence involving the initial recognition of LAM by a mannose receptor and the protein CD 14, LAM is first processed and then complexed with CD lb which is subsequently expressed on the surface of the antigen-presenting cell. Recognition by T- cell requires both the carbohydrate and lipid portion of the molecule and two distinct cell lines were shown to differentiate between structurally different LAM fragments. This suggests that there is significant amount of specificity in T-cell responses mediated by the
LAM-complexed CDl glycoproteins.
13 1.3 BIOLOGICAL FORMATION OF OLIGOSACCHARIDES
I.3.I GLYCOSIDASES AND GLYCOSYLTRANSFERASES
The biosynthesis of oligosaccharides is controlled by the combined action of two class of enzymes, the glycosyltransferases and the glycosidases/^ These classes of enzymes have complementary functions: the former enzymes add carbohydrates to oligosaccharide chains and the latter cleave them from these chains. The enzymes involved in the biosynthesis of LAM are mannosyl transferases and arabinofuranosyltransferases. The biosynthesis of LAM will be discussed after looking into the fundamental aspects of both classes of the enzymes mentioned above.
Glycosyltransferases catalyze the transfer of carbohydrates from activated carbohydrate donors to a hydroxyl group at the non-reducing end of a growing oligosaccharide as shown in Figure 1.6.'*’ Many of the activated carbohydrate donors in in vivo are nucleotide sugars. For example, in humans, the sugar nucleotides used are
derivatives of guanine (GDF-Fuc, GDP-Man), uridine (UDF-Gal ( 8 ), UDF-GlcNAc,
UDF-GalNAc. UDF-Xyl), and all are classified as sugar nucleotide diphosphate donors.
Cytosine (CMF-NeuAc, 9) is the only sugar nucleotide monophosphate donor."** Another type of donor employed for the syntheses catalyzed by many membrane-bound
glycosyltransferases is that of the dolichol phosphate sugars ( 1 0 ), themselves synthesized from a nucleotide phosphate sugar.'*'*
14 OH
-OH H' OH
OR OH
R = Glycoconjugate UDP-Gal, 8 OH OH
galactosyltransferase
. o - 'h o '- r
OR +
AcNH^ 7 ^ ^ - ^ ' ' HO OH OH
CMP-NeuAc. 9 O p
I / h o t
Dol-P-Man,10 n = 16-17
Figure 1.6 A) An example of a glycosyltransferase reaction and
B) other glycosyl donors
15 OH
Galactosidase OH OH
OR OH NHAc + • OH R = Glycoconjugate HO- T * ^ 0 R NHAc
Figure 1.7 An example of a glycosidase reaction
Glycosidases remove carbohydrate units from oligosaccharides, producing in most cases a reducing monosaccharide and a shortened oligosaccharide as shown in
Figure 1.7/" These enzymes play a significant role in the formation of glycoproteins in both the endoplasmic reticulum and Golgi apparatus.
1.3.2 SUGGESTED GLYCOSYLTRANSFERASE MECHANISMS
During the transfer of the carbohydrate from a sugar nucleotide catalyzed by the glycosyltransferase. the stereochemistry of the anomeric center of the sugar nucleotide can be preserved, that is, the enzymes transfers the carbohydrate with retention or inversion of configuration. Although no precise mechanisms are known, hypotheses exist.
16 % BH
O-R OR + U D P
ÜDP
Figure 1.8 Proposed mechanism for the glycosyltransferase mediated transfer
of a carbohydate with inversion of configuration
The simplest mechanism to explain inversion of configuration is a direct S^Z-type displacement of the nucleoside diphosphate (UDP) from the sugar nucleotide by the acceptor hydroxyl group.'' Possible assistance by a base on the enzyme to help deprotonate the incoming alcohol has also been proposed as shown in Figure 1.8.
To explain retention, a double displacement mechanism has been proposed.^
The first step would be the displacement of the UDP from the sugar nucleotide located in the active site thus forming a covalent glycosyl-enzyme intemediate. Transfer of the
17 UDF
UDP
i .
\ Z : : Z V ^
OR L r - R r
Figure 1.9 Proposed mechanism for the glycosyltransferase mediated transfer of a
carbohydrate with retention of configuration
18 sugar residue to the growing oligosaccharide would then be completed by expulsion of the enzyme from the glycosyl-enzyme intermediate by the OH of the acceptor residue
(Figure 1.9 Again, there is a possible assistance by a base on the enzyme to help deprotonate the incoming carbohydrate alcohol.
1.4 BIOSYNTHESIS OF LIPOARABINOMANNAN
1.4.1 MANNAN BIOSYNTHESIS
Initial insights into the assembly of the core mannan portions of LM/LAM was provided by studies on the assembly of the phosphatidylinositols (PlMs) found associated with the cell wall membrane,which constitute the core structures of this polymers.
RO OH
MO
Ri = palmitate OR3 HO Rg = tuberculostearate MO
11 R = H, PIM2 OH 12 R = a-D-Manp-( I—>2)- OH a-D-Manp-( I—>2)- OH a-D-Manp, PIM 5
Figure 1.10 Structure of Phosphotidylinositols PIM, and PIM,.
19 The formation of PI[‘‘*C]Man 2 (PIM^) from PI and GDP[‘‘*C]Man was demonstrated, and this intermediate was shown to be subsequently palmitoylated by palmitoyl CoA to give a mixture of acylated and non-acylated derivatives as shown in
Figure l.IO. It was initially believed that the immediate donor of all the mannose residues was GDP-Man in LM/LAM. However, identification of the presence of two
mannosyl phosphoprenols, 13 (C, 5 -Man) and 14 (Cjo-Man), in mycobacteria opened up the possibility that these species were also involved in mannan biosynthesis.
pH
HO
3-D-mannopyranosyl phosphooctahydroheptaprenol, 13
PH
HO
P-D-mannopyranosyl phosphodecaprenoi, 14
Figure 1.11 Structures of mannosyl phosphoprenols found in mycobacteria
20 The proposed pathway for lipomannan assembly in mycobacteria is shown in
Figure 1.12."^ The process is initiated by the conversion of PI to PIM, (11) by the addition of two mannose residues from GDP-Man. Transformation of this intermediate to larger PIM’s (e.g., 12) also uses a sugar nucleotide donor. Alternatively, conversion of
PIMi to a linear a (l-> 6 )-linked lipomannan is achieved by the transfer of mannose residues from Cjo-Man (14). The addition of a(l-^2)-branched mannose residues, is postulated to involve GDP-Man.
GDP-Man
PIM2 , 1 1 PIM5 , 12
OH PI
LAM [Man-a( l-) 6 )-Man|n-(^^|M 2
Linear-LM GDP-Man
Figure 1.12 Postulated pathways for the biosynthesis of the PIM’s, LM and LAM
21 None of the mannosyltransferases (ManT’s) involved in this process has been identified. However, based upon these biosynthetic studies, it appears that mycobacteria
have at least two such enzymes: an a( W 6 )-ManT, which uses Qo-Man (14) as a substrate and an a(l-^2)-M anT, which requires GDP-Man. It has been also suggested that 14 could have been formed from the biosynthetic reaction of decaprenolphosphate and GDP-Man.
1.4.2 ARABINAN BIOSYNTHESIS
The biogenesis of the arabinan portion of LAM has been shown to involve the transfer of
single arabinofuranosyl residues from P-D-arabonofuranosyl phosphodecaprenoi (C 5 0 -
Ara, 15) to an endogenous acceptor present in mycobacterial membrane preparations."
The nature of this acceptor, e.g., whether is it mature mannan or one devoid of the a (l->2)-linked branches, is unknown. The structural differences in the arabinan portions of LAM relative to AG suggest that there is a separate pathway by which these two polymers are synthesized. Nevertheless, C;o-Ara/has been shown to be a source of the majority of the arabinofuranosyl residues, suggesting that the enzymes are likely quite similar.
99 OH
(3-D-arabinofuranosyl phosphodecaprenoi. 15
Figure 1.13 Structure of P-D-arabonofuranosyl phosphodecaprenoi
1.4.3 ADDITION OF CAPPING SPECIES
To date, no studies have been carried out investigating the origin of the mannose or inositol phosphate caps or the pathways by which these groups are added to the termini of LAM. The mannose oligosaccharides present in ManLAM are a (1—>2)-linked (e.g.,
6 ). Considering the above proposed biosynthetic pathway for the assembly of the core mannan of LAM and, in particular, the origin of the a(l->2)-Man branches, it is
reasonable to postulate that the terminal oligosaccharides are synthesized by a a ( 1 -^ 2 )-
ManT enzyme that uses GDP-Man.
23 1.4.4 EVIDENCE FOR THE POSTULATED BIOSYNTHETIC
PATHWAYS
1. Formation of PI[‘'*C]Man, (FIM^) from PI and GDP[‘'*C]Man suggest that GDP-
Man is the source of man units of PIM^."'^
2. In the presence of C;Q-['''C]-Man (14). there was no PIM, formation but only LM
was formed. Whereas, in the presence of amphomycin, which inhibits the
biosynthesis of 14. only PIM; was formed which suggests that 14 is the source for
a. ( I—>6 )-Man-linked backbone in LM/LAM.'**"'
1.4.5 MODE OF ACTION OF ETHAMBUTOL
Ethambutol [(S.S')-2.2'-(ethylenediamino)di-I-butanoI] (EMB. 3, Page 5) was reported to have potent antimycobacterial activity in 1961.^** Ethambutol is only active against mycobacteria and. when coadministered with other antibiotics, it produces a synergistic effect, thus suggesting that the drugs acts by inhibiting the assembly of the cell wall. However, the structure of EMB is relatively simple and bears little resemblance to any cell wall component, which has complicated the identification of its precise target.
It has been demonstrated that the in vivo conversion of ['‘‘CJ-labeled glucose into arabinan was reduced soon after EMB is administered.^’ Recent studies have supported the idea that the primary effect of EMB is as an arabinofuranosyltransferase (AraT)
24 inhibitor. When the source of arabinofuranosyl residues, Cjo-Ara/ClS) was first identified, it was shown that the addition of EMB to growing cultures of M. smegmatis resulted in an accumulation of Cjo-Ara/."' Also, when radiolabeled C^-Ara/" was available, the additon of EMB to a mycobacterial membrane preparation resulted in reduced incorporation of the radiolabel to the polymer." Furthermore, administration of
EMB induced cleavage of arabinan residues both in LAM and .A.G leading to truncated structures.'^
The most interesting observation is that LAM and AG are differentially inhibited.
Inhibition of arabinan synthesis in AG occurs first, followed by LAM suggesting that distinct pathways are involved in the biosynthesis of both polymers. It has been postulated recently, that the primary site of EMB action is an a( W 3) AraT and not a (1^5) and P( W 2) AraT.'^*' It has further proposed that the differential inhibition of
AG over LAM is due to the fact that the latter has fewer a (1-^3) branch points than the former.
25 LAM
I ti-P-Afa/|-ri- 0 -Afa/ K ^ ------,,------a»0»Afa^ r| a»0»Ara/ pj g>0«Afa/ n ivO-Ara/ I ti-D-Ara^ |4~^0-Afa/ K ARA6INAN I n-O-Afa/ f~{ g-D-Afa/ \ _____ 2_____ S ____5______5 I |i-0-Afaf H g-O-Ara/ H a-0-A fâr|4 n-0-A râ/l- | a-Q-Ara/ |-| g-O-Ara
CORE
Ethambutol Inhibition
I H-0-Afa/|4 H-O-Ara/ K ^ ^ u-O -A ra / H u -O -A fa / H u O-Ara/ M ^O-Ara/ I frO-Ara/ tfg-O-Ara' ARABINAN I g-D-Afa» H ii-D-Afa/ | Truncated LAM
MANNAN CORE
AG
It-Q.Araf H frO-Ara/ K - f i-D>Ara/ H «i-0-Ara^ H a-C-Ara/ n u-O-Ara/ I t^O-Ara/l4ti-D-Ara/V^ ARABINAN n-D-Ara/ | 6 ? e I H-D-Ara< II------. a*D*Ara/ H u*0-Ara/ r) u-O-Ara/ H u-D-Ara/ I frO-Ara/ H ^0-Ara/ % GALACTAN
UNKER PEPTIDOGLYCAN
Figure 1.14 Inhibition of EMB and truncated LAM. structural comparison with AG
26 1.5 INHIBITORS OF GLYCOSYLTRANSFERASES (GT)
The aim of this thesis is to develop inhibitors of the mannosyltransferases involved in LAM biosynthesis. The development of specific glycosyltransferase inhibitors that inhibit enzymes that add terminal sugars has been hampered by a number of factors, among them a lack of knowledge of substrate specificity of the enzymes, and the time involved in synthesizing carbohydrate analogs via organic synthesis to probe this specificity."'*’^ Recent studies suggest that these enzymes will recognize structures considerably smaller than their natural structures. For most of the enzymes studied, it has been possible to replace large oligosaccharides (>5 monosaccharide units) with smaller
fragments (di- and trisaccharides) and still maintain activityThe use of smaller
fragments makes the synthesis of a number of substrate analogs much simpler. Hence, such substrate analogs can serve both as tools for probing the mechanism of biosynthesis and as templates for the design of potential drugs.
1.5.1 COMMON CARBOHYDRATE-PROTEIN INTERACTIONS
Information about the common structural motifs in the active sites of enzymes
that recognize carbohydrates would be useful in determining what substrate analogs to be
prepared as potential inhibitors of these enzymes. Carbohydrate-protein interactions can
be divided into two main classes: hydrogen bonding interactions and van der Waal’s
stacking interactions,"* Although hydrogen bonding interactions are most important for
polyhydroxylated structure of carbohydrates, usually only one or two of the four possible
27 hydroxyl groups on any given monosaccharide residue are critical for binding. That is, these hydroxyl groups are involved in an interaction such that removal of that hydroxyl group completely destroys the ability of the substrate to bind. Water molecules also contibute to hydrogen bonding interactions. In many crystal structures, water molecules
are involved in essential hydrogen bonding networks between the carbohydrate and the
protein. In some cases, water molecules have been found buried deep in the binding
pocket."'' Van der Waals stacking interactions result from the sandwiching of sugar
residues between aromatic amino acids, such as tryptophan, tyrosine, phenylalanine and
histidine. " Crystal structure data are vital for clear prediction of such interactions.
However, to date no crystal structures of mannosyltransferases have been reported.
Nevertheless, we can make use of the information available from the other
glycosyltransferases.
1.5.2 INFORMATION ON THE ACTIVE SITES OF
GLYCOSYLTRANSFERASES
Recently, the crystal structure of a well-studied enzyme, p-1,4-
galactosyltransferase (|34Gal-Tl) was reported.’’ This could serve as a useful model for
predicting the structures of other related glycosyltransferases. The enzyme P4Gal-Tl in
the presence of manganese acting as a cofactor, catalyzes the transfer of UDP-Gal to the
GlcNAc-R substrate to give rise to Gal-p-(l->4)-GlcNAc-R (Figure 1.15), which is a
core structure present in glycoproteins and glycosphingolipids. The crystal structure was
determined as a complex with UDP-Gal. The results of the crystal structure data
28 regarding the type of interactions involved in the binding of the donor UDP-Gal and the acceptor GlcNAc-R with p4Gal-Tl are discussed below/'
OH
H OH
NHAc
R = Glycoconjugate OHOH G lc N A c -R U D P -G al, 8
34G al-T l
OH OH
H -I- OH OR NHAc
OH OH Gal-p-(1->4)-GlcNAc-R UDF
Figure 1.15 Synthesis of Gal-P-(l—>4)-GlcNAc-R catalyzed by P4Gal-Tl.
29 The uracil ring of UDP-Gal is held in position by a stacking interaction between the Phe-226 side-chain and the Arg-191 side-chain. Out of the two phosphate esters of the UDP, only the |3-phosphate (see Figure 1.15) is apparently stabilized by hydrogen bonding with the amino group of Lys-279 and by a strong interaction mediated by a water molecule with the carboxyl group of Asp-254 side-chain. It was postulated that the
C-4 hydroxyl group of Glc-NAc is positioned close to the C-1 atom of the galactose unit of UDP-Gal and along with the Glu-317 residue and the Asp-318 carboxyl group side- chain. both Glu-317 and Asp-318 are believed to assist galactosyl transfer. In summary, the binding site has a number of charged amino acids including aspartates and glutamates that could ser\'e as the enzymatic nucleophile, (see Figure 1.8. 1.9)
The central importance of fucosylation of cell surface oligosaccharides to cell-cell interactions has prompted the mechanistic study of a (1—>3)-fucosyltransferase (FucT).’'
FucT catalyzes transfer of fucose from GDP-Fucose to LacNAc of sialyl LacNAc to form
Lewis X and sialyl Lewis X, respectively (Figure 1.16). A pH-rate profile study identified an ionizable catalytic group with a pK^ of 4.1, which suggest that a carboxylate group may be from Asp, which functions as a general base to deprotonate the C-3 hydroxyl group of LacNAc for attack on the activated GDP-Fuc. Hence, the presence of an amino acid with a carboxylate side-chain in the active site of the enzyme may be vital for the glycosyl transfer to occur, in case of other similar glycosyltransferases.
30 H OH HO OHOH OH OH OH ÛM
HO OR NHAc OH
R = Glycoconjugate
LacNAc
FucT
OH OH
HO OR OHNHAc
OH HO OH
‘NH,
OH OH
GDP
Figure 1.16 A representative example for a fucosyl transfer catalyzed by FucT.
31 1.5.3 N-ACETYLGALACTOSAMINYLTRANSFERASE INHIBITOR
Blood Group A N-acetylgalactosaminyltransferase (GalNAcT) catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc donor to the acceptor
[Fuc(i—>2)Gal(3-OR] to give rise to histo-blood group A determinant GalNAca-
(1^3)[Fuc(l->2)Gaip-ORl (Figure 1.17).’' It has been determined that 3-amino-3- deoxy-[Fuc(l—>2)Gal|3-0(CH2)7CHJ analog which has an amino substituent instead of the hydroxyl group to which GalNAc is transferred, is the first acceptor-based inhibitor of a mammalian glycosyltransferase with a nanomolar K,. The amino compound at physiological pH would be protonated. The inhibitory action was postulated to be due to a strong ionic interaction of the positively charged amine with the nearby negatively charged group resulting in a tightly held enzyme-substrate analog complex. This
inhibitor has shown to be active both in assays with purified enzymes as well as in cells.
32 OH OH
OR AcHN
H OH HO OH OH OH
R = Glycoconjugate U D P -G a lN A c Fuca-(1-^2)-Gal-p-0R
G a lN A c T
OH
OH H' AcHN IÎ f OR -q , P M / OH HO T OH OH OH UDP GalNAca-(1 ^3)-Fuca-(1 ^2)-Gal-p-0R
Figure 1.17 Synthesis of GalNAca-(l->3)[Fuc(1^2)Gaip-OR] catalyzed by GalNAcT
33 1.6 SCOPE OF PROJECT
As mentioned earlier, the mannan portions of LAM are biosynthesized by the tandem action of at least two mannosyltransferases, namely an a (1—»6)-ManT, which is required for the assembly of the mannan backbone and an a(l-^2)-ManT, responsible for the addition of mannose branches in the mannan core. It is plausible that a third enzyme, another a(l-^2)-ManT. is required for the addition of mannose caps found at the terminal ends of ManLAM. However, it is also possible that the organism possesses only one enzyme which can be responsible for the formation of all a(I->2)-mannosyl linkages.
1.6.1 DESIGN OF GLYCAN FRAGMENTS OF MANLAM
To date, there was no examples of mycobacterial mannosyltransferase inhibitors, and there is only very limited information available about these enzymes. Bearing this in mind, we decided to synthesize mannose oligosaccharides (Figure 1.18; 17-19) which are glycan fragments of ManLAM. Such compounds are potential substrates of a ( W 2)-
ManT which catalyzes the biosynthesis of mannose capping at the terminal of ends of
ManLAM. P-Arbinofuranoside 16. a potential substrate for the enzyme that holds the first mannosyl residue was also chosen as a target. Such oligosaccharide substrates can be used for the purification of the enzyme ct ( W 2)-ManT and in the development of assays for their activities. We chose hydrophobic octyl functionalized oligosaccharides
34 to simplify the enzymatic assays by allowing the use of reverse-phase (C[g) cartridges to separate and quantitate the product/^
p H HO' HO 1 HQ
HO
HO 16 17
p H HO'
HO' PH
HO' HO' HO' P
HO'
HO' HO'
HO'
H O P R Ck. HO P R
HO HO 19 18
Figure 1.18 Glycan fragments of ManLAM (R = (CHjjyCHj)
35 p H PH HO HO MO' HO"
GDP 0 —
HO
p H PH HO' HO HP" MO"
( GDP 0 —
HO
p H HP-
HP"
.0'
H O
H O H O ' HO
PRODUCT
Figure 1.19 Proposed schematic representation of the role of the substrate analog
36 As mentioned before, since the glycosyl transfer in the synthesis of these mannose caps proceeds with retention of configuration, which may involve formation of glycosyl- enzyme complex followed by attack of the OH of the acceptor residue with the expulsion of the enzyme. Based on this understanding, it can be postulated that the substrate analogs 16-19 and the donor GDP-Man could bind to the enzyme before the formation of the glycosy 1-enzyme intermediate as shown in Figure 1.19.
1.6.2 DESIGN OF MANNAN CORE INHIBITORS
As mentioned earlier, mannan core of LAM consists of an a ( 1—>6 )-linked linear mannan backbone attached to PIM. (II, Page 19) with a( W2)-Man branching. Our strategy was to synthesize series of structure-based analogs of type A and B (Figure 1.20) to understand major carbohydrate-protein interactions in the active site of these enzymes.
Such compounds are also potential inhibitors of manosyltransferases involved in building the mannan core structure in LAM.
1.6.3 MODIFICATIONS PROBING HYDROGEN BONDING
Substitutions of hydroxyl group by either H or F are sterically conservative modifications and can thus provide insights into whether the hydroxyl group removed was involved in a critical hydrogen bond interaction with the protein combining site.’^'^*
Deoxygenation provides information about whether that hydroxyl group is acting either as an acceptor or donor with the enzyme.
37 HO HO' HO^ HO- OH OH HO HO' un.
0 (CH2 )7CH3 0 (CH2)yCH] T y p e A T y p e B
20 X = OH 25 X = NHg, Y = F
21 X = OCH3 26 X = NHg, Y = NH2
22 X = H 27 X = F, Y = F
23 X = F 28 X = F, Y = NH2
24 X = NH2 29 X = OCH3 . Y = NH2
30 X = 0 CH3 , Y = F
Figure 1.20 Structures of analogs of type A and type B.
The amino compounds could be useful for probing the existence of a negatively charged amino acid residue in the active site. At physiological pH, the amine would be protonated, and therefore it can be expected that if there were a negatively charged group near the positively charged amine, a strong ionic interaction might result. Such a compound could serve as a potent inhibitor of the enzyme via a tightly held enzyme- 38 substrate analog complex. Strong inhibition by an amino functionalized analog has previously been demonstrated.'^
1.6.4 MODIFICATIONS PROBING STERIC INTERACTIONS
Méthylation of a hydroxyl group probes not only the hydrogen bonding
requirements of the enzyme, but also provides insight into whether the enzyme can
tolerate groups of larger steric bulk at that position.
1.6.5 DESIGN OF SUICIDE INHIBITORS
The criteria for a successful suicide inhibitor is that it should be a substrate analog
which the enzyme can recognize and it should have a active latent functional group
within the framework of the analog, with which the enzyme reacts to form a covalent
intermediate, resulting in modification of the active site. These approaches typically
involve alkylating or acylating agents that capitalize on the nucleophilic nature in the
active site of the enzyme.'** Bearing this in mind, we have designed inhibitors (Figure
1 .2 1 ) which could give information on mechanism of enzyme action.
39 HN
OH
HO' HO-
0 (CH2);CH3 0(CH2)7CH j 32 31
Figure 1.21 Structures of proposed suicide inhibitors
In terms of preparation, the chloroactamido disaccharide (32) analog can be synthesized from the amine (24) through a one step conversion. We believe the mannosyltransferases will recognize 32 and it can be expected that if there is a catalytically active base in the active site, this compound could serve as a covalent irreversible inhibitor. Displacement of the chloro group by the reactive base will therefore result in deactivation of the enzyme.
The azide (31) could also serve as a deactivating analog. When photolyzed, the compound would generate a reactive intermediate, namely a nitrene. This reactive intermediate would then insert into the protein, allowing the labeling of the active site with an acceptor substrate.
40 1.7 SYN TH ESIS OF O LIG O SA CCH A RID ES
1.7.1 GLYCOSIDE SYNTHESIS
The stereoselective construction of glycosidic bonds in oligosaccharides is one of the most challenging problems in carbohydrate synthesis. Although a great deal of work has been done in this area in the last 25 years, there still exist no reliably reproducible conditions that give predictable results for a broad range of target molecules. Each specific linkage to be synthesized must be addressed as an individual problem, and the reaction conditions tailored accordingly.’*^ The issue is complicated by the large number of linkages that are possible when one takes into account the identity of donor, the identity of the acceptor, the reaction conditions and the anomeric configuration of the glycosidic linkage desired.
In most of the cases, the first concern is stereocontrol. The preferred formation of the a or p glycoside is achieved with varying degrees of ease, although a number of strategies are commonly used to influence product ratios. Even if steroselectivity is possible, exclusive formation of one anomer over the other through chemical synthesis remains a challenging problem. This situation requires development of separation techniques to obtain the pure anomers.
The vast majority of glycoside synthesis involve an ionic mechanism. .As mentioned earlier, the idea is to find necessary conditions for the stereoselective
41 formation of the desired anomer. Bearing this in mind, three general strategies for glycoside synthesis can be envisioned.
1.7.2 ALKYLATION OF GLYCOSYL ALKOXIDES
This method involves alkylation of a glycosidic alkoxide directly via an 3^2 reaction with an alkyl iodide or a triflate as shown in Figure 1.22.’^“'^’ However due to the steric elements of the electrophile, this method is only applicable to primary alcohols and a few secondary alcohols:**' therefore it is of limited use in the construction of oligosaccharides. As expected, elimination reactions induced by the alkoxide is a major competing reaction.
RX
P = PROTECTING GROUP
Figure 1.22 Glycoside synthesis via S\2 reaction using a glycosyl alkoxide
42 1.7.3 ALKYLATION OF AGLYCOSIDIC ALKOXIDES
Alternatively, an S\2 reaction of an agiyconic alkoxide with a glycosyl halide can be employed as shown in Figure 1.23. However, this method is useful only in the synthesis of 0-aryl glycosides and thioglycosides." Alkoxides derived from alkyl alcohols are too basic resulting in elimination reaction leading to glycals.
RO
-X
P = PROTECTING GROUP
Figure 1.23 Glycoside synthesis via 5^2 reaction using a glycosyl halide
1.7.4 TRAPPING OF GLYCOSYL CATIONS WITH ALCOHOLS
The most effective method for glycosidic bond construction involves generation
of a cation from a glycosyl donor followed by trapping by an acceptor alcohol as shown
in Figure 1.24. The reactivity and stereoelectronic environment of the cation can be
tailored to favor a specific anomeric configuration.*'* Our research efforts are focussed
toward the synthesis of a-D-mannopyranosyl and p-arabinofiiranosyl glycosidic linkages. 43 Glyco^l Acceptor
OP Activator OP ROM po' ' PO PO OR OP OP X Glycosyl Donor P = PROTECTING GROUP
Figure 1.24 Glycoside synthesis via trapping of a glycosyl cation by an alcohol
1.7.5 1,2-TRANS MANNOPYRANOSYL LINKAGES
The synthesis of 1.2-trans-a linkage in case of mannopyranosyl systems has been extensively studied. The stereoselectivity of this type of linkage formation is well established and is favored by a neighboring group participation of an acetyl or a benzoyl group at the carbon adjacent to the anomeric carbon (C-2).
It has been established that the reaction proceeds with the formation of an
intermediate orthoester via an intramolecular reaction of the neighboring 2 -0 -acyl group to give the corresponding acetoxonium ion. A mechanistically related reaction involving a bromide 34. which on activation with Ag(I) salts underwent an acetoxonium ion formation followed by trapping of an alcohol to give orthoester 35. Upon treatment with
44 an acid, the orthoester 35 rearranged to give the 1,2-trans glycoside 36 as the major product as shown in Figure 1.25.*^
OAc AcO
I I
[A gi
OAc AcO
OR 36
Figure 1.25 Mechanism for orthoester 35 and the 1,2-trans mannoside 36.
45 Although, glycosyl bromides, chlorides, fluorides, acetates, sulfoxides have been widely used as glycosyl donors, we have chosen to use thioglycosides/' ** Activation of thioglycosides is achieved through a number of reagent combinations such as
NIS/AgOTf, NlS/TfOH, iodine dicollidine perchlorate (IDCP) and methyl triflate. These reactions are extremely fast and proceed via a glycosyl cation 37 intermediate. The thiophilic activator is needed to promote displacement of the thioglycoside moiety under the assistance from the anomeric oxygen, resulting in the resonance-stabilized carbocation 37 which is then trapped by the acceptor alcohol (Figure 1.26). It can be postulated that 37 can give rise to acyloxonium ion 38 via an intramolecular neighboring acyl group participation. The carbocation 38 can be trapped by the acceptor alcohol which will result in the formation of the orthoester 39. Under the acidic conditions, ortho ester 39 would give back the carbocation 38, which on nucleophilic attack by the alcohol at the anomeric center will give rise to a-D-mannopyranosyl linked product exclusively.
46 PO PO
,SR SR
THIOPHILE
OP 0' DP OP PO PO PO
38 37
-H^
R, OR;
^ OPw r p ' ^o V. -OP p 0 PO
-ROH OR, 39 1 / P = PROTECTING GROUP R = Alkyl or aryl Ri = alkyl or aryl
Figure 1.26 Mechanism of formation of glycosyl cations from a thioglycoside
47 Another class of donors which are known for its versatile application in oligosaccharide synthesis, are trichioroactamidates.*^ ’** These donors are activated by
Lewis acids such as TMSOTf and BFj-OEt, leading to glycosyl cation 37 as mentioned in the case of thioglycosides. Again, the mechanism of the glycoside formation from the cation 37 can be expected to be similar as mentioned above in case of thioglycosides. All our synthetic targets 17-32 (see Figures 1.18, 1.20) requires the formation of a a-D-
mannopyranosyl linkages and hence, a participating acyl group at the carbon adjacent to
anomeric center will be vital for the stereocontrolled synthesis of the above synthetic
targets.
OP OP P” ACTIVATION PO PO 37
NH
CCI] P = PROTECTING GROUP R-i = CH3 or Ph LA = LEWIS ACID
Figure 1.27 Mechanism of activation in case of trichloroacetimidates.
48 1.7.6 ROLE OF SUBSTITUENTS AND STEREOCONTROL
As we discussed earlier, the substituent at the carbon adjacent to the anomeric
center plays a significant role for the sterocontolled synthesis of 1 ,2 -trans linked oligosaccharides. In the presence of a non-participating group (e.g., benzyl group), the glycosylation reaction results in the formation of a and |3 mixture with the a-isomer usually being the major product.’*'* Hence, the stereocontrol synthesis of a-D- mannopyranosyl linked systems cannot be achieved stereoselectively in the absence of a participating group at C-2.
In case of 2-deoxy-2-fluoro-a-D-mannopyranosyl trichloroacetimidates,
substitution at the carbons C-3, C-4 and C - 6 influences the a:p ratio. It has been determined that the glycosylation of a trichloroacetimidate 40 with acetyl substituents as shown in resulted in exclusive formation of a-D-mannopyranosyl linked product.'*'*
Whereas, the glycosylation of similar trichloroacetimidate 41 with benzyl substituents resulted in a a and p mixture with the P-isomer being the major product. It has been postulated that the glycosylation reaction of donor 40 and a carbohydrate alcohol (ROH), under the activation by TMSOTf may undergo an in situ anomerization leading to the a- isomer. Under the same conditions, donor 41 could undergo a S^l reaction with direct inversion leading to P-isomer as shown in Figure 1.28. These results are found to be similar in the case of 2-azido-2-deoxy-a-D-mannopyranosyl trichloroacetimidates.
49 in situ anomerization S n2 P O ^ OR \ * PO- PO-2
40 P = Ac CCI3
41 P = Bn
Figure 1.28 Effect of substituents on sterocontrol of glycosylation of 2-deoxy
substituted trichloroacetimidates.
1.7.7 1,2-CIS GLYCOSYL LINKAGES
Contrary to the relative ease of formation of 1,2-trans glycosyl linkages, the synthesis of 1,2-cis linkages is non-trivial. From a synthetic standpoint, the most investigated example of the 1,2-cis linkage is that of P-mannopyranosides,’’ Numerous methods have been applied with limited success to achieve the synthesis of this linkage.
Usually an o/p mixture results which requires chromatographic separation.
50 1.7.8 1,2-CIS ARABINOFURANOSYL LINKAGES
As mentioned above, the synthesis of 1,2-cis linkages still remain a synthetic challenge, and relatively few reports describe the stereoselective preparation of p-D- arabinofuranosyl linkages. Earlier reports on the studies of methanolysis of 2-0- benzylated arabinofuranosyl chlorides 42 suggested the formation of methyl p-D- arabinofuranoside as the major product (Figure 1.29).''- On the basis of kinetic studies, it was proposed that these reactions proceed through an tight ion-pair formation leading to direct inversion in the absence of any participating group at the carbon adjacent to the anomeric center.
OCH3
CH3 OH
PO CH2CI2 PO C |S - PO 42
Figure 1.29 Methanolysis of a-D-arabinofuranosyl chloride 42.
The glycosylation method above do not work well with carbohydrate alcohols and hence, an efficient method for the synthesis of oligosaccharides comprised of P-D- arabinofuranosyl residues must be explored. An increasingly used approach used for the synthesis of 1,2-cis linkages in mannopyranosides is the Intramolecular Aglycone 51 Delivery (lAD) method.'^^ This methodology requires that the acceptor alcohol to be tethered to carbon adjacent to anomeric center of the donor species, followed by activation of the donor and the trapping of the developing carbocation as shown in Figure
1.30.
OR OR ACTIVATION PO PO P'
X
yv
OH -OP • OP
PO PO ,0 R ,0 R P' P
P = PROTECTING GROUP
Figure 1.30 General strategy of Intramolecular Aglycone Delivery
52 Recently this methodology was applied in the synthesis of P-D- arabinofuranosides. Thioglycoside 43 with a p-methoxybenzyl group (PMB) at 0-2 was used as the donor, the carbohydrate alcohol 44 was tethered to the donor on treatment with DDQ to give the intermediate 45 which upon thioglycoside activation underwent lAD resulting in the formation of p-D-arabinofuranoside 46 in 53% yield as shown in
Figure 1.31.'^
€H
TBDPSO—TBDPSO—I 0 —1 OBz DDQ Ck
SEt SEt OBn OBn OBn 45 43
53% IDCP HO— I OBz
OCgH^y O Bz TBDPSO—1 ,HQ a . '
OBn OBn 46
Figure 1.31 Synthesis of P-D-arabinofuranoside 46 via lAD methodology.
53 Though lAD methodology was successful, the reaction yields are poor in cases with secondary carbohydrate alcohols. A recent report, from our research group involving reaction of diol 48 with thioglycoside 49 at -78 °C, in the presence of NIS and
AgOTf, afforded the hexasaccharide 50 which on deprotection gave Ara^ 5 in excellent yields and with extremely high stereocontrol (Figure 1.32).'^^
54 BnO
48
BnO OBn NIS/AgOTf (8 1%) S C r
B nO Bn
B n O -" BnO B n O — I ^
BnO
BnO-^ ^ B n O -,
BnO DEPROTECTION
Figure 1.32 Synthesis of hexasaccharide Ara^
55 CHAPTER 2
SYNTHESIS OF GLYCAN FRAGMENTS OF MAN-LAM
2.1 IN TR O D U C TIO N
This chapter discusses the synthesis of terminal glycan fragments of ManLAM
(see Figure 1.5) utilizing conventional glycosylation methods. The stereoselective synthesis of P-D-arabinofuranosyl linkages is also described, which was crucial for the synthesis of the targets 16-19 (see Figure 1.18).
2.2 SYNTHETIC STRATEGY
The target mannopyranosyl oligosaccharides (17-19, Page 35) contain 1,2-trans glycosidic linkages, which can be synthesized in a stereocontrolled fashion using mannosyl donors with a participating group at C-2, as mentioned in chapter 1. From the outset, our plan was to utilize readily accessible synthetic building blocks, which in turn can be easily prepared from the commercially available starting materials. Thioglycosides were chosen as the glycosyl donors due to their hydrolytic stability as well as the number
56 of methods available for their activation/* Our strategy was to use two different protecting groups (P, PJ, where one (P,) can be removed selectively in the presence of the other (P). The acceptor alcohol that is liberated, can be glycosylated with the donor to give the desired glycosyl linkage as in Figure 2.1.
SELECTIVE pP DEPROTECTION pH OF Pi PO PP
OR PR
P, Pi = PROTECTING GROUPS PP R = glycosyl residues PO
SEt
p P
PP
P( p PP
OR
Figure 2.1 A representative example of our synthetic strategy
57 2.3 RETROSYNTHETIC ANALYSIS
We envisioned that oligosaccharides 17-19 could be synthesized straightforwardly from monsaccharide building blocks 51. 52. and 53. through a route involving the sequential addition of mannose residues to arabinofuranosyl-containing acceptors as shown in Figure 2.2.
OH HO' PAc Bm pH AcO' HO' AcO BnO Ac Bn
P P HI 51 SEt BnO' HO' BnO' Bn' PR
HO' P
HO' 71
BnO PR
Bn PAc
19 BnO HO Bni
SEt
BnO' PAc Bm PH BnO B nO ' pR Bn Bm PR SEt
BnP 53 69 BnO R = (CH2) tCH3
Figure 2.2 Retrosynthetic analysis
58 2.4 SYNTHESIS OF THIOGLYCOSIDE DONORS
The known thioglycoside donors 51 and 52 were prepared from commercially available D-mannose (54). Acétylation 54 of using acetic anhydride in the presence of
HCIO^ as a catalyst, followed by reaction with ethanethiol in the presence of BFj.OEt, afforded donor 51 as a crystalline solid in 70% yield over two steps (Figure 2.3).“
pA c PH Ad pA c AcO' AcO EtSH HO AcO Ad Ad OH SEt
Figure 2.3 Synthesis of thioglycoside 51
With 51 in hand, donor 52 could be prepared as outlined in Figure 2.4.
Peracetylated mannose 55 was converted to its bromide 56 using HBr/HOAc,“ which was in turn converted to orthoester 57, by treatment of crude 56 with methanol in the presence of lutidine.^^ The crystalline orthoester 57 prepared by this method gave exclusively one diastereomer in 75% yield over two steps. The orthoester fuctionality was found to be highly acid sensitive, deacetylation of 57 with NaOCHj in methanol followed by neutralization with pre-washed Amberlite resin gave significant amounts of side products. Hence, acetyl protecting groups were removed efficiently using NHj in 59 methanol followed by evaporation. This method did not require neutralization of the reaction prior to work-up. Benzylation under standard conditions afforded crystalline 58 in 80% yield over two steps.'^'* The orthoester 58 was then reacted with ethanethiol in the presence of BF^-OEt: as a Lewis acid catalyst but this method‘s' would require ethanethiol to be the solvent in order to minimize competing reaction with the methanol released from opening of the orthoester functionality. To avoid ethanethiol as the solvent, we treated 58 with HOAc which gave the diacetate 59. Treatment of 59 with a stoichiometric amount of ethanethiol in CH-CL, in the presence of excess of BFj.OEt, gave thioglycoside 52'" in 80% yield over two steps (Figure 2.4). The latter method involved easier purification process over the former one. in that large amounts of the odorous ethanethiol did not have to be used, and in turn disposed of.
60 QAc QAc AcO'
Aci H O A c OAc Br
(7 5 % ) CH 3OH, over two steps lutidine
OCH; OCH
Bm AcO'
BnO AcO Bm
2. BnBr, NaH
(8 0 % )
H O A c
QAc Bm OAc E tS H BnO BnO
BF3.0Et2 OAc SEt (8 0 % ) over two steps
Figure 2.4 Synthesis of thioglycoside 52
61 2.5 SYNTHESIS OF OCTYL p-D-ARABINOFURANOSIDE
Relatively little work has been done on the synthesis of furanosidic oligosaccharides and, since Fletcher and Glaudemans reported the preparation of methyl
P-D-arabinofuranoside over 30 years ago,*^' the stereoselective synthesis of linkages of this type has not been investigated. In this section, we will describe both our successful and unsuccessful attempts toward stereocontrolled synthesis of octyl P-D- arabinofuranoside.
2.5.1 .ATTEMPTED YblOTOj MEDIATED GLYCOSYLATION
Stereoselective synthesis of octyl P-D-ribofuranoside in the presence of Yb(OTf)s and methoxyacetic acid as catalysts has been reported (see Figure 2.5).'*''This glycosylation method proceeds efficiently with electron-donating alkyl protecting groups
(e.g., benzyl) rather than electron-withdrawing acyl groups (e.g.. acetyl).
Y b(0T f)3 BnO BnO— I ^ 0 (CH 2)7CH 3 M eOCHgCOOH OH CHaCCHglyOH BnO OBn BnO OBn (> 95%)
Figure 2.5 Stereoselective synthesis of octyl P-D-ribofuranoside
62 It was proposed that the reaction proceeds via an intermediate 61, which is an ester of methoxyacetic acid. Also, it was postulated that 61 could complex with [Yb] as shown in Figure 2.6, enabling the glycosylation to proceed with facial selectivity.'^^ The
reaction proceeded with gentle reflux in dry CH 2CI, for 2 to 4 hours in 90% yield.
BnO
Ph 61
BnO
61-Yb R = (CH2)7CH3
Figure 2.6 Pictorial representation of intemediate 61 complexed with [Yb]
This report prompted us to pursue the synthesis of octyl P-D-arabinofuranoside from commercially available 60 in the presence of Yb(OTf)] and methoxyacetic acid as catalysts as mentioned above. Unfortunately, our efforts toward glycosylation afforded the a-isomer as the major product as determined by ‘H-NMR spectroscopy with the a:P ratio being 8.5:1.5. The anomers can be distinguished by V hi.h:, which is small (0 Hz) for the a-anomer and larger (4 Hz) for the P-anomer.
63 Y b (0T f)3 OBn BnO BnO BnO OBn BnO- (CH2)tCH3 MeOCHgCOOH
CH3(CH2)7 0 H 0(CH2)tCH3 BnO BnO BnO (> 95%)
Figure 2.7 Attempted Yb(OTf), glycosylation of 60
2.5.2 ATTEMPTED GLYCOSYLATION WITH IMIDATE
Our idea was to use of arabinofuranosyl imidate 62 as a glycosyl donor with a non-participating group at C-2. Upon activation the imidate could give rise to P-D- arabinofuranosyl linkage via a S\2 reaction with octanol. The imidate 62 was easily prepared from 60 in 95 % yield with the a:P ratio being 9:1."" But the glycosylation of
62 with octanol proceeded again with «-selectivity, where the a : 3 ratio was determined, by ‘H-NMR. to be 19 : 1.
64 OBn OBn BnO BnO OBn
CCI3CN TMSOTf or B F 3.0 Et2 BnO (95%) (90%) R = (CH2 )tCH3
Figure 2.8 Attempted glycosylation of imidate 62
2.5.3 GLYCOSYLATION WITH ARABINOFURANOSYL CHLORIDE
Earlier reports on methanolysis of 2-0-benzylated arabinofuranosyl chlorides
{e.g., 63) demonstrated that methyl p-D-arabinofuranoside as the major product.'^' This prompted us to investigate this approach for the synthesis of octyl (3-D-arabinofuranoside.
Compound 60 was acetylated quantitatively with acetic anhydride and pyridine to give 60A.‘°‘ Treatment of the acetate 60A with HCl in dichloroethane provided the labile chloride 63 which was not isolated. Instead, immediately after its formation, 63 was reacted with octanol in the absence of a promoter to give the P-D-arabinofuranoside 64 as the only product in 58% yield. The formation of the a-anomer was not detected.
65 BnO BnO OBn AcgO, Py BnO OH OAc
( 100%) BnO BnO BOA 60
HCl
CICHgCHgCI
OBn BnO 0 (CH 2)tC H 3 BnO— BnO CHgCI;
CH3(CH2)7 0 H
(58% ) 63 over two steps
Figure 2.9 Synthesis of octyl 2.3.5-tri-O-benzyi-P-D-arabinofuranoside 64
It is tempting to speculate that the formation of P-glycosides from 63 proceeds through a direct 5^2 displacement reaction. However, previous studies on the
methanolysis of 2 -benzylated arabinofuranosyl chlorides have suggested these reactions proceed through an ion-pair S^l mechanism (see Figure 1.26)."” It should be noted that although the unpromoted glycosylation of these furanosyl halides proceeds efficiently with simple primary alcohols, attempts to react 63 with secondary carbohydrate alcohols produced no glycosides, only the hydrolysis product, 60. Therefore, while this method is
6 6 useful for the preparation of simple glycosides it appears to be of limited utility in oligosaccharide synthesis. Furthermore, it should also be mentioned that earlier work on the glycosylation of 63 in the presence of a promoter (Ag,0. AgCO^) results in the formation of the a-glycoside.“’^ This suggests that in the presence of a promoter, a naked carbocation forms and that the attack of the alcohol proceeds through the less sterically hindered face of the ring.
2.5.4 SYNTHESIS OF ACCEPTOR ALCOHOL 53
Our strategy for the synthesis of 53 (see Figure 2.2) was to start from 60 and to functionalize C-5 with an acetyl protecting group along with benzyl groups at C-2 and C-
3 before glycosylation with octanol. After glycosylation with the above method, the acetyl group could be removed from C-5 in the presence of benzyl groups to generate 53.
This approach was successful as illustrated in Figure 2.10.
Commercially available 2,3,5-tri-(9-benzyl-D-arabinofuranose 60 was treated
under acetolysis conditions which resulted in the simultaneous cleavage of the 0 - 5 benzyl ether and acétylation of the anomeric hydroxyl group. The product, 65, was
obtained in 8 6 % yield. Reaction of this diacetate with HCl in dichloroethane provided the
labile chloride 6 6 which was not isolated. As described above, 6 6 was reacted with octanol in the absence of a promoter to give the P-glycoside 67. The product was contaminated with traces of the a-isomer. However, separation could easily be achieved by first removing the acetyl group and then chromatography. The conversion of 65 to 53
67 proceeded in 35% overall yield.Despite this modest yield, the speed and ease with which 53 can be prepared by this route makes the method viable.
OBn OBn BnO AcO ACgO, HOAc I OH OAc
H-SO. BnO BnO 60 (86 %) 65
HCl
CICHgCHgCI
OBn AcO OBn AcO CHpCI
BnO 66
(35% ) N aO CHa over three steps
CH3 OH
HO
BnO
Figure 2.10 Synthesis of building block 53
68 2.6 SYNTHESIS OF OCTYL P-D-ARABINOFURANOSIDE (16)
With an efficient route to 64 in place, one of the targets, 16, could be easily synthesized. Hydrogenation of compound 64 in methanol afforded 16 in 82% yield as shown in Figure 2.11.
BnO 0 (CH 2)tC H 3 HO—1 HO 0(CH2)tCH3 BnO Hg, Pd/C -0 -
CH3 OH HO (82%) 16
Figure 2.11 Synthesis of octyl P-D-arabinofuranoside 16
2.7 SYNTHESIS OF OLIGOSACCHARIDES 17-19
With building blocks 51-53 in hand, the assembly of the oligosaccharides proceeded without serious difficulty. However, careful control of the temperature during the glycosylations was required in order to obtain good stereoselectivities. Despite the presence of a participating protecting group at C-2 in the donor, initiating the glycosylations at 0 °C or -10 °C resulted in the formation of significant amounts of the
undesired P-anomers. For example, in the case of glycosylation of 53 with 52 to give 6 8
69 (Figure 2.12), at 0 °C a 3:2 a:P ratio was obtained. When the same reaction was carried out at -10 °C, the a:P ratio was found to be 3:1. Ratios were determined by integration of signals in the ‘H NMR spectra of the products; either the anomeric hydrogen resonances or those arising from the acetate methyl group could be used for this purpose. The samples used for these NMR experiments were prepared by chromatographing the crude reaction mixtures and then pooling and concentrating all fractions containing either glycoside. In addition to lowering the overall yield, the formation of the P-glycosides greatly complicated purification as the two glycosides had very similar chromatographic mobilities in all solvent systems investigated. After exploring a number of reaction conditions, best results were obtained by starting the glycosylation at -40 '^C and allowing the solution to warm to 0 °C. Under these conditions, only trace amounts of the P- mannoside were formed, which could be separated from the desired compound by chromatography to give the product, which was pure by ‘H NMR spectroscopy.
Using these temperature conditions, reaction of alcohol 53 with thioglycoside 52
promoted by yV-iodosuccinimide and silver triflate afforded the protected disaccharide 6 8 in 82% yield. Deacetylation proceeded in 95% yield to give alcohol 69. A portion of this product was completely deprotected by hydrogenation providing 17 in 90% yield.
70 B n o rA OAc
OR NIS, AgOTf
CH2 CI2 BnO OR + Bm -40 X -> rt BnO (82%) 53 52 SEt 68 BnO R = (CHgjyCHs (95%) NaOCHa CH-QH
H c r \ pH BnO'^ OH HO"^ BnO' HO-\w ^ Bm OR H2 , Pd/C OR
CH3 OH 17 69 HO (90%) BnO R = (CH2)7CH3
Figure 2.12 Synthesis of disaccharide 17
The remainder of 69 was glycosylated, again with 52 under the same reaction conditions used for the synthesis of the disaccharide, to afford trisaccharide 70 in 87% yield. Treatment with sodium methoxide afforded a 96% yield of alcohol 71 which could then be converted, upon hydrogenation, to 18 (93%) (Figure 2.13)."’^
71 BnO- OAc
BnO' 52
NIS, AgOTf BnO' CHgCl 2 BnO Bm OR BnO (87%) BnO 70 A = (ChfejTCHs BnO
(96%) NaOCHs CH3 OH
OH MO OH BnO' BnO NO
BnO' O HO' O Ha Pd/C BnO HO Bm P R CH 3 OH OR (93%) 18 71
R = (CH2)7CH3 HO BnO
Figure 2.13 Synthesis of trisaccharide 18
Tetrasaccharide 19 was synthesized as shown in Figure 2.14. Its preparation involved first the reaction of trisaccharide 71 with the fully acylated mannose thioglycoside 51 to give 72 in 76% yield. Treatment with sodium methoxide followed by
72 hydrogenation afforded 19 in 78% yield. 103
OH OAc
BnO'
BnO' BnO' BnO'
BnO
BnO' -40 "C ^ BnO' BnO (76% ) OR pAc Act Act Act BnO
set NaOCl-fe CH 3OH
pH PH
BnO' P Hg, Pd/C BnO'
CH 3OH (78% ) over two steps BnO' Bn( PR PR Bnp
BnO R = (CHjyCHa HO
Figure 2.14 Synthesis of tetrasaccharide 19
73 2.8 NMR ANALYSIS
Determining the stereochemical outcome of these glycosylation reactions could be easily done by standard one-dimensional ‘’C and ‘H NMR experiments.The anomeric carbon of a-D-arabinofuranosides resonate in the range of 107-110 ppm, whereas the p-anomers appear between 97 and 104 ppm. Furthermore, V hi.h: is small
(0-2 Hz) for the a-anomers and larger (3-5 Hz) for the P-anomers.
One-bond carbon-hydrogen coupling constants ('7^ Hi) involving the anomeric carbon of the mannose residues in 17-19 were measured to prove glycoside stereochemistry.All ‘7ci.hi values were between 167-175 Hz, which are indicative of a-mannosides. This coupling is insensitive to anomeric stereochemistry in furanosides and therefore, the more reliable chemical shift and values mentioned above were used to prove the stereochemistry at that residue.'*’ Furthermore, the assignment of resonances in 17-19 were made by two dimensional homonuclear and heteronuclear shift correlation experiments such as ('H-'H) COSY, ('H-'H) TOCSY and ('H-''C) HSQC.
74 CHAPTER 3
SYNTHESIS OF MONO MODIFIED
MANNOSE DISACCHARIDES AS POTENTIAL
INHIBITORS OF LAM BIOSYNTHESIS
3.1 INTRODUCTION
This chapter discusses the synthesis of mono modified disaccharides of type A
(20-24. see Figure 3.1) utilizing conventional glycosylation methods. The conversion of a hydroxyl group to a methoxy, fluoro, deoxy and amino substituents is discussed.
75 T ype A
20 X = OH
21 X = OCH3
22 X = H
23 X = F
24 X = NH2
Figure 3.1 Type A disaccharides 20-24
3.2 RETROSYNTHETIC ANALYSIS
For the preparation of 20-22, we chose thioglycoside 52 and alcohol 75 as the building blocks for the glycosylation step as shown in Figure 3.2. Disaccharide 82 is a common intermediate for the synthesis of three disaccharides.
76 HO' OH BnO' HO BnO 8ni
OH OBn
HO' BnO
OR 8 2 OR R = (CH2)7CH3
20 X = OH 21 X = O C H3 2 2 X = H
OBn pAc HO' BnO' BnO BnO Bn( + OR 75
Figure 3.2 Retrosynthetic analysis for the disaccharides 20-22
The trichloroacetamidates, 76"^ and 77'“** were chosen as glycosyl donors for the synthesis of disaccharides 23 and 24 as shown in Figure 3.3.
77 AcOA f A c O '" '^ ^ --- AcoAw ^ A
76 X = F
77 X = N3 c^c
+ h o \ m A •iw« I \ \ OBn V^Di I OR R = (CH2)7CH3 BnO-V^ .X 23 X = F OR 24 X = NH2 75
Figure 3.3 Retrosvnthetic analysis for the disaccharides 23 and 24
3.3 SYNTHESIS OF ACCEPTOR ALCOHOL 75
Alcohol 75 was efficiently synthesized from D-mannose 54 in six steps (Figure
3.4). First. 54 was converted to peracetate 55 in 95% yield. Upon glycosylation with octanol in the presence of BFj-OEt, followed by acetyl deprotection with sodium methoxide in methanol 78 was obtained in 55% yield over two steps."” Selective 0-6 protection was achieved by reacting 78 with triphenylmethyl chloride in pyridine at 50
°C. this resulted in 79 as a yellow solid in 80% yield. Benzylation of 79 in dimethylformamide using benzyl bromide and sodium hydride afforded 80 as a colorless
78 oil in 79% yield. The acceptor alcohol 75 was prepared in 92% yield as a colorless oil by removing the trityl group from 80 with p-toluenesulfonic acid.
PAc
AcgO AcO Ad nuiU4 OAc (95%) 1. CH3(CH2)70H
(55%) Bp3.0Et2 2. NaOCHs CH 3 OH
pH PH HO TrCI, Py
(80%) 79 OR OR 78
(79%) BnBr, NaH
PBn PBn
BnO p-TsOH BnP Bn' Bm (92%) 80 PR 75 OR
R= (CH2 )tCH 3
Figure 3.4 Synthesis of acceptor alcohol 75
79 3.4 SYNTHESIS OF DISACCHARIDES 20-22
With building blocks 52 (prepared as discussed in section 2.4) and 75 in hand, the assembly of the disaccharides 20-22 proceeded without incident. The synthesis of 20 is illustrated in Figure 3.5. Excellent a-selectivity was achieved by starting the glycosylation reaction at -40 ^C and allowing it to react as it warmed to 0 °C. The
protected disaccharide. 81, was obtained in 8 6 % yield from the /V-iodosuccinimide/silver triflate promoted coupling of 52 and 75. Deacetylation proceeded in 96% yield to give the intermediate alcohol 82. A portion of this product was completely deprotected by hydrogenation providing, 20 in 91% yield.
80 BnO" O A c O A c B nO ' B nO ' -Q O B n B n O HO' Bn( B nO SEt O B n -O OR NIS, AgOTf B nO Bni C ltC l 2
OR
(86%)
(96% NaOCHa CH3 OH
OH OH HO' B nO '
HO B nO Ha Pd/c Bm
OH O B n
CH3 OH HO' B nO (91%) Bm
OR OR 20 R = (CHsVCHa
Figure 3.5 Synthesis of disaccharide 20
Disaccharides 21 and 22 are synthesized as outlined in Figure 3.6. Another portion of the intermediate 82 was methylated with methyl iodide in the presence of sodium hydride to give 83 in 87% yield. Hydrogenation of 83 provided 21 in 87% yield.
The remainder of 82 was converted in quantitative yield to its xanthate 84 by treatment
81 with carbon disulfide, sodium hydride and a catalytic amount of imidazole followed by methyl iodide. Barton deoxygenation of 84 with tributyltin hydride in the presence of catalytic amount of AIBN gave the C-2 deoxygenated compound 85 in 61% yield.
Hydrogenation of 85 provided 22 in 90% yield.
8 2 OH BnO' SCH BnO CSg. NaH, Im; B n O ' CH3I BnO Bni OBn OBn BnO (quant.) BnO
82 OR 84 OR
(87%: CH3I. NaH (61%) n-Bu3 SnH AIBN
OCR BnO' BnO' BnO BnO Bni
OBn OBn
BnO BnO Bn(
83 OR 85 OR
(87%) Ho. Pd/C (90%) Hg, Pd/c
PCH. HO' HO'
HO HO
OH OH
HO HO
OR 22 OR R = (CH2)7CH3 R = (CH2)7 CH3
Figure 3.6 Synthesis of disaccharides 21 and 22
83 3.5 SYNTHESIS OF TRICHLOROACETAMIDATE 76
Our initial approach to 76 involved D-glucal 8 6 as the starting material (Figure
3.7). Treatment of commercially available 8 6 with SelectFluor (87) in acetonitrile;water
(4:1). followed by acétylation with acetic anhydride in the presence of catalytic amount
of iodine"" afforded 8 8 in 20% yield as one among many products. Interestingly, when the same reaction was performed in DMFiwater (3:1) followed by acétylation with acetic anhydride and iodine, 89 was obtained in 53% yield as the major product. Although this
approach involved fewer reaction steps for the synthesis of 8 8 . the low reaction yield, the
difficulty involved in purification of the 8 8 from the other products,'" and the difficulty to reproduce the reaction yield.'" made this method less desirable.
A c O \
AcO ^ \ 1. 87 Ac 2. Ig, Ac^O OAc OAc 86 88 89
(70% ) 1. NHgNHg. HOAc y Cl over two steps 2. C C I3CN, C S 2CO 3
Ac F 87 AcO Ac
76 NH
CI3C
Figure 3.7 Fluorination of D-glucal 86 with selectfluor 87
84 We then turned our attraction to another route (Figure 3.8). The goal was to prepare methyl a-D-glucopyranoside derivative with a good leaving group (e.g., triflate) at C-2 and then carry out a displacement reaction with fluoride ion. The methyl glycoside would then be cleaved and converted to an imidate. The choice of protecting
groups was important. We chose a benzylidene acetal for the protection of C-4 and C- 6 .
The benzylidene protection provides rigidity to the pyranose ring and this is essential to prevent ring contraction to the furanose form,"' which can occur via the displacement of a leaving group at C-2 by the ring oxygen. Since fluoride anion is a good base, selective protection of 3-OH is necessary in order to minimize epoxide formation, which can occur by the intramolecular nucleophilic displacement between the alkoxide of 3-OH and the leaving group at C-2."'
Keeping in mind these issues, the trichloroacetimidate was successfully synthesized from 90 as outlined in Figure 3.8. Treatment of 90 with benzaldehyde in the presence of zinc chloride afforded cryatalline 91 in 87% yield.'" It has been determined that the 2-OH group of 91 is more nucleophilic than 3-OH and hence, standard benzylation of 91 resulted in the undesired 2-0-benzylated compound as the major product. This selectivity can be reversed by performing the benzylation in the presence of copper salts where Cu'* complexes with the anion of the 2-OH and thereby, deactivating benzylation at the C-2 site, but favoring 3-0 benzylation."■* Selective benzylation of 91 with benzyl bromide and sodium hydride, in the presence of cupric chloride gave 92 in 40% yield. Treatment of 92 with triflic anhydride in the presence of pyridine afforded triflate 93. Without purification, 93 was reacted with
85 tetrabutylammonium fluoride in tetrahydrofuran at 50 °C to afford 94 in 45% yield after two steps. Hydrogenation of 94 followed by acétylation with acetic anhydride and pyridine gave 95 in 90% yield over two steps. Acetolysis of 95 with acetic acid and
acetic anhydride in the presence of catalytic sulfuric acid at 60 °C afforded 8 8 in 90% yield. Selective deprotection of the anomeric acetate group was achieved by treatment of
8 8 with hydrazine acetate to give 96 in 85% yield.Trichloroacetimidate 76 was prepared in 82% yield from the reaction of 96 with trichloroacetonitrile in the presence of a catalytic amount of ceasium carbonate.'"
86 HO' PhCHO, ZnCl2
OH (87%) OH I 91 OOHa
(40%) BnBr, NaH CuCl2
Ph Tf2 0 , Py
Bn< OH I OOH3 9 3 OCH3 92
(45%) TBAF, THF over two steps
1. Ha Pd/c AcO Ac( 2. AC2 O, Py OCH 95 OCH; 94 (90%) over two steps (87%) HOAc, AC2 O Con. H 2 SO 4
Ac<
AcO 1. NH2 NH2 . HOAc Aci OAc 2. CCI3 CN, CS 2 CO 3 88 76 NH (70%) CI3 C
Figure 3.8 Synthesis of trichloroacetimidate 76
87 3.6 SYNTHESIS OF DISACCHARIDE 23
The synthesis of disaccharide 23 proceeded efficiently from the building blocks
75 and 76. Excellent a-selectivity (>95%) was achieved coupling trichloroactimidate 76 with the alcohol 75 in the presence of TM SOTf as a promoter at -10 °C and allowing it to react as it warmed to 0 °C. The protected disaccharide 97 was obtained in 89% yield.
Deacetylation proceeded in 8 6 % yield to provide the triol 98, which on hydrogenation afforded the deprotected 23 in 97% yield.
TM SO Tf
-10°C->0^C
97 OR
(86 %)
HO' HO HO Hg, Pd/c OH OBn CH 3OH HO Bn( (97% ) OR 23 OR R = (CH2)7CH3
Figure 3.9 Synthesis of disaccharide 23
88 3.7 SYNTHESIS OF TRICHLOROACETIMIDATE 77
Our strategy for the synthesis of trichloroacetimidate 77 was similar to that of 76, starting from methyl a-D-glucopyranoside. 90. Since the azide anion is a weak base, selective protection of the 3-OH of 91 is not required. As mentioned earlier, the azido group at C-2 of 77 could serve as a synthetic equivalent for amino functionality in the disaccharide 24. Azidonitration of D-glucal. 86 could also be used to prepare 77, but this reaction proceeds in low yield and produces both D-gluco and D-manno epimers which are difficult to separate.'”** Hence, we did not use this method.
The synthesis of 77 proceeded in 8 steps from 90 as illustrated in Figure 3.10.
Selective trillation of 2-OH group in 91 was achieved on treatment of 91 with triflic anhydride in the presence of pyridine, which gave triflate 99 in 94% yield."'Without purification. 99 was reacted with sodium azide in the presence of 15-crown-5 at 60 °C to afford 100 in 80% yield."' Triacetate 101 was prepared in 71% yield, by heating 100 in
80% aqueous HOAc at 80 °C (benzylidene deprotection), followed by acétylation with acetic anhydride and pyridine. Acetolysis of 101 with acetic acid and acetic anhydride in the presence of sulfuric acid as a catalyst at 60 °C afforded 102 in 80% yield. At this point, manipulations at the anomeric center were found to be non-trivial. For example,
selective deprotection of the anomeric acetate of 1 0 2 by treating with hydrazine acetate, as was done during the synthesis of 76, was unsuccessful. However, treatment of 102
with dichloromethyl methyl ether in the presence of fused zinc chloride gave the chloride
103 in 70% yield."* Chloride 103 was hydrolyzed with silver nitrate to 104 in 82% yield.
89 The trichloroactimidate 77 was prepared in 61% yield from the reaction of 104 with trichioroactonitrile, in the presence of catalytic sodium hydride.
Ph Ph TfgO, Py
OH (94%) 91 99
NaNj (80% ) 15-crow n-5
AcO' 1. 80% aq.HOAc AcO' Ac 2 . AC2O, Py 101 OCH (71% ) over two steps (80%)
AcO' AcO' AcO' AcO Ac
OAc 102 103 AgNOg (82% ) acetone : HgO AcO'
AcO A cO " \ ^ 3 CCI3CN, NaH AcO' Ac 77 NH (61%) CI3C 104
Figure 3.10 Synthesis of trichloroacetimidate 77
90 3.8 SYNTHESIS OF DISACCHARIDE 24
The synthesis of disaccharide 24 proceeded efficiently from the building blocks
75 and 77 as illustrated in Figure 3. II. Reaction of the trichloroactimidate 77 with the alcohol 75 in the presence of TMSOTf as a promoter afforded the protected disaccharide
105 in 70% yield. Excellent a-selectivity (>95%) was achieved by starting the glycosylation reaction at -10 °C and allowing it to react as it warmed to 0 °C, as mentioned above. Deacetylation of 105 proceeded in 87% yield to give the triol 106, which on hydrogenation in HO.Ac afforded 24 in 76% yield.
AcOA '*3 HO"^ P®" T M S O T f A, AcO
Ac 0 r \ PBn -1 0 ° C -^ 0 “C "H. 77 75 (70% ) CI3C 105 OR
(8 7 % )
HO' HO HO Hg, Pd/c OH OBn
HO H OAc BnO (76% ) Bni OR OR 24 106 R = (CH2)?CH3
Figure 3.11 Synthesis of disaccharide 24
91 3.9 NMR ANALYSIS
As mentioned in Chapter 2, one-bond carbon-hydrogen coupling constants
('•^ci.Hi) involving the anomeric carbon of the mannose residues in 20-24 were measured to prove glycoside stereochemistry."^’ All ‘/ clhi values were between 168-171 Hz, which are indicative of a-mannosides. Furthermore, the assignment of resonances in 20-24 were made by two dimensional homonuclear and heteronuclear shift correlation e.\periments such as ('H-'H) COSY, ('H-'H) TOCSY and ('H-'-'C) HSQC. The "F NMR chemical shifts and the coupling patterns will be discussed in Chapter 4.
3.9.1 EFFECT OF SUBSTITUTION ON "C CHEMICAL SHIFTS OF C-2'
The '^C chemical shifts of C-2' in case of deprotected compounds 20-24 are presented in Table 3.1. Chemical shifts of compounds 31 and 32 are also included for the purposes of comparision. The synthesis of 31 and 32 is described in Chapter 5.
3.10 BIOLOGICAL INVESTIGATIONS
The biological studies on disaccharides 20-24 as a(l->6 )-ManT substrates and inhibitors was described in Appendix A.
92 HO
OH
OR R = (CH2)tCH3
Compound No. Substituent, X ‘^C chemical shift at C-2' of C-2' (5c) ppm
23 F 90.0
21 OCH, 80.5
20 OH 70.4
31 N 3 65.3
32 NH(C0)CH:C1 54.4
24 NH, 53.6
22 H 37.2
Table 3.1 Comparison of ‘^C chemical shifts in compounds 20-24, 31 and 32
93 CHAPTER 4
SYNTHESIS OF DI-MODIFIED MANNOSE
DISACCHARIDES FOR THE INHIBITION OF
LAM BIOSYNTHESIS
4.1 INTRODUCTION
In this chapter, we will describe the methods for synthesis of the di modified disaccharides of type B (25-30, see Figure 4.1). Considering the structural similarity of
25-30 (C-2' and C-6 ' being functionally modified) with the disaccharide targets 21-24 (C-
2' modified) from Chapter 3, we decided to make use of the intermediates and methods developed in the previous chapter.
94 HO'
pH
HO
0(CH2)tCH3 T y p e B
25 X = NHz. Y = F
26 X = NHg, Y = NH 2
27 X = F, Y= F
28 X = F, Y = NHs
29 X = 0CH3, Y =NH2
30 X = OCH3 . Y = F
Figure 4.1 Type B disaccharides
4.2 RETROSYNTHETIC ANALYSIS FOR THE TARGETS 25-28
As mentioned in Chapter 3, the azido group was utilized as a synthetic equivalent
for amino functionality. The compounds 98 and 106 which are synthetic intemediates for
95 the synthesis of disaccharides 23 and 24, respectively, from Chapter 3, were used as precursors for the synthesis of our targets 25-28 as shown in Figure 4.2.
OH
HO
OR
113X = N3, Y = F 25 X = NHa, Y = F 116X = N3, Y = N3 26 X = NH2, Y = NH2 118 X = F, Y = F 27 X = F, Y = F 120 X = F, Y = N3 28 X = F, Y = NH2
HO'
HO'
OBn
BnO'
OR 98 X = F
106X = N3
Figure 4.2 Retrosynthetic analysis for 25-28
96 4.3 RETROSYNTHETIC ANALYSIS FOR THE TARGETS 29-30
We envisioned that the disaccharides 29 and 30 could be synthesized from monosaccharide building blocks 109'"^ and 75, followed by modifications at C-2' and C-
6 ' as outlined in Figure 4.3. The participating group at C-2 of the donor 109 could control a-selectivity during glycosylation with the acceptor 75. Moreover, the acyl
groups at C-2' and C- 6 ' in the disaccharide can be selectively removed in the presence of the benzyl protecting groups. The primary 6'-0H can be selectively protected with a
TBDPS functionality in the presence of a secondary 2'-0H. The TBDPS protection cannot be installed in the donor 109, because of its acid sensitivity and hence, it may be cleaved during the glycosylation step resulting in side products. Méthylation of 0-2 in
128 followed by removal of the TBDPS ether provides the common intermediate, 129.
This intermediate, in turn, can be functionally modified at C- 6 '.
97 OCH HO' OCH HO BnO
OH > OBn HO' BnO'
OR R = (CHglyCHg OR 129 29 X = OCH3, Y = NH2
30 X = OCH3, Y = F
OH TBDPSO' AcO' OBz BnO' BnO'
OBn OBn BnO BnO
OR OR 128 126
OBz
109 f
Figure 4.3 Retrosynthetic analysis for 29 and 30
98 4.4 SELECTIVE FLUORINATION AT C-6
The préparation of fluorinated carbohydrates via nucleophilic displacement of the corresponding primary sulfonate by fluoride ion is a well known reaction."^ However,
our desire was to selectively fluorinate C- 6 ' in the presence of secondary OH groups, from the precursors 98 and 106 for the synthesis of 25 and 27. Selective fluorination of primary alcohols been carried out with highly reactive DAST
(diethylaminosulfurtrifluoride) 108. The mechanism is shown in Figure 4.4.'-“
108
\ \ P
y ------.N— s -^ 0
+
R-F
Figure 4.4 Mechanism of fluorination with DAST reagent
99 Selective fluorination at 6 -OH in the presence of secondary OH groups has been reported.But such selectivity was not reported in case of disaccharide systems like
107. Reaction of 90 with excess DAST afforded mono fluorinated 110 in 8 8 % yield.'"'
However, reaction of methyl a-D-mannopyranoside 111 with excess DAST gave only a difluorinated product 112 in 80% yield. The two examples are shown in Figure 4.5.
OH
HO' DAST HO
HO- OH CH 2 CI2 OH OCH3 (88%)
OH HO'
HO'
1 1 2 OCH3
1— NEta DAST (80%)
1- 0 '
Figure 4.5 Some reported examples of DAST induced fluorination reactions
1 0 0 Difluorinated formation (112) in mannopyranosyl system could be avoided by protecting the 2-OH group (e.g., with a methyl group),which is in good agreement with the above proposed mechanism. This particular experimental observation prompted us to investigate selective fluorination of 6'-0H in 98 and 106, which contains either a fluoro or an azido substituent at C-2'. These substituents could play a vital role in
avoiding the possibility of difluorination and hence, selective fluorination at C- 6 ' can be accomplished.
4.5 SYNTHESIS OF DISACCHARIDE 25
The precursor 106 was prepared as described in Chapter 3, and its conversion to
25 is outlined in Figure 4.6. Reaction of 106 with excess DAST at -40 ^C in dichloromethane afforded the mono-lluorinated adduct 113 in 72% yield as the major product. Hydrogenation of 113 in HOAc with 10% Pd/C was unsuccessful. Thin layer chromatographic analysis showed several spots even after two days, and all of them gave the characteristic color with the ninhydrin reagent suggesting the presence of an amino functional group. At this point, considering the ease with which selective fluorination is achieved, we decided to reduce the azide first via Staudinger reaction with triphenylphosphine in a tetrahydrofuranrwater solvent system. Reaction of 113 with triphenylphosphine in THF:water (8:1) gave the amine. Without purification, the crude amine was treated with trifluoroacetic anhydride in the presence of pyridine followed by stirring with methanol (to remove any 0-trifluoroacetates). This afforded trifluoroacetamide 114 in 64% yield over two steps. Hydogenation of 114 in methanol
1 0 1 followed by treatment with aq. NaOH afforded 25 in 34% yield. The other major product of this reaction was a mono-O-benzylated amine. The molecular weight was determined by HRMS. However, we were unable to determine the position of the benzyl group by
NMR experiments. Further, hydrogenation of this product by-product provided additional product. In total, 64% yield of the product was obtained.
Our efforts to remove benzyl groups and to reduce the azide in one step worked only in case of 24 but did not work in any dimodified systems and hence, we have resorted to the alternate plan described above to synthesize 25, 26 and 28.
1 0 2 HO' HO DAST, -40 °C HO'
p B n (72%) OBn ^O BnO' BnO Bn(
OR OR 106 113
R = (CH o)tCH q (64%) 1. PPhg, THF, HgO over two steps 2. (CF3C 0 )2, Py
NHj
HO' 1. Hg, Pd/C HO'
2. aq. NaOH OBn HO ^O (64%) BnO over two steps Bni OR OR 114 R = (CH2)tCH3
Figure 4.6 Synthesis of disaccharide 25 involving selective tluorination at C- 6 '
4.6 SYNTHESIS OF DISACCHARIDE 26
The synthesis of disaccharide 26 from the precursor 106 was achieved successfully as outlined in Figure 4.7. Selective tosylation of 106 with p-toluenesulfonyl
103 chloride in pyridineiCH^CU (1:1) afforded 115 in 70% yield. Treatment of 115 with sodium azide and 15-crown-5 in DMF at 60 °C gave the diazide 116 in 85% yield.
Staudinger reaction of 116 with triphenylphosphine in THFiwater (8:1) gave the diamine, which was immediately treated with trifluoroacetic anhydride in the presence of pyridine.
Addition of methanol (to remove any 0-trifluoroacetates) followed by purification resulted in the di(trifluoroacetamide) 117 in 34% yield over two steps. Hydogenation of
117 in methanol followed by treatment with aq. NaOH afforded 26 in 61% yield over two steps. No mono-O-benzylated amine by-product formation was observed during the deprotection.
1 0 4 HO' T sO ' HO' TsCI, Py;CH 2Cl2 h o
OBn (70% ) OBn
BnO BnO Bni
OR OR 106 115
(85% ) NaNo, 15-crown-5
OF 1. PPhg, THF, H 2O HO' HO 2. (C F 3 6 0 )2 , Py H
OBn OBn (34% ) BnO over two steps BnO Bni
OR OR 117 116
1. H2 , Pd/C (61%) 2. aq. NaOH over two steps
HO
OH
HO
OR
R = (CH2 )7CH 3
Figure 4.7 Synthesis of disaccharide 26
105 4.7 SYNTHESIS OF DISACCHARIDE 27
Given that selective fluorination of the primary hydroxyl group was successful during the synthesis of 26, we undertook the synthesis of 27 from the precursor 98 as outlined in Figure 4.8. Fluorination of 98 with excess DAST at -40 °C afforded the mono fluorinated product 118 in 55% yield as the major product. Hydrogenation of 118 in
HOAc proceeded efficiently, resulting in 27 in 90% yield.
HO'
HO DAST, -40 =0 HO'
O Bn (55%) OBn
BnO' BnO Bn( Bn(
OR OR 118
(90%) Ho, Pd/C
HO
OH
HO'
OR 27
R = (CH2)7CH3
Figure 4.8 Synthesis of disaccharide 27 involving selective fluorination at C-6'
106 4.8 SYNTHESIS OF DISACCHARIDE 28
The synthesis of 28 proceeded successfully as outlined in Figure 4.9, using methods similar to those employed for the synthesis of 26. Selective tosylation of 98 with p-toluenesulfonyl chloride in pyridine and a catalytic amount of DMAP afforded
119 in 6 8 % yield. Treatment of 119 with sodium azide and 15-crown-5 at 60 °C gave the diazide 120 in 83% yield. Staudinger reaction of 120 with triphenylphosphine in
THFzwater (8:1) gave the amine, which was trifluoroacetylated by treatment with trifluoroacetic anhydride in pyridineiCHiCk followed by stirring with methanol (to cleave 0-trifluoroacetates). The trifluoroacetamide 121 was obtained in 83% yield over two steps. Hydogenation of 121 in HOAc followed by treatment with aq. NaOH afforded
28 in 47% yield over two steps. As in the syntheis of 25, a mono-O-benzylated amine was isolated, but was re-hydrogenated to give additionally 31%. with overall 78% yield over two steps.
107 HO' TsO' HO' TsCI, Py, DMAP
OBn (68 %) OBn
BnO BnO Bn(
OR OR 119 P = (CH2)yCH 3
(83%) NaN3 , 15-crown-5
hT\ 1. PPhg, THF, HgO HO HO 2. (CF3C0)2, H H' PyiCHgClg OBn BnO BnO ""^^— (83%) Bni B n O - V ^ .A over two steps OR OR 120 121
1.Hg, Pd/C (78%) 2. aq. NaOH over two steps
HO
OH
HO
OR
R = (CH2)tCH3
Figure 4.9 Synthesis of disaccharide 28
108 4.9 SYNTHESIS OF TRICHLOROACETIMIDATE 109
The synthesis of disaccharides 29 and 30 required the preparation of trichloroacetimidate 109. Trichloroacetimidate 109 was prepared from the benzylated orthoester 58, which was an intermediate in the synthesis of thioglycoside 52 (see Figure
2.4). Reaction of 58 with p-TsOH in dry CH.CU afforded 122 in 76% yield, via orthoester ring opening. At this point, we chose to use a benzoyl group at C-2 instead of an acetyl group. Deacetylation of 122 with sodium metho.xide, followed by treatment with benzoyl chloride in pyridine afforded 123 in 80% yield over two steps. Acetolysis of 123 with acetic anhydride and HOAc in the presence of a sulfuric acid catalyst afforded 124 in 63% yield.The anomeric acetate in 124 was selectively removed with
hydrazine acetate, which provided 125 in 8 6 % yield. Finally, 109 was prepared in 83% yield, by the reaction of 125 with trichloroactonitrile and DBU.'*'*
109 oc H, OAc p-TsO H
(76%) 1 2 2 0 CH3 58
(80%) 1. NaOCHg over two steps 2. BzCI, Py
OBz HOAc, A % 0 OBz Con. H 2SO 4
B nO —- (63%) 124 OAc 123 0 C H 3
(86 %) NH2NH 2. HOAc
OBz AcO' OBz Ac O' CCI3CN, DBU BnO' BnO' Bn Bn (83%) 109 NH
CI3C
Figure 4.10 Synthesis of trichloroacetimidate 109
1 1 0 4.10 SYNTHESIS OF DISACCHARIDES 29 AND 30
With the building blocks. 75 and 109 are in hand, the synthesis of the intermediate
129 proceeded as outlined in Figure 4.11. Reaction of the trichloroactimidate 109 with alcohol 75 in the presence of TMSOTf as a promoter afforded the protected disaccharide
126 in 91% yield. Excédent a-selectivity was achieved by starting the glycosylation reaction at -10 °C and allowing it to react as it warmed to 0 °C. Deacetylation of 126 resulted in the diol 127 in 96% yield. Reaction of 127 with TBDPSCl in the presence of imidazole, provided 128 in 74% yield, with the 6'-0H selectively protected. Méthylation of 128 was achieved, from the reaction with methyl iodide and sodium hydride. Without purification, the product was treated with solid TBAF in THF which afforded intermediate 129 in 67% yield over two steps.
I l l AcoA OBz HO^ P®" TMSOTf -R
Bni -10°C->0°C crA 9®" 75 (91% ) BnO' 109 V " R = (CH2)7CH3 Bm CI3C OR 126
(96% ) NaOCH3 CH3 0 H
OH OH TBOPSO' HO' BnO' BnO
OBn OBn
BnO (74% ) BnO' Bni
OR OR 128 127
1 .C H 3I, N a H (67% ) 2. TBAF, THF over two steps
PCH HO' BnO
OBn
BnO
OR 129 R = (CH2)7CH3
Figure 4.11 Synthesis of the intermediate 129
1 1 2 4.11 SYNTHESIS OF DISACCHARIDE 29
With a route to 129 in place, the disaccharide 29 was synthesized as shown in
Figure 4.12. In contrast to our prior studies, we chose the phthalimido group as a synthetic equivalent for an amino functional group. This functionality can be incorporated by a single-step Mitsunobu reaction of 129 with phthalimide. Reaction of
129 with phthalimide in the presence of DIAD and triphenylphosphine, afforded 130 in
85% yield. Treatment of 130 with ethylenediamine in «-butanol at 80 °C‘^ afforded the amine. Without further purification, the amine was hydrogenated in HOAc, which resulted in 29 in 84% yield over two steps.
1 1 3 OCH. HO' PhthI pCH BnO BnO PhthH, PPhg, DIAD Bm OBn PBn
BnO' (85%) BnO Bni
OR OR 129 130 R = (CH2)7CH3 R = (CHalrCHa
Ethylenediamine n-butanol
pCH. pCH HO BnO Bm pH PBn HO' (84%) BnO over two steps Bni OR OR R = (CH2)yCH3
= Phth
Figure 4.12 Synthesis of disaccharide 29
114 4.12 UNEXPECTED ANHYDRO SUGAR FORMATION
As mentioned above, selective fluorination of intermediates 98 and 106 proceeded successfully. However, our efforts to fluorinate intermediate 129 with DAST -40 =C, did not give the desired fluorinated compound. Instead, the reaction afforded 3,6-anhydro sugar 131 in 83% yield as shown in Figure 4.13. Hydrogenation of 131 resulted in disaccharide 33 in 65% yield. The structure of 131 was confirmed from the characteristic coupling constant, 7, ;- = 6.5 Hz, the absence of two benzylic protons and results from two-dimensional NMR experiments. The proposed mechanism for the anhydro sugar formation is shown in Figure 4.14.
OCHj
BnO" DAST, -40 °C BnO OCH 3 OBn (83%) OBn BnO’ OR OR 131 129 R = (CH2)tCH3 (65%) H2 . Pd/C
HO OCH 3
OH
OR 33
R = (CH2)tCH3
Figure 4.13 Unexpected anhydro sugar formation from the reaction of 129 with DAST
115 1 / OCH HO' OCH BnO Bni BnO -FT np 129 OR,
'P h OR, OCH, OR, OCH]
OBn
-Bn F
OR OCH,
OBn 131 0(CH3)7CH3 +
1 //°
Figure 4.14 Proposed mechanism for the formation of anhydro sugar 131
116 4.13 SYNTHESIS OF DISACCHARIDE 30
Since direct fluorination of 129 was unsuccessful, our alternate approach to 30, was to convert the 6'-0H to a triflate followed by a nucleophilic substitution reaction
with TBAF to incorporate the fluoro functionality at C- 6 '. Reaction of 129 with triflic anhydride in the presence of pyridine, resulted in the triflate, which was immediately
treated with TBAF in THF at 50 °C to afford 132 in 78% yield over two steps.
Hydrogenation of 132 in HOAc afforded 30 in 78% yield as shown in Figure 4.15.
OCH] HO' BnO BnO' I.TfgO, Py
pB n OBn BnO' BnO 2. TBAF, THF Bn( Bni (78%) OR OB over two steps 129 132
R = (CH2 )7 CH3 Hg, Pd/C, (78%) HOAc
OCH
HO'
OH
HO'
OR 30 R=(CH2)tCH3
Figure 4.15 Synthesis of disaccharide 30
117 4.14 NMR ANALYSIS
As mentioned in Chapter 3, one-bond carbon-hydrogen coupling constants
(‘7 ^ 1,hi) involving the anomeric carbon of the mannose residues in 25-30 and 33 were measured to prove glycoside stereochemistry.'"^ All ‘/ci.hi values were between 167-171
Hz, which are indicative of a-mannosides. Furthermore, the assignment of resonances in
25-30 and 33 were made by two dimensional homonuclear and heteronuclear shift correlation experiments such as ('H-'H) COSY, (‘H-'H) TOCSY and ('H-'^C) HSQC.
4.14.1 ‘’F NMR AND 'H-”F COUPLING CONSTANTS
The F-2' and F-6 ' can be easily distinguished from their chemical shifts (ôp) and their coupling patterns.'"" The '"F chemical shifts observed for all the fluorinated compounds are found to be between -203 to -208 ppm for F-2' and was -231 to -237 ppm
in case of F- 6 '. The coupling patterns for the deprotected compounds 23, 25, 27, 28 and
30 are described below in Table 4.1.
118 6' X
3 ' 2 ' 1
H0‘
6 Compoud F-2' F- ' Coupling A i2',F J H3 '.F •^ H 5 ’,F J H 6 a ',F H 6 b '.F No. (5p) (5 f) pattern ppm ppm Hz Hz Hz Hz Hz Hz 23 -204.7 ddd 7.2 48.7 29.7 ---
25 - -234.4 td - -- 25.9 47.1 47.1
27 - 207.6 - 236.8 ddd, td 7.1 49.4 33.0 25.9 47.1 47.1
28 - 207.3 - ddd 7.5 50.0 31.0 - - -
30 - -234.6 td - - - 25.9 46.7 46.7
Table 4.1 19 F chemical shifts and H- F coupling constants
119 4.14.2 EFFECT OF SUBSTITUTION ON "C CHEMICAL SHIFTS
The ‘^C chemical shifts for C-2' and C- 6 ' in case of deprotected compounds 25-30 are presented in Table 4.2. Compound 20 was also included for purposes of comparison.
HO'
O' HO'
0(CH2)7CH3
Compound No. X (C-2') Y (C- 6 ') ' ’’C chemical shift ‘^C chemical shift
of C-2' of C- 6 '
(6c) ppm (6 c) ppm 27 F F 91.0 82.0
28 F NH, 91.2 43.5
30 OCH, F 81.8 83.4
29 OCH, NH, 81.8 42.3
2 0 OH OH 70.4 60.3
25 NH, F 55.7 83.5
26 NH,NH, 55.1 41.8
Table 4.2 Comparison of ‘"C chemical shifts of compounds 20 and 25-30
1 2 0 CHAPTER 5
SYNTHESIS OF POTENTIAL SUICIDE INHIBITORS
OF MANNOSYLTRANSFERASES IN
LAM BIOSYNTHESIS
5.1 INTRODUCTION
As the synthesis of type A and type B inhibitors (see Figure 1.20) was successfully accomplished, we proceeded with the synthesis of suicide inhibitors 31 and
32 (see Figure 1.21). Compound 32 could inhibit by trapping a nucleophilic group at the active site of the enzyme via irreversible covalent bond formation as described in Chapter
1. Compound 31 could deactivate the enzyme by nitrene generation and insertion following photolysis.
121 5.2 RETRO SYNTHETIC ANALYSIS
Our strategy was to synthesize the disaccharide 31 from the building blocks 77 and 133 as outlined in Figure 5.1. Compound 32 could be synthesized from 31, by reducing the azido functionality to give the amine 24 followed by acylation reaction. We chose acyl protecting groups in 133 rather than benzyl groups, which can be easily removed in the presence of an azido group.
NH; HO' HO'
HO HO'
OH
HO HO'
OR OR 32 2 4 R = {CH2)tCH3
HoA pBz
\ 7 7 *^^NH 1 3 3 CI3C R = (CH2)tCH3 31
R = (CH2)tCH3
Figure 5.1 Retrosynthetic analysis for compounds 31 and 32
122 5.3 SYNTHESIS OF ACCEPTOR ALCOHOL 133
Alcohol 133 was efficiently synthesized from D-mannose 54 in six steps via intermediate 79. Benzoylation of 79 with BzCl in the presence of pyridine afforded the tri-O-benzylated compound, which was treated with p-toluenesulfonic acid without further purification, to afford 133 in 83% yield over two steps as shown in Figure 5.2.
OBz pH Figure 3.4 ^ 1. BzCI, Py BzO 54
OR 79 OR 133 (83%) R = (CH2)7CH3
Figure 5.2 Synthesis of building block 133
5.4 SYNTHESIS OF DISACCHARIDES 31 AND 32
With building blocks 77 and 133 in hand, the disaccharide 31 and 32 proceeded as outlined in Figure 5.3. Glycosylation of 77 with 133 in the presence of TMSOTf
afforded the protected disaccharide 134 in 6 6 % yield. Deacetylation of 134 with sodium
methoxide resulted in 31 in 8 8 % yield. Hydrogenation of a portion of 31 gave amino compound 24 in 90% yield. A portion of 24 was acylated with chloroacetyl chloride in 123 the presence of aq. NaHCOj to provide 32 in 17% yield. However, acylation with chioroacetic anhydride proceeded efficiently, resulting in 32 in 74% yield.
1 2 4 AcO AcO HOA 9Bz 77 T M S O T f OBz Bzi BzO -10 -4.0 °C 133 ° ’^ Bz( (66 %) R = (CH2)7CH3 OR 134
f88%1 N aO C Ha CH 3OH
HO' HO'
HO Ha, Pd/C
p H OH (90%) HO' HO
OR OR 24 R = (C H a)7 C H a
(CICHaC0)20 (74% aq. N aH C O a
Aci
AcO
HO' A d
HO' ■NH
OH CI3C
HO
OR R = (CHa)7CHa
Figure 5.3 Synthesis of disaccharides 31 and 32
125 5.5 SUMMARY
* Glycan fragments of Man-LAM were successfully synthesized along with the development of a method for the synthesis of P-D-arabinofuranosidic linkages.
Biological studies on the assembly of terminal mannose caps with the above synthetic
oligosaccharides are currently being investigated.
* Methodologies were developed for functional group modifications and utilized for
the synthesis of type A and type B disaccharides which are potential inhibitors of
mannosyltransferases involved in mycobacterial LAM biosynthesis. We also synthesized
two potential suicide inhibitors.
* All the tmal compounds were characterized completely using conventional
spectroscopic methods.
* Biological studies performed in collaboration with Dr. Besra and coworkers on
type A disaccharides with a(l-> 6 )-ManT suggested that four out of five disaccharides
were acceptors. Further studies are underway with a(l-»2)-ManT and with type B
disaccharides.
126 CHAPTER 6
EXPERIMENTAL SECTION
Optical rotations were measured at 22 ± 2 °C. Analytical TLC was performed on silica gel 60-F,;_, (0.25 mm, Merck). Spots were detected under UV light or by charring with 10% H,SOj in ethanol. Unless otherwise indicated, all reactions were carried out at room temperature and under a positive pressure of argon. Solvents were evaporated under reduced pressure and below 40 'C. Column chromatography was performed on silica gel or latrobeads. The ratio between silica gel and compound ranged from 100 to 50:1 (w/w).
‘H NMR spectra were recorded at 250. 300,400, 500 or 800 MHz, and first order proton
chemical shifts, 5^,, are referenced to either to TMS ( 6 ^ 0.0, CDClj) or HOD (5^ 4.78, D,0 and CD.QD). ‘^C NMR spectra were recorded at 75, 125.8 or 150.9 MHz and ‘^C chemical shifts, 5^, are referenced to either to TMS (5^ 0.0, CDClj), dioxane (Ô^ 67.4,
D,0) or CDjOD ( 6 (- 48.9). NMR spectra were recorded at 235.4 MHz, ‘‘'F chemical
shifts 5p are referenced to (5%) CFCI 3 in absolute ethanol as the external standard. The assignment of resonances in all the final deprotected compounds 16-33 were made by two-dimensional homonuclear and heteronuclear shift correlation experiments. One-bond carbon-hydrogen coupling constants involving the anomeric carbon of the mannose residues in case of deprotected oligosaccharides were measured to prove glycoside
1 2 7 stereochenisitry.‘°’ Elemental analyses were performed by Atlantic Microlab Inc., GA and the samples submitted for analyses were dried overnight under vacuum with phosphorus pentoxide at 56 °C (refluxing acetone). Fast atom bombardment mass spectra were recorded on samples suspended in thioglyceride matrix with a cesium gun. MALDl mass spectra were recorded on samples suspended in an a-hydroxycyanocinnamic acid matrix.
Electron spray ionisation (ESI) mass spectra were recorded on samples dissolved in (3:1
CH 3OH : H ,0).
l-0-Acetyl-2,3,5-tri-(?-benzyl-D-arabinofuranose (60A). This compound was prepared as previously described.
BnO 0 (CH 2)7 CH 3 B nO — 1 V
BnO g4
Octyl 2,3,5-tri-O-benzyl-P-D-arabinofuranoside (64) . HCl gas was bubbled through a solution of 60A (850 mg, 1.87 mmol) in dry dichloroethane (10 mL) for 30 min. After stirring for an additional 10 min, the reaction mixture was concentrated to
1 2 8 provide 2,3,5-tri-O-benzyl-a-D-arabinofuranosyl chloride 63 as a light yellow syrup.
Without further purification, the crude product was dissolved in dry CH^Cl, (15 mL) and octanol (0.35 mL, 2.24 mmol) was added. The reaction mixture was stirred for 60 min and then diluted with CH,CU (40 mL), before being washed with water (30 mL), a saturated NaHCOj solution (2 x 30 mL) and water (25 mL). The organic layer was dried
(Na^SOJ, filtered, and concentrated under vacuum to a light yellow syrup, which was purified by column chromatography (9:1 he.xaneiEtOAc) to give 64 (440 mg, 58%) as a colorless syrup. R, 0.66 (4:1 hexane:EtOAc); [al^ -49.6“ (c 1.4, CHCI 3); ‘H NMR (300
MHz, CDCl,) Sh 7.26-7.40 (m. 15 H, Ph), 4.90 (d, 1 H, 7,, = 4.0 Hz, H-1), 4.70 (d, 1
H. 7 = 11.7 Hz, C //,P h), 4.60-4.66 (m, 3 H, C//,Ph), 4.70 (s, 2 H, C //,Ph), 4.05-4.22
(m. 3 H. H-2, H-3. H-4), 3.68 (dt, 1 H, 7 = 6 .8 , 9.2 Hz, octyl OCH,), 3.50-3.58 (m, 2
H, H-5a, H-5b), 3.34 (dt, 1 H, 7 = 6 .8 , 9.2 Hz, octyl OCH,), 1.55-1.62 (m, 2 H, octyl
CH,), 1.29-1.35 (m, 10 H, octyl CH,), 0.91 (t, 3 H, 7= 6.4 Hz, octyl CH 3); "C NMR
(75.5 MHz, CDCI3) 5e 138.2, 138.1, 137.7, 128.3, 128.2, 128.2, 128.0, 127.8, 127.7,
127.6, 127.5, 127.5 (Ph). 100.5 (C-1), 84.2, 83.5, 80.1, 73.2, 72.7, 72.3, 72.2, 67.9
(ring and benzylic C, octyl OCH,), 31.8, 29.4. 29.3, 29.2, 26.2, 22.6 (octyl CH,), 14.0
(octyl CH 3). Anal. Calcd for C,jH ^ 0 5 (532.69): C. 76.66; H, 8.33. Found: C, 76.62; H,
8.36.
A cO —
l,5-Di-0-acetyI-2,3-dl-(?-benzyl-D-arabinofuranose ( 6 5 ) . To a solution of
60” (2.5 g, 5.95 mmol) in acetic anhydride (15 mL) was added a solution containing 6
1 2 9 drops of conc. H^SO^ in acedc anhydride (5 mL). The solution was stirred for 2 h, and was then poured into ice (100 g) and stirred for 2 h, before being extracted with CH^CL (3
X 30 mL). The organic layer was washed with water (30 mL) followed by a saturated
NaHCOj solution (2 x 30 mL) and water (30 mL). The organic extract was dried
(Na.SO^), filtered and concentrated to a light yellow syrup, which was purified by chromatography (4:1 toluene:EtOAc) to give 65 (2.1 g, 86%) as a colorless syrup in a 9:1 a:|3 ratio of anomers. R, 0.27 (4:1 toluene:EtOAc); [a]g -21.6“ (c 0.8, CHClj); 'H NMR
(250 MHz, CDCl;) 5„ 7.25-7.40 (m, Ph), 6.30 (d, 7,, = 4.2 Hz, H-lp), 6.23 (br. s, H- la), 6.67 (d, y = 11.8 Hz, CH.Ph) 4.50-4.69 (m. CH.Ph), 4.37 (ddd, 7,^, = 3.8, J, , =
5.8, 745b = 6.0 Hz, H-4a), 4.27 (dd, = 3.8, 7;,^b = ^ 19 Hz, H-5aa), 4.13 (dd, 7 4 5b
= 6.0, yjj jb = 1 1 . 9 Hz, H-5ba), 4.08 (d, 7, ^ = 2.3 Hz, H-2a), 3.89 (dd, 7,; = 2.3, 7 . 4 =
5.8 Hz, H-3a), 2.08 (s. a-OCOCH,), 2.06 (s, p-OCOCHj), 2.04 (s, P-OCOC// 3), 2.01
(s, a-OCOCH,): '"C NMR (75.5 MHz, CDCl;) 5^ 170.7, 169.9 (C=0), 137.7, 137.3,
137.1, 128.6, 128.2, 128.1, 128.0, 128.0 (Ph), 100.5 (C-la), 94.5 (C-lp), 8 6 .8 , 84.1,
83.6, 81.8, 81.2, 72.7, 72.3, 72.2, 65.2, 63.7 (ring and benzylic C), 21.3, 21.2, 20.8
(OCOCH;). Anal. Calcd for C^jHn^O; (414.22): C, 66.64: H, 6.33. Found: C, 66.40; H,
6.30.
BnO 0(CH2)7CH3
Octyl 2,3-di-O-benzyl-P-D-arabinofuranoside (53). HCl gas was bubbled through a solution of 65 (1.4 g, 3.38 mmol) in dry dichloroe thane (10 mL) for 30 min.
1 3 0 The reaction mixture was stirred for an additional 10 min and the solvent was concentrated
to provide 5-0-acetyl-2,3-di-C>-benzyi-a-D-arabinofuranosyl chloride 6 6 as a light yellow syrup. Without further purification, the crude product was dissolved in dry CH^CU (20 mL) and octanol (0.37 mL, 2.35 mmol) was added. After stirring for 90 min. the reaction mixture was diluted with CH,Ch (40 mL) and then washed with water (30 mL) followed by a saturated NaHCOj solution (2 x 30 mL) and water (25 mL). The organic layer was dried (Na,SO^). filtered and concentrated to give crude 67 as light yellow syrup. The product was dissolved in methanol (15 mL) and a few drops of 0.1 M NaOCHj was added and the reaction was stirred for 4 h. The reaction mixture was concentrated and the unreacted octanol was removed by azeotropic distillation with water (3 x 40 mL). The light yellow oil obtained was purified by chromatography (85:15 hexane: EtOAc) to obtain 53
(0.5 g. 35%) as a colorless oil. R, 0.31 (4:1 hexane:EtOAc): [ajp +14.5° (c 0.8. CHClj);
‘H NMR (200 MHz. CDCI3 ) Ô3, 7.25-7.39 (m. 15 H. Ph). 4.85 (d. 1 H. 7,. = 4.4 Hz. H-
1). 4.73 (d. 1 H. 7= 11.7 Hz. C7/,Ph). 4.60 (s. 2 H. C7/,Ph). 4.58 (d. 1 H. 7= 10.7 Hz.
CH,Ph). 4.31 (dd. 1 H. J.j = 5.8. J,, = 7.0 Hz. H-3). 4.00-4.10 (m. 2 H. H-2. H-4).
3.50-3.80 (m. 3 H. H-5a. H-5b, octyl OCH,). 3.39 (dt. 1 H. 7 = 6 .8 . 9.6 Hz. octyl
OCH,). 2.37 (dd. 1 H. Js^s.qh = -1-2. •fsb.s-oH ~ 7.7 Hz. 5-OH), 1.58-1.65 (m. 2 H. octyl
CH,). 1.23-1.29 (m. 10 H. octyl CH,). 0.87 (t. 3 H. 7= 6.1 Hz. octyl CH 3); ''C NMR
(75.5 MHz. CDCI3 ) 6 c 138.1. 137.7, 128.4. 128.1. 128.0, 127.8. 127.7 (Ph), 100.8 (C-
1). 84.5. 82.2, 81.2. 72.5. 72.4. 69.2. 63.8 (ring and benzylic C, octyl OCH,), 31.8,
29.6, 29.5, 29.4, 26.1. 22.6 (octyl CH ,), 14.1 (octyl C H 3). Anal. Calcd for C^^HjgOj
(414.22): C, 73.27; H. 8.65. Found: C, 73.37; H. 8.59.
1 3 1 HO—1 HO 0(CH2)7CH3
Octyl P-D-arabinofuranoside (16).'“^ To a solution of 53 (250 mg, 0.48 mmol) in methanol (10 mL) was added 10% Pd/C (70 mg). The reaction mixture was stirred under
H, atmosphere for 6 h at room temperature and then, filtered, washed with methanol (2 x 5 mL) and CH,CL (5 mL) and then concentrated under vacuum to obtain a 16 as colorless syrup (100 mg, 82%). R, 0.38 (1:4 toluene:EtOAc): [aj^ -29.3" (c 1.1, CHCl,); 'H NMR
(800 MHz, D;0) 6 h 4.90 (d, 1 H, 7 ,, = 4.7 Hz, H-1), 4.02 (dd, I H, 7 ,, = 4.7, J, . = 7.8
Hz, H-2), 3.93 (dd, 1 H. 7,, = 7.8, 7,, = 7.3 Hz, H-3), 3.79 (ddd, 1 H, 7j, = 7.3, 7,^, =
3.9, 7 , 5, = 7.0 Hz, H-4), 3.65-3.70 (m, 2 H, H-5a, octyl OCH,), 3.55 (dd, 1 H, 7, j, =
7.0, 7 ;, 5, = 11.9 Hz, H-5b), 3.42 (dt, 1 H, 7= 6 .6 , 9.5 Hz, octyl OCH,), 1.50-1.52 (m,
2 H, octyl CH,), 1.20-1.26 (m, 10 H, octyl CH,), 0.79 (t, 3 H, 7= 6 . 6 Hz, octyl CHj);
‘^C NMR (75.5 MHz, CDCl,) 101.1 (C-1), 82.3 (C-4), 78.2 (C-2), 75.8 (C-3), 69.2
(C-5), 63.0 (octyl OCH,), 31.7, 29.5, 29.2, 29.1, 25.8, 22.5, (octyl CH,), 14.0 (octyl
CH,). HR-FAB-MS calcd for C„H ,,0; [M + H]' 263.1859, found 263.1874.
QAc
BnO Bm
BnO
1 3 2 Octyl 2-0-acetyl-3,4, 6 -tri-O-benzyl-a-D-mannopy ranosyI-(l-> 5)-2,3-di-0- benzyl-P-D-arabinofuranoside ( 6 8 ).'°^ Thioglycoside 52*^' (230 mg, 0.43 mmol), alcohol 53 (170 mg, 0.39 mmol), and powdered 4 Â molecular sieves (300 mg) were placed in a round-bottom flask and dried overnight in vacuo. Diy CH,C1, (20 mL) was added and the mixture was cooled to -40 °C and stirred for 10 min. N-iodosuccinimide
(115 mg, 0.51 mmol) was added and the solution was stirred for 20 min before silver triflate (31 mg, 0.12 mmol) was added. The reaction mixture was allowed to gradually warm to 0 °C and then, triethylamine (0.5 mL) was added. The yellow solution was filtered, diluted with CHXU (20 mL), washed with saturated Na^S^Oj solution (2 x 20 mL) followed by brine (20 mL) and water (20 mL). After drying (Na,SOj), the organic layer was concentrated to a light yellow syrup, which was chromatographed (85:15 he.\ane:EtOAc) to give 6 8 (323 mg, 82%) as a colorless syrup. R,. 0.33 (4:1 hexane:EtOAc); [aj^ +3.4° (c 1.2, CHClj); 'H NMR (300 MHz, CDCI 3) 7.16-7.36 (m,
25 H, Ph). 5.38 (dd, 7,. ,. = 1.8, 7,.y = 3.0 Hz, H-2'), 4.41-4.90 (m, 12 H. H-1, H -1', 10
X C//,Ph), 3.60-4.10 (m, 10 H. H-2, H-3, H-4. H-5a, H-5b, H-3', H-4', H-5', H- 6 'a, octyl OCH,), 3.51 (dd, 1 H. 7;.,., = 5.6. = 10.1 Hz. H- 6 'b), 3.30 (dt, 1 H, 7= 6.9,
9.7 Hz, octyl O CH ,), 2.13 (s, 3 H, OCOCH 3), 1.56-1.64 (m, 2 H, octyl CH,),
1.21-1.29 (m, 10 H, octyl CH,), 0.87 (t, 3 H, 7= 6.7 Hz, octyl CH 3); '^C NM R (75.5
MHz, CDCI3) 6 c 170.3 (C=0), 138.5, 138.2, 138.1, 138.0, 137.9, 137.7. 128.4, 128.3,
128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.8, 127.7, 127.5, 127.5
(Ph), 100.5 (C-1), 98.1 (C-1'), 84.2, 83.3, 79.5, 78.3, 75.1, 74.2, 73.5, 72.4, 71.8,
71.6, 71.5, 70.1, 6 8 .6 , 68.5, 68.0 (ring and benzylic C, octyl OCH,), 31.8, 29.5, 29.4,
29.3, 26.2, 22.6 (octyl CH,), 21.2 (OCOCH 3), 14.1 (octyl C H 3). Anal. Calcd for
C,,H,,0,, (917.15): C, 73.34; H, 7.47. Found: C, 73.62; H, 7.40.
1 3 3 HO QH MO'
OR HQ
r = (CH2)7CH3 ho
Octyl a-D-mannopyranosyl-(1^5)-p-D-arabinofuranoside (17).'“^ A solution of
6 8 (210 mg, 0.23 mmol) was dissolved in methanol (20 mL) and then 2 drops of IM
NaOMe was added. After stirring overnight, the solution was neutralized with a minimum amount of Amberlite 118 resin and concentrated to a syrup, which was purified by chromatography (8:1 he.\ane:EtOAc) to give 69 (190 mg. 95%) as a colorless syrup. ‘H
NMR (300 MHz. CDCl,) 7.18-7.44 (m, 25 H. Ph). 4.98 (d. 7,,, = 1.4 Hz. H-L), 4.91
(d. 1 H. = 4.2 Hz. H-1). 4.87 (d. I H. 7 = 10.9 Hz. CH,Ph), 4.47-4.77 (m. 9 H.
CH,Ph), 3.64-4.24 (m. 11 H. H-2. H-3. H-4. H-5a. H-5b. H-2'. H-3'. H-4'. H-5'. H-
6 'a. octyl OCH,). 3.59 (dd. 1 H. 7,.^^ = 5.7.7^,,-^ = 10.2 Hz. H- 6 'b). 3.36 (dt, 1 H. 7 =
6.9. 9.7 Hz. octyl OCH,). 2.53 (br. s. 1 H. OH). 1.56-1.64 (m. 2 H. octyl CH,),
1.26-1.33 (m. 10 H, octyl CH,). 0.94 (t. 3 H. 7= 6 . 8 Hz. octyl CH,); '"C NMR (75.5
MHz. CDCl,) 5c 138.5. 138.2. 138.2, 138.0, 137.8. 128.6. 128.5, 128.4, 128.2, 128.0,
127.8, 127.7, 127.6 (Ph), 100.5 (C-l), 99.7 (C-l'), 84.3, 83.4, 80.3, 79.6, 75.2, 74.2,
73.5, 72.5, 72.5, 72.1, 71.3, 69.7, 6 8 .8 , 68.3. 68.2 (ring and benzylic C, octyl OCH,),
31.9, 29.6, 29.5. 29.4. 26.3. 22.8 (octyl CH,). 14.2 (octyl CHj).
To a solution of 69 (100 mg, 0.11 mmol) in methanol ( 8 mL), was added 10%
Pd/C (25 mg). The solution was stirred overnight under a H, atmosphere and then the catalyst was separated by filtration and washed with CHjOH (10 mL). After concentrating
1 3 4 the filtrate and washings, the product was purified by chromatography (4:1
CH^ClyCH^OH) on silica gel to give 17 (41 mg, 90%) as a foam. R,. 0.53 (4:1
CH,CL:CH,OH): [a]^ +2,9" (c 1.2, H.O); 'H NMR (800 MHz, D.O) 6 „ 4.91 (m, 1 H, H-
I), 4.84 (d, I H, y,.,. = 1.6 Hz, H-I'), 4.02-4.06 (m, 2 H, H-2, H-3), 3.91-3.94 (m, 1
H, H-4), 3.90 (dd, 1 H, 7, ,. = 1.6, J.-y = 3.3 Hz, H-2'), 3.70-3.81 (m, 4 H, H-5a, H-3',
H-6 'a, H-6 'b), 3.61-3.68 (m, 3 H, H-4', H-5b, octyl OCH,), 3.57 (ddd, 1 H, 7,.^ = 9.7, y,.,., = 2.1, 7;.,., = 5.0 Hz, H-5'), 3.42 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl OCH,), 1.58-1.65
(m, 2 H, octyl CH,), 1.23-1.29 (m, 10 H, octyl CH,), 0.87 (t, 3 H, 7 = 6.1 Hz, octyl
CH,); ‘^C NMR (125.8 MHz, D,0) 5^ 101.4 (C-l, '7^,^ =171.8 Hz), 100.3 (C-1',‘7c,h =
171.1 Hz), 80.0 (C-4), 76.8 (C-2), 75.3 (C-3), 73.3 (C-5'), 71.0 (C-3'), 70.1 (C-2'),
68.9 (octyl OCH,), 6 8 . 8 (C-5), 67.0 (C-4'), 61.2 (C-6 '), 31.7, 29.5, 29.2, 29.1, 25.8,
22.5 (octyl CH,), 14.0 (octyl CH,). HR-FAB-MS calcd for C„H, 7 0 ,u [M + H]"
425.2387, found 425.2386.
p A c
BnO
B nO '
ÇR
Octyl 2-0-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyI-(l->2)-3,4,6-tri-
0-benzyl-a-D-mannopyranosyl-(1^5)-2,3-di-0-benzyl-P-D-arabino- furanoside (70).'°^ Thioglycoside 52'‘ (120 mg, 0.22 mmol), disaccharide 69 (150
1 3 5 mg, 0.17 mmol), and powdered 4 Â molecular sieves (200 mg) were placed in a round- bottom flask and dried overnight in vacuo. Dry CH^CU (15 mL) was added and the mixture was cooled to -40 °C and stirred for 10 min. A(-iodosuccinimide (52 mg, 0.23 mmol) was added and the reaction mixture was stirred for 2 0 min before the addition of silver triflate (13 mg, 0.05 mmol). The solution was allowed to gradually warm to 0 °C and then, triethylamine (0.3 mL) was added. The yellow solution was filtered, diluted with
CH,CU (20 mL), washed with saturated Na^S^Oj solution (2 x 20 mL) followed by brine
(20 mL) and water (20 mL). After drying (Na^SOj, the organic layer was filtered and concentrated under vacuum to a light yellow syrup, which was chromatographed (85:15 hexane:EtOAc) to give 70 (130 mg, 87%) as a colorless syrup. R, 0.32 (4:1 he.\ane:EtOAc); [ajg +4.7“ (c 1.3. CHClj); ‘H NMR (250 MHz, CDClj) 6 ^ 7.13-7.37 (m,
40 H, Ph), 5.54 (dd, 1 H, 7, ,,.. = 1.7, J.-y = 3.0 Hz, H-2"), 5.11 (d, 1 H, 7,.,. = 1.7 Hz,
H-1"), 4.79-4.92 (m, 5 H, H-1, H-1', 3 x CH.Ph). 4.41-4.71 (m, 12 H, CH,Ph), 4.38
(d, 1 H ,7= 10.9 Hz, C//,Fh), 3.64-4.13 (m, 16 H, H-2, H-3, H-4, H-5a, H-5b, H-2',
H-3', H-4', H-5', H-6 'a, H-3", H-4", H-5", H-6 "a, H-6 "b, octyl OCH,), 3.47 (dd, 1 H,
7; = 5.6, = 10.0 Hz, H-6 'b), 3.30 (dt, 1 H, 7 = 6.9, 9.8 Hz, octyl OCH,), 2.13
(s, 3 H, OCOCHj) 1.56-1.64 (m, 2 H, octyl CH,), 1.21-1.29 (m, 10 H, octyl CH,),
0.88 (t, 3 H, 7 = 6.9 Hz, octyl CH 3); '"C NMR (75.5 MHz, CDCI3) 8 ^ 170.1 (C=0),
138.5, 138.4, 138.4, 138.3, 138.2, 138.1, 138.0, 137.7, 128.4, 128.3, 128.2, 128.2,
128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6, 127.6, 127.6, 127.5
127.4, 127.4, 127.4, 127.3, 127.3 (Ph), 100.4 (C-l), 99.5 (C-l'), 98.9 (C-l"), 84.3,
83.6, 79.9, 79.5, 78.2, 75.1, 74.5, 74.4, 74.2, 73.4, 72.4, 72.1, 72.0, 71.9, 71.8, 69.9,
69.0, 6 8 .8 , 68.7, 68.0 (ring and benzylic C, octyl OCH,), 31.8, 29.5, 29.4, 29.3, 26.2,
22.7 (octyl CH,), 21.2 (OCOCH 3), 14.1 (octyl CH 3). Anal. Calcd for CgjH^^Oi^
(1349.66); C, 73.86; H, 7.17. Found; C, 73.73; H, 7.04. HR-MALDI-MS calcd for
C,3H%0 ,g [M + Na]" 1371.6626, found 1371.6596.
1 3 6 HO OH
HO'
HO' HO'
I u n I
R = (CH2)7CH3lo I
O ctyl a-D-mannopyranosyi-(l—>2)-a-D-mannopy^anosyI-(l-^5)-p-D-a^abino- fu^anos^de (18).'“^ A solution of 70 (110 mg. 0.23 mmol) was dissolved in methanol
(20 mL) and then 2 drops of IM NaOMe was added. After stirring overnight, the solution was neutralized with a minimum amount of .Amberlite 118 H'' resin and concentrated to a syrup, then concentrated to a syrup, which was purified by chromatography (4:1 hexaneiEtOAc) to give 71 (100 mg, 96%) as a syrup. 'H NMR (300 MHz, CDClj) 5^
7.19-7.33 (m, 40 H. Ph), 5.14 (d. 1 H, 7,-,- = 1.6 Hz, H-1"). 4.94 (d, 1 H, 7,.,. = 1.6
Hz, H-1'), 4.41-4.86 (m 17 H, H-1, 16 x C//,Ph), 3.98-4.10 (m, 4 H), 3.60-4.15 (m,
17 H, H-2, H-3, H-4. H-5a. H-5b, H-2', H-3', H-4', H-5', H- 6 'a, H-2". H-3", H-4", H-
5", H-6 "a, H-6 "b, octyl OCH,), 3.47 (dd, 1 H, 7;.^ = 5.6, 7,.^,.^ = 10.0 Hz, H- 6 'b),
3.29 (dt, 1 H, 7 = 6.9, 9.8 Hz, octyl OCH,), 1.56-1.64 (m, 2 H, octyl CH,), 1.21-1.29
(m, 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 6.9 Hz, octyl CHj); ‘^C NMR (75.5 MHz, CDCy
5c 170.1 (C=0), 138.5, 138.4, 138.2, 138.1, 138.0. 137.9, 137.7, 137.6, 128.3, 128.3,
128.2, 128.2. 128.1. 128.0, 127.9, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 127.4,
127.3, 127.2, 127.2, 127.1 (Ph), 101.0 (C-l), 100.3 (C-l'), 98.9 (C-l"), 84.2, 83.5,
79.9, 79.8, 79.4, 74.9, 74.8, 74.7, 74.6, 74.5, 74.3, 74.2, 73.3, 72.2, 72.0, 71.5, 71.8,
1 3 7 69.9, 69.0, 68.4, 67.9 (ring and benzylic C, octyl OCH,), 31.7, 29.4, 29.3, 29.2, 26.0,
22.6 (octyl CH,), 14.0 (octyl CH,).
To a solution of 71 (100 mg, O.ll mmol) in methanol ( 8 mL) was added 10%
Pd/C (25 mg). The mixture was stirred overnight under an H, atmosphere and the catalyst was separated by filtration and washed with CH,C1, (10 mL). After concentrating the filuate and washings, the product was purified by chromatography (2:1 CH,C1,:CH,0H) on latrobeads to obtain 18 (38 mg, 93%) as a foam. R, 0.29 (2:1 CH,Cl,:CH,OH); [a],,
+5.2“ (c 0.9. H,0): 'H NMR (800 MHz, D,0) 6 „ 5.08 (s, I H, H-L), 4.96 (s, I H, H- l"). 4.93 (d. I H. 7,, = 4.5 Hz. H-l), 4.05 (dd, I H. 7,, = 4.6, 7,, = 8.0 Hz. H-3),
4.00-4.02 (m, 2 H. H-2. H-2"). 3.93 (ddd, I H, 7,, = 8.0, 7,,, = 3.3, 7,,, = 6.9 Hz, H-
4). 3.88-3.91 (m. 2 H. H-2', H-3'). 3.80-3.83 (m. 2 H, H- 6 'a. H-6 ''a), 3.79 (dd, I H,
7,.. j.. = 3.3. 7, = 9.6 Hz. H-3"). 3.63-3.75 (m. 5 H. H-5a, H-4', H-5", H- 6 'b, H- 6 "b),
3.54-3.61 (m, 4 H, H-5b, H-5', H-4". octyl OCH,). 3.44 (dt, I H, 7 = 7.0, 9.7 Hz, octyl
OCH,), 1.58-1.65 (m. 2 H. octyl CH,), 1.23-1.29 (m. 10 H, octyl CH,), 0.87 (t, 3 H, 7
= 6.1 Hz, octyl CH,); "C NMR (125.8 MHz, D,0) 8 ^ 102.8 (C -l', '7^,,, = 170.7 Hz),
101.4 (C -l, ‘7c.h = 174.4 Hz), 98.7 (C-l", '7c,h = 171.8 Hz), 80.0 (C-4), 79.2 (C-2'),
76.6 (C-3). 75.1 (C-2), 73.5 (C-5"), 73.3 (C-5'), 70.8 (C-3"), 70.6 (C-3'), 70.4 (C-2"),
69.1 (C-5). 69.0 (octyl OCH,). 67.3 (C-4'), 67.2 (C-4"), 61.6, 61.2 (C- 6 ', C- 6 "), 31.6,
29.2. 29.0, 29.0, 25.8. 22.5 (octyl CH,), 13.9 (octyl CH,). HR-FAB-MS calcd for
C„H,,0 , 5 [M + Na]* 609.2734, found 609.2717.
138 QAc
Bni
Bn(
□ n u Bni Bni QR BnO
FI - (CH glyCHg
Octyl 2,3,4,6-Tetra-0-acetyl-a-D-mannopyranosyI-(l->2)-3,4,6-tri-
0-benzyl-a-D-mannopyranosyl-(1^2)-3,4,6-tri-0-benzyI-a-D-manno- pyranosyl-{ 1^5)-2,3-di-0-benzyI-p-D-arabinofuranoside (72). Thioglycoside
51^" (35 mg. 0.09 mmol), trisaccharide 71 (80 mg, 0.06 mmol), and powdered 4 Â molecular sieves ( 1 2 0 mg) were placed in a round-bottom flask and dried overnight in vacuo. The above mixture was dissolved in dry CH^CU (10 mL) and the mixture was cooled to -40 °C and stirred for 10 min. yV-iodosuccinimide (20 mg, 0.09 mmol) was added and the reaction mixture was stirred for 20 min before the addition of silver triflate (5 mg, 0.02 mmol). The reaction mixture was allowed to gradually warm to 0 °C and then, triethylamine (0.3 mL) was added. The yellow solution was filtered, diluted with CH^CU
(15 mL), washed with a saturated Na^S^Oj solution (2 x 15 mL), followed by brine (20 mL) and water (20 mL). After drying (Na.SOJ, it was filtered and concentrated under vacuum to a light yellow syrup, which was purified by chromatography (85:15 hexane:EtOAc) to give 72 (75 mg, 76%) as a syrup. 0.37 (2:1 hexane:EtOAc); [a]p
139 + 11.4“ (c 0.9, CHCI 3); ‘H NMR (300 M Hz, CDCI3) 5„ 7.19-7.34 (m, 40 H, Ph),
5.45-5.15 (m, 4 H, H-L'", H-2'". H-3'", H-4'"), 4.95-4.40 (m, 19 H, H-l, H-l", H- l" , 16 X CH.Ph), 3.60-4.15 (m, 20 H, H-2, H-3, H-4, H-5a, H-5b, H-2', H-3', H-4',
H -5', H-6 'a, H-2", H-3", H-4", H-5", H-6 "a, H-6 "b. H-5'", H- 6 '"a, H-6 '"b, octyl
OCH,), 3.47 (dd, l H. 7 ;.^ = 5.6, 7,.^,3, = lO.O Hz, H-6 'b), 3.31 (dt. l H, 7= 6.9, 9.8
Hz, octyl OCH,), 2.11 (s, 3 H, OCOCH 3), 1.99 (s, 3 H, OCOCH 3), 1.98 (s, 3 H,
OCOCH 3), 1.95 (s, 3 H, OCOCH 3), 1.50-1.57 (m, 2 H, octyl CH,), 1.23-1.33 (m, 10
H, octyl CH,), 0.87 (t, 3 H. 7 = 6.3 Hz, octyl CH 3); "C NMR (75.5 MHz, CDCI3)
170.1, 169.8, 169.7, 169.6 (C=0), 138.5, 138.4, 138.4. 138.3, 138.1, 137.8, 128.9,
128.4, 128.3, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9. 127.9, 127.7, 127.6, 127.5,
(Ph). 100.5 (C-l'"), 100.3 (C-l"), 99.2 (C-l'), 98.9 (C-l), 84.3, 83.6, 79.8, 79.5, 79.3,
76.8, 75.2, 75.1, 75.0, 74.8, 74.7, 73.4, 73.3, 73.1, 72.5. 72.4, 72.3, 72.2, 72.1, 70.2,
69.6, 69.2, 69.1, 68.9, 68.0. 66.1 (ring and benzylic C, octyl OCH,), 31.9, 29.5, 29.5,
29.3, 26.2, 22.7 (octyl CH,), 20.9, 20.7, 20.6 (OCOCHJ, 14.1 (octyl CHJ. HR-
MALDl-MS calcd for C,,H „,0,JM + Na]" 1659.7441. found 1659.7504.
OH
OR HO
R = (Cht)7Ctt Hi
1 4 0 Octyl a-D-mannopyranosyI-(l-^2)-a-D-mannopyranosyI-(l->2)-a-D-manno- pyranosyl-(1^5)-P-D-ara-binofuranoside (19).A solution of 72 (75 mg, 0.05 mmol) was dissolved in methanol (10 mL) and then 3 drops of IM NaOMe was added.
After stirring overnight, the solution was neutralized with a minimum amount of Amberlite
118 H* resin and concentrated to a syrup, which was purified by chromatography (19:1
CHjCLcCHjOH) to obtain 73 ( 6 6 mg, 90%). To a solution of 73 ( 6 6 mg, 0.045 mmol) in
methanol ( 8 mL) was added 10% Pd/C (15 mg). The mixture was stirred overnight under an H, atmosphere and the catalyst was separated by filtration and washed with CHXL (10
mL). After concentrating the filtrate and washings, the product was purified by chromatography (2:1 CH.CL:CH)OH) on latrobeads to obtain 19 (29 mg, 78% from 72) as a foam. R, 0.25 (1:1 CHXL:CH,OH): [al^ +15.4" (c 1.4, H,0); 'H NMR (800 MHz,
D.O) 5h 5.22 (d, 1 H, 7,.,.. = 1.8 Hz, H-1"). 5.06 (d, 1 H, 7, ,. = 1.2 Hz, H -L), 4.98 (d,
1 H, 7,..= 1.8 Hz, H-L"), 4.93 (d, 1 H, 7,, = 4.6 Hz, H-l), 4.03-4.07 (m, 2 H, H-2,
H-2"), 3.99-4.02 (m, 2 H, H-3, H-2'"), 3.93 (ddd, 1 H, J, , = 7.1, 7, ,, = 3.0, 7, ,^ = 6.9
Hz. H-4), 3.87-3.90 (m, 3 H, H-2', H-3', H-3"), 3.81-3.84 (m, 3 H, H- 6 a', H-6 a", H-
6 a'"), 3.79 (dd, 1 H, J.-y = 3.3, Jy ,- = 9.6 Hz, H-3'"), 3.75-3.63 (m. 8 H, H-5a, H-4',
H6 b', H-4", H- 6 b", H-4'". H- 6 b'", octyl OCH,), 3.53-3.61 (m, 4 H, H-5b, H-5', H-5",
H-5'") 3.42 (dt, 1 H, 7 = 6 .6 , 9.5 Hz, octyl OCH,), 1,58-1.64 (m, 2 H, octyl CH,), 1.22-
1.29 (m, 10 H, octyl CH,), 0.86 (t, 3 H, 7 = 6.3 Hz, octyl CHj); ‘^C NMR (125.8 MHz,
D,0) 5c 102.7 (C-L", '7c.h = 169.3 Hz), 101.3 (C -l, '7 c.h = 174.5 Hz), 101.1 (C-l",
'7c.h = 167.8 Hz), 98.7 (C-L, ‘7 c.h = 170,0 Hz), 80.0 (C-4), 79.3 (C-2'), 79.3 (C-2"),
76.5 (C-2), 75.0 (C-3), 73,6 (C-5"), 73.5 (C-5'"), 73.3 (C-5'), 70.8 (C-3'"), 70.6 (C-
3"), 70.5 (C-2'"), 70.4 (C-3'), 69.1 (C-5), 69.0 (octyl OCH,), 67.5 (C-4'"), 67.2 (C-4'),
67.2 (C-4"), 61.6 (C- 6 '"), 61.5 (C- 6 "), 61.2 (C-6 '), 31.5, 29.1, 28.9, 28.8, 25.7, 22.4
(octyl CH ,), 13.8 (octyl CH j). HR-FAB-MS calcd for C^.H^^O,,, [M + Na]" 749.3443,
found 749.3470.
1 4 1 TiO' OH HO
OR
Octyl 6 -O-triphenylmethyl-a-D-mannopyranoside (79). To a solution of octyl a-
D-mannopyranoside 78(4.4 g. 15.2 mmol) in pyridine (50 mL), triphenylmethyl chloride (6.4 g. 22.8 mmol) was added. The solution was stirred overnight at 50 ^C. then cooled and the solvent was evaporated. Traces of pyridine were coevaporated with toluene
(3 .\ 50 mL). The crude brown residue was purified by chromatography (1:1
tolueneiEtOAc) on silica gel to give 79 (6.4 g, 80%) as light yellow solid. R, 0.42 (1:1
toluene:EtOAc): [ o t ] g +16.9° ( c 1.3, C H C I 3 ) ; ‘H NMR (500 MHz, C D 3 O D ) 7.50-7.51
(m. 5 H, Ph), 7.21-7.31 (m. 10 H, Ph), 4.83 (br. s, 1 H. H-l). 3.98 (dt, 1 H, 7= 7.0,
9.4 Hz. octyl O C H , ) . 3.83-3.86 (m, 2 H, H-5. H- 6 a), 3.70 (dd, 1 H, 7,^ = 3.4 Hz, 7,,,,
= 9.4 Hz, H- 6 b), 3.52 (dt, 1 H, 7 = 6.4, 9.5 Hz, octyl O C H , ) , 3.49 (t, 1 H, = 7,^ =
9.7 Hz, H-4), 3.33 (br. s, I H, H-2), 3.28 (dd, I H, 7 , 3 = 1.7 Hz. 7 3 ^ = 9.7 Hz, H-3),
1.68-1.75 (m, 2 H, octyl CH,), 1.45-1.50 (m, 2 H, octyl CH,), 1.27-1.39 (m, 8 H, octyl
CH,), 0.88 (t. 3 H. 7= 6.9 Hz, octyl CH 3); '"C N M R (125.8 MHz, CD 3OD) 5^ 147.8,
144.6, 129.0. 128.3, 127.7, 127.7, 127.0 (Ph), 100.4 (C-l), 86.7 (CPh 3), 72.8, 72.0,
71.2, 68.2, 67.5. 64.4 (ring and benzylic C, octyl OCH,), 32.0, 29.7, 29.6, 29.5, 26.5,
22.8 (octyl CH ,), 13.5 (octyl C H 3). HR-M ALDI-M S calcd for Cj^H^^O^ [M + Na]*
557.2879, found 557.2923.
1 4 2 T rO A
BnO Bm
8 0 OR
R = (CHjjyCHs
Octyl 2,3,4-tri-0-benzyl-6-0-triphenyImethyl-a-D-mannopyranoside (80).
To a solution of 79 (1.26 g, 2.36 mmol) in DMF (10 mL) at 0 °C, NaH (0.28 g, 11.8 mmol) was added with stirring. Benzyl bromide (1.1 mL, 9.2 mmol) was added after allowing the solution to warm to room temperature. The reaction mixture was stirred overnight and then, CH.QH (5 mL) was added, followed by stirring for 3 h. The solvent was evaporated and the residue was dissolved in CH,CL (30 mL). The organic layer was washed with water (2 x 25 mL), dried (Na,SO^), filtered and concentrated to a yellow oil.
The crude oil was purified by chromatography (toluene) on silica gel to give 80 (1.5 g,
79%) as a colorless oil. R, 0.37 (toluene); [a]p +18.7° (c 0.3, CHClj); ‘H NMR (500
MHz, CDCI3) 7.21-7.52 (m, 28 H, Ph), 6.89 (d, 2 H, 7= 7.0 Hz, Ph), 4.91 (br. s, 1
H, H-l), 4.84 (d, 1 H, y = 12.5 Hz, C//,Ph), 4.62-4.64 (m, 4 H, CH.Ph), 4.27 (d, 1 H, y = 10.4 Hz, CH,Ph), 4.00 (t, 1 H, J,, = = 9.6 Hz, H-4), 3.90 (dd, 1 H, y^,, = 2.9
Hz, y ^ ^ = 9 .7 Hz, H- 6 a), 3.81-3.82 (m, 2 H, H-2, H-5), 3.76 (dt, 1 H, y = 7.0, 9.6 Hz, octyl O CH ,), 3.50 (d, 1 H, J,, = 9.6 Hz, H-3), 3.41 (dt, I H, y = 6.4, 9.6 Hz, octyl
OCH,), 3.27 (dd, 1 H, y^.^ = 5.5 Hz, = 9.7 Hz, H- 6 b), 1.52-1.57 (m, 2 H, octyl
CH,), 1.26-1.28 (m, 10 H, octyl CH,), 0.86 (t, 3 H, y = 7.0 Hz, octyl CH,); '"C NMR
(125.8 MHz, CDCI 3) 6 c 144.7, 139.2, 139.2, 138.7, 129.4, 128.8, 128.7, 128.7, 128.2,
128.2, 128.0, 128.0, 127.9, 127.3 (Ph), 98.1 (C-l), 86.7 (CPh^), 80.9, 76.2, 75.7,
1 4 3 75.6, 73.2, 72.8, 72.4, 72.4, 67.9, 63.6 (ring and benzylic C, octyl OCH,), 32.3, 30.0,
29.9. 29.8, 26.7, 23.2 (octyl CH,), 14.6 (octyl CH,). Anal. Calcd for C,,H^O, (793.05):
C, 80.56; H, 7.51. Found: C, 80.34; H, 7.51.
QBn
OR
O ctyl 2 ,3 , 4 - tr i - 0 -b e n 2 yl-a-D-mannopyranoside (75). To a solution of 80 (4.71 g, 5.86 mmol) in CH,CI,:CH,OH (2:1. 60 mL) was added p-toluenesuifonic acid (l.O g,
5.26 mmol) and the mixture was stirred for 45 min. The reaction mixture was then diluted
with CH,C1, (40 mL). washed with a saturated NaHCO, solution (2 x 30 mL) and water
(50 mL). The organic extract was dried (Na,SOj. filtered, and concentrated to a light yellow oil which was purified by chromatography (4:1 toluene:EtOAc) on silica gel to give
75 (2.8 g, 92%) as a colorless oil. Rf 0.51 (1:1 toluene:EtOAc); [a]g +27.6° (c 0.7.
CHCl,); ‘H NMR (500 MHz, CDCl,) 5„ 7.24-7.36 (m, 15 H, Ph), 4.93 (d, I H, 7= 10.9
Hz, C//,Ph), 4.77 (d. 1 H, 7 = 12.4 Hz. C77,Ph), 4.61-4.69 (m, 4 H, H-l, C//,Ph), 3.98
(t. I H, 7,, = 7 , 5 = 9.5 Hz. H-4), 3.92 (dd, I H, 7,^ = 2.8 Hz. 7^^* = 9.4 Hz, H- 6 a),
3.76-3.82 (m, 3 H, H-2, H-3, H-5), 3.65 (dd, I H, 7 , 5^= 4.1 Hz, 7^^^ = 9.4 Hz, H- 6 b),
3.61 (dt, I H, 7 = 6 .8 , 9.5 Hz, octyl OCH,), 3.30 (dt, I H, 7 = 6.5, 9.5 Hz, octyl OCH,),
1.48-1.51 (m, 2 H, octyl CH,), 1.26-1.31 (m, 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 7.0
Hz, octyl CHj); ‘^C NMR (125.8 MHz, CDClj) 5,- 139.1, 139.0, 138.9, 128.9, 128.9,
128.6, 128.3, 128.2, 128.2, 128.1, 128.0 (Ph), 98.7 (C-l), 80.8, 75.7, 75.6, 75.5,
1 4 4 73.4, 72.8, 72.7, 68.2, 62.9 (ring and benzylic C, octyl OCH,), 32.3, 29.9, 29.9, 29.7,
26.6, 23.2 (octyl CH,), 14.6 (octyl CHj). Anal. Calcd for CjjH^^G^ (562.40): C, 74.70;
H, 8.24. Found: C, 74.65; H, 8.33.
Q A c BnO' BnO Bn'
Q Bn
B nO ' Bn'
OR
Octyl 2-0-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l—>6)-2,3,4-tri-
0-benzyl-a-D-mannopyranoside (81). Thioglycoside 52'" (337 mg, 0.63 mmol)
and alcohol 75 (272 mg, 0.49 mmol) were dried under vacuum with powdered 4 A
molecular sieves (300 mg) overnight. Dry CH,C1, (12 mL) was added and the mixture
was cooled to -40 °C and stirred for 10 min. A-iodosuccinimide (176 mg, 0.79 mmol)
was added and the solution was stirred for 20 min before silver triflate (40 mg, 0.16 mmol)
was added. The reaction mixture was allowed to gradually warm to 0 °C and then,
triethylamine (0.5 mL) was added. The yellow solution was filtered, diluted with CH,C1,
(25 mL), washed with a saturated Na,S,Oj solution (2 x 20 mL). followed by brine (20
mL) and water (20 mL). After drying (Na,SOJ, the organic layer was concentrated to a
brown syrup, which was chromatographed (4:1 hexane:EtOAc) on silica gel to give 81
(430 mg, 8 6 %) as a colorless syrup. 0.38 (4:1 hexane:EtOAc); [ a ] o -h40.5° ( c 0.8,
CHCl,); 'H NMR (500 MHz, CDCl,) 0„ 7.12-7.39 (m, 30 H, Ph), 5.50 (d, 1 H, 7,.,.=
2.0 Hz, H-2'), 4.97 (br. s, 1 H, H-l'), 4.91 (d, 1 H, 7= 11.2 Hz, C//,Ph), 4.85 (d, 1 H,
1 4 5 J = 10.9 Hz, Π,Ph), 4.81 (br. s, I H, H-l), 4.72 (s, 2 H, CH.Ph), 4.61-4.66 (m, 4 H,
7 = 11.3 Hz, Π,P h ), 4.49 (d, 2 H, 7 = 11.1 Hz, C77.Ph), 4.44 (t, 1 H, 7^^ = 7,^ = 9.5
Hz, H-4), 4.42-4.46 (m, 2 H, C//,Ph), 3.97 (dd, 1 H, 7,^ = 2.9 Hz, 7^^ = 9.3 Hz, H-
6 a), 3.88-3.91 (m, 4 H, H-2, H-5, H- 6 b, H-4'), 3.80 (dd, 1 H, = 1.7 Hz, 7,, ,,.= 9.7
Hz, H- 6 a'), 3.77-3.78 (m, 1 H, H-5'), 3.69-3.72 (m, 2 H, H-3', H- 6 b'), 3.57-3.68 (m, 2
H, H-3, octyl OCH,), 3.32 (dt, 1 H, 7 = 6.5, 9.8 Hz, octyl OCH,), 2.13 (s, 3 H,
OCOCT/j), 1.48-1.51 (m, 2 H, octyl CH,), 1.26-1.29 (m, 10 H, octyl CH,), 0.87 (t, 3 H,
7= 7.0 Hz, octyl CH,): '^C NMR (125.8 MHz, CDCl,) 8 ^ 170.7 (C=0), 139.2, 139.0,
139.0, 138.8, 138.4, 128.9, 128.8, 128.8, 128.7, 128.7, 128.3, 128.3, 128.2, 128.2,
128.1. 128.1, 128.0, 128.0, 127.9 (Ph), 98.5 (C-l), 98.1 (C-l'), 80.9, 78.3, 75.5, 75.5,
75.4, 75.2, 74.7, 73.8, 73.1, 72.5, 71.9, 71.9, 71.6, 69.3, 69.0, 68.1, 67.2 (ring and
benzylic C, octyl OCH,), 32.3, 29.9, 29.9, 29.7, 26.7, 23.1 (octyl CH,), 21.6
(OCOCHJ, 14.6 (octyl CH.). HR-MALDI-MS calcd for C^H,,0,, [M + Na|"
1059.5234, found 1059.5286.
QH BnO BnO' Bn
OA B nO 'V '-^^ B n O - ^ A
82 OR R = (CHslyCHs
Octyl 3,4,6-tri-0-benzyi-a-D-mannopyranosyI-(l-^6)-2,3,4-tri-(9-benzyI-a-
D-mannopyranoside (82). To a solution of 81 (270 mg, 0.27 mmol) in methanol (20
mL), was added 2 drops of IM NaOMe. After stirring overnight, the solution was
1 4 6 neutralized with a minimum amount of Amberlite 118 H* resin and concentrated to a syrup, which was purified by chromatography (2:1 hexanezEtOAc) on silica gel to give 16 (246 mg, 96%) as a colorless syrup. R^O.32 (2:1 hexane:EtOAc); ‘H NMR (500 MHz, CDClj)
5„ 7.21-7.38 (m, 28 H, Ph), 7.13-7.15 (m, 2 H, Ph), 5.09 (d, 1 H, 7, ,.= 1.1 Hz, H-l'),
4.91 (d. 1 H, 7= 11.0 Hz. CH.Ph), 4.85 (d. 1 H, 7 = 10.9 Hz. CH.Ph), 4.81 (d, 1 H, 7
= 10.9 Hz, CH.Ph), 4.80 (d, 1 H, 7 = 1.5 Hz, H -l), 4.68 (ABq, 2 H, 7 = 12.2 Hz, Au =
30.2 Hz, C//,Ph), 4.44-4.64 (m, 7 H, CH.Ph), 3.16-4.13 (m, 3 H, H-2, H-3, H-4, H-5,
H-6 a, H-6 b, H-2', H-3', H-4', H-5', H- 6 a', H-6 b', octyl OCH,), 3.32 (dt, 1 H, 7= 6 .6 ,
9.7 Hz, octyl OCH,), 2.36 (d, 1 H, 7= 2.7 Hz, OH), 1.48-1.51 (m, 2 H, octyl CH,),
1.26-1.30 (m, 10 H, octyl CH,), 0.87 (t, 3 H, 7= 7.1 Hz, octyl CHj); '^C NMR (125.8
MHz, CDCl,) 5c 139.0, 138.9, 138.8, 138.3. 129.0, 128.8, 128.8, 128.7, 128.4, 128.4,
128.3, 128.2, 128.2, 128.1, 128.0, 128.0 (Ph), 100.1 (C-l), 98.1 (C-l'), 80.8, 80.1,
75.6, 75.6, 75.5, 75.1, 74.7, 73.8, 73.2, 72.6, 72.0, 71.9, 71.5, 69.3, 68.5, 68.1, 66.7
(ring and benzylic C, octyl OCH,), 32.3, 30.0, 30.0, 29.7, 26.7, 23.2 (octyl CH,), 14.6
(octyl CH;).
HO OH
MO'
OH
HO'
Octyl a-D-mannopyranosyl-(l-^ 6 )-a-D-mannopyranoside (20). To a solution of
82 (100 mg, 0.10 mmol) in CHjOH (15 mL), was added 10% Pd/C (30 mg). The
1 4 7 solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CH 3OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (4:1 CHXLiCHjOH) on latrobeads to give 20 (41 mg, 91%) as a foam. R, 0.23 (4:1 CH,C 1,:CH 3 0 H); [ajp +27.3°
(c 0.7, H 3O); ‘H NMR (800 MHz, D,0) 6 » 4.91 (br. s, 1 H, H-l'), 4.74 (br. s, 1 H, H-
1), 3.91-3.93 (m, 1 H, H- 6 a), 3.90 (br. s, 1 H, H-2'), 3.81 (br. s, 1 H, H-2), 3.78 (d, 1
H, 11.9 Hz, H- 6 a'), 3.75 (dd, 1 H, J,.y= 2.0 Hz, Jy, = 9.5 Hz, H-3'), 3.69-3.71
(m, 2 H, H-4, H-6 b'), 3.65 (dd, 1 H, 7 , 3 = 3.0 Hz, J,, = 9.5 Hz, H-3), 3.55-3.60 (m, 5
H, H-5, H-6 b, H-4', H-5', octyl OCH,), 3.39 (dt, 1 H, 7 = 6.4, 9.3 Hz, octyl OCH,),
I.50-1.52 (m, 2 H, octyl CH,), 1.19-1.26 (m, 10 H, octyl CH,), 0.79 (t, 3 H, 7 = 7.2
Hz, octyl CH 3); "C NMR (150.9 MHz, D,0) 5^ 100.5 (C-l, '7^^ = 171.3 Hz), 100.0 (C-
1',‘7c,h= 170.1 Hz), 73.0 (C-5'), 71.6 (C-3), 71.4 (C-5), 71.1 (C-3'), 70.7 (C-2), 70.4
(C-2'), 68.2 (octyl OCH,), 67.0 (C-4), 6 6 . 8 (C-4'), 65.8 (C- 6 ), 61.3 (C-6 '), 32.0, 29.5,
29.5, 29.4, 26.3, 22.8 (octyl CH,), 14.1 (octyl CH 3). HR-FAB-MS calcd for C^^Hj^O,,
[M + H]" 455.2492. found 455.2479.
BnO' BnO' Bn O A ?Gn BnO Bn 83 R = (CHalrCHg
1 4 8 Octyl 2-0-methyI-3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l—>6)-2,3,4-tri-
0-benzyl-a-D-mannopyranoside (83). To a solution of 82 (126 mg, 0.14 mmol) in
DMF (5 mL), NaH (33 mg, 11.8 mmol) was added and the mixture was stirred for 15 min.
Methyl iodide (80 nL, 1.4 mmol) was added and the reaction mixture was stirred overnight
and then, CH 3OH (1.0 mL) was added, followed by stirring for I h. The reaction mixture
was concentrated and the residue was dissolved in CH,CU ( 2 0 mL), and then washed with
water (2 x 1 5 mL), dried (Na^SO^), filtered and concentrated to a light yellow syrup. The crude syrup was purified by chromatography (4:1 hexane:EtOAc) on silica gel to give 83
(120 mg, 87%) as a colorless syrup. R, 0.41 (4:1 hexane:EtOAc); [a]p +47.7° (c 1.0,
CHCI3); ‘H NMR (400 MHz, CDCI3) 7.11-7.37 (m, 30 H, Ph), 5.14 (d, I H,
1.4 Hz, H-L), 4.86 (d, 2 H, 7 = 10.9 Hz, C7/,Ph), 4.80 (d, 1 H. 7,,= 1.4 Hz, H-l),
4.73 (ABq, 2 H. 7= 12.3 Hz. Au = 28.7 Hz, CH.Ph), 4.55-4.68 (m, 6 H. C//,Ph), 4.46
(dd. 1 H, 7,.v= 1.4 Hz, 7,.3.= 2.0 Hz. H-2'), 4.43-4.44 (m, 2 H, C7/,Ph), 3.59-3.94 (m,
12 H, H-2, H-3, H-4, H-5, H-6 a, H-6 b, H-3'. H-4'. H-5'. H- 6 a'. H-6 b'. octyl OCH,),
3.42 (s, 3 H, OCH 3), 3.32 (dt, 1 H, 7 = 6.5. 9.6 Hz, octyl OCH,). 1.48-1.49 (m, 2 H,
octyl CH,), 1.26-1.29 (m. 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 7.0 Hz. octyl CH 3); ‘^C
NMR (125.8 MHz. CDCI 3) 6 c 139.2, 139.0. 139.0, 138.9, 138.9, 138.7. 128.8, 128.8,
128.7, 128.6, 128.5, 128.3. 128.3. 128.2, 128.1, 128.0, 128.0, 127.8 (Ph), 98.3 (C-l),
98.0 (C-L), 80.9. 79.6, 75.7, 75.6, 75.5, 75.2, 75.0, 73.8, 73.4, 72.7, 72.2, 72.1,
72.0, 69.6, 68.0, 66.5 (ring and benzylic C, octyl OCH,), 59.3 (OCH 3), 32.3, 29.9,
29.9, 29.7, 29.6, 23.1 (octyl CH ,), 14.6 (octyl C H 3). HR-MALDI-MS calcd for
C,3H,,0 ,| [M + N ar 1031.5285, found 1031.5265.
1 4 9 HcrA 9CH3
H o V ^ \
6A' - OHY" HO'
R = (CHzjyCHa
Octyl 2-0-methyi-a-D-mannopyranosyi-(1^6)-a-D-mannopyranoside (21).
To a solution of 83 (90 mg, 0.09 mmol) in CH 3OH (12 mL), was added 10% Pd/C (23 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CH 3OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (4:1 CH^CLcCHjOH) on latrobeads to give 21 (36 mg, 87%) as a foam. R, 0.57 (4:1 CH.ClyCHjOH); [aj^ +123°
(c 1.1, H.O); 'H NMR (800 MHz, D ,0) 8 ^ 4.90 (br. s, 1 H, H -L ), 4.70 (br. s, I H, H-
1), 3.84 (dd, 1 H, 7;^ = 3.8 Hz, 7,,,, = 10.8 Hz, H- 6 b), 3.78 (br. s, 1 H, H-2), 3.74
(dd, I H, 7;.^ = 2.5 Hz, 7 ,,.^ = 10.2 Hz, H-6 a'), 3.71-3.72 (m. I H, H-3'), 3.59-3.63
(m, 5 H, H-3, H-4, H-5, H-6 a, H-6 b'), 3.57 (dt, I H, 7 = 6.9, 9.5 Hz, octyl OCH,), 3.52
(ddd, 1 H, 7;.= 2.5 Hz, 7;.,^= 4.1 Hz, 7,.^ = 9.6 Hz, H-5'), 3.47 (d, I H, 7,- 3.= 2.4
Hz, H-2'), 3.44 (t, I H, 9.6 Hz, H-4'), 3.35-3.37 (m, 1 H, octyl OCH,),
3.34 (s, 3 H, OCH 3), 1.46-1.48 (m, 2 H, octyl CH,), 1.16-1.23 (m, 10 H, octyl CH,),
0.75 (t, 3 H, 7 = 7.1 Hz, octyl CH 3); '"C NMR (150.9 MHz, D,0) 0^ 100.4 (C-l, =
170.0 Hz), 96.8 (C-1','7c.h= 169.1 Hz), 80.5 (C-2'), 73.0 (C-5'), 71.5 (C-5), 71.4 (C-
3), 70.8 (C-3'), 70.6 (C-2). 68.2 (octyl OCH,), 67.5 (C-4'), 66.9 (C-4), 66.1 (C- 6 ), 61.4
(C-6 '), 59.3 (OCH 3), 31.9, 29.3, 29.3, 29.3, 26.2, 22.7 (octyl CH,), 14.1 (octyl CH 3).
1 5 0 HR-FAB-MS calcd for C„H,oO,, [M + H]' 469.2648, found 469.2672.
BnO" B nO Bn(
GBn
BnO '
Octyl 3,4,6-tri-0-benzyl-2-0-{methylthio)thiocarbonyi-a-D-mannopyrano- syl-(l—>6)-2,3,4-tri-0-benzyI-a-D-mannopyranoside (84). To a solution of 82
(300 mg. 0.30 mmol) in THE (20 mL), NaH (44 mg, 1.83 mmol) was added along with a catalytic amount of imidazole (10 mg). The mixture was stirred for 1 h, before carbon disulfide (230 |iL, 3.9 mmol) was added and the stirring continued for an additional hour.
Then methyl iodide (132 pT, 2.12 mmol) was added and the reaction mixture was stirred for 12 h. The solvent was evaporated and the residue was dissolved in CH,CL (25 mL), washed with water (20 mL), dried (Na^SOj, filtered, and concentrated to a yellow syrup.
The crude syrup was purified by chromatography (4:1 hexanezEtOAc) on silica gel to give
84 (324 mg, 99%) as a light yellow syrup. 0.46 (4:1 hexanezEtOAc); [aj^ +26.7° (c
1.7, CHClj); ‘H NMR (400 MHz. CDCI3) 5„ 7.16-7.38 (m. 30 H, Ph), 6.26 (t, 1 H, y, ,.
= J.jy= 2.0 Hz, H-2'), 5.11 (d. 1 H, 7,..= 2.0 Hz, H-l'), 4.91 (d, 1 H, J= 11.1 Hz,
CH.Ph), 4.87 (d, 1 H, 7 = 10.9 Hz, CH.Ph), 4.80 (d, 1 H, 7,,= 1.5 Hz, H-l), 4.61-
4.71 (m. 6 H. C//,Ph), 4.42-4.52 (m, 4 H, CH.Ph), 4.09 (dd, 1 H, = 3.0 Hz, 7^.,^
= 9.3 Hz, H- 6 b'), 3.98 (t, 1 H, 9.6 Hz, H-4'), 3.86-3.91 (m, 3 H, H-5, H-
151 6 b, H- 6 a'), 3.83 (t, 1 H, = = 1.5 Hz, H-2), 3.73-3.77 (m, 4 H, H-3, H-4, H-3%
H-5'), 3.65 (dd, I H, = 1.4 Hz, 7,^,, = 9.2 Hz, H- 6 a), 3.60 (dt, 1 H, 7= 6 .8 , 9.7
Hz, octyl OCH,), 3.31 (dt, 1 H, 7 = 6.5, 9.7 Hz, octyl OCHJ, 2.48 (s, 3 H, SCH,),
1.47-1.50 (m, 2 H, octyl CH,), 1.26-1.29 (m, 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 6.4
Hz, octyl CH.); ‘^C NMR (125.8 MHz, CDCl,) 5^ 215.5 (C=S), 139.0, 139.0, 138.8,
128.8, 128.8, 128.7, 128.6, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0
(Ph), 98.1 (C-1), 97.3 (C-1'), 80.9. 78.3, 77.2, 75.6, 75.5, 75.4, 75.2, 75.0, 73.9,
73.1. 72.5, 72.1, 72.0, 71.5, 69.3, 68.2. 67.1 (ring and benzylic C, octyl OCH,), 32.3,
29.9. 29.9. 29.8. 26.7. 23.2 (octyl CH,). 19.1 (SCH,), 14.6 (octyl CH,). HR-MALDI-
MS calcd for C„H,,0,,S,[M + Na|" 1107.4727. found 1107.4630.
B n0‘ BnO ' Bm 6A BnO' Bn 85 OR
R = (CH2 )tCH3
Octyl 3,4,6-tri-(?-benzyi-2-deoxy-a-D-ara^>i/io-hexopyranosyI-(l->6)-2,3,4- tri-O-benzyl-a-D-mannopyranoside (85). To a solution of 84 (296 mg, 0.27 mmol) in dry toluene (50 mL), was added tributylstannane (1.1 mL, 4.05 mmol) and AIBN (36 mg, 0.22 mmol). The reaction mixture was heated at reflux for 2 h and the solvent was evaporated. The crude syrup was purified chromatography (4:1 HexaneiEtOAc) on latrobeads to give 85 (160 mg, 61%). Rf 0.31 (4:1 hexane:EtOAc); [a]p +52.2° (c 0.8,
CHClj); ‘H NMR (500 MHz, CDClj) 6 ^ 7.14-7.37 (m, 30 H, Ph), 5.12 (d, 1 H,
152 3.4 Hz, H-1'), 4.93 (d, 1 H, 7= 11.2 Hz, CH.Ph), 4.88 (d, I H, 7= II.O Hz, C//,Ph),
4.81 (d, 1 H, 7,^= 1.2 Hz, H-1), 4.71 (ABq, 2 H, 7= 12.4 Hz, Au = 31.9 Hz, C//,Ph),
4.53-4.68 (m, 6 H, C//,Ph), 4.48 (d, 1 H, 7 = 11.0 Hz, CH.Ph), 4.43 (d, 1 H, 7 = 12.1
Hz, C//,Ph), 3.90-4.00 (m, 3 H, H-3, H-4. H- 6 b), 3.87 (dd, 1 H, 7,,^ = 4.6 Hz, 7^^^ =
11.3 Hz, H-6 b), 3.76-3.78 (m, 1 H, H-2), 3.74-3.75 (m, 1 H, H-5), 3.70 (dd, 1 H, 7,.^.
= 4.4 Hz, 7^,.6 b = 10.4 Hz, H - 6 b'), 3.57-3.67 (m, 4 H, H-3', H-4', H-5', octyl OCHO,
3.55 (dd. 1 H. 7,-6, = 1-6 Hz, 10.4 Hz, H-6 a'), 3.32 (dt, 1 H, 7= 6.5, 9.7 Hz, octyl OCH,), 2.37 (dd, 1 H. 7,,,^ = 4.9 Hz, 7,,,= 10.4 Hz, H-2'eq), 1.68 (td, 1 H,
7v„ 3 = 7v,„, = 12.7 Hz, 7y,m = 3.4 Hz, H-2'ax), 1.49-1.51 (m, 2 H, octyl CH,), 1.26-
1.30 (m, 10 H, octyl CH,), 0.87 (t, 3 H. 7= 7.2 Hz, octyl CH^); '^C NMR (125.8 MHz,
CDClj) 8 c 139.2. 139.1, 139.0, 138.6, 128.8. 128.8, 128.7, 128.4, 128.2, 128.2,
128.2, 128.1, 128.1, 128.0, 128.0. 127.9, 127.9 (Ph). 98.2 (C- 1 ), 98.1 (C-1'). 80.9,
78.7. 75.6, 75.5, 75.4, 75.3, 75.3, 73.9, 73.1, 72.6, 72.0, 72.0, 71.3, 69.3, 68.1, 66.4
(ring and benzylic C. octyl OCH,), 35.7 (C-2'), 32.3. 29.9. 29.9. 29.7. 26.6, 23.1 (octyl
CH,), 14.6 (octyl CH,). HR-MALDI-MS calcd for C^^H^PiJM + Na]" 1001.5180, found 1001.5177.
HO'
HO'
OH
HO
OR
Octyl 2-deoxy-a-D-arabino-hexopyranosyI-(l-»6)-a-D-mannopyranoside
(22). To a solution of 85 (100 mg, 0.10 mmol) in CH3OH ( 8 mL), was added 10% Pd/C 1 5 3 (20 mg). The solution was stirred overnight under an atmosphere and then the catalyst was separated by filtration and washed with CH 3 OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (4:1
CHXUrCHjOH) on latrobeads to give 22 (40 mg, 90%) as a foam. 0.53 (4:1
CH,CL:CH 3 0 H); [a]g +44.0° (c 1.0. H,0); 'H NMR (800 MHz, D,0) 5» 4.88 (d, 1 H,
3.2 Hz, H-1'). 4.70 (br. s, 1 H, H-1), 3.79-3.82 (m, 2 H, H- 6 b, H-3'), 3.77 (br. s. 1 H, H-2), 3.71 (d, 1 H.y,^.^.= 12.3 Hz, H- 6 a'), 3.64 (dd, 1 H, 7;.^ = 4.9 Hz, 7^.^
= 12.3 Hz, H-6 b'). 3.59-3.63 (m. 3 H, H-3, H-4, H-5), 3.55-3.57 (m, 2 H, H-5', octyl
OCH 3 ), 3.51 (dd, 1 H. 7 5 ,,= 1.8 Hz, 7 ^ ^ = 10.7 Hz, H- 6 a), 3.34-3.36 (m. 1 H, octyl
OCH,). 3.25 (t, 1 H, 7 3 .,.= 7 , 5 .= 9.5 Hz. H-4'). 2.03 (dd. 1 H, 7,.^ 3 .= 5.1 Hz, 7,„, v„ =
12.7 Hz. H-2'eq). 1.59 (td. 1 H. 7,,, 3 . = 7,,, ,„ = 12.7 Hz. 7,,^ , = 3.2 Hz. H-2'ax), 1.45-
1.48 (m. 2 H. octyl CH,). 1.15-1.22 (m. 10 H. octyl CH,), 0.74 (t. 3 H, 7 = 7.2 Hz, octyl CH,); ’’C NMR (150.9 MHz. D ,0) 6 ^ 100.5 (C-1. '7^^ = 168.9 Hz), 97.4 (C-
r.'7c,H= 169.5 Hz). 72.6 (C-5'), 71.6 (C-4'), 71.4 (C-5), 71.3 (C-3), 70.7 (C-2), 68.7
(C-3'), 68.2 (octyl OCH,). 66.9 (C-4). 65.6 (C- 6 ), 61.1 (C-6 '). 37.2 (C-2'), 32.0, 29.5,
29.4. 29.3, 26.2. 22.8 (octyl CH,). 14.1 (octyl CH 3 ). HR-FAB-MS calcd for C,qH 3 ^0 , 0
[M + H r 439.2543. found 439.2526.
A c c Q O \ ^ AcO ' I
BnO' B n
9 7 O R R = (C H 2)7CH 3
154 Octyl 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-D-mannopyranosyl-(l-^6)-
2,3,4-tri-O-benzyl-a-D-mannopyranoside (97). Trichloroacetimidate 76’“ (216 mg, 0.48 mmol) and alcohol 75 (223 mg, 0.40 mmol) were dried in vacuo with powdered
4 Â molecular sieves (300 mg) overnight. Dry CHXl, ( 8 mL) was added and the mixture was cooled to -10 °C with stirring. A solution of TMSOTf (20 |xL) in CH,CL (125 pL) was added dropwise to the reaction mixture and the stirring was continued for 2 h. The solution was neutralized by the addition of a saturated NaHCO^ solution (3 mL) and then,
CH,CU (30 mL) was added. The organic layer was washed with water (20 mL), dried
(Na,SOJ, filtered, and concentrated to a colorless syrup. The crude syrup was purified by chromatography (4:1 hexane:EtOAc) on silica gel to give 97 (302 mg, 89%) as a colorless syrup. R, 0.44 (4:1 hexane:EtOAc); [a]o +58.6° (c 1.3, CHCI 3 ); 'H NMR (500 MHz,
CDCl,) 6 h 7.25-7.36 (m, 15 H, Ph), 5.31 (t, 1 H, Jy , = J,-y=9.9 Hz, H-4'), 5.27 (ddd,
1 H. y, y = 2.0 Hz, y „ 3 p= 29.5 Hz, Jy , = 9.9 Hz, H-3'), 5.22 (dd, 1 H, 7,. ,.= 2.0 Hz.
7.5 Hz, H-1'), 4.98 (d, 1 H, 7 = 11.2 Hz, C//,Ph), 4.76 (dt, 1 H, 7,.,. = 7 ,-3 . = 2.0
Hz, 7 „ 3 p= 49.7 Hz, H-2'), 4.75-4.79 (m, 2 H. H-l, CH.Ph), 4.60-4.66 (m, 4 H,
C//,Ph). 4.19 (dd, 1 H, 7;.^ = 4.6 Hz, 7&, = 12 3 Hz, H- 6 b'), 4.11 (dd, 1 H, Jy,y =
2.1 Hz. 7^.,b = 12.3 Hz, H- 6 a'), 4.01-4.03 (m, 1 H, H-5'), 3.90-3.92 (m, 3 H, H-2, H-
4, H-5), 3.88 (dd, 1 H, 7,66=4.7 Hz, = 11.4 Hz, H- 6 b), 3.79 (dd, 1 H, 7,6,= 1.0
Hz, 76,6b = H.4 Hz, H- 6 a), 3.69-3.71 (m, 1 H, H-3), 3.60 (dt, 1 H, 7 = 6.7, 9.7 Hz, octyl OCH 3 ), 3.33 (dt, 1 H, 7 = 6.5, 9.7 Hz, octyl OCH,), 2.05 (s, 3 H, OCOC//,), 2.03
(s, 3 H, O CO C//3 ), 2.02 (s, 3 H, O COC//3 ), 1.50-1.53 (m, 2 H, octyl CH ,), 1.27-1,32
(m, 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 7.0 Hz, octyl CH,); '^C NMR (125.8 MHz,
CDCI3 ) 8 c 171.2, 170.2, 170.0 (C=0), 138.9, 138.8, 138.8, 128.8, 128.8, 128.8, 128.3,
128.1, 128.1, 128.1, 128.0 (Ph), 98.2 (C-1), 97.8 (C-1', 7c.i-,f= 29.0 Hz), 87.4 (C-2',
7c.,-j:= 179.5 Hz), 80.8, 75.5, 74.8, 73.3, 72.6, 72.2, 70.3 (ring and benzylic C), 70.1
(C-3', 7 c.3 p= 16.4 Hz), 68.9, 68.2, 67.2, 66.4, 62.5 (ring and octyl OCH,), 32.3, 29.8,
155 29.8, 29.7, 26.6, 23.1 (octyl CH.), 21.2, 21.1, 21.1 (OCOCH,), 14.5 (octyl CH,). ‘"F
NMR (235.4 MHz, CDCI3) 6 ^ - 203.2 (ddd, 1 F, Jh,-.f= 7.5 Hz, V f = 49.7 Hz, V p =
29.5 Hz, F-2'). HR-MALDI-MS calcd for C^.H^O.^F [M + Na]" 875.3994, found
875.4004.
HO" HO'
QBn
BnO'
OR
Octyl 2-deoxy-2-fluoro-a-D-mannopyranosyI-(l->6)-2,3,4-tri-0-benzyl-a-
D-mannopyranoside (98). To a solution of 97 (245 mg, 0.29 mmol) in CH 3 OH (15 mL), 5 drops of IM NaOMe was added. After stirring overnight, the solution was neutralized with a minimum amount of pre-washed Amberlite 118 H* resin and concentrated to a syrup, which was purified by chromatography (9:1 CH.Cl.iCHjOH) on latrobeads to give 98 (180 mg, 8 6 %) as a colorless syrup. R, 0.42 (9:1 CH.Cl.iCH^OH);
[aJo +56.3° (c 0.4, CHCI 3 ); 'H NMR (500 MHz, CDCI3 ) 0^ 7.24-7.25 (m, 15 H, Ph),
5.10 (dd, 1 H, y,.,.= 0.8 Hz, 7^, ^= 7.4 Hz, H-1'), 4.94 (d, 1 H, 7= 11.2 Hz, CH.Ph),
4.76 (d, 1 H, 7 , 3 = 1.2 Hz, H-1), 4.71 (d, 1 H, 7 = 12.3 Hz, CH.Ph), 4.65 (d, 1 H, 7 =
12.2 Hz, CH.Ph), 4.60-4.64 (m, 3 H, CH.Ph), 3.62-3.94 (m, 12 H, H-2, H-3, H-4, H-
5, H-6 a, H-6 b, H-2', H-3', H-4', H-5', H- 6 a', H-6 b'), 3.56 (dt, 1 H, 7= 6.5, 9.6 Hz, octyl OCH,), 3.30 (dt, 1 H, 7 = 6.5, 9.6 Hz, octyl OCH,), 1.47-1.49 (m, 2 H, octyl
CH,), 1.26-1.31 (m, 10 H, octyl CH,), 0.88 (t, 3 H, 7= 7.0 Hz, octyl CH 3 ); ‘^C NMR
156 (125.8 MHz, CDCI 3 ) Ôc 138.8, 138.8, 138.6, 128.9, 128.8, 128.3, 128.2, 128.1, 128.1
(Ph), 98.4 (C-l',yc-r.F= 29.4 Hz), 98.0 (C-1), 90.0 (C-2', 7^.^^= 173.9 Hz), 80.5, 75.5,
74.8, 73.1, 72.6. 72.5, 72.5, 71.9 (ring and benzylic C, octyl OCHO, 71.1 (C-3', Jc.y.F =
17.7 Hz), 68.2, 67.9, 67.1. 62.1 (ring and octyl OCH,), 32.3, 29.8, 29.8, 29.7, 26.6,
23.1 (octyl CH,), 14.6 (octyl CH 3 ). '"F NMR (235.4 MHz, CDCI3 ) 5^ - 206.0 (ddd, 1 F. y», F = 7.4 Hz. p = 49.5 Hz, y„ 3 .p = 33.0 Hz. F-2'). HR-MALDI-MS calcd for
C^H^jOi^F [M + Na]" 749.3677. found 749.3646.
HO
MO
OH
HO'
Octyl 2-deoxy-2-flucro-a-D-mannopyranosyi-(I^6)-a-D-inannopyranoside
(23). To a solution of 98 (120 mg. 0.17 mmol) in CH3OH (12 mL), was added 10%
Pd/C (40 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CH,OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (4:1
CH,C 1,:CH 3 0 H) on latrobeads to give 23 (75 mg, 97%) as a foam. R^ 0.60 (4:1
CH,C 1 ,:CH 3 0 H); [alp +187= (c 0.7, H,0); ‘H NMR (800 MHz, 0,0) 5^ 4.98 (d, 1 H, y„,..p= 7.2 Hz, H-1'), 4.72 (d, 1 H. 7 3 3 3 .^ = 4 8 . 7 Hz, H-2'), 4.73 (br. s. 1 H. H-1), 3.96-
3.98 (m, 1 H, H- 6 b), 3.85 (dd, 1 H, Jy,-= 8.1 Hz, 29.7 Hz, H -3'), 3.82 (br. s, 1
H, H-2), 3.79 (d, 1 H, 7,^.^ = 12.0 Hz, H- 6 a'), 3.71 (dd, 1 H, 7;.^= 4.0 Hz,
157 12.0 Hz, H-6 b'), 3.70 (t, I H, Jj, = J,, = 9.6 Hz, H-4), 3.64-3.66 (m, 3 H, H-3, H-4',
H-5'), 3.55-3.61 (m, 3 H, H-5, H-6 a, octyl OCH,), 3.36 (dt, 1 H, 7= 6.5, 9.4 Hz, octyl
OCH,), 1.49-1.51 (m, 2 H, octyl CH,), 1.20-1.26 (m, 10 H, octyl CH,), 0.80 (t, 3 H, 7
= 7.1 Hz, octyl CH,): ‘^C NMR (150.9 MHz, D,0) 5^ 100.6 (C-1, '7^^= 169.6 Hz), 97.3
(C-r,'7c.H= 169.5 Hz, 7c.,.f= 29.7 Hz ), 90.0 (C-2', 7^., ^ = 172.6 Hz), 73.1 (C-5'),
71.6 (C-4'), 71.4 (C-5), 70.7 (C-2). 70.2 (C-3', 17.2 Hz), 68.2 (octyl OCH,),
67.2 (C-3), 66.7 (C-4), 66.2 (C-6 ), 61.0 (C-6 '), 32.2, 29.8, 29.7, 29.6, 29.4, 22.8 (octyl
CH,). 14.2 (octyl CH,). 'T NMR (235.4 MHz, D,0) 5p - 204.7 (ddd, 1 F, 7^, ^= 7.2 Hz,
7„,.p= 48.7 Hz, 7„,.p= 29.7 Hz. F-2'). HR-FAB-MS calcd for for C,„H,,0,oF [M +
Nal'479.2268. found 479.2234.
AcO' lAcO' Ac cT \ 9®" BnO' Bn
105 OR R = (CH2 )7 CH3
Octyl 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-mannopyranosyl-(1^6)-
2,3,4-tri-O-benzyI-a-D-mannopyranoside (105). Trichloroacetimidate 77'“* (335 mg, 0.71 mmol) and alcohol 75 (335 mg, 0.60 mmol) were dried in vacuo with powdered
4 Â molecular sieves (300 mg) overnight. Dry CH,C1, ( 8 mL) was added and the mixture was cooled to -10 °C with stirring. A solution of TMSOTf (35 pL) in CH,C1, (125 |iL) was added dropwise to the reacdon mixture and the stirring was condnued for 2 h. The
soludon was neutralized by the addidon of a saturated NaHCO, soludon (0.5 mL) and
158 CHXU (40 mL) was added. The organic layer was washed with water (20 mL), dried
(Na^SOj, filtered and concentrated to a colorless syrup. The crude syrup was purified by chromatography (3:1 hexaneiEtOAc) on silica gel to give 105 (365 mg, 70%) as a colorless syrup. R., 0.43 (3:1 hexane:EtOAc); [a]g +42.1° (c 1.2, CHC1-); 'H NMR (500
MHz, CDClj) 5h 7.31-7.41 (m. 15 H, Ph), 5.43 (dd, 1 H. J,.y= 3.8 Hz, Jy ,-= 9.8 Hz,
H-3'), 5.34-5.35 (m, 1 H, H-5'), 5.33 (t, 1 H, 7,.,. = = 9.8 Hz, H-4'), 5.10 (d, 1 H, y, 1.7 Hz, H-l'), 5.05 (d, 1 H, 7 = 11.2 Hz. CH.Ph), 4.81 (d, I H, 7 = 12.2 Hz,
C//,Ph), 4.80 (d, 1 H, 7,,= 1.7 Hz. H-l), 4.73 (d, 1 H, 7= 12.4 Hz, C//,Ph), 4.68-
4.71 (m. 3 H. C//,Ph), 4.21 (dd, 1 H. 7;.^= 4.7 Hz, 7,,. ^ = 12.3 Hz, H-6 b'), 4.14 (dd,
1 H ,7,.^ = 2.3 Hz, 7^.^.= 12.3 Hz, H-6 a'), 4.10 (dd, 1 H. 7, ,= 1.7 Hz, 7,.,.= 3.8 Hz,
H-2'), 3.96-4.03 (m, 2 H. H-2, H-4), 3.92 (dd, 1 H, 7,^ = 4.7 Hz. 7^., = 11.6 Hz, H-
6 b), 3.82-3.83 (m, 1 H, H-3), 3.81 (dd, 1 H, 7 ,,,= 1.5 Hz, 7 ^ ^ = 11.6 Hz, H- 6 a),
3.75-3.78 (m, 1 H, H-5), 3.66 (dt, 1 H. 7 = 6 .8 , 9.6 Hz, octyl OCH,), 3.39 (dt, 1 H, 7 =
6.5, 9.7 Hz. octyl OCH,), 2.11 (s, 3 H. OCOCH^), 2.10 (s, 3 H, OCOCH,), 2.07 (s, 3
H, OCOCH,). 1.54-1.58 (m, 2 H, octyl CH,), 1.29-1.37 (m. 10 H, octyl CH,), 0.94 (t, 3
H, 7 = 7.1 Hz, octyl CH.); '-C NMR (125.8 MHz, CDCI 3 ) 6 ^ 171.2, 170.1, 170.0
(C =0). 138.9, 138.8. 138.8. 128.8, 128.8, 128.3, 128.1. 128.1, 128.0 (Ph), 98.6 (C-1),
98.3 (C-1'), 80.8, 75.5, 75.4, 74.8, 73.3, 73.3, 72.6, 72.2, 71.2, 68.9, 67.2, 66.5,
62.5, 62.0 (ring and benzylic C, octyl OCH,), 32.2, 29.8, 29.8, 29.7, 26.6, 23.1 (octyl
CH,), 21.2, 21.1, 20.9 (OCOCH3), 14.5 (octyl CH3). HR-ESI-MS calcd for for
C,,H„ 0 „N 3 [M + Nal*898.4097, found 898.4090.
159 HO' HO
OBn
BnO Bni
106 OR R — ^OHg/yOHg
Octyl 2-azido-2-deoxy-a-D-mannopyranosyI-(l-^6)-2,3,4-tri-0-benzy!-a-D- mannopyranoside (106). To a solution of 105 (380 mg, 0.43 mmol) in CH,OH (8 mL), 4 drops of IM NaOMe was added. After stirring overnight, the solution was neutralized with a minimum amount of pre-washed .Amberlite 118 H'" resin and concentrated to a syrup, which was purified by chromatography (1:1 he,\ane:EtOAc) on silica gel to give 106 (310 mg, 97%) as a colorless syrup. R, 0.54 (1:2 hexane:EtOAc);
[0 )0 + 5 9 . 1 ° (c 0.7, CHCI3 ); 'H NMR (500 MHz, CDCI3 ) 7.13-7.29 (m, 15 H, Ph),
4.90 (d, I H ,y , . 3 = 0 . 6 Hz, H-L), 4.87 (d, IH, 7=11.1 Hz, C//,Ph), 4.70 (d, I H, 7,,
= 1.6 Hz, H-l), 4.62 (ABq, 2 H, 7= 12.3 Hz, Au = 30.4 Hz, C7/,Ph), 4.52-4.53 (m, 2
H, C//,Ph), 4.51 (d, 1 H, 7= 11.4 Hz, C//,Ph), 3.48-4.03 (m. 12 H, H-2, H-3, H-4,
H-5, H-6 a, H-6 b, H-2', H-3', H-4', H-5', H- 6 a', H-6 b'), 3.41 (dt, 1 H, 7= 6.5, 9.7 Hz, octyl OCH 3 ), 3.23 (dt, 1 H, 7 = 6.5, 9.7 Hz, octyl OCH,), 1.40-1.42 (m, 2 H, octyl
CH,), 1.15-1.25 (m, 10 H, octyl CH,), 0.79 (t, 3 H, 7 = 7.1 Hz, octyl CH 3 ); '"C NMR
(125.8 MHz, CDCI3) 6 c 138.9, 138.8, 138.6, 128.9, 128.9, 128.8. 128.5, 128.3, 128.3,
128.2, 128.1 (Ph), 99.3 (C-L), 98.2 (C-1), 80.6, 75.6, 75.2, 74.9, 73.2, 72.7, 72.6,
72.1, 71.7, 68.3, 68.1, 67.2, 64.2, 62.3 (ring and benzylic C, octyl OCH,), 32.3, 29.9,
29.8, 29.7, 26.6, 23.1 (octyl CH,), 14.6 (octyl CH 3 ). HR-ESI-MS calcd for C „H ; 3 0 ,oN 3
[M + Na]" 772.3780, found 772.3788.
160 HO MHz
MO'
OH
HO'
Octyl 2-amino-2-deoxy-a-D-mannopyranosyi-(l-^6)-a-D-mannopyranoside
(24). To a solution of 106 (70 mg. 0.09 mmol) In HOAc (5 mL), was added 10% Pd/C
(25 mg). The solution was stirred overnight under an atmosphere and then the catalyst was separated by filtration and washed with CH-OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography ( 1 0 :2 :0 . 5
CHC1):CH)0H:(5N) aq.NH^OH) on latrobeads to give 23 (32 mg, 76%) as a colorless foam. R, 0.20 (10:4:1 CHC1):CH^0H:(5N) aq.NH.OH); [aj^ +35,5= (c 1.5, H,0); 'H
NMR (800 MHz, D,0) 5^ 4.77 (br. s, I H, H-L), 4.70 (d, 1 H, 7,^= 1.5 Hz, H-l), 3.88
(dd, 1 H, 7;* = 4.0 Hz, 7*,*= 10.9 Hz, H- 6 b), 3.84 (dd, 1 H, 7 .y = 4.3 Hz, Jy,.= 9.6
Hz, H-3'), 3.80 (dd, 1 H, 7, ,= 1.5 Hz, 7,, = 3.2 Hz, H-2), 3.74 (dd, 1 H, 7y*= 1.7
Hz, 7 * .* .= 12.0 Hz, H-6 a'), 3.69 (dd, 1 H, 7 ,.*.= 4.9 Hz, 7 * * . = 12.0 Hz, H-6 b'), 3.67
(t, 1 H, 7 j 4 = 7j^= 9.6 Hz, H-4), 3.63 (dd, 1 H, 7,j= 3.2 Hz, 7y^ = 9.6 Hz, H-3), 3.56-
3.61 (m, 4 H, H-5, H -5', H-6 a, octyl OCH,), 3.55 (t, 1 H, 7y^ = 7^y = 9.6 Hz, H-4'),
3.38 (dt, 1 H, 7= 6.4, 9.7 Hz, octyl OCH,), 3.17 (d, 1 H, 7y y= 4.3 Hz, H-2'), 1.47-
1.51 (m, 2 H, octyl CH,), 1.17-1.24 (m, 10 H, octyl CH,), 0.76 (t, 3 H, 7 = 6 . 8 Hz, octyl CH 3 ); ‘^C NMR (150.9 MHz, D,0) 5^ 100.0 (C-1, '7^^^= 169.7 Hz), 99.7 (C-L,
'7c.h= 168.4 Hz), 72.5, 71.1, 71.0 (C-3, C-5, C-5'), 70.2 (C-2), 70.0 (C-3'), 67.9 (octyl
161 OCH,), 66.4 (C-4), 66.1 (C-4'), 65.6 (C-6 ), 60.6 (C-6 '), 53.6 (C-2'), 31.5, 28.9, 28.9,
28.8, 25.7, 22.3 (octyl CH,), 13.6 (octyl CHj). HR-FAB-MS calcd for C,oH 3 , 0 ,oN [M + n r 454.2652, found 454.2667.
HO'
OBn
B n O Bn'
Octyl 2-azido-2,6-dideoxy-6-fIuoro-a-D-mannopyranosyl-(l->6)-2,3,4-tri-
O-benzyl-a-D-mannopyranoside (113). To a solution of 106 (150 mg, 0.20 mmol) in dry CH,C1, (3 mL) at -40 °C, DAST ( 140 pL, 1.1 mmol) was added. .After stirring for
2 h, methanol (1 mL) was added and the solution was concentrated. The residue was purified by chromatography (3:1 hexane:EtOAc) on silica gel to give 113 (110 mg, 72%) as a colorless syrup. R^ 0.65 (1:1 hexane:EtOAc); [a][, +43. T (c 0.5, CHCI 3 ); ‘H NMR
(500 MHz, CDCI3 ) 5„ 7.16-7.31 (m, 15 H, Ph), 4.94 (br. s, 1 H, H-l'), 4.88 (d, 1 H, 7 =
11.1 Hz, CH,Ph), 4.73 (br. s, 1 H, H-l), 4.64 (ABq, 2 H. 7= 12.1 Hz, Au = 30.4 Hz,
C//,Ph), 4.50-4.52 (m, 3 H, C//,Ph), 4.42 (ddd, 1 H, 3.4 Hz, 7,,.,^ = 10.1 Hz,
>^H6 b-.F= 46.7 Hz, H- 6 b'), 4.36 (dd, 1 H, 7^, sb = 10.1 Hz, 7„,,.p= 46.7 Hz, H- 6 a'), 3.84-
3.93 (m,5 H, H-2, H-4, H-3', H-4', H-5'), 3.71-3.72 (m, 1 H, H-2'), 3.53-3.62 (m, 4
H. H-3, H-5, H-6 a, H-6 b), 3.51 (dt, 1 H, 7 = 6.7, 9.4 Hz, octyl OCH,), 3.26 (dt, 1 H, 7
= 6.7, 9.4 Hz, octyl OCH,), 3.04 (br. s, 1 H, OH), 2.71 (br. s, 1 H, OH), 1.39-1.42 (m,
2 H, octyl CH,), 1.15-1.25 (m, 10 H, octyl CH,), 0.81 (t, 3 H, 7 = 7.0 Hz, octyl CH 3 );
162 '"c NMR (125.8 MHz, CDCI3) 138.8, 138.6, 138.4, 129.0, 128.9, 128.9, 128.6,
128.4, 128.3, 128.2 (Ph), 99.4 (C-1'), 98.0 (C-1), 82.4 (C- 6 ', 7^.6.F = 172.3 Hz), 80.5,
75.4, 75.1, 74.5. 73.2, 72.6, 71.8, 71.7 (ring and benzylic C, octyl OCH,), 71.7 (C-5',
7 c-5 '.f= 17.8 Hz), 68.4 (octyl OCH,), 67.4 (ring), 67.1 (C-4'. 7c-» p= 6 . 8 Hz), 63.8 (ring),
32.3, 29.8, 29.7, 26.6, 23.1 (octyl CH,), 14.6 (octyl CH,). ‘"F NMR (235.4 MHz,
CDCI3 ) Sp - 234.8 (td, 1 F, 7h,„f= 7„,b-p= 46.7 Hz, yH 3 p= 28.1 Hz, F- 6 '). HR-ESI-MS calcd for C^.HjP.NjF [M + Na]" 774.3736, found 774.3725.
I
HO'
6 A B nO Bm
114 OR R =(CH2)7CH3
Octyl 2,6-dideoxy-6-fluoro-2-trifluoroacetaniido-a-D-mannopyranosyI-
(I-^6)-2,3,4-tri-C?-benzyl-a-D-mannopyranoside (114). To a solution of 113
(150 mg, 0.20 mmol) in THF ( 8 mL), were added H,0 (1 mL) and PPh 3 (155 mg, 0.59 mmol). After stirring overnight, the reaction mixture was concentrated and dried under vacuum for 2 h. The crude amine was dissolved in pyridine (5 mL), and trifluoroacetic anhydride (3 mL) was added dropwise. After stirring for overnight, the reaction mixture was concentrated. The residue was dissolved in CH,C1, (25 mL), washed with ice-cold
IM HCl solution (2 x 15 mL) and a saturated NaHC 0 3 solution (15 mL). The organic extract was dried (Na,SOJ, filtered, and concentrated to a brown syrup. The crude syrup
1 6 3 was dissolved in methanol (10 mL) and stirred for 4 h and then concentrated. The residue was purified by chromatography (3:1 hexane:EtOAc) on silica gel to give 25 (105 mg,
64% over two steps) as a light yellow syrup. R, 0.63 (1:1 hexane:EtOAc); [a]g +51.9° (c
0.4, CHCI3 ); ‘H NMR (500 MHz, CDCI3 ) 6 » 7.30-7.43 (m, 15 H, Ph), 6.57 (d, 1 H, 7 =
9.1 Hz, N //C O C F 3 ), 5.03 (d, 1 H, 7= 11.2 Hz, C//,Ph), 5.00 (br. s, 1 H, H-l'), 4.86
(d, 1 H, 7 ,,= 1.2 Hz, H-l), 4.79-4.80 (m, 2 H, C//,Ph), 4.67-4.68 (m, 2 H, C//,Ph),
4.65 (d, 1 H. 7= 11.2 Hz, C//,Ph), 4.47-4.59 (m, 3 H, H-2', H- 6 a', H-6 b'), 4.12 (dd, 1
H, 7 , 3 .= 4.4 Hz, 7y,. = 9.8 Hz, H-3'), 3.96-3.99 (m, 3 H, H-2. H-3, H-5'). 3.74-3.84
(m, 4 H, H-4. H-5, H-6 a, H-6 b), 3.65 (dt, 1 H, 7 = 6.7, 9.6 Hz, octyl OCH,), 3.61 (t, 1
H, Jy,.= 7 , 3 = 9.8 Hz, H-4'), 3.38 (dt, 1 H, 7 = 6.7. 9.6 Hz, octyl OCH,), 3.13 (br. s, 1
H, OH), 3.03 (br. s, 1 H, OH), 1.55-1.58 (m, 2 H, octyl CH,), 1.33-1.40 (m, 10 H, octyl CH,). 0.95 (t, 3 H. 7 = 6 . 8 Hz, octyl CH 3 ); '^C NMR (125.8 MHz. CDCI 3 ) 5^ 158.5
(q, C=0, 7c=o.F = 37.9 Hz), 138.8, 138.7, 138.6, 128.9, 128.8, 128.7. 128.4, 128.3,
128.2. 128.2. 128.1 (Ph), 116.2 (q, CF., 7^^ = 288 Hz), 99.2 (C-1). 98.1 (C-1'), 82.1
(C-6 '. 7(. p = 172.2 Hz). 80.6. 75.4, 75.3, 74.8, 73.2, 72.5, 71.5 (ring and benzylic C, octyl OCH,). 71.2 (C-5'. 7,-.; ;= 17.8 Hz), 70.3, 68.3. 67.9 (ring and octyl OCH,), 66.7
(C-4'. 7c_, F= 6.9 Hz). 53.8 (C-2'). 32.3, 29.8. 29.8, 29.7. 26.6, 23.1 (octyl CH,), 14.5
(octyl CH,). '"F NMR (235.4 MHz, CDCI 3 ) 5p -74.6 (s, 3F. CF 3 ), -234.8 (td, 1 F, 7»^.^ =
Hz, 7 h5 -.p= 28.3 Hz, F- 6 '). HR-ESI-MS calcd for C.jHjjO.oNF, [M + Na]'
844.3654, found 844.3640.
1 6 4 NH
HO
HO'
Octyl 2-amino-2,6-dideoxy-6-fluoro-a-D-mannopyranosyl-(l—> 6 )-a-D - mannopyranoside (25). To a solution of 114 (92 mg, 0.11 mmol) in CHjOH (7 mL), was added 10% Pd/C (45 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CHjOH (10 mL). After concentrating the filtrate and the washings, the residue was redissolved in CHjOH (10 mL), aq. NaOH (1 mL, IM) was added and the solution was stirred overnight. The reaction mixture was neutralized with a minimum amount of pre-washed Amberlite 118
H* resin and concentrated. The product was purified by chromatography (10:2:0.5
CHClyCH^0H:(5N) aq.NH^OH) on latrobeads to give 25 (17 mg, 34%) as a foam. The
The mono benzylated amine (40 mg) isolated was dissolved in HOAc ( 6 mL) and 10%
Pd/C (20 mg) was added and the solution was stirred overnight under an H, atmosphere.
Filtration followed by concentration and purification as described above gave additional (15 mg, 30%) of the product. 0.62 (10:4:1 CHClyCH]0H:(5N) aq.NH.OH); [a]o +24.0°
(c 0.2, CH 3 OH); ‘H NMR (800 MHz, CD 3 OD) 5» 4.81 (br. s, 1 H, H-l'), 4.69 (d, 1 H,
1.7 Hz, H-l), 4.63 (ddd, I H, 4.4 Hz, 7,^.,,.= 10.2 Hz, 7H6b.F=47.1 Hz, H-
6 b'). 4.57 (ddd, I H, 7,.^,.= 1.7 Hz, 7^,.^-= 10-2 Hz, 7 „6 , 3 := 47.1 Hz, H- 6 a'), 3.87 (dd,
I H, 7 5 6b = 5.3 Hz, 76^66= 10 8 Hz, H-6 b), 3.82 (dd, I H, 7 , 3.= 4.2 Hz, J,.,. = 9.3 Hz,
H-3'), 3.78 (dd, I H, 7,,= 1.7 Hz, 7 ^3 = 3.0 Hz, H-2), 3.71 (d, I H, 10.8 Hz, H-
165 6 a), 3.69 (dt, I H ,J = 6.7, 9.6 Hz, octyl OCH,), 3.66 (dd, I H, J, - = 3.0 Hz, = 9.8
Hz, H-3), 3.62-3.64 (m, 3 H, H-4, H-5. H-5'), 3.58 (t, I H, 7,.;.= 9.7 Hz, H-4'),
3.40 (dt, I H, 7= 6.2, 9.6 Hz, octyl OCH,), 3.11 (d, 1 H, 7,.^ = 4.2 Hz, H-2'), 1.55-
1.60 (m, 2 H, octyl CH,), 1.28-1.40 (m. 10 H, octyl CH,), 0.90 (t, 3 H, 7 = 7.2 Hz, octyl CH;); "C NMR (150.9 MHz, CD.OD) 101.8 (C-1', = 168.6 Hz), 101.6 (C-
1 , '7e.H= 167.9 Hz). 83.5 (C- 6 ', 7^., ^= 170.9 Hz), 73.2 (C-3), 72.9 (C-5', 7c.;y== 17.6
Hz), 72.8 (C-2). 72.2 (C-5), 71.9 (C-3'), 6 8 . 6 (C-4), 68.5 (octyl OCH,), 66.7 (C- 6 ),
67.1 (C-4', 7c, p= 6 . 8 Hz), 55.7 (C-2'), 33.0, 30.6. 30.5, 30.4, 27.4, 23.7 (octyl CH,),
14.4 (octyl CH;). '"F NMR (235.4 MHz. CD;OD) 5p - 234.4 (td. 1 F, 7^6, p= 7»,^ ^= 47.1
Hz, 7 h5 -p= 25.9 Hz. F- 6 '). HR-ESI-MS calcd for for C,»H;,0,NF [M + H]' 456.2609. found 456.2599.
TsO'
HO'
O B n
B nO ' Bni
O ctyl 2-azido-2-deoxy-6-0-p-toluenesulfonyl-a-D-mannopyranosyl-(1^6)-
2,3,4-tri-O-benzyl-a-D-mannopyranoside (115). To a solution of 106 (450 mg,
0.60 mmol) in CH,C1, (5 mL) and pyridine (5 mL), p-toluenesulfonyl chloride (172 mg,
0.90 mmol) was added. After stirring overnight, the solution was concentrated under vacuum. The crude residue was purified by chromatography (3:1 hexaneiEtOAc) on silica gel to give 115 (375 mg, 70%) as a colorless syrup. Rp 0.61 (1:1 hexane:EtOAc); [a]^
166 +31.7° (c 0.4, CHCI3): ‘H NMR (500 MHz, CDCI3) 5„ 7.84 (d, 2 H, 7 = 12.0 Hz, tosyl
Ph), 7.29-7.43 (m, 17 H, Ph), 5.00 (d, 1 H, 7 = 12.0 Hz, C //,P h), 4.99 (d, 1 H, 7,-,.=
1.4 Hz, H-l'), 4.82 (d, 1 H, 7, ,= 1.6 Hz, H-l), 4.76 (ABq, 2 H, 7 = 12.1 Hz, Au = 41.3
Hz, C//,Ph), 4.68-4.69 (m, 2 H, C//,Ph), 4.63 (d, 1 H. 7= 11.4 Hz, C//,Ph). 4.31 (dd,
1 H, 7;.^.= 3.6 Hz, 7^.,b = 11.5 Hz. H- 6 b'), 4.18 (dd, 1 H, 7;.^= 1.0 Hz, 7^.,^ = H.5
Hz, H-6 a'), 4.01 (dd, 1 H, 7, , = 1.4 Hz, 7,...= 4.2 Hz, H-2'), 3.90-3.98 (m, 4 H, H-3,
H-4, H-3', H-5'), 3.81-3.82 (m, 1 H, H-2), 3.68-3.73 (m, 4 H, H-5, H- 6 a, H-6 b, H-4'),
3.63 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl OCH.), 3.38 (dt, 1 H, 7 = 6.5, 9.6 Hz. octyl OCH.),
2.48 (s, 3 H. tosyl CHj). 1.55-1.57 (m, 2 H, octyl CH.), 1.33-1.38 (m, 10 H, octyl
CH.), 0.94 (t, 3 H, 7 = 7.1 Hz, octyl CH,); "C NMR (125.8 MHz, CDCl,) 5^
145.0,138.8, 138.8, 138.7, 133.3, 130.2, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2,
128.2, 128.1,128.1 (Ph), 99.4 (C-1'), 98.2 (C-1), 80.6, 75.4, 75.3, 74.6, 73.3, 72.6,
72.1, 71.3. 70.8, 68.3, 68.1, 67.7, 67.5, 63.6 (ring and benzylic C, octyl OCH,), 32.3,
29.8, 29.8, 29.7, 26.6, 23.1 (octyl CH,), 22.1 (tosyl CH,), 14.5 (octyl CH,). HR-ESI-
MS calcd for C,,H,,0,,N.S [M + Na]" 926.3868, found 926.3850.
HO"
BnO"
Octyl 2,6-diazido-2,6-dideoxy-a-D-mannopyranosyl-(l->6)-2,3,4-tri-0- benzyi-a-D-mannopyranoside (116). To a solution of 115 (78 mg, 0.09 mmol) in
1 6 7 DMF (2 mL), were added NaNj (18 mg, 0.27 mmol) and 15-crown-5 (50 gL, 0.27 mmol). After stirring overnight at 60 °C, the solution was concentrated under vacuum. The crude residue was purified by chromatography (4:1 hexane:EtOAc) on silica gel to give
116 (60 mg, 85%) as a light yellow syrup. R,- 0.73 (1:1 hexane:EtOAc); [ajg +47.0° (c
1.1, CHCI3 ); 'H NMR (500 MHz, CDCI3 ) 6 3 , 7.24-7.40 (m, 15 H, Ph), 5.02 (d, 1 H, 7,.,.
= 1.3 Hz, H-l'), 4.95 (d, 1 H, 7= 11.1 Hz, C7/,Ph), 4.80 (d, 1 H, 7, ,= 1.6 Hz, H-l),
4.72 (ABq, 2 H, 7= 12.1 Hz, Au = 31.6 Hz, C//,Ph), 4.63-4.65 (m, 2 H. C//,Ph), 4.61
(d, 1 H, 7 = 11.1 Hz, CT/^Ph), 3.98-4.00 (m, 3 H, H-4, H-2', H-5'), 3.96 (dd, 1 H, 7 , 3 =
3.0 Hz, 7 , 3 = 9.4 Hz, H-3), 3.87 (dd, 1 H, J,-y= 5.0 Hz, Jy, = 9.4 Hz, H-3'), 3.79 (dd,
1 H, 7 , 3 = 1.6 Hz, 7 , 3 = 2.5 Hz, H-2), 3.71 (dd, 1 H, 1.7 Hz. 7^,^= Hz, H-
6 a), 3.66-3.69 (m, 2 H, H-5, H- 6 b), 3.58 (dt, 1 H, 7 = 6.7, 9.6 Hz, octyl OCH,), 3.57
(t, I H, 7 3 . 3 = 7 3 . 3 = 9.4 Hz, H-4'), 3.39 (dd, 1 H. Jy,^-= 2.6 Hz, 7 , 3 . ^ = 13.1 Hz, H-
6 b'), 3.37 (m, 2 H, H- 6 a', octyl CH,), 1.49-1.52 (m, 2 H, octyl CH,), 1.27-1.32 (m, 10
H. octyl CH,), 0.88 (t, 3 H, 7= 6 . 8 Hz, octyl CH,); '^C NMR (125.8 MHz, CDCI 3 ) 5^
138.4, 138.3, 138.1, 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.8, 127.8, 127.8,
127.7 (Ph), 99.0 (C-1), 97.7 (C-1'), 80.2, 75.1, 74.9, 74.2, 72.9, 72.2, 71.6, 71.6,
71.2, 6 8 .6 , 67.9, 67.2, 63.4 (ring and benzylic C, octyl OCH,), 51.3 (C- 6 ') 31.8, 29.4,
29.3, 29.2, 26.2, 22.7 (octyl CH,), 14.1 (octyl CH3). HR-ESI-MS calcd for C 3 ,H;3 0 ,Ng
[M -h Na]' 797.3844, found 797.3842.
168 H N ^ C F 3
pBn 1 A
117 OR R =(CH2)7CH3
Octyl 2,6-dideoxy-2,6-di-(trifluoroacetamido)-a-D-mannopyranosyl-
(l->6)-2,3,4 tri-O-benzyl-a D-mannopyranoside (117). To a solution of 116
(300 mg, 0.39 mmol) in THF (25 mL), were added H,0 (5 mL) and PPhj (720 mg, 2.7 mmol). After stirring overnight, the reaction mixture was concentrated and dried under vacuum for 2 h. The crude amine was dissolved in pyridine (5 mL), and trifluoroacetic anhydride (5 mL) was added dropwise. After stirring overnight, the reaction mixture was concentrated. The residue was dissolved in methanol (20 mL) and stirred for 4 h and then concentrated. The residue was taken in CH,CL (50 mL), washed with ice-cold IM HCl solution (2 X 30 mL) and water (30 mL). The organic extract was dried (Na.SO^), filtered, and concentrated to a brown syrup. The residue was purified by chromatography (3:1 hexane:EtOAc) on silica gel to give 117 (120 mg, 34% over two steps) as a light yellow syrup. RfO.73 (1:1 hexaneiEtOAc); [a]o+14.6° (c 0.3, CHClj); ‘H NMR (500 MHz,
CDClj) Ôh 7.31-7.42 (m, 15 H, Ph), 6.80 (d, 1 H, 7 = 7.0 Hz, NHCOCF,), 6.50 (d, 1 H,
7= 7.0 Hz, NT/COCFj), 5.08 (br. s, 1 H, H-l'), 5.05 (d, 1 H, 7 = 11.3 Hz, C7/,Ph),
4.86 (d, 1 H, 7, ,= 1.0 Hz, H -l), 4.75 (ABq, 2 H, 7 = 12.1 Hz, Au = 19.4 Hz, C //,P h),
4.67-4.69 (m. 2 H, C//,Ph), 4.63 (d, 1 H, 7 = 11.3 Hz, C//,Ph), 4.41 (d, 1 H, 7,-^.= 5.6
Hz, H-2'), 4.07-4.10 (m, 1 H, H-5'), 3.99 (dd, 1 H, 7,^ = 2.8 Hz, 7j, = 9.5 Hz, H-3),
1 6 9 3.93 (t, I H, •/ 3-.4 = ^ 4 -.5 = 9.4 Hz, H-4'), 3.88 (dd, 1 H, ^b- = 4.7 Hz, J6a\6b = 110 Hz,
H-6 b'), 3.83 (dd, 1 H, 7, ,= l.O Hz, 7,,= 2.8 Hz, H-2), 3.72-3.82 (m, 4 H, H-4. H-2',
H-3', H-6 a'), 3.65 (dt, 1 H, 7 = 6.7. 9.4 Hz, octyl OCH,), 3.39-3.41 (m, 3 H, H- 6 a, H-
6 b, octyl OCH,), 3.32 (dd, 1 H, 7,^ = 3.3 Hz, 7,^ = 9.5 Hz. H-5), 1.55-1.58 (m, 2 H, octyl CH ,), 1.32-1.37 (m, 10 H, octyl CH ,), 0.94 (t, 3 H, 7 = 6 . 6 Hz, octyl CHj); '^C
NMR (125.8 MHz, CDClj) 5^ 158.8 (q. C=0, 7^^ p = 37.4 Hz), 158.3 (q, C=0, 7^^p =
37.4 Hz), 138.8, 138.7, 138.6, 128.9, 128.9, 128.8, 128.4, 128.3, 128.3, 128.2, 128.2,
128.1 (Ph), 116.2 (q, CF„ 7 ^ = 287.4 Hz), 116.1 (q, CF,. 7^^ = 288.2 Hz), 98.4 (C-
1'), 98.2 (C-1), 80.6, 75.4, 75.2, 74.9, 73.2, 72.5, 71.5, 70.1, 69.4, 69.1, 68.3, 67.8
(ring and benzylic C, octyl OCH,), 53.8 (C-2'), 40.8 (C- 6 '), 32.3, 29.8, 29.8, 29.7,
26.6, 23.1 (octyl CH,), 14.5 (octyl CH,). HR-ESI-MS calcd for C,;H,,0,,N,F, [M +
Naj* 937.3681, found 937.3716.
NH,
H
HO
26 OR
R = (CH2)7CH3
Octyl 2,6-diamiiio-2,6-dideoxy-a-D-mannopyranosyI-(l-»6)-a-D- mannopyranoside (26). To a solution of 117 (102 mg, 0.11 mmol) in HOAc ( 6 mL)
10% Pd/C (35 mg) was added. The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CHjOH (10 mL). After concentrating the filtrate and the washings, the residue was redissolved in CH 3 OH (10
170 mL), aq. NaOH (1 mL, IM) was added and the reaction mixture was stirred overnight.
The solution was neutralized with a minimum amount of pre-washed Amberlite 118 H* resin and concentrated. The product was purified by chromatography (10:2:0.5
CHClyCH^0H:(5N) aq.NH^OH) on latrobeads to give 26 (30 mg, 61%) as a colorless solid. R, 0.47 (10:4:1 CHCl 3 :CH,0 H:(5 N) aq.NH.GH); [aj^ +64.5° (c 0.2, CH,OH); ‘H
NMR (800 MHz, D,0) 5» 5.07 (br. s, 1 H, H -l'), 4.78 (br. s, 1 H, H-l), 4.07 (dd, 1 H,
Jry= 4.7 Hz, Jyy= 9.7 Hz, H-3'), 3.92 (dd, 1 H, Hz, 7^,, = 11.2 Hz, H- 6 b),
3.87-3.90 (m. 2 H, H-2, H-5'), 3.76 (d, 1 H, 7 ^ ^ = 11.2 Hz, H- 6 a), 3.71-3.73 (m, 2 H,
H-3, H-5), 3.63-3.68 (m, 2 H, H-4, octyl OCH,), 3.63 (d, 1 H, Jyy = 4.7 Hz, H-2'),
3.49-3.50 (m, 1 H, octyl OCH,), 3.48 (t, I H, Jyy=J,.y= 9.7 Hz, H-4'), 3.42 (dd, 1 H,
Jy^.y= 2.3 Hz, = 13.4 Hz, H-6 a'), 3.12 (dd, 1 H, = 9.6 Hz, 7 ^.^.= 13.4 Hz,
H-6 b'), 1.52-1.56 (m. 2 H, octyl CH,), 1.20-1.30 (m, 10 H, octyl CH,), 0.78 (t, 3 H, 7 =
6 . 6 Hz, octyl CH^); ''C NMR (150.9 MHz, D,0) 6 ^ 101.4 (C-LVc» = 170.3 Hz), 98.2
(C-1','7c.h= 171.8 Hz), 72.5, 72.1 (C-3, C-5), 71.4 (C-2), 69.8 (C-5'), 69.6, 69.3 (C-
4', octyl OCH,), 6 8 . 6 (C-3'), 68.0 (C- 6 ), 67.9 (C-4), 55.1 (C-2'), 41.8 (C- 6 '), 32.5,
29.9, 29.9, 29.8, 26.8. 23.4 (octyl CH,), 14.8 (octyl CH^). HR-ESI-MS calcd for for
C,oH,oO,N,[M + H]"453.2812, found 453.2793.
A
)Bn o A
B n a v ^ * A
118 OR R = (CHzlyCHg
1 7 1 Octyl 2,6-dideoxy-2,6-difIuoro-a-D-mannopyranosyI-(l-^6)-2,3,4-tri-0- benzyl-a-D-mannopyranoside (118). To a solution of 98 (180 mg, 0.25 mmol) in dry CHnCT ( 6 mL) at -40 °C, DAST (130 pL. 1.0 mmol) was added. After stirring for 2 h, methanol (1 mL) was added and the solution was concentrated. The residue was purified by chromatography (3:1 he.xaneiEtOAc) on silica gel to give 118 (100 mg, 55%) as a colorless syrup. R,. 0.70 (1:1 he.xane:EtOAc); [al^-t-42.5° (c 1.4, CHCI 3 ); 'H NMR
(400 MHz, CDCI3 ) 5h 7.22-7.40 (m, 15 H, Ph), 5.15 (dd, 1 H, 7,.,.= 1.6 Hz, y„, p= 8.9
Hz, H-L), 4.92 (d, 1 H, 7= 11.4 Hz, CH,Ph), 4.77 (d, 1 H, 7,, = 1.5 Hz, H-l), 4.74
(d, 1 H, 7 = 12.2 Hz, CH.Ph), 4.61 -4.67 (m, 4 H, C//,Ph, H-2'), 4.59 (d, 1 H, 7 = 11.1
Hz, C//,Ph). 4.58 (ddd, 1 H, 7;.= 3.6 Hz, 7,,.,^ = 10.2 Hz, 7^,^ ^= 47.1 Hz, H-6 b'),
4.57 (ddd. 1 H, 7;.,,.= 1.5 Hz. 7 ,,.^ = 10.2 Hz, 7h,,-.p= 47.1 Hz, H- 6 a'), 3.91-3.99 (m, 3
H. H-3', H-4', H-5'), 3.66-3.76 (m, 6 H, H-2, H-3. H-4, H-5. H-6 a, H-6 b), 3.57 (dt, 1
H, 7 = 6.7. 9.6 Hz. octyl OCH,), 3.32 (dt, 1 H. 7 = 6.5, 9.6 Hz, octyl OCH,), 2.70 (br. s, 1 H, OH), 2.31 (br. s, 1 H, OH), 1.44-1.51 (m, 2 H. octyl CH,), 1.26-1.36 (m, 10 H. octyl CH ,). 0.88 (t. 3 H. 7 = 7.1 Hz, octyl CH 3 ); '^C NMR (100.6 MHz, CDCI3 ) 5^
138.4. 138.3, 138.2. 128.4. 127.9. 127.9. 127.8. 127.7. 127.7. 127.6 (Ph), 98.0 (C-L,
7c.,.F= 29.7 Hz ), 97.7 (C-1), 89.1 (C-2', 7c_,.^ = 174.3 Hz), 82.0 (C- 6 ', = 172.3
Hz), 80.1, 75.0, 74.9. 74.2, 72.8, 72.1, 71.5 (ring and benzylic C, octyl OCH,), 71.1
(C-5', 7c.j-.p= 17.7 Hz), 67.8 (octyl OCH,), 67.1 (C-4', 6 . 8 Hz). 66.9 (C- 6 ), 31.8,
29.4, 29.4, 29.2, 26.1, 22.7 (octyl CH,), 14.1 (octyl CH3). '"F NMR (235.4 MHz,
CDCI3) ôp - 205.6 (ddd, 1 F, 7h,-.p= 8.9 Hz, 7^,-^= = 49.4 Hz, 7 „ 3 j, = 32.9 Hz, F-2'), -
234.8 (td, 1 F, 7 3 3 ^ p = 7^^^. p = 47.1 Hz, Jny p= 28.2 Hz, F- 6 '). HR-ESI-MS calcd for
C,,H;^0,F, [M Na]' 751.3634. found 751.3607.
172 HO'
OH
HO'
Octyl 2,6-dideoxy-2,6-difiuoro-a-D-mannopyranosyI-(l-^6)-a-
D-mannopyranoside (27). To a solution of 118 (80 mg, 0.11 mmol) in HOAc ( 8 mL), was added 109c Pd/C (35 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CH^OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography
(5:1 CH.CLiCHjOH) on latrobeads to give 27 (45 mg, 90%) as a foam. 0.65 (4:1
CH,Cl,:CHjOH); [al^ +63.7° (c 0.3, CH^OH); ‘H NMR (800 MHz, CD^OD) 5„ 5.01 (d,
1 H,y„,.p=7.1 Hz, H-D, 4.70 (d, 1 H, 7,,= 1.0 Hz, H-l), 4.60-4.67 (m, 2 H, H-2',
H-6 b'), 4.58 (dd, 1 H, 7^.= 12.0 Hz, 7„,^-p= 47.1 Hz, H- 6 a'), 3.91 (dd, 1 H, 7;^ =
4.1 Hz, 7(,^f,b= 10.8 Hz, H- 6 b), 3.80 (ddd, 1 H, 7,.^^ = 3.6 Hz, 9.7 Hz, 7^^ p= 25.9
Hz, H-5'), 3.76-3.78 (m, 2 H, H-2, H-3'), 3.75 (d, 1 H, 7^^,^ = 10.9 Hz, H- 6 a), 3.70 (t,
1 H, ~ 7,.j.= 9.7 Hz, H-4'), 3.63-3.68 (m, 4 H, H-3, H-4, H-5, octyl OCH.), 3.40
(dt, 1 H, 7 = 6.2, 9.6 Hz, octyl OCH,), 1.55-1.61 (m, 2 H, octyl CH,), 1.29-1.40 (m, 10
H. octyl CH,), 0.90 (t, 3 H, 7 = 6.9 Hz, octyl CH,); ‘^C NMR (150.9 MHz, CD,OD) 5^
101.7 (C-1,'7c.h= 167.9 Hz), 98.8 (C-l',‘7c„= 170.8 Hz, 7^.,-^= 29.6 Hz ), 91.0 (C-2',
7c.,._p= 174.6 Hz), 82.0 (C- 6 ', 7^.6^= 171.6 Hz), 73.2 (C-5', 7c_y_p= 17.8 Hz), 73.1, 72.8
1 7 3 (C-3, C-5), 72.1 (C-2), 71.7 (C-3', 7^'^ = 17.4 Hz), 6 8 . 6 (C-4), 68.4 (octyl OCH,),
67.8 (C- 6 ), 67.4 (C-4', 7 ^.4 .;= 6.2 Hz), 33.0, 30.6, 30.5, 30.4, 27.4, 23.7 (octyl CH,),
14.4 (octyl CH 3 ). '"F NMR (235.4 MHz, CD 3 OD) 5p - 207.6 (ddd, F, 7^,.^= 7.1 Hz, 7h, p
= 49.4 Hz, 7 h3 p= 33.0 Hz, F-2'), - 236.8 (td, 1 F, 7y^.p = 7^^^p = 47.1 Hz, 7 ^3 .p = 25.9
Hz, F-6 '). HR-FAB-MS calcd for for C,oH 3^0 ,,F, [M 4 - Na]" 481.2225. found 481.2220.
T s O A '
pBn A 119 OR R = (CHzlyCHa
Octyl 2-deoxy-2-fluoro-6-0-p-toluenesuIfonyl-a-D-mannopyranosyl-(l->6)-
2,3,4-tri-O-benzyl-a-D-iiiannopyranoside (119). To a solution of 98 (255 mg,
0.35 mmol) in dry pyridine ( 8 mL), p-toluenesulfonyl chloride (330 mg, 1.70 mmol) and
DMAP (100 mg, 0.80 mmol) were added. .After stirring for 18 h, the solution was concentrated under vacuum. The crude residue was purified by chromatography (1:1 hexane:EtOAc) on silica gel to give 119 (209 mg, 6 8 %) as a colorless syrup. R, 0.64 (1:1 hexane:EtOAc); [a]o 4 -3 4 . 1 ° (c 1.1, CHCI3 ); 'H NMR (500 MHz, CDCI3 ) 7.84 (d, 2 H,
7=8.3 Hz, tosyl Ph), 7.29-7.42 (m, 17 H, Ph), 5.11 (dd, 1 H, 7,.,.= 1.8 Hz, 7^, ^= 7.2
Hz, H -l'), 4.99 (d, 1 H, 7 = 11.2 Hz, C //,P h), 4.81 (d, 1 H, 7 ,^ = 1.6 Hz, H -l), 4.79
(d, 1 H, 7 = 12.2 Hz, CT/.Ph). 4.75 (ddd, 1 H, 7, , = 1.8 Hz, J,-y= 3.8 Hz, 7hv.f= 49.4
Hz, H-2'), 4.69-4.71 (m, 3 H, C//,Ph), 4.61 (d, 1 H, 7= 11.2 Hz, CT/.Ph), 4.34 (dd, 1
H, 7 5 . 6 b = 2.9 Hz, 7b,. 6 b = 11.1 Hz, H- 6 b'), 4.18 (d, 1 H, = 1 1 . 1 Hz, H-6 a'), 3.96-
174 3.98 (m, 2 H, H-3', H-5'), 3.91 (ddd, I H, 4.1 Hz, = 10.7 Hz, H-3), 3.83 (br. s, I H, H-2), 3.79-3.81 (m, 3 H, H-4, H- 6 b, H-4'), 3.73 (dd, 1 H, 7;,, = 1.5 Hz, =
11.7 Hz, H-6 a), 3.69-3.70 (m, 1 H, H-5), 3.61 (dt, 1 H, 7= 6.7, 9.6 Hz, octyl OCH,),
3.37 (dt, 1 H, 7 = 6.5, 9.6 Hz, octyl OCH,), 2.48 (s, 3 H, tosyl CHj), 1.54-1.57 (m, 2
H, octyl CH,), 1.32-1.38 (m, 10 H. octyl CH,), 0.94 (t, 3 H, 7 = 7.1 Hz, octyl CHj); "C
NMR (125.8 MHz, CDCU) 5c 145.3,138.8, 138.8, 138.7, 133.3, 130.2, 128.8, 128.7,
128.6, 128.5, 128.4. 128.3, 128.3, 128.2, 128.1,128.1 (Ph), 98.4 (C-1', 7c., •,f = 29.4 Hz
), 98.1 (C-1), 89.3 (C-2'. 7c.v p = 174.6 Hz), 80.6, 75.4, 75.3, 74.7, 73.2, 72.5, 72.0
(ring and benzylic C), 71.1 (C-3', 7c.yp= 17.5 Hz), 70.6, 69.0, 68.2, 67.8, 67.3 (ring and benzylic C, octyl OCH,), 32.3, 29.8, 29.8, 29.7, 26.6, 23.1 (octyl CH,), 22.1 (tosyl
CH,), 14.5 (octyl CH,). "F NMR (235.4 MHz, CDCl,) 6 p - 205.6 (ddd, 1 F, 7.2
Hz, 7h, p = 49.4 Hz, 7,„.p= 33.0 Hz, F-2'). HR-ESI-MS calcd for C,gH,,0,,FS [M -t- Na]'
903.3765, found 903.3757.
N r \ A
)Bn o A ^
B n O V ii» *^ A
120 R = (CHalyCHa
Octyl 6-azido-2,6-dideoxy-2-fIuoro-a-D-mannopyranosyl-(l—>6)-2,3,4-tri-
O-benzyl-a-D-mannopyranoside (120). To a solution of 119 (209 mg, 0.24 mmol) in DMF (10 mL), were added NaN, (85 mg, 1.3 mmol) and 15-crown-5 (230 |xL, 1.3 mmol). After stirring overnight at 60 °C, the solution was concentrated under vacuum. The
1 7 5 residue was taken in CH^CU (2 x 40 mL), washed with water (30 mL), dried (Na^SO^), filtered, and concentrated to a yellow syrup. The crude syrup was purified by chromatography (3:1 hexane:EtOAc) on silica gel to give 120 (166 mg, 92%) as a light colorless syrup. 0.72 (1:1 hexane:EtOAc); [a]g +37.2° (c 0.6, CHCI 3 ): ‘H NMR (500
MHz, CDCIjIÔh 7.24-7.37 (m, 15 H, Ph), 5.14 (dd, 1 H, 7, ,.= 1.2 Hz, 7^,.^ = 7.2 Hz,
H -L), 4.94 (d. 1 H, 7 = 11.0 Hz, CH.Ph), 4.78 (d. 1 H, 7, , = 1.4 Hz, H-l), 4.74 (d, 1
H, 7 = 12.2 Hz, C//,Ph), 4.71 (ddd. 1 H, 7. ,. = 1.2 Hz, J. y= 2.0 Hz, 7^.^ = 49.4 Hz,
H-2'), 4.63-4.66 (m, 3 H. C//,Ph), 4.59 (d, 1 H, 7= 11.1 Hz, C//,Ph), 3.98 (dd, 1 H,
7;,y= 3.9 Hz,7,^,b= 11.4 Hz. H- 6 b), 3.90-3.94 (m, 2 H, H- 6 a, H-3'), 3.67-3.76 (m, 6
H. H-2, H-3. H-4, H-5, H-4', H-5'), 3.57 (dt, 1 H, 7= 6.7, 9.6 Hz, octyl OCH,), 3.45
(dd, 1 H. 2.5 Hz. 7,,= 13.2 Hz, H-6 a'), 3.39 (dd, 1 H, 7;.^ = 5.8 Hz, 7,,.^ =
13.2 Hz, H-6 b'), 3.32 (dt, I H. 7= 6 .6 , 9.6 Hz, octyl OCH,), 1.49-1.51 (m. 2 H, octyl
C H .), 1.27-1.33 (m, 10 H. octyl C H ,), 0.88 (t, 3 H, 7 = 7.1 Hz, octyl CHj): ‘^C NMR
(125.8 MHz, CDCI 3 ) 5c 138.4, 138.4, 138.3, 128.4, 128.3, 128.0, 127.9, 127.8, 127.8,
127.7 (Ph). 98.0 (C-1', 7c.,.p= 29.6 Hz ), 97.7 (C-1), 89.2 (C-2'. 7^.,,^ = 174.2 Hz),
80.1, 75.5, 75.4, 74.6, 73.2, 72.5, 72.0, 71.9 (ring and benzylic C, octyl OCH,), 71.3
(C- 3 '. 7 c.3 p= 17.7 Hz). 69.1, 68.3, 67.4 (ring and octyl OCH,). 51.3 (C- 6 ') 31.8, 29.4,
29.4, 29.2, 26.2, 22.7 (octyl CH,). 14.1 (octyl CH 3 ). "F NMR (235.4 MHz, CDCI3 ) 5p -
205.6 (ddd, 1 F, 7„, p= 7.2 Hz, 7^, p = 49.4 Hz, 7 „ 3 p= 30.6 Hz, F-2'). HR-ESI-MS calcd
for C ^H jA N jF [M + Na]" 774.3742, found 774.3787.
1 7 6 HO'
BnO-
Octyl 2,6-dideoxy-2-nuoro-6-trifIuoroacetamido-a-D-mannopyranosyl-
(l-^6)-2,3,4-tri-0-benzyi-a-D-mannopyranoside (121). To a solution of 120
(166 mg, 0.22 mmol) in THF ( 8 mL), were added H,0 (2 mL) and FPhj (173 mg, 0.66 mmol). After stirring overnight, the reaction mixture was concentrated and dried under vacuum for 2 h. The crude amine was dissolved in pyridine:CH,CU (1:1, 12 mL), and trifluoroacetic anhydride (0.6 mL) was added dropwise. After stirring overnight, the reaction mixture was concentrated. The residue was taken in CH,CL (30 mL), washed with ice-cold IM HCl solution (20 mL) and a saturated NaHCOj solution (10 mL). The organic extract was dried (Na^SO^), filtered, and concentrated to a brown syrup. The crude syrup was dissolved in methanol (TO mL) and stirred for 4 h and then concentrated.
The residue was purified by chromatography (1:1 hexane:EtOAc) on silica gel to give 121
(150 mg, 83% over two steps) as a light yellow syrup. 0.44 (1:1 hexane:EtOAc); [a]g
+39.4= (c 0.8, CHCI 3 ); ‘H NMR (500 MHz, CDCI3 ) 6 » 7.27-7.43 (m, 15 H, Ph), 6.57 (d,
1 H, 7 = 6.7 Hz, NHCOCF 3 ), 5.15 (dd, 1 H, 7, ,.= 2.1 Hz, 7^. ^ = 7.3 Hz, H-1'), 5.03
(d, 1 H, 7 = 11.2 Hz, CH.Ph), 4.83 (d, 1 H, 7,^ = 1.7 Hz, H-1), 4.81 (d, 1 H, 7 = 12.2
Hz, CH,Ph), 4.76 (dt, 1 H, 7,. ,.= 7v_,.= 2.1 Hz, 7h,- p= 49.8 Hz, H-2'), 4.71-4.73 (m, 3
177 H, CH^Ph), 4.65 (d, 1 H, 7 = 11.2 Hz, CH,Ph), 3.98-4.02 (m, 2 H, H-3', H- 6 a'), 3.94
(dd. 1 H, 7; = 4.5 Hz, 7^,.^-= 11.6 Hz, H-6 b'), 3.85-3.89 (m, 2 H, H-2, H-3), 3.74-
3.81 (m, 4 H, H-4, H-5, H- 6 a, H-6 b), 3.65 (dt, 1 H, 7 = 6.7, 9.6 Hz, octyl OCH,), 3.58
(t, 1 H, 7 3 .,.= 7, 5 -= 9.6 Hz, H-4'), 3.46 (dt, 1 H, 7,.^ = 4.5 Hz, 7, y = 9.6 Hz, H-5'),
3.38 (dt, l H, 7 = 6.5, 9.6 Hz, octyl OCH,), 1.56-1.59 (m, 2 H, octyl CH,), 1.29-1.41
(m, 10 H, octyl C H ,), 0.96 (t, 3 H, 7 = 7.1 Hz, octyl CH 3 ); ‘’C NMR (125.8 MHz.
CDCI3 ) 5c 158.5 (q, C=0, 7c.op= 37.2 Hz), 138.8, 138.8, 138.6. 128.9, 128.9, 128.7,
128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1 (Ph), 116.3 (q, CF 3 , 288 Hz),
98.4 (C-r, 7 c.,..f = 30.0 Hz ), 98.2 (C-1), 89.4 (C-2', 7^., ^ = 175.4 Hz), 80.6, 75.5.
75.4, 74.8. 73.3. 72.6, 72.0 (ring and benzylic C), 70.7 (C-3', Jc.y.F= 15.6 Hz), 70.6,
69.1, 68.3, 67.3, 65.5 (ring and benzylic C, octyl OCH,), 40.9 (C- 6 ') 32.3, 29.8, 29.8,
29.7. 26.6. 23.1 (octyl CH,), 14.5 (octyl CH,). '"F NMR (235.4 MHz, CDCI3 ) 5^ -74.6
(s, 3F. CF3 ), -205.6 (ddd. 1 F, 7^, .^= 7.3 Hz. 7^. ^ = 49.8 Hz, 7 ^ 3 = 30.0 Hz, F-2').
HR-ESI-MS calcd for C, 3 H„ 0 ,oNF, [M + Na]' 844.3660, found 844.3634.
HO' HO- OH
HO'
Octyl 6-amino-2,6-dideoxy-2-fluoro-a-D-mannopyranosyl-(l-»6)-a-D-
mannopyranoside (28). To a solution of 121 (140 mg, 0.17 mmol) in HOAc (10 tnL)
10% Pd/C (60 mg) was added. The solution was stirred overnight under an H, atmosphere
1 7 8 and then the catalyst was separated by filtration and washed with CH 3 OH (2 x 20 mL).
After concentrating the filtrate and the washings, the residue was redissolved in CHjOH
(10 mL), aq. NaOH (1.5 mL, IM) was added and the reaction mixture stirred overnight.
The solution was neutralized with a minimum amount of pre-washed Amberlite 118 H* resin and concentrated. The product was purified by chromatography (10:2:0.5
CHCl3 :CH 3 0 H:(5 N) aq.NH^OH) on latrobeads to give 28 (36 mg, 47%) as a colorless solid. The mono benzylated amine (40 mg) isolated was dissolved in HOAc (4 mL) and
10% Pd/C (20 mg) was added and the solution was stirred overnight under an H, atmosphere. Filtration followed by concentration and purification as mentioned above gave an additional (24 mg, 31%) of the product. R, 0.22 (10:4:1 CHCl 3 :CH 3 ÜH:(5 N) aq.NH.OH): [aj^ +62.4" (c 0.1, H,0): ‘H NMR (800 MHz. CDjOD) 5.00 (d, 1 H, y„,.p= 7.5 Hz, H-T). 4.75 (hr. s, 1 H, H-1), 4.63 (dd, 1 H, 7,.3 . = 2.6 Hz, 7^,:.F = 50.0
Hz, H-2'), 3.94 (dd, 1 H, 7 ;,, = 5.2 Hz. 7 ^ ,^ = 10.9 Hz, H- 6 b), 3.79 (d, 1 H, 7 ^ 3 = 1.4
Hz, H-2). 3.76 (dd, 1 H, 7,^= 1.3 Hz, 7^^= 10.9 Hz, H- 6 a), 3.74 (ddd, 1 H, 7 . 3 . =
2.6 Hz, Jy, = 9.7 Hz. 7 „ 3 p= 31.0 Hz. H-3'), 3.68 (dt, 1 H, 7 = 6.7, 9.4 Hz, octyl
OCH 3 ), 3.62-3.66 (m, 4 H, H-3, H-4, H-5, H-5'), 3.49 (t, 1 H, 7;., = 7,. 3 . = 9.7 Hz, H-
4'), 3.41 (dt, 1 H, 7= 6.2, 9.7 Hz, octyl OCH,), 3.07 (dd, 1 H, 7 , = 1.6 Hz. 7^.^.=
13.4 Hz, H-6 a'), 3.12 (dd, 1 H,7;„y= 7.8 Hz, 7,,.^-= 13.4 Hz, H- 6 b'), 1.55-1.61 (m, 2
H, octyl CH,), 1.29-1.40 (m, 10 H, octyl CH,), 0.89 (t, 3 H, 7 = 7.2 Hz, octyl CH 3 ); '^C
NMR (150.9 MHz, CD 3OD) 101.7 (C-1, '7^» = 167.9 Hz), 98.5 (C -l',‘7c,„ = 171.6
Hz, 7e.,-.F= 29.7 Hz ), 91.2 (C-2', 7^.,-^= 174.3 Hz), 74.0 (C-5'), 73.0 (C-5), 72.8 (C-3),
72.1 (C-2), 71.6 (C-3', 7 ^ 3 := 17.5 Hz), 70.3 (C-4'), 68.7 (octyl OCH,), 68.4 (C-4),
67.5 (C-6 ), 43.5 (C-6 '), 33.0, 30.6, 30.5, 30.4, 27.4, 23.7 (octyl CH,), 14.4 (octyl
CH,). 'T NMR (235.4 MHz, CD3OD) Sp - 207.3 (ddd, 1 F, 7.5 Hz, 50.0
Hz, 7 h3 -.p= 31.0 Hz, F-2'). HR-ESI-MS calcd for for C^oHjgO^NF [M + H]" 456.2609,
found 456.2611.
179 QBz AcO' BnO'
BnO'
Octyl 6-0-acety 1-2-0-benzoyI-3,4-di-O-benzyi-a-D-mannopy ranosy 1-
(1^6)-2,3,4-tri-0-benzyl-a-D-mannopyranoside (126). Trichloroacetimidate
106'-^ (1.67 g, 2.6 mmol) and alcohol 75 (1.20 g, 2.1 mmol) were dried in vacuo with powdered 4 Â molecular sieves (1.0 g) overnight. Dry CH,CU (35 mL) was added and the mixture was cooled to -10 °C with stirring. A solution of TMSOTf (135 joL) in CH,CL
(400 pL) was added dropwise to the reaction mixture and the stirring was continued for 2 h. The solution was neutralized by the addition of a saturated NaHCOj solution (1.0 mL) and CH^CL (2 x 50 mL) was added. The organic layer was washed with water (30 mL), dried (Na,SOj, filtered, and concentrated to a colorless syrup. The crude syrup was purified by chromatography (4:1 hexane:EtOAc) on silica gel to give 126 (2.0 mg, 91%) as a colorless syrup. R,- 0.55 (4:1 hexane:EtOAc); [aj^ 4-24.0° (c 0.4, CHClj); ‘H NMR
(500 MHz, CDCI3) Ôh 8.07 (dd, 2 H, 7 = 1.1, 8.4 Hz, Ph), 7.59 (tt, 1 H, 7 = 1.2, 7.7
Hz, Ph), 7.45 (t. 2 H, 7= 8.1 Hz, Ph), 7.16-7.40 (m, 25 H, Ph), 5.74 (dd, I H, 7,-r =
2.0 Hz, J..y= 3.0 Hz, H-2'), 5.06 (d, 1 H, 7,.,.= 2.0 Hz, H-1'), 4.93 (d, I H, 7= ll.l
Hz, C//,Ph), 4.87 (d, 1 H, 7= 11.0 Hz, CH.Ph), 4.82 (d, 1 H, 7, ,= 2.0 Hz, H-1), 4.76-
4.78 (m, 2 H, CH.Ph). 4.74 (d, 1 H, 7 = 11.3 Hz, CH.Ph), 4.62-4.63 (m, 2 H, C//,Ph),
4.57 (d, 1 H, 7= 11.0 Hz, C//,Ph), 4.49 (d, 1 H, 7 = 11.1 Hz, C77,Ph), 4.47 (d, 1 H, 7
180 = 11.3 Hz, C//,Ph), 4.29 (dd, 1 H, 7;-^= 2.1 Hz, 7^,.^.= 11.9 Hz, H- 6 a'), 4.24 (dd, 1
H, Js\6b = 3.8 Hz, J(,3,\6b = 1^9 Hz, H- 6 b'), 4.09 (dd, 1 H, 7^, 3 - = 3.0 Hz, J y = 8 . 6 Hz,
H-3'), 3.89-3.95 (m, 4 H, H-3, H- 6 a, H-6 b, H-4'), 3.86 (t, 1 H, J,, = 7,, = 9.8 Hz, H-
4), 3.79 (t, 1 H, J, , = J , 3 = 2.0 Hz, H-2), 3.70-3.74 (m, 2 H, H-5, H-5'), 3.59 (dt, 1 H,
J = 6 .8 , 9.7 Hz, octyl OCH,), 3.33 (dt, 1 H, 7 = 6.5, 9.7 Hz, octyl OCH,), 2.00 (s, 3 H,
OCOC//3), 1.47-1.50 (m, 2 H. octyl CH,), 1.24-1.29 (m, 10 H, octyl CH,). 0.87 (t, 3 H.
7= 7.1 Hz, octyl CHj); "C NMR (125.8 MHz, CDCI 3 ) 6 ^ 170.7, 165.3 (C=0), 138.5,
138.4, 138.4. 138.1, 137.7, 130.1, 129.9, 128.4, 128.3, 128.2, 128.1, 127.9, 127.7,
127.6, 127.6 (Ph), 97.9 (C-1), 97.6 (C-1'), 80.4, 77.8, 75.1, 74.9, 74.8, 74.6, 73.4,
72.7, 72.0, 71.5, 71.5, 69.8, 68.5, 67.7, 6 6 .8 , 63.3 (ring and benzylic C, octyl OCH,),
31.9, 29.4, 29.4, 29.2, 26.2, 22.7 (octyl CH,), 20.8 (OCOCH 3 ), 14.1 (octyl CH 3 ). HR-
ESl-MS calcd for for C^H. 3 0 , 3 [M -t- Na]" 1073.5027, found 1073.5011.
HO OH
BnO'
8 n'
6 A
BnO-W ^ \
127 OR R = (CH2)tCH3
Octyl 3,4,-di-0-benzyl-a-D-mannopyranosyl-(l->6)-2,3,4-tri-0-benzyl-a-
D-mannopyranoside (127). To a solution of 126 (1.90 g, 1.8 mmol) in methanol (50 mL), 10 drops of IM NaOMe was added. After stirring overnight, the solution was neutralized with a minimum amount of pre-washed Amberlite 118 H* resin and concentrated to a syrup, which was purified by chromatography (1:1 hexane;EtOAc) on
181 silica gel to give 127 (1.56 g, 96%) as a colorless syrup. 0.44 (1:1 hexane:EtOAc); ‘H
NMR (500 MHz, CDCI3) 6 ^ 7.20-7.48 (m, 25 H, Ph). 5.06 (d, 1 H, 7,-,.= 1.7 Hz, H-1'),
4.95 (d, 1 H, 7= 11.1 Hz, C//,Ph), 4.86 (d, 1 H, 7= 11.1 Hz. C//,Ph), 4.81 (d, 1 H,
7 ., = 1.7 Hz, H-1), 4.72 (ABq, 2 H, 7 = 12.3 Hz, Au = 27.8 Hz, CH .?h), 4.62-4.64 (m,
3 H, C H ,P hi 4.55 (ABq, 2 H, 7= 11.6 Hz, Au = 20.2 Hz, C H ,P h l 4.52 (d, 1 H, 7 =
12.0 Hz, C//,Ph), 4.05-4.06 (m, 1 H, H-2), 3.69-3.93 (m, 11 H, H-3, H-4, H-5, H- 6 a,
H-6 b, H-2', H-3', H-4', H-5', H- 6 a', H-6 b'), 3.58 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl
OCH,), 3.32 (dt, 1 H, 7 = 6 .6 , 9.7 Hz, octyl OCH,), 2.52 (d, 1 H, 7 = 1.8 Hz, OH), 2.05
(br. s. 1 H, OH), 1.48-1.51 (m, 2 H, octyl CH,), 1.26-1.31 (m, 10 H, octyl CH,), 0.87
(t, 3 H, 7 = 6.7 Hz, octyl CH 3 ); ‘^C NMR (125.8 MHz, CDCI 3) 5^ 138.9, 138.9, 138.2,
129.0, 128.8, 128.4, 128.4, 128.3, 128.2, 128.1, 128.1 (Ph), 99.9 (C-1), 99.1 (C-1'),
80.7, 79.9, 75.5, 75.5, 75.0, 74.4, 73.2, 72.5, 72.1, 72.0, 71.9. 68.5, 68.2, 66.7, 62.4
(ring and benzylic C, octyl OCH,), 32.3, 29.9, 29.9. 29.7, 26.6, 23.1 (octyl CH,), 14.6
(octyl CHj).
OH TBDPSO BnO" Bn P" BnO''V'^^ "S. BnO V i *^1 A
128 OR R = (CHgjyCHs
Octyl 3,4-di-0-benzyl-6-0-terf-butyIdiphenylsiIyI-a-D-mannopyranosyl-
(l->6)-2,3,4-tri-0-benzyI-a-D-mannopyranoside (128). To a solution of 127
(650 mg, 0.72 mmol) in DMF ( 6 mL), TBDPSCl (0.22 mL, 0.85 mmol) and imidazole (90
1 8 2 mg, 1.3 mmol) were added. After stirring overnight, the solution was concentrated under vacuum. The residue was taken in CH,CU (2 x 30 mL), washed with water (25 mL) and dried (Na^SO^), filtered, and concentrated to a colorless syrup. The crude syrup was purified by chromatography (4:1 hexaneiEtOAc) on silica gel to give 128 (610 mg, 74%) as a colorless syrup. R, 0.37 (4:1 hexane:EtOAc); [aj^+29.4° (c 0.5, CHClj); ‘H NMR
(400 MHz, CDCI3 ) 5h 7.73 (dd, 2 H, / = 1.0, 7.8 Hz, Ph), 7.68 (dd, 2 H, 7 = 1.0, 7.9
Hz. Ph), 7.15-7.40 (m. 36 H, Ph), 5.08 (br. s. 1 H. H-L), 4.88 (d, 1 H, 7= 11.0 Hz,
C H ,Phi 4.87 (d, 1 H, 7 = 11.0 Hz. CH,Ph), 4.81 (br. s, 1 H, H-1 ), 4.71 (ABq, 2 H, 7 =
12.3 Hz, Au = 24.0 Hz, CH,Ph), 4.60-4.64 (m, 4 H, CH,Ph), 4.56 (d, 1 H, 7 = 10.9 Hz.
CH,Ph), 4.49 (d. 1 H. 7 = 10.9 Hz, CH,Ph), 4.14 (br. s, 1 H, H-2), 3.80-3.97 (m, 7 H,
H-3. H-4, H-3', H-4'. H-5', H-6 a', H-6 b'), 3.77 (br. s, 1 H, H-2'), 3.68-3.71 (m, 3 H,
H-5, H-6 a, H-6 b), 3.59 (dt, 1 H, 7= 6.9, 9.2 Hz. octyl OCH,), 3.32 (dt, 1 H. 7= 6 . 8 ,
9.2 Hz, octyl OCH,), 2.30 (br. s, 1 H, OH), 1.48-1.51 (m, 2 H, octyl CH,), 1.19-1.31
(m, 10 H. octyl CH,), 1.04 (s, 9 H, te n -butyl CHj), 0.87 (t, 3 H, 7 = 7.1 Hz, octyl CHj);
'■’C NMR (125,8 MHz, CDCf) 5^ 138.5, 135.9, 135.7, 129.5, 128.5, 128.4, 128.3,
128.3, 128.3, 128.0, 127.8, 127.8, 127.8, 127.7, 127.6, 127.6, 127.5, 127.5 (Ph), 99.6
(C-1'), 97.6 (C-1), 80.3, 79.8, 75.1, 75.1, 75.0, 74.6, 74.1, 72.6, 72.3, 72.1, 71.6,
71.6, 68.2, 67.6, 66.0, 62.9 (ring and benzylic C, octyl OCH,), 31.9, 29.4, 29.4, 29.3
(octyl CH ,), 26.8 {len -butyl CH,), 26.2, 22.7 (octyl CH,), 19.3 (tert-butyl C(CH,),),
14.1 (octyl CH,). HR-ESI-MS calcd for C,,Hs,0,,Si [M + N a f 1165,5837, found
1165.5830.
1 8 3 HO OCH, BnO' Bn' 6 A ?Gn BnO' Bni
129 OR
R = (CH2)tC H3
O ctyl 3,4-di-0-benzyI-2-C>-methyl-a-D-mannopyranosyi-(l-4 6)-2,3,4-tri-0- benzyl-oc-D-mannopyranoside (129). To a solution of 128 (580 mg, 0.51 mmol) in
DMF (3 mL), NaH (60 mg, 2.5 mmol) was added at 0 °C. After stirring for 10 min, CH 3 I
(0.35 mL, 5.6 mmol) was added and the reaction mixture was stirred overnight. To tlie reaction mixture was added CH.OH (1 mL), followed by stirring for 1 h. The solvent was evaporated and the residue was dissolved in CHXU (2 x 25 mL), washed with water (25 mL), dried (Na,SO_,), filtered, and concentrated to a light yellow syrup, which was dried under vacuum for 4 h. The crude syrup was dissolved in THE (14 mL) and solid TBAF
(786 mg, 3.0 mmol) was added. After stirring for 48 h, the reaction mixture was concentrated and the residue was purified by chromatography (3:1 hexaneiEtOAc) on silica gel to give 129 (310 mg, 67% over two steps) as a colorless syrup. 0.41 (2:1 hexanezEtOAc): [0 ]^ +28.5° (c 0.2, CHCI3); ‘H NMR (500 MHz, CDCI3) 5^ 7.17-7.39
(m, 25 H, Ph), 5.09 (d, 1 H, 7,., = 1.6 Hz, H-1'), 4.92 (d, 1 H, 7= 11.0 Hz, Π,Ph),
4.89 (d, 1 H, 7= 11.0 Hz, Π,Ph), 4.81 (d, 1 H, 7, , = 1.6 Hz, H-1), 4.73 (ABq, 2 H, 7
= 12.2 Hz, Au = 36.0 Hz, C//,Ph), 4.63-4.66 (m, 3 H, C//,Ph), 4.60 (d, 1 H, 7 = 11.0
Hz, C7/,Ph), 4.58 (d, 1 H, 7 = 11.9 Hz, C/7,Ph), 4.46 (d, 1 H, 7= 11.0 Hz, C//,Ph),
184 3.87-3.94 (m, 4 H, H-2, H-3, H-3', H-5'), 3.83 (t, 1 H, = 7,^ = 9.5 Hz, H-4), 3.79
(t, l H ,7 ,.v = 1.6 Hz, 7 v 3.= 2.0 Hz, H-2'), 3.73 (dd, I H, 7;.,^-= 2.6 Hz, 11.7
Hz, H-6 b'), 3.61-3.71 (m, 4 H. H-5, H- 6 a, H-6 b, H- 6 b'), 3.58 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl OCH,), 3.41 (s, 3 H, OCHj), 3.33 (dt, 1 H, 7 = 6.5, 9.6 Hz. octyl OCH,), 1.92 (br. s, 1 H, OH), 1.49-1.51 (m. 2 H, octyl CH,), 1.26-1.32 (m, 10 H, octyl CH,), 0.88 (t, 3
H, 7 = 6 . 8 Hz, octyl CHj); ‘^C NMR (125.8 MHz, CDClj) 139.0, 138.9, 138.9,
138.6, 128.9, 128.8, 128.8, 128.4, 128.4, 128.3, 128.2, 128.2, 128.1, 128.1 (Ph), 98.3
(C-1'), 98.1 (C-1), 80.8, 79.4, 77.8, 75.6, 75.6, 75.6, 75.1, 75.0, 73.4, 72.6, 72.5,
72.1, 72.1, 68.1, 66.5, 62.7 (ring and benzylic C, octyl OCH,), 59.5 (OCH 3 ), 32.3,
29.8, 29.8, 29.7, 26.6, 23.1 (octyl CH,), 14.6 (octyl CH 3 ). HR-ESI-MS calcd for
C;,H,gO,, [M + Na]" 941.4816, found 941.4791.
Phthl OCH BnO'
BnO
Octyl 3,4-di-0-benzyi-6-deoxy-2-0-methyI-6-phthalimido-a-D- mannopyranosyl-(1^6)-2,3,4-tri-0-benzyI-a-D-mannopyranoside (130). To a solution of 129 (170 mg, 0.19 mmol), PPh 3 (100 mg, 0.38 mmol) and pthalimide (43 mg, 0.29 mmol) in dry THF ( 8 mL), was added DIAD (75 p.L, 0.38 mmol). After stirring overnight, the solution was concentrated under vacuum. The crude residue was purified by
185 chromatography (4:1 hexaneiEtOAc) on silica gel to give 130 (170 mg, 85%) as a colorless syrup. 0.34 (3:1 hexane:EtOAc); [a]g +16.5° (c 0.4, CHClj); 'H NMR (500
MHz, CDClj) 7.69-7.73 (m, 2 H, phthalimido), 7.56-7.59 (m, 2 H, phthalimido),
7.15-7.37 (m, 25 H, Ph), 5.04 (br. s, 1 H. H-1'), 5.03 (d, 1 H, 7= 11.0 Hz, C//,Ph),
4.77 (d, 1 H, y, ,= 2.0 Hz, H-1), 4.75 (d, 1 H, 7= 10.8 Hz, C//,Ph), 4.74 (d, 1 H, 7 =
12.2 Hz, CH.Ph), 4.71 (d, 1 H, 7 = 11.4 Hz, CH.Ph), 4.67 (d, 1 H, 7 = 12.2 Hz,
C//,Ph), 4.62-4.64 (m, 2 H, C//,Ph), 4.52 (d, 1 H, 7 = 12.0 Hz, C//,Ph), 4.51 (d, 1 H,
7 = 11.9 Hz, C //,Ph), 4.38 (d. 1 H. 7 = 10.8 Hz, C //,P h). 4.00-4.04 (m, 1 H, H-5'),
3.98 (dd. 1 H, 7,.^.= 7.1 Hz, 7 ,,.^ = 13.8 Hz, H- 6 b'), 3.90 (dd, 1 H, 7,.^. = 4.3 Hz,
7^.^. = 13.8 Hz, H- 6 a'), 3.83-3.88 (m, 2 H. H-3, H-3'), 3.81 (t, 1 H, 7y, = 7,.^ = 9.5
Hz, H-4'), 3.76 (d, 1 H, J.-y = 2.3 Hz, H-2'), 3.74 (t, 1 H, 7,, = 7,, = 2.0 Hz, H-2),
3.72 (t, 1 H, 7 ,, = 7 , 3 = 9.5 Hz, H-4), 3.54-3.59 (m, 3 H. H-5, H- 6 a, H-6 b), 3.51 (dt. 1
H, 7 = 6 .8 , 9.7 Hz, octyl OCH,), 3.33 (s, 3 H, OCH 3 ), 3.29 (dt, 1 H, 7= 6 .6 , 9.7 Hz, octyl OCH,), 1.45-1.47 (m, 2 H, octyl CH,), 1.24-1.32 (m, 10 H, octyl CH,), 0.87 (t, 3
H, 7= 6.9 Hz, octyl CH,); "C NMR (125.8 MHz, CDCI 3) 6 c 168.6 (C=0), 139.3,
139.0, 138.9, 138.5, 134.0, 132.5, 128.8, 128.8, 128.8, 128.6, 128.5, 128.5, 128.3,
128.1, 128.1, 128.0, 128.0, 127.7, 123.5 (Ph), 98.2 (C-1), 97.6 (C-1'), 80.7, 79.5,
77.5, 75.8, 75.6, 75.0, 74.9, 73.4, 73.4, 72.7, 72.4, 71.8, 71.8, 69.3, 68.0, 66.2 (ring and benzylic C, octyl OCH.), 59.1 (OCH 3), 32.3, 29.9, 29.7, 29.3, 26.6, 23.1 (octyl
CH.), 14.5 (octyl CH,). HR-ESI-MS calcd for C„H, 3 0 ,.N [M + Na]' 1070.5030. found
1070.4985.
1 8 6 OCH]
HO'
OH
HO'
OR
Octyl 6-amino-6-deoxy-2-0-methyI-a-D-mannopyranosyl-(l— > 6 )-a -D - mannopyranoside (29). To a solution of 130 (164 mg, 0.16 mmol) in n-butanol (18 mL), was added ethylenediamine (4 mL). After stirring overnight at 90 "C, the solution was cooled to room temperature and concentrated under vacuum. The residue was coevaporated with toluene (2 .\ 15 mL) and ethanol (20 mL) to obtain a yellow syrup. The crude amine was dissolved in HOAc (5 mL), and 10% Pd/C (35 mg) was added. The solution was stirred overnight under an H, atmosphere and the catalyst was separated by filtration and washed with CH 3OH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (10:2:0.5 CHCl3:CH3ÜH:(5N) aq.NH_,OH) on latrobeads to give 29 (34 mg, 84% over two steps) as a colorless solid.
0.63 (10:4:1 CHCl 3:CH3ÜH:(5N) aq.NH.OH); [al^ +49.4° (c 0.2, CH 3OH); ‘H NMR
(800 MHz, CD 3OD) 5h 5.05 (br. s, 1 H, H-1'), 4.75 (br. s, 1 H, H-1), 3.91 (dd, 1 H,
5.8 Hz, 10.8 Hz, H-6 b). 3.83-3.85 (m, 2 H, H-2, H-5'), 3.82 (d, 1 H,
= 10.8 Hz, H-6 a), 3.79 (dd, 1 H, J,.y= 3.4 Hz, Jy,.= 9.7 Hz, H-3'), 3.72-3.73 (m, 2 H,
H-5, octyl OCH 3), 3.70 (dd, 1 H, 7,^ = 4.8 Hz, Jy, = 9.6 Hz, H-3), 3.66 (t, 1 H, Jy^ =
J,y=9.6 Hz, H-4), 3.54-3.55 (m, 1 H, H-2'), 3.49 (t, 1 H, Jyy=Jy^.= 9.7 Hz, H-4'),
187 3.48 (s, 3 H, OCH 3 ), 3.45 (dt, 1 H, 7 = 6.2, 9.5 Hz, octyl OCH,), 3.39 (dd, I H, 7y^.=
2.9 Hz, 7,,.^.= 13.0 Hz, H- 6 a'), 3.03 (dd, 1 H. 7;.^-= 9.1 Hz, 7,,.^= 13.0 Hz, H- 6 b'),
1.58-1.64 (m, 2 H, octyl CH,), 1.31-1.44 (m, 10 H, octyl CH,), 0.92 (t, 3 H, 7 = 6 . 8
Hz, octyl CH 3 ); "C NMR (150.9 MHz. CD 3OD) 101.7 (C-1, '7^^ = 167.9 Hz), 98.2
(C -1','7c.h = 169.8 Hz), 81.8 (C-2'), 73.1 (C-3), 72.2 (C-5), 72.1 (C-3', C-5'), 70.3 (C-
4'), 70.2 (C-2), 68.7 (octyl OCH,), 6 8 . 6 (C-4), 67.7 (C-6 ), 59.3 (OCH3), 42.3 (C-6 '),
33.0, 30.6. 30.5. 30.4. 27.4. 23.7 (octyl CH,). 14.4 (octyl CH 3 ). HR-FAB-MS calcd for
C,,H^,0,„N [M 4- H]" 468.2808. found 468.2824.
OCH.
B n O ' Sri
BnO'
Octyl 3,4-di-0-benzyl-6-deoxy-6-fluoro-2-6)-methyl-a-D-raanno pyranosyI-(1^6)-2,3,4-tri-<9-benzyl-a-D-manncpyranoside (132). To an ice- cold solution of 129 (147 mg, 0.16 mmol) in dry CH,C1, (4 mL) and dry pyridine (130 pL. 1.6 mmol). triflic anhydride (50 pL, 0.32 mmol) was added dropwise. After stirring for 1 h. the solution was diluted with CH,C1, (2 x 20 mL), washed with ice-cold aqueous
IM HCl (20 mL) and then ice-cold water (20 mL). The organic extract was dried
(Na,SOJ, filtered and concentrated to a yellow syrup. The crude triflate was dissolved in
TBAF( IM) in THF and stirred at 50 °C overnight. The solution was concentrated and the residue was purified by chromatography (2:1 hexanerEtOAc) on silica gel to give 132 (115
1 8 8 mg, 78% over two steps) as a colorless syrup. 0.71 (2:1 hexanerEtOAc); [a]g +36.5°
(c 0.4, CHCI3 ); ‘H NMR (500 MHz, CDCI3) 6 ^ 7.17-7.39 (m, 25 H, Ph), 5.12 (d, 1 H, y, v= 2.0 Hz, H-1'), 4.93 (d, 1 H. 7= 11.0 Hz, C//,Ph), 4.89 (d, 1 H, 7 = 11.0 Hz,
C//,Ph). 4.80 (d. 1 H. 7 , 3 = 2.0 Hz, H-1), 4.76 (d, 1 H. 7= 12.2 Hz, C//,Ph), 4.69 (d,
1 H, 7= 12.2 Hz, C//,Ph), 4.64-4.67 (m. 4 H, C//,Ph), 4.57 (d, 1 H, 7 = 12.2 Hz,
C //,Ph), 4.52 (ddd, 1 H, 7 ,.^ = 3.6 Hz. 7,,.^ = 10.1 Hz, 7H,b,p= 48.1 Hz, H- 6 b'), 4.47
(d. 1 H, 7 = 11.0 Hz, C//,Ph), 4.46 (ddd, 1 H, 7,.^ = 1.4 Hz, 7,,.^ = 10.1 Hz,
48.1 Hz, H- 6 a'), 3.85-3.93 (m, 7 H, H-3, H-4, H-5, H- 6 b, H-3', H-4', H-5'), 3.79 (t, 1
H, 7 , . 3 = 7 3 3 = 2.0 Hz, H-2'), 3.70 (dd, 1 H, 7^^= 1.3 Hz, 7,,,, = 11.6 Hz, H-6 a), 3.67
(t, 1 H. 7, 3= 7 3 3 = 2.0 Hz, H-2), 3.58 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl OCH3), 3.42 (s, 3
H, OCH,), 3.33 (dt, 1 H, 7 = 6.5, 9.6 Hz, octyl OCH3), 1.48-1.51 (m, 2 H, octyl CH,),
I.26-1.31 (m, 10 H, octyl CH,), 0.88 (t, 3 H, 7= 6.7 Hz, octyl CH,); '^C NMR (100.6
MHz, CDCI3 ) 6 c 139.0, 138.9, 138.8, 138.6, 128.9, 128.8, 128.8, 128.7, 128.6, 128.5,
128.3, 128.3, 128.2, 128.1, 128.1, 128.0 (Ph), 98.4 (C- 1 ), 98.1 (C- 1 '), 82.7 (C- 6 ', 7^.
3 P= 172.7 Hz), 80.9, 79.4, 77.6, 75.7, 75.6, 75.6, 75.5 (ring and benzylic C), 74.1 (C-
4', 7c^ p= 6.4 Hz), 73.5, 72.7, 72.1, 72.0 (ring and benzylic C), 71.5 (C-5', Jc.5-,f= 18.2
Hz), 68.1, 6 6 . 6 (ring and octyl OCH 3 ), 59.4 (OCH 3), 32.3, 29.9, 29.7, 26.6, 23.1 (octyl
CH 3), 14.6 (octyl CH,). 'T NMR (235.4 MHz, CDCI 3) 5p - 231.9 (td, 1 F,
= 48.1 Hz, 7 h5 p = 28.0 Hz, F- 6 '). HR-ESI-MS calcd for C^^H.^O.oF [M + Na]"
943.4772, found 943.4863.
1 8 9 OCH
HO
HO"
Octyl 6-deoxy-6-fluoro-2-0-methyi-a-D-mannopyranosyl-(l->6)-a-
D-mannopyranoside (30). To a solution of 132 (100 mg, 0.11 mmol) in HOAc ( 6 mL), was added 10% Pd/C (40 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CHjOH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography (4:1 CH^CLiCH^OH) on latrobeads to give 30 (40 mg, 78%) as a foam.
Rf 0.74 (4:1 CHXLrCHjOH); [aj^ +59.7= (c 0.15, CH,OH); 'H NMR (800 MHz,
CDjODIÔh 5.02 (d, 1 H, 7, , = 1.3 Hz, H-1'), 4.72 (br. s, 1 H, H-1), 4.63 (ddd, 1 H,
7; = 4.2 Hz, 7^^ = 10.1 Hz, 7^^^ p = 46.7 Hz, H- 6 b'), 4.58 (ddd, 1 H, 7^^ = 1.1 Hz,
7,,.,,.= 10.1 Hz, 7h,,,p= 46.7 Hz, H- 6 a'), 3.91 (dd, 1 H, 7,,, = 5.1 Hz, 7^^ = 10.9 Hz,
H-6 b), 3.81 (br. s, 1 H, H-2), 3.79 (dd, 1 H, J..y= 3.4 Hz, 7y,.= 9.7 Hz, H-3'), 3.76 (d,
1 H ,7 ^ ,,= 10.9 Hz, H- 6 a), 3.73-3.74 (m, 1 H, H-5'), 3.72 (dt, 1 H, 7 = 6 .6 , 9.7 Hz, octyl OCH,), 3.65-3.69 (m, 3 H, H-3. H-4, H-5), 3.58 (t. 1 H, 7 y = 7,.^. = 9.7 Hz, H-
4'), 3.52 (dd, 1 H, 7,-,-= 1.3 Hz, 7, , = 3.4 Hz, H-2'), 3.49 (s, 3 H, OCH,), 3.43 (dt, 1
H, 7= 6.3, 9.7 Hz, octyl OCH,), 1.58-1.65 (m, 2 H, octyl CH,), 1.32-1.42 (m, 10 H, octyl CH,), 0.92 (t, 3 H, 7 = 7.2 Hz, octyl CH,); ‘^C NMR (150.9 MHz, CD,OD) 6 ^
101.7 (C-1, '7c_H= 167.9 Hz), 98.2 (C-1', ‘7c.h = 168.9 Hz), 83.4 (C- 6 ', 7^^.^ = 170.9
1 9 0 Hz), 81.8 (C-20, 73.2 (C-5), 73.1 (C-5', 7 c-5-.f= 18.5 Hz), 72.8 (C-3'), 72.4 (C-3), 72.1
(C-2), 6 8 . 6 (octyl OCH,). 68.5 (C-4), 67.7 (C-4', 7 ^.4 .? = 6 . 8 Hz), 67.6 (C-6 ), 59.3
(OCH,). 33.0, 30.6, 30.5, 30.4. 27.4, 23.7 (octyl CH,), 14.4 (octyl CH,). '"F NMR
(235.4 MHz, CDjOD) - 234.6 (td. 1 F, /H6a,F= ^ 6b.F= 46.7 Hz, y„,.p= 25.9 Hz, F- 6 ').
HR-ESI-MS calcd for for C„H,,0,^F [M + H]"493.2425, found 493.2415.
Bn( OCH Bni OBn Bni
OR
Octyl 3,6-anhydro-4-0-benzyl-2-0-methyl-a-D-mannopyranosyl-(l->6)-
2,3,4-tri-O-benzyi-a-D-mannopyranoside (131). To a solution of 129 (133 mg,
0.15 mmol) in dry CH,C1, (7 mL) at -40 °C, DAST (100 p.L. 0.76 mmol) was added.
After stirring for 6 h. was addedmethanol ( I mL) and the solution was concentrated. The residue was taken in CH,C1, (2 \ 20 mL), washed with water (20 mL) and dried (Na,SO^), filtered, and concentrated to a brown syrup. The crude syrup was purified by chromatography (3:1 hexane:EtOAc) on silica gel to give 131 (100 mg, 83%) as a light yellow syrup. R^ 0.53 (2:1 hexane:EtOAc): [ajg +29.5° (c 0.2, CHCl,); ‘H NMR (400
MHz, CDCl,) 0„ 7.21-7.37 (m, 20 H. Ph), 4.93 (d, 1 H, 7,.,.= 6.5 Hz, H-1'), 4.86 (d, 1
H .y = 10.6 Hz, C77,Ph), 4.81 (d, 1 H, 7, , = 1.5 Hz, H-1), 4.74 (d, 1 H, 7= 12.2 Hz,
C77,Ph), 4.73 (d, 1 H, 7 = 10.5 Hz, C//,Ph), 4.62-4.68 (m, 4 H, C77,Ph), 4.47 (d, 1 H,
7= 11.8 Hz, C//,Ph), 4.36 (t, 1 H, J^. = 7,^ = 2.0 Hz, H-2), 4.23-4,27 (m, 3 H, H-3',
191 H-4', H-5'), 3.87-3.94 (m, 4 H, H-3, H-4, H-5, H- 6 b'), 3.81 (dd, 1 H, 1.4 Hz,
V 6b = H O Hz, H- 6 a'), 3.73-3.76 (m, 2 H, H-6 a, H-6 b), 3.63 (dt, 1 H, 7 = 6 .8 , 9.6 Hz, octyl OCH,), 3.49 (dd, 1 H, 7, , = 6.5 Hz, 7,.= 1.2 Hz, H-2'), 3.41 (s, 3 H, OCH,),
3.32 (dt, 1 H, 7= 6 .6 , 9.6 Hz, octyl OCH,), 1.47-1.50 (m, 2 H, octyl CH,), 1.25-1.32
(m, 10 H, octyl CH,), 0.88 (t, 3 H, 7 = 7.1 Hz, octyl CH,); “'C NMR (125.8 MHz,
CDCl,) 5c 138.8, 138.7, 138.5, 137.4, 128.5, 128.3, 128,3, 128.2, 127.9, 127.8,
127.7, 127.6, 127.6, 127.5, 127.5 (Ph), 102.0 (C-1'), 97.6 (C-1), 80.1, 78.0, 77.3,
75.5, 75.2, 75.2, 74.9, 72.6, 72.6, 72.5, 71.7, 71.3, 69.7, 68.2, 67.6 (ring and benzylic
C, octyl OCH,), 59.0 (OCH,), 31.8, 29.4, 29.4, 29.2, 26.2, 22.7 (octyl CH,), 14.1
(octyl CH j). HR-ESI-MS calcd for C^yH^,0,^ [M + Na|* 833.4241, found 833.4232.
HQ OCH,
OH
OR
Octyl 3,6-anhydro-2-0-methyl-a-D-mannopyranosyI-(l—» 6 )-a-D - mannopyranoside (33). To a solution of 131 (94 mg, 0.12 mmol) in HOAc ( 8 mL), was added 10% Pd/C (35 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CHjOH (10 mL). After concentrating the filtrate and the washings, the product was purified by chromatography
(9:1 CH,CI,:CHjOH) on latrobeads to give 33 (34 mg, 65%) as a foam. R^ 0.36 (9:1
CH,Cl,:CHjOH); [a]^ +74.8° (c 0.2, CHjOH); ‘H NMR (800 MHz, CDjOD) 0» 4.89 (d.
192 I H, y,.,.= 6 . 6 Hz, H-1'), 4.73 (d, 1 H, /, ,= 1.7 Hz, H-I), 4.25 (t, I H,
2.6 Hz, H-5'), 4.22 (d, I H, Jy ,.= 6.1 Hz, H-3'), 4.20 (dd, I H, Jy , = 6.1 Hz, 7,.^.= 2.6
Hz, H-4'), 4.07 (dd, 1 H, 7,^ = 5.4 Hz, 7,^,^ = H.O Hz, H-6 b), 4.03 (d, 1 H, 7^.^.=
10.5 Hz, H-6 a'), 3.98 (dd, 1 H, 7^.= 2.6 Hz, 7,,.,^ = 10.5 Hz, H-6 b'), 3.88 (d. 1 H,
7^^= 11.0 Hz, H-6 a), 3.80 (dd, 1 H, 7, ,= 1.7 Hz, 7 ,,= 3.1 Hz, H-2), 3.74 (dt, 1 H, 7
= 6 .6 , 9.7 Hz. octyl OCH,), 3.68-3.71 (m. 3 H, H-3, H-4, H-5), 3.46 (d, 1 H. 7,., = 6 . 6
Hz. H-2'), 3.49 (s. 3 H, OCH,), 3.42 (dt. 1 H. 7= 6.3, 9.7 Hz. octyl OCH,), 1.58-1.62
(m. 2 H, octyl CH,), 1.32-1.41 (m, 10 H, octyl CH,), 0.92 (t, 3 H, 7 = 7.2 Hz, octyl
CH,); ‘-'C NMR (150.9 MHz. CD,OD) 102.8 (C-1', ‘7^,,, = 161.4 Hz), 101.6 (C-1,
'7c,h= 168.1 Hz), 79.5 (C-2'), 77.1 (C-4'), 76.9 (C-3'), 73.2 (C-5), 72.5 (C-3). 72.2 (C-
2). 72.0 (C-5'), 70.4 (C-6 '), 69.6 (C- 6 ), 6 8 . 6 (octyl OCH,), 6 8 . 6 (C-4). 58.8 (OCH,).
33.0. 30.6. 30.5. 30.4. 27.4. 23.7 (octyl CH,). 14.4 (octyl CH,). HR-ESI-MS calcd for
C,,H,sO,o [M -H N ar 473.2363. found 473.2325.
QBz
Octyl 2,3,4-tri-(9-benzoyl-a-D-mannopyranoside (133). To a solution of
79 (1.8 g, 3.4 mmol) in dry pyridine (15 mL), was added BzCl (1.6 mL, 13.6 mmol).
After stirring overnight, a saturated NaHCO, solution (25 mL) was added and the solution was stirred for 2 h. The reaction mixture was extracted with CH,C1, (2 x 50 mL) and the organic layer was washed with washed with aqueous IM HCl (2 x 30 mL) and water (30
1 9 3 mL). The organic extract was dried (Na^SOJ, filtered, and concentrated to a yellow syrup.
The crude syrup was dissolved in CH,CL:CH]OH (4:1, 30 mL), p-toluenesulfonic acid
(300 mg, 1.6 mmol) was added and the mixture was stirred overnight. The reaction
mixture was then diluted with CH,C1, (60 mL), washed with a saturated NaHCO^ solution
(2 X 30 mL) and water (30 mL). The organic extract was dried (Na,SO_,), filtered, and concentrated to a light yellow syrup which was purified by chromatography (4:1
hexane:EtOAc) on silica gel to give 133 (1.7 g, 83%) as a colorless syrup. R, 0.22 (4:1
hexane:EtOAc): [alp -76.1= (c 1.3, CHClj); 'H NMR (500 MHz, CDCI 3) 5^ 8.15 (dd, 2
H, y = 1 . 1 . 8 . 2 Hz, Ph), 8.03 (dd, 2 H, 7 = 1.1, 8.1 Hz, Ph), 7.66 (tt, 1 H, 7 = 1.2, 8 . 6
Hz, Ph), 7.28-7.56 (m, 10 H, Ph), 6.03 (dd, 1 H, 7 , 3 =3.4 Hz, 10.1 Hz, H-3),
5.88 (t, 1 H, 7 3 , = 7 , 3 = 10.1 Hz, H-4), 5.72 (dd, 1 H, 7, ,= 1.8 Hz, 7 , 3 = 3.4 Hz, H-2),
5.14 (d, 1 H, 7, ,= 1.8 Hz, H-1), 4.11 (tt, 1 H, 7;,b=3.7 Hz, 7 , 3 = 10.1 Hz, H-5). 3.81-
3.87 (m, 3 H, H- 6 a, H-6 b, octyl OCH,), 3.60 (dt, 1 H, 7 = 6 .6 , 9.6 Hz, octyl OCH,),
2.65 (br. s, 1 H, OH), 1.66-1.78 (m, 2 H, octyl CH,), 1.26-1.48 (m. 10 H, octyl CH,),
0.95 (t, 3 H, 7 = 6 . 8 Hz, octyl CH,); ‘‘C NMR (125.8 MHz, CDCI 3) 166.6, 166.5,
165.5 (C=0), 133.7, 133.5, 133.2, 130.0, 129.9, 129.7, 129.4, 128.8, 128.7, 128.5,
128.3 (Ph), 97.8 (C-1), 70.9, 70.8, 70.0, 68.7, 67.5, 61.5 (ring and octyl OCH,), 31.9,
29.4, 29.4, 29.3, 26.2, 22.7 (octyl CH,), 14.1 (octyl CH 3 ). Anal. Calcd for C 33H,qO,
(604.70): C, 69.52; H, 6.67. Found: C, 69.65; H, 6.70.
6 A BzO Bzi
134 OR R=(CH2)7CH3
194 Octyl 3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-mannopyranosyI-(l-^6)-
2,3,4-tri-0-benzoyl-a-D>mannopyranoside (134). Trichloroacetimidate 7 7 '°*
(520 mg, 1.1 mmol) and alcohol 133 (551 mg, 0.91 mmol) were dried in vacuo with
powdered 4 Â molecular sieves (1.0 g) overnight. Dry CHXh (15 mL) was added and the
mixture was cooled to -10 °C with stirring. A solution of TMSOTf (65 pL) in CH^Ch (200
laL) was added dropwise to the reaction mixture and the stirring was continued for 2 h.
The reaction mixture was neutralized by the addition of a saturated NaHCOj solution (1.0
mL) and CH,C1, (40 mL) was added. The organic layer was washed with water (20 mL),
dried (Na,SO^), filtered and concentrated to a yellow syrup. The crude syrup was purified
by chromatography (2:1 hexaneiEtOAc) on silica gel to give 134 (550 mg, 6 6 %) as a
yellow syrup. R, 0.47 (2:1 hexane:EtOAc); [ajg +1.1° (c 0.8, CHCI 3); ‘H NMR (500
MHz, CDCl.) 8.12 (dd, 2 H, V = 1.4, 7.1 Hz, Ph), 8.00 (dd, 2 H, 7 = 1.3, 6 . 6 Hz,
Ph), 7.82 (dd, 2 H, y = 1.2, 7.4 Hz, Ph), 7.63 (tt, 1 H, 7 = 1.2, 7.4 Hz, Ph), 7.24-7.56
(m, 8 H, Ph). 5.98 (t. 1 H, 7^, = 7,^ = 10.1 Hz, H-4), 5.89 (dd, 1 H. J., = 3.4 Hz, 7,, =
10.1 Hz, H-3), 5.69 (dd, 1 H. 7,,= 1.7 Hz. 7 , 3 = 3.4 Hz, H-2), 5.42 (dd, 1 H, 7yj.=
3.8 Hz, Jy,.= 9.9 Hz. H-3'), 5.29 (t, 1 H, Jy,-= J,.y= 9.9 Hz, H-4'), 5.07 (d, 1 H, 7,, =
1.7 Hz, H-1), 4.90 (d, 1 H, 7, , = 1.4 Hz, H-1'), 4.27-4.30 (m, 1 H, H-5'), 4.03-4.06
(m, 2 H, H-4. H-2'), 3.93 (dd, 1 H, 7,^= 4.7 Hz, 7^6b= H I Hz, H- 6 b), 3.88-3.91 (m,
2 H, H-6 a', H-6 b'), 3.82 (dt, 1 H, 7= 6.7, 9.6 Hz, octyl OCH,), 3.69 (dd, 1 H, =
2.4 Hz, 7 ^ = 11.1 Hz, H-6 a), 3.57 (dt, 1 H, 7= 6.7, 9.6 Hz, octyl OCH,), 2.11 (s, 3
H, OCOC 7 / 3), 2.07 (s, 3 H, O C O C // 3), 1.95 (s, 3 H, OCOCH^), 1.69-1.75 (m, 2 H,
octyl CH,), 1.24-1.45 (m, 10 H, octyl CH,), 0.89 (t, 3 H, 7= 7.1 Hz, octyl CH 3 ); '^C
NMR (125.8 MHz, CDCI 3) 5c 170.6, 169.9, 169.6, 165.6, 165.5 (C=0), 133.6, 133.6,
133.2, 129.9, 129.8, 129.7, 129.3, 129.1, 129.0, 128.8, 128.6, 128.3 (Ph), 98.1 (C-1'),
97.7 (C-1), 71.0, 70.8, 70.2, 69.3, 6 8 .8 , 6 8 .6 , 67.1, 66.5, 65.7, 62.0, 61.3 (ring and
octyl OCH,), 31.9, 29.4, 29.4, 29.3, 26.2, 22.7 (octyl CH,), 20.7, 20.6, 20.5
1 9 5 (O C O C //3), 14.1 (octyl C H 3 ) . HR-ESI-MS calcd for for [M-^Na]" 940.3480, found 940.3432.
hcA ^3 — L l
31 OR
R = (CH2 )tCH3
O ctyl 2-azido-2-deoxy-a-D-mannopyranosyI-(l—>6)-a-D-mannopyranoside
(31). To a solution of 134 (450 mg, 0.49 mmol) in CH 3OH (25 mL), 10 drops of IM
NaOMe was added. .-Xfter stirring overnight, the solution was neutralized with a minimum amount of pre-washed Amberlite 118 H* resin and concentrated to a syrup, which was purified by chromatography (4:1 CH,CL:CH 3 0 H) on latrobeads to give 31 (207 mg,
8 8 %) as a colorless solid. R, 0.67 (4:1 CH,C 1,:CH 3ÜH); [aj^ +18.0° (c 0.1, H^O); 'H
NMR (800 MHz, D,0) 4.85 (br. s. 1 H, H-T), 4.73 (br. s, 1 H, H-1), 4.00 (dd, 1 H,
Jyy= 3.3 Hz, Jyy= 8.1 Hz. H-3'), 3.94 (d, 1 H, = 3.3 Hz, H-2'), 3.89-3.90 (m, 1
H, H-6 b), 3.84 (br. s, 1 H, H-2), 3.78 (d, 1 H, 7,,.^= 11.9 Hz, H- 6 a'), 3.67-3.69 (m, 3
H, H-3, H-4, H-6 b'), 3.63 (d, 1 H, 7^^= 11.2 Hz, H- 6 a), 3.58-3.60 (m, 4 H, H-5, H-
4', H-5', octyl OCH,), 3.38 (dt. 1 H, 7= 6 .6 , 9.7 Hz, octyl OCH,), 1.52-1.54 (m, 2 H, octyl CH,), 1.20-1.24 (m, 10 H. octyl CH,), 0.80 (t, 3 H, 7= 7.0 Hz, octyl CHj); ‘^C
NMR (150.9 MHz, D,0) 6 ^ 101.6 (C-1. 171.5 Hz), 99.0 (C-1', ‘7c.h = 171.1 Hz),
74.2, 72.6, 72.1 (C-3, C-5, C-5'), 72.0 (C-3'), 71.7 (C-2), 69.2 (C-4'), 68.3 (octyl
OCH,), 67.9 (C-4), 67.1 (C- 6 ), 65.3 (C-2'), 62.3 (C-6 '), 33.2, 30.8, 30.7, 30.6, 27.5,
196 23.9 (octyl CH,), 15.2 (octyl CH 3 ). HR-FAB-MS calcd for C^gH^.O.oN^ [M + N a]'
502.2377, found 503.2360.
hcA " bx X 1pH
b s
32 OR R = (CHglyCHa
Octyl 2-chloroacetamido-2-deoxy-a-D-mannopyranosyl-(l-»6)-a-D- tnannopyranoside (32). To a solution of 31 (96 mg, 0.20 mmol) in CH3OH (5 mL), was added 10% Pd/C (20 mg). The solution was stirred overnight under an H, atmosphere and then the catalyst was separated by filtration and washed with CH 3OH ( 8 mL).
Concentrating the filtrate and the washings, gave the amine 24 (90 mg, 98%) as a colorless foam. The crude amine (60 mg, 0.13 mmol) was dissolved in methanol (4 mL), and then, solid NaHCO; (175 mg) and chloroacetic anhydride (300 mg, 2.0 mmol) were
added. After stirring for 2 days, the solution was filtered and the filtrate was concentrated.
The product was purified by chromatography (4:1 CH 3C 1,:CH 3 0 H) on latrobeads. Excess chloroacetic anhydride was removed by passing an aqueous solution of the product through
a C-18 coated silica gel cartridge; followed by elution with methanol. The methanol
fractions were concentrated to give 32 (50 mg, 74%) as a colorless solid. R^ 0.56 (4:1
CH,CL:CH 3 0 H); [a]^ +39.3° (c 0.2, CH 3OH); 'H NMR (800 MHz, D,0) 5„ 4.75 (br. s,
1 H, H-I'), 4.74 (br. s, I H, H-I), 4.37 (d, I H, 7,. 3.= 4.7 Hz, H-2'), 4.10 (ABq, 2 H, 7
197 = 13.7 Hz, Au = 19.2 Hz, -C(0)C//,C1), 4.01 (dd, I H, A y = 4.7 Hz, 7 y = 9.7 Hz, H-
3'), 3.91 (d, 1 H, 11.3 Hz, H- 6 b), 3.83-3.84 (m, 1 H, H-2), 3.79 (dd, 1 H, 7;.^- =
3.9 Hz, = 12.5 Hz, H- 6 b'), 3.76 (d, 1 H, 7 ,,.^ = 12.5 Hz, H- 6 a'), 3.72 (t, 1 H, 7^,
= 7 , 5 = 9.6 Hz, H-4), 3.66 (dd, 1 H. 7,^ = 2.7 Hz, J ,, = 9.6 Hz, H-3), 3.62-3.65 (m, 2
H, H-5, H-50, 3.58-3.59 (m, 2 H, H- 6 a, octyl OCH,), 3.56 (t, 1 H, 7y, = 7, y= 9.7 Hz,
H-4'), 3.37 (dt, 1 H, 7 = 6.3, 9.6 Hz, octyl OCH,), 1.48-1.51 (m, 2 H, octyl CH,), 1.18-
I.27 (m, 10 H, octyl CH,), 0.78 (t. 3 H. 7= ’’.I Hz, CH,); ”C NMR (150.9 MHz,
D,0) 5c 171.0 (C=0), 101.6 (C-1'. '7c,h= 168.6 Hz). 99.7 (C-1, ‘7 c.h = 174.1 Hz), 73.7
(C-5'), 72.6 (C-3), 71.9 (C-5), 71.8 (C-2), 70.7 (C-3'), 69.3 (octyl OCH,), 67.9 (C-4'),
67.9 (C-4), 6 6 . 8 (C-6 ), 61.8 (C- 6 '), 54.4 (C-2'), 43.8 (-C(0)C//,C1), 33.1, 30.7, 30.6,
30.5. 27.4, 23.9 (octyl CH,), 15.2 (octyl CH,). HR-FAB-MS calcd for C,,H,^0,,NC1 [M
+ N ar 552.2188, found 552.2159.
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206 APPENDIX A
PRELIMINARY BIOLOGICAL INVESTIGATIONS
A.l INTRODUCTION
In this section, we will describe the results from the biological screening of
disaccharides 20-24 as a ( l—>6 )-ManT substrates and inhibitors. The experiments were carried out by Dr. G. Besra and coworkers at New Castle University, United Kingdom.
Each disaccharide was incubated with ‘■*C,-labeled |3-D-mannopyranosyl phosphodecaprenol 14 (Cjo-Man) in the presence of membrane extracts from M. smegmatis. '‘‘C,-labeled 14 (C;o-Man) was in turn synthesized in situ from '■‘C,-labeled
GDP-Man and decaprenolphosphate (C 5 0 -P). Following incubation, residual 14 was
removed by passage of the incubation mixture through a ion-exchange cartridge and the radioactivity in the effluent was measured by scintillation counting.
207 OH
HO
GDP-Man ^op
Decaprenol phosphate
o
14 4-
GDP
* = ■ ' ' ♦ c
Figure A. I Synthesis of 14 (Cjo-Man) from GDP-Man
208 OH HO
MO'
+
HO' OH
HO'
OH
HO'
0(CH2);CH3 20 Example of a potential substrate
a (1-^6)-ManT
OH HO
MO'
OH
HO
= 14c OH
HO'
0(CH2)yCH3
Figure A.2 An illustrative example of a reaction of with ‘"‘C,-labeled 14 (Cjo-Man)
with a potential substrate 20 catalyzed by a (l-» 6 )-ManT
209 As mentioned in chapter 1, LAM from M. tuberculosis and M. smegmatis are identical and thus both organisms possess similar mannosyltransferase activities; the latter has been used as a model for M. tuberculosis because it is "fast growing” and is not pathogenic to humans. The experimental details and the results from biological studies are described below.
A.2 PREPARATION OF MEMBRANE FRACTIONS FROM M. smegmatis
M. smegmatis mc‘ 155 cells"'' were grown in Luna Bertoni broth (Gibco BRL,
Life technologies. Paisley, Scotland) to mid-log phase (about 16 hours), harvested and washed with phosphate buffered saline (pH 7.4) and stored at -20 °C. Approximately 5 g of wet weight cells were first washed and then re-suspended at 4 "C in buffer A containing 50 mM MOPS (4-morpholine propane sulfonic acid, adjusted to pH 7.9 with
KOH), 5 mM p-mercaptoethanol and 10 mM MgCL. After the addition of 150 pg of
Dnase I (Type IV. Sigma Chemical Co., St, Louis, MO) and 250 pg of Rnase
(Microsomal Nuclease [Sigma]), the cells were subjected to probe sonication (Soniprep
150; MSE Ltd,, Crawley, Sussex, United Kingdom; 1 cm probe, 10 Amplitude microns) at 4 =’C. The mycobacterial strains were sonicated for a total time of 10 min in 10 x 60 s cycles with 90 s cooling intervals between pulses. The resulting preparations were centrifuged initially at 27,000 x g for 20 min at 4 °C. Membranes and cytosolic fractions
were obtained by further centrifugation of the resulting supernatant fraction at 1 0 0 , 0 0 0 x g for 2 h at 4 °C, The cytosolic fractions (supernatants) were withdrawn and membranes
2 1 0 (pellets) were re-suspended in buffer A at 100 mg/mL as determined by a BCA Protein
Assay Reagent Kit (Pierce, Rockford, IL) with small aliquots (50 |aL) frozen at -20 °C.
4.2 DEVELOPMENT OF ACCEPTOR CELL-FREE ASSAY
M. smegmatis mc" 155 cells were grown in Luna Bertoni broth (Gibco BRL, Life technologies. Paisley, Scotland) and isolated membranes obtained (as described in previous section) and kept on ice and not subjected to any freeze/thaw cycles prior to assay. Table A.l illustrates the cell-free assay used to assess the acceptor specificities of the disaccharides 20-24 that utilize the mycobacterial (Cjo-Man).
2 1 1 Reagent Volume (pL)
MOPS (50 mM, pH 7.9) 2.5
ATP(1 mM) 5.0
DTT (2 mM) 2.5
M gC K d M) 0.5
N aF (l M) 1 . 0
GDP-["C] 12.5 (0.25 pCi)
C 5U-P ( 1 mM) 12.5
Synthetic acceptor 0.5 - 2.0 mM (20 mM stock solution)
Membranes 35.5 ( 15 mg/mL protein)
Table A. 1 Mannoside acceptor assay
The volumes of the assays was adjusted to 80 pL and performed in 1.5 mL
Eppendorf tubes. Once the reactions were started by the addition of the enzyme, the assays were incubated at 37 °C for I hour. After incubation for I h, the reactions were terminated by the addition of 533 uL of chloroform/methanol (1:1, v/v) and each mixture were transferred to a 13 x 100 mm glass tube. The lipids were then extracted for 1 h at room temperature. The tubes were centrifuged at 4000 rpm for 10 mins and the 212 supernatants were dried under a stream of nitrogen, re-dissolved in I mL of 50% aqueous ethanol and applied to a 1 mL Whatman strong anion exchange (SAX) cartridge previously equilibrated with 0.75 mL of 50% aqueous ethanol. The radiolabelled
products were eluted from the cartridges with 100% ethanol (3 mL) and dried under a stream of nitrogen. The radiolabelled products were then dissolved in 3 mL of n-butanol and 3 mL of water saturated with n-butanol. After mixing, the phases were separated by centrifugation and extracted once more with 3 mL of water saturated with n-butanol. The combined n-butanol phases were washed three times with 3 mL of water saturated with n-
butanol. dried under a stream of nitrogen and finally re-suspended in 300 pL of n-butanol
saturated with water 30 pL of this solution was used for TLC using
chloroform/methanol/IM ammonium acetate/(13 M) ammonia/water (180:140:9:9:23,
v/v/v/v/v) as the solvent system and the remainder was subjected to scintillation counting.
4.3 RESULTS
Disaccharides 20, 22, 23 and 24 (Page 38) acted as acceptors for 14 (Cjo-Man):
a (l—>6)-ManT activity of M. smegmatis. In addition, mixing experiments were
performed to establish if any of the disaccharides might act as competetive inhibitors.
However, no inhibition was observed for any of the above disaccharides; thus, studies
focussed towards their acceptor activities were performed using a range of acceptor
concentrations (0.5 - 2.0 mM) to determine their values (Figure A.2). The enzyme
a(l->6)-ManT did not recognize compound 21 which has a methoxy group at C-2'.
Hence, it can be postulated that a(l-46)-M anT might not tolerate any groups of larger
213 steric bulk at the C-2' position. However, all of the other compounds possessed a value that was similar to the parent compound. Knowing the type of acceptors recognized by the enzyme, we are looking forward to further investigations with type B disaccharides.
H O ^ H O ''^ ^ ---- HO-V^ A
r r
0(CH2)7CH3 0(CH2)7CH3 20 22
Km = 1.19 mM Km = 1.63 mM
HO' HO' NHj
HO HO
OH OH
HO HO'
0(CH2)7CH3 0(CH2)7CH3 23 24
Km = 1.76 mM Km = 1.33 mM
Figure A.3 Disaccharide acceptors and their values.
214 APPENDIX B
'H AND "C NMR SPECTRA OF THE UNKNOWN
COMPOUNDS
215 BnO ()(C H 2)7CH 3
BnO 0 4
VO
J"I
J w
7 50 7 .00 6 50 6 00 5 50 5 00 A 50 A 0 0 3 . 5 0 3 00 2 50 2.00 1.50 1 .00 .50 0,0 PPM m 3 O
o CD 2
Cà) J II
sf.JSe
2 2 382
L±JUS
217 OBn AcO
^O A c
BnO 65
OO (N
.. y\ _
'**'* T—^ 7 50 7 00 G 50 G 00 5 50 5 00 4 50 4 00 3 .5 0 3.00 g . 50 2 00 1 ,50 1 .00 PPM 'VrrTTT: «• * •^■TTTTyTT^ 1 T| tr]-|-]-ri I '1
AcO OBn
OAc
BnO
On CS
W (A#w #*W # w
I ;o mo 150 HO n o i?o n o loo 90 no jo no 50 *0 30 ?o 10 0 X o
w O X S' o X
tO îrr
--J-8 C :T(C
15;
- k . Taz! '>'■ C
I -
2 2 0 X o
o X
o wX i t
2 2 1 X o
X o
w O [OX O CJX
16.738 1.163 1.34 iJïS 0.472
0.291, 1.056. 0.267,
2.092
10.614
3.000
2 2 2 2 2 3 0(CH2)7CH3
BnO
/
tifJsV'yw L___ .à AÂ
? CÎ u I I I /' 0 00 7 ,SO 7 00 0 00 S 50 5 00 4 .5 0 4 00 3.50 3 00 2 50 2 00 1,50 1 00 PPM ,0 n
M jsa
<3 f
: :
- U jli _i3jas
225 Px
O)
0 . 0 6 9 0 . 0 7 1 , 0 . 8 6 0
0 . 1 4 6 0142^ 0 . 2 8 8 _ 0 . 2 8 9 ^ 0 . 0 7 5
0 . 1 5 0> 0 . 7 2 3
0 . 2 1 7
■ D T5 3
226 w o
9 x I
r 'P 6 1 1
I '0065 2
965? / 94/0 6 95ÎJ 5 39M 5 aa/4 6 9666 6 9657 ? 3420 8 i f
2 2 7 BnO'
BnO"
OR
70 V (N00 R = (CH2)7CH3g^r (N
n 01 r.Q *j 0 0 4 50 4 00 00 ? 50 ? 00 1 003 I .'i ;3W ; M m IY
B n O A O A c
B nO B nO
GnO-^n O - \ YO B nO ' B nO OR O 0\ 70 R = (CH;.)7CH3 'B n O
u
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13.831
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12 309 30 X 7 29 929 29 ’ *7 26 29 23 159
240 HO OBn
BnO ' BnO
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iâ ê»: :s as: :a
i:a *’C3 1:8 :W
T]7 C Mî V T* «96 «0 ? 7w T2 .•«
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32 331 • M 310 29 a;:
242 BnO B nO -
81 O R R = (CHgjyCHg
'il M 111.
IA s A «I m m ^
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O .O 3 3^ O J3
s-
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zs ::s
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x S ,0 3
1 9
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BnO-
82 OR R = (CH2)7CH3
VO X X o
X o X II o
O cf o X
1 . QOOO
1.4114
7.2242
I
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247 ■:C0.019
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71.090 70.717 70.437 £ 63.214 67.039 66.765 65.819 62.882 61.287
/ - 32.000 /) - 29.495 29.446 29.359 F 26.256 22.790
14.120
248 BnO BnO BnO O BnO BnO
U' ■A. /m OBn ÎÇÇ??!5ÇÇÇÇÇÇÇÇÇ55 ysgfrtrrrtcfffrcrrcre'C'C'SSïy: N//1
8 3 OR P = (CHgjyCHi
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p»« X o
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251 X o
X Q ■O I
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o
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61.j 5 /
31.897 29.340 F 29.250 26.184 22.7:'
14.065
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256 X I Q
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10.236
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2 5 7 ■100.45e f - 90.350
71.406 71.328 70.726 68.667 68.226 66.680 65.603 61.123
37.155 31.970 • 29.4429.446 29.41417 29 33 0 26.231 22.768
14.102
258 AcO AcO^ A cO - OBn
B nO ' BnO
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BnO
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262 % q
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32.189 29.749 29.652
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BnO B nO - 106 OR R = (CHjjrCHa
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31.452 26.96: 700
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294 to 00
2.272
11.580
3.302
295 33 II 0 1 O %
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14.409
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297 w w
298 3 2S6 i 4S2
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67.686 67.079 <2-1
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15.200
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73.740 72.649 71.950 71.825 70.693 69.255 67.935 67.875 66.836 61.798 54.378 43.812 33.137 30.690 30.614 30.511 27.399 23.883 15.205
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osdaai J t1r%iiiry ifintTf fiinlr " ii1‘ n iwiowi
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HO
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m
L A ; \ .li w NJ
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HOO OH PhlhN
R = iCHplyCH]
m r o ? >§2SS S 9 3 jt f: « S3
BiiO B nO - 130 OF' R = (CH ijyCHg
m •O
33 II Ô % 0 1 o 33
1 . 07: ^ 1.026^ jMæ 1 . 005/y O.ÎSlZ^ 0.933/
11.776
3.311
3 1 5 .551: 7;.079 70.319 70.037 66.700 68.602 67.711 59.471 49.422 49.280 49.138 46.996 48.854 48.712 48.570 42.250
32.975 30.597 30.482 30.372 27.400 23.688
14.398
316 BnO > BnO OBn
BnO ' BnO
1 3 2 OR R = 'CH2)7CH3
r-
I v l . J J. .o X
o wro •o O X
■'!i
» M *9 M 846?<9 M 666 a 64: es t96 <3 :09 3^ ?: «68
318 ■O %
33 II n w n o o o 31
■ O C G . Q= -A L
.075/
2.172
11.259
319 3 3 II 0 1 I o : X r
32.989 30.614 30.497 30.388 27.406 23.702
14.414
320 X o
2 365 1 376
3 2 1 o
X o
i
322 O)
:.6iQ)
'0 . 897 /
1.973
10.295
909
323 0 1
o X
;. .01 -C.4:4 09.919 6è.540 66.607 56.800 49.410 49.268 49.127 48.985 48.843 48.701 43.559
32.972 30.619 30.542 30.381 27.380 23.689
14.396
324