The 5-deoxy-5-methylthio-xylofuranose residue in mycobacterial lipoarabinomannan. Absolute stereochemistry, linkage position, conformation, and immunomodulatory activity

Article abstract of DOI:10.1021/ja057373q

Mycobacteria produce a cell-surface glycoconjugate, lipoarabinomannan (LAM), which has been shown to be a potent modulator of the immune response that arises from infection by these organisms. Recently, LAM from the human pathogens Mycobacterium tuberculo

Full text of DOI:10.1021/ja057373q

Published on Web 03/28/2006
The 5-Deoxy-5-methylthio-xylofuranose Residue in
Mycobacterial Lipoarabinomannan. Absolute Stereochemistry,
Linkage Position, Conformation, and Immunomodulatory
Activity
Maju Joe,† Daniel Sun,† Hashem Taha,† Gladys C. Completo,†
Joanne E. Croudace,‡ David A. Lammas,‡ Gurdyal S. Besra,§ and Todd L. Lowary*,†
Contribution from the Alberta Ingenuity Centre for Carbohydrate Science and Department of
Chemistry, The UniVersity of Alberta, Gunning-Lemieux Chemistry Centre Edmonton, Alberta,
T6G 2G2 Canada, Medical Research Council Centre for Immune Regulation, Birmingham
Medical School, Birmingham UniVersity, Edgbaston, Birmingham, B15 2TT, U.K., and
School of Biosciences, UniVersity of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Received October 28, 2005; E-mail: tlowary@ualberta.ca
Abstract: Mycobacteria produce a cell-surface glycoconjugate, lipoarabinomannan (LAM), which has been
shown to be a potent modulator of the immune response that arises from infection by these organisms.
Recently, LAM from the human pathogens Mycobacterium tuberculosis and M. kansasii has been shown
to contain an unusual 5-deoxy-5-methylthio-xylofuranose (MTX) residue as well as its corresponding oxidized
counterpart, 5-deoxy-5-methylsulfoxy-xylofuranose (MSX). To date, the absolute configuration of these
residues and their linkage position to the polysaccharide are unknown, as is their biological role. Through
the combined use of chemical synthesis and NMR spectroscopy, we have established that the MTX/MSX
residues in these glycoconjugates are of the D-configuration and that they are linked R-(1f4) to a
mannopyranose residue in the mannan portion of the glycan. Conformational analysis of the MTX/MSX
residue using NMR spectroscopy showed differences in ring conformation and as well as in the rotamer
populations about the C-4-C-5 bond, as compared to the parent compound, methyl R-D-xylofuranoside.
Two of the synthesized disaccharides, 3 and 34, were tested in cytokine induction assays, and neither led
to the production of TNF-R or IL-12p70. In contrast, both demonstrated modest inhibitory properties when
these same cytokines were induced using a preparation of Interferon-γ and Staphylococcus aureus Cowan
strain (SAC/IFN-γ). These latter observations suggest that this motif may play a role in the immune response
arising from mycobacterial infection.
Introduction
which serves as a formidable barrier to the passage of antibiotics
into the organism. In addition to its role as a permeability barrier,
Tuberculosis (TB) is the world’s most lethal bacterial disease,
killing more than 2 million people worldwide each year.1-3
Increased recent concern about the impact of this disease on
world health has resulted from the emergence4 of multidrug
resistant strains of Mycobacterium tuberculosis, the organism
that causes the disease, and difficulties in treating individuals
who have both TB and HIV.5 A hallmark of TB and other
mycobacterial diseases is the need for protracted treatments,
typically involving multiple antibiotics that must be taken over
several months.6 The need for this prolonged drug regimen is
due to the unusual structure7,8 of the mycobacterial cell wall,
it is now well-documented that mycobacterial cell wall com-
ponents act as immunomodulatory molecules, enabling the
organism to resist the immune system of the human host.9,10
The mycobacterial cell wall is rich in polysaccharides and
lipids.7,8 Among the many components that make up this
protective structure, the largest is an immense glycoconjugate,
the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex,
which is the major permeability barrier of the cell wall. Also
present in this macrostructure is another glycoconjugate, li-
poarabinomannan (LAM), a major antigenic species. Myco-
bacterial LAM has been implicated in a large, and increasing,
number of important immunological events.9,10 For example,
in the case of M. tuberculosis, it is believed that this poly-
saccharide is of critical importance in allowing the organism to
survive in host macrophages.
† The University of Alberta.
‡ Birmingham University.
§ University of Birmingham.
(1) Paolo, W. F., Jr.; Nosanchuk, J. D. Lancet Infect. Dis. 2004, 4, 287-293.
(2) Kremer, L.; Besra, G. S. Expert Opin. InVest. Drugs 2002, 11, 153-157.
(3) Coker, R. J. Trop. Med. Int. Health 2004, 9, 25-40.
(4) Nachega, J. B.; Chaisson, R. E. Clin. Infect. Dis. 2003, 36, S24-S30.
(5) De Jong, B. C.; Israelski, D. M.; Corbett, E. L.; Small, P. M. Annu. ReV.
Med. 2004, 55, 283-301.
(8) Lowary, T. L. Mycobacterial Cell Wall Components. In Glycoscience:
Chemistry and Chemical Biology; Fraser-Reid, B., Tatsuta, K., Thiem, J.,
Eds.; Springer-Verlag: Berlin, 2001; pp 2005-2080.
(9) Nigou, J.; Gilleron, M.; Puzo, G. Biochemie 2003, 85, 153-166.
(10) Briken, V.; Porcelli, S. A.; Besra, G. S.; Kremer, L. Mol. Microbiol. 2004,
53, 391-403.
(6) Bass, J. B., Jr.; Farer, L. S.; Hopewell, P. C.; Obrien, R.; Jacobs, R. F.;
Ruben, F.; Snider, D. E.; Thornton, G. Am. J. Respir. Crit. Care Med.
1994, 149, 1359-1374.
(7) Brennan, P. J. Tuberculosis 2003, 83, 91-97.
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J. AM. CHEM. SOC. 2006, 128, 5059-5072
5059

A R T I C L E S
Joe et al.
attachment site to the LAM was determined. This moiety has
also been found in M. kansasii, where it is attached not to the
mannopyranose capping residues but rather to the mannan
core.28 In addition to MTX, the corresponding sulfoxide,
5-deoxy-5-methylsulfoxy-xylofuranose (MSX), is also present
in these polysaccharides. The oxidation of MTX to MSX appears
not to be an enzymatic process because a 1:1 ratio of diaster-
eomeric sulfoxides is found.
The biological function of the MTX residue in LAM has not
been established, nor has the biosynthetic pathway by which it
is introduced into the polysaccharide. However, its distribution
across a range of mycobacterial strains suggests that it has an
important biological role. It is therefore of interest to determine
the absolute stereochemistry of this residue and to establish its
linkage to the polysaccharide. Furthermore, efficient access to
MTX-containing fragments of LAM is important as such
compounds will be of great use in studies focused on under-
standing the biological role of this motif. Described here is the
synthesis of a panel of MTX- and MSX-containing disaccha-
rides, which were used in NMR studies to demonstrate that these
monosaccharides have the D-configuration and that they are
attached to LAM via an R-(1f4)-linkage to a mannopyranose
residue. In addition, we have probed the conformation of the
MTX/MSX substituent and tested the ability of two of the
synthesized disaccharides to induce or suppress cytokine
production.
Figure 1. Schematic representation of the major structural domains in
mycobacterial LAM.
The fine structure of mycobacterial LAM is generally well
understood (Figure 1).9,10 At its core is a phosphatidylinositol
moiety to which is attached a mannan consisting of R-(1f6)
and R-(1f2)-linked mannopyranose residues. An arabinan
domain, composed of R-(1f5), R-(1f3), and â-(1f2)-linked
arabinofuranose residues, is attached to the mannan chain. This
arabinan is often further functionalized at its nonreducing
terminus with “capping” motifs of varying structure. In
M. tuberculosis, M. boVis, and M. aVium, the predominant
capping motifs are small R-(1f2)-linked mannopyranosyl
oligosaccharides, which, when present, give rise to a LAM
variant termed ManLAM.11,12 In contrast, in M. smegmatis, these
mannose caps are replaced with inositol phosphate moieties
providing a glycoconjugate called PILAM.13 At least some of
the immunomodulatory role of LAM has been ascribed to these
capping motifs.9,10
Over the past several years, the structures of LAM molecules
from a range of mycobacteria and other actinomycetes have
been reported14-24 and an impressive range of structural diversity
has been identified. Among these was the discovery that LAMs
from a number of M. tuberculosis strains contain a 5-deoxy-
5-methylthio-pentose residue. To date, this substituent has been
identified in both laboratory strains (H37Rv25 and H37Ra25),
as well as clinical isolates (CSU2025 and MT10326) of M.
tuberculosis. In the initial report describing this modification,25
its stereochemical identity was not elucidated, but it was
demonstrated that this motif is found linked to the manno-
pyranose capping residues. More recent work27 established that
this motif is a 5-deoxy-5-methylthio-R-xylofuranose (MTX)
residue, but neither the absolute configuration (D vs L) nor the
Results and Discussion
Approach. Through NMR spectroscopic investigations on
13C-labeled LAM from M. tuberculosis H37Ra, Treumann et
al. proposed that the MTX residue is linked to the mannopy-
ranose capping units.25 As part of these studies, an HMBC
experiment was carried out showing a correlation between the
anomeric hydrogen resonance of the MTX residue and a signal
at 77.0 ppm in the 13C NMR spectrum. Similarly, the anomeric
carbon resonance of the MTX residue correlated with a signal
at 3.77 ppm in the 1H NMR spectrum. These data suggest that
the linkage of the MTX to the mannose caps is via a secondary
hydroxyl group. Therefore, we selected as targets disaccharides
1-6 (Chart 1), which contain either a D- or L-MTX residue
(1-3 and 4-6, respectively) in an R-linkage to one of the three
secondary hydroxyl groups of methyl R-D-mannopyranoside.
We reasoned that after the synthesis of these six disaccharides,
comparison of their NMR data with that reported for this residue
in the native polysaccharide would allow us to establish not
only the absolute configuration of this modified pentose but
also its linkage position to the polysaccharide.
Synthesis. To synthesize these targets, we developed a
strategy in which the methylthio group would be introduced
near the end of the synthesis. This approach required the
preparation of a series of six protected disaccharides with a
leaving group at the primary position of the xylofuranose
residue. We envisioned that the five building blocks shown in
Chart 2 (7-11) could be used to assemble disaccharides 1-6.
Mannopyranosides 9-11 are known compounds and were
prepared as previously described.29 The tosylated thioglycosides
(11) Nigou, J.; Gilleron, M.; Cahuzac, B.; Bounery, J. D.; Herold, M.; Thurnher,
M.; Puzo, G. J. Biol. Chem. 1997, 272, 23094-23103.
(12) Khoo, K.-H.; Tang, J. B.; Chatterjee, D. J. Biol. Chem. 2001, 276, 3863-
3871.
(13) Khoo, K.-H.; Dell, A.; Morris, H. R.; Brennan, P. J.; Chatterjee, D. J. Biol.
Chem. 1995, 270, 12380-12389.
(14) Gue´rardel, Y.; Maes, E.; Elass, E.; Leroy, Y.; Timmerman, P.; Besra, G.
S. Locht, C.; Strecker, G.; Kremer, L. J. Biol. Chem. 2002, 277, 30635-
30648.
(15) Torrelles, J. B.; Khoo, K.-H.; Sieling, P. A.; Modlin, R. L.; Zhang, N.;
Marques, A. M.; Treumann, A.; Rithner, C. D.; Brennan, P. J.; Chatterjee,
D. J. Biol. Chem. 2004, 279, 41227-41239.
(16) Gibson, K. J. C.; Gilleron, M.; Constant, P.; Puzo, G.; Nigou, J.; Besra, G.
S. Biochem. J. 2003, 372, 821-829.
(17) Gibson, K. J. C.; Gilleron, M.; Constant, P.; Brando, T.; Puzo, G.; Besra,
G. S.; Nigou, J. J. Biol. Chem. 2004, 279, 22973-22982.
(18) Gibson, K. J. C.; Gilleron, M.; Constant, P.; Puzo, G.; Nigou, J.; Besra, G.
S. Microbiology 2003, 149, 1437-1445.
(19) Garton, N. J.; Gilleron, M.; Brando, T.; Dan, H.-H.; Gigue`re, S.; Puzo, G.;
Prescott, J. F.; Sutcliffe, I. C. J. Biol. Chem. 2002, 277, 31722-31733.
(20) Gilleron, M.; Garton, N. J.; Nigou, J.; Brando, T.; Puzo, G.; Sutcliffe, I.
C. J. Bacteriol. 2005, 187, 854-861.
(21) Sutcliffe, I. C. Antonie Van Leeuwenhoek 2000, 78, 195-201.
(22) Flaherty, C.; Sutcliffe, I. C. Syst. Appl. Microbiol. 1999, 22, 530-533.
(23) Flaherty, C.; Minnikin, D. E.; Sutcliffe, I. C. Zentralbl. Bakteriol. 1996,
285, 11-19.
(24) Gibson, K. J. C.; Gilleron, M.; Constant, P.; Sichi, B.; Puzo, G.; Besra, G.
S.; Nigou, J. J. Biol. Chem. 2005, 280, 28347-28356.
(25) Treumann, A.; Feng, X.; McDonnell, L.; Derrick, P. J.; Ashcroft, A. E.;
Chatterjee, D.; Homans, S. W. J. Mol. Biol. 2002, 316, 89-100.
(26) Ludwiczak, P.; Gilleron, M.; Bordat, Y.; Martin, C.; Gicquel, B.; Puzo, G.
Microbiology 2002, 148, 3029-3037.
(28) Gue´rardel, Y.; Maes, E.; Briken, V.; Chirat, F.; Leroy, Y.; Locht, C.;
Strecker, G.; Kremer, L. J. Biol. Chem. 2003, 278, 36637-36651.
(29) 9: Nashed, M. A.; Anderson, L. Tetrahedron Lett. 1976, 3503-3506. 10,
11: Koto, S.; Takenaka, K.; Morishima, N.; Sugimoto, A.; Zen, S. Bull.
Chem. Soc. Jpn. 1984, 57, 3603-3604.
(27) Turnbull W. B.; Shimizu, K. H.; Chatterjee, D.; Homans, S. W.; Treumann,
A. Angew. Chem., Int. Ed. 2004, 43, 3918-3922.
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5060 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006

5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
Chart 1
Chart 2
Scheme 2 a
a (a) H3BO3, AcOH, Ac2O, 50 °C then Ac2O, pyridine, rt, 90%. (b)
p-thiocresol, BF3‚Et2O, CH2Cl2, -20 °C, 75%. (c) NaOCH3, CH3OH,
CH2Cl2, rt, 84%. (d) TrCl, pyridine, 0 °C f rt f 40 °C, 89%. (e) NaH,
DMF, BnBr, 0 °C f rt, 80%. (f) cat. p-TsOH, CH3OH, CH2Cl2, rt, 81%.
(g) TsCl, pyridine, 0 °C f rt, 77%.
Scheme 1 a
(90%) using the boric acid-mediated approach developed by
Furneaux and co-workers.32 Peracetate 16, obtained as an ∼2:1
anomeric mixture, was converted to thioglycoside 17 in 75%
yield upon reaction with p-thiocresol and boron trifluoride
etherate. Deacetylation of 17 with sodium methoxide in
methanol provided, in 84% yield, triol 18, the enantiomer of
12. The synthesis of 8 from 18 was done via a sequence identical
to that used for the preparation of 7 from 12. Thus, tritylation
of 18 yielded 19 (89% yield), which was then benzylated
affording 20 in 80% yield. Cleavage of the trityl group in 20
provided alcohol 21, which was then tosylated affording
thioglycoside 8 in 62% yield over the two steps.
With sufficient quantities of building blocks 7-11 in hand,
their coupling to provide disaccharides proceeded without
significant problems. Shown in Scheme 3 is the synthesis of
disaccharides containing the D-enantiomer of MTX (1-3).
The first step toward disaccharide 1 involved the reaction of
thioglycoside 7 with mannopyranoside 9, in the presence of
N-iodosuccinimide and silver triflate. The product produced
from this reaction, disaccharide 22, was produced in 91% yield
a (a) TrCl, pyridine, 0 °C f rt f 40 °C, 91%. (b) NaH, DMF, BnBr, 0
°C f rt, 81%. (c) cat. p-TsOH, CH3OH, CH2Cl2, rt, 83%. (d) TsCl, pyridine,
0 °C f rt, 87%.
7 and 8, while not known, were straightforwardly synthesized
as described below.
The preparation of 7 (Scheme 1) began from thioglycoside
triol 12,30 which was tritylated and benzylated under conven-
tional conditions providing 14 in 74% yield over the two steps.
The trityl group was then cleaved (p-TsOH/CH3OH) affording
an 83% yield of alcohol 15. Subsequent tosylation of 15 yielded
7 in 87% yield.
The synthesis of the enantiomeric thioglycoside, 8, is il-
lustrated in Scheme 2. In the first step, L-xylose31 was converted
to the corresponding furanose tetraacetate 16 in excellent yield
(30) Tilekar, J. N.; Lowary, T. L. Carbohydr. Res. 2004, 339, 2895-2899.
(31) Ness, R. K. Methods Carbohydr. Chem. 1962, 1, 90-93.
(32) Furneaux, R. H.; Rendle, P. M.; Sims, I. M. J. Chem. Soc., Perkin Trans.
1 2000, 2011-2014.
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5061

A R T I C L E S
Scheme 3 a
Joe et al.
mized conditions.35,36 We are unsure as to the origin of the high
selectivities observed in glycosylations with 7 and 8 as compared
to other furanoside glycosylating agents containing nonpartici-
pating groups on O-2. It is plausible to speculate that the
R-xylofuranoside product is favored by the kinetic anomeric
effect,37 although in the absence of a detailed conformational
study of the putative oxocarbenium ion involved in these
reactions, this must remain only a hypothesis.
Because the separation of 22 from the corresponding â-isomer
was not possible, the mixture was submitted to the next reaction,
in which the methylthio group was introduced. This reaction
was done by heating 22 together with sodium thiomethoxide
and 18-Crown-6 in acetonitrile at reflux. The expected product,
23, was produced in 70% yield, again contaminated with traces
of its â-glycoside isomer. That the introduction of the methylthio
group had occurred was obvious from the NMR spectra of 23.
In the 1H NMR spectrum, the signals for the protons on C-5 of
the xylofuranose residue were significantly upfield (2.85 and
2.70 ppm) of their position in the 1H NMR spectrum of 22 (4.10
and 4.29 ppm). In addition, in the 13C NMR spectrum of 23,
the resonance for the xylofuranose C-5 appeared at 34.1 ppm,
consistent with its linkage to sulfur. Finally, as expected, a
methyl group bound to sulfur was apparent in both the 1H and
13C spectra (resonances as 2.16 and 16.5 ppm, respectively).
Similar features were observed in the NMR spectra for all
products of these substitution reactions.
With the methylthio group in place, the final step in the
synthesis of 1 was the cleavage of the benzyl ethers and the
benzylidene acetal, which was done by dissolving metal
reduction. Thus, treatment of a solution of 23 in THF at -78
°C with sodium and ammonia cleaved all protecting groups.
Following purification, disaccharide 1 was isolated in 61% yield.
The synthesis of 2 followed a similar sequence to that used
for the preparation of 1. Glycosylation of 10 with 7 promoted
by N-iodosuccinimide and silver triflate gave disaccharide 24,
as an inseparable mixture with the â-glycoside and small
amounts of hydrolyzed 7. The mixture was then subjected to
the thiolate substitution reaction, which gave, following chro-
matography, 25 as a pure compound in 53% overall yield from
10. Removal of the benzyl ethers upon treatment of 25 with
sodium and liquid ammonia in THF proceeded uneventfully,
yielding 2 in 64% yield.
The same series of transformations was used to convert 11
and 7 into disaccharide 3. The coupling of 11 and 7 under
standard conditions gave the expected disaccharide 26, which,
following chromatography, was also contaminated with traces
of hydrolyzed 7. This partially pure product was then reacted
with sodium thiomethoxide to give 27 in 66% yield from 11.
Disaccharide 3 was obtained in 89% yield upon treatment of
27 with sodium in liquid ammonia.
a (a) NIS, AgOTf, CH2Cl2, 0 ° f rt, 91%. (b) NaSCH3, 18-crown-6,
CH3CN, reflux, 70%. (c) Na, NH3, THF, -78 °C, 61%. (d) NIS, AgOTf,
CH2Cl2, 0 °C f rt, 73%. (e) NaSCH3, 18-crown-6, CH3CN, reflux, 72%.
(f) Na, NH3, THF, -78 °C, 64%. (g) NIS, AgOTf, CH2Cl2, 0 °C f rt,
89%. (h) NaSCH3, 18-crown-6, CH3CN, reflux, 76%. (i) Na, NH3, THF,
-78 °C, 89%.
as an inseparable 87:13 R:â mixture of glycosides. The
stereochemistry of the nascent glycosidic linkage could be
readily established by NMR spectroscopy. In the major product,
the coupling constant between H-1 and H-2 (3J1,2) in the
xylofuranose residue was 4.3 Hz as would be expected for a
1,2-cis furanoside.33 In contrast, in the minor isomer, H-1 of
the xylofuranose residue appeared as a singlet, consistent with
the 1,2-trans furanoside stereochemistry.33 Further support for
the anomeric stereochemistry of the xylofuranose residue was
obtained from the 13C NMR spectrum of the product. For the
major isomer, the anomeric carbon resonance appeared at 101.4
ppm, whereas in the minor isomer this resonance appeared at
106.1. Again, both of these data support the R-stereochemistry
of the major product.33 These same two NMR parameters were
used to establish the stereochemistry of the xylofuranosyl bond
in all the disaccharides synthesized.
The synthesis of disaccharides containing an L-MTX residue
(4-6) is shown in Scheme 4. The oligosaccharides were
synthesized via the same routes used for the preparation of 1-3,
by replacing donor 7 with 8. The protected disaccharides were
thus obtained in yields of 71-82% upon reaction of 8 with one
All glycosylations reported here were highly R-selective
providing, at worst, an 87:13 R:â ratio of glycosides. Indeed,
in some reactions, we were unable to isolate any of the
â-glycoside product. This high selectivity for the 1,2-cis
furanoside is in contrast to the synthesis of other 1,2-cis
furanosides (e.g., â-arabinofuranosides), which is often plagued
with modest anomeric selectivity,34 except under highly opti-
(35) Yin, H.; D’Souza, F. W.; Lowary, T. L. J. Org. Chem. 2002, 67, 892-
903.
(36) Lee, Y. J.; Lee, K.; Jung, E. H.; Jeon, H. B.; Kim, K. S. Org. Lett. 2005,
7, 3263-326.
(33) Cyr, N.; Perlin, A. S. Can. J. Chem. 1979, 57, 2504-2511.
(34) Yin, H.; Lowary, T. L. Tetrahedron Lett. 2001, 42, 5829-5832.
(37) Juaristi, E.; Cuevas, G. The Anomeric Effect; CRC Press: Boca Raton,
FL, 1995; pp 182-194.
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5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
Table 1. Comparison of NMR Chemical Shift Data for the 5-Deoxy-5-methylthio-xylofuranose Residue in 1-6 with Those Found in LAM
from M. tuberculosis H37Raa
1H
δ
(ppm)
13C
C-3
δ
(ppm)
C-4
compound
H-1
H-2
H-3
H-4
H-5
H-5’
SCH3
C-1
C-2
C-5
SCH3
1
2
3
4
5
6
5.30
5.36
5.41
5.25
5.27
5.21
5.40
4.21
4.20
4.21
4.19
4.20
4.20
4.21
4.27
4.29
4.26
4.30
4.31
4.28
4.26
4.40
4.43
4.38
4.47
4.47
4.47
4.38
2.69
2.69
2.68
2.68
2.68
2.68
2.68
2.80
2.81
2.80
2.79
2.80
2.80
2.80
2.18
2.17
2.18
2.16
2.16
2.16
2.21
105.8
105.4
105.3
103.0
103.4
104.6
105.2
80.4
80.4
79.4
80.0
80.2
79.6
79.4
78.5
78.5
78.4
78.3
78.4
78.2
78.3
80.6
80.6
80.6
80.4
80.2
80.3
80.5
35.6
35.6
35.8
35.6
35.7
35.8
35.8
17.9
17.8
17.8
17.7
17.7
17.8
17.4
experimentb
a NMR spectra were recorded in D2O, and chemical shifts are referenced to 3-(trimethylsilyl)-propionic acid, sodium salt at 0.0 ppm. b Taken from ref
25.
Scheme 4 a
Perusing these data it is possible to quickly determine that
the MTX residue in the polysaccharide is not of the L-
configuration. First, the anomeric hydrogen for this residue in
4-6 resonates between 5.21 and 5.27 ppm, whereas in the
polysaccharide the chemical shift for this hydrogen resonance
was reported to be 5.40 ppm, a difference of more than 0.13
ppm. Similarly, the chemical shift of the anomeric carbon
residue in 4-6 resonates between 103.0 and 104.6 ppm, which
is 0.6-2.2 ppm lower than that reported for the MTX substituent
in the polysaccharide. In contrast, the data for 1-3, which
contains an MTX residue with the D-configuration, matches the
polysaccharide data better. The MTX anomeric hydrogen
resonances in 1-3 are found between 5.30 and 5.41 ppm,
differing 0.01-0.1 ppm from the polysaccharide. The chemical
shift data for the anomeric carbon compare even better, with
these ranging from 105.3 to 105.8 ppm in 1-3 vs 105.2 in the
polysaccharide.
Having established the absolute stereochemistry of the MTX
substituent as D, we turned our attention to the position on the
mannose residue to which it was linked. Looking first at the
1H NMR data, the best fit to the polysaccharide is 3, the isomer
in which the linkage is R-(1f4). In particular, for the anomeric
hydrogen resonance, the chemical shift difference with the
polysaccharide is 0.1 ppm (1), 0.04 ppm (2), and 0.01 ppm (3).
The same conclusion can be drawn from the 13C NMR data.
The chemical shift of the anomeric carbon in 3 differed from
that reported for the polysaccharide by only 0.1 ppm, as
compared to 0.6 and 0.2 ppm for 1 and 2, respectively. However
most telling were the differences in the chemical shifts of the
MTX C-2 resonances. In 3, the value (79.4 ppm) matched that
of the polysaccharide exactly, while in 1 and 2, this resonance
was a full ppm more downfield, resonating at 80.4 ppm. Overall,
none of the chemical shift data for the polysaccharide differed
a (a) NIS, AgOTf, CH2Cl2, 0 °C f rt, 73%. (b) NaSCH3, 18-crown-6,
CH3CN, reflux, 71%. (c) Na, NH3, THF, -78 °C, 63%. (d) NIS, AgOTf,
CH2Cl2, 0 °C f rt, 82%. (e) NaSCH3, 18-crown-6, CH3CN, reflux, 70%.
(f) Na, NH3, THF, -78 °C, 65%. (g) NIS, AgOTf, CH2Cl2, 0 °C f rt,
71%. (h) NaSCH3, 18-crown-6, CH3CN, reflux, 77%. (i) Na, NH3, THF,
-78 °C, 67%.
1
from that of 3 by more than 0.03 ppm for the H data and 0.4
ppm for the 13C NMR data. The largest differences were seen
in the data for the methylthio group (0.03 and 0.4, respectively).
When these data are taken out of the comparison, the differences
between 3 and the polysaccharide differed by no more than 0.01
ppm for the 1H data and no more than 0.1 for the 13C data. We
are unsure as to why the data for the methylthio group in 3
agrees comparatively poorly with that reported for the poly-
saccharide, but we note that similarly poor agreement was seen
in the study establishing the xylo stereochemistry of this
substituent.27 Based on our analysis of these data, we propose
that the MTX substituent in M. tuberculosis has the D-
configuration and is linked R-(1f4) to a mannopyranose residue
present in the capping domains.
of acceptors 9-11. The resulting products 28, 30, and 32 were
then converted to the methylthio analogues 29, 31, and 33 in
70-77% yield and subsequently deprotected by dissolving metal
reduction, yielding 4-6 in 63-67% yield.
Determination of Absolute Stereochemistry and Linkage
Position of MTX Residue. Having synthesized oligosaccharides
1-6, we next carried out a series of two-dimensional NMR
experiments (COSY and HMQC) on each to fully assign all 1H
and 13C resonances for comparison with the data obtained for
the MTX residue present in mycobacterial LAM. The chemical
shift data of the MTX residue in 1-6 are provided in Table 1,
together with the data previously reported for this substituent
in M. tuberculosis H37Ra LAM.25
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5063

A R T I C L E S
Scheme 5 a
Joe et al.
Table 3. Comparison of NMR Chemical Shift Data for the
5-Deoxy-5-methylthio-xylofuranose Residue of 3 with Those Found
in LAM from M. kansasii (KanLAM)a
resonance
3
KanLAMb
H-1
H-2
H-3
H-4
H-5
H-5′
SCH3
5.24
3.90
3.98
4.18
2.70
2.53
2.12
5.23
3.90
3.99
4.18
2.70
2.53
2.10
a (a) 30% aqueous H2O2, rt, 81%.
C-1
C-2
C-3
C-4
C-5
SCH3
104.1
103.9
78.0
76.3
79.7
34.4
16.5
78.4
76.6
80.1
34.5
16.8
Table 2. Comparison of NMR Chemical Shift Data for the
Diastereomeric 5-Deoxy-5-methylsulfoxy-xylofuranose Residues in
34 with Those Found in LAM from M. tuberculosis H37Raa
resonance
34a
MSP-1b
34b
MSP-2b
H-1
H-2
H-3
H-4
H-5
H-5′
S(O)CH3
5.47
4.23
4.34
4.62
3.12
3.12
2.81
5.45
4.22
4.34
4.61
3.12
3.12
2.84
5.46
4.20
4.34
4.65
3.29
3.09
2.80
5.44
4.20
4.34
4.65
3.28
3.08
2.83
a NMR spectra were recorded in DMSO-d6, and chemical shifts are
referenced to the methyl group of the solvent at 2.52 ppm (1H) or 40.98
ppm (13C). b Taken from ref 28.
Chart 3
C-1
C-2
C-3
C-4
C-5
S(O)CH3
105.6
105.4
79.3
78.5
75.6
57.1
40.2
105.6
105.4
79.4
78.5
76.5
55.6
39.9
79.1
78.6
75.7
57.2
40.6
79.4
78.6
76.4
55.7
40.2
Conformation of the MTX Residue. In previous studies39,40
we completed a conformational analysis of the methyl R-D-
xylofuranoside (35, Chart 3), which showed that it differs from
many other furanosides in that it is relatively rigid. Using NMR
spectroscopy and computational chemistry we established that
the favored ring conformer is an envelope in which C-1 is
displaced below the plane (E1), which is very similar to the
conformation present in the crystal structure of 35.41 When
analyzing the NMR data for 3 and 34 it was immediately
apparent that the coupling constants of the MTX residue were
significantly different than those in 35 thus indicating differences
in conformation.
To obtain a more quantitative picture of these conformational
differences, we carried out PSUEROT42-44 calculations on the
MTX rings in 3, the diastereomers of 34, and the corresponding
methyl glycoside 36 (Chart 3, prepared as described in the
Supporting Information). The conformation of 36 was evaluated
to determine what, if any, role the aglycone plays in the
conformational equilibrium of the furanose ring. The PSEUROT
approach43 is a commonly used method for assessing the solution
conformation of five-membered rings and involves the measure-
ment of the three bond 1H-1H coupling constants (3JHH) of the
ring hydrogens and subsequent analysis of these data. The
program assumes a model in which two conformers are present,
one in the northern hemisphere of the pseudorotational wheel45
(Figure 2), the other in the southern hemisphere. These
a NMR spectra were recorded in D2O, and chemical shifts are referenced
to 3-(trimethylsilyl)-propionic acid, sodium salt at 0.0 ppm. b Taken from
ref 25.
Additional evidence for this assignment was obtained by
oxidizing 3 into the corresponding diastereomeric mixture of
sulfoxides upon treatment with hydrogen peroxide. As shown
in Scheme 5, the product was obtained in 81% yield. Compari-
son of the NMR data for 34 with that of the MSX residue in
the polysaccharide (Table 2) showed excellent agreement, thus
further bolstering support for the proposed MTX-R-(1f4)-
1
mannopyranose linkage. The H NMR data for the furanose
residue in 34 differed by no more than 0.03 ppm from the
polysaccharide, while for the 13C NMR data the chemical shifts
were all within 0.4 ppm of those reported. As was the case for
3, the worst agreement was seen for the resonance associated
with the methylsulfoxyl group.
As mentioned previously, in addition to being present in M.
tuberculosis LAM, the MTX residue has also been found in
LAM from M. kansasii (KanLAM).28 However, it was demon-
strated that in KanLAM the MTX residue is not attached via
the capping motifs of the polysaccharide, but rather to the
mannan core. To determine if the linkage position and absolute
stereochemistry of the M. kansasii MTX moiety is the same as
that in M. tuberculosis, the NMR data for 3 were compared to
those obtained for KanLAM (Table 3).38 As can be seen from
the table, there is good agreement between the data for 3 and
those for the polysaccharide, and thus we conclude that, like in
M. tuberculosis LAM, the MTX residue in KanLAM is also of
the D-configuration and is linked R-(1f4) to a mannopyranose
residue.
(39) Houseknecht, J. B.; Lowary, T. L.; Hadad, C. M. J. Phys. Chem. A 2003,
107, 372-378.
(40) Houseknecht, J. B.; Lowary, T. L.; Hadad, C. M. J. Phys. Chem. A 2003,
107, 5763-5777.
(41) Evdokimov, A.; Gilboa, A. J.; Koetzle, T. F.; Klooster, W. T.; Schulz, A.
J.; Mason, S. A.; Albinati, A.; Frolow, F. Acta Crystallogr. B 2001, 57,
213-220.
(42) PSEUROT 6.2 (1993), PSEUROT 6.3 (1999): van Wijk, J.; Haasnoot, C.
A. G.; de Leeuw, F. A. A. M.; Huckriede, B. D.; Westra Hoekzema, A.;
Altona, C. Leiden Institute of Chemistry, Leiden University.
(43) de Leeuw, F. A. A. M.; Altona, C. J. Comput. Chem. 1983, 4, 428-437.
(44) Altona, C. Recl. TraV. Chim. Pays-Bas 1982, 101, 413-433.
(45) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205-8212.
(38) In the work reported in ref 28, the NMR spectroscopy of the polysaccharide
was done using DMSO-d6 as the solvent. Therefore, we rerecorded the
NMR spectrum for 3 in DMSO-d6.
9
5064 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006

5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
Figure 3. Definition of gg, gt, and tg rotamers about the C-4-C-5 bond.
Table 5. C-4-C-5 Rotamer Populations for 3, 35, and 36a
compound
3
36
35
Xgg(%)
Xgt(%)
Xtg(%)
14
63
24
12
57
30
40
46
14
Figure 2. Pseudorotational wheel for a D-aldofuranose ring.
Table 4. Results of PSUEROT Calculations for 3 and 34-36a,b
a See Figure 3 for rotamer definitions.
compound
3
34a
34b
35c
36
interactions become more important, in turn favoring conforma-
tions (e.g., E) in which C-5 and OH-3 are staggered.
3
PN
14
50
20
43
14
45
324
8
13
48
%N
PS
Conformation about the C-4-C-5 Bond in the MTX
Residue. In addition to influencing the conformation of the five-
membered ring, the replacement of the C-5 hydroxyl group with
SCH3 is expected to alter rotamer populations about the C-4-
C-5 bond (Figure 3). Thus, through analysis of 3J4,5S and 3J4,5R
measured from the 1H NMR spectrum of 3 and 36 these rotamer
populations have been determined. Analysis of the coupling
constant data was done as outlined in the Experimental Section.
The C-4-C-5 rotamer populations for 3, 35, and 36 are
presented in Table 5. In the parent structure, 35, the two major
rotamers are gg and gt, conformers that are stabilized by a
gauche interaction with the ring oxygen.48 These two rotamers
are present in roughly equal amounts and predominate over the
tg conformer, in which the oxygen is trans to the ring oxygen.
In the methylthio substituted analogues 3 and 36 this distribution
is shifted. In particular, the population of the tg and gt
conformers increase at the expense of the gg rotamer. This
change is presumably driven by unfavorable steric interactions
between the ring and the comparatively bulky methylthio
substituent when adopting the gg conformation. Similarly, the
preference for the gt over tg rotamer is likely due to unfavorable
steric clashing between the methylthio group and the C-3
hydroxyl group. Previous conformational studies on 4′-thio-
nucleoside derivatives showed a similar increase in tg rotamer
when compared to their 4′-oxo counterparts.49 This conforma-
tional shift was ascribed, in part, to the preference for 1-alkoxy-
2-alkylthio ethane fragments to adopt trans rather than gauche
conformations50,51 and the same stereoelectronic effect may
contribute to the differences between rotamer populations in 3
and 36 compared to 35.
137
50
0.0
135
57
0.0
137
55
0.0
124
92
0.0
131
52
0.0
%S
RMSd
a Calculated using a constant Φm (Altona-Sundaralingam puckering
amplitude)
) ) Altona-Sundaralingam
40° for all compounds. b P
pseudorotational phase angle. c Taken from ref 40. d In Hz.
conformers, termed North (N) or South (S), equilibrate via
pseudorotation.46,47
The results of these PSEUROT analyses are provided in Table
4, where they are compared to the populations in the parent
structure 35. It is clear that the replacement of the C-5 hydroxyl
group with the 5-thiomethyl substituent (3, 36) or with the
corresponding sulfoxide (34) does alter the conformational
equilibrium of the furanose ring. In comparison to 35, the C-5
modified analogues are more flexible, all adopting roughly
equimolar mixtures of two conformers, as opposed to an
equilibrium in which a single conformer predominates. In
addition, this modification alters the conformers present in the
equilibrium mixture. Although the identity of the S conformer
remains approximately the same, shifting slightly south from
2
E1 toward T1 (P ) 124° f P ) 131°-137°), the change in
the N conformer is more dramatic, moving from approximately
1E (P ) 324°) to 3E (P ) 13°-20°). The origin of this
conformational shift is unclear; however, the observation that
3 and 36 have essentially identical conformer distributions rules
out the aglycone as a cause of these changes. Beyond that, it is
plausible to speculate that the conformational shift is driven by
eclipsing interactions between OH-3 and the substituent attached
to C-5. In the parent structure 35, in which the C-5 substituent
is OH, the predominant ring conformer is E1. The OH-3 and
C-5 are nearly perfectly eclipsed in this conformer, but the
energetic penalty for this negative interaction is apparently
compensated for by the pseudoaxial orientation of the OCH3
group, which maximizes the anomeric effect. In the minor
conformer of 35 (1E) these groups are also eclipsed. It could
be expected that as the size of the C-5 substituent is increased
(e.g., changing OH to SCH3 or S(O)CH3) these eclipsing
Effect of 3 and 34 on TNF-R and IL-12p70 Production.
The distribution of the MTX residue in a number of different
mycobacterial strains suggests that this motif has an important
biological function. However, to date, no role for this mono-
saccharide has been identified. Given its location in the capping
motif in LAM from M. tuberculosis we hypothesized that it
(48) Wolfe, S. Acc. Chem. Res. 1972, 5, 102-111.
(49) Crnugelj, M.; Dukhan, D.; Barascut, J.-L.; Imbach, J.-L.; Plavec, J. J. Chem.
Soc., Perkin Trans. 2 2000, 255-262.
(50) Yokoyama, Y.; Ohashi, Y. Bull. Chem. Soc. Jpn. 1998, 71, 1565-1571.
(51) Harada, T.; Yoshida, H.; Ohno, K.; Matsuura, H. Chem. Phys. Lett. 2002,
362, 453-460.
(46) Kilpatrick, J. E.; Pitzer, K. S.; Spitzer, R. J. Am. Chem. Soc. 1947, 69,
2483-2488.
(47) Pitzer, K. S.; Donath, W. E. J. Am. Chem. Soc. 1959, 81, 3213-3218.
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5065

A R T I C L E S
Joe et al.
Figure 4. (A) Average TNF-R production by THP-1 cells in response to IFNγ/SAC (8 h), following preincubation with synthetic/natural LAM derivatives
(24 h), (n ) 2). (B) Average IL-12p70 production by THP-1 cells in response to IFNγ/SAC (8 h), following preincubation with synthetic/natural LAM
a
derivatives (24 h), (n ) 3). Man ) ManLAM; Ara ) AraLAM.
may function as an immunomodulatory species and we thus
evaluated the ability of 3 and 34 to induce or inhibit the
production of the TNF-R and IL-12p70 using a human mono-
cytic cell line (THP-1). The results of these studies are
summarized in Figure 4.
As expected, treatment of THP-1 cells with a preparation of
Interferon-γ and Staphylococcus aureus Cowan strain (SAC/
IFN-γ) led to a strong production of both TNF-R (Figure 4a)
and IL-12p70 (Figure 4b). Neither 3 nor 34, when tested at
concentrations of 10 or 100 µg/mL, significantly induced the
production of these two cytokines. As a comparison, both
ManLAM and AraLAM were tested at 10 µg/mL and in line
with previous investigations10 also did not lead to TNF-R or
IL-12p70 induction. When 3 and 34 were tested as inhibitors
of the cytokine response induced by SAC/IFN-γ, modest levels
of inhibition were observed. For TNF-R (Figure 4a), 3 at a
concentration of 100 µg/mL led to a level of inhibition
comparable with ManLAM at 10 µg/mL, whereas 34 (at 10
µg/mL) was less effective and comparable to AraLAM at 10
µg/mL. These compounds were poorer inhibitors of IL-12p70,
with both 3 and 34 exerting only a very modest effect at either
10 or 100 µg/mL.
Because of the significant molecular weight differences
among 3, 34, and the two polysaccharides, we also carried out
assays in which the concentration of these compounds was kept
constant (see Figure S1 in Supporting Information). A concen-
tration of 5 µM was used in these assays, which is the
approximate molarity of a 10 µg/mL solution of ManLAM (mw
∼17 400). For the TNF-R assays, the trends were the same as
those shown in Figure 4a, i.e., a 5 µM concentration of 3
inhibited TNF-R production to a similar degree as a 5 µM
concentration of ManLAM. In addition, 34 was a weaker
9
5066 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006

5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
was added. The flask was warmed to rt and left open to the atmosphere
overnight to allow the NH3 to evaporate. The remaining solution was
concentrated, and the resulting residue was dissolved in a minimum
amount of CH3OH before being neutralized with glacial HOAc. The
solution was again concentrated, and the semisolid residue was purified
by column chromatography on Iatrobeads (85:15, CH2Cl2/CH3OH) to
afford 1 (6 mg, 61%) as a foam (data for major isomer). Rf 0.24
inhibitor than 3. The results with IL-12p70 (Figure S2) were
also similar to those shown in Figure 4b; neither 3 or 34 at 5
µM inhibited the production of the cytokine to the degree of
the same concentration of ManLAM. For IL-12p70, 3 had a
similar activity as that of AraLAM, whereas 34 was less active.
Finally, as controls we tested compounds 1, 2, 6, and 36 at
5 µM in both assays. In the case of TNF-R, none of these
compounds inhibited cytokine induction (Figure S1). Indeed,
each appeared to induce production of TNF-R to varying
degrees. For IL-12p70, all four of these compounds also
inhibited induction, but to a degree intermediate between 3 and
34. These results suggest that the inhibition of TNF-R by 3 and
34 is specific to the structures of the molecules, while for IL-
12p70 the effect is nonspecific.
1
(85:15, CH2Cl2/CH3OH); [R]D +75.2 (c 0.4, CH3OH); H NMR (500
MHz, D2O, δH) 5.30 (d, 1 H, J ) 4.5 Hz, H-1′), 4.93 (d, 1 H, J ) 1.7
Hz, H-1), 4.40 (ddd, 1 H, J ) 4.8, 5.0, 8.6 Hz, H-4′), 4.27 (dd, 1 H,
J ) 4.2, 4.5 Hz, H-3′), 4.21 (dd, 1 H, J ) 4.5, 4.5 Hz, H-2′), 3.99 (dd,
1 H, J ) 1.7, 3.4 Hz, H-2), 3.89 (dd, 1 H, J ) 1.9, 12.3 Hz, H-6), 3.85
(dd, 1 H, J ) 3.4, 9.7 Hz, H-3), 3.80 (dd, 1 H, J ) 5.6, 12.3 Hz, H-6),
3.71 (dd, 1 H, J ) 9.7, 9.7 Hz, H-4), 3.63-3.60 (m, 1 H, H-5), 3.42
(s, 3 H, OCH3), 2.80 (dd, 1 H, J ) 5.0, 13.8 Hz, H-5′), 2.69 (dd, 1 H,
J ) 8.6, 13.8 Hz, H-5′), 2.18 (s, 3 H, SCH3); 13C NMR (125 MHz,
D2O, δC) 105.8 (C-1′), 103.0 (C-1), 80.8 (C-2), 80.6 (C-4′), 80.4 (C-
2′), 78.5 (C-3′), 75.3 (C-5), 73.2 (C-3), 69.6 (C-4), 63.5 (C-6), 57.8
(OCH3), 35.6 (C-5′), 17.9 (SCH3). HRMS (ESI) calcd for (M + Na)
C13H24O9S 379.1033, found 379.1032.
Conclusions
In summary, through the combined use of chemical synthesis
and NMR spectroscopy, we have established that the 5-deoxy-
5-methylthio-xylofuranose (MTX) and 5-deoxy-5-methylsul-
foxy-xylofuranose (MSX) residues present in the LAM of M.
tuberculosis and M. kansasii are of the D-configuration and are
linked R-(1f4) to a mannopyranose residue in the glycan.
Conformational analysis of these residues indicated differences
in both ring conformation and rotamer populations about the
C-4-C-5 bond, as compared to the parent compound, methyl
R-D-xylofuranoside (35). Two of the synthesized disaccharides,
3 and 34, when tested in assays of cytokine induction did not
lead to production of TNF-R or IL-12p70; however, both
showed modest inhibitory properties when these cytokines were
induced using SAC/IFN-γ. These latter observations suggest
that this motif may play a role in the immune response arising
from mycobacterial infection.
Methyl 3-O-(5-Deoxy-5-methylthio-R-D-xylofuranosyl)-R-D-man-
nopyranoside (2). Prepared from 25 (24 mg, 0.03 mmol), liquid NH3
(20 mL), and sodium metal (80 mg) in THF (5 mL) as described for
1, to afford 2 (7 mg, 64%) as a foam. Rf 0.4 (85:15, CH2Cl2/CH3OH);
1
[R]D +106.6 (c 0.5, CH3OH); H NMR (500 MHz, D2O, δH) 5.36 (d,
1 H, J ) 4.5 Hz, H-1′), 4.76 (s, 1 H, H-1), 4.43 (ddd, 1 H, J ) 5.3,
5.0, 8.4 Hz, H-4′), 4.29 (dd, 1 H, J ) 4.0, 5.3 Hz, H-3′), 4.20 (dd, 1
H, J ) 4.5, 4.0 Hz, H-2′), 4.14-4.11 (m, 1 H, H-2), 3.92-3.86 (m, 2
H, H-3, H-6), 3.82-3.75 (m, 2 H, H-4, H-6), 3.69-3.65 (m, 1 H, H-5),
3.42 (s, 3 H, OCH3), 2.81 (dd, 1 H, J ) 5.0, 13.8 Hz, H-5′), 2.69 (dd,
1 H, J ) 8.4, 13.8 Hz, H-5′), 2.17 (s, 3 H, SCH3); 13C NMR (125
MHz, D2O, δC) 105.4 (C-1′), 103.5 (C-1), 81.6 (C-2), 80.6 (C-4′), 80.4
(C-2′), 78.5 (C-3′), 75.4 (C-5), 72.9 (C-3), 68.6 (C-4), 63.7 (C-6), 57.7
(OCH3), 35.6 (C-5′), 17.8 (SCH3). HRMS (ESI) calcd for (M + Na)
C13H24O9S 379.1033, found 379.1032.
Methyl 4-O-(5-Deoxy-5-methylthio-R-D-xylofuranosyl)-R-D-man-
nopyranoside (3). Prepared from 27 (0.39 g, 0.48 mmol), liquid NH3
(35 mL), and sodium metal (75 mg, 3.26 mmol) in THF (5 mL) as
described for 1, to afford 3 (0.15 g, 89%) as a foam; Rf 0.48 (85:15,
CH2Cl2/CH3OH); [R]D +109.5 (c 0.33, CH3OH); 1H NMR (500 MHz,
D2O, δH) 5.41 (d, 1 H, J ) 4.4 Hz, H-1′), 4.76 (s, 1 H, H-1), 4.38
(ddd, 1 H, J ) 5.0, 4.8, 8.4 Hz, H-4′), 4.26 (dd, 1 H, J ) 4.2, 5.0 Hz,
H-3′), 4.21 (dd, 1 H, J ) 4.4, 4.2 Hz, H-2′), 3.94-3.88 (m, 3 H, H-2,
H-4, H-6), 3.83-3.75 (m, 2 H, H-3, H-6), 3.72-3.66 (m, 1 H, H-5),
3.41 (s, 3 H, OCH3), 2.80 (dd, 1 H, J ) 4.8, 13.8 Hz, H-5′), 2.68 (dd,
1 H, J ) 8.4, 13.8 Hz, H-5′), 2.18 (s, 3 H, SCH3); 13C NMR (125
MHz, D2O, δC) 105.3 (C-1′), 103.7 (C-1), 80.6 (C-4′), 79.4 (C-2′), 78.4
(C-3′), 76.9 (C-2), 74.0 (C-5), 73.5 (C-3), 73.0 (C-4), 63.9 (C-6), 57.6
(OCH3), 35.8 (C-5′), 17.8 (SCH3). HRMS (ESI) calcd for (M + Na)
C13H24O9S 379.1033, found 379.1032.
Experimental Section
General Methods. Reactions were carried out in oven-dried
glassware. Reaction solvents were distilled from appropriate drying
agents before use. Unless stated otherwise, all reactions were carried
out with stirring at room temperature under a positive pressure of argon
and were monitored by TLC on silica gel 60 F254 (0.25 mm, E. Merck).
Spots were detected under UV light or by charring with acidified
p-anisaldehyde solution in ethanol. In the processing of reaction
mixtures, solutions of organic solvents were washed with equal amounts
of aqueous solutions. Organic solutions were concentrated under a
vacuum at <40 °C. All column chromatography was performed on
silica gel (40-60 µM) or Iatrobeads, which refers to a beaded silica
gel 6RS-8060, manufactured by Iatron Laboratories (Tokyo). In all cases
the ratio between adsorbent and crude product ranged from 100 to 50:1
(w/w). Optical rotations were measured at 22 ( 2 °C and in units of
degrees mL/g dm. 1H NMR spectra were recorded at 400 or 500 MHz,
and chemical shifts were referenced to either tetramethylsilane (0.0,
CDCl3), CD3OH (4.78, CD3OD) or 3-(trimethylsilyl)-propionic acid,
sodium salt (0.0, D2O). 13C NMR spectra were recorded at 100 or 125
MHz, and 13C chemical shifts were referenced to internal CDCl3 (77.23,
CDCl3), CD3OD (48.9, CD3OD) or 3-(trimethylsilyl)-propionic acid,
sodium salt (0.0, D2O). 1H data are reported as though they were first
order. Electrospray mass spectra were recorded on samples suspended
in mixtures of THF with CH3OH and added NaCl.
Methyl 2-O-(5-Deoxy-5-methylthio-R-L-xylofuranosyl)-R-D-man-
nopyranoside (4). Prepared from 29 (25 mg, 0.03 mmol), liquid NH3
(20 mL), and sodium metal (80 mg) in THF (5 mL) as described for
1, to afford 4 (8 mg, 63%) as a foam. Rf 0.39 (85:15, CH2Cl2/CH3-
OH); [R]D -13.4 (c 0.1, CH3OH); 1H NMR (500 MHz, D2O, δH) 5.25
(d, 1 H, J ) 4.4 Hz, H-1′), 4.88 (s, 1 H, H-1), 4.47 (ddd, 1 H, J ) 5.0,
4.9, 8.4 Hz, H-4′), 4.30 (dd, 1 H, J ) 4.9, 4.2 Hz, H-3′), 4.19 (dd, 1
H, J ) 4.2, 4.4 Hz, H-2′), 4.05-4.02 (m, 1 H, H-2), 3.88 (dd, 1 H, J
) 1.9, 12.0 Hz, H-6), 3.83 (dd, 1 H, J ) 3.5, 9.8 Hz, H-3), 3.80 (dd,
1 H, J ) 5.0, 12.0 Hz, H-6), 3.70 (dd, 1 H, J ) 9.8, 9.8 Hz, H-4),
3.65-3.60 (m, 1 H, H-5), 3.41 (s, 3 H, OCH3), 2.79 (dd, 1 H, J ) 5.0,
13.8 Hz, H-5′), 2.68 (dd, 1 H, J ) 8.4, 13.8 Hz, H-5′), 2.16 (s, 3 H,
SCH3); 13C NMR (125 MHz, D2O, δC) 103.0 (C-1′), 101.4 (C-1), 80.4
(C-4′), 80.0 (C-2′), 79.2 (C-2), 78.3 (C-3′), 75.4 (C-5), 72.8 (C-3), 69.7
(C-4), 63.3 (C-6), 57.7 (OCH3), 35.6 (C-5′), 17.7 (SCH3). HRMS (ESI)
calcd for (M + Na) C13H24O9S 379.1033, found 379.1031.
Methyl 2-O-(5-Deoxy-5-methylthio-R-D-xylofuranosyl)-R-D-man-
nopyranoside (1). Disaccharide 23 (21 mg, 0.03 mmol) was dissolved
in THF (5 mL), the solution was cooled to -78 °C, and then NH3 (20
mL) was condensed into the flask using a dry ice trap. Sodium metal
(80 mg) was added in three portions until a deep blue color persisted.
The solution was stirred for 1.5 h at -78 °C, and then CH3OH (2 mL)
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5067

A R T I C L E S
Joe et al.
Methyl 3-O-(5-Deoxy-5-methylthio-R-L-xylofuranosyl)-R-D-man-
nopyranoside (5). Prepared from 31 (32 mg, 0.04 mmol), liquid NH3
(25 mL), and sodium metal (80 mg) in THF (5 mL) as described for
1, to afford 5 (9 mg, 65%) as a foam. Rf 0.44 (85:15, CH2Cl2/CH3-
into ice water (30 mL) and extracted with CH2Cl2 (2 × 30 mL). The
combined CH2Cl2 extracts were washed with 7% aq CuSO4 solution
(3 × 75 mL) and water (1 × 75 mL) and then dried (Na2SO4) and
concentrated to a syrup that was purified by column chromatography
(4:1, hexanes/EtOAc) to afford 13 (2.12 g, 91%) as a syrup. Rf 0.5
(1;1, hexanes/EtOAc); [R]D -81.6 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3, δH) 7.53-7.40 (m, 8 H), 7.35-7.20 (m, 9 H), 7.10-7.14 (m,
2 H), 5.23 (d, 1 H, J ) 3.7 Hz), 4.34-4.28 (m, 2 H), 4.19 (dd, 1 H,
J ) 3.0, 5.1 Hz), 3.51 (dd, 1 H, J ) 4.6, 10.4 Hz), 3.32-3.27 (m, 2
H), 2.33 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC) 143.4 (3 C), 137.7,
132.3 (3 C), 130.6, 129.8 (3 C), 128.6 (4 C), 128.0 (4 C), 127.2 (3 C),
91.4, 87.6, 82.0, 80.2, 78.1, 62.9, 21.1. HRMS (ESI) calcd for (M +
Na) C31H30O4S 521.1757, found 521.1758.
1
OH); [R]D -18.4 (c 0.28, CH3OH); H NMR (500 MHz, D2O, δH)
5.27 (d, 1 H, J ) 4.4 Hz, H-1′), 4.80 (d, 1 H, J ) 1.8 Hz, H-1), 4.47
(ddd, 1 H, J ) 5.2, 5.6, 8.3 Hz, H-4′), 4.31 (dd, 1 H, J ) 4.6, 5.6 Hz,
H-3′), 4.20 (dd, 1 H, J ) 4.4, 4.6 Hz, H-2′), 4.12-4.10 (dd, 1 H, J )
1.8, 3.2 Hz, H-2), 3.94-3.87 (m, 2 H, H-3, H-6), 3.81-3.73 (m, 2 H,
H-4, H-6), 3.70-3.64 (m, 1 H, H-5), 3.42 (s, 3 H, OCH3), 2.80 (dd, 1
H, J ) 5.2, 13.8 Hz, H-5′), 2.68 (dd, 1 H, J ) 8.3, 13.8 Hz, H-5′),
2.16 (s, 3 H, SCH3); 13C NMR (125 MHz, D2O, δC) 103.4 (C-1′), 101.3
(C-1), 80.2(4) (C-4′), 80.2(1) (C-2′), 79.6 (C-2), 78.4 (C-3′), 75.3 (C-
5), 67.9(9) (C-3), 67.9(8) (C-4), 63.8 (C-6), 57.6 (OCH3), 35.7 (C-5′),
17.7 (SCH3). HRMS (ESI) calcd for (M + Na) C13H24O9S 379.1033,
found 379.1031.
p-Tolyl 2,3-Di-O-benzyl-5-O-trityl-1-thio-â-D-xylofuranoside (14).
To a solution of 13 (2.0 g, 4.0 mmol) in DMF (8 mL) at 0 °C was
added NaH (60% suspension in oil, 0.42 g, 10.42 mmol) in portions.
The mixture was stirred for 5 min before benzyl bromide (1.25 mL,
10.5 mmol) was added dropwise. After stirring for 4 h, the reaction
mixture was poured into ice water (80 mL) and extracted with CH2Cl2
(2 × 40 mL). The combined CH2Cl2 extracts were washed with water
(2 × 40 mL), dried (Na2SO4), and concentrated to a syrup that was
purified by column chromatography (12:1, hexanes/EtOAc) to afford
14 (2.2 g, 81%) as a syrup. Rf 0.46 (5.6:1, hexanes/EtOAc); [R]D -65.6
(c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, δH) 7.50-7.05 (m, 29 H),
5.34 (d, 1 H, J ) 2.8 Hz), 4.60 (d, 1 H, J ) 11.9 Hz), 4.52 (d, 1 H, J
) 12.2 Hz), 4.49 (d, 1 H, J ) 12.2 Hz), 4.40 (dd, 1 H, J ) 5.6, 10.6
Hz), 4.32 (d, 1 H, J ) 12.2 Hz), 4.10 (dd, 1 H, J ) 1.7, 1.7 Hz), 4.00
(dd, 1 H, J ) 1.7, 4.5 Hz), 3.60 (dd, 1 H, J ) 6.4, 9.6 Hz), 3.32 (dd,
1 H, J ) 5.5, 9.6 Hz), 2.31 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC)
144.1 (3 C), 137.7, 137.4, 137.1, 131.7, 131.6 (3 C), 129.6 (2 C), 128.8
(4 C), 128.5 (2 C), 128.3 (2 C), 128.2, 127.9, 127.8(4), 127.8(2), 127.7-
(4) (4 C), 127.7(2), 127.7, 127.6 (2 C), 127.3, 126.9 (3 C), 90.5, 86.8,
86.8, 81.6, 81.4, 72.0, 71.7, 62.5, 21.1. HRMS (ESI) calcd for (M +
Na) C45H42O4S 701.2696, found 701.2698.
Methyl 4-O-(5-Deoxy-5-methylthio-R-L-xylofuranosyl)-R-D-man-
nopyranoside (6). Prepared from 33 (32 mg, 0.04 mmol), liquid NH3
(30 mL), and sodium metal (90 mg) in THF (5 mL) as described for
1, to afford 6 (9 mg, 67%) as a foam. Rf 0.5 (85:15, CH2Cl2/CH3OH);
1
[R]D +1.3 (c 0.5, CH3OH); H NMR (500 MHz, D2O, δH) 5.21 (d, 1
H, J ) 4.4 Hz, H-1′), 4.77 (d, 1 H, J ) 1.8 Hz, H-1), 4.47 (ddd, 1 H,
J ) 5.2, 4.9, 8.6 Hz, H-4′), 4.28 (dd, 1 H, J ) 5.2, 4.6 Hz, H-3′), 4.20
(dd, 1 H, J ) 4.6, 4.4 Hz, H-2′), 3.99 (dd, 1 H, J ) 5.8, 3.4 Hz, H-2),
3.90-3.86 (m, 2 H, H-3, H-6), 3.85-3.76 (m, 2 H, H-4, H-6), 3.75-
3.71 (m, 1 H, H-5), 3.41 (s, 3 H, OCH3), 2.80 (dd, 1 H, J ) 4.9, 13.8
Hz, H-5′), 2.68 (dd, 1 H, J ) 8.6, 13.8 Hz, H-5′), 2.16 (s, 3 H, SCH3);
13C NMR (125 MHz, D2O, δC) 104.6 (C-1′), 103.6 (C-1), 80.3 (C-4′),
79.6 (C-2′), 78.6 (C-2), 78.2 (C-3′), 74.1 (C-5), 72.7 (C-3), 72.1 (C-4),
63.3 (C-6), 57.7 (OCH3), 35.8 (C-5′), 17.8 (SCH3). HRMS (ESI) calcd
for (M + Na) C13H24O9S 379.1033, found 379.1034.
p-Tolyl 2,3-Di-O-benzyl-5-O-toluenesulfonyl-1-thio-â-D-xylo-
furanoside (7). To a solution of 15 (1.1 g, 2.52 mmol) in pyridine (6
mL) at 0 °C was added toluenesulfonyl chloride (0.625 g, 3.28 mmol).
The reaction mixture was stirred at rt for 12 h and then poured into ice
water (40 mL) and extracted with CH2Cl2 (2 × 40 mL). The combined
CH2Cl2 extracts were washed with 7% aq. CuSO4 solution (3 × 75
mL) and water (1 × 75 mL) and then dried (Na2SO4) and concentrated
to a syrup that was purified by column chromatography (12:1, hexanes/
EtOAc) to afford 7 (1.29 g, 87%) as a syrup. Rf 0.38 (4:1, hexanes/
EtOAc); [R]D -70.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, δH)
7.80-7.75 (m, 2 H), 7.40-7.20 (m, 14 H), 7.10-7.05 (m, 2 H), 5.25
(d, 1 H, J ) 2.8 Hz), 4.56 (d, 1 H, J ) 11.8 Hz), 4.48 (dd, 2 H, J )
8.8, 11.8 Hz), 4.41-4.34 (m, 3 H), 4.32-4.25 (m, 1 H), 4.07-4.02
(m, 2 H), 2.40 (s, 3 H), 2.32 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC)
144.7, 137.5, 137.2, 137.1, 132.8, 131.9 (2 C), 130.9, 129.8 (2 C),
129.7 (2 C), 128.5 (2 C), 128.4(7) (2 C), 128.1, 128.0 (2 C), 127.8 (5
C), 90.8, 86.2, 81.4, 79.2, 72.1 (2 C), 68.2, 21.6, 21.1. HRMS (ESI)
calcd for (M + Na) C33H34O6S2 613.1689, found 613.1690.
p-Tolyl 2,3-Di-O-benzyl-1-thio-â-D-xylofuranoside (15). To a
solution of 14 (2.1 g, 3.09 mmol) in CH2Cl2/CH3OH (7:3, 30 mL) at
rt was added p-TsOH (40 mg). The mixture was stirred for 15 h,
neutralized with Et3N, and concentrated to a syrup that was purified
by column chromatography (4:1, hexanes/EtOAc) to afford 15 (1.12
g, 83%) as a syrup. Rf 0.21 (4:1, hexanes/EtOAc); [R]D -82.7 (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3, δH) 7.45-7.25 (m, 12 H), 7.15-
7.10 (m, 2 H), 5.32 (d, 1 H, J ) 4.0 Hz), 4.72 (d, 1 H, J ) 11.8 Hz),
4.60 (d, 1 H, J ) 11.8 Hz), 4.58 (d, 1 H, J ) 11.8 Hz), 4.45 (d, 1 H,
J ) 11.8 Hz), 4.27 (dd, 1 H, J ) 5.2, 10.5 Hz), 4.21-4.16 (m, 2 H),
3.92-3.82 (m, 2 H), 2.33 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC)
137.7, 137.4, 137.3, 132.2 (2 C), 130.6, 129.8 (2 C), 128.6 (2 C), 128.5
(2 C), 128.0(2), 128.0(1), 127.9 (2 C), 127.7 (2 C), 90.1, 86.5, 83.0,
81.1, 72.4, 72.2, 61.7, 21.1. HRMS (ESI) calcd for (M + Na) C26H28O4S
459.1600, found 459.1600.
p-Tolyl 2,3-Di-O-benzyl-5-O-toluenesulfonyl-1-thio-â-L-xylo-
furanoside (8). Prepared from 21 (0.9 g, 2.06 mmol) and toluene-
sulfonyl chloride (0.51 g, 2.68 mmol) in pyridine (6 mL) as described
for 7, to afford 8 (0.936 g, 77%) as a syrup. Rf 0.38 (4:1, hexanes/
EtOAc); [R]D +67.8 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, δH)
7.80-7.75 (m, 2 H), 7.40-7.20 (m, 14 H), 7.10-7.05 (m, 2 H), 5.25
(d, 1 H, J ) 2.8 Hz), 4.56 (d, 1 H, J ) 11.8 Hz), 4.48 (dd, 2 H, J )
8.8, 11.8 Hz), 4.41-4.34 (m, 3 H), 4.32-4.25 (m, 1 H), 4.07-4.02
(m, 2 H), 2.40 (s, 3 H), 2.32 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC)
144.7, 137.5, 137.2, 137.1, 132.8, 131.9 (2 C), 130.9, 129.8 (2 C),
129.7 (2 C), 128.5(2) (2 C), 128.4(7) (2 C), 128.1, 128.0, 127.9(6),
127.8 (5 C), 90.8, 86.2, 81.4, 79.2, 72.1 (2 C), 68.2, 21.6, 21.1. HRMS
(ESI) calcd for (M + Na) C33H34O6S2: 613.1689, found 613.1691.
p-Tolyl 5-O-Trityl-1-thio-â-D-xylofuranoside (13). To a solution
of 1230 (1.2 g, 4.67 mmol) in pyridine (8 mL) at rt was added DMAP
(0.183 g, 1.5 mmol) followed by trityl chloride (1.63 g, 5.84 mmol).
The reaction mixture was stirred at 45 °C for 14 h and then poured
1,2,3,5-Tetra-O-acetyl-L-xylofuranose (16). L-Xylose (4.17 g, 27.8
mmol), boric acid (3.8 g, 60.7 mmol), and acetic acid (95 mL) were
stirred at 50 °C for 1 h before acetic anhydride (95 mL) was added.
The mixture was heated at 50 °C for 16 h and then cooled to rt. The
boric acid was removed as trimethyl borate by the addition of methanol
(20 mL) and in vacuo concentration of the resulting mixture to 100
mL and then the addition of methanol (10 mL) and concentration in
vacuo to 50 mL (repeated twice). Acetic anhydride (100 mL) and
pyridine (100 mL) were added and the solution was stirred at rt for 2
h. Ice (∼250 g) was added, and the mixture was stirred for 1 h and
then extracted with CH2Cl2 (3 × 150 mL). The combined CH2Cl2
extracts were washed with 7% aq. CuSO4 solution (3 × 300 mL) and
water (2 × 250 mL) and then dried (Na2SO4) and concentrated to a
syrup that was purified by column chromatography (7:3, hexanes/
EtOAc) to afford 16 (7.96 g, 90%, R:â, 1:1.8) as a syrup. Rf 0.2 (7:3,
1
hexanes/EtOAc); H NMR (500 MHz, CDCl3, δH) 6.42 (d, 0.35 H, J
) 4.6 Hz), 6.10 (s, 0.65 H), 5.52 (dd, 0.35 H, J ) 6.5, 6.5 Hz), 5.36
9
5068 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006

5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
(dd, 0.65 H, J ) 1.7, 5.6 Hz), 5.30 (dd, 0.35 H, J ) 4.6, 6.2 Hz), 5.20
(d, 0.65 H, J ) 1.0 Hz), 4.67-4.60 (m, 1 H), 4.27-4.18 (m, 1.65 H),
4.12 (dd, 0.35 H, J ) 4.2, 12.2 Hz), 2.12 (s, 2H), 2.11 (s, 2 H), 2.09
(s, 3 H), 2.07 (s, 3 H), 2.06 (s, 2 H); 13C NMR (125 MHz, CDCl3, δC)
170.5, 170.3, 169.6, 169.5, 169.3, 169.2, 169.1, 98.8, 92.8, 79.9, 79.4-
(1), 75.3(9), 75.3, 74.3, 73.8, 62.3, 61.6, 21.0, 20.9, 20.8, 20.7, 20.6,
20.5, 20.4. HRMS (ESI) calcd for (M + Na) C13H18O9: 341.0843, found
341.0845.
p-Tolyl 2,3-Di-O-benzyl-1-thio-â-L-xylofuranoside (21). Prepared
from 20 (1.9 g, 2.80 mmol) and p-TsOH (40 mg) in CH2Cl2/CH3OH
(7:3, 30 mL) as described for 15, to afford 21 (0.99 g, 81%) as a syrup.
1
Rf 0.21 (4:1, hexanes/EtOAc); [R]D +89.7 (c 0.5, CHCl3); H NMR
(500 MHz, CDCl3, δH) 7.45-7.25 (m, 12 H), 7.15-7.10 (m, 2 H),
5.32 (d, 1 H, J ) 4.0 Hz), 4.72 (d, 1 H, J ) 11.8 Hz), 4.60 (d, 1 H, J
) 11.8 Hz), 4.58 (d, 1 H, J ) 11.8 Hz), 4.45 (d, 1 H, J ) 11.8 Hz),
4.27 (dd, 1 H, J ) 5.2, 10.5 Hz), 4.21-4.16 (m, 2 H), 3.92-3.82 (m,
2 H), 2.33 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC) 137.7, 137.4,
137.3, 132.2 (2 C), 130.6, 129.8 (2 C), 128.6 (2 C), 128.5 (2 C), 128.0-
(2), 128.0(1), 127.9 (2 C), 127.7 (2 C), 90.1, 86.5, 83.0, 81.1, 72.4,
72.2, 61.7, 21.1. HRMS (ESI) calcd for (M + Na) C26H28O4S:
459.1600, found 459.1601.
p-Tolyl 2,3,5-Tri-O-acetyl-1-thio-â-L-xylofuranoside (17). To a
solution of 16 (3.0 g, 9.43 mmol) in CH2Cl2 (60 mL) at -20 °C was
added p-thiocresol (1.29 g, 10.38 mmol) followed by BF3‚Et2O (2.96
mL, 23.58 mmol) dropwise over 6 min. The reaction mixture was stirred
at -20 °C for 6 h, neutralized (at -20 °C) with Et3N, and concentrated
to a syrup that was purified by column chromatography (4:1, hexanes/
EtOAc,) to afford 17 (2.3 g, 75%, â:R, 1:49) as a syrup. Rf 0.37 (7:3,
hexanes/EtOAc); data for major isomer; [R]D +83.8 (c 0.5, CHCl3);
1H NMR (400 MHz, CDCl3, δH) 7.44 (d, 2 H, J ) 8.1 Hz), 7.14 (d, 2
H, J ) 8.1 Hz), 5.30 (dd, 1 H, J ) 2.2, 5.1 Hz), 5.26 (dd, 1 H, J )
2.2, 3.3 Hz), 5.18 (d, 1 H, J ) 3.3 Hz), 4.45 (ddd, 1 H, J ) 5.1, 5.1,
6.5 Hz), 4.32 (dd, 1 H, J ) 5.1, 11.7 Hz), 4.24 (dd, 1 H, J ) 6.5, 11.7
Hz), 2.33 (s, 3 H), 2.09 (s, 3 H), 2.07 (s, 3 H), 2.05 (s, 3 H); 13C NMR
(100 MHz, CDCl3, δC) 170.5, 169.6, 169.2, 138.2, 133.3 (2 C), 129.7
(2 C), 129.3, 90.2, 80.4, 78.4, 75.2, 62.0, 21.1, 20.8, 20.7, 20.6. HRMS
(ESI) calcd for (M + Na) C18H22O7S 405.0978, found 405.0977.
Methyl
2-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-D-xylo-
furanosyl)-3-O-benzyl-4,6-O-benzylidene-R-D-mannopyranoside (22).
Thioglycoside 7 (0.21 g, 0.35 mmol) and alcohol 929 (0.11 g, 0.3 mmol)
were dried over P2O5 under a vacuum for 6 h and then dissolved in
CH2Cl2 (4 mL), and the resulting solution was cooled to 0 °C. Powdered
4 Å molecular sieves (75 mg) were added, and the suspension was
stirred for 20 min at 0 °C before N-iodosuccinimide (96 mg, 0.42 mmol)
and silver triflate (16 mg, 0.06 mmol) were added. The reaction mixture
was stirred for 15 min, neutralized with Et3N, diluted with CH2Cl2 (10
mL), and filtered through Celite. The filtrate was washed successively
with saturated aqueous sodium thiosulfate (3 × 15 mL) and water (1
× 15 mL) and then dried (Na2SO4) and concentrated to a syrup that
was purified by column chromatography (4:1, hexanes/EtOAc) to afford
22 (0.22 g, 91%), as a syrup. The product was an inseparable mixture
of isomers (R/â, 87:13), which was used in the next step; data provided
p-Tolyl 1-Thio-â-L-xylofuranoside (18). To a solution of 17 (2.0
g, 5.24 mmol) in CH2Cl2/CH3OH (7:3, 30 mL) was added NaOCH3
(0.16 g, 3.0 mmol). The mixture was stirred at room temperature for 7
h and then neutralized with glacial HOAc and concentrated to a syrup
that was purified by column chromatography (3:7, hexanes/EtOAc) to
afford 18 (1.13 g, 84%) as a syrup; Rf 0.22 (3:7, hexanes/EtOAc); [R]D
1
for major isomer. Rf 0.49 (7:3, hexanes/EtOAc); H NMR (500 MHz,
CDCl3, δH) 7.77 (d, 2 H, J ) 8.4 Hz), 7.50-7.20 (m, 22 H), 5.42 (d,
1 H, J ) 4.3 Hz), 5.27 (s, 1 H), 4.88 (d, 1 H, J ) 11.5 Hz), 4.82 (d,
1 H, J ) 11.3 Hz), 4.69 (d, 1 H, J ) 11.6 Hz), 4.64 (d, 1 H, J ) 1.6
Hz), 4.64 (d, 1 H, J ) 12.0 Hz), 4.48 (d, 1 H, J ) 11.9 Hz), 4.46-
4.39 (m, 1 H), 4.39-4.33 (m, 2 H), 4.29 (dd, 1 H, J ) 3.6, 11.0 Hz),
4.20 (d, 1 H, J ) 5.4 Hz), 4.13-4.07 (m, 2 H), 4.07-4.02 (m, 1 H),
3.96 (dd, 1 H, J ) 3.1, 9.8 Hz), 3.93 (dd, 1 H, J ) 4.3, 5.5 Hz), 3.75
(d, 2 H, J ) 7.1 Hz), 3.37 (s, 3 H), 2.43 (s, 3 H); 13C NMR (125 MHz,
CDCl3, δC) 144.7, 138.5, 138.1, 137.8, 137.7, 133.0, 129.7(4),
129.7(0), 128.8, 128.5, 128.3(7) (2 C), 128.3(5) (2 C), 128.3 (2 C),
128.1(8) (2 C), 128.1(5), 128.0, 127.9, 127.8, 127.6(9), 127.6(6),
127.5(9), 127.5(7), 127.5, 126.1, 126.0 (2 C), 101.4, 99.1, 97.5, 84.5,
81.4, 78.4, 74.4(4), 74.4(0), 72.5, 72.1(7), 72.1(5), 71.8, 68.9, 68.8,
64.1, 54.9, 21.6. HRMS (ESI) calcd for (M + Na) C47H50O12S 861.2915,
found 861.2912.
1
+151.2 (c 0.5, CH3OH); H NMR (500 MHz, CD3OD, δH) 7.40 (d, 2
H, J ) 8.2 Hz), 7.12 (d, 2 H, J ) 8.2 Hz), 5.06 (d, 1 H, J ) 3.7 Hz),
4.16-4.10 (m, 2 H), 4.06 (dd, 1 H, J ) 2.5, 3.7 Hz), 3.82 (dd, 1 H, J
) 4.3, 11.5 Hz), 3.74 (dd, 1 H, J ) 5.9, 11.5 Hz), 2.29 (s, 3 H); 13C
NMR (125 MHz, CD3OD, δC) 138.4, 133.3, 132.7 (2 C), 130.6 (2 C),
93.5, 83.9, 83.5, 77.9, 62.2, 21.1. HRMS (ESI) calcd for (M + Na)
C12H16O4S 279.0661, found 279.0659.
p-Tolyl 5-O-Trityl-1-thio-â-L-xylofuranoside (19). Prepared from
18 (1.05 g, 4.09 mmol), DMAP (0.123 g, 1.0 mmol), and trityl chloride
(1.425 g, 5.11 mmol) in pyridine (7 mL) as described for 13, to afford
19 (1.814 g, 89%) as a syrup. Rf 0.5 (1:1, hexanes/EtOAc); [R]D +88.6
(c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, δH) 7.53-7.40 (m, 8 H),
7.35-7.20 (m, 9 H), 7.10-7.14 (m, 2 H), 5.23 (d, 1 H, J ) 3.7 Hz),
4.34-4.28 (m, 2 H), 4.19 (ddd, 1 H, J ) 3.0, 2.2, 5.2 Hz), 3.51 (dd,
1 H, J ) 4.6, 10.4 Hz), 3.32-3.27 (m, 2 H), 2.33 (s, 3 H); 13C NMR
(125 MHz, CDCl3, δC) 143.4 (3 C), 137.7, 132.3 (3 C), 130.6, 129.8
(3 C), 128.6 (4 C), 128.0 (4 C), 127.2 (3 C), 91.4, 87.6, 82.0, 80.2,
78.1, 62.9, 21.1. HRMS (ESI) calcd for (M + Na) C31H30O4S 521.1757,
found 521.1753.
Methyl 2-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-D-xylo-
furanosyl)-3-O-benzyl-4,6-O-benzylidene-R-D-mannopyranoside (23).
To a solution of 22 (70 mg, 0.08 mmol) in CH3CN (2 mL) was added
18-crown-6 (20 mg) followed by sodium thiomethoxide (13 mg, 0.24
mmol). The reaction mixture was heated at reflux for 12 h and then
cooled to rt before being diluted with CH3CN (6 mL) and filtered
through Celite. The filtrate was concentrated to a syrup that was purified
by column chromatography (5.6:1, hexanes/EtOAc) to afford 23 (42
mg, 70%) as a syrup. The product was an inseparable mixture of isomers
(R/â, 87:13), which was used in the next step; data provided for major
isomer. Rf 0.39 (4:1, hexanes/EtOAc); 1H NMR (400 MHz, CDCl3, δH)
7.55-7.20 (m, 20 H), 5.46 (d, 1 H, J ) 4.4 Hz), 5.30 (s, 1 H), 4.90 (d,
1 H, J ) 9.3 Hz), 4.87 (d, 1 H, J ) 9.0 Hz), 4.75 (d, 1 H, J ) 1.6 Hz),
4.70 (dd, 2 H, J ) 7.5, 11.6 Hz), 4.54 (d, 1 H, J ) 11.9 Hz), 4.48-
4.40 (m, 2 H), 4.27 (dd, 2 H, J ) 4.7, 6.5 Hz), 4.22-4.16 (m, 2 H),
4.03-3.94 (m, 2 H), 3.78-3.74 (m, 2 H), 3.38 (s, 3 H), 2.85 (dd, 1 H,
p-Tolyl 2,3-Di-O-benzyl-5-O-trityl-1-thio-â-L-xylofuranoside (20).
Prepared from 19 (1.8 g, 3.60 mmol), NaH (0.374 g, 9.36 mmol), and
benzyl bromide (1.1 mL, 9.36 mmol) in DMF (9 mL) as described for
14, to afford 20 (1.96 g, 80%) as a syrup. Rf 0.46 (5.6:1, hexanes/
EtOAc,); [R]D +73.9 (c 1.2, CHCl3); 1H NMR (500 MHz, CDCl3, δH)
7.50-7.05 (m, 29 H), 5.34 (d, 1 H, J ) 2.8 Hz), 4.60 (d, 1 H, J )
11.9 Hz), 4.50 (d, 1 H, J ) 11.9 Hz), 4.48 (d, 1 H, J ) 12.2 Hz), 4.40
(dd, 1 H, J ) 5.7, 10.6 Hz), 4.32 (d, 1 H, J ) 12.2 Hz), 4.10 (dd, 1 H,
J ) 1.7, 1.7 Hz), 4.0 (dd, 1 H, J ) 1.7, 4.5 Hz), 3.60 (dd, 1 H, J )
6.4, 9.6 Hz), 3.32 (dd, 1 H, J ) 5.5, 9.6 Hz), 2.31 (s, 3 H, CH3); 13C
NMR (125 MHz, CDCl3, δC) 144.1 (3 C), 137.7, 137.4, 137.1, 131.7,
131.6 (3 C), 129.6 (2 C), 128.8 (4 C), 128.5 (2 C), 128.2(9) (2 C),
128.2(5), 127.9, 127.8(4), 127.8(2), 127.7(4) (4 C), 127.7(2), 127.7,
127.6 (2 C), 127.3, 126.9 (3 C), 90.5, 86.8, 86.8, 81.6, 81.4, 72.0, 71.7,
62.5, 21.1. HRMS (ESI) calcd for (M + Na) C45H42O4S 701.2696, found
701.2695.
J ) 5.1, 13.8 Hz), 2.70 (dd, 1 H, J ) 7.9, 13.8 Hz), 2.16 (s, 3 H); 13
C
NMR (125 MHz, CDCl3, δC) 138.6, 138.2, 138.1, 137.7, 128.8, 128.3-
(3) (3 C), 128.3(2) (3 C), 128.3 (2 C), 128.2 (2 C), 127.7 (4 C), 127.6-
(1), 127.6(0), 127.5 (2 C), 126.0, 101.7, 101.6, 101.2, 84.1, 82.1, 79.4,
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5069

A R T I C L E S
Joe et al.
77.3, 76.2, 75.7, 73.7, 72.0, 71.5, 68.7, 63.9, 54.8, 34.1, 16.5. HRMS
(ESI) calcd for (M + Na) C41H46O9S 737.2754, found 737.2750.
73.3, 73.1, 72.6, 72.4, 71.9, 71.8, 70.8, 70.5, 69.7, 69.1, 54.8, 21.6.
HRMS (ESI) calcd for (M + Na) C54H58O12S 953.3541, found
953.3540.
Methyl
3-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-D-xylo-
Methyl 4-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-D-xylo-
furanosyl)-2,3,6-tri-O-benzyl-R-D-mannopyranoside (27). Prepared
from 26 (0.7 g, 0.75 mmol), 18-crown-6 (60 mg), and sodium
thiomethoxide (0.16 g, 2.29 mmol) in CH3CN (14 mL) as described
for 23 to afford 27 (0.46 g, 76%) as a syrup; Rf 0.3 (4:1, hexanes/
EtOAc); [R]D +67.4 (c 0.3, CHCl3); 1H NMR (500 MHz, CDCl3, δH)
7.40-7.05 (m, 25 H), 5.55 (d, 1 H, J ) 4.4 Hz), 4.84 (d, 1 H, J ) 1.7
Hz), 4.74 (d, 1 H, J ) 12.5 Hz), 4.66 (d, 1 H, J ) 12.3 Hz), 4.64-
4.56 (m, 4 H), 4.54 (d, 1 H, J ) 11.8 Hz), 4.43 (d, 1 H, J ) 11.8 Hz),
4.40 (d, 1 H, J ) 11.8 Hz), 4.22 (d, 1 H, J ) 12.1 Hz), 4.14 (dd, 1 H,
J ) 9.6, 9.6 Hz), 4.10-4.03 (m, 2 H), 3.97-3.82 (m, 5 H), 3.72 (dd,
1 H, J ) 7.4, 10.7 Hz), 3.39 (s, 3 H), 2.68 (dd, 1 H, J ) 4.4, 13.8 Hz),
2.52 (dd, 1 H, J ) 6.3, 13.8 Hz), 2.06 (s, 3 H); 13C NMR (125 MHz,
CDCl3, δC) 138.7, 138.3, 138.1(4), 138.1, 137.7, 128.4(3), 128.3(8) (2
C), 128.3 (3 C), 128.2(8), 128.2(4) (2 C), 127.8, 127.7 (2 C), 127.6(7)
(2 C), 127.6 (3 C), 127.5(8) (2 C), 127.5 (2 C), 127.4, 127.3, 126.8 (2
C), 100.7, 98.5, 82.5, 81.7, 80.3, 77.2, 73.3, 73.2, 72.5, 72.4, 71.8(9),
71.8(8), 71.0, 70.6, 70.1, 54.8, 34.8, 16.6. HRMS (ESI) calcd for (M
+ Na) C48H54O9S 829.3380, found 829.3380.
furanosyl)-2,4,6-tri-O-benzyl-R-D-mannopyranoside (24). Prepared
from thioglycoside 7 (0.12 g, 0.2 mmol), alcohol 1029 (67 mg, 0.14
mmol), N-iodosuccinimide (55 mg, 0.24 mmol), and silver triflate (10
mg, 0.04 mmol) in CH2Cl2 (3 mL) as described for 22, to afford 24
(98 mg, 73%) as a syrup. The product 24 could not be completely
purified from ∼12% of the â-glycoside and some hydrolyzed donor
and hence was used as such for the next step; data provided for major
1
isomer. Rf 0.33 (4:1, hexanes/EtOAc); [R]D +67.5 (c 0.5, CHCl3); H
NMR (500 MHz, CDCl3, δH) 7.20 (d, 2 H, J ) 8.3 Hz), 7.40-7.14
(m, 25 H), 7.14-7.06 (m, 2 H), 5.20 (d, 1 H, J ) 4.2 Hz), 4.86 (d, 1
H, J ) 11.2 Hz), 4.82 (d, 1 H, J ) 11.6 Hz), 4.76 (d, 1 H, J ) 1.7
Hz), 4.69 (d, 1 H, J ) 8.4 Hz), 4.66 (d, 1 H, J ) 12.0 Hz), 4.60 (d, 1
H, J ) 12.0 Hz), 4.54 (d, 1 H, J ) 3.5 Hz), 4.51 (d, 1 H, J ) 11.3
Hz), 4.42 (d, 1 H, J ) 11.7 Hz), 4.38 (d, 1 H, J ) 8.1 Hz), 4.29-4.24
(m, 2 H), 4.18 (dd, 1 H, J ) 3.6, 10.5 Hz), 4.03 (dd, 2 H, J ) 3.2, 9.4
Hz), 4.00-3.94 (m, 1 H), 3.88-3.84 (m, 2 H), 3.80-3.70 (m, 3 H),
3.38 (s, 3 H), 2.40 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC) 144.6,
138.7, 138.6(9), 138.4, 137.6(0), 137.6, 133.0, 129.7, 128.6, 128.5,
128.4, 128.3(9) (2 C), 128.3(3) (2 C), 128.2(5), 128.2(4) (2 C), 128.2,
128.0, 127.9, 127.8, 127.7, 127.6(4) (2 C), 127.6(3) (2 C), 127.5(9) (2
C), 127.5(7) (2 C), 127.5(5), 127.3(9), 127.3(6), 127.2, 127.0, 101.9,
98.7, 82.8, 81.0, 80.1, 78.0, 74.6, 74.5, 74.4, 73.4, 72.6, 72.5, 72.3,
71.8, 69.4, 69.1, 54.9, 21.6. HRMS (ESI) calcd for (M + Na)
C54H58O12S 953.3541, found 953.3541.
Methyl
2-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-L-xylo-
furanosyl)-3-O-benzyl-4,6-O-benzylidene-R-D-mannopyranoside (28).
Prepared from thioglycoside 8 (0.12 g, 0.2 mmol), alcohol 929 (54 mg,
0.15 mmol), N-iodosuccinimide (0.54 g, 0.24 mmol), and silver triflate
(10 mg, 0.04 mmol) in CH2Cl2 (3 mL) as described for 22, to afford
28 (89 mg, 73%) as a syrup. Rf 0.24 (4:1, hexanes/EtOAc); [R]D -65.6
(c 0.5, CHCl3); 1H NMR (500 MHz, CDCl3, δH) 7.73 (d, 2 H, J ) 8.2
Hz), 7.50 (d, 2 H, J ) 8.2 Hz), 7.45-7.20 (m, 20 H), 5.58 (s, 1 H),
5.08 (d, 1 H, J ) 4.0 Hz), 4.70 (s, 1 H), 4.64 (s, 1 H), 4.65-4.54 (m,
3 H), 4.50 (d, 1 H, J ) 11.0 Hz), 4.46 (d, 1 H, J ) 11.9 Hz), 4.39 (dd,
1 H, J ) 5.8, 7.2 Hz), 4.25-4.07 (m, 5 H), 4.03 (dd, 1 H, J ) 4.2, 5.8
Hz), 3.92 (dd, 1 H, J ) 3.4, 10.0 Hz), 3.80-3.70 (m, 2 H), 3.34 (s, 3
H), 2.39 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC) 144.5, 138.5, 137.9,
137.8, 137.7, 133.1, 129.7 (2 C), 128.8, 128.4 (2 C), 128.3(5) (2 C),
128.3 (2 C), 128.1(2) (3 C), 128.1, 127.9 (3 C), 127.7 (2 C), 127.5,
127.5 (2 C), 127.4, 126.1 (2 C), 101.4, 99.1, 97.5, 84.5, 81.4, 78.4,
74.4(4), 74.4, 72.5, 72.1(7), 72.1(5), 71.8, 68.9, 68.8, 64.1, 54.9, 21.6.
HRMS (ESI) calcd for (M + Na) C47H50O12S 861.2915, found
861.2911.
Methyl 3-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-D-xylo-
furanosyl)-2,4,6-tri-O-benzyl-R-D-mannopyranoside (25). Prepared
from 24 (40 mg, 0.04 mmol), 18-crown-6 (10 mg), and sodium
thiomethoxide (8 mg, 0.12 mmol) in CH3CN (1 mL) as described for
23, to afford 25 (23 mg, 72%) as a syrup. Rf 0.38 (4:1, hexanes/EtOAc);
1
[R]D +62.1 (c 0.3, CHCl3); H NMR (500 MHz, CDCl3, δH) 7.46-
7.10 (m, 25 H), 5.34 (d, 1 H, J ) 4.1 Hz), 4.85 (d, 2 H, J ) 12.0 Hz),
4.76 (d, 2 H, J ) 12.0 Hz), 4.66 (d, 2 H, J ) 12.0 Hz), 4.62-4.50 (m,
4 H), 4.45 (d, 1 H, J ) 12.1 Hz), 4.36 (dd, 1 H, J ) 6.2, 12.6 Hz),
4.23 (dd, 1 H, J ) 5.2, 5.2 Hz), 4.12 (dd, 1 H, J ) 3.1, 9.4 Hz), 4.02
(dd, 1 H, J ) 9.4, 9.4 Hz), 4.00-3.95 (m, 2 H), 3.82-3.70 (m, 3 H),
3.36 (s, 3 H, OCH3), 2.75 (dd, 1 H, J ) 5.6, 13.8 Hz), 2.63 (dd, 1 H,
J ) 7.4, 13.8 Hz, H-5′), 2.08 (s, 3 H, SCH3); 13C NMR (125 MHz,
CDCl3, δC) 138.9, 138.8, 138.4, 138.0, 137.9, 128.4 (2 C), 128.3 (2
C), 128.2(4) (3 C), 128.2(3), 128.2(1), 127.7, 127.6(8) (2 C), 127.6(4)
(3 C), 127.6(3) (2 C), 127.5 (3 C), 127.4, 127.3, 127.2, 127.1 (2 C),
102.2, 99.0, 83.1, 82.0, 79.8, 78.2, 77.7, 74.7, 74.5, 73.4, 72.7, 72.5,
72.4, 71.9, 69.4, 54.8, 34.3, 16.6. HRMS (ESI) calcd for (M + Na)
C48H54O9S 829.3380, found 829.3383.
Methyl 2-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-L-xylo-
furanosyl)-3-O-benzyl-4,6-O-benzylidene-R-D-mannopyranoside (29).
Prepared from 28 (44 mg, 0.05 mmol), 18-crown-6 (10 mg), and sodium
thiomethoxide (10 mg, 0.18 mmol) in CH3CN (1 mL) as described for
23, to afford 29 (25 mg, 71%) as a syrup. Rf 0.33 (4:1, hexanes/EtOAc);
1
[R]D -54.1 (c 0.3, CHCl3); H NMR (500 MHz, CDCl3, δH) 7.55-
Methyl
4-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-D-xylo-
7.20 (m, 20 H), 5.58 (s, 1 H), 5.17 (d, 1 H, J ) 4.2 Hz), 4.82 (d, 1 H,
J ) 12.6 Hz), 4.77 (d, 1 H, J ) 12.6 Hz), 4.73-4.65 (m, 3 H), 4.64-
4.52 (m, 3 H), 4.35 (dd, 1 H, J ) 5.0, 6.6 Hz), 4.28-4.25 (m, 1 H),
4.24 (dd, 1 H, J ) 4.0, 9.3 Hz), 4.20 (dd, 1 H, J ) 9.3, 9.3 Hz), 4.10
(dd, 1 H, J ) 4.7, 4.7 Hz), 3.95 (dd, 1 H, J ) 3.4, 10.0 Hz), 3.80-
3.70 (m, 2 H), 3.35 (s, 3 H), 2.80 (dd, 1 H, J ) 5.6, 13.8 Hz), 2.65
(dd, 1 H, J ) 7.6, 13.8 Hz), 2.02 (s, 3 H); 13C NMR (125 MHz, CDCl3,
δC) 138.8, 138.2, 138.0, 137.7, 128.8, 128.4 (2 C), 128.3 (2 C), 128.2
(2 C), 128.1 (2 C), 128.0 (2 C), 127.9, 127.6(1), 127.5(5) (2 C), 127.3-
(3) (2 C), 127.3, 126.1 (2 C), 101.4, 99.0, 97.5, 84.8, 82.3, 78.6, 76.9,
74.6, 72.4, 72.2, 72.1, 71.9, 68.8, 64.1, 55.0, 34.1, 16.4. HRMS (ESI)
calcd for (M + Na) C41H46O9S 737.2754, found 737.2756.
furanosyl)-2,3,6-tri-O-benzyl-R-D-mannopyranoside (26). Prepared
from thioglycoside 7 (0.76 g, 1.29 mmol), alcohol 1129 (0.4 g, 0.86
mmol), N-iodosuccinimide (0.35 g, 1.56 mmol), and silver triflate (66
mg, 0.25 mmol) in CH2Cl2 (15 mL) as described for 22, to afford 26
(0.71 g, 89%) as a syrup. The product was contaminated with ∼5% of
hydrolyzed 7, and thus after characterization by NMR, the disaccharide
1
was used directly in the next step. Rf 0.28 (4:1, hexanes/EtOAc); H
NMR (500 MHz, CDCl3, δH) 7.69 (d, 2 H, J ) 8.3 Hz), 7.40-7.10
(m, 25 H), 7.05-7.00 (m, 2 H), 5.41 (d, 1 H, J ) 4.3 Hz), 4.83 (s, 1
H), 4.72 (d, 1 H, J ) 12.4 Hz), 4.65 (d, 1 H, J ) 12.2 Hz), 4.62-4.53
(m, 3 H), 4.50-4.44 (m, 2 H), 4.38-4.34 (m, 2 H), 4.16 (d, 1 H, J )
12.0 Hz), 4.13-3.98 (m, 3 H), 3.94-3.82 (m, 5 H), 3.76 (dd, 1 H, J
) 4.4, 6.7 Hz), 3.66 (dd, 1 H, J ) 1.5, 10.5 Hz), 3.55 (dd, 1 H, J )
7.3, 10.5 Hz), 3.39 (s, 3 H), 2.36 (s, 3 H); 13C NMR (125 MHz, CDCl3,
δC) 144.6, 138.6, 138.3, 138.1, 137.7, 137.5, 133.0, 129.6 (2 C), 128.4
(2 C), 128.3(4) (2 C), 128.3(0), 128.2(9) (3 C), 128.2, 127.9, 127.8 (2
C), 127.7(4), 127.7(0) (3 C), 127.6(8) (2 C), 127.6 (2 C), 127.5 (2 C),
127.4(3) (2 C), 127.4, 126.8 (2 C), 100.5, 98.4, 82.2, 80.7, 80.1, 74.1,
Methyl
3-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-L-xylo-
furanosyl)-2,4,6-tri-O-benzyl-R-D-mannopyranoside (30). Prepared
from thioglycoside 8 (170 mg, 0.29 mmol), alcohol 1029 (93 mg, 0.2
mmol), N-iodosuccinimide (78 mg, 0.35 mmol), and silver triflate (15
mg, 0.06 mmol) in CH2Cl2 (4 mL) as described for 22, to afford 30
(150 mg, 82%) as a syrup. The product was contaminated with ∼17%
of hydrolyzed 8, and thus after characterization by NMR, the disac-
9
5070 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006

5-Deoxy-5-methylthio-xylofuranose Residue
A R T I C L E S
charide was used directly in the next step. Rf 0.29 (4:1, hexanes/EtOAc);
1H NMR (500 MHz, CDCl3, δH) 7.72 (d, 2 H, J ) 8.4 Hz), 7.37-7.11
(m, 27 H), 5.14 (d, 1 H, J ) 4.0 Hz), 4.81 (d, 1 H, J ) 2.3 Hz), 4.80
(d, 1 H, J ) 11.2 Hz), 4.72-4.58 (m, 4 H), 4.57-4.38 (m, 6 H), 4.35-
4.26 (m, 1 H), 4.24-4.14 (m, 3 H), 4.01 (dd, 1 H, J ) 5.9, 10.6 Hz),
3.95 (dd, 1 H, J ) 4.0, 5.9 Hz), 3.90 (dd, 1 H, J ) 8.9, 8.9 Hz), 3.83
(dd, 1H, J ) 2.5, 2.5 Hz), 3.74-3.72 (m, 2H), 3.37 (s, 3 H), 2.40 (s,
3 H); 13C NMR (125 MHz, CDCl3, δC) 144.5, 138.6, 138.5, 138.2,
137.8, 137.7, 133.0, 129.7, 129.7, 128.5, 128.4(0) (2 C), 128.3(7) (3
C), 128.3(1), 128.3(0), 128.2(6) (2 C), 128.9(9), 127.9(5), 127.9 (2
C), 127.8 (2 C), 127.7, 127.6(5), 127.6(2) (2 C), 127.6, 127.5(7) (4
C), 127.4(3), 127.4(2), 98.7, 97.3, 83.2, 81.2, 75.7, 74.9, 74.6, 73.3,
72.6, 72.5, 72.5, 72.2, 71.7, 69.4, 68.7, 54.9, 21.6. HRMS (ESI) calcd
for (M + Na) C54H58O12S 953.3541, found 953.3545.
78.5, 74.8, 73.4, 72.9, 72.7, 72.6(5), 72.6, 72.0, 71.8, 69.5, 54.6, 34.2,
16.4. HRMS (ESI) calcd for (M + Na) C48H54O9S 829.3380, found
829.3382.
Methyl 4-O-(5-Deoxy-5-sulfoxymethyl-R-D-xylofuranosyl)-R-D-
mannopyranoside (34). To a solution of 3 (60 mg, 0.17 mmol) in
distilled water (0.3 mL) was added a solution of H2O2 (30% aq., 0.019
mL). The reaction mixture was stirred for 9 min at rt and then
lyophilized. The residue was purified by column chromatography on
Iatrobeads (85:15, CH2Cl2/CH3OH) to afford 34 (51 mg, 81%, 1:1
mixture of diastereomers) as a foam. Rf 0.12 (5.6:1, CH2Cl2/CH3OH);
1
[R]D +160.4 (c 0.3, CH3OH); H NMR (500 MHz, D2O, δH) 5.47 (d,
0.5 H, J ) 4.5 Hz, H-1′), 5.46 (d, 0.5 H, J ) 4.4 Hz, H-1′), 4.76 (s,
1H, H-1), 4.65 (ddd, 0.5 H, J ) 5.2, 4.4, 8.5 Hz, H-4′), 4.62 (ddd, 0.5
H, J ) 5.2, 4.6, 8.5 Hz, H-4′), 4.34 (dd, 1 H, J ) 5.2, 4.5 Hz, H-3′),
4.23 (dd, 1 H, J ) 4.5, 4.5 Hz, H-2′), 4.20 (dd, 1 H, J ) 4.4, 4.5 Hz,
H-2′), 3.94-3.85 (m, 3 H, H-2, H-3, H-6), 3.85-3.76 (m, 2 H, H-4,
H-6), 3.72-3.66 (m, 1 H, H-5), 3.41 (s, 3 H, OCH3), 3.29 (dd, 0.5 H,
J ) 4.4, 13.9 Hz, H-5′), 3.15-3.10 (m, 1.0 H, H-5′), 3.09 (dd, 0.5 H,
J ) 8.5, 13.9 Hz, H-5′), 2.81 (s, 1.5 H, S(O)CH3), 2.80 (s, 1.5 H, S(O)-
CH3); 13C NMR (125 MHz, D2O, δC) 105.6 (1 C, C-1′), 103.7 (1 C,
C-1), 79.4 (0.5 C, C-2′), 79.1 (0.5 C, C-2′), 78.6(0) (0.5 C, C-3′), 78.5-
(7) (0.5 C, C-3′), 77.0 (0.5 C, C-2), 76.8 (0.5 C, C-2), 76.4 (0.5 C,
C-4′), 75.7 (0.5 C, C-4′), 73.8(7) (0.5 C, C-5), 73.8(5) (0.5 C, C-5),
73.5(1) (0.5 C, C-3), 73.4(9) (0.5 C, C-3), 73.0 (1 C, C-4), 63.7(1)
(0.5 C, C-6), 63.6(9) (0.5 C, C-6), 57.6 (1 C, OCH3), 57.2 (0.5 C,
C-5′), 55.7 (0.5 C, C-5′), 40.6 (0.5 C, S(O)CH3), 40.2 (0.5 C, S(O)-
CH3). HRMS (ESI) calcd for (M + Na) C13H24O10S 395.0982, found
395.0984.
PSEUROT Calculations. All calculations were done with PSEUROT
6.3 following modification of the parameters provided for the xylo-
furanosyl ring. The electronegativities (in D2O) used were as follows:
1.25 for OH; 1.26 for OR; 0.68 for CH2OH; 0.62 for CH(OR); 0.0 for
H.52 For each endocyclic torsion angle, the parameters R and ꢀ were
set to 1 and 0, respectively. To translate the exocyclic H,H torsion
angles (ΦHH) into the endocyclic torsion angles (νi) that are used to
determine the pseudorotational phase angle (P), the program makes
use of the relationship: ΦHH ) Aνi + B. The values of A and B used
were those previously calculated for the methyl R-D-xylofuranoside.53
In all calculations the puckering amplitude, τm, was kept constant at
40°, the value found in the crystal structure of 35.41 These PSUEROT
calculations led to the identification of two different solutions, one of
which could be eliminated on the basis of the magnitude of the 3JC-1-H-4
in 36 (0.5 Hz), as described previously.39
Methyl 3-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-L-xylo-
furanosyl)-2,4,6-tri-O-benzyl-R-D-mannopyranoside (31). Prepared
from 30 (40 mg, 0.04 mmol), 18-crown-6 (10 mg), and sodium
thiomethoxide (10 mg, 0.18 mmol) in CH3CN (1 mL) as described for
23, to afford 31 (24 mg, 70%) as a syrup. Rf 0.28 (4:1, hexanes/EtOAc);
1
[R]D -20.5 (c 0.3, CHCl3); H NMR (500 MHz, CDCl3, δH) 7.40-
7.20 (m, 25 H), 5.25 (d, 1 H, J ) 4.1 Hz), 4.89 (d, 1 H, J ) 11.1 Hz),
4.84 (s, 1 H), 4.74 (d, 1 H, J ) 4.7 Hz), 4.72 (d, 1 H, J ) 4.9 Hz),
4.68 (d, 2 H, J ) 12.3 Hz), 4.63-4.45 (m, 5 H), 4.40 (dd, 1 H, J )
6.6, 12.9 Hz), 4.28-4.24 (m, 1 H), 4.24-4.19 (m, 1 H), 4.04 (dd, 1 H,
J ) 4.1, 4.2 Hz), 3.95 (dd, 1 H, J ) 8.9, 8.9 Hz), 3.91-3.86 (m, 1 H),
3.85-3.73 (m, 3 H), 3.37 (s, 3 H), 2.78 (dd, 1 H, J ) 6.4, 13.8 Hz),
2.61 (dd, 1 H, J ) 6.7, 13.8 Hz), 2.00 (s, 3 H); 13C NMR (125 MHz,
CDCl3, δC) 138.7, 138.5, 138.3, 138.1, 138.0, 128.4, 128.3(1) (3 C),
128.2(8) (4 C), 128.2 (2 C), 127.7(8), 127.7(6) (3 C), 127.7 (2 C),
127.6(8) (3 C), 127.6(6) (3 C), 127.6, 127.5(7), 127.4, 98.7, 97.2, 83.7,
82.3, 77.2, 75.4, 74.8, 74.5, 74.3, 73.3, 72.5, 72.4, 72.2, 71.7, 69.5,
54.9, 33.8, 16.3. HRMS (ESI) calcd for (M + Na) C48H54O9S 829.3380,
found 829.3381.
Methyl
4-O-(2,3-Di-O-benzyl-5-O-toluenesulfonyl-R-L-xylo-
furanosyl)-2,3,6-tri-O-benzyl-R-D-mannopyranoside (32). Prepared
from thioglycoside 8 (0.1 g, 0.17 mmol), alcohol 1129 (56 mg, 0.12
mmol), N-iodosuccinimide (45 mg, 0.2 mmol), and silver triflate (8
mg, 0.03 mmol) in CH2Cl2 (3 mL) as described for 22, to afford 32 (8
mg, 71%) as a syrup. Rf 0.29 (4:1, hexanes/EtOAc); [R]D -38.6 (c
1
0.5, CHCl3); H NMR (500 MHz, CDCl3, δH) 7.69 (d, 2 H, J ) 8.3
Hz), 7.39-7.11 (m, 27 H), 5.05 (d, 1 H, J ) 4.1 Hz), 4.76 (d, 1 H, J
) 1.9 Hz), 4.75-4.60 (m, 4 H), 4.55-4.36 (m, 6 H), 4.34-4.20 (m,
3 H), 4.19-4.13 (m, 2 H), 4.10 (dd, 1 H, J ) 4.1, 10.3 Hz), 3.90 (dd,
1 H, J ) 5.5, 10.3 Hz), 3.83 (dd, 1 H, J ) 3.1, 9.0 Hz), 3.80-3.66 (m,
3 H), 3.33 (s, 3 H), 2.36 (s, 3 H); 13C NMR (125 MHz, CDCl3, δC)
144.4, 138.5, 138.4, 138.3, 137.8, 137.7, 133.0, 129.7, 129.6, 128.5,
128.4 (3 C), 128.3(6), 128.3(2) (2 C), 128.3, 128.2(7), 128.0, 127.9 (2
C), 127.8(6) (2 C), 127.8, 127.7 (4 C), 127.6(4), 127.6(2), 127.5(8) (2
C), 127.5(5), 127.5(3) (2 C), 127.5, 99.4, 99.2, 83.4, 80.8, 78.5, 74.4,
73.9, 73.4, 72.8, 72.7, 72.7, 72.5, 71.9, 71.7, 69.2, 68.7, 54.8, 21.6.
HRMS (ESI) calcd for (M + Na) C54H58O12S 953.3541, found
953.3540.
Determination of C4-C5 Rotamer Populations. The rotamer
populations about the C4-C5 bond in the furanose residue in 3, 34-
1
36 were determined by analysis of the three bond H-1H coupling
constants between H4 and H5R (3J4,5R) and H4 and H5S (3J4,5S) using eqs
1-3, which were derived by taking into account the differences in
electronegativities between oxygen and sulfur. In assigning the
resonances arising from H5R and H5S, the assumption was made that
the chemical shift of H5S is greater than that of H5R, which is the case
in the parent glycoside, 35.54
Methyl 4-O-(2,3-Di-O-benzyl-5-deoxy-5-methylthio-R-L-xylo-
furanosyl)-2,3,6-tri-O-benzyl-R-D-mannopyranoside (33). Prepared
from 32 (47 mg, 0.05 mmol), 18-crown-6 (10 mg), and sodium
thiomethoxide (10 mg, 0.18 mmol) in CH3CN (1 mL) as described for
23, to afford 33 (31 mg, 77%) as a syrup. Rf 0.28 (4:1, hexanes/EtOAc);
2.0Xgg + 11.5Xgt + 3.9Xtg ) 3J4,5R
(1)
3.3Xgg + 2.6Xgt + 11.5Xtg ) 3J4,5S
Xgg + Xgt + Xtg ) 1
(2)
(3)
1
[R]D -28.8 (c 0.6, CHCl3); H NMR (500 MHz, CDCl3, δH) 7.40-
7.20 (m, 25 H), 5.19 (d, 1 H, J ) 4.1 Hz), 4.78-4.44 (m, 10 H), 4.41-
4.34 (m, 2 H), 4.23 (dd, 1 H, J ) 9.2, 9.2 Hz), 4.14 (dd, 1 H, J ) 6.2,
6.2 Hz), 3.92-3.86 (m, 2 H), 3.82-3.68 (m, 4 H), 3.35 (s, 3 H), 2.68
(dd, 1 H, J ) 5.9, 13.8 Hz), 2.52 (dd, 1 H, J ) 6.7, 13.8 Hz), 1.98 (s,
3 H); 13C NMR (125 MHz, CDCl3, δC) 138.8, 138.5, 138.4, 138.1,
137.9, 128.3(4), 128.3(3) (2 C), 128.2(6) (3 C), 128.2 (2 C), 127.8 (2
C), 127.7(9) (2 C), 127.7(1) (2 C), 127.7 (2 C), 127.6(2) (2 C), 127.5-
(8), 127.5(5) (2 C), 127.5 (2 C), 127.4(4), 127.4, 99.7, 99.3, 83.9, 81.8,
The results of these analyses were compared with the rotamer
populations found in 35, which were calculated using eqs 4-6.
1.1Xgg + 10.8Xgt + 4.2Xtg ) 3J4,5R
(4)
2.4Xgg + 2.9Xgt + 10.8Xtg ) 3J4,5S
Xgg + Xgt + Xtg ) 1
(5)
(6)
9
J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006 5071

A R T I C L E S
Joe et al.
The coefficients for eqs 1, 2, 4, and 5 were determined by calculating
(Invitrogen)) and plated into a 48-well plate (500 µL/well). Cells were
treated with either 3 or 34 (100 µg and 10 µg/mL) or AraLAM or
ManLAM (10 µg/mL) for 24 h and then stimulated for a further 8 h
with a combination of Staphylococcus aureus Cowan (SAC) (Pansorb-
inTM, Calbiochem, UK) and human IFNγ (1000 U/mL, Preprotech).
Following incubation, the supernatants were collected and stored in
200 µL aliquots (-80 °C) and analyzed by ELISA (R&D systems) for
IL-12p70 and TNF-R production.
3
the limiting JH,H for each rotamer using eq 7.52
3JH,H ) 14.63 cos2 θ - 0.78 cos θ + 0.60 +
[0.34 - 2.31 cos2 (êiθ +18.4øøiø)]øi (7)
∑
i
For eq 7, øi is the group electronegativity52 of the substituents along
the coupling pathway and êi ) +1 or -1 as previously defined.55 The
electronegativities used are as follows: 1.25 for OH; 1.26 for OR; 0.70
for SCH3, and 0.0 for H. The angles θ used in eq 7 were those of the
idealized staggered conformers (60°, -60°, and 180°).
Cytokine Induction Assays. THP-1 cells were resuspended at a
concentration of 1 × 106 cells/mL in RPMI 1640 + 10% FCS + 1%
GPS (200 mM penicillin/streptomycin (Sigma UK) + 2 mM l-glutamine
Acknowledgment. This work was supported by the Univer-
sity of Alberta, The Natural Sciences and Engineering Research
Council of Canada, and the Alberta Ingenuity Centre for
Carbohydrate Science. G.S.B. and D.A.L. acknowledge support
from the Medical Research Council (G9901077 and G0500590).
G.S.B. acknowledges support as a Lister-Jenner Research
Fellow.
Supporting Information Available: 1H and 13C NMR spectra
of all previously unreported compounds, and details on the
synthesis of 36. This material is available free of charge via
the Internet at http://pubs.acs.org.
(52) Altona, C.; Francke, R.; de Haan, R.; Ippel, J. H.; Daalmans, G. J.; Westra
Hoekzema, A. J. A.; van Wijk, J. Magn. Reson. Chem. 1994, 32, 670-
678.
(53) Houseknecht J. B.; Altona, C.; Hadad, C. M.; Lowary, T. L. J. Org. Chem.
2002, 67, 4647-4651.
(54) Serianni, A. S.; Barker, R. Can. J. Chem. 1979, 57, 3160-3167.
(55) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.; Altona,
C. Recl. TraV. Chim. Pays-Bas 1979, 98, 576-577.
JA057373Q
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5072 J. AM. CHEM. SOC. VOL. 128, NO. 15, 2006