Synthesis of galactofuranose-containing acceptor substrates for mycobacterial galactofuranosyltransferases

Article abstract of DOI:10.1021/jo800457j

(Chemical Equation Presented) The major structural component of the cell wall in Mycobacterium tuberculosis, infection by which causes tuberculosis, is the mycolyl-arabinogalactan (mAG) complex. This large glycoconjugates has at its core a backbone of ～30 D-galactofuranose (Galf) residues that are linked to peptidoglycan by way of a linker disaccharide containing L-rhamnose and 2-acetamido-2-deoxy-D-glucose. Recent studies have supported a model of galactan biosynthesis in which the entire structure is assembled by the action of two bifunctional galactofuranosyltransferases. These biochemical investigations were made possible, in part, by access to a panel of oligosaccharide fragments of the mAG complex (1-12), the synthesis of which we describe here. An early key finding in this study was that the iodine-promoted cyclization of galactose diethyl dithioacetal (19) in the presence of an alcohol solvent led to the formation Galf glycosides contaminated with no pyranoside isomer, thus allowing the efficient preparation of furanoside derivatives of this monosaccharide. The synthesis of disaccharide targets 1, 2, 11 and 12 proceeded without difficulty through the use of thioglycoside donors and octyl glycoside acceptors, both carrying benzoyl protection. In the synthesis of the tri- and tetrasaccharides 3-6, we explored routes in which the molecule was assembled from the reducing to nonreducing end, and the reverse. The latter approach was found to be preferable for the preparation of 6, and in the case of 3 and 4, this strategy allowed the development of efficient one-pot methods for their synthesis. We have also carried out the first synthesis of three mAG fragments (8-10) consisting of the linker disaccharide further elaborated with one, two or three Galf residues. A key step in the synthesis of these target compounds was the coupling of a protected linker disaccharide derivative (58) with a mono-, di-, or trigalactofuranosyl thioglycoside (17, 54, or 53, respectively).

Full text of DOI:10.1021/jo800457j

Synthesis of Galactofuranose-Containing Acceptor Substrates for
Mycobacterial Galactofuranosyltransferases
Gladys C. Completo and Todd L. Lowary*
Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, The UniVersity of
Alberta, Gunning-Lemieux Chemistry Centre, Edmonton, Alberta, T6G 2G2 Canada
tlowary@ualberta.ca
ReceiVed February 26, 2008
The major structural component of the cell wall in Mycobacterium tuberculosis, infection by which causes
tuberculosis, is the mycolyl-arabinogalactan (mAG) complex. This large glycoconjugates has at its core
a backbone of ∼30 D-galactofuranose (Galf) residues that are linked to peptidoglycan by way of a linker
disaccharide containing L-rhamnose and 2-acetamido-2-deoxy-D-glucose. Recent studies have supported
a model of galactan biosynthesis in which the entire structure is assembled by the action of two bifunctional
galactofuranosyltransferases. These biochemical investigations were made possible, in part, by access to
a panel of oligosaccharide fragments of the mAG complex (1-12), the synthesis of which we describe
here. An early key finding in this study was that the iodine-promoted cyclization of galactose diethyl
dithioacetal (19) in the presence of an alcohol solvent led to the formation Galf glycosides contaminated
with no pyranoside isomer, thus allowing the efficient preparation of furanoside derivatives of this
monosaccharide. The synthesis of disaccharide targets 1, 2, 11 and 12 proceeded without difficulty through
the use of thioglycoside donors and octyl glycoside acceptors, both carrying benzoyl protection. In the
synthesis of the tri- and tetrasaccharides 3-6, we explored routes in which the molecule was assembled
from the reducing to nonreducing end, and the reverse. The latter approach was found to be preferable
for the preparation of 6, and in the case of 3 and 4, this strategy allowed the development of efficient
one-pot methods for their synthesis. We have also carried out the first synthesis of three mAG fragments
(8-10) consisting of the linker disaccharide further elaborated with one, two or three Galf residues. A
key step in the synthesis of these target compounds was the coupling of a protected linker disaccharide
derivative (58) with a mono-, di-, or trigalactofuranosyl thioglycoside (17, 54, or 53, respectively).
Introduction
been and continue to be important targets for drug action.2 For
example, among the standard antibiotics used to treat tubercu-
losis, two, isoniazid and ethambutol, prevent the assembly of a
key structural element of the mycobacterial cell wall, the
mycolyl-arabinogalactan (mAG) complex.3
All mycobacteria, including the human pathogens Mycobac-
terium tuberculosis and Mycobacterium leprae, possess a unique
and particularly impermeable cell wall that provides the
organism with significant protection from its environment.1
Because of the role the cell wall plays in mycobacterial survival
and pathogenicity, enzymes involved in its biosynthesis have
The mAG is a large, structurally complex glycolipid that is
covalently attached to cell wall peptidoglycan via a phosphate
(2) (a) Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479–2513. (b) Sharma,
K.; Chopra, P.; Singh, Y. Exp. Opin. Ther. Targets 2004, 8, 79–9. (c) Kremer,
L.; Besra, G. S. Exp. Opin. InV. Drugs 2002, 11, 153–157. (d) Lowary, T. L.
Mini-ReV. Med. Chem. 2003, 3, 694–707.
(1) (a) Brennan, P. J. Tuberculosis 2003, 83, 91–97. (b) Lowary, T. L. In
Glycoscience: Chemistry and Chemical Biology; Fraser-Reid, B., Tatsuta, K.,
Thiem, J., Eds.; Springer: Berlin, 2001; pp 2005-2080.
10.1021/jo800457j CCC: $40.75
Published on Web 05/20/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 4513–4525 4513

Completo and Lowary
second Galf residues to the Rhap moiety and thus possesses
both Galf-ꢀ-(1f5)-Galf and Galf-ꢀ-(1f4)-Rhap transferase
activity. The second enzyme (GlfT2), arising in M. tuberculosis
from the expression of the Rv3808c gene,10,11 has both Galf-
ꢀ-(1f5)-Galf and Galf-ꢀ-(1f6)-Galf transferase activity and
adds the remaining Galf residues. As the donor species, both
enzymes use UDP-Galf, which is produced from UDP-Galp by
the action of UDP-Galp mutase.12
In previous studies, we7,11 and others10b,c,13 have used
synthetic fragments of the mAG complex to probe the specificity
of these galactofuranosyltransferases. We describe here the
synthesis of a panel of these compounds (Chart 1), including
disaccharides 1 and 2, trisaccharides 3 and 4, tetrasaccharides
5 and 6, and a series of compounds containing the linker
disaccharide motif (7-10). In addition, we report the preparation
of analogs of 1 and 2 in which the nonreducing Galf residue
has been replaced by an L-arabinofuranosyl moiety (11 and 12)
and also a one-pot approach to trisaccharides 3 and 4.
Compounds 11 and 12 were chosen for synthesis to probe the
effect that removing the terminal hydroxymethyl substituent in
disaccharides 1 and 2 had on recognition by GlfT2. The work
described here complements other previous investigations in
which di-13,14 and trisaccharide15 fragments of mycobacterial
galactan have been synthesized. To the best of our knowledge,
no syntheses of tetragalactofuranosyl fragments of this polysac-
charide (e.g., 5 and 6) have been reported, nor have tri-, tetra-
or pentasaccharide fragments containing the R-L-Rhap-(1f3)-
R-D-GlcpNAc motif (e.g., 8-10).
FIGURE 1. Schematic depiction of the mycobacterial mAG complex
highlighting the structure of the galactan and linker region. x ≈ 13-15.
(/) The three arabinan chains are proposed to be attached to the 8th,
10th and 12th Galf residues.5
linkage (Figure 1).1,4 From this phosphate moiety extends a
motif, termed the linker disaccharide, which has an R-L-
rhamnopyranosyl-(1f3)-2-acetamido-2-deoxy-R-D-glucopyra-
nosyl (R-L-Rhap-(1f3)-R-D-GlcpNAc) structure. The linker
disaccharide is further elaborated, at O-4 of the Rhap residue,
with a galactan composed of 30-35 alternating ꢀ-(1f5) and
ꢀ-(1f6) linked galactofuranosyl (Galf) units. Along this ga-
lactan backbone are three branch points to which are attached
the arabinan domains, each consisting of, on average, 31
arabinofuranosyl residues.5a A recent study, in which the
structure of the mAGP from Corynebacterium glutamicum was
investigated, clearly demonstrated that the three arabinan chains
are linked to O-5 of the 8th, 10th and 12th residues of the
galactan backbone.5b Given that corynebacteria and mycobac-
teria both produce a similar mAGP, it has therefore been
proposed5 that a similar number of arabinan domains, attached
via the same linkage points, decorate the mycobacterial galactan.
The final structural element of the mAG are mycolic acids, long-
chain branched lipids, which are esterified to the terminal ends
of the arabinan motifs.
Results and Discussion
Synthesis of Galactofuranosyl Oligomers 1-6 and Ara-
binofuranosyl-Containing Disaccharides 11 and 12. We chose
to synthesize all of the targets as octyl glycosides on the basis
of previous studies demonstrating that oligosaccharides contain-
(9) The nomenclature for these enzymes, GlfT1 and GlfT2, was recently
proposed (ref 7) and differs slightly from that used previously.
(10) (a) Mikusova´, K.; Yagi, T.; Stern, R.; McNeil, M. R.; Besra, G. S.;
Crick, D. C.; Brennan, P. J. J. Biol. Chem. 2000, 275, 33890–33897. (b) Kremer,
L.; Dover, L. G.; Morehouse, C.; Hitchen, P.; Everett, M.; Morris, H. R.; Dell,
A.; Brennan, P. J.; McNeil, M. R.; Flaherty, C.; Duncan, K.; Besra, G. S. J. Biol.
Chem. 2001, 276, 26430–26440. (c) Alderwick, L. J.; Dover, L. G.; Veerapen,
N.; Gurcha, S. S.; Kremer, L.; Roper, D. L.; Pathak, A. K.; Reynolds, R. C.;
Besra, G. S. Protein Expression Purif. 2008, 58, 332–341.
The biosynthesis of the mAG has been slowly unraveled over
the past several years,4,6 and while much remains to be
understood, a picture has emerged about how the galactan
portion of the molecule is assembled. On the basis of currently
available evidence, the proposed model involves only two
bifunctional galactofuranosyltransferases.7 One, encoded in M.
tuberculosis by the Rv3782 gene (GlfT1),8,9 adds the first and
(11) Rose, N. L.; Completo, G. C.; Lin, S.-J.; McNeil, M. R.; Palcic, M. M.;
Lowary, T. L. J. Am. Chem. Soc. 2006, 128, 6721–6729.
(12) Pedersen, L. L.; Turco, S. J. Cell. Mol. Life Sci. 2003, 60, 259–266.
(13) (a) Pathak, A. K.; Pathak, V.; Seitz, L.; Gurcha, S. S.; Besra, G. S.;
Riordan, J. M.; Reynolds, R. C. Bioorg. Med. Chem. 2007, 15, 5629–5650. (b)
Wen, X.; Crick, D. C.; Brennan, P. J.; Hultin, P. G. Bioorg. Med. Chem. 2003,
11, 3579–3587. (c) Pathak, A. K.; Besra, G. S.; Crick, D.; Maddry, J. A.;
Morehouse, C. B.; Suling, W. J.; Reynolds, R. C. Bioorg. Med. Chem. 1999, 7,
2407–2413. (d) Pathak, A. K.; Pathak, V.; Seitz, L.; Maddry, J. A.; Gurcha,
S. S.; Besra, G. S.; Suling, W. J.; Reynolds, R. C. Bioorg. Med. Chem. 2001, 9,
3129–3143.
(3) (a) Schroeder, E. K.; De Souza, O. N.; Santos, D. S.; Blanchard, J. S.;
Basso, L. A. Curr. Pharm. Biotechnol. 2002, 3, 197–225. (b) Belanger, A. E.;
Besra, G. S.; Ford, M. E.; Mikusova´, K.; Belisle, J. T.; Brennan, P. J.; Inamine,
J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11919–11924.
(4) (a) Crick, D. C.; Mahapatra, S.; Brennan, P. J. Glycobiology 2001, 11,
107R–118R. (b) Mahapatra, S.; Basu, J.; Brennan, P. J.; Crick, D. C. Structure,
Biosynthesis and Genetics of the Mycolic Acid-Arabinogalactan-Peptidoglycan
Complex. In Tuberculosis and the Tubercle Bacillus; Cole, S. T., Eisenach, K. D.,
McMurray, D. N., Jacobs, W. R., Jr., Eds.; American Society for Microbiology:
Washington, DC, 2005; pp 275-285.
(14) (a) Euzen, R.; Ferrie`res, V.; Plusquellec, D. Carbohydr. Res. 2006, 341,
2759–2768. (b) Sugawara, F.; Nakayama, H.; Ogawa, T. Agric. Biol. Chem.
1986, 50, 1557–156. (c) de Lederkremer, R. M.; Marino, C.; Varela, O.
Carbohydr. Res. 1990, 200, 227–235. (d) McAuliffe, J. C.; Hindsgaul, O. J.
Org. Chem. 1997, 62, 1234–1239. (e) Velty, R.; Benvegnu, T.; Gelin, M.; Privat,
E.; Plusquellec, D. Carbohydr. Res. 1997, 299, 7–14. (f) Choudhury, A. K.;
Roy, N. Carbohydr. Res. 1998, 308, 207–211. (g) Veeneman, G.; Notermans,
S.; Liskamp, R. M. J.; van der Marel, G. M.; van Boom, J. H. Recl. TraV. Chim.
Pays-Bas 1989, 108, 344–350. (h) van Heeswijk, W. A. R.; Visser, H. G. J.;
Vliegenthart, J. F. G. Carbohydr. Res. 1977, 59, 81–86. (i) Pathak, A. K.; El-
Kattan, Y. A.; Bansal, N.; Maddry, J. A.; Reynolds, R. C. Tetrahedron Lett.
1998, 12, 1497–1500. (j) Marino, C.; Varela, O.; de Lederkremer, R. M.
Carbohydr. Res. 1989, 190, 65–76.
(5) (a) Bhamidi, S.; Scherman, M. S.; Rithner, C. D.; Prenni, J. E.; Chatterjee,
D.; Khoo K.-H.; McNeil, M. R. J. Biol. Chem. 2008, 283, 12992–13000. (b)
Alderwick, L. J.; Radmacher, E.; Seidel, M.; Gande, R.; Hitchen, P. G.; Morris,
H. R.; Dell, A.; Sahm, H.; Eggeling, L.; Besra, G. S. J. Biol. Chem. 2005, 280,
32362–32371.
(6) Berg, S.; Kaur, D.; Jackson, M.; Brennan, P. J. Glycobiology 2007, 17,
35R–56R.
(7) Bela´nˇova´, M.; Dianisˇkova´, P.; Brennan, P. J.; Completo, G. C.; Rose,
N. L.; Lowary, T. L.; Mikusˇova´, K. J. Bacteriol. 2008, 190, 1441–1445.
(8) Mikusˇova´, K.; Bela´nˇova´, M.; Kordulakova, J.; Honda, K.; McNeil, M. R.;
Mahapatra, S.; Crick, D. C.; Brennan, P. J. J. Bacteriol. 2006, 188, 6592–6598.
(15) (a) Gandolfi-Donad´ıo, L.; Gallo-Rodriguez, C.; de Lederkremer, R. M.
J. Org. Chem. 2003, 68, 6928–6934. (b) Gandolfi-Donad´ıo, L.; Gallo-Rodriguez,
C.; de Lederkremer, R. M. Carbohydr. Res. 2008, Epub ahead of print; doi:
10.1016/j.carres.2008.01.024.
4514 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
CHART 1
CHART 2
ing long-chain aglycones are better substrates for mycobacterial
glycosyltransferases than glycosides of short-chain alcohols.16
In addition, the octyl group would facilitate enzyme assays by
allowing the use of reverse-phase cartridges to purify crude
incubation mixtures.17 On the basis of these considerations, we
chose the known13d octyl glycosides 13 and 14 (Chart 2) as
the building blocks for the “reducing” end of the targets. We
envisioned that thioglycosides 15-18 could be used to introduce
the other residues via an iterative process involving glycosylation
and selective deacetylation reactions.
In developing a route to 13-18, the first issue to address
was to identify a method for efficiently obtaining a galactose
intermediate in the furanose ring form. There are a variety of
processes reported for carrying out this transformation, with the
most common being Fischer glycosylation,18 which when carried
out under kinetic control affords the higher energy furanoside
isomers. However, in our hands, the use of this approach led to
mixtures of the furanosides contaminated with the more
thermodynamically stable pyranosides, which were difficult to
separate. Other methods for the preparation of galactofuranoside
derivatives include the reduction of galactonolactones,19 the
(16) Lee, R. E.; Brennan, P. J.; Besra, G. S. Glycobiology 1997, 7, 1121–
1128.
(17) Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycoconjugate
J. 1988, 5, 59–63.
(18) (a) Arasappan, A.; Fraser-Reid, B. Tetrahedron Lett. 1995, 36, 7967–
7070. (b) Ferrie`res, V.; Bertho, J.-N.; Plusquellec, D. Carbohydr. Res. 1998,
311, 25–35.
(19) Kohn, P.; Samaritano, R. H.; Lerner, L. M. J. Am. Chem. Soc. 1965,
87, 5469–5472.
J. Org. Chem. Vol. 73, No. 12, 2008 4515

Completo and Lowary
SCHEME 1
acid-catalyzed ring opening of 1,4-anhydrogalactopyranose
derivatives,20 the high-temperature benzoylation of galactose,21
anomeric alkylation,22 and the cyclization of open-chain S,O-
acetals14d or dithioacetals23 with mercuric salts. A recent paper
describes Fischer glycosylation of D-galacturonic acid in
methanol followed by the reduction of the product methyl
glycoside methyl ester, which affords good yields of galacto-
furanosides over the two-step process.24
NMR spectrum of 21, the anomeric hydrogen resonance
appeared as a singlet at 5.24 ppm, and in the 13C NMR spectrum
the anomeric carbon resonance appeared at 105.7 ppm. Both
are consistent with the ꢀ-galactofuranoside stereochemistry.29
We note that the stereoselectivity observed in this glycosylation
reaction is of the same magnitude as that reported previously
in cyclization of acyclic S,O-acetals.14d
With 21 in hand, reaction with trityl chloride in pyridine
followed by benzoyl chloride afforded the fully protected
derivative 22 in 87% overall yield over the two steps. The
required acceptor alcohols 13 and 14 were obtained upon
treatment of 22 with trifluoroacetic acid in wet dichloromethane,
as reported previously.13d Under these conditions, primary
alcohol 13 was obtained in 42% yield, while the secondary
alcohol 14, resulting from acid catalyzed migration of the O-5
benzoate ester, was isolated in 35% yield.
Preparation of Thioglycoside Donors. An analogous ap-
proach was used to synthesize thioglycosides 15-17 (Scheme
2). Dithioacetal 19 was converted into methyl glycoside 23 in
two steps and 71% yield upon reaction with iodine (2% by
weight) in methanol30 followed by benzoylation. As was the
case for the synthesis of 21, the glycosylation proceeded with
high ꢀ-selectivity (ꢀ:R ratio of 9:1). Methyl glycoside 23 was
next converted to the corresponding thioglycoside 17 upon
reaction with p-thiocresol and boron trifluoride etherate, which
afforded an 89% yield of the product. Transformation of 17 to
the remaining two galactofuranosyl thioglycoside donors, 15
and 16, was straightforward. Removal of the benzoate esters of
17 yielded the deprotected thioglycoside 24 (88%), which was
then tritylated (yielding 25) and benzoylated to give 26 in 70%
yield over the two steps. Subsequent treatment of 26 with
trifluoroacetic acid as described for the preparation of 13 and
14, yielded the expected thioglycoside alcohols 27 and 28 in
44% and 33% yields, respectively. The structures of 27 and 28
could be readily differentiated by NMR spectroscopy of the
products. In 27, the signal for the H-5 resonance is significantly
downfield (5.69 ppm) as would be expected as O-5 is acylated.
In contrast, in the NMR spectrum for 28, H-5 appears at 4.59
ppm. Reaction of 27 with acetic anhydride and pyridine
proceeded uneventfully affording a 90% yield of 16; similar
treatment of 28 yielded 15, also in 90% yield.
Previously,25 we explored a recently described variation of
the dithioacetal cyclization approach, in which galactose dithio-
acetals were cyclized with 1,3-dibromo-5,5-dimethylhydantoin.26
Although this reaction afforded the desired product, in our hands
the yield was modest and we therefore turned to an alternate
method, reported by Szarek and co-workers,27 which involves
treatment of the dithioacetal with an alcohol and iodine. An
attractive feature of the Szarek method is that the promoter is
inexpensive and relatively benign. The method was reported to
be highly selective for producing furanosides from dithioacetals
of glucose, arabinose and mannose; curiously, it has not, to the
best of our knowledge, been applied to the synthesis of
galactofuranose derivatives. As detailed below, we have found
that the iodine-promoted cyclization of galactose diethyl dithio-
acetal does indeed efficiently provide galactofuranosides inex-
pensively and free from contamination by galactopyranosides.
Preparation of Octyl Glycoside Acceptors. The synthesis
of 13 and 14 (Scheme 1) proceeded from dithioacetal 1928 by
reaction with iodine (5%, by weight) in n-octanol. Although
the original report with diothioacetals derived from other
monosaccharides used only 0.5% iodine when methanol was
used as the solvent,27 in n-octanol the reaction was very sluggish
at this concentration and hence a larger amount of the promoter
was used. Next, the crude product obtained after workup and
removal of the n-octanol was suspended in pyridine before
benzoyl chloride was added. While benzoylation was not
essential, doing so facilitated isolation of the product, 20, which
was obtained as a 9:1 ꢀ:R mixture. The ꢀ-glycoside was
successfully purified by chromatography and subsequent treat-
ment with sodium methoxide thus afforded 21 in 36% yield in
three steps from 19. The ꢀ-stereochemistry of the major product
1
could be readily established by NMR spectroscopy. In the H
(20) Kovensky, J.; Sinay¨, P. Eur. J. Org. Chem. 2000, 3523–2525.
(21) D’Accorso, N. B.; Thiel, I. M. E.; Schu¨ller, M. Carbohydr. Res. 1983,
124, 177–184.
The remaining thioglycoside, 18, was synthesized from the
known31 methyl glycoside 29 in 89% yield upon treatment with
p-thiocresol and boron trifluoride etherate (Scheme 3).
Synthesis of Disaccharides 1, 2, 11 and 12. Disaccharides
1 and 2 were synthesized by coupling either octyl glycoside 13
or 14 with thioglycoside 17 followed by deprotection of the
(22) Gola, G.; Libenson, P.; Gandolfi-Donad´ıo, L.; Gallo-Rodriguez, C.
ARKIVOC 2005, xii, 234–242.
(23) Green, J. W.; Pacsu, E. J. Am. Chem. Soc. 1938, 60, 2056–2057.
(24) Bordoni, A.; de Lederkremer, R. M.; Marino, C. Tetrahedron 2008,
64, 1703–1710.
(25) Bai, Y.; Lowary, T. L. J. Org. Chem. 2006, 71, 9658–9671.
(26) Madhusudan, S. K.; Misra, A. K. Carbohydr. Res. 2005, 340, 497–
502.
(29) Cyr, N.; Perlin, A. S. Can. J. Chem. 1979, 57, 2504–2511.
(30) The increased amount of iodine, compared to that originally reported
(ref 27) was to speed the completion of the reaction.
33.(31) Hatfield, R. D.; Helm, R. F.; Ralph, J. Anal. Biochem. 1991, 194, 25–
(27) Szarek, W. A.; Zamojski, A.; Tiwari, K. N.; Ison, E. R. Tetrahedron
Lett. 1986, 27, 3827–3830.
(28) Norris, P.; Horton, D. In PreparatiVe Carbohydrate Chemistry; Han-
essian, S., Ed.; Marcel Dekker: New York, 1997; pp 35-52.
4516 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
SCHEME 2
SCHEME 3
in 75% yield, alcohol 35. Under these conditions an isomeric
product, resulting from migration of the benzoyl group from
O-5 to O-6 (36, Chart 3), was also produced in 20% yield. Given
the efficiency of the TFA-promoted acyl migration during the
deprotection of 22 and 26,13d which was exploited in the
preparation of 13-16 (above), the formation of byproduct 36
under these acidic deacetylation conditions is perhaps not
unexpected. Regardless, isomers 35 and 36 could be easily
separated by chromatography and the latter could be deprotected
to afford 1. Subsequent reaction of 35 with 17 gave trisaccharide
37, which was then debenzoylated affording 3 in 70% yield
over the two steps.
resulting product as illustrated in Scheme 4. Thus, reaction of
14 with 17 in the presence of N-iodosuccinimide (NIS) and
silver triflate (AgOTf)32 afforded disaccharide 30 in 87% yield.
For this and the remainder of the glycosylation products
described in this paper, the anomeric stereochemistry of the
newly introduced Galf residue was determined by NMR
In a similar manner, trisaccharide 4 was synthesized by first
coupling 13 with 15 to give disaccharide 38 in 99% yield.
Selective deacetylation under acidic conditions provided 39 in
85% yield together with 10% of a compound believed to be
the benzoyl migration product, 40 (Chart 3). The lower amount
of benzoyl migration observed upon deacetylation of 38 as
opposed to 34 is presumably due to the tendency of acyl groups
to migrate to primary positions.34Because the product formed
initially from 36 has a benzoate ester at the primary position,
there is consequently little driving force for acyl migration.
Glycosylation of 39 with 17 followed by deprotection with
sodium methoxide afforded, over the two steps, an 81% yield
of target 4.
Synthesis of Tetrasaccharides 5 and 6. As illustrated in
Scheme 7, the synthesis of tetrasaccharide 5 was achieved from
disaccharide 35 by application of the same iterative approach
described above for the preparation of trisaccharides 3 and 4.
Thus, glycosylation of 35 with thioglycoside 15 proceeded to
give trisaccharide 42 in 78% yield. Deacetylation with acidic
methanol gave 43 in 86% yield together with 8% of a product
presumed to be the benzoyl migrated species, 44 (Chart 3),
which were separated by chromatography. The final monosac-
charide residue was introduced by the coupling of 43 with 17,
which afforded a 70% yield of tetrasaccharide 45. Deprotection
of 45 with sodium methoxide yielded 5 in 82% yield.
3
spectroscopy from the magnitude of the JH-1,H-2 and the
chemical shift of the resonance for the anomeric carbon. In all
3
cases, the JH-1,H-2 magnitude was between 0 and 2 Hz, while
the chemical shift of the anomeric carbon was between 104.0
and 106.8 ppm (107.9-110.6 ppm after full deprotection). These
1
13C and H NMR data both support the ꢀ-galactofuranoside
stereochemistry.29 Deprotection of 30 was achieved upon
treatment with sodium methoxide, which provided 1 in 90%
yield. Disaccharide 2 was synthesized via an identical series of
transformations: glycosylation of 13 with 17 gave disaccharide
31 in 89% yield, which was then deprotected affording 2 (87%).
The same general approach was also used to prepare the
L-arabinofuranosyl-containing disaccharides, 11 and 12 (Scheme
5). First, reaction of alcohol 14 with thioglycoside 18 upon
activation with NIS–AgOTf afforded disaccharide 32 (87%
yield), which was then deprotected giving 11 in 90% yield.
Application of the same transformations to 13 yielded 12 by
way of 33 in 82% yield overall yield.
Synthesis of Trisaccharides 3 and 4. The preparation of
trisaccharides 3 and 4 also began from octyl glycosides 13 and
14 as illustrated in Scheme 6. Reaction of 14 and 16 promoted
by NIS and AgOTf afforded the differentially protected disac-
charide 34 in 89% yield. The acetyl group was selectively
cleaved upon treatment with HCl in methanol,33 which provided,
(32) Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990,
31, 4313–4316.
(33) Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L. V.; Kochetkov,
N. K. Carbohydr. Res. 1983, 124, C8-C11.
(34) Haines, A. H. AdV. Carbohydr. Chem. Biochem. 1976, 33, 11–109.
J. Org. Chem. Vol. 73, No. 12, 2008 4517

Completo and Lowary
SCHEME 4
SCHEME 5
SCHEME 6
In contrast to the synthesis of the other oligosaccharides, the
preparation of 6 proved to be more problematic. Following the
same general approach used for the synthesis of 5, the coupling
of 39 with 16 initially produced the corresponding trisaccharide
46 together with the hydrolyzed donor, which coeluted during
purification by chromatography. This mixture of compounds was
then subjected to selective acetyl group deprotection to afford
pure alcohol 47 in 42% yield over two steps. The corresponding
acyl migration product (8%) was also observed as a side product.
However, with 47 in hand, all our attempts to further glycosylate
it with 17 failed to give any tetrasaccharide product. A variety
of reaction conditions were explored in an attempt to produce
the desired target from 47 and 17, but to no avail. We also
investigated if trichloroacetimidate 49 (Chart 4)35 could serve
as an effective donor for the preparation of 48 but were
unsuccessful, under a number of conditions. We do not know
the reasons why this stepwise approach, which had been
effective for the synthesis of 5, failed to produce 6. One
possibility is that trisaccharide alcohol 47 adopts a conformation
in which the reactive alcohol is somehow hindered, which in
turn substantially reduces its nucelophilicity.
Faced with this problem, we explored an alternative strategy
to 48, in which the molecule would be built from the nonre-
(35) Gallo-Rodriguez, C.; Gandolfi, L.; de Lederkremer, R. M. Org. Lett.
1999, 1, 245–247.
4518 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
CHART 3
SCHEME 7
CHART 4
An obvious advantage of this alternate strategy was that
it allowed us to access 6 successfully. Less obvious but still
important is that by building up the molecule from the
nonreducing to reducing end, it was possible to avoid the
need for selective deacetylation reactions at either O-5 or
O-6, which in all cases led to migration of benzoyl groups
to the adjacent positions (see Schemes 6 and 7). Therefore,
we also explored this approach to synthesize tetrasaccharide
5, and this was also successful. As illustrated in Scheme 9,
starting from imidate 49 and thioglycoside alcohol 28, the
thioglycoside disaccharide 54 was obtained in 86% yield.
Hydrolysis of the thioglycoside by NBS in wet ethyl acetate
gave 55 (72% yield), and in turn, imidate derivative 56 was
obtained in 79% yield upon reaction with trichloroacetonitrile
and DBU. TMSOTf-promoted glycosylation of alcohol 27
with this imidate yielded an 72% yield of trisaccharide 57.
The synthesis of 5 was then completed by reaction of 57
with 14 using NIS–AgOTf as the promoter, leading to a 70%
yield of the fully protected tetrasaccharide 45, which was
then deacylated using sodium methoxide in methanol yielding
5 in 82% yield. Overall, this route (Scheme 9) gave
ducing end to the reducing end. This approach, shown in Scheme
8, was successful and involved the use of thioglycoside alcohols
27 and 28, the synthesis of which was described earlier (see
Scheme 2). Thus, thioglycoside alcohol 27 was glycosylated
with trichloroacetimidate 49 promoted by TMSOTf, which gave
disaccharide 50, in 90% yield. This thioglycoside alcohol was
then hydrolyzed to produce an 87% yield of hemiacetal 51,
which was then converted to the corresponding trichloroace-
timidate 52 in 85% yield. With this disaccharide donor in hand,
it was used to glycosylate alcohol 28, thus affording trisaccharide
thioglycoside 53 in 76% yield. Finally, this thioglycoside was
activated using NIS–AgOTf in the presence of octyl glycoside
acceptor 13, giving the desired tetrasaccharide 48 in 75% yield.
Treatment with sodium methoxide then afforded a 70% yield
of 6.
J. Org. Chem. Vol. 73, No. 12, 2008 4519

Completo and Lowary
SCHEME 8
SCHEME 9
tetrasaccharide 5 in six steps and 20% yield from monosac-
charide building blocks, compared to six steps and 26% yield
for the approach shown in Schemes 6 and 7. While the first
approach proved slightly better in providing tetrasaccharide
5, the “non-reducing to reducing end” strategy may in some
cases have advantages in the synthesis of large galactofura-
nosides. This strategy relies on glycosylation reactions with
monosaccharide acceptors, which may be less prone to
conformational effects that reduce the nucleophilicity of the
acceptor alcohol. As the size of the chain increases such
conformational effects might be expected to increase.
One-Pot Syntheses of Trisaccharides 37 and 41. Our earlier
studies11 on one of the mycobacterial galactofuranosyltranseras-
es, GlfT2, revealed that trisaccharides 3 and 4 were optimal
substrates for this enzyme. In anticipation of needing to screen
a substantial number of inhibitors of the enzyme using a recently
4520 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
SCHEME 10
developed high-throughput assay for the enzyme,36 it was
necessary to have in place a robust and efficient route for the
preparation of a large amount of these compounds, which would
be needed in every inhibitor assay. Although the route illustrated
in Scheme 6 provided both targets in good overall yield, the
benzoyl migration that occurred upon deacetylation of disac-
charide intermediates 34 and 38 under acidic conditions was a
limitation of this approach. While this could, in theory, be solved
by replacing the acetyl fuctionality with another group (e.g.,
levulinoyl), which could be taken off under conditions to which
the benzoyl groups would be stable (hydrazine acetate or sodium
borohydride), we nevertheless were concerned that even under
these conditions some acyl migration would occur.34
Given the success we had in synthesizing tetrasaccharides 5
and 6 via the approach outlined in Schemes 8 and 9, in which
the molecules were assembled from the nonreducing to reducing
end, we viewed this as an attractive strategy for the efficient
synthesis of 3 and 4. In addition to eliminating the need for
selective deprotection reactions, we envisioned that these
reactions could be carried out in one-pot37 from trichloroace-
timidate 49, thioglycosides 27 or 28, and octyl glycosides 13
or 14, thus dramatically speeding the production of these
compounds. The one-pot method is made feasible as only two
glycosylation reactions are needed for the synthesis of 3 and 4,
and we had in hand two classes of donors, thioglycosides and
trichloroacetimidates, the latter of which could be selectively
activated in the presence of the former.
temperature. After 30-60 min, a new spot, disaccharide 50,38
was visible on TLC. Addition of octyl glycoside alcohol 14,
NIS and AgOTf led, after 15 min, to the formation of 37.
Following chromatography, 37 was obtained in 42% overall
yield from 28, which corresponds to an approximately 65% yield
in both glycosylation steps. Using a similar series of reactions,
trichloroacetimidate 49, thioglycoside alcohol 27 and octyl
glycoside 13 could be converted to thioglycoside 41, via
disaccharide 54, in 41% overall yield (Scheme 10). Trisaccha-
rides 37 and 41 obtained from these one-pot procedures were
deacylated affording 3 and 4 in 90% and 89% yields, respec-
tively. This route, in which the sugars are added from the
nonreducing end to the reducing end of the molecule has clear
advantages over the alternate approach we investigated earlier
(Scheme 6). Not only can the product be obtained rapidly (within
1 day) in a one-pot manner, but also the acyl migration observed
in the selective deacylation reactions (i.e., 38 f 39 + 40,
Scheme 6) is avoided.
Synthesis of Oligosaccharides Containing the Linker
Disaccharide (7-10). For the preparation of the oligosaccharide
targets with the linker disaccharide, 7-10, a convergent strategy
was adopted where an acceptor containing a suitably protected
R-Rhap-(1f3)-R-GlcpNAc moiety would be coupled with an
appropriate galactofuranose-containing thioglycoside, either a
mono-, di-, or trisaccharide. We therefore envisioned that the
step in which these two groups were joined would require
disaccharide 58 and either 17, 53, or 54 (Figure 2). The
preparation of thioglycosides 17, 53, and 54 was presented in
Schemes 2, 8, and 9, respectively. The synthesis of octyl
glycoside 58 is shown in Scheme 11.
The successful implementation of this approach is shown in
Scheme 10. Thus trisaccharide 37 (and in turn 3) could be
obtained by reacting trichloroacetimidate 49 with thioglycoside
alcohol 28 under the promotion of TMSOTf in CH2Cl2 at room
The route to 58 started from the known benzylidene protected
N-acetyl-glucosamine derivative 5939 (Scheme 11), which was
glycosylated with the previously reported rhamnose thioglyco-
side 60.40 This reaction provided an 89% yield of the expected
(36) Rose, N. L.; Zheng, R. B.; Pearcey, J.; Zhou, R.; Completo, G. C.;
Lowary, T. L. Carbohydr. Res. 2008, Accepted for publication; doi:10.1016/
j.carres.2008.03.023.
(37) (a) For some examples of one-pot approaches to oligosaccharides, see:
Huang, L. J.; Wang, Z.; Li, X. N.; Ye, X. S.; Huang, X. F. Carbohydr. Res.
2006, 341, 1669–1679. (b) Yu, B.; Yang, Z. Y.; Cao, H. Z. Curr. Org. Chem.
2005, 9, 179–194. (c) Huang, X. F.; Huang, L. J.; Wang, H. S.; Ye, X. S. Angew.
Chem., Int. Ed. 2004, 43, 5221–5224. (d) Codee, J. D. C.; van den Bos, L. J.;
Litjens, R. E. J. N.; Overkleeft, H. S.; van Boom, J. H.; van der Marel, G. A.
Org. Lett. 2003, 5, 1947–1950. (e) Zhang, Z. Y.; Ollmann, I. R.; Ye, X. S.;
Wischnat, R.; Baasov, T.; Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 734–753.
(f) Grice, P.; Ley, S. V.; Pietruszka, J.; Priepke, H. W. M.; Walther, E. P. E.
Synlett 1995, 781–784.
(38) The identity of the intermediates formed in these one-pot reactions was
confirmed by TLC comparison with authentic standards of 50 and 54, which
were obtained in the course of the synthesis of 5 and 6 (Schemes 8 and 9).
(39) Aguilera, B.; Romero-Ram´ırez, L.; Abad-Rodr´ıguez, J.; Corrales, G.;
Nieto-Sampedro, M.; Ferna´ndez-Mayoralas, A. J. Med. Chem. 1998, 41, 4599–
4606.
(40) Kihlberg, J. O.; Leigh, D. A.; Bundle, D. R. J. Org. Chem. 1990, 55,
2860–2863.
J. Org. Chem. Vol. 73, No. 12, 2008 4521

Completo and Lowary
FIGURE 2. Retrosynthetic analysis of 7-10.
SCHEME 11
disaccharide, 61. The stereochemistry of the rhamnopyranosyl
moiety in 61 was proven by measurement of the JC-1,H-1 for
well as tetrasaccharide 5, we were unable to prepare tetrasac-
charide 6 by this route, which may arise from conformational
features of the acceptor substrate that hinder the reactive alcohol
nucleophile in the final glycosylation step. A strategy in which
these compounds were assembled from the nonreducing to
reducing end was then investigated and was successfully
implemented for the synthesis of both tetrasaccharides 5 and 6.
In addition to allowing the preparation of these target molecules,
this approach also allowed us to eliminate the need for selective
deprotection of an acetyl group on O-5 or O-6 of the terminal
Galf residue in the oligosaccharide intermediates. This was a
key feature of our initial strategy, which in all cases led to some
O-5 f O-6 (or O-6 f O-5) benzoyl group migration, thus
reducing yields and complicating purifications. Furthermore, this
strategy enabled a one-pot synthesis of trisaccharides 3 and 4.
Finally, four oligosaccharides (7-10) that contain the linker
disaccharide region of the AG and up to three Galf residues
were synthesized. The work described here represents the first
preparation of three of these mAG fragments (8-10), which
were obtained by the coupling of a key R-Rhap-(1f3)-R-
GlcpNAc disaccharide intermediate with a donor containing one,
two or three Galf residues (17, 54 or 53, respectively).
Oligosaccharides 1-12 have already found significant applica-
tion in probing mycobacterial AG biosynthesis7,11 and further
studies with these glycans are ongoing.
1
this residue, which revealed a value of 170 Hz, consistent with
the proposed R-stereochemistry.41 A portion of 61 was treated
with aqueous acetic acid at 70 °C and then with sodium
methoxide thus giving disaccharide 7 in 55% yield. The
remainder of 61 was deacetylated upon treatment with sodium
methoxide to give triol 62 in 72% yield. Subsequent treatment
of 62 with dimethoxypropane and camphorsulfonic acid afforded
58 in 86% yield.
With all the building blocks in hand, the preparation of 8-10
was straightforward as shown in Scheme 12. Thus, glycosylation
of 58 with 17, 54, or 53 gave the expected oligosaccharide
products 63-65 in 75-85% yield. Each of these compounds
was then deprotected in two steps: removal of the acetal
protecting groups under acidic conditions followed by deben-
zoylation under basic conditions. These reactions afforded 8-10
in 62-89% yield.
Conclusions
In conclusion, we describe here the synthesis of a panel of
oligosaccharides (1-12) that have found application as sub-
strates for galactofuranosyltransferases involved in the biosyn-
thesis of mycobacterial AG.7,11 An important element of this
work was the use of an iodine-promoted cyclization27 of
galactose diethyl dithioacetal (19) for the preparation of Galf
derivatives (e.g., 13-17), which were required building blocks
for all of the target molecules, except 7. The major advantages
of this approach for accessing the thermodynamically least stable
ring form of this sugar include the use of inexpensive reagents
and the preparation of furanosides in the apparent total absence
of pyranosides.
With these building blocks in hand, the preparation of the
disaccharides proceeded without difficulty. In the synthesis of
the tri- and tetrasaccharides 3-6, our initial strategy involved
building up the molecules from the reducing to nonreducing
end. While this allowed us to obtain trisaccharides 3 and 4, as
Experimental Section
General Procedure for Deprotection of Intermediates Leading
to 1-6, 11 and 12. The starting material was dissolved in 3:1
MeOH-CH2Cl2 (10-50 mL) followed by dropwise addition of
NaOMe in MeOH (0.1 M) until the pH of the solution was 12.
The reaction mixture was stirred for 4 h and was neutralized
by the careful addition of Amberlyst-15 (H+) cation exchange resin.
The solution was filtered and the filtrate concentrated to give a
syrupy residue, which was then purified by chromatography. The
solvent was evaporated and the residue was redissolved in water
before filtration through a Sep-pak C-18 cartridge.
General Procedure for Deprotection of Intermediates Leading
to 7-10. The starting material was dissolved in 4:1 HOAc-H2O
(10-30 mL) and heated at 70 °C for 4 h. The solvent was
(41) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293–297.
4522 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
SCHEME 12
evaporated upon completion of the reaction. The residue was
dissolved in MeOH (10-50 mL) followed by dropwise addition
of NaOMe in MeOH (0.1 M) until the pH of the solution was 12.
The reaction mixture was then stirred at room temperature for 4 h
and was neutralized by the addition of Amberlyst-15 (H+) cation
exchange resin. The solution was filtered and the filtrate concen-
trated to give a syrupy residue. The resulting crude product was
then purified by chromatography. The solvent was evaporated and
the residue was redissolved in water before filtration through a Sep-
pak C-18 cartridge.
Octyl ꢀ-D-Galactofuranosyl-(1f5)-ꢀ-D-galactofuranoside (1).
Using the general deprotection procedure, octyl glycoside 30 (92.0
mg, 0.078 mmol) gave a crude product which was purified by
column chromatography (5:1 CH2Cl2-MeOH). The solvent was
evaporated and the residue was redissolved in water before filtration
through a Sep-pak C-18 cartridge to afford 1 as a clear, colorless
oil (32.0 mg, 90%). The data for this compound matched that
previously reported.13d
Octyl ꢀ-D-Galactofuranosyl-(1f6)-ꢀ-D-galactofuranoside (2).
Using the general deprotection procedure, octyl glycoside 31 (37.1
mg, 0.032 mmol) gave a crude product, which was purified by
column chromatography (5:1 CH2Cl2-MeOH). The solvent was
evaporated and the residue was redissolved in water before filtration
through a Sep-pak C-18 cartridge to furnish 2 as a colorless oil
(12.5 mg, 87%). The data for this compound matched that
previously reported.13d
Octyl ꢀ-D-Galactofuranosyl-(1f6)-ꢀ-D-galactofuranosyl-(1f5)-
ꢀ-D-galactofuranoside (3). Using the general deprotection procedure,
octyl glycoside 37 (45.4 mg, 0.027 mmol) gave a crude product,
which was purified by column chromatography (3:1 CH2Cl2-
MeOH). The solvent was evaporated and the residue was redis-
solved in water before filtration through a Sep-pak C-18 cartridge
to furnish trisaccharide 3 as a clear, colorless oil (15.2 mg, 90%).
Rf 0.21 (3:1 CH2Cl2-MeOH); [R]D -93.0 (c 1.0, MeOH); 1H NMR
(500 MHz, D2O, δH): 5.23 (d, 1 H, J ) 1.3 Hz, H-1′), 5.04 (d, 1
H, J ) 1.5 Hz, H-1′′), 4.98 (d, 1 H, J ) 2.0 Hz, H-1), 4.14-3.94
(m, 9 H, H-2′′, H-2′, H-3′, H-2, H-3, H-4, H-4′, H-3′′, H-4′′), 3.90
(dd, 1 H, J ) 7.0, 3.5 Hz, H-6a′′′), 3.61-3.75 (m, 3 H, H-5′, H-5′′,
H-6b′), 3.74-3.61 (m, 6 H, H-5, H-6a, octyl OCH2, H-6b′, H-6b′′,
H-6b), 3.54 (app dt, 1 H, J ) 10.0, 7.5 Hz, octyl OCH2), 1.60-1.37
(m, 2 H, octyl CH2), 1.37-1.25 (m, 10 H, octyl CH2), 0.88 (t, 3 H,
J ) 7.0 Hz, octyl CH3); 13C NMR (125 MHz, D2O, δC): 108.7
(C-1′), 108.0 (C-1), 107.9 (C-1′′), 83.9 (C-4′′), 83.8 (C-4), 82.3
(C-4′), 82.1 (C-2′), 81.8 (C-2), 81.2 (C-2′′), 77.7 (C-3′), 77.6 (C-
3′′), 77.3 (C-3), 76.8 (C-5), 71.7 (C-5′), 70.4 (C-5′′), 70.2 (C-6′),
69.6 (octyl OCH2), 63.8 (C-6), 61.9 (C-6′′), 32.0 (octyl CH2), 29.5
(octyl CH2), 29.3 (octyl CH2), 29.2 (octyl CH2), 26.1 (octyl CH2),
22.9 (octyl CH2), 14.5 (octyl CH3). ESI-MS m/z calcd for [M +
Na]+ C26H48O16Na: 639.2837. Found: 639.2835.
Octyl ꢀ-D-Galactofuranosyl-(1f5)-ꢀ-D-galactofuranosyl-(1f6)-
ꢀ-D-galactofuranoside (4). Using the general deprotection pro-
cedure, octyl glycoside 41 (60.3 mg, 0.036 mmol) gave a crude
product, which was purified by column chromatography (3:1
CH2Cl2–MeOH). The solvent was evaporated and the residue was
redissolved in water before filtration through a Sep-pak C-18
cartridge to furnish 4 as a clear, colorless oil (20.0 mg, 89%). Rf
1
0.24 (3:1 CH2Cl2–MeOH); [R]D -88.0 (c 0.9, MeOH); H NMR
(500 MHz, D2O, δH): 5.21 (d, 1 H, J ) 1.9 Hz, H-1′′), 5.00 (s, 1
H, H-1′), 4.98 (d, 1 H, J ) 2.2 Hz, H-1), 4.16 (dd, 1 H, J ) 4.0,
1.9 Hz, H-2′′), 4.14-4.13 (m, 2 H, H-2′, H-3′), 4.10-4.04 (m, 4
H, H-3′′, H-4′, H-3, H-2), 4.02 (dd, 1 H, J ) 4.0, 3.0 Hz, H-4′′),
3.96-3.94 (m, 3 H, H-4, H-5′, H-5), 3.86-3.71 (m, 6 H, H-6a,
H-6b, H-6a′, H-6b′, H-6a′′, H-5′′), 3.66 (dd, 1 H, J ) 12.0, 8.0 Hz,
H-6b′′), 3.60 (app dt, 1 H, J ) 10.0, 7.0 Hz, octyl OCH2), 3.55
(app dt, 1 H, J ) 10.0, 7.0 Hz, octyl OCH2), 1.60-1.37 (m, 2 H,
octyl CH2), 1.37-1.25 (m, 10 H, octyl CH2), 0.88 (t, 3 H, J ) 7.0
Hz, octyl CH3); 13C NMR (125 MHz, D2O δC): 108.6 (C-1′), 108.0
(C-1), 107.9 (C-1′′), 83.5 (C-4′), 83.3 (C-4), 82.7 (C-4′′), 82.1 (C-
J. Org. Chem. Vol. 73, No. 12, 2008 4523

Completo and Lowary
2′′), 82.0 (C-2′), 81.9 (C-2), 77.5 (C-3), 77.4 (C-3′′), 77.38 (C-3),
76.8 (C-5′), 71.4 (C-5), 70.2 (C-5′′), 70.1 (C-6), 69.4 (octyl OCH2),
63.8 (C-6′′), 61.9 (C-6′), 32.0 (octyl CH2), 29.6 (octyl CH2), 29.3
(octyl CH2), 29.3 (octyl CH2), 26.1 (octyl CH2), 22.9 (octyl CH2),
14.5 (octyl CH3). ESI-MS m/z calcd for (M + Na) C26H48O16Na:
639.2837. Found: 639.2835.
(s, 3 H, C(dO)CH3), 1.64-1.60 (m, 2 H, octyl CH2), 1.38-1.23
(m, 13 H, octyl CH2, H-6′), 0.85 (t, 3 H, J ) 7.0 Hz, octyl CH3);
13C NMR (125 MHz, D2O, δC): 170.9 (CdO), 102.1 (C-1′), 97.8
(C-1), 80.4 (C-2′), 72.9 (C-4′), 72.7 (C-3′), 71.6 (C-4), 71.1 (C-3),
69.7 (C-5′), 69.2 (C-5), 69.0 (octyl OCH2), 61.4 (C-6), 54.2 (C-2),
32.1 (octyl CH2), 29.44 (octyl CH2), 29.40 (2 × octyl CH2), 26.3
(octyl CH2), 23.0 (octyl CH2), 22.8 (C(dO)CH3), 17.4 (C-6′), 14.3
(octyl CH3). ESI-MS m/z calcd for [M + Na]+ C22H41NO10Na:
502.2623. Found: 502.2621.
Octyl ꢀ-D-Galactofuranosyl-(1f5)-ꢀ-D-galactofuranosyl-(1f6)-
ꢀ-D-galactofuranosyl-(1f5)-ꢀ-D-galactofuranoside (5). Using the
general deprotection procedure, octyl glycoside 45 (11.0 mg, 0.005
mmol) gave a crude product, which was purified by column
chromatography (3:1 CH2Cl2-MeOH). The solvent was evaporated
and the residue was redissolved in water before filtration through
a Sep-pak C-18 cartridge to furnish 5 as a clear, colorless oil (3.3
mg, 82%). Rf 0.21 (3:1 CH2Cl2–MeOH); [R]D -67.4 (c 0.2, MeOH);
1H NMR (600 MHz, D2O, δH): 5.23 (d, 1 H, J ) 1.8 Hz, H-1′′′),
5.22 (d, 1 H, J ) 2.0 Hz, H-1′), 5.01 (s, 1 H, H-1′′), 4.96 (s, 1 H,
J ) 1.0 Hz, H-1), 4.14-3.94 (m, 12 H, H-2′, H-2′′, H-2′′′, H-3′,
H-3′′, H-3′′′, H-2, H-3, H-4, H-4′, H-4′′, H-4′′′), 3.61-3.75 (m, 7
H, H-5′, H-5′′, H-6a′, H-6a′′, H-6a′′′, H-6b′′′, H-5′′′), 3.74-3.61 (m,
6 H, H-5, H-6a, octyl OCH2, H-6b′,H-6b′′, H-6b), 3.54 (app dt, 1 H,
J ) 12.0, 7.5 Hz, octyl OCH2), 1.60-1.37 (m, 2 H, octyl CH2),
1.37-1.25 (m, 10 H, octyl CH2), 0.88 (t, 3 H, J ) 7.0 Hz, octyl
CH3); 13C NMR (125 MHz, D2O δC): 108.7 (C-1′′′), 108.0, 107.9
(C-1′′, C-1′), 107.9 (C-1), 83.9 (C-4′′), 85.5 (C-4′′′), 82.7 (C-2′′′),
82.3 (C-2′′), 82.1 (C-4, C-4′), 82.00 (C-2′), 81.9 (C-2), 77.6 (C-
3′′′), 77.5 (C-3′), 77.4 (C-3′′,C-3), 76.8 (C-5′′), 76.7 (C-5,C-5′′′),
71.4 (C-5′), 70.3 (C-6′), 69.5 (octyl OCH2), 63.7 (C-6), 62.0 (C-
6′′), 61.9 (C-6′′′), 32.0 (octyl CH2), 30.7 (octyl CH2), 29.6 (octyl
CH2), 29.3 (octyl CH2), 26.1 (octyl CH2), 22.9 (octyl CH2), 14.3
(octyl CH3). ESI-MS m/z calcd for [M + Na]+ C32H58O21Na:
801.3362. Found: 801.3363.
Octyl ꢀ-D-Galactofuranosyl-(1f4)-r-L-rhamnopyranosyl-(1f3)-
2-acetamido-2-deoxy-r-D-glucopyranoside (8). Using the general
deprotection procedure, octyl glycoside 63 (57.3 mg, 0.05 mmol)
gave a crude product, which was purified by column chromatog-
raphy (4:1 CH2Cl2-MeOH). The solvent was evaporated and the
residue was redissolved in water before filtration through a Sep-
pak C-18 cartridge to afford trisaccharide 8 as a white amorphous
solid (27.2 mg, 89%). Rf 0.36 (3:1 CH2Cl2-MeOH); [R]D -13.2
1
(c 0.1, MeOH); H NMR (500 MHz, D2O, δH): 5.29 (br s, 1 H,
H-1′′), 4.87 (s, 1 H, H-1′), 4.80 (d, 1 H, J ) 3.5 Hz, H-1), 4.11
(dd, 1 H, J ) 4.0, 1.8 Hz, H-2′′), 4.08-3.99 (m, 4 H, H-3′′, H-4′′,
H-5′, H-2), 3.88-3.48 (m, 13 H, H-3, H-4, H-5, H-6a, H-6b, H-2′,
H-3′, H-4′, H-5′′, H-6a′′, H-6b′′, octyl OCH2), 2.06 (s, 3 H,
C(dO)CH3), 1.64-1.60 (m, 2 H, octyl CH2), 1.38-1.23 (m, 13
H, octyl CH2, H-6′), 0.85 (t, 3 H, J ) 7.0 Hz, octyl CH3); 13C
NMR (125 MHz, D2O, δC): 174.9 (CdO), 109.1 (C-1′′), 101.9 (C-
1′), 97.7 (C-1), 83.6 (C-4′′), 82.0 (C-2′′), 80.5 (C-2′), 78.7 (C-4′),
77.6 (C-3′′), 72.9 (C-4), 71.9 (C-3′), 71.5 (C-3), 71.4 (C-5′′), 69.1
(C-5′), 69.0 (octyl OCH2), 68.3 (C-5), 63.7 (C-6′′), 61.4 (C-6), 54.2
(C-2), 32.1 (octyl CH2), 29.42 (octyl CH2), 29.40 (2 × octyl CH2),
26.3 (octyl CH2), 22.9 (octyl CH2), 22.8 (C(dO)CH3), 17.4 (C-
6′), 14.3 (octyl CH3). ESI-MS m/z calcd for [M + Na]+
C28H51NO15Na: 664.3151. Found: 664.3153.
Octyl ꢀ-D-Galactofuranosyl-(1f6)-ꢀ-D-galactofuranosyl-(1f5)-
ꢀ-D-galactofuranosyl-(1f6)-ꢀ-D-galactofuranoside (6). Using the
general deprotection procedure, octyl glycoside 48 (25.0 mg, 0.01
mmol) gave a crude product, which was purified by column
chromatography (3:1 CH2Cl2-MeOH). The solvent was evaporated
and the residue was redissolved in water before filtration through
a Sep-pak C-18 cartridge to furnish 6 as a clear, colorless oil (5.4
mg, 70%). Rf 0.22 (3:1 CH2Cl2-MeOH,); [R]D -143.3 (c 0.1,
MeOH); 1H NMR (600 MHz, D2O, δH): 5.22 (d, 1 H, J ) 1.3 Hz,
H-1′′), 5.04 (d, 1 H, J ) 1.5 Hz, H-1′′′), 5.00 (s, 1 H, H-1′), 4.95
(s, 1 H, H-1), 4.14-3.94 (m, 14 H, H-2′′, H-2′′′, H-2′, H-3′, H-3′′,
H-3′′′,H-4′, H-3, H-2, H-4′′, H-4, H-5′, H-5, H-4′′′), 3.90-3.71 (m,
10 H, H-6a, H-6b, H-6a′, H-6b′, H-6a′′, H-5′′, H-6a′′′, H-5′′′, H-6b′′′,
H-6b′′), 3.60 (app dt, 1 H, J ) 10.0, 7.0 Hz, octyl OCH2), 3.55
(app dt, 1 H, J ) 10.0, 7.0 Hz, octyl OCH2), 1.60-1.57 (m, 2 H,
octyl CH2), 1.37-1.25 (m, 10 H, octyl CH2), 0.88 (t, 3 H, J ) 7.0
Hz, octyl CH3); 13C NMR (125 MHz, D2O, δC): 108.8 (C-1′′′),
108.7 (C-1′), 108.0 (C-1), 107.9 (C-1′′), 83.9 (C-4′′′), 83.83 (C-
2′′′), 83.81 (C-4′), 83.3 (C-4), 82.8 (C-4′′), 82.1 (C-2′′), 81.9 (C-
2), 77.6 (C-3′′′), 77.6 (C-3′), 77.5 (C-3′′), 77.4 (C-3), 76.7 (C-5′),
71.72 (C-5′′′), 71.69 (C-5), 70.4 (C-5′′), 70.0 (C-6′′), 69.4, (octyl
OCH2), 63.6 (C-6), 63.6 (C-6′′′), 61.9 (C-6′), 32.0 (octyl CH2), 29.0
(octyl CH2), 29.6 (octyl CH2), 29.3 (octyl CH2), 26.1 (octyl CH2),
22.9 (octyl CH2), 14.5 (octyl CH3). ESI-MS m/z calcd for [M +
Na]+ C32H58O21Na: 801.3362. Found: 801.3363.
Octyl ꢀ-D-Galactofuranosyl-(1f5)-ꢀ-D-galactofuranosyl-(1f4)-
r-L-rhamnopyranosyl-(1f3)-2-acetamido-2-deoxy-r-D-glucopyra-
noside (9). Using the general deprotection procedure, octyl glycoside
64 (60.0 mg, 0.04 mmol) gave a crude product, which was purified
by column chromatography (4:1 CH2Cl2-MeOH). The solvent was
evaporated and the residue was redissolved in water before filtration
through a Sep-pak C-18 cartridge to afford tetrasaccharide 9 as a
white amorphous solid (21.5 mg, 68%). Rf 0.21 (3:1
CH2Cl2-MeOH); [R]D -11.7 (c 0.2, MeOH); 1H NMR (600 MHz,
D2O, δH): 5.27 (d, 1 H, J ) 1.8 Hz, H-1′′), 5.22 (d, 1 H, J ) 1.8
Hz, H-1′′′), 4.86 (d, 1 H, J ) 1.8 Hz, H-1′), 4.80 (d, 1 H, J ) 3.6
Hz, H-1), 4.15 (dd, 1 H, J ) 3.6, 1.8 Hz, H-2′′′), 4.13-4.02 (m, 7
H, H-3′′′, H-4′′′, H-2′′, H-3′′, H-4′′, H-5′, H-2), 3.96 (app t, 1 H, J
) 8.5 Hz, H-3), 3.89-3.63 (m, 15 H, H-4, H-5, H-6a, H-6b, H-2′,
H-3′, H-4′, H-5′′, H-6a′′, H-6b′′, H-5′′′, H-6a′′′, H-6b′′′, 2 × octyl
OCH2), 2.06 (s, 3 H, C(dO)CH3), 1.64-1.60 (m, 2 H, octyl CH2),
1.38-1.23 (m, 13 H, octyl CH2, H-6′), 0.85 (t, 3 H, J ) 7.0 Hz,
octyl CH3); 13C NMR (125 MHz, D2O, δC): 174.9 (CdO), 109.3
(C-1′′), 108.1 (C-1′′′), 102.1 (C-1′), 97.7 (C-1), 83.5 (C-4′′), 82.5
(C-2′′′), 82.4 (C-4′′′), 82.1 (C-2′′), 80.5 (C-2′), 79.2 (C-4′), 77.4
(C-3′′), 77.3 (C-3′′′), 76.6 (C-3′), 72.9 (C-4), 71.9 (C-3), 71.5 (C-
5′′), 71.4 (C-5′′′), 69.2 (C-5′), 69.0 (octyl OCH2), 68.2 (C-5), 63.7
(C-6′′), 62.1 (C-6′′′), 61.4 (C-6), 54.2 (C-2), 32.0 (octyl CH2), 29.42
(octyl CH2), 29.40 (octyl CH2), 29.4 (octyl CH2), 26.3 (octyl CH2),
22.9 (octyl CH2), 22.8 (C(dO)CH3), 17.4 (C-6′), 14.3 (octyl CH3).
ESI-MS m/z calcd for [M + Na]+ C34H61NO20Na: 826.3679. Found:
826.3682.
Octyl r-L-Rhamnopyranosyl-(1f3)-2-acetamido-2-deoxy-r-D-
glucopyranoside (7). Using the general deprotection procedure, octyl
glycoside 61 (58.0 mg, 0.08 mmol) gave a crude product, which
was purified by column chromatography (10:1 CH2Cl2-MeOH).
The solvent was evaporated and the residue was redissolved in water
before filtration through a Sep-pak C-18 cartridge to afford
disaccharide 7 as a white amorphous solid (22.0 mg, 55%). Rf 0.54
Octyl ꢀ-D-Galactofuranosyl-(1f6)-ꢀ-D-galactofuranosyl-(1f5)-
ꢀ-D-galactofuranosyl-(1f4)-r-L-rhamnopyranosyl-(1f3)-2-aceta-
mido-2-deoxy-r-D-glucopyranoside (10). Using the general depro-
tection procedure, octyl glycoside 65 (46.0 mg, 0.02 mmol) gave
a crude product, which was purified by column chromatography
(4:3:3:2 EtOAc-AcOH-CH3OH-H2O). The solvent was evapo-
rated and the residue was redissolved in water and lyophilized. The
residue obtained was filtered through a Sep-pak C-18 cartridge to
afford pentasaccharide 10 as a white amorphous solid (12.0 mg,
1
(6:1 CH2Cl2-MeOH); [R]D -20.1 (c 1.0, MeOH); H NMR (500
MHz, D2O, δH): 4.86 (d, 1 H, J ) 1.6 Hz, H-1′), 4.82 (d, 1 H, J )
3.6 Hz, H-1), 4.05 (dd, 1 H, J ) 10.4, 3.6 Hz, H-2), 3.98-3.68
(m, 1 H, H-5′), 3.86 (dd, 1 H, J ) 10.0, 2.2 Hz, H-3′), 3.81-3.68
(m, 6 H, H-3, H-5, H-6a, H-4, octyl OCH2, H-2′), 3.57-3.48 (m,
2 H, H-6b, octyl OCH2), 3.45 (app t, 1 H, J ) 10.0 Hz, H-4′), 2.06
1
62%). [R]D -2.9 (c 0.1, MeOH); H NMR (600 MHz, D2O, δH):
4524 J. Org. Chem. Vol. 73, No. 12, 2008

Galactofuranose-Containing Acceptor Substrates
5.26 (d, 1 H, J ) 1.8 Hz, H-1′′), 5.20 (d, 1 H, J ) 1.8 Hz, H-1′′′),
5.04 (d, 1 H, J ) 1.8 Hz, H-1′′′′), 4.86 (s, 1 H, H-1′), 4.79 (br s,
1 H, H-1), 4.14-3.94 (m, 13 H, H-2′′′, H-2′′, H-3′′′′, H-2′′′′, H-2,
H-3′′, H-3′′′, H-4′′′, H-4′′′′, H-5′, H-5′′, H-3, H-4), 3.89-3.45 (m,
17 H, H-2′, H-3′, H-4′, H-4′′, H-6a, H-6b, H-5, H-6a′′, H-6b′′, H-5′′′,
H-6a′′′, H-6b′′′, H-5′′′′, H-6a′′′′, H-6b′′′′, 2 × octyl OCH2), 2.06 (s,
3 H, C(dO)CH3), 1.64-1.60 (m, 2 H, octyl CH2), 1.38-1.23 (m,
13 H, octyl CH2, H-6′), 0.85 (t, 3 H, J ) 7.0 Hz, octyl CH3); 13C
NMR (125 MHz, D2O, δC): 174.9 (CdO), 109.3 (C-1′′), 108.7 (C-
1′′′′), 108.1 (C-1′′′), 102.1 (C-1′), 97.7 (C-1), 83.84 (C-4′′), 83.80
(C-4′′′′), 82.6 (C-2′′′), 82.5 (C-4′′′), 82.1 (C-2′′), 81.8 (C-2′′′′), 80.5
(C-2′), 79.2 (C-4′), 77.6 (C-3′′), 77.5 (C-3′′′), 77.4 (C-3′′′′), 76.5
(C-3′), 72.9 (C-4), 71.9 (C-3), 71.7 (C-5′′′′), 71.5 (C-5′′), 70.4 (C-
5′′′), 70.2 (C-6′′′′), 69.2 (C-5′), 69.0 (octyl OCH2), 68.3 (C-5), 63.6
(C-6′′), 62.1 (C-6′′′), 61.4 (C-6), 54.2 (C-2), 32.0 (octyl CH2), 29.4
(octyl CH2), 29.4 (octyl CH2), 29.4 (octyl CH2), 26.3 (octyl CH2),
22.9 (octyl CH2), 22.8 (C(dO)CH3), 17.4 (C-6′), 14.3 (octyl CH3).
ESI-MS m/z calcd for [M + Na]+ C40H71NO25Na: 988.4207. Found:
988.4211.
Octyl r-L-Arabinofuranosyl-(1f5)-ꢀ-D-galactofuranoside (11).
Using the general deprotection procedure, octyl glycoside 32 (53.0
mg, 0.05 mmol) gave a crude product, which was purified by
column chromatography (6:1 CH2Cl2-MeOH). The solvent was
evaporated and the residue was redissolved in water before filtration
through a Sep-pak C-18 cartridge to afford 11 as clear, colorless
oil (19.3 mg, 90%). Rf 0.31 (4:1 CH2Cl2-MeOH,); [R]D -95.0 (c
1.0, MeOH); 1H NMR (500 MHz, CD3OD, δH): 5.19 (d, 1 H, J )
0.5 Hz, H-1′), 4.82 (d, 1 H, J ) 1.8 Hz, H-1), 4.10 (dd, 1 H, J )
8.7, 5.0 Hz, H-4′), 4.04 (dd, 1 H, J ) 6.5, 3.0 Hz, H-3), 4.01 (dd,
1 H, J ) 3.0, 0.5 Hz, H-2′), 3.98 (dd, 1 H, J ) 6.5, 4.1 Hz, H-4),
3.92-3.89 (m, 3 H, H-6a, H-2, H-5), 3.94 (d, 1 H, J ) 5.0, 3.0 Hz,
H-3′), 3.88-3.77 (m, 4 H, H-6b, H-5a′, octyl OCH2, H-5b′), 3.54
(app dt, 1 H, J ) 9.5, 7.0 Hz, octyl OCH2), 1.60-1.37 (m, 2 H,
octyl CH2), 1.37-1.25 (m, 10 H, octyl CH2), 0.88 (t, 3 H, J ) 7.0
Hz, octyl CH3); 13C NMR (125 MHz,CD3OD δC): 109.4 (C-1′),
109.1 (C-1), 86.7 (C-4′), 83.8 (C-4), 83.7 (C-2), 82.5 (C-2′), 78.9
(C-3), 78.8 (C-3′), 77.2 (C-5), 68.9 (octyl OCH2), 63.3 (C-5′), 62.7
(C-6), 33.0 (3 × octyl CH2), 30.72 (octyl CH2), 30.70 (octyl CH2),
27.3 (octyl CH2), 14.4 (octyl CH3). ESI-MS m/z calcd for [M +
Na]+ C19H36O10Na: 447.2250. Found: 447.2250.
Octyl r-L-Arabinofuranosyl-(1f6)-ꢀ-D-galactofuranoside (12).
Using the general deprotection procedure, octyl glycoside 33 (80.6
mg, 0.077 mmol) gave a crude product, which was purified by
column chromatography (6:1 CH2Cl2-MeOH). The solvent was
evaporated and the residue was redissolved in water before filtration
through a Sep-pak C-18 cartridge to afford 12 as a clear, colorless
oil (30.0 mg, 92%). Rf 0.31 (4:1 CH2Cl2-MeOH,); [R]D -90.0 (c
1.0, MeOH); 1H NMR (500 MHz, CD3OD, δH): 4.91 (s, 1 H, H-1′),
4.82 (d, 1 H, J ) 1.5 Hz, H-1), 3.99-3.95 (m, 3 H, H-3, H-2′,
H-5), 3.94 (d, 1 H, J ) 1.5 Hz, H-2), 3.88-3.77 (m, 4 H, H-4,
H-4′, H-3′, H-5a′), 3.78-3.64 (m, 3 H, H-6a, octyl OCH2, H-6b),
3.54 (dd, 1 H, J ) 11.0, 3.0 Hz, H-5b′), 3.40 (app dt, 1 H, J ) 9.5,
7.0 Hz, octyl OCH2), 1.60-1.37 (m, 2 H, octyl CH2), 1.37-1.25
(m, 10 H, octyl CH2), 0.88 (t, 3 H, J ) 7.0 Hz, octyl CH3); 13C
NMR (125 MHz, CD3OD, δC): 110.1 (C-1′), 109.3 (C-1), 86.0 (C-
2′), 84.6 (C-2), 83.6 (C-4′), 83.2 (C-4), 78.8 (C-3′), 78.8 (C-3),
71.0 (C-5), 70.6 (C-5′), 68.9 (octyl OCH2), 63.1 (C-6), 33.0 (octyl
CH2), 30.7 (octyl CH2), 30.6 (octyl CH2), 30.4 (octyl CH2), 27.3
(octyl CH2), 23.7 (octyl CH2), 14.5 (octyl CH3). ESI-MS m/z calcd
for [M + Na]+ C19H36O10Na: 447.2250. Found: 447.2249.
Acknowledgment. This work was supported by the Alberta
Ingenuity Centre for Carbohydrate Science and the Natural
Sciences and Engineering Research Council of Canada. We
thank Mr. Ravi Sankar Loka for technical assistance.
Supporting Information Available: Details on the synthesis
and data for additional new compounds not included above; 1H
and 13C NMR spectra of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO800457J
J. Org. Chem. Vol. 73, No. 12, 2008 4525