Synthesis of Oligosaccharide Fragments of Mannosylated Lipoarabinomannan Appropriately Functionalized for Neoglycoconjugate Preparation

Article abstract of DOI:10.1081/CAR-120025322

The synthesis of a panel of oligosaccharides that are fragments of mannosylated lipoarabinomannan from Mycobacterium tuberculosis is reported. The compounds were prepared as their 8-aminooctyl glycosides to enable their easy incorporation into neoglycoconjugates.

Full text of DOI:10.1081/CAR-120025322

This article was downloaded by: [Dalhousie University]
On: 09 October 2014, At: 07:51
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lcar20
Synthesis of Oligosaccharide Fragments of
Mannosylated Lipoarabinomannan Appropriately
Functionalized for Neoglycoconjugate Preparation
Rajendrakumar Reddy Gadikota a , Christopher S. Callam a , Ben J. Appelmelk b & Todd L.
Lowary a
a Department of Chemistry , Ohio State University , 100 West 18th Avenue, Columbus, Ohio,
43210, USA
b Department of Medical Microbiology , Vrije University, Medical School , Amsterdam, The
Netherlands
Published online: 23 Aug 2006.
To cite this article: Rajendrakumar Reddy Gadikota , Christopher S. Callam , Ben J. Appelmelk & Todd L. Lowary
(2003) Synthesis of Oligosaccharide Fragments of Mannosylated Lipoarabinomannan Appropriately Functionalized for
Neoglycoconjugate Preparation , Journal of Carbohydrate Chemistry, 22:6, 459-480, DOI: 10.1081/CAR-120025322
To link to this article: http://dx.doi.org/10.1081/CAR-120025322
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 6, pp. 459–480, 2003
Synthesis of Oligosaccharide Fragments of Mannosylated
Lipoarabinomannan Appropriately Functionalized for
Neoglycoconjugate Preparation#
Rajendrakumar Reddy Gadikota,1 Christopher S. Callam,1
Ben J. Appelmelk,2 and Todd L. Lowary1,
*
1Department of Chemistry, Ohio State University, Columbus, Ohio, USA
2Department of Medical Microbiology, Vrije University, Medical School,
Amsterdam, The Netherlands
ABSTRACT
The synthesis of a panel of oligosaccharides that are fragments of mannosylated
lipoarabinomannan from Mycobacterium tuberculosis is reported. The compounds
were prepared as their 8-aminooctyl glycosides to enable their easy incorporation into
neoglycoconjugates.
Key Words: Immunology; Mycobacteria; Lipoarabinomannan; LAM; ManLAM.
#This article appeared in the Journal of Carbohydrate Chemistry, Volume 22, Issues 3 & 4,
pp. 149–170, 2003 and is being reprinted due to errors in the original version.
*
Correspondence: Todd L. Lowary, Department of Chemistry, Ohio State University, 100 West
18th Ave., Columbus, OH 43210, USA; E-mail: lowary.2@osu.edu.
459
DOI: 10.1081/CAR-120025322
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

460
Gadikota et al.
INTRODUCTION
All mycobacteria, including the human pathogen Mycobacterium tuberculosis,
synthesize a complex cell wall structure that is composed in large part of two
polysaccharides, an arabinogalactan (AG) and a lipoarabinomannan (LAM).[1,2] The
AG is covalently bound to branched chain lipids, the mycolic acids, to form the
mycolyl–arabinogalactan complex, which is the major structural component of the cell
wall. Within this glycolipid complex is interspersed the major antigenic component of
the cell wall, the LAM.[3] A number of immunomodulatory events are known to
involve LAM, including the inhibition of protein kinase activities,[4] the inhibition
of macrophage activation,[5] the neutralization of potentially cytotoxic oxygen free
radicals,[6] the induction of cytokines,[7–9] and the induction of collagenases that
destroy the extracellular matrix of the lung.[10] It is also known that T-cells recognize
LAM via major histocompatibility complex (MHC)-independent presentation path-
ways.[11,12]
The structure of LAM consists of a phosphatidylinositol moiety that is non-
covalently attached to the cytoplasmic membrane of the organism through its lipid
portion.[3,13] A polysaccharide comprised of mannopyranosyl and arabinofuranosyl
residues is attached to the inositol moiety and this glycan chain is terminated at the
non-reducing end with the hexasaccharide 1 (Scheme 1). In some mycobacterial
strains, hexasaccharide 1 is found unsubstituted, while in others this motif is further
glycosylated with short mannopyranosyl oligosaccharides to provide mannosylated-
lipoarabinomannan or ManLAM, 2.[3,13] It has been suggested that the terminal man-
nopyranosyl residues of ManLAM (the mannose ‘‘caps’’) are involved in the initial
stages of infection by adhering to human cells through their interaction with mannose
binding proteins.[14,15]
It was recently shown that one of the major antibodies generated against
mycobacterial LAM (CS-35) binds hexasaccharide 3 and, to a lesser degree,
tetrasaccharide 4 (Scheme 1).[16] Interestingly, this antibody did not recognize another
oligosaccharide fragment of 3, tetrasaccharide 5. These investigations not only clarified
the epitope bound by this antibody, but also demonstrated that the glycan portion of
hexasaccharide 3 is a potential hapten for the generation of an anti-tuberculosis
Scheme 1.

Synthesis of Oligosaccharide Fragments
461
Scheme 2.
vaccine.[17] These findings prompted our interest in determining the oligosaccharide
structures preferentially recognized by other antibodies generated against mycobacterial
LAM. In particular, we were curious as to if any of these antibodies recognized
ManLAM structural motifs.
In order to efficiently complete these investigations, it was necessary to have
access to oligosaccharide fragments of LAM and ManLAM containing a functional
group that could be used for the conjugation of these compounds either to an ELISA
plate (for immunoassays) or to a protein carrier (for vaccine generation). We describe
here the synthesis of a panel of oligosaccharides, (6–13, Schemes 1 and 2) that contain
an 8-aminooctyl aglycone. This aglycone was chosen because the amino group in
the products can be readily used as a reactive functionality for the generation of
neoglycoconjugates.[18–20] The potential of hexasaccharide 1 in vaccine development (see
above) prompted us to synthesize 6. Compounds 7–13 were of interest as they represent
structures that are expressed at the periphery of ManLAM and thus are likely to be the
motifs that are recognized by antibodies other than CS-35.
RESULTS AND DISCUSSION
We envisioned that the oligosaccharide targets could all be synthesized from eight
monosaccharide building blocks 14–21 (Scheme 3). Thioglycoside donors 17–21 are
known and were synthesized as previously reported from either D-arabinose or
D-mannose.[21–25] The preparation of the acceptors 14–16 is detailed below. In
designing these syntheses, we chose to use an azido functional group as the precursor
to the amino group present in the targets.
Synthesis of Monosaccharides 14 and 15
The synthesis of the arabinofuranoside acceptors 14 and 15 commenced from
glycosyl bromide 22[26] (Figure 1). Glycosylation of 8-azidooctanol[27] with 22 was
achieved upon reaction with iodine[28] in acetonitrile, which produced a 3:1 :b mixture
of glycosides 23 in 75% combined yield. The isomers were not separated but were

462
Gadikota et al.
Scheme 3.
instead debenzoylated to give an inseparable a:b mixture of deprotected glycosides 24.
Treatment of 24 with t-butyldiphenylchlorosilane in pyridine afforded the corre-
sponding 5-O-t-butyldimethylsilyl ethers 25 and 26, which were separated by chro-
matography and isolated in 67% and 21% yield, respectively. Differentiation of 25 and
1
1
26 was readily done by H and 13C NMR spectroscopy.[29] In the H NMR spectrum of
a-glycoside 25 the anomeric hydrogen appeared as a singlet, while in the spectrum of
3
b-glycoside 26 this hydrogen appeared as a doublet with JH1,H2 = 4.3 Hz. The values
are consistent with the assigned structures as are the chemical shifts of the anomeric
carbons in the 13C NMR spectrum (108.6 ppm for 25, 101.1 ppm in 26). Benzylation
of 25 gave 27 and then the silyl ether was removed to give 15 in 89% yield over
two steps. Identical transformations were used to convert diol 26 into 14 in 85%
overall yield.
Synthesis of 7–10
With these building blocks in hand, we were able to proceed with the synthesis of
the targets. Monosaccharide 7 was obtained in two steps from 26 as illustrated in
Figure 1. (a) HO(CH2)8N3, I2, CH3CN, rt, 75%. (b) NaOCH3, CH3OH, CH2Cl2, rt, 97%, (c) t-
BuPh2SiCl, pyridine, 0°C to rt, 67% 25, 21% 26. (d) BnBr, NaH, DMF, 0°C to rt, 96%. (e) n-
Bu4NF, THF, 93%. (f) BnBr, NaH, DMF, 0°C to rt. (g) n-Bu4NF, THF, 85% (2 steps from 26).

Synthesis of Oligosaccharide Fragments
463
Figure 2. (a) n-Bu4NF, THF, rt, 88%. (b) Ph3P, H2O, THF, 0°C to rt, 77%.
Figure 2. First, the silyl ether in 26 was cleaved by treatment with n-Bu4NF, which
afforded 29 in 88% yield. Reduction of the azido group with triphenylphosphine and
water provided a 77% yield of 7.
The preparation of oligosaccharides 8–10 is illustrated in Figure 3.[30] All
glycosylation reactions with the thioglycoside donors were carried out in dichloro-
methane using activation by N-iodosuccinimide and silver triflate. Glycosylation of 14
with thioglycoside 20 provided a 79% yield of disaccharide 30, which was sub-
sequently deacetylated upon treatment with sodium methoxide to give alcohol 31 (96%
yield). A portion of 31 was converted to target 8, in 76% yield, by hydrogenolysis of
the benzyl ethers and simultaneous reduction of the azido group. The remainder of 31
was glycosylated, again with 20, to give trisaccharide 32 in 79% yield; the acetate ester
Figure 3. (a) 20, N-iodosuccinimide, AgOTf, CH2Cl2, 0°C, 79% (for 14), 79% (for 31). (b)
NaOCH3, CH3OH, CH2Cl2, rt, 96% (for 30), 93% (for 32). (c) H2, Pd/C, CH3OH, rt, 76% (for 31),
65% (for 33), 66% (for 35). (d) 21, N-iodosuccinimide, AgOTf, CH2Cl2, 0°C. (e) NaOCH3,
CH3OH, CH2Cl2, rt, 82% (over 2 steps from 33).

464
Gadikota et al.
was then cleaved providing a 93% yield of 33. Reaction of 33 with hydrogen and Pd/C
gave target 9 in 65% yield. Alternatively, coupling of 33 with thioglycoside 21 gave an
impure tetrasaccharide (34) that was deacetylated to give 35 in 82% overall yield.
Reduction of the azide and removal of the benzyl ethers in 35 was achieved in a single
step (H2, Pd/C) giving tetrasaccharide 10 (66% yield). In the course of our
investigations, we also prepared an alternate protected tetrasaccharide derivative (36),
through the coupling of 33 with thioglycoside 20. However, although the product could
be synthesized without difficulty, removal of the benzyl groups in the product was
extremely sluggish and complete cleavage of all the benzyl ethers was never possible,
even under forcing conditions. Fortunately, we found that this problem could be
avoided through the use of 21 as the reagent for the introduction of the terminal
mannose residue into the tetrasaccharide. The anomeric stereochemistry in the mannose
1
residues was confirmed through measurement of the JC1,H1 magnitudes in
oligosaccharides 8–10, which were in the range of 167.3–172.0 Hz, consistent with
the a-mannopyranose stereochemistry.[31]
Synthesis of Oligosaccharides 11–13
Oligosaccharides 11–13 were synthesized (Figure 4) via routes analogous to
those used for the preparation of 8–10. Thus, 8-azidooctanol was glycosylated with
20 to give the a-mannoside 37, which was then converted to 16 by reaction with
sodium methoxide. This alcohol was then either deprotected and the azide reduced
(affording 11 in 82% yield) or converted to disaccharide 38 (in 86% yield) upon
reaction with 20 and N-iodosuccinimide and silver triflate. Removal of the acetate
ester in 38 yielded an 98% yield of 39, which was converted (in 83% yield) to
trisaccharide 40 by glycosylation with 21. Disaccharide 39 was also the precursor to
12, which was obtained in 80% yield upon treatment with hydrogen and palladium on
Figure 4. (a) HO(CH2)8N3, N-iodosuccinimide, AgOTf, CH2Cl2, 0°C, 90%. (b) NaOCH3,
CH3OH, CH2Cl2, rt, 99% (for 37), 98% (for 38), 98% (for 40). (c) H2, Pd/C, CH3OH, rt, 82% (for
16), 80% (for 39), 70% (for 41). (d) 20, N-iodosuccinimide, AgOTf, CH2Cl2, 0°C, 86%. (e) 21,
N-iodosuccinimide, AgOTf, CH2Cl2, 0°C, 83%.

Synthesis of Oligosaccharide Fragments
465
carbon. Conversion of trisaccharide 40 into 13 was achieved in two steps by standard
means, providing the product in 68% yield. As was done for 8–10, the stereo-
chemistry of the mannopyranose residues in 11–13 was established by measurement
1
of the JC1,H1 magnitudes.
Synthesis of Hexasaccharide 6
For the synthesis of hexasaccharide 6, we used a modification of the approach[32]
we have previously used to prepare its methyl glycoside counterpart, 3 (Figure 5).
Alcohol 15 was glycosylated with thioglycoside 17 to give a siloxane-protected
disaccharide, which was immediately debenzoylated affording 42 in 72% yield over the
two steps. Conversion of 42 into diol 43 was achieved in two steps and 62% overall
yield by benzylation and then cleavage of the siloxane protecting group with n-Bu4NF.
Coupling of 43 with an excess of 18 afforded tetrasaccharide 44 (81% yield), which
was subsequently reacted with sodium methoxide to give 45 (85% yield). The
introduction of the b-arabinofuranoside residues was achieved in a highly stereo-
selective manner by reaction of 45 at low temperature with thioglycoside 19 using
N-iodosuccinimide and silver triflate promotion.[32] The product, 46, was produced in
81% yield. We first attempted to convert 46 directly into target 6, by reaction with H2
Figure 5. (a) 17, N-iodosuccinimide, AgOTf, CH2Cl2, 0°C. (b) NaOCH3,CH3OH, CH2Cl2, rt,
72% (2 steps). (c) BnBr, NaH, DMF, rt. (d) n-Bu4NF, THF, rt, 62% (2 steps). (e) 18, N-
iodosuccinimide, AgOTf, CH2Cl2, 0°C, 81%. (f) NaOCH3, CH3OH, rt, 85%. (g) 19, N-
iodosuccinimide, AgOTf, CH2Cl2, À 78°C, 81%. (h) H2, Pd/C, CH3OH, rt, 0%. (i) Ph3P, H2O,
THF, 0°C to rt, 78%. (j) Trifluoroacetic anhydride, pyridine, rt. (k) H2, Pd/C, CH3OH, rt, 65% (2
steps). (l) NH3, CH3OH.

466
Gadikota et al.
and Pd/C, but were unsuccessful. The products produced, even at long reaction times,
had one or more benzyl groups still in place. We were therefore forced to take a more
circuitous route, as illustrated in Figure 5. First the azido group was reduced (in 78%
yield) to the amine via a Staudinger reaction. The amino group in the product (47) was
protected as an N-trifluoroacetamide and the benzyl groups were removed by
hydrogenolysis to give hexasaccharide 48 in 65% overall yield. Treatment of 48 with
ammonia in methanol afforded the target 6 in 53% yield.
CONCLUSION
In summary, we describe here the synthesis of a number of oligosaccharide
fragments of mycobacterial LAM and ManLAM functionalized with an amino group
that will allow for their ready incorporation into neoglycoconjugates. These compounds
were prepared, for the most part, without incident. However, in the synthesis of 6 and
10, the removal of all of the benzyl ether protecting groups at the end of the synthesis
was problematic and slight modification of the synthetic routes was required. The use
of these glycans in probing the selectivity of antibodies directed against mycobacterial
LAM and in vaccine generation is currently in progress (a preliminary report of these
investigations has appeared in Ref. [33]).
EXPERIMENTAL
Optical rotations were measured at 22 2°C. Analytical TLC was performed on
silica gel 60-F254 (0.25 mm, Merck). Spots were detected under UV light or by charring
with 10% H2SO4 in ethanol. Unless otherwise indicated, all reactions were carried out
at room temperature and under positive pressure of argon. Solvents were evaporated
under reduced pressure and below 40°C. Column chromatography was performed on
silica gel or Iatrobeads. Iatrobeads refers to a beaded silica gel 6RS-8060, which is
manufactured by Iatron Laboratories (Tokyo). The ratio between silica gel and
1
compound ranged from 100 to 50:1 (w/w). H NMR spectra were recorded at 400 or
500 MHz, and first order proton chemical shifts dH are referenced either to TMS (dH
0.0, CDCl3) or HOD (dH 4.78, D2O). 13C NMR spectra were recorded at 100 or 125
MHz and 13C chemical shifts dC are referenced either to TMS (dC 0.0, CDCl3) or
dioxane (dC 67.4, D2O). One-bond carbon–hydrogen coupling constants involving the
anomeric carbon of the mannose residues were measured where appropriate to prove
glycoside stereochemistry. Electrospray mass spectra were recorded on samples sus-
pended in mixtures of THF and CH3OH with added NaCl.
8-Aminooctyl 5-O-{3,5-di-O-[2-O-( -D-arabinofuranosyl)- -D-arabinofura-
nosyl]- -D-arabinofuranosyl}- -D-arabinofuranoside (6). A solution of 48 (65 mg,
0.062 mmol) dissolved in NH3-saturated CH3OH (5 mL) was stirred for 36 h and then
concentrated. Purification of the product on Iatrobeads (H2O/CH3OH, 1:1) gave 6 (31
1
mg, 53%) as an oil: Rf 0.11 (CH2Cl2/CH3OH, 1:2); [a]D + 22.3° (c 1.0, H2O); H NMR
(400 MHz, D2O, d) 5.29 (s, 1 H), 5.19 (s, 1 H), 5.18 (s, 1 H), 5.14–5.12 (m, 2 H), 4.95
(s, 1 H), 4.23–3.63 (m, 32 H), 2.66 (dd, 2 H, J = 6.8, 6.8 Hz), 1.61–1.53 (m, 4 H),

Synthesis of Oligosaccharide Fragments
467
1.14–1.02 (m, 8 H); 13C NMR (100 MHz, D2O, d) 107.7, 107.5, 106.0 (2), 101.1,
101.0, 87.4, 87.1, 83.2, 82.4, 81.9, 81.2, 76.6, 75.2, 74.5, 69.0, 63.3, 60.9, 39.9, 28.9,
28.5, 28.4, 27.0, 25.8, 25.4. HRMS (ESI) Calcd for [C38H67NO25]Na+: 960.3894.
Found: 960.3819.
8-Aminooctyl -D-arabinofuranoside (7). To a solution of 29 (300 mg, 0.99
mmol), in THF:water (10 mL, 10:1) cooled to 0°C was added Ph3P (1.03 g, 3.9 mmol).
The reaction mixture was stirred for 10 h while warming to rt and then concentrated to
an oil, which was purified by chromatography (CH2Cl2/CH3OH, 10:1) to give 7 (212
1
mg, 77%) an oil: Rf 0.1 (CH2Cl2/CH3OH, 10:1); [a]D À 32.1° (c 0.8, H2O); H NMR
(400 MHz, D2O, d) 4.85 (d, 1 H, J = 4.8 Hz), 3.97 (dd, 1 H, J = 4.6, 7.8 Hz), 3.73–
3.71 (m, 1 H), 3.65–3.59 (m, 2 H), 3.49 (dd, 1 H, J = 7.2, 10.1 Hz), 3.46–3.35 (m, 1
H), 2.64 (dd, 2 H, J = 6.8, 6.8 Hz), 1.48–1.38 (m, 4 H), 1.20–1.18 (m, 8 H); 13C NMR
(100 MHz, D2O, d) 101.4, 82.2, 76.7, 75.2, 68.8, 63.7, 40.4, 29.7, 28.8, 28.7, 26.4,
26.1, 25.6. HRMS (ESI) Calcd for [C13H27NO5]Na+: 300.1781. Found: 300.1767.
8-Aminooctyl 5-O-( -D-mannopyranosyl)- -D-arabinofuranoside (8). To a
solution of 31 (210 mg, 0.22 mmol) in CH3OH (20 mL), was added 10% Pd/C (50
mg). The solution was stirred overnight under a H2 atmosphere and then the catalyst
was filtered. The filtrate was concentrated to a residue that was purified by
chromatography on Iatrobeads (CH2Cl2/CH3OH, 1:1) to give 8 (76 mg, 76%) as an
1
oil: Rf 0.2 (CH2Cl2/CH3OH, 1:1); [a]D + 6.3° (c 1.1, H2O); H NMR (400 MHz, D2O,
d) 4.90 (d, 1 H, J = 4.3 Hz), 4.81 (d, 1 H, J = 0.8 Hz), 4.03–3.87 (m, 2 H), 3.86–3.76
(m, 2 H), 3.74–3.47 (m, 8 H), 3.27 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 2.91 (d, 2 H,
J = 6.8, 6.8 Hz), 1.58–1.49 (m, 4 H), 1.30–1.21 (m, 8 H); 13C NMR (100 MHz, D2O,
d) 101.2, 100.1 (1JC1-H1 = 168.1 Hz), 81.3, 76.3, 74.7, 73.1, 70.8, 70.4, 68.9, 68.6, 66.9,
61.2, 39.8, 29.0, 28.6, 27.0, 25.8, 25.5, 25.4. HRMS (ESI) Calcd for [C19H37NO10]Na+:
462.2309. Found: 462.2311.
8-Aminooctyl 5-O-[2-O-( -D-mannopyranosyl)- -D-mannopyranosyl]- -D-arabi-
nofuranoside (9). Debenzylation of trisaccharide 33 (220 mg, 0.163 mmol) was
carried out in CH3OH (20 mL) with 10% Pd/C (50 mg) as described for the synthesis
of 8. The product was purified by chromatography on Iatrobeads (CH2Cl2/CH3OH, 1:1)
to give 9 (66 mg, 65%) as an oil: Rf 0.15 (CH2Cl2/CH3OH, 1:1); [a]D + 8.1° (c 1.0,
1
H2O); H NMR (400 MHz, D2O, d) 5.09 (s, 1 H), 4.98 (d, 1 H, J = 1.1 Hz), 4.95(d, 1
H, J = 4.3 Hz), 4.08–4.02 (m, 3 H), 3.95–3.79 (m, 6 H), 3.76–3.56 (m, 9 H), 3.46
(ddd, 1 H, J = 5.5, 5.5, 1.6 Hz), 2.96 (dd, 2 H, J = 6.1, 6.1 Hz), 1.63–1.56 (m, 4 H),
1.39–1.30 (m, 8 H); 13C NMR (100 MHz, D2O, d) 102.7, 101.3 (1JC1-H1 = 169.0 Hz),
99.9 (1JC1-H1 = 170.1 Hz), 80.0, 77.1, 76.4, 74.8, 73.6, 73.2, 70.7, 70.5, 70.3, 68.9,
68.8, 67.3, 67.2, 61.5, 61.2, 39.9, 29.1, 28.6, 28.5, 27.0, 25.9, 25.5. HRMS (ESI) Calcd
for [C25H47NO15]Na+: 624.2837. Found: 624.2809.
8-Aminooctyl 5-O-{2-O-[2-O-( -D-mannopyranosyl)- -D-mannopyranosyl]- -D-
mannopyranosyl}- -D-arabinofuranoside (10). Debenzylation of tetrasaccharide 35
(150 mg, 0.99 mmol) was carried out in CH3OH (20 mL) with 10% Pd/C (40 mg) as
described for the synthesis of 8. The product was purified by chromatography on
Iatrobeads (CH2Cl2/CH3OH, 1:2) to give 10 (50 mg, 66%) as an oil: Rf 0.12 (CH2Cl2/

468
Gadikota et al.
1
CH3OH, 1:2); [a]D + 21.3° (c 1.0, H2O); H NMR (400 MHz, D2O, d) 5.22 (d, 1 H,
J = 0.7 Hz), 5.07 (s, 1 H), 4.99 (d, 1 H, J = 0.8 Hz), 4.96 (d, 1 H, J = 4.3 Hz), 4.05–
3.29 (m, 24 H), 2.95 (dd, 2 H, J = 6.8, 6.8 Hz), 1.58–1.52 (m, 4 H), 1.29–1.22 (m,
8 H); 13C NMR (100 MHz, D2O, d) 102.6, 101.3 (1JC1-H1 = 167.9 Hz), 101.0 (1JC1-
= 170.1 Hz), 98.5 (1JC1-H1 = 168.9 Hz), 80.0, 79.3, 78.9, 76.4, 74.9, 73.6, 73.2, 70.7,
H1
70.5, 70.3, 69.0, 68.8, 67.4, 67.1, 62.9, 61.5, 61.4, 61.2, 39.9, 29.0, 28.6, 28.4, 27.0,
25.9, 25.5. HRMS (ESI) Calcd for [C31H57NO20]Na+: 786.3366. Found: 786.3368.
8-Aminooctyl -D-mannopyranoside (11). To a solution of 16 (150 mg, 0.23
mmol) dissolved in CH3OH (10 mL) was added 10% Pd/C (40 mg). The resulting
mixture was placed under a H2 atmosphere and allowed to stir at rt for 10 h. The
mixture was subsequently filtered through Celite and concentrated under reduced
pressure. The product was purified by chromatography on Iatrobeads (CHCl3/CH3OH,
1:3) to give 11 (60 mg, 82%) as an oil: Rf 0.11 (CHCl3/CH3OH, 1:3); [a]D + 6.3° (c
1
1.0, H2O); H NMR (500 MHz, D2O, d) 5.06 (s, 1 H), 4.05–3.29 (m, 8 H), 2.95 (dd, 2
H, J = 6.7, 6.7 Hz), 1.58–1.53 (m, 4 H), 1.29–1.22 (m, 8 H); 13C NMR (125 MHz,
D2O, d) 100.2, 82.3, 76.8, 75.3, 70.7, 68.9, 63.7, 40.3, 29.7, 28.8, 28.6, 26.4, 26.1, 25.6.
HRMS (ESI) Calcd for [C14H29NO6]Na+: 330.1893. Found: 330.1880.
8-Aminooctyl 2-O-( -D-mannopyranosyl)- -D-mannopyranoside (12). Deben-
zylation and reduction of disaccharide 39 (150 mg, 0.14 mmol) was carried out in
CH3OH (10 mL) with 10% Pd/C as described for the synthesis of 11. The product was
purified by chromatography on Iatrobeads (CHCl3/CH3OH, 1:3) to give 12 (52 mg,
1
80%) as an oil: Rf 0.09 (CHCl3/CH3OH, 1:3); [a]D + 7.1° (c 1.0, H2O); H NMR (500
MHz, D2O, d) 5.09 (s, 1 H), 5.01 (s, 1 H), 4.08–4.02 (m, 3 H), 3.95–3.78 (m, 6 H),
3.76–3.56 (m, 3 H), 2.96 (dd, 2 H, J = 6.7, 6.7 Hz), 1.58–1.52 (m, 4 H), 1.29–1.20
(m, 8 H); 13C NMR (125 MHz, D2O, d) 101.3, 100.1, 80.1, 76.8, 76.1, 73.6, 73.2, 70.7,
70.3, 68.8, 67.2, 67.1, 61.3, 39.3, 29.1, 28.6, 28.5, 27.1, 26.1, 25.5. HRMS (ESI) Calcd
for [C19H37N11]Na+: 478.2264. Found: 478.2271.
8-Aminooctyl 2-O-[2-O-( -D-mannopyranosyl)- -D-mannopyranosyl]- -D-man-
nopyranoside (13). Debenzylation and reduction of trisaccharide 41 (160 mg, 0.13
mmol) was carried out in CH3OH (10 mL) with 10% Pd/C as described for the
synthesis of 11. The product was purified by chromatography on Iatrobeads (CHCl3/
CH3OH, 1:3) to give 13 (56 mg, 70%) as an oil: Rf 0.10 (CHCl3/CH3OH, 1:3);
1
[a]D + 8.9° (c 1.0, H2O); H NMR (500 MHz, D2O, d) 5.13 (d, 1 H, J = 0.7 Hz), 5.09
(s, 1 H), 5.01 (s, 1 H), 4.08–4.02 (m, 6 H), 3.98–3.73 (m, 8 H), 3.76–3.53 (m, 4 H),
2.98 (dd, 2 H, J = 6.7, 6.7 Hz), 1.58–1.51 (m, 4 H), 1.29–1.22 (m, 8 H); 13C NMR
(125 MHz, D2O, d) 101.3, 101.0, 99.8, 80.1, 79.3, 78.8, 76.8, 76.4, 74.8, 73.6, 73.2,
70.7, 70.5, 70.3, 69.0, 68.8, 67.4, 67.1, 63.8, 40.4, 29.8, 28.7, 28.6, 26.4, 26.1, 25.6.
HRMS (ESI) Calcd for [C25H47NO16]Na+: 640.2793. Found: 640.2781.
8-Azidooctyl 2,3-di-O-benzyl- -D-arabinofuranoside (14). Diol 26 (1.4 g, 2.58
mmol) was benzylated with benzyl bromide (920 mg, 5.4 mmol) and NaH (155 mg,
6.46 mmol) in DMF (15 mL) as described for the preparation of 27. Following workup,
the crude dibenzyl ether (28) was dissolved in THF, the solution cooled to 0°C under
argon atmosphere and n-Bu4NF (3.1 mL of a 1.0 M solution in THF, 3.1 mmol) was

Synthesis of Oligosaccharide Fragments
469
added. The reaction mixture was stirred for 2 h and then concentrated to an oil that was
purified by chromatography (hexane/EtOAc, 6:1) to give 14 (1.05 g, 85%) as an oil: Rf
1
0.35 (hexanes/EtOAc, 2:1); [a]D + 17.6° (c 0.9, CHCl3); H NMR (400 MHz, CDCl3,
d) 7.36–7.17 (m, 10 H), 4.85 (d, 1 H, J = 4.8 Hz), 1.70 (d, 1 H, J = 11.9 Hz), 4.62–
4.55 (m, 3 H), 4.28 (dd, 1 H, J = 6.9, 5.9 Hz), 4.08–4.03 (m, 3 H), 3.75–3.67 (m, 2
H), 3.56 (dd, 1 H, J = 4.7, 5.9 Hz), 3.36 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.22 (dd, 2 H,
J = 6.8, 6.8 Hz), 1.64–1.53 (m, 4 H), 1.35–1.25 (m, 8 H); 13C NMR (100 MHz,
CDCl3, d) 138.5, 138.1, 129.4, 129.3, 128.8, 128.4, 128.3, 128.2, 128.1, 101.2, 84.9,
82.6, 81.6, 73.0, 72.9, 69.4, 64.3, 51.8, 29.9, 29.6, 29.4, 29.2, 27.0, 26.4. HRMS (ESI)
Calcd for [C27H37N3O5]Na+: 506.2625. Found: 506.2624.
8-Azidooctyl 2,3-di-O-benzyl- -D-arabinofuranoside (15). To a solution of 27
(4.8 g, 6.6 mmol) in THF (50 mL) cooled to 0°C, was added n-Bu4NF (8.0 mL of a 1.0
M solution in THF, 8.0 mmol) under an argon atmosphere. The reaction mixture was
stirred for 1 h and then concentrated to an oil, which was purified by chromatography
(hexane/EtOAc, 6:1) to give 15 (2.99 g, 93%) as an oil: Rf 0.4 (hexanes/EtOAc, 2:1);
1
[a]D + 33.2° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3, d) 7.37–7.25 (m, 10 H), 5.08
(s, 1 H), 4.61–4.49 (m, 4 H), 4.14–4.10 (m, 1 H), 4.03–3.97 (m, 2 H), 3.84–3.82 (m,
1 H), 3.72–3.61 (m, 2 H), 3.39 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.24 (dd, 2 H, J = 6.8,
6.8 Hz), 1.62–1.55 (m, 4 H), 1.37–1.32 (m, 8 H); 13C NMR (100 MHz, CDCl3, d)
138.8, 137.8, 135.2, 128.9, 128.8, 128.3, 128.2, 128.1(2), 106.6, 88.5, 83.1, 82.2, 72.7,
72.3, 68.0, 62.6, 51.8, 29.9, 29.6, 29.5, 29.2, 27.0, 26.4. HRMS (ESI) Calcd for
[C27H37N3O5:]Na+506.2625. Found: 506.2624.
8-Azidooctyl 3,4,6-tri-O-benzyl- -D-mannopyranoside (16). To a solution of 37
(2.3 g, 3.6 mmol) in CH3OH (10 mL) and CH2Cl2 (10 mL) was added NaOCH3 (1 mL,
1M solution in CH3OH). The solution was stirred at rt for 2 h, then neutralized with
acetic acid and concentrated. The product was purified by chromatography (hexanes/
EtOAc 10:1) to yield 16 (2.2 g, 99%) as an oil: Rf 0.21 (hexanes/EtOAc 4:1);
1
[a]D + 39.9° (c 1.0, CHCl3); H NMR (500 MHz, CDCl3, d) 7.42–7.23 (m, 15 H), 4.96
(d, 1 H, J = 0.8 Hz), 4.89 (d, 1 H, J = 10.6 Hz), 4.79–4.70 (m, 3 H), 4.60 (d, 1 H,
J = 10.6 Hz), 4.57 (d, 1 H, J = 10.7 Hz), 4.09 (dd, 1 H, J = 3.0, 1.8 Hz), 3.97–3.72
(m, 6 H), 3.48 (ddd, 1 H, J = 6.6, 6.6, 9.6 Hz), 3.29 (dd, 2 H, J = 6.9, 6.9 Hz), 1.67–
1.59 (m, 4 H), 1.41–1.33 (m, 8 H); 13C NMR (125 MHz, CDCl3, d) 138.7, 138.6,
128.9, 128.8, 128.7, 128.4, 128.3, 128.2 (2), 128.1, 128.0, 99.6, 80.9, 75.6, 74.8, 73.9,
72.4, 71.5, 69.5, 68.9, 68.1, 51.9, 29.8, 29.7, 29.5, 29.3, 27.1, 26.5. HRMS (ESI) Calcd
for [C35H45N3O6]Na+: 626.3200. Found: 626.3201.
8-Azidooctyl 2,3,5-tri-O-benzoyl- / -D-arabinofuranoside (23). To a solution
of freshly prepared glycosyl bromide 22 (11.0 g, 20.9 mol) and 8-azidooctanol (4.3 g,
25.1 mmol) in CH3CN (75 mL) was added I2 (8.0 g, 63.0 mmol). After stirring for 3 h,
the reaction mixture was diluted with a saturated aq. Na2S2O3 solution and CH2Cl2.
The organic layer was washed with water, dried, concentrated to an oil and purified by
chromatography (hexane/EtOAc, 8:1) to give 23 (10.2 g, 75%) as an oil: Rf 0.35
1
(hexanes/EtOAc, 10:1); H NMR (400 MHz, CDCl3, d) 8.10–8.00 (m, 6 H), 7.56–7.29
(m, 9 H), 6.00–5.98 (m, 0.25 H), 5.58 (d, 0.75 H, J = 4.8 Hz), 5.30 (s, 0.75 H), 5.47–
5.46 (m, 0.5 H), 5.29 (s, 0.75 H), 4.88–4.57 (m, 3 H), 3.81–3.78 (m, 1 H), 3.56–3.15

470
Gadikota et al.
(m, 3 H), 1.67–1.10 (m, 12 H); 13C NMR (100 MHz, CDCl3, d) 166.6, 166.5, 166.3
(2), 166.1, 165.8, 133.9 (2), 133.8, 133.4 (2), 130.3 (2), 130.2 (2), 130.1, 129.6 (2),
129.5, 128.9, 128.8, 128.7, 106.1, 101.1, 82.4, 81.4, 79.2, 78.8, 78.2, 77.2, 69.0, 67.9,
66.4, 64.2, 51.8, 29.9, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.0, 26.9, 26.4, 26.2. HRMS
(ESI) Calcd for [C34H37N3O8]Na+: 638.2478. Found: 638.2463.
8-Azidooctyl / -D-arabinofuranoside (24). To a solution of 23 (9.5 g, 14.7
mmol) in CH3OH (75 mL) and CH2Cl2 (25 mL) was added 1.0 M CH3ONa in CH3OH
(2 mL). After stirring for 4 h, the reaction mixture was neutralized with pre-washed
Amberlite IR-120(H+) resin, filtered, and concentrated. The residue was purified by
chromatography (CH2Cl2/CH3OH 15:1) to give 24 (4.31 g, 97%) as an oil: Rf 0.25
1
(hexanes/EtOAc, 1:2); H NMR (400 MHz, CDCl3, d) 4.98 (s, 0.7 H), 4.90 (d, 0.3 H,
J = 4.3 Hz), 4.11–3.68 (m, 7 H), 3.44–3.24 (m, 4 H), 1.63–1.56 (m, 4 H), 1.38–1.32
(m, 8 H); 13C NMR (100 MHz, CDCl3, d) 108.2, 101.6, 86.5, 82.6, 79.7, 78.4, 77.1,
69.6, 68.1, 63.3, 61.8, 51.8, 29.9, 29.8, 29.5, 29.4, 29.1, 27.0, 26.3, 26.2. HRMS (ESI)
Calcd for [C13H25N3O5]Na+: 326.1686. Found: 326.1691.
8-Azidooctyl 5-O-tert-butyldiphenylsilyl- -D-arabinofuranoside (25) and 8-
azidooctyl 5-O-tert-butyldiphenylsilyl- -D-arabinofuranoside (26). To a solution
of 24 (4.0 g, 13.2 mmol) in pyridine (30 mL) cooled to 0°C was added tert-
butylchlorodiphenylsilane (4.2 g, 15.3 mmol). The reaction mixture was allowed to stir
for 10 h while warming to rt. The solution was then diluted with CH2Cl2 and washed
successively with 2% aq. HCl, a saturated aq. solution of NaHCO3, and water. The
organic layer was dried, concentrated, and the resulting residue was purified by
chromatography (hexane/EtOAc, 4:1) to give 25 (4.77 g, 67%) and 26 (1.48 g, 21%) as
1
an oil. 25: Rf 0.5 (hexanes/EtOAc, 2:1); [a]D + 19.2° (c 1.1, CHCl3); H NMR (400
MHz, CDCl3, d) 7.70–7.65 (m, 4 H), 7.45–7.25 (m, 6 H), 5.08 (s, 1 H), 4.20–4.10 (m,
3 H), 4.02 (d, 1 H, J = 11.8 Hz), 3.82 (dd, 1 H, J = 2.2, 11.4 Hz), 3.77–3.71 (m, 3 H),
3.46 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.24 (dd, 2 H, J = 6.8, 6.8 Hz), 1.65–1.54 (m, 4
H), 1.36–1.10 (m, 8 H) 1.09–1.05 (m, 9 H); 13C NMR (100 MHz, CDCl3, d) 136.0 (2),
132.2, 132.1, 130.6, 130.4, 128.4, 128.3, 108.6, 87.9, 78.6, 78.4, 67.9, 64.4, 51.8, 29.8,
29.5, 29.4, 29.2, 27.0 (2), 26.4, 19.4. HRMS (ESI) Calcd for [C29H43N3O5Si]Na+:
564.2864. Found: 564.2837. 26: Rf 0.35 (hexanes/EtOAc, 2:1); [a]D + 14.7° (c 1.0,
1
CHCl3); H NMR (400 MHz, CDCl3, d) 7.69–7.66 (m, 4 H), 7.42–7.25 (m, 6 H), 4.90
(d, 1 H, J = 4.3 Hz), 4.11–4.02 (m, 2 H), 3.90 (dd, 1 H, J = 5.8, 11.6 Hz), 3.79–3.67
(m, 5 H), 3.38 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.23 (dd, 2 H, J = 6.8, 6.8 Hz), 1.58–
1.47 (m, 4 H), 1.34–1.24 (m, 8 H) 1.09–1.06 (m, 9 H); 13C NMR (100 MHz, CDCl3,
d) 135.9, 133.6, 130.2, 130.1, 128.1, 101.1, 82.3, 78.4, 78.2, 68.7, 65.9, 51.8, 29.8,
29.6, 29.4, 29.2 (2), 27.2, 27.0, 26.3, 19.6. HRMS (ESI) Calcd for [C29H43N3O5Si]Na+:
564.2864. Found: 564.2831.
8-Azidooctyl 5-O-tert-butyldiphenylsilyl-2,3-di-O-benzyl- -D-arabinofuranoside
(27). To a slurry of hexane-washed NaH (711 mg, 29.5 mmol) in DMF (10 mL)
cooled to 0°C, was added a solution of 25 (4.0 g, 7.3 mmol) in DMF (10 mL). Benzyl
bromide (3.14 g, 18.4 mmol) was added dropwise to this solution and the mixture was
stirred for 1 h while warming to rt before CH3OH (2 mL) was added. The reaction
mixture was poured into ice water and was extracted into diethyl ether. The combined

Synthesis of Oligosaccharide Fragments
471
ethereal extracts were dried, filtered, and concentrated to a residue, which was purified
by chromatography (hexane/EtOAc, 10:1) to give 27 (5.11 g, 96%) as an oil: Rf 0.45
1
(hexanes/EtOAc, 10:1); [a]D + 42.1° (c 1.2, CHCl3); H NMR (400 MHz, CDCl3, d)
7.69–7.65 (m, 4 H), 7.40–7.24 (m, 16 H), 5.03 (s, 1 H), 4.58–4.48 (m, 4 H), 4.17–
4.13 (m, 1 H), 4.05–4.03 (m, 2 H), 3.84–3.67 (m, 3 H), 3.41 (ddd, 1 H, J = 6.8, 6.8,
1.6 Hz), 3.20 (dd, 2 H, J = 6.8, 6.8 Hz), 1.63–1.53 (m, 4 H), 1.36–1.32 (m, 8 H),
1.04–1.00 (m, 9 H); 13C NMR (100 MHz, CDCl3, d) 138.5, 138.5, 138.2, 136.1 (2),
133.9, 130.0 (2), 128.8, 128.7, 128.2 (2), 128.1, 128.0 (3), 106.6, 88.9, 83.6, 82.5, 72.4,
72.2, 68.0, 64.0, 51.8, 30.0, 29.7, 29.5, 29.2, 27.3, 27.1, 26.5, 19.7. HRMS (ESI) Calcd
for [C43H55N3O5Si]Na+: 744.3803. Found: 744.3747.
8-Azidooctyl -D-arabinofuranoside (29). Glycoside 26 (800 mg, 1.47 mmol)
was desilylated with n-Bu4NF (1.8 mL of a 1.0 M solution in THF, 1.8 mmol) as
described for the synthesis of 15. The product was purified by chromatography
(hexane/EtOAc, 1:2) to give 29 (390 mg, 88%) as an oil: Rf 0.23 (hexanes/EtOAc, 1:2);
1
[a]D À 14.7° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3, d) 4.92 (d, 1 H, J = 4.8 Hz),
4.16 (dd, 1 H, J = 6.9, 7.0 Hz), 4.15–3.92 (m, 2 H), 3.91–3.89 (m, 1 H), 3.80–3.64
(m, 3 H), 3.50 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.26 (dd, 2 H, J = 6.8, 6.8 Hz), 3.16
(br. d, 1 H, J = 1.0 Hz), 2.74 (br. s, 1 H), 1.62–1.56 (m, 4 H), 1.40–1.32 (m, 8 H); 13C
NMR (100 MHz, CDCl3, d) 101.6, 82.7, 78.7, 76.2, 69.6, 63.4, 51.8, 29.9, 29.6, 29.4,
29.1, 27.0, 26.2. HRMS (ESI) Calcd [C13H25N3O5]Na+: 326.1700. Found: 326.1701.
8-Azidooctyl 5-O-(2-O-acetyl-3,4,6-tri-O-benzyl- -D-mannopyranosyl)-2,3-di-O-
benzyl- -D-arabinofuranoside (30). Thioglycoside 20 (1.31 g, 2.19 mmol), alcohol
˚
14 (800 mg, 1.82 mmol), and powdered 4 A molecular sieves (1.0 g) were dried
overnight in vacuo and then CH2Cl2 (40 mL) was added. The suspension was cooled to
0°C and stirred for 10 min. N-iodosuccinimide (490 mg, 2.18 mmol) was added and the
reaction was stirred for 20 min before silver triflate (99 mg, 0.38 mmol) was added.
The reaction mixture was stirred for 1 h and then Et3N (1 mL) was added. The
resulting yellow solution was filtered, diluted with CH2Cl2, washed with a saturated aq.
Na2S2O3 solution and then brine and water. After drying, the organic layer was
concentrated to an oil, which was purified by chromatography (hexanes/EtOAc, 10:1)
to give 30 (1.37 g, 79%) as an oil; Rf 0.43 (hexanes/EtOAc, 4:1); [a]D + 22.6° (c 1.1,
1
CHCl3); H NMR (400 MHz, CDCl3, d) 7.34–7.15 (m, 25 H), 5.39 (d, 1 H, J = 0.8
Hz), 4.86–4.82 (m, 3 H), 4.69–4.44 (m, 9 H), 4.12 (dd, 1 H, J = 5.7, 5.9 Hz), 4.05–
4.38 (m, 4 H), 3.83–3.64 (m, 6 H), 3.51 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.31 (d, 2 H,
J = 6.8, 6.8 Hz), 2.13 (s, 3 H), 1.68–1.51 (m, 4 H), 1.33–1.26 (m, 8 H); 13C NMR
(100 MHz, CDCl3, d) 170.8, 138.9, 138.6, 138.5, 138.3, 138.1, 128.8 (2), 128.7 (2),
128.5, 128.4, 128.3(2), 128.2, 128.1, 128.0(2), 100.9, 98.5, 84.6, 83.7, 79.9, 78.7, 75.6,
74.6, 73.9, 72.8 (2), 72.2, 72.0, 70.5, 69.1, 69.0, 68.5, 51.8, 29.9, 29.7, 29.5, 29.2, 27.1,
26.5, 21.5. HRMS (ESI) Calcd for [C56H67N3O11]Na+: 980.4667. Found: 980.4695.
8-Azidooctyl 5-O-(3,4,6-tri-O-benzyl- -D-mannopyranosyl)-2,3-di-O-benzyl- -D-
arabinofuranoside (31). Disaccharide 30 (1.2 g, 1.25 mmol) was deacetylated as
described for the preparation of 24 using 1.0 M NaOCH3 in CH3OH (1 mL) and
CH3OH (10 mL). The product was purified by chromatography (hexanes/EtOAc, 6:1)
to give 31 (1.1 g, 96%) as an oil: Rf 0.3 (hexanes/EtOAc, 4:1); [a]D + 36.1° (c 1.3,

472
Gadikota et al.
CHCl3); 1H NMR (400 MHz, CDCl3, d) 7.35–7.16 (m, 25 H), 4.92 (d, 1 H,
J = 4.3Hz), 4.80 (d, 1 H, J = 11.3 Hz), 4.68–4.48 (m, 9 H), 4.13 (dd, 1 H, J = 5.9, 6.0
Hz), 4.05–4.00 (m, 3 H), 3.88–3.86 (m, 3 H), 3.70–3.64 (m, 5 H), 3.52 (dd, 1 H,
J = 6.8, 6.8), 3.30 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.21 (dd, 2 H, J = 6.8, 6.8 Hz),
1.68–1.52 (m, 4 H), 1.32–1.24 (m, 8 H); 13C NMR (100 MHz, CDCl3, d) 138.3, 138.1,
137.8, 137.7, 128.5, 128.3 (2), 128.0, 127.8 (2), 127.7 (2), 127.6 (2), 127.5, 100.4, 99.5,
84.2, 83.3, 80.2, 79.5, 75.0, 74.1, 73.4, 72.4 (2), 72.0, 71.2, 69.6, 68.7, 68.2, 68.1, 51.4,
29.4, 29.3, 29.1, 28.8, 26.6, 26.0. HRMS (ESI) Calcd for [C54H65N3O10]Na+: 938.4562.
Found: 938.4597.
8-Azidooctyl 2,3-di-O-benzyl-5-O-[2-O-(2-O-acetyl-3,4,6-tri-O-benzyl- -D-man-
nopyranosyl)-3,4,6-tri-O-benzyl- -D-mannopyranosyl]- -D-arabinofuranoside (32).
Disaccharide 31 (300 mg, 0.32 mmol) was glycosylated with thioglycoside 20 (250
˚
mg, 0.42 mmol) as described for the preparation of 30 using powdered 4 A molecular
sieves (400 mg), N-iodosuccinimide (88 mg, 0.39 mmol) and silver triflate (25 mg,
0.09 mmol) in CH2Cl2 (20 mL). The product was purified by chromatography
(hexanes: EtOAc, 10: 1) to give 32 (360 mg, 79%) as an oil: Rf 0.4 (hexanes/EtOAc,
1
6:1); [a]D + 22.8° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3, d) 7.34–7.19 (m, 40
H), 5.53 (dd, 1 H, J = 1.8, 2.9 Hz), 5.09 (d, 1 H, J = 0.8 Hz), 4.90 (d, 1 H, J = 0.8 Hz),
4.86–4.80 (m, 3 H), 4.68–4.35 (m, 14 H), 4.08–3.88 (m, 9 H), 3.79–3.64 (m, 7 H),
3.45 (dd, 1 H, J = 6.8, 6.8 Hz), 3.28 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.18 (dd, 2 H,
J = 6.8, 6.8 Hz), 2.10 (s, 3 H), 1.57–1.50 (m, 4 H), 1.31–1.26 (m, 8 H); 13C NMR
(100 MHz, CDCl3, d) 170.5, 138.9, 138.7, 138.6, 128.8, 128.7 (2), 128.6 (2), 128.4 (2),
128.3, 128.2 (2), 128.1, 128.0(2), 127.9, 127.7, 100.9, 100.8, 99.9, 84.7, 79.9, 78.6,
75.5, 75.4, 74.9, 74.6, 73.8 (2), 72.5, 72.3, 69.1, 68.4, 51.8, 30.1, 29.9, 29.7, 29.5, 27.1,
26.5, 21.5. HRMS (ESI) Calcd for [C83H95N3O16]Na+: 1412.6604. Found: 1412.6630.
8-Azidooctyl 2,3-di-O-benzyl-5-O-[2-O-(3,4,6-tri-O-benzyl- -D-mannopyra-
nosyl)-3,4,6-tri-O-benzyl- -D-mannopyranosyl]- -D-arabinofuranoside (33). Tri-
saccharide 32 (300 mg, 0.21 mmol) was deacetylated as described for the
preparation of 24 using 1.0 M NaOCH3 in CH3OH (0.5 mL) and CH3OH (10 mL).
The product was purified by chromatography (hexanes/EtOAc, 4:1) to give 33 (271 mg,
1
93%) as an oil: Rf 0.31 (hexanes/EtOAc, 3:1); [a]D + 31.1° (c 1.2, CHCl3); H NMR
(400 MHz, CDCl3, d) 7.50–7.07 (m, 40 H), 5.14 (d, 1 H, J = 1.7 Hz), 4.85 (d, 1 H,
J = 1.6 Hz), 4.83–4.46 (m, 18 H), 4.11–4.01 (m, 5 H), 3.93–3.64 (m, 12 H), 3.45 (dd,
1 H, J = 1.5, 1.9 Hz), 3.29 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.17 (dd, 2 H, J = 6.8, 6.8
Hz), 1.72–1.26 (m, 12 H); 13C NMR (100 MHz, CDCl3, d) 139.0 (2), 138.8, 138.7,
138.6, 138.5, 138.4, 128.9, 128.8 (2), 128.7 (2), 128.4, 128.3 (3), 128.1, 128.0, 127.8
(2), 101.5, 100.9, 99.4, 84.7, 84.0, 80.4, 80.3, 80.0, 75.5, 75.4, 75.2, 75.1, 74.7, 73.8,
72.8 (2), 72.7, 72.6, 72.1, 69.4, 69.0, 68.4, 51.8, 29.9, 29.7, 29.5, 29.2, 27.1, 26.5.
HRMS (ESI) Calcd for [C81H93N3O15]Na+: 1370.6498. Found: 1370.6403.
8-Azidooctyl 5-O-[3,4,6-tri-O-benzyl-2-O-[3,4,6-tri-O-benzyl-2-O-( -D-manno-
pyranosyl)- -D-mannopyranosyl]- -D-mannopyranosyl]-2,3-di-O-benzyl- -D-arabi-
nofuranoside (35). Trisaccharide 33 (200 mg, 0.15 mmol) was glycosylated with
thioglycoside 21 (100 mg, 0.22 mmol), as described for the preparation of 30 using
˚
powdered 4 A molecular sieves (500 mg), N-iodosuccinimide (49 mg, 0.22 mmol) and

Synthesis of Oligosaccharide Fragments
473
silver triflate (10 mg, 0.03 mmol) in CH2Cl2 (15 mL). The product was purified by
chromatography (hexanes/EtOAc, 9:1) to give a tetrasaccharide (34), which was conta-
minated with succinimide. The crude trisaccharide was therefore dissolved in CH3OH
(30 mL) and 1.0 M NaOCH3 in CH3OH (0.5 mL) was added dropwise. After stirring
for 4 h, the reaction mixture was neutralized with Amberlite IR-120 (H+) resin, filtered,
and concentrated. The residue was purified by chromatography (hexanes/EtOAc, 4:1) to
give 35 (180 mg, 82%) as an oil: Rf 0.25 (hexanes/EtOAc, 3:1); [a]D + 22.9° (c 1.1,
1
CHCl3); H NMR (400 MHz, CDCl3, d) 7.32–7.12 (m, 40 H), 5.13 (s, 1 H), 5.06 (s, 1
H), 4.93 (s, 1 H), 4.84 (d, 1 H, J = 4.3 Hz), 4.83–4.39 (m, 20 H), 4.09–4.35 (m, 24 H),
3.28 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.17 (dd, 2 H, J = 6.8, 6.8 Hz), 1.54–1.48 (m, 4
H), 1.28–1.25 (m, 8 H); 13C NMR (100 MHz, CDCl3, d) 139.0, 138.6, 138.5, 138.2,
129.0, 128.8(2), 128.7(2), 128.4, 128.3 (2), 128.2 (2), 128.0, 127.9, 127.8, 101.9, 101.1,
100.9, 99.4, 84.7, 84.0, 80.0, 79.9, 75.6, 75.4, 75.3, 73.8, 73.6, 73.3, 72.8 (2), 72.5,
72.1, 71.5, 70.6, 69.6 (2), 68.4, 66.8, 61.3, 51.8, 29.9, 29.7, 29.5, 29.2, 27.1, 26.5.
HRMS (ESI) Calcd for [C87H103N3O20]Na+: 1532.7027. Found: 1532.7156.
8-Azidooctyl 2-O-acetyl-3,4,6-tri-O-benzyl- -D-mannopyranoside (37). 8-azi-
dooctanol (0.59 g, 3.6 mmol) was glycosylated with 20 (2.5 g, 4.2 mmol) as described
˚
for the preparation of 30 using powdered 4 A molecular sieves (1.0 g),
N-iodosuccinimide (0.9 g, 4.2 mmol) and silver triflate (100 mg, 0.42 mmol) in
CH2Cl2 (50 mL). The product was purified by chromatography (hexanes/EtOAc 10:1)
to yield 37 (2.3 g, 90%) as an oil: Rf 0.46 (hexanes/EtOAc 4:1); [a]D + 58.1° (c 1.0,
1
CHCl3); H NMR (500 MHz, CDCl3, d) 7.41–7.23 (m, 15 H), 5.43 (dd, 1 H, J = 3.2,
1.8 Hz), 4.92 (d, 1 H, J = 10.6 Hz), 4.88 (d, 1 H, J = 1.6 Hz), 4.77 (d, 1 H, J = 10.6
Hz), 4.76 (d, 1 H, J = 10.6 Hz), 4.61–4.53 (m, 3 H), 4.05 (dd, 1 H J = 9.3, 3.4 Hz),
9.94 (dd, 1 H, J = 3.9, 3.9 Hz), 3.88–3.85 (m, 2 H), 3.79–3.70 (m, 2 H), 3.47 (ddd, 1
H, J = 6.6, 6.6, 9.6 Hz), 3.29 (dd, 2 H, J = 6.9, 6.9 Hz), 2.21 (s, 3 H), 1.67–1.59 (m, 4
H), 1.41–1.32 (m, 8 H); 13C NMR (125 MHz, CDCl3, d) 170.9, 138.8, 138.7, 138.4,
128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0 (2), 98.2, 78.8, 75.7, 74.8, 73.8,
72.2, 71.8, 69.4, 69.3, 68.4, 51.9, 29.8, 29.7, 29.5, 29.3, 27.1, 26.5, 21.6. HRMS (ESI)
Calcd for [C37H47N3O7]Na+: 668.3306. Found: 668.3317.
8-Azidooctyl 3,4,6-tri-O-benzyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl- -D-manno-
pyranosyl)- -D-mannopyranoside (38). Alcohol 16 (1.3 g, 2.2 mmol) was glyco-
sylated with 20 (1.5 g, 2.6 mmol) as described for the preparation of 30 using
˚
powdered 4 A molecular sieves (1.0 g), N-iodosuccinimide (0.58 g, 2.6 mmol) and
silver triflate (70 mg, 0.26 mmol) in CH2Cl2 (50 mL). The product was purified by
chromatography (hexanes/EtOAc 10:1) to yield 38 (1.9 g, 86%) as an oil: Rf 0.26
1
(hexanes/EtOAc 3:1); [a]D + 18.9° (c 1.0, CHCl3); H NMR (500 MHz, CDCl3, d)
7.42–7.29 (m, 30 H), 5.63 (dd, 1 H, J = 3.2, 1.8 Hz), 5.17 (d, 1 H, J = 0.8 Hz), 4.96–
4.92 (m, 3 H), 4.79–4.72 (m, 5 H), 4.65–4.48 (m, 5 H), 4.09–4.04 (m, 4 H), 4.01–
3.77 (m, 7 H), 3.68 (ddd, 1 H, J = 3.6, 3.6, 6.6 Hz), 3.37–3.29 (m, 3 H), 2.20 (s, 3 H),
1.68–1.56 (m, 4 H), 1.42–1.34 (m, 8 H); 13C NMR (125 MHz, CDCl3, d) 170.6, 139.0,
138.9, 138.8, 138.4, 128.8 (2), 128.7 (3), 128.6, 128.5, 128.2, 128.1, 128.0 (2), 127.9
(3), 127.8, 100.0, 99.1, 80.3, 78.6, 75.6, 75.5, 75.4, 74.8, 73.8, 73.7, 72.4, 72.3, 72.2,
69.8, 69.6, 69.2, 68.1, 51.8, 29.8, 29.7, 29.5, 29.3, 27.1, 26.5, 21.6. HRMS (ESI) Calcd
for [C64H75N3O12]Na+: 1100.5242. Found: 1100.5243.

474
Gadikota et al.
8-Azidooctyl 3,4,6-tri-O-benzyl-2-O-(3,4,6-tri-O-benzyl- -D-mannopyranosyl)- -
D-mannopyranoside (39). Disaccharide 38 (1.2 g, 1.1 mmol) was deacetylated as
described for the preparation of 16 using 1.0 M NaOCH3 in CH3OH (1 mL) and CH3OH
(10 mL). The product was purified by chromatography (hexanes/EtOAc 10:1) to yield
39 (1.1 g, 98%) as an oil: Rf 0.41 (hexanes/EtOAc 2:1); [a]D + 61.4° (c 1.0, CHCl3); 1H
NMR (500 MHz, CDCl3, d) 7.39–7.24 (m, 30 H), 5.21 (d, 1 H, J = 0.9 Hz), 4.97 (d, 1
H, J = 0.8 Hz), 4.91 (d, 1 H, J = 10.6 Hz), 4.86 (d, 1 H, J = 10.6 Hz), 4.77–4.55 (m, 11
H), 4.19 (dd, 1 H, J = 2.3, 1.8 Hz), 4.09–3.76 (m, 10 H), 3.64 (ddd, 1 H, J = 3.6, 3.6,
6.6 Hz), 3.34–3.27 (m, 3 H), 1.67–1.59 (m, 4 H), 1.41–1.33 (m, 8 H); 13C NMR (125
MHz, CDCl3, d) 138.9, 138.7, 138.4, 128.9, 128.8 (2), 128.7 (3), 128.4, 128.3, 128.2 (2),
128.1, (3), 127.8, 127.7, 101.5, 99.2, 80.5, 80.3, 75.6, 75.5, 75.4, 75.3, 74.9, 73.8, 73.7,
72.7, 72.5, 72.2, 71.9, 69.8, 69.6, 69.0, 68.1, 51.9, 29.8, 29.7, 29.5, 29.3, 27.1, 26.5.
HRMS (ESI) Calcd for [C62H73N3O11]Na+: 1058.5137. Found: 1058.5099.
8-Azidooctyl 3,4,6-tri-O-benzyl-2-O-[3,4,6-tri-O-benzyl-2-O-(2-3,4,6-tetra-O-
acetyl- -D-mannopyranosyl)- -D-mannopyranosyl]- -D-mannopyranoside (40).
Alcohol 39 (0.80 g, 0.77 mmol) was glycosylated with 21 (0.42 g, 0.93 mmol)
˚
as described for the preparation of 30 using powdered 4 A molecular sieves (1.0 g),
N-iodosuccinimide (0.22 g, 0.93 mmol) and silver triflate (100 mg, 0.01 mmol) in
CH2Cl2 (50 mL). The product was purified by chromatography (hexanes/EtOAc 10:1) to
yield 40 (0.88 g, 83%) as an oil: Rf 0.13 (hexanes/EtOAc 1:1); [a]D + 98.5°(c 0.9,
1
CHCl3); H NMR (500 MHz, CDCl3, d) 7.41–7.23 (m, 30 H), 5.49–5.44 (m, 2 H),
5.34–5.24 (m, 2 H), 4.96 (dd, 1 H, J = 1.6, 2.8 Hz), 4.90–4.87 (m, 3 H), 4.76–4.54 (m
10 H), 4.19–4.16 (m, 2 H), 4.06–3.75 (m, 13 H), 3.61 (ddd, 1 H, J = 3.6, 3.6, 6.6 Hz),
3.30–3.26 (m, 3 H), 2.17 (s, 3 H), 2.07 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H), 1.67–1.59 (m,
4 H), 1.41–1.32 (m, 8 H); 13C NMR (125 MHz, CDCl3, d) 170.9, 170.2, 170.1, 170.0,
169.9, 139.0, 138.8 (2), 138.7, 128.9, 128.8 (2), 128.7 (2), 128.5, 128.4, 128.3, 128.1,
128.0 (2), 127.9 (2), 127.8, 127.7, 100.8, 99.6, 99.1, 80.1, 79.6, 76.1, 75.6, 75.4, 75.3,
73.7, 73.5, 72.8, 72.7, 72.6, 72.2, 70.0, 69.9, 69.6, 69.3, 68.1, 66.5, 62.7, 51.9, 29.8, 29.7,
29.5, 29.2, 27.1, 26.5, 21.3, 21.1, 21.0. HRMS (ESI) Calcd for [C76H91N3O20]Na+:
1388.6088. Found: 1388.6099.
8-Azidooctyl 3,4,6-tri-O-benzyl-2-O-[3,4,6-tri-O-benzyl-2-O-( -D-mannopyra-
nosyl)- -D-mannopyranosyl]- -D-mannopyranoside (41). Trisaccharide 40 (500
mg, 0.36 mmol) was deacetylated as described for the preparation of 16 using 1.0 M
NaOCH3 in CH3OH (1 mL) and CH3OH (10 mL) and CH2Cl2 (10 mL). Theproduct
was purified by chromatography (hexanes/EtOAc 10:1) to yield 41 (430 mg, 98%) as
an oil: Rf 0.21 (hexanes/EtOAc 4:1); [a]D + 39.9° (c 1.0, CHCl3);1H NMR (500 MHz,
CDCl3, d) 7.37–7.23 (m, 30 H), 5.17 (s, 1 H), 5.09 (s, 1 H), 4.92 (d, 1 H, J = 0.7 Hz),
4.86 (d, 1 H, J = 10.7 Hz), 4.83 (d, 1 H, J = 10.7 Hz), 4.72 (d, 1 H, J = 10.7 Hz),
4.68–4.48 (m, 8 H), 4.11–4.09 (m, 2 H), 3.99–3.92 (m, 6 H), 3.84–3.52 (m, 14 H),
3.29–3.25 (m, 3 H), 1.64–1.58 (m, 2 H), 1.52–1.50 (m, 2 H), 1.37–1.29 (m, 10 H);
13C NMR (125 MHz, CDCl3, d) 139.0, 138.8, 138.7, 138.6, 128.9, 128.8, 128.7 (3),
128.4, 128.2, 128.1 (2), 127.9 (2), 127.8 (2), 101.8, 101.1, 99.1, 80.0, 78.9, 75.8, 75.5,
75.4 (4), 73.7, 73.6, 73.1, 72.8, 72.7, 72.5, 72.2, 72.1, 69.8, 69.7, 68.1, 67.3, 61.6, 51.8,
29.8, 29.6, 29.4, 29.3, 27.1, 26.4. HRMS (ESI) Calcd for [C68H83N3O16]Na+:
1220.5665. Found: 1220.5618.

Synthesis of Oligosaccharide Fragments
475
8-Azidooctyl 5-O-[3,5-O-(1,1,3,3-tetraisopropylsiloxane-1,3-diyl)- -D-arabino-
furanosyl]-2,3-di-O-benzyl- -D-arabinofuranoside (42). Alcohol 15 (1.5 g, 3.1
mmol) was glycosylated with thioglycoside 17 (2.5 g, 3.91 mmol), as described for
˚
the preparation of 30 using powdered 4 A molecular sieves (1.0 g), N-iodosuccinimide
(1.04 g, 4.6 mmol) and silver triflate (150 mg, 0.58 mmol) in CH2Cl2 (30 mL). The
product was purified by chromatography (hexanes/EtOAc, 15:1) to yield the product as
an oil, which was contaminated with a p-thiocresol-based impurity. Therefore, a
solution of the crude product in CH2Cl2 (25 mL) and CH3OH (75 mL) was treated with
0.1 M NaOCH3 in CH3OH (2 mL). After stirring for 4 h, the reaction mixture was
neutralized with prewashed Amberlite IR-120 (H+) resin, filtered, and concentrated.
The resulting residue was purified by chromatography (hexanes/EtOAc, 6:1) to give 42
1
(1.99 g, 72%) as an oil: Rf 0.28 (hexanes/EtOAc, 6:1); [a]D + 31.2°(c 1.1, CHCl3; H
NMR (400 MHz, CDCl3, d) 7.36–7.23 (m, 10 H), 5.01 (s, 1 H), 4.95 (d, 1 H, J = 0.8
Hz), 4.14–3.68 (m, 16 H), 3.25 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.20 (dd, 2 H, J = 6.8,
6.8 Hz), 1.59–1.55 (m, 4 H), 1.31–1.28 (m, 8 H) 1.10–1.00 (m, 28 H); 13C NMR (100
MHz, CDCl3, d) 138.8, 137.7, 129.4, 128.9, 128.8, 128.6, 128.5, 128.4, 128.1 (2),
125.7, 108.4, 106.4, 88.6, 83.3, 82.5, 81.6, 80.9, 76.4, 72.5, 72.4, 67.8, 67.4, 61.7, 51.8,
31.0, 29.8, 29.5, 29.2, 27.0, 26.4, 18.4 (2), 17.8, 17.7, 17.5 (2), 17.4, 13.9, 13.5, 13.4,
13.2, 12.9. HRMS (ESI) Calcd for [C44H71N3O10]Na+: 880.4570. Found: 880.4573.
8-Azidooctyl 5-O-(2-O-benzyl- -D-arabinofuranosyl)-2,3-di-O-benzyl- -D-arabi-
nofuranoside (43). Disaccharide 42 (1.8 g, 2. 0 mmol) was benzylated with benzyl
bromide (0.4 mL, 2.6 mmol) and NaH (120 mg, 5. 0 mmol) in DMF (10 mL) as
described for the preparation of 27. Following workup, the crude product was dissolved
in THF (75 mL) and the solution was cooled to 0°C before n-Bu4NF (4.8 mL of a 1.0
M solution in THF, 4.8 mmol) was added under an argon atmosphere. The reaction
mixture was stirred for 2 h and then concentrated; the product was purified by chro-
matography (hexane/EtOAc, 5:1) to give 43 (880 mg, 62%) as an oil: Rf 0.21 (hexanes/
1
EtOAc, 3:1); [a]D + 11.3° (c 0.9, CHCl3); H NMR (400 MHz, CDCl3, d) 7.37–7.25
(m, 15 H), 5.12 (s, 1 H), 4.99 (s, 1 H), 4.59–4.39 (m, 6 H), 4.19–4.16 (m, 1 H), 4.10–
3.64 (m, 10 H), 3.35 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.22 (dd, 2 H, J = 6.8, 6.8 Hz),
1.60–1.54 (m, 4 H), 1.35–1.31 (m, 8 H); 13C NMR (100 MHz, CDCl3, d) 138.0, 137.7,
137.4, 128.9 (2), 128.8, 128.5, 128.4 (2), 128.2 (2), 106.4, 105.5, 87.8, 87.4, 87.2, 84.0,
80.8, 75.6, 72.4, 72.3, 72.0, 67.9, 66.1, 63.0, 51.8, 29.8, 29.6, 29.5, 29.2, 27.0, 26.4.
HRMS (ESI) Calcd for [C39H51N3O9]Na+: 728.3517. Found: 728.3469.
8-Azidooctyl 5-O-[3,5-di-O-(2-O-benzoyl-3,5-di-O-benzyl- -D-arabinofura-
nosyl)-2-O-benzyl- -D-arabinofuranosyl]-2,3-di-O-benzyl- -D-arabinofuranoside
(44). Disaccharide 43 (800 mg, 1.13 mmol) was glycosylated with thioglycoside 18
˚
(1.6 g, 2.96 mmol), as described for the preparation of 30 using powdered 4 A
molecular sieves (1.0 g), N-iodosuccinimide (660 mg, 2.94 mmol) and silver triflate (87
mg, 0.33 mmol) in CH2Cl2 (20 mL). The product was purified by chromatography
(hexanes/EtOAc, 6:1) to give 44 (1.41 g, 81%) as an oil: Rf 0.31 (hexanes/EtOAc, 4:1);
[a]D + 42.3° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, d) 7.94–7.90 (m, 4 H),
7.35–7.17 (m, 41 H), 5.47 (s, 1 H), 5.36 (s, 1 H), 5.31 (s, 2 H), 5.15 (s, 1 H), 5.00 (s, 1
H), 4.78 (d, 1 H, J = 11.3 Hz), 4.71 (d, 1 H, J = 11.3 Hz), 4.60–4.30 (m, 17 H), 4.15–
3.87 (m, 6 H), 3.70 (dd, 1 H, J = 5.9, 2.6 Hz), 3.62–3.52 (m, 7 H), 3.36 (ddd, 1 H,

476
Gadikota et al.
J = 6.8, 6.8, 1.6 Hz), 3.26 (dd, 2 H, J = 6.8, 6.8 Hz), 1.58–1.51 (m, 4 H), 1.30–1.20
(m, 8 H); 13C NMR (100 MHz, CDCl3, d) 165.7 (2), 138.6 (2), 138.3, 138.2, 130.3,
130.2, 128.8 (3), 128.7 (2), 128.4, 128.3 (2), 128.2, 128.1, 128.0 (3), 106.8, 106.7,
106.5, 106.0, 89.1, 88.7, 84.0, 83.6, 82.9, 82.7, 82.6, 82.1, 80.6, 73.8 (2), 72.8, 72.5 (2),
72.4, 72.3, 69.8, 69.5, 68.1, 51.9, 30.0, 29.7, 29.5, 29.3, 27.1, 26.5. HRMS (ESI) Calcd
for [C91H99N3O19]Na+: 1561.6798. Found: 1561.6824.
8-Azidooctyl 5-O-[3,5-di-O-(3,5-di-O-benzyl- -D-arabinofuranosyl)-2-O-benzyl-
-D-arabinofuranosyl]-2,3-di-O-benzyl- -D-arabinofuranoside (45). Tetrasaccharide
44 (1.35 g, 0.88 mmol) was debenzoylated as described for the preparation of 24 using 1.0
M NaOCH3 in CH3OH (0.5 mL) in CH2Cl2 (15 mL) and CH3OH (10 mL). The product
was purified by chromatography (hexanes/EtOAc, 3:1) to give 45 (980 mg, 85%) as an oil:
Rf 0.29 (hexanes/EtOAc, 2:1); [a]D + 17.6° (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3,
d) 7.30–7.21 (m, 35 H), 5.13 (s, 1 H), 5.07 (s, 1 H), 5.04 (s, 1 H), 4.97 (s, 1 H), 4.60–3.80
(m, 29 H), 3.68–3.66 (m, 3 H), 3.55–3.54 (m, 2 H), 3.44–3.36 (m, 2 H), 3.27 (ddd, 1 H,
J = 6.8, 6.8, 1.6 Hz), 3.20–3.16 (m, 3 H), 1.58–1.54 (m, 4 H), 1.30–1.26 (m, 8 H); 13C
NMR (100 MHz, CDCl3, d) 138.8, 138.3 (2), 137.9 (2), 128.9 (2), 128.8, 128.7, 128.4 (2),
128.3 (2), 128.2 (3), 128.1, 128.0 (2), 109.6, 107.6, 106.5, 106.4, 85.0, 83.5 (2), 82.8, 80.3,
78.9, 78.8, 74.0, 72.7, 72.5, 72.3, 72.2, 70.2, 70.1, 68.0, 66.1, 51.8, 29.9, 29.6, 29.5, 29.2,
27.0, 26.4. HRMS (ESI) Calcd for [C77H91N3O17]Na+: 1352.6240. Found: 1352.6174.
8-Azidooctyl 5-O-{3,5-di-O-[2-O-(2,3,5-tri-O-benzyl- -D-arabinofuranosyl)-3,5-
di-O-benzyl- -D-arabinofuranosyl]-2-O-benzyl- -D-arabinofuranosyl]-2,3-di-O-ben-
zyl- -D-arabinofuranoside (46). Tetrasaccharide 45 (500 mg, 0.37 mmol) and
thioglycoside 19 (593 mg, 1.12 mmol) were dissolved in CH2Cl2 (30 mL) and
˚
powdered 4 A molecular sieves (2.0 g) were added. The reaction mixture was cooled to
À 78°C and stirred for 20 min before the addition of N-iodosuccinimide (25 mg, 1.1
mmol) and silver triflate (28 mg, 0.10 mmol). After being stirred for 2 h at À 78°C,
Et3N was added until the solution changed from red to yellow and then the reaction
mixture was diluted with CH2Cl2 and filtered through Celite. The filtrate was washed
in succession with a saturated aqueous solution of Na2S2O3, water, and brine, before
being dried (Na2SO4) and concentrated. Chromatography of the resulting residue
(hexanes/EtOAc, 10:1) gave 46 (65 mg, 81%) as an oil: Rf 0.45 (hexanes/EtOAc, 4:1);
1
[a]D + 26.2° (c 0.9, CHCl3); H NMR (400 MHz, CDCl3, d) 7.29–7.16 (m, 65 H), 5.11
(s, 2 H), 5.09–5.07 (m, 2 H), 4.95 (d, 1 H, J = 4.8 Hz), 4.93 (s, 1 H), 4.61–4.05 (m, 45
H), 3.99–3.51 (m, 12 H), 3.21 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.19 (dd, 2 H, J = 6.8,
6.8 Hz), 1.56–1.54 (m, 4 H), 1.28–1.26 (m, 8 H); 13C NMR (100 MHz, CDCl3, d)
138.7 (2), 138.2, 138.1, 128.3 (3), 128.7 (2), 128.6, 128.4, 128.2 (2), 128.1 92), 128.0
(2), 127.9, 107.0, 106.6 106.5, 105.7, 100.7, 100.4, 89.0, 86.4, 86.2, 84.6, 84.5 (2),
84.4, 83.6, 83.5 (2), 82.1, 81.6, 80.6, 80.5, 80.4, 80.3, 73.7 (2), 73.5, 73.4, 72.7 (2),
72.6, 72.4 (2), 72.2, 70.4, 70.3, 68.0, 66.4, 65.8, 51.8, 29.9, 29.7, 29.5, 29.2, 27.1, 26.5.
HRMS (ESI) Calcd for [C129H143N3O25]Na+: 2157.9936. Found: 2157.9967.
8-Aminooctyl 5-O-{3,5-di-O-(2-O-[2,3,5-tri-O-benzyl- -D-arabinofuranosyl]-3,5-
di-O-benzyl- -D-arabinofuranosyl)-2-O-benzyl- -D-arabinofuranosyl]-2,3-di-O-ben-
zyl- -D-arabinofuranoside (47). To a solution of 46 (300 mg, 0.14 mmol) in
THF:water (10 mL, 10:1) was added Ph3P (73 mg, 0.27 mmol) at 0°C. The reaction

Synthesis of Oligosaccharide Fragments
477
mixture was stirred for 10 h while warming to rt and then concentrated to an oil, which
was purified by chromatography (CH2Cl2/CH3OH, 10:1) to give 47 (235 mg, 78%) as
1
an oil: Rf 0.20 (CH2Cl2/CH3OH, 8:1); [a]D + 33.1°(c 1.1, CHCl3); H NMR (400 MHz,
CDCl3, d) 7.35–7.10 (m, 65 H), 5.16 (s, 1 H), 5.15 (s, 1 H), 5.11–5.10 (m, 2 H), 4.98
(d, 1 H, J = 4.8 Hz), 4.96 (s, 1 H), 4.62–4.30 (m, 34 H), 4.09–3.88 (m, 16 H), 3.67–
3.47 (m, 12 H), 3.32 (ddd, J = 6.8, 6.8, 1.6 Hz), 3.16 (dd, 2 H, J = 6.8, 6.8 Hz), 1.52–
1.27 (m, 12 H); 13C NMR (100 MHz, CDCl3, d) 138.8 (2), 138.6, 138.3, 129.0, 128.9
(3), 128.8, 128.7, 128.5 (2), 128.4, 128.3, 128.2 (2), 128.1 (2), 128.0, 107.2, 106.6 (2),
105.9, 100.9, 100.6, 89.1, 84.6, 84.5, 83.7, 83.6(3), 73.8, 73.6, 73.5, 72.8(2), 72.7 (2),
72.6, 72.5 (2), 72.3, 70.5, 68.1, 66.4, 42.3, 30.9, 29.9, 29.8, 28.1, 27.3, 26.6. HRMS
(ESI) Calcd for [C129H146N3O25]Na+: 2110.0212. Found: 2110.0141.
8-Trifluoroacetamidooctyl 5-O-(3,5-di-O-(2-O-( -D-arabinofuranosyl)- -D-ara-
binofuranosyl)- -D-arabinofuranosyl}- -D-arabinofuranoside (48). To a solution
of 47 (200 mg, 0.09 mmol) in pyridine (5 mL) was added trifluoroacetic anhydride
(0.04 mL, 2.8 mmol). The reaction mixture was stirred for 24 h, then diluted with
CH2Cl2, washed with water, and concentrated. Purification of the product by
chromatography (CH2Cl2/CH3OH, 10:1) gave an oil, which was immediately dissolved
in CH3OH (10 mL) and then 10% Pd/C (30 mg) was added. The solution was stirred
overnight under a H2 atmosphere and then the catalyst was filtered away. The filtrate
was concentrated to a residue, which was purified by chromatography on latrobeads
(CH2Cl2/CH3OH, 1:1) to give 48 (64 mg, 65%) as an oil: Rf 0.12 (CH2Cl2/
1
CH3OH,5:1)); [a]D + 7.1° (c 0.4, H2O); H NMR (400 MHz, D2O, d) 5.16 (s, 1 H),
5.12 (s, 1 H), 5.07 (d, 1 H, J = 4.8 Hz), 5.02–5.01 (m, 2 H), 4.92 (s, 1 H), 4.20–3.49
(m, 32 H), 3.25 (ddd, 1 H, J = 6.8, 6.8, 1.6 Hz), 3.21 (dd, 2 H, J = 6.8, 6.8 Hz), 1.52–
1.49 (m, 4 H), 1.25–1.18 (m, 8 H); 13C NMR (100 MHz, D2O, d) 179.0, 107.5, 106.0,
105.8, 101.1, 100.9, 87.4, 87.1, 83.2, 83.1, 82.9, 82.3, 82.1, 81.9, 81.6, 79.5, 76.6, 75.0,
74.5, 74.4, 68.9, 63.3, 63.2, 60.9, 40.1, 28.9, 28.6, 28.5, 28.0, 26.1, 25.4. HRMS (ESI)
Calcd for [C40H66F3NO26]Na+: 1056.3717. Found: 1056.3672.
ACKNOWLEDGMENTS
This work was supported by The National Institutes of Health. CSC’s contributions
to this work were supported by an American Chemical Society Division of Organic
Chemistry Fellowship sponsored by Aventis Pharmaceuticals.
REFERENCES
1. Brennan, P.J.; Nikaido, H. The envelope of mycobacteria. Ann. Rev. Biochem.
1995, 64, 29.
2. 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; 2005–2080.
3. Chatterjee, D.; Khoo, K.H. Mycobacterial lipoarabinomannan: an extraordinary
lipoheteroglycan with profound physiological effects. Glycobiology 1998, 8, 113.

478
Gadikota et al.
4. Knutson, K.L.; Hmama, Z.; Herrera-Velit, P.; Rochford, R.; Reiner, N.E.
Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine
dephosphorylation and inhibition of mitogen-activated protein kinase in human
mononuclear phagocytes. J. Biol. Chem. 1998, 273, 645.
5. Sibley, L.D.; Hunter, S.W.; Brennan, P.J.; Krahenbuhl, J.L. Mycobacterial lipo-
arabinomannan inhibits gamma interferon-mediated activation of macrophages.
Infect. Immun. 1998, 56, 1232.
6. Chan, J.; Fan, X.; Hunter, S.W.; Brennan, P.J.; Bloom, B.R. Lipoarabinomannan, a
possible virulence factor involved in persistence of Mycobacterium tuberculosis
with macrophages. Infect. Immun. 1991, 59, 1755.
7. Roach, T.I.A.; Barton, C.H.; Chatterjee, D.; Blackwell, J.M. Macrophage
activation—lipoarabinomannan from avirulent and virulent-strains of Mycobacte-
rium tuberculosis differentially induces the early genes FOS, KC, JE, and tumor
necrosis-factor-a. J. Immunol. 1993, 150, 1886.
8. Yoshida, A.; Koide, Y. Arabinofuranosyl-terminated and mannosylated lipoarabi-
nomannans from Mycobacterium tuberculosis induce different levels of interleukin-
12 expression in murine macrophages. Infect. Immun. 1997, 65, 1953.
9. Zhang, Y.; Broser, M.; Cohen, H.; Bodkin, M.; Law, K.; Reibman, J.; Rom, W.M.
Enhanced interleukin-8 release and gene expression in macrophages after exposure
to Mycobacterium tuberculosis and its components. J. Clin. Invest. 1995, 95, 586.
10. Chang, J.C.; Wysocki, A.; Tchou-Wong, K.M.; Moskowitz, N.; Zhang, Y.; Rom,
W.N. Effect of Mycobacterium tuberculosis and its components on macrophages
and the release of matrix metalloproteinases. Thorax 1996, 51, 306.
11. Sieling, P.A.; Chatterjee, D.; Porcelli, S.A.; Prigozy, T.I.; Mazzaccaro, R.J.;
Soriano, T.; Bloom, B.R.; Brenner, M.B.; Kronenberg, M.; Brennan, P.J.; Modlin,
R.L. CD1-restricted T-cell recognition of microbial lipoglycan antigens. Science
1995, 269, 227.
12. Porcelli, S.A.; Morita, C.T.; Modlin, R.L. T-cell recognition of non-peptide
antigens. Curr. Opin. Immunol. 1996, 8, 510.
13. Besra, G.S.; Morehouse, C.B.; Rittner, C.M.; Waechter, C.J.; Brennan, P.J.
Biosynthesis of mycobacterial lipoarabinomannan. J. Biol. Chem. 1997, 272,
18460.
14. Schlesinger, L.S.; Hull, S.R.; Kaufman, T.M. Binding of the terminal mannosyl
units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to
human macrophages. J. Immunol. 1994, 52, 4070.
15. Schlesinger, L.S.; Kaufman, T.M.; Iyer, S.; Hull, S.R.; Marchiando, L.K.
Differences in mannose receptor-mediated uptake of lipoarabinomannan form
virulent and attenuated strains of Mycobacterium tuberculosis by human
macrophages. J. Immunol. 1996, 157, 4568.
16. Kaur, D.; Lowary, T.L.; Vissa, V.D.; Crick, D.C.; Brennan, P.J. Characterization
of the epitope of anti-lipoarabinomannan antibodies as the terminal hexaarabino-
furanosyl motif of mycobacterial arabinans. Microbiology UK 2002, 148, 3059.
17. Hamasur, B.; Kallenius, G.; Svenson, S.B. Synthesis and immunologic character-
isation of Mycobacterium tuberculosis lipoarabinomannan specific oligosaccharide-
protein conjugates. Vaccine 1999, 17, 2853.
18. Kosma, P.; Reiter, A.; Hofinger, A.; Brade, L.; Brade, H. Synthesis of

Synthesis of Oligosaccharide Fragments
479
neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci
lipopolysaccharides. J. Endotoxin Res. 2000, 6, 57.
19. Kamath, V.P.; Diedrich, P.; Hindsgaul, O. Use of diethyl squarate for the coupling
of oligosaccharide amines to carrier proteins and characterization of the resulting
neoglycoproteins by MALDI-TOF mass spectrometry. Glycoconj. J. 1996, 13, 315.
20. Bovin, N.V.; Korchagina, E.Y.; Zemlyanukhina, T.V.; Byramova, N.E.; Galanina,
O.E.; Zemlyakov, A.E.; Ivanov, A.E.; Zubov, V.P.; Mochalova, L.V. Synthesis of
polymeric neoglycoconjugates based on N-substituted polyacrylamides. Glycoconj.
J. 1993, 10, 142.
21. D’Souza, F.W.; Ayers, J.D.; McCarren, P.R.; Lowary, T.L. Arabinofuranosyl
oligosaccharides from mycobacteria: synthesis and effect of glycosylation on ring
conformation and hydroxymethyl group rotamer populations. J. Am. Chem. Soc.
2000, 122, 1251.
22. Gadikota, R.R.; Callam, C.S.; Wagner, T.; Del Fraino, B.; Lowary, T.L.
Anhydrosugars in glycoside bond synthesis. Highly stereocontrolled synthesis of
oligosaccharides containing a- and b-arabinofuranosyl linkages. J. Am. Chem. Soc.
2003, 125, 4155.
23. Yin, H.; D’Souza, F.W.; Lowary, T.L. Arabinofuranosides from mycobacteria:
synthesis of a highly branched hexasaccharide and related fragments containing
b-arabinofuranosyl residues. J. Org. Chem. 2002, 67, 892.
24. Zhang, Y.M.; Mallet, J.M.; Sina¨y, P. Glycosylation using a one-electron-transfer,
homogeneous reagent—application to an efficient synthesis of the trimannosyl core
of N-glycosylproteins. Carbohydr. Res. 1992, 236, 73.
25. Dasgupta, F.; Garegg, P.J. Synthesis of ethyl and phenyl 1-thio-1,2-trans-D-
glucopyranosides from the corresponding per-O-acetylated glycopyranoses having
a 1,2-trans configuration using ferric chloride as a promoter. Acta Chem. Scand.
1989, 43, 471.
26. Fletcher, H.G. The anomeric tri-O-benzoyl-D-arabinofuranosyl bromides. Methods
Carbohydr. Chem. 1963, 2, 228.
27. Biogegrain, R.; Castro, B.; Selve, C. Alkoxyphosphonium salts. XIII. Selective
monoactivation of symmetric biprimary 1,n-diols in nucleophilic substitution
reactions. Tetrahedron Lett. 1975, 2529.
28. Kartha, K.P.R.; Field, R.A. Iodine and its interhalogen compounds: versatile
reagents in carbohydrate chemistry V. Synthesis of 1,2-trans-linked 1-thioglyco-
sides from the per-O-acetylated glycoses. J. Carbohydr. Chem. 1998, 17, 693.
29. Mizutani, K.; Kasai, R.; Nakamura, M.; Tanaka, O.; Matsuura, H. NMR spectral
study of a-L-arabinofuranosides and b-L-arabinofuranosides. Carbohydr. Res. 1989,
185, 27.
30. Subramaniam, V.; Lowary, T.L. Synthesis of oligosaccharide fragments of
mannosylated lipoarabinomannan from Mycobacterium tuberculosis. Tetrahedron
1999, 55, 5965.
31. Bock, K.; Pedersen, C. Carbon-13-hydrogen coupling constants in hexopyranoses.
J. Chem. Soc., Perkin Trans. 2 1974, 293.
32. D’Souza, F.W.; Lowary, T.L. The first total synthesis of a highly branched
arabinofuranosyl hexasaccharide found at the nonreducing termini of mycobacterial
arabinogalactan and lipoarabinomannan. Org. Lett. 2000, 2, 1493.

480
Gadikota et al.
33. Appelmelk, B.J.; Lowary, T.L.; Hokke, C.H.; Kolk, A.H.J.; Nigou, J.; Gadikota,
R.R.; Maaskant, J.J.; Kewalapat, V.; Van Heijst, J.; Vandenbroucke-Grauls,
C.M.J.E. Fifth International Conference on the Pathogenesis of Mycobacterial
Infections, Stockholm, Sweden, June, 2002.
Received October 25, 2002
Accepted February 5, 2003