Synthesis of branched Man5 oligosaccharides and an unusual stereochemical observation

Article abstract of DOI:10.1021/jo7013824

(Chemical Equation Presented) Branched mannopentaoses were synthesized through two routes. While assembly from the nonreducing end to the reducing end was more convergent with fewer intermediate steps, two diastereomers were obtained. On the other hand, s

Full text of DOI:10.1021/jo7013824

processing, and tumor migration.18-23 Therefore, efficient
syntheses of branched oligomannoses can provide much needed
materials to facilitate their biological studies.
Synthesis of Branched Man5 Oligosaccharides
and an Unusual Stereochemical Observation
Nardos Teumelsan and Xuefei Huang*
There are two general approaches to assemble branched oligo-
mannoses such as Man5 1. With the first approach, synthesis
is carried out from the nonreducing end to the reducing end
(route a in Figure 1).9,23-28 R-1,2-Linked dimannoside 4 will
be synthesized first with the R linkage typically controlled by
a participating neighboring group. The dimannoside will then
be used in glycosylation of a diol acceptor 5 to produce penta-
saccharide 8. With a mannosyl moiety already installed on the
axially oriented O-2 of mannoside 4, one cannot rely on
neighboring group participation to control stereochemistries of
the two newly formed linkages in 8. The R linkages should be
favored due to the anomeric effect as well as steric hindrance
posed by the mannoside moiety on the axial O-2. As an
alternative, Man5 can be assembled from the reducing end to
the nonreducing end (route b in Figure 1).8,29-34 For this
approach, a selectively removable protective (PG) group such
as acetate must be installed on O-2 of the mannosyl donor 2.
Upon formation of branched trisaccharide 6, the protective group
will be removed to generate a diol trisaccharide acceptor 7.
Repeating the double glycosylation with mannosyl donor 2 will
lead to pentasaccharide 8. Stereochemical outcome of the
glycosylations can be controlled by neighboring group participa-
Department of Chemistry, The UniVersity of Toledo, 2801 West
Bancroft Street, MS 602, Toledo, Ohio 43606
xuefei.huang@utoledo.edu
ReceiVed June 26, 2007
Branched mannopentaoses were synthesized through two
routes. While assembly from the nonreducing end to the
reducing end was more convergent with fewer intermediate
steps, two diastereomers were obtained. On the other hand,
synthesis from the reducing end to the nonreducing end
yielded the mannopentaose with the desired stereochemistry
as a single isomer. Our results suggest that it is still
challenging to reliably predict stereochemical outcome of a
glycosylation reaction. It is necessary to thoroughly char-
acterize anomeric configurations of newly formed glycosidic
linkages in complex oligosaccharide synthesis.
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HIV is one of the most devastating modern diseases. The
availability of an effective anti-HIV vaccine may be a good
approach to prevent this worldwide epidemic.1-4 One of the
promising targets for immunogen design is the carbohydrate
moieties on HIV-1 envelop protein gp120,5-9 as validated by a
potent neutralizing human monoclonal antibody 2G12.10-13
The epitope structures of 2G12 have recently being character-
ized to be a series of branched oligomannose residues, as
represented by mannopentaose (Man5) 1.11-13 Oligomannoses
are also primary targets for cyanovirin-N (CVN),14-16 an
example of HIV envelop binding lectins.17 Besides their
relationship to HIV, branched oligomannoses are integral
structural components of asparagine-linked glycan (N-glycan),
one of the major types of postsynthetic protein modifications.18
N-Glycans are involved in many fundamental biological pro-
cesses such as cell differentiation, viral infection, nascent protein
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10.1021/jo7013824 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/16/2007
8976
J. Org. Chem. 2007, 72, 8976-8979

FIGURE 1. Two general approaches for the synthesis of branched mannopentaoses.
tion through this route. Although both of these approaches have
been applied to the assembly of branched oligomannosides,
except for a few examples,28-31 stereochemical proofs of the
newlyformedglycosidiclinkagesareoftennotpresented.8,9,23-27,32-34
Synthesis of the diol acceptor 11 started from the known
azidopropyl R-mannoside 1249,50 (Scheme 1). Selective protec-
tion of the 3 and 6 hydroxyl groups led to disilyl ether 13 in
66% yield.11 Benzylation of 13 followed by removal of the tert-
butyldimethylsilyl (TBDMS) ethers gave acceptor 11. The
presence of free 3 and 6-OH in 11 was ascertained by NMR
analysis of its acetylation product.
The majority of glycosylation reactions were performed by
adding a promoter to a mixture of glycosyl donor and acceptor.35
Alternatively, a glycosyl donor can be preactivated in the ab-
sence of an acceptor.36-42 This can often lead to unique
stereochemical outcomes36,41 and chemoselectivities.37-40 Re-
cently, we adapted the preactivation scheme into a chemose-
lective glycosylation method, where a thioglycosyl donor is
preactivated in the absence of the acceptor.43-46 Upon adding
a thioglycosyl acceptor, nucleophilic addition of the acceptor
to the activated donor yields a disaccharide product containing
a thioaryl aglycon, which can be directly activated for the next
round of glycosylation. Because donor activation and addition
of acceptor are performed in two distinct steps, the anomeric
reactivity of the donor does not need to be higher than that of
the acceptor37,38,43-47 as required by the traditional reactivity-
based armed-disarmed chemoselective glycosylation.48 We
envision that we can apply our preactivation-based method to
convergently assemble Man5 using building blocks 9,43 10,43
and 11.
SCHEME 1. Synthesis of Diol Acceptor 11
Preactivation of thiomannosyl donor 9 by promoter p-
TolSOTf,41,43,51 formed in situ through reaction of p-TolSCl and
AgOTf, was followed by addition of acceptor 10 and a sterically
bulky base 2,4,6-tri-tert-butylpyrimidine (TTBP),52 leading to
disaccharide 14 in 63% yield (Scheme 2). The structure of 14
was confirmed by one-bond coupling constants between the
anomeric carbon and hydrogen atoms (1JCH ) 172.5 and 171.5
Hz).53,54 In addition, the presence of carbonyl groups in 14 was
determined by 13C NMR (δ ) 170.4 ppm). The anomeric
reactivity of donor 9 is lower than that of acceptor 10 due to
the presence of acetate on O-2 of 9. With the traditional armed-
disarmed chemoselective glycosylation approach,48 direct gly-
cosylation of acceptor 10 by donor 9 was not possible.9,11
Instead, the thioglycosyl donor had to be hydrolyzed into a
hemiacetal followed by conversion into trichloroacetimidate
donor.11 Selective activation of the imidate donor over the
thioglycosyl acceptor then gave thioglycosyl disaccharide. The
usage of the preactivation procedure enables us to bypass the
(35) Wang, Y.; Ye, X.-S.; Zhang, L.-H. Org. Biomol. Chem. 2007, 5,
2189-2200 and references cited therein.
(36) Kim, J.-H.; Yang, H.; Park, J.; Boons, G.-J. J. Am. Chem. Soc. 2005,
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T.; Yoshida, J.-I. Chem.sEur. J. 2005, 11, 6159-6194.
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Romagnoli, B. Tetrahedron 2003, 59, 7983-7996.
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326.
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4589-4614.
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2001, 40, 414-417.
(41) Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321-8348.
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Soc. 1989, 111, 6881-6882.
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Ed. 2004, 42, 5221-5224.
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J. Org. Chem, Vol. 72, No. 23, 2007 8977

SCHEME 2. Synthesis of Man5 from the Nonreducing End
to the Reducing End
pentamannoside 15R in 60% yield, which was found to have
identical polarity, NMR, and molecular weight as 15R synthe-
sized from the nonreducing end to the reducing end (Scheme
2). No 15â was obtained through this route, demonstrating that
anomeric configurations of 15R are stable under the reaction
conditions.
Deprotection of pentamannoside 15R was quite straightfor-
ward (Scheme 3b). Removal of the two acetates by sodium
methoxide followed by Staudinger reduction of the azido moiety
and catalytic hydrogenation46 produced fully deprotected Man5
18 in 75% overall yield for the three steps. The aminopropyl
moiety at the reducing end can be utilized for future biocon-
jugation and the development of anti-HIV vaccine studies.
The fact that the major product synthesized from the
nonreducing end to the reducing end direction is 15â is
surprising considering that (1) reaction is carried out in diethyl
ether, an ethereal solvent well-known to favor the generation
of thermodynamically more stable, axial glycoside product,55-57
and (2) the anomeric effect and the steric hindrance posed by
the bulky mannoside moiety on the axial O-2 of disaccharide
14 should disfavor the equatorial glycoside. Crich and co-
workers have developed a powerful methodology for â-man-
noside formation using 4,6-benzylidene-protected mannosyl
donors.41,47 R-Mannosyl triflate was identified as the resting state
of the reactive intermediate,58 with SN2-like displacement of
the anomeric triflate through an exploded transition state leading
to the â product.59 Benzylidene moiety was found to be crucial
to conformationally lock the pyranoside ring into chair confor-
mation, disfavoring oxacarbenium ion formation, while electron-
withdrawing protective groups on O-2 were shown to have a
similar effect.60 The generation of Man5 15â in our synthesis
may be rationalized by the formation of R-mannosyl triflate
intermediate 19 upon activation of disaccharide 14. With the
presence of the carbohydrate moiety on O-2, which can be
viewed as an electron-withdrawing61 nonparticipating group, the
R-mannosyl triflate intermediate 19 may be stabilized. The O-2
mannosyl unit on 19 can rotate around the O-2 and C-2 bond,
with the large pyranoside ring pointing away from the anomeric
position. This can reduce the steric hindrance of the â face posed
by the pyranoside ring, allowing formation of the â linkage.
The formation of R linkage may be disfavored due to the
mismatch62 of 19 with the acceptor at the transition state.63
SCHEME 3. Synthesis of Man5 from the Reducing End to
the Nonreducing End
need for aglycon transformation, thus simplifying synthetic
designs and improving overall synthetic efficiencies.
With disaccharide 14 prepared, double glycosylation of diol
acceptor 11 by 14 was performed with the p-TolSCl/AgOTf
promoter system (Scheme 2). Two compounds were formed in
a ratio of 3:7 with slight polarity difference shown by TLC.
After careful silica gel chromatography separation, to our
surprise, the two products were found to have identical
molecular weights corresponding to pentamannoside, indicating
that they are diastereomers. The less polar minor component
1
15R was obtained in 14% yield with JCH between anomeric
carbons and hydrogens determined to be 169.9, 170.7, 170.9,
171.6, and 171.6 Hz, confirming that all five glycosidic linkages
are R.53,54 The more polar major products 15â (33%) have 1JCH
between anomeric carbons and hydrogens of 162.4, 162.9, 172.8,
173.6, and 173.6 Hz, suggesting that both of the newly formed
glycosidic linkages are â! Glycosylation of acceptor 11 by 14
using N-iodosuccinimide and triflic acid as the promoter11 failed
to yield any pentamannosides in our hands. Instead, multiple
decomposition products were observed presumably due to acid
sensitivity of the oligosaccharide products.
Our results suggest that factors controlling stereochemistry
of glycosylation especially in the absence of neighboring group
(55) Chiba, H.; Funasaka, S.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 2003,
76, 1629-1644.
(56) Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M. Tetrahedron
Lett. 2002, 43, 5573-5577.
In order to confirm our structure assignment, we investigated
the alternative route for Man5 construction (Scheme 3a). Double
glycosylation of diol 11 by donor 9 provided trimannan 16 in
60-63% yield. 13C NMR of 16 has two resonances at 170.4
and 170.6 ppm, and 1JCH values between anomeric protons and
carbons are 169.5, 172.1, and 172.1 Hz, proving it is not an
orthoester and both new glycosidic linkages are R. Removal of
the acetates with NaOMe produced trisaccharide diol 17 in 77%
yield. Double glycosylation of diol 17 by donor 9 led to
(57) Wulff, G.; Rohle, G. Angew. Chem., Int. Ed. 1974, 13, 157-170.
(58) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997, 119, 11217-11223.
(59) Crich, D.; Chandrasekera, S. Angew. Chem., Int. Ed. 2004, 43,
5386-5389.
(60) Crich, D.; Picione, J. Org. Lett. 2003, 5, 781-784.
(61) Zhang, Z.; Ollman, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong,
C.-H. J. Am. Chem. Soc. 1999, 121, 734-753.
(62) Fraser-Reid, B.; Lo´pez, J. C.; Go´mez, A. M.; Uriel, C. Eur. J. Org.
Chem. 2004, 1387-1395.
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30, 180-183.
8978 J. Org. Chem., Vol. 72, No. 23, 2007

6H), 3.87-3.94 (m, 5H), 3.97-4.03 (m, 4H), 4.21-4.26 (m, 3H),
4.32-4.43 (m, 7H), 4.44-4.65 (m, 15H), 4.66-4.75 (m, 2H),
4.77-4.85 (m, 4H), 5.06 (s, 1H), 5.13-5.15 (m, 1H), 5.50 (s, 1H),
5.57 (d, J ) 1.8 Hz, 1H), 7.08-7.34 (m, 70H); 13C NMR (150
MHz, CDCl3) δ 21.40, 21.43, 22.9, 28.8, 29.9, 30.5, 48.5, 64.5,
68.5, 68.8, 69.0, 69.07, 69.09, 69.5, 69.7, 71.5, 72.0, 72.08, 72.14,
72.21, 72.28, 72.33, 72.5, 72.8, 73.4, 73.52, 73.55, 74.2, 73.6, 74.5,
74.84, 74.87, 74.93, 75.04, 75.24, 75.26, 75.32, 75.9, 76.0, 77.0,
77.2, 77.5, 77.7, 78.3, 78.8, 82.5, 97.0 (1JH1,C1 ) 162.4 Hz), 99.1
participation are still not well understood. Although assembly
of branched oligomannosides from the direction of the nonre-
ducing end to the reducing end is a popular approach, caution
needs to be taken regarding stereochemical outcome of the
reaction. Anomeric effect and steric hindrance are not depend-
able control of stereochemistry. For the development of a
reliable and general automated glycosylation method, stereo-
chemical control remains a significant challenge.
(1JH1,C1 ) 173.6 Hz), 99.5 (1JH1,C1 ) 173.6 Hz), 100.9 (1JH1, C1
)
162.9 Hz), 101.1 (1JH1, C1 ) 172.8 Hz), 127.26, 127.36, 127.39,
127.55, 127.61, 127.65, 127.67, 127.69, 127.72, 127.76, 127.82,
127.86, 127.89, 127.94, 127.97, 128.09, 128.11, 128.18, 128.20,
128.25, 128.28, 128.32, 128.34, 128.38, 128.39, 128.44, 128.48,
128.49, 128.50, 128.54, 128.56, 128.58, 128.61, 128.63, 128.66,
128.69, 128.75, 128.82, 128.86, 138.17, 138.23, 138.35, 138.38,
138.42, 138.45, 138.57, 138.61, 138.7, 138.8, 139.1, 170.3, 170.4.
MS (ESI) m/z calcd for C135H145N3NaO28 [M + Na]+ 2279.0; found
2279.8. HRMS: m/z calcd for C135H145N3NaO28 [M +Na]+
2278.9912; found 2278.9900. 15R: [R]25D +17 (c ) 1.0, CH2Cl2);
1H NMR (600 MHz, CDCl3) δ 1.64-1.66 (m, 2H), 2.10 (s, 6H),
3.18-3.34 (m, 4H), 3.45-3.49 (t, J ) 11.4 Hz, 2H), 3.58-3.67
(m, 8H), 3.77-3.81 (m, 4H), 3.84-3.85 (m, 2H), 3.88-4.07 (m,
12H), 4.24-4.27 (d, J ) 10.8 Hz, 1H), 4.34-4.47 (m, 8H), 4.50-
4.69 (m, 15H), 4.73 (s, 1H), 4.78-4.87 (m, 4H), 4.93 (s, 1H), 5.06-
Experimental Section
3-Azidopropyl 2-O-acetyl-3,4,6-tri-O-benzyl-D-mannopyra-
nosyl-R-(1f2)-3,4,6-tri-O-benzyl-D-mannopyranosyl-R-(1f3)-
(2-O-acetyl-3,4,6-tri-O-benzyl-D-mannopyranosyl-R-(1f2)-3,4,6-
tri-O-benzyl-D-mannopyranosyl-R-(1f6))-2,4-di-O-benzyl-R-D-
mannopyranoside (15R) and 3-Azidopropyl 2-O-acetyl-3,4,6-tri-
O-benzyl-D-mannopyranosyl-R-(1f2)-3,4,6-tri-O-benzyl-D-
mannopyranosyl-â-(1f3)-(2-O-acetyl-3,4,6-tri-O-benzyl-D-
mannopyranosyl-R-(1f2)-3,4,6-tri-O-benzyl-D-mannopyranosyl-
â-(1f6))-2,4-di-O-benzyl-R-D-mannopyranoside (15â). Donor 14
(0.180 g, 0.175 mmol) and acceptor 11 (0.4 equiv, 0.033 g, 0.070
mmol) were azeotropically dried over toluene together three times
and mixed with freshly activated molecular sieves (MS 4 Å) (200
mg) in diethyl ether (5 mL). The mixture was stirred at room
temperature for 30 min and cooled to -78 °C, which was followed
by addition of TTBP (0.043 g, 0.175 mmol) and a solution of
AgOTf (0.135 g, 0.53 mmol, 3 equiv) dissolved in Et2O (2 mL)
without touching the wall of the flask. After 5 min, orange colored
p-TolSCl (25 µL, 0.175 mmol) was added through a microsyringe.
Since the reaction temperature was lower than the freezing point
of p-TolSCl, p-TolSCl was added directly into the reaction mixture
to prevent it from freezing on the flask wall. The characteristic
yellow color of p-TolSCl in the reaction dissipated rapidly within
a few seconds, indicating depletion of p-TolSCl. The reaction
mixture was warmed to -20 °C under stirring over 90 min. The
reaction was quenched with triethylamine, diluted with CH2Cl2 (30
mL), and filtered over Celite. The Celite was further washed with
CH2Cl2 until no organic compounds were observed in the filtrate
by TLC. All CH2Cl2 solutions were combined and washed twice
with saturated aqueous solution of NaHCO3 (20 mL) and twice
with water (10 mL). The organic layer was collected and dried
over Na2SO4. After removal of the solvent, the yellowish residue
was purified by silica gel chromatography (toluene/CH2Cl2/acetone
5.06 (m, 2H), 5.16 (s, 1H), 5.51 (s, 2H), 7.09-7.32 (m, 70H); 13
C
NMR (150 MHz, CDCl3) δ 21.4, 29.0, 48.5, 64.5, 66.7, 68.4, 68.8,
69.0, 69.1, 69.8, 71.6, 72.0, 72.11, 72.19, 72.25, 72.9, 73.4, 73.56,
73.60, 74.2, 74.4, 74.6, 74.7, 74.8, 74.9, 75.1, 75.3, 75.4, 77.0, 77.3,
77.6, 77.8, 78.0, 78.3, 78.4, 79.4, 79.9, 97.2 (1JH1, C1 ) 169.9 Hz),
99.3 (1JH1, C1 ) 170.9 Hz), 99.5 (1JH1, C1 ) 171.6 Hz), 99.8 (1JH1, C1
) 171.6 Hz), 101.4 (1JH1, C1 ) 170.7 Hz), 127.2, 127.5, 127.70,
127.78, 127.84, 127.95, 127.99, 128.02, 128.29, 128.42, 128.47,
128.49, 128.55, 128.59, 128.71, 138.24, 138.27, 138.37, 138.44,
138.46, 138.51, 138.63, 138.72, 138.78, 138.80, 170.37, 170.40.
HRMS: m/z calcd for C135H145N3NaO28 [M +Na]+ 2278.9912;
found 2278.9949.
Acknowledgment. We are grateful for financial support from
the University of Toledo, the National Institutes of Health (R01-
GM-72667), and a Research Supplement to Promote Diversity
in Health-Related Research (R01 GM72667-S1).
Supporting Information Available: Experimental procedures
for synthesis of compounds 11, 14, 15R, 16, 17, and 18. Selected
1H, 13C, and 2D NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
) 1.5:0.02:0.02) to give pentasaccharides 15R (22 mg) and 15â
1
(51.5 mg) in 47% yield. 15â: [R]25 +16 (c ) 1.0, CH2Cl2); H
D
NMR (600 MHz, CDCl3) δ 1.20-1.24 (m, 3H), 1.51-1.52 (m,
2H), 2.07 (s, 3H), 2.09 (s, 3H), 3.30-3.34 (m, 2H), 3.42-3.44
(m, 1H), 3.49-3.61 (m, 7H), 3.65-3.72 (m, 4H), 3.78-3.84 (m,
JO7013824
J. Org. Chem, Vol. 72, No. 23, 2007 8979