A linear synthesis of branched high-mannose oligosaccharides from the HIV-1 viral surface envelope glycoprotein gp120

Article abstract of DOI:10.1002/1099-0690(200203)2002:5<826::AID-EJOC826>3.0.CO;2-2

Described is a linear solution-phase synthesis of the HIV-1 viral surface envelope glycoprotein gp120 high-mannose nonasaccharide pentyl glycoside. Envisioning the automated solid-phase assembly of complex carbohydrates, the synthesis of the nonasaccharide and the related tri- and hexamannosides demonstrates the facile assembly of highly branched structures in a stepwise fashion incorporating monosaccharide building blocks. A differentially protected core trisaccharide was prepared and further elongated in two high-yielding tri-mannosylations to furnish the triantennary structure. The tri-, hexa-, and nonamannoside n-pentyl glycosides obtained via the described synthesis are currently being used for detailed study of the carbohydrate protein interactions responsible for binding of the anti-HIV protein cyanovirin-N to the glycoprotein gp120. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200203)2002:5<826::AID-EJOC826>3.0.CO;2-2

FULL PAPER
A Linear Synthesis of Branched High-Mannose Oligosaccharides from the
HIV-1 Viral Surface Envelope Glycoprotein gp120
Daniel M. Ratner,[a] Obadiah J. Plante,[a] and Peter H. Seeberger*[a]
Keywords: High-Mannose / Nonasaccharide / Cyanovirin / Oligosaccharides / HIV
Described is a linear solution-phase synthesis of the HIV-1
high-yielding tri-mannosylations to furnish the triantennary
viral surface envelope glycoprotein gp120 high-mannose structure. The tri-, hexa-, and nonamannoside n-pentyl gly-
nonasaccharide pentyl glycoside. Envisioning the automated
solid-phase assembly of complex carbohydrates, the syn-
cosides obtained via the described synthesis are currently
being used for detailed study of the carbohydrate protein in-
thesis of the nonasaccharide and the related tri- and hexa- teractions responsible for binding of the anti-HIV protein cy-
mannosides demonstrates the facile assembly of highly anovirin-N to the glycoprotein gp120.
branched structures in a stepwise fashion incorporating
monosaccharide building blocks. A differentially protected
core trisaccharide was prepared and further elongated in two
(
Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
the development of potential HIV preventatives and pos-
sibly even therapeutics. Access to synthetically derived man-
Carbohydrates play a vital role in HIV retroviral patho- nosides as synthetic tools has therefore become particularly
genesis. The function of HIV-1 surface envelope glycopro- important in order to probe CVN-gp120 binding.
tein gp120 in helper T lymphocyte (Th) infection has been
The total synthesis of high-mannose type cell surface gly-
understood by biologists for some time.[1,2] Glycoprotein cans, that are found throughout nature as N-linked glyco-
gp120 is highly glycosylated, containing up to 24 N-linked conjugates, has been explored for the past two decades.[9Ϫ12]
high-mannose carbohydrates, which compose 50% of the A synthesis of the high-mannose core structures of Man9,
molecular mass of the glycoprotein.[3] Gp120 mediates viral isolated from calf thyroglobul, was reported by Ogawa in
fusion with the CD4 receptor on the surface of Th cells. 1981; a decade before the role of such structures in HIV
HIV fusion and subsequent lymphocyte infection occur pathogenesis was discovered.[9] Two successful and highly
upon binding of the glycoprotein and the CD4 receptor.
convergent syntheses of the HIV-1 nonamannoside struc-
A novel protein has been recently discovered that exhibits ture by Fraser-Reid[13] and Ley[14Ϫ16] were recently com-
potent anti-HIV activity. Isolated from the blue-green algae pleted. However, the convergent nature of these solution-
Nostoc elliposporum, cyanovirin-N (CVN) was found to phase syntheses does not allow for their application to
bind gp120, inactivating HIV, and preventing lymphocyte automated solid-phase synthesis.
infection.[2,4Ϫ7] CVN is a novel, monomeric, 11-kDa viruci-
Herein we describe a linear synthesis of the pentyl non-
dal protein. Both natural and recombinant forms of the amannoside 2 and the related hexamannoside 3 and trim-
protein have been shown to irreversibly inactivate a wide annoside 4 structures (Figure 1). The synthetic strategy was
variety of HIV strains while exhibiting minimal toxicity to planned and executed with automated solid-phase synthesis
host cells. The mode of HIV inactivation by CVN has been in mind. Using three monomer building blocks the non-
studied and the protein’s affinity for the gp120 high-man- amannoside was assembled in four glycosylation events.
nose structure Man9 1 was established (Figure 1).[8] This af- Two sequential tri-mannosylation reactions, with a single
finity appears to be the mechanism through which CVN mannosyl donor, allowed for access to the completed struc-
prevents gp120 from interacting with the CD4 receptor of ture in a minimal number of steps. The target structures
the host lymphocyte. A detailed understanding of the spe- currently serve as tools in biophysical studies focusing on
cific interaction between CVN and Man9 1 could lead to the elucidation of cyanovirin-N-binding to branched high-
mannose structures.
[a]
Department of Chemistry, Massachusetts Institute
of Technology,
18-211, 77 Massachusetts Ave., Cambridge, MA 02139, USA
Results and Discussion
Fax: (internat.) ϩ 1-617/258-7247
E-mail: seeberg@mit.edu
Our retrosynthetic analysis of Man9 analog 2 (Scheme 1)
was guided by our long-term goal of developing a synthetic
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
826
WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0305Ϫ0826 $ 17.50ϩ.50/0 Eur. J. Org. Chem. 2002, 826Ϫ833

Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120
FULL PAPER
Figure 1. High-mannose oligosaccharide targets
strategy that could be applied to the solid support and even-
tually automated. We planned to obtain the fully protected
nonamannoside 5 via simultaneous tri-mannosylation of a
hexasaccharide triol derived from 6. This hexasaccharide
would in turn be prepared by the tri-mannosylation of a
trisaccharide core triol ensuing from 7. The differentially
protected trisaccharide 7 would be constructed from three
protected monosaccharide building blocks 8, 9, and 10. The
stepwise nature of this strategy would facilitate access to a
triply branched nonasaccharide using just four glycosyl-
Scheme 1. Retrosynthesis of nonamannoside 5
ations. A solution-phase synthesis of the β-pentenyl glyco- may allow for attachment of the products of our synthetic
side analog of the natural structure was selected for our studies to a linker, fluorescent tag, or biomarker.
studies, based on our strategy for solid phase oligosaccha-
Initially, reliable synthetic access to the monosaccharide
ride synthesis that furnishes the pentenyl glycoside upon building blocks had to be established. The core 3,6-differen-
cleavage from the solid-support.[17] In addition to func- tially protected β-mannoside 8 was the most synthetically
tioning as glycosyl donors, n-pentenyl glycosides are versa- challenging building block to be procured. Since glycals
tile handles, that can be converted into a range of functional have proven to be useful intermediates in oligosaccharide
groups by transformation of the terminal olefin to a car- assembly[21Ϫ22] we utilized glycals for the preparation of β-
boxylic acid, aldehyde, ester, thioether, thioester, or hy- mannoside 8. The synthesis of 8 commenced with benzyl-
droxyl group.[18Ϫ20] In this fashion the n-pentenyl moiety ation of known glycal 11[23] to yield differentially protected
Eur. J. Org. Chem. 2002, 826Ϫ833
827

D. M. Ratner, O. J. Plante, P. H. Seeberger
FULL PAPER
Scheme 3. Assembly of core trimannoside 7
donor 9 afforded disaccharide 15 (Scheme 3). Removal of
the C6 silyl ether was accomplished in high yield using
aqueous trifluoroacetic acid in THF to furnish disaccharide
acceptor 16. Installation of the second mannose building
block on C6 that will serve as root for branches D2 and D3
Scheme 2. Synthesis of the core β-mannoside 8
glucal 12 (Scheme 2). Stereospecific epoxidation of glycal required the use of a mannose donor that did not utilize a
12 by treatment with dimethyldioxirane, followed by open- C2 participating ester group but rather a permanently
ing of the 1,2-anhydrosugar with 4-penten-1-ol in the pres- blocked benzyl ether. Nevertheless, complete α-selectivity
ence of zinc chloride yielded β-glucoside 13 containing an was achieved in the TBDMSOTf catalyzed glycosylation of
unprotected C2 hydroxyl group. Inversion of the C2 ste- disaccharide acceptor 16 with mannosyl donor 10 to yield
reocenter was achieved via an oxidation-reduction se- the fully-protected core trisaccharide 7. Precedence for α-
quence. Oxidation of the C2 hydroxyl under selectivity has been established previously in the case of
PfitznerϪMoffatt conditions (Ac2O/DMSO) was followed similar mannosyl donors containing non-participating pro-
by stereoselective reduction with sodium borohydride and tecting groups on C2.[26Ϫ27] Treatment of trisaccharide 7
benzylation to furnish the fully protected β-mannoside 8. with sodium methoxide accomplished the simultaneous
Selective removal of the halobenzyl ether was accomplished cleavage of one C2 acetate and two benzoates to provide
by palladium-catalyzed amination with N-methyl aniline trisaccharide triol 17 in good yield (89%).
followed by treatment with dichloroacetic acid to afford
Simultaneous extension of the D1, D2, and D3 branches
from the core trisaccharide was accomplished via two se-
monosaccharide acceptor 14.[23]
At this stage only two other mannosyl monosaccharide quential tri-mannosylations with donor 9 (Scheme 4). Man-
building blocks were required for the completion of the nosylation of 17 with donor 9 (4.5 equivalents) yielded
nonamannoside; one with a temporary protecting group on hexasaccharide 6 in a single step in 94% yield. Deprotection
C2 (9), and the other incorporating temporary protecting with sodium methoxide produced hexasaccharide triol 18.
groups on C3 and C6 (10). The C3 hydroxyl of the reducing Trimannosylation of 18 using 9 furnished the fully pro-
end mannose is the point of attachment of a linear strand tected high-mannose nonasaccharide 5 in 80% yield. In just
of α-(1Ǟ2) linked mannoses (branch D1). The C6 hydroxyl three steps the initial trisaccharide 7 tripled in size to nona-
of the core n-pentenyl-β-mannoside is the origin of a 3,6- saccharide 5 with 75% overall yield. Liberation of 5 from
differentiated α-mannose that serves as the core for two all protecting groups in two steps by treatment with sodium
branches (D2 and D3) of the triantennary structure (Fig- methoxide to afford triol 19, followed by Pd-catalyzed hy-
ure 1). Differentially protected mannopyranosyl trichlo- drogenation yielded the desired high-mannose pentyl gly-
roacetimidate 9[24] as well as mannosyl donor 10[25] were coside 2 (Man9). Fully deprotected hexasaccharide 3 and
prepared using known procedures. Synthesized on multi core trisaccharide 4 pentyl glycosides were prepared in sim-
gram scale, 9 was about to serve as the source of seven of ilar fashion from 17 and 18 (Scheme 4). The three high-
the nine mannoses in the final Man9 structure.
mannose oligosaccharides 2, 3, and 4 are currently used in
With the three monosaccharide building blocks 8, 9, and biophysical studies to better understand the carbohydrate
10 in hand the assembly of larger structures began. We first protein interactions between cyanovirin-N and gp120. Re-
focused on the preparation of core trimannoside 7. The α- sults of these studies are expected to elucidate the formation
selective mannosylation of the C3 hydroxyl of 14 with of very tight carbohydrate-protein interactions that form
828
Eur. J. Org. Chem. 2002, 826Ϫ833

Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120
FULL PAPER
Scheme 4. Assembly of nonasaccharide 6
neutral alumina and copper(II) oxide columns prior to use. Analyt-
ical thin-layer chromatography was performed on E. Merck silica
gel 60 F254 plates (0.25 mm). Compounds were visualized by dip-
ping the plates in a cerium sulfate-ammonium molybdate solution,
followed by heating. Liquid chromatography was performed using
forced flow of the indicated solvent on Silicycle Inc. silica gel
(230Ϫ400 mesh). 1H NMR spectra were obtained on either a
Bruker Avance 400 (400 MHz) or Varian VXR-500 (500 MHz) and
are reported in parts per million (δ) relative to chloroform (δ ϭ
the basis for novel HIV preventatives and possibly consti-
tute a general principle for protein-carbohydrate interac-
tions.
Conclusion
In summary, we have completed a linear synthesis of the
high-mannose nonasaccharide pentyl glycoside 2, and the
trimannoside 4 and hexamannoside 3 structures, using just 7.26). Coupling constants (J) are reported in Hz. 13C NMR spectra
were recorded on either a Bruker Avance 400 (100 MHz) or a
Varian VXR-500 (125 MHz) and are reported in δ relative to
CDCl3 (δ ϭ 77.23) as an internal reference. IR spectra were ob-
tained on a PerkinϪElmer 1600 series FTIR spectrometer. ESI
Mass spectrometry was performed on a Bruker Daltonics Apex 3
Tesla Fourier Transform Mass Spectrometer. MALDI-TOF mass
spectra were obtained on a PerSpective Biosystems Voyager Elite
DE Spectrometer using 2,5-dihydroxybenzoic acid or α-cyano-4-
hydroxycinnamic acid as the matrix.
three monomeric glycosyl building blocks. Construction of
triantennary nonasaccharide 2 was achieved in four high-
yielding glycosidation events; construction of a differenti-
ated core trisaccharide followed by two sequential triman-
nosylations. Access to these synthetic structures has permit-
ted further study of the anti-HIV micobicide activity exhib-
ited by cyanovirin-N. The strategy described here for the
solution phase assembly of three large oligosaccharides
constitutes the basis for the solid phase and eventually the
automated synthesis of branched high-mannose motifs.
1,5-Anhydro-4-O-benzyl-3-O-(4-bromobenzyl)-2-deoxy-6-O-tri-
isopropylsilyl-D-arabino-hex-1-enitol (12): 1,5-anhydro-3-O-(4-bro-
mobenzyl)-2-deoxy-6-O-triisopropylsilyl--arabino-hex-1-enitol 11
(7.80 g, 16.5 mmol) was dissolved in DMF (125 mL) and cooled
on an ice-bath to 0 °C. Sodium hydride (0.80 g, 60% in mineral oil,
20 mmol) was carefully added to the solution, and stirred for
Experimental Section
All commercial materials were used without further purification, 20 min at 0 °C. Benzyl bromide (3.39 g, 19.9 mmol) was added to
unless otherwise noted. Dichloromethane (CH2Cl2) and diethyl the reaction mixture and slowly warmed to room temperature for
ether (Et2O) were purchased from J. T. Baker (CycletainerTM) and 2 h. Methanol (3 mL) was slowly added to quench the reaction,
passed through neutral alumina columns prior to use. Toluene was
which was further diluted in 150 mL water. The solution was ex-
tracted with diethyl ether (3 ϫ 300 mL). After concentration in
purchased from J. T. Baker (CycletainerTM) and passed through
Eur. J. Org. Chem. 2002, 826Ϫ833
829

D. M. Ratner, O. J. Plante, P. H. Seeberger
FULL PAPER
vacuo the resulting residue was purified by flash column chroma- 7.17Ϫ7.29 (m, 7 H), 7.36 (d, J ϭ 8.4 Hz, 2 H). 13C NMR (CDCl3):
tography on silica gel (2Ǟ5% EtOAc/hexanes) to afford 8.60 g δ ϭ 12.4, 18.4, 29.1, 30.7, 63.0, 68.9, 69.1, 71.0, 74.3, 75.6, 76.8,
(93%) of 12 as a clear oil. [α]2D4 ϭ Ϫ19.6 (c ϭ 1.1, CH2Cl2). IR
82.0, 99.8, 115.2, 122.0, 128.2, 128.5, 128.9, 129.9, 131.9, 137.5,
1
(thin film): ν˜ ϭ 942, 2865, 1647, 1240, 1101, 682 cmϪ1. H NMR
138.5, 138.8. ESI MS m/z (M ϩ Naϩ) calcd. 685.2530, found
(CDCl3): δ ϭ 1.07Ϫ1.10 (m, 21 H), 3.93Ϫ4.07 (m, 4 H), 4.18Ϫ4.20 685.2539. 4-Pentenyl 4-O-benzyl-3-O-(4-bromobenzyl)-6-O-triiso-
(m, 1 H), 4.52 (d, J ϭ 12.2 Hz, 1 H), 4.59 (d, J ϭ 11.9 Hz, 1 H), propylsilyl-β--mannopyranoside (1.0 g, 1.5 mmol) was azeotrop-
4.78Ϫ4.85 (m, 3 H), 6.41 (dd, J ϭ 1.5, 6.1 Hz, 1 H), 7.20Ϫ7.22 (d, ically dried with toluene (3 ϫ 3 mL) and dissolved in DMF
J ϭ 8.2 Hz, 2 H), 7.30Ϫ7.36 (m, 5 H), 7.44Ϫ7.46 (d, J ϭ 8.2 Hz, (15 mL). The solution was cooled to 0 °C and sodium hydride
2 H). 13C NMR (CDCl3): δ ϭ 12.2, 18.2, 18.2, 62.0, 70.0, 74.1, (90 mg, 60% in mineral oil, 1.81 mmol) was carefully added and
74.2, 76.0, 78.3, 99.5, 121.6, 128.0, 128.1, 128.6, 129.5, 137.7, 138.6, the mixture was warmed to room temperature. Benzyl bromide (214
145.1. ESI MS m/z (M ϩ Naϩ) calcd. 583.1849, found 583.1838.
µL, 1.81 mmol) was added to the solution, and stirred for 2 h. The
reaction was diluted with diethyl ether (100 mL), washed with
water (100 mL), followed by extraction of the combined aqueous
phase with diethyl ether (50 mL). The combined organic phase was
washed with sat. aqueous NaHCO3 (100 mL), water (100 mL),
brine (100 mL), dried (Na2SO4), and concentrated to give a thick
oil in vacuo. The residue was purified by flash column chromatog-
raphy on silica gel (5 Ǟ 20% EtOAc/hexanes) to afford 1.17 g (98%)
of 8. [α]2D4 ϭ Ϫ54.0° (c ϭ 1.2, CH2Cl2). IR (thin film): ν˜ ϭ 2940,
4-Pentenyl 4-O-Benzyl-3-O-(4-bromobenzyl)-6-O-triisopropylsilyl-β-
D-glucopyranoside (13): Glucal 12 (1.82 g, 3.23 mmol) was dissolved
in CH2Cl2 (6 mL) and cooled to 0 °C. A 0.08  solution of di-
methyldioxirane in acetone (48.5 mL, 3.88 mmol) was added and
the reaction was stirred for 15 min. After the solvent was removed
the remaining residue was dried in vacuo for 1.5 h and sub-
sequently dissolved in CH2Cl2 (10 mL). The solution was cooled to
Ϫ78 °C followed by the addition of 4-penten-1-ol (1.61 mL,
16.15 mmol). A 1.0  solution of ZnCl2 in diethyl ether (3.55 mL,
3.55 mmol) was added and the reaction was warmed slowly to
room temperature and stirred over 16 h. The reaction was diluted
with EtOAc (200 mL) and washed with sat. aqueous NaHCO3 (2
ϫ 100 mL), water (2 ϫ 100 mL), and brine (2 ϫ 100 mL) and dried
(Na2SO4). The organic phase was concentrated in vacuo and the
resulting residue purified by flash column chromatography on silica
gel (15% EtOAc/hexanes) to afford 1.86 g (87%) of 13 as a clear
oil. [α]2D4 ϭ Ϫ20.8 (c ϭ 1.3, CH2Cl2). IR (thin film): ν˜ ϭ 456, 2941,
2865, 1115, 1069, 689 cmϪ1. 1H NMR (CDCl3): δ ϭ 0.89Ϫ1.07 (m,
21 H), 1.61Ϫ1.68 (m, 2 H), 2.02Ϫ2.08 (m, 2 H), 2.27Ϫ2.28 (m, 1
H), 3.21Ϫ3.25 (m, 1 H), 3.51Ϫ3.60 (m, 4 H), 3.76Ϫ3.92 (m, 3 H),
4.15 (d, J ϭ 7.5, 1 H), 4.63 (d, J ϭ 11.6, 1 H), 4.79 (d, J ϭ 12.5,
1 H), 4.82 (d, J ϭ 11.6, 1 H), 4.81 (d, J ϭ 11.4, 1 H), 4.89Ϫ4.98
(m, 2 H), 5.69Ϫ5.79 (m, 1 H), 7.16Ϫ7.26 (m, 7 H), 7.36 (d, J ϭ
6.5 Hz, 2 H). 13C NMR (CDCl3): δ ϭ 12.2, 18.2, 29.0, 30.5, 62.6,
69.2, 74.4, 75.1, 75.3, 76.4, 77.4, 84.6, 102.6, 115.1, 121.7, 128.0,
128.1, 128.7, 129.8, 131.7, 138.0, 138.3, 138.5. ESI MS m/z (M ϩ
Naϩ) calcd. 685.2530, found 685.2532.
1
2865, 1454, 1361, 1107, 1070, 696 cmϪ1. H NMR (CDCl3): δ ϭ
1.04Ϫ1.16 (m, 21 H), 1.72Ϫ1.79 (m, 1 H), 2.18 (m, 2 H), 3.28Ϫ3.32
(m, 1 H), 3.43Ϫ3.51 (m, 2 H), 3.89Ϫ4.05 (m, 5 H), 4.68 (d, J ϭ
11.0 Hz, 1 H), 4.85Ϫ5.08 (m, 5 H), 5.82Ϫ5.92 (m, 1 H), 7.17 (d,
J ϭ 8.4 Hz, 2 H), 7.28Ϫ7.49 (m, 12 H). 13C NMR (CDCl3): δ ϭ
12.2, 18.2, 29.2, 30.6, 63.3, 69.1, 70.7, 73.7, 74.0, 75.0, 75.4, 77.4,
82.6, 102.0, 115.1, 121.7, 127.5, 127.9, 128.2, 128.4, 128.6, 129.4,
131.6, 137.6, 138.5, 138.7, 139.1. ESI MS m/z (M ϩ Naϩ) calcd.
775.3000, found 775.3020.
4-Pentenyl 2,4-Di-O-benzyl-6-O-triisopropylsilyl-β-D-mannopyrano-
side (14): Mannoside 8 (0.51 g, 0.67 mmol) was azeotropically dried
with toluene (3 ϫ 3 mL) then for 1 h in vacuo. The residue was
dissolved in toluene (2 mL) followed by the addition of N-methyl
aniline (86 mg, 0.805 mmol). An oven-dried Schlenk flask was
evacuated and backfilled with argon (5 ϫ). The flask was charged
with Pd2(dba)3 (12.3 mg, 2 mol %), (o-biphenyl)P(tBu)2 (4 mol %),
and NaOtBu (0.090 g, 0.94 mmol), evacuated and backfilled with
argon (5 ϫ). A rubber septum was installed and the aryl bromide/
amine solution was added via cannula. The flask was sealed using
a Teflon screwcap and the reaction mixture was heated to 80 °C
with stirring. After 18 h, the reaction was cooled to room temper-
4-Pentenyl 2,4-Di-O-benzyl-3-O-(4-bromobenzyl)-6-O-triisopropylsi-
lyl-β-D-mannopyranoside (8): Glucoside 13 (0.56 g, 0.85 mmol) was ature, diluted with diethyl ether (20 mL), filtered through a pad of
azeotropically dried with toluene (3 ϫ 3 mL) and dissolved in di- celite, and concentrated in vacuo. The crude product was purified
methyl sulfoxide (3.5 mL). Acetic anhydride (1.75 mL) was added
and the reaction was allowed to stir 24 h at room temperature.
After the solvent was removed in vacuo, addition of CH2Cl2
by flash column chromatography on silica gel (2 Ǟ 5% EtOAc/
hexanes) to yield 0.483 g (92%) of 4-pentenyl 4-O-benzyl-3-O-[4-
(N-methyl-N-phenylamino)benzyl]-6-O-triisopropylsilyl-β--
(20 mL) was followed by washing with water (2 ϫ 20 mL) and dry- mannopyranoside. [α]2D4 ϭ Ϫ52.6 (c ϭ 1.5, CH2Cl2). IR (thin film):
ing of the organic phase (Na2SO4). After concentration in vacuo
the residue was dissolved in 1:1 CH2Cl2/MeOH (10 mL) and cooled
ν˜ ϭ 2940, 2865, 1595, 1497, 1105, 1067, 696 cmϪ1
.
1H NMR
(CDCl3): δ ϭ 1.10Ϫ1.16 (m, 21 H), 1.71Ϫ1.79 (m, 2 H), 2.15Ϫ2.21
to 0 °C. NaBH4 (0.161 g, 4.25 mmol) was slowly added and the (m, 2 H), 3.29Ϫ3.35 (m, 4 H), 3.43Ϫ3.49 (m, 1 H), 3.55 (dd, J ϭ
reaction was stirred 16 h at room temperature. CH2Cl2 (100 mL) 3.0, 9.4 Hz, 1 H), 3.90Ϫ4.06 (m, 5 H), 4.40 (s, 1 H), 4.47 (d, J ϭ
was added and the organic phase was washed with water (100 mL), 11.5 Hz, 1 H), 4.54 (d, J ϭ 11.5 Hz, 1 H), 4.67 (d, J ϭ 10.9 Hz, 1
1% aqueous citric acid (2 ϫ 100 mL), sat. aqueous NaHCO3 H), 4.89Ϫ5.09 (m, 5 H), 5.82Ϫ5.93 (m, 1 H), 6.97Ϫ7.08 (m, 4 H),
(100 mL), brine (100 mL), and dried (Na2SO4). The organic phase 7.23Ϫ7.37 (m, 13 H), 7.51 (d, J ϭ 6.9 Hz, 2 H). 13C NMR (CDCl3):
was dried in vacuo to give a clear oil and purified by flash column δ ϭ 12.2, 18.2, 29.2, 30.6, 40.5, 63.4, 69.0, 71.5, 73.7, 74.1, 75.0,
chromatography on silica gel (6Ǟ7% EtOAc/hexanes) to afford 75.4, 82.6, 102.0, 115.1, 120.6, 120.9, 121.6, 127.4, 127.8, 128.2,
0.42 g (74%) of the desired 4-pentenyl 4-O-benzyl-3-O-(4-
128.4, 128.5, 129.2, 129.4, 131.0, 138.5, 138.9, 139.3, 148.9, 149.3.
bromobenzyl)-6-O-triisopropylsilyl-β--mannopyranoside. [α]2D4
ϭ
ESI MS m/z (M ϩ Naϩ) calcd. 802.4479, found 802.4473. The
˜
Ϫ32.0 (c ϭ 1.3, CH2Cl2). IR (thin film): ν ϭ 3439, 2940, 2864, aminated product (68 mg, 0.088 mmol) was dissolved in CH2Cl2
1638, 1105, 1069, 683 cmϪ1. 1H NMR (CDCl3): δ ϭ 0.97Ϫ1.05 (m, (3 mL) followed by the addition of dichloroacetic acid (72 µL,
2 H), 1.60Ϫ1.67 (m, 21 H), 2.04 (m, 2 H), 2.26 (d, J ϭ 2.9 Hz, 1
0.88 mmol), resulting in a transparent blue color. The reaction was
H), 3.15Ϫ3.19 (m, 1 H), 3.40Ϫ3.46 (m, 2 H), 3.78Ϫ3.92 (m, 4 H), stirred for 30 min at room temperature then diluted with CH2Cl2
3.98 (m, 1 H), 4.32 (d, J ϭ 0.9 Hz, 1 H), 4.53- 4.68 (m, 3 H), 4.80 (20 mL). Washing with sat. aqueous NaHCO3 (2 ϫ 30 mL), brine
(d, J ϭ 10.9 Hz, 1 H), 4.87Ϫ4.97 (m, 2 H), 5.68Ϫ5.78 (m, 1 H), (30 mL), was followed by drying (Na2SO4) and concentration in
830
Eur. J. Org. Chem. 2002, 826Ϫ833

Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120
FULL PAPER
vacuo. The crude product was purified by flash column chromato-
graphy on silica gel (2 Ǟ 5% EtOAc/ toluene) to afford 46 mg cooled to Ϫ20 °C for 15 min, followed by the addition of
TBDMSOTf (4 µL, 0.018 mmol), and stirred for 30 min at Ϫ20 °C.
1H NMR The reaction was quenched by the addition of Et3N (50 µL), and
(CDCl3): δ ϭ 0.94Ϫ1.07 (m, 21 H), 1.62Ϫ1.67 (m, 2 H), 2.03Ϫ2.08 dried in vacuo. The crude product was purified by flash column
ϫ 3 mL) and dissolved in diethyl ether (2 mL). The solution was
(90%) of 14. [α]2D4 ϭ Ϫ31.4° (c ϭ 1.3, CH2Cl2). IR (thin film): ν ϭ
446, 2940, 2865, 1734, 1455, 1249, 1085, 695 cmϪ1
˜
.
(m, 2 H), 2.48 (d, J ϭ 9.7 Hz, 1 H), 3.17Ϫ3.20 (m, 1 H), 3.35Ϫ3.41
chromatography on silica gel (5 Ǟ 10% EtOAc/toluene) to afford
(m, 1 H), 3.53Ϫ3.64 (m, 2 H), 3.73 (d, J ϭ 3.1 Hz, 1 H), 3.83Ϫ3.96 120 mg (93%) of trisaccharide 7. [α]2D4 ϭ ϩ9.1 (c ϭ 1.0, CH2Cl2).
Ϫ1
˜
IR (thin film): ν ϭ 917, 1721, 1452, 1270, 1097, 1070, 698 cm .
(m, 3 H), 4.39 (d, J ϭ 0.4 Hz, 1 H), 4.53Ϫ4.56 (m, 2 H), 4.80 (d,
J ϭ 11.1 Hz, 1 H), 4.87Ϫ5.00 (m, 3 H), 5.69Ϫ5.80 (m, 1 H), 1H NMR (CDCl3): δ ϭ 1.42Ϫ1.51 (m, 2 H), 1.88Ϫ1.94 (m, 2 H),
7.17Ϫ7.32 (m, 10 H). 13C NMR (CDCl3): δ ϭ 12.5, 18.2, 29.2, 1.97 (s, 3 H), 3.21Ϫ3.27 (m, 2 H), 3.46Ϫ3.53 (m, 2 H), 3.65Ϫ4.03
30.6, 63.2, 69.1, 74.1, 74.6, 75.0, 76.7, 76.9, 77.9, 101.7, 115.0, (m, 12 H), 4.16Ϫ4.35 (m, 5 H), 4.40Ϫ4.62 (m, 10 H), 4.73Ϫ4.85
127.9, 128.2, 128.5, 138.4, 138.7, 138.8. ESI MS m/z (M ϩ Naϩ) (m, 5 H), 5.11 (s, 1 H), 5.40Ϫ5.42 (m, 1 H), 5.55Ϫ5.64 (m, 2 H),
calcd. 607.3425, found 607.3440.
6.83Ϫ6.86 (m, 1 H), 6.95Ϫ7.25 (m, 36 H), 7.31Ϫ7.49 (m, 4 H),
7.92Ϫ7.98 (m, 4 H). 13C NMR (CDCl3): δ ϭ 21.5, 29.3, 30.6, 64.0,
66.5, 69.3, 69.4, 69.6, 70.4, 72.5, 72.5, 72.5, 73.6, 73.8, 74.4, 74.6,
74.8, 75.2, 75.4, 75.5, 75.7, 76.1, 77.1, 77.8, 78.4, 80.7, 98.6, 100.1,
101.9, 115.2, 127.6, 127.7, 127.8, 128.0, 128.1, 128.2, 128.2, 128.4,
128.5, 128.6, 128.7, 128.7, 128.8, 128.8, 128.9, 128.9, 130.2, 130.5,
130.5, 133.3, 133.5, 137.9, 138.3, 138.3, 138.6, 138.8, 139.0, 139.0,
166.0, 166.8, 170.4. ESI MS m/z (M ϩ Naϩ) calcd. 1475.6130,
found 1475.6141.
4-Pentenyl
2-O-Acetyl-3,4,6-tri-O-benzyl-α-
D
-mannopyranosyl-
(1Ǟ3)-2,4-di-O-benzyl-6-O-triisopropylsilyl-β-
D-mannopyranoside
(15): Mannosyl trichloroacetimidate 9 (65 mg, 0.10 mmol) and gly-
cosyl acceptor 14 (30 mg, 0.051 mmol) were azeotropically dried
with toluene (3 ϫ 3 mL) and dissolved in CH2Cl2 (1 mL). The solu-
tion was cooled to Ϫ20 °C for 15 min, followed by the addition
TBDMSOTf (2.4 µL, 0.010 mmol), and stirred for 30 min at Ϫ20
°C. The reaction was quenched by addition of Et3N (50 µL), and
dried in vacuo. The crude product was purified by flash column
chromatography on silica gel (2 Ǟ 5% EtOAc/toluene) to afford
54 mg (99%) disaccharide 15. [α]2D4 ϭ Ϫ15.6 (c ϭ 1.4, CH2Cl2). IR
4-Pentenyl 3,4,6-Tri-O-benzyl-α-
D-mannopyranosyl-(1Ǟ3)-[2,4-di-
O-benzyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-benzyl-β-
D
D
-manno-
pyranoside (17): Trisaccharide 7 (100 mg, 0.0687 mmol) was dis-
solved in CH2Cl2/MeOH (4 mL, 1:1). A solution of sodium me-
thoxide in MeOH (450 µL, 25% w/v, 2 mmol) was added and the
reaction was heated on an oil-bath to 45 °C for 1.5 h. The reaction
was quenched with DOWEX-50 W-hydrogen strongly acidic ion-
exchange resin, filtered, and dried in vacuo. The resulting crude
product was purified by flash column chromatography on silica gel
(10 Ǟ 40% EtOAC/toluene) to afford 73 mg (89%) of trisaccharide
(thin film): ν˜ ϭ 940, 2865, 1745, 1235, 1078, 697 cmϪ1. H NMR
1
(CDCl3): δ ϭ 0.93Ϫ1.04 (m, 21 H), 1.57Ϫ1.64 (m, 2 H), 1.99Ϫ2.06
(m, 5 H), 3.13Ϫ3.17 (m, 1 H), 3.27Ϫ3.33 (m, 1 H), 3.51Ϫ3.52 (m,
2 H), 3.54Ϫ3.93 (m, 9 H), 4.29 (s, 1 H), 4.35Ϫ4.70 (m, 8 H), 4.79
(d, J ϭ 11.1 Hz, 1 H), 4.87Ϫ4.95 (m, 3 H), 5.12 (d, J ϭ 1.2 Hz, 1
H), 5.43Ϫ5.44 (m, 1 H), 5.69Ϫ5.79 (m, 1 H), 7.04Ϫ7.32 (m, 25 H).
13C NMR (CDCl3): δ ϭ 12.5, 18.2, 21.2, 29.2, 30.6, 63.0, 69.0,
69.1, 69.2, 72.1, 72.2, 73.6, 73.8, 73.9, 74.5, 75.0, 75.3, 77.2, 77.7,
78.3, 80.6, 99.9, 101.7, 114.9, 127.2, 127.6, 127.7, 127.8, 127.9,
127.9, 128.2, 128.3, 128.4, 128.4 128.5, 128.6, 138.1, 138.3, 138.4,
138.4, 138.8, 139.2, 170.5. ESI MS m/z (M ϩ Naϩ) calcd.
1081.5468, found 1081.5428.
17. [α]2D4 ϭ 11.0 (c ϭ 1.3, CH2Cl2). IR (thin film): ν ϭ 470, 2922,
˜
1453, 1364, 1072, 697 cmϪ1. 1H NMR (CDCl3): δ ϭ 1.50Ϫ1.56 (m,
2 H), 1.84 (br. s, 1 H), 1.94Ϫ2.00 (m, 2 H), 2.23 (br. s, 2 H),
3.20Ϫ3.29 (m, 2 H), 3.49Ϫ3.54 (m, 2 H), 3.55Ϫ3.83 (m, 13 H),
3.86Ϫ3.92 (m, 3 H), 4.25Ϫ4.58 (m, 10 H), 4.64Ϫ4.76 (m, 3 H),
4.83Ϫ4.91 (m, 4 H), 5.11 (d, J ϭ 0.97 Hz, 1 H), 5.18 (d, J ϭ 1.2 Hz,
1 H), 5.61Ϫ5.72 (m, 1 H), 7.05Ϫ7.30 (m, 35 H). 13C NMR
(CDCl3): δ ϭ 29.3, 30.6, 62.7, 66.5, 69.1, 69.4, 69.7, 71.7, 72.2,
72.2, 72.6, 73.0, 73.7, 75.2, 75.4, 75.7, 76.1, 76.9, 77.6, 77.9, 79.1,
80.3, 80.6, 98.0, 101.7, 102.1, 115.3, 127.8, 127.9, 127.9, 128.0,
128.1, 128.1, 128.1, 128.1, 128.3, 128.3, 128.4, 128.6, 128.7, 128.7,
128.8, 128.9, 129.0, 129.0, 138.2, 138.2, 138.4, 138.4, 138.4, 138.6,
138.9, 138.9, 139.0. ESI MS m/z (M ϩ Naϩ) calcd. 1225.5500,
found 1225.5497.
4-Pentenyl
2-O-Acetyl-3,4,6-tri-O-benzyl-α-
D-mannopyranosyl-
(1Ǟ3)-2,4-di-O-benzyl-β-D-mannopyranoside (16): To a solution of
disaccharide 15 (0.108 g, 0.103 mmol) in THF (2 mL), water
(2 mL) and trifluoroacetic acid (0.8 mL) were added. The turbid
white solution was stirred for 2 h at room temperature. The reac-
tion was diluted with diethyl ether (30 mL) and washed with sat.
aqueous NaHCO3 (2 ϫ 20 mL), brine (20 mL), dried (Na2SO4),
and concentrated. The residue was purified by flash column chro-
matography on silica gel (10 Ǟ 30% EtOAc/hexanes) to afford
84 mg (91%) of disaccharide 16. [α]2D4 ϭ Ϫ24.0 (c ϭ 0.5, CH2Cl2).
4-Pentenyl
(1Ǟ2)-3,4,6-tri-O-benzyl-α-
3,4,6-tri-O-benzyl-α- -mannopyranosyl-(1Ǟ3)-[2-O-acetyl-3,4,6-tri-
O-benzyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-benzyl-α- -manno-
pyranosyl-(1Ǟ6)]-2,4-di-O-benzyl-β- -mannopyranoside (6): Trisac-
2-O-Acetyl-3,4,6-tri-O-benzyl-α-
D-mannopyranosyl-
Ϫ1
IR (thin film): ν ϭ 443, 2089, 1639, 1234, 698 cm
.
1H NMR
˜
D
-mannopyranosyl-(1Ǟ3)-[2-O-acetyl-
(CDCl3): δ ϭ 1.70Ϫ1.78 (m, 2 H), 2.12Ϫ2.22 (m, 6 H), 3.31Ϫ3.39
(m, 1 H), 3.41Ϫ3.44 (m, 1 H), 3.63Ϫ3.69 (m, 2 H), 4.75Ϫ3.43 (m,
9 H), 4.43Ϫ4.73 (m, 8 H), 4.78Ϫ4.84 (m, 2 H), 4.91 (d, J ϭ
11.0 Hz, 1 H), 4.97Ϫ5.09 (m, 3 H), 5.26 (d, J ϭ 1.5 Hz, 1 H),
D
D
D
D
charide acceptor 17 (25 mg, 0.021 mmol) and mannosyl trichlo-
roacetimidate 9 (60 mg, 0.093 mmol, 4.5equiv.) were combined, az-
eotropically dried with toluene (3 ϫ 3 mL) and dissolved in CH2Cl2
(2 mL). The solution was cooled to Ϫ20 °C for 15 min, followed
by the addition of TMSOTf (2.2 µL, 0.012 mmol). The reaction
mixture was stirred and warmed to room temperature over 40 min.
The reaction was quenched by the addition of Et3N (100 µL), and
dried in vacuo. The crude product was purified by flash column
5.54Ϫ5.55 (m, 1 H), 5.80Ϫ5.90 (m, 1 H), 7.17Ϫ7.42 (m, 25 H). 13
C
NMR (CDCl3): δ ϭ 21.2, 29.0, 30.4, 62.3, 68.9, 69.2, 69.7, 72.1,
72.3, 74.4, 74.4, 75.0, 75.4, 76.0, 77.5, 78.1, 80.0, 99.8, 101.8, 115.4,
127.6, 127.7, 127.8, 127.8, 127.8, 128.0, 128.0, 128.1, 128.3, 128.4,
128.2, 128.6, 128.6, 137.9, 137.9, 138.2, 138.3, 138.6, 138.7, 170.5.
ESI MS m/z (M ϩ Naϩ) calcd. 925.4133, found 925.4141.
4-Pentenyl
(1Ǟ3)-[2,4-di-O-benzyl-3,6-di-O-benzoyl-α-
(1Ǟ6)]-2,4-di-O-benzyl-β- -mannopyranoside (7): Mannosyl trichlo-
2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-
D
-mannopyranosyl- chromatography on silica gel (2 Ǟ 20% EtOAc/toluene) to afford
D
51 mg (94%) of hexasaccharide 6. [α]2D4 ϭ ϩ34.9 (c ϭ 1.3, CH2Cl2).
1
roacetimidate 10 (94 mg, 0.15 mmol) and disaccharide 15 (80 mg,
IR (thin film): ν˜ ϭ 029, 2916, 1744, 1235, 1076, 736, 697 cmϪ1. H
0.088 mmol) were combined, azeotropically dried with toluene (3
NMR (CDCl3): δ ϭ 1.56Ϫ1.60 (m, 2 H), 2.01Ϫ2.04 (m, 2 H), 2.09
Eur. J. Org. Chem. 2002, 826Ϫ833
831

D. M. Ratner, O. J. Plante, P. H. Seeberger
FULL PAPER
(s, 3 H), 2.13 (s, 3 H), 2.16 (s, 3 H), 3.19Ϫ3.22 (m, 1 H), 3.30Ϫ3.36 tional 1.5 h in vacuo and dissolved in diethyl ether (2 mL). The
(m, 2 H), 3.54Ϫ3.62 (m, 7 H), 3.67Ϫ3.72 (m, 6 H), 3.78Ϫ3.87 (m, solution was cooled to Ϫ20 °C for 15 min, followed by the addition
6 H), 3.89Ϫ3.99 (m, 10 H), 4.00Ϫ4.07 (m, 2 H), 4.09Ϫ4.13 (m, 1
of TMSOTf (1 µL, 0.005 mmol), and stirred for 30 min. The reac-
H), 4.23Ϫ4.28 (m, 2 H), 4.34Ϫ4.37 (m, 1 H), 4.39Ϫ4.50 (m, 10 H), tion was quenched by the addition of Et3N (50 µL), and dried in
4.53Ϫ4.59 (m, 6 H), 4.61Ϫ4.68 (m, 6 H), 4.73 (app. s, 1 H), 4.76 vacuo. The crude product was purified by flash column chromato-
(d, J ϭ 2.3 Hz, 1 H), 4.81Ϫ4.89 (m, 5 H), 4.90Ϫ4.94 (m, 3 H), graphy on silica gel (2 Ǟ 18% EtOAc/toluene) affording 24 mg
4.98Ϫ5.03 (m, 3 H), 5.20Ϫ5.24 (m, 2 H), 5.52Ϫ5.53 (m, 3 H),
(80%) of nonasaccharide 5. [α]2D4 ϭ ϩ24.7 (c ϭ 0.9, CH2Cl2). IR
Ϫ1
5.70Ϫ5.77 (m, 1 H), 7.10Ϫ7.40 (m, 80 H). 13C NMR (CDCl3): δ ϭ (thin film): ν ϭ 029, 2864, 1744, 1453, 1137, 1056, 736, 697 cm
.
˜
21.8, 21.9, 29.6, 31.0, 66.1, 66.3, 68.4, 68.7, 68.8, 69.0 69.4, 69.5, 1H NMR (CDCl3): δ ϭ 1.48Ϫ1.55 (m, 2 H) 1.94Ϫ1.98 (m, 2 H),
71.0, 71.4, 71.7, 72.0, 72.0, 72.0, 72.1, 72.3, 72.4, 72.6, 73.4, 73.4, 2.08 (s, 3 H), 2.12 (s, 3 H), 2.13 (s, 3 H), 3.13Ϫ3.18 (m, 2 H),
73.5, 73.6, 74.1, 74.2, 74.7, 74.9, 75.0, 75.2, 75.2, 75.3, 75.4, 77.5,
77.6, 77.8, 78.2, 78.4, 79.8, 81.7, 97.0, 98.4, 99.6, 100.0, 101.2, 3.55Ϫ3.67 (m, 10 H), 3.73Ϫ3.78 (m, 4 H), 3.82Ϫ3.94 (m, 21 H),
101.7, 114.9, 127.4, 127.4, 127.5, 127.5, 127.6, 127.6, 127.6, 127.6, 3.97Ϫ4.06 (m, 5 H), 4.08Ϫ4.13 (m, 4 H), 4.16Ϫ4.22 (m, 3 H),
3.26Ϫ3.30 (m, 1 H), 3.35Ϫ3.46 (m, 5 H), 3.50Ϫ3.52 (m, 3 H),
127.7, 127.7, 127.7, 127.7, 127.8, 127.8, 127.9, 127.9, 127.9, 128.0, 4.30Ϫ4.43 (m, 12 H), 4.45Ϫ4.51 (m, 9 H), 4.54Ϫ4.55 (m, 6 H),
128.0, 128.1 128.2, 128.3, 128.3, 128.4, 128.4, 128.4, 128.4, 128.5, 4.56Ϫ4.58 (m, 4 H), 4.60Ϫ4.61 (m, 2 H), 4.63Ϫ4.68 (m, 4 H),
128.5, 128.5, 128.6, 128.6, 128.7, 128.7, 129.2, 138.0, 138.0, 138.2,
4.71Ϫ4.73 (m, 1 H), 4.74Ϫ4.77 (m, 2 H), 4.80Ϫ4.89 (m, 7 H),
138.3, 138.4, 138.4, 138.5, 138.5, 138.5, 138.7, 138.8, 138.9, 139.0, 4.95Ϫ5.00 (m, 2 H), 5.08Ϫ5.11 (m, 3 H), 5.14 (m, 1 H), 5.17 (m,
170.3, 170.3, 170.4; HSQC anomeric cross-peaks (CDCl3) δ (4.23 1 H), 5.23Ϫ5.24 (m, 1 H), 5.52Ϫ5.55 (m, 4 H), 5.60Ϫ5.69 (m, 1
ϫ 101.8), (4.93 ϫ 97.0), (5.02 ϫ 98.4), (5.06 ϫ 99.5), (5.21 ϫ 99.9), H), 7.00Ϫ7.02 (m, 1 H), 7.07Ϫ7.34 (m, 124 H). 13C NMR (CDCl3):
(5.22 ϫ 101.1). ESI MS m/z (M ϩ Naϩ) calcd. 2648.1622, found δ ϭ 20.5, 21.0, 21.3, 22.0, 29.1, 30.3, 66.6, 66.6, 66.6, 66.7, 68.2,
2648.1530.
68.2, 68.8, 68.8, 68.8, m68.9, 69.0, 69.2, 69.2, 69.2, 69.4, 70.5, 70.8,
70.9, 71.4, 71.4, 71.9, 72.0, 72.0, 72.1, 72.2, 72.3, 72.7, 72.8, 73.3,
73.3, 73.4, 73.5, 73.5, 74.2, 74.9, 74.9, 75.2, 75.2, 75.2, 75.4, 75.4,
75.5, 77.4, 78.3, 78.4, 79.1, 79.2, 79.5, 79.9, 80.1, 82.2, 97.0, 99.3,
99.5, 99.5, 99.7, 100.7, 101.1, 101.4, 101.4, 115.0, 125.7, 126.2,
126.7, 126.6, 127.3, 128.0, 128.0, 128.3, 128.7, 129.3, 129.8, 130.3,
131.3, 132.5, 171.0, 171.0, 171.1; HSQC anomeric cross-peaks
(CDCl3) δ (4.16 ϫ 102.0), (4.86 ϫ 97.0), (5.00 ϫ 99.6), three (5.10
ϫ 99.6), (5.12 ϫ 100.7), (5.18 ϫ 101.2), (5.21 ϫ 101.3). ESI MS
m/z (M ϩ Naϩ) calcd. 3944.7437, found 3944.7440.
4-Pentenyl 3,4,6-Tri-O-benzyl-α-D-mannopyranosyl-(1Ǟ2)-3,4,6-tri-
O-benzyl-α-
mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-benzyl-α-
(1Ǟ6)]-2,4-di-O-benzyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-
benzyl-β- -mannopyranoside (18): Hexasaccharide (46 mg,
D
-mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-benzyl-α-
D-
D
-mannopyranosyl-
D
D
6
0.018 mmol) was azeotropically dried with toluene (3 ϫ 3 mL) and
dissolved in CH2Cl2 (1.5 mL). MeOH (5 mL) was added followed
by a solution of sodium methoxide in MeOH (120 µL, 25%
w/v, 0.525 mmol). The reaction was stirred for 1 h, quenched with
DOWEX-50 W-hydrogen strongly acidic ion-exchange resin, fil-
4-Pentenyl 3,4,6-Tri-O-benzyl-α-
-mannopyranosyl-(1Ǟ2)-3,4,6-tri-O-benzyl-α-
mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-benzyl-α- -mannopyranosyl-
(1Ǟ2)-3,4,6-tri-O-benzyl-α- -mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-
ϩ34.3 (c ϭ 1.0, CH2Cl2). IR (thin film): ν ϭ 448, 3029, 2916, 1453, benzyl-α- -mannopyranosyl-(1Ǟ2)-3,4,6-tri-O-benzyl-α- -manno-
1053, 697 cmϪ1 1H NMR (CDCl3): δ ϭ 1.43Ϫ1.53 (m, 2 H),
pyranosyl-(1Ǟ6)]-2,4-di-O-benzyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-
1.89Ϫ1.95 (m, 2 H), 2.07Ϫ2.28 (br. s, 3 H), 3.10Ϫ3.13 (m, 1 H), di-O-benzyl-β- -mannopyranoside (19): Nonasaccharide 5 (14 mg,
D-mannopyranosyl-(1Ǟ2)-3,4,6-tri-
tered, and dried in vacuo. The resulting residue was purified by O-benzyl-α-
flash column chromatography on silica gel (5 Ǟ 20% EtOAc/tolu-
ene) to afford 46 mg (quant.) of hexasaccharide triol 18. [α]2D4
D
D-
D
ϭ
D
˜
D
D
.
D
D
3.19Ϫ3.24 (m, 1 H), 3.33Ϫ3.36 (m, 1 H), 3.43Ϫ3.62 (m, 13 H), 0.0035 mmol) was dissolved in a mixture of CH2Cl2/MeOH (3 mL,
3.64Ϫ3.81 (m, 14 H), 3.82Ϫ3.92 (m, 3 H), 3.99Ϫ4.02 (m, 4 H), 1:2) and cooled to 0 °C. A solution of sodium methoxide in MeOH
4.13 (app. s, 1 H), 4.22Ϫ4.30 (m, 3 H), 4.35Ϫ4.40 (m, 8 H), (35 µL, 25% w/v) was added and the reaction was slowly warmed
4.45Ϫ4.51 (m, 12 H), 4.53Ϫ4.65 (m, 4 H), 4.69Ϫ4.75 (m, 5 H),
to room temperature over 1 h, quenched with DOWEX-50 W-hy-
4.78Ϫ4.83 (m, 3 H), 4.86Ϫ4.97 (m, 2 H), 5.02 (app. s, 1 H), 5.11 drogen strongly acidic ion-exchange resin, filtered, and dried in va-
(app. s, 1 H), 5.16 (app. s, 1 H), 5.57Ϫ5.67 (m, 1 H), 6.81Ϫ7.48 (m, cuo. The resulting residue was purified by flash column chromatog-
80 H). 13C NMR (CDCl3): δ ϭ 14.9, 21.8, 22.2, 69.4, 69.5, 69.7, raphy on silica gel (5 Ǟ 30% EtOAc/toluene) affording 12 mg
69.9, 70.1, 71.9, 72.0, 72.0, 72.3, 72.5, 72.6, 72.7, 72.8, 73.0, 73.2,
73.9, 74.0, 74.1, 74.2, 74.6, 74.9, 74.9, 75.2, 75.4, 75.5, 75.7, 75.8,
75.9, 76.2, 77.9, 78.4, 78.5, 80.3, 80.5, 80.8, 82.5, 97.5, 100.5, 101.8,
(90%) of nonasaccharide triol 19. 1H NMR (CDCl3): δ ϭ
1.37Ϫ1.43 (m, 2 H), 1.85Ϫ1.88 (m, 2 H), 2.23 (br. s, 1 H), 2.29 (br.
s, 2 H), 3.07Ϫ3.27 (m, 3 H), 3.30Ϫ4.27 (m, 56 H), 4.28Ϫ4.66 (m,
101.8, 102.1, 102.1, 115.4, 127.9, 128.0, 128.1, 128.1, 128.2, 128.3, 41 H), 4.70Ϫ4.77 (m, 9 H), 4.79 (d, J ϭ 10.5 Hz, 1 H), 4.94 (app.
128.3, 128.4, 128.4, 128.4, 128.5, 128.5, 128.6, 128.6, 128.8, 128.9, s, 1 H), 5.02 (app. s, 1 H), 5.08Ϫ5.09 (m, 3 H), 5.13 (m, 2 H),
128.9, 129.0, 129.0, 129.0, 129.1, 129.1, 129.1, 129.2, 129.3, 138.6,
138.7, 138.7, 138.8, 138.8, 138.9, 139.0, 139.0, 139.1, 139.3, 139.4,
139.5. ESI MS m/z (M ϩ Naϩ) calcd. 2522.1305, found 2522.1307.
5.51Ϫ5.60 (m, 1 H), 6.92Ϫ7.44 (m, 125 H).
n-Pentyl α-D-Mannopyranosyl-(1Ǟ3)-[α-D-mannopyranosyl-(1Ǟ6)]-
β-D-mannopyranoside (4): Activated palladium on carbon (100 mg,
4-Pentenyl
(1Ǟ2)-3,4,6-tri-O-benzyl-α-
benzyl-α-
α- -mannopyranosyl-(1Ǟ2)-3,4,6-tri-O-benzyl-α-
pyranosyl-(1Ǟ3)-[2-O-acetyl-3,4,6-tri-O-benzyl-α-
pyranosyl-(1Ǟ2)-3,4,6-tri-O-benzyl-α-
2,4-di-O-benzyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-benzyl-β-
mannopyranoside 5. Hexasaccharide 18 (19 mg, 0.0075 mmol) and
mannosyl trichloroacetimidate 9 (36 mg, 0.056 mmol, 7.5equiv.)
were azeotropically dried with toluene (3 ϫ 3 mL), dried an addi-
2-O-Acetyl-3,4,6-tri-O-benzyl-α-
D
-mannopyranosyl-
10%) was suspended in ethanol (10 mL) and exposed to an atmo-
sphere of hydrogen gas (balloon). After 30 min, trisaccharide triol
D
-mannopyranosyl-(1Ǟ2)-3,4,6-tri-O-
D
-mannopyranosyl-(1Ǟ3)-[2-O-acetyl-3,4,6-tri-O-benzyl- 17 (70 mg, 0.058 mmol) in EtOAc (5 mL) was added by cannula
D
D
-manno- and stirred for 48 h under an atmosphere of hydrogen. The product
D
-manno- was filtered through celite, dried in vacuo, to afford 6 mg (79%)
1
D
-mannopyranosyl-(1Ǟ6)]- trisaccharide 4. [α]2D4 ϭ ϩ35.0 (c ϭ 0.4, H2O/EtOH 1:1). H NMR
D
D
-
(D2O): δ ϭ 0.70Ϫ0.73 (m, 3 H), 1.15Ϫ1.17 (m, 4 H), 1.43Ϫ1.46
(m, 2 H), 3.37Ϫ3.40 (m, 1 H), 3.47Ϫ3.83 (m, 19 H), 3.90Ϫ3.91 (m,
1 H), 3.98 (d, J ϭ 1.6 Hz, 1 H), 4.52 (app. s, 1 H), 4.75 (app. s, 1
H), 4.94 (app. s, 1 H). 13C NMR (D2O): δ ϭ 13.6, 22.1, 27.2, 28.7,
832
Eur. J. Org. Chem. 2002, 826Ϫ833

Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120
FULL PAPER
61.2, 65.8, 66.1, 67.0, 67.1, 70.2, 70.3, 70.4, 70.6, 70.7, 72.9, 73.6,
DCIF Mercury 300 was provided by NSF (Award# CHE-9808061)
74.4, 81.1, 99.7, 100.0, 102.7. MALDI-TOF m/z (M ϩ Naϩ) calcd. and NSF (Award# DBI-9729592).
595.22, found 596.77.
[1]
E. O. Freed, M. A. Martin, J. Biol. Chem. 1995, 270,
23883Ϫ23886.
C. A. Bewley, K. R. Gustafson, M. R. Boyd, D. G. Covell, A.
Bax, G. M. Clore, A. M. Gronenborn, Nature Structural Bio-
logy 1998, 7, 571Ϫ578.
T. Feizi, ‘‘Glycobiology of AIDS,’’ Carbohydrates in Chemistry
and Biology Wiley-VCH 2000, v.4, 851Ϫ863.
M. R. Boyd, K. R. Gustafson, J. B. McMahon, R. H. Shoe-
maker, B. R. O’Keefe, T. Mori, R. J. Gulakowski, L. Wu, M.
I. Rivera, C. M. Laurencot, M. J. Currens, J. H. Cardellina II,
R. W. Buckheit, P. L. Nara, L. K. Pannell, R. C. Sowder II, L.
E. Henderson, Antimicrobial Agents and Chemotherapy 1997,
41, 1521Ϫ1530.
T. Mori, M. R. Boyd, Antimicrobial Agents and Chemotherapy
2001, 45, 664Ϫ672.
B. R. O’Keefe, S. R. Shenoy, D. Xie, W. Zhang, J. M. Muschik,
M. J. Currens, I. Chaiken, M. R. Boyd, Mol. Pharm. 2000,
58, 982Ϫ992.
C. A. Bewley, J. Am. Chem. Soc. 2001, 123, 1014Ϫ1015.
C. A. Bewley, S. Otero-Quintero, J. Am. Chem. Soc. 2001,
123, 3892Ϫ3902.
T. Ogawa, K. Kiyoaki, K. Sasajima, M. Matsui, Tetrahedron
1981, 37, 2779Ϫ2786.
T. Ogawa, K. Sasajima, Carbohydr. Res. 1981, 93, 53Ϫ66.
T. Ogawa, T. Nukada, Carbohydr. Res. 1985, 136, 135Ϫ152.
T. Ogawa, S. Nakabayashi, T. Kitajima, Carbohydr. Res. 1983,
114, 225Ϫ236.
J. R. Merritt, E. Naisang, B. Fraser-Reid, J. Org. Chem. 1994,
59, 4443Ϫ4449.
P. Grice, S. V. Ley, J. Pietruszka, H. Osborn, H. Priepke, S.
Warriner, Chem. Eur. J. 1997, 3, 431Ϫ440.
S. V. Ley, D. K. Baeschlin, D. J. Dixon, A. C. Foster, S. J. Ince,
H. W. M. Priepke, D. J. Reynolds, Chem. Rev. 2001, 101,
53Ϫ80.
P. Grice, S. V. Ley, J. Pietruszka, H. Priepke, Angew. Chem. Int.
Ed. Engl. 1996, 35, 197Ϫ200.
R. B. Andrade, O. J. Plante, L. G. Melean, P. H. Seeberger,
Org. Lett. 1999, 1, 1811Ϫ1814.
J. R. Allen, C. R. Harris, S. J. Danishefsky, J. Am. Chem. Soc.
2001, 123, 1890Ϫ1897.
B. Fraser-Reid, U. E. Udodong, Z. Wu, H. Ottosson, J. R.
Merritt, C. S. Rao, C. Roberts, R. Madsen, Synlett 1992,
927Ϫ942.
T. Buskas, E. Soderberg, P. Konradsson, B. Fraser-Reid, J. Org.
Chem. 2000, 65, 958Ϫ963.
S. J. Danishefsky, M. T. Bilodeau, Angew. Chem. Int., Ed. Engl.
1996, 35, 1380Ϫ1419.
K. K. Liu, S. J. Danishefsky, J. Org. Chem. 1994, 59,
1892Ϫ1894.
O. J. Plante, S. L. Buchwald, P. H. Seeberger, J. Am. Chem.
Soc. 2000, 122, 7148Ϫ7149.
J. Rademann, A. Geyer, R. R. Schmidt, Angew. Chem. Int. Ed.
1998, 37, 1241Ϫ1245.
T. G. Mayer, B. Kratzer, R. R. Schmidt, Angew. Chem. Int. Ed.
Engl. 1994, 33, 2177Ϫ2181.
W. Frick, A. Bauer, J. Bauer, S. Wied, G. Müller, Biochem.
1998, 37, 13421Ϫ13436.
n-Pentyl α-
[α- -mannopyranosyl-(1Ǟ3)-[α-
mannopyranosyl-(1Ǟ6)]-β- -mannopyranoside (3): Activated palla-
D
-Mannopyranosyl-(1Ǟ2)-α-
D-mannopyranosyl-(1Ǟ3)-
D
D
-mannopyranosyl-(1Ǟ6)]-α-D-
[2]
D
dium on carbon (50 mg, 10%) was suspended in ethanol (5 mL)
and exposed to an atmosphere of hydrogen gas (balloon). After
30 min, hexasaccharide triol 18 (35 mg, 0.014 mmol) in EtOAc
(2 mL) was added by cannula and stirred for 48 h under an atmo-
sphere of hydrogen. The product was filtered through celite, dried
in vacuo, to afford 12 mg (81%) hexasaccharide 3. [α]2D4 ϭ ϩ54.5
[3]
[4]
1
(c ϭ 0.5, H2O/EtOH 1:1). H NMR (D2O): δ ϭ 0.70Ϫ0.74 (m, 3
H), 1.15Ϫ1.17 (m, 4 H), 1.43Ϫ1.46 (m, 2 H), 3.38Ϫ3.43 (m, 1 H),
3.46Ϫ3.89 (m, 33 H), 3.89Ϫ3.92 (m, 2 H), 3.96Ϫ3.97 (m, 2 H),
4.00 (app. s, 1 H), 4.51 (app. s, 1 H), 4.72 (app. s, 1 H), 4.75 (app.
s, 1 H), 4.89 (app. s, 1 H), 4.98 (app. S, 1 H), 5.19 (app. s, 1 H),.
MALDI-TOF m/z (M ϩ Naϩ) calcd. 1081.38, found 1082.54.
[5]
[6]
n-Pentyl α-D-Mannopyranosyl-(1Ǟ2)-α-D-mannopyranosyl-(1Ǟ2)-α-
[7]
[8]
D-mannopyranosyl-(1Ǟ3)-[α-
D
-mannopyranosyl-(1Ǟ2)-α-D-manno-
pyranosyl-(1Ǟ3)-[α-
pyranosyl-(1Ǟ6)]-α-
D
D
-mannopyranosyl-(1Ǟ2)-α-
-mannopyranosyl-(1Ǟ6)]-β-
D
-manno-
-manno-
[9]
D
pyranoside (2): Activated palladium on carbon (50 mg, 10%) was
suspended in ethanol (5 mL) and exposed to an atmosphere of hy-
drogen gas (balloon). After 30 min, nonasaccharide triol 19 (12 g,
0.0031 mmol) in EtOAc (2 mL) was added by cannula and stirred
for 48 h under an atmosphere of hydrogen. The product was sepa-
rated by filtration through celite and dried in vacuo to yield 4 mg
(88%) of fully deprotected nonasaccharide 2. [α]2D4 ϭ ϩ38.0 (c ϭ
[10]
[11]
[12]
[13]
[14]
[15]
1
0.05, H2O/EtOH 1:1). H NMR (D2O): δ ϭ 0.71Ϫ0.74 (m, 3 H),
1.16Ϫ1.18 (m, 5 H), 1.44Ϫ1.46 (m, 2 H), 3.38Ϫ3.40 (m, 1 H),
3.46Ϫ3.52 (m, 5 H), 3.52Ϫ3.57 (m, 5 H), 3.58Ϫ3.66 (m, 12 H),
3.68Ϫ3.72 (m, 6 H), 3.73Ϫ3.76 (m, 6 H), 3.79Ϫ3.82 (m, 3 H),
3.84Ϫ3.87 (m, 4 H), 3.91Ϫ3.99 (m, 7 H), 4.00 (app. s, 1 H), 4.51
(app. s, 1 H), 4.72 (app. s, 1 H), 4.89 (app. s, 3 H), 5.00 (app. s, 1
H), 5.16 (app. s, 1 H), 5.19 (app. s, 1 H), 5.26 (app. s, 1 H). 13C
NMR (CDCl3): δ ϭ 13.4, 21.9, 23.3, 27.5, 28.4, 61.1, 61.1, 61.2,
61.2, 65.6, 65.6, 65.7, 65.7, 65.7, 65.9, 66.9, 67.1, 69.7, 70.1, 70.3,
70.3, 70.3, 70.3, 70.3, 70.5, 71.2, 72.8, 73.3, 73.3, 74.2, 78.6, 78.8,
78.8, 79.1, 98.2, 99.7, 99.9, 100.8, 100.8, 100.9, 102.4, 102.4, 102.4;
HSQC anomeric cross-peaks (CDCl3) δ (4.51 ϫ 99.9), (4.72 ϫ
99.8), three (4.89 ϫ 102.4), (5.00 ϫ 98.1), (5.16 ϫ 100.8), (5.19 ϫ
100.8), (5.26 ϫ 100.9). MALDI-TOF m/z (M ϩ Naϩ) calcd.
1567.54, found 1569.09.
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Acknowledgments
Financial support from: the donors of the Petroleum ether Re-
search Fund, administered by the ACS (ACS-PRF 34649-G1) for
partial support of this research; the Mizutani Foundation for Gly-
coscience; the Kenneth M. Gordon Scholarship Fund (Fellowship
for O.J.P.); the NIH (Biotechnology Training Grant for O.J.P. and
D.M.R.); and Pfizer (ACS Division of Organic Chemistry-Pfizer
Fellowship for O.J.P.) is gratefully acknowledged. Funding for the
MIT-DCIF Inova 500 was provided by NSF (Award# CHE-
9808061). Funding for the MIT-DCIF Avance (DPX) 400 was pro-
vided by NIH (Award# 1S10RR13886Ϫ01). Funding for the MIT-
W. Frick, G. Mueller, Inositolglycans Having Insulin-Like Ac-
tion, U.S. Patent 6,004,938, Dec. 21, 1999.
Received October 22, 2001
[O01511]
Eur. J. Org. Chem. 2002, 826Ϫ833
833