Chemical synthesis of N-linked glycans carrying both mannose-6-phosphate and GlcNAc-mannose-6-phosphate motifs

Article abstract of DOI:10.1021/jo2010999

Mannose-6-phosphate (M6P) containing N-linked glycans are the essential targeting signals for hydrolases sorting in eukaryotic cells. To facilitate their structural and binding analyses, a highly efficient and convergent method has been developed to prepa

Full text of DOI:10.1021/jo2010999

Article
pubs.acs.org/joc
Chemical Synthesis of N-Linked Glycans Carrying Both Mannose-6-
phosphate and GlcNAc-Mannose-6-phosphate Motifs
Yunpeng Liu and Gong Chen*
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
S
* Supporting Information
ABSTRACT: Mannose-6-phosphate (M6P) containing N-linked glycans are
the essential targeting signals for hydrolases sorting in eukaryotic cells. To
facilitate their structural and binding analyses, a highly efficient and convergent
method has been developed to prepare complex N-linked glycans with well-
defined M6P and N-acetylglucosamine (GlcNAc)-M6P motifs, a newly
identified binding element for M6P receptors. The GlcNAc-M6P motif was
stereoselectively installed at the late stage of the synthesis. Sequential
deprotection of benzyl and acetate groups provided the fully deprotected N-
glycans in excellent yield.
sized that GlcNAc-M6P could be a key element necessary to
control the diversion of hydrolases at the trans Golgi network.
Therefore, a much more complex ternary coding system
consisting of M, M6P, and Glc-M6P might exist for the
hydrolase sorting system. To test this hypothesis via structural
and binding analyses, we need to obtain enough homogeneous
N-glycan materials with various well-defined GlcNAc-M6P and
M6P modification patterns. Herein, we report the first chemical
synthesis of GlcNAc-M6P-containing N-glycans, which pro-
ceeds in a highly efficient fashion.
INTRODUCTION
■
Mannose-6-phosphate (M6P)-containing N-linked glycans are
the essential targeting signals for the transport of hydrolases
and other M6P-containing proteins in eukaryotic cells (Figure
1A).1 In the ER-Golgi network, hydrolases become distinct
from the other N-glycoproteins by acquiring M6P residues in a
two-step process: First, the GlcNAc-phosphotransferase trans-
fers N-acetylglucosamine-1-phosphate to mannose residues on
a N-glycan to generate a phosphodiester intermediate GlcNAc-
M6P. Second, α-N-acetyl-glucosaminidase removes the N-
acetylglucosamine residue to generate the M6P phosphomo-
noester (Figure 1B).2 Depending on the M6P-based binding
signals embedded in N-glycans, M6P receptors (MPRs) can
guide the transport of hydrolases to different destinations,
mainly lysosomes or extracellular matrix. Because of the
complexity caused by the inherent heterogeneity and multi-
valence of M6P N-glycans, the detailed mechanism of this
universal M6P-coded protein sorting process is still poorly
understood.3 The conventional binding model relies on a
simple binary coding system (M and M6P) where M6P serves
as the sole positive binding element for MPRs. However, recent
studies from Dahms and co-workers provided critical insights to
the functional roles of the GlcNAc-M6P motif.4 They
discovered that M6P receptor homology (MRH) domain 5
of the cation-independent (CI)-MPR provides a previously
unnoticed weak binding site for GlcNAc-M6P (Figure 1B).
More surprisingly, MRH domain 5 shows a stronger binding
affinity for GlcNAc-M6P as compared to M6P.5 Even though
GlcNAc-M6P-containing N-glycans were found in secreted
hydrolases, they were mostly ignored due to their unknown
binding abilities.6 Inspired by Dahms's discovery, we hypothe-
RESULTS AND DISCUSSION
■
Phosphor-containing N-glycans pose a greater synthetic
challenge when compared to unmodified N-glycans.7,8
Synthetic routes have to be delicately orchestrated and
executed to accommodate the labile phosphor moieties. In
our previous exploration of the chemical synthesis of
multiphosphorylated N-glycans, a late stage phosphorylation
strategy was successfully employed to install two benzyl-
protected phosphate groups on an octasaccharide substrate
using the phosphoramidite chemistry.9 Accordingly, such a late
stage phosphorylation strategy was adopted for the synthesis of
GlcNAc-M6P-containing N-glycans. To synthesize complex
GlcNAc-M6P N-glycans, we first need to develop a highly
efficient and robust procedure to install GlcNAc to a
monomeric mannopyranose substrate through the phospho-
diester linkage in a stereoselective manner (Scheme 1).
Received: June 10, 2011
Published: September 28, 2011
© 2011 American Chemical Society
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Figure 1. M6P N-glycans for hydrolase sorting. (A) A representative structure N-glycan carrying M6P and/or GlcNAc-M6P motifs. (B) Biosynthesis
of M6P. (C) A schematic structure of a homodimeric cation-independent MPR: Each octahedron is a MPH domain, and domains 3 and 9 are known
binding sites for M6P, while domain 5 was recently revealed as a GlcNAc-M6P binding site.
a
Scheme 1. Synthesis of GlcNAc-M6P Disaccharide
a
Reagents and conditions: (a) (i-Pr2N)2PCl, DIPEA, CH2Cl2, MS 4 Å, rt, 32%. (b) BnOH, 1H-tetrazole, CH2Cl2, rt, 36%. (c) (i) 4 (2 equiv), 6, 1H-
tetrazole, CH2Cl2, MS 4 Å, rt; (ii) t-BuO2H, 90% over two steps. (d) H2, Pd/C, MeOH, rt. (e) NH2NH2·H2O, MeOH; rt, 67% over steps d and e.
Among the various options for the construction of the
GlcNAc-M6P phosphodiester linkage, the disconnection
between the C6-OH group on the mannopyranose and the
phosphorus (P) group linked to C1α-OH of GlcNAc looked
particularly attractive. An O-(P-X) (X as a leaving group) group
can be first stereoselectively installed on the C1-OH of GlcNAc,
which then reacts as an electrophilic phosphorylation reagent
with the C6-OH group of a mannopyranose. If successful, the
difficult C1α-O-P stereochemistry can be pre-established in a
simple intermediate, and the overall sequence to synthesize
complex N-glycans will also be more convergent. The blueprint
of this approach had been elegantly demonstrated by Boons
and co-workers in the synthesis of GlcNAc-P-serine motifs
bearing a similar α phosphodiester linkage between the C1 of
GlcNAc and the β-OH group of serine.10 Using this strategy,
GlcNAc-P-N(i-Pr)2 4 was prepared from GlcNAc 2 in
moderate yield and excellent α stereoselectivity, which reacted
with the β-OH group of protected serine to form the desired
phosphotriester upon oxidation with tBuO2H. Initially, we
explored a new phosphoramidite reagent 5, a Bn-protected
version of reagent 4, for the synthesis of GlcNAc-M6P because
of its facile deprotection by catalytic hydrogenolysis. However,
this compound failed to undergo the desired phosphorylation
with mannopyranose due to intramolecular cyclization
presumably caused by the increased nucleophilicity of the
NHAc group (Scheme 1). To our delight, compound 4 reacted
well with monomeric mannopyranose 6 to give GlcNAc-M6P 7
in excellent yield under the tetrazole-promoted coupling and
tBuO2H-mediated oxidation condition. Following Boons's
procedure, a clean deprotection of GlcNAc-M6P 7 was
achieved to provide the disaccharide 9 in a two-step sequence:
first removal of the OBn groups via catalytic hydrogenolysis,
followed by removal of the OAc groups with NH2NH2·H2O in
MeOH. It is noteworthy that, in contrast to the labile Bn-
protected phosphotriester linkage, phosphodiester linkage was
stable under the above nucleophilic conditions.
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a
Scheme 2. Synthesis of the Hybrid N-Glycan 20
a
Reagents and conditions: (a) (i) NIS, TfOH, Et2O, MS 4 Å, −20 °C−rt, 92%; (ii) PhBCl2, Et3SiH, CH2Cl2, −78 °C, 92%. (b) (i) TABF, THF, rt,
90%; (ii) Ac2O, Py, rt, 95%. (c) 12, 14 (2 equiv), NIS, AgOTf, Et2O, MS 4 Å, −20 °C−rt. (d) (i) Ethylenediamine, n-BuOH, then Ac2O, Py, rt,
∼50% over steps c and d−i; (ii) CH3COSH, CHCl3, reflux, 82%. (e) (i) NaOMe, MeOH, rt, >95%; (ii) dibenzyl N,N-diisopropylphosphoramidite
(3 equiv), 1H-tetrazole; (iii) mCPBA, 95%, over two steps. (f) (i) TABF, THF, rt, >95%; (ii) 4 (2 equiv), 1H-tetrazole, CH2Cl2, MS 4 Å; then t-
BuO2H, >95%, over two steps. (g) H2, Pd/C, MeOH, rt. (h) NH2NH2·H2O, MeOH, rt, 81%, over two steps g and h.
With this highly efficient GlcNAc-M6P construction method
in hand, we set out to synthesize complex N-glycans carrying
GlcNAc-M6P and/or M6P motifs. An octasaccharide skeleton
as shown in structure 1 (Figure 1A) was chosen as our target.4
To allow for different modes of phosphorylation at the late
stage of synthesis, two mannose branches bearing differentially
protected C6-OH groups with OTIPS and OAc needed to be
built into the skeleton (compound 16, Scheme 2).
Following our previous synthetic route for the bis-M6P N-
glycan, Man2 compound 10 carrying the O-TIPS group was
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a
Scheme 3. Synthesis of the Bis GlcNAc-M6P N-Glycan 24
a
Reagents and conditions: (a) (i) NaOMe, MeOH, rt, >95%; (ii) TABF, THF, rt, >95%. (b) 4 (5 equiv), 1H-tetrazole, CH2Cl2, MS 4 Å; then t-
BuO2H, >95%, over two steps. (c) H2, Pd/C, MeOH, rt. (d) NH2NH2·H2O, MeOH, rt, 87%, over steps c and d.
first installed to the central trisaccharide 11 under the
promotion of NIS and TfOH, followed by selective benzylidene
opening, to give pentasaccharide 12 in good yield.9 The TIPS
group of Man3 branch 13, used in our previous synthesis,9 was
converted to the OAc group. The resulting trisaccharide 14 was
installed onto 12 under the promotion of NIS and AgOTf.
Subsequently, the Phth groups were converted to the NHAc
groups; the anomeric azido group was transformed to NHAc by
AcSH11 to provide octasaccharide 16 with two different masked
phosphorylation sites. The OAc group of 16 was then removed
by the treatment with NaOMe in MeOH; the unmasked OH
group was phosphorylated with dibenzyl N,N-diisopropylphos-
phoramidite, followed by tBuO2H oxidation, to place the first
Bn-protected phosphoester motif in 17. The TIPS group of 17
was then removed by TBAF. To our delight, installation of the
GlcNAc phosphotriester on the octasaccharide substrate
proceeded cleanly with 2 equiv of reagent 4 under the standard
coupling/oxidation conditions. Following the sequential
deprotection of OBn and OAc groups of 18, analytically pure
product 20 carrying both M6P and GlcNAc-M6P motifs was
obtained in nearly quantitative yield. The stereochemistry of
Following the same late stage phosphorylation strategy, N-
glycan 24 carrying two GlcNAc-M6P motifs, a compound
found in secreted hydrolases,6 was also synthesized. As shown
in Scheme 3, both OAc and OTIPS groups of compound 15
were removed to provide intermediate 21. Installation of two
GlcNAc phosphotriester linkages on 21 proceeded cleanly with
5 equiv of phosphoramidite 4 under the standard phosphor-
ylation condition to give compound 22. Final deprotection of
OBn and OAc groups of 22 provided the decasaccharide 24 in
excellent yield.
CONCLUSION
■
In summary, we developed a highly efficient and convergent
method for the synthesis of GlcNAc-M6P motif. Following a
late stage phosphorylation strategy, we have achieved the first
syntheses of complex N-glycans carrying well-defined GlcNAc-
M6P and/or M6P motifs. We expect this strategy would allow
us to prepare a series of N-glycans with various glycoforms and
phosphoforms for detailed structural and binding analyses with
CI-MPRs.
EXPERIMENTAL SECTION
Allyl 6-[Benzyl-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-
glucopyranosyl)]-phosphonato-2,3,4-tri-O-benzyl-α-D-manno-
■
1
C1α of GlcNAc in 20 was confirmed by H NMR (see the
Supporting Information).
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pyranoside (7). To a mixture of compounds 410 (120 mg, 0.2
mmol) and 612 (50 mg, 0.1 mmol) in CH2Cl2 (4 mL) under an
atmosphere of N2 was added 1H-tetrazole solution (1.0 mL, 0.45 M
solution in CH3CN). The reaction mixture was stirred at room
temperature for 12 h and then cooled to −40 °C. t-BuO2H (47 μL,
70% solution in water) was then added and stirred for 2 h at −40 °C.
The solvent was then removed under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
(EtOAc/Hex, 4/1). This product was further purified by size-exclusion
column chromatography over Sephadex LH-20 (MeOH/CH2Cl2, 1/1)
to give compound 7 as a yellow oil (75 mg, 90%). Rf = 0.4 (EtOAc/
Hex, 4/1). [α]D20 25 (c 0.44, CH2Cl2). 1H NMR (CDCl3, 400 MHz):
δ 7.25−7.32 (m, 20 H), 6.07 (d, 1 H, J = 9.3 Hz, NH), 5.70 (dd, J =
3.3 Hz, J1 = 5.8 Hz), 5.85 (m, 1H), 5.06−5.22 (m, 6 H), 4.86−4.96
(m, 2 H), 4.72 (s, 1 H), 4.56−4.68 (m, 4 H), 4.34−4.43 (m, 2 H),
4.28−4.31 (m, 1 H), 4.09−4.15 (m, 2 H), 3.88−4.03 (m, 5 H), 3.79−
3.82 (m, 2 H), 2.00 (s, 3 H, Ac), 1.99 (s, 3 H, Ac), 1.97 (s, 3 H, Ac),
1.82 (s, 3 H, Ac). 13C NMR (CDCl3, 75 MHz): δ 171.5, 171.0, 170.6,
169.5, 138.5, 133.8, 129.1, 128.8, 128.7, 128.4 (2 C), 128.2, 128.0,
117.8, 97.7, 80.3, 77.7, 75.6, 74.8, 74.3, 73.1, 72.5, 70.7, 69.9, 68.6,
67.7, 67.6, 61.5, 35.6, 23.3, 21.0, 20.9. 31P NMR (CDCl3, 146 MHz): δ
−1.82, −1.94 (diastereomers). IR (NaCl, cm−1): 2942, 1755, 1244,
1453, 1098. HRMS (ESI+) calcd for C83H94NO15S+ [M + NH4 ],
1376.6344; found, 1376.6349. Compound 25 (100 mg, 0.07 mmol)
was dissolved in anhydrous pyridine (2 mL) and Ac2O (2 mL). After it
was stirred at room temperature for 12 h, the solution was diluted with
EtOAc and washed with aqueous HCl (1 M), saturated NaHCO3, and
brine. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified via silica gel column
chromatography (EtOAc/Hex, 1/4) to give compound 14 as a yellow
+
20
oil (98 mg, 95%). Rf = 0.67 (EtOAc/Hex, 1/2). [α]D 17.9 (c 2.6,
CH2Cl2). 1H NMR (CDCl3, 300 MHz): δ 7.14−7.33 (m, 45 H), 5.32
(s, 1 H), 5.30 (s, 1 H), 4.98 (s, 1 H), 4.91 (d, 1 H, J = 10.9 Hz), 4.82
(d, 1 H, J = 10.4 Hz), 4.36−4.66 (m, 14 H), 4.20−4.28 (m, 4 H),
3.97−4.06 (m, 3 H), 3.69−3.89 (m, 13 H), 2.41−2.60 (m, 2 H), 1.97
(s, 3 H, Ac), 1.14−1.19 (m, 3 H). 13C NMR (CDCl3, 100 MHz): δ
171.4, 139.1 (2 C), 138.9, 138.8 (2 C), 138.7, 138.2, 128.9, 128.8,
128.7, 128.6, 128.5, 128.4, 128.3, 128.2 (2 C), 128.1, 128.0 (2 C),
127.9 (2 C), 127.8, 127.6, 100.4, 97.9, 81.4, 80.8, 80.6, 80.3, 77.7, 76.2,
75.8, 75.7, 75.4, 75.1, 74.8, 73.8, 73.5, 72.8, 72.5, 72.3, 72.1, 71.7, 71.5,
70.3, 69.5, 67.1, 63.9, 25.6, 21.4, 15.3. IR (NaCl, cm−1): 2917, 1453,
1104. HRMS (ESI+) calcd for C85H96NO16S+ [M + NH4 ], 1418.6450;
+
found, 1418.6461.
Acetamido (6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-mannopyra-
nosyl)-(1→6)-O-[2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
(1→3)-O]-(2,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-
[(2,3,4-tri-O-benzyl-6-O-triisopropylsilyl-α-D-mannopyrano-
syl)-(1→2)-O-(3,4,6-O-tribenzyl-α-D-mannopyranosyl)-(1→3)-
O]-(2,4-di-O-benzyl-β-D-mannopyranosyl)-(1→4)-O-(2-acetami-
do-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-O-2-
acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside
(16). A mixture of donor 14 (470 mg, 0.335 mmol), acceptor 129
(525 mg, 0.234 mmol), and MS (4 Å, 500 mg) in anhydrous Et2O (6
mL) was stirred at room temperature under argon for 2 h. NIS (75.0
mg, 0.335 mmol) and AgOTf (5.0 mg, 0.112 mmol) were added at
−20 °C. The reaction mixture was stirred at −20 °C for 0.5 h before it
was quenched with a few drops of triethylamine. The resulting mixture
was filtered through Celite. The filtrate was diluted with CH2Cl2 and
washed with saturated NaHCO3 and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified via silica
gel chromatography (EtOAc/Hex, 1/4) to give the crude intermediate
product 15. To the suspension of compound 15 in n-BuOH (5 mL)
was added ethylenediamine (2.5 mL) at room temperature. The
mixture was stirred at 90 °C under Ar for 12 h and then concentrated
in vacuo. The residue was azeotroped with toluene for 3 times and was
then dissolved in anhydrous pyridine (5 mL) and Ac2O (5 mL). After
it was stirred at room temperature for 12 h, the solution was diluted
with EtOAc and washed with aqueous HCl (1 M), saturated NaHCO3,
and brine. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified via silica gel column
chromatography (EtOAc/Hex, 1/1) to give compound 26 as a yellow
oil (410 mg, ∼50% over three steps, see the Supporting Information
1028, 959. HRMS (ESI+) calcd for C51H64N2O17P+ [M + NH4 ],
+
1007.3943; found, 1007.3923.
Propyl 6-(2-Acetamido-2-deoxy-α-D-glucopyranosyl)-phos-
phonato-1-α-D-mannopyranoside (9). The phosphoester com-
pound 7 (60 mg, 0.06 mmol) was dissolved in MeOH (2 mL), and
Pd/C (100 mg, 10 wt %) was added. The resulting suspension was
placed under an atmosphere of H2. The reaction mixture was stirred
1
for 24 h. The progress of the reaction was monitored by H NMR
spectroscopy, which showed the disappearance of Ar-H peaks at δ H
=
7.40−7.15 ppm. The reaction mixture was filtered through a
polytetrafluoroethylene (0.2 μM) membrane filter, which, upon
concentration, gave the fully debenzylated phosphodiester intermedi-
ate 8. Compound 8 was then dissolved in MeOH (1 mL),
NH2NH2·H2O (0.24 mL) was added to the solution at room
temperature, and the reaction mixture was stirred for 8 h. The solvent
was then removed under reduced pressure, and the crude product was
purified via size-exclusion column chromatography (Sephadex G-25,
eluent H2O) to give compound 9 as a white amorphous powder (20
20
1
mg, 67%). [α]D 20.4 (c 0.49, H2O). H NMR (500 MHz; D2O): δ
5.39 (dd, 1 H, J1,2 = 2.9 Hz, J1,P = 6.7 Hz, H-1, α isomer)1, 4.78 (s, 1
H), 3.98−4.08 (m, 2 H), 3.85−3.89 (m, 2 H), 3.78−3.80 (m, 2 H),
3.70−3.75 (m, 3 H), 3.67−3.68 (m, 2 H), 3.58−3.60 (m, 1 H), 3.41−
3.50 (m, 2 H), 1.98 (s, 3 H, Ac), 1.52−1.56 (m, 2 H), 0.82−0.85 (m, 3
H). 13C NMR (D2O, 75 MHz): δ 174.9, 100.0, 94.4, 73.3, 71.8, 70.9,
70.8, 70.4, 70.0, 69.8, 66.6, 64.9, 60.6, 54.1, 22.4, 22.2, 10.2. 31P NMR
(D2O, 146 MHz): δ −1.99. IR (NaCl, cm−1): 3337, 1654, 1034.
HRMS (ESI+) calcd for C17H33NO14P+ [M + H+], 506.1639; found,
506.1624.
20
for its structure). Rf = 0.21 (EtOAc/Hex, 1/1). [α]D −4.6 (c 0.65,
CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.05−7.42 (m, 105 H), 6.22
(d, 1 H, J = 8.3 Hz, NH), 5.31 (s, 1 H), 5.20 (s, 1 H), 5.02−5.11 (m, 3
H), 4.70−4.94 (m, 10 H), 4.18−4.70 (m, 38 H), 3.35−4.14 (m, 43 H),
3.09−3.11 (m, 2 H), 1.94 (s, 3 H, Ac), 1.88 (s, 3 H, Ac), 1.59 (s, 3 H,
Ac), 0.99 (brs, 21 H). 13C NMR (CDCl3, 100 MHz): δ 171.0, 170.4 (2
C), 139.1, 139.0, 138.8, 138.7, 138.6, 138.5, 138.4, 138.2, 137.8, 128.7,
128.6, 128.4 (3 C), 128.3 (2 C), 128.2 (2 C), 128.1 (3 C), 127.9,
127.7 (2 C), 127.5, 127.4, 127.0, 102.0, 101.7, 100.0, 99.8, 98.7, 98.3,
98.1, 88.4, 82.7, 80.4, 80.2, 79.6, 79.5, 78.8, 78.6, 78.0, 77.3, 76.4, 75.9,
75.2, 75.1, 74.9, 74.8, 74.7, 74.4, 74.3, 74.2 (2 C), 73.9, 73.7, 72.7, 72.6,
72.3, 72.1, 71.9, 71.6, 71.5, 70.4, 70.0, 69.9, 69.1, 68.9, 68.3, 66.6, 66.2,
64.4, 63.6, 62.6, 55.3, 51.7, 23.4, 23.2, 21.0, 18.1, 12.0. IR (NaCl,
Ethanethiol (6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-mannopyra-
nosyl)-(1→6)-O-[2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)-
(1→3)-O]-2,4-di-O-benzyl-1-α-D-mannopyranoside (14). To the
solution of compound 139 (2.05 g, 1.35 mmol) in THF (30 mL) was
added TBAF/THF (1 M, 4.05 mL, 4.05 mmol) at room temperature.
After it was stirred at room temperature for 10 h, the reaction mixture
was concentrated in vacuo. The resulting residue was purified via silica
gel flash chromatography (EtOAc/Hex, 1/3) to give compound 25 as
a yellow oil (1.67 g, 90%, see the Supporting Information for its
20
structure). Rf = 0.24 (EtOAc/Hex, 1/2). [α]D 18.3 (c 0.6, CH2Cl2).
1H NMR (CDCl3, 400 MHz): δ 7.15−7.33 (m, 45 H), 5.31 (s, 1 H),
5.29 (s, 1 H), 4.96 (s, 1H), 4.90 (d, 1 H, J = 11.0 Hz), 4.83 (d, 1 H, J =
10.6 Hz), 4.55−4.68 (m, 9 H), 4.41−4.52 (m, 4 H), 4.26−4.29 (m, 3
H), 3.95−4.06 (m, 3 H), 3.84−3.93 (m, 9 H), 3.68−3.79 (m, 6 H),
2.46−2.58 (m, 2 H), 1.16−1.21 (m, 3 H). 13C NMR (CDCl3, 100
MHz): δ 138.7 (2 C), 138.6, 138.5, 138.3, 137.8, 137.7, 128.5, 128.4,
128.3 (3 C), 128.2, 128.1, 127.9, 127.8, 127.7 (2 C), 127.6 (2 C),
127.5, 127.3, 127.2, 127.1, 99.9, 97.7, 81.2, 80.3, 80.2, 79.9, 77.2, 75.7,
75.4, 75.2, 75.1, 75.0, 74.9, 74.8, 74.7, 73.4, 73.1, 72.7, 72.2, 72.0, 71.9,
71.8, 71.1, 71.0, 69.3, 66.8, 62.3, 25.3, 15.0. IR (NaCl, cm−1): 2907,
cm−1): 2864, 2113, 1454, 1054. HRMS (ESI+) calcd. for
+
C
210H240N6O41Si2+ [M + NH4 + H+], 1765.8351; found, 1765.8373.
To a stirred solution of compound 26 (560 mg, 0.16 mmol) in CHCl3
(5 mL) was added 2,6-lutidine (1.1 mL, 8.0 mmol) followed by
dropwise addition of thioacetic acid (563 μL, 8.0 mmol).13 The
reaction mixture was stirred at reflux and monitored by TLC. After
completion of the reaction, the solvent was evaporated, and the residue
was dried under vacuum. The residue was purified by a silica gel flash
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Article
column chromatography (MeOH/CH2Cl2, 1/50) to give compound
16 as a yellow oil (450 mg, 82%). Rf = 0.35 (MeOH/CH2Cl2, 1/30).
[α]D20 −1.8 (c 0.55, CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.21−
7.52 (m, 105 H), 5.38 (s, 1 H), 5.18−5.30 (m, 3 H), 5.09 (s, 1 H),
4.80−5.00 (m, 9 H), 4.30−4.76 (m, 38 H), 3.50−4.19 (m, 42 H),
3.30−3.48 (m, 2 H), 3.10−3.21 (m, 2 H), 2.00 (s, 3 H, Ac), 1.96 (s, 3
H, Ac), 1.80 (s, 3 H, Ac), 1.64 (s, 3 H, Ac), 1.06 (brs, 21 H). 13C
NMR (CDCl3, 100 MHz): δ 172.1, 171.0, 170.9, 170.1, 139.4, 139.2,
139.0, 138.8, 138.7 (2 C), 138.5, 138.4, 138.3, 138.2, 138.1, 138.0,
137.8, 137.6, 128.8, 128.7, 128.5, 128.4 (2 C), 128.3 (3 C), 128.2 (2
C), 128.1, 128.0, 127.9, 127.7 (2 C), 127.6 (2 C), 127.5, 127.4 (2 C),
127.3 (2 C), 127.2, 127.1, 127.0, 126.9, 126.8, 126.6, 101.9, 101.7,
100.0, 98.6, 98.3, 98.1, 82.9, 80.4, 80.2, 79.7, 79.5, 78.7, 78.4, 77.3,
75.9, 75.2, 75.1, 74.9, 74.6, 74.3, 74.2, 73.9, 73.6, 73.4 (2 C), 73.1, 73.0,
72.5, 72.3, 72.2, 72.1, 71.9, 71.6, 71.4, 70.4, 70.0, 69.8, 69.1, 68.4, 67.8,
66.7, 66.0, 64.4, 63.6, 62.6, 55.4, 52.7, 23.4, 23.3, 23.0, 20.9, 18.1, 12.0.
78.8, 78.5, 77.3, 75.8, 75.2, 75.0, 74.8, 74.6, 74.2, 73.9, 73.4, 73.0, 72.5,
72.3, 72.1, 72.0, 71.5, 71.4, 70.7, 70.4, 69.8, 69.5, 69.4, 69.0, 68.3, 67.7,
66.8, 66.5, 66.1, 29.7, 23.4, 23.0, 18.0, 12.0. 31P NMR (CDCl3, 146
MHz): δ −1.06. IR (NaCl, cm−1): 2923, 1659, 1454, 1052. HRMS
(ESI+) calcd for C224H255N4O44PSi2+ [M + NH4 + H+], 1882.8700;
+
found, 1882.8684.
Acetamido (6-Phosphato-α-D-mannopyranosyl)-(1→6)-O-[α-
D-mannopyranosyl-(1→6)-O]-(α-D-mannopyranosyl)-(1→3)-O-
[6-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-phosphonato-
(α-D-mannopyranosyl)-(1→2)-O-(α-D-mannopyranosyl)-(1→3)-
O]-(β-D-mannopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-D-
glucopyranosyl)-(1→4)-O-2-acetamido-2-deoxy-β-D-glucopyr-
anoside (20). To the solution of compound 17 (68 mg, 0.018
mmol) in THF (2 mL) was added TBAF/THF (1 M, 363 μL, 0.363
mmol) at room temperature. After it was stirred at room temperature
for 10 h, the reaction mixture was concentrated in vacuo. The resulting
residue was purified via silica gel flash chromatography (MeOH/
CH2Cl2, 1/50) to give compound 28 as an oil (64 mg, >95%, see the
Supporting Information for its structure). Rf = 0.33 (MeOH/CH2Cl2,
1/30). [α]D20 −2.3 (c 0.43, CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ
6.99−7.44 (m, 115 H), 5.24 (s, 1 H), 5.21 (s, 1 H), 4.71−5.10 (m, 17
H), 4.18−4.70 (m, 35 H), 3.40−4.15 (m, 45 H), 3.28−3.33 (m, 2 H),
3.06−3.13 (m, 2 H), 1.89 (s, 3 H, Ac), 1.72 (s, 3 H, Ac), 1.57 (m, 3 H,
Ac). 13C NMR (CDCl3, 75 MHz): δ 172.1, 170.9, 170.1, 139.4, 139.1,
138.7, 138.6, 138.5, 138.4, 138.2, 138.0, 137.9, 137.7, 136.0, 135.9,
135.8, 128.8, 128.7, 128.5, 128.4 (2 C), 128.3, 128.2, 128.1, 128.0 (2
C), 127.9 (2 C), 127.8, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0,
101.8, 101.1, 100.0, 99.6, 98.2, 98.0, 82.4, 80.2, 80.0, 79.9, 79.8, 79.5,
78.6, 78.3, 77.2, 75.9, 75.8, 75.2, 75.1, 75.0, 74.6, 74.2, 74.1, 73.6, 73.4
(2 C), 73.3, 73.0, 72.9, 72.4, 72.2, 72.1, 71.9, 71.3, 70.3, 69.3, 69.2,
69.1, 69.0, 66.6, 62.1, 29.7, 23.4, 23.0. 31P NMR (CDCl3, 146 MHz): δ
IR (NaCl, cm−1): 2933, 1664, 1053. HRMS (ESI+) calcd for
+
C
212H244N4O42Si2+ [M + NH4 + H+], 1773.8451; found, 1773.8427.
Acetamido (2,3,4-Tri-O-benzyl-6-dibenzylphosphonato-α-D-
mannopyranosyl)-(1→6)-O-[(2,3,4,6-tetra-O-benzyl-α-D-man-
nopyranosyl)-(1→3)-O]-(2,4-di-O-benzyl-α-D-mannopyrano-
syl)-(1→6)-O-[(2,3,4-tri-O-benzyl-6-O-triisopropylsilyl-α-D-
mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-benzyl-α-D-mannopyr-
anosyl)-(1→3)-O]-(2,4-di-O-benzyl-β-D-mannopyranosyl)-(1→
4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyrano-
syl)-(1→4)-O-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluco-
pyranoside (17). Compound 16 (440 mg, 0.125 mmol) was
dissolved in MeOH/CH2Cl2 (2/1, 10 mL) at room temperature.
NaOMe in MeOH was added until the pH of the reaction mixture
reached 10. After it was stirred at room temperature for 12 h, the
solution was neutralized with ion-exchange resin (H+), filtered, and
concentrated in vacuo. The residue was purified via silica gel
chromatography (MeOH/CH2Cl2, 1/50) to give the desired product
27 as a yellow oil (420 mg, >95%, see the Supporting Information for
−0.77. IR: υ 2925, 1659, 1454, 1027 cm−1. HRMS (ESI+) calcd for
+
C
215H235N4O44P2+ [M + NH4 + H+], 1804.8033; found, 1804.7988.
To a mixture of compounds 28 (66 mg, 0.018 mmol) and 4 (21 mg,
0.036 mmol) in CH2Cl2 (4 mL) under an atmosphere of nitrogen was
added 1H-tetrazole solution (176 μL, 0.45 M solution in acetonitrile).
The reaction mixture was stirred at room temperature for 12 h and
cooled to −40 °C, and t-BuO2H (92 μL, 70% solution in water) was
added and stirred for another 2 h. The solvent was removed under
reduced pressure, and the resulting residue was purified by silica gel
column chromatography (MeOH/CH2Cl2, 1/50). This product was
further purified by size-exclusion column chromatography over
Sephadex LH-20 (MeOH/CH2Cl2, 1/1) to give compound 18 as a
20
its structure). Rf = 0.35 (MeOH/CH2Cl2, 1/30). [α]D 28.0 (c 0.5,
CH2Cl2). 1H NMR (CDCl3, 300 MHz): δ 7.01−7.45 (m, 105 H), 5.32
(s, 1 H), 5.20 (s, 1 H), 5.12 (s, 1 H), 5.03 (s, 1 H), 4.92−4.95 (m, 2
H), 4.76−4.88 (m, 6 H), 4.64−4.71 (m, 7 H), 4.30−4.60 (m, 26 H),
4.10−4.30 (m, 6 H), 3.83−4.10 (m, 15 H), 3.40−3.82 (m, 28 H),
3.31−3.37 (m, 2 H), 3.07−3.15 (m, 2 H), 1.89 (s, 3 H, Ac), 1.74 (s, 3
H, Ac), 1.57 (s, 3 H, Ac), 1.00 (m, 21 H). 13C NMR (CDCl3, 100
MHz): δ 170.7, 169.6, 168.8, 137.8 (2 C), 137.6, 137.4, 137.3, 137.2,
137.1 (2 C), 136.9 (2 C), 136.7, 136.6, 136.5, 127.4 (2 C), 127.2,
127.1, 126.9 (2 C), 126.8, 126.7 (2 C), 126.5 (2 C), 126.4, 126.3 (2
C), 126.2, 126.1 (2 C), 125.9, 125.8, 125.7, 125.6, 125.4, 100.6, 100.3,
98.5, 97.2, 96.6, 95.5, 81.3, 79.1, 78.8, 78.7, 78.1, 77.8, 77.5, 77.2, 76.0,
74.6, 74.4, 73.9, 73.7, 73.5, 73.4, 73.3, 73.1, 72.9, 72.5, 72.3, 72.1, 72.0,
71.7, 71.1, 71.0, 70.7, 70.6, 70.4, 70.3, 70.2, 69.0, 68.4, 67.8, 66.9, 66.4,
64.8, 64.6, 61.2, 60.9, 54.2, 51.4, 22.1, 21.7, 16.7, 10.7. IR (NaCl,
20
yellow oil (60 mg, >95%). Rf = 0.37 (MeOH/CH2Cl2, 1/30). [α]D
3.57 (c 1.12, CH2Cl2). 1H NMR (CDCl3, 600 MHz): δ 7.03−7.28 (m,
120 H), 5.95 (d, 1 H, J = 6.0 Hz, NH), 5.68 (dd, 1 H, J1,2 = 3.2 Hz, J1,P
= 5.7 Hz, H-1, α isomer), 5.29 (brs, 2 H), 5.10−5.15 (m, 5 H), 4.10−
5.09 (m, 11 H), 4.74−4.82 (m, 5 H), 4.57−4.71 (m, 6 H), 4.18−4.55
(m, 31 H), 3.82−4.32 (m, 22 H), 3.35−3.81 (m, 26 H), 3.25−3.33 (m,
1 H), 3.16−3.20 (m, 1 H), 3.11−3.14 (m, 1 H), 1.94 (s, 3 H, Ac), 1.93
(s, 3 H, Ac), 1.89 (s, 3 H, Ac), 1.77 (s, 3 H, Ac), 1.71 (s, 3 H, Ac), 1.70
(s, 3 H, Ac), 1.57 (s, 3 H, Ac). 13C NMR (CDCl3, 75 MHz): δ 171.0,
170.0, 169.9, 169.4, 169.1, 168.1, 138.2, 138.1, 137.7, 137.6, 137.4,
137.2, 137.1, 136.8, 134.9, 134.8, 134.1, 128.7, 127.8, 127.6, 127.4,
127.3, 127.2, 127.1, 127.0 (2 C), 126.9, 126.8, 126.7, 126.6, 126.5,
126.4, 126.3, 126.2, 126.1, 126.0, 125.8, 100.9, 100.2, 99.0, 98.8, 98.2,
97.4, 97.1, 95.3, 79.2, 78.9, 78.7, 78.4, 77.8, 77.5, 76.3, 75.0, 74.8, 74.1,
73.8, 73.6, 73.3, 72.7, 72.5, 72.3, 72.0, 71.8, 71.5, 71.0, 70.9, 70.8, 70.3,
69.4, 69.2, 68.9, 68.6, 68.3, 68.1, 67.6, 66.2, 65.3, 65.1, 60.0, 54.7, 51.7,
50.8, 28.7, 22.5, 22.4, 22.0, 21.8, 19.6, 19.5. 31P NMR (CDCl3, 146
MHz): δ −0.98, −1.95. IR (NaCl, cm−1): 2923, 1748, 1665, 1454,
cm−1): 2917, 1659, 1454, 1051. HRMS (ESI+) calcd for
210H242N4O41Si2+ [M + NH4 + H+], 1752.8398; found, 1752.8414.
+
C
A mixture of compound 27 (72 mg, 0.02 mmol), 1H-tetrazole (222
μL, 0.45 M in CH3CN, 0.1 mmol), and 4 Å molecular sieves (50 mg)
in anhydrous CH2Cl2 (3 mL) was stirred at room temperature under
Ar for 2 h. Dibenzyl N,N-diisopropylphosphoramidite (27 μL, 0.08
mmol) was then added, and the reaction mixture was stirred at room
temperature for another 3 h. Subsequently, mCPBA (18.0 mg, 0.08
mmol) was added; the solution was stirred at room temperature for
another 1 h. The reaction was quenched by the addition of a few drops
of triethylamine and filtered. The filtrate was concentrated in vacuo,
and the resulting residue was purified by silica gel flash
chromatography (MeOH/CH2Cl2, 1/50) to give product 17 as a
1047. HRMS (ESI+) calcd for C236H261N5O55P2 [M + NH4 + H+],
2054.3655; found, 2054.3630. Phosphoester 18 (36 mg, 0.009 mmol)
was dissolved in MeOH (2 mL), and Pd/C (50 mg, 10 wt %) was
added. The resulting suspension was placed under an atmosphere of
H2. The reaction mixture was stirred at room temperature under H2
atmosphere for 24 h. The progress of the reaction was monitored by
1H NMR spectroscopy, which showed the disappearance of Ar-H
2+
+
20
yellow oil (74 mg, 95%). Rf = 0.33 (MeOH/CH2Cl2, 1/30). [α]D
−1.8 (c 0.55, CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.00−7.45 (m,
115 H), 5.37 (s, 1 H), 5.32 (s, 1 H), 4.60−5.23 (m, 22 H), 4.15−4.60
(m, 31 H), 3.35−4.15 (m, 45 H), 3.15−3.20 (m, 2 H), 1.93 (s, 3 H,
Ac), 1.78 (s, 3 H, Ac), 1.63 (s, 3 H, Ac), 1.01 (brs, 21 H). 13C NMR
(CDCl3, 100 MHz): δ 172.3, 171.3, 170.6, 139.2, 139.1, 138.9, 138.6,
138.5, 138.3, 138.2, 138.0, 137.9, 137.8, 135.7, 128.7, 128.5, 128.4,
128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.5, 127.2, 126.9, 126.6,
102.0, 101.6, 100.0, 99.7, 98.5, 98.3, 98.2, 82.8, 80.4, 80.2, 79.9, 79.4,
peaks at δ = 7.40−7.15 ppm. The reaction mixture was filtered
H
through a polytetrafluoroethylene (0.2 μM) membrane filter to give
8687
dx.doi.org/10.1021/jo2010999|J. Org. Chem. 2011, 76, 8682−8689

The Journal of Organic Chemistry
Article
the fully debenzylated phosphoester intermediate 19. NH2NH2·H2O
(0.24 mL) was added to a solution of 19 in MeOH (1 mL), and the
reaction mixture was stirred for 8 h. The solvent was removed under
reduced pressure, and the crude product was purified by size-exclusion
column chromatography over Sephadex G-25 (eluent: H2O) to give
compound 20 as a white amorphous powder upon freeze drying (13
mg, 81%). [α]D20 15 (c 0.4, H2O). 1H NMR (600 MHz; D2O): δ 5.33
(dd, 1 H, J1,2 = 3.2 Hz, J1,P = 7.1 Hz, H-1, α isomer), 5.22 (s, 1 H),
5.07 (s, 1 H), 4.89 (d, 1 H, J = 9.7 Hz), 4.86 (s, 1 H), 4.79 (s, 1 H),
4.75 (s, 1 H), 4.65 (s, 1 H), 4.44−4.47 (m, 1 H), 4.05−4.15 (m, 1 H),
3.98−4.04 (m, 1 H), 3.40−3.95 (m, 52 H), 1.92 (s, 3 H, Ac), 1.91 (s, 3
H, Ac), 1.84 (s, 3 H, Ac), 1.75 (s, 3 H, Ac). 13C NMR (D2O, 125
MHz): δ 181.4, 174.9, 174.7, 174.5, 102.3, 101.1, 100.8, 100.7, 100.0,
99.8, 99.5, 93.9, 93.8, 80.8, 79.2 (2 C), 79.1, 78.5, 78.2, 76.1, 74.4, 74.2,
73.9, 73.5, 73.3, 73.0, 72.6, 72.2, 72.0, 71.9, 71.7, 70.3 (2 C), 70.2 (2
C), 70.1, 70.0, 69.8, 69.4, 69.3, 66.9, 66.5, 66.4, 66.1, 65.9, 65.7, 65.6,
65.1, 62.7, 61.0, 60.7, 60.2, 59.8, 59.7, 55.0, 53.9, 53.8, 53.7, 23.2, 22.1,
22.0, 21.9, 21.8, 21.6. 31P NMR (D2O, 146 MHz): δ −0.72, 4.76. IR
(NaCl, cm−1): 3292, 1061. HRMS (ESI−) calcd for C62H104N4O52P2−
[M − 2H+], 899.2546; found, 899.2545.
6.8 Hz), 6.05 (d, 1 H, J = 8.6 Hz), 5.69 (s, 2 H), 3.31−5.69 (m, 114
H), 1.98 (s, 3 H, Ac), 1.95 (s, 3 H, Ac), 1.94 (s, 3 H, Ac), 1.93 (s, 3 H,
Ac), 1.89 (s, 3 H, Ac), 1.85 (s, 3 H, Ac), 1.80 (s, 3 H, Ac), 1.72 (s, 3 H,
Ac), 1.67 (s, 3 H, Ac), 1.64 (s, 3 H, Ac), 1.57 (s, 3 H, Ac). 13C NMR
(CDCl3, 75 MHz): δ 171.4, 170.9, 169.7, 169.6, 169.4, 169.3, 169.0,
168.9, 168.8, 168.0, 167.9, 138.1, 137.9, 137.5, 137.4 (2 C), 137.3,
137.1, 137.0, 136.9, 136.8, 136.7, 136.5, 134.2, 134,1 (2 C), 134.0,
127.6, 127.5, 127.3, 127.2, 127.1, 127.0 (2 C), 126.8 (2 C), 126.7,
126.6, 126.5, 126.4, 126.3 (2 C), 126.2, 126.1, 126.0, 125.9, 125.8,
125.6, 125.4, 100.6, 100.0, 98.7, 98.1, 98.0, 97.3, 95.3, 95.2, 95.1, 81.6,
81.0 (2 C), 78.7, 78.6, 78.2, 77.4, 77.3, 76.1, 74.7, 74.6, 74.1, 74.0, 73.9,
73.8, 73.7, 73.7, 73.6, 73.5, 73.4, 73.3, 73.1, 73.0, 72.4, 72.2, 72.1, 71.9,
71.5, 71.3, 71.2, 71.0, 70.8 (2 C), 70.6, 70.1, 69.8, 69.3, 69.2, 69.1, 68.7,
68.6, 68.4, 68.3, 67.8, 67.3, 66.5, 66.2, 66.0, 65.8, 65.6, 60.0, 59.9, 54.3,
51.5, 50.5, 50.3 (2 C), 50.2, 22.2 (2 C), 21.8, 21.6 (2 C), 19.5, 19.4,
19.3 (2 C). 31P NMR (CDCl3, 146 MHz): δ −1.99. IR (NaCl, cm−1):
3286, 2939, 1751, 1666, 1041. HRMS (ESI+) calcd for
243H273N6O63P2 [M + NH4 + H+], 2173.8977; found, 2173.8948.
2+
+
C
Acetamido 6-(2-Acetamido-2-deoxy-α-D-glucopyranosyl)-
phosphonato-(α-D-mannopyranosyl)-(1→6)-O-[α-D-mannopyr-
anosyl-(1→6)]-(α-D-mannopyranosyl)-(1→3)-O-[6-(2-acetami-
do-2-deoxy-α-D-glucopyranosyl)-phosphonato-(α-D-manno-
pyranosyl)-(1→2)-O-(α-D-mannopyranosyl)-(1→3)-O]-(β-D-
mannopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-D-gluco-
pyranosyl)-(1→4)-O-2-acetamido-2-deoxy-β-D-glucopyrano-
side (24). Compound 22 (50 mg, 0.01 mmol) was dissolved in
MeOH (2 mL), and Pd/C (50 mg, 10 wt %) was added. The resulting
suspension was placed under an atmosphere of H2. The reaction
mixture was stirred at room temperature for 24 h. The progress of the
Acetamido (2,3,4-Tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-
O-[2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-(1→3)-O]-(2,4-
di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-[(2,3,4-tri-O-ben-
zyl-α-D-mannopyranosyl)-(1→2)-O-(3,4,6-O-tribenzyl-α-D-man-
nopyranosyl)-(1→3)-O]-(2,4-di-O-benzyl-β-D-mannopyrano-
syl)-(1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluco-
pyranosyl)-(1→4)-O-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-
D-glucopyranoside (21). To the solution of compound 27 (68 mg,
0.02 mmol, derived from 16) in THF (2 mL) was added TBAF/THF
(1 M, 100 μL, 0.1 mmol) at room temperature. After it was stirred at
room temperature for 10 h, the reaction mixture was concentrated in
vacuo and purified via silica gel flash chromatography (MeOH/
CH2Cl2, 1/50) to give compound 21 as a yellow oil (64 mg, >95%). Rf
1
reaction was monitored by H NMR spectroscopy, which showed the
disappearance of Ar-H peaks at δ = 7.40−7.15 ppm. The reaction
H
mixture was filtered through a polytetrafluoroethylene (0.2 μM)
membrane filter to give the fully debenzylated intermediate 23.
NH2NH2·H2O (0.24 mL) was added to the solution of compound 23
in MeOH (1 mL), and the reaction mixture was stirred at room
temperature for 8 h. The solvent was removed under reduced pressure,
and the crude product was purified by size-exclusion column
chromatography over Sephadex G-25 (eluent: H2O) to give 24 as a
white amorphous powder upon freeze drying (20 mg, 87%). [α]D20 23
20
1
= 0.35 (MeOH/CH2Cl2, 1/30). [α]D −4.5 (c 0.44, CH2Cl2). H
NMR (CDCl3, 400 MHz): δ 7.00−7.50 (m, 105 H), 5.28 (s, 1 H),
5.20 (s, 1 H), 5.11 (s, 1 H), 5.08 (s, 1 H), 4.93−5.05 (m, 4 H), 4.78−
4.88 (m, 6 H), 4.20−4.73 (m, 35 H), 4.09−4.17 (m, 3 H), 3.83−4.03
(m, 14 H), 3.42−3.77 (m, 28 H), 3.28−3.34 (m, 2 H), 3.07−3.14 (m,
2 H), 1.89 (s, 3 H, Ac), 1.72 (s, 3 H, Ac), 1.56 (s, 3 H, Ac). 13C NMR
(CDCl3, 75 MHz): δ 172.0, 170.9, 170.0, 139.2, 139.1, 138.7, 138.6,
138.5 (2 C), 138.4, 138.3, 138.2, 138.0 (2 C), 137.9, 137.8, 137.7,
128.7, 128.6, 128.5, 128.4, 128.3 (2 C), 128.2, 128.1, 127.8, 127.6,
127.5, 127.4 (2 C), 127.2, 127.1, 127.0, 126.8, 101.8, 101.1, 100.0,
99.8, 97.9, 97.8, 82.3, 80.2, 80.0, 79.8, 79.4, 79.1, 78.7, 78.5, 77.2, 75.9,
75.7, 75.3, 75.0, 74.9, 74.7, 74.6, 74.3, 74.1, 73.6, 73.4, 73.3, 73.0, 72.9,
72.4, 72.2, 72.0, 71.8, 71.7, 71.6, 70.3, 69.5, 69.1, 68.2, 67.7, 66.1, 62.2,
55.4, 52.7, 29.7, 23.4, 23.0. IR (NaCl, cm−1): 2924, 1654, 1051. HRMS
(ESI) calcd for C201H222N4O412+ [M + NH4+ + H+], 1674.7732; found,
1674.7770.
1
(c 0.65, H2O). H NMR (500 MHz; D2O): δ 5.40−5.41 (m, 2 H),
5.30 (s, 1 H), 5.15 (s, 1 H), 4.97 (d, 1 H, J = 9.8 Hz), 4.94 (s, 1 H),
4.86 (s, 1 H), 4.82 (s, 1 H), 4.52−4.55 (m, 1 H), 4.17 (s, 1 H), 3.46−
4.20 (m, 60 H), 2.00 (s, 9 H, Ac), 1.93 (s, 6 H, Ac). 13C NMR (D2O,
75 MHz): δ 175.3, 175.1, 175.0, 174.9, 102.8, 101.5 (2 C), 101.2,
100.4 (2 C), 99.9, 94.5, 94.4 (2 C), 79.6, 79.0, 78.6, 76.5, 76.5, 74.8,
74.4, 74.0, 73.7, 73.4, 73.3, 72.4, 72.1, 71.0, 70.8, 70.7, 69.9, 69.8, 67.3,
66.8, 66.4, 66.1, 65.5, 64.9, 61.5, 61.2, 60.7, 60.6, 60.2, 55.4, 54.0 (2 C),
22.5, 22.4, 22.3 (2 C). 31P NMR (D2O, 146 MHz): δ −1.23, −1.31. IR
(NaCl, cm−1): 3324, 1646, 1031. HRMS (ESI−) calcd for
C70H117N5O57P2 [M − 2H+], 1000.7948; found, 1000.7927.
2−
Acetamido 6-[Benzyl-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-phosphonato]-2,3,4-tri-O-benzyl-
(α-D-mannopyranosyl)-(1→6)-O-[(2,3,4,6-tetra-O-benzyl-α-D-
mannopyranosyl)-(1→3)-O]-(2,4-di-O-benzyl-α-D-mannopyra-
nosyl)-(1→6)-O-{6-[benzyl-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-phosphonato-(2,3,4-tri-O-benzyl-
α-D-mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-benzyl-α-D-manno-
pyranosyl)-(1→3)-O}-(2,4-di-O-benzyl-β-D-mannopyranosyl)-
(1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyr-
anosyl)-(1→4)-O-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-
glucopyranoside (22). To a mixture of compounds 21 (65 mg, 0.02
mmol) and 4 (57 mg, 0.1 mmol) in CH2Cl2 (4 mL) under an
atmosphere of nitrogen was added 1H-tetrazole solution (260 μL, 0.45
M solution in acetonitrile). The reaction mixture was stirred at room
temperature for 12 h and then cooled to −40 °C, and t-BuO2H (27
μL, 70% solution in water) was added and stirred for another 2 h. The
solvents were then removed under reduced pressure, and the residue
was purified by silica gel column chromatography (MeOH/CH2Cl2, 1/
50). The resulting product was further purified by size-exclusion
column chromatography over Sephadex LH-20 (MeOH/CH2Cl2, 1/1)
to give compound 22 (75 mg, >95%) as an oil. Rf = 0.36 (MeOH/
ASSOCIATED CONTENT
* Supporting Information
■
S
General experimental information, structures of all intermedi-
ates, and NMR spectra for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: guc11@psu.edu.
■
ACKNOWLEDGMENTS
We gratefully acknowledge The Pennsylvania State University
for financial support of this work.
■
REFERENCES
■
20
1
CH2Cl2, 1/30). [α]D 9.6 (c 2.7, CH2Cl2). H NMR (CDCl3, 500
MHz): δ 7.78 −8.02 (4 H), 7.03−7.49 (m, 111 H), 6.28 (d, 1 H, J =
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