Synthesis of biantennary complex-type nonasaccharyl asn building blocks for solid-phase glycopeptide synthesis

Article abstract of DOI:10.1021/jo200149d

The biantennary complex-type N-glycans bearing LacNAc and LacdiNAc as the nonreducing end motif were synthesized in a protected form suitable to use in the Fmoc solid-phase peptide synthesis studies. Two approaches for the nonasaccharide synthesis were examined by taking advantage of the highly β-selective glycosylation with GlcNTCA (N-phenyl)trifluoroacetimidate. An earlier approach, which involved the reaction of the trisaccharide donor (Gal-GlcNTCA-Man) and trisaccharide acceptor (Man-GlcNPhth₂-N ₃), produced a mixture of nonasaccharide isomers. On the other hand, mannosylation of the trisaccharide acceptor (Man-GlcNPhth₂-N ₃) stereoselectively afforded the known pentasaccharide (Man ₃-GlcNPhth₂-N₃), which was reacted with the disaccharyl glycosyl donor (Gal-GlcNTCA or GalNTCA-GlcNTCA) to produce the desired nonasaccharide as a single stereoisomer. Selective dephthaloylation followed by N-acetylation furnished the GlcNAc₂ functionality. The resulting nonasaccharyl azides were condensed with Fmoc-Asp(OPfp)-OBu ^t or Fmoc-Asp(OPfp)-OPac in the presence of Ph(CH₃) ₂P and HOOBt. Finally, the Zn reduction and cleavage of the tert-butyl ester or Zn reduction alone produced the targeted nonasaccharyl Asn building blocks.

Full text of DOI:10.1021/jo200149d

ARTICLE
pubs.acs.org/joc
Synthesis of Biantennary Complex-Type Nonasaccharyl Asn Building
Blocks for Solid-Phase Glycopeptide Synthesis
Masashi Hagiwara,† Mizuki Dohi,† Yuko Nakahara,†,‡ Keiko Komatsu,† Yuya Asahina,† Akiharu Ueki,†
Hironobu Hojo,*,† Yoshiaki Nakahara,*,† and Yukishige Ito‡
†Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka-shi,
Kanagawa 259-1292, Japan
‡RIKEN, Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
S Supporting Information
b
ABSTRACT: The biantennary complex-type N-glycans bear-
ing LacNAc and LacdiNAc as the nonreducing end motif were
synthesized in a protected form suitable to use in the Fmoc
solid-phase peptide synthesis studies. Two approaches for the
nonasaccharide synthesis were examined by taking advantage of
the highly β-selective glycosylation with GlcNTCA (N-phenyl)tri-
fluoroacetimidate. An earlier approach, which involved the reaction
of the trisaccharide donor (Gal-GlcNTCA-Man) and trisaccharide
acceptor (Man-GlcNPhth2-N3), produced a mixture of nonasac-
charide isomers. On the other hand, mannosylation of the
trisaccharide acceptor (Man-GlcNPhth2-N3) stereoselectively af-
forded the known pentasaccharide (Man3-GlcNPhth2-N3), which
was reacted with the disaccharyl glycosyl donor (Gal-GlcNTCA or GalNTCA-GlcNTCA) to produce the desired nonasaccharide as a
single stereoisomer. Selective dephthaloylation followed by N-acetylation furnished the GlcNAc2 functionality. The resulting
nonasaccharyl azides were condensed with Fmoc-Asp(OPfp)-OBut or Fmoc-Asp(OPfp)-OPac in the presence of Ph(CH3)2P and
HOOBt. Finally, the Zn reduction and cleavage of the tert-butyl ester or Zn reduction alone produced the targeted nonasaccharyl Asn
building blocks.
reaction (Scheme 1). In addition, the adoption of the N-tri-
chloroacetyl group, which could be easily converted to an
N-acetyl group by Zn reduction, made it possible to achieve
the highly convergent synthesis of the O-glycoamino acid. The
powerful stereocontrolling ability of the N-trichloroacetyl group
used in the construction of the β-LacNAc glycoside prompted us
to apply this method to the synthesis of the complex type
N-glycoasparagines 1 and 2 (Figure 1).
In the last two decades, many synthetic investigations have
been directed to the N-glycan derivatives. Chemical and che-
moenzymatic syntheses of the biantennary complex-type N-
glycan framework carrying an R-(2f6)- or R-(2f3)-disialo
motif have been reported.6 However, only a limited number of
research groups have realized the further transformation of the
synthetic full-size N-glycan into a glycosylated oligopeptide7,8
because considerable processes are required to produce a
sufficient quantity of such an N-glycan sample. Alternative ap-
proaches utilizing the N-glycans derived from natural sources
have also been demonstrated,9ꢀ11 but the natural N-glycans obtain-
able in quantity are so far structurally restricted.12 Therefore, the
1. INTRODUCTION
Insights into the molecular mechanisms of such significant
glycoprotein-mediated biological events including cancer metas-
tasis, apoptosis, and microbe infection are expected to produce
many improvements in diagnostic or therapeutic approaches.
However, only a limited amount of glycoprotein samples is
available from natural sources, and the attached oligosaccharides
are frequently heterogeneous. Thus, a large number of studies
have been directed toward establishing a reliable synthetic
method for glycopeptides including those as large as glycopro-
teins since chemically synthesized homogeneous samples are
useful to study the biological functions of not only the attached
oligosaccharides but also the protein portions.1,2
Among these studies, we have synthesized both the N- and O-
glycan-linked glycopeptides using the benzyl-protected oligosac-
charyl amino acids as a building block for the Fmoc solid-phase
peptide synthesis (SPPS). The benzyl groups were ultimately
removed under the conditions of low-acidity TfOH with mini-
mum rupture of the acid-labile glycosidic linkages.3
Recently, we accomplished the highly stereoselective synthesis
of a series of LacNAc-containing O-glycans3ꢀ5 based on the use
of the benzyl- and/or benzylidene-protected N-trichloroacetyl-
lactosaminyl fluoride as a glycosyl donor in the key glycosylation
Received: January 27, 2011
Published: May 25, 2011
r
2011 American Chemical Society
5229
dx.doi.org/10.1021/jo200149d J. Org. Chem. 2011, 76, 5229–5239
|

The Journal of Organic Chemistry
ARTICLE
Scheme 1. Synthetic Route for O-Glycoamino Acid Using the Key LacNAc Donor
Scheme 2. Retrosynthetic Pathway to 3 by the First Plan
trisaccharide 814 and 2 equiv of the LacNAc-Man donor 7 (see
Scheme 2). The β-mannoside synthesis of the trisaccharide 8 was
facilitated using Crich’s method (Scheme 3).15,16 The mannosyl
thioglycoside 1117 was activated with 1-benzenesulfinyl piper-
idine (BSP), 2,4,6-tri-tert-butylpyrimidine (TTBP), and Tf2O at
ꢀ60 °C and reacted with the disaccharide 12 for 2 days. The
product 13 was obtained as a mixture of stereoisomers (β/R =
7/1). The major isomer was separated from the minor one by
recycling HPLC, which enables the fraction containing the
desired product to return to the same column and repeat the
cycles until the separation with the minor component becomes
perfect. The highly pure compound 13 was obtained in 73%
Figure 1. Structures of Asn-linked nonasaccharides 1 and 2 and
synthetic targets 3 and 4.
development of an efficient method to synthesize the glycoaspar-
agine units with diverse N-glycan structures is still requested.
In this paper, we describe our synthetic approach to the
complex type N-glycans 3 and 4 (Figure 1) suitably protected
for SPPS. Although the latter glycoform (4: LacdiNAc sub-
stitution) appears only as a minor modification of the complex-
type N-glycan, a unique sex-dependent biological function of the
glycoprotein has been reported.13 The LacdiNAc N-glycan identified
as a female-specific glycoform of glycodelin (GdA: a glycoprotein
secreted in human amniotic fluid) potentially inhibits spermꢀoocyte
binding, whereas the male-specific glycodelin (GdS: secreted in
seminal plasma) carrying the high-mannnose type and standard
complex type N-glycans has no such inhibiting effect.
18
yield. Then it was reduced with Et3SiH and PhBCl2 to
regioselectively cleave the benzylidene acetal. The MPM group
was then removed to afford 8 in a 69% overall yield. The
trisaccharyl donor (7s) was synthesized by the Cp2Zr(ClO4)2-
mediated glycosidation19 of the known LacNTCA fluoride 9f5 and
1020 with a high yield and stereoselectivity (85%; β/R = 98/2).
The β-selective synthesis of 7s was also achieved by the reaction of
10 and (N-phenyl)trifluoroacetimidate 9i derived from the corre-
sponding hemiacetal5 (79%; β/R = 99/1).
Glycosylation of 8 with 7s (2 equiv) was promoted by NIS and
TfOH in CH2Cl2 to afford the nonasaccharide 17, which was
isolated by gel permeation chromatography in 66% yield. How-
ever, the 1H and 13C NMR spectra of the nonasaccharide showed
’ RESULTS AND DISCUSSION
On the basis of the merits of convergent synthesis, our first
synthetic plan for 3 was the condensation between the known
5230
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
Scheme 3. Syntheses of the Trisaccharyl Intermediates 7 and 8a
a Reagents and conditions: (a) BSP, TTBP, Tf2O, CH2Cl2, ꢀ60 °C, 2 days, 73%; (b) Et3SiH, PhBCl2, MS4A, CH2Cl2, ꢀ78 °C, 0.5 h; (c) 90% TFA aq,
CH2Cl2, ꢀ10 °C, 0.5 h, 95% (2 steps); (d) Cp2ZrCl2, AgClO4, MS4A, CH2Cl2, ꢀ60 to ꢀ50 °C, 3 h, 85% (β/R = 98/2); (e) TMSOTf, MS AW300,
CH2Cl2, ꢀ78 to ꢀ40 °C, 2 h, 79% (β/R = 99/1); (f) NBS, acetone aq, rt, 3.5 h, 85%; (g) DAST, CH2Cl2, rt, 0.5 h, quant.
Scheme 4. Synthesis of Nonasaccharide 5a
a Reagents and conditions: (a) Cp2ZrCl2, AgClO4, MS4A, CH2Cl2, ꢀ15
°C, 2 h, 83%; (b) 30% H2O2, LiOH, THF, 0 °C, 3 d, 75%; (c) 9i,
TMSOTf, MS AW300, CH2Cl2, ꢀ40 °C, 2 h, 85%; (d) 1. (CH2NH2)2,
BuOH, 2. Ac2O, CH2Cl2, MeOH, 75%.
Figure 2. Analysis of nonasaccharide samples of 17 prepared from
(a) 7s and 8 and (b) 9i and 16 by the recycling HPLC system. The major
peak shown as 1st cycle was collected and returned to the same column.
The sample flowed through the same column repeatedly, which was
shown by the cycle number. Conditions: column: Inertsil SIL-100A (20
mm ꢁ 250 mm) at a flow rate of 9.5 mL/min using (a) 30% EtOAc/
hexane and (b) 35% EtOAc/hexane as eluents.
catalytic amount of TMSOTf at ꢀ40 °C for 2.5 h to give the desired
17 as a single isomer in 85% yield. The homogeneity of the sample
was determined by analysis using recycling HPLC (Figure 2b) as well
as by the clearly split nine signals of anomeric carbon in the 13C NMR
spectrum. The newly generated GlcNTCA linkages were determined
as β on the basis of the 1JCH values (166.3 and 165.5 Hz at 97.6 and
97.8 ppm, respectively).23
the heterogeneous property of the product. Through careful
examination of the product by recycling HPLC, it turned out that
the product consisted of at least three major components as
shown in Figure 2a. A similar result was obtained when acceptor
8 was glycosylated with the fluoride donor 7f. By these glycosyla-
tion methods, we could not attain a high R-selectivity, although
several examples have previously been reported for the related R-
mannnoside synthesis of an N-glycan using the nonparticipating
2-O-glycosylated mannnose donors of trichloroacetimidate6aꢀc
and thioglycoside.21 Therefore, we altered the synthetic route to
the nonasaccharide (Scheme 4).
Having obtained the desired nonasaccharide with high stereo-
selectivity, the N-phthaloyl protecting groups were selectively
converted to N-acetyl groups before condensing with the amino
acid moiety. Compound 17 was heated with a large excess of
1,2-diaminoethane in BuOH for 2 h, and the extracted inter-
mediate was heated in toluene overnight to complete cleavage
of the phthalimido groups. The prolonged reaction in the
presence of 1,2-diaminoethane caused a considerable decrease
in the yield of the desired diamino compound. Conversion to
the diacetamide was then performed by acetylation with Ac2O
to give 5 in 75% yield.
Mannosylation of 8 with 2-O-acetyl-3,4,6-tri-O- benzyl-R-D-man-
nopyranosyl fluoride 1422 stereoselectively gave the R,R-dimannosy-
lated pentasaccharide 15,14 which was readily deacetylated to the diol
16. The glycosylation of 16 with 9i (3.4 equiv) was promoted using a
Since a stereocontrolled route to the nonasaccharide frame-
work has been established, we next directed our attention to the
synthesis of the LacdiNAc-containing congener as shown in
5231
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
Scheme 5. Synthesis of Nonasaccharide 28a
a Reagents and conditions: (a) NaH, CH2dCHCH2Br, DMF, 0 °C, 2 h, 87%; (b) 1. Zn, AcOH, CH2Cl2, rt, 0.5 h, 2. Cl3CCOCl, pyridine, CH2Cl2, 0 °C,
1 h, 77%; (c) TBAF, AcOH, THF, rt, 20 h, 96%; (d) ClC(dNPh)CF3, K2CO3, acetone, rt, 1 h, 88%; (e) TMSOTf, MS AW300, CH2Cl2, ꢀ78 to ꢀ40
°C, 2.5 h, 94%; (f) TBAF, AcOH, THF, rt, 20 h, 96%; (g) ClC(=NPh)CF3, K2CO3, acetone, rt, 1 h, 96%; (h) 16, TMSOTf, MS AW300, CH2Cl2, ꢀ78 to
ꢀ40 °C, overnight, 69%; (i) 1. (CH2NH2)2, BuOH, 2. Ac2O, CH2Cl2, MeOH, 52%.
Scheme 5, before conjugation with an amino acid and dechlor-
ination of the trichloroacetyl groups. The disaccharide inter-
mediate for LacdiNAc was synthesized using the GalNTCA and
GlcNTCA derivatives. The hydroxyl group at the 3-position of
tert-butyldiphenylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-β-D-
galactopyranoside 1824 was protected as an allyl ether by taking
into account the potential glycan elongation. Recently, we found
that the allyl group was concomitantly removed when a synthetic
glycopeptide was deprotected under the low-acidity TfOH
conditions.25 The rather unstable compound 19 bearing azide
and allyl groups in a molecule was immediately reduced by Zn/
AcOH and then trichloroacetylated to give 20 (77%), which was
converted to 22 (84%) through desilylation and imidate forma-
tion. The glycosylation of 2326 with 22 stereoselectively pro-
duced the disaccharide 24 (94%), which was converted into the
glycosyl donor 26 (92% in 2 steps). Crucial glycosylation of 16
required increased equivalences of the donor 26 to be 5 and
TMSOTf to be 0.25, in addition to the longer reaction time
(overnight) in contrast to the smooth coupling between 9i and
16. On the basis of monitoring the reaction by TLC, the
oxazoline intermediate derived from 26 seemed more stable
than that from 9i. The product 27 was obtained in 69% yield, and
the 1JCH value (both 166.3 Hz at 97.7 and 97.9 ppm for C-1e and
C-1 h) supported the β-configuration of the newly formed
glycosidic linkages. Conversion of the diphthalimide 27 to the
diacetamide 28 (52%) was less efficient than the preparation of 5,
when similar conditions were used.
O1-tert-butyl O4-pentafluorophenyl aspartate [Fmoc-Asp(OPfp)-
OBut] and 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HO-
OBt) in 98% aq. THF.
Among the tested phosphines (Ph(CH3)2P, [(Et)3P],
[(n-Bu)3P], Ph3P) using a monosaccharide model, the use of
Ph(CH3)2P effectively minimized the side products derived from
the dechlorination of the trichloroacetyl group. The desired
glycoamino acid 29 was obtained in 84% yield. In addition,
two fractions were separated by HPLC as the more hydrophilic
side products (9 and 2%, respectively, based on the HPLC
analysis). Their structures were determined by HRMS to be
monodechlorinated and didechlorinated compounds, respec-
tively. Similarly, the glycoamino acid derivative 30 was obtained
by the condensation of 28 and O1-phenacyl O4-pentafluorophe-
nyl aspartate [Fmoc-Asp(OPfp)-OPac] in 78% yield (Scheme 6).
The trichloroacetamide groups were then converted into the
acetamide groups by the reported procedure using microwave
irradiation with Zn and AcOH in EtOAc, and the dechlorinated
product 31 was obtained from 29 in 86% yield.5 After the
cleavage of the tert-butyl ester in 31 by treatment with aq.
TFA, the title compound 3 (71%) was successfully obtained.
In the case of compound 30, the reduction of the trichloroace-
tamide group and the cleavage of Pac ester were simultaneously
achieved by treatment with Zn and AcOH in EtOAc, and the
LacdiNAc congener 4 was successfully obtained in 45% yield.
In conclusion, we have synthesized two nonasaccharyl aspar-
agines of the biantennary N-glycan, each containing LacNAc and
LacdiNAc as the nonreducing end motifs. The synthesis was
achieved by combination of the N-phthaloylated and N-trichlor-
oacetylated glucosamine derivatives, both promoting the exclusive
1,2-trans-glycoside formation. Selective conversion of phthalimide
Conjugation of the synthetic nonasaccharides, 5 and 28, with
a properly protected aspartic acid derivative was performed by
a modified “Staudinger Ligation”.27 The nonasaccharyl azide 5
was treated with dimethylphenylphosphine [Ph(CH3)2P],
5232
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
Scheme 6. Synthesis of Nonasaccharyl Asn Building Blocksa
a Reagents and conditions: (a) Ph(CH3)2P, Fmoc-Asp(OPfp)-OBut, HOOBt, 98% THF aq, rt, 2 h, 29: 84%; (b) Ph(CH3)2P, Fmoc-Asp(OPfp)-OPac,
HOOBt, 98% THF aq, rt, 2 h, 30: 83%; (c) Zn, AcOH, EtOAc, microwave (150 W), 1 h, 31: 86%, 4: 45%; (d) 80% TFA aq, 3: 71%.
to acetamide was necessary before coupling with the base-labile N-
Fmoc aspartic acid derivative, in which the dephthaloylation condi-
tions were carefully explored to avoid any side reactions on the
susceptible trichloroacetyl groups. Coupling of the nonasaccharides
with aspartic acid derivatives was successfully promoted under the
modified Staudinger reaction by selecting Ph(CH3)2P. The benzyl-
protected 3 and 4 would be used for the solid-phase synthesis of
glycopeptides carrying N-glycan, which would contribute to the
biological studies to discriminate the functions between the LacNAc
and LacdiNAc N-glycans. Syntheses of these glycopeptides are
currently being investigated.
MS 4A (6.42 g) in anhydrous CH2Cl2 (35 mL) was cooled at ꢀ60 °C for
30 min. The mixture was stirred for a further 20 min after addition of
Tf2O (0.33 mL, 1.98 mmol). A solution of 12 (0.88 g, 0.89 mmol) in
anhydrous CH2Cl2 (10 mL) was slowly added to the mixture through a
cannula. Then the mixture was allowed to stir for 2 days at the
temperature, before the reaction was quenched by addition of satd
NaHCO3 aq. The mixture was diluted with CHCl3 and filtered through
Celite. The organic layer of the combined filtrate and CHCl3 was
successively washed with satd NaHCO3 aq, water, and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
chromatographed on silica gel with tolueneꢀEtOAc (9:1) to give
trisaccharides as a mixture (1.30 g). Separation of the isomers was
performed by recycling HPLC to give 13 (0.94 g, 73%). The R-isomer
was not obtained as a homogeneous sample even by the recycling HPLC.
’ EXPERIMENTAL SECTION
1
General. Specific rotation values were at 20 ( 2 °C for solutions in
CHCl3. 1H and 13C NMR spectra were recorded on a 400 or 600 MHz
spectrometer. HꢀH and CꢀH COSY spectra were measured to confirm
the NMR peak assignments. Chemical shifts are expressed in parts per
million downfield from the signal for internal Me4Si for solutions in
CDCl3. For description of the NMR data, each sugar residue in
oligosaccharide is indicated by an alphabetical mark as shown in Figure 1.
Recycling HPLC was performed with a recycling preparative HPLC.
MALDI TOF mass spectra were obtained using 2,5-dihydroxybenzoic
acid as a matrix. High-resolution mass spectra were obtained with a
Fourier transform ion cyclotron resonance mass spectrometer.
2-O-Benzyl-4,6-O-benzylidene-3-O-(4-methoxyphenyl)methyl-β-
D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
β-D-glucopyranosyl Azide 13. A stirred mixture of 11 (1.02 g, 1.79
mmol), BSP (0.56 g, 2.67 mmol), TTBP (0.89 g, 3.58 mmol), and dried
Compound 13: [R]D þ0.3 (c 1.3). Rf 0.38 (9:1 tolueneꢀEtOAc). H
NMR: δ 7.87ꢀ7.21 (m, 20H, Ar), 6.95ꢀ6.75 (m, 12H, Ar), 5.49 [s, 1H,
PhCH(O)2<], 5.29 (d, 1H, J = 8.0 Hz, H-1b), 5.16 (d, 1H, J = 9.3 Hz,
H-1a), 4.90ꢀ4.82 (m, 4H, ꢀCH2Ar ꢁ 4), 4.66 (d, 1H, J = 12.0 Hz, ꢀ
CH2Ar), 4.56 (s, 1H, H-1c), 4.67ꢀ4.48 (m, 5H, ꢀCH2Ar ꢁ 5), 4.40
(d, 1H, J = 12.4 Hz, ꢀCH2Ar), 4.37 (d, 1H, J = 12.2 Hz, ꢀCH2Ar),
4.28ꢀ4.02 (m, 8H), 3.78 (s, 3H, ꢀOCH3), 3.72 (d, 1H, J = 3.2 Hz, H-2c),
3.62ꢀ3.49 (m, 3H), 3.45ꢀ3.37 (m, 4H), 3.20 (m, 1H), 3.12 (m, 1H,
H-5c). 13C NMR: δ85.8 (C-1a, 1JCH = 165.1 Hz), 97.3 (C-1b, 1JCH = 167.2
1
Hz), 101.5 (C-1c, JCH = 168.5 Hz), 102.0 [PhCH<]. Anal. Calcd for
C84H79N5O18: C, 69.75; H, 5.50; N, 4.84. Found: C, 69.85; H, 5.54; N, 4.80.
2,4-Di-O-benzyl-β-D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl Azide 8. To a stirred mixture
of 13 (338 mg, 0.23 mmol) and dried MS 4A (1.7 g) in anhydrous
CH2Cl2 (20 mL) were successively added Et3SiH (0.11 mL, 0.70 mmol)
5233
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
and PhBCl2 (0.11 mL, 0.82 mmol) at ꢀ78 °C. The mixture was stirred
for 1 h at that temperature. The reaction was quenched by adding Et3N
(1.7 mL) and MeOH (3 mL). The resulting mixture was stirred for 30
min at room temperature, diluted with CHCl3, and filtered through
Celite. The combined filtrate and washings were successively washed
with satd NaHCO3 aq, water, and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The residue was dissolved in CH2Cl2 (8 mL)
andstirredwith90% aq. TFA(8mL) atꢀ10 °C for 30 min. Then the acidic
mixture was carefully neutralized with satd NaHCO3 aq and extracted with
EtOAc. The organic layer was successively washed with satd NaHCO3 aq,
water, and brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was chromatographed on silica gel with tolueneꢀEtOAc (4:1)
to afford 8 (294 mg, 95%), NMR data of which were superimposable on
those of the authentic sample prepared previously.14 [R]D þ0.3 (c 1.3). Rf
0.32 (3:1 tolueneꢀEtOAc). 1H NMR: δ 5.30 (d, 1H, J = 7.8 Hz, H-1b),
5.17 (d, 1H, J = 9.3 Hz, H-1a), 5.17 (s, 1H, H-1c). 13C NMR: d 101.1
(C-1c), 96.9 (C-1b), 85.4 (C-1a). MALDI TOF MS: calcd for
C76H73N5O17 (MþNa)þ, m/z 1350.50. Found: 1350.60.
Method B (Reaction of 9i and 10). A stirred mixture of 9i (102 mg,
85 μmol), 10 (48 mg, 89 μmol), and dried MS AW300 (300 mg) in
anhydrous CH2Cl2 (3 mL) was cooled at ꢀ78 °C. Then 2% TMSOTf/
CH2Cl2 (39 μL, 4.3 μmol) was added to the mixture. The stirring was
continued for 1.5 h at that temperature and for 0.5 h at ꢀ40 °C. The
reaction was quenched by addition of Et3N, and the insoluble material
was filtered off. The filtrate was concentrated in vacuo. The residue was
chromatographed on Bio-Beads and then on silica gel as described above
to give 7s (105 mg, 79%, R/β = 1/99). The minor isomer was separated
by HPLC and identified by the mass spectrum.
2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-3,6-di-O-ben-
zyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-
benzyl-R-D-mannopyranosyl Fluoride 7f. Compound 7s (210 mg,
0.14 mmol) was dissolved in 85% aq. acetone (2.7 mL). N-Bromosucci-
mide (NBS, 133 mg, 0.75 mmol) was added to the stirred solution,
which was stirred at room temperature for 2.5 h. The reaction mixture
was diluted with EtOAc, washed with 5% aq. Na2S2O3, water, and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with hexaneꢀEtOAc
(7:3ꢀ3:2) to afford trisaccharyl hemiacetal (168 mg, 85%). Rf 0.41
(3:2 hexaneꢀEtOAc). MALDI TOF MS: calcd for C83H86Cl3NO16 [M þ
Na]þ, m/z 1480.58. Found: 1480.50. To a stirred solution of the
hemiacetal in anhydrous CH2Cl2 (5.2 mL) was added (diethylam-
ino)sulfur trifluoride (DAST, 28 μL, 0.21 mmol) at 0 °C. The mixture
was stirred for 0.5 h, and the reaction was quenched by adding satd
NaHCO3 aq. The mixture was diluted with EtOAc, successively washed
with satd NaHCO3 aq, water, and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude product was chromatographed on silica gel
with tolueneꢀEtOAc (9:1) to give 7f [R-fluoride (151 mg, 90%) and β-
fluoride(13mg, 8%)].r-Fluoride:Rf 0.41 (3:2 tolueneꢀEtOAc). 1HNMR:
δ 5.58 (brd, 1H, J = 50.7 Hz, H-1a), 5.20 (d, 1H, J = 8.3 Hz, H-1b), 4.48 (d,
1H, J = 7.3 Hz, H-1c). MALDI TOF MS: calcd for C83H86Cl3NO16 [M þ
Na]þ, m/z 1482.50. Found: 1482.54. β-Fluoride: Rf 0.27 (3:2 tolueneꢀ
EtOAc). 1H NMR: δ 5.31 (brd, 1H, J = 51.7 Hz, H-1a), 5.09 (d, 1H, J = 8.3
Hz, H-1b), 4.43 (d, 1H, J = 7.3 Hz, H-1c). MALDI TOF MS: calcd for
C83H86Cl3NO16 [M þ Na]þ, m/z 1482.50 (96%). Found: 1482.61.
2-O-Acetyl-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1f3)-[2-O-acet-
yl-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-
β-D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimi-
do-β-D-glucopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido
-β-D-glucopyranosyl Azide 15. A mixture of Cp2ZrCl2 (497 mg, 1.70
mmol), AgClO4 (500 mg, 2.41 mmol), and dried MS 4A (1.2 g) in
anhydrous CH2Cl2 (4 mL) was stirred under Ar at room temperature for 30
min and then cooled at ꢀ60 °C. To the stirred mixture was added a mixture
of 8 (263 mg, 0.20 mmol) and 14 (293 mg, 0.59 mmol) in anhydrous
CH2Cl2 (8 mL) through a cannula. Then the temperature was raised to
ꢀ15 °C. The mixture was stirred for 2 h at the temperature before the
reaction was quenched by addition of satd NaHCO3 aq. The mixture was
diluted with EtOAc and filtered through Celite. The organic layer was
successively washed with satd NaHCO3 aq, water, and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on Bio-Beads S-X1 with toluene to afford 15 (373 mg,
83%). The NMR data of 15 were identical with those of the sample reported
2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-3,6-di-O-ben-
zyl-2-deoxy-2-trichloroacetamido-D-glucopyranosyl (N-Phenyl)tri-
fluoroacetimidate 9i. A mixture of known hemiacetal5 (87 mg, 85
μmol), (N-phenyl)trifluoroacetimidoyl chloride (35 mg, 170 μmol), and
K2CO3 (24 mg, 170 μmol) in anhydrous acetone (1 mL) was stirred at
room temperature for 1 h. Then the insoluble material was filtered off
through Celite, and the filtrate was concentrated in vacuo. The residue was
chromatographed on silica gel with tolueneꢀEtOAc (49:1) to quantita-
tively afford 9i (102 mg) as a mixture of stereoisomers. Rf 0.28 (49:1
tolueneꢀEtOAc). 1H NMR:δ7.36ꢀ7.16 (m, 32H, Ar), 7.07 (t, 1H, J = 7.5
Hz, Ar), 6.76 (d, 2H, J = 7.8 Hz, Ar), 6.61 (d, 1H, J = 7.6 Hz, ꢀNH), 4.14
(brt, 1H, J = 9.2 Hz, H-4a), 3.91 (d, 1H, J = 2.7 Hz, H-4b). Anal. Calcd for
C64H62Cl3F3N2O11: C, 64.14; H, 5.21; N, 2.34. Found: C, 64.24; H, 5.27;
N, 2.36.
Thiophenyl 2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-
(1f2)-3,4,6-tri-O-benzyl-1-thio-R-D-mannopyranoside 7s. Method
A (Reaction of 9f and 10). A mixture of Cp2ZrCl2 (121 mg, 0.41
mmol), AgClO4 (171 mg, 0.82 mmol), and dried MS 4A (380 mg) in
anhydrous CH2Cl2 (4.6 mL) was stirred under Ar at room temperature
for 30 min and then cooled at ꢀ60 °C. To the stirred mixture was added
a mixture of 9f (215 mg, 0.21 mmol) and 10 (124 mg, 0.23 mmol) in
anhydrous CH2Cl2 (5 mL) though a cannula. The resulting mixture was
stirred at ꢀ60 °C for 1 h and then at ꢀ50 °C for 2.5 h before the reaction
was quenched by addition of sat. NaHCO3 aq. The mixture was diluted
with EtOAc and filtered through Celite. The organic layer was succes-
sively washed with satd NaHCO3 aq, water, and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on Bio-Beads S-X1 with toluene and then on silica gel
with hexaneꢀEtOAc (4:1ꢀ3:1) to afford 7s (276 mg, 85%, R/β =
1/49). The minor R-isomer was separated by HPLC and identified by
the mass spectrum. Compound 7s: [R]D þ38.6 (c 1.1). Rf 0.35 (8:3
hexaneꢀEtOAc). 1H NMR: δ 7.45ꢀ7.10 (m, 51H, Ar, ꢀNH), 5.54 (d,
1H, J = 1.5 Hz, H-1a), 5.23 (d, 1H, J = 8.3 Hz, H-1b), 5.06 (d, 1H,
J = 10.2 Hz, ꢀCH2Ph), 4.98 (d, 1H, J = 11.2 Hz, ꢀCH2Ph), 4.85ꢀ4.72
(m, 6H, ꢀCH2Ph), 4.56ꢀ4.37 (m, 11H, H-1c, H-2a, H-3b, ꢀCH2Ph),
4.27 (d, 1H, J = 10.2 Hz, ꢀCH2Ph), 4.25 (m, 1H, H-5a), 4.24 (d, 1H, J =
11.7 Hz, ꢀCH2Ph), 4.03 (brt, 1H, J = 8.8, 9.3 Hz, H-4b), 3.92ꢀ3.63 (m,
8H, H-3a, H-4a, H-5a, H-6a, H-6a, H-2c, H-4c, H-6c, H-6c), 3.54ꢀ3.51
1
previously.14 Rf 0.46 (4:1 tolueneꢀEtOAc). H NMR: δ 5.49 (brs, 1H,
H-2e), 5.31 (brs, 1H, H-2d), 5.21 (d, 1H, J = 7.8 Hz, H-1b), 5.14 (s, 1H,
H-1e), 5.13 (d, 1H, J = 7.8 Hz, H-1a), 4.84 (s, 1H, H-1d), 2.09 (s, 3H,
ꢀCOCH3), 1.79 (s, 3H, ꢀCOCH3). 13C NMR: δ 102.2 (C-1c), 99.9 (C-
1d), 98.5 (C-1e), 97.2 (C-1b), 85.8 (C-1a), 21.3 and 21.0 (ꢀCOCH3).
MALDI TOF MS: calcd for C134H133N5O29 [M þ Na]þ, m/z 2298.91.
Found: 2297.93.
(m, 2H, H-5b, H-6b), 3.44ꢀ3.34 (m, 4H, H-2b, H-6b, H-3c, H-5c). 13
C
NMR δ: 102.8 (C-1c), 97.0 (C-1b), 92.2 (-CCl3), 86.2 (C-1a). MALDI
TOF MS: calcd for C89H92Cl3NO15S [MþNa]þ, m/z 1574.51 (100%).
Found: 1573.12. Anal. Calcd for C89H92Cl3NO15S: C, 68.87; H, 5.84; N,
0.90. Found: C, 68.82; H, 5.90; N, 0.96.
3,4,6-Tri-O-benzyl-R-D-mannopyranosyl-(1f3)-[3,4,6-tri-O-benno-
pyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-gluco-
pyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyra-
nosyl Azide 16. To a solution of 15 (130 mg, 57 μmol) in THF (32 mL)
r-isomer: Rf 0.35 (8:3 hexaneꢀEtOAc). MALDI TOF MS: Found:
1573.09.
5234
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
were added 30% H2O2 (6.5 mL, 80.3 mmol) and 1 N LiOH aq (6.5 mL, 6.5
mmol) at 0 °C. The mixture was stirred for 18 h at 0 °C. Then the mixture
was diluted with EtOAc, successively washed with aq Na2S2O3, water, and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was purified by two chromatographies on silica gel with tolue-
neꢀEtOAc (9:1ꢀ7:3) and then with hexaneꢀEtOAc (3:2ꢀ1:1) to afford
16 (91 mg, 72%). [R]D þ19.9 (c 0.5). Rf 0.36 (7:3 tolueneꢀEtOAc). 1H
NMR: δ 5.24 (m, 1H, H-1b), 5.15 (s, 1H, H-1e), 5.14 (d, 1H, J = 7.8 Hz,
H-1a), 4.81 (s, 1H, H-1d), 4.59 (s, 1H, H-1c). 13C NMR: δ 101.7, 101.5,
and 99.8 (C-1c, C-1d, C-1e), 97.1 (C-1b), 85.5 (C-1a). MALDI TOF MS:
calcd for C130H129N5O27 [M þ Na]þ, m/z 2215.88 (100%). Found:
2215.99. Anal. Calcd for C130H129N5O27: C, 71.18; H, 5.93; N, 3.19.
Found: C, 70.92; H, 6.00; N, 3.16.
2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-
benzyl-R-D-mannopyranosyl-(1f3)-[2,3,4,6-tetra-O-benzyl-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-
(1f6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1f4)-3,6-di-O-benzyl-
2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1f4)-3,6-di-D-benzyl-
2-deoxy-2-phthalimido-β-D-glucopyranosyl Azide 17. Method A
(Reaction of 7s and 8). A mixture of 7s (97 mg, 62 μmol), 8 (42
mg, 32 μmol), N-iodosuccimide (NIS, 22 mg, 98 μmol), and dried MS
3A (130 mg) in anhydrous CH2Cl2 (1 mL) was stirred at ꢀ20 °C under
Ar for 30 min. To the mixture was added a solution of TfOH (0.83 μL,
9 μmol) in anhydrous CH2Cl2 (0.1 mL). The reaction mixture was
stirred at ꢀ20 °C for 1 h before quenching with satd NaHCO3 aq. The
mixture was diluted with EtOAc and filtered through Celite. The filtrate
was washed with aq NaS2O3, water, and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude product was chromato-
graphed on Bio-Beads S X1 in toluene to afford a fraction composed of
nonasaccharides (88 mg). The separated fraction was concentrated in
vacuo, and the residue was analyzed by recycling HPLC with a column of
Inertsil SIL-100A (20 mm ꢁ 250 mm) in hexaneꢀEtOAc (13:7). After
five times recycling elution, the chromatogram showed three major
components.
Method B (Reaction of 7f and 8). A mixture of Cp2ZrCl2 (55 mg,
187 μmol), AgClO4 (75 mg, 362 μmol), and dried MS 4A (190 mg) in
anhydrous CH2Cl2 (0.4 mL) was stirred under Ar at room temperature
for 0.5 h and then cooled at ꢀ40 °C. To the stirred mixture was slowly
added a mixture of 7f (132 mg, 90 μmol) and 8 (55 mg, 41 μmol) in
anhydrous CH2Cl2 (1.1 mL). The mixture was stirred at the temperature
for 0.5 h, and then the temperature was raised to ꢀ20 °C during 1.5 h.
Finally, the mixture was stirred at 0 °C for 1.5 h before quenching with
satd NaHCO3 aq. The mixture was diluted with EtOAc and filtered
through Celite. The organic layer was successively washed with satd
NaHCO3 aq, water, and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude product was chromatographed on Bio-
Beads S-X1 in toluene to afford a nonasaccharide fraction (73 mg).
Recycling HPLC demonstrated a pattern of the chromatogram for this
sample similar to that obtained by method A.
Method C (Reaction of 9i and 16). A stirred mixture of 9i (1.40 g,
1.17 mmol), 16 (0.75 g, 0.34 mmol), and dried MS AW300 (10 g) in
anhydrous CH2Cl2 (30 mL) was cooled at ꢀ40 °C. Then TMSOTf (30
μL, μmol) was added to the mixture. The stirring was continued for 2 h
at that temperature. The reaction was quenched by addition of satd
NaHCO3 aq, and the insoluble material was filtered off. The filtrate was
concentrated in vacuo. The residue was chromatographed on Bio-Beads
S-X1 in toluene and then on silica gel with hexaneꢀEtOAc (3:2) to give
17 (1.23 g, 85%) as a single stereoisomer. Homogeneity of the product
was evidenced by recycling HPLC. [R]D þ17.8 (c 0.8). Rf 0.36 (7:5
tolueneꢀEtOAc). 1H NMR: δ 5.18 (d, 1H, J = 7.8 Hz, H-1b), 5.12 (d,
1H, J = 8.8 Hz, H-1a). 13C NMR: δ 102.7 and 102.5 (1JCH = 160.6 Hz,
C-1f and C-1i), 101.8 (1JCH = 158.9 Hz, C-1c), 100.0 (1JCH = 173.0 Hz)
and 98.8 (1JCH = 168.3 Hz) (C-1d and C-1 g), 97.8 (1JCH = 165.5 Hz)
and 97.6 (1JCH = 166.3 Hz) (C-1 h and C-1e), 96.7 (1JCH = 168.0 Hz,
C-1b), 92.4 and 92.2 (ꢀCCl3), 85.3 (1JCH = 170.5 Hz, C-1a). Anal.
Calcd for C242H241Cl6N7O47: C, 69.00; H, 5.77; N, 2.33. Found: C,
68.99; H, 5.86; N, 2.38.
2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-
O-benzyl-R-D-mannopyranosyl-(1f3)-[2,3,4,6-tetra-O-benzyl-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyran-
osyl-(1f6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1f4)-2-aceta-
mido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f4)-2-aceta-
mido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl Azide 5. A mix-
ture of 17 (245 mg, 58 μmol) and 1,2-diaminoethane (785 μL, 11.7
mmol) in n-BuOH (5 mL) was heated at 90 °C for 2 h. After cooling, the
mixture was diluted with EtOAc and washed with water until the
washings became neutral pH. The organic extract was washed with
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was dissolved in toluene (10 mL), heated at 90 °C overnight, and
then concentrated in vacuo. The crude diamino product was stirred with
Ac2O (1 mL) in a mixture of CH2Cl2 (2 mL) and MeOH (4 mL) at
room temperature for 0.5 h. The reaction mixture was concentrated in
vacuo, and the volatile materials were removed by coevaporation with
toluene. The crude product was chromatographed on silica gel with
hexaneꢀEtOAc (2:3) to give 5 (75%). [R]D þ1.9 (c 2.3). Rf 0.28 (2:3
1
hexaneꢀEtOAc). H NMR: δ 1.64 (s, 3H, ꢀCOCH3), 1.40 (s, 3H,
ꢀCOCH3). 13C NMR: δ 102.8 and 102.6 (C-1f and C-1i), 101.0, 100.0,
99.6, 99.0, 97.9, and 97.2 (C-1b, C-1c, C-1d, C-1e, C-1 g, C-1 h), 92.6
and 92.3 (-CCl3), 88.0 (C-1a), 23.2 (ꢀCOCH3), 22.7 (ꢀCOCH3).
MALDI TOF MS: calcd for C230H241Cl6N7O45 [M þ Na]þ, m/z
4059.1. Found: 4060.9. Anal. Calcd for C230H241Cl6N7O45: C, 68.44; H,
6.02; N, 2.43. Found: C, 68.09; H, 6.05; N, 2.42.
tert-Butyldiphenylsilyl 3-O-Allyl-2-azido-4,6-O-benzylidene-2-deoxy-β-
D-galactopyranoside 19. To a stirred mixture of 18 (1.46 g, 2.7 mmol) and
60% NaH/mineral oil (0.16 g, 4.1 mmol) in anhydrous DMF (25 mL) was
added allyl bromide (0.36 mL, 4.1 mmol). The mixture was stirred at room
temperature for 1.5 h. Then the reaction was quenched carefully by adding a
few pieces of ice, and the mixture was concentrated in vacuo. The residue
was extracted with EtOAc, successively washed with water and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on silica gel with tolueneꢀEtOAc (39:1ꢀ19:1) to give
19 (1.36 g, 87%). [R]D þ27.0 (c 0.5). Rf 0.44 (19:1 tolueneꢀEtOAc). 1H
NMR: δ 7.80 (m, 2H, Ar), 7.73 (m, 2H, Ar), 7.53 (m, 2H, Ar), 7.43ꢀ7.31
(m, 9H, Ar), 5.92 (m, 1H, ꢀCHdCH2), 5.47 [s, 1H, PhCH<], 5.30
(dq, 1H, J = 1.5, 17.1 Hz, ꢀCHdCH2), 5.17 (dd, 1H, J = 1.5, 10.2 Hz,
ꢀCHdCH2), 4.40 (d, 1H, J = 7.8 Hz, H-1), 4.20ꢀ4.10 (m, 2H,
ꢀCH2CHdCH2), 4.07 (d, 1H, J = 2.9 Hz, H-4), 3.95 (dd, 1H, J = 1.5,
12.2 Hz, H-6), 3.89ꢀ3.83 (m, 2H, H-2, H-6), 3.21 (dd, 1H, J = 3.4, 10.2 Hz,
H-3), 2.94 (d, 1H, J = 1.0 Hz, H-5), 1.13 (s, 9H, But). Anal. Calcd for
C32H37N3O5Si: C, 67.22; H, 6.52; N, 7.35. Found: C, 67.45; H, 6.57; N, 7.33.
tert-Butyldiphenylsilyl 3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-tri-
chloroacetamido-β-D-galactopyranoside 20. A mixture of 19 (200
mg, 0.35 mmol), powdered Zn (875 mg, 13.4 mmol), and AcOH
(0.4 mL, 7.0 mmol) in CH2Cl2 (8.7 mL) was stirred at room
temperature for 40 min. The mixture was diluted with CHCl3 and
filtered through Celite. The filtrate was successively washed with satd
NaHCO3 aq and brine, dried over anhydrous Na2SO4, and concentrated
in vacuo. The residue was dissolved in pyridine (3.5 mL) and stirred with
trichloroacetyl chloride (59 μL) at 0 °C for 30 min. Most of the pyridine
was evaporated in vacuo, and the residue was extracted with EtOAc. The
extract was washed with water and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The crude product was purified by column
chromatography on silica gel with tolueneꢀEtOAc (29:1) to afford 20
(187 mg, 77%). [R]D þ16.7 (c 0.5). Rf 0.32 (19:1 tolueneꢀEtOAc). 1H
5235
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
NMR: δ 7.76 (m, 2H, Ar), 7.65 (m, 2H, Ar), 7.53 (m, 2H, Ar),
7.40ꢀ7.30 (m, 9H, Ar), 6.92 (d, 1H, J = 6.8 Hz, ꢀNH), 5.86 (m, 1H,
ꢀCHdCH2), 5.50 [s, 1H, PhCH<], 5.24 (brd, 1H, J = 17.4 Hz,
ꢀCHdCH2), 5.16 (d, 1H, J = 7.8 Hz, H-1), 5.14 (m, 1H, CHd
CH2), 4.17 (d, 1H, J = 3.4 Hz, H-4), 4.13ꢀ4.08 (m, 2H, H-3,
ꢀCH2CHdCH2), 4.02 (dd, 1H, J = 5.8, 12.7 HZ, ꢀCH2CHdCH2),
3.97 (dd, 1H, J = 1.0, 12.2 Hz, H-6), 3.93ꢀ3.86 (m, 2H, H-2, H-6), 3.09
(s, 1H, H-5), 1.09 (s, 9H, But). 13C NMR: δ 100.9 [PhCH(O)2<], 94.2
(C-1)1, 92.5 (ꢀCCl3). Anal. Calcd for C34H38Cl3NO6Si: C, 59.09; H,
5.54; N, 2.03. Found: C, 59.16; H, 5.55; N, 2.07.
H-4a, H-4b, H-6b), 4.14ꢀ4.00 (m, 3H, H-3b, ꢀCH2CHdCH2),
3.92ꢀ3.71 (m, 4H, H-6b, H-3a, H-2a, H-2b), 3.52 (dd, 1H, J = 2.9,
11.2 Hz, H-6a), 3.30 (brd, 1H, J = 10.3 Hz, H-6a), 3.31 (s, 1H, H-5b),
3.00 (brd, 1H, J = 8.8 Hz, H-5a), 1.04 (s, 9H, But). 13C NMR: δ 100.9
[PhCH<], 97.8 (C-1b), 95.1 (C-1a), 92.5 (ꢀCCl3), 92.3 (ꢀCCl3).
MALDI TOF MS: calcd for C56H60Cl6N2O11Si [M þ Na]þ, m/z
1199.20 (100%). Found: 1199.28. Anal. Calcd for C56H60Cl6N2O11Si:
C, 57.10; H, 5.13; N, 2.38. Found: C, 57.08; H, 5.12; N, 2.36.
In a more mobile fraction, the R-isomer (8 mg, 3%) was also obtained.
1
Rf 0.48 (3:2 hexaneꢀEtOAc). H NMR: δ 5.68 (d, 1H, J = 3.9 Hz,
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-D-ga-
lactopyranose 21. To a stirred mixture of 20 (216 mg, 0.18 mmol) and
AcOH (105 μL, 1.8 mmol) in freshly distilled THF (2 mL) was added 1
M tetra-n-butylammonium fluoride/THF (TBAF, 3.5 mL, 3.5 mmol) at
0 °C. Then the mixture was stirred at room temperature for 20 h. THF
was evaporated in vacuo, and the product was extracted with EtOAc. The
extract was washed with water and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The residue was chromatographed on silica
gel with tolueneꢀEtOAc (1:1) to give 21 (166 mg, 96%). Rf 0.35 (1:1
tolueneꢀ-EtOAc). 1H NMR: δ 7.53ꢀ7.51 (m, 2H, Ar), 7.38ꢀ7.30 (m,
3H, Ar), 6.90 (d, 1H, J = 8.3 Hz, ꢀNH), 5.88 (m, 1H, ꢀC HdCH2), 5.53
[s, 1H, PhCH<], 5.45 (brt, 1H, J = 3.4 Hz, H-1), 5.29 (dd, 1H, J = 1.5,
17.1 Hz, ꢀCHdCH2), 5.18 (dd, 1H, J = 1.5, 10.2 Hz, ꢀCHdCH2), 4.47
(m, 1H, H-2), 4.26ꢀ4.14 (m, 3H, H-4, H-6, ꢀCH2CHdCH2),
4.05ꢀ3.97 (m, 3H, H-6, ꢀCH2CHdCH2, ꢀOH), 3.82 (dd, 1H, J =
2.9, 10.7 Hz, H-3), 3.81 (brs, 1H, H-5). Anal. Calcd for C18H20Cl3NO6:
C, 47.75; H, 4.45; N, 3.09. Found: C, 47.85; H, 4.51; N, 3.12.
H-1b), 5.02 (d, 1H, J = 7.3 Hz, H-1a). MALDI TOF MS: m/z 1200.16.
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-D-glucopyranose 25. To a stirred mixture of 24 (102 mg, 86 μmol)
and AcOH (49 μL, 860 μmol) in freshly distilled THF (1 mL) was added 1
M tetra-n-butylammonium fluoride/THF (0.34 mL, 340 μmol) at 0 °C.
Then the mixture was stirred at room temperature for 20 h. THF was
evaporated in vacuo, and the product was extracted with EtOAc. The extract
was washed with water and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica gel with
tolueneꢀEtOAc (7:1ꢀ2:1) to give 25 (77 mg, 96%) as an R-anomer-rich
1
mixture (R/β = 4.4/1). Rf 0.38 (1:1 hexaneꢀEtOAc). H NMR (R-
anomer): δ 7.41ꢀ7.14 (m, 15H, Ar), 6.83 (d, 1H, J = 7.3 Hz, ꢀNH),
6.77 (d, 1H, J = 7.8 Hz, ꢀNH), 5.89 (m, 1H, ꢀCHdCH2), 5.52 [s, 1H,
PhCH<], 5.39 (d, 1H, J = 3,4 Hz, H-1a), 5.32 (dq, 1H, J = 1.5, 17.1 Hz,
ꢀCHdCH2), 5.17 (dd, 1H, J = 1.5, 10.3 Hz, ꢀCHdCH2), 5.01 (d, 1H. J =
8.3 Hz, H-1b). Anal. Calcd for C40H42Cl6N2O11: C, 51.14; H, 4.51; N, 2.98.
Found: C, 51.15; H, 4.47; N, 2.92.
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-D-ga-
lactopyranosyl (N-Phenyl)trifluoroacetimidate 22. A mixture of 21
(373 mg, 0.83 mmol), (N-phenyl)trifluoroacetimidoyl chloride (343 mg,
1.65 mmol), and K2CO3 (570 mg, 4.13 mmol) in anhydrous acetone (8 mL)
was stirred at room temperature for 1 h. Then the mixture was diluted with
EtOAc, and the insoluble material was filtered off through Celite. The filtrate
was concentrated in vacuo to the residue, which was chromatographed on
silica gel with tolueneꢀEtOAc (29:1) to quantitatively afford 22 (455 mg,
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-D-glucopyranosyl (N-phenyl)trifluoroacetimidate 26. A mixture
of 25 (74 mg, 80 μmol), (N-phenyl)trifluoroacetimidoyl chloride (33
mg, 158 μmol), and K2CO3 (55 mg, 398 μmol) in anhydrous acetone
(1 mL) was stirred at room temperature for 1 h. Then the mixture was
diluted with EtOAc, and the insoluble material was filtered off through
Celite. The filtrate was concentrated in vacuo to the residue, which was
chromatographed on silica gel with tolueneꢀEtOAc (9:1ꢀ7:1) to give
26 (85 mg, 96%) as a mixture of isomers. Rf 0.41 and 0.34 (4:1
tolueneꢀEtOAc). 1H NMR (major isomer): δ 7.41ꢀ7.18 (m, 17H, Ar),
7.09 (t, 1H, J = 7.3 Hz, Ar), 6.84 (d, 1H, J = 7.8 Hz, ꢀNH), 6.74 (d, 2H, J =
7.8 Hz, Ar), 6.51 (d, 1H, J = 7.3 Hz, ꢀNH), 5.90 (m, 1H, ꢀCHdCH2),
5.54 [s, 1H, PhCH<], 5.32 (dd, 1H, J = 1.5, 17.1 Hz, ꢀCHdCH2), 5.22
(dd, 1H, J= 1.0, 10.5 Hz, ꢀCHdCH2), 5.08 (d, 1H, J = 8.3 Hz, H-1b), 3.15
(s, 1H, H-5b). Anal. Calcd for C48H46Cl6F3N3O11: C, 51.91; H, 4.17; N,
3.78. Found: C, 51.95; H, 4.28; N, 3.71.
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannop-
yranosyl-(1f3)-[3-O-allyl-4,6-O-benzylidene-2-deoxy-2-trichloroa-
cetamido-β-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-
trichloroacetamido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-
D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl
Azide 27. A stirred mixture of 26 (243 mg, 219 μmol), 16 (92 mg, 42
μmol), and dried MS AW300 (180 mg) in anhydrous CH2Cl2 (2 mL)
was cooled at ꢀ78 °C. Then 10% TMSOTf/CH2Cl2 (38 μL, 21 μmol)
was added to the mixture, which was stirred for 2 days at at ꢀ40 °C. The
reaction was quenched by addition of satd NaHCO3 aq, and the
insoluble material was filtered off. The filtrate was concentrated in
vacuo. The residue was chromatographed on Bio-Beads S-X1 in toluene
to give a nonasaccharide fraction, which was further purified by chroma-
tography on silica with hexaneꢀCHCl3ꢀEtOAc (2:1:1) to give 27 (117
mg, 69%). [R]D þ5.1 (c 0.5). Rf 0.22 (1:1 hexaneꢀEtOAc). 1H NMR: δ
1
88%). Rf 0.44 (9:1 tolueneꢀEtOAc). H NMR: δ 7.52 (m, 2H, Ar),
7.38ꢀ7.23 (m, 5H, Ar), 7.11 (t, 1H, J = 7.8 Hz, Ar), 6.80 (d, 2H, J = 7.8 Hz,
Ar), 6.66 (d, 1H, J =6.8 Hz, ꢀNH), 5.93 (m, 1H, ꢀCHdCH2), 5.60 [s, 1H,
PhCH<], 5.34 (dd, 1H, J = 1.5, 17.4 Hz, ꢀCHdCH2), 5.26 (dd, 1H, J = 1.0,
10.2 Hz, ꢀCHdCH2), 4.69 (m,1H, H-2), 4.42ꢀ4.25 (m, 3H, H-6,
ꢀCH2CHdCH2), 4.10ꢀ4.04 (m, 2H, H-3, H-4), 3.93 (dd, 1H, J = 2.4,
10.7 Hz, H-6), 3.79 (brs, 1H, H-5). Anal. Calcd for C26H24Cl3F3N2O6: C,
50.06; H, 3.88; N, 4.49. Found: C, 50.16; H, 3.95; N, 4.40.
tert-Butyldiphenylsilyl 3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-tri-
chloroacetamido-β-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-
deoxy-2-trichloroacetamido-β-D-glucopyranoside 24. A stirred mix-
ture of 22 (153 mg, 0.25 mmol), 23 (151 mg, 0.20 mmol), and dried MS
AW300 (610 mg) in anhydrous CH2Cl2 (6 mL) was cooled at ꢀ78 °C.
Then 10% TMSOTf/CH2Cl2 (20 μL, 0.01 mmol) was added to the
mixture. The stirring was continued for 1 h at that temperature and for
1.5 h at ꢀ40 °C. The reaction was quenched by addition of satd
NaHCO3 aq; the mixture was diluted with EtOAc; and the insoluble
material was filtered off. The combined filtrate and washings were
washed with water and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica gel
with hexaneꢀEtOAc (4:1ꢀ2:1) to give 24 (224 mg, 94%). [R]D þ4.0
(c 1.0). Rf 0.40 (3:2 hexaneꢀEtOAc). 1H NMR: δ 7.68 (d, 2H, J = 6.8
Hz, Ar), 7.61 (d, 2H, J = 6.8 Hz, Ar), 7.60ꢀ7.09 (m, 21H, Ar), 6.83 (d,
1H, J = 8.3 Hz, ꢀNHa), 6.79 (d, 1H, J = 7.3 Hz, ꢀNHb), 5.86 (m, 1H,
ꢀCHdCH2), 5.51 [s, 1H, PhCH<], 5.27 (dd, 1H, J = 1.5, 17.1 Hz,
ꢀCHdCH2), 5.17 (brd, 1H, J = 10.7 Hz, ꢀCHdCH2), 5.07 (d, 1H, J =
10.7 Hz, ꢀCH2Ph), 5.03 (d, 1H, J = 8.3 Hz, H-1b), 4.80 (d, 1H, J = 7.3
Hz, H-1a), 4.56 (d, 1H, J = 10.7 Hz, ꢀCH2Ph), 4.49 (d, 1H, J = 11.7 Hz,
ꢀCH2Ph), 4.36 (d, 1H, J = 11.7 Hz, ꢀCH2Ph), 4.28ꢀ4.19 (m, 3H,
5236
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
7.64ꢀ7.44 (m, 9H, Ar), 7.34ꢀ6.76 (m, 86H, Ar, ꢀNH ꢁ 3), 6.67ꢀ6.55
(m, 7H, Ar, ꢀNH), 5.86ꢀ5.74 (m, 2H, ꢀCHdCH2 ꢁ 2), 5.43 [s, 1H,
PhCH(O)2<], 5.40 [s, 1H, PhCH(O)2<], 5.22 (brd, 1H, J = 17.0 Hz, ꢀ
CHdCH2), 5.20(brd, 1H,J= 17.1 Hz, ꢀCHdCH2),5.18ꢀ4.91 (m, 9H, ꢀ
CHdCH2 ꢁ 2, H-1b, H-1a, H-1d, H-1e, H-1h, ꢀCH2Ph ꢁ 2), 4.49 (s, 1H,
H-1 g), 4.46 (s, 1H, H-1c), 2.88 and 2.74 (2s, 2H, H-5f and H-5i). 13CNMR:
δ 102.1 (1JCH = 159.7 Hz, C-1c), 101.1 [PhCH<], 100.6 (1JCH = 172.9 Hz)
and 99.0 (1JCH = 170.5 Hz) (C-1d and C-1g), 98.3 and 98.2 (1JCH = 163.8
Hz, C-1f and C-1i), 97.9 and 97.7 (1JCH = 166.3 Hz, C-1h and C-1e), 97.1
(1JCH = 168.0 Hz, C-1b), 92.8 and 92.7 (ꢀCCl3), 85.7 (1JCH = 165.5 Hz,
C-1a). Anal. Calcd for C210H209Cl12N9O47: C, 62.49; H, 5.22; N, 3.12.
Found: C, 62.20; H, 5.22; N, 3.05.
ꢀCOCH3), 1.33 (s, 9H, But). HRMS: calcd for C253H266Cl6N6O50Na2
[M þ 2Na]2þ, m/z 2224.3212 (100%). Found: 2224.3228.
In the two more mobile fractions separated by the HPLC, monode-
chlorinated and didechlorinated compounds were identified by HRMS
analysis. HRMS for (ꢀCl þ H): calcd for C253H267Cl5N6O50Na2 [M þ
2Na]2þ, m/z 2207.3421 (100%). Found: 2207.3458. HRMS for (ꢀ2Cl
þ 2H): calcd for C253H268Cl4N6O50Na2 [M þ 2Na]2þ, m/z 2189.8607
(100%). Found: 2189.8645.
N2-(9-Fluorenylmethoxycarbonyl)-N4-{3-O-allyl-4,6-O-benzyli-
dene-2-deoxy-2-trichloroacetamido-β-D-galactopyranosyl-(1f4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-
(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1f3)-[3-O-allyl-4,6-
O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-galactopyranosyl-
(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopy-
ranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1f6)]-2,4-
di-O-benzyl-β-D-mannopyranosyl-(1f4)-2-acetamido-3,6-di-O-ben-
zyl-2-deoxy-β-D-glucopyranosyl-(1f4)-2-acetamido-3,6-di-O-ben-
zyl-2-deoxy-β-D-glucopyranosyl}-L-asparagine Phenacyl Ester 30. A
mixture of 28 (45 mg, 12 μmol), Fmoc-Asp(OPfp)-OPac (8.4 mg,
13 μmol), HOOBt (2.2 mg, 13 μmol), and Ph(CH3)2P (2.0 μL, 14 μmol)
in 98% aq THF (0.2 mL) was stirred at room temperature for 5 h under Ar.
Then the mixture was diluted with EtOAc, washed successively with
saturated aq NH4Cl, saturated aq NaHCO3, water, and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was chroma-
tographed on Bio-Beads SX1 in tolueneꢀEtOAc (1:1), followed by HPLC
purification by InertSIL 100A (10 ꢁ 250 mm) using hexaneꢀEtOAc (1:3)
as an eluent to give 30 (43 mg, 83%). [R]D ꢀ1.7 (c 0.9). Rf 0.13 (3:7
hexaneꢀEtOAc). 1H NMR: δ 7.83 [d, 2H, J = 7.3 Hz, Ar (Fmoc)], 7.74 [d,
2H, J = 7.8 Hz, Ar (Fmoc)], 6.18 (d, 1H, J = 8.8 Hz, Asn-RNH), 5.94ꢀ5.84
(m, 2H, ꢀCHdCH2 ꢁ 2), 5.52 [s, 1H, PhCH<], 5.50 [s, 1H, PhCH<],
5.30 (brd, 2H, J = 17.0 Hz, ꢀCHdCH2), 2.99 and 2.84 (2s, 2H, H-5f and
H-5i), 2.78 (dd, 1H, J = 4.1, 16.4 Hz, Asn-βH), 1.62 (s, 3H, ꢀCOCH3),
The crude heptasaccharides (33 mg) derived by the incomplete
glycosylation were also obtained by the Biobeads column separation.
3-O-Allyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyra-
nosyl-(1f3)-[3-O-allyl-4,6-O-benzylidene-2-deoxy-2-trichloroace-
tamido-β-D-galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-
mannopyranosyl-(1f6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-(1f4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy -β-D-glucopyranosyl Azide 28.
Compound 27 (34 mg, 8.4 μmol) was converted to 28 (18 mg, 52%) by
dephthaloylation and acetylation, in a similar manner as described for 5.
[R]D ꢀ9.4 (c 0.9). Rf 0.49 (1:4 hexaneꢀEtOAc). 1H NMR: δ 6.85 (d,
1H, J = 7.3 Hz, ꢀNH), 6.61 (d, 1H, J = 7.3 Hz, ꢀNH), 6.16 (d, 1H, J =
8.8 Hz, ꢀNH), 5.94ꢀ5.83 (m, 2H, ꢀCHdCH2 ꢁ 2), 5.52 [s, 1H,
PhCH<], 5.50 [s, 1H, PhCH<], 5.30 (brd, 2H, J = 17.1 Hz, ꢀCHd
CH2), 5.19 (brd, 2H, J = 10.2 Hz, ꢀCHdCH2), 2.98 and 2.86 (2s, 2H,
H-5f and H-5i), 1.73 (s, 3H, ꢀCOCH3), 1.49 (s, 3H, ꢀCOCH3). 13
C
NMR: δ 101.1 (C-1c), 100.9 [PhCH(O)2<], 100.2, 99.8, 98.9, 98.2,
97.5, and 97.4 (C-1d, C-1g, C-1f, C-1i, C-1h, C-1e, and C-1b), 92.6,
92.5, 92.4, and 92.3 (ꢀCCl3), 88.1 (C-1a), 23.3 (ꢀCOCH3), 23.0
(ꢀCOCH3). Anal. Calcd for C198H209Cl12N9O45: C, 61.61; H, 5.46; N,
3.27. Found: C, 62.00; H, 5.27; N, 3.05.
1.52 (s, 3H, ꢀCOCH3). Anal. Calcd for C225H232Cl12N8O51 5H2O: C,
3
61.70; H, 5.57; N, 2.56. Found: C, 61.90; H, 5.81; N, 2.43.
N2-(9-Fluorenylmethoxycarbonyl)-N4-{2,3,4,6-tetra-O-benzyl-β-D-
galactopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-
glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-
(1f3)-[2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-2-acet-
amido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f2)-3,4,6-tri-
O-benzyl-R-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-β-D-manno-
pyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluco-
pyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluco-
pyranosyl}-L-asparagine tert-Butyl Ester 31. A mixture of 29 (170 mg,
39 μmol), powdered Zn (380 mg, 5.8 mmol), and AcOH (0.4 mL,
7.0 mmol) in EtOAc (15 mL) was stirred under microwave irradiation at
150 W for 1.5 h. The microwave machine was controlled to keep gentle
refluxing during this period. After cooling, the insoluble materials were
removed by filtration, and the filtrate was concentrated in vacuo. The
residual crude product was purified by chromatography on silica gel with
1% CH3OH/CHCl3 and then by HPLC with Mightysil Si 60 (10 ꢁ 250
mm) in 1% CH3OH/CHCl3 to afford 31 (139 mg, 86%). [R]D þ14.2
N2-(9-Fluorenylmethoxycarbonyl)-N4-{2,3,4,6-tetra-O-benzyl-β-D-
galactopyranosyl-(1f4)-3,6-di-O-benzyl-2-deoxy-2-trichloroaceta-
mido-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopy-
ranosyl-(1f3)-[2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-
(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1f6)]-2,4-di-O-ben-
zyl-β-D-mannopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-
deoxy-β-D-glucopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-
deoxy-β-D-glucopyranosyl}-L-asparagine tert-Butyl Ester 29. A mix-
ture of 5 (236 mg, 59 μmol), Fmoc-Asp(OPfp)-OBut (53 mg, 92 μmol),
HOOBt (18 mg, 110 μmol), and Ph(CH3)2P (15 μL, 105 μmol) in 98%
aq. THF (6 mL) was stirred at room temperature for 3 h under Ar. Then
the mixture was diluted with EtOAc, washed with water and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was
chromatographed by HPLC using a gel-permeation column (JAIGEL
2H, 20 ꢁ 600 mm) with CHCl3 (3.5 mL/min) and then a column
(Inertsil SIL100A, 20 ꢁ 250 mm) with hexaneꢀEtOAc (35:65, 5 mL/
min) to give 29 (216 mg, 84%). [R]D þ12.4 (c 2.8). Rf 0.44 (95:5
tolueneꢀEtOH). 1H and 13C NMR spectra in CDCl3 showed multiple
signals due to the presence of conformational isomers. 1H NMR: δ 7.74
[brd, 2H, J = 7.6 Hz, Ar (Fmoc)], 7.58 [brd, 2H, J = 7.3 Hz, Ar (Fmoc)],
2.80 (dd, 1H, J = 3.9, 16.1 Hz, Asn-βH), 2.62 (dd, 1H, J = 3.4, 16.1 Hz,
Asn-βH), 1.71 and 1.68 (2s, 3H, ꢀCOCH3), 1.54, 1.53, 1.50, and 1.49
(4s, 3H, ꢀCOCH3), 1.41 (s, 9H, But). 13C NMR: δ 102.8 and 102.7 (C-
1f and C-1i), 101.2, 100.0, 99.6, 99.4, 99.0, 98.4, and 97.2 (C-1b, C-1c,
C-1d, C-1e, C-1g, C-1h), 92.6 and 92.4 (ꢀCCl3), 79.9 (C-1a), 42.6
[ꢀC(CH3)3], 27.9 [ꢀC(CH3)3], 23.2 (ꢀCOCH3), 22.7 (ꢀCOCH3).
1H NMR (DMSO-d6): δ 1.71 (s, 3H, ꢀCOCH3), 1.69 (s, 3H,
1
(c 2.4). Rf 0.23 (2% CH3OH/CHCl3). H NMR (600 MHz, DMSO-
d6): δ 5.08 (brs, 1H, H-1d), 4.92 (brt, 1H, H-1a), 2.60 (br, 1H, Asn-βH),
2.46 (br, 1H, Asn-βH), 1.77 (s, 3H, ꢀCOCH3), 1.75 (s, 3H, ꢀ
COCH3), 1.72 (s, 3H, ꢀCOCH3), 1.71 (s, 3H, ꢀCOCH3), 1.33
(s, 9H, But). 13C NMR (150 MHz, DMSO-d6): δ 102.2 and 102.0 (C-1f
and C-1i), 100.7 (C-1c), 100.4 and 99.8 (ꢁ 2) (C-1e, C-1h, and C-1b), 99.4
(C-1d), 97.3 (C-1g), 78.4 (C-1a), 27.5 [ꢀC(CH3)3], 23.0, 22.9 (ꢁ 2), and
22.7 (ꢀCOCH3 ꢁ 4). HRMS: calcd for C253H272N6O50Na2 [M þ
2Na]2þ, m/z 2120.9388 (100%). Found: 2120.9370.
N2-(9-Fluorenylmethoxycarbonyl)-N4-{2-acetamido-3-O-allyl-4,6-
O-benzylidene-2-deoxy-β-D-galactopyranosyl-(1f4)-2-acetami-
do-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-
benzyl-R-D-mannopyranosyl-(1f3)-[2-acetamido-3-O-allyl-4,6-O-
5237
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
benzylidene-2-deoxy-β-D-galactopyranosyl-(1f4)-2-acetamido-3,6-
di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-
D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-β-D-mannopyranosyl-
(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-
(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl}-L-
asparagine 4. A mixture of 30 (12 mg, 2.8 μmol), powdered Zn (280
mg, 4.3 mmol), and AcOH (0.28 mL, 4.9 mmol) in EtOAc (3.0 mL) was
stirred under microwave irradiation at 150 W for 1 h. The microwave
machine was controlled to keep gentle refluxing during this period. After
cooling, the insoluble materials were removed by filtration, and the
filtrate was concentrated in vacuo. The residual crude product was purified by
preparative thin-layer chromatogoraphy with CHCl3ꢀCH3OH (95:5) con-
taining 1% AcOH to give 4 (4.8 mg, 45%). [R]D ꢀ2.34 (c 0.75). Rf 0.34
(CHCl3ꢀCH3OH (95:5) containing 1% AcOH). 1H NMR (DMSO-d6): δ
7.89 [d, 2H, J = 7.3 Hz, Ar (Fmoc)], 7.70 [d, 2H, J = 7.8 Hz, Ar (Fmoc)],
5.92ꢀ5.84 (m, 2H, ꢀCHdCH2 ꢁ 2), 5.63 [s, 1H, PhCH<], 5.61 [s, 1H,
PhCH<], 1.91, 1.90, 1.88 (s, 6H, ꢀCOCH3 ꢁ 6), 1.76, 1.72 (s, 12H,
Dr. T. Nakamura for HRMS, and staff of the analysis laboratory
for elemental analysis at Riken. We also thank Tokai University
for their support by a grant-aid for high-technology research.
’ REFERENCES
(1) Hojo, H.; Nakahara, Y. Biopolymers 2007, 88, 308–324.
(2) Brik, A.; Ficht, S.; Wong, C.-H. Curr. Opin. Chem. Biol. 2006,
10, 638–644.
(3) (a) Takano, Y.; Habiro, M.; Someya, M.; Hojo, H.; Nakahara, Y.
Tetrahedron Lett. 2002, 43, 8395–8399. (b) Takano, Y.; Kojima, N.;
Nakahara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron 2003, 59, 8415–8427.
(4) Nakahara, Y.; Ozawa, C.; Tanaka, E.; Ohtsuka, K.; Takano, Y.;
Hojo, H.; Nakahara, Y. Tetrahedron 2007, 63, 2161–2169.
(5) Ueki, A.; Nakahara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron 2007,
63, 2170–2181.
(6) (a) Ogawa, T.; Sugimoto, M.; Kitajima, T.; Sadozai, K. K.;
Nukada, T. Tetrahedron Lett. 1986, 27, 5739–5742. (b) Unverzagt, C.
Angew. Chem., Int. Ed. Engl. 1996, 35, 2350–2353. (c) Seifert, J.;
Lergenm€uller, M.; Ito, Y. Angew. Chem., Int. Ed. 2000, 39, 531–534.
(d) Wu, B.; Hua, Z.; Warren, J. D.; Ranganathan, K.; Wan, Q.; Chen, G.;
Tan, Z.; Chen, J.; Endo, A.; Danishefsky, S. J. Tetrahedron Lett. 2006,
47, 5577–5579. (e) Sun, B.; Srinivasan, B.; Huang, X. Chem.—Eur.
J. 2008, 14, 7072–7081.
(7) (a) Unverzagt, C. Tetrahedron Lett. 1997, 38, 5627–5630.
(b) Mezzato, S.; Schaffrath, M.; Unverzagt, C. Angew. Chem., Int. Ed.
2005, 44, 1650–1654.
(8) (a) Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. J. Am. Chem. Soc.
2004, 126, 736–738. (b) Wu, B.; Tan, Z.; Chen, G.; Chen, J.; Hua, Z.;
Wan, Q.; Ranganathan, K.; Danishefsky, S. J. Tetrahedron Lett. 2006,
47, 8009–8011. (c) Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto,
K.; Zhang, X.; Wang, L. X. J. Am. Chem. Soc. 2009, 131, 2214–2223.
(9) Meinjohanns, E.; Meldal, M.; Paulsen, H.; Dwek, R. A.; Bock, K.
J. Chem. Soc., Perkin Trans 1 1998, 549–560.
(10) Yamamoto, N.; Ohmori, Y.; Sakakibara, T.; Sasaki, K.; Juneja,
L. R.; Kajihara, Y. Angew. Chem., Int. Ed. 2003, 42, 2537–2540.
(11) (a) Haneda, K.; Inazu, T.; Mizuno, M.; Iguchi, R.; Yamamoto,
K.; Kumagai, H.; Aimoto, S.; Suzuki, H.; Noda, T. Bioorg. Med. Chem.
Lett. 1998, 8, 1303–1306. (b) O’Conner, S. E.; Pohlmann, J.; Imperiali,
B.; Saskiawan, I.; Yamamoto, K. J. Am. Chem. Soc. 2001, 123, 6187–6188.
(12) Preparation of diverse complex-type N-glycans by chemo-
enzymatical degradation processes has been reported. Kajihara, Y.;
Suzuki, Y.; Yamamoto, N.; Sasaki, K.; Sakakibara, T.; Juneja, L. R.
Chem.—Eur. J. 2004, 10, 971–985.
ꢀCOCH3 ꢁ 4). HRMS: calcd for C217H237N8O50Na3 [M ꢀ H þ 3Na]2þ
m/z 1912.7998 (100%). Found: 1912.8007.
,
N2-(9-Fluorenylmethoxycarbonyl)-N4-{2,3,4,6-tetra-O-benzyl-β-
D-galactopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-
glucopyranosyl-(1f2)-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-
(1f3)-[2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl-(1f4)-2-aceta-
mido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1f2)-3,4,6-tri-
O-benzyl-R-D-mannopyranosyl-(1f6)]-2,4-di-O-benzyl-β-D-man-
nopyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluc-
opyranosyl-(1f4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-
glucopyranosyl}-L-asparagine 3. A solution of 31 (40 mg, 10 μmol) in
80% aq TFA (1 mL) was stirred at room temperature for 3 h. The solvent
was evaporated in vacuo, and the residue was purified by HPLC on a
column of Mightysil Si 60 (10 ꢁ 250 mm) with 1% AcOH/2%
CH3OH/CHCl3 to give 3 (28 mg, 71%). [R]D þ10.1 (c 1.5). Rf 0.23
(3% CH3OH/CHCl3). 1H NMR (600 MHz, DMSO-d6): δ 5.09 (brs,
1H, H-1d), 4.93 (brt, 1H, J = 9.0 Hz, H-1a), 2.63 (dd, 1H, J = 4.1, 15.8
Hz, Asn-βH), 2.49 (brd, 1H, Asn-βH), 1.76 (s, 3H, ꢀCOCH3), 1.74
(s, 3H, ꢀCOCH3), 1.71 (s, 6H, ꢀCOCH3 ꢁ 2). 13C NMR (150 MHz,
DMSO-d6): δ 102.0 and 101.9 (C-1f and C-1i), 100.7 (C-1c), 100.3 and
99.7 (ꢁ 2) (C-1e, C-1h, and C-1b), 99.3 (C-1d), 97.3 (C-1g), 78.4
(C-1a), 22.8, 22.7 (ꢁ 2), and 22.5 (ꢀCOCH3 ꢁ 4). HRMS: calcd for
C249H264N6O50Na2 [M þ 2Na]2þ, m/z 2092.9075 (100%). Found:
2092.9079; calcd for C249H263N6O50Na3 [M ꢀ H þ 3Na]2þ, m/z
2103.8985 (100%). Found: 2103.8991.
(13) For a review on the glycoforms and functions of glycodelin:
Lapid, K.; Sharon, N. Glycobiology 2006, 16, 39R–45R.
(14) Matsuo, I.; Nakahara, Y.; Ito, Y.; Nukada, T.; Nakahara, Y.;
Ogawa, T. Bioorg. Med. Chem. 1995, 3, 1455–1463.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra for
S
b
(15) (a) Crich, D.; Sun, S. J. Org. Chem. 1996, 61, 4506–4507.
(b) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997, 119, 11217–11223.
(c) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020.
(16) The closely related trisaccharides were synthesized by the
R-triflate-mediated mannnosylation. (a) Dudkin, V. Y.; Crich, D. Tetra-
hedron Lett. 2003, 44, 1787–1789. (b) Dudkin, V. Y.; Miller, J. S.;
Danishefsky, S. J. Tetrahedron Lett. 2003, 44, 1791–1793.
(17) Crich, D.; Li, H. M.; Yao, Q. J.; Wink, D. J.; Sommer, R. D.;
Rheingold, A. L. J. Am. Chem. Soc. 2001, 123, 5826–5828.
(18) Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41, 5547–
5551.
new compounds and the result of the Low-TfOH treatment of
compounds 3 and 4. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel.: þ81-463-58-1211 (Ex. 4173). Fax: þ81-463-50-2075.
E-mail: yonak@keyaki.cc.u-tokai.ac.jp or hojo@keyaki.cc.u-to-
kai.ac.jp
(19) Suzuki, K.; Maeta, H.; Matsumoto, T. Tetrahedron Lett. 1989,
30, 4853–4856.
(20) Zang, Y.-M.; Mallet, J.-M.; Sina€y, P. Carbohydr. Res. 1992,
236, 73–88.
(21) Matsuo, I.; Totani, K.; Tatami, A.; Ito, Y. Tetrahedron 2006,
62, 8262–8277.
(22) Cumpstey, I.; Fairbanks, A. J.; Redgrave, A. J. Org. Lett. 2001,
3, 2371–2374.
’ ACKNOWLEDGMENT
This work was supported by a grant-in-aid for creative
scientific research (17GS0420) from the Japan Society for the
Promotion of Science. We thank Dr. H. Koshino for HRNMR,
5238
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239

The Journal of Organic Chemistry
ARTICLE
(23) (a) Bock, K.; Lundt, I.; Pedersen, C. Tetrahedron Lett. 1973,
14, 1037–1040. (b) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2
1974, 293–297.
(24) Nakahara, Y.; Iijima, H.; Shibayama, S.; Ogawa, T. Carbohydr.
Res. 1991, 216, 211–225.
(25) Ueki, A.; Takano, Y.; Kobayashi, A.; Nakahara, Y.; Hojo, H.;
Nakahara, Y. Tetrahedron 2010, 66, 1742–1759.
(26) Ueki, A.; Hirota, M.; Kobayashi, Y.; Komatsu, K.; Takano, Y.;
Iwaoka, M.; Nakahara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron 2008,
64, 2611–2618.
(27) (a) Doores, K. L.; Miura, Y.; Dwek, R. A.; Rudd, P. M.; Elliot,
T.; Davis, B. G. Chem. Commun. 2006, 1401–1403. (b) Inazu, T.;
Kobayashi, K. Synlett 1993, 869–870.
5239
dx.doi.org/10.1021/jo200149d |J. Org. Chem. 2011, 76, 5229–5239