Rapid assembly of oligosaccharides: A highly convergent strategy for the assembly of a glycosylated amino acid derived from PSGL-1

Article abstract of DOI:10.1021/jo901135k

(Chemical Equation Presented) P-Selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are vascular adhesion molecules that play an important role in the recruitment of leukocytes to inflamed tissue by establishing leukocyte-endothelial and leukocyte-plat

Full text of DOI:10.1021/jo901135k

pubs.acs.org/joc
Rapid Assembly of Oligosaccharides: A Highly Convergent Strategy for
the Assembly of a Glycosylated Amino Acid Derived from PSGL-1
Yusuf Vohra, Therese Buskas, and Geert-Jan Boons*
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602
gjboons@ccrc.uga.edu
Received May 28, 2009
P-Selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are vascular adhesion molecules that play
an important role in the recruitment of leukocytes to inflamed tissue by establishing leukocyte-
endothelial and leukocyte-platelet interaction. P-Selectin binds to the amino-terminus of PSGL-1
through recognition of a sialyl Lewisx (SLex) moiety linked to a properly positioned core-2 O-glycan
and three tyrosine sulfate residues. We have developed a highly convergent synthesis of the PSGL-1
oligosaccharide linked to threonine based on the use of trichoroacetimidate donors and thioglycosyl
acceptors that give products that can immediately be employed in a subsequent glycosylation step
without the need for protecting group manipulations. Furthermore, by employing one-pot multistep
glycosylation sequences the number of purification steps could be minimized. The process of
oligosaccharide assembly was further streamlined by combining protecting group manipulations
and glycosylations as a one-pot multistep synthetic procedure. The resulting PSGL-1 oligosaccharide
is properly protected for glycopeptide assembly. It is to be expected that the strategic principles
employed for the synthesis of the target compound can be applied for the preparation of other
complex oligosaccharides of biological and medical importance.
Introduction
organ-transplant rejection, arthritis, sickle cell anemia, and
tumor metastasis.4-6
The selectins are a family of three Ca2þ-dependent mem-
brane-bound glycoproteins that mediate the adhesion of
leukocytes and platelets to vascular surfaces.1,2 Several
studies have demonstrated that they play important
roles in inflammation, immune responses, hemeostasis, and
wound repair.3 Selectins also contribute to a broad spect-
rum of diseases such as arteriosclerosis, thrombosis,
Although there are many candidates for selectin ligands,
only P-selectin glycoprotein ligand-1 (PSGL-1) has clearly
been demonstrated to mediate the adhesion of leukocytes to
selectins under flow. P-Selectin binds to the amino-terminus
of PSGL-1 through recognition of a sialyl Lewisx (SLex)
moiety linked to a properly positioned core-2 O-glycan and
three tyrosine sulfate residues.7,8
Inhibitors of selectins may possess therapeutic properties
for the treatment of a number of diseases.9 In this respect, a
recombinant truncated form of a PSGL-1 immunoglobulin
fusion protein has already demonstrated effectiveness as
*To whom correspondence should be addressed. Fax: þ1-706-542-4412.
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J.; Camphausen, R. T.; Widom, A.; Xia, L.; Kazazian, H. H.; Schaub, R. G.;
McEver, R. P.; Wagner, D. D. Nat. Med. 2003, 9, 1020–1025.
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Varki, A.; Mcever, R. P. J. Cell Biol. 1992, 118, 445–456.
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6064 J. Org. Chem. 2009, 74, 6064–6071
Published on Web 07/16/2009
DOI: 10.1021/jo901135k
r
2009 American Chemical Society

Vohra et al.
JOCArticle
such an inhibitor.10-13 This glycoprotein can, however, only
be produced in mammalian cells that are cotransfected with
fucosyl- and core-2 GlcNAc transferases, making produc-
tion of even small amounts of glycoprotein difficult. The
Davis laboratory is beginning to address these problems by
employing a PSGL-1 mimetic by incorporation of azidoho-
moalanine and cysteine in PSGL-1 using E. coli B834 as a
Met-auxotrophic expression system and selective attach-
ment of sialic acid containing oligosaccharides and a sulfated
tyrosine mimic by employing the thiol and azide of the
protein as chemical tags.14 Also, it has been shown that
conjugation of sialyl Lewisx and tyrosine sulfates to a poly-
acrylamide gave a polymer with high affinity of L-selectin.15
The N-terminal glycosulfopeptide of PGSL-1 has been
obtained by chemoenzymatic approaches.14,16,17 In these
procedures, a glycosulfopeptide that contains an N-acetyl
galactosamine linked to a threonine moiety was chemically
assembled. Subsequently, glycosyl transferases were em-
ployed to assemble the complete oligosaccharide. The pro-
blems of this approach include difficulties of preparing
sufficient quantities of glycosyltransferases, which often
require a eukaryotic cell expression system and the need of
expensive sugar nucleotides or the use of a complicated in
situ recycling system. Furthermore, the high selectivity of
glycosyltransferases also complicates the preparation of
analogues that may exhibit more desirable pharmacological
properties.
oligosaccharide of PSGL-1.24-30 In this respect, Kunz and
co-workers have reported the chemical synthesis of a prop-
erly protected oligosaccharide of PSGL-1, which was at-
tached to threonine for the preparation of a glycopeptide.26
Although synthetic problems such as anomeric selectivity
and the acid and base sensitivity of the PSGL-1 glycopeptide
were addressed by employing properly protected saccharide
building blocks, the synthetic approach suffered from poor
regioselectivity in key glycosylations and a need for replace-
ment of protecting groups at an advanced stage of synthesis.
It is to be expected that a highly convergent approach for the
synthesis of PSGL-1 will make it possible to prepare a wide
range of glycopeptides structural analogues for structure
activity relationships. Furthermore, it may offer an oppor-
tunity to make mimetics that have improved pharmacoki-
netic properties.
As part of a program to prepare the PGSL-1 analogues
with improved properties, we report here a highly efficient
and convergent synthesis of a properly protected oligosac-
charide of PSGL-1 linked to threonine (1) that is appro-
priately protected for solid-phase glycopeptide synthesis.
Key features of the approach include an orchestrated use
of thioglycosides and trichoroacetimidates31 for oligosac-
charide assembly, which minimized protecting group manip-
ulations and made it possible to employ one-pot multistep
glycosylations. The process of oligosaccharide assembly was
further streamlined by combining protecting group manip-
ulations and glycosylations as a one-pot multistep synthetic
procedure.22,23,32-34 It is to be expected that the strategic
principles employed for the synthesis of the target compound
can be applied for the preparation of other complex oligo-
saccharides of biological and medical importance.
Recent progress in chemical oligosaccharide synthesis is
beginning to provide opportunities for the efficient and
large-scale synthesis of complex oligosaccharides18-23 and
several laboratories are pursuing the preparation of the
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The synthesis of target compound 1 is complicated by the
fact that O-glycosylated peptides are sensitive to acidic and
basic conditions. In addition, sufficient quantities of such a
complex glycosylated amino acid for glycosulfopeptide
assembly can only be obtained by employing a highly con-
vergent synthetic strategy, which uses properly pro-
tected monosaccharide building blocks that can be as-
sembled into the target by using a minimal number of
synthetic steps. In this respect, strategies such as chemose-
lective, orthogonal, two-directional, and one-pot multistep
glycosylations22,23,32-34 have engendered an increased effi-
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ber of protecting group manipulations on advanced
intermediates. Furthermore, combining protecting group
manipulations with glycosylations as a one-pot procedure
can further expand the scope of these procedures.35-39
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It was envisaged that 1 could be prepared by a combina-
tion of chemo- and regioselective glycosylations and one-pot
multistep protocols that combine glycosylations and protect-
ing group manipulations. Thus, the core-2 disaccharide
linked to a properly protected threonine (Thr) was prepared
by a chemoselective glycosylation of trichloroacetimidate 2
with thioglycoside 3 to give a disaccharide, which immedi-
ately can be activated with a thiophilic reagent for coupling
with threonine acceptor 440 (Figure 1). Removal of the
benzylidene acetal of the resulting compound will give an
acceptor for a regioselective coupling with a properly pro-
tected SLex derivative. It was envisaged the latter compound
could be obtained by chemoselective glycosylations and one-
pot reactions by using compounds 5,41 6,42 and 7.
Unfortunately, coupling of galactosyl trichloroacetimi-
date 2a43 with thioglycosyl acceptor 3a44 in the presence of
TMSOTf led to a complex mixture of products that included
the required disaccharide, the corresponding ortho-ester,45
and a thiophenyl galactoside derived from aglycon-trans-
fer46 (Scheme 1). It is well-known that the use of C-2 benzoyl
esters will suppress ortho-ester formation; however, this
protecting group is not compatible with glycopeptide synth-
esis because the rather strong basic conditions required for
its removal47 will result in β-elimination of the O-glycopep-
tide linkage. It has been shown that a 2,5-difluorobenzoyl
ester (dFBz) is an efficient neighboring group participant
that suppresses ortho-ester formation.48,49 This protecting
group has, however, the advantage that it can be re-
moved under mild basic conditions without affecting threo-
nine and serine glycosides. Thus, dFBz-protected glycosyl
donor 2b,48,49 having a 2,5-difluorobenzoyl (dFBz) ester at
C-2 and acetyl esters at C-3, C-4, and C-6, was coupled with
thioglycosyl acceptor 3a50 by using TMSOTf as the catalyst.51
Although ortho-ester formation was suppressed, the aglycon-
transfer byproduct was still formed. Recently, it was reported
that aglycon transfer of thioglycosyl acceptors can be
avoided by employing a 2,6-dimethylthiophenyl glycoside.52
The rationale of this observation is that the bulky 2,6-
dimethylthiophenyl hinders reaction with an activated glycosyl
donor, thereby reducing aglycon transfer. Indeed, trimethylsi-
lyl triflate (TMSOTf) promoted glycosylation of 2b with 3b52
gave the corresponding disaccharide 8 in an excellent yield of
90% as only the β-anomer. Next, the core-2 O-glycan 9 was
obtained in high yield with exclusively R-selectivity by a
FIGURE 1. Target molecule and building blocks.
diphenyl sulfoxide/triflic anhydride-mediated glycosylation53
of thioglycoside 8 with threonine derivative 4.
Having established efficient reaction conditions for the
synthesis of 9, attention was focused on its preparation by a
one-pot procedure. Thus, coupling of galactosyl trichloroa-
cetimidate 2b with the galactosyl acceptor 3b in the presence
of TMSOTf followed by activation of the resulting thio-
disaccharide 8 by addition of diphenyl sulfoxide and triflic
anhydride in the presence of DTBMP53 and coupling with
threonine 4 gave oligosaccharide 9 in an overall yield of 61%.
Finally, glycosyl acceptor 10 was obtained by the removal
of the benzylidene acetal of 9 by using aqueous acetic acid
at 70 °C.
The next stage of the synthesis entailed the preparation of
properly protected SLex glycosyl donor 15 for coupling with
glycosyl acceptor 10 to give protected PSGL-1 16. Com-
pound 15 was prepared from the readily available saccharide
building blocks 5, 6, and 7 (Schemes 2 and 3). Thus, fucosyl
trichloroacetimidate 5 was coupled with phenyl thioglycosyl
acceptor 6 by using TMSOTf as the promoter to give the
corresponding disaccharide, exclusively as the R-anomer,
which was then treated with triethylsilane and trifluoro-
methanesulfonic acid (TfOH) for regioselective opening of
the benzylidene acetal39,54 to provide glycosyl acceptor 11 in
an overall yield of 84% with excellent stereo- and regioselec-
tivity. The regioselectivity of the latter reaction was con-
firmed by acetylation of compound 11 and the 1H NMR of
the resulting derivative showed a significant downfield shift
for H-4 (4.94 ppm).
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Glycosyl donor 7 could be obtained in facile manner from
the known disaccharide 1255 by a four-step reaction se-
quence. Thus, hydrogenation of 12 over Pd/C to remove
the benzylidene acetal was followed by acetylation of the
hydroxyls of the resulting compound 13 to give 14 in a quan-
titative overall yield. Next, the anomeric trimethylsilylethyl
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SCHEME 1. One-Pot Three-Component Reactiona
aSDMP = 2,6-dimethylthiophenyl, dFBz = 2,5-difluorobenzyl.
SCHEME 2. One-Pot Glycosylation Followed by Reductive
Opening of the Benzylidene Acetal
hydroxyl of 10. The latter was also supported by Nuclear
Overhauser Enhancement Spectroscopy (NOESY), which
revealed cross peaks between the H-1 of β-GluNTroc and
H-6a and H-6b of the R-GalN3 moiety. Furthermore, due to
effective neighboring group participation of the N-Troc
group of 15 only the β-glycoside was formed, which was
confirmed by a large coupling constant between H-1 and H-2
(10.0 Hz).
Finally, the Troc and the azido moiety of 16 were con-
verted into acetamido functions by reduction with Zn/
CuSO4 in a mixture of THF, acetic acid, and acetic anhy-
dride56 to give the target compound 1. It is envisaged that the
benzyl ester of compound 1 can be removed by performing
hydrogenolysis over palladium in a mixture of isopropanol
and pyridine.57 The benzyl ethers in the glycan will be
removed after glycopeptide assembly by using the previously
described “low TfOH” method.58,59
In conclusion, a properly protected PSGL-1 oligosac-
charide linked to threonine has been described that is
appropriately protected for glycosulfopeptide assembly. A
highly convergent strategy utilizing six strategically pro-
tected building blocks, combined with one-pot chemo- and
regioselective glycosylations was employed to minimize the
number of protecting group manipulations and purifications
during oligosaccharide assembly. The longest linear se-
quence entailed only seven chemical steps and gave the target
compounds in an excellent overall yield of 17%. Previous
moiety of 14 was cleaved by treatment with trifluoroacetic
acid in dichloromethane and the resulting lactol was con-
verted into trichloroacetimidate 7 by reaction with trichlor-
oacetonitrile and DBU in dichloromethane.
Next, a TMSOTf-mediated coupling of trichloroactimi-
date 7 with 11 gave the properly protected SLex tetrasac-
charide 15 in good yield. Up to this stage of the synthesis, the
thiophenyl moiety of 15 has functioned as an effective
anomeric protecting group. However, in the next step it
was activated with the thiophilic promoter NIS/TfOH for
coupling with 10 to give the hexasaccharide 16 in a yield of
55%. As expected, no glycosylation of the less reactive C-4
hydroxyl of 11 was observed, which was confirmed by a
range of two-dimensional NMR experiments. Thus, Hetero-
nuclear Multiple Bond Correlation NMR Spectroscopy
(HMBC) of 16 showed a cross peak between H-1 of
β-GluNTroc (4.62 ppm) and C-6 of R-GalN3 (69.3 ppm),
confirming that the glycosylation had occurred at the C-6
(56) Winans, K. A.; King, D. S.; Rao, V. R.; Bertozzi, C. R. Biochemistry
1999, 38, 11700–11710.
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2004, 5, 1228–1236.
(58) Tam, J. P.; Heath, W. F.; Merrifield, R. B. J. Am. Chem. Soc. 1986,
108, 5242–5251.
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Tetrahedron Lett. 2002, 43, 8395–8399.
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SCHEME 3. Synthesis of Target Molecule
(170 mg, 90%) as a white foam. Analytical data for 8:Rf 0.35 (hexanes:
EtOAc, 2:1, v:v); 1H NMR (500 MHz, CDCl3) δ 7.52-6.907 (m, 6H,
0
aromatic), 5.49 (dd, 1H, J1 ,2 =8.2 Hz, J2 ,3 =10.2 Hz, H-2 ), 5.43 (s,
0
0
0
0
0
0
1H, CHPh), 5.38 (br d, 1H, J=3.2Hz, H-4 ), 5.12(dd, 1H, J3 ,4 =3.1
0
0
0
0
0
0
Hz, J2 ,3 =10.5 Hz, H-3 ), 4.96 (d, 1H, J1 ,2 =7.9 Hz, H-1 ), 4.18 (m,
2H, H-1, H-4), 4.15-4.05 (m, 3H, H-6a, H-6a0, H-6b0), 3.91-3.85 (m,
2H, H-50, H-6b), 3.72 (t, 1H, J1,2=J2,3=9.9 Hz, H-2), 3.46 (dd, 1H,
J3,4=3.1 Hz, J2,3=9.9 Hz, H-3), 3.17 (br s, 1H, H-5), 2.51 (s, 6H, 2 ꢀ
CH3,SDMP),2.11(s,3H,COCH3),1.98(s,3H,COCH3),1.87(s,3H,
COCH3) ppm; 13C from HSQC (125.7 MHz, CDCl3) δ 102.3 (C-10),
101.1 (CHPh), 89.4 (C-1), 80.9 (C-3), 75.3 (C-4), 71.4 (C-50), 71.2
(C-30), 70.3 (C-20), 70.0 (C-5), 69.6 (C-6), 67.3 (C-40), 62.4 (C-2), 61.8
(C-60), 22.9 (CH3-SDMP), 21.0, 20.9, 20.8 (3 ꢀ OAc); HR-MALDI-
ToF/MS m/z calcd for C40H41F2N3O13S [MþNa]þ 864.2226, found
864.2231.
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-azido-4,6-O-benzyli-
dene-2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-
β-D-galactopyranosyl)-r-D-galactopyranosyl]-L-threonine Benzyl
Ester (9). Method A:. A mixture of disaccharide donor 8
˚
(170 mg, 0.20 mmol), Ph2SO (114 mg, 0.56 mmol), and 4 A
MS in CH2Cl2 (5 mL) was placed under an atmosphere of argon
and stirred at room temperature for 1 h. The reaction mixture
was then cooled to -60 °C after the addition of 2,6-di-tert-butyl-
4-methylpyridine (124 mg, 0.60 mmol). Stirring was continued
for 10 min at the same temperature followed by the addition of
Tf2O (47 μL, 0.28 mmol). Stirring was continued for another
15 min at the same temperature followed by the addition of a
solution of threonine acceptor 4 (173 mg, 0.40 mmol) in CH2Cl2
(2 mL). The temperature of the reaction mixture was raised to
0 °C over a period of 1 h. The progress of reaction was monitored
by TLC and MALDI-ToF MS. The reaction mixture was
diluted with CH2Cl2 (30 mL), filtered, and washed with satu-
rated aq NaHCO3 solution (15 mL), water (15 mL), and brine
(15 mL). The organic layer was dried (MgSO4) and filtered, and
the filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexanes:EtOAc, 2:1, v:v)
to afford compound 9 (156 mg, 70%) as a white amorphous
solid.
Method B:. A mixture of galactosyl acceptor 3b (65 mg, 0.16
mmol), galactosyl trichloroacetaimidate donor 2b (120 mg, 0.20
˚
mmol), and 4 A MS in CH2Cl2 (2 mL) was placed under an
atmosphere of argon and stirred at room temperature for 1 h.
The reaction mixture was then cooled to -60 °C. TMSOTf
(0.016 mmol, 0.16 M solution in CH2Cl2) was added and stirring
was continued for 1 h at the same temperature. The reaction
mixture was then cooled to -78 °C followed by addition of
Ph2SO (88 mg, 0.44 mmol) and 2,6-di-tert-butyl-4-methylpyr-
idine (112 mg, 0.55 mmol). After stirring for 10 min at the same
temperature, Tf2O (37 μL, 0.22 mmol) was added followed by
increasing the temperature to -60 °C over a period of 15 min.
The reaction mixture was again cooled to -78 °C followed by
addition of a solution of threonine acceptor 4 (100 mg, 0.23
mmol) in CH2Cl2 (1 mL). The temperature of the reaction
mixture was raised to 0 °C over a period of 1 h. The progress
of the reaction was monitored by TLC and MALDI-ToF MS.
The reaction mixture was diluted with CH2Cl2 (20 mL), filtered,
and washed with saturated aq NaHCO3 solution (10 mL),
water (10 mL), and brine (10 mL). The organic layer was
dried (MgSO4) and filtered, and the filtrate was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexanes:EtOAc, 2:1, v:v) to afford compound
9 (106 mg, 61%) as a white amorphous solid. Analytical data for
9: Rf 0.25 (hexanes:EtOAc, 2:1, v:v); 1H NMR (500 MHz,
CDCl3) δ 7.72-6.87 (m, 21H, aromatic), 5.66 (d, 1H, J=9.4,
attempts to chemically synthesize the PSGL-1 oligosacchar-
ide suffered from extensive replacement of protecting groups
at advanced stages of the synthesis and poor regioselectivities
in crucial glycosylation steps compromising the poor overall
yield of the target compound.26 It is to be expected that the
strategic principles employed for the synthesis of 1 will be
relevant for the synthesis of many other complex oligosac-
charides of biological and medical importance.
Experimental Section
2,6-Dimethylphenyl [2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(3,4,6-
tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-β-D-galactopyranosyl)]-1- thio-r-
D-galactopyranoside (8). A mixture of galactosyl acceptor 3b (93 mg,
0.22 mmol), galactosyl trichloroacetaimidate donor 2b (200 mg, 0.34
˚
mmol), and 4 A MS in CH2Cl2 (3 mL) was placed under an atmo-
sphere of argon and stirred at room temperature for 1 h. The reaction
mixture was then cooled to -78 °C. TMSOTf (0.023 mmol, 0.23 M
solution in CH2Cl2) was added and the temperature was raised to
-15 °C with stirring over a period of 2 h. The progress of the reaction
was monitored by TLC and MALDI-ToF MS. The reaction mixture
was diluted with CH2Cl2 (20 mL), filtered, and washed with saturated
aq NaHCO3 solution(10mL), water(10mL), andbrine(10mL).The
organic layer was dried (MgSO4) and filtered, and the filtrate was
concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexanes:EtOAc, 2:1, v:v) to afford compound 8
NHFmoc), 5.51-5.45 (m, 2H, CHPh, H-20), 5.40 (br d, 1H, H-
0
40), 5.15 (dd, 1H, J3 ,4 = 3.2 Hz, J2 ,3 =10.2 Hz, H-3 ), 5.10 (br t,
0
0
0
0
2H, CH2, Bn), 4.88 (d, 1H, J1 ,2 = 7.9 Hz, H-10), 4.83 (d, 1H,
0
0
J1,2 = 3.5, H-1), 4.46-4.25 (m, 5H, OCHCH3 threonine,
6068 J. Org. Chem. Vol. 74, No. 16, 2009

Vohra et al.
JOCArticle
CH2CH-Fmoc, CHCOOBn threonine, H-4), 4.21-4.08 (m, 4H,
H-6a0, CH2CH-Fmoc, H-6b0, H-6a), 3.96-3.92 (m, 3H, H-6b,
H-3, H-50), 3.69 (dd, 1H, J1,2=3.5 Hz, J2,3=10.8 Hz, H-2), 3.56
(br s, 1H, H-5), 2.11 (s, 3H, OCH3), 1.97 (s, 3H, COCH3), 1.87
(s, 3H, COCH3), 1.23 (d, 3H, OCHCH3 threonine) ppm; 13C
from HSQC (125.7 MHz, CDCl3) δ 102.3 (C-10), 100.8 (CHPh),
99.4 (C-1), 76.2 (OCHCH3 threonine), 75.9 (C-4), 75.8 (C-3),
71.2 (C-50), 71.1 (C-30), 70.3 (C-20), 69.3 (C-6), 68.0 (CH2Ph),
67.6 (CH2CH-Fmoc), 67.3 (C-40), 63.7 (C-5), 61.5 (C-60), 59.5
(C-2), 58.9 (CHCOOBn threonine), 47.4 (CH2CH-Fmoc), 21.0,
20.9, 20.7 (3 ꢀ OAc), 19.0 (OCHCH3 threonine); HR-MALDI-
ToF/MS m/z calcd for C58H56F2N4O18 [M þ Na]þ 1157.3455,
found 1157.3460 [M þ Na]þ.
(m, 2H, H-4, H-5), 3.31 (m, 1H, H-2), 2.13 (s, 3H, COCH3), 1.98
(s, 3H, COCH3), 1.12 (d, 3H, J=6.6 Hz, CH3 fucose) ppm; 13C from
HSQC (125.7 MHz, CDCl3) δ 98.6 (C-10), 85.9 (C-1), 83.8 (C-3),
78.7 (C-5), 74.1-73.6 (2 ꢀ CH2Ph, CH2Troc), 73.8 (C-20), 71.2
(C-30), 71.0 (C-4), 70.3 (C-40), 70.2 (C-6), 66.0 (C-50), 55.7 (C-3),
21.0, 20.8 (2ꢀOAc), 16.4 (C-60); HR-MALDI-ToF/MS m/z calcd
for C39H44Cl3NO12S [MþNa]þ 878.1547, found 878.1543.
2-(Trimethylsilyl)ethyl [Methyl 5-(N-acetylacetamido)-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyrano-
sylonate]-(2f3)-O-β-D-galactopyranoside (13). To a solution of 12
(250 mg, 0.283 mmol) in CH2Cl2:MeOH (30:1, v:v; 15 mL) under
an argon atmosphere was added Pd, 10 wt % on activated carbon
(150 mg), then the mixture was stirred for 20 min at room
temperature. The argon was replaced with H2(g), and the reaction
was stirred for 8 h. The solution was diluted with CH2Cl2 (50 mL)
and filtered through Celite. The solvent was removed by evapora-
tion under reduced pressure and the residue was purified by silica
gel column chromatography (toluene:acetone, 5:2, v:v) to afford
compound 13 (220 mg, 98%) as a white amorphous solid.
Analytical data for 13: Rf 0.48 (toluene:acetone, 1:1, v:v); 1H
NMR (500 MHz, CDCl3) δ 5.52 (ddd, 1H, H-40), 5.34 (dd, 1H, H-
80), 5.15 (dd, 1H, J=8.2 Hz, H-70), 4.94 (d, 1H, J=10.3 Hz, H-60),
4.42 (d, 1H, J=7.6 Hz, H-1), 4.33 (dd, 1H, H-9a0), 4.21 (t, 1H, J=
10.2 Hz, H-50), 4.12 (dd, 1H, J2,3=9.4 Hz, J3,4=3.1 Hz, H-3), 4.08
(dd, 1H, H-9b0), 4.02 (m, 1H, OCHH), 3.91 (dd, 1H, H-6), 3.92-
3.82 (m, 5H, H-6a, H-6b,COOCH3), 3.70-3.60 (m, 3H, H-4, H-2,
OCHH), 3.55 (t, 1H, H-5), 2.86 (dd, 1H, H-30eq), 2.35, 2.28 (2s,
6H, N(COCH3)2), 2.10, 2.09, 2.00, 1.97 (4s, 12H, 4 ꢀ COCH3),
1.93 (dd, 1H, H-30ax), 1.13-0.85 (m, 2H, CH2SiMe3), 0.01 (s, 9H,
Si(CH3)3); 13C from HSQC (125.7 MHz, CDCl3) δ 102.7 (C-1),
77.2 (C-3), 73.8 (C-5), 70.4 (C-60), 69.5 (C-2), 68.9 (C-4), 68.7 (C-
80), 67.3 (CH2CH2SiMe3), 66.9 (C-70), 66.8 (C-40), 62.6 (C-6), 62.2
(C-90), 56.9 (C-50), 53.7 (COOCH3), 38.6 (C-30), 28.3, 26.3 (NAc2),
21.4, 21.1, 21.0 (3 ꢀ OAc), 18.3 (CH2SiMe3); HR-MALDI-ToF/
MS m/z calcd for C33H53NO19Si [M þ Na]þ 818.2879, found
818.2880.
2-(Trimethylsilyl)ethyl [Methyl 5-(N-acetylacetamido)-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-glycero-r-D-galacto-2-nonulopyranosy-
lonate]-(2f3)-O-(2,4,6-tri-O-acetyl-β-galactopyranoside) (14). Com-
pound 13 (215 mg, 0.270 mmol) was dissolved in pyridine (10 mL)
and acetic anhydride (5 mL) and the reaction was stirred for 14 h at
room temperature. The solvent was removed by coevaporation with
toluene (3 ꢀ 50 mL). Silica gel column chromatography (hexanes:
EtOAc, 1:1, v:v) of the residue afforded compound 14 (246 mg, 99%)
as a white solid. Analytical data for 14:Rf 0.55 (hexanes:EtOAc, 1:3, v:
v); 1H NMR (500 MHz, CDCl3) δ 5.53-5.47 (m, 2H, H-40, H-80),
5.14 (dd, 1H, H-70, J=9.3 Hz, 2.4 Hz), 4.98-4.94 (m, 2H, H-3, H-4),
4.58-4.53 (m, 3H, H-2, H-60, H-1), 4.28-4.25 (m, 2H, H-50, H-9a0),
4.08-3.98 (m, 3H, H-6a, H-6b, H-9b0), 3.97-3.92 (dt, 1H,
OCHHCH2Si(CH3)3), 3.85 (s, 3H, COOCH3), 3.83 (t, 1H, H-5),
3.59-3.54 (dt, 1H, OCHHCH2Si(CH3)3), 2.63 (dd, 1H, J=5.4 Hz,
12.7 Hz, H-30eq), 2.32, 2.25 (2s, 6H, N(COCH3)2), 2.17, 2.15, 2.04,
2.01, 2.00, 1.99, 1.91 (7s, 21H, 7 ꢀ COCH3), 1.60 (t, 1H, J=12.2 Hz,
H-30ax), 1.01-0.86(m,2H,CH2Si(CH3)3),0.00(s,9H,Si(CH3)3);13C
from HSQC (125.7 MHz, CDCl3) δ 100.8 (C-1), 71.8 (C-2), 70.6
(C-5), 70.4 (C-3), 69.6 (C-60), 67.9 (C-4), 67.8 (C-40), 67.6
(CH2CH2SiMe3), 67.4 (C-70), 67.3 (C-80), 62.7 (C-6), 62.4 (C-
90), 56.5 (C-50), 53.3 (COOCH3), 38.7 (C-30), 28.4, 27.0 (NAc2),
22.0-20.6 (7 ꢀ OAc), 18.4 (CH2SiMe3), 1.3 (SiMe3); HR HR-
MALDI-ToF/MS m/z calcd for C39H59NO22Si [M þ Na]þ
944.3196, found 944.3194.
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-azido-2-deoxy-3-O-(3,4,-
6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-β-D-galactopyranosyl)-r-D-gal-
actopyranosyl]-L-threonine Benzyl Ester (10).A solution of compound 9
(80 mg, 0.072 mmol) in 5 mL of 70% aq acetic acid was heated at 70 °C
for 3 h. The progress of the reaction was monitored by TLC and
MALDI-ToF MS. The reaction was cooled to rt and concentrated by
coevaporation with toluene in vacuo. The residue was purified by silica
gel column chromatography (hexanes:EtOAc, 1:2, v:v) to afford
compound 10 (59 mg, 92%) as a white amorphous solid. Analytical
data for 10: Rf 0.25 (hexanes:EtOAc, 1:2, v:v); 1H NMR (500 MHz,
CDCl3) δ 7.72-6.94 (m, 16H, aromatic), 5.60 (d, 1H, J=9.5 Hz,
0
0
00
0
0
NHFmoc), 5.50-5.46 (q, 1H, J1 ,2 =8.6 Hz, J3 ,2 =10.3 Hz, H-20),
0
0
00
00
0
5.40 (br d, 1H, H-4 ), 5.16 (q, 1H, J3 ,4 =2.9 Hz, J2 ,3 =10.3 Hz, H-3 ),
0
0
0
5.12-5.01 (dd, 2H, CH2Ph), 4.81 (d, 1H, J1 ,2 =7.8 Hz, H-1 ), 4.76 (d,
1H, J1,2 = 3.4 Hz, H-1), 4.41-4.35 (m, 3H, OCHCH3 threonine,
CHCOOBn threonine, CHH-Fmoc), 4.25-4.21 (m, 1H, CHH-
Fmoc), 4.16-4.05 (m, 4H, H-6a0, H-6b0, H-4, CH2CH-Fmoc),
3.96 (m, 1H, H-50), 3.91-3.88 (dd, 1H, J3,4=2.7 Hz, J2,3=10.7
Hz, H-3), 3.83-3.70 (m, 3H, H-5, H-6a, H-6b), 3.46-3.44 (dd,
1H, J1,2=3.7 Hz, J2,3=10.5 Hz, H-2), 2.75 (br s, 1H, C4-OH),
2.32 (br s, 1H, C6-OH), 2.13 (s, 3H, OCH3), 2.00 (s, 3H,
COCH3), 1.90 (s, 3H, COCH3), 1.24 (d, 3H, OCHCH3) ppm;
13C from HSQC (125.7 MHz, CDCl3) δ 101.8 (C-10), 99.2 (C-1),
78.1 (C-3), 76.2 (OCHCH3 threonine), 71.7 (C-50), 70.7 (C-30),
69.8 (C-20), 69.7 (C-5), 69.3 (C-4), 67.8 (CH2Ph), 67.4 (CH2CH-
Fmoc), 67.2 (C-40), 62.7 (C-6), 61.6 (C-60), 59.0 (C-2), 58.7
(CHCOOBn threonine), 47.4 (CH2CH-Fmoc), 20.8, 20.7, 20.6
(3 ꢀ OAc), 18.6 (OCHCH3 threonine); HR-MALDI-ToF/MS
m/z calcd for C51H52F2N4O18 [M þ Na]þ 1069.3142, found
1069.3140.
Phenyl 3,4-Di-O-acetyl-2-O-benzyl-6-deoxy-5-methyl-r-L-fu-
copyranosyl-(1f3)-2-(2,2,2-trichloroethoxy)carbonylamino-6-O-ben-
zyl-2-deoxy-1-thio-β-D-glucopyranoside (11). A mixture of glycosyl
acceptor 6 (50 mg, 0.09 mmol), trichloroacetimidate donor 5 (63 mg,
˚
0.13 mmol), and 4 A MS in CH2Cl2 (1 mL) was placed under an
atmosphere of argon and stirred at room temperature for 1 h. The
reaction mixture was then cooled to 0 °C. TMSOTf (0.018 mmol,
0.18 M solution in CH2Cl2) was added and stirring was continued for
30 min at the same temperature. The reaction mixture was then
cooled to -78 °C followed by addition of TfOH (23 μL, 0.26 mmol)
and triethylsilane (48 μL, 0.30 mmol). The reaction mixture was then
stirred at -78 °C for 30 min. The progress of the reaction was moni-
tored by TLC and MALDI-ToF MS. The reaction was quenched by
the addition of pyridine (25 μL) and MeOH (0.2 mL), diluted with
CH2Cl2 (20 mL), and washed with saturated aq NaHCO3 solution
(10 mL), water (10 mL), and brine (10 mL). The organic layer was
dried (MgSO4) and filtered, and the filtrate was concentrated in
vacuo. The residue was purified by silica gel column chromatography
(hexanes:EtOAc, 2:1, v:v) to afford compound 11 (67 mg, 84%) as a
white amorphous solid. Analytical data for 11: Rf 0.40 (hexanes:
EtOAc, 1:1, v:v); 1H NMR (500 MHz, CDCl3) δ7.50-7.17 (m, 15H,
aromatic), 5.46 (d, 1H, J=6.6 Hz, NHTroc), 5.30-5.27 (m, 2H, H-
30, H-40), 5.09 (m, 2H, H-1, H-10), 4.70-4.57 (m, 6H, 2 ꢀ CH2, Bn,
OCH2CCl3), 4.42 (m, 1H, H-50), 3.89-3.83 (m, 3H, H-20, H-3, H-
6a), 3.78-3.75 (dd, 1H, H-6b), 3.68 (br s, 1H, C4-OH), 3.58-3.55
Methyl 5-(N-Acetylacetamido)-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-D-glycero-r-D-galacto-2-nonulopyranosylonate-(2f3)-O-(2,4,6-
tri-O-acetyl-β-D-galactopyranosyl)trichloroacetimidate (7). TFA
(2 mL) was added to a solution of compound 14 (240 mg, 0.260
mmol) in CH2Cl2 (10 mL) at 0 °C and the reaction was stirred for 4
h at the same temperature. The solvent was removed by coeva-
poration with toluene (5ꢀ20 mL). The residue was purified by
J. Org. Chem. Vol. 74, No. 16, 2009 6069

Vohra et al.
JOCArticle
silica gel column chromatography (hexanes:EtOAc, 2:5, v:v).
Trichloroacetonitrile (130 μL, 1.26 mmol) and 1,8-diazabicyclo-
[5.4.0]undec-7-ene (14 μL, 94.8 μmol) were added to a solution of
methyl 5-(N-acetylacetamido)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D-glycero-R-D-galacto-2- nonulopyranosylonate-(2f3)-O-(2,4,6-
tri-O-acetyl-β-D-galactopyranoside) (207 mg, 0.252 mmol) in
CH2Cl2 (5 mL). The reaction mixture was stirred for 1 h and
then concentrated in vacuo. Silica gel column chromatography
(hexanes:EtOAc, 1:2, v:v) of the syrup afforded compound 7 (220
mg, 87% over two steps, 3:2 R:β) as a white foam. Analytical data
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-azido-2-deoxy-3-
O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-β-D-galactopy-
ranosyl)-6-O-(O-methyl 5-(N-acetylacetamido)-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyranosylo-
nate)-(2f3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1f4)-
O-[(3,4-di-O-acetyl-2-O-benzyl-r-L-fucopyranosyl)-(1f3)]-O-(6-
O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranosyl)-r-D-galactopyranosyl]-L-threonine Benzyl Es-
ter (16). A mixture of disaccharide acceptor 10 (22 mg, 0.025
mmol) and tetrasaccharide donor 15 (32 mg, 0.019 mmol) in
CH2Cl2 (2 mL) was placed under an atmosphere of argon and
1
for 7: Rf 0.30 (hexanes:EtOAc, 1:2, v:v); H NMR (500 MHz,
˚
CDCl3) δ 8.65 (s, 1H, NH), 8.61 (s,1H, NH), 6.48 (d, 1H, J1,2=3.8
Hz, H-1R), 5.93 (d, 1H, J1,2=8.2Hz, H-1β), 5.56-5.50 (m, 2H, H-
40R, H-80R), 5.37-5.35 (m, 2H, H-40β, H-80β), 5.27-5.24 (m, 2H,
H-2R, H-2β), 5.15-5.12 (m, 2H, H-70R, H-70β), 5.04 (br d, 1H, H-
4β), 4.99 (dd, 1H, J=3.4 Hz, 10.5 Hz, H-3R), 4.77 (dd, 1H, J=3.4
Hz, 10.0 Hz, H-3β), 4.62 (m, 2H, H-60R, H-60β), 4.32-4.28 (m,
2H, H-50R, H-6a), 4.23-4.06 (m, 4H, H-50β, H-6b, H-90aR, H-
90bR), 4.02-3.95 (m, 2H, H-90aβ, H-90bβ), 3.88 (s, 3H, COO-
CH3R), 3.85 (s, 3H, COOCH3β), 2.70 (dt, 1H, H-30eq), 2.35, 2.33
(2s, 6H, NCOCH3R), 2.28, 2.27 (2s, 6H, NCOCH3β), 2.16-1.93
(7s, 21H, 7ꢀCOCH3), 1.68 (dd, 1H, H-30ax); 13C from HSQC
(125.7 MHz, CDCl3) δ 96.3 (C-1β), 94.2 C-1R), 72.0 (C-5), 71.2
(C-3β), 69.8 (C-60), 68.4 (C-2), 68.3 (C-3R), 67.9 (C-40R), 67.8 (C-
80R), 67.5 (C-4β), 67.3 (C-40β), 67.2 (C-80β), 67.1 (C-70), 62.5 (C-
6), 62.3 (C-90β), 61.8 (C-90R), 56.6 (C-50R), 56.1 (C-50β), 53.3
(COOCH3), 38.9 (C-30), 28.4, 28.3 (NAc2R), 26.9, 26.8 (NAc2β),
23.0-21.8 (7ꢀOAc R/β);
stirred at room temperature with 4 A MS for 1 h. The reaction
mixture was then cooled to 0 °C. N-Iodosuccinimide (22 mg,
0.096 mmol) and TfOH (0.019 mmol, 0.20 M solution in
CH2Cl2) were added sequentially and the mixture was was
stirred for 1 h at the same temperature. The progress of the
reaction was monitored by TLC and MALDI-ToF MS. The
reaction was diluted with CH2Cl2 (10 mL), filtered, and washed
with saturated aq NaHCO3 solution (10 mL), water (10 mL),
and brine (10 mL). The organic layer was dried (MgSO4) and
filtered, and the filtrate was concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexanes:
EtOAc, 1:2, v:v) to afford compound 16 (29 mg, 55%) as a
white amorphous solid. Analytical data for 16: Rf 0.30
(hexanes:EtOAc, 1:2, v:v); 1H NMR (500 MHz, CDCl3) δ
7.77-6.42 (m, 26H, 3ꢀBn, Fmoc, dFBz), 5.92-5.88 (m, 2H,
H-400000, H-800000), 5.84 (t, 1H, H-20), 5.74 (m, 2H, H-3000, H-4000),
5.61 (d, 1H, J=3 Hz, H-1000), 5.47-5.41 (m, 4H, H-20000, H-700000
,
Phenyl [O-Methyl 5-(N-acetylacetamido)-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyranosylonate]-(2f3)-
O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1f4)-O-[(3,4-di-O-
acetyl-2-O-benzyl-r-L-fucopyranosyl)-(1f3)]-O-[6-O-benzyl-2-
deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopy-
ranoside] (15). A mixture of disaccharide acceptor 11 (30 mg, 0.035
mmol) and trichloroacetimidate donor 7 (50 mg, 0.052 mmol) in
CH2Cl2 (4 mL) was placed under an atmosphere of argon and
H-40, H-40000), 5.34 (m, 1H, H-5000), 5.27 (m, 1H, H-30), 5.19 (d,
1H, J=8.1 Hz, H-10000), 5.04-4.99 (m, 3H, H-600000, H-30000, CO-
OCHHPh), 4.95 (d, 1H, OCHHPh), 4.86-4.77 (m, 3H, CO-
OCHHPh, COOCHHCCl3, OCHHPh), 4.73-4.49 (m, 9H,
COOCHHCCl3, H-6a0000
, ,
OCHHPh, H-100, H-1, H-500000
OCHCH3 threonine, OCHHPh, H-6b0000), 4.44-4.18 (m, 9H,
CH2Fmoc, H-9a00000, H-9b00000, H-50000, CHCOOBn threonine, H-
10, H-300, H-400), 4.15-3.79 (m, 11H, H-6a, H-6b, H-2000, H-6a0, H-
6b0, CH2CHFmoc, H-5, H-4, H-6a0, H-6b0, H-3), 3.77 (s, 3H,
COOCH3), 3.46 (m, 4H, H-200, H-2, H-50, H-500), 2.85 (dd, 1H,
H-300000), 2.26, 2.22 (2s, 6H, 2 ꢀ NCOCH3), 1.87 (m, 1H, H-300000),
1.88-1.65 (11s, 33H, 11 ꢀ COCH3), 1.60 (d, 1.623H, J=6.5 Hz,
CH3 fucose), 1.57 (s, 3H, COCH3), 1.34 (m, 3H, CH3 threonine)
ppm; 13C from HSQC (125.7 MHz, CDCl3) δ 101.6 (C-10), 100.7
(C-100), 100.5 (C-10000), 99.9 (C-1), 97.4 (C-1000), 78.4 (C-3), 76.5
(OCHCH3 threonine), 75.6 (C-50’), 74.9 (C-2000), 74.8 (C-30’), 74.7
(C-400), 74.5 (CH2Ph), 73.6 (CH2Ph), 73.1 (CH2Troc), 72.6 (C-
4000), 72.4 (C-30000), 71.7 (C-50), 71.5 (C-50000), 71.1 (C-30), 71.0 (C-
20000), 70.8 (C-700000), 70.6 (C-3000), 70.5 (C-600000), 70.3 (C-20), 69.9
(C-5), 69.3 (C-6), 69.1 (C-600), 68.1 (C-4), 67.9 C-40000), 67.7
(CH2Fmoc), 67.6 (COOCH2Ph threonine), 67.5 (C-40), 67.4 (C-
400000), 67.3 (C-800000), 65.2 (C-5000), 62.5 (C-900000), 62.1 (C-60000), 61.6
(C-6’), 59.5 (OCHCH3 threonine), 59.3 (C-2), 58.9 (C-200), 56.3
(C-500000), 52.9 (COOCH3), 47.6 (CH2CHFmoc), 39.0 (C-300000),
21.0, 20.9 (NAc2), 20.8-20.1 (12 ꢀ OAc), 18.9 (CH3 threonine),
˚
stirred at room temperature with 4 A MS for 1 h. The reaction
mixture was then cooled to 0 °C. TMSOTf (3.0 μmol, 0.035 M
solution in CH2Cl2) was added and stirring was continued for 1 h at
the same temperature. The progress of reaction was monitored by
TLC and MALDI-ToF MS. The reaction was quenched by the
addition of pyridine (25 μL), diluted with CH2Cl2 (10 mL), filtered,
and washed with saturated aq NaHCO3 solution (10 mL), water
(10 mL), and brine (10 mL). The organic layer was dried (MgSO4)
and filtered, and the filtrate was concentrated in vacuo. The residue
was purified by silica gel column chromatography (CHCl3:EtOAc,
1:1, v:v) to afford compound 15 (35 mg, 61%) as a white
amorphous solid. Analytical data for 15: Rf 0.25 (acetone:toluene,
1
1:3, v:v); H NMR (500 MHz, CDCl3) δ 7.43-7.13 (m, 15H,
aromatic), 5.56-5.51 (m, 2H, H-8000, H-4000), 5.32 (d, 1H, J=7.3
Hz, NHTroc), 5.27 (br s, 1H, H-400), 5.21-5.14 (m, 3H, H-1, H-100,
H-300), 4.99 (d, 1H, J=1.0 Hz, H-40), 4.91-4.86 (m, 2H, H-500, H-
0
20), 4.83 (d, 1H, J1 ,2 =8.2 Hz, H-1 ), 4.79 (d, 1H, CHHPh), 4.68-
0
0
4.90 (m, 7H, CH2Ph, CHHPh, CH2CCl3, H-30, H-600), 4.29 (t,
16.5 (C-6000); HR-MALDI-ToF/MS m/z calcd for C118H135
Cl3F2N6O51 [M þ Na]þ 2617.7086, found 2617.7091.
-
0
0
0
0
0
0
0
J
4 ,5 =J5 ,6 =10.1 Hz, H-5 ), 4.23-4.14 (m, 4H, H-6a , H-6b , H-
9a000, H-3), 4.03 (m, 1H, H-9b000), 3.96 (t, 1H, J3,4=J5,4=9.0 Hz, H-
4), 3.87-3.80 (m, 5H, COOCH3, H-6a, H-6b), 3.78 (m, 1H, H-50),
3.52(br d, 1H, H-5), 3.10 (br m, 1H, H-2), 2.62 (m, 1H, H-3a000),
2.33, 2.26 (2s, 6H, 2ꢀNCOCH3), 2.15-1.92 (9s, 27H, 9ꢀCOCH3),
1.62 (m, 1H, H-3b000), 1.17 (d, 3H, J=6.6 Hz, CH3 fucose) ppm; 13C
from HSQC (125.7 MHz, CDCl3) δ 99.6 (C-10), 97.7 (C-100), 84.5
(C-1), 79.7 (C-5), 75.7 (C-3), 74.6 (CH2Ph), 74.4 (C-200), 73.9 (C-4),
73.2 (CH2Ph), 72.3 (C-400), 71.8 (C-30), 71.2 (C-50), 70.8 (C-500),
70.6 (C-300), 69.7 (C-6000), 68.7 (C-6), 67.8 (C-40), 67.5 (C-8000), 67.4
(C-7000), 67.3 (C-4000), 64.8 (C-20), 62.4 (C-9000), 62.0 (C-60), 57.9 (C-
2), 56.2 (C-5000), 53.4 (COOCH3), 38.7 (C-3000), 28.6, 27.2 (NAc2),
21.6-20.5 (9ꢀOAc), 16.3 (C-6’’); HR-MALDI-ToF/MS m/zcalcd
for C73H89Cl3N2O33S [MþNa]þ 1681.4032, found 1681.4029.
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-(N-acetamido)-2-
deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-β-D-gal-
actopyranosyl)-6-O-(O-methyl 5-(N-acetylacetamido)-4,7,8,9-tet-
ra-O-acetyl-3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyranosy-
lonate)-(2f3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1f4)-
O-[(3,4-di-O-acetyl-2-O-benzyl-r-L-fucopyranosyl)-(1f3)]-O-(6-O-
benzyl-2-deoxy-2-(N-acetamido)-β-D-glucopyranosyl)-r-D-gala-
ctopyranosyl]-L-threonine Benzyl Ester (1). Zn dust (400 mg,
6.12 mmol) and saturated aq CuSO4 (25 μL) were added to a
solution of 16 (20 mg, 7.70 μmol) in THF (3 mL), Ac2O (2 mL),
and AcOH (1 mL) and the reaction was stirred at rt for 3 h. The
reaction mixture was filtered and coevaporated with toluene
(3 ꢀ 5 mL). The residue was purified by silica gel column
6070 J. Org. Chem. Vol. 74, No. 16, 2009

Vohra et al.
JOCArticle
chromatography (CHCl3:acetone, 2/1, v/v) to afford com-
pound 1 (11.5 mg, 60%) as a white amorphous solid. Analytical
Hz, CH3 fucose) ppm; 13C from HSQC (150.9 MHz, CDCl3) δ
104.3 (C-10), 104.1 (C-100), 102.2 (C-10000), 101.9 (C-1), 98.6 (C-
1000), 81.0 C-3), 77.6 (C-500), 77.4 (OCHCH3 threonine), 77.3 (C-
300), 76.3 (C-2000), 76.1 (C-4000), 75.1 (CH2Ph), 74.6 (C-4000), 74.2
(C-30000), 73.8 (CH2Ph), 73.6 (C-50000), 73.5 (C-50), 73.4 (C-30),
73.1 (C-20000), 72.8 (C-20), 72.5 (C-3000), 72.3 (C-600000), 72.2 (C-6),
72.1 (C-5), 71.4 (C-4), 71.2 (C-600), 70.4 (C-800000), 70.2 (C-40000),
70.0 (C-40), 69.8 (C-700000), 69.5 (COOCH2Ph), 69.2 (C-400000),
69.1 (CH2CHFmoc), 66.4 (C-5000), 64.7 (C-900000), 64.1 (C-60000),
63.9 (C-60), 61.8 (OCHCH3 threonine), 58.6 (C-200), 58.5 (C-
500000), 55.2 (COOCH3), 50.5 (C-2), 49.8 (CH2CHFmoc), 41.1
(C-300000), 29.8, 28.4 (NAc2), 25.5-22.4 (12 ꢀ OAc, 2 ꢀ NHAc),
21.4 (CH3 threonine), 18.3 (C-6000); HR-MALDI-ToF/MS m/z
calcd for C119H140F2N4O51 [M þ Na]þ 2501.8350, found
2501.8353.
1
data for 1: Rf 0.30 (CHCl3:acetone, 2/1, v/v); H NMR (600
MHz, acetone-d6) δ 7.75-7.08 (m, 26H, 3ꢀBn, Fmoc, dFBz),
6.99 (d, 1H, NH, GlcNAc), 6.51 (d, 1H, NH, GalNAc), 6.42 (d,
1H, NHFmoc, threonine), 5.5 (m, 1H, H-800000), 5.45 (m, 1H, H-
400000), 5.35 (d, 1H, J=3.7 Hz, H-1000), 5.29 (d, 1H, J=3.5 Hz,
H-40), 5.25 (dd, 1H, J=8.1 Hz, J = 10.5 Hz, H-20), 5.19 (br d,
1H, J = 2.6 Hz, H-4000), 5.12-5.08 (m, 3H, H-3000, H-700000, H-30),
4.99 (br d, 1H, J = 3.7 Hz, H-40000), 4.97-4.93 (dd, 2H, CO-
OCH2Ph, threonine), 4.88 (q, 1H, H-5000), 4.84-4.81 (m, 2H,
H-20000, H-10000), 4.79 (d, 1H, J=8.1 Hz, H-100), 4.67 (d, 2H, 2ꢀ
CHHPh), 4.60 (m, 1H, H-30000), 4.57 (d, 1H, J=2.8 Hz, H-1),
4.55 (dd, 1H, H-600000), 4.49 (d, 1H, J=7.5 Hz, H-100), 4.45 (d, 1H,
CHHPh), 4.38 (d, 1H, CHHPh), 4.35 (m, CHHFmoc), 4.27-
4.22 (m, 2H, CHHFmoc, H-2), 4.20 (m, 1H, H-500000), 4.15-4.05
(m, 8H, OCHCH3 threonine, H-6a0000, CHCOOBn threonine,
CHFmoc, H-6b0000, H-9a00000, H-50 H-6a0), 4.03-3.91(m, 5H, H-
400, H-6b0, H-9b00000, H-4, H-300), 3.87-3.81 (m, 3H, H-6a00, H-200,
H-5), 3.76-3.73 (m, 6H, H-50000, COOCH3, H-6b00, H-3), 3.70
(dd, 1H, H-2000), 3.54 (m, 2H, H-6a, H-6b), 3.47 (m, 2H,
C4-OH, H-500), 2.48 (dd, 1H, H-300000), 2.24, 2.20 (2s, 6H,
NAc2), 2.14-1.74 (14s, 42H, 12 ꢀ OAc, 2 ꢀ NHAc), 1.43 (t,
1H, H-300000), 1.16 (d, 3H, CH3 threonine), 1.03 (d, 3H, J=6.6
Acknowledgment. The authors would like to thank Dr.
Christian Heiss for help with elucidating NMR spectra. This
work was supported by NIH NIGM grant No. R01-
GM065248 and R01-GM61761.
Supporting Information Available: 1H NMR spectra and
HSQC of all synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 16, 2009 6071