Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides

Article abstract of DOI:10.1021/ja015570t

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interestfor development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (Ga1NAcα-Ser/Thr) and T [Galβ(1→3)Ga1NAcα-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Acα(2→6)Ga1NAcα-Ser/Thr] and 2,3-sialyl-T [Neu5Acα(2→3)Ga1β(1→3)Ga1NAcα-Ser/ Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Ga1β1→3(G1cNAcβ1→6)Ga1NAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUCl, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.

Full text of DOI:10.1021/ja015570t

VOLUME 123, NUMBER 45
NOVEMBER 14, 2001
© Copyright 2001 by the
American Chemical Society
Chemoenzymatic Synthesis of Sialylated Glycopeptides Derived from
Mucins and T-Cell Stimulating Peptides
Shaji K. George,† Tilo Schwientek,‡ Bjo1rn Holm,† Celso A. Reis,‡,§ Henrik Clausen,‡ and
Jan Kihlberg*,†
Contribution from the Department of Organic Chemistry, Umeå UniVersity, SE-901 87 Umeå, Sweden,
School of Dentistry, UniVersity of Copenhagen, No¨rre Alle´ 20, DK-2200 Copenhagen N, Denmark, and
Institute of Molecular Pathology and Immunology, UniVersity of Porto, IPATIMUP,
Rua Dr. Roberto Frias s/n, 4200 Porto, Portugal
ReceiVed January 22, 2001
Abstract: The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed
on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides
carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy
for their synthesis is reported. Building blocks corresponding to the Tn (GalNAcR-Ser/Thr) and
T [Galâ(1f3)GalNAcR-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were
prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give
the sialyl-Tn [Neu5AcR(2f6)GalNAcR-Ser/Thr] and 2,3-sialyl-T [Neu5AcR(2f3)Galâ(1f3)GalNAcR-Ser/
Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore
carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from
the solid phase. In the same manner, the core 2 trisaccharide [Galâ1f3(GlcNAcâ1f6)GalNAc] was
incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase.
The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the
mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.
Introduction
acetylgalactosamine to serines and threonines by a family of
N-acetylgalactosaminyltransferases, which show overlapping but
somewhat different specificities with regard to peptide se-
quence.5 When the mucin MUC1 is produced by the normal
human mammary gland, galactosyl residues are added to
the N-acetylgalactosamine moieties to form the type 1 core
Galâ1f3GalNAcR-Ser/Thr (also called the T antigen). The type
1 core, in turn, acts as a substrate for core 2 â1,6GlcNAc
transferases, so that the type 2 core trisaccharide [Galâ1f
3(GlcNAcâ1f6)GalNAc] is formed.3,4 Finally, oligo N-acetyl-
Most epithelial cells produce mucins, i.e. glycoproteins in
which the polypeptide backbone consists of highly conserved
tandem repeats with complex carbohydrates linked to multiple
serine and threonine residues.1-4 Biosynthesis of mucins occurs
in the Golgi apparatus and is initiated by addition of N-
† Umeå University.
‡ University of Copenhagen.
§ University of Porto.
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10.1021/ja015570t CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/20/2001

11118 J. Am. Chem. Soc., Vol. 123, No. 45, 2001
George et al.
lactosamine moieties are added, after which addition of sialic
acid or fucose residues terminates growth of the mucin linked
oligosaccharide structure.
less complex Tn and T antigens has been described by several
groups during recent years (reviewed in refs 11, 12, and 26).
However, incorporation of sialic acid in glycoconjugates by
chemical means is a substantially more demanding task due to
poor control of the anomeric configuration of the sialic acid
residue and the requirement for multistep synthetic schemes.27,28
This may explain why only a few building blocks corresponding
to the sialyl-Tn29-33 and 2,3-sialyl-T34,35 antigens have been
prepared chemically and then employed in glycopeptide syn-
thesis. Recently, enzymatic synthesis of 2,3-sialyl-T antigen
building blocks has been described,36,37 but the carboxyl group
of the sialic acid residue and the different hydroxyl groups must
be protected before these building blocks can be used for
synthesis of glycopeptides.38 Herein we describe the use of an
alternative approach for preparation of glycopeptides in which
building blocks corresponding to the simple Tn and T antigens
are first assembled into glycopeptides on the solid phase.
Sialyltransferases and core 2 â1,6GlcNAc transferases, which
operate with complete regio- and stereocontrol, are then used
to convert these readily available glycopeptides into more
complex ones that contain the sialyl-Tn and 2,3-sialyl-T
antigens, as well as the core 2 trisaccharide. This cassette-like
strategy13,32 is illustrated by synthesis of glycopeptides from
the tandem repeat domain of the mucin MUC1, as well as
neoglycosylated derivatives of a T cell stimulating viral peptide.
To accomplish syntheses of the target glycopeptides novel routes
to Tn and T antigen building blocks have also been developed.
In epithelial cancers, such as those affecting the breast, ovary,
lung, and colon, low expression of core 2 â1,6GlcNAc trans-
ferases together with increased levels of sialyltransferases
result in that mucins display simpler O-linked carbohydrates.3,4
The Tn (GalNAcR-Ser/Thr), T [Galâ(1f3)GalNAcR-Ser/Thr],
sialyl-Tn [Neu5AcR(2f6)GalNAcR-Ser/Thr] and 2,3-sialyl-T
[Neu5AcR(2f3)Galâ(1f3)GalNAcR-Ser/Thr] antigens are im-
portant examples of such saccharides which constitute tumor-
associated carbohydrate antigens.3,6-10 The presence of these
antigens on the surface of common human malignant tumors
has led to intense studies directed toward development of
synthetic carbohydrate-based anticancer vaccines.11,12 Recently,
studies in mice showed that short Tn-based glycopeptides
coupled to the protein KLH induced a strong IgM response and
a moderate IgG response, both of which were reactive to colon
cancer cells.12,13 Moreover, a sialyl-Tn KLH conjugate vaccine
used in combination with cyclophosphamide was indicated to
have a therapeutic effect when evaluated in clinical trials
involving breast cancer patients.14-16 These vaccines elicit an
antibody response, but it would also be an advantage if T cells
could be directed to tumor-associated carbohydrate antigens.
Studies performed during recent years have indeed shown that
T cells can recognize glycopeptides bound by MHC molecules
on the surface of antigen-presenting cells.17-20 Interestingly, in
one case a set of T cells reactive only to the carbohydrate moiety
of a neoglycopeptide antigen was obtained, suggesting that it
may be possible to target T cells to carbohydrate antigens on
tumor cells.21
Results and Discussion
Synthesis of building blocks corresponding to the Tn
(GalNAcR-Thr) and T [Galâ(1f3)GalNAcR-Thr] antigens
started from 2 which was obtained by silylation of azido
galactose 1.39 N-Bromosuccinimide/tetrabutylammonium triflate
promoted40 glycosylation of Fmoc-threonine allyl ester (3) with
thioglycoside 2 gave 4 (50%, Scheme 1). The Tn building block
6, which carries acid labile protective groups on the carbohydrate
moiety, was then obtained by reductive acetylation of the azido
Today, the most general synthetic route to O-linked glyco-
peptides employs NR-Fmoc protected glycosylated amino acids
as building blocks in stepwise solid-phase peptide synthesis.22-25
Synthesis of building blocks corresponding to the structurally
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Tetrahedron 1995, 51, 4923-4932.

Chemoenzymatic Synthesis of Sialylated Glycopeptides
J. Am. Chem. Soc., Vol. 123, No. 45, 2001 11119
Scheme 3a
Scheme 1a
a Reagents (yields): (a) TBDMSOTf, pyridine, room temperature
(93%); (b) NBS, QOTf, CH2Cl2, -28 °C (50%); (c) AcSH, pyridine,
room temperature (80%); (d) (PPh3)4Pd(0), N-methylaniline, THF, room
temperature (85%).
Scheme 2a
a Reagents (yields): (a) Fmoc solid-phase peptide synthesis (23%
for 12, 19% for 13); (b) R-G-Y-V-Y-X-G-L (X ) homo-
cysteine), Cs2CO3, DMF, room temperature (74% for 16, 58% for 17).
2-bromoethyl glycosides 14 and 15,46,47 into the two selected
peptide backbones (Scheme 3). The first peptide represents the
tandem repeat domain of the mucin MUC1 in which the
glycosylated threonine is located in the center of the immuno-
dominant region.3 The second peptide is an analogue of a class
I MHC restricted epitope from vesicular stomatitis virus
nucleocapsid protein,48 where the native Gln6 has been replaced
by homocysteine. Incorporation of the Tn and T antigen building
blocks 6 and 11 in the first peptide to give glycopeptides 12
and 13 was accomplished by Fmoc solid phase peptide synthesis
on a TentaGel resin, using conditions reported previously by
us.31,49 Neoglycopeptides 16 and 17, which carry the carbohy-
drate moieties of the Tn and T antigens, were prepared in
solution by a convergent strategy involving akylation of the
homocysteine residue of the viral peptide with 2-bromoethyl
glycosides 14 and 15.46
Having sufficient quantities of the Tn and T glycopeptides
in hand, enzymatic extension of the carbohydrate moieties to
provide the sialyl-Tn and 2,3-sialyl-T antigens, as well as the
core 2 trisaccharide, was investigated (Schemes 4-6). First,
recombinant mouse N-acetylgalactosamine R2-6 sialyltrans-
ferase50 (ST6GalNAc-I) expressed in insect cells5 was used to
convert Tn glycopeptides 12 and 16 into the corresponding
sialyl-Tn analogues (Scheme 4). On a semipreparative scale,
incubation of 12 and 16 (each 0.5 mg) with recombinant
ST6GalNAc-1 and CMP-Neu5Ac at pH 6.0 gave sialylated
glycopeptides 18 and 19 with >90-95% conversion of 12 and
a Reagents (yields): (a) 80% aqueous TFA, 0 °C (97%); (b) R,R-
dimethoxytoluene, p-TsOH (cat.), CH3CN, room temperature (87%);
(c) AgOTf, CH2Cl2-toluene (1:1), -30 °C (59%); (d) AcSH, pyridine,
room temperature (86%); (e) (PPh3)4Pd(0), N-methylaniline, THF, room
temperature (88%).
group with thioacetic acid in pyridine,41 followed by (PPh3)4-
Pd(0) catalyzed42 cleavage of the allyl ester. Glycoside 4 also
served as a precursor for synthesis of the T antigen building
block 11 (Scheme 2). Unfortunately, attempted cleavage of the
TBDMS group in 4 with tetrabutylammonium fluoride in acetic
acid,13 or using zinc tetrafluoroborate,43 failed. Instead, hy-
drolysis of both the TBDMS and the benzylidene group with
aqueous TFA followed by reprotection with R,R-dimethoxy-
toluene gave 7. Silver triflate mediated glycosylation of 7 with
peracetylated galactosyl bromide 8 proceeded with excellent
stereoselectivity to afford â-glycoside 9 as the only product
(59%). Reductive acetylation and deprotection of the allyl ester
was performed as for 4 to afford T building block 11,44,45 which
is ready for use in solid-phase peptide synthesis.
(46) Bengtsson, M.; Broddefalk, J.; Dahme´n, J.; Henriksson, K.; Kihlberg,
J.; Lo¨nn, H.; Srinivasa, B. R.; Stenvall, K. Glycoconj. J. 1998, 15, 223-
231.
(47) George, S. K.; Holm, B.; Reis, C. A.; Schwientek, T.; Clausen, H.;
Kihlberg, J. J. Chem. Soc., Perkin Trans. 1 2001, 880-885.
(48) Fremont, D. H.; Matsumura, M.; Stura, E. A.; Peterson, P. A.;
Wilson, I. A. Science 1992, 257, 919-927.
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1999, 64, 8948-8953.
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Tachida, Y.; Narimatsu, H.; Tsuji, S. J. Biochem. (Tokyo) 2000, 127, 845-
854.
After preparing the two building blocks we turned our
attention to incorporating them, as well as the Tn and T antigen
(41) Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53,
1580-1582.
(42) Ciommer, M.; Kunz, H. Synlett 1991, 593-595.
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Lett. 2000, 41, 3127-3130.

11120 J. Am. Chem. Soc., Vol. 123, No. 45, 2001
George et al.
Scheme 4a
Scheme 6a
a Reagents (conversion): (a) UDP-GlcNAc, core 2 O-glycan â1-6
N-acetylglucosaminyltransferase (C2GnT3), calf intestinal phosphatase,
MES buffer (100 mM, pH 6.5) containing EDTA, D-galactono-1,5-
lactone, and 2-acetamido-2-deoxy-D-glucono-1,5-lactone, 37 °C, ∼24
h for 22 and <3 h for 23 (>95% conversion of both 13 and 17).
cells), calf intestinal phosphatase, and CMP-Neu5Ac at pH 6.5
gave 20 and 21 with almost quantitative conversion (>95%) of
the starting materials on a semipreparative scale (0.5 mg). When
13 and 17 were sialylated on a larger scale (2.0 and 3.5 mg,
respectively) glycopeptides 20 and 21 could be isolated in 94
and 64% yields, respectively, after purification by reversed-
phase HPLC. This reveals that ST3Gal-I, just as ST6GalNAc-
I, accepts glycopeptide substrates in which the carbohydrate
moiety does not need to be linked via serine or threonine to the
peptide backbone.
a Reagents (conversion and yield): (a) CMP-Neu5Ac, R2-6-sialyl-
transferase (ST6GalNAc-I), Bis-Tris buffer (20 mM, pH 6.0), 37 °C,
<6 h (for 18: >95% conversion of 12, 74% isolated yield; for 19:
90-95% conversion of 16).
Scheme 5a
Finally, recombinant human core 2 O-glycan â1-6 N-
acetylglucosaminyltransferase52 (C2GnT3), expressed in insect
cells, was used to convert the T antigen of glycopeptides 13
and 17 into the core 2 trisaccharide (Scheme 6). Initial studies
with mucin derived glycopeptide 13 led to the formation of a
3:1 mixture of the expected product 22 and glycopeptide 12
which carries the Tn antigen. This was assumed to be due to a
competing â-galactosidase activity in the C2GnT3 enzyme
preparation leading to degradation of 13 in parallel with
conversion of the T antigen into the core 2 trisaccharide.
However, by inclusion of the galactosidase inhibitor, D-galac-
tono-1,5-lactone, as well as the hexosaminidase inhibitor
2-acetamido-2-deoxy-D-glucono-1,5-lactone, degradation of 13
could be avoided. In the presence of these two inhibitors 13
and 17 could be transformed to 22 and 23, respectively, by
incubation with C2GnT3, UDP-GlcNAc, and calf intestinal
phosphatase. On a semipreparative scale (0.6 mg) both 13 and
17 were completely converted to 22 and 23, as determined by
nanoscale reversed-phase HPLC in combination with MALDI-
TOF mass spectrometry. Interestingly, conversion of neoglyco-
peptide 17 into 23 with C2GnT3 was found to be substantially
faster than transformation of mucin-derived 13. This is in
contrast to use of the R2-6 and R2-3 sialyltransferases, where
conversion of mucin glycopeptides 12 and 13 was faster than
for neoglycopeptides 16 and 17. The observation that all three
transferases were able to glycosylate neoglycopeptides 16 and
17 suggests that these enzymes may find wide use for synthesis
of (neo)glycopeptides with substantial structural variation in the
peptide moiety.
a Reagents (conversion and yield): (a) CMP-Neu5Ac, R2-3-sialyl-
transferase (ST3Gal-I), calf intestinal phosphatase, Tris-HCl buffer (25
mM, pH 6.5), 37 °C, <3 h (For 20: >95% conversion of 13, 94%
isolated yield; for 21: ∼95% conversion of 17, 64% isolated yield).
16, as determined by nanoscale reversed-phase HPLC51 in
combination with MALDI-TOF mass spectrometry. When
glycopeptide 12 was sialylated on a preparative scale (5.0 mg)
18 was obtained in 74% yield after purification by reversed-
phase HPLC. To the best of our knowledge this is the first report
describing the preparation of glycopeptides which contain the
sialyl-Tn antigen based on enzymatic incorporation of the sialic
acid moiety. Moreover, synthesis of 19 reveals that glyco-
peptides with nonnatural linkages between GalNAcR and the
peptide moiety can be sialylated by ST6GalNAc-I.
Previously, chemoenzymatic approaches to glycopeptides
have predominantly concerned synthesis of N-linked glyco-
peptides53-56 and glycopeptides which carry the sialyl Lewis
Synthesis of glycopeptides 20 and 21 reveals that enzymatic
R-(2f3)-sialylation of glycopeptides which contain the T
antigen is also facile (Scheme 5). Incubation of either of
glycopeptides 13 or 17 with human core 1-specific R2-3
sialyltransferase (ST3Gal-I,37 obtained by expression in Sf9
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Chemoenzymatic Synthesis of Sialylated Glycopeptides
J. Am. Chem. Soc., Vol. 123, No. 45, 2001 11121
X tetrasaccharide, or fragments thereof.57-61 As described herein
chemoenzymatic synthesis is an attractive approach also for
preparation of glycopeptides which contain the tumor associated
sialyl-Tn and 2,3-sialyl-T antigens, as well as the core 2
trisaccharide. As revealed by the examples given above, both
natural and nonnatural glycopeptides can be prepared since the
glycosyl transferases show little, if any, specificity for the
peptide moiety. The approach is based on the fact that cassette-
like building blocks corresponding to the Tn and T antigens
can be readily prepared via chemical synthesis. Such building
blocks allow site-specific incorporation of the carbohydrate
moieties at selected positions in a polypeptide, in contrast to
enzymes belonging to the family of GalNAc-transferases5 which
constitute potential alternatives. Introduction of sialic acid, which
is difficult when performed chemically at the building block
level, is then carried out in solution after cleavage of the
glycopeptide from the solid phase. Performing the enzymatic
step in solution avoids problems associated with penetration of
the enzyme into the cross-linked solid support,62 as well as
incompatibilities between enzyme and solid support or the linker
used as attachment for the glycopeptide.57 Studies directed
toward chemoenzymatic synthesis of more complex mucin-
derived glycopeptides, as well as attempts to use the glyco-
peptides described herein for development of cancer vaccines,
are underway in our laboratories.
4-Methylphenyl 2-azido-2-deoxy-1-thio-â-D-galactopyranoside was
prepared from 3,4,6-tri-O-acetyl-2-azido-2-deoxy-R-D-galactopyranosyl
bromide63,64 as described previously.31 2,3,4,6-Tetra-O-acetyl-â-D-
galactopyranosyl bromide 8 was prepared from peracetylated galactose
by treatment with HBr in HOAc/Ac2O at 0 °C. l-Arginylglycyl-L-
tyrosyl-L-valyl-L-tyrosyl-L-homocysteinylglycyl-L-leucine46 was pre-
pared manually by Fmoc solid-phase synthesis in a mechanically
agitated reactor with standard conditions.65 2-Bromoethyl 2-acetamido-
2-deoxy-R-D-galactopyranoside (14) and 2-bromoethyl 2-acetamido-
2-deoxy-3-O-â-D-galactopyranosyl-R-D-galactopyranoside (15) were
prepared as described.47 Recombinant mouse N-acetylgalactosamine
R2-6 sialyltransferase50 (ST6-GalNAc-I) was expressed in insect cells.
The enzyme was purified by sequential ion-exchange chromatographies
on Amberlite (IRA95, Sigma) and SP-sepharose (Sigma) essentially
as described.5 Purified samples were concentrated in a centrifugation
cartridge (Sigma) before use. Human core 1-specific R2-3 sialyltrans-
ferase66 (ST3Gal-I) was expressed and partially purified as described.37
Final purification of ST3Gal-I was performed on MiniS (PC3.2/3) with
use of the Smart system (Pharmacia). Recombinant human core 2
O-glycan â1-6 N-acetylglucosaminyltransferase (C2GnT3) was ex-
pressed in insect cells and purified by sequential ion-exchange
chromatography as described.52 Purified samples were concentrated in
Biomax-10 Ultrafree cartridges (Millipore) before use.
4-Methylphenyl 2-Azido-4,6-O-benzylidene-2-deoxy-1-thio-â-D-
galactopyranoside (1). A solution of 4-methylphenyl 2-azido-2-deoxy-
1-thio-â-D-galactopyranoside31 (2.98 g, 9.6 mmol) and R,R-dimethoxy-
toluene (2.00 g, 13.1 mmol) in acetonitrile (60 mL) was stirred at room
temperature in the presence of a catalytic amount of p-toluenesulfonic
acid (monohydrate, 50 mg) for 2 h. The solution was concentrated and
flash column chromatography of the residue (toluene/EtOAc, 9:1f3:
Experimental Section
1
2) gave 1 (3.69 g, 97%) as a white solid. H and 13C NMR data were
General Methods and Materials. All reactions were carried out
under an inert nitrogen atmosphere with dry solvents, under anhydrous
conditions, unless otherwise stated. CH2Cl2 was dried by distillation
from calcium hydride whereas THF and toluene were distilled from
sodium benzophenone. Pyridine was dried over 4 Å molecular sieves.
DMF was distilled and then dried over flame dried 3 Å molecular sieves.
Organic solutions were dried over anhydrous Na2SO4 before being
concentrated. TLC was performed on silica gel 60 F254 (Merck) with
detection by UV light or charring with aqueous sulfuric acid (10%).
Flash column chromatography was performed on silica gel (Matrex,
60 Å, 35-70 µm, Grace Amicon) with distilled solvents. Analytical
reversed-phase HPLC was performed on a Kromasil C-8 column (250
× 4.6 mm, 5 µm, 100 Å), eluted with a linear gradient of MeCN in
H2O containing 0.1% TFA (flowrate of 1.5 mL/min, detection at 214
nm). Preparative reversed-phase HPLC was performed on a Kromasil
C-8 column (250 × 20 mm2, 5 µm, 100 Å) with the same solvent
system (flowrate of 11 mL/min, detection at 214 nm).
consistent with those reported previously.39
4-Methylphenyl 2-Azido-4,6-O-benzylidene-3-O-tert-butyldimeth-
ylsilyl-2-deoxy-1-thio-â-D-galactopyranoside (2). tert-Butyldimeth-
ylsilyl triflate (1.73 g, 1.5 mL, 6.55 mmol) was added dropwise to an
ice cold and stirred solution of 1 (1.47 g, 3.68 mmol) in pyridine (15
mL), containing a catalytic amount of DMAP (15 mg). After 2 h, the
solution was allowed to attain room temperature and was then stirred
for a further 24 h. It was diluted with methanol (25 mL) and the solvents
were evaporated under vacuum after which the residue was dissolved
in CH2Cl2 (150 mL). The solution was washed with cold aqueous
saturated NaHCO3 (50 mL), followed by brine (50 mL), and then dried.
Concentration followed by flash column chromatography of the residue
(toluene/EtOAc, 100:1) gave 2 (1.75 g, 93%) as a white amorphous
solid. [R]D20 -40.8° (c 0.99, CHCl3); 1H NMR (CDCl3) δ 7.60 (2H, d,
ArH), 7.47-7.36 (5H, m, ArH), 7.01 (2H, d, ArH), 5.59 (1H, s, CHPh),
4.43-4.35 (2H, m, H-1 and H-6), 4.05-3.96 (2H, m, H-3 and H-6),
3.66-3.58 (2H, m, H-4 and H-2), 3.47 (1H, m, H-5), 2.32 (3H, s,
ArCH3), 0.88 (9H, s, t-BuSi), 0.13 and 0.09 (6H, 2s, Si(CH3)2); 13C
NMR (CDCl3) δ 138.4, 137.8, 134.3, 129.7, 128.9, 128.0, 126.6, 126.3,
100.7, 85.4, 75.6, 74.4, 69.8, 69.3, 61.7, 25.6, 21.3; HRMS (FAB):
calcd for C26H35N3O4SSi (M + H+) 514.2196, found 514.2194.
Nr-Fluoren-9-ylmethoxycarbonyl-L-threonine Allyl Ester (3).
Compound 3 was prepared by modification of a known procedure.67
Thus, a solution of Fmoc-Thr-OH (2.0 g, 5.9 mmol) in aqueous ethanol
(80%, 17 mL) was titrated with a solution of Cs2CO3 (1.0 g, 3.1 mmol,
25% in water) to pH 7 with bromothymol blue as indicator. The solvents
were evaporated, the residue was co-concentrated with absolute ethanol
(4 × 25 mL) and then dried under vacuum overnight. The cesium salt
thus obtained was suspended in dry DMF (20 mL), cooled to 0 °C,
and treated with allyl bromide (7.08 g, 58.6 mmol) by dropwise addition
over 10 min. After 30 min the solution was allowed to attain room
1
The H and 13C NMR spectra for 2-11 were recorded at 400 and
100 MHz, respectively, for solutions in CDCl3 [residual CHCl3 (δH
7.26 ppm) or CDCl3 (δC 77.0 ppm) as internal standard], CD3OD
[residual CD2HOD (δH 3.35 ppm) or CD3OD (δC 49.0 ppm) as internal
standard], or in a 1:1 mixture of CD3OD and CDCl3 [residual CD2HOD
(δH 3.35 ppm) or CD3OD (δC 49.0 ppm) as internal standard] or DMSO-
d6 [residual (CH3)2SO (δH 2.50 ppm) or (CH3)2SO (δC 39.5 ppm) as
internal standard] at 298 K. Proton resonances were assigned from
appropriate combinations of COSY, NOESY, and TOCSY experiments.
Optical rotations were recorded on a Perkin-Elmer 343 polarimeter.
(56) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.;
Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc. 1999, 121, 284-
290.
(57) Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766-8776.
(58) Leppa¨nen, A.; Mehta, P.; Ouyang, Y.-B.; Ju, T.; Helin, J.; Moore,
K. L.; van Die, I.; Canfield, W. M.; McEver, R. P.; Cummings, R. D. J.
Biol. Chem. 1999, 274, 24838-24848.
(63) Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244-
1251.
(59) Sallas, F.; Nishimura, S.-I. J. Chem. Soc., Perkin Trans. 1 2000,
2091-2103.
(64) Broddefalk, J.; Nilsson, U.; Kihlberg, J. J. Carbohydr. Chem. 1994,
13, 129-132.
(60) Koeller, K. M.; Smith, M. E. B.; Huang, R.-F.; Wong, C.-H. J.
Am. Chem. Soc. 2000, 122, 4241-4242.
(65) Holm, B.; Broddefalk, J.; Flodell, S.; Wellner, E.; Kihlberg, J.
Tetrahedron 2000, 56, 1579-1586.
(61) Matsuda, M.; Nishimura, S.-I.; Nakajima, F.; Nishimura, T. J. Med.
Chem. 2001, 44, 715-724.
(66) Chang, M. L.; Eddy, R. L.; Shows, T. B.; Lau, J. T. Glycobiology
1995, 5, 319-325.
(62) Meldal, M.; Auzanneau, F.-I.; Hindsgaul, O.; Palcic, M. M. J. Chem.
Soc., Chem. Commun. 1994, 1849-1850.
(67) Wang, S.-S.; Gisin, B. F.; Winter, D. P.; Makofske, R.; Kulesha, I.
D.; Tzougraki, C.; Meienhofer, J. J. Org. Chem. 1977, 42, 1286-1290.

11122 J. Am. Chem. Soc., Vol. 123, No. 45, 2001
George et al.
temperature and stirring was continued for a further 3 h. The solids
were filtered off, the filtrate was concentrated, and the residue was
suspended in water. The aqueous phase was extracted with CH2Cl2 (3
× 25 mL) and the combined organic layers were dried and concentrated.
Flash column chromatography of the residue (toluene/EtOAc, 1:1) gave
342,68 (2.0 g, 90%) as a white amorphous solid.
amorphous pale yellow solid. [R]D20 +123° (c 0.72, CHCl3); 1H NMR
(CDCl3:CD3OD) δ 7.82 (2H, d, ArH), 7.71 (3H, m, ArH), 7.55 (3H,
m, ArH), 7.47-7.29 (8H, m, ArH), 5.61 (1H, s, CHPh), 4.99 (1H, d,
J ) 3.0 Hz, H-1), 4.65-4.48 (2H, m, CHCH2OCO), 4.48-4.36 (2H,
m, H-2 and H-â), 4.35-4.12 (4H, m, CHCH2OCO, H-6, H-6′, and
H-4), 3.99 (1H, m, H-R), 3.79 (1H, bs, H-5), 1.99 (3H, s, NHCOCH3),
1.25 (3H, d, J ) 6.2 Hz, γ-CH3), 0.89 (9H, s, t-BuSi), 0.11 (6H, s,
Si(CH3)2); 13C NMR (CDCl3:CD3OD) δ 173.4, 172.6, 158.8, 145.0,
144.8, 142.4, 139.1, 129.7, 128.8, 128.6, 128.0, 127.2, 125.9, 125.8,
120.8, 120.8, 101.9, 101.2, 79.1, 78.8, 78.5, 77.3, 76.6, 70.2, 69.6, 67.5,
64.4, 59.6, 56.7, 48.3, 26.1, 23.3, 19.4, 18.8, -3.9; HRMS (FAB): calcd
for C40H50N2O10SiNa (M + Na+) 769.3132, found 769.3121. Anal.
Calcd for C40H50N2O10Si: C 63.3; H 6.8; N 3.8. Found: C 63.6; H
6.8; N 3.8.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-(2-azido-4,6-O-benzylidene-
2-deoxy-r-D-galactopyranosyl)-L-threonine Allyl Ester (7). An ice
cold solution of TFA in water (25 mL, 80%) was added to an ice cold
and stirred solution of 4 (1.24 g, 1.61 mmol) in CH2Cl2 (5 mL) during
5 min. After 8 h at 0 °C, cold CH2Cl2 (50 mL) was added and the
solution was neutralized with cold saturated aqueous NaHCO3. The
phases were separated and the aqueous phase was successively extracted
with CH2Cl2 (3 × 50 mL) and EtOAc (3 × 50 mL). The combined
organic phases were washed with brine (50 mL), dried, and concen-
trated. Flash column chromatography of the residue (CHCl3/MeOH,
9:1) gave NR-fluoren-9-ylmethoxycarbonyl-3-O-(2-azido-2-deoxy-R-D-
galactopyranosyl)-L-threonine allyl ester (881 mg, 97%) as a gum. A
solution of NR-fluoren-9-ylmethoxycarbonyl-3-O-(2-azido-2-deoxy-R-
D-galactopyranosyl)-L-threonine allyl ester (760 mg, 1.33 mmol), R,R-
dimethoxytoluene (407 mg, 2.67 mmol), and a catalytic amount of
p-toluenesulfonic acid (monohydrate, 30 mg) in CH3CN (18 mL) was
stirred at room temperature for 24 h. Concentration of the mixture
followed by flash column chromatography of the residue (toluene/
EtOAc, 5:1) gave 7 (762 mg, 84% from 4) as a white amorphous solid.
[R]D20 +110° (c 0.90, CHCl3); 1H NMR (CDCl3) δ 7.77 (2H, d, ArH),
7.64-7.60 (2H, m, ArH), 7.51-7.46 (2H, m, ArH), 7.45-7.30 (7H,
m, ArH), 5.95 (1H, m, OCH2CHdCH2), 5.74 (1H, d, J ) 9.4 Hz,
NHFmoc), 5.59 (1H, s, CHPh), 5.34 (1H, dd, J ) 17.1, 1.1 Hz,
OCH2CHdCH2), 5.28 (1H, dd, J ) 10.4, 1.1 Hz, OCH2CHdCH2),
5.07 (1H, d, J ) 3.4 Hz, H-1), 4.71 (2H, d, J ) 5.9 Hz, OCH2CHd
CH2), 4.59-4.41 (3H, m, CHCH2OCO, H-R and H-â), 4.37 (1H, m,
FmocCH2), 4.33-4.20 (3H, m, H-6, CHCH2OCO and H-4), 3.80 (1H,
bs, H-5), 3.62 (1H, dd, J ) 10.6 and 3.5 Hz, H-2), 2.47 (1H, d, J )
11.0 Hz, 3-OH), 1.33 (3H, d, J ) 6.4 Hz, γ-CH3); 13C NMR (CDCl3)
δ 169.9, 156.8, 143.9, 143.7, 141.3, 137.8, 131.3, 129.4, 129.0, 129.0,
128.6, 128.6, 128.4, 128.2, 127.7, 127.7, 127.1, 127.1, 126.2, 120.0,
119.9, 119.3, 101.3, 99.6, 76.4, 75.3, 69.1, 66.5, 63.3, 61.2, 58.8, 47.2,
18.8; HRMS (FAB): calcd for C35H36N4O9Na (M + Na+) 679.2380,
found 679.2368.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-(2-azido-4,6-O-benzylidene-
3-O-tert-butyldimethylsilyl-2-deoxy-r-D-galactopyranosyl)-L-threo-
nine Allyl Ester (4). A solution of N-bromosuccinimide (600 mg, 3.37
mmol) and tetrabutylammonium triflate40 (328 mg, 0.84 mmol) in
CH2Cl2 (30 mL) was added, dropwise, during 5 min to a stirred solution
of 2 (1.72 g, 3.35 mmol) and 3 (2.68 g, 7.03 mmol) in CH2Cl2 (70
mL) at -28 °C. The solution was stirred at this temperature until TLC
indicated completion of the reaction (toluene/EtOAc, 10:1, RF ) 0.3).
The reaction was quenched by adding triethylamine (2.5 mL) and the
solution was allowed to attain room temperature. Concentration
followed by flash column chromatography of the residue (toluene/
EtOAc, 10:1, containing 0.5% triethylamine) gave 4 (1.28 g, 50%) as
20
1
a colorless amorphous solid. [R]D +98.0° (c 1.1, CHCl3); H NMR
(CDCl3) δ 7.79 (2H, d, ArH), 7.67-7.59 (2H, m, ArH), 7.55-7.49
(2H, m, ArH), 7.45-7.31 (7H, m, ArH), 6.01-5.89 (1H, m, OCH2CHd
CH2), 5.85 (1H, d, J ) 9.3 Hz, NH-R), 5.39 (1H, dd, J ) 17.2, 1.4 Hz,
OCH2CHdCH2), 5.27 (1H, dd, J ) 10.4, 1.2 Hz, OCH2CHdCH2),
5.04 (1H, d, J ) 3.6 Hz, H-1), 4.69 (2H, d, J ) 5.9 Hz, OCH2CHd
CH2), 4.50 (1H, dd, J ) 9.9, 6.6 Hz, CHCH2OCO), 4.46 (1H, dd, J )
6.5, 2.4 Hz, H-â), 4.41 (1H, dd, J ) 9.3, 2.4 Hz, H-R), 4.36-4.25
(3H, m, CHCH2OCO, H-6 and H-4), 4.14-4.05 (3H, m, CHCH2OCO,
H-6 and H-3), 3.79-3.71 (2H, m, H-2 and H-5), 3.28 (3H, d, J ) 6.4
Hz, γ-CH3), 0.95 (9H, s, t-BuSi), 0.22 and 0.17 (6H, 2s, Si(CH3)2);
13C NMR (CDCl3) δ 176.5, 170.1, 143.8, 141.4, 137.7, 133.9, 131.5,
130.2, 129.0, 128.2, 127.8, 127.2, 127.1, 126.1, 125.3, 125.2, 120.1,
119.4, 100.7, 99.7, 76.5, 76.1, 69.4, 68.7, 67.4, 66.6, 63.6, 61.0, 58.9,
47.3, 28.7, 25.8, 19.0, 18.1, 4.2, 4.6; HRMS (FAB): calcd for
C41H51N4O9Si (M + H+) 771.3425, found 771.3400.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-(2-acetamido-4,6-O-ben-
zylidene-3-O-tert-butyldimethylsilyl-2-deoxy-r-D-galactopyranosyl)-
L-threonine Allyl Ester (5). Freshly distilled (5 times) thioacetic acid
(8 mL) was added dropwise to a stirred solution of 4 (285 mg, 370
µmol) in pyridine (8 mL) at 0 °C and the solution was allowed to attain
room temperature. After 4 h, toluene (20 mL) was added and the
solvents were evaporated. The residue was co-concentrated twice from
toluene. Flash column chromatography of the residue (toluene/EtOAc,
6:2) gave 5 (233 mg, 80%) as a white amorphous solid. [R]D20 +89.6°
(c 0.66, CHCl3); 1H NMR (DMSO-d6) δ 7.91 (1H, d, ArH), 7.73 (1H,
d, ArH), 7.60 (1H, d, J ) 9.7 Hz, NH-R), 7.47-7.23 (12H, m, ArH
and NHCOCH3), 5.94-5.81 (1H, m, OCH2CHdCH2), 5.83 (1H, s,
CHPh), 5.33 (1H, dd, J ) 17.2, 1.6 Hz, OCH2CHdCH2), 5.25 (1H,
dd, J ) 10.4, 1.5 Hz, OCH2CHdCH2), 4.68 (1H, d, J ) 3.8 Hz, H-1),
4.56-4.47 (4H, m, OCH2CHdCH2 and CHCH2OCO), 4.37-4.24 (3H,
m, CHCH2OCO, H-R and H-â), 4.18 (1H, m, H-4), 4.12-4.99 (3H,
m, H-2 and H-6), 3.88 (1H, dd, J ) 10.9, 3.5 Hz, H-3), 3.68 (1H, bs,
H-5), 1.81 (3H, s, NHCOCH3), 1.10 (1H, d, J ) 6.4 Hz, γ-CH3), 0.80
(9H, s, t-BuSi), 0.03 and 0.02 (6H, 2s, Si(CH3)2); 13C NMR (DMSO-
d6) δ 169.7, 168.7, 156.7, 143.7, 143.6, 140.8, 140.7, 138.4, 131.9,
128.8, 128.6, 128.2, 127.9, 126.6, 127.0, 125.9, 125.1, 120.2, 120.1,
118.6, 99.7, 99.6, 75.6, 74.4, 68.5, 67.9, 65.5, 65.2, 62.8, 58.5, 48.7,
46.8, 25.8, 22.9, 18.8, 17.8, -4.4, -4.6; HRMS (FAB): calcd for
C43H54N2O10SiNa (M + Na+) 809.3445, found 809.3438.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-[2-azido-4,6-O-benzylidene-
2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-r-D-
galactopyranosyl]-L-threonine Allyl Ester (9). A solution of silver
triflate (165 mg, 642 µmol) in toluene (3 mL) was added dropwise to
a suspension of 7 (300 mg, 457 µmol), 2,3,4,6-tetra-O-acetyl-R-D-
galactopyranosyl bromide 8 (245 mg, 596 mmol), and crushed
molecular sieves (flame dried, 4 Å, 400 mg) in CH2Cl2 (6 mL) at -30
°C. After 1 h, the reaction was quenched by addition of pyridine (1
mL) while keeping the temperature below -30 °C. After 5 min, the
mixture was allowed to attain room temperature and the solids were
removed by filtration (Hyflow, Supercel) and washed with CH2Cl2 (5
× 10 mL). The combined filtrates were washed with a mixture of
aqueous Na2S2O3 (0.5 M) and saturated aqueous NaHCO3 (1:1, 2 ×
12 mL), dried, and concentrated. Flash column chromatography of the
residue (toluene/EtOAc, 3:1) gave 9 (265 mg, 59%) as a white
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-(2-acetamido-4,6-O-ben-
zylidene-3-O-tert-butyldimethylsilyl-2-deoxy-r-D-galactopyranosyl)-
L-threonine (6). A solution of allyl ester 5 (120 mg, 153 µmol),
(PPh3)4Pd(0) (16 mg, 14 µmol), and N-methylaniline (62 mg, 580 µmol)
in THF (4 mL) was stirred at room temperature for 1 h in the absence
of light. The solution was diluted with EtOAc (50 mL) and washed
with saturated aqueous ammonium chloride (5 mL) and the aqueous
phase was extracted with EtOAc (2 × 15 mL). The organic phases
were combined, dried, and concentrated. Flash column chromatography
of the residue (CHCl3/MeOH, 40:1) gave 6 (97 mg, 85%) as an
20
1
amorphous solid. [R]D +51.7° (c 1.00, CHCl3); H NMR (CDCl3) δ
7.78 (2H, d, ArH), 7.66-7.60 (2H, m, ArH), 7.56-7.50 (2H, m, ArH),
7.45-7.30 (7H, m, ArH), 5.96 (1H, m, OCH2CHdCH2), 5.75 (1H, d,
J ) 9.5 Hz, NHFmoc), 5.57 (1H, s, CHPh), 5.43 (1H, m, H-4′), 5.41-
5.26 (3H, m, OCH2CHdCH2 and H-2′), 5.10-5.05 (1H, m, H-3′), 5.04
(1H, d, J ) 3.1 Hz, H-1), 4.82 (1H, d, J ) 8.0 Hz, H-1′), 4.70 (2H, m,
OCH2CHdCH2), 4.54 (1H, dd, J ) 10.3, 6.8 Hz, CHCH2OCO), 4.50
(1H, m, H-â), 4.46 (1H, m, H-R), 4.41 (1H, m, H-4), 4.36 (1H, m,
(68) de la Torre, B. G.; Torres, J. L.; Bardaji, E.; Clape´s, P.; Xaus, N.;
Jorba, X.; Calvet, S.; Albericio, F.; Valencia, G. J. Chem. Soc., Chem.
Commun. 1990, 965-967.

Chemoenzymatic Synthesis of Sialylated Glycopeptides
J. Am. Chem. Soc., Vol. 123, No. 45, 2001 11123
CHCH2OCO), 4.32-4.18 (3H, m, H-6, CHCH2OCO and H-6′), 4.18-
4.10 (1H, m, H-6′), 4.10-4.00 (2H, m, H-3 and H-6), 3.99-3.93 (1H,
m, H-5′), 3.84 (1H, dd, J ) 10.9, 3.6 Hz, H-2), 3.73 (1H, m, H-5),
2.18, 2.07, 2.05, and 2.00 (12H, 4s, 4 Ac), 1.35 (3H, d, J ) 6.2 Hz,
γ-CH3); 13C NMR (CDCl3) δ 170.3, 170.3, 170.1, 169.9, 169.4, 156.7,
143.8, 143.6, 141.3, 137.5, 131.2, 128.2, 127.8, 127.1, 127.0, 126.1,
125.1, 125.1, 120.0, 119.5, 102.4, 100.7, 99.7, 76.2, 75.8, 75.6, 71.0,
70.9, 69.1, 68.6, 67.3, 66.9, 66.6, 63.5, 61.3, 59.1, 58.7, 53.8, 47.1,
29.2, 20.7, 20.6, 18.9; HRMS (FAB): calcd for C49H54N4O18Na (M +
Na+) 1009.3331, found 1009.3337.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-[2-acetamido-4,6-O-ben-
zylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-
r-D-galactopyranosyl]-L-threonine Allyl Ester (10). Freshly distilled
(5 times) thioacetic acid (5 mL) was added dropwise to an ice cold
solution of 9 (210 mg, 213 µmol) in pyridine (5 mL) and the solution
was allowed to attain room temperature. After 8 h, the solvents were
evaporated and the residue was co-concentrated with toluene (3 × 20
mL). Flash column chromatography of the residue (toluene/EtOAc, 3:2)
gave 10 (184 mg, 86%) as a white amorphous solid. [R]D20 +73.1° (c
0.70, CHCl3); 1H NMR (CDCl3) δ 7.78 (2H, d, ArH), 7.65-7.58 (2H,
m, ArH), 7.54 (2H, dd, ArH), 7.45-7.30 (7H, m, ArH), 5.88 (1H, m,
OCH2CHdCH2), 5.72 (1H, d, J ) 9.2 Hz, NHCOCH3), 5.64 (1H, d, J
) 9.3 Hz, NHFmoc), 5.56 (1H, m, H-4′), 5.39-5.24 (2H, m,
OCH2CHdCH2), 5.20 (1H, dd, J ) 10.4, 7.9 Hz, H-2′), 5.00 (1H, m,
H-3′), 4.98 (1H, d, J ) 2.9 Hz, H-1), 4.77 (1H, d, J ) 7.7 Hz, H-1′),
4.73-4.46 (3H, m, H-2 and H-R), 4.36-4.18 (2H, m, H-â and H-4),
4.01-3.86 (2H, m, H-5′ and H-3), 2.16 (3H, s, NHCOCH3), 2.15, 2.04,
1.98, 1.95, and 1.90 (15H, 5s, 5 Ac), 1.29 (3H, d, J ) 6.4 Hz, γ-CH3);
13C NMR (CDCl3) δ 170.6, 170.5, 170.3, 170.2, 169.8, 169.5, 156.5,
143.6, 141.3, 137.5, 130.8, 128.9, 128.2, 127.9, 127.1, 126.2, 124.9,
120.1, 120.1, 120.0, 101.0, 100.7, 100.4, 76.4, 75.5, 73.8, 71.0, 70.8,
69.1, 68.8, 67.0, 66.4, 63.6, 61.4, 58.6, 48.0, 47.2, 20.7, 20.6, 18.7;
HRMS (FAB): calcd for C51H58N2O19Na (M + Na+) 1025.3532, found
1025.3551.
Nr-Fluoren-9-ylmethoxycarbonyl-3-O-[2-acetamido-4,6-O-ben-
zylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-
r-D-galactopyranosyl]-L-threonine (11). A solution of allyl ester 10
(160 mg, 160 µmol), (PPh3)4Pd(0) (19 mg, 16 µmol), and N-
methylaniline (62 mg, 580 µmol) in THF (4 mL) was stirred at room
temperature for 1 h in the absence of light. The solution was diluted
with EtOAc (50 mL) and washed with saturated aqueous ammonium
chloride (5 mL). The aqueous phase was extracted with EtOAc (2 ×
15 mL) and the combined organic phases were dried and then
concentrated. Flash column chromatography of the residue (CHCl3/
MeOH/AcOH, 92:7:1) gave 11 (135 mg, 88%) as an amorphous pale
yellow solid. [R]D20 +98.4° (c 0.63, CHCl3); 1H NMR (CDCl3) δ 7.82
(1H, d, ArH), 7.69 (1H, d, ArH), 7.56-7.51 (2H, m, ArH), 7.47-7.30
(8H, m, ArH), 5.61 (1H, s, CHPh), 5.39 (1H, m, H-4′), 5.16 (1H, dd,
J ) 10.5, 8.0 Hz, H-2′), 5.04-4.86 (2H, m, H-3′ and H-1), 4.73 (1H,
d, J ) 7.9 Hz, H-1′), 4.61 (2H, m, H-2 and H-R), 4.46-4.42 (1H, m,
H-â), 3.89 (1H, dd, J ) 11.3, 3.13 Hz, H-3), 3.78-3.71 (1H, m, H-5),
2.18, 2.06, 2.04, 1.99, and 1.98 (15H, 5s, 5CH3CO), 1.25 (1H, d, J )
6.4 Hz, γ-CH3); 13C NMR (CDCl3) δ 173.6, 171.8, 171.3, 170.3, 170.1,
169.4, 157.1, 143.4, 143.2, 140.8, 137.2, 128.3, 127.6, 127.4, 127.2,
126.6, 125.7, 124.3, 119.4, 101.3, 100.2, 99.5, 70.6, 69.9, 68.6, 68.3,
66.6, 66.1, 62.9, 60.7, 57.9, 46.8, 21.9, 19.9, 19.8, 19.7, 19.6, 18.2;
HRMS (FAB): calcd for C48H54N2O19Na (M + Na+) 985.3219, found
985.3248. Anal. Calcd for C48H54N2O19: C 59.8; H 5.7; N 2.9. Found
C 59.3; H 5.7; N 2.8.
equiv) and 1-hydroxybenzotriazole (HOBt, 6 equiv) in dry DMF.
Acylations were monitored by using bromophenol blue as indicator69
(the color of the resin changes from blue to yellow). NR-Fmoc
deprotections were performed by a flow of piperidine in DMF (20%)
for 3 min and then by shaking for 7 min. Before and after treatment
with piperidine the resin was washed 5 times with DMF. Glycosylated
amino acid building blocks 6 (70 mg, 94 mmol) and 11 (110 mg, 114
mmol) were activated in dry DMF (1 mL) at room temperature during
35 min by addition of 1,3-diisopropylcarbodiimide (1.1 equiv) and
1-hydroxy-7-azabenzotriazole (HOAt, 3 equiv), respectively. The
activated esters were then coupled to the peptide resins during 24 h.
After coupling of building blocks 6 and 11, unreacted peptide NR amino
groups were capped by addition of a 1:1 mixture of acetic anhydride
and DMF.
After completion of the synthesis, the resin was washed with CH2Cl2
(5 times) and dried under vacuum. Cleavage from the resin and removal
of acid labile protective groups was performed with TFA/H2O/
thioanisole/ethanedithiol (87.5:5:5:2.5, 20 mL/200 mg of resin) for
3-3.5 h, followed by filtration. Acetic acid (15 mL) was added to the
filtrate which was then concentrated. The residue was co-concentrated
several times with acetic acid until it formed a thin film. It was then
washed with diethyl ether (3 times), dissolved in a mixture of water
and acetic acid (6:1), and freeze-dried. Purification by preparative
reversed-phase HPLC gave glycopeptides 12 and 13.
L-Alanyl-L-histidinylglycyl-L-valyl-L-threonyl-L-seryl-L-alanyl-L-
prolyl-L-aspartyl-3-O-(2-acetamido-2-deoxy-r-D-galactopyranosyl)-
L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolylglycyl-L-seryl-L-thre-
onyl-L-alanyl-L-prolyl-L-prolyl-L-alanine (12). Synthesis, cleavage of
the resin-bound glycopeptide (78 mmol) with simultaneous deprotection,
and then purification by reversed-phase HPLC (gradient 0f100%
CH3CN in H2O, both containing 0.1% TFA, during 80 min), according
to the general procedure, gave 12 (35 mg, 78% peptide content, 23%
overall yield). MS (FAB): calcd for C91H145N27O34 (M + H)+ 2161,
found 2162. Amino acid analysis: Ala 5.00 (5), Arg 1.00 (1), Asp
1.02 (1), Gly 2.08 (2), His 1.01 (1), Pro 5.00 (5), Ser 1.99 (2), Thr
2.91 (3), Val 1.00 (1).
L-Alanyl-L-histidinylglycyl-L-valyl-L-threonyl-L-seryl-L-alanyl-L-
prolyl-L-aspartyl-3-O-(2-acetamido-2-deoxy-3-O-â-D-galactopyrano-
syl-r-D-galactopyranosyl)-L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-
prolylglycyl-L-seryl-L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-
alanine (13). Synthesis, cleavage of the resin-bound glycopeptide (90
µmol), with simultaneous deprotection, and then purification by
reversed-phase HPLC (gradient 0f100% CH3CN in H2O, both
containing 0.1% TFA, during 60 min), according to the general
procedure, gave the target glycopeptide which carried O-acetyl groups
on the Galâ-moiety [56 mg, MS (FAB): calcd (M + H)+ 2490.14 ,
found 2492.14]. Methanolic sodium methoxide (233 µL, 0.2 M) was
added to a solution of this O-acetylated glycopeptide (23 mg) in
methanol (23 mL) and the solution was stirred under nitrogen for 1 h.
It was neutralized with a solution of acetic acid in methanol (2:5, dry
pH paper) and concentrated under vacuum. The residue was purified
by reversed-phase HPLC (gradient 0f100% CH3CN in H2O, with 0.1%
TFA, during 60 min) to give 13 (16 mg, 75% peptide content, 19%
overall yield). MS (ES): calcd for C97H155N27O39 2322.1 (M)+, found
2322.2. Amino acid analysis: Ala 5.13 (5), Arg 1.01 (1), Asp 1.02
(1), Gly 2.03 (2), His 1.02 (1), Pro 4.78 (5), Ser 2.02 (2), Thr 2.98 (3),
Val 1.02 (1).
Tn neoglycopeptide 16. l-Arginylglycyl-L-tyrosyl-L-valyl-L-tyrosyl-
L-homocysteinylglycyl-L-leucine (15 mg, 13 µmol) was added to a
mixture of 2-bromoethyl glycoside 14 (15 mg, 46 µmol) and cesium
carbonate (50 mg, 154 µmol) in dry DMF (1.3 mL) under a nitrogen
atmosphere. The solution was stirred at room temperature until
analytical reversed-phase HPLC (gradient 0f100% CH3CN in H2O,
both containing 0.1% TFA, during 60 min, RT ) 17.2 min) indicated
that the peptide was consumed. The reaction was quenched by addition
of 0.1% aqueous TFA (15 mL) and the mixture was freeze-dried.
Purification of the residue by preparative reversed-phase HPLC
(gradient 0f100% CH3CN in H2O, both containing 0.1% TFA, during
General Procedure for Solid-Phase Synthesis of Glycopeptides
12 and 13. A TentaGel S Trt-Ala-Fmoc resin (Rapp Polymere,
Germany) was used for the synthesis of glycopeptides 12 and 13. NR-
Fmoc-amino acids (Bachem, Switzerland) with the following side chain
protecting groups were used in the synthesis: 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl (Pmc) for arginine; triphenylmethyl (Trt) for
histidine, and tert-butyl (tBu) for aspartic acid, serine, and threonine.
DMF was distilled before being used.
Glycopeptides 12 and 13 were synthesized with 78 and 90 mmol of
resin, respectively. Couplings were performed manually in a mechani-
cally agitated reactor. Fmoc amino acids (4 equiv) were activated as
benzotriazolyl esters by using 1,3-diisopropylcarbodyimide (DIC, 3.9
(69) Flegel, M.; Sheppard, R. C. J. Chem. Soc., Chem. Commun. 1990,
536-538.

11124 J. Am. Chem. Soc., Vol. 123, No. 45, 2001
George et al.
Table 1. 1H NMR Data (δ, ppm) for Glycopeptide 18 in Water
Table 2. 1H NMR Data (δ, ppm) for Glycopeptide 20 in Water
Containing 10% D2Oa,b
Containing 10% D2Oa,b
residue NH
HR
Hâ
1.42
3.21, 3.17
Hγ
others
residue NH HR
Hâ
Hγ
others
Ala1
His2
4.01
4.63
Ala1
His2
Gly3
Val4
Thr5
Ser6
Ala7
Pro8
Asp9
4.08 1.49
8.42 (H2), 7.24 (H4)
8.95 4.69 3.29, 3.23
8.56 (H4), 7.30 (H2)
Gly3
Val4
8.62 3.91c
8.25 4.19
8.48 4.37
8.45 4.40
8.45 4.55
8.59 4.69
8.74 4.46
8.47 4.46
8.62 3.91c
8.24 4.46
8.34 4.32
8.64 4.54
8.65 3.98c
2.06
0.89c
1.15
8.26 4.23 2.10
8.47 4.42 4.22
8.44 4.44 3.84c
8.47 4.55 1.36
0.96, 0.95
1.20
Thr5
4.17
Ser6
3.79c
1.31
Ala7
Asp9
Thr10d
Arg11
Gly15
Ser16
Thr17
Ala18
2.72, 2.56
4.27
4.40 2.28, 2.00 1.87c
3.81 and 3.66 (d)
1.24
8.58 4.74 2.81, 2.64
1.79, 1.67 1.66c 3.17 (δ), 7.34 (NH)
Thr10d 8.85 4.54 4.44
1.23
Arg11 8.42 4.48 1.85, 1.76 1.73c
7.52, 6.90, 6.62 (NH);
3.23 (δ)c
3.76 and 3.61 (δ)
3.87, 3.83
4.17
1.29
Pro12 b
Ala13 8.63 4.62 1.38
Pro14 b 4.43 2.29b
4.36 2.29b
1.15
3.80 and 3.66 (δ)
a Recorded at 600 MHz, 278 K, and pH 5.6 with H2O as internal
standard (δH 4.98 ppm). b Resonances that could not be unambiguously
assigned are not reported. This includes all resonances of Pro8, Pro12-
Pro14, and Pro19-Ala21. c Degeneracy has been assumed. d Chemical
shifts (δ, ppm) for the Neu5AcR(2f6)GalNAc moietysGalNAc: 4.79
(H-1), 3.99 (H-2), 3.89 (H-4), 3.82 (H-3). Neu5Ac: 4.07 (H-8), 3.89
and 3.49 (H-9,9′), 3.79 (H-5), 3.64 (H-6), 3.59 (H-4), 2.64 (H-3eq),
1.61 (H-3ax).
Gly15 8.65 3.98c
Ser16
8.27 4.52 3.93, 3.88
Thr17 8.33 4.37 4.22
Ala18 8.42 4.61 1.35
1.21
Pro19
4.72 2.38, 1.92 2.05c
4.38 2.30b
3.81 and 3.65 (δ)
3.83 and 3.65 (δ)
Pro20 b
Ala21 8.11 4.10 1.34
a Recorded at 600 MHz, 279 K, and pH 5.4 with H2O (δH ) 4.95)
as internal standard. b All proline resonances could not be unambigu-
ously assigned due to overlap. c Degeneracy has been assumed.
d Chemical shifts (δ, ppm) for the Neu5AcR(2f3)Galâ(1′3)GalNAc
moietysGalNAc: 8.11 (NH), 4.84 (H-1), 4.21 (H-2), 4.02 (H-3), 2.02
(Ac). Gal: 4.51 (H-1), 4.06 (H-3), 3.50 (H-2). Neu5Ac: 8.14 (NH),
3.85 (H-5), 3.67 (H-4), 3.61 (H-6), 2.75 (H-3eq), 1.78 (H-3ax).
60 min) gave 16 (14 mg, 77% peptide content, 74% yield). HRMS
(FAB): calcd for C53H83N12O17S (M + H+) 1191.5720, found
1191.5728. Amino acid analysis: Arg 0.98 (1), Gly 1.99 (2), Leu 1.01
(1), Tyr 1.98 (2), Val 1.04 (1).
T neoglycopeptide 17. L-Arginylglycyl-L-tyrosyl-L-valyl-L-tyrosyl-
L-homocysteinylglycyl-L-leucine (12 mg, 13 µmol) was added to a
mixture of 2-bromoethyl glycoside 15 (10 mg, 20 µmol) and cesium
carbonate (35 mg, 107 µmol) in dry DMF (1.0 mL) under a nitrogen
atmosphere. The mixture was stirred at room temperature until analytical
reversed-phase HPLC (gradient 0f100% CH3CN in H2O, both
containing 0.1% TFA, during 60 min, RT ) 16.9 min) indicated that
the peptide was consumed. The reaction was quenched by addition of
0.1% aqueous TFA (15 mL) and the mixture was freeze-dried.
Purification of the residue by preparative reversed-phase HPLC
(gradient 0f100% CH3CN in H2O, both containing 0.1% TFA, during
60 min) gave 17 (10 mg, 81% peptide content, 58% yield). HRMS
(FAB): calcd for C59H93N12O22S (M + H+) 1353.6248, found
1353.6215. Amino acid analysis: Arg 1.00 (1), Gly 1.99 (2), Leu 1.03
(1), Tyr 2.00 (2), Val 0.99 (1).
mass spectrometry indicated 90-95% conversion of 16. Purification
by HPLC on a Zorbax 300SB-C3 column (9.4 × 250 mm) with a
gradient of 0f90% CH3CN in water, both containing 0.1% TFA, gave
19 (∼250 mg) after freeze-drying; MS (MALDI-TOF) calcd for
C64H100N13O25S (M+) 1483 , found 1483.
L-Alanyl-L-histidinylglycyl-L-valyl-L-threonyl-L-seryl-L-alanyl-L-
prolyl-L-aspartyl-3-O-[(5-acetamido-3,5-dideoxy-D-glycero-r-D-
galacto-2-nonulopyranosylonic acid)-(2f3)-O-â-D-galactopyranosyl-
(1f3)-O-(2-acetamido-2-deoxy-r-D-galactopyranosyl)]-L-threonyl-
L-arginyl-L-prolyl-L-alanyl-L-prolylglycyl-L-seryl-L-threonyl-L-alanyl-
L-prolyl-L-prolyl-L-alanine (20). Purified ST3Gal-I (20 µL, ≈2 mU)
was added to 13 (0.50 mg, 0.16 µmol based on 75% peptide content)
in 25 mM Tris-HCl buffer (pH 6.5, 1.0 mL) containing CMP-Neu5Ac
(2 mM), Triton X-100 (0.1%), and calf intestinal phosphatase (2 mU).
The solution was then incubated at 37 °C for 3 h, after which analysis51
by nanoscale reversed-phase HPLC in combination with MALDI-TOF
mass spectrometry indicated >95% conversion of 13. This procedure
was repeated so that 2.0 mg (0.64 µmol) of 13 was sialylated.
Purification by HPLC on a Zorbax 300SB-C3 column (9.4 × 250 mm)
using a gradient of 0f90% CH3CN in water, both containing 0.1%
TFA, gave 20 (2.0 mg, 79% peptide content, 94% yield) after freeze-
drying. 1H NMR data, see Table 2; MS (ES) calcd for C108H173N28O47
(M + H+) 2614.2, found 2614.5. Amino acid analysis: Ala 4.90 (5),
Arg 1.00 (1), Asp 1.02 (1), Gly 2.02 (2), His 1.03 (1), Pro 5.13 (5),
Ser 1.97 (2), Thr 2.90 (3), Val 1.04 (1).
L-Alanyl-L-histidinylglycyl-L-valyl-L-threonyl-L-seryl-L-alanyl-L-
prolyl-L-aspartyl-3-O-[2-acetamido-2-deoxy-6-O-(5-acetamido-3,5-
dideoxy-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-r-D-
galactopyranosyl]-L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-
prolylglycyl-L-seryl-L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-
alanine (18). Glycopeptide 12 (0.50 mg, 0.18 µmol based on 78%
peptide content) was added to purified ST6GalNAc-I (∼2 mU) in 20
mM Bis-Tris buffer (pH 6.0, 1.0 mL) containing CMP-Neu5Ac (2mM),
EDTA (20 mM), and dithiothreitol (1 mM). The solution was then
incubated at 37 °C for 6 h, after which analysis51 by nanoscale reversed-
phase HPLC in combination with MALDI-TOF mass spectrometry
indicated >95% conversion of 12. This procedure was repeated so that
5.0 mg (1.8 µmol) of 12 was sialylated. Purification by HPLC on a
Zorbax 300SB-C3 column (9.4 × 250 mm) with a gradient of 0f90%
CH3CN in water, both containing 0.1% TFA, gave 18 (4.5 mg, 73%
peptide content, 74% yield) after freeze-drying. 1H NMR data, see Table
1; MS (MALDI TOF) calcd for C102H162N28O42 (M)+ 2451 , found 2451.
Amino acid analysis: Ala 4.99 (5), Arg 1.01 (1), Asp 1.02 (1), Gly
1.98 (2), His 0.99 (1), Pro 5.08 (5), Ser 1.98 (2), Thr 2.93 (3), Val
1.01 (1).
2,3-Sialyl-T Neoglycopeptide 21. Purified ST3Gal-I (20 mL, ≈2
mU) was added to neoglycopeptide 17 (0.50 mg, 0.30 µmol based on
81% peptide content) in 25 mM Tris-HCl buffer (pH 6.5, 1.0 mL)
containing CMP-Neu5Ac (2 mM), Triton X-100 (0.1%), and calf
intestinal phosphatase (10 mU). The solution was then incubated at 37
°C for 3 h, after which analysis51 by nanoscale reversed-phase HPLC
in combination with MALDI-TOF mass spectrometry indicated ∼95%
conversion of 17. This procedure was repeated so that 3.5 mg (2.1
mmol) of 17 was sialylated. Purification by HPLC on a Zorbax 300SB-
C3 column (9.4 × 250 mm) with a gradient of 0f90% CH3CN in
water, both containing 0.1% TFA, gave 21 (2.9 mg, 77% peptide
Sialyl-Tn Neoglycopeptide 19. Neoglycopeptide 16 (0.50 mg, 0.32
µmol based on 77% peptide content) was added to purified ST6GalNAc-I
(∼2mU) in 20 mM Bis-Tris buffer (pH 6.0, 1.0 mL) containing CMP-
Neu5Ac (2mM), EDTA (20 mM), and dithiothreitol (1 mM). The
solution was then incubated at 37 °C for 6 h, after which analysis51 by
nanoscale reversed-phase HPLC in combination with MALDI-TOF
1
content, 64% yield) after freeze-drying. H NMR data, see Table 3;

Chemoenzymatic Synthesis of Sialylated Glycopeptides
J. Am. Chem. Soc., Vol. 123, No. 45, 2001 11125
Table 3. 1H NMR Data (δ, ppm) for Glycopeptide 21 in Water
µL of a 0.4 mM solution in water) were added. After incubation for
18 h analysis51 by nanoscale reversed-phase HPLC in combination with
MALDI-TOF mass spectrometry indicated >95% conversion of 13.
This procedure was repeated so that 0.60 mg (0.19 mmol) of 13 was
converted. Purification by HPLC on a Zorbax 300SB-C3 column (9.4
× 250 mm) with a gradient of 0f90% CH3CN in water, both containing
0.1% TFA, gave 22 (∼600 µg) after freeze-drying. MS (ES) calcd for
Containing 10% D2Oa
residue NH
Arg
HR
3.99
Hâ
1.88b
Hγ
1.62b
others
3.16b (Hδ,δ′);
7.19 (CH2NH)
Gly
Tyr
Val
Tyr
8.78 4.02, 3.90
8.43 4.48
7.97 3.94
2.94, 2.85
1.83
3.03, 2.87
7.03 and 6.74 (ArH)
7.16 and 6.78 (ArH)
C
105H169N28O44 (M + H+) 2526.2, found 2526.5.
0.82, 0.78
Core 2 Neoglycopeptide 23. Purified C2GnT3 (30 µL, ≈ 1.7 mU)
8.49 4.40
Hcyc 8.37 4.42
2.04, 1.81 2.52, 2.36 2.72b (SCH2CH2O);
3.80and 3.60
was added to neoglycopeptide 17 (100 µg, 60 nmol based on 81%
peptide content) in 100 mM MES buffer (pH 6.5, 0.50 mL) containing
UDP-GlcNAc (2 mM), EDTA (2 mM), D-galactono-1,5-lactone (5
mM), and 2-acetamido-2-deoxy-D-glucono-1,5-lactone (2 mM). Calf
intestinal phosphatase (100 mU) was added after 1 h incubation. The
solution was then incubated at 37 °C for 3 h, after which analysis51 by
nanoscale reversed-phase HPLC in combination with MALDI-TOF
mass spectrometry indicated >95% conversion of 17. This procedure
was repeated so that 0.6 mg (3.6 µmol) of 17 was converted. Purification
by HPLC on a Zorbax 300SB-C3 column (9.4 × 250 mm) using a
gradient of 0f90% CH3CN in water, both containing 0.1% TFA, gave
23 (∼400 µg) after freeze-drying. MS (ES) calcd for C67H106N13O27S
(M + H+) 1556.7, found 1556.8.
(SCH2CH2O)
Gly
Leu
7.01 3.80b
7.90 4.19
1.57b
1.57
0.88 and 0.84
(γ,γ′Me)
a Recorded at 500 MHz, 278 K, and pH 5.4 with H2O (δH 4.98 ppm)
as internal standard. b Degeneracy has been assumed. c Chemical shifts
(δ, ppm) for the Neu5AcR(2f3)Galâ(1f3)GalNAc moietysNeu5Ac:
8.15 (NH), 3.82 (H-5), 3.64 (H-4), 3.57 (H-6), 2.71 (H-3eq), 2.00 (Ac),
1.76 (H-3ax). Gal: 4.47 (H-1), 4.03 (H-3), 3.50 (H-2). GalNAc: 8.21
(NH), 4.87 (H-1), 4.27 (H-2), 4.19 (H-4), 3.98 (H-3), 1.98 (Ac).
MS (ES) calcd for C70H110N13O30S (M+) 1643.7, found 1643.6. Amino
acid analysis: Arg 1.00 (1), Gly 2.01 (2), Leu 1.01 (1), Tyr 2.00 (2),
Val 0.99 (1).
Acknowledgment. This research was supported by a post-
doctoral research fellowship to S.K.G. from the program
“Glycoconjugates in Biological Systems” (GLIBS) sponsored
by the Swedish Foundation for Strategic Research. Financial
support from the EU Biotech 5th Framework and by FCT
(Sapiens 36376/99) is also acknowledged.
L-Alanyl-L-histidinylglycyl-L-valyl-L-threonyl-L-seryl-L-alanyl-L-
prolyl-L-aspartyl-3-O-[2-acetamido-6-O-(2-acetamido-2-deoxy-â-D-
glucopyranosyl)-3 O-(â-D-galactopyranosyl)-2-deoxy-r-D-galacto-
pyranosyl]-L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolylglycyl-L-
seryl-L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-alanine (22). Purified
C2GnT3 (30 µL, ≈ 1.7 mU) was added to 13 (100 µg, 32 nmol based
on 75% peptide content) in 100 mM MES buffer (pH 6.5, 0.50 mL)
containing UDP-GlcNAc (2 mM), EDTA (2 mM), D-galactono-1,5-
lactone (5 mM), and 2-acetamido-2-deoxy-D-glucono-1,5-lactone (2
mM). Calf intestinal phosphatase (100 mU) was added after 1 h
incubation. The solution was then incubated at 37 °C for 6 h, after
which additional C2GnT3 (10 µL, ≈ 0.6 mU) and UDP-GlcNAc (2
Supporting Information Available: 1H and 13C NMR
1
spectra for compounds 2, 4, 5, 7, 9, and 10 and tabulated H
NMR data for glycopeptides 13, 16, and 17 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA015570T