Chemoenzymatic synthesis of derivatives of a T-cell-stimulating peptide which carry tumor-associated carbohydrate antigens

Article abstract of DOI:10.1039/b009567m

The Tn (GalNAcα-Ser/Thr), T[Galβ(1→3)GalNAcβ-Ser/Thr], sialyl-Tn[Neu5Acα(2→6)GalNAcα-Ser/Thr] and 2,3-sialyl-T[Neu5Acα(2→3)Galβ(1→3)GalNAcα-Ser/Thr] antigens are examples of tumor-associated carbohydrate antigens expressed by epithelial cancers. We now describe the preparation of 2-bromoethyl glycosides corresponding to the Tn and T antigens in one and five chemical steps (51 and 15% total yield), respectively, starting from N-acetylgalactosamine. The 2-bromoethyl Tn and T glycosides were used to alkylate a homocysteine residue incorporated in a peptide that is able to bind to class I MHC molecules on antigen-presenting cells. The two neoglycopeptides were then converted into glycopeptides which carry the sialyl-Tn and 2,3-sialyl-T antigens by using recombinant sialyltransferases. Interestingly, the sialyltransferases were able to sialylate the Tn and T carbohydrate moieties even though they were linked to the peptide backbone via a spacer instead of being attached to serine or threonine. The four glycopeptides will be used in studies directed towards inducing a carbohydrate-specific T cell response against the Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens.

Full text of DOI:10.1039/b009567m

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Chemoenzymatic synthesis of derivatives of a T-cell-stimulating
peptide which carry tumor-associated carbohydrate antigens
Shaji K. George,a Björn Holm,a Celso A. Reis,bc Tilo Schwientek,b Henrik Clausenb and
Jan Kihlberg*a
1
a Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden
b School of Dentistry, University of Copenhagen, Nörre Allé 20, DK-2200 Copenhagen N,
Denmark
c Institute of Molecular Pathology and Immunology of the University of Porto, IPATIMUP,
Rua Dr. Roberto Frias s/n, 4200 Porto, Portugal
Received (in Cambridge, UK) 27th November 2000, Accepted 1st February 2001
First published as an Advance Article on the web 2nd March 2001
The Tn (GalNAcα-Ser/Thr), T [Galβ(1→3)GalNAcα-Ser/Thr], sialyl-Tn [Neu5Acα(2→6)GalNAcα-Ser/Thr]
and 2,3-sialyl-T [Neu5Acα(2→3)Galβ(1→3)GalNAcα-Ser/Thr] antigens are examples of tumor-associated
carbohydrate antigens expressed by epithelial cancers. We now describe the preparation of 2-bromoethyl
glycosides corresponding to the Tn and T antigens in one and five chemical steps (51 and 15% total yield),
respectively, starting from N-acetylgalactosamine. The 2-bromoethyl Tn and T glycosides were used to alkylate
a homocysteine residue incorporated in a peptide that is able to bind to class I MHC molecules on antigen-
presenting cells. The two neoglycopeptides were then converted into glycopeptides which carry the sialyl-Tn
and 2,3-sialyl-T antigens by using recombinant sialyltransferases. Interestingly, the sialyltransferases were able to
sialylate the Tn and T carbohydrate moieties even though they were linked to the peptide backbone via a spacer
instead of being attached to serine or threonine. The four glycopeptides will be used in studies directed towards
inducing a carbohydrate-specific T cell response against the Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens.
(KLH) or to an immunogenic synthetic lipopeptide, either as
Introduction
individual glycosylated amino acids or after incorporation in
short peptides. Studies in mice revealed that short Tn-based
glycopeptides coupled to KLH induced a strong IgM response
and a moderate IgG response, both of which were reactive to
colon cancer cells.25 Moreover, a sialyl-Tn KLH conjugate
vaccine used in combination with cyclophosphamide was indi-
cated to have a therapeutic affect when evaluated in clinical
trials involving breast cancer patients.26,27 These vaccines elicit
an antibody response, but it would also be an advantage if T
cells could be directed to tumor-associated carbohydrate anti-
gens. T cells are known to recognize short peptides presented by
major histocompatibility complex (MHC) molecules, but recent
studies have also shown that glycopeptides can be recognized in
a carbohydrate-specific manner.11,28 Interestingly, one study per-
formed in mice revealed that immunization with neoglyco-
peptides elicited a set of γ/δ cytotoxic T cells that were able to
kill target cells expressing the same carbohydrate moiety in
glycolipid form.29 This study was based on the model carbo-
hydrate galabiose [Galα(1→4)Galβ] which was linked to pep-
tides known to be bound by class I MHC molecules. To allow
investigations of whether it is possible to use this approach to
direct T cells to the tumor-associated Tn, T, sialyl-Tn and 2,3-
sialyl-T antigens we have now prepared 2-bromoethyl glyco-
sides corresponding to the Tn and T antigens. These building
blocks were then used in chemoenzymatic synthesis of four
glycosylated derivatives of a peptide from vesicular stomatitis
virus nucleocapsid protein, which binds to class I MHC
molecules.
Most epithelial cells produce mucins, which are a family of
glycoproteins that are either secreted or exposed to the exterior
in membrane-associated form.1–3 Mucins have a rod-like
appearance and are composed of a polypeptide backbone con-
sisting of highly conserved tandem repeats, in which multiple
serine and threonine residues carry complex carbohydrates. In
epithelial cancers, such as those affecting the breast, ovary,
stomach, and colon, mucins express simpler O-linked carbo-
hydrates which are regarded as tumor-associated carbohydrate
antigens.4–8 The Tn (GalNAcα-Ser/Thr),
T [Galβ(1→3)-
GalNAcα-Ser/Thr], sialyl-Tn [Neu5Acα(2→6)GalNAcα-Ser/
Thr] and 2,3-sialyl-T [Neu5Acα(2→3)Galβ(1→3)GalNAcα-
Ser/Thr] antigens are important examples of such antigens.
In view of their importance as tumor-associated antigens
substantial efforts have been focused on chemical synthesis of
glycopeptides containing the Tn, T, sialyl-Tn and 2,3-sialyl-T
antigens. As for all O-linked glycopeptides the most general
synthetic route in use today employs Nα-Fmoc-protected glyco-
sylated amino acids as building blocks in stepwise assembly,
preferably on solid phase.9–12 Synthesis of building blocks
corresponding to the structurally less complex Tn and T anti-
gens has been described by several groups during recent years
(reviewed in ref. 13). However, incorporation of sialic acid in
glycoconjugates by chemical means is a demanding task,14,15
which may explain why only a few syntheses of building blocks
corresponding to the sialyl-Tn16–19 and 2,3-sialyl-T19–21 antigens
have been reported. Recently, a few enzymatic syntheses of
the 2,3-sialyl-T antigen have been described,22,23 but since the
carboxy group of the sialic acid residue in the resulting building
blocks is unprotected they cannot be used for synthesis of
glycopeptides.
Results and discussion
Synthetic vaccines based on the tumor-associated Tn or
sialyl-Tn antigens have been developed during recent years.7,24,25
In these vaccines the antigens were coupled to an immuno-
logical carrier protein such as keyhole limpet hemocyanin
Coupling of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α--galacto-
pyranosyl bromide30,31 to 2-bromoethanol under promotion by
silver perchlorate and silver carbonate was investigated in an
initial attempt to obtain an α-linked 2-bromoethyl glycoside of
880
J. Chem. Soc., Perkin Trans. 1, 2001, 880–885
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b009567m

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Scheme 1 Reagents and conditions (and yields): i) 2-Bromoethanol, HCl (g), 55–60 ЊC (52%); ii) α,α-dimethoxytoluene, p-TsOH (cat.), CH3CN, rt
(98%); iii) AgOTf, CH2Cl2–toluene (1 : 1), Ϫ25 ЊC (50%); iv) 80% aq. TFA, 0 ЊC (70%); v) NaOMe in MeOH, rt (84%).
N-acetylgalactosamine, i.e. an analogue of the Tn antigen. This
approach was not successful since a mixture of the desired
α-glycoside and the corresponding β-glycoside that could not
be separated by flash column chromatography was obtained.
Instead, it was found that 2-bromoethyl glycoside 2 could be
synthesized from N-acetyl--galactosamine 1 in a Fischer
glycosidation (Scheme 1). This was achieved by heating of 1 in
2-bromoethanol at 60 ЊC in the presence of a catalytic amount
of hydrogen chloride to give 2 in 52% yield.32 By performing the
reaction under thermodynamic control a very short route to the
desired α-glycoside 2 was thus found, in spite of the presence
of a participating acetamido group at C-2 of N-acetylglucos-
amine.
Conversion of 2 into an analogue of the T antigen requires
regioselective β-galactosylation of HO-3 (Scheme 1). In
order to facilitate this HO-4 and HO-6 in 2 were first protected
by treatment with α,α-dimethoxytoluene to afford 3 (98%).
Formation of the β-galactosidic bond in derivatives of the
T antigen is usually performed by employing derivatives of
N-acetylgalactosamine as glycosyl acceptors, in which the
acetamido group is masked as an azido group.13 As compared
with the acetamido group, the azido group reduces the steric
hindrance for glycosylation at the adjacent HO-3 and also
increases the nucleophilicity of this hydroxy group by avoid-
ing an unfavourable hydrogen-bonding pattern.33,34 After
investigation of different glycosyl donors and promoters for
Scheme 2 Reagents and conditions (and yields): i) Cs2CO3, DMF,
glycoside synthesis it was found that glycosylation of 3 could
be carried out directly using per-acetylated galactosyl brom-
ide 4 to give the β-linked disaccharide 5 in 50% yield. Cleav-
age of the 4,6-benzylidene group in 5 was then achieved by
using an ice-cold solution of aq. trifluoroacetic acid (TFA) to
give 6 (70%). Finally, the acetyl groups in 6 were removed by
treatment with methanolic sodium methoxide to give fully
unprotected T antigen analog 7 (84%), after purification by
reversed-phase HPLC.
Peptide 8 is an analog of an immunodominant, class I MHC
restricted epitope from vesicular stomatitis virus nucleocapsid
protein,35 in which glutamine at position 6 has been replaced by
homocysteine (Schemes 2 and 3). Compound 8 was prepared by
Fmoc solid-phase synthesis using conditions described previ-
ously,36 and was then purified by reversed-phase HPLC. Alkyl-
ation of the sulfhydryl group in 8 was accomplished by reaction
with the unprotected 2-bromoethyl glycosides 2 and 7 using
caesium carbonate as base.37 The reaction was carried out in
dry N,N-dimethylformamide (DMF) under an inert atmos-
phere of nitrogen in order to avoid oxidative dimerization of 8.
Alkylation of 8 was monitored by analytical reversed-phase
HPLC and was generally found to be complete within an
hour. The reaction was then quenched by addition of aq. TFA.
Freeze-drying and purification by preparative reversed-phase
HPLC gave neoglycopeptides 9 and 11 (74 and 58% yield,
rt (74%); ii) CMP-Neu5Ac, α-2,6 sialyltransferase (ST6-GalNAc-I),
Bis-Tris buffer (20 mM; pH 6.0), 37 ЊC (≈40%).
respectively), which carry the carbohydrate moieties of the Tn
and T antigens. The structures of glycopeptides 9 and 11 were
verified by amino acid analysis, mass spectrometry and 1H
NMR spectroscopy (Tables 1 and 2).
Recombinant mouse N-acetylgalactosamine α2-6 sialyl-
transferase38 (ST6-GalNAc-I) was expressed in insect cells and
purified by ion-exchange chromatography.39 ST6-GalNAc-I
was then used to transform 9 into 10, which carries the carbo-
hydrate moiety of the sialyl-Tn antigen (Scheme 2). This was
achieved by incubating the enzyme with 9 and CMP-Neu5Ac
(CMP = cytosine monophosphate) in Bis-Tris buffer (pH 6.0) at
37 ЊC. After 6 h, analysis40 by nano-scale reversed-phase HPLC
in combination with MALDI-TOF mass spectrometry indi-
cated 90–95% conversion of 9 into 10. When the sialylation was
performed on a semipreparative scale (0.5 mg of 9) glyco-
peptide 10 was obtained in ≈40% yield after purification by
reversed-phase HPLC and freeze-drying. This result reveals
that ST6-GalNAc-I can act on substrates in which the N-acetyl-
galactosamine residue being sialylated is linked to a different
entity than serine or threonine.
Human core 1-specific α2-3 sialyltransferase41 (ST3-Gal-I)
was obtained by expression in Sf9 cells followed by purification
J. Chem. Soc., Perkin Trans. 1, 2001, 880–885
881

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Table 1 1H NMR chemical shifts for glycopeptide 9a
Residue
NH
Hα
Hβ
Hγ
Others
Arg
Gly
Tyr
Val
Tyr
Hcyc
Gly
Leu
3.99
4.00, 3.90
4.46
3.91
4.37
1.87b
1.61b
3.15 (H-δ, -δЈ), 7.20, 6.90 and 6.45 (NH)
7.01 and 6.75 (ArH)
8.78
8.42
7.96
8.48
8.37
7.00
7.90
2.92, 2.84
1.82
3.02, 2.86
1.80, 2.02
0.81, 0.76
2.52, 2.38
1.55
7.15 and 6.75 (ArH)
4.36
2.70b (SCH2CH2O), 3.78 and 3.56 (SCH2CH2O)
3.76b
4.16
1.58b
0.87 and 0.83 (γ-, γЈ-Me)
a Spectra were recorded at 500 MHz for a solution in H2O–D2O (9 : 1; pH 5.8) at 278 K using water (δH 4.98) as internal standard. b Degeneracy has
been assumed. c Chemical shifts (δ) for the GalNAc moiety in 9: 8.14 (NH), 4.85 (H-1), 4.09 (H-2), 3.84 (H-3), 1.96 (NHCOCH3).
Table 2 1H NMR chemical shifts for glycopeptide 11a
Residue
NH
Hα
Hβ
Hγ
Others
Arg
Gly
Tyr
Val
Tyr
Hcyc
Gly
Leu
3.93
3.97, 3.87
4.46
3.92
4.37
1.81b
1.56b
3.11b (H-δ, -δЈ), 7.20 (CH2NH)
8.74
8.38
7.93
8.45
8.34
7.08
7.86
2.90, 2.81
1.79
2.99, 2.84
1.99, 1.78
6.99 and 6.77 (ArH)
0.76b
7.12 and 6.74 (ArH)
4.38
2.49, 2.34
1.53
2.73b (SCH2CH2O), 3.79 and 3.59 (SCH2CH2O)
3.76b
4.16
1.53b
0.83b (γ-, γЈ-Me)
a Spectra were recorded at 500 MHz for a solution in H2O–D2O (9 : 1; pH 5.8) at 278 K using water (δH 4.98) as internal standard. b Degeneracy has
been assumed. c Chemical shifts (δ) for the Galβ(1→3)GalNAc moiety in 11: 8.19 (NH), 4.81 (H-1), 4.37 (H-1Ј), 4.27 (H-2), 3.97 (H-3), 3.85 (H-4Ј),
3.67 (H-4), 3.58 (H-5), 3.56 (H-3Ј), 3.47 (H-2Ј), 1.96 (NHCOCH3).
by ion-exchange and gel-filtration chromatography.23 Purified
ST3-Gal-I was used for sialylation of neoglycopeptide 11 to
give 12, which carries the 2,3-sialyl-T antigen (Scheme 3). The
performing repeated sialylations on a semipreparative scale
(7 × 0.5 mg of 11) glycopeptide 12 could be obtained in ≈70%
yield after purification by HPLC and freeze-drying. Assign-
ment of the 1H NMR spectrum of 12 (Table 3) revealed NOEs
between H-3 of the galactose moiety and both H-3ax and H-3eq
of the sialic acid residue, thereby confirming that sialylation
had occurred on HO-3 of the galactose moiety as expected.
A side-product (≈20%) with a mass 16 daltons higher than that
of 12 was also obtained in the sialylation. This was believed
to originate from oxidation of the sulfur atom in the linker
between the carbohydrate and peptide moieties of 12, a sugges-
1
tion that was further supported by H NMR chemical-shift
differences for the resonances in the linker of the side-product
as compared with glycopeptide 12.
Conclusions
The Tn, T, sialyl-Tn and 2,3-sialyl-T antigens are important
tumor-associated antigens in epithelial cancers affecting the
breast, ovary, stomach, and colon.4–8 Herein we have described
the preparation of 2-bromoethyl glycosides 2 and 7, which
correspond to the Tn and T antigens, in one and five chemical
steps, respectively. The 2-bromoethyl glycosides were then con-
verted into neoglycopeptides 9 and 11 by chemical methods.
Finally, enzymatic sialylation of 9 and 11 gave glycopeptides 10
and 12, which carry carbohydrate moieties corresponding to the
sialyl-Tn and 2,3-sialyl-T antigens. Chemical incorporation of
sialic acid in glycoconjugates is generally regarded as a demand-
ing task.14,15 This is supported by the fact that only a few, multi-
step syntheses of sialyl-Tn16–19 and 2,3-sialyl-T19–21 antigen
building blocks using organochemical methods have been
reported in the literature. Moreover, only in one case was such a
2,3-sialyl-T building block incorporated in a glycopeptide.20 As
demonstrated in the present paper, chemical synthesis of glyco-
peptides that carry the simpler Tn and T antigens, followed by
enzymatic sialylation to give the more complex sialyl-Tn and
2,3-sialyl-T antigens, constitutes a more efficient route to these
glycopeptides. It is interesting to note that the sialyltransferases
employed in this study were able to sialylate the Tn and T anti-
gens even though they were linked to the peptide backbone
Scheme 3 Reagents and conditions (and yields): i) Cs2CO3, DMF,
rt (58%); ii) CMP-Neu5Ac, α-2,3 sialyltransferase (ST3-Gal-I), calf
intestinal phosphatase, Tris-HCl buffer (25 mM; pH 6.5), 37 ЊC (68%).
sialylation was performed at 37 ЊC in Tris-HCl buffer (pH 6.5)
containing CMP-Neu5Ac as sialic acid donor. Calf intestinal
phosphatase was added in order to prevent product inhibition
of the sialyltransferase by CMP. After 3 h, nano-scale reversed-
phase HPLC in combination with MALDI-TOF mass
spectrometry40 indicated ≈95% conversion of 11 into 12. By
882
J. Chem. Soc., Perkin Trans. 1, 2001, 880–885

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Table 3 1H NMR chemical shifts for glycopeptide 12a
Residue
NH
Hα
Hβ
Hγ
Others
Arg
Gly
Tyr
Val
Tyr
Hcyc
Gly
Leu
3.99
4.02, 3.90
4.48
3.94
4.40
1.88b
1.62b
3.16b (H-δ, -δЈ), 7.19 (CH2NH)
7.03 and 6.74 (ArH)
8.78
8.43
7.97
8.49
8.37
7.01
7.90
2.94, 2.85
1.83
3.03, 2.87
2.04, 1.81
0.82, 0.78
2.52, 2.36
1.57
7.16 and 6.78 (ArH)
4.42
2.72b (SCH2CH2O), 3.80 and 3.60 (SCH2CH2O)
3.80b
4.19
1.57b
0.88 and 0.84 (γ-, γЈ-Me)
a Spectra were recorded at 500 MHz for a solution in H2O–D2O (9 : 1; pH 5.4) at 278 K using water (δH 4.98) as internal standard. b Degeneracy has
been assumed. c Chemical shifts (δ) for the Neu5Acα(1→3)Galβ(1→3)GalNAc moiety in 12. Neu5Ac: 8.15 (NH), 3.82 (H-5), 3.64 (H-4), 3.57 (H-6),
2.71 (H-3eq), 2.00 (NHCOCH3), 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).
via a spacer, instead of being attached to serine or threonine.
Chemoenzymatic synthesis thus appears to be able to provide
access to both natural glycopeptides, i.e. those having the
carbohydrate moieties linked to serine or threonine, and to
neoglycopeptides which contain the tumor-associated sialyl-Tn
and 2,3-sialyl-T antigens.
in a centrifugation cartridge (Sigma) before use. Human core
1-specific α2–3 sialyltransferase41 (ST3-Gal-I) was expressed
and partially purified as described.23 Final purification of
ST3-Gal-I was performed on MiniS (PC3.2/3) using the
Smart system (Pharmacia).
2-Bromoethyl 2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 2
A mixture of N-acetyl--galactosamine 1 (Sigma, 1.50 g,
6.75 mmol) and 2-bromoethanol (20 g, 160 mmol) containing
2% of HCl (gas) was stirred at 55–60 ЊC for 5 h. The mixture
was allowed to attain room temperature and the solvents were
evaporated off. The residue was co-concentrated several times
from toluene. Flash column chromatography of the residue
(CH2Cl2–MeOH 65 : 20) gave 2 (1.14 g, 52%) as a white
amorphous solid, [α]D20 ϩ141.8 (c 0.53, methanol); 1H NMR (400
MHz; CD3OD) δ 4.91 (1H, d, J 3.8 Hz, H-1), 4.17 (1H, dd, J 11,
3.7 Hz, H-2), 3.88 (1H, m, OCH2CH2Br), 3.80 (1H, m, H-5),
3.81–3.77 (1H, m, H-4), 3.72–3.65 (1H, m, H-3), 3.65–3.56 (1H,
m, OCH2CH2Br), 3.65–3.56 (2H, m, H2-6), 3.48 (2H, m,
BrCH2), 1.79 (3H, s, CH3CO); 13C NMR (100 MHz; CD3OD)
δC 172.5, 98.0, 71.5, 69.0, 68.2, 68.0, 61.2, 50.0, 30.2,
21.2; HRMS (FAB) Calc. for C10H18BrNO6ؒNa: 350.0215
(M ϩ Naϩ). Found: m/z, 350.0211.
Experimental
General methods and materials
All reactions were carried out under a nitrogen atmosphere and
anhydrous conditions using dry solvents unless otherwise
stated. CH2Cl2 was dried by distillation from calcium hydride
whereas THF and toluene were distilled from sodium benzo-
phenone ketyl. Pyridine was dried over 4 Å molecular
sieves. DMF was distilled and then dried over flame-dried 3 Å
molecular sieves. TLC was performed on silica gel 60 F254
(Merck) with detection by UV light, or by charring with aq.
sulfuric acid (10%). Flash column chromatography was per-
formed on silica gel (Matrex, 60 Å; 35–70 µm, Grace Amicon)
with distilled solvents. Organic solutions were dried over
anhydrous Na2SO4 before being concentrated.
1
The H and 13C NMR spectra for compounds 2, 3, and 5–7
were recorded at 298 K with a Bruker DRX-400 spectrometer at
400 MHz and 100 MHz, respectively. NMR samples of these
compounds were dissolved in CDCl3 [residual CHCl3 (δH 7.26)
or CDCl3 (δC 77.0) as internal standard] or CD3OD [residual
CD2HOD (δH 3.35), CD3OD (δC 49.0) as internal standard]. 1H
NMR spectra of glycopeptides 9, 11 and 12 were recorded with
a Bruker ARX-500 spectrometer for solutions in a 9 : 1 mixture
of water and D2O [H2O (δH 4.98) as internal standard] at 278 K.
Proton resonances for 9, 11 and 12 were assigned from COSY,
NOESY and TOCSY experiments. Optical rotations were
recorded on a Perkin-Elmer 343 polarimeter and [α]D-values are
given in 10Ϫ1 deg cm2 gϪ1. 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 water contain-
ing 0.1% TFA (flow-rate of 1.5 mL minϪ1; detection at 214
nm). Preparative reversed-phase HPLC was performed on a
Kromasil C-8 column (250 × 20 mm; 5 µm; 100 Å) using the
same solvent system (flow-rate of 11 mL minϪ1; detection at
214 nm).
2-Bromoethyl 2-acetamido-4,6-O-benzylidene-2-deoxy-ꢀ-D-
galactopyranoside 3
A solution of 2 (302 mg, 0.92 mmol), α,α-dimethoxytoluene
(281 mg, 1.84 mmol) and a catalytic amount of toluene-p-
sulfonic acid (p-TsOH) (17 mg) in acetonitrile (17 mL) was
stirred at room temperature for 24 h. The reaction was
quenched by addition of triethylamine (0.1 mL), the solvents
were evaporated off, and the residue was dried under vacuum.
Flash column chromatography of the residue (EtOAc–MeOH
96 : 4) afforded 3 (357 mg, 98%) as an amorphous white solid;
[α]D20 ϩ114.3 (c 0.64, CHCl3); 1H NMR (CDCl3) δ 7.53–7.35 (5H,
m, ArH), 5.93 (1H, d, J 8.9 Hz, AcNH), 5.58 (1H, s, PhCH),
5.05 (1H, d, J 3.6 Hz, H-1), 4.48 (1H, ddd, J 10.9, 9.0, 3.6 Hz,
H-2), 4.27 (1H, dd, J 12.6, 1.6 Hz, H-4), 4.24 (1H, m, H-3),
4.10–4.03 (2H, m, H-5 and OCH2CH2Br), 3.85–3.78 (3H, m,
OCH2CH2Br and H2-6), 3.56 (1H, t, J 5.1 Hz, CH2Br), 3.55
(1H, t, J = 5.1 Hz, CH2Br), 2.1 (3H, s, CH3CO); 13C NMR (400
MHz; CDCl3) δC 171.2, 137.2, 129.0, 128.0, 126.1, 101.2, 98.7,
75.5, 69.4, 69.2, 68.0, 63.4, 50.2, 31.2, 23.5; HRMS (FAB) Calc.
for C17H23BrNO6: 416.0709 (M ϩ Hϩ). Found: m/z, 416.0708.
2,3,4,6-Tetra-O-acetyl-β--galactopyranosyl bromide 4 was
prepared from per-acetylated galactose by treatment with
HBr in HOAc–Ac2O at 0 ЊC. -Arginylglycyl--tyrosyl--valyl-
-tyrosyl--homocysteinylglycyl--leucine37
8 was prepared
2-Bromoethyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosyl)-ꢀ-D-galacto-
pyranoside 5
manually by Fmoc solid-phase synthesis in a mechanically
agitated reactor using conditions described previously.36
Recombinant mouse N-acetylgalactosamine α2–6 sialyltrans-
ferase38 (ST6-GalNAc-I) was expressed in insect cells. The
enzyme was purified by sequential ion-exchange chromato-
graphy on Amberlite (IRA95, Sigma) and SP-sepharose (Sigma)
essentially as described.39 Purified samples were concentrated
A solution of silver triflate (465 mg, 1.81 mmol) in toluene (10
mL) was added dropwise to a suspension of 3 (500 mg, 1.2
mmol), 2,3,4,6-tetra-O-acetyl-α--galactopyranosyl bromide 4
(738 mg, 1.80 mmol) and crushed molecular sieves (flame-dried,
J. Chem. Soc., Perkin Trans. 1, 2001, 880–885
883

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4 Å; 1.0 g) in CH2Cl2 (10 mL) at Ϫ25 ЊC. After 1 h, the reaction
was quenched by addition of pyridine (1 mL) while keeping
the temperature at Ϫ25 ЊC. After 5 min the mixture was allowed
to attain room temperature, and the solids were removed by
filtration (Hyflow, Supercel) and washed with CH2Cl2 (30 mL).
The combined filtrates were washed with a mixture of aq.
Na2S2O3 (0.5 M) and saturated aq. NaHCO3 (1 : 1; 20 mL),
dried and concentrated. Repeated flash column chromato-
graphy of the residue (EtOAc–MeOH 100 : 0.5) afforded 5 as a
white amorphous solid (447 mg, 50%), [α]D20 ϩ94.4 (c 0.45,
CHCl3); 1H NMR (400 MHz; CDCl3) δ 7.52 and 7.50 (2H, 2d,
ArH), 7.37–7.30 (3H, m, ArH), 5.76 (1H, d, J 8.8 Hz, NH), 5.55
(1H, s, PhCH), 5.36 (1H, dd, J 3.4, 1.1 Hz, H-4Ј), 5.17 (1H, dd,
J 10.4, 7.9 Hz, H-2Ј), 5.15 (1H, d, J 3.6 Hz, H-1), 4.97 (1H, dd,
J 10.4, 3.5 Hz, H-3Ј), 4.75 (1H, d, J 7.9 Hz, H-1Ј), 4.64 (1H, m,
H-2), 4.29 (1H, m, H-4), 4.24–4.06 (5H, m, H2-6, -6Ј and H-5Ј),
4.03 (1H, m, OCH2CH2Br), 3.99 (1H, dd, J 5.9, 0.9 Hz, H-3),
3.80 (1H, m, OCH2CH2Br), 3.75 (1H, br s, H-5), 3.57 (2H, m,
CH2Br), 2.13, 2.05, 2.00, 1.97 and 1.94 (15H, 5s, CH3CO); 13C
NMR (CDCl3) δC 170.1, 166.5, 166.5, 165.2, 137.1, 133.5,
129.9, 129.8, 129.7, 129.6, 128.6, 128.5, 128.3, 128.3, 128.2,
127.8, 125.6, 100.2, 98.5, 93.9, 72.7, 71.7, 69.2, 69.0, 68.7, 68.3,
68.0, 66.8, 63.2, 62.4, 47.7, 31.8, 30.7, 29.0, 23.3, 22.6; HRMS
(FAB) Calc. for C31H41BrNO15: 746.1660 (M ϩ Hϩ). Found:
m/z, 746.1677.
2-bromoethyl glycoside 2 (15 mg, 46 µmol) and caesium
carbonate (50 mg, 154 µmol) in dry DMF (1.3 mL) under
nitrogen. The solution was stirred at room temperature until
analytical reversed-phase HPLC (gradient 0→100% CH3CN in
water, both containing 0.1% TFA, during 60 min; tR 17.2 min)
indicated that 8 was consumed. The reaction was quenched by
addition of 0.1% aq. TFA (15 mL) and the mixture was freeze
dried. Purification of the residue by preparative reversed-phase
HPLC (gradient 0→100% CH3CN in water, both containing
0.1% TFA, during 60 min) gave 9 (14 mg, 77% peptide content,
1
74% yield) as an amorphous white solid. H NMR data, see
Table 1; HRMS (FAB) Calc. for C53H83N12O17S: 1191.5720
(M ϩ Hϩ). Found: m/z, 1191.5728; amino acid analysis: Gly
1.99 (2), Val 1.04 (1), Leu 1.01 (1), Tyr 1.98 (2), Arg 0.98 (1).
Sialyl-Tn neoglycopeptide 10
Neoglycopeptide 9 (0.50 mg, 0.42 µmol) was added to purified
ST6-GalNAc-I (≈2 mU) in 20 mM Bis-Tris buffer (pH 6.0; 1.0
mL) containing CMP-Neu5Ac (2 mM), EDTA (20 mM) and
dithiothreitol (1 mM). The solution was then incubated at 37 ЊC
for 6 h, after which analysis40 by nano-scale reversed-phase
HPLC in combination with MALDI-TOF mass spectrometry
indicated 90–95% conversion of 9. Purification by HPLC on a
Zorbax 300SB-C3 column (9.4 × 250 mm) using a gradient of
0→90% CH3CN in water, both containing 0.1% TFA, gave 10
(250 µg, ≈40%) as an amorphous white solid after freeze-
drying; MS (MALDI-TOF, linear mode, 2,5-dihydroxybenzoic
acid) Calc. for C64H99N13O25S: 1482 (Mϩ). Found: M, 1483.
2-Bromoethyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-
ꢁ-D-galactopyranosyl)-ꢀ-D-galactopyranoside 6
An ice-cold solution of TFA in water (10 mL; 80%) was added
during 5 min to a solution of 5 (204 mg, 0.27 mmol) in CH2Cl2
(5 mL) at 0 ЊC. After stirring of the mixture for 1 h, toluene
(25 mL) was added and the mixture was concentrated. Flash
column chromatography of the residue (EtOAc–MeOH 100 : 1)
gave 6 (143 mg, 70%) as an amorphous white solid, [α]D20 ϩ54.0
T neoglycopeptide 11
Peptide 8 (12 mg, 13 µmol) was added to a mixture of a
2-bromoethyl glycoside 7 (10 mg, 20 µmol) and caesium
carbonate (35 mg, 107 µmol) in dry DMF (1.0 mL) under
nitrogen. The mixture was stirred at room temperature until
analytical reversed-phase HPLC (gradient 0→100% CH3CN in
water, both containing 0.1% TFA, during 60 min, tR 16.9 min)
indicated that 8 was consumed. The reaction was quenched by
addition of 0.1% aq. TFA (15 mL) and the mixture was freeze
dried. Purification of the residue by preparative reversed-phase
HPLC (gradient 0→100% CH3CN in water, both containing
0.1% TFA, during 60 min) gave 11 (10 mg, 81% peptide content,
1
(c 0.62, CHCl3); H NMR (CDCl3) δ 6.04 (1H, d, J 8.9 Hz,
NH), 5.35 (1H, m, H-4Ј), 5.15 (1H, dd, J 10.2, 7.9 Hz, H-2Ј),
4.99 (1H, dd, J 10.5, 3.2 Hz, H-3Ј), 4.90 (1H, d, J 2.7 Hz, H-1),
4.66 (1H, d, J 7.7 Hz, H-1Ј), 4.54 (1H, m, H-2), 4.24–3.99 (4H,
m, H-6, -6Ј, OCH2CH2Br and H-5), 3.99–3.87 (2H, m, H-6 and
-6Ј), 3.87–3.72 (2H, m, OCH2CH2Br and H-3), 3.49 (2H, m,
CH2Br), 2.13, 2.05, 2.03, 1.99 and 1.95 (15H, 5s, 5 × CH3CO);
13C NMR (CDCl3) δC 170.5, 170.2, 170.0, 169.8, 101.1, 97.9,
77.9, 70.8, 70.5, 69.7, 69.5, 68.7, 67.8, 66.9, 62.5, 61.3, 47.9,
31.6, 20.6, 20.5, 20.5, 20.4, 20.3; HRMS (FAB) Calc. for C24H36-
BrNO15ؒNa: 680.1166 (M ϩ Naϩ). Found: m/z, 680.1161.
1
58% yield) as an amorphous white solid. H NMR data, see
Table 2; HRMS (FAB) Calc. for C59H93N12O22S: 1353.6248
(M ϩ Hϩ). Found: m/z, 1353.6215; amino acid analysis: Gly
1.99 (2), Val 0.99 (1), Leu 1.03 (1), Tyr 2.00 (2), Arg 1.00 (1).
2-Bromoethyl 2-acetamido-2-deoxy-3-O-ꢁ-D-galactopyranosyl-
ꢀ-D-galactopyranoside 7
2,3-Sialyl-T neoglycopeptide 12
A solution of 6 (140 mg, 0.286 mmol) in methanol (6 mL) was
treated with methanolic NaOMe (0.2 M; 0.28 mL). After 1 h
the solution was neutralized by addition of acetic acid in
methanol (10%) and was then concentrated. The residue was
purified using reversed-phase HPLC (gradient 0→100%
CH3CN in water, both containing 0.5% TFA, during 60 min; tR
7.65 min) followed by freeze drying to give 7 (86 mg, 84%) as a
Purified ST3-Gal-I (20 µL, ≈2 mU) was added to neoglyco-
peptide 11 (0.50 mg, 0.37 µmol) 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 ЊC for 3 h, after which analysis40
by nano-scale reversed-phase HPLC in combination with
MALDI-TOF mass spectrometry indicated ≈95% conversion
of 11. This procedure was repeated so that 3.5 mg (2.6 µmol) of
11 was sialylated. Purification by HPLC on a Zorbax 300SB-C3
column (9.4 × 250 mm) using a gradient of 0→90% CH3CN in
water, both containing 0.1% TFA, gave 12 (2.9 mg, 68%) as an
amorphous white solid after freeze-drying. 1H NMR data,
see Table 3; MS (ES) Calc. for C70H109N13O30S: 1643.7 (Mϩ).
Found: m/z, 1643.6.
1
white amorphous solid, [α]D20 ϩ92.8 (c 0.53, MeOH); H NMR
(CD3OD) δ 4.92 (1H, d, J 3.8 Hz, H-1), 4.43 (1H, dd, J 7.3, 3.7
Hz, H-2), 4.40 (1H, d, J 7.5 Hz, H-1Ј), 4.18 (1H, m, H-4), 4.00
(1H, m, OCH2CH2Br), 3.98–3.88 (2H, m, H-5 or -5Ј and -3),
3.85–3.75 (2H, m, H-4Ј and OCH2CH2Br), 3.75–3.68 (4H, m,
H2-6 and -6Ј), 3.62–3.55 (2H, m, CH2Br), 3.55–3.49 (2H, m,
H-2Ј and -5Ј or -5), 3.45 (1H, dd, J 9.7, 3.3 Hz, H-3Ј), 2.00 (3H,
s, CH3CO); 13C NMR (CDCl3) δC 174.3, 106.5, 99.4, 79.0, 76.9,
74.9, 72.7, 72.6, 70.4, 69.6, 63.0, 62.8, 50.4, 31.9, 23.0; HRMS
(FAB) Calc. for C16H28BrNO11ؒNa: 512.0743 (M ϩ Naϩ).
Found: m/z, 512.0749.
Acknowledgements
This article is dedicated (by J. K.) to the memory of Göran
Magnusson, an outstanding scientist, supervisor and a wise
mentor. This research was supported by a postdoctoral research
fellowship to S. K. G. from the program ‘Glycoconjugates
Tn neoglycopeptide 9
Peptide 8 (15 mg, 13 µmol) was added to a mixture of
884
J. Chem. Soc., Perkin Trans. 1, 2001, 880–885

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21 S. Komba, M. Meldal, O. Werdelin, T. Jensen and K. Bock, J. Chem.
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.
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