Efficient chemoenzymatic synthesis of O-linked sialyl oligosaccharides

Article abstract of DOI:10.1021/ja017881+

The tumor associated Tn (GalNAcα(1-1)-Thr/Ser)- and T (Galβ(1-3)-GalNAcα(1-1)Thr/Ser)-antigens and their sialylated derivatives are present on the surface of many cancer cells. Preparative synthesis of these sialylated T- and Tn-structures has been achieved mainly from a chemical synthetic approach due to the lack of the required glycosyltransferases. We demonstrate a flexible and efficient chemoenzymatic approach for using recombinant sialyltransferases including a chicken GalNAcα2,6-sialyltransferase (chST6GalNAc I) and a porcine Galβ(1-3)GalNAcα-2,3-sialyltransferase (pST3Gal I). Using these enzymes, the common O-linked sialosides Neu5Acα(2-6)GalNAcα(1-1)Thr, Galβ(1-3)[Neu5Acα(2-6)] GalNAcα(1-1)Thr, Neu5Acα(2-3)Galβ(1-3)GalNAcα(1-1)Thr, and Neu5Acα(2-3)Galβ(1-3)[Neu5Acα(2-6)]GalNAcα(1-1)Thr were readily prepared at preparative scale. The chST6GalNAc I was found to require at least one amino acid (Thr/Ser) for optimal activity, and is thus an ideal catalyst for synthesis of synthetic glycopeptides and glycoconjugates with O-linked glycans.

Full text of DOI:10.1021/ja017881+

Published on Web 04/30/2002
Efficient Chemoenzymatic Synthesis of O-Linked Sialyl
Oligosaccharides
Ola Blixt, Kirk Allin, Laura Pereira, Arun Datta, and James C. Paulson*
Contribution from the The Scripps Research Institute, Department of Molecular Biology,
MEM-L71, 10550 North Torrey Pines Road, La Jolla, California 92037
Received December 26, 2001
Abstract: The tumor associated Tn (GalNAcR(1-1)-Thr/Ser)- and T (Galâ(1-3)-GalNAcR(1-1)Thr/Ser)-
antigens and their sialylated derivatives are present on the surface of many cancer cells. Preparative
synthesis of these sialylated T- and Tn-structures has been achieved mainly from a chemical synthetic
approach due to the lack of the required glycosyltransferases. We demonstrate a flexible and efficient
chemoenzymatic approach for using recombinant sialyltransferases including a chicken GalNAcR2,6-
sialyltransferase (chST6GalNAc I) and a porcine Galâ(1-3)GalNAcR-2,3-sialyltransferase (pST3Gal I). Using
these enzymes, the common O-linked sialosides Neu5AcR(2-6)GalNAcR(1-1)Thr, Galâ(1-3)[Neu5AcR(2-
6
6
)]GalNAcR(1-1)Thr, Neu5AcR(2-3)Galâ(1-3)GalNAcR(1-1)Thr, and Neu5AcR(2-3)Galâ(1-3)[Neu5AcR(2-
)]GalNAcR(1-1)Thr were readily prepared at preparative scale. The chST6GalNAc I was found to require
at least one amino acid (Thr/Ser) for optimal activity, and is thus an ideal catalyst for synthesis of synthetic
glycopeptides and glycoconjugates with O-linked glycans.
Introduction
also targets for immunotherapy. Indeed, clinical trials have been
reported with vaccine-induced antibody responses against the
Carbohydrate groups of glycoproteins exhibit tremendous
structural diversity and are major components of the outer
surface of animal cells. The two major classes are N-linked to
asparagine and O-linked to threonine and serine. While the core
regions of these carbohydrate groups have elements common
to many cells, the terminal sequences vary in a tissue and cell-
type specific manner in both developing and mature organisms.
The most common O-linked carbohydrates are based on core
structures represented by the Tn (GalNAcR(1-1)Thr/Ser)- and
T (GalR(1-3)GalNAcR(1-1)Thr/Ser)-antigens (Scheme 1). Si-
sialyl-Tn-antigen Neu5AcR(2-6)GalNAcR(1-1)Thr/Ser.1
1-13
In addition to their roles as antigens, the sialylated T- and
Tn-antigens have been implicated in a variety of other biological
functions. The sequence Neu5AcR(2-3)Galâ(1-3)[Neu5AcR(2-
6
)]GalNAc has been documented to be a cell surface receptor
14-16
1-3
mediating attachment of several bacteria and virus pathogens.
Members of the Siglec family of cell adhesion molecules have
been demonstrated to use them as carbohydrate ligands and to
4
distinguish between glycans containing N-acetylneuraminic acid
7-19
(
Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).1
alylated versions of these O-antigens are expressed at low levels
by many normal tissues, but become highly expressed in many
types of human malignancies including colon, breast, pancreas,
Neu5AcR2-3 sialylated T-antigen has also recently been shown
+
20
to be critical for normal development of CD8 T cells.
5,6
Despite the importance of these sialylated O-linked glycans,
methods for their efficient preparation have been limiting.
ovary, stomach, lung adenocarcinomas, and myelogenous
7
leukemias. The abnormal expression distinguishes them as
tumor associated antigens, and the specific biosynthetic pathway,
based on the in vitro activities of recombinant glycosyltrans-
ferases, has been previously discussed.8-10 These antigens are
(
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*
To whom correspondence should be addressed. Phone: 858-784-9633.
M. Cancer Immunol. Immunother. 1996, 42, 303-9.
Fax: 858-784-9690. E-mail: jpaulson@scripps.edu.
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Smolenski, M.; Samuel, J.; Nabholtz, J. M.; Longenecker, B. M. Cancer
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10.1021/ja017881+ CCC: $22.00 © 2002 American Chemical Society
J. AM. CHEM. SOC. 2002, 124, 5739-5746
9
5739

A R T I C L E S
Blixt et al.
Scheme 1. Biosynthesis and Structure of O-Linked Sialosides
Strategies for pure chemical synthesis of O-linked glycans must
address a host of issues, including appropriate blocking and
deprotection steps, and the formation of the glycosidic bond of
the 2-keto sugar N-acetylneuraminic acid, which is still con-
sidered one of the most difficult problems in synthetic carbo-
hydrate chemistry.21 Despite these obstacles, several groups have
for a chemoenzymatic synthesis strategy since the sialic acids
are the most difficult sugars to handle in a chemical synthesis,
and sialic acids are normally added as terminal sugars in the
last steps of a biosynthetic pathway.
Several groups have reported the successful synthesis of R2-3
sialylated T-antigen, Neu5AcR(2-3)Galâ(1-3)GalNAcR(1-1)Thr,
through a chemoenzymatic strategy employing sialyltransferase
developed elegant solutions to the synthesis of the sialylated
derivatives of the Tn- and T-antigens.2
2-29
Nontheless, the
39-41
42
ST3Gal I
George et al.
or by transglycosidation using a sialidase.
complexity of the synthetic strategies restricts the ultimate scale
and overall yields of the desired compounds, and consequently
limits the availability of these compounds to the biomedical
community.
43,44
recently reported the semipreparative chemo-
enzymatic synthesis of sialyl-Tn glycopeptide and a synthetic
analogue of sialyl-Tn Neu5AcR(2-6)GalNAcR(1-spacer-1)Thr/
Ser using a murine ST6GalNAc I.
Chemoenzymatic synthesis employing glycosyltransferases
offers advantages for efficient production of sialosides, owing
to the stereo- and regioselective catalysis and lack of blocking
In this report, we document the successful high yield
expression of a chicken ST6GalNAc I, and present a detailed
analysis of its substrate specificity, relevant for its use in the
synthesis of all four possible sialyl-T- and sialyl-Tn-antigens
in preparative scale.
30-33
or deprotection steps for the addition of sialic acid.
While
the scale of the reactions can be limited by the availability of
the appropriate glycosyltransferase, use of recombinant glyco-
3
1,34-38
syltransferases to a large extent obviates this limitation.
Results and Discussion
A typical strategy for chemoenzymatic synthesis of oligosac-
charides involves chemical synthesis of a simple glycoside that
serves as a substrate for final steps involving the enzymatic
addition of one or more sugar units. Sialosides are ideally suited
Synthesis Strategy. As shown in Scheme 1, four sialylated
O-linked glycans are derived from the Tn- and T-antigens. The
primary decision in the synthesis strategy is which structures
should be prepared chemically. Reliable procedures for the
chemical synthesis of Tn- and T-antigens have been reported.
Thus, it was determined to prepare these compounds chemically,
deprotect, and add the sialic acids enzymatically.
(
21) Okamoto, K.; Goto, T. Tetrahedron 1990, 46, 5835-57.
45-48
(
22) Gottfried, A. W.; Schmidt, R. R. Angew. Chem., Int. Ed. 2001, 40, 2654-
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For synthesis of the critical sialic acid linkages, two recom-
binant sialyltransferases are required (Scheme 1), one that
synthesizes the Neu5AcR(2-3)Galâ(1-3)GalNAcR(1-1)Thr/Ser
sequence, and another that synthesizes the Neu5AcR(2-6)-
GalNAcR(1-1)Thr/Ser sequence. The former sequence is ef-
ficiently produced by the sialyltransferase ST3Gal I, and its
2
06-14.
(
25) Sames, D.; Chen, X. T.; Danishefsky, S. J. Nature 1997, 389, 587-91.
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2.
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5740 J. AM. CHEM. SOC.
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VOL. 124, NO. 20, 2002

O-Linked Sialyl Oligosaccharides
A R T I C L E S
production in insect cells using a bacculovirus expression system
has been previously reported.39,49,50
There were several options for production of the Neu5AcR-
2-6)GalNAcR(1-1)Thr/Ser linkage. An R2-6-sialyltransferase
(
from Photobacterium damsela was demonstrated to be useful
in the large scale synthesis of the oligosaccharide portion of
sialyl-Tn, Neu5AcR(2-6)GalNAc. However, because it also
transfers sialic acid to terminal galactose, and does not transfer
to substituted GalNAc, it is not useful for the synthesis of R2-6
sialylated T-antigens Galâ(1-3)[Neu5AcR(2-6)]GalNAcR(1-1)-
Thr.3
0,51,52
Six members of the vertebrate GalNAc-R2-6-sialyl-
transferase family have been cloned, which, in principle, can
synthesize the Neu5AcR(2-6)GalNAc linkage.9
,53-63
Of these,
ST6GalNAc I has the broadest substrate specificity, transferring
sialic acid in R2-6 linkage to the GalNAc residue in the Tn-,
T-, and R2-3 sialylated T-antigen structures. To date, ST6GalNAc
Figure 1. Plasmid construct for transfection.
I has been cloned and expressed from human, mice, and
chicken.9
,54,63
The enzymatic feature of chST6GalNAc I from
(Pharmingen, San Diego, CA) which contained an ampicillin-
these three species differs significantly with respect to their use
as a synthetic catalyst. Indeed, relative to a reference acceptor
substrate, asialo-bovine submaxillary mucin (Galâ(1-3)GalNAc),
a GalNAcRThr-glycopeptide, was a good acceptor (160%)
resistant gene and a polyhedrin promoter (Figure 1). The plasmid
DNA was used for recombinant baculovirus preparation using
the BaculoGold transfection kit (Pharmingen, San Diego, CA).
The enzyme was expressed at a level of approximately 10
units/L media. The soluble recombinant enzyme from 1 L
culture supernatant was purified as described in the Experimental
Section by affinity chromatography on CDP-hexanolamine-
Sepharose. The typical yield was approximately 8 U of
chST6GalNAc I.
Acceptor Specificity of ST6GalNAc I. A general concern
about the use of mammalian glycosyltransferases for synthesis
of oligosaccharides is that their stringent acceptor specificity
may limit their utility as a synthetic tool. In the case of
chST6GalNAc I, the natural substrates are oligosaccharides
attached to glycoproteins, not the small molecule glycosides
targeted for synthesis. To evaluate the utility of chST6GalNAc
I for preparative synthesis of sialylated Tn- and T-antigens, and
to determine to what extent the oligosaccharide substrate could
be modified and still be recognized as an acceptor, a panel of
different synthetic precursors of R-N-acetylgalactosamine (R-
GalNAc) was prepared (Table 1).
9
for hST6GalNAc-I, but was a poor acceptor (<1%) for
mST6GalNAc I.55 Preliminary analysis with the chST6GalNAc
I showed that it also transferred well to GalNAcRThr substrates.
For this reason, chST6GalNAc I was chosen as the target
enzyme for synthesis of O-linked sialosides.
Preparation
of
Recombinant
Sialyltransferase
62
chST6GalNAc I. chST6GalNAc I plasmid DNA, pCEB-1800,
was used for production of recombinant protein in a Sf9
baculovirus system. To obtain expression of a secretable form
of chST6GalNAc I, the expression vector was constructed by
6
4
subcloning the dog proinsulin signal sequence (ins ss) from
the pGIR201Nhe3′ vector into the mammalian expression vector
pcDNA3 generating pcDNAins. Thus, as described in the
Experimental Section, the PCR-amplified chST6GalNAc I gene
was cloned into this expression vector, and the resulting fusion
construct was subcloned into the pVL1392 shuttle vector
(
(
(
(
(
(
(
49) Williams, M. A.; Kitagawa, H.; Datta, A. K.; Paulson, J. C.; Jamieson, J.
C. Glycoconjugate J. 1995, 12, 755-61.
2-Azidoethyl R-N-acetyl galactopyranoside 1 and its (2-N-
acetylethyl)-derivative 2 were prepared from N-acetylgalac-
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1
0.
6
5
51) Yamamoto, T.; Nakashizuka, M.; Kodama, H.; Kajihara, Y.; Terada, I. J.
Biochem. 1996, 120, 104-10.
tosmine and 2-azidoethanol using a Lewis acid as a catalyst
66
as reported previously. Compounds 3c-7c were prepared from
52) Kajihara, Y.; Yamamoto, T.; Nagae, H.; Nakashizuka, M.; Sakakibara, T.;
Terada, I. J. Org. Chem. 1996, 61, 8632-5.
the N-(9-fluorenylmethyloxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-
azido-2-deoxy-R-D-galactopyranosyl)-L-threonine phenacylester
(3a) and N-(9-fluorenylmethyloxycarbonyl)-O-[4,6-di-O-2-
azido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-
R-D-galactopyranosyl]-L-threonine phenacylester (7a) glyco-
53) Samyn-Petit, B.; Krzewinski-Recchi, M. A.; Steelant, W. F.; Delannoy,
P.; Harduin-Lepers, A. Biochim. Biophys. Acta 2000, 1474, 201-11.
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Itzkowitz, S. H.; Nishimura, S.; Tsuji, S. Biochem. Biophys. Res. Commun.
2
000, 272, 94-7.
47,48
peptide building blocks.
Complete deprotection with Zemplen
(
56) Okajima, T.; Chen, H. H.; Ito, H.; Kiso, M.; Tai, T.; Furukawa, K.; Urano,
T. J. Biol. Chem. 2000, 275, 6717-23.
deacetylation followed by treatment with acetic anhydride gave
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S.; Kurosawa, N.; Liu, H.; Pircher, H.; Tsuji, S. J. Biol. Chem. 1999, 274,
R
the methylester of N -acetyl-L-threonine-glycosides 3c and 7c,
1
1958-67.
respectively. N-Fluorenylmethyloxycarbonyl (Fmoc) protecting
group could be preserved by carefully controlled deacetylation
(
(
(
(
(
(
(
58) Kurosawa, N.; Inoue, M.; Yoshida, Y.; Tsuji, S. J. Biol. Chem. 1996, 271,
1
5109-16.
59) Okajima, T.; Fukumoto, S.; Ito, H.; Kiso, M.; Hirabayashi, Y.; Urano, T.;
Furukawa, K.; Furukawa, K. J. Biol. Chem. 1999, 274, 30557-62.
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J. Biol. Chem. 1996, 271, 7450-9.
+
(
pH < 9) followed by neutralization with Dowex (H ) to give
6
7
the amino acid glycoside 6 (72%). Compound 3a was
deacetylated to give 4 (78%). The remaining azido functional
62) Kurosawa, N.; Kojima, N.; Inoue, M.; Hamamoto, T.; Tsuji, S. J. Biol.
Chem. 1994, 269, 19048-53.
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J. AM. CHEM. SOC.
9
VOL. 124, NO. 20, 2002 5741

A R T I C L E S
Blixt et al.
Table 1. chST6GalNAc I Activity of R-D-Galacto Derivatives
nmol/mg min-
1
compound
R
1
R
2
R
3
R
4
R
5
cpm
D-GalNAc
H
H
H
H
H
H
H
NHAc
NHAc
NHAc
NHAc
N3
0
65
121
0.00
0.06
1
2
c
N3
H
H
H
NHAc
NHAc
NHAc
NHAc
NHFmoc
NHAc
H
0.11
3
4
5
6
7
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
CH3
CH3
CH3
CH3
CH3
9133
8.60
3043
2.90
NH2
1421
1.30
a
NHAc
NHAc
13 277
12 760
12.50
12.00
Galâ1,3
a
Product was isolated by using Sep-Pak (C18) cartridges as described.77
group was further reduced by hydrogenolysis to the aminogly-
coside 5 (82%).
Thus, enzymatic sialylation of 3c and 7c with chST6GalNAc
I enzyme (0.6-2 U) and CMP-Neu5Ac (1.5 equiv) was
efficiently carried out to give, after purification, the STn (8,
349 mg, 97%)- and sialyl(2,6)-T-epitopes (10, 74 mg, 93%),
respectively. The enzyme showed a strict specificity for the
GalNAc residue on the T-epitope. A 2.5-fold excess of donor,
increased amount of enzyme, together with longer reaction times
did not incorporate any additional sialic acid to the terminal
galactose residue on compound 10 (data not shown). Sialyl-
(2,3)-T-epitope (9, 78 mg, 95%) was prepared from 3c with
the porcine ST3Gal I enzyme. Compound 9 was further
elongated with chST6GalNAc I to generate the di-sialyl-T-
epitope (11, 77 mg, 73%). CMP-Neu5Gc donor could also be
effectively utilized by the chST6GalNAc I, and Neu5Gc-Tn-
epitope (12, 49 mg, 87%) was isolated. The enzymatic products
were subject to ion-exchange and size-exclusion chromatography
for purification. All enzymatic reactions could easily be scaled
up.
The specificity of the chST6GalNAc I was evaluated using
the purified recombinant enzyme and the prepared R-galacto-
pyranoside-epitopes in Table 1. The assays were performed with
14
14
CMP-[C ]Neu5Ac, and the relative transfer of [C ]Neu5Ac
to the corresponding N-acetylgalactoside acceptor was measured
as described in the Experimental Section. Neither free N-
acetylgalactosamine (GalNAc), nor the R-glycosides 1 and 2,
served as suitable acceptor substrates for the chST6GalNAc I
enzyme (Table 1), indicating that a R-glycosidic linkage alone
is not sufficient. Kurosawa et al.63 previously reported low
activity toward R-glycosides of GalNAc-benzyl and GalNAc-
(
1-1)SerNAc for chST6GalNAc I. However, as shown in Table
, GalNAc- or (Galâ(1-3)GalNAc- R-linked to the hydroxyl
group of the carboxymethylester derivative of L-ThrNAc (3c,
1
7c) gave a rate of transfer 80-200-fold higher than to compound
2
. For this reason, the carboxymethylester-ThrNAc was used
as the standard aglycon in O-linked oligosaccharide synthesis.
Moreover, replacement of the N-acetyl group on GalNAc with
N3 or NH2 reduced transfer, but not to the extent of precluding
preparative synthesis. Additionally, replacement of ThrNAc with
ThrFmoc enhanced activity adding an important synthetic
dimension for preparation of glycosylated amino acids for
Conclusion
Enzyme-catalyzed sialylation to produce sialylated oligosac-
charides has been established as a powerful tool in the field of
carbohydrate chemistry. Overexpression and purification of the
chST6GalNAc I has enabled a detailed acceptor specificity study
and evaluation of its potential for preparative synthesis of
sialylated T- and Tn-structures. The specificity study shows that
by stripping down the natural glycoprotein acceptor to a single
amino acid residue R-O-linked to the glycosidic acceptor, the
chST6GalNAc I enzyme could still effectively be used for
synthesis of O-linked sialoside epitopes with different func-
tionalities. The purified recombinant chST6GalNAc I together
with pST3Gal I are shown to be important tools for simple and
efficient preparation of O-linked sialoside epitopes.
68
peptide synthesis.
Synthesis of Sialylated Tn- and T-antigens. With the
substrate specificity of the target glycosides in hand,
chST6GalNAc I was used in preparative chemoenzymatic
synthesis. Five different O-linked sialoside epitopes containing
the amino acid aglycon, threonine, were synthesized in high
yield and high purity (Figure 2). The highly expensive nucleotide
donor, CMP-Neu5Ac, required for the enzymatic transfer of
N-acetyl neuraminic acid (Neu5Ac), was prepared crude from
CTP and Neu5Ac and was catalyzed by the bacterial fusion
protein ST3-/CMP-Neu5Ac-synthetase.31 The crude nucleotide
sugar was used in the sialylation reactions after removal of
Experimental Section
General Method. Solvent concentration was performed under
reduced pressure at <40 °C bath temperature. NMR spectra were
recorded at 30 °C using a Varian Unity Inova 500 spectrometer. The
following reference signals were used: acetone 2.225 ppm ( H in D O),
2
2
acetone 29.7 ppm ( C in D O). MALDI-FTMS spectra were recorded
3
4
1
fusion protein by 10 000 MW cutoff membrane filter. CMP-
13
Neu5Gc was synthesized from Neu5Gc69 and CTP as described
with an IonSpec Ultima FTMS (IonSpec Corp., Irvine, CA) using
dihydroxybenzoic acid as matrix. Thin-layer chromatography was
performed on Kieselgel 60 F254 Fertigplatten (Merck, Darmstadt,
Germany). After development with appropriate eluants, spots were
visualized by UV light and/or by dipping in 5% sulfuric acid in ethanol,
followed by charring. Water was produced from a NanoPure Infinity
for CMP-Neu5Ac above.
(
68) Lepp a¨ 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, 24838-48.
(69) Kuboki, A.; Okazaki, H.; Sugai, T.; Ohta, H. Tetrahedron 1997, 53, 2387-
4
00.
5742 J. AM. CHEM. SOC.
9
VOL. 124, NO. 20, 2002

O-Linked Sialyl Oligosaccharides
A R T I C L E S
Figure 2. Enzymatic preparation of O-linked sialosides. Reagents and conditions: (a) chST6GalNAc I (2 U), CMP-Neu5Ac (1.5 equiv); (b) pST3Gal I (0.6
U), CMP-Neu5Ac (1.4 equiv); (c) chST6GalNAc I (0.6 U), CMP-Neu5Ac (1.5 equiv); (d) (i) pST3Gal I (1.2 U), CMP-Neu5Ac (1.5 equiv), (ii) chST6GalNAc
I (1 U), CMP-Neu5Ac (1.5 equiv); (e) chST6GalNAc I (1 U), CMP-Neu5Gc (1.5 equiv).
Ultrapure water system (Barnstead/Thermolyne, Dubuque, IA). The
cDNA of chST6GalNAc I was kindly provided by Dr. S. Tsuji, RIKEN,
Wako, Japan. Plasmid DNA for pGIR201Nhe3′ containing proinsulin
signal sequence was kindly provided by Dr. K. Drickamer, University
of Oxford, Oxford, U.K. All other chemicals substrates were purchased
from Sigma Chemical Co. (St. Louis, MO). Ovine submaxillary mucin
was prepared as described. CMP[ C]NeuAc were purchased from
Du Pont-New England Nuclear (Boston, MA). The radioactive
nucleotide-sugar was diluted with unlabeled CMP-Neu5Ac-sugar
in the presence of T7 DNA polymerase (unmodified with 3′-5′
exonuclease activity). This treatment deleted the unique restriction sites
BamHI and EcoRI besides KpnI and EcoRV from the modified
pcDNA3, herefrom denoted as pcDNA3mod. A dog proinsulin signal
sequence was then subcloned into this modified vector to obtain
pcDNAins which was finally confirmed by double-stranded sequencing.
For subcloning the secretable form of chST6GalNAc-I into pcDNAins
vector, two restriction sites BamHI and EcoRI were introduced into its
cDNA by polymerase chain reaction (PCR) as follows: a forward
primer, CGGGATCCCAAGACTGAGCCACAGTGGGAT (5′-3′; nt.
725-745), with an internal BamHI site (underlined) and a reverse
primer, CGGAATTCTCAGGATCTCTGGTAGAGCTTC (5′-3′; nt
1711-1732), with an internal EcoRI site (underlined) were used for
amplification using the cDNA for the chST6GalNAc I previously
7
0
14
(Sigma) to the desired specific radioactivity.
Production of Recombinant chST6GalNAc I. Construction of
Recombinant Secretable chST6GalNAc I. For expression of a
secretable form of chST6GalNAc I, the mammalian expression vector
pcDNA3 (Invitrogen, San Diego, CA) was used after modifications as
7
1
72
described earlier. In brief, plasmid DNA for the vector pcDNA3 was
subcloned in pBluescript as template. The conditions for PCR were
digested with KpnI and EcoRV and then ligated using T4 DNA ligase
(
71) Datta, A. K.; Chammas, R.; Paulson, J. C. J. Biol. Chem. 2001, 276, 15200-7.
(70) Hill, H. D. J.; Reynolds, J. A.; Hill, R. L. J. Biol. Chem. 1977, 252, 3791-8.
(72) Tsuji, S.; Datta, A. K.; Paulson, J. C. Glycobiology 1996, 6, 5-7.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 20, 2002 5743

A R T I C L E S
4 °C, 30 s, 56 °C, 1 min, and 73 °C, 2 min for 20 cycles. The gel
Blixt et al.
9
terminated by the addition of water (0.7 mL). The reaction mixtures
were then passed through Pasteur pipet columns of Dowex resin (1-
analysis showed the generation of a single band of about 1 kb. This
band was purified using Geneclean II (Bio 101, San Diego, CA),
digested with BamHI and EcoRI, and then subcloned into similarly
digested mammalian expression vector pcDNAins described above. The
sequence of the resulting expression vector, termed sST6GalNAc I,
2-
X8, 200-400 mesh, PO
4
form, 0.5 × 5 cm, 1 mL). The columns
were washed with water (1.3 mL), the effluents were directly collected
in scintillation vials, and the radioactivity was assayed as described
above.
7
3
was confirmed by dideoxy double-stranded sequencing of the entire
subcloned fragment, including the restriction sites used.
General Procedure for Preparation of Crude CMP-Neu5Ac and
CMP-Neu5Gc. Cytidine 5′triphosphate (CTP) (980 mg, 1.72 mmol)
and N-acetyl neuraminic acid or N-glycolyl neuraminic acid (500 mg,
1.62 mmol) were dissolved in Tris-HCl (100 mL, 10 mM, pH 8.5),
2
containing MgCl (20 mM). ST3-CMPNeu5Ac-synthetase fusion
69
Expression of chST6GalNAc I Using Baculoviral System. For
expression, the cDNA for the chST6GalNAc I was released from the
above constructed sST6GalNAc I by digestion with EcoRI and NotI.
The fragment was purified by gel electrophoresis and ligated into
similarly digested pVL1392 (Pharmingen, San Diego, CA). Transfor-
mation, plasmid DNA isolation, and other molecular biological
techniques were done following the standard protocols (Pharmingen).
The sequence of the resulting expression vector, termed pVL-
sST6GalNAc I, was confirmed by dideoxy double-stranded sequencing73
of the entire subcloned fragment, including the restriction sites used.
To produce the Baculo viral stock, the plasmid DNA was used for
transfection of Sf9 using the BaculoGold Technique (Pharmingen) and
following the protocol of the supplier (Pharmingen). For protein
protein (300 U) was added, and the reaction proceeded at room
temperature for 1 h. The pH was kept constant (8.5) with NaOH (2
i
M). When all CTP was consumed according to TLC ( PrOH/H
2
O/NH
3
,
6:3:2 by vol), the reaction mixture was filtered through a 10 000 MW
cutoff membrane to remove the fusion protein, and the filtrate was
lyophilized. Prior to use, the crude residue was dissolved in water, and
the pH was set to 7.0 by HCl (2 M). The approximate concentration of
nucleotide-sugar was determined by absorbance (A271).
2-Azidoethyl N-Acetyl-2-deoxy-r-D-galactopyranoside (1). N-
Acetyl galactosamine (300 mg, 1.36 mmol) was dissolved in 2-azido-
ethanol (2 mL), and boron trifluoridetherate (1 mL, 7.0 mmol) was
added. The mixture was stirred at room temperature for 2 h. The reaction
was neutralized with sodium hydroxide (10 M) and loaded onto a
6
expression, High Five cells (Invitrogen) were kept up to 6 × 10 cells/
mL density at 27 °C in Insect Select media (BioWhitticker) using a
6
refrigerated shaker incubator. Cell density was brought to 2 × 10 with
n
fresh media (350 mL) using a 1 L fernback baffled shaker flask, and
Bacula virus stock was added at a MOI of 5. After 96 h or 70% viability,
the cells were harvest by centrifugation at 5000g, and the recombinant
protein from the supernatant was purified as follows.
Sephadex G15 (5 × 140 cm) column and eluted with 5% BuOH.
Fractions containing 1 were collected, lyophilized, and a second round
of purification was performed. Isolated fractions gave 1 (309.7 mg,
1
78%). H NMR (D
2
O): δ 2.05 (s, 3H, CH
), 3.75-3.77 (m, 2H, 6a, 6b), 3.67, 3.91 (2 m, 2H,
), 3.94 (dd, 1H, H-3), 3.99 (t, 1H, H-5), 4.00 (d, 1H, H-4),
4.18 (dd, 1H, H-2), 4.97 (dd, 1H, H-1, J1,2 4.0 Hz). 13C NMR (D
O):
3
CONH), 3.49-3.54 (m,
Affinity Purification of chST6GalNAc I. The clarified supernatant
was diluted by 1/10 vol cacodylic acid (500 mM, pH 6.5) and loaded
onto a cytidine-5′-diphospho-(CDP)-hexanolamine column (5 mL)
2 3
2H, OCH CH2N
OCH CH N
2
2 3
2
(
Calbiochem Inc., San Diego, CA) equilibrated with cacodylic acid
δ 21.5, 49.3, 49.8, 60.7, 66.0, 67.0, 67.3, 70.6, 96.5, 174.0. Maldi-FT-
MS: expected, m/z ) 291.1299 (M + H); found, m/z ) 291.1295.
2-N-Acetylethyl N-Acetyl-2-deoxy-r-D-galactopyranoside (2). 1
(100.0 mg, 345 µmol) and Pd/C (10%, 20 mg) were taken up in
buffer (50 mM, pH 6.5) containing sodium chloride (100 mM). The
column was washed with cacodylic acid buffer (50 mM, pH 6.5) until
the absorbance (A280) reached background level, and the recombinant
protein was eluted with cacodylic acid buffer (50 mM, pH 6.5)
containing sodium chloride (1.5 M). Fractions (1 mL) containing
chST6GalNAc I activity were collected and pooled and finally diluted
with glycerol (1 vol). The enzyme was stored at least 6 months at -20
2
methanol (10 mL) and hydrogenated with H at room temperature and
atmospheric pressure. The mixture was filtrated, washed with methanol
(5 mL), and acetic anhydride (0.3 mL) was added. After 10 min at
room temperature, the mixture was evaporated to dryness, dissolved
°
C without loss of any activity. The typical yield of enzyme from 1 L
in H
(Sephadex G15 1.6 × 140 cm) to give 2 (93.2 mg, 87%). H NMR
(D O): δ 2.01 (s, 3H, CH CONH), 2.04 (s, 3H, CH CONH), 3.43-
2
O (2 mL), and further purified by gel filtration chromatography
1
of culture fluid was 8 U with a specific activity of 1 U/mg protein.
Assay for chST6GalNAc I. Enzyme activity was performed as
2
3
3
described previously7 in 2-(N-morpholino)ethanesulfonic acid buffer
4
3.50 (m, 2H, OCH CH N ), 3.74-3.78 (m, 2H, 6a, 6b), 3.54, 3.78 (2
2
2
3
(
MES) (100 µL, 50 mM, pH 6.5) containing asialo ovine submaxillary
m, 2H, OCH
2
CH
2
N
3
), 3.87 (dd, 1H, H-3), 3.92 (t, 1H, H-5), 3.98 (d,
13
mucin (80 µg), sodium chloride (100 mM), diluted purified enzyme (5
1H, H-4), 4.16 (dd, 1H, H-2), 4.89 (dd, 1H, H-1, J1,2 4.0 Hz). C NMR
14
(D O): 21.3, 21.4, 49.2, 60.7, 65.8, 67.3, 68.0, 70.0, 70.5, 96.6, 173.8,
µL), bovine serum albumin (1 mg/mL), and CMP[ C]Neu5Ac (90 µM,
final specific activity 2.7 Ci/mol) at 37 °C for 30 min. The reaction
was quenched by freezing, and the thawed sample was loaded onto a
gel filtration column (Sephadex G50 0.7 × 13 cm) and eluted with 0.1
M sodium chloride. The flow-through was directly collected in
scintillation vials, and the radioactivity was assayed by liquid scintil-
lation counting (Beckman LS counter). Values (µmol transferred/min
incubation) obtained in this standard assay were multiplied by the factor
2
174.0. MALDI-FT-MS m/z calcd for (M + Na), 329.1319; found,
329.1322. N-(9-Fluorenylmethyloxycarbonyl)-O-(3,4,6-tri-O-acetyl-
2-azido-2-deoxy-r-D-galactopyranosyl)-L-threonine Phenacylester
47,48,76
(3a). This was prepared as described previously.
3a: 8.0 g (75%).
1
H NMR (CDCl
3
): δ 1.44 (d, 3H, H-γ), 1.63, 2.05, 2.07 (s, 3H, CH
3
-
CO), 2.18 (s, 3H, CH
(Fmoc)), 4.29 (t, 1H, H-5), 4.32 (m, 1H, CH (Fmoc)), 4.35, 4.42 (m,
2H, 6a, 6b), 5.34 (t, 1H, H-3), 5.48 (d, 1H, H-4), 4.57 (d, 1H, H-R,
3
CONH), 3.80 (dd, 1H, H-2), 4.13 (d, 1H, CH
2
7
5
5
.5, derived from kinetic analysis to give units of enzyme. One unit
(U) of enzyme activity is defined as the amount of enzyme that catalyzes
J
R,â 2.0 Hz), 4.64 (m, 1H, H-â), 5.54 (d, 1H, H-1, J1,2 3.5 Hz), 5.93 (d,
the transfer of 1 µmol of Neu5Ac from CMP-Neu5Ac to asialo ovine
submaxillary mucin per min at saturating substrate concentrations.
1H, NH (thr)), 5.32, 5.60 (d, 2H, CHa/b (phenacyl)), 7.2-8.0 (m, Ar).
C NMR (CDCl ): δ 19.1, 20.6, 47.0, 58.2, 58.4, 61.8, 66.7, 66.9,
3
13
6
1
7.5, 67.6, 68.4, 76.1, 98.1, 120-144, 156.8, 169.8, 169.9, 170.0, 170.3,
91.5. MALDI-FT-MS m/z calcd for (M + Na), 795.2484; found,
Enzyme activity for different acceptors was assayed at 37 °C for 30
min in a final volume of 100 µL containing cacodylic acid (50 mM,
pH 6.5), CMP[ C]Neu5Ac (0.5 mM, final specific activity 0.70 Ci/
1
4
795.2479.
N-(9-Fluorenylmethyloxycarbonyl)-O-(2-acetamido-3,4,6-tri-O-
mol), acceptor (1 mM), and diluted enzyme (3.8 mU). Reactions were
acetyl-2-deoxy-r-D-galactopyranosyl)-L-threonine Phenacylester (3b).
(
(
(
73) Sanger, F.; NicklenN, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A.
977, 74, 5463-7.
74) Sadler, J. E.; Rearick, J. I.; Paulson, J. C.; Hill, R. L. J. Biol. Chem. 1979,
54, 4434-43.
75) Sadler, J. E.; Rearick, J. I.; Hill, R. L. J. Biol. Chem. 1979, 254, 5934-41.
1
(76) F u¨ gedi, P.; Garegg, P.; L o¨ nn, H.; Norberg, T. Glycoconjugate J. 1987, 4,
97-109.
2
(77) Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycoconjugate J.
1988, 5, 49-63.
5
744 J. AM. CHEM. SOC. VOL. 124, NO. 20, 2002
9

O-Linked Sialyl Oligosaccharides
A R T I C L E S
This was prepared as described previously.47,48 3b: 3.5 g (88%). H
NMR (CDCl ): δ 1.44 (d, 3H, H-γ), 1.63, 2.01, 2.04 (s, 9H, CH CO),
.18 (s, 3H, CH CONH), 4.10, 4.15 (m, 2H, 6a, 6b), 4.30 (t, 1H, H-5),
.30 (m, 1H, CH (Fmoc)), 4.47 (d, 2H, CH (Fmoc)), 4.54 (m, 1H,
1
γ-CH
), 2.06 (s, 3H, CH
CONH), 2.15 (s, 3H, CH
3
3
3
CONH), 3.74 (s,
3
3
3H, COOMe), 3.74-3.76 (m, 2H, 6a, 6b), 3.87 (dd, 1H, H-3), 3.94 (d,
1H, H-4), 4.01 (t, 1H, H-5), 4.08 (dd, 1H, H-2), 4.28 (m, 1H, CH
2
4
3
2
2
(Fmoc)), 4.48 (q, 1H, â-CH), 4.54 (d, 2H, CH (Fmoc)), 4.64 (d, 1H,
H-â), 4.57 (d, 1H, H-R, JR,â 2.0 Hz), 4.68 (dd, 1H, H-2), 5.18 (dd, 1H,
H-3), 5.28, 5.60 (d, 2H, CHa/b (phenacyl)), 5.42 (d, 1H, H-1, J1,2 3.5
Hz), 5.44 (d, 1H, H-4), 5.74 (d, 1H, NH (thr)), 6.01 (d, 1H, NH (Ac),
R-CH, JR,â 2.0 Hz), 4.89 (d, 1H, H-1, J1,2 4.0 Hz), 7.2-8.0 (m, Ar).
13
C NMR (D O): δ 19.3, 23.2, 49.3, 52.8, 51.2, 60.1, 62.8, 67.7, 69.6,
2
70.3, 72.9, 76.5, 100.6, 120-145, 159.2, 172.4, 173.9. MALDI-FT-
MS m/z calcd for (M + Na), 581.2106; found, 581.2129.
N-(9-Fluorenylmethyloxycarbonyl)-O-[4,6-di-O-acetyl-2-azido-2-
deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-R-D-galac-
topyranosyl]-L-threonine Phenacylester (7a). This was prepared as
1
3
7
4
1
.2-8.0 (m, Ar). C NMR (CDCl ): δ 18.5, 20.6, 20.7, 22.8, 47.1,
7.4, 58.4, 62.2, 66.6, 67.2, 67.5, 67.5, 68.6, 76.7, 99.9, 120-144, 156.6,
70.3, 170.3, 170.4, 170.8, 171.0, 191.1. MALDI-FT-MS m/z calcd
3
for (M + Na), 811.2685; found, 811.2687.
N-(Acetyl)-O-(2-acetamido-2-deoxy-r-D-galactopyranosyl)-L-thre-
onine Methylester (3c). 3b (3.5 g, 4.4 mmol) was dissolved in MeOH
described previously.4
7,48,76
7a: 0.80 g (39%). H NMR (CDCl
1.42 (d, 3H, H-γ), 1.59, 1.97, 2.03, 2.04, 2.06, 2.14 (s, 18H, CH
2.15 (s, 3H, CH CONH), 3.92 (t, 1H, H-5′), 4.02 (dd, 1H, H-2), 4.13
(d, 1H, CH (Fmoc)), 4.10, 4.17 (m, 2H, 6a′, 6b′), 4.29 (t, 1H, H-5),
1
): δ
CO),
3
3
(70 mL) and NaOMe (0.5 M, 15 mL). The mixture was stirred at room
3
temperature for 30 min. The reaction was treated with Ac O (10 mL)
2
2
and stirred for 10 min. The mixture was neutralized with sodium
methoxide (0.5 M) and concentrated by evaporation. The residue was
loaded onto a Sephadex G15 (5 × 140 cm), and the compound was
4.32 (m, 1H, CH (Fmoc)), 4.00, 4.20 (m, 2H, 6a, 6b), 5.01 (dd, 1H,
H-3′), 5.36 (d, 1H, H-4′), 5.34 (t, 1H, H-3), 5.49 (d, 1H, H-4), 4.56 (d,
1H, H-R, JR,â 2.0 Hz), 4.61 (m, 1H, H-â), 4.74 (d, 1H, H-1′, J1,2 7.5
Hz), 5.18 (dd, 1H, H-2′), 5.50 (d, 1H, H-1, J1,2 4.0 Hz), 5.91 (d, 1H,
n
eluted with 5% BuOH. Fractions containing the product were collected
1
NH (thr)), 5.35, 5.57 (d, 2H, CHa/b (phenacyl)), 7.2-8.0 (m, Ar). 13
NMR (CDCl ): δ 19.1, 20.5, 20.6, 20.6, 20.7, 47.1, 58.5, 60.3, 60.9,
and lyophilized to give 3c (798 mg, 47%) as a white powder. H NMR
C
(
D
2
O): δ 1.27 (d, 3H, γ-CH
CH CONH), 3.74 (s, 3H, COOMe), 3.74-3.76 (m, 2H, 6a, 6b), 3.87
dd, 1H, H-3), 3.94 (d, 1H, H-4), 4.01 (t, 1H, H-5), 4.08 (dd, 1H, H-2),
.48 (q, 1H, â-CH), 4.64 (d, 1H, R-CH, JR,â 2.0 Hz), 4.89 (d, 1H, H-1,
3
), 2.06 (s, 3H, CH
3
CONH), 2.15 (s, 3H,
3
3
62.9, 66.7, 67.3, 67.7, 68.8, 69.3, 70.7, 74.8, 76.0, 98.9, 101.4, 120-
144, 156.7, 169.4-170.4, 191.5. MALDI-FT-MS m/z calcd for (M +
Na), 1083.3329; found, 1083.3353.
(
4
J
1
3
1,2 4.0 Hz). C NMR (D
2
O): δ 17.5, 21.1, 21.6, 49.3, 52.4, 56.7,
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-acetamido-4,6-di-O-acetyl-
2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-r-D-ga-
lactopyranosyl]-L-threonine Phenacylester (7b). This was prepared
6
0.7, 66.9, 67.9, 70.7, 74.7, 98.1, 171.6, 173.8, 174.3. MALDI-FT-
MS m/z calcd for (M + Na), 401.1530; found, 401.1524.
N-(Acetyl)-O-(2-azido-2-deoxy-r-D-galactopyranosyl)-L-threo-
nine Methylester (4). 3a (150 mg, 0.19 mmol) was dissolved in MeOH
as described previously.4 7b: 0.62 g (88%). H NMR (CDCl
7,48
1
): δ
CO),
CONH), 3.76 (t, 1H, H-5′), 4.58 (dd, 1H, H-2), 4.13
2
(Fmoc)), 4.02, 4.17 (m, 2H, 6a′, 6b′), 4.27 (t, 1H, H-5),
3
1.42 (d, 3H, H-γ), 1.59, 1.97, 2.03, 2.04, 2.06, 2.14 (s, 18H, CH
2.15 (s, 3H, CH
(d, 1H, CH
3
(2 mL) and NaOMe (0.5 M, 0.5 mL). The mixture was stirred at room
3
temperature for 30 min. The reaction was treated with Ac O (0.3 mL)
2
and stirred for 10 min. The mixture was neutralized with Dowex resin
4.32 (m, 1H, CH (Fmoc)), 4.00, 4.20 (m, 2H, 6a, 6b), 5.01 (dd, 1H,
H-3′), 5.27 (d, 1H, H-4′), 4.04 (t, 1H, H-3), 5.14 (d, 1H, H-4), 4.56 (d,
1H, H-R, JR,â 2.0 Hz), 4.61 (m, 1H, H-â), 4.89 (d, 1H, H-1′, J1,2 7.5
Hz), 5.01 (dd, 1H, H-2′), 5.49 (d, 1H, H-1, J1,2 4.0 Hz), 6.26 (d, 1H,
+
(1 × 8, 200-400 mesh, H ), filtered, and concentrated. The residue
was redissolved in water and loaded onto a SepPak C18 column (0.5
g). The column was washed with water (2 vol), and the compound
was eluted with 10-60% MeOH. Appropriate fractions were collected
NH (Ac), 5.59 (d, 1H, NH (thr)), 5.28, 5.60 (d, 2H, CHa/b (phenacyl)),
1
13
and evaporated to give 4 (55 mg, 78.8%). H NMR (D
2
O): δ 1.29 (d,
7.2-8.0 (m, Ar). C NMR (CDCl
3
): δ 17.5, 20.5, 20.5, 20.6, 20.7,
3
(
1
H, γ-CH
m, 2H, 6a, 6b), 3.84 (s, 3H, COOMe), 3.99 (dd, 1H, H-3), 4.00 (d,
H, H-4), 4.02 (t, 1H, H-5), 4.48 (q, 1H, â-CH), 4.67 (d, 1H, R-CH,
3
), 2.12 (s, 3H, CH
3
CONH), 3.58 (dd, 1H, H-2), 3.74-3.76
22.9, 47.1, 49.0, 58.6, 60.8, 62.9, 66.8, 66.9, 67.2, 68.0, 68.6, 69.3,
70.4, 70.7, 72.8, 76.9, 99.4, 101.2, 120-144, 156.5, 169.4-170.4,
192.6. MALDI-FT-MS m/z calcd for (M + Na), 1099.353; found,
1099.3530.
1
3
JR,â 2.0 Hz), 5.10 (d, 1H, H-1, J1,2 4.0 Hz). C NMR (D
2
O): δ 17.1,
2
1.1, 52.6, 56.9, 59.7, 60.6, 67.0, 68.3, 70.8, 75.7, 98.8, 171.6, 174.2.
N-(Acetyl)-[2-acetamido-2-deoxy-3-O-(â-D-galactopyranosyl)-r-
D-galactopyranosyl]-L-threonine Methylester (7c). Acetylated dis-
accharide 7b (580 mg, 538 µmol) was dissolved in MeOH (10 mL)
and NaOMe (2 mL, 0.5 M). After reaction at room temperature for 12
MALDI-FT-MS m/z calcd for (M + Na), 581.2106; found, 581.2129.
N-(Acetyl)-O-(2-amino-2-deoxy-r-D-galactopyranosyl)-L-threo-
nine Methylester (5). 4 (25 mg, 74 µmol) was treated with PdC (10%)
in methanol (5 mL) and hydrogenated with H
2
at room temperature
2
h, Ac O (1.5 mL) was then added to the mixture. After 10 min of
and atmospheric pressure. The mixture was filtrated and concentrated
reaction, the mixture was neutralized with sodium methoxide (0.5 M)
and concentrated by evaporation. The residue was loaded onto a
Sephadex G15 (5 × 140 cm), and the compound was eluted with 5%
1
to give the amino glycoside 5 (21.0 mg, 82%). H NMR (D
2
O): δ
1
3
3
.30 (d, 3H, γ-CH ), 2.11 (s, 3H, CH CONH), 3.07 (dd, 1H, H-2),
3
3
n
.74-3.76 (m, 2H, 6a, 6b), 3.84 (s, 3H, COOMe), 3.95 (d, 1H, H-4),
.97 (dd, 1H, H-3), 4.03 (t, 1H, H-5), 4.40 (q, 1H, â-CH), 4.64 (d, 1H,
BuOH. Appropriate fractions were collected and lyophilized to give
1
7c (200 mg, 71%) as a white powder. H NMR (D
2
O): δ 1.27 (d, 3H,
CONH), 3.52 (dd,
1
3
R-CH, JR,â 2.0 Hz), 5.04 (d, 1H, H-1, J1,2 4.0 Hz). C NMR (D
2
O): δ
3
γ-CH ), 2.04 (s, 3H, CH
3
CONH), 2.15 (s, 3H, CH
3
1
1
3
7.1, 21.1, 50.4, 52.6, 56.4, 60.7, 67.9, 70.9, 75.3, 76.4, 98.6, 171.8,
74.1. MALDI-FT-MS m/z calcd for (M + H), 337.1605; found,
37.1605.
1H, H-2′), 3.62 (dd, 1H, H-3′), 3.65 (dd, 1H, H-5′), 3.74 (s, 3H,
COOMe), 3.74-3.76 (m, 2H, 6a, 6b), 3.74-3.76 (m, 2H, H-6a′, H-6b′),
3.91 (d, 1H, H-4′), 4.01 (dd, 1H, H-3), 4.05 (t, 1H, H-5), 4.22 (d, 1H,
H-4), 4.24 (dd, 1H, H-2), 4.47 (dd, 1H, H-1′, J1,2 7.5 Hz), 4.49 (q, 1H,
â-CH), 4.65 (d, 1H, R-CH, JR,â 2.0 Hz), 4.88 (dd, 1H, H-1, J1,2 4.0
N-(9-Fluorenylmethyloxycarbonyl)-O-(2-acetamido-2-deoxy-r-D-
galactopyranosyl)-L-threonine Methylester (6). 3b (100 mg, 126.8
µmol) was dissolved in MeOH (10 mL), and a few drops of NaOMe
Hz). 13C NMR (D
O): δ 17.5, 21.1, 21.6, 48.1, 52.4, 56.7, 60.6, 60.7,
2
(
0.5 M) were added until pH indicated not more than 9. The mixture
68.1, 68.2, 70.0, 70.4, 72.0, 74.5, 74.7, 76.0, 98.3, 104.1, 171.7, 173.7,
174.3. MALDI-FT-MS m/z calcd for (M + Na), 563.2059; found,
563.2073.
was stirred at room temperature for 30 min, and the reaction was
+
neutralized with Dowex (1 × 8, 200-400 mesh, H ), filtered, and
concentrated. The residue was redissolved in EtOAc and MeOH (1:1,
N-(Acetyl)-(sodium[5-acetamido-3,5-dideoxy-D-glycero-r-D-galacto-
2-nonulo-pyranosyl]-onate-6-O-(2-acetamido-2-deoxy-r-D-galacto-
pyranosyl))-L-threonine Methylester (8). Tn-antigen 3c (201 mg, 0.55
mmol) was mixed with crude CMP-Neu5Ac (0.82 mmol, pH 7.0).
Water (10 mL) and chST6GalNAc I (2 U) were added, and the reaction
1
0 mL) and loaded onto a Silica column (1.5 × 10 cm) packed in
EtOAc and eluted with EtOAc (2 vol), EtOAc/MeOH (5/1, 2 vol), and
EtOAc/MeOH (1/1). Fractions containing the product were pooled and
concentrated to give 6 (50 mg, 72%). H NMR (D
1
2
O): δ 1.27 (d, 3H,
J. AM. CHEM. SOC.
9
VOL. 124, NO. 20, 2002 5745

A R T I C L E S
Blixt et al.
was kept at 37 °C overnight. The reaction was completed after adding
72.0, 72.0, 74.5, 75.1, 76.1, 98.3, 99.8, 104.2, 171.6, 173.1, 173.7, 174.3,
174.5. MALDI-FT-MS m/z calcd for (M + Na), 854.3013; found,
854.3000.
1
0% extra CMP-Neu5Ac and an additional 6 h of incubation. The
mixture was filtered through Dowex 1 × 8-200 (1.5 × 7 cm,
phosphate) and washed with 4 vol of water. The filtrate was lyophilized,
and the residue was loaded onto Sephadex G15 (5 × 160 cm) eluted
Di-sialyl-T-epitope (11). T-antigen 7c (50.0 mg, 95 µmol) was
mixed with crude CMP-Neu5Ac (143 µmol, pH 7.0), pST3Gal-I (1.2
U) was added, and the mixture was reacted at 37 °C for 4 h. When
n
with 5% BuOH. Appropriate fractions were collected and lyophilized
1
to give the disaccharide 8 (349 mg, 97%). H NMR (D
2
O): δ 1.30 (d,
CONH′), 2.05 (s,
CONH), 2.71 (dd, 1H, H-3′eq), 3.58
TLC (6:3:3:2, EtOAc, MeOH, HOAc, H O by vol) showed complete
2
3
H, γ- CH
3
), 1.68 (t, 1H, H-3′ax), 2.03 (s, 3H, CH
3
consumption of the disaccharide, the mixture was loaded onto a gel
n
3
H, CH CONH), 2.15 (s, 3H, CH
3
3
filtration column (Sephadex 1.5 × 160 cm) eluted with 5% BuOH.
(
dd, 1H, H7′), 3.67 (d, 1H, H-4′), 3.70 (dd, 1H, H-6′), 3.74 (s, 3H,
COOMe), 3.64-4.10 (m, 2H, 6a, 6b), 3.65-3.90 (m, 2H, H-9′), 3.82
dd, 1H, H-5′), 3.83 (t, 1H, H-5), 3.86 (dd, 1H, H-3), 3.88 (m, 1H,
H-8′), 3.99 (d, 1H, H-4), 4.07 (dd, 1H, H-2), 4.45 (q, 1H, â-CH), 4.64
Appropriate fractions were concentrated to dryness. The residue (142
mg) was dissolved in crude CMP-NeuAc pH 7.0 as described above,
and chST6GalNAc-I (1 U) was added. The mixture was reacted at 37
°C for 24 h. When completed, the mixture was passed through a Dowex
1 × 8-200 (phosphate, 1 × 4 cm) column and lyophilized. The residue
was then loaded onto a Sephadex G15 (2.5 × 160 cm) eluted with 5%
(
(
d, 1H, R-CH, JR,â 2.0 Hz), 4.86 (dd, 1H, H-1, J1,2 4.0 Hz). 13C NMR
O): δ 17.5, 21.1, 21.5, 21.6, 39.5, 49.3, 51.3, 52.3, 56.7, 62.1, 63.3,
6.8, 67.6, 67.7, 68.0, 72.0, 75.2, 98.3, 99.7, 172.7, 173.7, 174.3, 174.5.
(D
2
n
6
BuOH. Appropriate fractions was collected and lyophilized to give
1
MALDI-FT-MS m/z calcd for (M + Na), 692.2485; found, 692.2506.
Sodium N-(Acetyl)-[5-acetamido-3,5-dideoxy-D-glycero-r-D-ga-
lacto-2-nonulo-pyranosyl]-onate-3-O-(2-acetamido-2-deoxy-3-O-(â-
D-galactopyranosyl)-r-D-galactopyranosyl)-L-threonine Methylester
11 (76.7 mg, 73%). Selected H NMR (D O): δ 1.30 (d, 3H, γ-CH ),
2
3
1.66 (t, 1H, H-3′′ ), 1.78 (t, 1H, H-3′′′ ), 2.03 (s, 3H, CH CONH),
ax
ax
3
2.03 (s, 3H, CH CONH′′), 2.03 (s, 3H, CH CONH′′′), 2.16 (s, 3H, CH -
3
3
3
CONH), 2.72 (dd, 1H, H-3′′ ), 2.73 (dd, 1H, H-3′′′ ), 3.53 (d, 1H,
eq
eq
(
9). T-antigen 7c (53.0 mg, 0.10 mmol) was mixed with crude CMP-
H-2′), 3.65 (dd, 1H, H-5′), 3.66 (d, 1H, H-4′′), 3.69 (d, 1H, H-4′′′),
3.73 (s, 3H, COOMe), 3.70-4.12 (m, 2H, 6a, 6b), 3.75-3.80 (m, 2H,
6a′, 6b′), 3.98 (d, 1H, H-4′), 3.99 (dd, 1H, H-3), 3.82 (t, 1H, H-5),
4.22 (dd, 1H, H-2), 4.07 (dd, 1H, H-3′), 4.22 (d, 1H, H-4), 4.48 (q,
1H, â-CH), 4.52 (d, 1H, H-1′, J1,2 8.0 Hz), 4.66 (d, 1H, R-CH, JR,â 2.0
Neu5Ac (0.14 mmol, pH 7.0). Water (10 mL) and pST3Gal-I (0.6 U)
were added, and the mixture was reacted at 37 °C for 14 h. When
TLC (6:3:3:2, EtOAc, MeOH, HOAc, H O by vol) showed complete
2
consumption of the disaccharide, the mixture was passed through a
Hz), 4.87 (d, 1H, H-1, J1,2 4.0 Hz). Selected 13C NMR (D O): δ 17.5,
Dowex 1 × 8-200 (1.5 × 4 cm, phosphate) column, and the filtrate
2
was lyophilized. The residue was purified on a gel filtration column
21.1, 21.5, 21.6, 21.7, 39.6, 48.0, 51.1, 51.3, 52.3, 56.7, 60.4, 62.1,
63.3, 66.8, 67.6, 67.7, 67.8, 68.0, 68.3, 69.0, 69.1, 71.0, 71.2, 71.4,
72.0, 72.2, 74.3, 75.2, 75.3, 76.4, 98.4, 99.8, 104.1, 171.6, 173.3, 173.9,
174.2, 174.3, 174.5. MALDI-FT-MS m/z calcd for (M + Na),
1145.3967; found, 1145.3991.
n
(
Sephadex 2.5 × 160 cm) eluted with 5% BuOH. Appropriate fractions
containing the trisaccharide were collected and lyophilized to give sialyl
1
(
2,3)-T-epitope 9 (78.0 mg, 95%). H NMR (D
CH ), 1.78 (t, 1H, H-3′′ax), 2.03 (s, 3H, CH CONH′′), 2.04 (s, 3H, CH
CONH), 2.15 (s, 3H, CH CONH), 2.75 (dd, 1H, H-3′′eq), 3.54 (d, 1H,
H-2′), 3.59 (dd, 1H, H7′′), 3.64 (dd, 1H, H-5′), 3.66 (d, 1H, H-4′′),
2
O): δ 1.26 (d, 3H, γ-
3
3
3
-
3
Sodium N-(Acetyl)-[5-glycolylamido-3,5-dideoxy-D-glycero-r-D-
galacto-2-nonulo-pyranosyl]-onate-6-O-(2-acetamido-2-deoxy-r-D-
galactopyranosyl))-L-threonine Methylester (12). Tn-antigen 3c (30.0
mg, 79.3 µmol) was mixed with crude CMP-Neu5Gc (119 µmol, pH
3
6
1
4
.68 (dd, 1H, H-6′′), 3.74 (s, 3H, COOMe), 3.74-3.76 (m, 2H, 6a,
b), 3.74-3.76 (m, 2H, 6a′, 6b′), 3.65-3.90 (m, 2H, H-9′′), 3.85 (dd,
H, H-5′′), 3.89 (m, 1H, H-8′′), 3.92 (d, 1H, H-4′), 4.01 (dd, 1H, H-3),
.04 (t, 1H, H-5), 4.07 (dd, 1H, H-2), 4.07 (dd, 1H, H-3′), 4.22 (d, 1H,
7
.0). Water (10 mL) and chST6GalNAc-I (1 U) were added, and the
reaction was kept at 37 °C for 24 h. The reaction was completed after
adding 10% extra CMP-Neu5Gc and an additional 6 h of incubation.
The mixture was filtered through Dowex 1 × 8-200 (1.5 × 7 cm,
phosphate) and washed with 4 vol of water. The filtrate was lyophilized,
and the residue was loaded onto Sephadex G15 (5 × 160 cm) eluted
H-4), 4.50 (q, 1H, â-CH), 4.53 (d, 1H, H-1′, J1,2 7.5 Hz), 4.65 (d, 1H,
R-CH, JR,â 2.0 Hz), 4.89 (dd, 1H, H-1, J1,2 4.0 Hz). 13C NMR (D
O):
δ 17.5, 21.1, 21.5, 21.7, 39.3, 48.1, 51.1, 52.4, 56.6, 60.5, 60.7, 62.1,
6
9
2
6.8, 67.6, 67.8, 68.0, 68.3, 70.5, 71.4, 72.2, 74.4, 74.8, 75.1, 76.3,
8.3, 99.0, 104.1, 171.6, 173.3, 173.9, 174.3, 174.5. MALDI-FT-MS
n
with 5% BuOH. Appropriate fractions were collected and lyophilized
m/z calcd for (M + Na), 854.3013; found, 854.2991.
1
to give the disaccharide 12 (49.0 mg, 87%). H NMR (D
d, 3H, γ-CH ), 1.69 (t, 1H, H-3′ax), 2.05 (s, 3H, CH CONH), 2.15 (s,
CONH), 2.73 (dd, 1H, H-3′eq), 3.58 (dd, 1H, H7′), 3.77 (dd,
2
O): δ 1.30
Sodium N-(Acetyl)-[5-acetamido-3,5-dideoxy-D-glycero-r-D-ga-
lacto-2-nonulo-pyranosyl]-onate-6-O-(2-acetamido-2-deoxy-3-O-(â-
D-galactopyranosyl)-r-D-galactopyranosyl)-L-threonine Methylester
(
3
3
3
1
2
1
4
4
H, CH
3
H, H-4′), 3.65 (dd, 1H, H-6′), 3.75 (s, 3H, COOMe), 3.64-4.10 (m,
H, 6a, 6b), 3.65-3.90 (m, 2H, H-9′), 3.90 (dd, 1H, H-5′), 3.83 (dd,
H, H-5), 3.86 (dd, 1H, H-3), 3.88 (m, 1H, H-8′), 4.00 (d, 1H, H-4),
(10). T-antigen 7c (50.0 mg, 95.0 µmol) was mixed with crude CMP-
NeuAc (143 µmol, pH 7.0). Water (10 mL) and chST6GalNAc-I (0.6
U) were added, and the mixture was reacted at 37 °C for 14 h. When
TLC (6:3:3:2, EtOAc, MeOH, HOAc, H
.07 (dd, 1H, H-2), 4.13 (s, 2H, HOCH
.64 (d, 1H, R-CH, JR,â 2.0 Hz), 4.87 (d, 1H, H-1, J1,2 4.0 Hz).
O): δ 17.5, 21.0, 21.6, 39.6, 49.3, 51.0, 52.3, 56.7, 60.4,
2
CONH), 4.49 (q, 1H, â-CH),
O by vol) showed complete
13
2
C
consumption of the disaccharide, the mixture was passed through a
Dowex (1 × 8-200 mesh, 1.5 × 4 cm, phosphate) column, lyophilized,
and loaded onto a gel filtration column (Sephadex 2.5 × 160 cm) eluted
NMR (D
2
6
1
2.0, 63.3, 66.8, 67.4, 67.6, 68.0, 69.3, 71.3, 71.8, 75.1, 98.2, 99.8,
71.6, 172.8, 173.7, 174.3, 175.2. MALDI-FT-MS m/z calcd for (M +
n
with 5% BuOH. Appropriate fractions containing the trisaccharide were
Na), 708.2434; found, 708.2460.
concentrated to dryness to give sialyl (2,6)-T-epitope 10 (75.0 mg, 96%).
1
H NMR (D
2
O): δ 1.30 (d, 3H, γ- CH
CONH′′), 2.03 (s, 3H, CH CONH), 2.15 (s, 3H, CH
.70 (dd, 1H, H-3′′eq), 3.50 (d, 1H, H-2′), 3.58 (dd, 1H, H7′′), 3.63
3
), 1.66 (t, 1H, H-3′′ax), 2.03 (s,
Acknowledgment. This work was funded in part by the grants
from NIGMS (GM60938) and the NCI (CA46462). The
Swedish Institute (SI) is acknowledged for financial support for
O.B. The authors also acknowledge Yuzuru Ikehara for critical
reading of the manuscript.
3
2
H, CH
3
3
3
CONH),
(dd, 1H, H-5′), 3.70 (dd, 1H, H-4′′), 3.68 (dd, 1H, H-6′′), 3.74 (s, 3H,
COOMe), 3.70, 4.13 (m, 2H, 6a, 6b), 3.74-3.76 (m, 2H, 6a′, 6b′),
3
3
.65-3.90 (m, 2H, H-9′′), 3.86 (dd, 1H, H-5′′), 3.89 (m, 1H, H-8′′),
.90 (d, 1H, H-4′), 3.99 (dd, 1H, H-3), 3.90 (t, 1H, H-5), 4.23 (dd, 1H,
1
Supporting Information Available: Copies of H NMR,
H-2), 3.61 (dd, 1H, H-3′), 4.24 (d, 1H, H-4), 4.48 (q, 1H, â-CH), 4.46
d, 1H, H-1′, J1,2 8.0 Hz), 4.65 (d, 1H, R-CH, JR,â 2.0 Hz), 4.86 (d, 1H,
H-1, J1,2 4.0 Hz). 13C NMR (D
O): δ 17.5, 21.1, 21.5, 21.7, 39.6, 48.1,
1.3, 52.3, 56.7, 60.5, 62.1, 63.3, 67.7, 68.1, 68.1, 69.1, 70.0, 71.2,
spectra for compounds 8, 9, 10, 11, and 12 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
(
2
5
JA017881+
5746 J. AM. CHEM. SOC.
9
VOL. 124, NO. 20, 2002