Chemo-enzymatic synthesis of glycopolymers and sequential glycopeptides bearing lactosamine and sialyl Lewisx unit pendant chains

Article abstract of DOI:10.1039/b001612h

A variety of glycoconjugates bearing either N-acetyllactosamine or sialyl Lewis^x units have been synthesised in a chemo-enzymatic way. This includes the synthesis of glycopolymer copolymerised with acrylamide and whose glucosamine unit was substituted with different kinds of side-chain. Glycopeptides with different spacer-arm glucosamine units have also been prepared and polymerised. The sugar chain was then elongated using glycosyl transferases to afford the novel sequential glycopeptides. In these cases, the polymeric sugar cluster effect led to enzymatic glycosylation with high efficiency. Nevertheless, some differences have been noticed depending on the reaction conditions used for each substrate.

Full text of DOI:10.1039/b001612h

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Chemo-enzymatic synthesis of glycopolymers and sequential
glycopeptides bearing lactosamine and sialyl Lewisx unit pendant
chains
1
Florence Sallas and Shin-Ichiro Nishimura*
Laboratory of Bioorganic Chemistry and Glycoclusters, Graduate School of Science,
Hokkaido University, Kita Ku, Kita 10, Nishi 8, Sapporo, 060-0810, Japan
Received (in Cambridge, UK) 28th February 2000, Accepted 19th April 2000
Published on the Web 5th June 2000
A variety of glycoconjugates bearing either N-acetyllactosamine or sialyl Lewisx units have been synthesised
in a chemo-enzymatic way. This includes the synthesis of glycopolymer copolymerised with acrylamide and
whose glucosamine unit was substituted with different kinds of side-chain. Glycopeptides with different spacer-arm
glucosamine units have also been prepared and polymerised. The sugar chain was then elongated using glycosyl
transferases to afford the novel sequential glycopeptides. In these cases, the polymeric sugar cluster effect led to
enzymatic glycosylation with high efficiency. Nevertheless, some differences have been noticed depending on the
reaction conditions used for each substrate.
Introduction
In recent years, it has become popular to use enzymes for the
construction of complex oligosaccharides1 and glycopoly-
mers.2,3 Indeed, more and more enzymes such as glycosyl trans-
ferases and sugar nucleotide donors are now commercially
available. Although this method offers several obvious advan-
tages over the usual chemical one (such as stereo- and regio-
selectivity, specificity and shorter reaction pathways), the use of
enzymes can become uncertain in some cases due to their speci-
ficity towards their natural substrate or their sensitivity to the
reaction conditions. Furthermore, several studies have already
shown that the sugar nucleotide donors can be modified to
some extent without losing their donor properties for the
enzyme,1 but it is still of interest to get information about sub-
strate specificity when working with an enzyme and chemically
synthesised molecules as potential artificial acceptors. On the
other hand, the chemistry of glycoconjugates and glycopoly-
mers is now being developed much further.4 Due to the poly-
meric glycoside cluster effect,5 enzymes can be very useful in
this area as they allow a high rate of glycosylation. As a part of
our present project concerning the chemo-enzymatic synthesis
of glycoconjugates as tools for glycotechnology6 it is of the
utmost importance to develop well-defined and easily handled
protocols leading to a high rate of glycosylation in high yield.
In this field, we wish to report, the synthesis of a variety of
new glycoconjugate bearing LacNAc, sialyl α-(2,6)-LacNAc
and sialyl Lewisx pendant chains. While the glycopolymers and
Scheme 1 Reagents and conditions: i, BF3ؒEt2O, 4 Å molecular sieves,
sequential glycopeptides7 were prepared chemically, the
subsequent elongation of the sugar chain was carried out
enzymatically with good to high efficiency.
CH2Cl2, 0 ЊC→rt, 24 h; ii, ethylenediamine, n-butanol, 70 ЊC, 24 h then
pyridine–Ac2O, rt, 24 h; iii, MeONa, MeOH, rt, 2 h; iv, H2, Pd–C,
MeOH, rt, 4 h; v, CH2᎐᎐CHCOCl, triethylamine, MeOH, 0 ЊC, 3 h.
Zemplen conditions and reduction of the azido group for 5 or
Z-deprotection for 9 and 10 with hydrogen and palladium–
carbon afforded the free amino intermediates which were both
used for the synthesis of glycopolymers and sequential
glycopeptides.
Results and discussion
The synthetic strategy involved at first the preparation of
glucosamine pyranoside intermediates 4, 7 and 8. These were
prepared using well-known glycosidation reactions either from
N-phthalimidoglucosaminidylimidate8 and azido(ethoxy)-
ethanol 29 in the presence of boron trifluoride–diethyl ether
(Scheme 1) or from 2-methyl-4,5-dihydro-(3,4,6-tri-O-acetyl-
1,2-dideoxy-α--glucopyranoso)[2,1-d ]-1,3-oxazole10 and ami-
noalkyl alcohol in the presence of camphor-10-sulfonic acid
as a promoter (Scheme 2). Subsequent deacetylation using
Synthesis of glycopolymers
Two kinds of glycopolymers were prepared: acrylamide
copolymers 12 and 13 and homopolymer 14. The synthesis of
12 and 14 involved first the reduction of the azido group of 5
DOI: 10.1039/b001612h
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were also important parameters for these reactions, two kinds
of acrylamide copolymers were synthesised with different
spacer-arms and different sugar densities. Thus, radical copoly-
merisation of monomer 6 was carried out in a mixture of
deaerated DMSO–water with 4 equivalents of acrylamide,
in the presence of TEMED and APS as initiators, and led to
acrylamide glycopolymer 12 bearing an ethylene glycol based
spacer moiety in 77% yield. The average molecular weight of 12
was estimated to be 135000 using the gel permeation chrom-
atography (GPC) method. The ratio of glycomonomer to acry-
lamide in 12 was estimated to be 1:4 from the integration of 1H
NMR data. The synthesis of copolymer 13 started with the
Z-N-protected N-acetylglucosamine derivative 10. Removal of
the Z group followed by N-acryloylation under the conditions
described for 6 provided the polymerisable N-acryloylated
N-acetylglucosamine derivative 11 in 41% yield (Scheme 2).
Derivative 11 was then copolymerised using 9 equivalents of
acrylamide under the conditions described above for 12
(Scheme 3). Acrylamide glycopolymer 13 was thus obtained in
89% yield. Its molecular weight was estimated to be over
380000 using the GPC method according to the previous
report.5 These values were the upper limit of our GPC method
and thus it was not possible to get a calibration curve for higher
molecular weight polymers. The ratio of glycomonomer to
acrylamide was estimated to be 1:9 from the integration of
1H NMR data. These polymers were large enough to allow a
positive cluster effect for further enzymatic reactions.
Scheme 2 Reagents and conditions: i, HO-[CH2]n-NHZ, CSA, (CH2)2-
Cl2, 70 ЊC, 3 h; ii, MeONa, MeOH, 2 h, rt; iii, H2, Pd–C, MeOH–
AcOHcat, rt, 3 h then CH2᎐᎐CHCOCl, triethylamine, MeOH, 0 ЊC, 3 h.
to get the free amino derivative. Then, N-acryloylation of
this derivative was performed using acryloyl chloride and tri-
ethylamine in methanol at 0 ЊC11 to afford the polymerisable
monomer 6 in 68% yield (Scheme 1). Subsequent radical
homopolymerisation with N,N,NЈ,NЈ-tetramethylethylene-
diamine (TEMED) and ammonium persulfate (APS) as pro-
moters in deaerated water for 24 h at room temperature
proceeded smoothly to give homopolymer 14 in 50% yield
(Scheme 3). The average number of repeating units in 14 was
evaluated to be 8 using MALDI-TOF spectroscopy.
Synthesis of sequential glycopeptides
In order to evaluate the influence of the nature of the support
towards the enzymatic glycosylation reactions, the protected
tripeptidic sequence Fmoc-Asp-Gly-Ser(OBn)2 was prepared.
This sequence was chosen because it is the primary recognition
motif required for the biosynthesis of N-linked glycoproteins
process to be initiated. The synthesis of this tripeptide is shown
in Scheme 4. It was obtained in 5 steps from a Boc and benzyl
Scheme 4 Reagents and conditions: i, DPPA, Et3N, 0 ЊC→rt, 15 h
(83%); ii, HCl 4 M–dioxane, 0 ЊC, 3 h; iii, DPPA–Et3N, 0 ЊC→rt, 15 h
(59%); iv, TFA, rt, 2 h (90%).
protected serine derivative 15. The coupling reactions between
the partially protected amino acid residues were achieved using
diphenylphosphoryl azide (DPPA) as a convenient activator of
the carboxy group. Finally, the tert-butyl deprotection of
the β-carboxy function of the aspartic residue with TFA at
room temperature led to the partially protected tripeptidic
intermediate 18 required for the synthesis of glycopeptides. In
this synthesis, two different kinds of protective groups were
employed, first Boc and then the base-labile Fmoc because of
the well-known sensitivity of the glycosidic bond to acidic
conditions.
Scheme 3 Reagents and conditions: i, acrylamide, TEMED, APS,
DMSO–H2O (5:1), 24 h, rt; ii, TEMED, APS, H2O, 24 h, rt.
As it was demonstrated that the efficiency of sugar elong-
ation reactions with glycosyl transferases is strongly dependent
on the sugar density and its distribution on the primer,11 and as
it was suggested that the length and flexibility of the spacer-arm
For the coupling reaction between the monosaccharidic and
the peptidic part, the EEDQ (ethyl 2-ethoxy-1,2-dihydroquino-
line-1-carboxylate) method was employed (Scheme 5). EEDQ is
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Scheme 5 Reagents and conditions: i, H2, Pd–C, MeOH, rt, 4 h (for 5) or H2, Pd–C, MeOH–AcOHcat, rt, 3 h (for 9 and 10); ii, EEDQ, ethanol–
benzene (1:1), rt, 24 h; iii, piperidine, DMF, 2 h, rt then H2, Pd–C, AcOH–H2O (8:2), rt, 24 h.
a selective activator of the carboxy groups of peptides. This
reaction proceeded smoothly in a mixture of ethanol–benzene at
room temperature and led to the corresponding glycopeptide
after purification by simple silica gel chromatography. Thus,
protected glycopeptides 19, 20 and 21 were obtained in 77, 59
and 52% yields respectively. Finally, 19, 20 and 21 were fully
deprotected first with piperidine for removal of the Fmoc
group, and then with hydrogen and palladium–carbon in a mix-
ture of acetic acid–water (8:2) to remove the benzyl protecting
groups. This afforded the fully deprotected glycopeptide
“primer” 22 (with a propyl chain), 23 (with a hexyl chain), and
24 (with a hydrophilic chain) in 61, 84 and 77% yields respec-
tively (Scheme 5) after simple purification by gel filtration
chromatography (G-10) using deionized water as eluent. The
polymerisation of these compounds was carried out in DMSO,7
at room temperature in the presence of a slight excess of DPPA
and triethylamine under a nitrogen atmosphere. The reaction
usually lasted 4 days (Scheme 6). An increase in the reaction
time did not lead to longer glycopeptides. The sequential glyco-
peptides 25, 26 and 27 were easily purified by gel filtration
chromatography (G-25) with deionized water as eluent and
were thus obtained in 73, 65 and 66% isolated yield. The aver-
age number of repeating units in each compound was evaluated
using MALDI-TOF spectroscopy and was found to be from 2
to 4 for 25 (M 1222–2444) and 26 (M 1158–2316) and from 2 to
5 for 27 (M 1076–2687).
Enzymatic modifications of glycopolymers
The use of enzymes in organic synthesis and for sugar elong-
ation in the construction of complex oligosaccharidic sequences
is already well-documented.1 Moreover, it has been demon-
strated that enzymatic reaction with glycosyl transferases can
occur with high yield using synthetic water-soluble polymer
supports.11,12 This is due to the polymeric sugar cluster effect
which promotes the successful binding of the sugar residues
with enzymes. Furthermore, the extent of galactosylation
depends strongly on the sugar density along the polymer and on
the length and flexibility of the spacer arm between the mono-
saccharidic unit and the polymeric backbone. Consequently we
chose to perform enzymatic galactosylation reactions with
homopolymer 14 having a hydrophilic spacer, with acrylamide
copolymer 12 having the same side chain and with acrylamide
copolymer 13 having a hexylamido side chain as substrates for
the galactosyl transferase. Thus, first homopolymer 14 was used
as substrate and the enzymatic galactosylation reaction was
carried out as previously described11 using only a slight excess
of uridine-5Ј-diphosphogalactose (UDP-Gal, 1.3–1.5 equiv.)
and galactosyl transferase from bovine milk in HEPES (N-(2-
hydroxyethyl)piperazine-N-ethanesulfonic acid) buffer at pH 6.
As no galactosylation occurred, the same reaction was
attempted using as an acceptor the acrylamide copolymer 12
whose sugar density is lower. In that latter case as well, no
galactosylation occured. Finally the amount of UDP-Gal was
increased to 3 equivalents (Scheme 7) and thus galactosylated
acrylamide copolymer 28 and homopolymer 30 were obtained
in 77 and 50% yield respectively. The extent of galactosylation
was estimated from the integration of 1H NMR data to be 50%
in both cases.
The length of the sequential glycopeptides in our case might
be due to the structure of the peptidic sequence itself. Although
it was shown that long sequential glycopeptides could be
obtained with a hydrophobic sequence,7 in our case, the
sequence was very hydrophilic. This fact could be a factor
preventing us from getting longer sequential glycopeptides.
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efficient conditions to get polymeric sialylated compounds in
good yields. The reaction proceeded smoothly in sodium
cacodylate buffer in the presence of Triton X-100 at pH 6.
Three equivalents of CMP-NANA were used together with
α-(2,3)-sialyl transferase at 37 ЊC for 5 days. These very simple
conditions afforded the fully sialylated polymer 31 in 75% yield.
The sialylation rate was estimated to be quantitative from the
integration of 1H NMR data.
Finally, derivative 31 was fucosylated using α-(1,3) fucosyl
transferase V and GDP-fucose in HEPES buffer11 to give the
expected glycopolymer 32 bearing the multiple sialyl Lex
antenna (Scheme 8). In this latter case, the extent of fucosyl-
ation was estimated to be 50% from NMR data and the isolated
yield was only 21%.
Enzymatic modifications of sequential glycopeptides
As it cannot be denied that the nature of the polymeric support
is of no importance in considering the substrate specificity of
an enzyme, subsequent galactosylation reactions were carried
out with the sequential glycopeptides 25, 26 and 27 (Scheme 9).
A similar chemo-enzymatic approach has already been used by
Baisch and Öhrlein to get sialyl Lewisx glycopeptides.13 Thus,
galactosylation of 27 was first achieved using 3 equivalents of
UDP-galactose and β-(1,4)-galactosyl transferase in HEPES
buffer (50 mM, pH 7) containing manganese chloride (Method
I). The solution was incubated at 37 ЊC for 24 hours. The galac-
tosylated derivative 33 was then easily purified by Sephadex
G-25 gel chromatography using deionized water as eluent.
From the 1H NMR data, the galactosylation rate was estimated
to be 70%. In order to improve the galactosylation efficiency,
the conditions of the reaction were improved according to a
slightly modified protocol of that published by Roy et al. for
dendritic molecules.14 The reaction was then carried out in
sodium cacodylate buffer (50 mM) and in the presence of
MgCl2 and MnCl2 at pH 7.5 and 25 ЊC for 72 hours. Addition
of UDP-Gal took place at 3 different stages along the reaction
(Method II). These conditions furnished a fully galactosylated
derivative 33Ј in 84% yield. The same conditions were then
employed for the enzymatic galactosylation of the other glyco-
peptides 25 and 26. Thus, galactosylated derivatives 34 and 35
were obtained in 55 and 62% yield respectively. The estimated
extent of galactosylation was estimated to be 80% for both
compounds.
Scheme 6 Reagents and conditions: i, DPPA, Et3N, DMSO, rt, 4 d.
To confirm the influence of the side chain on the efficiency
of enzymatic galactosylation, the acrylamide copolymer 13
with a hexylamido side chain as the hydrophobic linker was
then used as the substrate for enzymatic galactosylation. The
reaction was conducted as described above for 28 using only
a slight excess of UDP-Gal (1.3 equiv.). In that case, a fully
galactosylated copolymer 29 was readily obtained as proven by
1H NMR spectra. Indeed, the peak at 3.28 ppm corresponding
to H-4 of the N-acetylglucosamine unit disappeared com-
pletely, while a peak corresponding to the anomeric proton of
the galactose unit appeared at 4.35 ppm. Furthermore, the
integration corresponding to H-1 and H-1Ј showed two protons
confirming the full substitution of the glucosamine units in the
polymer. From these results, it can be assumed that, in this case,
besides the sugar density, the structure and the flexibility of the
linker between the N-acetylglucosamine unit and its polymeric
anchor are the most important parameters for enzyme effi-
ciency. Although the hydrophilic spacer-arm allowed more
flexibility, the presence of heteroatoms, more so than the sugar
density, seemed to be a limiting factor for these molecules to be
good substrates for galactosyl transferase. Indeed, the extent of
galactosylation is the same for the homo- and copolymer despite
the presence of an excess of galactosyl donor. As the reaction is
carried out in aqueous media, the possibility of a network of
ionic bonds leading to cyclisation of the side chain and also the
possibility of weak interactions between the side chain and
the enzyme (preventing it from interacting properly with its
substrate) have to be taken into consideration.
These reactions demonstrate once again that whatever the
nature of the backbone, glycosyl acceptors with a hydrophilic
linker are not suitable substrates for the galactosyl transferase,
unlike those with an aliphatic linker.
In the next step, still with the aim of getting multivalent sialyl
Lewisx derivatives that are well-known to be highly efficient as
selectin inhibitors, the sialylation reaction at position 3 was
attempted with the N-acetyllactosamine unit compounds with
either an acrylamide or peptidic support. It is already shown
that α-(2,3)-sialyl transferase is suitable for modifying chem-
ically prepared small molecules of biological interest15 and
glycopolymers.11 Several sets of conditions including those
described by Yamada et al.11a or those involving an excess of
CMP-NANA and/or the use of Bovine Serum Albumin (BSA),
MnCl2 in solution in cacodylate buffer at different concen-
trations and pH values, failed to provide any sialylated deriv-
ative. Finally, α-(2,3)-sialylation of compound 33Ј was achieved
in sodium cacodylate buffer (50 mM, pH 6) containing 0.1%
Triton X-100. It required 3 equivalents of CMP-NANA and 0.3
U of α-(2,3)-sialyl transferase. After 5 days of incubation at
37 ЊC and purification using G-25 gel filtration, the sialylated
compound 37 was obtained in 47% yield. Its 1H NMR spectra
showed clearly a doublet of doublets at 2.64 ppm correspond-
ing to H-3eq of the sialyl residue. The extent of sialylation was
estimated to be almost quantitative.
In the next step, galactosylated copolymer 29 was sialylated
using α-(2,3)-sialyl transferase from rat liver. As the conditions
described by Yamada et al.11a (sodium cacodylate buffer, pH
7.4, MnCl2, Triton CF-54) for the sialylation of glycopolymers
failed to provide any sialylated compound in our cases, a new
set of conditions was employed. These are new, simple and
In the above case, as in the case of glycopolymers, no calf
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Scheme 7 Reagents and conditions: i, UDP-Gal (3 equiv. for 12 and 14, 1.3 equiv. for 13), α-lactalbumin (0.2 g dmϪ3), β-(1,4)-galactosyl transferase
(1 U), HEPES buffer (pH 6.5, 50 mM, 1 cm3), 37 ЊC, 72 h.
Scheme 8 Reagents and conditions: i, CMP-NANA (3 equiv.), α-(2,3)-sialyl transferase (0.3 U), sodium cacodylate buffer (pH 6.0, 50 mM), 0.1%
Triton X-100, 37 ЊC, 5 d. then GDP-Fucose (2 equiv.), α-(1,3)-fucosyl transferase V (10 mU), HEPES buffer (pH 7.5, 100 mM), NaN3, MnCl2ؒ4H2O,
37 ЊC, 3 d.
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Scheme 9 Reagents and conditions: i, Method I: UDP-Gal (3 equiv.), α-lactalbumin (0.2 g dmϪ3), β-(1,4)-galactosyl transferase (1 U), MnCl2 (5
mM), HEPES buffer (pH 7.0, 50 mM, 1 cm3), 37 ЊC, 26 h; Method II: UDP-Gal (3 equiv.), α-lactalbumin (0.2 g dmϪ3), β-(1,4)-galactosyl transferase
(1 U), MnCl2 (25 mM), MgCl2 (5 mM), dithiothreitol (0.2 mM), sodium cacodylate buffer (pH 7.50, 50 mM, 1 cm3), 37 ЊC, 72 h; ii, BSA, NaN3,
MnCl2, CMP-NANA (3 equiv.), calf intestinal alkaline phosphatase, α-(2,6)-sialyl transferase (1 U), sodium cacodylate buffer (pH 7.40, 50 mM),
37 ЊC, 48 h.
intestinal alkaline phosphatase was added to the reaction
mixture. Although it was shown that this enzyme improved
the yield of the galactosyl transferase reaction,16 in our reaction,
no significant difference was noticed. This can be explained by
the polymeric nature of the support we used. Indeed, the high
concentration of sugar units along the polymer chain led to a
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Subsequently, compound 37 was used to obtain the expected
multivalent sialyl Lewisx derivative. The reaction was carried
out as described by a previous report for acrylamide glyco-
polymers.11 It took place in 100 mM HEPES buffer containing
manganese chloride tetrahydrate and sodium azide at pH 7.5.
Two equivalents of GDP-fucose were used and the reaction
proceeded smoothly at 37 ЊC for 3 days in the presence of 10
mU of fucosyl transferase V and led to the N-acetyllactos-
aminyl derivative 38 bearing sialyl Lewisx pendant chains in
a quantitative yield (Scheme 10). As our aim is to produce
tools for glycotechnology using easily handled and efficient
protocols, the sialylation of compound 33Ј in position 6 was
also performed. This kind of derivative is also known to be an
influenza virus inhibitor. The reaction was carried out using
2 equivalents of CMP-NANA and 0.1 U of α-(2,6)-sialyl trans-
ferase in sodium cacodylate buffer (pH 7.4, 50 mM) at 37 ЊC for
48 h (Scheme 9). After purification by gel filtration chrom-
1
atography, H NMR spectroscopy of compound 36 showed
that the derivative had been sialylated in position 6 to an extent
of 70%. A doublet of doublets corresponding to H-3eq appeared
at 2.53 ppm. The presence of the sialyl residue was confirmed
by its 13C NMR spectra.
Conclusions
Several water-soluble glycopolymers and sequential glyco-
peptides bearing GlcNAc units have been synthesised
chemically and their sugar chain was then elongated enzym-
atically. All of these compounds but one turned out to be very
good substrates for galactosyl transferase and high degrees of
galactosylation were obtained together with good yields of N-
acetyllactosaminated derivatives. This demonstrates that the
methodology set up by Yamada et al. for galactosylation of
glycopolymers can be easily and efficiently extended to sequen-
tial glycopeptides. Although this method could not be extended
to the sialylation of sequential glycopeptides in position 3,
simple and efficient new conditions were investigated that easily
afforded this important intermediate in good yield in the con-
struction of multivalent sialyl Lewisx derivatives. This improved
methodology led to the construction of polymeric derivatives
32 and 38 bearing a multivalent sialyl Lewisx pattern after fuco-
sylation was applied. On the other hand, sequential glycopep-
tide 36 was successfully sialylated in position 6 using α-(2,6)-
sialyl transferase. These kinds of compounds are expected to be
useful tools in the study of sugar–lectin interactions and as
selectin inhibitors.
Experimental
General procedure
Unless otherwise stated all commercially available solvents
and reagents were used without further purification. CHCl3,
dichloroethane, DMF, dimethyl sulfoxide and MeOH were
stored over molecular sieves 3 Å before use. Et3N and pyridine
were stored over NaOH pellets. MS 4 Å were dried under
reduced pressure at ca. 100 ЊC before use. Reactions were moni-
tored by thin layer chromatography (TLC) on precoated plates
of silica gel 60F254 (layer thickness 0.25 mm; E. Merck, Darm-
stadt). Column chromatography was performed on silica gel
(silica gel 60N; 100–210 µm, Kanto Chemical Co., Inc.). Gel
filtration chromatography was performed using Sephadex
G-10, Ϫ15 or Ϫ25 (Pharmacia Biotech.). Polymer detection
polymer was performed using a UV detector (EYELA 9900) at
λ = 220 nm. Average molecular weights of glycopolymers were
estimated by gel permeation chromatography (GPC) with an
Asahipak GS-50 column, and pullulans (5.8, 12.2, 23.7, 48.0,
100.0, 186.0, and 380K; Shodex Standard P-82) were used as
standards. Enzymatic reactions were also performed using
commercially available materials. Galactosyl transferase from
bovine milk (Sigma) and sialyl transferase from rat liver
Scheme 10 Reagents and conditions: i, CMP-NANA (3 equiv.), α-(2,3)-
sialyl transferase (0.3 U), sodium cacodylate buffer (pH 6.0, 50 mM),
0.1% Triton X-100, 37 ЊC, 5 d. then GDP-fucose (2 equiv.), α-(1,3)-
fucosyl transferase V (10 mU), HEPES buffer (pH 7.5, 100 mM),
NaN3, MnCl2ؒ4H2O, 37 ЊC, 3 d.
stronger association constant between sugar residues on the
polymer and the enzyme.11 This might accelerate the total reac-
tion rate between donor and acceptor substrates.
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(Sigma) were used without further purification. Sugar com-
position of glycopolymers was determined from the integration
data of the 1H NMR spectra. Proton NMR spectra were
pressure. The residue was purified by column chromatography
(SiO2, chloroform–methanol = 90:10) to give 5.
To a solution of 5 (0.25 g, 0.66 mmol) in methanol (4 cm3)
was added a suspension of Pd–C (20 mg) in methanol (1 cm3).
The reaction mixture was stirred at room temperature for 3 h
under hydrogen, then the Pd–C was filtered off over Celite and
the filtrate was evaporated under reduced pressure. The free
amino compound thus obtained was freeze-dried and used
without further purification.
recorded at 400 MHz using Me4Si as internal standard and 13
C
NMR spectra were recorded at 100 MHz on a JEOL lambda
400 spectrometer. FAB mass spectra were recorded on a JEOL
JMS-SX102A spectrometer. Matrix-associated laser desorption
ionization–time of flight–mass spectrometry (MALDI-TOF-
MS) was performed on a Laser mat 2000 mass spectrometer.
Abbreviations used for MALDI-TOF matrices are: 3-AQ, 3-
aminoquinoline; cinnamic acid, α-cyano-4-hydroxycinnamic
acid.
To a solution of the crude amine (0.232 g, 0.66 mmol) in
methanol (5 cm3), under nitrogen and cooled to below 0 ЊC,
were added triethylamine (0.184 cm3, 1.32 mmol) and acryloyl
chloride (0.107 cm3, 1.32 mmol). The reaction mixture was
stirred for 4 h at room temperature then the solvent was evap-
orated under reduced pressure. The residue was purified
by column chromatography (SiO2, chloroform–methanol =
80:20). The monomer-containing fractions were recombined,
evaporated under reduced pressure and freeze-dried to give 6
(0.182 g, 68%) as a white powder (Found: C, 48.6; H, 7.7; N,
6.7. C17H30O9N2ؒ0.8H2O requires C, 48.5; H, 7.6; N, 6.65%);
δH (400 MHz; D2O): 1.93 (3H, s, COCH3), 3.32–3.48 (5H, m,
N-CH2, 4-H, 5-H and 3-H), 3.53–3.70 (11H, m, 5 × O-CH2 and
2-H), 3.80–3.93 (2H, m, 6-Ha and 6-Hb), 4.45 (1H, d, J 8.06,
Trichloroacetimidoyl 3,4,6-tri-O-acetyl-2-N-phthalimido-2-
deoxy-β--glucopyranoside 1, 2-[2-(2-azidoethoxy)ethoxy]-
ethanol
2 and 2-methyl-4,5-dihydro(3,4,6-tri-O-acetyl-1,2-
dideoxy-α--glucopyranoso)[2,1-d]-1,3-oxazole were synthe-
sised as previously described.8–10
Synthesis of the monosaccharidic part
2-[2-(2-Azidoethoxy)ethoxy]ethyl
phthalimido-2-deoxy-ꢀ-D-glucopyranoside 3. To a solution of
trichloroacetimidoyl 3,4,6-tri-O-acetyl-2-N-phthalimido-2-
3,4,6-tri-O-acetyl-2-N-
deoxy-β--glucopyranoside 1 (2.85 g, 4.92 mmol) in dichloro-
methane (10 cm3) containing molecular sieves 4 Å (3 g) and
under nitrogen was added 2-[2-(2-azidoethoxy)ethoxy]ethanol 2
(2.6 g, 14.76 mmol). The solution was cooled to below 0 ЊC and
BF3ؒEt2O (1.28 cm3, 4.92 mmol) was added. The solution was
stirred for 24 h under nitrogen, then triethylamine (1 cm3) was
added and the solution was stirred for another 15 min at room
temperature and then diluted with chloroform. The organic
layer was washed with icy saturated aq. NaHCO3 (100 cm3) and
brine (100 cm3), then dried (MgSO4) and evaporated under
reduced pressure. The residue was purified by column chrom-
atography (SiO2, toluene–ethyl acetate = 80:20) to afford 3
(2.38 g, 79%) as a colourless oil; δH (400 MHz; CDCl3): 1.86–
2.11 (9H, all s, 3 × COCH3), 3.30–3.54 (10H, m, OCH2), 3.69
(1H, m, 5-H), 3.89 (2H, m, CH2-N3), 4.17 (1H, dd, J 2.29 and
12.21, 6-Hb), 4.32 (2H, m, 6-Ha, 2-H), 5.16 (1H, t, J 9.92, 4-H),
5.43 (1H, d, J 8.55, 1-H), 5.80 (1H, dd, J 9 and 10.83, 3-H), 7.79
(4H, m, Phthal.).
1-H), 5.66 (1H, dd, J 1.46 and 10.36, CH᎐CH ), 6.07–6.22 (2H,
᎐
2
m, CH᎐CH ); m/z (FABϩ): 407 (21%, [M ϩ H]ϩ); (Found:
᎐
2
M ϩ H, 407.2042. C17H30O9N2 requires M ϩ H, 407.2028).
3-[(N-Benzyloxycarbonyl)amino]propyl 3,4,6-tri-O-acetyl-2-
N-acetamido-2-deoxy-ꢀ-D-glucopyranoside 7. To a solution of 2-
methyl(3,4,6-tri-O-acetyl-1,2-dideoxy-α--glucopyrano)[2,1-d ]-
2-oxazole (4.23 g, 12.84 mmol) in dichloroethane (50 cm3)
was added 3-N-[(benzyloxy)carbonyl]aminopropanol (6.7 g,
32.0 mmol) and camphor-10-sulfonic acid until pH 2–3. The
reaction mixture was heated at 70 ЊC for 3 h and then allowed to
cool at room temperature. The solvent was evaporated under
reduced pressure. The mixture was extracted with chloroform
(3 × 100 cm3) and the recombined organic layers were washed
with saturated aq. NaHCO3 (100 cm3) and brine (100 cm3) and
then dried (MgSO4). The solvent was evaporated under reduced
pressure and the resulting material was purified by column
chromatography (SiO2, toluene–ethyl acetate = 70:30) to afford
7 (3.20 g, 46%) as a pale yellow powder; δH (400 MHz, CDCl3):
1.77, 1.84 (2H, m, CH2), 1.94, 2.03, 2.07 (12H, all s,
4 × COCH3), 3.55 and 3.94 (2H, m, OCH2), 3.56 (1H, m, 5-H),
3.96 (1H, ddd, J 8.9, 2-H), 4.12 (1H, dd, J 2.3 and 12.2, 6-Ha),
4.23 (1H, dd, J 4.5 and 12.2, 6-Hb), 4.31 (1H, d, J 8.3, 1-H), 4.95
(1H, m, NHCOO), 5.0–5.16 (4H, m, 4-H and PhCH2), 6.38
(1H, d, J 8.6, NH), 7.31–7.40 (5H, m, C6H5).
2-[2-(2-Azidoethoxy)ethoxy]ethyl
3,4,6-tri-O-acetyl-2-N-
acetamido-2-deoxy-ꢀ-D-glucopyranoside 4. To a solution of 3
(0.911 g, 1.5 mmol) in n-butanol (6 cm3) was added ethylenedi-
amine (0.202 cm3, 3 mmol). The mixture was heated at 70 ЊC
under a nitrogen atmosphere for 24 h. The white precipitate that
appeared was filtered off and the filtrate was evaporated under
reduced pressure. After drying overnight under vacuum, the
crude compound was solubilized in a mixture of pyridine and
acetic anhydride (2:1, 15 cm3) and was stirred at room temper-
ature for 24 h. The solvents were evaporated under reduced
pressure and the residual material was poured into ice water (50
cm3) and extracted with chloroform (3 × 50 cm3). The com-
bined extracts were washed with saturated aq. NaHCO3 (50
cm3) and brine (50 cm3), then dried (MgSO4) and evaporated
under reduced pressure. The residue was purified by column
chromatography (SiO2, chloroform–methanol = 99:1) to afford
4 (0.318 g, 63%) as a white powder; δH (400 MHz; CDCl3): 1.97–
2.08 (12H, s, 4 × COCH3), 3.50–3.89, (13H, m, OCH2 and 5-H),
3.89 (1H, m, 5-H), 4.09–4.14 (2H, m, 6-Hb and 2-H), 4.25 (1H,
dd, J 4.4 and 11.72, 6-Ha), 4.77 (1H, d, J 8.79, 1-H), 5.06–5.16
(2H, m, 3-H and 4-H), 6.27 (1H, d, J 9.53, NH).
6-[(N-Benzyloxycarbonylamino]hexyl 3,4,6-tri-O-acetyl-2-N-
acetamido-2-deoxy-ꢀ-D-glucopyranoside 8. This compound
was obtained using the way described above for 7 starting
from 2-methyl-4,5-dihydro-(3,4,6-tri-O-acetyl-1,2-dideoxy-α--
glucopyranoso)[2,1-d]-1,3-oxazole and 6-[N-(benzyloxy)carb-
onylamino]hexanol (3.43 g, 46%); δH (400 MHz, CDCl3): 1.35
and 1.50 (8H, m, CH2), 1.93, 2.02, 2.03 and 2.08 (12H, all s,
4 × COCH3), 3.19 (2H, m, N-CH2), 3.46 and 3.80 (2H, m,
OCH2), 3.63 (1H, ddd, 2-H), 4.09 (1H, dd, J 2.3 and 12.2, 6-Ha),
4.25 (1H, dd, J 4.5 and 12.2, 6-Hb), 4.63 (1H, d, J 8.2, 1-H), 4.87
(1H, br s, NHCOO), 5.06 (1H, t, J 9.6, 4-H), 5.12 (2H, dd,
J 16.2, PhCH2), 5.29 (1H, t, J 9.6, 3-H), 5.93 (1H, d, J 8.6, NH),
7.30–7.37 (5H, m, Ph).
3-[(N-Benzyloxycarbonyl)amino]propyl
2-N-acetamido-2-
2-[2-(2-Acrylamidoethoxy)ethoxy]ethyl
2-N-acetamido-2-
deoxy-ꢀ-D-glucopyranoside 9. To a solution of 7 (3.2 g, 5.94
mmol) in methanol (10 cm3) was added sodium methoxide (0.32
g). The solution was stirred at room temperature for 2 h then
neutralized with DOWEX 50-X8 resin. The resin was filtered
off and filtrate was evaporated to dryness. The resulting
material was purified by column chromatography (SiO2,
deoxy-ꢀ-D-glucopyranoside 6. To a solution of 4 (0.3 g, 0.59
mmol) in methanol (5 cm3) was added sodium methoxide
(30 mg). The reaction mixture was stirred at room temperature
for 3 h, then neutralized with DOWEX-50W X8 resin. The resin
was filtered off and the filtrate was evaporated under reduced
2098
J. Chem. Soc., Perkin Trans. 1, 2000, 2091–2103

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chloroform–methanol = 80:20) to afford 9 as a slightly yellow
powder (1 g, 41%); δH (400 MHz, DMSO-d6): 1.52 (2H, m,
CH2), 1.73 (3H, s, COCH3), 2.97 (4H, m, 6-Ha, 6-Hb, N-CH2),
3.58–3.69 (2H, m, 2-H and 3-H), 4.17 (1H, d, J 8.30, 1-H), 4.49
(1H, t, J 5.86, 4-H), 4.86 (1H, d, J 5.37, O-CH2), 4.95 (3H, m,
COO-CH2-C6H5 and O-CH2), 7.15 (1H, br t, NH), 7.22–7.32
(5H, m, C6H5), 7.60 (1H, d, J 9.03, NH).
CH), 3.28 (1H, br s, 4-H), 4.33 (1H, d, J 8.54, 1-H); δC (100
MHz; D2O): 23.0 (COCH3), 25.5–29.3 (CH2, hex), 35.3 (CH2),
40.2 (N-CH2), 42.8 (CH), 56.3 (2-C), 61.4 (6-C), 70.6 (OCH2),
71.1 (4-C), 74.6 (3-C), 76.6 (5-C), 101.8 (1-C), 175.1 (COCH3),
180.2 (CONH2). M > 380,000 (GPC method).
Homopolymer 14 from monomer 6. H2O (3 cm3) was deaer-
ated for 1 h with a water aspirator. Monomer 6 (0.168 g, 0.414
mmol) was added along with TEMED (0.0.31 cm3, 0.207
mmol) and APS (0.047 g, 0.207 mmol). The solution was stirred
for 24 h at room temperature under nitrogen and directly
purified by gel filtration chromatography (Sephadex G-25,
42 × 2 cm) and eluted with water. Homopolymer 14 (0.083 g,
50%) was freeze-dried and obtained as a white powder; δH (400
MHz; D2O): 1.94 (3H, s, CH3), 3.34–3.89 (18H, m, 2-H, 3-H, 4-
H, 5-H, 6-H, O-(CH2)2 and NH-CH2), 4.45 (1H, d, J 8.06, 1-H);
δC (100 MHz; D2O): 25.5 (COCH3), 42.1 (N-CH2), 44.5
(CH), 58.5 (2-C), 63.8 (6-C), 71.7–73.0 (OCH2), 73.1 (4-C),
76.9 (3-C), 79.4 (5-C), 103.9 (1-C), 176.5 (COCH3); TOF-
MASS (negative mode, 3-AQ): 956.0 [2M ϩ matrix]ϩ, 1365.0
[3M ϩ 3H ϩ matrix]ϩ, 1787.7 [4M ϩ matrix ϩ H2O]ϩ, 2174.0
6-(Acrylamido)hexyl 2-N-acetamido-2-deoxy-ꢀ-D-glucopyr-
anoside 11. To a solution of 8 (3.2 g, 5.51 mmol) in methanol
(10 cm3) was added sodium methoxide (0.32 g). The solution
was stirred at room temperature for 2 h then neutralized with
DOWEX 50-X8 resin. The resin was filtered off and the filtrate
was evaporated to dryness. The resulting material was purified
by column chromatography (SiO2, chloroform–methanol =
80:20) to afford 10 as a slightly yellow powder (1.25 g, 50%). To
a solution of 10 (1.25 g, 2.75 mmol) in methanol (7 cm3) con-
taining a catalytic amount of acetic acid was added 10% Pd–C
(0.245 g) in suspension in the same solvent (3 cm3). The reaction
mixture was stirred under hydrogen, at room temperature for
4 h then Pd–C was filtered off over Celite and the filtrate was
evaporated under reduced pressure. The free amino compound
thus obtained was freeze-dried and used without further purifi-
cation. To a solution of crude amine (0.2 g, 0.625 mmol) in
methanol (5 cm3), under nitrogen and cooled to below 0 ЊC, was
added triethylamine (0.147 cm3, 1.25 mmol) and acryloyl
chloride (0.102 cm3, 1.25 mmol). The reaction mixture was
stirred at room temperature for 4 h, and then the solvent was
removed under reduced pressure. Monomer 11 was purified by
column chromatography (SiO2, chloroform–methanol = 80:20)
and the pure compound was freeze-dried (0.097 g, 41%)
(Found: C, 50.6; H, 8.3; N, 6.9. C17H30O9N2ؒ1.5H2O requires C,
50.9; H, 8.3; N, 7.0%); δH (400 MHz, D2O): 1.15–1.41 (8H, m,
4 × CH2), 1.77 (3H, s, COCH3), 3.04–3.09 (2H, m, N-CH2),
3.37–3.47 (2H, m, 6-Ha and 6-Hb), 3.63–3.70 (3H, m, 2-H, 3-H
and 5-H), 4.23 (1H, d, J 8.32, 1-H), 4.52 (1H, t, J 5.95, 4-H),
4.89 (1H, m, O-CH2), 4.97 (1H, m, O-CH2), 5.54 (1H, dd, J 2.37
[5M ϩ matrix]ϩ,
2586.6
[6M ϩ 6H ϩ matrix]ϩ,
2993.2
[7M ϩ 7H ϩ matrix]ϩ, 3394.3 [8M ϩ 2H ϩ matrix]ϩ.
Synthesis of the peptidic part
Boc-Ser-(OBn)2 15. Boc-Ser-(OBn) (2 g, 6.77 mmol) was
dissolved in 10% aqueous methanol (10 cm3) and caesium carb-
onate (1.10 g, 3.385 mmol) was added. The clear solution was
evaporated under reduced pressure several times with methanol
in order to remove H2O, then DMF (10 cm3) was added. The
reaction mixture was cooled to below 0 ЊC and benzyl bromide
(1.2 cm3, 10.15 mmol) was added. The mixture was stirred at
room temperature overnight then DMF was evaporated under
vacuum. The residue was extracted with chloroform (3 × 100
cm3) and the organic layers were washed with water (100 cm3)
then dried (MgSO4) and evaporated under reduced pressure.
The residue was purified by column chromatography (SiO2,
100% hexane) to give the title compound 15 (2.6 g, 100%) as a
colourless oil; δH (400 MHz; CDCl3): 1.44 (9H, s, C(CH3)3),
3.68 (1H, dd, J 9.46, Serβ), 3.89 (1H, dd, J 9.15, Serβ), 4.41–
4.51 (3H, m, Serα, O-CH2-C6H5), 5.12–5.24 (2H, m, COO-CH2-
Ph), 5.43 (1H, d, J 8.59, NH), 7.20–7.31 (10H, m, 2 × C6H5).
and 10.07, CH᎐CH2cis), 6.02 (1H, dd, J 2.33 and 17.09,
᎐
CH᎐CH
), 6.18 (1H, m, CH᎐CH ); m/z (FABϩ): 375 (10%,
᎐
2
᎐
2trans
M ϩ Hϩ); (Found: M ϩ H, 375.2140. C17H30O7N2 requires
M ϩ H, 375.2131).
Synthesis of polymers
Boc-Gly-Ser-(OBn)2 16. To a solution of 15 (2.6 g, 6.75
mmol) in dioxane (50 cm3), cooled to below 0 ЊC, was added
HCl (4 M) in dioxane (50 cm3). The reaction mixture was stirred
at room temperature for 3 h, and then the solvent was evap-
orated under reduced pressure. The free amino benzylated
serine derivative crystallized from diethyl ether as a white
powder (1.5 g, 69%) and was used without further purification.
A solution of this free amino derivative (1.5 g, 4.66 mmol)
and Boc-Glycine (1.23 g, 6.99 mmol) in DMF (20 cm3) was
cooled to below 0 ЊC. DPPA (1.7 cm3, 7.92 mmol) and triethyl-
amine (0.975 cm3, 6.99 mmol) were added and the solution was
stirred for 2 h at 0 ЊC then at room temperature overnight.
DMF was evaporated under vacuum and the residue was
extracted with chloroform (3 × 100 cm3). The extracts were
washed successively with 5% citric acid (100 cm3), 5% NaHCO3
(100 cm3), and brine (100 cm3), then dried (MgSO4) and evap-
orated under reduced pressure. The resulting material was
purified by column chromatography (SiO2, hexane–ethyl
acetate = 70:30) to afford the dipeptide 16 (1.7 g, 83% ) as a
colourless oil; δH (400 MHz; CDCl3): 1.43 (9H, s, C(CH3)3),
3.67 (1H, dd, J 3.2 and 9.61, Serβ), 3.82 (1H, br d, Glyα), 3.91
(1H, dd, J 3.05 and 9.61, Serβ), 4.41 (2H, dd, J 12.21 and 16.78,
O-CH2-C6H5), 4.79 (1H, m, Serα), 5.16 (2H, m, COO-CH2-Ph),
6.95 (1H, d, J 7.93, NH), 7.18–7.32 (10H, m, 2 × C6H5).
Acrylamide copolymer 12 from monomer 6. A mixture of
DMSO–H2O (5:1, 3 cm3) was deaerated for 1 h with a water
aspirator. Monomer 6 (0.130 g, 0.32 mmol) and acrylamide
(0.091 g, 1.28 mmol) were added with TEMED (0.024 cm3, 0.16
mmol) and APS (0.0365 g, 0.16 mmol). The solution was stirred
for 24 h at room temperature under nitrogen and directly
purified by gel filtration chromatography (Sephadex G-25,
41 × 2 cm) and eluted with deionized water. Copolymer 12 (0.17
g, 77%) was freeze-dried and obtained as a white powder;
δH (400 MHz; D2O): 1.5–1.62 (12H, br m, CH2), 1.90 (3H, s,
COCH3), 2.05–2.20 (2H, br m, CH), 3.31 (1H, br s, 4-H), 4.42
(1H, d, J 8.24, 1-H); δC (100 MHz; D2O): 23.02 (COCH3), 35.5
(CH2 ), 39.6 (CH), 43.0 (N-CH2), 56.2 (2-C), 61.5 (6-C), 69.4–
70.4 (OCH2), 70.6 (4-C), 74.6 (3-C), 76.7 (5-C), 101.9 (1-C),
175.2 (COCH3), 180.2 (CONH2). M ~ 135,000 (GPC method).
Acrylamide copolymer 13 from monomer 11. A mixture of
DMSO–H2O (5:1, 3 cm3) was deaerated for 1 h with a water
aspirator. Monomer 11 (0.09 g, 0.24 mmol) and acrylamide
(0.0685 g, 0.96 mmol) were added followed by TEMED (0.018
cm3, 0.12 mmol) and APS (0.0275 g, 0.12 mmol). The solution
was stirred for 48 h at room temperature under nitrogen and
directly purified by gel filtration chromatography (Sephadex
G-25, 42 × 2 cm) and eluted with deionized water. Copolymer
13 (0.08 g, 89%) was freeze-dried and obtained as a white
powder; δH (400 MHz; D2O): 1.16 (4H, br s, 4 × CH2), 1.48–
1.60 (21H, br m, CH2), 1.87 (3H, s, CH3), 2.0–2.14 (15H, br m,
Fmoc-Asp(OtBu)-Gly-Ser-(OBn)2 17. To a solution of 16
(1.6 g, 3.61 mmol) in dioxane (30 cm3), cooled to below 0 ЊC,
J. Chem. Soc., Perkin Trans. 1, 2000, 2091–2103
2099

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was added HCl (4 M) in dioxane (30 cm3). The reaction mixture
was stirred at room temperature for 3 h, then the solvent was
evaporated under reduced pressure. The free amino dipeptide
crystallized from diethyl ether as a white powder (1.09 g, 80%)
and was used without further purification.
Asp), 39.8 (CH2-NH), 42.9 (Cα, Gly), 47.0 (CH, Fmoc), 53.0
(Cα Ser), 56.1 (2-C), 61.5 (6-C), 67.2, 68.5 (CH2, Fmoc,
O-CH2C6H5), 70.1 (O-CH2), 73.0 (4-C), 74.7 (3-C), 75.9 (5-C),
101.5 (1-C), 119.9–128.5 (2 × C6H5), 135.2–143.8 (CHAr,
Fmoc), 156.4 (OCONH), 170.1(CO); m/z (FABϩ): 1014 [M ϩ
H]ϩ (Found: M ϩ H, 1014.4330. C52H63O16N5 requires M ϩ H,
1014.4347).
A solution of this dipeptide (1.09 g, 2.89 mmol) and Fmoc-
Asp(OtBu) (1.43 g, 3.47 mmol) in DMF (20 cm3) was cooled to
below 0 ЊC. DPPA (0.811 cm3, 3.76 mmol) and triethylamine
(0.484 cm3, 3.47 mmol) were added and the solution was stirred
for 2 h at 0 ЊC and then at room temperature overnight. DMF
was evaporated under vacuum and the residue was extracted
with chloroform. The extracts were washed with 5% citric
acid (100 cm3), and brine (100 cm3), then dried (MgSO4) and
evaporated under reduced pressure. The resulting material was
purified by column chromatography (SiO2, toluene–ethyl acet-
ate = 70:30) to afford the tripeptide 17 (1.26 g, 59%) as a white
powder (Found: C, 68.6; H, 6.4; N, 5.5. C42H45O9N3 requires C,
68.6; H, 6.2; N, 5.7%); δH (400 MHz; CDCl3): 1.42 (9H, s,
C(CH3)3), 2.65 (1H, dd, J 5.2 and 17.8, Aspβ), 2.90 (1H, dd,
J 6.0 and 17.2, Aspβ), 3.67 (1H, dd, J 3.2 and 9.60, Serβ), 3.89
(1H, dd, J 3.2 and 9.6, Serβ), 3.94–4.03 (2H, m, Glyα), 4.2 (1H,
t, J 6.7, CH Fmoc), 4.44 (5H, m, Aspα, CH2 Fmoc, O-CH2-
C6H5), 4.79 (1H, m, Serα), 5.15 (2H, m, COO-CH2-Ph), 5.85
(1H, d, J 8.4, NH), 6.81 (1H, d, J 7.60, NH), 7.02 (1H, br d,
NH), 7.18–7.80 (18H, m, CHAr, Fmoc and 2 × C6H5); m/z
(FABϩ): 736 [M ϩ H]ϩ, 680 [M ϩ H Ϫ t-Bu]ϩ (Found: M ϩ H,
736.3248. C42H45O9N3 requires M ϩ H, 736.3234).
Fmoc-Asp(CONH-[CH2]6-O-GlcNac)-Gly-Ser-(OBn)2
20.
This compound was obtained via the general procedure
described above starting from the amino derivative from 10
(0.181 g, 0.57 mmol) and acid 18 (0.5 g, 0.74 mmol). The residue
was purified by column chromatography (SiO2, chloroform–
methanol = 90:10 then 85:15) to afford the protected glyco-
peptide 20 (0.344 g, 62%) as a white powder (Found: C, 62.0; H,
6.4; N, 6.7. C52H63O14N5ؒ1.5H2O requires C, 61.9; H, 6.7; N,
6.9%); δC (100 MHz; DMSO-d6): 23.1 (COCH3), 25.2, 26.2,
29.0, 29.1 (4 × CH2, hexyl), 37.6 (Cβ, Asp), 38.6 (CH2-NH),
41.9 (Cα, Gly), 46.6 (CH, Fmoc), 51.8 (Cα, Asp), 52.5 (CαSer),
55.4 (2-C), 61.0 (6-C), 65.8, 66.05, 68.2 (CH2 Fmoc, O-CH2-
C6H5, COO-CH2-C6H5), 69.1 (Cβ, Ser), 70.6 (OCH2), 72.2 (4-
C), 74.3 (3-C), 77.0 (5-C), 101.0 (1-C), 120.15–128.4 (2 × C6H5),
135.85–143.8 (CHAr, Fmoc), 155.8 (OCONH), 168.9, 169.0,
169.05, 169.9, 171.5 (CO); m/z (FABϩ): 982 [M ϩ H]ϩ (Found:
M ϩ H, 982.4474. C52H63O14N5 requires M ϩ H, 982.4450).
Fmoc-Asp(CONH-[CH2]3-O-GlcNac)-Gly-Ser-(OBn)2
21.
This compound was obtained via the general procedure
described above starting from the amino derivative derived
from 9 (0.2 g, 0.72 mmol) and acid 18 (0.636 g, 0.93 mmol). The
residue was purified by column chromatography (SiO2,
chloroform–methanol = 95:5 then 90:10) to afford the pro-
tected glycopeptide 21 (0.4 g, 59%) as a white powder (Found:
C, 61.2; H, 6.2; N, 7.15. C49H57O14N5ؒ1H2O requires C, 61.4; H,
6.2; N, 7.3%); δC (100 MHz; DMSO-d6): 23.05 (COCH3), 29.1
(CH2, propyl), 35.9 (Cβ, Asp), 37.6 (CH2 -NH), 41.9 (Cα, Gly),
46.6 (CH, Fmoc), 51.8 (Cα, Asp), 52.4 (Cα, Ser), 55.4 (2-C),
61.05 (6-C), 65.8, 66.1, 66.4 (CH2, Fmoc, O-CH2-C6H5, COO-
CH2-C6H5), 69.1 (Cβ, Ser), 70.6 (OCH2), 72.2 (4-C), 74.3 (3-C),
76.9 (5-C), 101.2 (1-C), 120.1–143.8 (CHAr, Fmoc and
2 × C6H5), 155.8 (OCONH), 169.1, 169.15, 169.45, 169.9,
171.55 (CO); m/z (FABϩ): 940 [M ϩ H]ϩ (Found: M ϩ H,
940.4004. C49H57O14N5 requires M ϩ H, 940.3980).
Fmoc-Asp-Gly-Ser-(OBn)2 18. Tripeptide 17 (1.26 g, 1.70
mmol) in solution in TFA (12 cm3) was stirred at room temper-
ature for 2 h. TFA was evaporated under reduced pressure and
the residue was purified by column chromatography (SiO2,
chloroform–methanol = 95:5) to afford the free β-carb-
oxytripeptide 18 (1.04 g, 90%) as a white powder (Found: C,
65.8; H, 5.6; N, 6.0. C38H37O9N3ؒ0.8H2O requires C, 65.75; H,
5.6; N, 6.05%); δH (400 MHz; DMSO-d6): 2.62 (1H, dd, J 3.17
and 16.6, Aspβ), 3.55 (1H, dd, J 4.15 and 9.52, Serβ), 3.67–3.83
(3H, m, Serβ and Glyα), 4.13–4.43 (6H, m, CH Fmoc, CH2
Fmoc, Aspα, O-CH2-C6H5), 4.55 (1H, br m, Serα), 5.06 (2H,
dd, J 4.15 and 16.84, COOCH2C6H5), 7.16–7.83 (18H, m,
CHAr, Fmoc and 2 × C6H5), 8.11 (1H, br t, NH), 8.28 (1H, d,
J 7.8, NH); δC (100 MHz; DMSO-d6): 36.3 (Cβ, Asp), 41.9
(Cα, Gly), 46.6 (CH, Fmoc), 51.45 (Cα, Asp), 52.5 (Cα Ser),
65.85, 66.1 (CH2 Fmoc, COOCH2-C6H5), 69.2 (Cβ, Ser), 72.25
(O-CH2-C6H5), 120.2–128.4 (2 × C6H5), 135.85–143.8 (CHAr,
Fmoc), 156.0 (OCONH), 169.0, 169.9, 171.25, 171.8 (CO); m/z
(FABϩ): 680 [M ϩ H]ϩ (Found: M ϩ H, 680.2612. C38H37O9N3
requires M ϩ H, 680.2608).
General procedure for the full deprotection of glycopeptides
The Fmoc amino protecting group of the glycopeptides 19, 20
or 21 was removed using 10% piperidine in DMF. The solution
was stirred for 2 h at room temperature, then DMF was evap-
orated in vacuo. The residual material was decanted 3 times
with a hexane–diethyl ether mixture (3:1) to remove the di-
benzofulvene and dried overnight under vacuum. It was used
for the next step without further purification.
Crude free amino compound was dissolved in a mixture of
H2O and acetic acid (2:8, 3 cm3), and 10% Pd–C (50–70% w/w)
in suspension in the same solvent (2 cm3) was added. The
reaction mixture was stirred at room temperature, under hydro-
gen for 24 h. Then, the Pd–C was removed by filtration over
Celite and the filtrate was evaporated under reduced pressure.
The residue was directly subjected to gel filtration chrom-
atography (Sephadex G-10, 41 × 1 cm) using H2O as eluent.
The fractions containing the compound were recombined, con-
centrated under reduced pressure and freeze-dried to afford the
deprotected glycopeptide 22, 23 or 24 as a white powder.
Synthesis of glycopeptides
General procedure. The free amino derivatives required were
first obtained from derivative 5 by reduction of the azido group
or from derivatives 9 or 10 by removal of the Z protecting
group. Then, to a solution of these crude amino sugars and
tripeptide 18 (1.3 equiv.) in a mixture of ethanol and benzene
(1:1, 10 cm3) was added EEDQ (1.4 equiv.). The reaction
mixture was stirred for 24 h at room temperature under
nitrogen, and then the solvents were evaporated under reduced
pressure. The residues were purified by column chromatography
to afford the protected glycopeptides 19, 20 or 21.
Fmoc-Asp(CONH-[CH2]2-O-[CH2O]2-GlcNac)-Gly-Ser-
(OBn)2 19. This compound was obtained via the general pro-
cedure described above starting from the amino derivative
afforded by 5 (0.07 g, 0.2 mmol) and acid 18 (0.177 g, 0.26
mmol). The residue was purified by column chromatography
(SiO2, chloroform–methanol = 90:10) to afford the protected
glycopeptide 19 (0.155 mg, 77%) as a white powder (Found: C,
58.45; H, 6.1; N, 6.5. C52H63O16N5ؒ3H2O requires C, 58.5; H,
5.9; N, 6.6%); δC (100 MHz; CDCl3): 23.4 (COCH3), 38.4 (Cβ,
Asp(CONH-[CH2]2-O-[CH2O]2-GlcNac)-Gly-Ser 22. Com-
pound 19 (155 mg, 0.15 mmol) was used to prepare compound
22 (64 mg, 68.5%) via the general procedure described above
(Found: C, 42.05; H, 6.6; N, 9.9. C23H41O14N5ؒ2.5H2O requires
C, 42.1; H, 7.05; N, 10.7%); δC (100 MHz; D2O): 22.9 (COCH3),
36.2 (Cβ, Asp), 39.7 (CH2-NH), 43.2 (Cα, Gly), 50.8 (Cα, Asp),
2100
J. Chem. Soc., Perkin Trans. 1, 2000, 2091–2103

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56.2 αSer), 57.7 (2-C), 61.4 (Cβ, Ser), 62.6 (6-C), 69.4–70.4
(OCH2), 70.6 (4-C), 74.6 (3-C), 76.6 (5-C), 101.8 (1-C), 170.1,
170.8, 171.0, 175.2, 176.5 (CO); m/z (FABϩ): 612 [M ϩ H]ϩ
(Found: M ϩ H, 612.2719. C23H41O14N5 requires M ϩ H,
612.2728).
procedure described above. The average number of repeating
units was found to be from n = 2 to n = 5; δC (100 MHz; D2O):
22.85 (COCH3), 29.05 (CH2, propyl), 37.0 (Cβ, Asp), 43.4 (Cα,
Gly), 51.6 (Cα, Asp), 56.3 (Cα, Ser), 57.6 (2-C), 61.45 (Cβ, Ser),
62.5 (6-C), 68.4 (OCH2), 70.6 (4-C), 74.5 (3-C), 76.6 (5-C),
101.9 (1-C), 172.1, 172.2, 172.6, 172.9, 175.3 (CO); TOF-MASS
(positive mode, cinnamic acid): 1059.4 [2M ϩ H Ϫ H2O]ϩ,
1579.9 [3M ϩ 2H Ϫ 2H2O]ϩ, 2098.9 [4M ϩ 3H Ϫ 3H2O]ϩ,
2619.9 [5M ϩ 4H Ϫ 4H2O]ϩ.
Asp(CONH-[CH2]6-O-GlcNac)-Gly-Ser 23. Compound 20
(270 mg, 0.28 mmol) was used to prepare compound 23 (134
mg, 84%) via the general procedure described above (Found: C,
43.9; H, 6.7; N, 10.8. C23H41O12N5ؒ3H2O requires C, 43.6; H,
7.5; N, 11.0%); δC (100 MHz; D2O): 22.9 (COCH3), 25.5, 26.4,
28.9, 29.2 (4 × CH2, hexyl), 36.3 (Cβ, Asp), 40.2 (CH2NH), 43.2
(Cα, Gly), 50.9 (Cα, Asp), 56.3 (Cα, Ser), 57.8 (2-C), 61.4 (Cβ,
Ser), 62.7 (6-C), 70.6 (OCH2), 71.1 (4-C), 74.5 (3-C), 76.6 (5-C),
101.8 (1-C), 170.1, 170.4, 170.9, 175.1, 176.6 (CO); m/z (FABϩ):
580 [M ϩ H]ϩ (Found: M ϩ H, 580.2845. C23H41O12N5 requires
M ϩ H, 580.2830).
Enzymatic elongation of the primer polymer
General procedure for the enzymatic galactosylation reaction.
To acrylamide copolymer 12 or 13 or homopolymer 14 in
HEPES buffer (pH 6, 50 mM, 1 cm3) were added successively
UDP-galactose (3 equiv. for 12 and 14, 1.3 equiv. for 13), α-
lactalbumin (0.2 g dmϪ3) and β-(1,4)-galactosyl transferase
(1 U). The solution was incubated at 37 ЊC for 72 h. The mix-
ture was directly subjected to gel filtration chromatography
(Sephadex G-25, 42 × 2 cm) and eluted with water. The
fractions containing galactosylated polymer were recombined,
concentrated under reduced pressure and freeze-dried to afford
the copolymer derivatives 28, 29 or the homopolymer 30 as
white powders.
Galactosylation of copolymer 12. Copolymer 12 (10 mg,
~ 0.014 mmol GlcNAc) was used to prepare galactosylated
copolymer 28 (10.4 mg, 84%, galactosylation extent: 50%) via
the general procedure described above; δH (400 MHz; D2O):
1.86 (3H, s, CH3), 3.28 (1H, br s, 4-H), 4.30 (1H, d, J 7.81, 1-H),
4.39 (1H, br t, 1Ј-H); δC (100 MHz; D2O): 23.0 (COCH3), 36.6
(CH2), 39.7 (CH2NH), 42.3–42.9 (CH), 55.7, 56.2 (2-C and 2-C
non substituted units), 60.8, 61.5, 61.8 (6-C, 6-C non substi-
tuted units, 6Ј-C), 69.3 (4Ј-C), 70.2–70.5 (OCH2 ), 71.7 (4-C non
substituted), 73.2 (2Ј-C), 74.6 (3-C), 75.5 (5Ј-C), 76.1 (3Ј-C),
76.6 (5-C), 79.1 (4-C), 101.8 (1-C), 103.6 (1Ј-C), 175.2
(COCH3), 177.4 (CO), 180.2 (CONH2).
Asp(CONH-[CH2]3-O-GlcNac)-Gly-Ser 24. Compound 21
(0.407 g, 0.43 mmol) was used to prepare compound 24 (0.142
g, 61%) via the general procedure described above (Found: C,
41.4; H, 6.6; N, 11.6. C20H35O12N5ؒ2.5H2O requires C, 41.2; H,
6.9; N, 12.0%); δC (100 MHz; D2O): 22.8 (COCH3), 28.9 (CH2,
propyl), 36.3 (Cβ, Asp), 37.1 (CH2-NH), 43.2 (Cα, Gly), 50.8
(Cα, Asp), 56.25 (Cα, Ser), 57.7 (2-C), 61.4 (Cβ, Ser), 62.6
(6-C), 68.5 (OCH2), 70.6 (4-C), 74.4 (3-C), 76.6 (5-C), 101.85
(1-C), 170.0, 170.5, 170.9, 175.3, 176.5 (CO); m/z (FABϩ): 538
[M ϩ H]ϩ (Found: M ϩ H, 538.2377. C20H35O12N5 requires
M ϩ H, 538.2360).
General procedure for the polymerisation of glycopeptides
A solution of glycopeptide 22, 23 or 24 (0.07 to 0.15 mmol) in
DMSO (0.2 to 0.6 cm3) was kept under nitrogen and at room
temperature for a few minutes before DPPA (0.084 to 0.18
mmol) and triethylamine (0.084 to 0.18 mmol) were added. The
reaction mixture was stirred vigorously at room temperature
and under nitrogen for 4 days, and was then directly subjected
to gel filtration chromatography (Sephadex G-25, 42 × 2 cm)
using water as eluent. The fractions containing the compound
were recombined, concentrated under reduced pressure and
freeze-dried to afford the sequential glycopeptides 25, 26 or 27
as white powders. The average number of repeating units was
determined using MALDI-TOF mass spectrometry.
Galactosylation of copolymers 13. Copolymer 13 (10 mg,
~ 0.01 mmol GlcNAc) was used to prepare galactosylated
copolymer 29 (11.6 mg, 100%, galactosylation extent: 100%) via
the general procedure described above; δH (400 MHz; D2O):
1.55 (4H, br s, CH2, hexyl), 1.86 (3H, s, CH3), 4.34 (2H, m, 1-H
and 1Ј-H); δC (100 MHz; D2O): 22.8 (COCH3), 25.4–29.1 (CH2,
hexyl), 35.5 (CH2), 40.1 (CH2NH), 42.4–42.7 (CH), 55.9 (2-C),
60.9 (6-C), 61.6 (6Ј-C), 69.1 (OCH2), 72.9 (2Ј-C), 76.1 (3Ј-C),
79.2 (4-C), 101.9 (1-C), 103.6 (1Ј-C), 180.2 (CONH2).
Polymerisation of 22. Compound 22 (45.2 mg, 0.074 mmol)
was used to prepare compound 25 (33 mg, 73%) via the general
procedure described above. The average number of repeating
units was found to be from n = 2 to n = 4; δC (100 MHz; D2O):
23.1 (COCH3), 37.5 (Cβ, Asp), 39.85 (CH2-NH), 43.6 (Cα,
Gly), 51.6 (Cα, Asp), 56.3 (2-C), 61.6 (Cβ, Ser), 62.0 (6-C),
69.6–70.5 (OCH2), 71.8 (4-C), 74.7 (3-C), 76.7 (5-C), 101.8
(1-C), 172.3, 172.4, 172.9, 173.1, 175.3 (CO); TOF-MASS
(positive mode, cinnamic acid): 1223.2 [2M ϩ H]ϩ, 1833.8
[3M ϩ H]ϩ, 2446.4 [4M ϩ 2H]ϩ, 3058.0 [5M ϩ 3H]ϩ.
Galactosylation of homopolymer 14. Homopolymer 14 (10
mg, ~ 0.025 mmol GlcNAc) was used to prepare galactosylated
homopolymer 30 (7.1 mg, 50%, galactosylation extent: 50%) via
the general procedure described above; δH (400 MHz; D2O):
1.87 (3H, s, CH3), 3.27 (1H, br s, 4-H), 4.29 (1H, d, 1-H), 4.38
(1H, br t, 1Ј-H); δC (100 MHz; D2O): 23.1 (COCH3), 39.6 (CH2-
NH), 55.7 (2-C), 60.8 (6-C), 61.5 (6Ј-C), 69.3 (4Ј-C), 69.7–70.7
(OCH2), 71.7 (4-C, non substituted), 73.2 (2Ј-C), 74.6 (3-C),
75.5 (5Ј-C), 76.1 (3Ј-C), 76.7 (5-C), 79.1 (4-C), 101.6 (1-C),
103.6 (1Ј-C), 175.0 (COCH3), 176.9 (CO).
α-(2,3)-Sialylation of copolymer 29. To a solution of 29
(3 mg, ~ 0.006 mmol LacNAc) in sodium cacodylate buffer (50
mM, pH 6.0, 1 cm3) containing 0.1% Triton X-100 were added
successively cytidine 5Ј-monophospho-N-acetylneuraminic
acid (11 mg, 0.018 mmol), and α-(2,3)-sialyl transferase (0.3 U).
The reaction mixture was incubated at 37 ЊC for 5 days, then
directly subjected to gel filtration chromatography (G-25,
42 × 2 cm, eluent H2O) to afford the sialylated glycopolymer 31
(6.5 mg, 75%) as a white powder after freeze-drying; δH (400
MHz; D2O): 1.11 (4H, br s, CH2), 1.82 (6H, br s, 2 × CH3), 2.54
(1H, dd, J 4.58 and 12.36, 3-Heq), 3.27 (1H, br s, 4-H), 4.33 (2H,
m, 1-H and 1Ј-H); δC (100 MHz; D2O): 22.7, 23.0 (2 × COCH3),
25.4–30.9 (CH2, hexyl), 35.6–36.6 (CH2), 40.1, 40.3 (3Љ-C, CH2-
NH), 42.05–42.8 (CH), 52.3 (5Љ-C), 55.8 (2-C), 60.7 (6-C), 61.7
(6Ј-C), 63.2 (9Љ-C), 68.1 (4Ј-C), 68.8 (7Љ-C), 69.1 (4Љ-C), 69.8–
Polymerisation of 23. Compound 23 (66.5 mg, 0.11 mmol)
was used to prepare compound 26 (43 mg, 65%) via the general
procedure described above. The average number of repeating
units was found to be from n = 2 to n = 4; δC (100 MHz; D2O):
22.9 (COCH3), 25.4, 26.4, 29.0, 29.2 (CH2, hexyl), 37.3 (Cβ,
Asp), 40.1 (CH2-NH), 43.4 (Cα, Gly), 51.5 (Cα, Asp), 56.3 (Cα,
Ser), 56.6 (2-C), 61.4 (Cβ, Ser), 61.6 (6-C), 70.6 (OCH2), 71.05
(4-C), 74.5 (3-C), 76.5 (5-C), 101.8 (1-C), 171.9, 172.2, 172.3,
173.0, 175.1 (CO); TOF-MASS (positive mode, cinnamic acid):
1141.2 [2M ϩ H Ϫ H2O]ϩ, 1703.9 [3M ϩ H Ϫ 2H2O]ϩ, 2264.4
[4M ϩ 2H Ϫ 3H2O]ϩ.
Polymerisation of 24. Compound 24 (68 mg, 0.13 mmol) was
used to prepare compound 27 (45 mg, 66%) via the general
J. Chem. Soc., Perkin Trans. 1, 2000, 2091–2103
2101

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78.9 (4Ј-C, 7Љ-C, 4Љ-C, 2Ј-C, 8Љ-C, 3-C, 6Љ-C, 5-C, 5Ј-C,
3Ј-C, 4-C), 100.5 (2Љ-C), 101.8 (1Ј-C), 103.3 (1-C), 174.6–180.2
(CO).
Galactosylation of 26 (Method II). Compound 26 (12.4 mg,
0.021 mmol GlcNAc) was used to prepare 35 (10 mg, 63%) via
the procedure described above for 33Ј. The reaction mixture
was incubated for 72 h and UDP-Gal was added after 22 and
26 h of incubation respectively; δC (100 MHz; D2O): 22.9
(COCH3), 25.45, 26.4, 29.0, 29.2 (CH2, hexyl), 40.2 (N-CH2),
43.4 (Cα, Gly), 51.5 (Cα, Asp), 55.8 (Cα, Ser), 56.3 (2-C),
60.8 (Cβ, Ser), 61.5 (6-C), 61.7 (6Ј-C), 69.3 (OCH2), 71.4 (4-C,
non substituted), 71.7 (4Ј-C), 73.2 (2Ј-C), 74.5 (3-C), 75.4
(5Ј-C), 76.1 (3Ј-C), 76.6 (5-C), 79.1 (4-C), 101.8 (1-C), 103.6
(1Ј-C), 172.25–175.0 (CO); TOF-MASS (positive mode,
cinnamic acid): 1303.4 [2M ϩ 1Gal Ϫ H2O]ϩ, 1465.2 [2M ϩ
2Gal Ϫ 2H2O]ϩ, 2029.5 [3M ϩ 2Gal Ϫ 2H2O]ϩ, 2188.8 [3M ϩ
3Gal Ϫ 2H2O]ϩ.
Sialyl Lewisx derivative 32. To a solution of HEPES buffer
(100 mM, 20 cm3) were added MnCl2ؒ4H2O (10 mM, 36.9 mg)
and NaN3 (1 mM, 1.3 mg). The resulting solution was adjusted
to pH 7.5. To a solution of 31 (6.5 mg, ~ 0.0044 mmol
SialLacNAc) in the buffer described above (1 cm3) were added
successively cytidine 5Ј-monophospho-N-acetylneuraminic
acid (8.4 mg, 0.013 mmol), and α-(1,3)-fucosyl transferaseV (10
mU). The reaction mixture was incubated at 37 ЊC for 3 days,
then directly subjected to gel filtration chromatography (G-25,
42 × 2 cm, eluent H2O) to afford the sialyl Lewisx glycopolymer
32 (1.5 mg, 21%) as a white powder after freeze-drying; δH (400
MHz; D2O, 70 ЊC): 1.48 (3H, d, J 6.26, CH3 Fuc), 1.63 (4H,
br s, CH2), 2.34 (6H, s, 2 × CH3), 3.07 (1H, br dd, 3Љ-Heq), 3.46
(2H, m, CH2), 4.83 (1H, m, 1-H Fuc).
ꢁ-2,6-Sialylation of 33Ј. A solution of BSA (20 mg), NaN3
(9.34 mg) and MnCl2 (312 mg) in sodium cacodylate buffer (50
mM, 10 cm3) was adjusted to pH 7.40. To this solution (1 cm3)
was added successively 33Ј (8.54 mg, ~ 0.012 mmol LacNAc),
cytidine 5Ј-monophospho-N-acetylneuraminic acid (15 mg,
0.024 mmol), calf intestinal alkaline phosphatase (10 U) and
α-(2,6)-sialyl transferase (0.1 U). The reaction mixture was
incubated at 37 ЊC for 48 h, and then directly subjected to gel
filtration chromatography (G-25, 42 × 2 cm, eluent H2O) to
afford the sequential sialylated glycopeptide 36 (7 mg, 58%) as
a white powder after freeze-drying; δC (100 MHz; D2O): 22.7,
23.3 (2 × COC H3), 29.0 (CH2, propyl), 36.9 (Cβ, Asp), 40.8
(CH2-NH), 43.3 (Cα, Gly), 48.2 (3Љ-C), 51.5, 51.7 (Cα, Asp, Cα,
Ser), 55.6 (2-C), 61.1 (6-C), 61.8 (Cβ, Ser), 63.3 (9Љ-C), 64.0
(6Ј-C), 68.4 (OCH2), 68.9–69.2 (4Ј-C, 4Љ-C, 2Љ-C), 72.4 (8Љ-C),
71.4 (2Ј-C), 73.1 (3-C), 73.2 (6Љ-C), 73.3 (5Ј-C), 75.5 (3Ј-C), 74.4
(5-C), 75.2 (4-C), 100.85 (2Љ-C), 101.6 (1-C), 104.2 (1Ј-C), 134.1
(COOH), 174.3, 175.6 (CO).
Enzymatic elongation of sequential glycopeptides
Galactosylation of 27 (Method I). To a solution of 27 (16 mg,
~0.03 mmol GlcNAc) in HEPES buffer (50 mM, pH 7, 1 cm3)
containing MnCl2 (5 mM) were added successively UDP-
galactose (54.6 mg, 0.09 mmol), α-lactalbumin (0.2 g dmϪ3) and
β-(1,4)-galactosyl transferase (1 U). The reaction mixture was
then incubated at 37 ЊC for 26 h and then directly subjected to
gel filtration chromatography (G-25, 42 × 2 cm, eluent H2O) to
afford the galactosylated sequential glycopeptide 33 (20 mg,
96%) as a white powder after freeze-drying; δC (100 MHz;
D2O): 22.9 (COCH3), 29.05 (CH2, propyl), 37.05 (Cβ, Asp),
43.4 (Cα, Gly), 51.6 (Cα, Asp), 55.8 (Cα, Ser), 56.3 (Cα, Ser),
60.75 (Cβ, Ser), 61.3 (6-C), 61.7 (6Ј-C), 68.4 (OCH2), 69.2
(4Ј-C), 71.7 (4-C, non substituted), 73.2 (2Ј-C), 74.5 (3-C), 75.5
(5Ј-C), 76.05 (3Ј-C), 76.55 (5-C), 79.1 (4-C), 101.8 (1-C),
103.6 (1Ј-C), 172.2–175.3 (CO); TOF-MASS (positive mode,
cinnamic acid): 1220.8 [2M ϩ 1Gal Ϫ H2O]ϩ, 1381.0 [2M ϩ
2Gal Ϫ H2O]ϩ, 1902.7 [3M ϩ 2Gal Ϫ 2H2O]ϩ, 2064.0 [3M ϩ
3Gal Ϫ 2H2O]ϩ.
ꢁ-2,3-Sialylation of 33Ј. To a solution of 33Ј (3 mg, ~ 0.0043
mmol LacNAc) in sodium cacodylate buffer (50 mM, pH 6.0,
0.5 cm3) containing 0.1% Triton X-100 were added successively
cytidine 5Ј-monophospho-N-acetylneuraminic acid (8 mg,
0.013 mmol), and α-(2,3)-sialyl transferase (0.3 U). The reac-
tion mixture was incubated at 37 ЊC for 5 days and then directly
subjected to gel filtration chromatography (G-25, 42 × 2 cm,
eluent H2O) to afford the sequential sialylated glycopeptide 37
(2 mg, 47%) as a white powder after freeze-drying; δH (400
MHz; D2O): 1.65 (3H, m, 3-Hax, CH2), 1.92 (6H, br s, 2 × CH3),
2.69 (1H, m, Aspβ, 3-Heq), 3.09 (1H, br dd, Aspβ), 4.39 (2H, m,
1-H and 1Ј-H).
Galactosylation of 27 (Method II). To a solution of 27 (16
mg, ~0.03 mmol GlcNAc) in sodium cacodylate buffer (50 mM,
pH 7.5, 1 cm3) containing MgCl2 (5 mM), MnCl2 (25 mM) and
dithiothreitol (0.2 mM) were added successively UDP-galactose
(36.4 mg, 0.06 mmol), α-lactalbumin (0.2 g dmϪ3) and β-(1,4)-
galactosyl transferase (1 U). The reaction mixture was then
incubated at 25 ЊC for 21 h, then UDP-Gal (9.5 mg, 0.015
mmol) was added. More UDP-Gal (9.5 mg, 0.015 mmol) was
added after 25 h of incubation. The reaction mixture was incu-
bated for 47 more hours and then directly subjected to gel filtra-
tion chromatography (G-25, 42 × 2 cm, eluent H2O) to afford
the galactosylated sequential glycopeptide 33Ј (17.3 mg, 84%)
as a white powder after freeze-drying; δC (100 MHz; D2O): 22.9
(COCH3), 29.0 (CH2, propyl), 37.0 (Cβ, Asp), 43.3 (Cα, Gly),
51.4 (Cα, Asp), 55.8 (Cα, Ser), 56.5 (2-C), 60.8 (Cβ, Ser), 61.4
(6-C), 61.7 (6Ј-C), 68.4 (OCH2), 69.2 (4Ј-C), 71.7 (2Ј-C), 73.1 (3-
C), 73.2 (5Ј-C), 75.5 (3Ј-C), 76.1 (5-C), 79.1 (4-C), 101.8 (1-C),
103.6 (1Ј-C), 172.1–175.2 (CO); TOF-MASS (positive mode,
cinnamic acid): 1381.0 [2M ϩ 2Gal Ϫ H2O]ϩ, 2064.0 [3M ϩ
3Gal Ϫ 2H2O]ϩ, 2747.0 [4M ϩ 4Gal Ϫ 3H2O]ϩ.
Sialyl Lewisx derivative 38. To a solution of HEPES buffer
(100 mM, 20 cm3) were added MnCl2ؒ4H2O (10 mM, 36.9 mg)
and NaN3 (1 mM, 1.3 mg). This resulting solution was adjusted
to pH 7.5. To a solution of 37 (1 mg, ~ 0.001 mmol
SialLacNAc) in the buffer described above (0.5 cm3) were added
successively GDP-fucose (2 mg, 0.003 mmol), and α-(1,3)-
fucosyl transferaseV (10 mU). The reaction mixture was incu-
bated at 37 ЊC for 3 days then directly subjected to gel filtration
chromatography (G-25, 42 × 2 cm, eluent H2O) to afford the
sialyl Lewisx sequential glycopeptide 38 (1.1 mg, 100%) as a
white powder after freeze-drying; δH (400 MHz; D2O): 1.18
(3H, d, J , CH3 Fuc), 1.80 (3H, m, 3-Hax, CH2), 2.05 (6H, br s,
2 × CH3), 2.77 (1H, m, Aspβ, 3-Heq), 3.09 (1H, br dd, Aspβ),
4.39 (2H, m, 1-H and 1Ј-H).
Galactosylation of 25 (Method II). Compound 25 (10 mg,
0.016 mmol GlcNAc) was used to prepare 34 (7 mg, 55%) via
the procedure described above for 33Ј. The reaction mixture
was incubated for 45 h and UDP-Gal was added after 18 h and
22 h of incubation respectively; δC (100 MHz; D2O): 22.9
(COCH3), 39.7 (CH2-NH), 43.3 (Cα, Gly), 51.4 (Cα, Asp), 55.7
(Cα, Ser), 56.2 (2-C), 60.7 (Cβ, Ser), 61.4 (6-C), 61.7 (6Ј-C), 68.4
(OCH2), 69.2 (4Ј-C), 71.6 (2Ј-C), 73.2 (4-C, non substituted),
74.6 (3-C), 75.5 (5Ј-C), 76.1 (3Ј-C), 76.6 (5-C), 79.1 (4-C), 101.7
(1-C), 103.6 (1Ј-C), 172.1–175.2 (CO).
Acknowledgements
We would like to thank Ms H. Matsumoto and Ms A. Maeda
(Center for Instrumental Analysis, Hokkaido University)
for elemental analysis measurements and Ms S. Oka and
Ms N. Hazama (Center for Instrumental Analysis, Hokkaido
University) for FABMS measurements. We are grateful to
Dr K. Yamada for useful discussions. This work was partly
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