Formation of lactones from sialylated MUC1 glycopeptides

Article abstract of DOI:10.1039/b514918e

The tumor-associated carbohydrate antigens T_N, T, sialyl T _N and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Form

Full text of DOI:10.1039/b514918e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Formation of lactones from sialylated MUC1 glycopeptides
Maciej Pudelko,a Anna Lindgren,a Tobias Tengel,b Celso A. Reis,c Mikael Elofsson*a and Jan Kihlberg*a,d
Received 24th October 2005, Accepted 22nd December 2005
First published as an Advance Article on the web 19th January 2006
DOI: 10.1039/b514918e
The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins
in several epithelial cancers. This has stimulated studies directed towards development of
glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid
residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur
for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and
tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have
now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found
that the model compound sialyl T benzyl glycoside 6, and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-
Ala 7 from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen,
lactonized during treatment with glacial acetic acid. Compound 6 gave the 1ꢀꢀ → 2ꢀ lactone as the major
product and the corresponding 1ꢀꢀ → 4ꢀ lactone as the minor product. For glycopeptide 7 the 1ꢀꢀ → 4ꢀ
lactone constitued the major product, whereas the 1ꢀꢀ → 2ꢀ lactone was the minor one. When lactonized
7 was dissolved in water the 1ꢀꢀ → 4ꢀ lactone underwent slow hydrolysis, whereas the 1ꢀꢀ → 2ꢀ remained
stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide 8 did
not lactonize in glacial acetic acid.
side chains. Termination of the chain growth is accomplished by
Introduction
addition of sialic acid or fucose, or by sulfation.6 In epithelial
Mucins are members of an expanding family of large multi-
functional glycoproteins present on the surface of many ep-
tumor cells, low expression of core 2 b1,6GlcNAc transferases
combined with elevated levels of sialyl transferases give simplified
ithelial cells. Their main function is to provide lubrication and
carbohydrate patterns which may be accessible to the immune
moisturisation of the surfaces of the epithelial tissues as well
as protection against invasion of pathogenic microorganisms
and mechanical injury.1 Mucins are in general defined by the
presence of a substantial amount of carbohydrates attached
as O-glycans to threonines and serines and also by a high
content of proline in their protein backbone. Of special interest
is the membrane-bound mucin MUC1,2,3 which is a heavily O-
glycosylated, high-molecular weight glycoprotein present between
many epithelia and their extracellular environments including
those of the mammary gland, uterus, and gastrointestinal tract.
Its protein backbone consists of repeating units of 20 amino
acids with the sequence HGVTSAPDTRPAPGSTAPPA,4 bearing
five potential O-glycosylation sites. When MUC1 is produced
by the mammary gland, galactosyl residues are attached to N-
acetylgalactosamine on Ser/Thr leading to the core 1 structure
Galb(1 → 3)GalNAc a-Ser/Thr known as the T antigen. The
T antigen serves as a substrate for the core 2 b1,6GlcNAc
transferases forming the trisaccharide Galb(1 → 3)(GlcNAcb(1 →
6))GalNAc.2,5 The latter can be further extended by adding N-
acetyllactosamine to produce more complex and branched glycan
system. The TN (GalNAca-Ser/Thr), T (Galb(1 → 3)GalNAca-
Ser/Thr), 2,6-sialyl-TN (Neu5Aca(2 → 6)GalNAca-Ser/Thr)
and 2,3-sialyl-T (Neu5Aca(2 → 3)Galb(1 → 3)GalNAca-
Ser/Thr) structures constitue tumor-associated antigens. The
presence of these antigens on the surface of common malignant tu-
mors has stimulated intense studies directed towards development
of synthetic carbohydrate-based anticancer vaccines.7–9
For glycolipids, formation of intramolecular lactones is known
to occur between sialic acid residues and suitably placed hydroxyl
groups in neighboring galactose moieties.10–12 It has been suggested
that such lactones are more immunogenic and tumor-specific as
compared to their native, open form and that they therefore have
potential as immunogenes in development of cancer vaccines.13
Since glycopeptides containing the 2,6-sialyl-TN and 2,3-sialyl-T
antigenes could potentially form lactones in acidic environment,
there is a possibility that these structures could be immunological
analogues to ganglioside lactones and constitue a starting point
for development of glycopeptide based anticancer vaccines. In this
article we describe synthesis and lactonization studies of the 2,6-
sialyl-TN and 2,3-sialyl-T antigens attached to a peptide from the
MUC1 repeating unit. Methods for synthesis of tumor-associated
glycopeptides from mucins have been reviewed recently.14–17 In
spite of the recent, successful chemical sialylation used in the
aOrganic Chemistry, Department of Chemistry, Umea˚ University, SE-901
87, Umea˚, Sweden. E-mail: mikael.elofsson@chem.umu.se, jan.kihlberg@
chem.umu.se
18,19
syntheses of building blocks corresponding to the 2,6-sialyl-TN
bDepartment of Medicinal Biochemistry and Biophysics, Umea˚ University,
SE-901 87, Umea˚, Sweden
and 2,3-sialyl-T20 antigens, low yields and poor stereoselectivity
are often encountered due to the lack of neighboring group
assistance on the sialyl donor. A convenient alternative is the
chemoenzymatic approach to sialylated T and TN antigens, using
cInstitute of Molecular Pathology & Immunology, University of Porto, 4200,
Porto, Portugal
dAstraZeneca R & D Mo¨lndal, SE-431 83, Mo¨lndal, Sweden
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various enzymes which are readily available today. Depending on
their specificity, these enzymes catalyze the transfer of a sialic
acid moiety from cytidine-5ꢀ-monophospho-N-acetylneuraminic
acid (CMP-Neu5Ac) in an a-selective fashion to a particu-
lar hydroxyl group of galactose residues. Consequently sialyl
transferases were succesfully employed in the synthesis of wide
range of oligosaccharides21 and glycopeptides22–24 operating with
complete stereo- and regiocontrol. We therefore decided to use a
chemoenzymatic approach for synthesis of the glycopeptides used
in this article.
lation of azide group, and deprotection of the tert-butyl ester
afforded the T building block 225 in 35% yield (Scheme 1). Both
building blocks are suitable for use in solid-phase peptide synthesis.
After the synthesis of building blocks 1 and 2 we turned
our attention to solid-phase synthesis of glycopeptide 3 and 4
(Scheme 1), that are based on the tandem repeating unit from the
mucin MUC1 with the glycosylated threonine located in the center
of the immunodominant region.5 Incorporation of the two units 1
and 2 was performed using the standard Fmoc protocol for solid-
phase peptide synthesis on Tentagel and ArgoGel resins. In the
synthesis of 3 the acid labile protective groups originating from
building block 1 were removed during the cleavage from the solid
support, whereas glycopeptide 4 was deacetylated after cleavage
from the resin using conditions previously described by us.23,26
With sufficient amounts of glycopeptides 3 and 4 in hand we
turned our interest towards the extension of the carbohydrate
side-chains by means of enzymatic sialylation. We thought that
benzyl T glycoside 527 could serve as a good model compound
for sialylation using commercially available recombinant a2,3OST
sialyl transferase from rat liver. Following standard sialylation
procedures,28,29 saccharide 5 was incubated with the sialic acid
donor CMP-NeuAc and recombinant a2,3OST in buffer at pH 6
in preparative scale (Scheme 2). It was found that the enzyme
Results and discussion
Chemoenzymatic synthesis of glycopeptides
Synthesis of the TN building block 1 (Scheme 1), carrying acid
labile protective groups on the carbohydrate moiety, was accom-
plished from 4-methylphenyl 2-azido-2-deoxy-1-thio-b-D-galacto-
pyranoside, according to a previously published procedure.23
Silver triflate mediated glycosylation of Fmoc-3-O-(2-azido-4,6-
O-benzylidene-2-deoxy-a-D-galactopyranosyl)-L-threonine tert-
butyl ester with peracetylated galactosyl bromide, reductive acety-
Scheme 2 Enzymatic sialylation of benzyl glycoside 5 and glycopeptides
3 and 4. Reagents and conditions: (i) CMP-Neu5Ac (1.75 equiv), a2,3OST,
0.25 M sodium cacodylate buffer (pH 6) containing 0.1% Triton X-100,
37 ◦C. (ii) CMP-Neu5Ac (2 mM), ST6GalNAc-I, 20 mM Bis-Tris buffer
(pH 6.5) containing EDTA (20 mM) and dithiothreitol (1 mM), 37 ◦C.
Scheme 1 Solid-phase synthesis of glycopeptides 3 and 4. Reagents and
conditions: (i) glycopeptide synthesis according to the Fmoc protocol.
(ii) Deacetylation with NaOMe/MeOH, 20 mM.
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transferred a sialic acid unit regio- and stereoselectively onto the 3-
OH group of the terminal galactose moiety. After purification on a
Waters Sep-Pak C18 solid-phase extraction column and reversed-
phase HPLC the sialylated T benzyl glycoside 6 was produced in
72% isolated yield. In the sialylation more than 95% of the sub-
strate 5 was consumed. The identity of compound 6 was confirmed
by electrospray (ES) and fast atom bombardment (FAB) mass
spectrometry and its 1H NMR data were in agreement with those
published.30 This result promped us to investigate enzymatic sia-
lylation of the T glycopeptide 4 and also of the corresponding TN
glycopeptide 3 (Scheme 2). The synthesis of glycopeptide 7 from 4
revealed that a-(2 → 3)-sialylation of glycopeptides which bear the
T antigen is facile. Glycopeptide 4 was sialylated as described for 5
and the sialyl T glycopeptide 7 was isolated in 60% yield with ap-
proximately 90% conversion of the starting material as determined
by reversed-phase HPLC. When glycopeptide 3 was sialylated
on preparative scale using recombinant ST6GalNAc-I31 sialyl
transferase, 8 was obtained in 60% yield after purification. The
identity of the sialylated glycopeptides 7 and 8 was confirmed by
ES mass spectrometry and 1H NMR spectroscopy (Tables 1 and 2).
In conclusion this chemoenzymatic synthesis approach effectively
produced sialylated derivatives suitable as lactonization substrates.
The most characteristic NMR features were significant high
shifts (Table 1) for H-2 in galactose and H-4 of Neu5Ac in the
lactone 9a, and for H-4 in the galactose moiety of the lactone 9b
as compared with sialyl T glycoside 6. Although all asignments
were based on spectra in acetic acid-d4, these type of large
downfield shifts are comparable to those of lactone units acting
as intermediates in the synthesis of sialyl LewisX derivatives,35 the
gangliosides GM1, GM3, and GM4,
10–12 and a glycosylated heptapep-
tide corresponding to the human M blood group determinant.36
Formation of intramolecular esters in sialyl T antigen glycoside
6 promped us to further investigate lactonization in sugar units
of sialyl T and sialyl TN glycopeptides 7 and 8. First, sialyl TN
glycopeptide 8 was incubated in acetic acid-d4 at ambient temper-
ature for 14 days (Scheme 4). The reaction was monitored every
second day using a set of standard 1D and 2D NMR experiments.
However during the course of the reaction no changes in the
NMR spectra were observed. This suggests that the formation
of a seven-membered ring between the sialic acid carboxyl group
and C4–OH in galactosamine is not facile. Thereafter compound
7 was subjected to the lactonization conditions as decribed above
during a time frame of 17 days (Scheme 3). The advance of the
reaction was monitored each day using mainly 1D 1H NMR
experiments in combination with COSY, ROESY and TOCSY
experiments. After a period of 11 days no further alterations in
Formation of lactones
1
the H NMR spectra were observed suggesting the culmination
of the process. Although we were able to detect two different
products by analysis of the NMR spectra recorded in acetic acid-
d4, it turned out to be difficult to assign resonances to a particular
product. In order to make an accurate assignment the solvent
was changed to DMSO-d6. From a combination of hetero- and
homonuclear experiments in DMSO-d6, we could identify the two
different products; the major product being the 1ꢀꢀ → 4ꢀ lactone
10b with the corresponding 1ꢀꢀ → 2ꢀ lactone 10a being formed as
Lactonization studies of sialic acid residues of certain free
oligosaccharides, e.g. 3ꢀ-sialyllactose32 and several gangliosides
12
11
10
33
34
including GM1
,
GM3
,
GM4
,
GD1a
,
and GD1b have been
described. To the best of our knowledge lactonization of sialylated
glycopeptides has not been investigated. Two methods have been
applied in order to obtain intramolecular esters of different
gangliosides; incubation in glacial acetic acid11 or treatment
with dicyclohexylcarbodiimide in anhydrous DMSO.12 Under
these conditions the ester linkage is often produced in almost
quantitative yield. In this study we decided to use acetic acid
incubation because of its ease of handling and the simplistic
experimental procedure.
First, we turned our attention to the lactonization of free sialyl
T antigen benzyl glycoside 6. Compound 6 was dissolved in glacial
deuterated acetic acid and allowed to incubate for 14 days at room
temperature. The reaction was monitored every second day with
1H NMR spectroscopy. Two lactones, 9a and 9b were produced
in a 3 : 2 ratio (Scheme 3). In 9a and 9b the ester linkage was
formed between the carboxyl group of N-acetylneuraminic acid
and the C2–OH and C4–OH of galactose moiety, respectively. The
1
conversion of 6 to 9a and 9b was complete according to the H
NMR spectra.
Scheme 4 Lactonization of sialylated glycopeptide 8. Reagents and
conditions: (i) glacial acetic acid (AcOH-d4), RT.
Scheme 3 Lactonization of sialylated benzyl glycoside 6 and sialylated glycopeptide 7. Reagents and conditions: (i) glacial acetic acid (AcOH-d4), RT.
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Table 1 1H NMR chemical shifts (d, ppm) for the the saccharide 6a, O-linked glycopeptides 7b and 8c and the lactones 9a,a 9b,a 10a,d and 10bd
6
7
8
9a
9b
10a
10b
Benzyl
H-a
H-b
4.61–4.64
4.85–4.88
7.39–7.52
—
—
—
—
—
—
n.d.e
n.d.e
—
—
—
—
—
—
n.d.e
n.d.e
Ar
7.41–7.55
7.41–7.55
GalNHAc
NHAc
H-1
H-2
H-3
n.d.e
5.14
4.64
4.17
4.43
7.81
3.98
4.19
3.73
n.d.e
7.57
3.94
4.05
3.82
3.57
n.d.e
5.17
4.68
4.17
4.47
n.d.e
5.17
4.68
4.17
4.47
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
n.d.e
H-4
Gal
H-1
H-2
H-3
4.70
3.82
4.26
4.14
4.48
3.50
4.01
3.71
—
—
—
—
4.88
5.08
4.12
4.17
4.74
3.85
4.32
5.51
4.51
5.19
4.10
n.de
4.48
n.d e
4.06
5.26
H-4
Neu5Ac
NHAc
H-3ax
H-3eq
H-4
H-5
H-6
n.d.e
2.94
2.07
4.12
3.83
n.d.e
8.04
1.76
2.72
3.65
3.82
n.d.e
8.01
1.64
2.66
3.65
3.80
n.d.e
n.d.e
2.74
1.96
4.52
4.22
n.d.e
n.d.e
2.98
2.12
4.13
n.d.e
n.d.e
n.d.e
1.54
2.30
4.13
3.50
n.d.e
n.d.e
1.54
2.27
4.14
3.53
3.24
a Recorded at 400 MHz, 298 K, with CD3COOD (dH = 11.59) as internal standard. b Recorded at 500 MHz, 298 K, with H2O (dH = 4.75) as internal
standard containing 20% D2O. c Recorded at 500 MHz, 298 K, with H2O (dH = 4.75) as internal standard containing 10% D2O. d Recorded at 500 MHz,
298 K, with DMSO-d6 (dH = 2.50) as internal standard. e Not determined.
a minor product (ratio 7 : 3, Table 1). The relative ratio was based
on the volumes of two crosspeaks derived from H-3 of neuraminic
acid (Fig. 1). The key feature which allowed us to identify 1ꢀꢀ → 4ꢀ
lactone 10b was the downfield peak at 5.26 ppm which was shown
to be H-4 in the galactose moiety, in comparison with 3.71 ppm
of H-4 of galactose in sialylated glycopeptide 7. Downfield shifts
of the same magnitude were also observed for lactones formed
of lactones 10a and 10b are derived from observed crosspeaks
in COSY and TOCSY experiments. These lactonization effects
are probably due to the reorientation of Neu5Ac carboxyl group
after esterification and to the alteration in electronegativity along
the Neu5Ac backbone. Although it was possible to identify two
different peptide chains in post-lactonized mixture, we could not
attribute them to a particular saccharide lactone.
Somewhat surprisingly the benzyl glycoside 6 gave approxi-
mately equal amounts of the 1ꢀꢀ → 2ꢀ lactone 9a and the 1ꢀꢀ →
4ꢀ lactone 9b while the 1ꢀꢀ → 4ꢀ lactone 10b dominated when
glycopeptide 7 was lactonized. For lactones obtained from benzyl
glycoside 6 this might be explained by the fact that no steric
interactions occur between the sialic acid unit and the benzyl
group, allowing the two possible lactones to be formed in relatively
similar amounts. Construction of a space-filling model of the 1ꢀꢀ →
2ꢀ lactone 10a reveals that the Neu5Ac ring lies in a plane
perpendicular to the plane of galactose–galactosamine moiety,
hence interacting with the peptide chain. This could be the
explanation for the fact that 10a was produced as the minor
product. On the other hand a model of the major 1ꢀꢀ → 4ꢀ
lactone 10b shows that Neu5Ac backbone lies in the same plain as
galactose and galactosamine, producing almost no contact with
the peptide moiety.
10
during synthesis of GM4 and sialyl LewisX lactones.37,38 The high
shift peak at 5.19 ppm in lactone 10a was assigned to H-2 in
the galactose residue as compared to the peak at 3.50 ppm of
H-2 in the non-lactonized glycopeptide 7, thus showing extensive
deshielding of the ester linkage. This large downfield shift of H-2
in galactose was also observed for 1ꢀꢀ → 2ꢀ lactones in GM1 and
12
11
GM3 gangliosides and for intermediate products in the synthesis
of the sialyl LeX trisaccharide.39 As stated above, the assignment
Hydrolysis of lactone products 10a and 10b
It has been suggested that lactones originating from gangliosides
are more immunogenic than the gangliosides themselves, implying
that the lactones might be valuable as immunogens for vaccination
against tumors.13 However sialic acid lactones might not be
sufficiently stable to be therapeutically useful. Having now in
our hands the mixture of the two glycopeptide lactones 10a and
10b, we were therefore interested in investigating their hydrolytic
Fig. 1 Part of the TOCSY spectrum at the beginning of the hydrolysis.
Two crosspeaks derived from H-3 equatorial (e) and axial (a) in the sialic
acid residues of lactones 10a and 10b. (The spectrum was recorded in H2O
containing 10% D2O).
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Table 2 1H NMR chemical shifts (d, ppm) for the amino acids in
glycopeptides 7a and 8b
period of 30 days, as determined by 1H NMR spectroscopy. This
remarkable stability might be due to the highly rigid structure of
10a where the Neu5Ac ring lies in a plane perpendicular to the
plane of the galactose moiety and lactone ring. Since lactone 10a
showed reasonable stability in an aqueous environment, we suggest
that it could serve as a good immunogen also under physiological
conditions.
Lactams, which are hydrolytically more stable and structurally
similar to lactones, should be good substitutes. The synthesis
of ganglioside lactams corresponding to GM2, GM3 and GM4
ganglioside lactones has been reported previously.40–42 They proved
to be very stable upon storage with only minor hydrolysis occuring
in D2O at 37 ◦C during one month. It was also found that
7
8
Ala1
NH
a-H
b-H
Pro2
a-H
b-H
c-H
d-H
Asp3
NH
a-H
b-H
Thr4
NH
a-H
b-H
c-H
Arg5
NH
a-H
b-H
c-H
d-H
d-NH
Pro6
a-H
b-H
c-H
d-H
Ala7
NH
a-H
b-H
8.030
4.329
1.521
n.d.d
4.328
1.512
4.460
4.461
1.866, 2.308
1.874, 2.322
2.007c
2.013c
3.604, 3.690
3.596, 3.698
8.628
4.813
2.794, 2.914
8.635
4.822
2.812, 2.920
antibodies raised towards GM3-lactam cross-reacted with the GM3
lactone in vitro.43
-
8.640
4.504
4.366
1.230
8.378
4.496
4.270
1.259
In conclusion, a chemoenzymatic approach has been used to
efficiently prepare sialylated T and TN glycopeptides derived from
the mucin MUC1. By using these glycopeptides the formation of
intramolecular lactones in sialylated glycopeptides was examined
for the first time. The sialyl T glycopeptide formed 1ꢀꢀ → 4ꢀ and
1ꢀꢀ → 2ꢀ lactones upon treatment with acetic acid. The 1ꢀꢀ → 2ꢀ
lactone, in contrast to the 1ꢀꢀ → 4ꢀ lactone, showed significant
stability in aqueous solution, implying its potential for use as
immunogen in cancer vaccines. No lactones were formed from the
sialyl TN glycopeptide, even after prolonged treatment with acetic
acid.
8.334
4.534
8.389
4.552
1.688, 1.820
1.668, 1.821
1.668c
1.668c
3.193c
3.194c
7.232
7.170
4.332
4.314
1.897, 2.293
1.898, 2.296
2.031c
2.020c
3.595, 3.725
3.596, 3.725
Experimental
8.456
4.186
1.377
8.437
4.185
1.380
General methods and materials
All reactions were carried out under an inert nitrogen atmosphere
using dry, freshly distilled solvents under anhydrous conditions,
unless otherwise stated. CH2Cl2 was distilled from calcium hy-
dride. Organic solutions were dried over Na2SO4 before being
concentrated. TLC was performed on Silica Gel F254 (Merck)
with detection by UV light and by charring with 10% sulfuric
acid. Flash column chromatography was performed on Silica Gel
a Recorded at 500 MHz, 298 K, with H2O (dH = 4.75) as internal standard
containing 20% D2O. b Recorded at 500 MHz, 298 K, with H2O (dH = 4.75)
as internal standard containing 10% D2O. c Degeneracy has been assumed.
d Not determined.
stability. The mixture of 10a and 10b was dissolved in H2O/D2O
and the solution was kept at ambient temperature for 30 days
with NMR experiments being run every second day. We found
that the 1ꢀꢀ → 4ꢀ lactone 10b was completely hydrolysed to give
the sialyl T peptide 7 after 9 days (Fig. 2). Interestingly, the 1ꢀꢀ →
2ꢀ lactone 10a showed extensive stability compared to the 1ꢀꢀ →
4ꢀ lactone 10b as it remained in the aqueous solution even after a
˚
(Matrex, 60 A, 35–70 lm, Grace Amicon). Preparative reversed-
phase HPLC was performed on a Kromasil C-8 column (250 ×
˚
20 mm, 5 lm, 100 A), eluted with a linear gradient of MeCN
in H2O containing 0.1% TFA, with a flowrate of 11 mL min−1
and detection at 214 nm. Analytical HPLC was performed on
a Beckman System Gold HPLC, using a Kromasil C-8 column
˚
(250 × 4.6 mm, 5 lm, 100 A), with the same eluent flowrate of
1.5 mL min−1 and detection at 214 nm.
1H and 13C NMR spectra were recorded on a Bruker
DRX-400 spectrometer and a Bruker AMX2-500 spectrometer
(Massachusetts, USA). All NMR experiments were conducted
at 298 K using CDCl3 [residual CHCl3 at 7.26 ppm (dH)],
DMSO-d6 [residual DMSO-d5 at 2.50 ppm (dH) and 39.60 ppm
(dC)], CD3CO2D [residual CD3CO2DH at 11.59 ppm (dH) and
20.0 ppm (dC)] or CD3OD [residual CD2HOD at 3.35 ppm (dH)].
The chemical shift of the water signal was used as a reference
for compounds dissolved in water and calibrated to 4.75 ppm.
The 13C HSQC spectra were calibrated using the gyromagnetic
ratio for carbon.44 The spectra used for resonance assignments
included phase sensitive DQF-COSY,45 TOCSY,46 ROESY47
and gradient enhanced HSQC.48 The DIPSI pulse sequence
Fig. 2 Crosspeak derived from the residual lactone 10a after 9 days in
water. (The spectrum was recorded in H2O containing 10% D2O).
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with a spin lock time of 45 or 75 ms was used in the TOCSY
experiments whereas the ROESY spectra were recorded with a
mixing time of 200 ms. Mass spectra were recorded on a Waters
Micromass ZQ using positive electrospray ionization (ES+).
High-resolution fast atom bombardment mass spectra (HRMS)
were recorded with a JEOL SX102 A mass spectrometer. Ions
for FABMS were produced by a beam of xenon atoms (6 keV)
from a matrix of glycerol and thioglycerol. (2-acetamido-2-
deoxy-3-O-b-D-galactopyranosyl-a-D-galactopyranosyl)-O-benzyl
ether (three times), dissolved in a mixture of water and acetic acid
(6 : 1), and then freeze-dried.
Deprotection of acetates was performed with methanolic
sodium methoxide solution (0.02 M, 3 mL/8 mg in 8 mL
MeOH), under nitrogen atmosphere for several hours, with careful
monitoring on HPLC. Solutions were neutralized with acetic acid
and concentrated under vacuum. The residues were then purified
by reversed-phase HPLC.
R
glycoside 5 (benzyl T antigen) was purchased from Calbiochemꢀ
L-Alanyl-L-prolyl-L-aspartyl-3-O-(2-acetamido-2-deoxy-3-O-b-
D-galactopyranosyl-a-D-galactopyranosyl)-L-threonyl-L-arginyl-L-
prolyl-L-alanine amide (4). Synthesis, cleavage of the resin-bound
glycopeptide, and then purification by reversed-phase HPLC
(gradient 0 → 80% CH3CN in H2O, both containing 0.1% TFA,
during 60 min) gave the acetylated target glycopeptide [22 mg, MS
(ES): calcd 1343.3, found 1343.4]. The acyl protected glycopeptide
(8 mg) was subjected to deacetylation with methanolic sodium
methoxide which was carried out for 6 h (careful monitoring with
HPLC). Purification by preparative HPLC (gradient 0 → 30%
CH3CN in H2O, with 0.1% TFA, during 60 min) gave 4 (4.6 mg,
25% overall yield). MS (ES): calcd for C44H74N12O20 1091.522 m/z
(M + H)+, observed 1091.647.
(Germany). The TN antigen Na-fluoren-9-ylmethoxycarbonyl-3-
O-(2-acetamido-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-
deoxy-a-D-galactopyranosyl)-L-threonine 1 was prepared as des-
cribed previously.23 The threonine-based T antigen, Na-fluoren-
9-ylmethoxycarbonyl-O-[O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-acetyl-b-D-galacto-
pyranosyl)-(1ꢀ
→
3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-a-D-
galactopyranosyl]-L-threonine was synthesised accordning to
previously reported procedure.25 Glycosylated building blocks
had data in agreement with the cited references.
General procedure for solid-phase peptide synthesis
L-Alanyl-L-prolyl-L-aspartyl-3-O-(2-acetamido-2-deoxy-a-D-
galactopyranosyl)-L-threonyl-L-arginyl-L-prolyl-L-alanine amide
(3). Synthesis, cleavage of the resin-bound glycopeptide together
with deprotection of acid labile protective groups on sugar moiety,
and then purification by reversed-phase HPLC (isocratic elution
5% CH3CN in H2O, both containing 0.1% TFA, during 30 min)
gave the target glycopeptide 3 (8.8 mg, 24% yield). MS (FAB):
calculated for C38H64N12O15 929.468 m/z (M + H)+, observed
929.447.
Glycopeptides were prepared under conditions identical to those
described previously.49 A Tentagel S NH2 resin (Rapp Poly-
mere, Germany) functionalized with the Rink amide linker
{p-[a-fluoren-9-ylmethoxyformamido)-2,4-dimethoxybenzyl]-
R
phenoxyacetic acid}50,51 B(achem AG, Switzerland) and ArgoGelꢀ-
Rink-NH-Fmoc resin (Argonaut Technologies Inc.) were used in
the synthesis of glycopeptides. Na-Fmoc-amino acids (Neosystem,
France and Bachem, Switzerland) with the following side
chain protecting groups were used in the synthesis: 2,2,5,7,8-
pentamethylchroman-6-sulfonyl (Pmc) for arginine and tert-butyl
(tBu) for aspartic acid. DMF was distilled before being used.
Couplings were performed manually in a mechanically agitated re-
actor. Fmoc amino acids (4 equiv) were activated as benzotriazolyl
esters by using 1,3-diisopropylcarbodiimide (DIC, 3.9 equiv) and
1-hydroxybenzotriazole (HOBt, 6 equiv) in dry DMF. Acylations
were monitored by using bromophenol blue (BFB, 2 mM solution
in DMF) as indicator52 (the colour of the reaction mixture changes
from blue to yellow). Na-Fmoc deprotections were effected by a
flow of 20% solution of piperidine in DMF for 3 min and futher
by shaking for 7 min. Before and after treatment with piperidine
solution the resin was washed five times with DMF. The threonine-
based building blocks: 1 (1 equiv, 40 lmol) and 2 (1.04 equiv,
52.2 lmol) were activated in distilled DMF (1 mL) at room temper-
ature during 1–2 min by addition of 1,3-diisopropylcarbodiimide
(1.1 equiv) and 1-hydroxy-7-azabenzotriazole (HOAt, 3.3 equiv).
The activated esters were then coupled to the peptides resin
during 24 h.
After completion of the synthesis, the resin carrying the
protected glycopeptide was washed with CH2Cl2 (five times)
and dried under vacuum. Cleavage from the resin and re-
moval of acid labile protective groups was performed with
TFA/H2O/thioanisole/ethanedithiol (87.5 : 5 : 5 : 2.5, 20 mL/
200 mg of resin) for 3.5 h, followed by filtration. Acetic acid
(10 mL) was added to filtrate which was then concentrated, and
acetic acid was added again (3 × 10 mL) followed by concentration
after each addition. The residue was triturated with cold ethyl
General procedure for enzymatic sialylation of the compounds 4
and 5
Soluble recombinant form of a2,3OST Spodoptera frugiperda
(76–81 m units, b-D-galactosyl-b-1,3-N-acetyl-b-D-galactosamine-
R
a-2,3-sialyltransferase, Calbiochemꢀ, Germany) from rat liver
(≥1 unit mg−1 protein) was used for sialylation of glycopeptide 4
and benzyl T glycoside 5. The reactions were performed in 0.25 M
sodium cacodylate buffer (2 mL, pH 6) containing 0.1% Triton X-
100 and CMP-b-D-sialic acid disodium salt donor (1.75 equiv,
R
Calbiochemꢀ, Germany). The solution was then incubated at
37 ◦C for 1–2 days. Progress of the reaction was monitored by
TLC. Purification on pre-conditioned Waters Sep-Pak Vac 12cc
C-18 solid-phase extraction column (2 g) and then by preparative
reversed-phase HPLC gave sialylated compounds.
Benzyl [(5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-
nonulopyranosylonic acid)-(2 → 3)-O-b-D-galactopyranosyl]-(1 →
3)-2-acetamido-2-deoxy-a-D-galactopyranoside (6). The reaction
progress was monitored with TLC (EtOH : AcOH : MeOH : H2O
6 : 3 : 3 : 2). Purification on Waters Sep-Pak C18 solid-phase
extraction column with gradient elution 0 → 50% MeOH in H2O
and futher by preparative HPLC (gradient 0 → 30% CH3CN in
H2O during 30 min) gave 6 (2.4 mg, 72% yield). MS (FAB): calcd
for C32H48N2O19 m/z (M + 2Na–H)+ 809.2568, observed 809.2565.
1H NMR data was in agreement with that published previously.30
718 | Org. Biomol. Chem., 2006, 4, 713–720
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L-Alanyl-L-prolyl-L-aspartyl-3-O-[(5-acetamido-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2 → 3)-O-b-D-
galactopyranosyl-(1 → 3)-O-(2-acetamido-2-deoxy-a-D-galacto-
pyranosyl)]-L-threonyl-L-arginyl-L-prolyl-L-alanine amide (7).
Synthesis, purification on Waters Sep-Pak C18 solid-phase extrac-
tion column with gradient elution 0 → 50% MeOH in H2O and
futher by preparative HPLC (gradient 0 → 10% CH3CN in H2O
during 30 min, then 10 → 100% during 30 min) gave 7 (3 mg, 60%
yield). MS (ES): calcd for C55H91N13O28 m/z (M + H)+ 1382.618,
observed 1382.617.
4 S. J. Gendler, C. A. Lancaster, J. T. Papadimitriou, T. Duhig, N. Peat,
J. Burchell, L. Pemberton, E. N. Lalani and D. Wilson, J. Biol. Chem.,
1990, 265, 15285–15293.
5 J. Taylor-Papadimitriou, J. Burchell, D. W. Miles and M. Dalziel,
Biochim. Biophys. Acta, 1999, 1455, 301–313.
6 I. Brockhausen, Biochem. Soc. Trans., 2003, 31, 318–325.
7 S. J. Danishefsky and J. R. Allen, Angew. Chem., Int. Ed., 2000, 39,
836–863.
8 T. Toyokuni and A. K. Singhal, Chem. Soc. Rev., 1995, 231–242.
9 G. Ragupathi, D. M. Coltart, L. J. Williams, F. Koide, E. Kagan, J.
Allen, C. Harris, P. W. Glunz, P. O. Livingston and S. J. Danishefsky,
Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 13699–13704.
10 T. Terabayashi, T. Ogawa and Y. Kawanishi, J. Biochem., 1990, 107,
868–871.
L-Alanyl-L-prolyl-L-aspartyl-3-O-[(5-acetamido-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2 → 6)-O-(2-
acetamido-2-deoxy-a-D-galactopyranosyl)]-L-threonyl-L-arginyl-L-
prolyl-L-alanine amide (8). Sialylation of 3 was essentially
performed as previously described:23 compound 3 (3 mg) was
added to purified ST6GalNAc-I (12 m units)53 in 20 mM Bis-Tris
buffer (pH 6.5) containing CMP-b-D-sialic acid disodium salt
donor (2 mM), EDTA (20 mM), and dithiothreitol (1 mM). The
11 R. K. Yu, T. A. W. Koerner, S. Ando, H. C. Yohe and J. H. Prestegard,
J. Biochem., 1985, 98, 1367–1373.
12 G. Kirschner, G. Fronza, H. Egge, R. Ghidoni, D. Acquotti, G.
Tettamanti and S. Sonnino, Glycoconjugate J., 1985, 2, 343–354.
13 G. A. Nores, T. Dohi, M. Taniguchi and S.-I. Hakomori, J. Immunol.,
1987, 139, 3171–3176.
14 M. Meldal, in Neoglycoconjugates: Preparation and Applications, ed.
Y. C. Lee and R. T. Lee, Academic Press, Inc., San Diego, 1994,
pp. 145–198.
15 T. Norberg, B. Lu¨ning and J. Tejbrant, Methods Enzymol., 1994, 247,
◦
87–106.
solution was then incubated at 37 C for 6 h, and monitoring of
sialylation was performed by nanoscale reverse-phase HPLC54 in
combination with MALDI-TOF mass spectrometry. Purification
by preparative HPLC (gradient 0 → 80% CH3CN in H2O in
60 min) gave 8 (2.37 mg, 60% yield). MS (ES): calcd for
C49H81N13O23 m/z (M)+ 1220.24, observed 1220.25.
16 C. Brocke and H. Kunz, Bioorg. Med. Chem., 2002, 10, 3085–3112.
17 G.-J. Boons and A. V. Demchenko, Chem. Rev., 2000, 100, 4539–
4565.
18 C. Brocke and H. Kunz, Synthesis, 2004, 525–542.
19 M. Elofsson, L. A. Salvador and J. Kihlberg, Tetrahedron, 1997, 53,
369–390.
20 S. Dziadek, C. Brocke and H. Kunz, Chem. Eur. J., 2004, 10, 4150–4162.
21 O. Blixt, K. Allin, L. Pereira, A. Datta and J. C. Paulson, J. Am. Chem.
Soc., 2002, 124, 5739–5746.
22 C. Unverzagt, S. Kelm and J. C. Paulson, Carbohydr. Res., 1994, 251,
285–301.
23 S. K. George, T. Schwientek, B. Holm, C. A. Reis, H. Clausen and J.
Kihlberg, J. Am. Chem. Soc., 2001, 123, 11117–11125.
24 Y. Takano, N. Kojima, Y. Nakahara, H. Hojo and Y. Nakahara,
Tetrahedron, 2003, 59, 8415–8427.
25 N. Mathieux, H. Paulsen, M. Meldal and K. Bock, J. Chem. Soc.,
Perkin Trans. 1, 1997, 2359–2368.
26 P. Sjo¨lin, M. Elofsson and J. Kihlberg, J. Org. Chem., 1996, 61, 560–
565.
27 Y. C. Lee, N. Kojima, E. Wada, N. Kurosawa, T. Nakaoka, T.
Hamamoto and S. Tsuji, J. Biol. Chem., 1994, 269, 10028–10033.
28 B. Ernst and R. Oehrlein, Glycoconjugate J., 1999, 16, 161–170.
29 O. Schwardt, G.-P. Gao, T. Visekruna, S. Rabbani, E. Gassmann and
B. Ernst, J. Carbohydr. Chem., 2004, 23, 1–26.
General procedure for the lactonization
The sialylated benzyl glycoside 6 and glycopeptides 7 and 8 were
dissolved in CD3COOD and incubated at ambient temperature.
The reactions were monitored using mainly 1H NMR spectroscopy
over a period of 14 days in order to verify completion. The product
mixture 10a and 10b was freeze-dried several times from acetic
acid, dissolved in DMSO-d6 and the set of NMR experiments
were recorded, which allowed us to elucidate their structures.
Hydrolysis of mixture of lactones 10a and 10b
The lactones 10a and 10b were freeze-dried several times from
DMSO and dissolved in D2O. Hydrolysis was performed in NMR
tube at ambient temperature and several NMR experiments were
run every second day during 30 days in order to verify the reaction
progress. The NMR spectra in DMSO-d6 were also recorded after
multiple lyophilisation from water.
30 J. Xia, J. L. Alderfer, C. F. Piskorz, R. D. Locke and K. L. Matta,
Carbohydr. Res., 2000, 328, 147–163.
31 N. Kurosawa, S. Takashima, M. Kono, Y. Ikehara, M. Inoue, Y.
Tachida, H. Narimatsu and S. Tsuji, J. Biochem. (Tokyo), 2000, 127,
845–854.
32 W. A. Bubb, T. Saito, I. Arai, T. Urashima and T. Nakamura, Carbohydr.
Res., 2000, 329, 471–476.
33 G. Fronza, G. Kirschner, D. Acquotti, R. Bassi, L. Tagliavacca and S.
Sonnino, Carbohydr. Res., 1988, 182, 31–40.
34 D. Acquotti, G. Fronza, L. Riboni, S. Sonnino and G. Tettamanti,
Glycoconjugate J., 1987, 4, 119–127.
Acknowledgements
35 K. C. Nicolaou, C. W. Hummel, N. J. Bockovich and C.-H. Wong,
J. Chem. Soc., Chem. Commun., 1991, 870–872.
This work was funded by the EU Prime Boost grant (QLRT-
2001-02010). Celso A. Reis was supported by FCT (POCTI/
CBO/44598/02) and the Association for International Cancer
Research (Grant 05–088).
36 Y. Nakahara, H. Iijima and T. Ogawa, Tetrahedron Lett., 1994, 35,
3321–3324.
37 U. Sprengard, G. Kretzschmar, E. Bartnik, C. Hu¨ls and H. Kunz,
Angew. Chem., Int. Ed. Engl., 1995, 34, 990–993.
38 W. Stahl, U. Sprengard, G. Kretzschmar and H. Kunz, Angew. Chem.,
Int. Ed. Engl., 1994, 33, 2096–2098.
39 S. J. Danishefsky, J. Gervay, J. M. Peterson, F. E. McDonald, K. Koseki,
D. A. Griffith, T. Oriyama and S. P. Marsden, J. Am. Chem. Soc., 1995,
117, 1940–1953.
References
1 Y. S. Kim, J. Gum and I. Brockhausen, Glycoconjugate J., 1996, 13,
693–707.
40 A. K. Ray, A. Nilsson and G. Magnusson, J. Am. Chem. Soc., 1992,
114, 2256–2257.
41 M. Wilstermann, L. O. Kononov, U. Nilsson, A. K. Ray and G.
Magnusson, J. Am. Chem. Soc., 1995, 117, 4742–4754.
2 F.-G. Hanisch and S. Mu¨ller, Glycobiology, 2000, 10, 439–449.
3 J. Taylor-Papadimitriou, J. M. Burchell, T. Plunkett, R. Graham, I.
Correa, D. Miles and M. Smith, J. Mammary Gland Biol. Neoplasia,
2002, 7, 209–221.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 713–720 | 719
©

View Article Online
42 M. Wilstermann and G. Magnusson, J. Org. Chem., 1997, 62, 7961–
7971.
48 L. E. Kay, P. Keifer and T. Saarinen, J. Am. Chem. Soc., 1992, 114,
10663–10665.
43 K. Ding, A. Rosen, A. K. Ray and G. Magnusson, Glycoconjugate J.,
49 J. Broddefalk, M. Forsgren, I. Sethson and J. Kihlberg, J. Org. Chem.,
1992, 9, 303–306.
1999, 64, 8948–8953.
44 D. S. Wishart, C. G. Bigam, J. Yao, F. Abildgaard, H. J. Dyson, E.
Oldfield, J. L. Markley and B. D. Sykes, J. Biomol. NMR, 1995, 6,
135–140.
45 M. Rance, O. W. Sorensen, G. Bodenhausen, G. Wagner, R. R. Ernst
and K. Wu¨thrich, Biochem. Biophys. Res. Commun., 1983, 117, 479–
485.
50 H. Rink, Tetrahedron Lett., 1987, 28, 3787–3790.
51 M. S. Bernatowicz, S. B. Daniels and H. Ko¨ster, Tetrahedron Lett.,
1989, 30, 4645–4648.
52 M. Flegel and R. C. Sheppard, J. Chem. Soc., Chem. Commun., 1990,
536–538.
53 N. T. Marcos, S. Pinho, C. Grandela, A. Cruz, B. Samyn-Petit, A.
Harduin-Lepers, R. Almeida, F. Silva, V. Morais, J. Costa, J. Kihlberg,
H. Clausen and C. A. Reis, Cancer Res., 2004, 64, 7050–7057.
54 J. Gobom, E. Nordhoff, E. Mirgorodskaya, R. Ekman and P.
Roepstorff, J. Mass Spectrom., 1999, 34, 105–116.
46 L. Braunschweiler and R. R. Ernst, J. Magn. Reson., 1983, 53, 521–
528.
47 A. A. Bothner-By, R. L. Stephens, J.-M. Lee, C. D. Warren and R. W.
Jeanloz, J. Am. Chem. Soc., 1984, 106, 811–813.
720 | Org. Biomol. Chem., 2006, 4, 713–720
This journal is
The Royal Society of Chemistry 2006
©