Chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein

Article abstract of DOI:10.1016/S0040-4039(00)02286-3

Two types of maleimidosugars as thiol-selective carbohydrates, 1-maleimidosugars and acetyl-linked maleimidosugars, were efficiently synthesized. They were coupled to glutathione, Fas peptide and bovine serium albumin (BSA) to prepare the corresponding glycosylated peptides and a protein via stable thioether linkages in a chemoselective manner.

Full text of DOI:10.1016/S0040-4039(00)02286-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1325–1328
Chemoselective ligation of maleimidosugars to peptides/protein
for the preparation of neoglycopeptides/neoglycoprotein
Injae Shin,* Hyuk-jun Jung and Myung-ryul Lee
Department of Chemistry, Yonsei University, Seoul 120-749, South Korea
Received 15 November 2000; revised 6 December 2000; accepted 8 December 2000
Abstract—Two types of maleimidosugars as thiol-selective carbohydrates, 1-maleimidosugars and acetyl-linked maleimidosugars,
were efficiently synthesized. They were coupled to glutathione, Fas peptide and bovine serium albumin (BSA) to prepare the
corresponding glycosylated peptides and a protein via stable thioether linkages in a chemoselective manner. © 2001 Elsevier
Science Ltd. All rights reserved.
It is known that oligosaccharides in glycoconjugates
such as glycoproteins and glycolipids play important
roles in many biological processes and thus their bio-
logical functions have been extensively investigated by
chemists, biochemists and biologists.1 In general, glyco-
proteins in eukaryotic cells are expressed as heteroge-
neous mixtures of glycoforms, namely, proteins bearing
heterogeneous carbohydrate moieties. As a conse-
quence, their purification from natural sources is
difficult and their biological roles in glycoproteins
remain elusive. To better understand the molecular
basis of oligosaccharides and to develop glycoproteins
as potential pharmaceutical agents, it is imperative to
readily access glycoproteins with well-defined oligosac-
charide chains.
Several approaches to introduce carbohydrate moieties
into proteins or peptides at specific positions via nonna-
tive glycosidic linkage have been reported.2 For exam-
ple, iodoacetamide-containing sugars were employed
for site-specific glycosylation of a cysteine residue in
proteins/peptides.3 Carbohydrates bearing an asymmet-
ric disulfide linkage were also used as thiol-reactive
reagents.4 O-Linked N-acetylgalactosaminyl peptides
after treatment with galactose oxidase were coupled to
aminooxy sugars to prepare O-linked glycopeptides.5 In
a different approach, a lysine residue was glycosylated
using a designed helix–loop–helix motif.6
In an attempt to develop new methodology to prepare
homogeneous glycoproteins, we have investigated the
OH
O
OAc
O
O
(a) 0.018 M NaOMe
MeOH, 15 min
O
RO
HO
RO
AcO
N
N
X
or (b) TEA/H2O/MeOH
(3 : 1 : 1)
OH
OAc
O
O
2
1
30 min
R = H
R = Ac
Gal(OH)4-1-β-
Glc(OH)4-1-β-
Glc(OH)4-1-α-
Gal(OAc)4-1-β-
Glc(OAc)4-1-β-
Glc(OAc)4-1-α-
Scheme 1.
Abbreviations: DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide; ESI MS, electrospray ionization mass spectrometry; TBSOTf,
tert-butyldimethylsilyl trifluoromethanesulfonate; TEA, triethylamine; TFA, trifluoroacetic acid; TMSOTf, trimethylsilyl trifluoromethanesul-
fonate.
Keywords: carbohydrates; thioethers; glycopeptides/glycoproteins; glycosylations.
* Corresponding author. Tel.: 82-2-2123-2631; fax: 82-2-364-7050; e-mail: injae@alchemy.yonsei.ac.kr
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02286-3

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I. Shin et al. / Tetrahedron Letters 42 (2001) 1325–1328
chemoselective ligation of carbohydrates containing a
maleimide group to peptides or proteins. Maleimide
functionality has been widely used for the selective
modification of thiol in the presence of other nucleo-
philes such as amine, alcohol, carboxylate and
guanidine.7 Two types of thiol-reactive carbohydrates,
1-maleimidosugars (7) and acetyl-linked maleimidosug-
ars (10), were synthesized to produce neoglycosylated
peptides/proteins. For the preparation of anomeric
maleimidosugars (7), we first made attempts to remove
acetyl groups in peracetylated maleimidosugars 18 under
mild basic conditions such as 0.018 M NaOMe and
TEA–H2O–MeOH (3:1:1) (Scheme 1).4a However, it was
found that a maleimide group was hydrolyzed with
concomitant deacetylation under these conditions.
Therefore, we protected hydroxyl groups by an acid-
labile group such as TBS or TMS. Acetohalosugars 3
were converted to the corresponding acetylated glycosyl
azides 4 by substitution of chloro (3a) and bromo (3b,
c) groups with azide under biphasic conditions (Scheme
2). Removal of acetyl groups and reprotection of the
exposed alcohols by TBS or TMS with TBSOTf or
TMSOTf in the presence of DMAP gave TBS or TMS-
protected sugars. However, it was discovered that a
TMS protecting group was not suitable since it was
partially cleaved during the next reduction of azide with
Pd/C and H2. Reduction of TBS-protected azide 5 and
reaction of the resultant glycosylamines with maleic
anhydride in THF afforded amic acids 6 in moderate to
high yields. Cyclization of amic acids 6 with hexa-
methyldisilazane (HMDS) in the presence of ZnCl29 and
a subsequent deprotection of TBS by CH2Cl2–TFA–
H2O (10:5:1) furnished the desired products 7. On the
other hand, we also prepared acetyl-linked maleimido-
sugars (10) that could be synthesized by a simple reac-
tion step. One-pot amination of carbohydrates10 and
a subsequent coupling with pentafluorophenyl N-
maleoylacetate11 in DMF produced the acetyl-linked
maleimidosugars 10 in 54–95% yield (Scheme 3).
We then examined the potential of synthesized maleimi-
dosugars as thiol-reactive oligosaccharides to generate
glycosylated peptides/protein. First, glutathione (g-
GluCysGly) was efficiently glycosylated at a cysteine
residue to yield the corresponding carbohydrate-ad-
ducts 7a1–7c1 and 10a1–10c1 by 1 molar equiv. of
7a–c and 10a–c, respectively, in H2O (Table 1).12,13
Next, a synthetic Fas peptide (Ac-VARLSCKSVNAQ-
NH2, Table 1) was also glycosylated according to the
similar procedure. The interaction between glyco-
proteins Fas and FasL is known to be involved in
apoptosis (programmed cell-death) and thus has been
extensively studied.14 However, the function and nature
of oligosaccharide chains on Fas and FasL remain
unclear. Based on preliminary mutagenesis studies, it
appears that Ser4 corresponds to an O-glycosylation
site in the Fas protein.15 We synthesized a Fas peptide
using standard solid phase peptide synthesis and re-
acted with 1 molar equiv. of 7a–c and 10a–c in
DMSO–H2O to give the corresponding glycosylated
products. Characterization of the Fas peptide by ESI
MS following ligation revealed selective incorporation
of the oligosaccharides into the Fas peptide.16 The
thiol-selective glycosylation was investigated using a
Fas peptide whose thiol was blocked by 5-thio-2-ni-
trobenzoic acid (TNB). The TNB-protected peptide was
not glycosylated, suggesting that maleimidosugars are
thiol-specific.
Bovine serum albumin (BSA), possessing a single re-
duced cysteine at position 58, which provides a unique
site for attachment of thiol-reactive maleimidosugars,
was employed as a model protein. BSA was incubated
with 40 molar equiv. of 7 and 10 in 50 mM sodium
phosphate buffer (pH 7). Subsequently, the reaction
mixture was passed through gel filtration column (PD-
10, Amersham Pharmacia) to remove an excess of
maleimidosugars. The resultant glycosylated products
of BSA were characterized by ESI MS, which gave a
OAc
O
R3
OAc
O
R3
1. NaOMe, MeOH
NaN3, TBAHS
R2
AcO
R2
N3
AcO
2. TBSOTf, pyr
DMAP
sat. NaHCO3/CH2Cl2
(quant)
R1
R
1X
(50 - 80%)
4
3
a. GlcNAc: R1 = NHAc, R2 = OAc, R3 = H, X = Cl
b. Gal : R1 = OAc, R2 = H, R3 = OAc, X = Br
c. Mal : R1 = OAc, R2 = Glc(OAc)4-α-, R3 = H, X = Br
a. GlcNAc: R1 = NHAc, R2 = OAc, R3 = H
b. Gal : R1 = OAc, R2 = H, R3 = OAc
c. Mal : R1 = OAc, R2 = Glc(OAc)4-α-, R3 = H
1. Pd/C, H2
THF
OTBS
O
1. HMDS/ZnCl2
Benzene, DMF
R3
O
OH
O
R3
OTBS
O
R3
R2
O
R2
HO
R2
TBSO
NH
N
TBSO
2. THF
O
N3
2. CH2Cl2/TFA/H2O
(10 : 5 : 1)
R1 HO2C
R1
R1
O
(30 - 40%)
O
(50 - 90%)
6
7
5
O
a. GlcNAc: R1 = NHAc, R2 = OTBS, R3 = H
b. Gal : R1 = OTBS, R2 = H, R3 = OTBS
a. GlcNAc: R1 = NHAc, R2 = OH, R3 = H
b. Gal : R1 = OH, R2 = H, R3 = OH
c. Mal : R1 = OTBS, R2 = Glc(OTBS)4-α-, R3 = H
c. Mal : R1 = OH, R2 = Glc(OH)4-α-, R3 = H
Scheme 2.

I. Shin et al. / Tetrahedron Letters 42 (2001) 1325–1328
1327
O
OH
OH
O
N
CO2Pfp
OH
O
sat. NH4HCO3
O
R2O
HO
O
H
N
R2O
HO
O
O
OH
R2O
HO
N
NH2
42 oC, 36 h
(quant)
R1
DMF
(54 - 95%)
R1
R1
O
8
9
10
a. GlcNAc: R1 = NHAc, R2 = H
b. Lac : R1 = OH, R2 = Gal-β-
c. Cell : R1 = OH, R2 = Glc-β-
Scheme 3.
Table 1. Chemoselective ligation of maleimidosugars to peptides and a protein
Reaction time
Product
Maleimidosugar
Protein/Peptide
(min)
γ-Glu Cys Gly
1. γ-Glu Cys Gly
7a ~ 7c
7a1~ 7c1
30 ~ 60
30
O
S
10a ~ 10c
10a1~ 10c1
N Sugar
O
Ac-VARLSCKSVNAQ-NH2
7a2 ~ 7c2
2. Ac-VARLSCKSVNAQ-NH2
7a ~ 7c
30 ~ 60
30
O
S
10a2 ~ 10c2
10a ~ 10c
N
Sugar
Sugar
O
3. BSA - Cys58
BSA - Cys58
S
7a ~ 7c
7a3 ~ 7c3
30 ~ 60
30
O
10a ~ 10c
10a3 ~ 10c3
N
O
The experimental details are described in the text.
mass change of 322.3, 228.7 and 487.7 following reac-
tion with 7a–c, and 386.3, 477.1 and 536.1 with 10a–c,
respectively. Chemoselective ligation of maleimidosug-
ars to a cysteine residue in BSA was also investigated
using TNB-protected BSA. It was found that BSA
blocked by TNB at a cysteine residue was not glycosy-
lated. These results suggest that a single carbohydrate
moiety was incorporated into a cysteine in BSA. We
believe that this methodology may be useful in the
synthesis of a variety of glycoconjugates.
2. Reviews: (a) Stowell, C. P.; Lee, Y. C. Adv. Carbohydr.
Chem. Biochem. 1980, 37, 225; (b) Lemieux, G. A.;
Bertozzi, C. R. TIBTECH. 1998, 16, 506.
3. (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1991, 32,
6793; (b) Wong, S. Y. C.; Guile, G. R.; Dwek, R. A.;
Arsequell, G. Biochem. J. 1994, 300, 843.
4. (a) Macindoe, W. M.; van Oijen, A. H.; Boons, G.-J.
Chem. Commun. 1998, 847; (b) Davis, B. G.; Maughan,
M. A. T.; Green, M. P.; Ullman, A.; Jones, J. B.2
Tetrahedron: Asymmetry 2000, 11, 245.
5. Rodriguez, E. C.; Winans, K. A.; King, D. S.; Bertozzi,
C. R. J. Am. Chem. Soc. 1997, 119, 9905.
6. Andersson, L.; Stenhagen, G.; Baltzer, L. J. Org. Chem.
1998, 63, 1366.
Acknowledgements
7. Hermanson, G. T. Bioconjugate Techniques; Academic
Press: New York, 1996; p. 148.
8. Shin, I.; Jung, H.-j.; Cho, J. Bull. Korean Chem. Soc.
2000, 21, 845.
9. Reddy, P. Y.; Kondo, S.; Fujita, S.; Toru, T. Synthesis
1998, 999.
This work was supported by a grant from the Korea
Research Foundation (1999-015-DP0218), Ministry of
Education, Korea. I.S. thanks the Korea Basic Science
Institute for synthesizing Fas peptide and providing us
with mass spectra of glycosylated peptides and BSA.
10. Lubineau, A.; Auge´, J.; Drouillat, B. Carbohydr. Res.
1995, 266, 211.
11. Oishi, T.; Kagawa, K.; Fujimoto, M. Macromolecules
1993, 26, 24.
References
12. The progress of ligation reactions was directly monitored
by a decrease in absorbance at 220 nm or the unreacted
1. (a) Dwek, R. A. Chem. Rev. 1996, 96, 683; (b) Varki, A.
Glycobiology 1993, 3, 97.
.

1328
I. Shin et al. / Tetrahedron Letters 42 (2001) 1325–1328
SH was determined by 5,5%-dithiobis(2-nitrobenzoic acid)
calcd for C28H45N5O19S [M+H]+ 786.4, found 786.4.
14. Nagata, S.; Golstein, P. Science 1995, 267, 1449.
15. The N-terminal dipeptide, Val-Ala, is included in the
C-terminus of the lead sequence of the Fas protein.
16. Selected data for 7a2 (ESI): calcd for C66H115N21O24S
[M+H]+ 1616.8, found 1616.5. 7b2: calcd for
C64H110N20O24S [M]+ 1575.9, found 1575.5. 7c2: calcd for
C70H120N20O29S [M]+ 1737.1, found 1737.4. 10a2 (ESI):
calcd for C68H118N22O25S [M+H]+ 1673.8, found 1673.7.
10b2: calcd for C72H125N21O30S [M+H]+ 1794.8, found
1794.5. 10c2: calcd for C72H125N21O30S [M+H]+ 1794.8,
found 1794.9.
(DTNB) using the Ellman method. After different time
intervals, an aliquot of the reaction mixture was removed
and reacted with DTNB, and UV absorbance at 412 nm
was recorded. Ellman, G. L. Arch. Biochem. Biophys.
1959, 82, 70.
13. Selected data for 7a1 (ESI): calcd for C22H35N5O13S
[M+H]+ 608.4, found 608.5. 7b1: calcd for C20H31N4O13S
[M+H]+ 567.5, found 567.4. 7c1: calcd for C26H41N4O18S
[M+H]+ 729.6, found 729.5. 10a1 (ESI): calcd for
C24H38N6O14S [M+H]+ 665.4, found 665.4. 10b1: calcd
for C28H45N5O19S [M+H]+ 786.4, found 786.4. 10c1:
.
.