From disulfide- to thioether-linked glycoproteins

Article abstract of DOI:10.1002/anie.200704381

(Chemical Presented) Strengthening the bond: The introduction of a thiol tag in combination with chemoselective ligation to form a disulfide-linked bioconjugate is a selective and useful method for site-selective protein glycosylation. The phosphine-mediated desulfurization of such glycoconjugates to their reductant-resistant thioether-linked counterparts completes a convergent, site-selective synthesis of thioether-linked glycoproteins (see scheme).

Full text of DOI:10.1002/anie.200704381

Communications
DOI: 10.1002/anie.200704381
Protein Modification
From Disulfide- to Thioether-Linked Glycoproteins**
Gonçalo J. L. Bernardes, Elizabeth J. Grayson, Sam Thompson, Justin M. Chalker,
James C. Errey, Farid El Oualid, Timothy D. W. Claridge, and Benjamin G. Davis*
The presence of carbohydrate structures on proteins has been
estimated to occur in 50% of eukaryotic cells[1] and is linked
to several biological events,[2] such as the regulation of cell
signaling,[3] cellular differentiation,[4] and immune response.[5]
In nature, glycoproteins are found as heterogeneous mixtures,
which complicates their characterization and functional
determination.[6] Better access to homogeneous glycoproteins
and their mimics is likely to improve our understanding of
their roles.
present the synthesis of thioether-linked glycoconjugates
from these readily synthesized disulfide-linked precursors.
Important examples of the contraction of disulfide and
peroxide linkages upon treatment with sources of PIII are
known;[20] however, the generality of such transformations
remains to be established. Our research in this area was
motivated by a then surprising reaction of the sugar disulfide
1 with tributylphosphine to afford thioglycoside 2 in 74%
yield (Scheme 1). Inversion at the anomeric center suggested
Naturally occurring protein and peptide glycans are
predominantly linked to an asparagine or serine/threonine
residue, and many glycopeptide syntheses are based on the
introduction of mimics of such tethers.[2] It was not until 1971
that a natural S-glycosidic linkage was identified on a
peptide:[7] Löte and Weiss isolated octa- and decapeptides
in which galactose and glucose, respectively, were attached to
the side chain of an N-terminal cysteine residue.[8] Several
methods have since been developed for the synthesis of S-
linked glycopeptides:[9] the conjugate addition of a glycosyl
thiol to a dehydroalanine-containing peptide,[10] the reaction
of a glycosyl thiol with a b-bromoalanine moiety,[11] and the
rearrangement of an allylic selenenylsulfide.[12] However, to
date no chemical method has been applied to the synthesis of
S-linked glycoproteins.[13] Importantly, S-linked glycopeptides
display enhanced chemical[14] and enzymatic[15] stability
relative to their native congeners. A process for desulfurizing
disulfide-linked glycoproteins to provide thioether-linked
homologues would allow ready access not only to this class
of natural products but also to novel glycoproteins.
Scheme 1. Desulfurization of a thioglycoside. Bn=benzyl.
attack at the disulfide bond followed by an SN2-like reaction
of the resulting thiophosphonium salt (see Scheme S1 in the
Supporting Information).[21] We considered that phosphines
might mediate an analogous reaction in which a single sulfur
atom is lost from a disulfide[22] glycoprotein to give the
corresponding S-linked glycoprotein (Scheme 2). This trans-
formation would provide controlled access to a thioether
linkage resistant to reduction and less prone to enzymatic
degradation than the corresponding disulfide linkage.
We have described previously the use of glycomethane-
thiosulfonates (glycoMTS),[16] glycophenylthiosulfonates
(glycoPTS),[17] and glycoselenenylsulfides (glycoSeS)[18,19] as
efficient chemoselective reagents for the synthesis of disul-
fide-linked glycopeptides and glycoproteins. Herein we
[*] G. J. L. Bernardes, S. Thompson, J. M. Chalker, Dr. J. C. Errey,
Dr. F. El Oualid, Dr. T. D. W. Claridge, Prof. B. G. Davis
Chemistry Research Laboratory
Scheme 2. Strategy for the site-selective synthesis of thioether-linked
glycoproteins.
Department of Chemistry, University of Oxford
12 Mansfield Road, Oxford OX13TA (UK)
Fax: (+44)1865-285002
Dr. E. J. Grayson
Department of Chemistry, University of Durham
South Road, Durham DH13LE (UK)
Disulfide-linked glycosyl amino acid and glycopeptide
model substrates were constructed by using glycoPTS[17] and
glycoSeS[18] methods, the reagents for which were prepared
from the parent carbohydrates (Glc, Gal, Fuc, GlcNAc) in
good yields (see Supporting Information). Initially, we used
the glucosyl amino acid 3 as a model substrate with
tributylphosphine (1.2 equiv) as the reducing agent and
methanol as the solvent. The desulfurized product 4 was
obtained in 8% yield as a mixture of diastereoisomers
[**] We thank the Fundaç¼o para a CiÞncia e a Tecnologia, Portugal
(G.J.L.B.), the Rhodes Trust (J.M.C), and the European Commission
(F.E.O.) for financial support and the Daphne Jackson Trust for a
returner’s fellowship funded by the Royal Commission for the
Exhibition of 1851 (E.J.G.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
Table 1: Desulfurization of disulfide glycosyl amino acids.[a]
dehydroalanine would provide 4
(see Scheme S3 in the Supporting
Information).
The recovery of reduced cys-
teine and glycosyl thiols prompted
us to use more polarized, electron-
rich phosphines, which would attack
the disulfide to give a phosphonium
ion that was less susceptible to
hydrolysis. Accordingly, the use of
hexamethylphosphorous triamide
(HMPT) afforded thioether 4 in an
improved yield of 41%. Under
Entry Substrate
PX3 (equiv)
t [h] Product
Yield [%][b]
1
2
3
4
PBu3 (1.2)
6
3
3
3
8
41
71
73
P(NMe2)3 (1.2)
P(NMe2)3 (2.2)
P(NEt2)3 (2.2)
optimized
reaction
conditions
(HMPT (2.2 equiv), degassed anhy-
drous methanol, room tempera-
ture),[24] 3 was converted into the
S-glycosyl amino acid 4 in 71%
yield.[25]
5
P(NMe2)3 (2.2)
3
61
6
7
8
P(NMe2)3 (2.2)
P(NEt2)3 (2.2)
P(NMe2)3 (2.2)
3
3
5
69
72
47[c]
The reaction was found to be
broadly tolerant with respect to the
sugar moiety (Glc, Gal, Fuc,
GlcNAc) and the protecting group
(Bn/Ac) and afforded the desired
desulfurized products in good yields
(61–75%; Table 1, entries 3–12)
with retention of the anomeric con-
figuration. The reaction could even
be performed in aqueous solvents
(Table 1, entries 8 and 10). The
standard reaction conditions were
fully compatible with the use of
unprotected glycosyl amino acids:
Compounds 15 and 17, accessed by
the glycoSeS method,[18] underwent
desulfurization to give 16 and 18 in
good yields (Table 1, entries 13 and
14).
The compatibility of the method
with glycopeptides and other amino
acid residues was also shown. The
glucotripeptide 19 and galactotetra-
peptide 21, both of which were
prepared by using glycoPTS[17]
reagents, were desulfurized success-
fully with HMPT to give the corre-
sponding S-linked glycopeptides 20
and 22 in 72 and 67% yield, respec-
tively (Scheme 3).[26]
9
10
P(NMe2)3 (2.2)
P(NMe2)3 (2.2)
3
7
75
9[d]
11
P(NMe2)3 (2.2)
3
70
12
13
14
P(NMe2)3 (2.2)
P(NMe2)3 (2.0)
P(NMe2)3 (2.0)
3
3
3
73
68
74
[a] Unless otherwise noted, reactions were carried out in degassed anhydrous methanol at room
temperature; see the Supporting Information for details. [b] The product was obtained as a 1:1 mixture
of diastereomers in all cases, except in entry 12 (d.r. 6:5). [c] The reaction was carried out in methanol/
water (2:1, v/v). [d] The reaction was carried out in methanol/water (1:1, v/v).
Next, we investigated the
applicability of this novel ligation
(Table 1, entry 1). Thus, the mechanism for the reaction of 3
appeared to differ from that observed for the reaction of 1.
The retention of the anomeric configuration suggests that the
stereodivergence arises from the racemization of cysteine.[23]
This hypothesis can be rationalized mechanistically by
invoking a dehydroalanine intermediate formed by the
disulfide cleavage of 3 and b elimination of the resulting
thiophosphonium salt. The redelivery of the glycosyl thiol to
strategy to the conversion of disulfide-linked glycoproteins
into thioether-linked glycoproteins. The disulfide-linked glu-
cosyl protein 23 was constructed from a mutant of the serine
protease subtilisin Bacillus lentus SBL-S156C, which contains
a single cysteine residue, by using the glycoPTS method.[17]
HMPT-mediated desulfurization converted 23 into the thio-
ether-linked glycoprotein 24 (Figure 1).[27,28] The treatment of
the newly formed thiother-containing protein 24 with tris(2-
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Communications
Figure 1. ESIMS spectrum of S156C-S-Glc(OAc)4 (see the Supporting Information for full reaction details).
[2] a) A. Varki, Glycobiology 1993, 3, 97 – 130; b) R. A. Dwek,
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[13] Recently, a biosynthetic route to a dehydroalanine-containing
protein that serves as a Michael acceptor for thiol nucleophiles,
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Scheme 3. Extension of desulfurization to the synthesis of S glycopep-
tides.
carboxyethyl)phosphine (TCEP) showed it to be resistant to
reduction. When the disulfide-linked protein 23 was treated
with TCEP, rapid reduction to the free thiol protein SBL-
S156C was observed.
In summary, we have developed a desulfurization reaction
that enables the conversion of readily synthesized disulfide-
linked glycosyl amino acids, glycopeptides, and glycoproteins
into the corresponding thioether-linked glycoconjugates. The
reaction is compatible with a variety of protected and
unprotected sugar residues, different amino acid residues,
and an aqueous environment, and proceeds with retention of
the anomeric configuration. We also applied this ligation
method to protein modification in the first example of the
chemical conversion of a surface-exposed cysteine residue to
provide the corresponding S-linked glycoprotein.
[14] B. Capon, Chem. Rev. 1969, 69, 407 – 498.
[15] Z. J. Witczak, R. Chhabra, H. Chen, X. Q. Xie, Carbohydr. Res.
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Jones, Tetrahedron: Asymmetry 2000, 11, 245 – 262.
[17] D. P. Gamblin, P. Garnier, S. J. Ward, N. J. Oldham, A. J.
Fairbanks, B. G. Davis, Org. Biomol. Chem. 2003, 1, 3642 – 3644.
[18] D. P. Gamblin, P. Garnier, S. van Kasteren, N. J. Oldham, A. J.
Fairbanks, B. G. Davis, Angew. Chem. 2004, 116, 846 – 851;
Angew. Chem. Int. Ed. 2004, 43, 828 – 833.
[19] G. J. L. Bernardes, D. P. Gamblin, B. G. Davis, Angew. Chem.
2006, 118, 4111 – 4115; Angew. Chem. Int. Ed. 2006, 45, 4007 –
4011.
[20] a) D. N. Harpp, J. G. Gleason, J. P. Snyder, J. Am. Chem. Soc.
1968, 90, 4181 – 4182; b) D. N. Harpp, J. G. Gleason, J. Org.
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[21] The electron-withdrawing nitro group was necessary for the
formation of the product in useful yields. When unsubstituted
thiophenol was used instead of p-nitrothiophenol as a compo-
nent of the disulfide substrate, the thioglycoside was obtained in
low yield, and a large amount of the anomeric thiol was formed
(see the Supporting Information).
Received: September 22, 2007
Published online: February 14, 2008
Keywords: disulfides · glycoproteins · glycosylation ·
.
protein modifications · thioethers
[1] R. Apweiler, H. Hermjakob, N. Sharon, Biochim. Biophys. Acta
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[22] A disulfide contraction of this type was observed recently for a
disulfide derivative of a protected cysteine residue: D. Crich, V.
Krishnamurthy, F. Brebion, M. Karatholuvhu, V. Subramanian,
K. T. Hutton, J. Am. Chem. Soc. 2007, 129, 10282 – 10294.
[23] Cysteine epimerization, as opposed to carbohydrate anomeriza-
tion, was established through the complete assignment of the
NMR spectrum, including the critical observation of two distinct
signals for 1-Hb.
[24] Similar yields were observed with hexaethylphosphorus tria-
mide; however, the purification of the product was more
cumbersome than when HMPT was used.
[25] Crossover experiments on differentially protected glycosyl
cysteine derivatives under the optimized reaction conditions
revealed that glycosyl exchange takes place and thus lent
support for a dehydroalanine intermediate. 1H NMR spectro-
scopic analysis allowed the direct observation of dehydroalanine
formation and consumption (see the Supporting Information for
details). The low diastereoselectivity in the subsequent con-
jugate addition is consistent with that previously reported in
reference [10].
[26] Other a centers in the peptide did not undergo epimerization,
which suggests that the observed epimerization at the a center of
cysteine derives largely, if not exclusively, from dehydroalanine
formation and not from deprotonation by HMPT.
[27] The diastereoselectivity of the reaction in Figure 1 will depend
on the stereochemical environment of C156. Conjugate addition
to dehydroalanine-containing peptides has been shown to be
largely sequence dependent, with a diastereomeric ratio of
> 85:15 estimated for certain scaffolds: a) Ref. [10]; b) U.
Schmidt, E. ꢀhler, Angew. Chem. 1976, 88, 54 ;Angew. Chem.
Int. Ed. Engl. 1976, 15, 4 2.
[28] Modification at C156 was corroborated by trypsin digestion and
MALDI MS analysis (see the Supporting Information).
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