Glyco-SeS: Selenenylsulfide-mediated protein glycoconjugation - A new strategy in post-translational modification

Article abstract of DOI:10.1002/anie.200352975

Site-selective glycosylation by Se-S-mediated ligation has led to the efficient formation of a wide variety of conjugates 1 without the need for a large excess of the carbohydrate reagent. By this convergent method it was possible to introduce a heptasaccharide glycan selectively, and to perform a multiple site-selective chemical glycosylation of protein. A chemically Cysglycosylated glycoprotein was elaborated enzymatically.

Full text of DOI:10.1002/anie.200352975

Angewandte
Chemie
Communications
Precise mimics of post-translationally modified proteins can
be constructed by using a highly selective, selenenylsulfide-mediated
method. On the following pages B. G. Davis and co-workers describe
the use of this approach for the glycosylation of a protein with a
heptasaccharide glycan and for a multiple site-selective, chemical
protein glycosylation.
Angew. Chem. Int. Ed. 2004, 43, 827
DOI: 10.1002/anie.200352975
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
827

Communications
Glycoproteins
cysteine modification to create a linkage, for example, the use
of a 5-nitropyridine-2-sulfenyl-activated N-acetylglucosamine
(GlcNAc),[23] or glycosyl iodoacetamides.[16] However, these
methods have so far been limited to the addition of a single
GlcNAc monosaccharide[16,23] and can be plagued by a lack of
selectivity (modification of histidine residues) and/or incom-
plete reactions.[16] Furthermore, no multiple site-selective
glycoconjugation has been demonstrated, a weakness that was
highlighted recently.[24]
Inspired by the occurrence, albeit rare, of selenenylsulfide
proteins in Nature as selective electrophilic moieties,[25,26] we
report herein a selenenylsulfide-mediated protein glycocon-
jugation,[27] which allows glycoconjugation with mono- and
oligosaccharides of up to seven saccharide units in size at
single and multiple sites in a variety of proteins. Two parallel
strategies were investigated in which the protein cysteine
residue plays potentially contrasting electrophilic and nucle-
ophilic roles (Scheme 1). In the first approach A, a cysteine-
Glyco-SeS: Selenenylsulfide-Mediated Protein
Glycoconjugation—A New Strategy in Post-
Translational Modification**
David P. Gamblin, Philippe Garnier,
Sander van Kasteren, Neil J. Oldham,
Antony J. Fairbanks, and Benjamin G. Davis*
The co- and post-translational glycosylation[1] of proteins[2,3] is
a key factor in protein folding and stability,[4] and plays a
major role in essential biological processes, such as cell
signaling and regulation,[5,6] development,[7] and immunity.[8]
The study of these events is made difficult by the fact that
glycoproteins occur naturally as mixtures of so-called glyco-
forms,[9] which possess the same peptide backbone but differ
in both the nature and the site of glycosylation. Furthermore,
since protein glycosylation is not under direct genetic control,
the expression of therapeutic glycoproteins in mammalian
cell cultures leads to heterogeneous mixtures of glycoforms.[10]
The ability to synthesize homogeneous glycoprotein glyco-
forms is therefore a prerequisite not only for purposes of
accurate investigation, but is of increasing importance for the
preparation of therapeutic glycoproteins, which are currently
marketed as multiglycoform mixtures (e.g. erythropoietin[11,12]
and interleukins[13]).
Several chemical synthetic strategies have been developed
to this effect.[14–18] We showed previously that the combined
use of site-directed mutagenesis and chemoselective glyco-
conjugation could be used for site-selective protein glyco-
conjugation.[19,20] In this approach, a cysteine residue is
introduced through mutagenesis to generate a protein nucle-
ophile with a single free thiol, which is subsequently modified
chemoselectively with electrophilic thiol-specific carbohy-
drate reagents, such as glycosyl methanethiosulfonates[19,20] or
glycosyl phenylthiosulfonates.[21] This method makes possible
the introduction of spacer-linked free and protected glycans
alike, but only allows the preparation of directly linked free
glycosides after enzymatic deprotection on protein,[22] thus
potentially limiting its utility. Several other methods have
been developed for the synthesis of glycoproteins based on
Scheme 1. Two potential parallel glycoconjugation strategies. The
protein cysteine plays either an electrophilic role (A) or a nucleophilic
role (B).
containing protein is converted into the corresponding
(phenylselenenyl)sulfide; the electrophilic character of the
[28]
À
sulfur atom in the resulting S Se bond renders it suscep-
[*] D. P. Gamblin, Dr. P. Garnier, S. van Kasteren, Dr. N. J. Oldham,
Dr. A. J. Fairbanks, Dr. B. G. Davis
tible to nucleophilic substitution by 1-thio mono- or oligo-
saccharides. In the opposite approach B, 1-thio mono- or
oligosaccharides are first converted into their selenenylsulfide
analogues, which can subsequently be coupled to a cysteine
residue. Thus, the cysteine residue this time acts as a
Dyson Perrins Laboratory, University of Oxford
South Parks Road, Oxford, OX1 3QY (UK)
Fax: (+44)1865-275674
E-mail: ben.davis@chem.ox.ac.uk
À
[**] This work was supported by the EPSRC (D.P.G.) and Glycoform
(D.P.G., P.G., S.v.K.). We thank Dr. B. O'Neil, Dr. T. D. W. Claridge,
and Dr. J. Kirkpatrick for invaluable technical support. We thank the
EPSRC for access to the Mass Spectrometry Service at Swansea and
the Chemical Database Service at Daresbury and for the award of a
DTA studentship (D.P.G.). We also thank Dr. Mario Polywka for
stimulating discussions and Susan M. Hancock for providing SSbG
mutants.
nucleophile. Importantly, the exquisite selectivity of S Se
chemistry would obviate all need for protecting groups during
glycoconjugation.
The representative monosaccharides glucose (Glc), gal-
actose (Gal), and N-acetylglucosamine (GlcNAc), and oligo-
saccharides (trisaccharides) Glca(1,4)-Glca(1,4)-Glc (7) and
Glca(1,4)-(Glca(1,4))5-Glc (10) were chosen for the glyco-
conjugation reactions. To evaluate the feasibility of approach
A, the glycosyl halides 1a–c were converted into the
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
828
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200352975
Angew. Chem. Int. Ed. 2004, 43, 828 –828

Angewandte
Chemie
corresponding monosaccharide glycosyl-b-thiols 4a (Glc), 4b
(Gal), and 4c (GlcNAc) through treatment with thiourea to
afford the corresponding isothiouronium salts,[29] followed by
mild hydrolysis with sodium metabisulfite and ZØmplen
deacetylation (Scheme 2).[30] Following essentially similar
procedures to those for the monosaccharides, the deprotected
oligosaccharide thiols 4d and 4e were prepared from the
Single-site glycoconjugation was explored by using the
model cysteine-containing protein serine protease subtilisin
Bacillus lentus mutant S156C[19,31] (SBL-Cys156, 12; Table 1).
The protein 12 was treated with phenylselenenyl bromide
(PhSeBr)[32,33] to give the corresponding selenenylsulfide 13,
which was subsequently treated with the deprotected 1-thio
monosaccharides 4a–c to afford the corresponding glycosy-
lated proteins 14a (SBL-Glc), 14b (SBL-Gal), and 14c (SBL-
GlcNAc) in quantitative yields as confirmed by ESI-MS
(Table 1).[34] The power of this method was
such that we were also able to glycosylate 12
quantitatively with the bulky trisaccharide
4d and even with the heptasaccharide 4e. To
our knowledge the latter carbohydrate is the
largest to have been used in a convergent
site-selective protein glycoconjugation to
date (Figure 1). In all cases quantitative
protein glycoconjugation occurred rapidly
(within 60 min)[35] with a low ratio of reagent
to protein (typically 10–20 equiv of thiol,
and as little as 1 equiv in some cases).[36]
Moreover, the compatibility of this proce-
dure with deprotected thio sugars removes
the need for a postmodification deprotec-
tion step.
maltotriose
(Scheme 2).
7 and the maltoheptaose 10, respectively
To demonstrate the applicability of this
methodology to other proteins, and to carry
out multiple-site glycoconjugations, we con-
structed a mutant of the thermophilic b-
glycosidase from the archeon Sulfolobus
solfataricus, which contains two cysteine
residues (SSbG-Cys344Cys432, 15). The
doubly glycosylated protein 17 (SSbG-
[Glc]2) was obtained upon activation of 15
with PhSeBr, followed by treatment with the
Glc thiol 4a, as shown by ESI-MS (Table 1;
m/z: calcd: 57775; found: 57760[37]). The
three glycosylation sites in 12 (SBL-Cys156)
and 15 (SSbG-Cys344Cys432) are found in a
wide variety of protein structures and envi-
ronments with different levels of exposure.
This approach based on the modification of
electrophilic cysteine residues can thus be
used to prepare glycoproteins from very
different proteins, and is not limited to
single-site glycoconjugation, but is also
amenable to multiple site-selective glyco-
conjugation. The site-selective modification
of electrophilic protein residues is a rare but
potentially powerful approach given the
scarcity of other competing electro-
philes.[15,38]
To probe approach B (Scheme 1) and
the potential of a cysteine residue as a
nucleophile, a series of protected glyco-SeS
reagents were prepared readily from the
thiols 3a–c used above (Scheme 2). The
protected glycosyl (phenylselenenyl)sulfides
(glyco-SeS) 5a–c were obtained from the
Scheme 2. Synthesis of protected and deprotected glycosylation reagents: a) thiourea, ace-
tone, reflux; b) Na2S2O5, CH2Cl2, water, 508C; c) NaOMe, MeOH; d) PhSeBr; e) NaOAc,
Ac2O, reflux; f) HBr (33%), AcOH, CH2Cl2; g) KSAc, acetone; h) thiourea, acetone, Bu4NI
(0.1 equiv), reflux.
Angew. Chem. Int. Ed. 2004, 43, 828 –828
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
829

Communications
Table 1: Glycoconjugation of 12 and 15 after activation with PhSeBr.[a]
Sugar thiol
Protein
Product
14a
Conversion [%][b]
ESI-MS
Found (calcd)
Glcb-SH 4a
Galb-SH 4b
GlcNAcb-SH 4c
SBL-SSePh
SBL-SSePh
SBL-SSePh
>95
>95
>95
26908 (26909)
26908 (26909)
26944 (26950)
14b
14c
GlcaGlcaGlcb-SH 4d
SBL-SSePh
SBL-SSePh
14d
14e
>95
>95
27228 (27233)
27878 (27881)
Glca(Glca)5Glcb-SH 4e
Glcb-SH 4a
SSbG-(SSePh)2
17
>95
57760 (57775)
[a] Reagents: thiol (20 equiv), CHES (70 mm), MES (5 mm), CaCl2 (2 mm), pH 9.5. [b] Conversion determined by ESI-MS.
Figure 1. Deconvoluted ESI mass spectrum of 14e (SBL-Glc(Glc)5Glc).
reaction of the acetylated thiols 3a–c with PhSeBr. Their
deprotected counterparts 6a–c were obtained by using the
same method following ZØmplen deacetylation.[39] The pro-
tected and deprotected trisaccharides glyco-SeS 5d and 6d,
respectively, were also prepared by this method (Scheme 2).
The protected and deprotected glyco-SeS reagents 5a–c
and 6a–c were investigated in the glycoconjugation of the
representative thiol EtSH (18) and dipeptide 19. The
corresponding protected glycoconjugate mixed disulfides
21a–c and 22a–c and their deprotected counterparts 25a,
25c, 26a, and 26c were all obtained in excellent yields, with
complete retention of the anomeric b stereochemistry
(Table 2 and Table 3). These novel glyco-SeS reagents were
also investigated for their ability to glycosylate the model
protein 12. Again the glycoconjugation was quantitative, and
the high purity of the eight glycoproteins 14a–d and 23a–d
formed was confirmed by ESI-MS and Ellman titration.[40]
The glycoprotein products 14a–d were identical to those
synthesized by using strategy A. To demonstrate the versa-
tility of this methodology, we extended it to a larger cysteine-
containing protein, bovine serum albumin (BSA-Cys58, 20).
The protein 20 was glycosylated successfully with both the
protected and the deprotected Glc-SeS reagents 5a and 6a to
afford 24 and 27, respectively, and thus a third family of
glycoproteins.
To further elucidate the mechanism of glycoconjugation
with these glyco-SeS reagents, a time–course study of the
reaction of 12 with the deprotected triose reagent 6d was
conducted, and the reaction conditions were explored
(Figure 2). To our surprise, analysis by mass spectrometry
830
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 828 –828

Angewandte
Chemie
Table 2: Glycoconjugation with protected glyco-SeS reagents.
EtSH (18)[a]
Dipeptide 19[b]
Product Yield [%]
SBLCys156 (12)[c]
BSA-Cys58 (20)[c]
[d]
[d]
Product
Yield [%]
Product
Conv. [%]
Product
Conv. [%]
Glc(Ac)4-SSePh 5a
Gal(Ac)4-SSePh 5b
GlcNAc(Ac)3-SSePh 5c
Glc(Ac)4Glc(Ac)3Glc(Ac)3-SSePh 5d
21a
21b
21c
–
82
82
93
–
22a
22b
22c
–
75
93
88
–
23a
23b
23c
23d
>95
>95
>95
90
24
[e]
–
>95
–
–
–
–
[a] Et3N, CH2Cl2, room temperature, glyco-SeS/18 1:1. [b] Et3N, CH2Cl2/MeOH (20:1), room temperature, glyco-SeS/19 3:1. [c] CHES (70 mm), MES
(5 mm), CaCl2 (2 mm), pH 9.5, 10–75 equivalents of glyco-SeS. Different quantities of the reagent were used typically to increase rate and/or
convenience. Although prolonged exposure of glyco-SeS reagents to aqueous conditions caused decomposition in some cases, the reactions
described were fast enough to minimize decomposition. In all cases no side reactions with protein were observed. [d] Conversion (Conv.) was
determined by ESI-MS. [e] Dash (–) indicates reaction was not studied.
Table 3: Glycoconjugation with deprotected glyco-SeS reagents.
EtSH (18)[a]
Product Yield [%]
Peptide 19[b]
Product Yield [%]
SBLCys156 (12)[c]
Product Conv. [%]
BSA-Cys58 (20)[c]
Product Conv. [%]
Glc-SSePh 6a
Gal-SSePh 6b
GlcNAc-SSePh 6c
GlcGlcGlc-SSePh 6d
25a
[d]
25c
–
90
–
77
–
26a
–
26c
–
91
–
77
–
14a
14b
14c
14d
>95
>95
>95
>95
27
–
–
>95
–
–
–
–
[a] Et3N, CH2Cl2, room temperature, glyco-SeS/18 1:1. [b] Et3N, CH2Cl2/MeOH (20:1), room temperature, thioselenide/19 3:1. [c] 70 mm CHES, 5 mm
MES, 2 mm CaCl2, pH 9.5, 10–150 equivalents of glyco-SeS, conversion determined by ESI-MS. Different quantities of the reagent were used typically
to increase rate and/or convenience. Although prolonged exposure of glyco-SeS reagents to aqueous conditions caused decomposition in some cases,
the reactions described were fast enough to minimize decomposition. In all cases no side reactions with protein were observed. [d] Dash (–) indicates
reaction was not studied.
showed the rapid formation of 13 (SBL-SePh; less than
1 min), followed by the subsequent formation of the glyco-
sylated protein 14d (Figure 2). This result suggests that an
electrophilic glycoconjugation mechanism dominates regard-
less of the strategy (A or B) used. Thus, these two strategies
may be used in a complementary manner, thereby increasing
the flexibility of the glyco-SeS approach. We suggest that in
strategy B an initial nucleophilic attack at the selenium center
by the protein thiol generates 13 and liberates the sugar thiol
4d, which is subsequently able to proceed exactly as in
strategy A to displace SePh and form the glycosylated protein
14d (SBL-GlcGlcGlc). Consistent with this mechanism, the
use of a large excess of the glyco-SeS reagent was detrimental
to the reaction, as the thiols released were then trapped by
this reagent to give symmetrical disulfides. The reaction is
pH-dependent: when carried out at pH 7.5 or pH 8.5 for-
mation of the SBL-SePh intermediate 13 is observed, but just
10% conversion into the glycosylated protein 14d occurs.
Higher conversion is only observed at pH 9.5.[41] This
observation is also consistent with the proposed mechanism
of nucleophilic attack by the thiol 4d (or the corresponding
thiolate). These results show that glyco-SeS reagents in
strategy B effectively act as the source of both the selenating
reagent and the sugar thiol, thereby offering a convenient
alternative to the two-step use of PhSeBr and a sugar thiol in
strategy A.
Finally, to show the stability of the newly formed disulfide
linkage towards enzymatic carbohydrate extension and to
show that disulfide-linked glycoproteins may be processed by
glycosyltransferases, this new glycoconjugation method was
coupled with enzymatic carbohydrate extension (Figure 3).
Inhibited GlcNAc-S-S-SBL was incubated with UDP-galac-
tose (UDP-Gal) in the presence of b-1,4-galactosyltransfer-
ase,[42] which is known to catalyze the selective formation of
the Galb(1,4)-GlcNAc linkage.[43] The full conversion of the
GlcNAc-S-S-SBL protein 14c into the Galb(1,4)-GlcNAc-S-
S-SBL 28 was confirmed by ESI-MS (Figure 3), thereby
further extending the utility of the glyco-SeS method.
In conclusion, we have described the synthesis of glycosyl
selenenylsulfides (glyco-SeS), a novel class of glycosylating
agents, and their use not only for the glycoconjugation of
simple thiols and peptides (EtSH and dipeptides), but also of
proteins. Whereas most site-specific glycoconjugation meth-
ods take advantage of the nucleophilic character of cysteine
Angew. Chem. Int. Ed. 2004, 43, 828 –828
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
831

Communications
thiols (for example with glycosyl maleimides[44] or iodoacet-
amides[16,45]), our methodology is based upon a rarely
exploited[15,38] but nonetheless powerful electrophilic glyco-
conjugation mechanism. In particular, this approach allows
for the preparation of fully deprotected glycoconjugates and
glycoproteins. We have demonstrated multiple site-selective
glycoconjugation, which potentially provides access to poly-
valent neoglycoproteins with control over valency,[24] the
coupling of a heptasaccharide, and enzymatic elongation on a
disulfide-linked glycoprotein after cysteine modification.[15]
This methodology, combined with site-directed mutagenesis,
allows the rapid, site-selective glycoconjugation of very
different proteins with different fully deprotected carbohy-
drates, and the use of a low reagent-to-protein ratio. We are
currently investigating the application of this novel selene-
nylsulfide methodology to other kinds of post-translational
modification.
Received: September 29, 2003 [Z52975]
Published Online: January 21, 2004
Keywords: carbohydrates · glycoproteins · glycosylation ·
protein modifications · selenium
Figure 2. Deconvoluted ESI mass spectra showing glycoconjugation of
.
12 (SBLCys156; 26708 Da) with 6d (GlcGlcGlc-SeS; 20 equiv) in a
buffer solution (CHES (70 mm), MES (5 mm), CaCl2 (2 mm); pH 9.5)
after 1 min, 10 min, 30 min, 60 min, 90 min, 150 min. The seleneny-
lated protein 13 (SBL-SePh; 26864 Da) is formed rapidly, followed by
the glycoprotein 14d (SBL-GlcGlcGlc; 27226 Da).
[1] The Gene Ontology Consortium has defined biological-process
term-number GO:0006486 (protein amino acid glycosylation) as
Figure 3. Chemical glycoconjugation of 12 (SBLCys156) with GlcNAc to form 14c (GlcNAc-SBL; 2 mm), followed by the addition of a second
monosaccharide unit (Gal) mediated by galactosyltransferase (Gal-T) to give 28 (Galb(1,4)-GlcNAc-SBL): deconvoluted ESI mass spectra showing
14c (GlcNAc-SBLCys156) before (top spectrum) and after (bottom spectrum) inactivation with phenylmethanesulfonyl fluoride (PMSF) and enzy-
matic galactosylation.
832
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 828 –828

Angewandte
Chemie
“The addition of a sugar unit to a protein amino acid, for
example, the addition of glycan chains to protein.” (see Genome
Res. 2001, 11, 1425). Herein we use the broader term glyco-
conjugation to refer to the general process of addition of a
glycosyl-unit-containing moiety to another moiety through a
covalent linkage.
would then feed into the same glycosylation sequence and
À
ultimately lead to the required disproportionation of S Se to
À
S S.
[29] W. A. Bonner, J. E. Kahn, J. Am. Chem. Soc. 1951, 73.
[30] B. D. Johnston, B. M. Pinto, J. Org. Chem. 2000, 65, 4607.
[31] G. DeSantis, P. Berglund, M. R. Stabile, M. Gold, J. B. Jones,
Biochemistry 1998, 37, 5968.
[2] R. A. Dwek, Chem. Rev. 1996, 96, 683.
[32] O. Behaghel, H. Seibert, Justus Liebigs Ann. Chem. 1932, 65,
812.
[33] H. Rheinboldt, E. Giesbrecht, Justus Liebigs Ann. Chem. 1950,
568, 198.
[34] Representative glycosylation procedure: The protein 12
(SBLCys156; 1 mg) was dissolved in a buffer solution (1 mL;
CHES (70 mm), MES (5 mm), CaCl2 (2 mm); pH 9.5). The thio
sugar 4a (Glc-SH; 20 equiv) was added as a solution in water to
the solution of the protein, and the mixture was placed on an
end-over-end rotator. After 1 h the reaction was analyzed by
mass spectrometry.
[3] J. B. Lowe, J. D. Marth, Annu. Rev. Biochem. 2003, 72, 643.
[4] A. J. Parodi, Annu. Rev. Biochem. 2000, 69, 6 9.
[5] A. Helenius, M. Aebi, Science 2001, 291, 2364.
[6] R. Kannagi, Curr. Opin. Struct. Biol. 2002, 12, 599.
[7] R. S. Haltiwanger, Curr. Opin. Struct. Biol. 2002, 12, 593.
[8] J. B. Lowe, Cell 2001, 104, 809.
[9] T. W. Rademacher, R. B. Parekh, R. A. Dwek, Annu. Rev.
Biochem. 1988, 57, 785.
[10] The recent preparation of homogeneous N-glycosylated protein
in yeast represents a remarkable achievement in this regard; see:
S. R. Hamilton, P. Bobrowicz, B. Bobrowicz, R. C. Davidson, H.
Li, T. Mitchell, J. H. Nett, S. Rausch, T. A. Stadheim, H.
Wischnewski, S. Wildt, T. U. Gerngross, Science 2003, 301,
1244. Elegant in vitro misacylated tRNA systems may also
facilitate powerful biological glycoprotein synthesis; see: R. R.
Schmidt, J. C. Castro-Palomino, O. Retz, Pure Appl. Chem. 1999,
71, 729.
[35] A comparable glycosylation with iodoacetamides may take as
long as 24 h: see reference [16].
[36] Considerably smaller quantities of the reagent (1–20 equiv) are
required than typically used in protein glycosylation or protein
modification (often of the order of 1000 equiv); see: B. G. Davis,
Curr. Opin. Biotechnol. 2003, 14, 379. For more information, see
Supporting Information.
[37] For further details, see Supporting Information.
[38] K. L. Kiick, E. Saxon, D. A. Tirrell, C. R. Bertozzi, Proc. Natl.
Acad. Sci. USA 2002, 99, 19.
[39] Attempted deprotection of the acetylated Glyco-SeS reagents
only resulted in degradation.
[40] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Featherstone,
Biochem. Pharmacol. 1961, 7, 88.
[11] R. S. Rush, P. L. Derby, D. M. Smith, C. Merry, G. Rogers, M. F.
Rohde, V. Katta, Anal. Chem. 1995, 67, 1442.
[12] L. C. Wasley, G. Timony, P. Murtha, J. Stoudemire, A. J. Dorner,
J. Caro, M. Krieger, R. J. Kaufman, Blood 1991, 77, 2624.
[13] C. King, R. Mueller Hoenger, M. Malo Cleary, K. Murali-
Krishna, R. Ahmed, E. King, N. Sarvetnick, Nat. Med. 2001, 7,
206.
[14] B. G. Davis, Chem. Rev. 2002, 102, 579.
[41] For further details, see Supporting Information.
[42] The glycoprotein 14c (GlcNAc-S-S-SBL; 3 mg, as a solution in
aqueous buffer (1 mgmLÀ1)) was deactivated with PMSF
(500 equiv, as a solution in acetonitrile (100 mgmLÀ1)) at room
temperature. The protein was purified (Sephadex G-25 PD-10
column), lyophilized, and redissolved in sodium cacodylate
(0.1m)/MnCl2 buffer (0.05m, 1.0 mL). The mixture was incu-
bated for 40 min with UDP-Gal (1.6m m) and recombinant
bovine beta-1,4-galactosyltransferase (100 mU, Calbiochem)
prior to analysis by mass spectrometry.
[15] H. Liu, L. Wang, A. Brock, C. H. Wong, P. G. Schultz, J. Am.
Chem. Soc. 2003, 125, 1702.
[16] D. Macmillan, R. M. Bill, K. A. Sage, D. Fern, S. L. Flitsch,
Chem. Biol. 2001, 8, 133.
[17] Y. Shin, K. A. Winans, B. J. Backes, S. B. H. Kent, J. A. Ellman,
C. R. Bertozzi, J. Am. Chem. Soc. 1999, 121, 11684.
[18] K. Witte, P. Sears, R. Martin, C.-H. Wong, J. Am. Chem. Soc.
1997, 119, 2114.
[19] B. G. Davis, R. C. Lloyd, J. B. Jones, J. Org. Chem. 1998, 63, 9614.
[20] B. G. Davis, M. A. T. Maughan, M. P. Green, A. Ullman, J. B.
Jones, Tetrahedron: Asymmetry 2000, 11, 245.
[43] F. L. Schanbacher, K. E. Ebner, J. Biol. Chem. 1970, 245, 5057.
[44] I. Shin, H.-j. Jung, M.-r. Lee, Tetrahedron Lett. 2001, 42, 1325.
[45] L. A. Marcaurelle, C. R. Bertozzi, J. Am. Chem. Soc. 2001, 123,
1587.
[21] D. P. Gamblin, P. Garnier, S. J. Ward, N. J. Oldham, A. J.
Fairbanks, B. G. Davis, Org. Biomol. Chem. 2003, 1, 3642.
[22] B. G. Davis, R. C. Lloyd, J. B. Jones, Bioorg. Med. Chem. 2000, 8,
1527.
[23] W. M. Macindoe, A. H. van Oijen, G.-J. Boons, Chem. Commun.
1998, 847.
[24] B. T. Houseman, M. Mrksich, Top. Curr. Chem. 2002, 218, 1.
[25] K. Anestꢀl, E. S. J. ArnØr, J. Biol. Chem. 2003, 278, 15966.
[26] L. Zhong, E. S. J. ArnØr, A. Holmgren, Proc. Natl. Acad. Sci.
USA 2000, 97, 5854.
[27] The reactions of thiols with unsymmetrical disulfides, in contrast
to selenenylsulfides, are often slow and nonselective, typically
require a large excess of the thiol, and lead to the release of
thiol(ate), which may serve to cleave any disulfide formed.
À
Reactions with selenenylsulfides are typically fast, favor S S
À
formation from S Se through disproportionation, and release a
selenate, which does not compete in the reaction.
À
[28] For examples of the behavior of S as an electrophile in S Se-
containing compounds, see: G. Bergson, G. Nordstrom, Ark.
Kemi 1961, 17, 569; J. L. Kice, T. W. S. Lee, J. Am. Chem. Soc.
1978, 100, 5094; H. Fischer, N. Dereu, Bull. Soc. Chim. Belg.
1987, 96, 757. This reaction may compete with reaction at the
electrophilic Se atom. However, reaction at the Se center would
simply lead to in situ generation of glyco-SeS reagents, which
Angew. Chem. Int. Ed. 2004, 43, 828 –833
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
833