Glycosyl phenylthiosulfonates (glyco-PTS): novel reagents for glycoprotein synthesis.

Article abstract of DOI:10.1039/b306990g

Controlled site-selective glycosylation can be achieved by combining site-directed cysteine mutagenesis with chemical modification of the introduced thiol; a new class of more efficient chemoselective reagents, glycosyl phenylthiosulfonates, allow rapid glycosylations of representative simple thiols, peptides and proteins.

Full text of DOI:10.1039/b306990g

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Glycosyl phenylthiosulfonates (Glyco-PTS): novel reagents for
glycoprotein synthesis†‡
David P. Gamblin, Philippe Garnier, Sarah J. Ward, Neil J. Oldham, Antony J. Fairbanks* and
Benjamin G. Davis*
Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford, UK OX1 3QY.
E-mail: Ben.Davis@chem.ox.ac.uk; Antony.Fairbanks@chem.ox.ac.uk;
Fax: ϩ44 1865 275674; Tel: ϩ44 1865 275652
Received 19th June 2003, Accepted 28th July 2003
First published as an Advance Article on the web 11th August 2003
Controlled site-selective glycosylation can be achieved by
combining site-directed cysteine mutagenesis with chemical
modification of the introduced thiol; a new class of
more efficient chemoselective reagents, glycosyl phenyl-
thiosulfonates, allow rapid glycosylations of representative
simple thiols, peptides and proteins.
modification monitoring would also be welcome. We con-
sidered that simple alteration of the methyl group in the
methanethiosulfonate (MTS) aglycon to a phenyl group to form
the novel class of phenylthiosulfonates (PTS) might provide
solutions or advantages in all of these respects. In particular,
the removal of the potentially acidic methyl group site α to the
sulfone unit (pKa ∼ 14)12 might enhance stability both during
preparation and reaction. Moreover, the introduction of
this UV-active chromophore into the aglycon would also
advantageously allow direct monitoring of reagent formation
and use in, for example, protein glycosylation.
Representative mono- and oligo-saccharide glyco-PTS
reagents 2a–d were readily synthesized by displacement of
halide from the appropriate glycosyl halides13,14 using
sodium phenylthiosulfonate (NaPTS),15 in acetonitrile at 70 ЊC
in the presence of 0.1 equiv. of tetrabutylammonium halide
(Scheme 2).
The glycosylation§ of proteins plays a vital role in their bio-
logical behaviour, destination and stability.1 The chemical syn-
thesis of glycoproteins offers certain key advantages,2 not least
of which is more ready access to pure glycoprotein glycoforms.3
Several alternative and complementary strategies that provide
access to pure glycoforms have been described.2,4 We have pre-
viously described the first examples of a site-selective glyco-
sylation process that combines site-directed mutagenesis with
chemoselective modification.4b In this two-step strategy a
cysteine is introduced through mutagenesis at a preselected
position in a given protein to create a protein with a single free
thiol, which is then chemoselectively modified with a thiol-
selective carbohydrate reagent (Scheme 1) thereby allowing full
control of both site and sugar. This early system exploited
glycosyl methanethiosulfonate (glyco-MTS) reagents5 1 due to
the strong history of use of methanethiosulfonates (MTS) as
potent protein modifying reagents.6–10 However, during the
course of our work on glyco-4b,5 and other10 MTS reagents we
have considered methods for increasing their utility yet further
and describe here our synthesis of an improved class of
carbohydrate thiosulfonates, the glycosyl phenylthiosulfonates
(glyco-PTS) 2.
Scheme 1
Leaving group alteration is a tried-and-tested method for
tuning the reactivity of electrophilic structures.11 We therefore
focussed on the aglycon group of our glyco-MTS reagents to
improve their use. Shortcomings of MTS reagents include
occasionally moderate yields and difficulties in their prepar-
ation and occasional instability under the basic conditions
in which they are often used. Additionally, any benefits in the
form of enhanced rate and efficiency of reaction and in situ
Scheme 2 Reagents and conditions: (i) ref. 13 for 4a,c; ref. 14 for 4b;
Ac2O, NaOAc then HBr, AcOH for 4d; (ii) NaSSO2Ph, CH3CN, 70 ЊC,
0.1 equiv. Bu4NBr for 2a,c, Bu4NI for 2d.
† This is one of a number of contributions from the current members
of the Dyson Perrins Laboratory to mark the end of almost 90 years of
organic chemistry research in that building, as all its current academic
staff move across South Parks Road to a new purpose-built laboratory.
‡ Electronic supplementary information (ESI) available: experimental
procedures, characterization, protein ESI-MS spectra and crystal data.
See http://www.rsc.org/suppdata/ob/b3/b306990g/
NaPTS is more conveniently prepared than sodium
methanethiosulfonate.15 In all cases, syntheses of the glyco-PTS
reagents 2 were achieved in superior yields to corresponding
syntheses of glyco-MTS reagents 1 (Table 1). Moreover, the
costs of the starting materials of these reagents are lower than
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T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Table 1 Comparison of the synthesis and glycosylation reactions of glyco-MTS 1 and glyco-PTS 2 reagents
Protein
Protein
EtSH (5)
Peptide (6)
(7, SBL-Cys156)
(8, BSA-Cys58)
Preparationa
glycosylationb
glycosylationc
glycosylationd
glycosylationd
Glycosylating
reagent
Total yield
(%)
Yield
(%)
Time/
h
Yield
(%)
Time/
h
Conversion
(%)
Time/
min
Conversion
(%)
Time/
min
Steps
g
Glc(Ac)4β-MTS 1a
Glc(Ac)4β-PTS 2a
Glc(Bn)4β-MTS 1b
Glc(Bn)4β-PTS 2b
Gal(Ac)4β-MTS 1c
Gal(Ac)4β-PTS 2c
Glc(Ac)4α(1,4)Glc(Ac)3α(1,4)-
Glc(Ac)3β-PTS 2d
46e
64
3
3
5
5
3
3
3
96f
82
78f
95
83
91
93
3
1
15
1.5
1
1
1
62f
99
65
82
—
95
74
5
5
4
5
—
2
3
100e
100
—
—
—
50e
30
—
—
—
30
30
—
—
30
—
—
—
30
—
100
—
43f
67
—
—
47
65
60
100
100
100
—
a From the corresponding parent carbohydrate -glucose (Glc), -galactose (Gal) or Glcα(1,4)Glcα(1,4)Glc according to Scheme 2; b Et3N, DCM,
RT, 1 equiv. of thiosulfonate; c Et3N, DCM/MeOH (20 : 1), RT, 1 equiv. of thiosulfonate; Peptide [P]-Cys-Ser-OMe, [P] = Ac except for reaction with
2d where [P] = Boc; d 70 mM CHES, 5 mM MES, 2 mM CaCl2 pH 9.5 or 50 mM TrisؒHCl, pH 7.7, RT, ∼30 equiv. for 1, ∼10 equiv. for 2a,c ϩ 7,
∼20 equiv. for 2a,c ϩ 8, ∼40 equiv. for 2d ϩ 7. Conversion was determined by HPLC, A280 and ESI-MS; e Taken from refs. 4b and 5; f Taken from
ref. 21; g — indicates reaction not studied rather than an absence of reaction.
those for 1 by almost 10-fold.16 Excellent (>99% β for 2a,c,d
due to anchimeric assistance of O-2 acetate)17 or workable (3 : 1
α : β for 2b due to solvent participation)18 β stereoselectivity
was observed. The structure, and importantly the anomeric
configuration, of 2a was further confirmed by X-ray crystallo-
graphy (Fig. 1).¶
Cys156) and bovine serum albumin (8, BSA-Cys58). The high
purity of the glycoprotein products was confirmed by ESI-MS,
Ellman’s titration19 and PAGE.
Pleasingly, the glyco-PTS reagents 2a,c exhibited enhanced
stability and efficiency under reaction conditions and this
allowed the use of reduced ratio of reagent to protein (∼10–20
equivalents||) as compared with the corresponding glyco-MTS
1 reagents (∼30 equivalents)5 and the recovery of unused
reagent. Post-glycosylation deprotection of these peracetylated
glycoproteins, which has been demonstrated by us previously,5
also allows access to deacetylated variants.
As a further, more stringent test of the utility of the glyco-
PTS method, we investigated the use of the Glcα(1,4)-
Glcα(1,4)Glcβ-trisaccharide PTS 2d (Scheme 2). This allowed
the successful glycosylation not only of thiols 5 and 6 but also
of protein 7 to create 7d (Fig. 2). To the best of our knowledge,
this is one of the largest carbohydrates to have been used in a
convergent and site-selective protein glycosylation to date.20
Fig. 1 Crystal structure of 2a.¶
Glyco-PTS reagents 2a–c were investigated in the glyco-
sylation of representative thiols, peptides and proteins 5–8
under basic conditions. We were delighted that in most cases
glyco-PTS reagents proved superior to glyco-MTS reagents and
yielded glycosylated products 5a–c–6a–c in 82–99% yields
(Scheme 3, Table 1), and in all cases with absolute control of
anomeric stereochemistry.
Fig. 2 ESI-MS spectrum of 7d (Found 27654, Theoretical 27649).
Finally, we explored the potential of the PhSO2– group in
glyco-PTS reagents 2a–d as an in situ indicator of protein glyco-
sylation. Advantageously the PhSO2– chromophore displays a
maximum in the UV spectrum at ∼265 nm that is not obscured
by either protein or buffer associated chromophores (Fig. 3).
The PhSO2– moiety is present in both glyco-PTS reagent and in
Ϫ
the PhSO2 that is the by-product of glycosylation but the
associated extinction coefficients differ sufficiently (e.g. ε = 2308
M
Ϫ1 cmϪ1 for 2c, ε = 986 MϪ1 cmϪ1 for PhSO2Ϫ) for the rate of
protein glycosylation to be monitored. Initial results indicate
that glycosylations with 2a–d are typically complete within
20 min. Moreover, rates of glycosylation appear to vary with
the size (e.g. 2d vs. 2a) of the carbohydrate introduced during
protein glycosylation. This interestingly raises the possibility
that the steric bulk of the carbohydrate could be used to control
the kinetics of protein glycosylation. Further kinetic analyses
of these glycosylation reactions are under investigation and will
be published in due course.**
Scheme 3 Reagents and conditions: (i) for 5 DCM, Et3N; for 6 MeOH/
DCM, Et3N; for 7 70 mM CHES, 5 mM MES, 2 mM CaCl2, pH 9.5; for
8 50 mM TrisؒHCl, pH 7.7.
Notably, these useful glyco-PTS reagents allowed quanti-
tative glycosylation in aqueous buffer solution (HEPES, CHES,
MES or Tris pH 7–9.5) of the model single thiol-containing
proteins, subtilisin Bacillus lentus mutant S156C (7, SBL-
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¶ Crystal data for 2a: C20H24O11S2, M = 504.52, monoclinic, space group
P21, a = 10.7470(3), b = 8.3113(2), c = 13.5613(4) Å, β = 100.4321(12)Њ,
V = 1191.3 Å3, Z = 2, T = 150 K, µ = 0.280 mmϪ1, reflections measured =
10203, unique reflections = 4759, Rint = 0.024, R = 0.0417, wR = 0.0465.
CCDC reference number 213324. See http://www.rsc.org/suppdata/ob/
b3/b306990g/ for crystallographic data in CIF or other electronic
format.
|| It should be noted that 10–20 equivalents of reagent represents a
stoichiometry that is well below that typically used in protein modifi-
cations, which are often of the order of 1000 equivalents. See B. G.
Davis, Curr. Opin. Biotechnol., 2003, 14, in press.
** These investigations include the potential use of other thiosulfonates
such as para-nitrophenylthiosulfonates (pNPTS).
1 R. A. Dwek, Chem. Rev., 1996, 96, 683.
2 B. G. Davis, Chem. Rev., 2002, 102, 579.
3 T. W. Rademacher, R. B. Parekh and R. A. Dwek, Annu. Rev. Bio-
chem., 1988, 57, 785.
Fig. 3 UV Monitoring of the PTS group: the presence of the 265 nm
PhSO2– chromophore in the PTS group allows the monitoring of its
presence and hence glycosylation. This is illustrated by UV absorbance
spectra of solutions of 2c (black) and NaPTS (grey) with 6c (1.85 mM
of each component in 1 : 1 pH 7 HEPES : CH3CN) which show reduced
absorbance at 265 nm for the PhSO2 anion as compared with glyco-
PTS 2c.
4 For leading and recent additional examples see: (a) K. Witte,
P. Sears, R. Martin and C.-H. Wong, J. Am. Chem. Soc., 1997, 119,
2114; (b) B. G. Davis, R. C. Lloyd and J. B. Jones, J. Org. Chem.,
1998, 63, 9614; (c) Y. Shin, K. A. Winans, B. J. Backes, S. B. H. Kent,
J. A. Ellman and C. R. Bertozzi, J. Am. Chem. Soc., 1999, 121,
11684; (d ) D. Macmillan, R. M. Bill, K. A. Sage, D. Fern and S. L.
Flitsch, Chem. Biol., 2001, 8, 133; (e) H. Liu, L. Wang, A. Brock,
C.-H. Wong and P. G. Schultz, J. Am. Chem. Soc., 2003, 125, 1702.
5 B. G. Davis, M. A. T. Maughan, M. P. Green, A. Ullman and
J. B. Jones, Tetrahedron: Asymmetry, 2000, 11, 245.
6 G. L. Kenyon and T. W. Bruice, Methods Enzymol., 1977, 47, 407.
7 R. Wynn and F. M. Richards, Methods Enzymol., 1995, 251, 351.
8 P. Berglund, G. DeSantis, M. R. Stabile, X. Shang, M. Gold,
R. R. Bott, T. P. Graycar, T. H. Lau, C. Mitchinson and J. B. Jones,
J. Am. Chem. Soc., 1997, 119, 5265.
Ϫ
In summary, this communication describes the synthesis of
glyco-PTS reagents 2a–d of double utility not only as protein,
peptide and thiol glycosylating reagents but also as reagents
that may be used for the preparation of glycosyl disulfide glyco-
syl donors such as 5a–d.21 Moreover, the successful use of phe-
nylthiosulfonates as protein modifying reagents, demonstrated
here for carbohydrate modifications, highlights the potentially
broader utility of other PTS reagents as improved variants of
their useful MTS counterparts.6,7
9 G. DeSantis and J. B. Jones, Curr. Opin. Biotechnol., 1999, 10, 324.
10 K. Matsumoto, B. G. Davis and J. B. Jones, Chem. Eur. J., 2002, 8,
4129.
Acknowledgements
11 For select examples see: J. I. Brauman, W. N. Olmstead and
C. A. Lieder, J. Am. Chem. Soc., 1974, 96, 4030; P. J. Stang and
A. G. Anderson, J. Org. Chem., 1976, 41, 781; M. J. Gresser
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G. M. L. Lin, Tetrahedron Lett., 1978, 949; M. J. Pellerite and
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Perkin Trans. 2, 1981, 221; K. B. Sloan and S. A. M. Koch, J. Org.
Chem., 1983, 48, 3777.
We thank the EPSRC (D. P. G., S. J. W.) and Glycoform for
funding (D. P. G., P. G.); Mario Polywka for useful discussions;
Barbara O’Dell, Tim D. Claridge, Andrew R. Cowley, Joanna
Kirkpatrick for invaluable technical support and the EPSRC
for access to the Mass Spectrometry Service at Swansea, the
Chemical Database Service at Daresbury and for the award of
quota (S. J. W.) and DTA (D. P. G.) studentships.
12 R. G. Pearson and R. L. Dillon, J. Am. Chem. Soc., 1953, 75,
2439.
13 P. G. Scheurer and F. J. Smith, J. Am. Chem. Soc., 1954, 76,
Notes and references
3224.
§ The Gene Ontology Consortium has defined biological process term
number GO:0006486 protein amino acid glycosylation as “The addition
of a sugar unit to a protein amino acid, e.g. the addition of glycan
chains to proteins.” [see Genome Res., 2001, 11, 1425.]. In this
communication we similarly use the term glycosylation to refer to the
general process of addition of a glycosyl unit to another moiety via a
covalent linkage. Glycation has also been suggested by the IUPAC-IUB
Joint Commission on Biochemical Nomenclature (JCBN) [see Eur. J.
Biochem., 1986, 159, 1; 1989, 185, 485; Glycoconjugate J., 1986, 3, 123;
J. Biol. Chem., 1987, 262, 13; Pure Appl. Chem., 1988, 60, 1389; Amino
Acids Pept., 1990, 21, 329; and in Biochemical Nomenclature and
Related Documents, 2nd edition, Portland Press, London, 1992, pp. 84–
89] as a general term for the product of all reactions that covalently link
a sugar starting molecule to a protein or peptide.
14 H. P. Wessel and D. Bundle, J. Chem. Soc., Perkin Trans. 1, 1985,
2251.
15 T. G. R. Sato, Y. Takakawa and S. Takizawa, Synthesis, 1980, 615.
16 2003 preparation costs: 1 g of NaMTS £11.02, 1 g of NaPTS
£1.21.
17 G. Wulff and G. Röhle, Angew. Chem., Int. Ed. Engl., 1974, 13, 157.
18 A. Marra, J. Esnault, A. Veyrières and P. Sinaÿ, J. Am. Chem. Soc.,
1992, 114, 6354.
19 G. L. Ellman, K. D. Courtney, V. Andres and R. M. Featherstone,
Biochem. Pharmacol., 1961, 7, 88.
20 For one other convergent trisaccharide-protein glycosylation see ref.
4e.
21 B. G. Davis, S. J. Ward and P. M. Rendle, Chem. Commun., 2001,
189.
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