Facile conversion of cysteine and alkyl cysteines to dehydroalanine on protein surfaces: Versatile and switchable access to functionalized proteins

Article abstract of DOI:10.1021/ja800800p

An efficient and robust oxidative elimination of cysteine to dehydroalanine has been discovered. The reaction is induced by O-mesitylenesulfonylhydroxylamine (MSH) and is compatible with methionine. The key elimination has been executed on protein surfaces and allows ready access to different post-translationally modified proteins through conjugate addition of sulfur nucleophiles to dehydroalanine. Treatment of the resulting thioether with MSH results in regeneration of dehydroalanine, allowing a "functional switch" by subsequent addition of a different thiol. Copyright

Full text of DOI:10.1021/ja800800p

Published on Web 03/22/2008
Facile Conversion of Cysteine and Alkyl Cysteines to
Dehydroalanine on Protein Surfaces: Versatile and Switchable
Access to Functionalized Proteins
Gonçalo J. L. Bernardes, Justin M. Chalker, James C. Errey, and Benjamin G. Davis*
Chemistry Research Laboratory, Dept. of Chemistry, UniVersity of Oxford, 12 Mansfield Road,
Oxford OX1 3TA, U.K.
Received February 10, 2008; E-mail: ben.davis@chem.ox.ac.uk
Scheme 1. Methionine Recovery from Iminosulfonium Salt
Site-selective chemical modification of proteins has emerged
as a versatile strategy for modulating macromolecular function and
understanding post-translational processing.1 Dehydroalanine (Dha)
is a unique chemical handle for such modifications. Synthetically,
Dha can be accessed by oxidative elimination of alkylated
derivatives of cysteine (Cys)2 and selenocysteine.3 Recently,
biosynthetic incorporation of selenocysteine derivatives into pep-
tides4 and proteins5 and conversion to Dha was reported. These
strategies, however, rely on peroxide-induced oxidative elimination
that compromises sensitive side chains such as methionine (Met).4
Direct chemical conversion of a natural amino acid to Dha would
be generally useful in protein modification provided such a reaction
proceeded rapidly in aqueous media while preserving all other
amino acid side chains. Cys is an attractive precursor to Dha
because of the unique reactivity of the side chain thiol. While the
ꢀ-elimination of dialkylated sulfonium derivatives of Cys is known,
their use on proteins is limited by competing oxazoline formation,
long reaction times, and nonselective alkylation.6 Instead we sought
other Cys derivatives that could undergo similar elimination yet
avoid such problems of selectivity and efficiency.
To ascertain whether MSH could be used in the presence of
Met, 4 was treated with MSH. TLC indicated rapid, complete
conversion of thioether, potentially to 5, but subsequent treatment
with thiol (DTT) and K2HPO3—conditions encountered in the
conjugate addition of a thiol to Dha, an ultimate goal of this
study—regenerated the Met derivative 4 in excellent yield (Scheme
1). No sulfoxide or sulfone formation was detected, even in aqueous
media.10,11 With a Met-compatible conversion of Cys to Dha in
hand, we explored its application to protein modification.
The serine protease mutant subtilisin Bacillus lentus (SBL)
S156C contains a single, surface-exposed Cys and was selected
as a model for the use of MSH on protein surfaces.12 Much to our
delight, treatment of this protein with MSH at 4 °C resulted in
rapid and complete conversion of Cys156 to Dha156 (eq 2).
Elimination was verified by ESI-MS (34 Da decrease from 6; 7:
26681 calculated, 26681 found); absence of free thiol in 7 was
verified by modified Ellman’s assay.10 Phosphate buffer (50 mM)
at pH 8.0 was optimal for the elimination. Organic and carbonate
buffers led to incomplete conversion, reflecting MSH sensitivity
to certain bases.10 Importantly, no oxidation at any of three Met
residues was observed.13
We explored O-mesitylenesulfonylhydroxylamine (MSH, 1)7 as
a potential reagent in the oxidative elimination of Cys to Dha. While
the transformation of sulfenamides to alkenes has not been reported,
our motivation derived from the base-induced elimination of
sulfilimines formed from thioethers using MSH.8 It might seem
that the reaction of MSH with thioethers does not bode well for
preserving Met in the proposed oxidative elimination, but it is
known that sulfilimines can undergo reversion to the native
thioether in the presence of certain bases.8b,9 Furthermore, oxidative
elimination of Cys was expected to be faster than Met since the
former proceeds through the removal of an acidic R proton while
the latter through the less labile ꢀ proton.
A simple amino acid model was investigated to test the strategy
outlined above. Treatment of Boc-Cys methyl ester 2 with MSH
resulted in conversion to the Dha derivative 3 and the disulfide of
2 in 48 and 45% yield, respectively. Remarkably, the reaction was
complete in less than a minute at room temperature in aqueous
DMF and could be conducted in open air. Isolated disulfide did
not react with MSH, ruling out disulfide as an intermediate, and
was avoided by a reverse addition (2 to excess MSH). Under such
conditions, Boc-Dha methyl ester 3 is obtained in near quantitative
yield (eq 1).
The presence of Dha was further corroborated by conversion to
several thioether derivatives by conjugate addition of thiol nucleo-
philes (Table 1). Phosphorylation14 and glycosylation15 are the most
prevalent post-translational modifications, mediating a wide range
of cellular processes. Phospho- and glycocysteine derivatives were
accessed by the efficient addition of the corresponding thiols to
Dha protein 7 (entries 1–3). Importantly, SBL remained catalyti-
cally active after two-step modification to thioether protein (kcat
)
5.7 s-1; KM ) 0.72 mM for 9).10,16 Peptide conjugation (entry 4),
of relevance to synthetic vaccine design,17 and methyl lysine
analogues18 (entries 5–7) were also readily accessed. The latter
provide efficient access to the full series of a poorly understood
post-translational modification of histone proteins involved in
encoding cellular epigenetic status.19 Finally, the natural (S)-farnesyl
linkage was installed (entry 8). The incorporation of farnesyl and
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10.1021/ja800800p CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Synthesis of Post-Translationally Modified Proteinsa
Scheme 2. MSH-Induced Oxidative Elimination Of Cys Thioethers:
A Functional Switch on Protein Surfaces
its utility in peptide synthesis has not escaped our attention.
Applications to this end, expanded chemistry at Dha, and mecha-
nistic studies are currently under investigation.
Acknowledgment. We gratefully acknowledge FCT, Portugal
(G.J.L.B.) and the Rhodes Trust (J.M.C.) for financial support. We
thank Professor Steven Ley for helpful discussions.
Supporting Information Available: Full experimental details
a See Supporting Information for full details. b Determined by
ESI-MS analysis. c Na+ adduct.
1
including H and 13C NMR data for new compounds and ESI-MS
spectra for all protein modifications. This material is available free of
charge via the Internet at http://pubs.acs.org.
other hydrophobic polyprene units onto proteins is critical in
membrane localization and protein-protein interactions.20 While
diastereoselectivity of thiol conjugate addition in simple Dha
peptides is typically low,21 the outcome on a protein surface will
be highly dependent on sequence22 and local geometry of the
protein. Furthermore, diastereoselectivity may be inconsequential
when only a reliable, well-defined conjugation strategy is required
or when one modified protein diastereoisomer interacts stereospe-
cifically with enzyme or receptor.
To demonstrate thioether stability, SBL-S-GlcNAc 9 was
subjected to 10 mM glutathione, the natural cellular redox buffer.
Whereas the disulfide counterpart underwent rapid reduction,
thioether 9 exhibited the expected resilience (S23, Supporting
Information).10 This stability, however, does not imply permanence.
The same method for eliminating Cys to Dha was used for
converting such (S)-alkyl cysteine thioethers to Dha; the mechanism
is likely a syn-elimination of the sulfilimine. This strategy was
applied to (S)-ethyl cysteine 16 as well as its protein counterpart
17 (Scheme 2). Regeneration of Dha allows different modifications
in a subsequent step, in this case glycosylation of 7 with GlcNAc-
thiol. This alkyl cysteine nonpermanence is distinguished from other
temporary protein modifications in that it is not due to inherent
reversibility or instability of the linkage, but rather a consequence
of unique reactivity of both cysteine and (S)-alkyl cysteine to MSH.
This “functional switch” allows rapid, controlled conversion
between desired protein modifications.
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