Enabling olefin metathesis on proteins: Chemical methods for installation of S-allyl cysteine

Article abstract of DOI:10.1039/b908004j

Multiple, complementary methods are reported for the chemical conversion of cysteine to S-allyl cysteine on protein surfaces, a useful transformation for the exploration of olefin metathesis on proteins.

Full text of DOI:10.1039/b908004j

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Enabling olefin metathesis on proteins: chemical methods for installation
of S-allyl cysteinew
Justin M. Chalker, Yuya A. Lin, Omar Boutureira and Benjamin G. Davis*
Received (in Cambridge, UK) 22nd April 2009, Accepted 7th May 2009
First published as an Advance Article on the web 27th May 2009
DOI: 10.1039/b908004j
Multiple, complementary methods are reported for the chemical
conversion of cysteine to S-allyl cysteine on protein surfaces, a
useful transformation for the exploration of olefin metathesis on
proteins.
chemical methods for the conversion of cysteine to Sac on
proteins: direct allylation of cysteine with allyl chloride and
allylation by an allyl selenenylsulfide rearrangement. We
demonstrate that these methods are cysteine selective
and provide metathesis-active Sac-containing proteins.
Moreover, the electrophilic allylation and allylic selenenyl-
sulfide rearrangement methods provide a single diastereomer
of S-allyl cysteine, unlike the nucleophilic addition to
dehydroalanine. Finally, the methods are mild, efficient, and
easily accomplished without denaturing the protein.
The direct allylation of the amino acid cysteine7,14
and cysteine-containing peptides15,16 with allyl halides is
well-known and the allylated products have been coupled to
proteins through linkers15 or by native chemical ligation.17
However, to the best of our knowledge, direct allylation of
cysteine on protein surfaces has not been reported. Perhaps
the limited solubility of allyl halides in water or potential
complications in selectivity have dissuaded such efforts. To
investigate this transformation, we chose a single-cysteine
mutant of subtilisin from Bacillus lentus (SBL-S156C)
as a model protein. The single-cysteine construct simplifies
reaction analysis and the protease activity of the enzyme can
be assayed to ensure that the protein is not denatured during
the modification. Allylation was attempted simply by treating
a sample of SBL-S156C in pH 8.0 phosphate buffer with a
solution of allyl chloride in DMF. We discovered that the total
amount of DMF need not exceed 5% of the total volume of
buffer to achieve complete homogeneity; this level of DMF is
sufficiently low to be compatible with most proteins. Within
30 minutes at 37 1C full conversion to allylated protein 2 was
observed (Scheme 2). Consumption of free thiol, and thus
reaction at cysteine, was further verified by modified Ellman’s
assay (see ESIw).13,18 The selective allylation of cysteine was
observed even when a large excess of allyl chloride was used
Selective modification of biomolecules is a long-standing goal
in chemical biology. In particular, precise chemical modification
of proteins allows investigation of enzymatic and cellular
activities and controlled alteration of macromolecular
function.1–4 As part of our continuing effort to expand the
repertoire of selective methods for protein modification, we
have set out to render transformations generally useful in
organic synthesis as reliable methods for protein modification.5,6
Among these reactions, olefin metathesis has emerged as a
viable candidate for selective carbon–carbon bond formation
on protein surfaces.7,8
Aqueous olefin metathesis has made strides in recent years,9
facilitating ambitious applications of this reaction in chemical
biology.8,10 Complementing these advances is a maturing
understanding of substrate reactivity in olefin metathesis and
especially cross-metathesis.11 For the purpose of protein
modification this understanding is critical since it will guide
the selection of the amino acid to be modified as well as its
metathesis partner and any intervening linker. In our exploratory
work in aqueous cross-metathesis, we discovered that
allyl sulfides are privileged substrates and undergo rapid
cross-metathesis in the presence of Hoveyda–Grubbs second
generation catalyst 1.7,12 Taking advantage of the enhanced
reactivity of allyl sulfides in olefin metathesis, we used the
normally non-proteinogenic amino acid S-allyl cysteine (Sac)
as the reactive handle for site-specific ligation of carbohydrates
and poly(ethylene glycol) to protein surfaces. Sac was initially
incorporated chemically into the model protein by the
nucleophilic 1,4-addition of allyl thiol to dehydroalanine
(Dha), which in turn was installed through the oxidative
elimination of cysteine using O-mesitylenesulfonylhydroxylamine
(MSH)13 (Scheme 1).
While these first examples of cross-metathesis are very
promising, the full scope of olefin metathesis for protein
modification is yet to be determined. We considered that
multiple, complementary routes to Sac would facilitate such
investigations. We herein report alternative and selective
Chemistry Research Laboratory, University of Oxford, 12 Mansfield
Road, Oxford, UK OX1 3TA. E-mail: Ben.Davis@chem.ox.ac.uk;
Fax: +44 (0)1865 275674; Tel: +44 (0)1865 275652
w Electronic supplementary information (ESI) available: Full experi-
mental details, including 1H and 13C NMR spectra for all compounds
and ESI-MS for all protein samples. See DOI: 10.1039/b908004j
Scheme 1 Nucleophilic route to S-allyl cysteine.
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Scheme 2 Electrophilic route to S-allyl cysteine.
Scheme 4 Attempted desulfurization with TPPTS.
(41000 equivalents)—a testament to the unique nucleophilicity
of cysteine. Furthermore, a protocol with pre-reduction of
cysteine with dithiothreitol (DTT) was also demonstrated,
a useful option for cysteines prone to oxidation. Finally,
peptidase activity of the allylated protein was retained, as
evidenced by liberation of p-nitroaniline upon treatment with
the peptide suc-AAPF-pNA (see ESIw).19
While the allylation of our model protein with allyl chloride
is a selective and direct route to S-allyl cysteine, it is possible
that, as with other alkylating reagents,20 proteins containing
strongly basic lysine or histidine residues may be allylated
non-selectively. This selectivity problem may also be more
pronounced when the target cysteine is hindered. It is therefore
useful to have an allylation method that is specific for cysteine.
The formation and dechalcogenative rearrangement of allyl
disulfides and allyl selenenylsulfides is such a transformation
(Scheme 3).
Seminal reports by Hoefle and Baldwin21 and Sharpless and
Lauer22 on the rearrangement and reduction of allylic
disulfides and allylic diselenides, respectively, inspired the
Crich laboratory to use this chemistry as a cysteine-specific
ligation strategy. Crich and co-workers have demonstrated the
utility of this methodology in the specific allylation of cysteine
in peptides up to 10 residues long in methanol or aqueous
acetonitrile.23–26 These transformations are efficient in aqueous
media23,25,26 since polar solvents stabilize the pericyclic
transition state of the rate-determining rearrangement step.27
The ease and specificity of disulfide and selenenylsulfide
formation at cysteine and the ample precedence for the
reductive rearrangement motivated us to explore the potential
of this transformation on proteins as a route to S-allyl
cysteine.
peptidic systems in less polar solvents.23,25,26 Unfortunately
only reduced product was observed (Scheme 4). Perhaps the
unsubstituted allylic disulfide is too exposed and TPPTS too
nucleophilic, resulting in direct attack on the disulfide rather
than the thiosulfoxide product of rearrangement. While
screening other phosphines may rescue this disulfide route to
S-allyl cysteine, we instead turned to the analogous allyl
selenenylsulfide system since loss of selenium is far more facile
than the desulfurization attempted above and in many cases
does not require phosphine,24,25 an important feature for
proteins with natural disulfides.
Allyl selenocyanate and Se-allyl selenosulfate are both
suitable reagents for the conversion of cysteine to its Se-allyl
selenenylsulfide.24,25 We opted to use allyl selenocyanate (6)
since previous reports indicated higher overall yields of
allylated peptides using allylic selenocyanates than when the
allylic selenosulfates were employed.25 Gratifyingly, when
SBL-S156C was treated with 6 at room temperature, smooth
conversion to S-allyl cysteine was observed. LC-MS analysis
revealed rapid formation of Se-allyl selenenylsulfide/S-allyl
selenosulfide intermediates (MS calculated: 26 834; observed:
26 834) and spontaneous loss of selenium over the course of
1 h (MS calculated: 26 755; observed 26 755) (Scheme 5).
Full conversion was observed and the specificity for cysteine
was verified by modified Ellman’s assay (see ESIw).13,18
Allylated protein 2 was an active peptidase, reflecting the mild
nature of this allylation method. This is the first demonstration
of the Se-allyl selenenylsulfide reductive rearrangement on
proteins, a result that bodes well for the use of this ligation
method in the synthesis of other lipidated proteins.
For our purpose, the overriding motivation for exploring
methods for allylating cysteine is to provide flexible and
efficient access to proteins suitable for olefin metathesis. As a
demonstration of S-allyl cysteine reactivity in cross-metathesis
and further corroboration of the formation of S-allyl cysteine
by the transformations described above, the allylated proteins
were used in a model cross-metathesis with allyl alcohol. Full
conversion was observed after two hours at room temperature
The allyl disulfide was investigated first. Treating
SBL-S156C with allyl phenylthiosulfonate (3)28 gave the
expected allyl disulfide 4. To promote the desired desulfurization,
4 was treated with the trisodium salt of triphenylphosphine-
3,30,300-trisulfonic acid (TPPTS, 5). TPPTS was chosen since
it is a water-soluble derivative of triphenylphosphine, the
phosphine used by Crich to promote desulfurization in
using catalyst
1 under previously optimized conditions
(see ESIw).7 As expected, no difference in cross-metathesis
reactivity was observed between the sample obtained from
allyl chloride and the sample obtained from allyl selenocyanate
(Scheme 6).
In summary, we have investigated multiple chemical methods
for the conversion of cysteine to S-allyl cysteine on proteins.
The versatile chemistry of cysteine29 allows mechanistic
divergence in these strategies: the electrophilic allylation with
allyl chloride and the reductive sigmatropic rearrangement of
the Se-allyl selenenylsulfide of cysteine fully complement
the nucleophilic addition of allyl thiol to dehydroalanine.
Scheme 3 Dechalcogenative rearrangement of allyl disulfides and
allyl selenenylsulfides.
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Scheme 5 Cysteine-specific allylation by reductive rearrangement of Se-allyl selenenylsulfide.
8 Y. A. Lin, J. M. Chalker and B. G. Davis, ChemBioChem, 2009,
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Scheme 6 Model cross-metathesis at S-allyl cysteine.
Together, these methods provide three modes of flexible entry
to protein substrates suitable for olefin metathesis. This
flexibility is important when adapting these reactions to a
protein of interest. For instance, allyl thiol adds efficiently to
dehydroalanine but might reduce natural disulfides; in this
case allyl chloride and allyl selenocyanate would be more
suitable. For a hindered cysteine, allyl chloride is more likely
to allylate non-selectively; in this case allyl thiol addition to
dehydroalanine (if there are no disulfides) and allylation
with allyl selenocyanate should be the methods of choice.
Regardless of the synthetic route, access to S-allyl cysteine-
containing proteins is useful to elucidate the full scope of olefin
metathesis as a protein conjugation method. The potential of
olefin metathesis in bioconjugation8,10,30 is driving our current
research and progress to this end will be reported in due
course.
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We acknowledge our sources of generous financial
support: Rhodes Trust (J.M.C.); EPSRC (Y.A.L.); European
Commission (O.B.).
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