Bromomaleimides: New reagents for the selective and reversible modification of cysteine

Article abstract of DOI:10.1039/b915136b

Bromomaleimides react rapidly and selectively with cysteine to afford thiomaleimides which can be cleaved with a phosphine to regenerate the cysteine or treated with a base to afford dehydroalanine. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b915136b

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Bromomaleimides: new reagents for the selective and reversible
modification of cysteinew
Lauren M. Tedaldi, Mark E. B. Smith, Ramiz I. Nathani and James R. Baker*
Received (in Cambridge, UK) 24th July 2009, Accepted 3rd September 2009
First published as an Advance Article on the web 16th September 2009
DOI: 10.1039/b915136b
Bromomaleimides react rapidly and selectively with cysteine to
afford thiomaleimides which can be cleaved with a phosphine
to regenerate the cysteine or treated with a base to afford
dehydroalanine.
C-3 and cleave the cysteine, reversing the modification
(Scheme 1).
To the best of our knowledge the reaction of mono-
halomaleimides with thiols has never been reported before. We
synthesised bromomaleimide 2 and bromo-N-methylmaleimide
3 by bromination of the corresponding maleimides and
Reagents that can selectively modify individual amino acids
are of widespread use in the study of proteins. They allow us to
mimic post-translational modification, to install tags such as
fluorophores for visualisation or biotin for immobilisation and
purification, to manipulate protein therapeutics, and broadly
1
6
subsequent elimination of HBr. We chose the commercially
available N-Boc-Cys-OMe 4 as the cysteine model system on
which to react the maleimides. Treatment of N-Boc-Cys-OMe
4 with bromomaleimide 2 resulted in a rapid (o1 minute)
and extremely clean reaction. The product isolated was the
expected thiomaleimide 5 in quantitative yield (Scheme 2).
The same result was found with the N-methyl variant 3 with a
slightly lower yield of 84% obtained. Sodium acetate was
employed to neutralise the HBr, although this was not essential
in the case of 5 which was stable to the highly acidic conditions.
We also carried out the reaction of N-Boc-Cys-OMe 4 with
bromomaleimide 2 in conditions suitable for a protein; 5%
1
,2
to enable bioconjugation. Cysteine is often the most nucleo-
philic residue in a protein, and as such is generally regarded
1
the easiest to modify in a selective manner.
,3,4
One of the most widely used reactive motifs for cysteine
3
modification are maleimides. Maleimides are extremely
5
cysteine selective, undergoing rapid conjugate additions via
,6
7
,8
the thiolate of the cysteine residue.
Notably however
maleimides react irreversibly and thus cannot be cleaved in
subsequent steps to regenerate the free cysteine. Such
reversibility could be extremely useful as it would allow the
modification to be temporary. Applications for reversible
cysteine modification include temporarily blocking an active
2
DMF–H O, again isolating 5 in 97% yield.
Intrigued by the rapid nature of the reaction we performed a
competition experiment to reveal the relative reactivity of
bromomaleimide compared to maleimide itself. N-Boc-Cys-
OMe 4 was treated with a 1 : 1 mixture of bromomaleimide 2
and maleimide 7. The result was a 7 : 3 mixture of the two
products 5 and 8, showing that bromomaleimide reacts faster
with cysteine than maleimide (Scheme 3).
9
10
cysteine residue, protein purification, quantitative proteomic
1
1
12
analysis,
probing binding sites,
14
enabling structural
1
studies and in drug delivery. Currently reversible cysteine
3
modification can only be carried out using reagents that
5
1
form disulfides such as methanethiosulfonates.
The
To check that this was the kinetic and not the thermo-
dynamic outcome we carried out two experiments that
confirmed the irreversible nature of the reactions. The addition
of N-Boc-Cys-OMe 4 to bromomaleimide 2 followed after
10 min by maleimide 7 resulted exclusively in thiomaleimide 5.
In contrast addition of N-Boc-Cys-OMe to maleimide 7,
followed after 10 min by bromomaleimide 2 afforded exclusively
succinimide 8.
disulfides can subsequently be cleaved with reagents such as
DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine)
to regenerate the cysteine. However cleavable disulfide
reagents can be unstable, particularly with respect to disulfide
1
1
scrambling. We report herein on the bromomaleimides, the
first of a new class of reagents for the selective and reversible
modification of cysteine.
We postulated that incorporation of a leaving group on the
maleimide double bond would enable an addition–elimination
sequence to occur on reaction with cysteine. The product
would be a thiomaleimide 1, which retains a double bond, in
contrast to the saturated succinimide product obtained from
the addition of a thiol to maleimide. The thiomaleimide would
have a new reactivity profile which could be exploited in
subsequent steps. Of particular interest to us was the possibility
that a nucleophile could add in another conjugate addition to
We then carried out experiments to model how selective
these bromomaleimides would be for cysteine over other
nucleophiles. A simple competition experiment between
cysteine and propylamine was informative, with only the
cysteine–maleimide adduct isolated in quantitative yield
(Scheme 4). None of 9 was observed highlighting the excellent
selectivity for thiol over amine nucleophiles. We also
Department of Chemistry, University College London, 20 Gordon St,
London, UK. E-mail: j.r.baker@ucl.ac.uk; Fax: +44 (0)2076797463;
Tel: +44 (0)2076792653
w Electronic supplementary information (ESI) available: Full
experimental details on all experiments. See DOI: 10.1039/b915136b
Scheme 1 Proposed outcome for reaction of a cysteine residue with a
bromomaleimide, and the reversal of the process by a nucleophile.
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Scheme 2 Rapid reaction of bromomaleimides with N-Boc-Cys-
Scheme 5 TCEP (tris(2-carboxyethyl)phosphine) cleavage of the
OMe.
thiomaleimide.
Dha residues are found in a number of naturally occurring
peptides, thought to provide rigidity to the peptide backbone
and a reactive site for nucleophilic attack that is central
1
7–20
to function.
Davis et al. have recently demonstrated
the importance of the Cys to Dha conversion, reporting
2
O-mesitylenesulfonylhydroxylamine (MSH) to carry it out.
1
Scheme 3 Competition experiment between bromomaleimide and
maleimide for N-Boc-Cys-OMe.
The authors highlight the significance of avoiding unselective
modification of other amino acid residues, such as methionine
2
which is modified by peroxide based methods. A potential
2
confirmed that bromomaleimide does not react with methionine.
Treatment of bromomaleimide with N-Boc-Met-OMe resulted
in no reaction after 2 days. This is in line with the proposed
mechanism for cysteine addition to maleimides which involves
drawback of MSH however is that it is explosive, and thus
23
difficult to handle.
In their model study Davis et al. use N-Boc-Cys-OMe 4,
treating it with MSH (10 equivalents) and K CO , to afford
2
7
3
the thiolate rather than the thiol.
21
Dha 10. We were guided by these conditions and thus treated
N-Boc-Cys-OMe 4 with bromomaleimide 2 (1 equivalent),
We then moved on to investigate the reactivity of these
new thiomaleimide adducts with nucleophiles. Treatment of
the thiomaleimide 5 with hexanethiol or another equivalent of
N-Boc-Cys-OMe 4 resulted in no reaction after 2 h. This result
indicates that thiomaleimides of this type are much less
reactive to thiol conjugate additions than the bromomaleimides.
This is likely due to a combination of electronics—the
sulfur atom mesomerically releasing electron density into the
maleimide ring—and sterics.
followed by K
2 3
CO . We were pleased to observe after 2 h
the complete conversion to Dha 10, isolable in 93% yield
24
(
Scheme 6). The mixture turns yellow over the course of the
reaction and it is proposed that this is due to the thiolate 11,
although this by-product has not been isolated. This Cys
to Dha conversion can also be carried out with the
N-methylmaleimide 3 to afford Dha 10 in 76% yield.
Interestingly in this N-methyl example the reaction occurs in
just 10 min which we suggest indicates that the thiomaleimide
In contrast treatment of thiomaleimide 5 with water
2
soluble phosphine TCEP in 5% DMF–H O (sodium
5
is deprotonated under the reaction conditions hindering the
phosphate buffered, pH 8) resulted in a clean reaction to
afford N-Boc-Cys-OMe 4, in 98% yield (Scheme 5). We used
this buffer system as we wanted to employ conditions suitable
rate of the elimination.
We are currently exploring this reaction further, however a few
points should be noted at this stage. The reaction does not work
well in water; while Dha 10 is observed to form by TLC analysis, it
can only be obtained impure and in low yields (o5%). We believe
the problems with water stem from the known susceptibility of
for peptides and proteins. The [a]
D
for 4 confirmed that no
racemisation had taken place in this cleavage. With this
convenient cleavage demonstrated we have illustrated the
potential of bromomaleimide as a reversible modifying reagent
for cysteine. In addition to applications in protein labelling
bromomaleimides have potential applications in the temporary
modification of thiols generally, for example as protecting
groups in synthesis.
7
maleimides to hydrolyse at pH 4 8. Currently therefore this
method is limited to methanol as the solvent of choice. We believe
we will be able to solve this in the future by developing base stable
analogues to the bromomaleimides.
Another goal of our research has been to employ bromo-
maleimide to enable the conversion of cysteine to dehydroalanine
(
Dha). We envisaged this to be possible as the bromomaleimide
is serving to convert the thiol into a good leaving group in the
form of a thiomaleimide.
Scheme 4 Competition experiment between N-Boc-Cys-OMe and
Scheme 6 Thiomaleimides are converted to Dha on treatment with
propylamine for bromomaleimide.
base.
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In conclusion we have demonstrated that bromomaleimides
react rapidly and selectively with cysteine. In contrast to
maleimides bromomaleimides can be employed for reversible
cysteine modification as the thiomaleimide conjugate can be
cleaved with TCEP. Furthermore these reagents can be
employed for the conversion of cysteine to dehydroalanine.
We envisage that variously functionalised maleimides
incorporating a leaving group will provide an array of reagents
for the reversible modification and protection of cysteine. We
are currently investigating this further, along with the application
of these maleimides to proteins, and will report in due course.
We would like to thank Prof. Steve Caddick for his
input into this project and Prof. Kevin Booker-Milburn and
Dr Hugh Britton for helpful discussions.
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