Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

Article abstract of DOI:10.1039/c4cc02107j

Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds.

Full text of DOI:10.1039/c4cc02107j

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Aryloxymaleimides for cysteine modification,
disulfide bridging and the dual functionalization
of disulfide bonds†
Cristina Marculescu,a Hanno Kossen,a Rachel E. Morgan,a Patrick Mayer,a
Sally A. Fletcher,a Berend Tolner,b Kerry A. Chester,b Lyn H. Jonesc and
James R. Baker*a
Cite this: Chem. Commun., 2014,
50, 7139
Received 20th March 2014,
Accepted 14th May 2014
DOI: 10.1039/c4cc02107j
www.rsc.org/chemcomm
Tuning the properties of maleimide reagents holds significant promise in than thiosuccinimide 2. Such next generation maleimide reagents
expanding the toolbox of available methods for bioconjugation. Herein enable new applications including reversible cysteine modification,2
we describe aryloxymaleimides which represent ‘next generation the construction of triconjugates,2b,3 and the bridging of disulfide
maleimides’ of attenuated reactivity, and demonstrate their ability bonds.2b,4
to enable new methods for protein modification at disulfide bonds.
We envisaged that the choice of leaving group on the maleimide
core could result in reagents with finely tuned properties. For
The maleimide motif 1 is a highly reactive conjugate acceptor and example the reactivity could be controlled, in a manner similar
can be employed effectively in selective reactions with thiols, such as to the use of iodo-, bromo- and chloroacetamides as varyingly
cysteine residues in proteins.1 As a result it is one of the most widely reactive reagents for selective cysteine modification.5 In addition
used functional groups in bioconjugation (Scheme 1). We have to guiding the reactivity, the choice of leaving group could
recently described bromomaleimides 3 as the parent members of also control other properties of the reagents, such as aqueous
a new class of reagents, which can be termed ‘next generation solubility. Herein we report on the aryloxymaleimides, as NGMs
maleimides’ (NGMs). The incorporation of the leaving group leads to of attenuated reactivity, and demonstrate application of these
a switch in the mechanism that occurs upon reaction with a thiol, reagents in cysteine modification and in new modes of conjugation
from an addition reaction to an addition–elimination reaction. of disulfide bonds.
The result is the formation of a thiomaleimide product 4, rather
Having previously shown that thiophenol served as a suitable
leaving group in dithiophenolmaleimide reagents,4b we postulated
that phenol may do like-wise. Furthermore we considered that
the increased mesomeric contribution from the oxygen into the
maleimide ring would lead to reagents of reduced electrophilicity.
The synthesis of aryloxymaleimides 6–8 was carried out by simple
addition of the corresponding phenol to N-methyl bromomaleimide 5
(Scheme 2).
Addition of these reagents to a model protein, the single
cysteine mutant (L111C) of the SH2 domain of the Grb2 adaptor
protein 9,2b was monitored over 2 hours and compared to the
use of bromomaleimide 5 (Fig. 1). All three aryloxymaleimides
underwent the desired addition–elimination reaction to afford
thiomaleimide conjugate 10, at a reduced rate compared to
bromomaleimide 5.2b As expected, the incorporation of electron
Scheme 1 Cysteine labelling of proteins using maleimide 1 and next
generation maleimide (NGM) 3.
a Department of Chemistry, University College London, 20 Gordon St, London, UK.
E-mail: j.r.baker@ucl.ac.uk; Tel: +44 (0)2076792653
b UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK
c Pfizer, Chemical Biology Group, BioTherapeutics Chemistry,
WorldWide Medicinal Chemistry, 610 Main Street, Cambridge, MA 02139, USA
† Electronic supplementary information (ESI) available: Experimental procedures
and full spectroscopic data are available. See DOI: 10.1039/c4cc02107j
Scheme 2 Synthesis of monoaryloxymaleimides 6–8.
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Fig. 1 Addition of aryloxymaleimides to Grb2 SH2 (L111C), with a graph of
conversion over time measured by LCMS.
withdrawing groups on the aryloxy ring (7 and 8) led to an accelera-
tion in rate compared to the unsubstituted phenoxymaleimide 6. It is
notable that the aryloxymaleimides are still considerably more
reactive than iodoacetamide, another commonly employed cysteine
reactive reagent, which underwent o5% reaction with this protein
Scheme 3 Succinimide bridging of the disulfide bond in somatostatin.
after 30 min under the same conditions.2b Furthermore the reaction extremely high selectivity for disulfide bridging. To prove that
could be accelerated by increasing the number of equivalents of succinimide bridging had reached completion, product 14 was
phenoxymaleimide 6 to ten, leading to complete conversion to treated with 1 equiv. of maleimide. No reaction was observed by LC-
the product in just 10 min. These results demonstrate that MS analysis after 5 min at room temperature, proving the absence
phenoxymaleimides represent cysteine modification reagents of detectable amounts of free thiol.
of attenuated reactivity, a feature we postulated could be useful
in affording improved selectivity in certain bioconjugations.
We also found that, unlike bromomaleimides,4b aryloxym-
aleimides are resistant to cross-reactivity with the reducing agent
A particular regioselectivity problem had become apparent in our TCEP and therefore that the succinimide bridging could be
studies on disulfide bridging for which we envisaged that the carried out in situ. The optimised in situ conditions involve
aryloxymaleimides could offer an ideal solution. We were interested addition of 1.5 equiv. aryloxymaleimide to the peptide followed
in attempting to use bromomaleimide to bridge a disulfide bond in by addition of the equimolar amount of the reducing agent TCEP
the peptide hormone somatostatin 11, in a manner related to our leading to quantitative conversion after 2 h. In situ protocols are
work with dibromomaleimide.2b,4b This would afford a saturated valuable in disulfide bridging as they limit the amount of time
dithiosuccinimide 14 (presumed to be as a mixture of diastereo- the peptide or protein lacks its bridging functionality and thus
mers), in contrast to the dithiomaleimides generated by dibromo- reduces the propensity to aggregate or unfold.4b,6
maleimide bridging. Such dithiosuccinimides would be expected to
A selection of derivatised aryloxymaleimides (Fig. 2) were then
have different properties to dithiomaleimides (e.g. reduced reactivity synthesised (see ESI,† Scheme S1) to explore the scope of this
with thiols)2b,3b and could serve as a complementary approach. succinimide bridging. Notably a new, scalable synthesis of bromom-
However, addition of bromomaleimide 5 to reduced somatostatin aleimide was developed to allow convenient access to these molecules.
12 led to a mixture of the desired bridged adduct 14 and the bis- A water soluble aryloxy motif was incorporated in 16 to investigate the
addition product 15 (Scheme 3). Thus addition of bromomaleimide to possibility of organic solvent free labelling. A ‘miniPEG’ phenoxym-
the second thiol in intermediate 13 was competing with the bridging aleimide 17 was developed as a model for PEGylation of proteins, and
reaction. What was required was an NGM analogue which rhodamine phenoxymaleimide 18 for fluorescent labelling.
underwent a slower reaction with cysteine, to ensure that the
The use of the water soluble aryloxymaleimide 16 successfully
cyclisation of 13 would take place before the second maleimide enabled the labelling of somatostatin under organic solvent free
addition. The aryloxymaleimides appeared to fit this specifica- conditions. It is noteworthy that this is the first example of a
tion. Indeed treatment of reduced somatostatin 12 with phenoxy- maleimide reagent incorporating a water solubilising group which
maleimide 6 led after 1 h to quantitative conversion to the is released upon conjugation, and further exemplifies the enhanced
succinimide bridged somatostatin conjugate 14. Increasing the versatility of NGMs over traditional maleimides.
amount of phenoxymaleimide to 10 equiv. in this reaction still
To demonstrate that the succinimide bridging was also applicable
afforded none of the bis-labelled product 15, demonstrating the to larger proteins, chemical modification of an antibody fragment
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conjugate addition to generate acyclic thiomaleimide 13 (see
Scheme 3), and thus 13 and 14 are in fact in equilibrium.
Thiomaleimide 13 is then cleaved by GSH in a manner consistent
with our previous results,9 via a conjugate addition–elimination
sequence. The difference in thiol stability of the somatostatin–
succinimide 14 to the scFv-succinimide 20 we postulate to be
attributable to the extent of intramolecular interactions that are
present constraining these structures. In the case of somatostatin
the formation of acyclic thiomaleimide intermediate 13 can be
followed by a shift in conformation reducing the rate of rebridging
and allowing a significant amount of this intermediate to form in
solution. In contrast in the case of the scFv the extensive inter-
actions between the variable regions of the light and heavy chain
hold the cysteines in close proximity, precluding the formation of
significant amounts the acyclic thiomaleimide intermediate
required for thiol mediated cleavage.
Fig. 2 Aryloxymaleimide derivatives.
Notably employing N-phenyl phenoxymaleimide to bridge
somatostatin led to the resultant conjugate which underwent
rapid hydrolysis of the succinimide bridge (consistent with the
equivalent N-phenyl maleimide bridge,4a and see ESI,† Fig. S2),
affording a succinamic acid which was completely stable to
thiols; this represents a general method of producing completely
thiol stable conjugates.
Whilst the focus of this study was investigating the use of
phenoxymaleimides in succinimide bridging, we realised that the
reversibility of the bridging offered an interesting possibility for dual
labelling of disulfide bonds. We envisaged that addition of a second
electrophile could lead to interception of intermediate 13, attaching a
second tag. Dual labelling of a reduced disulfide bond is extremely
difficult to achieve using traditional reagents as it relies on a
Scheme 4 Succinimide bridging of ds-scFv shMFE.
was investigated. A substantial portion of all biotechnology products significant difference in reactivity between the two cysteines.10
under development are antibodies or antibody fragments,7 and Methods for the site-selective dual-labelling of peptides are widely
homogeneous conjugation technologies have a key role to play in sought, to allow controlled introduction of multiple functionalities.11
realising the full potential in this area.6,8 Treatment of cystine For example, the incorporation of two different fluorophores allows
stabilized anti-carcinoembryonic antigen single chain Fv fragment FRET studies while the attachment of an in vivo stabilising group (e.g.
(anti-CEA ds-scFv) 196 with N-miniPEG phenoxymaleimide 17 and a PEG) and a drug can prospectively enable therapeutic applications.
benzeneselenol as the reducing agent6 led to quantitative conversion
to the conjugate 20 (Scheme 4). The same outcome was observe using 6 (R1 = Me) afforded the succinimide bridged somatostatin 14 which
Thus treatment of reduced somatostatin 12 with phenoxymaleimide
N-methyl phenoxymaleimide 6 to afford conjugate 21.
was then treated, without purification, with monobromomaleimide
We have previously demonstrated that maleimide bridges are
susceptible to thiol mediated cleavage at high concentration of
thiols, such as the mM concentrations found in the cytoplasm of
cells.2b,6 We wanted to test the stability of the succinimide bridge
to thiols, to investigate if a different reactivity profile was
demonstrated. Treatment of succinimide bridged scFv conjugate
21 with 100 equiv. GSH (7 mM, pH 7.4) led to no reaction even
after 48 h at room temperature. Thus the succinimide bridge in
this scFv is completely resistant to thiol mediated cleavage in
contrast to the maleimide bridge.
Returning to somatostatin however, it became apparent that
this thiol stability is variable depending on the exact nature of the
disulfide bridge. A comparison of maleimide and succinimide
bridged somatostatin revealed that both were cleaved at similar
rates by GSH under cytoplasm mimicking conditions to release the
free peptide 12 (see ESI,† Fig. S1). We propose that the mechanism
for the thiol cleavage of the succinimide bridge starts with a retro-
Scheme 5 Dual modification of somatostatin (conditions: 1. 1 h, r.t.,
pH 6.4; 2. 2 h, 37 1C, pH 6.4).
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(R2 = H) (Scheme 5). The reaction required warming to 37 1C for 2 h to
ensure complete conversion to the dual modified peptide 22. To
confirm the formation of two cleavable thiomaleimides the conjugate
was treated with 2-mercaptoethanol (100 equiv.) to quantitatively
convert to reduced somatostatin. To demonstrate that this approach
could be employed to access dual functionalised peptides, we con-
structed miniPEG–fluorescein and rhodamine–fluorescein bis-
conjugates (23 and 24). Notably other electrophiles can also be
employed in the second modification step; for example maleimide
was employed affording a dual conjugate in which one of the tags was
cleavable by thiols and the other was not. Trypsin digest of 25
confirmed that the reaction led, as expected, to a mixture of the two
possible regioisomers (see ESI,† Fig. S3).
In conclusion, by varying the leaving groups on a maleimide core,
a range of next generation maleimides can be produced with tuneable
properties. Here a selection of aryloxymaleimides have been demon-
strated to serve as efficient cysteine labelling reagents of attenuated
reactivity. This enables the aryloxymaleimides to be employed in
succinimide bridging, offering a novel strategy for disulfide labelling.
The reversibility of the succinimide bridge in the peptide also enabled
a one-pot dual labelling strategy to be developed.
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