ChemComm
Cite this: Chem. Commun., 2011, 47, 8781–8783
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COMMUNICATION
Bromopyridazinedione-mediated protein and peptide bioconjugationw
a
a
a
b
Vijay Chudasama, Mark E. B. Smith, Felix F. Schumacher, Danai Papaioannou,
b
a
a
Gabriel Waksman, James R. Baker and Stephen Caddick*
Received 12th May 2011, Accepted 20th June 2011
DOI: 10.1039/c1cc12807h
Bromopyridazinedione-mediated bioconjugation to a cysteine
containing protein and a disulfide containing peptide is described.
The conjugates are cleavable in an excess of thiol, including
cytoplasmically-relevant concentrations of glutathione, and show
a high level of hydrolytic stability. The constructs have the
potential for four points of chemical attachment.
Following on from this success, we are interested to determine
whether the ability to construct reversible constructs is
something that is restricted to maleimides or whether other
cyclic or acyclic haloeneamide systems will afford alternative
opportunities.
We report herein the use of both monobromo- and dibromo-
,2-dihydro-pyridazine-3,6-diones (MBPDs 1 and DBPDs 2)
Fig. 1) to assemble cleavable bioconjugates. The bioconjugates
1
(
The published sequence of the human genome revealed
encoding for only one tenth of the total proteome. The role
of protein post-translational modification has therefore
become of intense interest in understanding complex, physio-
logical processes. Combining synthetic organic chemistry with
molecular biology has enabled the construction of a range of
proteins and peptides as probes to aid in this venture. This
has allowed the introduction of synthetic post-translational
generated demonstrate exceptional hydrolytic stability with
the potential for four points of chemical attachment. We
have previously shown that hydrolytic stability is crucial in
preserving the thiol-cleavable property of bromomaleimide-
13d
linked bioconjugates.
Our initial studies focused on the functionalisation of a
single cysteine mutant (L111C) of the SH2 domain of the Grb2
13a
adapter protein 3, a protein that does not otherwise contain
1
–3
4–6
7
modifications and a variety of fluorescent, radiolabelled
8
any cysteine residues, with pyridazinedione (PD) 4 (100 mol
eq., 37 1C, 16 h). No reaction was seen to occur (Scheme 1).
The absence of reactivity observed with PD-4 may be ratio-
nalised by the acidic nature of its hydrazide protons. At pH 8,
PD-4 will predominantly exist in an unreactive, anionic
–10
and affinity tags
into a variety of protein and peptide
structures. The selective modification of cysteine remains a
popular method for achieving protein modification. This is
due to its low natural abundance, high nucleophilicity and
the ease with which cysteine can be introduced at desired
locations in protein sequences using modern molecular bio-
14
form. However, treatment of protein 3 with PD-5 (100 mol
eq., 37 1C, 16 h) yielded quantitative conversion to the desired
conjugate 6 (Scheme 1). As this protein sequence contains
eight lysine residues, the reaction with PD-5 demonstrates
remarkable selectivity for cysteine as was evidenced following
an absence of reaction with Ellman’s reagent. To our knowl-
edge, this is the first reported example of a pyridazinedione
being used to modify cysteine.
1
1–12
logy methods.
In our laboratory, we have recently become interested in the
construction of protein bioconjugates that are cleavable in a
reducing environment. We believe that such an approach
could deliver reversible affinity or fluorescent labels, or con-
structs cleavable under cytoplasmic conditions with potential
as prodrugs. Maleimides are known to react rapidly and
selectively, but irreversibly, with thiols. However, we have
recently demonstrated an approach to reversible cysteine
bioconjugation using bromomaleimides, where retention of
the maleimide double bond allows cleavage of the constructs
Encouraged by the successful functionalisation of protein 3
with PD-5, we sought to synthesise and evaluate MBPD-7 and
DBPD-8 as reagents for the reversible bioconjugation of
proteins. Synthesis of both 7 and 8 was achieved in high yield
by the initial condensation of maleic anhydride with diethyl-
hydrazine followed by sequential dibromination/elimination
steps (Scheme 2).
1
3
via conjugate addition reaction. Using bromomaleimides we
have been able to demonstrate the modular construction of
complex bioconjugates, without requirement for reagent pre-
activation, that have three points of chemical attachment.
a
Department of Chemistry, University College London, 20 Gordon
Street, London, WC1H 0AJ, UK. E-mail: s.caddick@ucl.ac.uk;
Tel: +44 (0)20 3108 5071
Institute of Structural and Molecular Biology at UCL/Birkbeck,
b
Malet Street, London, WC1E 7HX, UK
w Electronic supplementary information (ESI) available: Full experi-
mental details and characterisation. See DOI: 10.1039/c1cc12807h
Fig. 1 (Di)bromo-1,2-dihydro-pyridazine-3,6-diones.
Chem. Commun., 2011, 47, 8781–8783 8781
This journal is c The Royal Society of Chemistry 2011