Correction: Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Correction for 'Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis' by Maurício Morais et al., Org. Biomol. Chem., 2017, 15, 2947-2952, DOI: .

Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to 'lock' the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.

A novel synthetic chemistry approach to linkage-specific ubiquitin conjugation.

Ubiquitination is of great importance as the post-translational modification of proteins with ubiquitin, or ubiquitin chains, facilitates a number of vital cellular processes. Herein we present a facile method of preparing various ubiquitin conjugates under mild conditions using michael acceptors based on dibromo-maleimides and dibromo-pyridazinediones.

A platform for efficient, thiol-stable conjugation to albumin's native single accessible cysteine.

Herein we report the use of bromomaleimides for the construction of stable albumin conjugates via conjugation to its native, single accessible, cysteine followed by hydrolysis. Advantages over the classical maleimide approach are highlighted in terms of quantitative hydrolysis and absence of undesirable retro-Michael deconjugation.

Homogeneous bispecifics by disulfide bridging.

We report on a chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics.

Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment.

The rapidly increasing interest in the synthesis of antibody-drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin-antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case.

Bromo- and thiomaleimides as a new class of thiol-mediated fluorescence 'turn-on' reagents.

Bromo- and thiomaleimides are shown to serve as highly effective quenchers of a covalently attached fluorophore. Reactions with thiols that lead to removal of the maleimide conjugation, or detachment of the fluorophore from the maleimide, result in 'turn-on' of the fluorescence. These reagents thus offer opportunities in thiol sensing and intracellular reporting.

Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging.

The advent of Adcetris™ and Kadcyla™, two recently FDA-approved antibody-drug conjugates (ADCs), in the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively, worldwide. Despite these successes many new ADCs fail at various stages of development, often due to shortcomings in the methods used for their assembly. To address this problem we have developed next generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow the efficient conjugation of small molecules to antibodies, without the need for engineering of the target antibody. The method is site-specific and generates near homogeneous products in good yields. Moreover, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry (drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when compared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and simple platform for the controlled assembly of ADCs from an antibody.

Reversible protein affinity-labelling using bromomaleimide-based reagents.

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics.

A mild synthesis of N-functionalised bromomaleimides, thiomaleimides and bromopyridazinediones.

Bromomaleimides are useful building blocks in synthesis and powerful reagents for the selective chemical modification of proteins. A mild new synthesis of these reagents is described, along with the convenient transferability of the approach to dithiomaleimides and bromopyridazinediones.

Bioconjugation of green fluorescent protein via an unexpectedly stable cyclic sulfonium intermediate.

Smooth converter: Bioconjugation of superfolder GFP involving the formation of an unusually stable, and unprecedented, cyclic sulfonium species is described. This sulfonium can undergo smooth reaction with a range of nucleophiles to give sulfur-, selenium- and azide-modified GFP derivatives in high conversions.

In situ maleimide bridging of disulfides and a new approach to protein PEGylation.

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.

Protein modification, bioconjugation, and disulfide bridging using bromomaleimides.

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

Non-alpha-hydroxylated aldehydes with evolved transketolase enzymes.

Transketolase mutants previously identified for use with the non-phosphorylated aldehyde propanal have been explored with a series of linear and cyclic aliphatic aldehydes, and excellent stereoselectivities observed.

Development of chemical probes: toward the mode of action of a methylene-linked di(aryl acetate) E1.

Analogues of the novel inhibitor of the PI3-K/PKB pathway, 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1), have been prepared and preliminary SAR performed. This established that at least one of the chloroethyl para-substituents could be removed or modified and the ability to inhibit PKB/Akt activation retained. Synthetic methodologies were then developed to methylene-linked aryl acetates for use as molecular probes to identify the target of compound E1.

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde.

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.

Directed evolution of transketolase activity on non-phosphorylated substrates.

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.

A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy.

Although recent methods for the engineering of antibody-drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.

Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody-drug conjugate (ADC).

Herein we report the use of next generation maleimides (NGMs) for the construction of a potent antibody-drug conjugate (ADC) via functional disulfide bridging. The linker has excellent stability in blood serum and the ADC, armed with monomethyl auristatin E (MMAE), shows excellent potency and cancer cell selectivity in vitro.