The use of bromopyridazinedione derivatives in chemical biology

Tools that facilitate the chemical modification of peptides and proteins are gaining an increasing amount of interest across many avenues of chemical biology as they enable a plethora of therapeutic, imaging and diagnostic applications. Cysteine residues and disulfide bonds have been highlighted as appealing targets for modification due to the highly homogenous nature of the products that can be formed through their site-selective modification. Amongst the reagents available for the site-selective modification of cysteine(s)/disulfide(s), pyridazinediones (PDs) have played a particularly important and enabling role. In this review, we outline the unique chemical features that make PDs especially well-suited to cysteine/disulfide modification on a wide variety of proteins and peptides, as well as provide context as to the problems solved (and applications enabled) by this technology

Recent advances in N- and C-terminus cysteine protein bioconjugation

Advances in the site-specific chemical modification of proteins, also referred to as protein bioconjugation, have proved instrumental in revolutionary approaches to designing new protein-based therapeutics. Of the sites available for protein modification, cysteine residues or the termini of proteins have proved especially popular owing to their favorable properties for site-specific modification. Strategies that, therefore, specifically target cysteine at the termini offer a combination of these favorable properties of cysteine and termini bioconjugation. In this review, we discuss these strategies with a particular focus on those reported recently and provide our opinion on the future direction of the field

Site-selective multi-porphyrin attachment enables the formation of a next-generation antibody-based photodynamic therapeutic

Herein we present a significant step towards next-generation antibody-based photodynamic therapeutics. Site-selective modification of a clinically relevant monoclonal antibody, with a serum-stable linker bearing a strained alkyne, allows for the controlled Cu-free “click” assembly of an in vitro active antibody-based PDT agent using a water soluble azide porpyhrin

Trifunctional Dibromomaleimide Reagents Built Around A Lysine Scaffold Deliver Site-selective Dual-modality Antibody Conjugation

We describe the synthesis and application of a selection of trifunctional reagents for the dual-modality modification of native, solvent accessible disulfide bonds in trastuzumab. The reagents were developed from the dibromomaleimide (DBM) platform with two orthogonal clickable functional groups built around a lysine core. We also describe the development of an aryl diselenide additive which enables antibody disulfide reduction in 4 minutes and a rapid overall reduction-bridging-double click sequence

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

Synthesis of an Amphiphilic Miktoarm Star Terpolymer for Self-Assembly into Patchy Polymersomes

Herein we report the synthesis of an amphiphilic miktoarm star terpolymer and combine it with an equivalent diblock copolymer to form polymersomes with controlled surface topology. The three branches are ligated onto a central maleimide moiety in a reaction sequence that exploits various “click” chemistries. The final star was self-assembled with a linear block copolymer to generate a “patchy” surface on vesicles

Enabling the formation of native mAb, Fab ' and Fc-conjugates using a bis-disulfide bridging reagent to achieve tunable payload-to-antibody ratios (PARs)

Either as full IgGs or as fragments (Fabs, Fc, etc.), antibodies have received tremendous attention in the development of new therapeutics such as antibody-drug conjugates (ADCs). The production of ADCs involves the grafting of active payloads onto an antibody, which is generally enabled by the site-selective modification of native or engineered antibodies via chemical or enzymatic methods. Whatever method is employed, controlling the payload-antibody ratio (PAR) is a challenge in terms of multiple aspects including: (i) obtaining homogeneous protein conjugates; (ii) obtaining unusual PARs (PAR is rarely other than 2, 4 or 8); (iii) using a single method to access a range of different PARs; (iv) applicability to various antibody formats; and (v) flexibility for the production of heterofunctional antibody-conjugates (e.g. attachment of multiple types of payloads). In this article, we report a single pyridazinedione-based trifunctional dual bridging linker that enables, in a two-step procedure (re-bridging/click), the generation of either mAb-, Fab′-, or Fc-conjugates from native mAb, (Fab′)2 or Fc formats, respectively. Fc and (Fab′)2 formats were generated via enzymatic digestion of native mAbs. Whilst the same reduction and re-bridging protocols were applied to all three of the protein formats, the subsequent click reaction(s) employed to graft payload(s) drove the generation of a range of PARs, including heterofunctional PARs. As such, exploiting click reactivity and/or orthogonality afforded mAb-conjugates with PARs of 6, 4, 2 or 4 + 2, and Fab′- and Fc-conjugates with a PAR of 3, 2, 1 or 2 + 1 on-demand. We believe that the homogeneity, novelty and variety in accessible PARs, as well as the applicability to various antibody-conjugate formats enabled by our non-recombinant method could be a suitable tool for antibody-drug conjugates optimisation (optimal PAR value, optimal payloads combination) and boost the development of new antibody therapeutics (Fab′- and Fc-conjugates)

Functionalisation of ethereal-based saturated heterocycles with concomitant aerobic C–H activation and C–C bond formation

With an ever-growing emphasis on sustainable synthesis, aerobic C–H activation (the use of oxygen in air to activate C–H bonds) represents a highly attractive conduit for the development of novel synthetic methodologies. Herein, we report the air mediated functionalisation of various saturated heterocycles and ethers via aerobically generated radical intermediates to form new C–C bonds using acetylenic and vinyl triflones as radical acceptors. This enables access to a variety of acetylenic and vinyl substituted saturated heterocycles that are rich in synthetic value. Mechanistic studies and control reactions support an aerobic radical-based C–H activation mechanism

Dual reactivity disulfide bridging reagents; enabling new approaches to antibody fragment bioconjugation

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a ‘stable-labile’ design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation. By incorporating thioesters into these bridges, we demonstrate that they are primed for native chemical ligation (NCL) with N-terminal cysteines; offering an alternative to the requirement for C-terminal thioesters for use in such ligations. Alternatively, the use of hydrazine as the ligating nucleophile enables a separate cargo to be attached to each cysteine residue, which are exploited to insert variably cleavable linkers. These methodologies are demonstrated on an antibody fragment, and serve to expand the scope of disulfide bridging strategies whilst offering a convenient route to the construction of multifunctional antibody fragment conjugates

Synthesis of a novel HER2 targeted aza-BODIPY–antibody conjugate: synthesis, photophysical characterisation and in vitro evaluation

We herein report the synthesis and analysis of a novel aza-BODIPY–antibody conjugate, formed by controlled and regioselective bioconjugation methodology. Employing the clinically relevant antibody, which targets HER2 positive cancers, represents the first example of an antibody targeting strategy for this class of near-IR emitting fluorophore. The NIR fluorescence and binding properties were validated through in vitro studies using live cell confocal imaging