Vijay Chudasama

Hydroacylation of α,β-unsaturated esters via aerobic C–H activation

The development of methods for carbon–carbon bond formation under benign conditions is an ongoing challenge for the synthetic chemist. In recent years there has been considerable interest in using selective C–H activation as a direct route for generating reactive intermediates. In this article, we describe the use of aldehyde auto-oxidation as a simple, clean and effective method for C–H activation, resulting in the generation of an acyl radical. This acyl radical can be used for carbon–carbon bond formation and herein we describe the application of this method for the hydroacylation of α,β-unsaturated esters without the requirement of additional catalysts or reagents. This methodology generates unsymmetrical ketones, which have been shown to have broad use in organic synthesis. The development of benign methods for carbon–carbon bond formation is a continuing challenge. Here, a simple procedure for the hydroacylation of α,β-unsaturated esters is described, in which auto-oxidation of aldehydes and subsequent acyl radical addition to α,β-unsaturated esters occurs without the need for additional reagents.

Recent advances in the construction of antibody–drug conjugates

Antibody-drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.

Acid-cleavable thiomaleamic acid linker for homogeneous antibody–drug conjugation

Homogeneous antibody–drug conjugation is affected using a novel thiomaleamic acid linker that is stable at physiological temperature and pH, but quantitatively cleaves at lysosomal pH to release the drug payload.

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.

Motional timescale predictions by molecular dynamics simulations: case study using proline and hydroxyproline sidechain dynamics.

We propose a new approach for force field optimizations which aims at reproducing dynamics characteristics using biomolecular MD simulations, in addition to improved prediction of motionally averaged structural properties available from experiment. As the source of experimental data for dynamics fittings, we use 13C NMR spin‐lattice relaxation times T1 of backbone and sidechain carbons, which allow to determine correlation times of both overall molecular and intramolecular motions. For structural fittings, we use motionally averaged experimental values of NMR J couplings. The proline residue and its derivative 4‐hydroxyproline with relatively simple cyclic structure and sidechain dynamics were chosen for the assessment of the new approach in this work. Initially, grid search and simplexed MD simulations identified large number of parameter sets which fit equally well experimental J couplings. Using the Arrhenius‐type relationship between the force constant and the correlation time, the available MD data for a series of parameter sets were analyzed to predict the value of the force constant that best reproduces experimental timescale of the sidechain dynamics. Verification of the new force‐field (termed as AMBER99SB‐ILDNP) against NMR J couplings and correlation times showed consistent and significant improvements compared to the original force field in reproducing both structural and dynamics properties. The results suggest that matching experimental timescales of motions together with motionally averaged characteristics is the valid approach for force field parameter optimization. Such a comprehensive approach is not restricted to cyclic residues and can be extended to other amino acid residues, as well as to the backbone. Proteins 2014; 82:195–215.

Bromopyridazinedione-mediated protein and peptide bioconjugation

Bromopyridazinedione-mediated bioconjugation to a cysteine containing protein and a disulfide containing peptide is reported. These bioconjugates exhibit excellent hydrolytic stability and cleave in an aqueous, thiol-mediated reducing environment.

Bromomaleimide-Linked Bioconjugates Are Cleavable in Mammalian Cells

Bromomaleimides have recently been shown to act as small, versatile scaffolds for the controlled assembly of thiolated biomolecules.[1–4] They present three points of chemical attachment, thereby allowing the modular construction of multifunctional bioconjugates. The bromomaleimide scaffold introduces no new chiral centres to the final construct and, in contrast to disulfide linkages, bromomaleimides can be used to link two unactivated biomolecules of equal concentration with minimal formation of homodimeric side products.2 Bromomaleimide adducts have been shown to dissociate in vitro in the presence of reducing agents to liberate the composite thiols.2 The cytoplasm of cells contains 1–10 mm reduced glutathione,5 thus raising the possibility that bromomaleimide conjugates could be cleavable in vivo. If this could be demonstrated, then a number of medical and academic applications can be envisaged that combine in vivo cleavage with multiple points of scaffold attachment. However, a number of factors might inhibit cytoplasmic cleavage, in particular pH-dependant3 and protease-catalysed amide bond hydrolysis. Using a series of bromomaleimide-linked green fluorescent protein (GFP)–rhodamine conjugates, designed as FRET pairs, we demonstrate herein that bromomaleimide-linked bioconjugates cleave in the cytoplasm of mammalian cells. Rhodamine–maleimide derivatives 4–6 were generated by condensation of the relevant maleic anhydride with a common intermediate, 3 (Scheme 1). Scheme 1 Synthesis of rhodamine-bromomaleimides. a) (COCl)2, 20 °C, 15 h; b) piperidin-4-yl carbamic acid tert-butyl ester (10.4 equiv), CsCO3 (10.4 equiv), CH2Cl2, 20 °C, 24 h, 71 % (2 steps); c) TFA/CH2Cl2 (1:1), 20 °C, 5 h, 100 %; d) ... The two native cysteines in wild-type superfolder GFP,6 C48 and C70, were shown to be inaccessible to maleimide functionalisation under our reaction conditions (see Section 4 in the Supporting Information). A GFP with a free, accessible thiol close to its fluorophore (GFP-SH) was generated by introducing an S147C mutation into superfolder GFP. The mutated GFP-SH produces a similar emission spectrum to wild-type superfolder GFP. The folded protein was shown to be resistant to disulfide-mediated dimerisation, thus allowing GFP-SH to be conjugated to maleimides without the need for reducing agents (see Section 5 in the Supporting Information). Compounds 4–6 were attached to GFP-SH as described in the Supporting Information, Section 5. Stoichiometric addition of rhodamine–maleimide was confirmed by mass spectrometry. The emission spectra of the resultant constructs 7–10 are illustrated in Figure 1. In each case, addition of rhodamine–maleimide was shown to result in efficient quenching of GFP fluorescence. Little increase in emission at 590 nm was seen. Figure 1 Emission spectra of superfolder GFP, the mutant GFP-SH and the rhodamine conjugates; compounds 7–9 (0.85 μM), compound 10 (0.425 μM; λex=494 nm). Cleavage of compounds 7–10 by a physiologically relevant concentration of reduced glutathione (1 mM) was monitored in vitro by dual-channel measurement of the GFP and rhodamine emission intensities upon excitation of GFP at 494 nm (Figure 2). Figure 2 In vitro cleavage of the rhodamine–GFP conjugates. Glutathione (1 mM) was added to compounds A) 7, B) 8, C) 9 (0.85 μM each) and D) 10 (0.43 μM). GFP was excited at 494 nm; GFP emission (515 nm, green) and rhodamine emission (590 ... As expected, the GFP emission intensity associated with 7 did not change upon addition of glutathione, whereas that associated with 8–10 increased. The data fitted well to a single exponential increase in free GFP concentration over time, suggesting first-order kinetics with respect to conjugates 8–10 under these conditions. Rate constants of 1.9 s−1 for 8, 26 s−1 for 9 and 2.8 s−1 for 10 were calculated. For compounds 7–9, little change in apparent rhodamine emission (590 nm) was seen during the experiments. For compound 10, a decrease in rhodamine emission was observed; this suggesting that FRET is disrupted. The efficient quenching of GFP fluorescence allowed us to use the ratio of GFP/rhodamine emission intensities as a quantitative measure of cleavage during subsequent microinjection experiments. Cleavage of constructs 8–10 was also confirmed by mass spectrometry under analogous reaction conditions (see Section 7 in the Supporting Information). Cleavage of constructs 8–10 in human cells was demonstrated by microinjection into live, cultured HeLa cells, followed by time-lapse fluorescence microscopy. GFP was excited by using a 450–490 nm filter, and GFP and rhodamine emission were measured simultaneously by using an image splitter that contained GFP (500–550 nm) and rhodamine (575–630 nm) emission filters (Figures 3 and ​and44). Figure 3 In-cell cleavage of compound 9 following microinjection into HeLa cells. The image was cast onto the upper and lower halves of the CCD sensor by using an image splitter (Optical Insights). Top: GFP emission, bottom: rhodamine emission. A) DIC image immediately ... Figure 4 In-cell cleavage of compounds 7–10 in HeLa cells. The GFP/rhodamine emission ratio and exponential fits were calculated as described in the Supporting Information. In each case, both the absolute GFP intensity post-microinjection (see Section 8 in the Supporting Information) and ratiometric data fitted well to a single exponential increase, thus showing that bromomaleimide-linked constructs are cleaved in cells. The order of reactivity is the same as that observed in vitro (see Section 9 in the Supporting Information); this suggests that the mechanism of cleavage in cells is the same as the mechanism in vitro. The ratio of GFP/rhodamine emission intensity at equilibrium is consistent with the ratio observed for cleaved constructs deposited directly onto glass slides (see Section 10 in the Supporting Information). This suggests that complete cleavage of the bromomaleimide linkages occurs in HeLa cells. Disassembly of the fully conjugated dibromomaleimide (10) was also successfully demonstrated in COS-7 cells (see Section 8 in the Supporting Information), thus highlighting that observed in cell cleavage is not restricted to HeLa cells. In summary, we have demonstrated that bromomaleimide-linked conjugates can be cleaved with first-order kinetics both in vitro and in cells. Dibromomaleimide derivatives (9 and 10) cleave at a faster rate than monobromomaleimide construct 8. Maleimide construct 7 is stable to thiol-promoted cleavage. We envisage that bromomaleimides have potential as scaffolds for the synthesis of a variety of multifunctional reagents. We believe that the FRET construct 9 has a specific application in the screening of attached thiolated cell-penetrating structures capable of successfully delivering protein cargo to the cytoplasm. More broadly, we believe that bromomaleimide scaffolds provide a potential core structure for the facile and versatile construction of targeted therapeutics that will cleave to release their cargo once internalised into the cytoplasm. We shall report on the further development and application of this methodology in due course.

Reversible protein affinity-labelling using bromomaleimide-based reagents

A mild and highly efficient, reversible protein biotinylation method using a hydrolytically stable linker and mild disassembly conditions is described.

Functionalisation of aldehydes via aerobic hydroacylation of azodicarboxylates 'on' water.

Herein we report the functionalisation of aldehydes via hydroacylation of azodicarboxylates. A range of functionalised aldehydes are employed as the limiting reagent including chiral non-racemic aldehydes bearing α-stereocentres which are functionalised giving access to enantiomerically pure products. The resultant hydrazides can be employed as acyl donors in the synthesis of amides.

Pyridazinediones deliver potent, stable, targeted and efficacious antibody–drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody

Herein we report the use of pyridazinediones to functionalise the native solvent accessible interstrand disulfide bonds in trastuzumab with monomethyl auristatin E (MMAE). This method of conjugation delivers serum stable antibody–drug conjugates (ADCs) with a controlled drug loading of 4. Moreover, we demonstrate that the MMAE-bearing ADCs are potent, selective and efficacious against cancer cell lines in both in vitro and in vivo models.