Rapid and robust cysteine bioconjugation with vinylheteroarenes

Abstract

Methods for residue-selective and stable modification of canonical amino acids enable the installation of distinct functionality which can aid in the interrogation of biological processes or the generation of new therapeutic modalities. Herein, we report an extensive investigation of reactivity and stability profiles for a series of vinylheteroarene motifs. Studies on small molecule and protein substrates identified an optimum vinylheteroarene scaffold for selective cysteine modification. Utilisation of this lead linker to modify a number of protein substrates with various functionalities, including the synthesis of a homogeneous, stable and biologically active antibody-drug conjugate (ADC) was then achieved. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology.

Fig. 1. Use of maleimides, divinylpyrimidines and vinylheteroarenes for post-translational protein modification.

Fig. 2. Stability comparison of pyrimidine 5, triazine 6, and succinimide 7 in NaPi (pH 7.4, 50 mM) and CD₃CN, in the presence of 1-thioglycerol (150 mM). Average of two replicates; error bars represent standard deviation of the mean.

Fig. 3. Modification of human serum albumin (HSA) using vinylpyrimidine and vinyltriazine linkers. The modification of HSA with a variety of functional units including alkyne reactive handle, fluorophore and biotin was successfully achieved.

Fig. 4. The modification of mAb1 with linker 2 and 8. The samples were deglycosylated and reduced prior to LCMS analysis.

Fig. 5. (a) On-protein copper catalysed azide–alkyne cycloaddition (CuAAC) reactions to functionalise alkynyl antibody mAb1-8. This antibody was successfully modified with biotin 12, Alexa Fluor 488 fluorophore 13 and arylsulfate-MMAE 14. (b) Deconvoluted mass spectrum of mAb1-8-12 heavy chain. (c) Deconvoluted mass spectrum of mAb1-8-14 heavy chain. Species with 46 878 Da correspond to light chain dimer. (d) UV-vis spectrum of unmodified mAb1, mAb1-8, and mAb1-8-13. Bioconjugate mAb1-8-13 displayed a fluorophore-to-antibody ratio of 1.9.

Fig. 6. The in vitro evaluation of ADC mAb1-8-14 in HER2-positive cells (SKBR3) and HER2-negative cells (MCF7). Viability data shows the mean of three independent experiments and error bars represent standard error of the mean.

Fig. 7. SDS-PAGE analysis of MCF7 cell lysate labelling studies. Cell lysates were first labelled with varying concentrations of probe 8, and further modified with Alexa Fluor 488 azide 13via a CuAAC reaction. For cysteine blocking studies, cell lysates were first pre-incubated with iodoacetamide. (a) In-gel fluorescence of cell lysates. (b) Coomassie staining of cell lysates. All lanes were prepared under reducing conditions. Molecular weight ladder in kDa. Refer to ESI Section 8 for further details.

Fig. 8. Determination of plasma stability by SDS-PAGE for (a) mAb2-8-13 and (b) mAb2-15-13, which are bioconjugates synthesised by reduction of interchain disulfides of native trastuzumab mAb2, followed by bioconjugation with linker 8 or 15, respectively, and functionalisation with Alexa Fluor 488 13. In-gel fluorescence analysis displays no transfer of fluorescence for vinylpyrimidine-derived conjugate mAb2-8-13. In contrast, fluorescence transfer to serum proteins (indicated by the beige box) was observed for maleimide-derived mAb2-15-13. All lanes were prepared under reducing conditions. (c) UV-vis spectra for mAb2, mAb2-8-13, and mAb2-15-13.