We next examined the warhead reactivity against a panel of amino acid derivatives (Ser, His, Tyr, Lys and Cys) in aqueous conditions (Scheme 4). NH sulfoximine 5a gave complete recovery of the starting material for all except for cysteine which showed complete conversion (64% isolated yield). When switching to the more reactive N-mesyl BCB-sulfoximine 6d low consumption of the BCB substrate was observed after 18 h with several amino acid nucleophiles. On the other hand, the reaction with cysteine gave complete conversion. A competition experiment reacted mesyl-protected BCB sulfoximine 6d with cysteine and lysine methyl esters in a 1:1 ratio. The thiol nucleophile outcompeted the three NH2 sites (56% vs 15% yield by 1 H NMR). The rates of reaction of four representative BCB compounds to conjugation with glutathione were established using a qIT (quantitative irreversible tethering) assay over a range of concentrations and benchmarked against that of acrylamides (Figure ). BCB-sulfoximines bearing electron-withdrawing groups demonstrated intrinsic reactivities (6d, 6e, 6i, t1/2 (GSH) = 1.2-13 h), comparable to that of a moderately reactive acrylamide (t1/2 (GSH) = 9.4 h). This moderate electrophilicity positions BCB-sulfoximines within a promising range for biological applications. We next examined whether the BCB-sulfoximines would label proteins in a cysteine-selective manner under biocompatible conditions. The GSH rate data was used to estimate appropriate BCB concentrations to apply in protein models over a 24 h reaction period. A series of BCB-sulfoximines was then reacted with recombinantly expressed CDK2 (cyclin-dependent kinase 2) possessing a single surface cysteine C177 (CDK2 WT), and its Cys-deficient mutant CDK2 C177A as a model system to confirm reactivity and Cys selectivity of the compounds (Figure ). CDK2 WT adducts with the N-acetyl (6c), and N-mesyl BCBs (6d) were observed in high percentage conversions. CDK2 C177A did not react with any of the BCB sulfoximines tested nor with Cysselective electrophile iodoacetamide control (Fig S15 ). This is notably different reactivity to that reported for a targeted vinyl sulfone CDK2 inhibitor (see Figure ) which displayed selectivity for a reactive lysine residue. Treatment with the less reactive NH, NMe and NPh sulfoximines resulted in no observed reactivity with the protein. We prepared BCB sulfoximine 6i containing a propargyl carbamate to model a fully functionalized probe, exploiting the N-vector. This displayed a useful level of intrinsic reactivity with CDK2 with 50% conversion in a 24 h period to the mono-labelled protein and no reaction with CDK2 C177A (Figure ). warhead selectivity is ideally governed by non-covalent interactions and thus electrophiles which demonstrate differences in labelling selectivity when appended to varied scaffolds are promising for use within covalent ligand discovery. Alkynefunctionalised BCB-sulfoximine probes were incubated with HEK293 cell lysates (100 µM, 3h), conjugated to TAMRA-azide using copper-catalyzed azide alkyne cycloaddition (CuAAC) chemistry, and fluorescent bands visualized via gel scanning (See SI for full details). Pleasingly, in this preliminary study, BCBsulfoximines with variation in N-functionality (N-propargyl or Npropargyl carbamate) and in arene substitution labelled different proteins (See Fig S21) indicating that proteome selectivity can be manipulated. Finally, we envisaged that the BCB-sulfoximines would provide promising warheads for use within enantioprobe pair screening studies due to the central stereogenic center and the ability to access analogues without the need for downstream warhead installation. Cravatt has demonstrated the value of using enantioprobe pairs within ABPP experiments, as factors which distort proteome-selectivity results are controlled for, allowing high confidence in the identification of ligandable cysteines. In a preliminary study, enantioprobe pair (S)-11 and (R)-11 displayed enantioselective protein labelling when incubated with HEK 293 cell lysates (10 µM, 3 h), as visualized by clear differences in the fluorescence intensity of select bands (Figure ).