Figure 1: Identification of a suitable carbonylacrylic derivative for reaction with Cys.
(a) Reaction of 2a with N-Boc-Cys-OMe 1 provides the thioether product 3a in 99% yield after 2 min. (b) Reaction of 2a in an equimolar mixture of 1 and N-Boc-Lys-OMe 4 shows complete Cys chemoselectivity. (c) Relative reaction rates at 25 °C (krel) calculated for the conjugate addition of methyl thiolate to maleimide and 3-carbonylacrylic acid derivatives 2a–d. (d) Reaction of derivatives 2b–d with 1. (e) Experimental determination of the second-order rate constant for the addition of compound 2c to 1. See the Supplementary Methods for additional details and kinetics data for compound 2a. (f) Kinetic assay for the stability of chromogenic thiol adducts 5 and 6 at physiological pH. The absorbance of 5 at 410 nm is constant over time, indicating no release of 4-nitrophenolate. In contrast, for 6, a steady increase in absorbance over time was observed. It is important to note that although both compounds absorb at 410 nm, we have set the initial absorbance to zero for clarity. The original data for 5 (A0h=0.45 nm, A5h=0.45 nm) and for 6 (A0h=0.23 nm, A5h=0.35 nm) is presented in the Supplementary Fig. 7. NR, no reaction; RT, room temperature.
