Scheme 1. Chemical synthesis of probe A, intermediate 2 and conjugation optimization. (a) Synthesis of probe A: (i) BnOH, EDC·HCl, DMAP, and CH₂Cl₂; (ii) NaBH₄ and MeOH; (iii) MsCl, Et₃N, and CH₂Cl₂; (iv) LiBr and DMF; (v) NaOH and THF; (vi) T3P, DIPEA, and EtOAc/DMF; (vii) LiHMDS and toluene; (viii) Na₂CO₃ and MeOH/H₂O; (ix) T3P, DIPEA, and DMF; (x) LiOH and MeOH/H₂O (1 : 1); (xi) amine-derivatized beads, PyBOP, HOBt, DIPEA, and DMF. (b) Optimization of thiol conjugation with the probe under different conditions. Compound 1a was treated with N-acetyl-l-cysteine (T2) as a test substrate under six conditions to obtain the conjugated product TI2 with or without the carbamate hydrolysis by-product TC2. (c) Synthesis of simplified probe 2. Reaction conditions: (i) T3P, DIPEA, and DMF.