Synthesis of the fluorescent β1 selective proteasome probe 23. Reagents and conditions: (i) 1% TFA in DCM, 30 min, 3×. (ii) TMS–diazomethane (2 equiv.), 15 min, 90% from Fmoc-Nle-resin. (iii) Hydrazine monohydrate (60 equiv.), MeOH, reflux, 1.5 h, quant. (iv) (a) HCl (2.8 equiv.), tBuONO (1.1 equiv.), DMF–EtOAc (1: 1, v/v), −25 °C, 4 h. (b) TFA·H-Leu-epoxyketone (1.1 equiv.), DiPEA (3.8 equiv.), −25 °C to RT, 15 h, 23%. (v) 22, CuSO4 (10 mol%), sodium ascorbate (15 mol%), Tol.–H2 O–tBuOH (1: 1: 1, v/v/v), 80 °C, 22 h, 65%.