Scheme 1.

Lys(mPEG₁₀)-βAla-Val-Cit-PAB-MMAE (6). SPPS: Coupling of acids: 4 eq. acid, 4 eq. TBTU, 4 eq. DIPEA, DMF. Fmoc deprotection: 20% piperidine in DMF with 0.1 M HOBt. Mtt deprotection: 1% TFA in DCM. Coupling of mPEG₁₀-CO₂H: 1.66 eq. PEG-linker, 1.66 eq. DIPEA, 1.66 eq. HOAt, 1.5 eq. HATU, DMF. Cleavage: TFA:H₂O:TIPS (95:2.5:2.5, v/v/v). (i) 1.1 eq. DBCO-C6-NHS ester, 3 eq. DIPEA, DMF, rt, 3 h, 55.5%. (ii) 1 eq. Boc₂O, 2.5 eq. Na₂CO₃, H₂O, THF, rt, o.n., 84%. (iii) 2 eq. PAB-OH, 2 eq. EEDQ, DCM:MeOH (2:1, v/v), rt, 24 h, 91%. (iv) 3 eq. 4-nitrophenyl carbonate, 2.25 eq. DIPEA, DMF, rt, 5 h, 95%. (v) 1 eq. MMAE, 3 eq. HOAt, 1.5 eq. DIPEA, DMF, rt, o.n., quantitative. (vi) Dry TFA, dry DCM, 0 °C, 25 min, 68%. (vii) 1.3 eq. 2, 1.3 eq. PyAOP, 3 eq. HOAt, 3 eq. DIPEA, DMF, rt, 2 h, 50.6%.