Figure 5. Semisynthesis of full-length sumoylated histone H2B(A117C).

(a) Synthesis of photoprotected auxiliary 3. (i) CH₃C(O)SH, K₂CO₃, THF, 8 h, 25 °C. (ii) HCl, CH₃OH, 6 h, 60 °C, 75% (2 steps). (iii) N-(2-bromoethoxy)phthalimide, Et₃N, DMSO, 4 h, 25 °C, 74%. (iv) H₂NNH₂, CHCl₃, 1 h, 25 °C, 98%. (b) (i) Site-specific coupling of 3 to H2B(117–125)A117C Lys120 followed by acidolytic release of the unprotected peptide, 4, from the solid-phase. (ii) Expressed protein ligation of 4 with H2B(1–116)-α-thioester to generate full-length H2B(A117C) with protected auxiliary at Lys120, 5. (iii) photolytic removal of the auxiliary protecting group to give H2B(A117C) with unprotected auxiliary at Lys120, 6. (iv) Expressed protein ligation of 6 with SUMO-3(2–91)C47S-α-thioester to generate sumoylated H2B(A117C) 7, with retention of the ligation auxiliary. (v) Selective removal of the ligation auxiliary with 150 mM MPAA under non-denaturing conditions to yield sumoylated H2B(A117C) 8. ivDde=1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl group. PDB codes, 1KX5 (H2B) and 1U4A (SUMO-3).