To synthesize a minimized Max version, we initially divided the DNA binding domain of Max (13-93) into two peptide segments (Figure ): Max(13-51)-NHNH2 and Cys-Max(53-93). Ala52 was temporarily mutated into Cys to allow native chemical ligation (NCL) at this site, which could be converted back to Ala after NCL via a final desulfurization step. All peptide segments were prepared parallelly using stepwise Fmoc-SPPS with the selective incorporation of the desired single-point mutation, as described in Figure (SI section 4). We obtained 18 Max(13-51)-NHNH2 segments (1-18) and Having all segments, we ligated each Max(13-51)-NHNH2 segment (1-18) with the Cys-Max(53-93) partner (19, 21, or 22) under standard NCL conditions (Figure ). In brief, Max(13-51)-NHNH2 segments (1-18) were converted into acyl-azide using NaNO2 in 6 M Gun•HCl, 0.2 M Na2HPO4 buffer, pH 3.0 at -15 ℃. Then, 4-mercaptophenylacetic acid (MPAA) was added for in situ thioesterification, followed by the addition of relevant peptide segments (19, 21, or 22) in the presence of tris(2-carboxyethyl)phosphine (TCEP). The reaction pH was adjusted to 7.2 and kept at room temperature for 1 hour (SI section 5). The progress of the ligation reaction was analyzed by LCMS. Subsequently, the crude ligation reaction was desalted to enable a one-pot desulfurization reaction in the presence of the radical initiator 2,2'-Azobis [2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), TCEP, and glutathione (GSH) to convert Cys52 at the ligation junction to the native Ala (SI section 5). Following the RP-HPLC purification step, all mutated Max (Max1-19) analogs were obtained in 10-34% isolated yields after the four synthetic steps. In addition, following the same synthetic procedure, we synthesized the wild-type Max (13-93) analog (WT Max) as a control. The identity and purity of all 20 synthetic Max analogs were confirmed by LCMS (SI Figures ).