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Synthesis of Ins-SA via trypsin mediated ligation and cleavage. Cleavage of insulin to DOI was completed by dissolving trypsin in pH 7.5 buffer. This mixture was added to native insulin dissolved in 1:1 (v/v) DMSO:1,4-butanediol, 3 mL total. Ligation of octapeptide to DOI was completed by dissolving 1 mg/μmol trypsin in pH 7.6 buffer. This mixture was added to native insulin dissolved in 1:1 (v/v) DMSO:1,4-butanediol, 348 μL total.
