Reissmann S, Greiner G
Institute of Biochemistry and Biophysics, Friedrich Schiller University of Jena, Germany.
Int J Pept Protein Res. 1992 Aug;40(2):110-3. doi: 10.1111/j.1399-3011.1992.tb01457.x.
The combined use in peptide synthesis of the Fmoc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver esterase, carboxypeptidase A and alpha-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring the protected peptides into solution. To solve both problems we used dimethylformamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficiently pure products.
由于在碱性条件下以及通过催化氢化会导致Fmoc基团消除，因此在肽合成中Fmoc基团与甲酯、苄酯或对硝基苄酯的联合使用并不实用。然而，在某些情况下，肽的溶液合成需要这些组合。为此，我们研究了一些三肽和四肽酯的酶促水解。水解在pH控制下进行。我们测定了嗜热菌蛋白酶、猪肝酯酶、羧肽酶A和α-胰凝乳蛋白酶对羧基的脱保护作用。主要问题是抑制肽键的蛋白水解降解，并使受保护的肽溶解于溶液中。为了解决这两个问题，我们使用二甲基甲酰胺和二甲基亚砜作为助溶剂。在各种助溶剂浓度下估算酯解活性与蛋白水解活性之间的比率。该方法的优点是避免了碱性不稳定侧链（如天冬酰胺、谷氨酰胺）的副反应、碱不稳定保护基团的裂解以及碱性皂化引起的消旋作用。酶促脱保护后采用高效液相色谱、高效薄层色谱和滴定法进行分析。在制备规模上，该方法能获得良好的产率和足够纯的产物。
