Clapés P, Adlercreutz P, Mattiasson B
Department of Biotechnology, University of Lund, Sweden.
J Biotechnol. 1990 Sep;15(4):323-38. doi: 10.1016/0168-1656(90)90090-x.
Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).
肽合成是在水含量低的多种有机溶剂中进行的。酶从缓冲水溶液中沉积在载体材料硅藻土上。水蒸发后，生物催化剂悬浮在反应混合物中。以Z-苯丙氨酸甲酯和亮氨酸酰胺之间的胰凝乳蛋白酶催化反应作为模型反应。在所使用的条件下（[Z-苯丙氨酸甲酯]0≤40 mM，[亮氨酸酰胺]0/[Z-苯丙氨酸甲酯]0 = 1.5），反应对Z-苯丙氨酸甲酯为一级反应。pH 7.8的Tris缓冲液是制备生物催化剂时最好使用的缓冲液。在与水混溶的溶剂中，反应速率随水含量的增加而增加，但由于Z-苯丙氨酸甲酯的竞争性水解，肽的最终产率降低。在与水混溶的溶剂中，乙腈是最合适的，在4%（体积）水的情况下产率为91%。在与水不混溶的溶剂中，在超过水饱和度0%至2%（体积%）的范围内加水对反应速率和产物分布影响很小。反应速率与溶剂的log P值相关性良好。在乙酸乙酯中获得了最高产率（93%）；在这种溶剂中反应也很快。在大多数使用的反应条件下，反应产物是稳定的；形成的肽的二次水解通常可以忽略不计。所提出的方法是动力学控制的肽合成（给出高反应速率）和热力学控制的肽合成（给出稳定的反应产物）的结合。
