单酶体系中转胺酶催化的反应平衡消除研究

Transaminases are widely used for the preparation of bioactive pharmaceutical intermediates due to their high stereo-selectivity, readily available substrate resources and tailored amino donors. Nevertheless, the intrinsic equilibrium during reaction leading to low conversions of substrates when transaminase is employed as the biocatalyst. Generally, the equilibrium favors substrates over products. This project aims at designing amino donors based on reaction mechanism and screening more than one amino donors for puepose of shifting the equilibrium to product side in an one-enzyme system. When the amino donors obtained are applied in one-enzyme system, the pH can thus be made at the optimum conditions for the employed transaminase while not a compromized one usually employed in muti-enzyme systems, which will faciliate transaminase to display its best performance and reduce the enzyme loading, no additional enzyme(s) and expensive coenzyme(s) such as NAD(P)H and NAD(P)+ are required for shifting the equilibrium to product side or totally replacing it. Which will benefit for the preparation of the desired products at low cost. This project is supposed to lay the foundation for theoretical and pratical preparation of the desired chiral amines,followed by is further exploration for the feasibility for green industrial applications.

转氨酶因其手性选择性高，底物酮来源广泛且氨基供体根据需要可选等优点近年来被广泛应用于各种生物活性药物关键中间体-手性胺的制备中。但是转氨酶催化的反应中存在的内在反应平衡而导致底物酮转化率低,通常情况下，平衡有利于底物而不利于产物的生成。本项目拟根据转胺酶催化机理特点设计并筛选一种以上的特殊氨基供体，利用该类氨基供体，单酶体系中能有效促使反应向产物方向转移或消除内在的反应平衡；将所获氨基供体应用于单转氨酶体系中的反应中。体系可设置为转氨酶最优反应条件，有利于酶发挥其最佳催化性能，减小酶使用量，且不需要额外的生物酶和价格昂贵的辅酶就能成功促使反应向产物方向转移或消除单转氨酶体系中的反应平衡，可大大降低目标产物的制备成本。为规模化制备目标手性胺类化合物奠定理论和实践基础。并进一步探讨其应用于其他底物制备手性胺（氨基酸）的可能性。

本项目主要研究在利用转胺酶催化的单酶体系中和无昂贵辅酶NAD(P)H/NAD(P)+的情况下，如何有效的转移/消除反应平衡的问题。本项目拟根据转胺酶催化机理筛选系列特殊胺基供体，体系可设置为转胺酶最优反应条件，有利于酶发挥其最佳催化性能，减小酶使用量的同时无需昂贵的辅酶就能达到消除/转移反应平衡的目的。项目执行过程中，筛选到了几种胺基供体，用于在转胺酶催化的单酶反应体系中，胺基供体的副产物能有效的进行反应转换为不再参与到转胺反应中的稳定的化合物。并且，根据副产物醛的特点，我们研究出了两种在转胺酶相同催化体系条件下，将醛转移出反应体系的有效方法。其中之一是将醛绿色转化为相应的羧酸，另一种是合成了系列选择性的非均相催化剂，能在转胺酶催化体系同样条件下，选择性将醛还原为相应的醇。并且进一步结合醛转化的反应体系，实现了单酶体系中将副产物酮进行有效的消除（醛氧化为羧酸去除反应平衡，和醛还原为醇去除反应平衡），达到了转移反应平衡的问题。另外，根据氨基受体的特点，我们开发了几种绿色反应制备相应的羰基酮酸（酰胺）类化合物的的绿色反应工艺，后续将基于现有研究的基础，进一步接近工业化利用的目的。