Univ Rennes, ENSCR, CNRS, ISCR (Institut des Sciences Chimiques de Rennes) - UMR 6226, F-35000 Rennes, France.
L'Institut Agro - Montpellier SupAgro, Univ Montpellier, INRA, CIRAD - UMR IATE, F-34060 Montpellier, France.
Biomacromolecules. 2020 Jul 13;21(7):2874-2883. doi: 10.1021/acs.biomac.0c00593. Epub 2020 Jun 30.
The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of β-substituted β-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol ( = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 °C). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of ()- and ()-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.
使用安全的天然催化剂，如酶，进行β-取代β-内酰胺（如苄基丙交酯）的开环聚合（ROP）是一个重要的目标，因为由此得到的（共）聚合物具有生物医学应用。然而，使用此类体系制备具有良好定义的聚合材料需要了解酶-底物相互作用。在这种情况下，我们研究了脂肪酶催化苄基丙交酯的 ROP 机制，因为它似乎与广泛描述的其他内酯（如ε-己内酯、丙交酯和丙交酯）的机制不同。在脂肪酶/酰基转移酶 CpLip2 及其丝氨酸敲除（丝氨酸 KO）突变体（CpLip2_180A）存在下，酶催化的 ROP 导致聚（苄基丙交酯）（PMLABe）以单苄基富马酸酯端基终止，单体转化率高于 70%，重均摩尔质量约为 3600 g/mol（= 1.42）。另一方面，当聚合反应在失活的 CpLip2（在 100°C 下加热）存在下进行时，仅观察到不到 7%的 MLABe 转化率和没有聚合物形成。此外，在咪唑存在下，一种催化组氨酸的合成模拟物，导致以单苄基富马酸酯端基终止的 PMLABe。相反，CpLip2 和 CpLip2_180A 存在下，苄基二甲基丙交酯（diMeMLABe）的酶催化 ROP 以及咪唑存在下其化学 ROP 均未成功。总之，所有这些结果表明，脂肪酶催化的丙交酯聚合是通过内酯环上的两个“酸性”质子之一被催化三联体中的组氨酸夺取而发生的，导致相应的单苄基富马酸酯负责聚合剩余的单体。最后，CpLip2 和 DFT 量子化学计算的分子建模突出了单体在 CpLip2 催化位点的定位，表明（）-和（）-MLABe 与酶的催化组氨酸相互作用，优先与丝氨酸形成氢键，其中一个“酸性”质子与 MLABe 结合，从而支持催化组氨酸在聚合此类环状内酯中的重要作用。
