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Nat. Catal | 人工酶中两个非生物催化位点同时作用的协同催化反应

发布: 2022-07-22 1,101阅读 208评论

本周为大家介绍一篇2020年发表在Nature Catalysis的文章：人工酶中两个非生物催化位点同时作用的协同催化反应。人工酶是蛋白质和非生物催化基团的杂交，其开发面临的最大挑战是如何设计催化活性位点。作者介绍了一种人工酶的设计，它由两个不同的非生物催化部分组成，并可以协同作用，获得高活性和对映体选择性(高达99%e.e.)。

1.1基于LmrR蛋白设计的人工酶

该人工酶的设计基于乳球菌多药耐药调节因子（LmrR）作为蛋白支架。此蛋白是一个小型的同形二聚体，在二聚体界面上有一个异常大的疏水性空腔，空腔中心有两个相互平行色氨酸残基可以通过π堆积作用去结合外来平面分子（图1a）。

该课题组之前报道过运用LmrR蛋白结合金属辅助因子如菲咯啉铜Cu(1,10-phenanthroline)(NO3)2来构建人工金属酶，实现了吲哚和烯酮的不对称加成反应。其中，菲咯啉铜作为路易斯酸来活化烯酮（图1b）。

同时，作者以扩展遗传密码方法引入对氨基苯丙氨酸(pAF)。在这里，pAF的苯胺侧链被用作亲核催化剂(图1c)。

结合以上想法，作者将这两种人工酶设计策略结合在一个蛋白质分子中，以创建具有两个可以同时作用的非天然催化位点的人工酶。在本研究中，作者使用了突变体 LmrR_V15pAF，它在第15位含有pAF残基。其中，pAF是间接引入的：通过使用pEVOL-pAzF质粒引入对叠氮苯丙氨酸（pAzF），然后在纯化过程中用TCEP将叠氮还原为胺。再通过向LmrR_V15pAF蛋白缓冲液中加入Cu(ll)-phen，经过超分子自组装结合得到路易斯酸位点。

在作者的设计中，α,β-不饱和醛可以通过与苯胺侧链形成亚胺离子中间体而被活化，而烯醇盐则通过路易斯酸Cu(ll)-phen络合物活化酮而形成，从而通过协同催化得到迈克尔加成产物（图1d）。亲核试剂结合的铜络合物将结合在两个平行的色氨酸残基之间，可以将烯醇盐递送至活化的烯酮的一个优选的前手性面，从而可以实现高的立体选择性。

1.2 新型人工酶LmrR_V15pAF/Cu(ll)-phen协同催化的Michael加成反应

         为了检测这种新型人工酶的催化性能，研究者选用了一种反应活性非常低的咪唑酮（1a）作为迈克尔供体，其可以在路易斯酸的作用下烯醇化。然后加成到丙烯醛（2a）上得到迈克尔加成产物（3a）。结果显示当只有一个催化位点时，反应的不能进行（Table 1,entry 1-2）。当LmrR_V15pAF与Cu(ll)-phen的协同组合时，该反应能够得到36%的收率和 86%的e.e.值（Table 1,entry 5）。同时LmrR_V15pAF与Cu(NO3)2的结合仅10%的e.e.值，这也说明铜配合物与两个中心色氨酸残基的结合对于反应的对映选择性十分重要（Table 1,entry 4）。

1.3 人工酶的反应条件优化及诱变研究

         接着，作者用巴豆醛（2b）代替丙烯醛作为迈克尔受体进行了该反应，得到含两个手性中心的产物（3b），具有优良的对映选择性。结果显示将中心色氨酸残基突变为丙氨酸时，得到了较低的产率和e.e.，证实精确定位Cu(ii)结合烯醛对于激活烯醛的重要性（Table 2,entry 9）。同时作者评估了在M8位置突变对该反应的效果，筛选得到的突变体LmrR_V15pAF_M8L能够同时提升反应收率和立体选择性（Table 2, entry 14）。另外作者发现催化剂用量降至0.5 mol%也不会影响反应的立体选择性（Table 2, entries 6–8, 16 and 17)。

1.4 底物适用性范围研究

此人工酶能够兼容一系列的不饱和醛和咪唑酮底物。除了芳香环取代的不饱和醛会导致反应对映选择性的降低外（Table 3, entries 3-4），其他烷基取代的不饱和醛和各种取代的咪唑酮底物均能得到非常优异的立体选择性。并且相比于LmrR_V15pAF，突变体LmrR_V15pAF_M8L能够得到相似或者更好的收率和对映选择性(3b, 3c, 3e, 3f, 3g 和 3h)。

总结：作者提出了一种人工酶的设计，它是由遗传编码的非天然催化基团pAF残基与催化活性Cu(ii)复合物的协同组合产生的，该复合物是通过在LmrR的二聚体界面的疏水腔中的超分子结合而引入的。该设计的两个关键要素是LmrR疏水腔的混杂性和两个整合的非生物催化位点的物理分离和明智定位，避免了与单个催化组分不相容相关的问题，同时允许亲核试剂有效和选择性地接近活化的亲电试剂。这种方法的有效性在水中的不对称迈克尔加成反应中得到了证明，给出了优异的立体选择性和反应性。最后，这项研究显示了两种非生物催化基团的协同组合是应用到包含单一非生物催化位点的人工酶催化领域的。

 

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