Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Darmstadt, Germany.
Chemistry. 2011 Feb 25;17(9):2623-32. doi: 10.1002/chem.201002942. Epub 2011 Feb 2.
The majority of prokaryotic drugs are produced in glycosylated form, with the deoxygenation level in the sugar moiety having a profound influence on the drug's bioprofile. Chemical deoxygenation is challenging due to the need for tedious protective group manipulations. For a direct biocatalytic de novo generation of deoxysugars by carboligation, with regiocontrol over deoxygenation sites determined by the choice of enzyme and aldol components, we have investigated the substrate scope of the F178Y mutant of transaldolase B, TalB(F178Y), and fructose 6-phosphate aldolase, FSA, from E. coli against a panel of variously deoxygenated aldehydes and ketones as aldol acceptors and donors, respectively. Independent of substrate structure, both enzymes catalyze a stereospecific carboligation resulting in the D-threo configuration. In combination, these enzymes have allowed the preparation of a total of 22 out of 24 deoxygenated ketose-type products, many of which are inaccessible by available enzymes, from a [3×8] substrate matrix. Although aliphatic and hydroxylated aliphatic aldehydes were good substrates, D-lactaldehyde was found to be an inhibitor possibly as a consequence of inactive substrate binding to the catalytic Lys residue. A 1-hydroxy-2-alkanone moiety was identified as a common requirement for the donor substrate, whereas propanone and butanone were inactive. For reactions involving dihydroxypropanone, TalB(F178Y) proved to be the superior catalyst, whereas for reactions involving 1-hydroxybutanone, FSA is the only choice; for conversions using hydroxypropanone, both TalB(F178Y) and FSA are suitable. Structure-guided mutagenesis of Ser176 to Ala in the distant binding pocket of TalB(F178Y), in analogy with the FSA active site, further improved the acceptance of hydroxypropanone. Together, these catalysts are valuable new entries to an expanding toolbox of biocatalytic carboligation and complement each other well in their addressable constitutional space for the stereospecific preparation of deoxysugars.
大多数原核药物以糖基化形式产生，糖部分的脱氧水平对药物的生物特性有深远影响。由于需要繁琐的保护基团操作，化学脱氧具有挑战性。为了通过卡罗利加作用直接生物催化从头生成去氧糖，通过选择酶和醛缩酶组分来实现脱氧位置的区域控制，我们研究了大肠杆菌来源的转醛醇酶 B 的 F178Y 突变体 TalB(F178Y)和果糖 6-磷酸醛缩酶 FSA 的底物范围，分别作为醛缩接受体和供体，针对一系列不同脱氧的醛和酮。与底物结构无关，两种酶均催化立体特异性卡罗利加作用，导致 D-赤型构型。组合使用时，这些酶总共可以从[3×8]底物矩阵中制备 24 种去氧酮型产物中的 22 种，其中许多产物是可用酶无法获得的。尽管脂肪族和羟基脂肪族醛是很好的底物，但 D-乳醛被发现是一种抑制剂，可能是由于无活性的底物与催化 Lys 残基结合。鉴定出 1-羟基-2-烷酮部分是供体底物的共同要求，而丙酮和丁酮则无活性。对于涉及二羟丙酮的反应，TalB(F178Y)被证明是优越的催化剂，而对于涉及 1-羟基丁酮的反应，FSA 是唯一的选择；对于使用羟基丙酮的转化，TalB(F178Y)和 FSA 都适用。在 TalB(F178Y)的远距离结合口袋中的 Ser176 进行结构指导的突变，类似于 FSA 活性位点，进一步提高了对羟基丙酮的接受能力。总之，这些催化剂是卡罗利加生物催化不断扩大的工具包中的有价值的新成员，并且在其可寻址的结构空间中互补，用于立体特异性制备去氧糖。
