Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima.
Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima.
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DOI：
10.1021/acs.joc.8b00517
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发表时间：
2018-07-20
期刊：
影响因子：
--
通讯作者：
Arnold FH
中科院分区：
文献类型：
--
作者：
Boville CE;Romney DK;Almhjell PJ;Sieben M;Arnold FH
随着化学家们努力减少对有机溶剂的依赖，转而使用更环保的水性介质，酶在合成中的应用越来越广泛。考虑到这一点，我们先前致力于工程改造色氨酸合成酶β-亚基（TrpB），用于生产非经典氨基酸，这些氨基酸先前是通过涉及水敏感性试剂的多步路线合成的。这种酶平台被证明对合成氨基酸色氨酸（Trp）的类似物是有效的，这些类似物经常用于药物合成以及化学生物学。然而，某些有价值的化合物，如蓝色荧光氨基酸4-氰基色氨酸（4-CN-Trp），即使在高温（75°C）下也只能以低产率制备。在这里，我们描述了来自海栖热袍菌的TrpB的工程改造，其将4-CN-Trp的合成从24%提高到78%产率。值得注意的是，尽管最终的酶保持了高的热稳定性（T50 = 93°C），但其温度曲线发生了变化，使得在约37°C下观察到高反应性（76%产率），从而为体内4-CN-Trp生产创造了可能性。这些改善并不特定于4-CN-Trp;其他Trp类似物在较低温度下的活性增强也得到了证实。
The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogs of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75°C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability (T50 = 93°C), its temperature profile is shifted, such that high reactivity is observed at ~37°C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogs.
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