Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C?H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C?H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

A Chemoenzymatic Approach to the Synthesis of Glycopeptide Antibiotic Analogues

Glycopeptide antibiotics (GPAs) are important antibiotics that are highly challenging to synthesise due to their unique and heavily crosslinked structure. Given this, the synthetic production and diversification of this key compound class remains impractical. Furthermore, the possibility of biosynthetic reengineering of GPAs is not yet feasible since the selectivity of the biosynthetic crosslinking enzymes for altered substrates is largely unknown. We show that combining peptide synthesis with enzymatic cyclisation enables the formation of novel examples of GPAs and provides an indication of the utility of these crucial enzymes. By accessing the biosynthetic process in vitro, we identified peptide modifications that are enzymatically tolerated and can also reveal the mechanistic basis for substrate intolerance where present. Using this approach, we next specifically activated modified residues within GPAs for functionalisation at previously inaccessible positions, thereby offering the possibility of late‐stage chemical functionalisation after GPA cyclisation is complete.     

细胞色素P450酶催化的烷烃选择性羟化

碳氢键选择氧化是合成化学领域的重要课题,其中烷烃选择性羟化反应更是面临着化学选择性、区域选择性和立体选择性等多重挑战.细胞色素P450酶广泛分布于动植物和微生物体内,是公认的多功能生物氧化催化剂. P450酶对惰性C—H键的选择性氧化具有独特优势,在催化烷烃选择性羟化反应方面拥有巨大潜力.本综述简述了P450单加氧酶及其催化烷烃选择性羟化的反应机理,梳理了来自CYP153家族、CYP52家族和其他家族的天然P450酶催化各类烷烃底物的氧化反应和选择性,讨论了理性设计和定向进化策略在开发烷烃羟化P450突变酶过程中的经典案例,介绍了底物工程、诱饵分子、双功能小分子协同催化等几种化学活化P450酶的策略及其在烷烃羟化上的应用,探讨了P450酶在烷烃选择性羟化方面所面临的挑战和解决途径,并展望了其应用前景.     

A Simple Biosystem for the High-Yielding Cascade Conversion of Racemic Alcohols to Enantiopure Amines

The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing, requiring simple and efficient solutions. Herein, we report the development of a cascade biotransformation to aminate racemic alcohols. This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize a racemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine, and isopropylamine to recycle PMP and NAD⁺ cofactors via the reversed cascade reactions. The concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs, an enantioselective BmTA, and isopropylamine. A biosystem containing isopropylamine and E. coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72–99 % yield and 98–99 % ee, providing with a simple and practical solution to this type of reaction.     

Enzymatic Desymmetrising Redox Reactions for the Asymmetric Synthesis of Biaryl Atropisomers

Atropisomeric biaryls carrying ortho‐hydroxymethyl and formyl groups were made enantioselectively by desymmetrisation of dialdehyde or diol substrates. The oxidation of the symmetrical diol substrates was achieved using a variant of galactose oxidase (GOase), and the reduction of the dialdehydes using a panel of ketoreductases. Either M or P enantiomers of the products could be formed, with absolute configurations assigned by time‐dependent DFT calculations of circular dichroism spectra. The differing selectivities observed with different biaryl structures offer an insight into the detailed structure of the active site of the GOase enzyme.     

A Promiscuous De Novo Retro‐Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5‐8, designed and evolved as a retro‐aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X‐ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.     

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

The self‐sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in‐chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω‐7 position. It is the first reported P450 to give regioselective in‐chain ω‐7 hydroxylation of C10–C16 n‐alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5‐dodecanol and 7‐tetradecanol. It shows more than 70 % regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high‐value flavour compound δ‐dodecalactone via two routes: 1) conversion of dodecanoic acid into 5‐hydroxydodecanoic acid (24 % regioselectivity), which at low pH lactonises to δ‐dodecalactone, and 2) conversion of 1‐dodecanol into 1,5‐dodecanediol (55 % regioselectivity), which can be converted into δ‐dodecalactone by horse liver alcohol dehydrogenase.     

Cortistatin类型天然产物的不对称形式全合成:金催化串联Semi-Pinacol重排反应策略

本工作详细报道了Cortistatin类型天然产物不对称形式全合成的研究路线.以近期作者发展的金催化串联semi-pinacol重排反应构建[3,2,1]七元氧桥环结构的方法学为基础,进一步将其应用于复杂体系,高效构建了Cortistatin类型天然产物独特的七元氧桥环核心骨架,从而完成了该类型天然产物的不对称形式全合成.