Abstract Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non‐complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2‐amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on β‐OH amino acids while avoiding activity on l‐threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2‐amino alcohols in a one‐pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C−C bond forming cascades.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/anie.202212637

Engineering Enzyme Substrate Scope Complementarity for Promiscuous Cascade Synthesis of 1,2‐Amino Alcohols

Multi-enzyme biocatalytic cascades are emerging as practical routes for the synthesis of complex bioactive molecules. However, the relative sparsity of water-stable carbon electrophiles limits the synthetic complexity of molecules made from such cascades. Here, we develop a chemoenzymatic platform that leverages styrene oxide isomerase (SOI) to covert readily accessible aryl epoxides into α-aryl aldehydes through a Meinwald rearrangement. These unstable aldehyde intermediates are then intercepted with a C-C bond forming enzyme, ObiH, that catalyzes a transaldolase reaction with l-threonine to yield synthetically challenging β-hydroxy-α-amino acids. Co-expression of both enzymes in E. coli yields a whole cell biocatalyst capable of synthesizing a variety of stereopure non-standard amino acids (nsAA) and can be produced on gram-scale. We used isotopically labelled substrates to probe the mechanism of SOI, which we show catalyzes a concerted isomerization featuring a stereospecific 1,2-hydride shift. The viability of in situ generated α-aryl aldehydes was further established by intercepting them with a recently engineered decarboxylative aldolase to yield γ-hydroxy nsAAs. Together, these data establish a versatile method of producing α-aryl aldehydes in simple, whole cell conditions and show that these intermediates are useful synthons in C‒C bond forming cascades.

Efficient chemoenzymatic synthesis of α-aryl aldehydes as intermediates in C-C bond forming biocatalytic cascades.

Condensed phosphates are a critically important class of molecules in biochemistry, with a myriad of derived structures being known. Moreover, non-natural analogues are important for various applications, such as single molecule real time DNA sequencing. Often, such analogues contain more than three phosphate units in their oligophosphate chain. Consequently, investigations into phosphate reactivity enabling new ways of phosphate functionalization and oligophosphorylation are an essential endeavour in the field. Here, we scrutinize the potential of phosphates to act as arynophiles, paving the way for follow-up oligophosphorylation reactions. The aryne phosphate reaction is a powerful tool to - depending on the perspective - (oligo)phosphorylate arenes or arylate (oligo-cyclo)phosphates. Based on Kobayashi-type o-silylaryltriflates, the aryne phosphate reaction enables rapid entry into a broad spectrum of arylated products, like monophosphates, diphosphates, phosphodiesters and polyphosphates. The synthetic potential of these new transformations is demonstrated by efficient syntheses of nucleotide analogues and an unprecedented one-flask octaphosphorylation.

The Aryne Phosphate Reaction

Pyridoxal‐5’‐phosphate (PLP)‐dependent enzymes have garnered interest for their ability to synthesize non‐standard amino acids (nsAAs). One such class of enzymes, O‐acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l‐cysteine. Here, we examine the β‐substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l‐mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non‐native reactivity with a variety of O‐nucleophiles. Optimization of reaction conditions for carboxylate and phenolate substrates led to distinct conditions that were leveraged for the preparative‐scale synthesis of nsAAs. We further show this enzyme is capable of C−C bond formation through a β‐alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.

Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

Practical Synthesis of Tenofovir Alafenamide Fumarate Inspired by New Retrosynthetic Disconnection Featuring a Novel Carbon–Phosphorus Bond Construction Methodology

Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally-simple and well-characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β-hydroxy-α-amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an l-threonine transaldolase, to achieve selective milligram-scale synthesis of a diverse array of non-standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re-entry into the catalytic cycle. ObiH-catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β-hydroxy-α-amino acids possessing broad functional-group diversity. Furthermore, we demonstrated that ObiH-generated β-hydroxy-α-amino acids could be modified through additional transformations to access important motifs, such as β-chloro-α-amino acids and substituted α-keto acids.

Scalable and selective β-hydroxy-α-amino acid synthesis catalyzed by promiscuous L-threonine transaldolase ObiH

Synthetic methodology utilizing two aryne intermediates (i.e., a formal benzdiyne) enables the rapid generation of structurally complex molecules with diverse functionality. This report describes the sequential generation of two ortho-benzyne intermediates for the synthesis of 2,3-disubstituted aryl phosphonates. Aryl phosphonates have proven useful in medicinal chemistry and materials science, and the reported methodology provides a two-step route to functionally dense variants by way of 3-phosphonyl benzyne intermediates. The process begins with regioselective trapping of a 3-trifloxybenzyne intermediate by an O-silyl phosphite in an Abramov-like reaction to bond the strained Csp carbons with phosphorus and silicon. Standard aryne-generating conditions follow to convert the resulting 2-silylphenyl triflate into a 3-phosphonyl benzyne, which readily reacts with numerous aryne trapping reactants to form a variety of 2,3-difunctionalized aryl phosphonate products. DFT computational studies shed light on important mechanistic details and revealed that 3-phosphonyl benzynes are highly polarizable. Specifically, the distortion in the internal bond angles at each of the Csp atoms was strongly influenced by both the electronegativity of the phosphonate ester groups as well as the dielectric of the computational solvation model. These effects were verified experimentally as the regioselectivity of benzyl azide trapping increased with more electronegative esters and/or increasingly polar solvents. Conversely, replacing the conventional solvent, acetonitrile, with nonpolar alternatives provided attenuated or even inverted selectivities. Overall, these studies showcase new reactivity of benzyne intermediates and extend the aryne relay methodology to include organophosphonates. Furthermore, this work demonstrates that the regioselectivity of aryne trapping reactions could be tuned by simply changing the solvent.

Sierra A. Thein

Papers

Scalable and selective β-hydroxy-α-amino acid synthesis catalyzed by promiscuous L-threonine transaldolase ObiH

The Aryne-Abramov Reaction as a 1,2-Benzdiyne Platform for the Generation and Solvent-Dependent Trapping of 3-Phosphonyl Benzynes.