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 βhydroxy-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-αamino acids and substituted α-keto acids.

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 convert readily accessible aryl epoxides into α-aryl aldehydes through 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 a gram scale. We used isotopically labeled substrates to probe the mechanism of SOI, which we show to catalyze 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, wholecell conditions and show that these intermediates are useful synthons in C−C bond forming cascades.

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.

Compound 22 was incorrectly drawn as the isomer being derived from 1-napthaldehyde, when it was instead derived from 2-napthaldehyde. Compound 15 should also be drawn as a carboxylate, and not a carboxylic acid. A corrected Figure 2 is provided. A correction to the corresponding text has also been made on Page 4, paragraph 2: "Instead, to further probe the limits of the ObiH active site, we sought to challenge the enzyme with sterically bulky aromatic aldehydes to generate napth-1-ylserine ( 22) and biphenylserine (23) (Figure 2)." Should state: "Instead, to further probe the limits of the ObiH active site, we sought to challenge the enzyme with sterically bulky aromatic aldehydes to generate napth-2-ylserine (22) and biphenylserine (23) (Figure 2).