Efficient Chemoenzymatic Synthesis of α-Aryl Aldehydes as Intermediates in C–C Bond Forming Biocatalytic Cascades

Abstract

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.

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

“…The SOI‐ObiH cascade was previously characterized to produce 21 c with a 65 : 35 d.r. with an anti‐ relationship between the γ‐Me and β‐OH groups [31] . Serendipitously, Rgn TDC NMY was highly selective for the minor syn diastereomer, exclusively producing the corresponding syn 1,2‐amino alcohol (Figure S13).…”

Engineering enzyme substrate scope complementarity for promiscuous cascade synthesis

“…with an anti-relationship between the γ-Me and β-OH groups. 31 Serendipitously, RgnTDC NMY was highly selective for the minor syn diastereomer, exclusively producing the corresponding syn 1,2-amino alcohol (Fig S13). Although the activity with some of these substrates is low, further improvements could be obtained through additional rounds of directed evolution.…”

“…Therefore, we introduced a third enzyme, styrene oxide isomerase (SOI), to produce these compounds in situ from the corresponding epoxides. We recently reported the cascade synthesis of β-OH amino acids using SOI and ObiH, 31 which drew on the strong work of Li et al 32,33 With just 0.1% w/v whole cells bearing SOI, the α-arylaldehydes successfully entered the ObiH-RgnTDC NMY cascade on analytical scale (Fig 4b). We found that RgnTDC NMY has relatively low activity on these challenging substrates; nevertheless, the three-enzyme cascade furnished diverse benzylic 1,2-amino alcohols with excellent e.r.…”

“…Recent work by our lab and others has characterized the activity and specificity of a promiscuous and highly stereoselective LTTA, ObiH. [28][29][30][31] ObiH (also known as ObaG) catalyzes the formation of β-OH amino acids from diverse aryl, α-aryl, and aliphatic aldehydes (Fig 2a). However, no amino acid decarboxylase has been shown to have a scope that encompasses the wide array of amino acids produced via ObiH.…”

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

“…We sought to develop a robust and highly generalizable C−C bond forming biocatalytic cascade to form 1,2‐amino alcohols from achiral aldehydes using an l ‐threonine transaldolase (LTTA) and an amino acid decarboxylase. Recent work by our lab and others has characterized the activity and specificity of a promiscuous and highly stereoselective LTTA, ObiH [28–31] . ObiH (also known as ObaG) catalyzes the formation of β‐OH amino acids from diverse aryl, α‐aryl, and aliphatic aldehydes (Figure 2a).…”