Cutting Long Syntheses Short: Access to Non‐Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol Lyase

Abstract

Abstract:The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this C À C bond forming enzyme.The best variant M379V transformed, for example, o-cresol, o-methoxyphenol and o-chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various ltyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (> 97%) in just a single green step starting from pyruvate and commercially available phenol derivatives.

Targeted Rediscovery and Biosynthesis of the Farnesyl‐Transferase Inhibitor Pepticinnamin E

“…To identify the position of methylation, the methylated l ‐DOPA product from a 10 mL overnight Pcm10 reaction was purified and subjected to 1 H NMR analysis (Figure S3). The 1 H shifts are consistent with those reported for 4‐methyl‐ l ‐DOPA, but differ from those of 3‐methyl‐ l ‐DOPA . This result indicated that Pcm10 methylates l ‐DOPA at the 4‐hydroxy position, consistent with the structure of pepticinnamin E. No methyltransfer activity was observed when Pcm10 was incubated with l ‐tyrosine, thus suggesting that hydroxylation of l ‐tyrosine occurs prior to 4‐methylation.…”

Efficient production of phenylpropionic acids by an amino‐group‐transformation biocatalytic cascade

“…To efficiently introduce amino groups to convert phenols, pyruvate, and ammonia into ( S )‐α‐amino acids (Figure b), we chose the variant M379V of the recombinant TPL from Citrobacter freundii ( Cf TPL M379V ) because of its broad substrate scope (Seisser et al, ). The gene of Cf TPL M379V was cloned in plasmid pET28a and expressed in E. coli BL21, designated as E. coli 01 (0.086 U/mg cells, and 0.039 mg TPL proteins/mg cells; Table ).…”

“…They are of great importance in the fields of fine chemicals synthesis, cosmetics, and pharmaceutical manufacturing (Busto et al, ; Busto, Simon, Richter, & Kroutil, ; Hou et al, ; Lütke‐Eversloh, Santos, & Stephanopoulos, ; Xue, Cao, & Zheng, ). For example, tyrosine derivatives play prominent roles in the synthesis of pharmaceuticals (Dennig, Busto, Kroutil, & Faber, ; Seisser et al, ). Among them, ʟ‐tyrosine is used as a dietary supplement and an important precursor of thyroxine, adrenaline, and 3,4‐dihydroxy‐ʟ‐phenylalanine (ʟ‐DOPA).…”

“…. For example, tyrosine derivatives play prominent roles in the synthesis of pharmaceuticals (Dennig, Busto, Kroutil, & Faber, 2015;Seisser et al, 2010). Among them, ʟ-tyrosine is used as a dietary supplement and an important precursor of thyroxine, adrenaline, and 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA).…”

Biocatalytic Methods for CC Bond Formation

“…The enzyme was applied in the reverse direction for the synthesis of tyrosine derivatives 144 . The variant M379V was the most active mutein for the preparation of 3′‐substituted tyrosines derived from ortho ‐substituted phenols (Scheme ) 216…”