Transketolase Catalyzed Synthesis of N‐Aryl Hydroxamic Acids

Abstract

Hydroxamic acids are metal‐chelating compounds that show important biological activity including anti‐tumor effects. We have recently engineered the transketolase from Geobacillus stearothermopilus (TKgst) to convert benzaldehyde as a non‐natural acceptor substrate. Realizing the structural and electronic similarity to nitrosobenzene, we studied the TK‐catalyzed conversion of nitrosoarenes to yield N‐arylated hydroxamic acids. Here we demonstrate that wild‐type and variants of this versatile TKgst enzyme indeed induce the rapid biocatalytic conversion of variously p‐, m‐ and o‐substituted nitrosoarenes to produce a variety of corresponding N‐aryl hydroxamic acids via creation of a carbon‐nitrogen instead of a carbon‐carbon bond. Further structural modifications can be introduced by varying the donor component, such as hydroxypyruvate or pyruvate.

Hydroxamate Assays for High‐Throughput Screening of Transketolase Libraries Against Arylated Substrates

“…To aid the solubility of the aromatic substrates, DMSO (20%) was chosen as the reaction co‐solvent. Acetone had proven superior for the preparative scale synthesis of HA, [12] but was dismissed here because acetone might etch the surface of polysterene microtiter plates and cause unreliable readings over time.…”

“…The TK gst L382 N/D470S variant (short: N/S) was used, which previously was identified for its apparent best reactivity with benzaldehyde (3) and preparative utility in the synthesis of N-aryl HA. [12] Standard HA have an extraordinary ability to chelate iron (III) ions. In particular, in alkaline media the dissociable HA with a pK a of ~9 favors a stable hexacoordinate 3:1 liganded structure.…”

“…Specifically, a microtiter plate was used that contained a collection of 96 positive hits from the L382X/D470X mutant library that were found active against propanal, [6] and also was previously screened against 3. [7] To test for the anticipated general applicability of the assay, three differently substituted nitrosoarenes with varying constitutional and electronic features were used that had shown good activity in preparative syntheses, [12] Some preliminary tests were run to determine the minimum lysate quantities for reaching sufficient conversion toward HA formation at short incubation times. For 1 c, the rates of different variants were well discernable with normal lysates, while for 1 b and 1 d, the lysates were diluted 1:1 prior to use.…”

“…HA products readily form metal chelate complexes, which can be used for analytical purposes in case of the deep red color of the iron(III) complex. [12] A critical challenge for projects in directed evolution is the need for a robust, specific, sensitive, and efficient assay method for the high-throughput screening of large libraries of mutants to identify new catalyst variants with novel or improved properties. [13] Several methods for measuring TK activity have been hitherto proposed.…”

Hot off the Press

“…Using the structural similarity of nitrosobenzene for benzaldehyde, a transketolase from Geobacillus stearothermopilus (TK gst ) has been used as a biocatalyst for the synthesis of N -aryl hydroxamic acids. 31 A range of substituted nitrosoarenes were found to undergo the N -acyl condensation process with hydroxypyruvate, allowing rapid access to various N -aryl hydroxamic acids (Scheme 5). A two-step bio-organocatalytic cascade process has been developed for the preparation of 2-substituted piperidines (Scheme 6).…”