ATP Regeneration System in Chemoenzymatic Amide Bond Formation with Thermophilic CoA Ligase

Abstract

CoA ligases are enzymes catalyzing the ATP‐dependent addition of coenzyme A to carboxylic acids in two steps through an adenylate intermediate. This intermediate can be diverted by a nucleophilic non enzymatic addition of amine to get the corresponding amide for synthetic purposes. To this end, we selected thermophilic CoA ligases to study the conversion of various carboxylic acids into their amide counterparts. To limit the use of ATP, we implemented an ATP regeneration system combining polyphosphate kinase 2 (PPK2 Class III) and inorganic pyrophosphatase. Suitability of this system was illustrated by the lab‐scale chemoenzymatic synthesis of N‐methylbutyrylamide in 77 % yield using low enzyme loading and 5 % molar ATP.

Carboxylic Acid Reductase Can Catalyze Ester Synthesis in Aqueous Environments

“…[15] However, CAR has the disadvantage of using costly ATP as a co-substrate which requires further process optimization in the future, possibly using an ATP regenerating system to convert AMP back to ATP by a low cost polyphosphate. [23] In comparison to the production of amides previously reported, [13] without adjustment of reaction buffer to imidazole, esterification behaves poorly (less than 1 % conversion of methyl benzoate compared to 11 % conversion of benzamide). However, with imidazole enhancement, methyl cinnamate was produced up to 41 % in 24 hours (Table 1) compared to 22 % conversion of cinnamamide at 24 hours.…”

Carboxylic Acid Reductase Can Catalyze Ester Synthesis in Aqueous Environments

“…A plausible explanation could be that CAR does not contain a catalytic triad like lipase or other transferase enzymes, where the amino acids of the catalytic triad can also promote the nucleophilic attack of water [15] . However, CAR has the disadvantage of using costly ATP as a co‐substrate which requires further process optimization in the future, possibly using an ATP regenerating system to convert AMP back to ATP by a low cost polyphosphate [23] …”

Recent developments in catalytic amide bond formation

“…At the moment however, such methods lack broad scope (as might be expected of enzymatic methods), often not tolerating even secondary amines [32,33] and require activated AMP-esters which hydrolyze under the reaction conditions [34] and can require large excesses to achieve desirable yields. While recent reports have sought to address both issues, [35][36][37] such technologies can be presently of interest as complements to SPPS in the form of alternative strategies to native chemical ligation or cyclization. [38][39][40] Another alternative approach to strictly catalytic methods is the recycling of reagents and solvents.…”