Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

Abstract

A set of phosphatases was evaluated for their potential to catalyze the regio‐ and stereoselective phosphorylation of alcohols using a high‐energy inorganic phosphate donor, such as di‐, tri‐ and polyphosphate. Parameters such as type and amount of phosphate donor and pH of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester. Diols were monophosphorylated with high selectivities. This biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale (6.86 g) in a flow setup with immobilized phosphatase.

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters

“…All enzymes were tested with a broad range of substrates (Fig. ) including variously substituted primary ( 1a–11a ) as well as secondary alcohols ( 12a–16a ) in the transphosphorylation mode under standard reaction conditions (Tasnádi et al, ). Substrates bearing a sec ‐OH moiety ( 12a–16a ) were not accepted by PhoC‐Mm G92D/I171T and NSAP‐Eb 11‐mutants.…”

“…Disodium α‐glycerophosphate hydrate ( 3b ) was purchased from TCI. 4‐Hydroxybutyl phosphate barium salt ( 1b ) and 2‐(acryloyloxy)ethyl phosphate barium salt ( 2b ) were prepared according to a literature procedure (Tasnádi et al, ,), racemic 2‐hydroxypropyl phosphate ( 6b ) was synthetized as reported (Lampson and Lardy, ). Ni‐NTA column for His‐tag purification was from GE Healthcare Life Sciences, Strep ‐Tactin Superflow High Capacity column for Strep‐tag purification was from IBA Solutions.…”

“…Despite the modest efficiency of ATP‐recycling, several preparative‐scale processes have been developed using hexokinase and glycerol kinase, although only sugar‐like molecules and derivatives of glycerol were well accepted (Chenault et al, ; Crans and Whitesides, ,; Drueckhammer and Wong, ). A modern and simpler alternative to kinases employs phosphatases in the synthesis (transphosphorylation) direction by using cheap inorganic phosphate donors, such as pyrophosphate (PP i ), triphosphate (PPP i ) and polyphosphoric acid (polyP) (Asano et al, ; Médici et al, ; Pradines et al, ; Tasnádi et al, ,; van Herk et al, ). The broad substrate‐scope and the simplified (cofactor‐independent) protocol make phosphatases suitable candidates for large‐scale production (Scheme ).…”

“…The broad substrate‐scope and the simplified (cofactor‐independent) protocol make phosphatases suitable candidates for large‐scale production (Scheme ). To overcome undesired product hydrolysis, several techniques were recently introduced, such as continuous flow production (Babich et al, ,), reaction engineering (Tasnádi et al, ,) and protein engineering (Suzuki et al, ). During the early 2000's, the group of Asano developed a robust catalyst for the large‐scale production of inosine 5′‐monophosphate (5′‐IMP), a known umami taste enhancer (Behrens et al, ).…”

“…In line with our recent interest in phosphorylation of diverse alcohols and diols, (Tasnádi et al, ,) we decided to investigate variants of PhoC‐Mm and NSAP‐Eb with substrates other than nucleosides and develop a generally applicable and robust biocatalyst for the production of phosphoric acid monoesters. Single and double mutants of PhoC‐Mm (G92D and G92D‐I171T) were able to produce and maintain high product levels with a range of primary alcohols, allowing easy scale‐up.…”