Synthesis of N_ω‐Phospho‐l‐arginine by Biocatalytic Phosphorylation of l‐Arginine

Abstract

The Nω‐phospho‐l‐arginine energy‐buffering system is mainly present in invertebrates for regulating energy requirements when it is highly needed, such as in the flight muscles of an insect or when energy supply fluctuates, as in the medically important protozoa Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. The lack of availability of this important metabolite was due to a tedious chemical procedure, by which Nω‐phospho‐l‐arginine was prepared in over 5 reaction steps in a low yield. Therefore, we aimed at improving the synthetic methodology for the preparation of this important metabolite. As site‐ and enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites, a stable and selective arginine kinase has been selected for the selective phosphorylation of l‐arginine. The Arg gene has been cloned and expressed in E. coli and a highly active arginine kinase has been prepared. A simple synthesis of Nω‐phospho‐l‐arginine has been developed by arginine kinase catalyzed phosphorylation of l‐arginine combined with the recycling of the phosphorylating agent ATP by using the phosphoenolpyruvate/pyruvate kinase system. After standard work‐up, the desired product Nω‐phospho‐l‐arginine was obtained in gram quantities and in one step.

Route selection and reaction engineering for sustainable metabolite synthesis

“…4) from suitably selected and easily available starting compounds using isolated recombinant enzymes. [74][75][76][77][78][79][80] For diverse metabolites with very long and complex biosynthetic pathways, the reduction of complexity by…”

“…4) from suitably selected and easily available starting compounds using isolated recombinant enzymes. 74–80…”

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

“…Suitable methods with the necessary high information content, such as MS and NMR, for the analysis of the biocatalytic transformations and products are therefore a key success factor, both for the development and in-process control in the production phase (Jani et al, 2008;Gauss et al, 2014;Matsubara et al, 2014;Schoenenberger et al, 2017b).…”

Biocatalysis in the Swiss Manufacturing Environment

“…The exploration of the biocatalytic space has been very useful for the synthesis of metabolites by a systems biocatalysis approach [41]. Selective enzyme-catalyzed Oand N-phosphorylations have been demonstrated as versatile platform technologies and provide various advantages over classical chemical phosphorylations as demonstrated by Sigma-Aldrich/Merck KGaA for the manufacturing of a large variety of phosphorylated metabolites [42][43][44][45][46][47][48][49]. Selective enzymatic water elimination reactions exhibiting full conversion enabled a simple synthetic access to 2-keto-3-deoxysugar acids from easily accessible sugar acids using dehydratases [50][51][52].…”