- Synthesis of Nω-Phospho-l-arginine by Biocatalytic Phosphorylation of l-Arginine
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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.
- Schoenenberger, Bernhard,Wszolek, Agata,Milesi, Thomas,Brundiek, Henrike,Obkircher, Markus,Wohlgemuth, Roland
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- Thiophosphorylation of free amino acids and enzyme protein by thiophosphoramidate ions
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In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of 31P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of 31P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His26) or lysine (Lys33) side-chain.
- Ruman, Tomasz,Dlugopolska, Karolina,Jurkiewicz, Agata,Rut, Dagmara,Fraczyk, Tomasz,Ciesla, Joanna,Les, Andrzej,Szewczuk, Zbigniew,Rode, Wojciech
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experimental part
p. 74 - 80
(2010/05/17)
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