Michael Podeschwa

Stereoselective synthesis of several azido/amino- and diazido/diamino-myo-inositols and their phosphates from p-benzoquinone.

A practical route is described for the preparation of azido-myo-inositols, amino-myo-inositols and azido-conduritol B derivatives. Starting from p-benzoquinone, optically pure compounds in both forms can be prepared via enzymatic resolution of a derived diacetoxy conduritol B derivative. Selective introduction of nitrogen-containing functional groups in four of the six possible positions in the cyclitol moiety is followed by further functionalization to yield the target compounds.

The acarbose-biosynthetic enzyme AcbO from Actinoplanes sp. SE 50/110 is a 2-epi-5-epi-valiolone-7-phosphate 2-epimerase

The C7‐cyclitol 2‐epi‐5‐epi‐valiolone is the first precursor of the cyclitol moiety of the α‐glucosidase inhibitor acarbose in Actinoplanes sp. SE50. The 2‐epi‐5‐epi‐valiolone becomes phosphorylated at C7 by the ATP dependent kinase AcbM prior to the next modifications. Preliminary data gave evidences that the AcbO protein could catalyse the first modification step of 2‐epi‐5‐epi‐valiolone‐7‐phosphate. Therefore, the AcbO protein, the encoding gene of which is also part of the acbKMLNOC operon, was overproduced and purified. Indeed the purified protein catalysed the 2‐epimerisation of 2‐epi‐5‐epi‐valiolone‐7‐phosphate. The chemical structure of the purified reaction product was proven by nuclear magnetic resonance spectroscopy to be 5‐epi‐valiolone‐7‐phosphate.

Stereo- and regiospecificity of yeast phytases–chemical synthesis and enzymatic conversion of the substrate analogues neo- and l-chiro-inositol hexakisphosphate

Phytases are enzymes that catalyze the hydrolysis of phosphate esters in myo-inositol hexakisphosphate (phytic acid). The precise routes of enzymatic dephosphorylation by phytases of the yeast strains Saccharomyces cerevisiae and Pichia rhodanensis have been investigated up to the myo-inositol trisphosphate level, including the absolute configuration of the intermediates. Stereoselective assignment of the myo-inositol pentakisphosphates (D-myo-inositol 1,2,4,5,6-pentakisphosphate and D-myo-inositol 1,2,3,4,5-pentakisphosphate) generated was accomplished by a new method based on enantiospecific enzymatic conversion and HPLC analysis. Via conduritol B or E derivatives the total syntheses of two epimers of myo-inositol hexakisphosphate, neo-inositol hexakisphosphate and L-chiro-inositol hexakisphosphate were performed to examine the specificity of the yeast phytases with these substrate analogues. A comparison of kinetic data and the degradation pathways determined gave the first hints about the molecular recognition of inositol hexakisphosphates by the enzymes. Exploitation of the high stereo- and regiospecificity observed in the dephosphorylation of neo- and L-chiro-inositol hexakisphosphate made it possible to establish enzyme-assisted steps for the synthesis of D-neo-inositol 1,2,5,6-tetrakisphosphate, L-chiro-inositol 1,2,3,5,6-pentakisphosphate and L-chiro-inositol 1,2,3,6-tetrakisphosphate.

Identification of a 1-epi-valienol 7-kinase activity in the producer of acarbose, Actinoplanes sp. SE50/110

In the biosynthesis of the C7‐cyclitol moiety, valienol, of the α‐glucosidase inhibitor acarbose in Actinoplanes sp. SE50/110 various cyclitol phosphates, such as 1‐epi‐valienol‐7‐phosphate, are postulated precursors. In the cell extracts of Actinoplanes SE50/110 we found a new kinase activity which specifically phosphorylates 1‐epi‐valienol; other C7‐cyclitol analogs were only weakly or not phosphorylated. The purified product of the kinase reaction turned out to be 1‐epi‐valienol‐7‐phosphate in analyses by nuclear magnetic resonance spectroscopy. The enzyme seems not to be encoded by an acb gene and, therefore, plays a role in a salvage pathway rather than directly in the de novo biosynthesis of acarbose.