A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

Abstract

The chemoenzymatic route to 2-deoxy-2-propionamido- d-mannose ( 1b), 2-butyramido-2-deoxy- d-mannose ( 2b) and 2-deoxy-2-phenylacetamido- d-mannose ( 3b) involved N-acylation of 2-amino-2-deoxy- d-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido- d-glucose ( 1a), 2-butyramido-2-deoxy- d-glucose ( 2a) and 2-deoxy-2-phenylacetamido- d-glucose ( 3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6- O-phospho- d-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy- d-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 °C and 8.0–9.0, respectively. 2-Acetamido-2-deoxy-6- O-phospho- d-glucose and 2-acetamido-2-deoxy-6- O-sulfo- d-glucose but not 2-acetamido-2-deoxy-1- O-phospho- d-glucose or 2-acetamido-2-deoxy- d-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al 3+, Ca 2+, Co 2+, Cu 2+, Mn 2+ or Zn 2+ and activated by 1 mM Mg 2+. The concentrated enzyme is highly stable at 4 °C in the presence of 0.1 M ammonium sulfate.