| Part I. The synthesis of two complementary trisaccharides of hyaluronan is described. Construction of the target molecules was achieved by a combination of the phenyl sulfoxide and trichloroacetimidate glycosylation methodologies. This represents the first report of the β-methyl derivatives, which are the smallest fragments incorporating all the structural features of polymeric hyaluronan. Next, the conversion of methyl carbamate to the corresponding free amine is described for a series of 2-amino-2-deoxy-D-glucosamine derivatives. Cleavage of methoxycarbonyl group with methyl trichlorosilane and triethylamine in dry THF at 60°C and subsequent aqueous hydrolysis provides the free amine in 54 to 93% yields. In addition, the selective cleavage of methyl carbamates with methyl trichlorosilane in the presence of Troc or azido groups affords selectively, orthogonal N-protected carbohydrates. Finally, this methodology is applied to the gram-scale synthesis of the β(1,3)- and β(1,4)-linked disaccharides of hyaluronan.;Part II. A manganese binding site has been creating in CcP corresponding to the Mn-binding domain in MnP by site directed mutagenesis. Several spectroscopic techniques including paramagnetic NMR and EPR were employed to characterize the CcP mutant (MnCcP). The data is consistent with a newly formed metal site that binds a single Mn2+ near the heme periphery. Wild-type CcP exhibits none of the spectroscopic features as MnCcP. The new binding site provides a five-fold increase in Mn-oxidizing activity in MnCcP over that of WTCcP. |
