Sequencing Microbial Copolymers of 3-Hydroxybutyric and 3-Mercaptoalkanoic Acids by NMR, Electrospray Ionization Mass Spectrometry, and Size Exclusion Chromatography NMR

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amts. of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatog. NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry anal. of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. 13C NMR spectroscopy of intact polymer samples, with values of av. block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, compn. anal. by 1H NMR of fractions obtained by size exclusion chromatog. showed significant variations with mol. wt., revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The exptl. NMR carbonyl dyad signal intensities were satisfactorily matched by a random seq