(R,S)-[Carboxyl-¹⁴C]–cis-3-hydroxyproline was synthesized from S-[carboxyl-¹⁴C] proline. The oxygen functionality at the three position was obtained by acetylation of 1,2-dehydroproline methyl ester using lead tetraacetate. Reduction of the imine with sodium borohydride gave predominately (R,S)-[caboxyl-¹⁴C]-cis-3-acetoxyproline which was hydrolyzed with hydrochloric acid and purified by ion-exchange chromatography and recrystallization. In order to determine if cis-3-hydroxyproline is a precursor for the dehydroproline moietyof virginiamycin M1, (R,S)-[carboxyl-¹⁴C]-cis-3-hydroxyproline and S-[3,4-³H] proline with a ³H/¹⁴C of 9 were fed simultaneously to a virginiamycin producing strain of Streptomyces. The resulting antibiotic had a ³H/¹⁴C ratio of 41.3. The proline portion of the antibiotic had a ratio of 19.9. Therefore, 45% of the cis-3-hydroxyproline was incorporated, and cis-3-hydroxyproline is a precursor to the dehydroproline moiety. / Master of Science

Some aspects of the biosynthesis of three amino acid residues in virginiamycin S₁ have been studied in Streptomyces virginiae by the incorporation of amino acid precursors labeled with either radioactive or stable isotopes. L-Lysine-U-¹⁴C was incorporated into both the 4-oxo-L-pipecolic acid and 3-hydroxypicolinic acid residues. The formation of the heterocyclic ring of both of these amino acids was shown to occur with retention of the nitrogen from the ε-amino group of lysine, as shown by the incorporation of DL-lysine-6-¹³C-6-¹⁵N. In addition, the 3-hydroxypicolinic acid residue incorporated deuterium from (2RS, 5R)-lysine-5-d₁, but not from (2RS, 5S)-lysine-5-d₁. This finding indicates that the 5-pro-(R) hydrogen of L-lysine is retained during the biogenesis of 3-hydroxypicolinic acid. In the conversion of L-phenylalanine to L-phenylglycine, the amino group moves to the benzylic position. This process could proceed either by an intermolecular mechanism, in which the original nitrogen is lost, or by an intramolecular mechanism, in which that nitrogen is retained. Administration of (RS)-phenylalanine-3-¹³C-¹⁵N resulted in its incorporation with loss of the labeled nitrogen. The process therefore occurs by an intermolecular mechanism. / Ph. D.

The biosynthesis of virginiamycin S₁, a macrocyclic peptidolactone antibiotic, was studied by growing a strain of Streptomyces virginiae in a complex medium and observing the incorporation of radiolabeled compounds into the antibiotic. These studies have established several of the biosynthetic precursors of virginiamycin S₁. L-(U-14C)-Proline and L-(U-14C)-threonine were effectively incorporated into the respective amino acid components in the antibiotic. N-Methyl-L-phenylalanine was shown to arise from L-(U-14C)-phenylalanine and L-(methyl-14C)-methionine. L-(U-14C)-Phenylalanine was also efficiently incorporated into L-phenylglycine. The origin of the remaining three components was less clear. A small amount of L-(U-14C)-threonine was observed in D-α-aminobutyric acid. A biosynthetic pathway is known between these two amino acids which suggests that L-threonine may be the biosynthetic precursor of D-α-aminobutyric acid. Both L-(U-14C)-aspartic acid and L-(U-14C)-lysine were incorporated into 4-oxo-L-pipecolic acid and 3-hydroxypicolinic acid. A biosynthetic pathway was hypothesized to explain these results. / Master of Science