Biosynthesis and characterization of [15N]actinomycin D and conformational analysis by nitrogen-15 nuclear magnetic resonance.

Abstract

We describe the production and characterization of actinomycin D labeled with 15N at all twelve nitrogen positions. Cultures of Streptomyces parvulus were incubated in the presence of racemic [15N]glutamic acid and, following an initial delay, labeled antibiotic was produced. Evidence is presented that the D enantiomorph of glutamic acid was ultimately used for actinomycin biosynthesis. The 15N NMR spectrum at 10.14 and 20.47 MHz of the labeled drug in CDCl3 is presented. All nitrogens except the phenoxazone chromophore nitrogen are inverted when spectra are obtained under broad-band proton irradiation conditions. All 15N resonances have been assigned, and the proton-nitrogen one-bond coupling constants were determined in CDCl3 to be 92.5 +/- 0.3 Hz for the valine and threonine amide protons by both 1H and 15N NMR. 15N NMR spectra were also obtained in dimethyl sulfoxide, methanol, and water in order to probe solvent interactions with the peptide nitrogens and carbonyl groups. Large downfield shifts (greater than 5 ppm) were seen for the Pro, sarcosine, and methylvaline resonances when the solvent was changed from dimethyl sulfoxide to water. Smaller downfield shifts were observed for the Val and Thr peaks. These results are discussed in terms of a model for the solution conformation of the actinomycin pentapeptide rings based on different hydrogen-bonding interactions in the monomer in organic solvents and the dimer which is formed in water.