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.

Intramolecular interactions, mesomerism and dynamics in actinomycin D studied by 15N NMR spectroscopy

“…Assuming that all the available proton-donating groups actually form intermolecular hydrogen bridges to MezSO molecules, then an upfield shift of the proline signals as compared to their position in the other polar solvents is to be expected. Actually, a value very close to that reported for the chloroform solution is observed [12]. A similar behaviour is observed for the sarcosine and methylvaline resonances, in line with the loss of H bridges at the corresponding carbonyl groups.…”

“…In this case, the asymmetry of the chromophore ring system very probably precludes a complete overlap of both nitrogen resonances. This interpretation of the MezSO-induced changes in chemical shift is at variance with the one put forward by Shafer and co-workers [12].…”

“…The presence of such a conformation is supported by the 15N chemical shift values reported by Shafer and co-workers [12] for the ('H6)MezS0 solution of actinomycin D. MezSO is an extremly good H-bond acceptor and will compete effectively with other proton acceptor groups for the available acid protons in the actinomycin D molecule. Assuming that all the available proton-donating groups actually form intermolecular hydrogen bridges to MezSO molecules, then an upfield shift of the proline signals as compared to their position in the other polar solvents is to be expected.…”

“…We therefore synthesized ' 5N-labelled actinomycin D microbiologically and studied its conformation in solution by NMR techniques. For production of the drug the species Streptomyces parvulus was chosen, as it produces actinomycin D with more than 95% purity [l].Recently, a study on the conformation of 15N-labelled actinomycin D in solution was published [12]. The use of an NMR spectrometer with low magnetic field strength pre-…”

“…Recently, a study on the conformation of 15N-labelled actinomycin D in solution was published [12]. The use of an NMR spectrometer with low magnetic field strength pre-…”