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Biosynthesis and characterization of [15N]actinomycin D and conformational analysis by nitrogen-15 nuclear magnetic
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.
Insights
This study produced actinomycin D labeled with nitrogen-15 (15N). Researchers confirmed the D-enantiomer of glutamic acid is used in biosynthesis and characterized the drug's structure and solvent interactions using NMR.
Area of Science:
- Biochemistry
- Organic Chemistry
- Spectroscopy
Background:
- Actinomycin D is a potent antibiotic with a complex structure.
- Understanding its biosynthesis and conformation is crucial for drug development.
Purpose of the Study:
- To synthesize and characterize actinomycin D fully labeled with nitrogen-15 (15N).
- To investigate the stereochemistry of glutamic acid incorporation in biosynthesis.
- To elucidate the solution conformation of actinomycin D using NMR spectroscopy.
Main Methods:
- Incubation of Streptomyces parvulus with racemic [15N]glutamic acid.
- Production and isolation of 15N-labeled actinomycin D.
- Nuclear Magnetic Resonance (NMR) spectroscopy (15N and 1H NMR) in various solvents (CDCl3, DMSO, methanol, water).
Main Results:
- Successful synthesis of actinomycin D labeled at all twelve nitrogen positions.
- Confirmation that the D-enantiomer of glutamic acid is utilized in actinomycin biosynthesis.
- Assignment of all 15N resonances and determination of 1H-15N coupling constants.
- Observation of significant solvent-dependent shifts in peptide resonances, indicating conformational changes.
Conclusions:
- The study provides a detailed characterization of 15N-labeled actinomycin D.
- The findings offer insights into the stereospecificity of actinomycin biosynthesis.
- NMR data supports a model of actinomycin D solution conformation influenced by solvent polarity and hydrogen bonding.