The reaction of anthramycin with DNA. Proton and carbon nuclear magnetic resonance studies on the structure of the anthramycin-DNA adduct.

Nuclear magnetic resonance techniques are used to confirm the points of attachment of anthramycin to DNA. Using 13C NMR spectroscopy, the C-11 resonance of anthramycin is shown to undergo a 16-ppm upfield shift upon formation of a covalent bond with DNA, indicative of an aminal linkage at that position. The site of attachment on the DNA is determined using the self-complementary oligodeoxyribonucleotide d-(ApTpGpCpApT) as a DNA model. Proton NMR, both in H2O and D2O solutions, provides a direct characterization of the anthramycin-oligonucleotide adduct. Upon covalent attachment to the duplex, a loss in the helical symmetry is observed, resulting in a doubling of several of the oligonucleotide resonances. Examination of the data confirms that the point of attachment of the anthramycin to the d-(ApTpGpCpApT) is at the guanine-NH2-position, consistent with the model proposed by Hurley and Petrusek (Hurley, L. H., and Petrusek, R. L. (1979) Nature (Lond.) 282, 529-531) and Petrusek et al. (Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., and Hurley, L. H. (1981) Biochemistry 20, 1111-1119).

Chemical conversion of anthramycin 11-methyl ether to didehydroanhydroanthramycin and its utilization in studies of the biosynthesis and mechanism of action of anthramycin.

Reaction of anthramycin 11-methyl ether (AME) with trifluoroacetic acid results in formation of (1,11a)-didehydroanhydroanthramycin (DAA). Anthramycin biosynthetically labelled from DL-[3'RS(3'-3H)]; DL-[3'S(3'-3H)] and DL-[3'R(3'-3H)] tyrosine each lose approximately 50% of their tritium during this conversion to DAA confirming the labelling pattern of 3'-tritiated species of tyrosine in AME. As expected negligible losses of tritium occurred from AME biosynthetically labelled fron L-[2- or 6-3H] or L-[3- or 5-3H]tyrosine. DAA did not form a stable adduct with DNA in accord with the postulated mechanism of action of anthramycin.

Proof for the biosynthetic conversion of L-[indole-15N]tryptophan to [10-15N]anthramycin using (13C, 15N) labelling in conjunction with 13C-NMR and mass spectral analysis.

Using 13C-NMR and mass spectral analysis we have demonstrated that the N-10 nitrogen of anthramycin is biosynthetically derived from the indole-nitrogen of tryptophan. Our experimental approach was to bring a 15N atom, which is derived from L-[indole-15N]tryptophan, and a 13C atom which is derived from DL-[1-13C]tyrosine, into adjacent positions of anthramycin. From resonance intensities and 13C-15N spin-spin coupling in the 13C-NMR spectrum of didehydroanhydroanthramycin, a derivative of anthramycin, we could then determine the 13C enrichment at C-11 and the proportion of 13C bonded to 15N at N-10. These results when combined with mass spectral analysis and isotopic dilution measurements proved that the indole nitrogen of tryptophan was completely retained at N-10 of anthramycin.

Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthetic conversion of tyrosine to the C2- and C3-proline moieties of anthramycin, tomaymycin, and sibiromycin.

This paper descirbes biosynthetic labeling experiments on the conversion of tyrosine to the C2- and C3-proline units of anthramycin, tomaymycin, and sibiromycin. The biosynthetic fate of all of the aromatic and side-chain hydrogens has been determined in each antibiotic by using dual tagged (3H/14C) and 2H-labeled tyrosine molecules. In addition, experiments uing [15N]tyrosine and the tritiated D and L isomers of tyrosine have shed some light on the biochemical reactions which take place at tha alpha position of tyrosine. On the basis of results of all these experiments, a biosynthetic scheme had been proposed to rationalize the apparent inconsistencies which occur between the results for the three antibiotics. This scheme proposes that a common main pathway involving proximal extradiol cleavage of Dopa and condensation to form the pyrrolo ring leads ultimately to a C-7 branch point compound. Parallel pathways from this central branch point compound lead by well-known biochemical transformations to the C2-and C3-proline units of anthramycin, tomaymycin, and sibiromycin. The reactions in these parallel pathways are suggested to be "cosmetic or after events".