Actinomycin D, ¹H NMR studies on intramolecular interactions and on the planarity of the chromophore

Abstract

The conformation of actinomycin D in acetone and chloroform solution at different temperatures has been studied by 'H NMR spectroscopy.At lower temperature the resonances due to the two chromophoric amino protons were observed. These signals exhibit very different resonance positions indicating a severely hindered rotation of the 2-amino group and the presence of a hydrogen-bond connecting the 2-amino and the I-carbonyl groups.In 'H NMR spectra of partially "N-enriched actinomycin D, the lJN.fl coupling constants at the 2-amino group were determined and a strong sp2 character for the 2-amino nitrogen was deduced. The strong amide character of the 2-amino group is caused by mesomerism involving the I-carbonyl group.The amino proton signals are sensitive indicators for differences in the spatial relationship of the diverse parts of the actinomycin molecule.At lower temperatures a simultaneous and selective broadening of the CI ring threonine and valine amide proton signals as well as of the 2-amino group resonance was observed, indicating the presence of one dynamic process in the molecule which slows down upon temperature reduction. A swinging motion of the N(10) nitrogen through the chromophore plane would explain this observation.The interpretation of these results requires the presence of a non-planar chromophoric system in the actinomycin molecule in acetone and chloroform solution. The possible implications of this non-planarity for the intercalation process and for the biological activity of the drug are discussed.Actinomycin D is the best studied representative of a family of chromopeptides of bacterial origin, which has aroused interest because of its antineoplastic activity [I, 21. The biological activity seems to be correlated with the ability of actinomycins to form intercalation complexes with DNA, whereby the DNA-dependent RNA synthesis is inhibited. The formation of the DNA-actinomycin complex requires the presence of certain structural features in the actinomycin molecule (cf. Fig. I), e.g. both lactone rings have to be intact [2].Actinomycin derivatives in which the 2-amino group on the

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

“…Assuming stronger hydrogen bonds at lower temperature, the stabilization of polarized structures with a negatively charged 3-0x0 group will lead to a more deshielded N10 nucleus. This concept is supported by the temperature dependence of the H7 proton resonance [8] ; with lower temperature this signal shifts downfield, while the H8 resonance exhibits a very slight upfield shift. Structures (b), (d) and (f) in Fig.…”

“…This X-ray data profoundly influenced the interpretation of subsequent NMR studies of the actinomycin D conformation in solution [3,4]. The dynamics and flexibility inherent to the free actinomycin D molecule in solution are of interest, as conformational changes in the peptide moieties of the drug are assumed to account for the small dissociation rate constant measured for the DNAactinomycin-D complex [5, 71. In a recent 'H NMR study on the secondary structure of the actinomycin D molecule in organic solvents, an intramolecular interaction involving the 2-amino and the C1 carbonyl groups was derived [8]. This interaction explains the increased molecular symmetry derived by 'H NMR Abbreviations.…”

“…The absence of any hydrogen bond at the N10 position has already been mentioned and, hence, the stabilization of a negatively charged N10 atom by such an interaction is not possible. The conjugation of the 2-amino group with the N10 atom or with the C9 carbonyl group may be possible, but does not seem to be of great influence in the actinomycin chromophore [8]. Resonance structures (c) and (d) in Fig.…”

“…To explain this observation, a swinging motion of the N10 nitrogen through the chromophore's plane was suggested [8].…”

“…'H NMR studies of actinomycin D in acetone [8] have shown the presence of a dynamic process affecting u-ring proton amide resonances and the amino proton signal (B).…”