ROLE OF D-VALINE RESIDUES IN THE ANTITUMOR DRUG ACTINOMYCIN-D - REPLACEMENT OF D-VALINES WITH OTHER D-AMINO ACIDS CHANGES THE DNA-BINDING CHARACTERISTICS AND TRANSCRIPTION INHIBITORY ACTIVITIES

D-valine analogues of the antitumor drug actinomycin D, in which D-val ine residues were replaced with D-threonine, D-tyrosine, D-phenylalani ne, and D-O-methyltyrosine residues, have been totally synthesized. Th e crystal structure of the D-O-methyltyrosine analogue has been determ ined (a = b = 21.352(6), c = 44.525(9) Angstrom space group P4(1)2(1)2 ; R = 0.19 for 803 out of 1114 reflections at 1.8 Angstrom resolution data). Replacements of D-valines did not change the overall conformati on of the molecule, and the substituted groups were located on the sid e opposite to the DNA binding site, suggesting that the analogues can bind intercalatively at 5'-GC-3' sequences of DNA like actinomycin D d oes. In the crystals, the analogue molecules constitute a tight dimer, and a pair of stacked chromophores of the dimer was further sandwiche d by two methoxyphenyl groups of neighboring molecules. These strong a romatic-aromatic stacking forces among the molecules appear to reduce very much the water solubility of the aromatic analogues. The characte ristics of binding of the analogues to various DNA's including d(GAAGC TTC)(2), d(GTTGCAAC)(2), poly(dA-dT), poly(dG-dC), and calf thymus DNA have been examined by using the visible spectrum methods. Difference spectra of actinomycin D and the analogues with oligonucleotides indic ated that the analogues bind intercalatively to the DNA, as actinomyci n D does, but the association constants were reduced to approximately one-half that of actinomycin D. The spectra of the aromatic analogues titrated with calf thymus DNA indicated that the aromatic analogues bo und somehow differently to the longer DNA's. A simple profile analysis of the spectra suggested that the aromatic analogues bound to calf th ymus DNA not only with intercalation, as actinomycin D does, but also with side binding. Nevertheless, the association constants of the arom atic analogues to calf thymus DNA with the intercalation mode were fou nd to be quite similar to those of the short oligonucleotides. This co nclusion has been supported by the melting behaviors of the DNA with t he aromatic analogues, in which the melting curves of the analogues we re superimposable on the melting curve of DNA with actinomycin D, sugg esting that the aromatic analogue molecules were intercalated into the DNA. The inhibitory activities of actinomycin D and analogues on RNA polymerase in vitro were examined using calf thymus DNA and E. coli RN A polymerase. All actinomycin D analogues severely inhibited RNA synth esis at relatively low drug concentrations. In general, inhibitory act ivities of the analogues on the RNA synthesis were found to be correla ted with those of DNA binding characteristics. However, the analogue i n which D-phenylalanine replaced D-valines inhibited RNA synthesis mor e strongly than actinomycin D itself, but this-analogue bound to the D NA's much more weakly than actinomycin D. In this study, the D-valine residues in the cyclic depsipeptides of actinomycin D were found not t o be directly involved in DNA binding, but this amino acid residue was found to be an important biological modulator of the antibiotic. Alth ough the D-valine is a hydrophobic amino acid residue, this amino acid residue appears to play an important role in increasing the water sol ubility of the antibiotic. Replacements of D-valine residues reduced d rastically the water solubility of the analogues, and consequently; th is physical character of the analogues reduced their capacities for bi nding to DNA. As a result, the biological activities of the analogues were generally decreased.