A new series of reductive amination derivatives of daunorubicin: syntheses, partition coefficients, and DNA binding.

A series of daunorubicin derivatives were prepared by sodium cyanoborohydride reductive amination of daunorubicin with appropriate amines. All derivatives were found to bind quite strongly to DNA and viscosity increases with linear DNA indicated that each formed an intercalation complex. A range of octanol-aqueous buffer partition coefficients was obtained, around the values of daunorubicin and dauxorubicin hydrochloride, by varying the character of the starting amine. All monoamine derivatives had activity against P388 leukemia in mice which was similar to that of daunorubicin. A diamine derivative had reduced activity against P388. Several anthracyclines administered as DNA complexes had similar activity against P388 but significantly reduced toxicity compared to the uncomplexed compounds. For anthracyclines which bind strongly to DNA, optimum activity against P388 leukemia in mice seems to be centered on compounds with octanol-buffer partition coefficients in the range of 0.5-0.8.

Visualization of drug-nucleic acid interactions at atomic resolution. IX. Structures of two N,N-dimethylproflavine: 5-iodocytidylyl (3'-5') guanosine crystalline complexes.

This paper describes two complexes containing N,N-dimethylproflavine and the dinucleoside monophosphate, 5-iodocytidylyl (3'-5') guanosine (iodoCpG). The first complex is triclinic, space group P1, with unit cell dimensions a = 11.78 A, b = 14.55 A, c = 15.50 A, alpha = 89.2 degrees, beta = 86.2 degrees, gamma = 96.4 degrees. The second complex is monoclinic, space group P21, with a = 14.20 A. b = 19.00 A, c = 20.73 A, beta = 103.6 degrees. Both structures have been solved to atomic resolution and refined by Fourier and least squares methods. The first structure has been refined anisotropically to a residual of 0.09 on 5,025 observed reflections using block diagonal least squares, while the second structure has been refined anisotropically to a residual of 0.13 on 2,888 reflections with full matrix least squares. The asymmetric unit in both structures contains two dimethylproflavine molecules and two iodoCpG molecules; the first structure has 16 water molecules (a total of 134 non-hydrogen atoms), while the second structure has 18 water molecules (a total of 136 non-hydrogen atoms). Both structures demonstrate intercalation of dimethylproflavine between base-paired iodoCpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along b and a axes, respectively. The basic structural feature of the sugar-phosphate chains accompanying dimethylproflavine intercalation in both structures is the mixed sugar puckering pattern: C3' endo (3'-5') C2' endo. This same structural information is again demonstrated in the accompanying paper, which describes a complex containing dimethylproflavine with deoxyribo-CpG. Similar information has already appeared for other "simple" intercalators such as ethidium, acridine orange, ellipticine, 9-aminoacridine, N-methyl-tetramethylphenanthrolinium and terpyridine platinum. "Complex" intercalators, however, such as proflavine and daunomycin, have given different structural information in model studies. We discuss the possible reasons for these differences in this paper and in the accompanying paper.

Stereospecific binding of diastereomeric peptides to salmon sperm deoxyribonucleic acid. Further evidence for partial intercalation.

A series of diastereomeric dipeptide amides, containing an N-terminal L-lysyl residue and a C-terminal L- or D-amino acid with a derivatized aromatic ring on the side chain, was synthesized to determine the dependence of (1) the chirality of the N-terminal amino acid alpha-carbon and (2) the length of the N-terminal amino acid side chain for intercalation of the aromatic ring. The nature of the complex between the peptide and DNA (i.e., electrostatic, intercalative, or a combination of these) was determined by UV and CD studies, viscometric titrations, and 1H NMR studies. The results of these studies reveal distinct differences in the binding site of the aromatic rings of the various peptides. In particular, the results suggest that the alpha- and epsilon-amino groups of the lysyl residue bind electrostatically to adjacent phosphates on the DNA backbone in a stereospecific manner. As a result of this stereospecificity, the aromatic rings of the peptides with the L-L designation point toward the DNA helix, while those of the peptides of the L-D designation point away from the helix. This is completely consistent with previously reported work [Gabbay, E.J., Adawadkar, P. D., & Wilson, W. D. (1976) Biochemistry 15, 146; Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S., & Wilson, W. D. (1976) Biochemistry 15, 152]. The results also indicate a great dependence on the length of the side chain for intercalation of the aromatic ring. Specifically, if the side chain is long enough, and flexible enough, the aromatic ring can fully or partially intercalate, regardless of the chirality of the N-terminal amino acid alpha-carbon. However, if the side chain is too short, only partial intercalation is observed for peptides of the L-D designation, and no intercalation is observed for peptides of the L-D designation.

Interactions of some novel amide-linked bis(acridines) with deoxyribonucleic acid.

A novel series of bis(acridines) has been synthesized in which the two potential intercalating chromophores are separated by symmetrical amide-linked chains varying in both length and conformational flexibility. By comparison to a monointercalating adduct, mono- vs. bis-intercalative behavior has been established for the bis(acridines). Spectrophotometric (visible, circular dichroism, and fluorescence) and viscometric (linear sonicated and closed circular superhelical DNA) experiments indicate that a highly rigid 8.8 A separated bis(acridine) monointercalates, whereas the longer and more flexible bis(acridines) are capable of bis-intercalation. In addition, spectrophotometric studies suggest a correlation between the tendency of intramolecular association and the ability to bis-intercalate. The results are in agreement with predictions based on the neighbor-exclusion principle and indicate that connecting chain rigidity is capable of playing a determining role in the mono- vs. bis-intercalation mechanism.

Nitroaniline diamine.poly(dA-dT) complexes: 1H and 19F NMR parameters for full intercalation of aromatic rings into DNA.

High-resolution proton, fluorine, and phosphorus NMR studies have been undertaken on complexes of methyl- and trifluoromethyl-substituted nitroaniline diamines with the synthetic DNA poly(dA-dT) in 10 mM buffer solution. We demonstrate full intercalation of the nitroaniline group of these reporter molecules between base pairs, based on large upfield proton shifts (1.3-1.7 ppm) at all four aromatic proton markers on complex formation. The temperature and pH dependences of the thymidine H-3 Watson-Crick proton chemical shift and line width require the formation of intact and stable base pairs in this intercalative complex in solution. The 19F chemical shift of the trifluoromethyl-labeled nitroaniline diamine shifts downfield by approximately 2 ppm on formation of the synthetic DNA complex and most likely reflects the nonpolar environment of the aromatic ring when sandwiched between base pairs. A sequence specificity in the binding of the nitroaniline dication to poly(dA-dT) is implied by the observation of two partially resolved 31P resonances with the phosphodiester at the intercalation site shifting downfield by approximately 0.4 ppm on complex formation.

Interaction specificities of a platinum metallointercalation reagent to DNA.

The interaction specificity of salmon sperm DNA with 2-hydroxyethanethionlato(2,2',2''-terpyridine)platinum(II),PtTS has been studied. The results of 1H and 13C nuclear magnetic resonance, flow dichroism and circular dichroism studies are found to be consistent with an intercalation mode of binding as has been proposed earlier by lippard and coworkers.

Interaction specificity of the anthracyclines with deoxyribonucleic acid.

The interaction specificity of salmon sperm DNA with various derivatives of daunorubicin has been studied. The results of binding, viscometric, 1H nuclear magnetic resonance (NMR), flow dichroism, DNA template inhibition, rates of dissociation, and circular dichroism studies are found to be consistent with an intercalation mode of binding of the anthracycline ring as has been shown by other investigators. Moreover, it is observed that (i) strength of binding, (ii) the ease of dissociation of DNA-anthracycline complexes, and (iii) the degree of inhibition of the DNA-dependent RNA polymerase are dependent on the presence of the amino sugar moiety of daunoseamine. The results are consistent with specific H bonding of the amino group of the sugar moiety with DNA as has been suggested earlier by Pigram et al. (Pigram, W.J., Fuller, W., and Hamilton, L.D. (1972), Nature (London), New Biol. 235, 17). Peptide derivatives substituted at the amino sugar function of daunorubicin lower the affinity of the drug to DNA and presumably interfere with the "full insertion" of the anthracycline drugs between base pairs of DNA. The significance of these findings in relation to the biological efficacy of daunorubicin and related derivatives as antileukemic agents is discussed.

Sturcture-activity relationship of daunorubicin and its peptide derivatives.

The synthesis of potentially specific antitumor peptide derivatives of daunorubicin is presented. The interaction specificites of the drugs with nucleic acids have been examined via stop-flow kinetics as well as the inhibition of DNA template activity. It is found that the biological activity of the daunorubicin derivatives against the mouse P388 tumor is directly proportional to RNA polymerase inhibition and inversely proportional to the rate of dissociation of the DNA complex. It is concluded that the biological efficacy of drugs which act at the replicative and transcriptional level may be estimated by the more rapid in vitro techniques provided that problems of permeability, solubility, stability, etc., in vivo are not encountered.

Stereospecific binding of diastereomeric peptides to salmon sperm DNA.

Studies of the interaction specificities of L-lysyl-L-phenylalaninamide (1) and the diastereomeric dipeptide amide, L-lysyl-D-phenylalaninamide (2), with salmon sperm DNA reveal distinct differences in the binding site of the aromatic ring of the phenylalanine residue. The results of 1H nuclear magnetic resonance (NMR), spin-lattice relaxation rates, viscometric, and flow dichroism studies indicate the aromatic ring of 1 is "partially" inserted between base pairs of DNA whereas the aromatic ring of 2 points outward toward the solution. The terminal L-lysyl residue presumably interacts stereospecifically with DNA helix thus dictating the positioning of the aromatic ring of the C-terminal phenylalanine residue. In the accompanying paper (E. J. Gabbay et al. (1976), Biochemistry, following paper in this issue), the interaction of several oligopeptide amides (containing the N-terminal L-Lys-L-Phe residue) with DNA is examined. The results are found to be consistent with stereospecific binding of the terminal L-lysyl residue, and in addition, the evidence suggests that oligopeptides may bind to DNA via a modified single-stranded beta-sheet structure which is wrapped around the nucleic acid helix in a manner similar to that described by M. H. F. Wilkins (1956), Cold Spring Harbor Symp. Quant. Biol. 21, 75).

The interaction specificity of peptides with DNA. Evidence for peptide beta-sheet-DNA binding.

Proton magnetic resonance studies (1H NMR) of the interaction of oligopeptide amides of defined sequence (and containing the amino acid, phenylalanine) with salmon sperm DNA are reported. The extent of upfield chemical shifts, deltasigma, and signal line broadening of the aromatic protons (in the presence of excess DNA) are found to depend on the primary sequence and stereochemistry of alpha carbons of the amino acids in the oligopeptide amides. The results obtained with 21 different di-, tri-, tetra-, penta- and hexapeptide amides are found to be consistent with a model whereby the peptide assumes a slightly modified single-stranded beta-sheet structure which is wrapped around the nucleic acid helix in a manner similar to that described by M. H. F. Wilkins (1956), Cold Spring Harbor Symp. Quant. Biol. 21, 75).

Evidence for fidelity of chromatin reconstitution.

Several lines of evidence are presented which support the contention that chromatin may be dissociated, fractionated, and reconstituted without altering the compositional, structural, or transcriptional integrity of the genome. The similar compositions of native and reconstituted chromatins are suggested by the absence of significant differences in their protein/DNA ratios and in the polyacrylamide gel electrophoretic profiles of their histones and nonhistone chromosomal proteins. Criteria for fidelity of genome structure in reconstituted chromatin include binding of reporter molecules with specificity for the minor groove of DNA, binding of histones, number of sites available for addition of nucleotides, and circular dichroism spectra. When the transcriptional activities of native and reconstituted chromatins were compared under conditions where reinitiation is prohibited, significant changes were not observed. Taken together, the present results strongly suggest, but do not conclusively establish, fidelity of chromatin reconstitution.

Binding of manganese(II) to DNA and the competitive effects of metal ions and organic cations. An electron paramagnetic resonance study.

The binding of manganese(II) to DNA was studied by monitoring the concentration of free Mn2+ by electron paramagnetic resonance (EPR). It was found that the association constnat of the Mn-DNA complex depends upon the degree of saturation. The competitive effects of magnesium, calcium, sodium, and a number of organic cations including the antibiotic drug daunomycin were analyzed and the parameters describing the cation-DNA interaction were evaluated. It was found that the association constant as well as the parameter describing its dependence upon the degree of saturation decrease along the series Mn, Mg, Ca, Na. Differences in the extent of interaction with the base nitrogens (N-7) are suggested as the possible mechanisms leading to these observations. The EPR spectrum of the manganese-DNA complex was found to be similar to that of manganese-nucleotide complexes suggesting a similar mode of coordination. A comparison of the results of competitive and direct binding studies reveals some salient features of the small molecule-DNA interaction and leads to the conclusion that manganese binds at the major groove of the DNA helix.

The Alkali Ion-DNA Interaction as Reflected in the Nuclear Relaxation Rates of Na and Rb.

Longitudinal relaxation rates of (23)Na and (87)Rb in aqueous solutions of DNA were measured and interpreted in terms of contributions of "bound" and free species. The frequency dependence of the (23)Na relaxation rate sets an upper limit of 5.5 nsec for the correlation time characteristic of the alkali ion-DNA interaction. The plausible description of the alkali ion-DNA interaction emerging from the results is one in which the "bound" ions comprise a highly mobile ionic cloud around the lattice of phosphate groups with a population characterized by an apparent dissociation constant of 10.9 mM. Any specific binding to phosphate groups involving severe restrictions of motional freedom must be much (at least two orders of magnitude) weaker and of a very short duration (<5.5 nsec).