Analogue Of Actinomycin Research Articles

Application of 7-amino-actinomycin D for the fluorescence microscopical analysis of DNA in cells and polytene chromosomes.

The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands of Chironomus thummi thummi and Drosophila lummei (Hackman). 7-Amino-actinomycin D has been shown to bind to DNA-containing structures, thereby inducing in them a bright red fluorescence. No specific fluorescence has been found in RNA-containing structures treated with this fluorescent probe. The fluorescence pattern of some regions of polytene chromosomes with a known nucleotide composition was analysed. It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of the Drosophila lummei toromere structure. Data indicating a nonlinear dependence between the fluorescence intensity of a stained chromosome region and the GC content in its DNA have been obtained. The influence of DNA nucleotide composition in a chromosome region on the fluorescence of 7-amino-actinomycin D is discussed. In combination with quinacrine staining and the Feulgen fluorescence reaction, treatment with 7-amino-actinomycin D provides useful information about the distribution of GC base pairs in the chromosome region under study.

DNA binding studies of 7-bulky-substituted actinomycin analogues.

The DNA binding properties of several 7-substituted aralkylaminoactinomycin D analogues have been studied by spectrophotometry, DNA melting temperature studies, DNA-drug dissociation studies, and circular dichroism. Despite the presence of such bulky groups as 2-pyrrolylmethylamino or 3,4-dichlorobenzylamino at the 7 position, these analogues bind to DNA, inhibit RNA synthesis, and exhibit antitumor activity. A model is proposed for the interaction of the pyrrolyl analogue with phosphate groups of the DNA binding site, explaining the increased binding affinity for DNA of this actinomycin D analogue.

High-resolution proton nuclear magnetic resonance analysis of conformational properties of biosynthetic actinomycin analogues.

High-resolution proton nuclear magnetic resonance (1H NMR) has been used to study the conformation and dynamics of biosynthetic analogues of actinomycin D that have been substituted at one or both of the 3'-amino acids with azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline for one or both of the prolines. The results of measurements made in organic solvents indicate that the amino acid residues on the alpha and beta pentapeptide lactone rings experience different magnetic environments in all the analogues studied. Analysis of the J alpha NH coupling constants and temperature coefficients for the valine and threonine amide protons indicates that substitution at the 3' position has little effect on the conformational and hydrogen-bonding properties of the amino acids at the 1' or 2' position. Examination of the low-field position of the H alpha proton of the 3'-amino acid reveals that the biosynthetic substitutions were made at a unique site on either the alpha or the beta pentapeptide ring and that only one conformation of proline, azetidine-2-carboxylic acid, or pipecolic acid is observed. In D2O, the dynamics of the prolines appear to differ; one remains in a preferred conformation while the other fluctuates at a rate that is intermediate on the NMR time scale. A possible relationship between the conformational and dynamic properties and the DNA binding and kinetic properties is discussed.

Role of actinomycin pentapeptides in actinomycin-deoxyribonucleic acid binding and kinetics.

Results are reported on equilibrium and kinetic experiments probing the DNA binding properties of a series of actinomycin analogues differing at the 3'-amino acid position. While the parent compound, actinomycin D, contains proline at this position on both pentapeptide lactone rings, the analogues under consideration here contain either azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline on one or both pentapeptide rings. This study extends our earlier results on doubly substituted analogues [Shafer, R.H., Burnett, R. R., & Mirau, P.A. (1980) Nucleic Acids Res. 8, 1121]. DNA binding constants were determined from Scatchard plots constructed from visible absorption data and covered the range of (0.3-9) X 10(6) M-1 for the whole series of analogues. The thermal denaturation temperature of calf-thymus DNA was increased by 3-17 degrees C. DNA dissociation kinetics, along with enthalpies and entropies of activation, were also determined. The time constant for the slowest dissociation process ranged from 278 to 10 900 s. The strongest DNA binding analogue, in terms of the largest binding constant, the largest increase in DNA thermal denaturation temperature, and the slowest DNA dissociation rate, was actinomycin V, which has 4-ketoproline in the beta peptide ring, while the weakest DNA binding analogue has pipecolic acid on both peptide rings. Evidence is presented for one peptide ring exerting a greater influence than the other in the interaction with DNA. Also, the possible role of cis-trans isomerization about one or two peptide bonds in determining the slow DNA binding kinetics is discussed.

ChemInform Abstract: A PHENAZINE ANALOGUE OF ACTINOMYCIN D

AbstractEin Analogon (VI) von Actinomycin D (VII) wird ausgehend von dem Brombenzol (I) über das Diphenylamin (IIIa) und das Cyclisierungsprodukt (IVa) dargestellt.

A phenazine analogue of actinomycin D.

An analogue of actinomycin D (1), in which the phenoxazone chromophore has been replaced by a phenazine, has been synthesized and characterized. Although this compound (2) lacks the 2-amino group and does not possess the quinoid structure of 1, it does bind to DNA, but less tightly than either 1 or the 2-deamino derivative of 1. NMR and CD spectra indicate that the peptide conformations in 2 are approximately as in 1; there was no apparent asymmetry of the two peptide rings. Compound 2 inhibited nucleic acid synthesis in L1210 cell cultures more effectively than does 2-deaminoactinomycin D, but about one-tenth as well as does actinomycin D.

ChemInform Abstract: SYNTHESIS AND EVALUATION OF BIS‐DIPEPTIDE AND BIS‐TRIPEPTIDE ANALOGS OF ACTINOMYCIN D

AbstractVerschiedene Bis‐di‐ und ‐tri‐peptide in der Actinomycin‐Reihe werden hergestellt, indem z.B. aus dem Nitro‐benzamid (II) über (III) und (IV) die Verbindung (Ib), aus (III) über (V) die Verbindungen (Id)‐(Ig) und aus (VI) die Verbindung (Ie) hergestellt werden.

Synthesis and evaluation of bis-dipeptide and bis-tripeptide analogues of actinomycin D.

Six-bis-dipeptide analogues of actinomycin D, all containing two threonyl-D-valine side chains, were prepared. Also two bis-tripeptide analogues containing an additional proline or oxoproline residue were synthesized. None of the compounds bound to DNA in a manner similar to actinomycin D. This lack of strong intercalative binding emphasizes the importance of the pentapeptidolactone side chains in the binding of actinomycin D to DNA and also highlights the deficiences inherent in using only small nucleotide sequences in investigating drug-DNA binding. None of the analogues tested showed any antitumor activity, although actinocylbis(threonyl-D-valine methyl ester) did show 10% of the antibacterial activity of actinomycin D vs. Bacillus subtilis.

Synthesis and some properties and antitumor effects of the actinomycin lactam analog, (di(1-L-alpha, beta-diaminopropionic))actinomycin D1.

A lactam analog of actinomycin D (AMD) has been synthesized as a potential antitumor chemotherapeutic agent. Both L-threonine residues were replaced by L-alpha,beta-diaminopropionic acid. Starting with Nalpha-benzyloxycarbonyl-Nbeta-tert-butyloxycarbonyl-L-alpha,beta-diaminopropionic acid methyl ester hydrochloride the linear intermediate Nalpha-benzyloxycarbonyl-Nbeta-(tert-butyloxycarbonylsarcosyl-L-N-methylvalyl)-L-alpha,beta-diaminopropionyl-D-valyl-L-proline p-nitrophenyl ester was prepared by conventional methods of peptide synthesis in solution. Selective cleavage of the Nbeta-tert-butyloxycarbonyl group and lactam formation afforded the desired cyclic pentapeptide derivative. The chromophore precursor, Nalpha-(2-nitro-3-benzyloxy-4-methylbenzoyl) substituent, was introduced via its symmetric anhydride. Catalytic reduction followed by ferricyanide-mediated phenoxazinone formation provided the lactam analog, [di(1'-L-alpha,beta-diaminopionic acid)]actinomycin D ([Dpr1]2-AMD). Its binding to natural and synthetic DNA and that of an analogous L-threo-alpha,beta-diaminobutyric acid containing lactam ([Dbu1]2-AMD) compared with the binding of AMD (in which the peptides are in lactone form) was studied by circular dichroic (CD) spectroscopy. The visible and uv CD spectra of free AMD differed from those of the free lactam analogs, indicating that the asymmetric environment of the pentapeptide rings in the region of the chromophore differs in free actinomycin lactone and lactams. In the presence of calf thymus DNA, PM2 DNA, and the synthetic d(A-T)-like copolymers containing 2,6-diaminopurine (DAP), poly[d(DAP-T)], and poly[d(DAP-A-T)], the rotational strengths of the optically active transitions in the visible region of the actinomycins increased, and the CD spectra in the presence of the various DNA duplexes were qualitatively similar. The CD spectra of bound actinomycin lactams resembled the spectrum of bound AMD. This suggests that the lactone and lactam actinomycins acquire a similar environment when bound to DNA. [Dpr1]2-AMD was less cytotoxic than AMD in antibacterial assays but exhibited somewhat higher toxicity in mice than AMD. At optimal dose levels the lactam analog had little or no antitumor activity in three murine tumor systems.

Binding of actinomycin chromophore analogues to DNA.

Five chromophore analogues of actinomycin, viz. phenoxazone (1), aminophenoxazone (2), N-acetylaminophenoxazone (3), N-methylaminophenoxazone (4), and actinocin (5), were synthesized. Of these compounds, only 2 and 4 were found to interact with DNA. The binding constants for these compounds, obtained by spectrophotometric titration, were of the same order of magnitude as that obtained with actinomycin-DNA interaction. The major difference between the binding of actinomycin and of the analogues to DNA was the lability of the complex of the latter compounds in the presence of Mg2+ ions. These observations have been interpreted to indicate intercalation of the chromophore analogues between successive base pairs of DNA. The spectra of actinocin were unaffected by the addition of DNA. The spectral changes indicative of an interaction between actinocin and DNA observed at low concentration of Mg2+ ions were abolished at high concentrations of these ions. The implications of these observations to the mechanism of actinomycin binding to DNA are discussed.

Synthesis of an actinomycin analog with substituted lactone oxygens (1',1'-bis(L-,-diaminopropionic acid))-actinomycin C (C 1).

A synthesis of a lactam analog of actinomycin D is described in which the threonine residues have been replaced by L‐α, β‐diaminopropionic acid. The key reaction, cyclization of the pentapeptide intermediate, was carried by a nitrophenyl ester condensation between the proline and sarcosine residues. [I′, I′‐Bis (L‐α, β‐diaminopropionic acid)]‐actinomycin D exhibited potent antibacterial activity.

Synthesis of some simple actinomycin analogs

AbstractThe synthesis of some simple actinomycin analogs, using 3‐nitrosalicylic acid as a starting material, is reported.

Interaction between Model Compounds of Actinomycin D and DNA: Physicochemical Studies

THE remarkable inhibitory effect of actinomycin D on DNA dependent RNA synthesis has been correlated with its ability to interact in a specific way with the DNA template1. The nature of the sites of interaction has been investigated by physicochemical and biochemica1 methods2–4. For this purpose several chemical modifications of the actinomycin have been tried. Studies of simple chemical analogues of actinomycin which lack the peptide lactone moiety have, however, probably been discouraged by the great decrease in water solubility which is produced by the removal of the non-phenoxazone region of the molecule. Previous experiments in our laboratory have provided useful information on the interaction between DNA and polycyclic aromatic hydrocarbons5,6, which are sparingly soluble in water, through the introduction of a technique based on solubilization phenomena. This method has been used in the present work in order to study the interaction of DNA with two model compounds containing the chromophore group of actinomycin: These compounds were prepared from 2-methoxy-toluic acid (Aldrich) through either five or six steps, respectively7–9. Their solubilities in DNA solutions and in water were determined according to the technique described earlier5,6. Fine crystals of AMD 1 and AMD 2 were equilibrated with solutions of DNA (Sigma, type 1) or with doubly distilled water for 3 days at 25° C by gentle shaking in the dark.

Inhibition by Actinomycin D of Oxidation-dependent Biosynthesis in Sarcoma 37 Ascites Cells

Abstract When Sarcoma 37 ascites cells were incubated in the presence of actinomycin D, protein synthesis was inhibited. The inhibitory effect could be prevented or relieved by glucose or by a variety of glycolyzable substrates. The inhibitory effect was independent of the concentration of the labeled amino acid precursor. Under conditions inhibitory to protein synthesis, no significant inhibition of respiration or of amino acid uptake was observed. Actinomycin D inhibited amino acid incorporation into ribosomal and soluble protein, as well as into soluble ribonucleic acid, and these inhibitory effects were all abolished by the presence of glucose. Three analogues of actinomycin D, which were inactive as inhibitors of RNA synthesis, were inactive as inhibitors of protein synthesis in the present studies. Actinomycin D also inhibited the incorporation of mevalonic acid into sterols, and the inhibition was relieved by glucose. Although the characteristics of these inhibitions suggested an interference with the availability of oxidative energy, measurements of levels of adenine nucleotides and of incorporation of 32P-labeled orthophosphate into ATP failed to show an effect of the antibiotic on oxidative phosphorylation.

The synthesis of actinomycin analogues. II. Actinocylgramicidin S.

The synthesis of an analogue of actinomycin, in which the peptide moiety of the natural antibiotic is replaced by the sequence of gramicidin S, is described.

THE SYNTHESIS OF ACTINOMYCIN ANALOGUES. I. 3-BENZYLOXY-4-METHYL-2-NITROBENZOYL-L-THREONINE AND RELATED COMPOUNDS.

THE SYNTHESIS OF ACTINOMYCIN ANALOGUES. I. 3-BENZYLOXY-4-METHYL-2-NITROBENZOYL-L-THREONINE AND RELATED COMPOUNDS.

Synthesis of Actinomycin Analogs. I.

In the course of investigation on the synthesis of analog of actinomycin, L-threonine, O-benzyl-L-serine, and methyl esters of glycine, DL-alanine, L-threonine, DL-threonine, and O-benzyl-L-serine were acylated with 2-nitro-3-methoxybenzoyl chloride or azide. Some of these products were catalytically hydrogenated to 2-amino-3-methoxybenzoyl compounds. N-(2-Nitro-3-methoxybenzoyl)-glycyl-DL-valine methyl ester was synthesized from N-(2-nitro-3-methoxybenzoyl)-glycyl azide.