Synthesis and CMR analysis of phenoxazinone derivatives related to the actinomycin D chromophore

Synthesis of 2-deamino- and 2-deamino-2-nitro-chromophores of actinomycin was accomplished by reaction of nitrous acid in fluoroboric acid via diazotization of the 2-amino group in the chromophore. Structural assignments were made by cmr and pmr.     

Synthesis of analogs of actinomycin

D, L-Valine derivatives of actinomycin and its chlorine analog have been synthesized. The replacement of the methyl groups in positions 4 and 6 of the chromophore of actinomycin by chlorine has practically no effect on the absorption spectrum of compounds of this class.For part I, see [5].     

Synthesis of analogs of actinomycin

Chlorine-containing analogs of the actinomycin chromophore-2-amino-4, 6-dichloro-3-phenoxazone-1,9-dicarboxylic acid and its methyl ester-have been synthesized.     

Synthesis of some simple actinomycin analogs

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

Synthesis of actinomycin D analogs: XXIII. Actinocin derivatives containing azacrown fragments

A number of actinocin amides containing residues of aza-15-crown-5 and aza-18-crown-6 where crown fragments were separated from the heterocyclic chromophore by the residues of ω-amino acids were obtained as actinomycin D models.     

On the formation of “symmetrical” phenoxazinone derivative related to the actinomycin D chromophore

In studies related to the synthesis of 2-deamino chromophores of actinomycin by reaction of nitrous acid in fluoroboric acid via diazotization of the 2-amino group in the chromophore, the unknown fluorescent product is identified as the symmetrical phenoxazinone by cmr and pmr.     

Synthesis and photochemistry of pH-sensitive GFP chromophore analogs

GFP chromophore analogs (7a-e, 8, and 10a,b) containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-methyl-2-pyridyl-groups were synthesized and their fluorescence spectra recorded in the pH range 1-7. NMR studies showed that protonation of 8 (2-thienyl system) inhibited photoisomerization (Z-E) about the exocyclic double bond but that protonation of 7c (E + Z) (2-pyrryl system) gave only 7cE. Fluorescence studies revealed enhancement of fluorescence intensity of 7c and 7b,e (furyl system) below pH 2.5 and gave a similar result for 10a (pyridyl system) below pH 6. Quantum yields at pH 1 were low, probably due to excited state proton transfer (ESPT).     

N-substitution in the 2-amino- and 2,7-diaminophenoxazin-3-one ring system. Application to the preparation of actinomycin D analogs

A method is described for the direct N²-monoalkylation of the 2-aminophenoxazin-3-one system by treatment with dimethylsulfonium methylide in DMSO-THF, which acts as a base by abstracting a proton from the 2-amino function to form a stabilized anion, which is then alkylated by reaction with an alkyl halide. Selective N⁷-monoalkylation of the 2,7-diaminophenoxazin-3-one system can be accomplished by reaction with aromatic aldehydes in glacial acetic acid to give Schiff bases, which are then reduced with dimethylamine borane. These reactions have been applied to the preparation of N²-benzyl- and 7-benzylaminoactinomycin D.     

Facile synthesis of 4-arylidene-5-imidazolinones as synthetic analogs of fluorescent protein chromophore

A facile and effective synthesis for a wide variety of 4-arylidene-5-imidazolinone derivatives was developed. 4-Arylidene-5-oxazolinones were prepared by Erlenmeyer azlactone synthesis from N-acylglycines and arylaldehydes. The ring-opening reactions of the 4-arylidene-5-oxazolinones with primary amines afforded 2-acylamino-3-arylacrylamides in excellent yields. A new dehydrative cyclization of the 2-acylamino-3-arylacrylamides in pyridine under reflux furnished the corresponding 4-arylidene-5-imidazolinones in good yields.     

Structure–affinity relationships for the binding of actinomycin D to DNA

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin–d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino- purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure–affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in the design of novel sequence-specific DNA-binding agents.     

Excited state properties of the chromophore of the asFP595 chromoprotein: 2D and 3D theoretical analyses

The ground and excited state properties (e.g., the intramolecular charge and energy transfer, and electron‐hole coherence) of the chromophore of the asFP595 chromoprotein from Anemonia sulcata in the neutral and anionic forms are theoretically studied with quantum chemistry methods. The ground‐state properties of the asFP595 in the neutral and anionic forms, such as the alternations of the bond lengths and the Mulliken charge distributions, are compared. The calculated transition energies of the asFP595 in the neutral and anionic form are consistent with the experimental results. To study the excited state properties of the asFP595 chromophore, the energies and densities of highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs), as well as the CI main coefficients, are compared between the two forms. The intramolecular charge and energy transfer in the neutral and anionic forms are investigated and compared with the three‐dimensional (3D) real‐space analysis methods, including the strength and orientation of the transition dipoles with transition density, and the orientation and result of the intramolecular charge transfer with charge difference density. The electron‐hole coherence and delocalization on the excitation are studied with the 2D real‐space analysis method of the transition density matrix. In all, the calculated results are remain in good agreement with the experimental data, and the theoretical analysis results supported the proposed models in the experiment. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

EPR study on the photosensitized generation of reactive oxygen species by actinomycin D

Actinomycin D (AMD) is an anticancer antibiotic that can bind selectively to both double-stranded and single-stranded DNA, and this binding greatly enhances DNA photosensitization. Using electron paramagnetic resonance (EPR) in combination with spin trapping techniques, a systematic study was carried out on the reactive oxygen species generated in the photosensitization process of AMD. It was found that 1O2 and O2- are important reactive intermediates either insolution or in DNA complexes, and the generation of these species is in competition. This finding suggests that the photodynamic action of AMD proceeds via two pathways: energy transfer (type Ⅰ mechanism) and electron transfer (type Ⅱ mechanism). 1O2 is the main product formed via energy transfer reaction in solution while electron transfer between the excited states of AMD and DNA becomes the predominant pathway in DNA complexes.     

Excited state relaxation dynamics of model green fluorescent protein chromophore analogs: evidence for cis-trans isomerism

Two green fluorescent protein (GFP) chromophore analogs (4Z)-4-(N,N-dimethylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DMPI) and (4Z)-4-(N,N-diphenylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DPMPI) were investigated using femtosecond fluorescence up-conversion spectroscopy and quantum chemical calculations with the results being substantiated by HPLC and NMR measurements. The femtosecond fluorescence transients are found to be biexponential in nature and the time constants exhibit a significant dependence on solvent viscosity and polarity. A multicoordinate relaxation mechanism is proposed for the excited state relaxation behavior of the model GFP analogs. The first time component (τ(1)) was assigned to the formation of twisted intramolecular charge transfer (TICT) state along the rotational coordinate of N-substituted amine group. Time resolved intensity normalized and area normalized emission spectra (TRES and TRANES) were constructed to authenticate the occurrence of TICT state in subpicosecond time scale. Another picosecond time component (τ(2)) was attributed to internal conversion via large amplitude motion along the exomethylenic double bond which has been enunciated by quantum chemical calculations. Quantum chemical calculation also forbids the involvement of hula-twist because of high activation barrier of twisting. HPLC profiles and proton-NMR measurements of the irradiated analogs confirm the presence of Z and E isomers, whose possibility of formation can be accomplished only by the rotation along the exomethylenic double bond. The present observations can be extended to p-HBDI in order to understand the role of protein scaffold in reducing the nonradiative pathways, leading to highly luminescent nature of GFP.     

Retention behaviour of crown ethers and actinomycin D in reversed-phase HPLC

Summary Retention of crown ethers in reverse-phase HPLC has been determined by their bonding ability with cations present in the eluent. The dependence of retention of crown ethers on cation concentration exhibits an inflection and makes it possible to calculate stability constant for the crown ether-cation complex. It is shown that in 75% MeOH retention of antitumor antibiotic, actinomycin D, depends on [Na⁺] and not on [K⁺] at concentrations of K⁺ from 5×10^–7 to 10^–1 mol l^–1. Hence, actinomycin D may be classified as an ionophore-antibiotic.     

New analogs of D-homoequilenine with substituents in the D ring

Stereoselectivity of reaction with Raney nickel of D-homoestra-1,3,5(10),8,14-pentaenes containing one or two methyl groups in position 16 was investigated. The reaction direction is governed by the orientation of the substituent at C^17a. The signals in the ¹H and ¹³C NMR spectra of four synthesized compounds were completely assigned. Criteria for evaluation of the character of rings junction in analogs of D-homoequilenine were suggested. 16,16-Dimethyl-3-methoxy-D-homo-13α-estra-1,3,5(10),6,8-pentaen-17a-one was subjected to X-ray diffraction analysis.     

Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor

Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.     

First total synthesis of versicotide D and analogs

The first total synthesis of cyclotetrapeptide versicotide D has been achieved in 21% overall yield using solid phase peptide synthesis and solution cyclization. In addition, in the search for candidates of antimalarial new drugs, one cyclic tetrapeptide analog which differs in the sequence, and four cyclic pentapeptide containing N-methyl amino acids, were prepared. The obtained compounds were evaluated against P. falciparum 3D7. Versicotide D showed low micromolar antiplasmodial activity.     

A 13C NMR study of actinomycin D and related model peptides

¹³C n.m.r. pulsed Fourier transform spectra were measured and interpreted for actinomycin D and for two related peptide derivatives: BOC-Pro-Sar-OCH₃ and BOC-Sar-Meval-OCH₃. Actinomycin D, specifically enriched with ¹³C in the chromophoric C-methyl and in the peptide N-methyl carbons, was produced biosynthetically. Enrichment of specific N-methyl carbons in the model peptides was effected synthetically. Spectral assignments relied on the use of the enriched samples and particularly on off-resonance and selective low power ¹H decoupling experiments. Poor correlation was observed between some of the ¹³C chemical shifts in the model compounds and those of the analogous carbons in actinomycin D. Ten of our ¹³C assignments for actinomycin D differ from those published by Hollstein, Breitmaier and Jung.     

Antifungal effects of actinomycin D on Verticillium dahliae via a membrane-splitting mechanism

Antifungal bioassays led to the isolation of actinomycins D and A₁ from Streptomyces luteus TRM45540 collected from Norpo in Xinjiang, and these compounds were identified by nuclear magnetic resonance spectroscopy. The antifungal activity of actinomycin D was higher than that of actinomycin A₁. Actinomycin D clearly inhibited the spore germination, hyphal growth and biomass accumulation of Verticillium dahliae in a dose-dependent manner. Flow cytometric analysis with propidium iodide, total ergosterol measurement, cell leakage and scanning electron microscopy experiments demonstrated that the plasma membrane of this fungus was damaged by actinomycin D, resulting in swollen cells and cellular content leakage. Transmission electron microscopy revealed that parts of the plasma membrane infolded after being treated with actinomycin D. The antifungal activity of actinomycin D damaged the fungal plasma membrane of V. dahliae via a membrane-splitting mechanism, which provided new insights into the functional mechanism of actinomycin D.     

On the chromophore of polyacrylonitrile

Further evidence is presented to show that the chromophore of polyacrylonitrile is formed by polymerization of the nitrile groups, rather than by dehydrogenation of the polymer backbone. The polyimine structure formed is resistant to hydrolysis; hence, the nitrogen content of hydrolyzed polymer increases with increased chromophore formation.