Theoretical CD studies of polypeptide helices: examination of important electronic and geometric factors

An improved model for calculating the CD of polypeptides has been developed. Excited state wavefunctions were derived from CNDO/S (complete neglect of differential overlap, spectroscopic) calculations on N-methylacetamide. Four discrete peptide-localized transitions were employed: pi 0 pi* (NV1), pi* + pi* (NV2), n pi*, and n' pi*. Inclusion of the pi + pi transition (lambda 0 = 140 nm) significantly improves the accuracy of the calculated CD spectra in the 180-250-nm region. Spectra were computed for various helical structures, including right-handed alpha-, alpha II-, omega-, pi-, 3(10-), and poly (proline) I-helices, and the left-handed poly (proline) II-helix. Sensitivity to changes in the peptide backbone geometry and chain length are examined. Electronic factors such as ground-state charge distribution, hybridization effects, and basis set deorthogonalization have been investigated. The nonconservative nature of the poly (Pro) I and II CD spectra is reproduced, and the helix band present in earlier exciton calculations on the alpha-helix has been diminished.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.     

Refractive index effects on the oscillator strength and radiative decay rate of 2,3-diazabicyclo[2.2.2]oct-2-ene.

The photophysical properties of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) were determined in 15 solvents, two supramolecular hosts (cucurbit[7]uril and beta-cyclodextrin) as well as in the gas phase. The oscillator strength and radiative decay rate of DBO as a function of refractive index i.e. polarizability have been analyzed. The oscillator strength increases by a factor of 10 upon going from the gas phase to the most polarizable carbon disulfide, while the corresponding radiative decay rates increase by a factor of 40. There is a good empirical correlation between the oscillator strength of the weakly allowed n,pi* transition of DBO and the reciprocal bulk polarizability, which can be employed to assess the polarizability of unknown microheterogeneous environments. A satisfactory correlation between the radiative decay rate and the square of the refractive index is also found, as previously documented for chromophores with allowed transitions. However, the correlation improves significantly when the oscillator strength is included in the correlation, which demonstrates the importance of this factor in the Strickler-Berg equation for chromophores with forbidden or weakly allowed transitions, for which the oscillator strength may be strongly solvent dependent. The radiative decay rate of DBO in two supramolecular assemblies has been determined, confirming the very low polarizability inside the cucurbituril cavity, in between perfluorohexane and the gas phase. The fluorescence quantum yield of DBO in the gas phase has been remeasured (5.1 +/- 0.5%) and was found to fall one full order of magnitude below a previously reported value.     

UV Raman studies of peptide conformation demonstrate that betanova does not cooperatively unfold.

We used UV resonance Raman spectroscopy (UVRR) excited within the peptide bond pi --> pi* electronic transitions and within the aromatic amino acid pi --> pi* electronic transitions to examine the temperature dependence of the solution conformation of betanova, a 20-residue beta-sheet polypeptide [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256]. The 206.5 nm excited UVRR enhances the amide vibrations and demonstrates that betanova has a predominantly beta-sheet structure between 5 and 82 degrees C. The 229 nm excited UVRR, which probes the tyrosine and tryptophan side chain vibrations, shows an increase in the solvent exposure of the tryptophan side chains as the temperature is increased. Our results are consistent with the existence of an intermediate state similar to that calculated by Bursulaya and Brooks [Bursulaya, B. D., and Brooks, C. L. (1999) J. Am. Chem. Soc. 121, 9947-9951] and exclude the previously proposed two-state cooperative folding mechanism. Betanova's structure appears to be molten globule over the 3-82 degrees C temperature range of our study.     

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.     

Denaturation of Nucleic Acids Induced by Intercalating Agents. Biochemical and Biophysical Properties of Acridine Orange-DNA Complexes

Abstract

At high binding denstities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar-and base-composition) of nucleic acids.     

Formation of a DNA monofunctional cis-platinum adduct cross-linking the intercalating drug N-methyl-2,7-diazapyrenium.

Our purpose was to better understand the mutual influence of cis-diamminedichloroplatinum (II) (cis-DDP) and intercalating drugs in their interactions with DNA. The present study deals with the intercalating drug N-methyl-2,7-diazapyrenium (MDAP). Two sets of experiments have been performed. In one set, the reaction between cis-DDP and nucleic acid was carried out in the presence of MDAP. The main adduct is a guanine residue chelated by platinum to a MDAP residue. It has the same spectroscopic properties as the synthesized compound cis-[Pt (NH3)2 (N7-d-guanosine) (N7-MDAP)] , the structure of which has been determined by 1H NMR. This adduct was only formed with double-stranded nucleic acids which reveals the importance of DNA matrix in orienting favorably the reactants. In the second set of experiments, the triamine complex cis-[Pt(NH3)2 (MDAP)CI]++ was reacted with the nucleic acids. At molar ratios drug over nucleotide residue equal or less than 0.10, all the added triamine complexes bind by covalent coordination to double-stranded nucleic acids. With natural DNA, the major adduct is cis-[Pt(NH3)2(d-guanosine) (MDAP)] . Thus the same adduct is formed on one hand in the reaction between DNA, MDAP and cis-DDP and on the other hand in the reaction between the triamine complex and DNA. The triamine complex offers the possibility to study the biological role of the new adduct.     

Interactions between purine derivatives: Electronic spectral studies. II. Excition interactions in the dimer of 6-methylpurine in aqueous solution

From a study of the concentration dependence of the ultraviolet absorption spectra of aqueous solutions of 6-methylpurine, the spectra of monomeric and dimeric species have been derived. The dichroic spectrum of 6-methylpurine in stretched poly(vinyl alcohol) films has been determined. The absorption envelope of 6-methylpurine up to 44,000 cm^?1 appears to include a low-intensity nitrogen (π*,n) transition at the low wavenumber tail and two additional electronic transitions of dominant intensities. Both the experimental dichroic ratios and theoretical calculations suggest that the letter two transitions are in-plane (π*,π) types. Analysis of the dimer species spectrum in terms of exciton interactions between molecules in “sandwich” conformation has been carried out. The interaction between the moments of the lowest energy (π*,π) transitions of the molecule in the dimer seems to result in transfer rates of energy of the order of 10¹³ sec^?1 between the two molecules.     

Circular dichroism of nucleic acid monomers. II. Derivation and application to polymers of a consistent set of guanine and cytosine transition parameters

An approximate semiempirical procedure has been developed in order to derive nucleic acid monomer π → π* electronic transition moment parameters. Using the approximate procedure, guanine (G) and cytosine (C) transition moment parameters have been derived from agreement found between calculated weight-averaged and measured CD spectra of cyclic-GMP and cyclic-CMP. The derived base transition moment parameters have been assessed in CD spectral calculations on some G- and C-containing nucleic acids for which reasonably good structural information exists. An attempt was also made at evaluating the likely CD spectral contributions of G and C electric n → π* transition moments whose magnitudes were taken to be the maximum expected. Overall, the results indicate that the derived G and C π → π* transition moment parameters are more successful in nucleic acid CD spectral calculations than those used in previous DeVoe theory CD calculations. In addition, the results indicate that electric n → π* transitions may be of importance in understanding nucleic acid monomer CD spectra but appear to be relatively unimportant in understanding nucleic acid polymer CD spectra. It is concluded that the derived G and C π → π* parameters are more useful in DeVoe theory CD calculations than parameters used previously.     

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.     

Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

Two G-quadruplex forming sequences, 5'-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5'-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge-charge repulsion. Contrary to that, we found that attachment of the 5'-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation-forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed.     

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin--an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe(2)(CO(3))(2)BTf], [Fe(2)(C(2)O(4))(2)BTf], [Cu(2)(CO(3))(2)BTf] and [Cu(C(2)O(4))BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal-oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C(2)O(4))BTf], the mixed-anion complexes [Cu(2)(CO(3))(C(2)O(4))BTf] and [Fe(2)(CO(3))(C(2)O(4))BTf] and the mixed-metal complex [FeCu(C(2)O(4))(2)BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p(pi)-d(pi*) CT band for iron(III)-BTf and the hypsochromic shift of the p(pi)-d(sigma*) CT band for copper(II)-BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin--a serum transferrin--very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.     

Effects of the substitution positions of Br group in intercalative ligand on the DNA-binding behaviors of Ru(II) polypyridyl complexes

Two new polypyridyl ligands containing substituent Br at different positions in the phenyl ring, PBIP [PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline], OBIP [OBIP=2-(2-bromophenyl)imidazo[4,5-f]1,10-phenanthroline] and their Ru(II) complexes, [Ru(phen)2PBIP]2+ 1, [Ru(phen)2OBIP]2+ 2 (phen=1,10-phenanthroline), have been synthesized and characterized. The binding strength of the two complexes to calf thymus DNA (CT DNA) was investigated with spectrophotometric methods, viscosity measurements, as well as equilibrium dialysis and circular dichroism spectroscopy. The theoretical calculations for these two complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that the Br group substituting H at different positions of the phenyl ring in the intercalated ligand has significant effects on the spectral properties and the DNA-binding behaviors of Ru(II) complexes. Both the complexes can bind to CT DNA in intercalative mode and interact with CT DNA enantioselectively. Moreover, complex 1 can bind to CT DNA more strongly than complex 2, and complex 2 can become a much better candidate as an enantioselective binder to CT DNA than complex 1. The theoretical calculations show that both intercalative ligands, PBIP and OBIP, in these two complexes are essentially planar, and the obtained electronic structures of the complexes can be used to explain reasonably some of their experimental regularities or trends. Such experimental and theoretical information will be useful in design of novel probes of nucleic acid structures.     

Calculation of translational friction and intrinsic viscosity. I. General formulation for arbitrarily shaped particles.

A general method for calculating translational friction and intrinsic viscosity is developed through exploiting relations between hydrodynamics and electrostatics. An approximate relation xi = 6 pi eta 0C between the translational friction coefficient xi of a particle (eta 0: solvent viscosity) and its capacitance C was derived previously. This involved orientationally preaveraging the Oseen tensor, but the result was found to be very accurate. Based on preaveraging, we find that the intrinsic viscosity [eta] of a particle can be estimated from its polarizability alpha through [eta] = 3/4 alpha + 1/4 Vp, where Vp is the volume of the particle. Both the capacitance and the polarizability can be obtained in a single calculation using the boundary-element technique. An efficient approach is thus found for estimating [eta], a quantity that is very useful in practice because of its sensitivity to particle shape but is notoriously difficult to calculate. Illustrative calculations on ellipsoids, cylinders, and dumbbells demonstrate both the accuracy of the approximate relations and the efficiency of the present method.     

Alkyl phenols and derivatives from Tapirira obtusa

From the bark of Tapiria obtusa, six alkyl phenol derivatives were isolated: 1-hydroxy-3-[(Z)-7'-nonadecenyl]-benzene, 1-hydroxy-3-[(Z)-7'-heptadecenyl]-benzene, 1-hydroxy-3-[14'-phenyltetradecyl]-benzene, and 1-hydroxy-3-[16'-phenyltetradecyl]-benzene, and their possible biogenetic precursors, 1-(16'-phenyl-12'Z-hexadecenyl)-4-Z-cyclohexene-(1S*,3S*)-diol and (4S*,6S*)-dihydroxy-6-(14'Z-nonadecenyl)-2-cyclohexenone. The structures of these compounds were elucidated by chemical and spectroscopic analysis, (4S*,6S*)-Dihydroxy-6-(14'Z-nonadecenyl)-2-cyclohexenone showed cytotoxic activity.     

A comparison of the intercalative binding of non-reactive benzo[a]pyrene metabolites and metabolite model compounds to DNA

The reversible DNA physical binding of a series of non-reactive metabolites and metabolite model compounds derived from benzo[a]pyrene (BP) has been examined in UV absorption and in fluorescence emission and fluorescence lifetime studies. Members of this series have steric and pi electronic properties similar to the highly carcinogenic metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and the less potent metabolite 4,5-epoxy-4,5-dihydrobenzo(a)pyrene (4,5-BPE). The molecules examined are trans-7,8-dihydroxy-7,8-dihydrobenzo[a]-pyrene (7,8-di(OH)H2BP), 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (tetrol) 7,8,9,10-tetrahydrobenzo[a]pyrene (7,8,9,10-H4BP), pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (4,5-di(OH)H2BP) and 4,5-dihydrobenzo[a]pyrene (4,5-H2BP). In 15% methanol at 23 degrees C the intercalation binding constants of the molecules studied lie in the range 0.79-6.1 X 10(3) M-1. Of all the molecules examined the proximate carcinogen 7,8-di(OH)-H2BP is the best intercalating agent. The proximate carcinogen has a binding constant which in UV absorption studies is found to be 2.8-6.0 times greater than that of the other hydroxylated metabolites. Intercalation is the major mode of binding for 7,8-di(OH)H2BP and accounts for more than 95% of the total binding. Details concerning the specific role of physical bonding in BP carcinogenesis remain to be elucidated. However, the present studies demonstrate that the reversible binding constants for BP metabolites are of the same magnitude as reversible binding constants which arise from naturally occurring base-base hydrogen bonding and pi stacking interactions in DNA. Furthermore, previous autoradiographic studies indicate that in human skin fibroblasts incubated in BP, pooling of the unmetabolized hydrocarbons occurs at the nucleus. The high affinity of 7,8-di(OH)H2BP for DNA may play a role in similarly elevating in vivo nuclear concentrations of the non-reactive proximate carcinogen.     

Three sesquiterpene hydrocarbons from the roots of Panax ginseng C.A. Meyer (Araliaceae)

The volatile constituents of the roots of Panax ginseng C.A. Meyer have been investigated after hydrodistillation and analysed by means of different analytical methods. Besides several compounds already known three sesquiterpene hydrocarbons have been isolated from the essential oil. Structure elucidation of the bicyclic panaxene as well as of the tricyclic panaginsene and ginsinsene was performed by MS and NMR. They have been identified as (1R*,2S*,5S*)-2-ethenyl-1(1-methylethenyl)-2,6,6-trimethylbicyclo[3.2.0]heptane (panaxene), (1S*,8S*,11R*)-4,7,7,11-tetramethyltricyclo[6.3.0.0(1,5)]undec-4-ene (panaginsene) und (1R*,6R*,7R*)-3,7,10,10-tetramethyltricyclo[4.3.2.0(2,6)]undec-2-ene (ginsinsene).     

Interactions between purine derivatives: Electronic spectral studies. I. Electronic transitions in caffeine monomer and exciton coupling in caffeine dimer

The concentration dependence of the ultraviolet absorption spectrum of aqueous solutions of caffeine has been studied. Individual species spectra have been derived for the monomer, dimer, and tetramer of caffeine. The emission spectrum of caffeine in aqueous solution and the dichroic spectra in oriented poly(vinyl alcohol) and polyethylene films have been measured. The long-wavelength tail of the absorption spectrum of caffeine in non-polar environment has been found to incorporate at least one carbonyl(π*, n) transition. Dichroic spectral data and molecular orbital calculations have been used to assign transition moment directions to the (π*,π) transitions. The lowest energy (π*,π) transition, responsible for the near-ultraviolet absorption peak in aqueous solution of caffeine, has been used for the study of degenerate exciton interactions in the dimeric species of caffeine. Assuming that the caffeine molecules in the dimer are stacked in parallel planes, theoretical calculations of the ground-state interactions and of the degenerate exciton interactions have been combined with experimental data and a unique model for the dimer of caffeine has been derived. The transfer rate of energy between the molecules in the dimer is of the order of 10¹³_S^?1.