Interaction of berberine chloride with deoxyribonucleic acids: evidence for base and sequence specificity

The interaction of berberine chloride with natural and synthetic DNAs of differing base composition and sequences was followed by various spectroscopic and viscometric studies. The binding of berberine chloride was characterized by hypochromism and bathochromism in the absorption bands, enhancement of fluorescence intensity, stabilization against thermal denaturation, perturbations in the circular dichroic spectrum, increase in the contour length of sonicated rod-like DNA and induction of unwinding-rewinding process of covalently closed superhelical DNA, depending on the base composition and sequences of base pairs. Binding parameters determined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicated a very high specificity of berberine to AT-rich DNAs and alternate AT polymer. Fluorescence quantum yield was maximum for the complexes with AT-rich DNAs and alternate AT polymer. Taken together, these results suggest that berberine chloride exhibits considerable specificity towards alternating AT polymer and binds to AT-rich DNAs by a mechanism of classical intercalation.     

A systematic approach toward the analysis of drug-DNA interactions using Raman spectroscopy: the binding of metal-free bleomycins A(2) and B(2) to calf thymus DNA

Bleomycins A(2) and B(2) are the two active components in the antineoplastic drug Blenoxane. DNA is targeted by this drug in cancer cells and the mode of action of this drug involves DNA binding. Ambiguity exists as to the way in which bleomycin binds to DNA. Raman spectroscopy was used to examine both calf thymus DNA and a bleomycin/DNA complex at two temperatures. A curvefitting technique was applied to these spectra for a spectral region obscured by many overlapping bands associated with the nucleotide bases in order to derive information about frequencies, bandwidths, and intensities of the vibrational modes in this region. This allowed identification and analysis of bands associated with specific assigned nucleotide base residues. Upon binding of bleomycin, several significant changes in bandwidth, intensities, and frequencies relative to uncomplexed DNA were observed consistently at both higher (30 degrees C) and lower (19 degrees C) temperature. The data presented here support at least a partial intercalation mode of binding for bleomycin that is temperature dependent and more pronounced at the more physiologically relevant temperature of 30 degrees C.     

Effects of sequence and length on imino proton exchange and base pair opening kinetics in DNA oligonucleotide duplexes.

The base catalysed imino proton exchange in DNA oligonucleotides of different sequences and lengths was studied by 1H-NMR saturation recovery experiments. The self-complementary sequences studied were GCGCGAATTCGCGC (I), CGCGAATTCGCG (II), GCGAATTCGC (III), and CGCGATCGCG (IV). The evaluation of base pair lifetimes was made after correction for the measured 'absence of added catalyst' effect which was found to be characterized by recovery times of 400-500 ms for the AT base pairs and 250-300 ms for the GC base pairs at 15 degrees C. End effects with rapid exchange is noticeable up to 3 base pairs from either end of the duplexes. The inner hexamer cores GAATTC of sequences I-II show similar base pair lifetime patterns, around 30 ms for the innermost AT, 5-10 ms for the outer AT and 20-50 ms for the GC base pairs at 15 degrees C. The shorter sequences III and particularly IV show much shorter lifetimes in their central AT base pairs (11 ms and 1 ms, respectively).     

Is the bithiazole moiety of bleomycin a classical intercalator?

Bleomycin is a widespread anticancerous drug, the biological activity of which having been extensively studied. Its metal ion-chelating portion has been shown to cleave DNA whereas the role of the bithiazole moiety is still questionable. In order to elucidate this problem some 2', 4-disubstituted bithiazoles structurally related to the "tripeptide S" moiety of bleomycin were synthesized and their interaction with DNA was studied using delta Tm, fluorescence, EPR and viscometry techniques. The results of delta Tm and fluorescence quenching determinations were in favour of a binding of the bithiazole part by an intercalation process. Nevertheless, the use of the spin-label probes indicated only a partial intercalation of the ring between the base-pairs. Moreover, viscometry data which clearly exhibited a slight decrease of DNA length in the presence of bithiazole derivative led to the proposal of a binding model involving a partial insertion of a thiazole ring which wedges in between the bases at a bending point of DNA.     

Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA

The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Interactions of molecules with nucleic acids. XII. Theoretical model for the interaction of a fragment of bleomycin with DNA

An intercalation model of a complex between DNA and a bleomycin fragment (BLMF), consisting of the bithiazole core and an amide and a protonated amino substituent, is presented. The model, which shows a preference for BLMF with the protonated amine in the minor groove and the acetyl terminal inserted into either the minor and major grooves, respectively, agrees with recently obtained nmr data. The selection of sites I and II, which have the smallest unwinding of the three theoretical intercalation sites, is consistent with the experimental unwinding angle of 12°. The bithiazole moiety stacks between two base pairs of the double helix, while the protonated substituent interacts ionically with the negatively charged regions of the backbone in the minor groove of the DNA. The protonated amine also forms an intramolecular hydrogen bond with the carbonyl oxygen of the amide group on the same substituent. Analysis of drug complexes with different base-pair sequences reveal four energetically defined groups. The relative energy of the dimer duplex complexes of BLMF correlates with bleomycin's observed base-sequence specificity upon cleavage. The most stable intercalation complexes form adjacent to the bases cleaved most readily. This correlation suggests a primary connection between intercalation and cleavage. A model cleavage site based on these preliminary theoretical calculations and the experimental observations is proposed. It consists of an intercalation site in a trimer duplex. Pyrimidine(p)purine sequences are the predominant sites for intercalation, and the base adjacent to the site at the (3′) end is cleaved.     

1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects

Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.     

Intercalation of bulky Delta,Delta- and Lambda,Lambda-bis-Ru(II) complex between DNA base pairs

Interaction of Δ,Δ- and Λ,Λ-bis-Ru(II) complexes with native DNA was investigated by isotropic absorption and polarized spectroscopy including circular and linear dichroism (CD and LD). Despite the steric hindrance originating from its four bulky phenanthroline ligands at both ends of the molecule, this molecule rapidly intercalates between DNA base pairs. Intercalation was judged by large hypochromism and red shift in the UV-visible absorption spectra in the absorption region of the bridging moiety as well as in the metal-to-ligand-charge transfer absorption region. Further support for the intercalation is found in the fact that the magnitude of negative reduced LD signal in the absorption region of the bridging moiety was comparable to that of the DNA absorption region, indicating that the bridge connecting the two Ru(II) complexes is nearly parallel to the DNA base planes. No difference in the binding mode between the two enantiomers was observed. In the presence of either bis-Ru(II) complex, ethidium bromide, a classical intercalator, can intercalate into the empty sites but was not able to replace the Ru(II) complexes. Near the saturation, ground state interaction between ethidium and bis-Ru(II) complex was evident by LD.     

Intercalation of daunomycin into stacked DNA base pairs. DFT study of an anticancer drug

We have computationally studied the intercalation of the antitumor drug daunomycin into six stacks of Watson-Crick DNA base pairs (i.e., AT-AT, AT-TA, GC-AT, CG-TA, GC-GC, GC-CG) using density functional theory (DFT). The proton affinity of the DNA intercalator daunomycin in water was computed to be 159.2 kcal/mol at BP86/TZ2P, which is in line with the experimental observation that daunomycin is protonated under physiological conditions. The intercalation interaction of protonated daunomycin with two stacked DNA base pairs was studied through a hybrid approach in which intercalation is treated at LDA/TZP while the molecular structure of daunomycin and hydrogen-bonded Watson-Crick pairs is computed at BP86/TZ2P. We find that the affinity of the drug for the six considered base pair dimers decreases in the order AT-AT > AT-TA > GC-AT > GC-TA > GC-CG > GC-GC, in excellent agreement with experimental data on the thermodynamics of the interaction between daunomycin and synthetic polynucleotides in aqueous solution. Our analyses show that the overall stability of the intercalation complexes comes mainly from pi-pi stacking but an important contribution to the computed and experimentally observed sequence specificity comes from hydrogen bonding between daunomycin and hetero atoms in the minor groove of AT base pairs.     

DNA binding properties of the marine sponge pigment fascaplysin

Association of fascaplysin with double-stranded calf thymus DNA was investigated by means of isothermal titration calorimetry, absorption spectroscopy, and circular dichroism. The UV spectroscopic data could be well interpreted in terms of a two-site model for the binding of fascaplysin to DNA revealing affinity constants of K1 = 2.5 x 10(6) M(-1) and K2 = 7.5 x 10(4) M(-1) (base pairs of DNA). Based on the typical change observed in the absorption and circular dichroism spectra, intercalation of fascaplysin is regarded as the major binding mode. The calorimetric titration curves showed an exothermic reaction which was exhausted at a 2:1 base pair/drug; ratio. This finding is in agreement with an intercalation model comprising nearest neighbor exclusion. In addition, significantly weaker non-intercalative DNA interactions can be observed at high drug concentration. By comparison of all these data with the binding behavior of known intercalating agents, it is concluded that fascaplysin intercalates into DNA.     

紫外线B区对小牛胸腺DNA损伤的拉曼光谱研究

用拉曼光谱检测了远紫外区UVB(280m～320nm)对小牛胸腺DNA的损伤,并对这个区段紫外线对DNA的不同照射时间时的损伤特征进行了比较分析。实验中所用的紫外线强度与太阳光强度相当。结果表明,辐照3h以内时,UVB对DNA的构型有较明显的损伤,这可能是受到嘧啶碱基损伤的影响。相对而言,UVB对脱氧核糖和碱基的损伤要严厉得多。就碱基对的受损伤程度来说,嘧啶碱受损最严重,部分证明了环丁烷嘧啶二聚体和6-4光产物的形成。经过3h UVB照射,AT碱基对和和胞嘧啶环的堆叠程度有所瓦解,一些碱基对受到修饰。而一些拉曼特征峰强度的反复波动,则说明了长时间的紫外照射可以导致部分DNA光复活的产生。进一步分析发现,UVB以一种较快的方式对DNA产生损伤。     

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.     

Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd.

Interactions of divalent metal cations (Mg2+, Ca2+, Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) with DNA have been investigated by laser Raman spectroscopy. Both genomic calf-thymus DNA (> 23 kilobase pairs) and mononucleosomal fragments (160 base pairs) were employed as targets of metal interaction in solutions containing 5 weight-% DNA and metal:phosphate molar ratios of 0.6:1. Raman difference spectra reveal that transition metal cations (Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) induce the greatest structural changes in B-DNA. The Raman (vibrational) band differences are extensive and indicate partial disordering of the B-form backbone, reduction in base stacking, reduction in base pairing, and specific metal interaction with acceptor sites on the purine (N7) and pyrimidine (N3) rings. Many of the observed spectral changes parallel those accompanying thermal denaturation of B-DNA and suggest that the metals link the bases of denatured DNA. While exocyclic carbonyls of dT, dG, and dC may stabilize metal ligation, correlation plots show that perturbations of the carbonyls are mainly a consequence of metal-induced denaturation of the double helix. Transition metal interactions with the DNA phosphates are weak in comparison to interactions with the bases, except in the case of Cu2+, which strongly perturbs both base and phosphate group vibrations. On the other hand, the Raman signature of B-DNA is largely unperturbed by Mg2+, Ca2+, Sr2+, and Ba2+, suggesting much weaker interactions of the alkaline earth metals with both base and phosphate sites. A notable exception is a moderate perturbation by alkaline earths of purine N7 sites in 160-base pair DNA, with Ca2+ causing the greatest effect. Correlation plots demonstrate a strong interrelationship between perturbations of Raman bands assigned to ring vibrations of the bases and those of bands assigned to exocyclic carbonyls and backbone phosphodiester groups. However, strong correlations do not occur between the Raman phosphodioxy band (centered near 1092 cm-1) and other Raman bands, suggesting that the former is not highly sensitive to the structural changes induced by divalent metal cations. The structural perturbations induced by divalent cations are much greater for > 23-kilobase pair DNA than for 160-base pair DNA, as evidenced by both the Raman difference spectra and the tendency toward the formation of insoluble aggregates. In the presence of transition metals, aggregation of high-molecular-weight DNA is evident at temperatures as low as 11 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.     

Mixed ligand copper(II) complexes of phenanthroline/bipyridyl and curcumin diketimines as DNA intercalators and their electrochemical behavior under Nafion and clay modified electrodes

The mode of binding of copper(II) mixed ligand complexes of phen/bpy and Knoevenagel condensate of curcumin (4-salicylidene-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) and 4-X-anilines with herring sperm DNA has been investigated using spectral and electrochemical techniques in Tris-HCl buffer pH 7.1. On titration with DNA, usual hypochromism and unusual (large) red shift (30-35 nm) for some of these complexes were observed in their absorption spectra of intense intraligand (IL) pi-pi* transition around 420 nm. Variations in the absorbance due to their interaction with DNA on time scale were also investigated, under fixed concentrations of complex and DNA. On interaction with DNA, the quasi-reversible CuII/I redox couple slightly improves its reversibility with considerable decrease in current intensity. The intercalation of these copper complexes into the DNA base pairs was also investigated by gel retardation assay method. All the experimental results indicate that the phen mixed copper(II) complexes intercalate more effectively into the DNA base pairs than their bpy counterparts. Significant differences in the redox behavior of these copper(II) complexes under electrochemically modified GC electrodes with Nafion and K10 Montmorillonite clay have also been investigated and discussed.     

Selective DNA Binding of (N-alkylamine)-Substituted Naphthalene Imides and Diimides to G+C-rich DNA

Abstract

Alkylamine-substituted naphthalene imides and diimides bind DNA by intercalation and have applications as anticancer agents. The unique structures of these imides in which two adjacent carbonyl groups lie coplanar to an extended aromatic ring system allow the possibility of sequence-selective interactions between the intercalated chromophore and guanine amino groups situated in the DNA minor groove. The binding affinities of N-[3- (dimethylamino)propyl amine]-1,8-naphthalenedicarboxylic imide (N-DMPrNI) and N, N′- bis[3,3′-(dimethylamino)propylamine]-naphthalene-1,4,5,8-tetracarboxylic diimide (N- BDMPrNDI) for natural DNAs of differing base composition were determined spectroscopically and by equilibrium dialysis. In agreement with the above proposition, binding studies indicated that both the naphthalene imide and diimide strongly prefer to intercalate into steps containing at least one G:C base pair. The dependencies of association constants on DNA base composition are consistent with a requirement for one G:C pair in the binding site of the monoimide, and two G:C pairs in binding sites of the diimide. These selectivities are comparable to or exceed that of actinomycin D, a classic G:C-selective drug. Protection footprinting with DNase I confirmed that the naphthalene monoimide (N-DMPrNI) prefers to bind adjacent to G:C base pairs, with a most consistent preference for “mixed” steps containing both a G:C and an A:T pair, excepting GA:TC. Several 5-CG-3′ steps were also good binding sites as indicated by nuclease protection, but few GC:GC or GG:CC steps were protected. The naphthalene diimide inhibited DNase I digestion, but did not yield a footprint. The base recognition ability and versatile chemistry make naphthalene imides and diimides attractive building blocks for design of highly sequence-specific, DNA-directed drug candidates including conjugated oligonucleotides or oligopeptides.     

Investigation of DNA dynamics and drug-DNA interaction by steady state fluorescence anisotropy.

We have used steady-state fluorescence polarization anisotropy (FPA) of ethidium probe molecules bound to DNA to investigate DNA-DNA interactions and the effect of high densities of intercalating drugs on the internal motions of DNA responsible for depolarization of the ethidium fluorescence. To calibrate the method, we examined the effect of DNA length on (FPA) using DNA varying in size from 10-150 base pair. The association of approximately 30 base pair DNA at high concentrations was then detected by its effect on (FPA). With sample concentrations approaching those commonly used in various physical experiments (NMR, Raman) significant DNA-DNA interactions are observed. With high molecular weight DNA (greater than 500 base pair), the limiting value of the (FPA) (0.23) is due to internal motions of the DNA (and bound chromophores). The (FPA) of ethidium probe molecules (1 drug/200 base pair) is unaffected by the addition of high levels (1 drug/2 base pair) proflavine. This indicates that either the elastic properties of DNA are unaffected by high densities of intercalated drug or that the depolarization of the ethidium fluorescence is due to highly localized motions of the base pairs that are unperturbed by binding of drugs at neighboring sites.     

Classification of CD and absorption spectra in the Soret band of H(2)TMPyP bound to various synthetic polynucleotides

The binding mode of porphyrins, namely meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (H(2)TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H(2)TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.     

Glycosaminoglycan and DNA Binding Induced Intra‐ and Intermolecular Exciton Coupling of the bis‐4‐Aminoquinoline Surfen

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High‐affinity (K_a ~ 10⁷ M^‐1) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right‐handed heparin template, binding of surfen to the left‐helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf‐thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists. Chirality 27:605–612, 2015. © 2015 Wiley Periodicals, Inc.