Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine

2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems. To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments. The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T. Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS). This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them. Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated. At pH near 4, the majority of species shows C protonated and P deprotonated. Finally, at pH values near 3, the majority of species shows both protonated C and P. These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence including the P x T mismatch, only one transition was observed at neutral pH. However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.     

Three-way multivariate curve resolution applied to speciation of acid-base and thermal unfolding transitions of an alternating polynucleotide.

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.     

Resolution of parallel and antiparallel oligonucleotide triple helices formation and melting processes by multivariate curve resolution

A procedure is described for the complete resolution of concentration profiles of oligonucleotide triplexes as a function of pH and temperature. The pH and temperature ranges at which triplexes are present and the relative concentrations of all the species involved in acid-base and conformational equilibria are successfully estimated from Multivariate Curve Resolution analysis of UV absorbance spectra recorded along acid-base titrations and melting experiments of single stranded, hairpin and their mixtures. The dependence of formation constants upon pH was successfully estimated. The hairpin h26 (5'-GAAGGAGGAGA-TTTT-TCTCCTCCTTC-3'), and the single stranded oligonucleotides s11CT (5'-CTTCCTCCTCT-3'), s11AG (5'-AGAGGAGGAAG-3') and s11TG (5'-TGTGGTGGTTG-3') were synthesized and their protonation and conformational equilibria were studied in detail. The procedure was shown to be especially useful for the study of triplexes with a low hypochromism upon formation.     

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-β-cyclodextrin

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy was synthesized by C₆-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.     

Design of histidine containing peptides for better understanding of their coordination mode toward copper(II) by CD spectroscopy

The systematic investigation of the copper(II) complexes of tripeptides Xaa-Xaa-His, Xaa-His-Xaa and His-Xaa-Xaa, where Xaa=Gly or Ala was performed by combined pH-metry, spectrophotometry, CD and in part EPR spectroscopy. The matrix rank analysis of the spectral data revealed the number of the coloured and optically active species as a basis for the solution speciation. A critical evaluation on the speciation and solution structure of the complexes formed is presented on the basis of their d-d band optical activity. The replacement of a Gly residue with the chiral Ala amino acid allowed us to gain decisive information on the solution structure of the complexes by CD spectroscopy. It was shown that the tripeptides with histidine in the third position formed CuH(-2)L species with (NH(2), 2N(-), ImN - where Im stands for imidazole) coordination sphere as a major species, and only the macrochelated CuL complexes as minor species around pH 5.0. In copper(II)-Xaa-His-Xaa tripeptide systems the CuH(-1)L (NH(2), N(-), ImN) is the most stable species at physiological pH, but the vacant fourth site around copper(II)ions is offered for further deprotonation, most probably resulting in mixed hydroxo species at low (<5 x 10(-4)M) metal ion concentrations, while a tetrameric complex is dominant when the copper concentration exceeds 3 x 10(-3)M. The histamine type coordination mode in CuL and CuL(2) complexes of His-Xaa-Xaa ligands predominates at low pH. The structural consequences drawn from the CD spectra for the mono and bis parent complexes were supported by theoretical calculations. CD spectra strongly suggest the participation of the imidazole nitrogen both in the Cu(2)H(-2)L(2) and CuH(-2)L complexes.     

High resolution derivative denaturation profiles of DNA in the presence of copper(II) ions

Derivative denaturation profiles of calf thymus DNA in the presence of copper(II) ions have been directly obtained from high resolution thermal denaturation profiles recorded in an isoabsorbance wavelength of the AT and GC hyperchromic spectra. The analysis of the very sensitive profiles provides further evidence that the melting temperature (Tm) of DNA decreases in the presence of stoichiometric ratio of copper(II) ions to nucleotide. Also, evidence is given of peculiar behaviour at higher temperatures where a new melting transition is observed. This phenomenon could be in line with the presence of bridging of DNA single strands by copper ions which are disrupted when the temperature is raised.     

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 degrees C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 microM. At higher concentrations of copper(II) ions (c>700 microM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.     

A new, model-free calculation method to determine the coordination modes and distribution of copper(II) among the metal binding sites of multihistidine peptides using circular dichroism spectroscopy

A new calculation method to determine microscopic protonation processes from CD spectra measured at different pH and Cu(II):ligand ratios was developed and used to give the relative binding strengths for the three histidines of hsPrP(84-114), a 31-mer polypeptide modeling the N-terminal copper(II) binding region of human (homo sapiens) prion protein. Mutants of hsPrP(84-114) with two or one histidyl residues have also been synthesized and their copper(II) complexes studied by CD spectroscopy. The 1-His models were analyzed first, and the molar CD spectra for the different coordination modes on the different histidines were calculated using the general computational program PSEQUAD. These spectra were deconvoluted into the sum of Gaussian curves and used as a first parameter set to calculate the molar spectra for the different coordination modes (3N and 4N coordination) and coordination positions (His85, His96 and His111) of the 2-His peptides. The calculation method therefore does not require the direct use of CD spectra measured in the smaller peptide models. This is a significant improvement over earlier calculation methods. In the same runs, the stepwise deprotonation pK(mic) values were refined and the pH-dependent distribution of copper(II) between the two histidines was determined. The results revealed the high, but different copper(II) binding affinities of the three separate histidines in the following order: His85 < His96His111. The calculation also showed that molar CD spectra which belong to the same coordination mode and coordination position in different ligands have very similar transition energies but different intensities. For this reason, direct transfer of molar CD spectra between different ligands may be a source of error, but the pK(mic) values and the copper(II) binding preferences are transferable from the 2-His peptides to the 3-His hsPrP(84-114).     

Systematical investigation of binding interaction between novel ruthenium(II) arene complex with curcumin analogs and ctDNA

In this study, the interaction between a novel ruthenium(II) arene complex with curcumin analogs and calf thymus DNA (ctDNA) was investigated systematically by viscosity measurement, the DNA melting approach, multispectroscopic techniques and electrochemical methods. The absorption spectra of the ctDNA–drug complex showed a slight red shift and a weak hypochromic effect. The relative viscosity and melting temperature of ctDNA increased on addition of the drug. The evidence obtained from fluorescence competitive experiments indicated that the binding mode of the drug with ctDNA was intercalative. Using acridine orange (AO) as a fluorescence probe, the drug statically quenched the fluorescence of the ctDNA–AO complex, and hydrogen bonding and van der Waals interactions played vital roles in the binding interaction between the drug and ctDNA. The influences of ionic strength, chemical denaturants and pH on the binding interaction were also investigated. Circular dichroism and Fourier transform infrared spectra suggested that this drug might bond with the G–C base pairs of ctDNA and the right‐handed B‐form helicity of ctDNA remained after drug binding. The intercalative binding between the drug and ctDNA was further investigated using electrochemical techniques. All these results suggested that the biological activity of ctDNA was affected by ruthenium(II) arene complex with curcumin analogs. Copyright © 2016 John Wiley & Sons, Ltd.     

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

Copper(II) complexation by D-glucosamine. Spectroscopic and potentiometric studies

The Cu(II) complex formation equilibria of D- glucosamine were studied in aqueous solution by potentiometric and spectroscopic (ESR, CD, absorption spectra) techniques. All data agree that two major species are formed in the pH region 6–9 involving two D-glucosamine ligand molecules bound to the cupric ion via NH₂(CuL₂) or NH₂ and O^? (CuH_?2L₂). In the latter case deprotonated hydroxyls were found to be very effective coordination sites for Cu(II) giving rise to chelate complexes. On the contrary, no complex formation was observed for the Cu(II) N-acetyl-D-glucosamine system.     

Multispectroscopic studies on the interaction of a copper(ii) complex of ibuprofen drug with calf thymus DNA

The interaction of copper(II)–ibuprofenato complex with calf thymus DNA (ct-DNA) has been explored following, UV-visible spectrophotometry, fluorescence measurement, dynamic viscosity measurements, and circular dichroism spectroscopy. In spectrophotometric studies of ct-DNA it was found that [Cu(ibp)₂]₂ can form a complex with double-helical DNA. The association constant of [Cu(ibp)₂]₂ with DNA from UV-Vis study was found to be 6.19 × 10⁴ L mol^?1. The values of K_f from fluorescence measurement clearly underscore the high affinity of [Cu(ibp)₂]₂ to DNA. The experimental results showed that the conformational changes in DNA helix induced by [Cu(ibp)₂]₂ are the reason for the fluorescence quenching of the DNA-Hoechst system. In addition, the fluorescence emission spectra of intercalated methylene blue (MB) with increasing concentrations of [Cu(ibp)₂]₂ represented a significant increase of MB intensity as to release MB from MB-DNA system. The results of circular dichroism (CD) suggested that copper(II)–ibuprofenato complex can change the conformation of DNA. In addition, the results of viscosity measurements suggest that copper(II)–ibuprofenato complex may bind with non-classical intercalative mode. From spectroscopic and hydrodynamic studies, it has been found that [Cu(ibp)₂]₂ interacts with DNA by partial intercalation mode which contains intercalation and groove properties.     

A soft-modeling approach to interpret thermodynamic and conformational transitions of polynucleotides

Multivariate outputs from the experimental monitoring of biochemical processes are usually difficult to interpret applying methods based on a priori chemical models. Curve resolution methods are model-free procedures, generally known as soft-modeling methods, which obtain the concentration profiles and instrumental responses of each individual species involved in a multivariate monitored process without making any kind of external assumption. Of the curve resolution methods available, the alternating least squares (ALS) is proposed here because of its ability to operate on one or on several matrices. Furthermore, ALS allows the introduction of information related to the internal data structure and to the general features of the concentration profiles and instrumental responses through the input of suitable constraints in the iterative resolution procedure. The ALS potential is tested on several data sets coming from the multivariate spectrometric monitoring of polyuridylic (polyU), polycytidylic (polyC), and polyadenylic (polyA) protonation equilibria in dioxane/water 30% (v/v). Information concerning the evolution of the concentration profiles and the spectra of each individual species involved in the acid-base equilibria, the presence and pattern of polyelectrolyte effects, and the presence of conformational transitions associated or not with the proton uptake process is presented.     

Investigation of the Interaction Between Human Serum Albumin and Antitumor Palladium(II) Complex Containing 1,10-Phenanthroline and Dithiocarbamate Ligands

The interaction between [Pd(But-dtc)(phen)]NO₃ (where But-dtc = butyldithiocarbamate and phen = 1,10-phenanthroline) with HSA (Human Serum Albumin) was investigated by applying fluorescence, UV–Vis and circular dichroism techniques under physiological conditions. The results of fluorescence spectra indicated that the Pd(II) complex could effectively quench the fluorescence intensity of HSA molecules via static mechanism. The number of binding sites and binding constant of HSA–Pd(II) complex were calculated. Analysis of absorption titration data on the interaction between Pd(II) complex and HSA revealed the formation of HSA–Pd(II) complex with high-binding affinity. Thermodynamic parameters indicated that hydrophobic forces play a major role in this interaction. Furthermore, CD measurements were taken to explore changes in HSA secondary structure induced by the Pd(II) complex.     

Copper(II) complexes of carnosine, glycylglycine, and glycylglycine-imidazole mixtures

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.     

Syntheses and characterization of copper complexes with the ligand 2-aminoethyl(2-pyridylmethyl)-1,2-ethanediamine (apme)

Copper(I)/(II) complexes with the ligand 2-aminoethyl(2-pyridylmethyl)1,2-ethanediamine (apme, abbreviated as PDT in the literature as well) were prepared and characterized. Crystal structures of the copper(I) complexes, [Cu₂(apme)₂]X₂ (1, 2; X = ClO₄, CF₃SO₃), showed that they are dinuclear, in contrast to the trigonal bipyramidal copper(II) complexes [Cu(apme)Cl]BPh₄ (3) and [Cu(apme)(DMF)](BPh₄)₂ (4). 1 and 2 could be investigated in solution by NMR spectroscopy and 3 and 4 by cyclovoltammetry. From the results of these studies it is clear that in solution equilibria between the dinuclear complexes 1/2 and another species exist, most likely the monomeric [Cu(apme)CH₃CN]⁺. Time-resolved UV/vis spectra at low temperatures allowed the spectroscopic detection of dioxygen adduct complexes as reactive intermediates during the oxidation of 1/2 with dioxygen that seem to play an important role in copper enzymes such as peptidylglycine--hydroxylating monooxygenase (PHM).     

Interaction of Cu(II) 3,5-diisopropylsalicylate with human serum albumin - an evaluation of spectroscopic data

The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II) (3,5-DIPS) , that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 M aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl , CuSO , or Cu(II) (3,5-DIPS). Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II) (3,5-DIPS) to HSA at pH 7.5 similarly resulted in spectra that suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS) , or Cu(II) (3,5-DIPS) complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper. © Rapid Science 1998.