DNA-based phosphane ligands

In order to expand the repertoire of DNA sequences specifically interacting with transition metals, we report here the first examples of DNA sequences carrying mono- and bidentate phosphane ligands as well as P,N-ligands. Aminoalkyl-modified oligonucleotides have been reacted at predetermined internal sites with carboxylate derivatives of pyrphos, BINAP and phosphinooxazoline (PHOX) 2 b-d. Carbodiimide coupling in the presence of N-hydroxysuccinimide provided the DNA-ligand conjugates in 38-78 % yield. Phosphane-containing oligonucleotides and their phosphane sulfide analogues were characterized by mass spectrometry (MALDI-TOF and FT-ICR-ESI) and their stability after purification and isolation was systematically investigated. While DNA-appended pyrphos ligand was quickly oxidized, BINAP and PHOX conjugates showed high stabilities, making them useful precursors for incorporation of transition metals into DNA.     

Temperature induced hyperchromism exhibited by Hoechst 33258: evidence of drug aggregation from UV-melting method

UV-thermal denaturation is a simple optical method widely employed for determination of DNA stability and interaction with ligands. Thermal denaturation of DNA and DNA-ligand complex is usually monitored at 260 nm. These data are generally presented as a function of the absorption increase of DNA alone with no consideration of the temperature dependent hyperchromism of the free ligand. Since not every ligand has absorption at 260 nm, usually this property of the ligand is ignored. Here, we report the temperature dependent hyperchromicity exhibited by Hoechst 33258 in the presence and absence of DNA. The presence of Hoechst, added to the duplex (monophasic profile, T(m)=75 degrees C) in various ratios generates a new transition at lower temperature displaying biphasic thermal transition profiles. We attributed this new transition (hyperchromic), a mere contribution from Hoechst, which might exist in aggregated forms. The extent of drug aggregation/self-association is concentration dependent. We suggest that prior to UV-melting studies the thermal dependence of the free ligand should be investigated.     

Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry of Pt(II) and Pd(II) complexes

In this work, we have analysed matrix-assisted laser desorption and ionisation time-of-flight (MALDI-TOF) mass spectra of [PtCl₂(en)], [PtCl₂(dach)] and [PdCl(dien)]Cl acquired either with 2,5-dihydroxybenzoic acid (DHB) or α-cyano-hydroxycinnamic acid (CHCA) as matrices. For certain experiments, small amounts of trifluoro acetic acid (TFA) or higher concentration of inorganic salts (NaCl or KCl) was added to the matrix solution. The majority of peaks arising from the Pt(II) and Pd(II) complexes could be identified, but certain ions detectable in the spectra were generated upon ligand loss. Additionally, the analysis of Pt(II) complexes was also possible in the presence of a higher salt content, which is a commonly used analysis condition for the samples of biological origin. While DHB appears to be the best suited for MALDI-TOF mass spectrometric analysis of Pt(II) complexes, CHCA seems to be a better matrix for Pd(II) complex used in this study. On the other hand, small amounts of TFA improve the spectra quality of Pt(II) complexes, but lead most probably to the degradation of Pd(II) complex. Taken together, we have demonstrated that the analysis of metallo-drugs using MALDI-TOF MS, though accompanied with certain identification problems, is easy and reliable. On the other hand, having in mind that some complexes (i.e. a combination of a particular transition metal/ligand) cannot be analysed under conditions usually applied for others, we deem it necessary to find out the best conditions for MALDI-TOF MS analysis of each metal complex.     

Differences in affinity of arylstilbazolium derivatives to tetraplex structures

Research into the interactions of small molecules (ligands) with DNA is a very important field of biochemistry. A ligand interacts with a DNA structure in many ways, depending on the structural features of the ligand (the presence of rings, substituent groups, length of bonds, etc.) or nucleic acid (number and association of strands, base sequence etc.). This study reports on an investigation of the preferential binding of arylstilbazolium ligands to a four-stranded DNA. For this purpose, an equilibrium dialysis was used. Equilibrium dialysis is a versatile method which enables many DNA structures to be investigated at the same time. A dozen different DNA structures of (single-stranded, double-stranded (duplex), triple-stranded (triplex), and four-stranded (tetraplex)) were involved in experiments with each ligand. Following the dissociation of DNA-ligand complexes by SDS, the concentration of the ligand bound was calculated from fluorescence and absorbance calibration curves. As a result, the amount of the ligand bound was directly related to the ligand-binding affinity. Equilibrium dialysis was used as a powerful tool to indicate which of the arylstilbazolium ligands investigated was the best therapeutic agent targeting G-quadruplex. Arylstilbazolium derivatives demonstrated strong interactions with the DNA samples used in the assay. The most interesting finding was a selective, preferential binding of anthryl derivative to c-MYC DNA (c-MYC is a DNA sequence that appears in an oncogene). Furthermore, as this derivative binds preferentially to one of the triplexes investigated, it can find an application in the TFO-triplex forming oligonucleotides which are used in gene therapy.     

Structural characterization of synthetic poly(ester amide) from sebacic acid and 4-amino-1-butanol by matrix-assisted laser desorption ionization time-of-flight/time-of-flight tandem mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) was employed to analyze a poly(ester amide) sample (PEA-Bu) from the melt condensation of sebacic acid and 4-amino-1-butanol. In particular, we investigated the fragmentation pathways, the ester/amide bond sequences and the structure of species derived from side reactions during the synthesis. MALDI-TOF/TOF-MS/MS analysis was performed on cyclic species and linear oligomers terminated by dicarboxyl groups, carboxyl and hydroxyl groups and diamino alcohol groups. The sodium adducts of these oligomers were selected as precursor ions. Different end groups do not influence the fragmentation of sodiated poly(ester amide) oligomers and similar series of product ions were observed in the MALDI-TOF/TOF-MS/MS spectra. According to the structures of the most abundant product ions identified, the main cleavages proceed through a beta-hydrogen-transfer rearrangement, leading to the selective scission of the --O--CH2-- bonds. Abundant product ions originating from --CH2--CH2-- (beta-gamma) bond cleavage in the sebacate moiety were also detected. Their formation should be promoted by the presence of an alpha,beta-unsaturated ester or amide end group. MALDI-TOF/TOF-MS/MS provided structural information concerning the ester/amide sequences in the polymer chains. In the MALDI-TOF/TOF-MS/MS spectra acquired, using argon as the collision gas, of cyclic species and linear oligomers terminated by diamino alcohol groups, product ions in the low-mass range, undetected in the mass spectra acquired using air as the collision gas, proved to be diagnostic and made it possible to establish the presence of random sequences of ester and amide bonds in the poly(ester amide) sample. Furthermore, MALDI-TOF/TOF-MS/MS provided useful information to clarify the structures of precursor ions derived from side reactions during the synthesis.     

Cooperativity during binding of a ligand to a multidentate oligomer

A matrix method to describe the equilibrium binding of a ligand by a multidentate oligomer with a system of binding centers differing in the affinity for the binding ligand has been proposed. The example of a complexation process simulating the formation of a complex between an oligonucleotide and zinc phthalocyanine ZnPc is considered. With the use of the proposed method, diagrams of the relative contents of different forms of DNA-ligand complexes have been constructed and analyzed. It is shown that the cooperativity of complexation has a considerable effect on the equilibrium of the ligand with the system of nonequivalent centers of binding. The experimental data on changes in the intensity of luminescence in the course of binding between zinc tetrakis(diisopropylguanidinio)phthalocyanine and a DNA molecule with the nucleotide sequence GTTA(GAGTTA)4GG have been analyzed.     

Calculation of equilibrium formation constants of complexes with a polydentate oligomer

A methodology of the calculation of ligand-oligomer complex formation and cooperative binding constants by the matrix method is described. Theoretical analysis of the equilibrium binding between a ligand and a polydentate oligomer having a system of sites with different ligand affinities is performed. The simulated equilibrium complex formation between the oligonucleotide G4-DNA and porphyrin TMPyP4 was used to construct and analyze diagrams of the relative fraction of DNA-ligand complexes by the matrix method. Cooperative ligand binding to a system of nonequivalent sites can be established by chemometric analysis and rigid modeling of spectrophotometric titration data (in terms of the chemical equilibrium model).     

MALDI-TOF mass spectrometric analysis for the characterization of the 5,10,15,20-tetrakis- (m-hydroxyphenyl)bacteriochlorin (m-THPBC) photoproducts in biological environment

Photoproducts formation upon irradiation (739 nm) of 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (m-THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption spectroscopy and MALDI-TOF mass spectrometry. The experiments were performed with a freshly prepared PBS-HSA solution of m-THPBC and with a PBS-HSA m-THPBC solution incubated for 6 h at 37 degrees C. The incubation of m-THPBC solution leads to the dye monomerisation, whereas in the freshly prepared solution, m-THPBC is under an aggregated form. Regardless of the incubation condition, photobleaching experiments carried out by absorption spectroscopy demonstrate the degradation of the photosensitizer and its phototransformation in m-THPC. Moreover, m-THPC was the sole photoproduct detected using absorption spectroscopy. Together with a degradation of m-THPBC and formation of m-THPC, MALDI-TOF mass spectrometry evidenced several other photoinduced modifications. Photoproducts such as dihydroxy m-THPBC and dihydroxy m-THPC were detected in both conditions; however, the formation of hydroxylated photoproducts was significantly greater in incubated solution. In addition, small molecules arising from the degradation of the photosensitizer and identified as dipyrin derivatives and dipyrrolic synthon were observed.     

Determination by MALDI-TOF MS of the structures obtained from polytransesterification of diethyl 2,6-pyridinedicarboxylate and poly(ethylene glycol)

Polyesters from reaction of diethyl 2,6-pyridinedicarboxylate with poly(ethylene glycol) 1000 were synthesized through a polytransesterification process using titanium(IV) alkoxide as catalyst. The resulting polyesters were characterized by size-exclusion chromatography (SEC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The structures of the polyesters were established and revealed the formation of several cyclic and linear populations of varying size. Detailed analysis of the MALDI-TOF mass spectra of the obtained polyesters has revealed the presence of linear polycondensates bearing various end groups among which those attributed to an exchange reaction between alkyl groups of the catalyst and ester groups of the polycondensates.     

Site-specific DNA photocleavage by rhodium intercalators analyzed by MALDI-TOF mass spectrometry

The DNA photocleavage reaction of mismatch-selective Rh complexes has been analyzed by MALDI-TOF mass spectrometry as well as gel electrophoresis analysis of radioactively tagged oligonucleotides. Analogous results are obtained with these two techniques showing site-specific cleavage neighboring the mismatch to yield primarily 5'- and 3'-phosphate termini. Additional intermediates and products are observed, however, using MALDI-TOF analysis. MALDI-TOF mass spectrometry is seen to be particularly powerful in the analysis of DNA cleavage by site-specific molecules. The method requires no radioactive labeling, only little material, and analysis can be accomplished within minutes. Moreover, this mass spectral analysis of DNA cleavage yields direct information regarding products rather than simply the base pair site of cleavage.     

Equilibrium, kinetic and HPLC study of the reactions between platinum(II) complexes and DNA constituents in the presence and absence of glutathione

The complex formation equilibria of [Pt(SMC)(H(2)O)(2)](+) and [Pt(terpy)H(2)O](2+), where SMC =S-methyl-L-cysteine and terpy = 2,2':6',2"-terpyridine, with some biologically relevant ligands such as inosine (INO), inosine-5'-monophosphate (5'-IMP), guanosine-5'-monophosphate (5'-GMP) and glutathione (GSH) were studied. The stoichiometry and stability constants of the complexes formed are reported, and the concentration distribution of the various complex species have been evaluated as a function of pH. Also the kinetics and mechanism of the complex formation reactions were studied as a function of nucleophile concentration and temperature. For the complex [Pt(SMC)(H(2)O)(2)](+), two consecutive reaction steps, which both depend on the nucleophile concentration, were observed under all conditions. The negative entropies of activation support an associative complex formation mechanism. Reaction of guanosine-5'-monophosphate (5'-GMP) with Pt(II) complexes was carried out in the presence and absence of glutathione (GSH) at neutral pH. The rate constants clearly showed a kinetic preference toward GSH at neutral pH. The reactions were also monitored by HPLC. However, only a small amount of coordinated 5'-GMP was detected in the HPLC trace. The products were isolated and characterized by MALDI-TOF mass spectrometry.     

类黄酮化合物对G-四链体及双链DNA选择性的电喷雾质谱研究

利用电喷雾质谱(ESI-MS)研究了4种常见的类黄酮化合物芦丁、 槲皮素、 葛根素和柚皮苷与2种不同形态结构的G-四链体DNA和3种双链DNA的非共价相互作用, 比较了这些小分子化合物与不同形态结构DNA结合的强弱及形成复合物的化学计量. 结果表明, 芦丁和槲皮素对G-四链体DNA具有一定的选择性, 同时它们对双链DNA的选择性也较高; 而葛根素和柚皮苷对G-四链体DNA仅显示了较低的选择性.     

Evaluation of the quantitativeness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an equimolar mixture of uniform poly(ethylene glycol) oligomers

Quantitativeness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was elucidated using an equimolar mixture of uniform poly(ethylene glycol) (PEG) oligomers with no molecular weight distributions. Uniform PEG oligomers with degrees of polymerization n = 6-40 were separated from commercial PEG samples by preparative super-critical fluid chromatography. MALDI-TOF mass spectra of an equimolar mixture of the uniform PEG oligomers were recorded by adding a mixture of 2,5-dihydroxybenzoic acid as a matrix reagent and four chlorinated salts, i.e. LiCl, NaCl, KCl and RbCl. Remarkable non-quantitative effects were observed in the MALDI-TOF mass spectra in both the lower and higher molecular mass regions. At higher molecular masses greater than about 10(3), PEG oligomers with larger molecular mass yielded lower spectral intensities irrespective of the species of adduct cations and higher laser powers induced larger decreases in mass spectral intensities with the increase in their molecular masses. On the other hand, in the lower molecular mass region, less than about 10(3), the observed non-quantitative effect greatly depends on the species of adduct cations, indicating that the stability of the PEG-cation complex affects the MALDI-TOF mass spectral intensities of uniform PEG oligomers.     

Photoinduced in situ generation of a DNA-binding benzothiazoloquinolinium derivative

A styrylbenzothiazole substrate that has no significant affinity towards DNA was transformed photochemically into a DNA-binding benzothiazoloquinolinium ion. The photoreaction may be performed directly in the presence of DNA thus establishing the DNA-ligand interaction in situ with temporal control.     

Synthesis and characterization of iridium(III) cyclometalated complexes with oligonucleotides: insights into redox reactions with DNA

Heteroleptic cyclometalated complexes of Ir(III) containing the dipyridophenazine ligand are synthesized through the direct introduction of a functionalized dipyridophenazine ligand onto a bis(dichloro)-bridged Ir(III) precusor and characterized by 1H NMR, mass spectrometry, as well as spectroscopic and electrochemical properties. The excited state of the Ir(III) complexes have sufficient driving force to oxidize purines and to reduce pyrimidine nucleobases. Luminescence and EPR measurements of the Ir(III) complex with an unmodified dppz bound to DNA show the formation of a guanine radical upon irradiation, resulting from an oxidative photoinduced electron-transfer process. Evidence is also obtained indirectly for reductive photoinduced electron transfer from the excited complex to the thymine base in DNA. We have also utilized cyclopropylamine-substituted nucleosides as ultrafast kinetic traps to report transient charge occupancy in oligonucleotides when DNA is irradiated in the presence of noncovalently bound complexes. These experiments establish that the derivatized Ir(III) complexes, with photoactivation, can trigger the oxidation of guanine and the reduction of cytosine.     

Investigation of the interaction between 1-hydroxyethane-1,1'-diphosphonic acid (HEDP) and uranium(VI)

A detailed study, using a panel of spectroscopic analytical methods, of the complexation between 1-hydroxyethane-1,1'-diphosphonic acid (HEDP) and uranyl ion (UO(2)(2+)) is reported. Results suggest that the metal complex is present as only 1:1 (metal/ligand) species at low concentration (<10(-)(4) M). The conditional constants of this complex were determined at various pH using time-resolved laser-induced fluorescence (TRLIF) and electrospray ionization mass spectrometry (ESI-MS). Further investigations indicate the presence of a 1:2 (metal/ligand) complex at higher concentrations ( approximately 10(-)(2) M). Selectivity studies as well as structural aspects are presented.     

Derivatisation of peptides with osmium tetroxide, 2,2′-bipyridine: capillary electrophoretic and MALDI-TOF mass spectrometric study

Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2′-bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI-TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and methionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI-TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins.     

DNA interaction studies of Cu(II), Co(II), and Ni(II) chelates derived from schiff base ligand

Chelates of the type M(L)₂ {where, M ?= ?Co(II), Ni(II) and Cu(II), and L ?= ?3-{(E)-[(2-hydroxy-3-methoxyphenyl)methylidene]amino}pyridin-4(1H)-one)} were synthesized by using the Schiff base ligand in the stochiometric ratio 2:1 (L:M) and Schiff base ligand (L) was synthesized by simple condensation between 2-hydroxy-3-methoxybenzaldehyde with 3-aminopyridin-4-ol. The structure and formation of synthesized compounds were established by different analytical and spectroscopic methods like, elemental analysis, UV- spectroscopy, FT-IR, Proton and Carbon NMR, mass spectrometry and Powder XRD. Further, the synthesized chelates screened for the DNA binding studies of Calf Thymus (CT)-DNA by exploiting electronic absorption spectra, relative viscosity measurements and thermal denaturation methods. The proposed DNA binding mode supports the enhancement in the binding activity of the complexes in presence of newly synthesized ligand. The cleavage activities of the PUC-18 DNA in the presence and the absence of the complexes were recorded with the help of gel-electrophoresis. The cleavage experiment results reveals that all the synthesized chelates can cleave pUC-18 DNA effectively.     

Inhibition of Thermus thermophilus HB8 thioredoxin activity by platinum(II)

A 1:1 thioredoxin-Pt(bpy) complex was prepared by adding [Pt(bpy)(en)]Cl(2)(bpy = 2,2'-bipyridine, en = ethylenediamine) to Thermus thermophilus HB8 thioredoxin in pH 8 phosphate buffer. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) and UV spectra of indicate the formation of Pt(bpy)(cys-Ala-Pro-cys-containing peptide fragment). These findings suggest that the Pt(bpy)(2+) unit binds to the active site of thioredoxin. The thioredoxin-platinum complex has no catalytic activity for the reduction of glutathione disulfide in the presence of NADPH and thioredoxin reductase, so that the platinum complex functions as an inhibitor.     

The effect of charge on the volume change of DNA binding with intercalator DAPP

To study the effect of ligand charge on DNA-ligand binding, we measured the difference in volume change of the noncovalent complex formed between calf thymus DNA and the charged and uncharged form of 3,8-diamino-6-phenylphenanthridine (DAPP). We found that the volume change for binding with the charged DAPPH+ is about 7.8 +/- 1.5 cm3 mol(-1) more positive than with the neutral DAPP. We hypothesize that this large difference in interaction volume originates from partial desolvation of the charge of DAPPH+ when it is bound to DNA.