A fluorescence study of the molecular interactions of harmane with the nucleobases, their nucleosides and mononucleotides

Fluorescence binding studies of harmane to the elemental components of the nucleic acids were undertaken to investigate the origin of the interaction between the drug and DNA. Most of the tested substrates have been found to induce hypochromism in the absorption spectrum of harmane and to quench its fluorescence. The quenching process induced by the nucleobases and their nucleosides is mainly due to the formation of ground state 1:1 complexes. However, in the case of the mononucleotides a dynamic quenching component is also observed. This quenching component is likely due to the excited state interaction of harmane with the phosphate group of the nucleotides. UV-vis spectral changes and quenching measurements have been used to quantify the ground state association constants of the complexes and the quenching rate constants.     

Synthesis and evaluation of diazine containing bioisosteres of (-)-ferruginine as ligands for nicotinic acetylcholine receptors

In this structure-affinity relationship (SAFIR) study, the bioisosteric potential of diazines in the field of ferruginine-type nAChR ligands was investigated. Novel enantiopure analogues of (-)-Ferruginine (3) such as 6-8 were synthesized utilizing enantiomerically pure N-protected (+)-2-tropanone 9 from the 'chiral pool' as versatile chiral building block and a palladium-catalyzed Stille cross-coupling of the tributylstannyl diazines 12, 14 and 16 with the vinyl triflate 11 of (+)-2-tropanone 9. The structures of the novel diazine analogues 6-8 of (-)-ferruginine (3) were assigned on the basis of spectral data, that of ligand 7 being additionally verified by X-ray crystallography. The bioisosteric replacement of the acetyl moiety as structural part of the lead compound 3 with the pyridazine, pyrimidine and pyrazine nucleus resulted in ligands with high to moderate affinity for the central alpha4beta2 and remarkably low affinity for the alpha7* nAChR subtypes. Among the compounds synthesized and tested, 7 was the most active one with K(i)=3.7 nM (alpha4beta2). Compared with the lead 3, this value represents a 30-fold improvement in the affinity for the alpha4beta2 subtype combined with a substantially improved selectivity ratio between the alpha4beta2 and alpha7* subtypes.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with pyrimidine nucleosides and their derivatives.

1. In aqueous and non-aqueous solutions, copper(II) interacts with the N-3 of cytidine but not with the carbonyl group oxygens of pyrimidine nucleosides. 2. In aqueous solution, copper(II) interacts with the phosphate group and ribose of pyrimidine nucleotides, and additionally with N-3 of 5'-CMP. 3. Broadening of resonance signals of the H-5 proton of 5'-UMP and C-5 of 5'-UMP and 5'-TMP results probably from the interaction between metal ion and the phosphate group situated in direct vicinity of the above atoms. 4. In the copper(II)-pyrimidine nucleotide complexes in solid state, copper is coordinated with the phosphate group, and in 5'-CMP additionally with the pyrimidine moiety of the nucleotide.     

The synthesis and behaviour of pyrazine mononuclear carbonyl complexes of Rh(I), Ir(I), Ru(II) and Os(II)

The synthesis in high yields and the dissociative behaviour in the solid state and in solution of the mononuclear complexes [cis-M(CO)₂Cl(pyz)] (M=Rh, Ir; PYZ=pyrazine) and [fac-M(CO)₃Cl₂(pyz)] (M=Ru, Os) are reported. The mononuclear complexes of Rh and Ir are relatively labile with respect to pyrazine release. Particularly in the case of rhodium they generate even in the solid state the corresponding dinuclear complexes [cis-Cl(CO)₂M(pyz)cis-M(CO)₂Cl] (M=Rh, Ir). The ¹H NMR spectra of these mononuclear Rh and Ir complexes in CHCl₃ solution show, at 25 and 60 °C, respectively, a fast and reversible dissociation of metal coordinated pyrazine, which is hindered by lowering the temperature. Crystallographic aspects of [cis-Ir(CO)₂Cl(pyz)] have been investigated via single crystal X-ray diffraction. The mononuclear complexes of Ru and Os are more stable. In the solid state they do not rearrange, with release of pyrazine, to generate the related dimeric complexes with pyrazine as bridge. In solution, at room temperature, they do not dissociate quickly, although a mixture of monomeric and dimeric pyrazine complexes (ratio monomer to dimer 9:1 and 15:1 for Ru and Os, respectively) is slowly formed by a process which is reverted by addition of excess pyrazine, as expected for a dissociative equilibrium.     

Evidence for specific complex formation between alpha-melanocyte stimulating hormone and 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin using near infrared Fourier transform Raman spectroscopy

The cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) and its 2 and 4 electron oxidation products 7,8-dihydro-L-biopterin and L-biopterin have been shown to form 1:1 complexes with the thirteen amino acid peptide alpha-melanocyte stimulating hormone (alpha-MSH). Hydrogen bonding to the pyrimidine ring of the cofactor has been established for glu(5) and his(6) of the hormone using Near Infrared Fourier Transform Raman spectroscopy. Binding of these pterins primarily involves the pyrimidine ring, although with the reduced pterins, 7,8-dihydro-L-biopterin and 6BH(4), there is evidence for pi orbital interaction with the pyrazine ring. It is proposed that this pi orbital interaction with the reduced biopterins and alpha-MSH could provide the basis for the observed stability of these pterins to oxidation by either molecular oxygen or photooxidation by UVB (290-320 nm) light. Our results suggest that the formation of the alpha-MSH/6BH(4) complex could play a major role in the control of all 6BH(4) dependent processes.     

Exciplexes or ground state complexes of (dibenzoylmethanato)boron difluoride and benzene derivatives? A study of their optical properties revisited via liquid state investigations and structure calculations.

(Dibenzoylmethanato)boron difluoride (DBMBF2) was found to form with benzene (B) and its methylated derivatives (MB) in cyclohexane (cHex) two types of ground state complexes. The first complexes with low stoichiometries 2 : 1, 1 : 1 and 1 : 2 do not fluoresce when they are excited. On the other hand, the ground state complexes with high stoichiometry, DBMBF2-(B)(n) or DBMBF2-(MB)(n) (with n> 2), exhibit a strong fluorescence in their excited states. These findings differ from the previous results, where the strongly fluorescing complexes have been argued to be the 1 : 1 and 1 : 2 exciplexes, complexes of the singlet excited state of DBMBF2 with one or two B or MB molecules. These differences are discussed in terms of the solute-solute and solute-solvent interactions when DBMBF2 and MB are solutes in cyclohexane or when MB is a co-solvent of cyclohexane in binary mixtures of DBMBF2. We also argue that the use of well-suited analytical methods is important for the determination of the nature of the various complexes. Furthermore, to understand the nature of the interactions between benzene and DBMBF2 molecules, we attempt to predict the sites of interaction between DBMBF2 and benzene molecules by determining theoretically the structure of the 1 : 1 complex.     

Molecular Orbital Study of the Magnetic Properties of Pyrazine- and Pyrimidine-Bridged Copper(II) Complexes

Magnetic interactions in the three copper(II)-complex polymers, [Cu(PZ)(NO₃)₂]_n, [Cu(PM)(NO₃)₂(H₂O)₂]_n, and [Cu(PM)₂(NO₃)₂]_n are discussed on the basis of extended Hückel calculations inthe formulas PZ and PM stand for pyrazine and pyrimidine, respectively. Interactions between the Cu-3d orbitals and the lone-pair orbitals of pyrazine and pyrimidine are analyzed from the viewpoint of `through-space' and `through-bond' interactions using binuclear complexes to model the three copper(II) polymers. Three conclusions can be drawn from the orbital interaction analysis: (1) in the first polymer, a superexchange pathway is formed with the bond of Cu–-N and the through-bond interaction between the lone pairs of the nitrogen atoms of pyrazine will lead to an antiferromagnet state; (2) in the second polymer a superexchange pathway is formed with the bond of Cu–-N and the through-space interaction between the lone pairs of the nitrogen atoms of pyrimidine, and as a result an antiferromagnetic state will be preferred; and (3) in the third polymer., there is no effective pathway in respect of overlap interaction and the HOMO and the LUMO are actually degenerate, and thus a ferromagnetic state will arise. The band structures are analyzed to characterize the magnetic properties of the antiferromagnetic polymers, [Cu(PZ)(NO₃)₂]_n and [Cu(PM)(NO₃)₂(H₂O)₂], and the ferromagnetic polymer, [Cu(PM)₂(NO₃)₂]_n.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission

The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.     

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.     

An FT-IR and XPS study of copper(II)–diazine polymers

Solid copper(II) complexes have been obtained following reaction of copper chloride with pyridazine (pdz) and pyrimidine (pym) ligands. Elemental analyses, diffuse reflectance Fourier-Transform and X-ray photoelectron spectral data recorded from these compounds are consistent with polymeric structures of the general formulae Cu(pdz)Cl₂ and Cu(pym)Cl₂. X-ray photoelectron data also indicates that the surface composition of each of these complexes closely resembles that of the bulk material.     

Effect of solvents,solvent mixture and silver nanoparticles on photophysical properties of a ketocyanine dye

The effect of solvents of varying polarity and hydrogen bonding ability, solvent mixture and silver nanoparticles on the photophysical properties of a ketocyanine dye, 2,5‐di[(E)‐1‐(4‐diethylaminophenyl) methylidine]‐1‐cyclopentanone (2,5‐DEAPMC), is investigated at room temperature. Solvent effect is analyzed using Lippert–Mataga bulk polarity function, Reichardt's microscopic solvent polarity parameter, and Kamlet's and Catalan's multiple linear regression approaches. The spectral properties better follow Reichardt's microscopic solvent polarity parameter than the Lippert–Mataga bulk polarity function. This indicates that both general and specific solute–solvent interactions are operative. Kamlet's and Catalan's multiple linear regression approaches indicate that polarizability/dipolarity solvent influences are greater than hydrogen bond donor and hydrogen bond acceptor solvent influences. The solvatochromic correlations are used to estimate excited state dipole moment using the experimentally determined ground state dipole moment. The excited state dipole moment of the dye is found to be larger than its corresponding ground state dipole moment and ground and excited state dipole moments are not parallel, but subtend an angle of 77°. The absorption and emission spectra are modulated in the presence silver nanoparticles. The fluorescence of 2,5‐DEAPMC is quenched by silver nanoparticles. The possible fluorescence quenching mechanisms are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.     

A combined spectroscopic and TDDFT investigation of the solute‐solvent interactions of two coumarin derivatives

The UV/Vis absorption and fluorescence characteristics of 3‐cyano‐7‐hydroxycoumarin [ CHC ] and 7‐amino‐4‐methyl‐3‐coumarinylacetic acid [ AMCA‐H ] were studied at room temperature in several neat solvents and binary solvent mixtures of 1,4‐dioxane/acetonitrile. The effects of solvent on the spectral properties are analyzed using single and multi‐parameter solvent polarity scales. Both general solute/solvent interactions and hydrogen bond interactions are operative in these systems. The solvation of CHC and AMCA‐H dyes in 1,4‐dioxane/acetonitrile solvent mixtures has been studied. The solutes CHC and AMCA‐H are preferentially solvated by acetonitrile and a synergistic effect is observed for both molecules in dioxane/acetonitrile solvent mixtures. In addition, using the solvatochromic method the ground‐ and the excited‐state dipole moments of both the dyes were calculated. The ground‐ and excited‐state dipole moments, absorption and emission maxima and HOMO–LUMO gap were also estimated theoretically using B3LYP/6–311+ G (d,p) level of theory in the gaseous phase, dioxane and acetonitrile solvents. Furthermore, changes in dipole moment values were also calculated using the variation of Stokes shift with the molecular–microscopic empirical solvent polarity parameter ( ). The observed excited‐state dipole moments are larger than their ground‐state counterparts, indicating a substantial redistribution of the electron densities in a more dipolar excited state for both coumarins investigated.     

Resonance Raman and lifetime studies on regioselectively deuteriated ruthenium(II) polypyridyl complexes.

Two series of ruthenium(II) polypyridyl complexes [Ru(bipy)(2)(phpytr)](+) and [Ru(bipy)(2)(phpztr)](+) (where Hphpytr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyridine and Hphpztr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyrazine) are examined by electrochemistry, UV/Vis, emission, resonance Raman, transient resonance Raman and transient absorption spectroscopy, in order to obtain a more comprehensive understanding of their excited state electronic properties. The interpretation of the results obtained is facilitated by the availability of several isotopologues of each of the complexes examined. For the pyridine-1,2,4-triazolato based complex the lowest emissive excited state is exclusively bipy based, however, for the pyrazine based complexes excited state localisation on particular ligands shows considerable solvent and pH dependency.     

Effect of water molecules on the fluorescence enhancement of Aflatoxin B1 mediated by Aflatoxin B1:beta-cyclodextrin complexes. A theoretical study.

In order to explain the observed fluorescence enhancement of Aflatoxin B1 (AFB1) when forming AFB1:beta-cyclodextrin (AFB1:beta-CD) inclusion complexes, we have performed a theoretical (quantum chemistry calculations) study of AFB1 and AFB1:beta-CD in vacuum and in the presence of aqueous solvent. The AM1 method was used to calculate the absorption and emission wavelengths of these molecules. With the help of density functional theory (DFT) and time-dependent DFT (TDDFT) vibrational frequencies and related excitation energies of AFB1 and AFB1.(H2O)m = 4,5,6,11 were calculated. On the basis of these calculations we propose a plausible mechanism for the fluorescence enhancement of AFB1 in the presence of beta-CD: (1) before photoexcitation of AFB1 to its S1 excited state, there is a vibrational coupling between the vibrational modes involving the AFB1 carbonyl groups and the bending modes of the nearby water molecules (CG + WM); (2) these interactions allow a thermal relaxation of the excited AFB1 molecules that results in fluorescence quenching; (3) when the AFB1 molecules form inclusion complexes with beta-CD the CG + WM interaction decreases; and (4) this gives rise to a fluorescence enhancement.     

Influence of solvent and of cation size on the conformations of lasalocid A-lanthanide(III) ion complexes: circular dichroism and fluorescence studies

The interaction of lanthanide(III) nitrates (La3+ to Lu3+) with the carboxylic ionophore lasalocid A (LS) has been studied by circular dichroism (CD) and fluorescence spectroscopic techniques in acetonitrile and in methanol. Analysis of the CD data in acetonitrile has revealed the coexistence of both 1:1 (ionophore:cation) and 2:1 complexes in solution. For 1.22 A greater than ionic radius greater than 1.13 A, 1:1 complexes are preferred, and for 1.13 A greater than ionic radius greater than 1.03 A, 2:1 complexes are preferred. Induced CD bands for Ln3+ ions have been observed upon binding to LS in acetonitrile. The LS-Ln3+ complexes are less stable in methanol than in acetonitrile. CD spectral changes showed that the conformations of the complexes in methanol are different from those in acetonitrile. The complexes have rather open conformations in methanol compared to those in acetonitrile. The results underscore the importance of ionic radius, solvent environment, and ionization state of LS in determining the conformations of the ionophore-cation complexes.     

Intramolecular exciplexes based on benzoxazole: photophysics and applications as fluorescent cation sensors.

Exciplex behaviour of three benzoxazole derivatives has been detected and intensively investigated by means of steady-state and time-resolved fluorescence techniques and transient absorption spectroscopy. The fluorescence of these compounds shows the properties which are typical for the excited state charge transfer complexes (exciplexes). Besides of the short wavelength fluorescence, which is similar in spectral distribution to the fluorescence of the electron acceptor (2-p-tolyl-benzoxazole), the red shifted, broad and structureless emission band is observed in solvents of low and medium polarity. The detailed analysis of the fluorescence data shows that the ratio of the CT and LE fluorescence initially increases with increasing solvent polarity, achieves a maximum, and drops for more polar solvents (epsilon(s) = 7). Similar behaviour is observed for the exciplex fluorescence lifetimes. The overall fluorescence and the relative intersystem crossing quantum yields show the decrease of these values with increasing solvent polarity. These observations have been explained on the basis of Marcus-type theory for nonradiative charge transfer rate constants. Increasing solvent polarity strongly accelerates the back electron transfer process which recovers the whole molecule in the ground state. The probability of the compact exciplex formation (i.e. sandwich-type structures) depends on solvent viscosity and degree of freedom of the bending of the saturated linker. The compound containing crown ether as a donor subunit may be used as a fluorescent indicator of inorganic cations (barium and lithium). We found an effective complexation of the compound in the ground state with barium and lithium cations. The complex is also stable in the excited state which manifests itself in strong increase of the fluorescence intensity.