Calorimetric evidence for conformational transitions of RNase A in the presence of cytidine 2′,3′-cyclic phosphate

Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3′-monophosphate (3′CMP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2′,3′-cyclic phosphate (cCMP) as a substrate, and 3′CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3′CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points (T_m) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3′CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3′CMP indicates single DSC profile and monophasic binding isotherm.     

Semiempirical studies on the interactions of dialkyldi(aquo)tin cations, [R2Sn(H2O)2]2+, with selected nucleotides

Semiempirical calculations have been carried out on the interactions of [R₂Sn(H₂O)₂]²⁺, [R = H(CH₂)_n: n = 1–8], mainly with five nucleotides, 5′‐adenosine monophosphate (5′‐AMP), but also with guanosine 5′‐monophosphate (5′‐GMP), cytidine 5′‐monophosphate (5′‐CMP), uridine‐5′‐monophosphate (5′‐UMP) and inosine 5′‐monophosphate (5′‐IMP). The preferred sites of interaction were calculated to be the ribose O2 and O3 hydroxyl oxygens and/or the phosphate oxygens, with the nitrogen sites in the bases the least attractive to the tin compounds. This is in general agreement with experimental findings. Structures of the 1:1 coordination complexes vary from distorted tetrahedral, to distorted trigonal pyramidal to distorted octahedral geometries. Copyright © 2007 John Wiley & Sons, Ltd.     

Cytidylate cyclase activity: identification of cytidine 3',5'-cyclic monophosphate and four novel cytidine cyclic phosphates as biosynthetic products from cytidine triphosphate

Identification of cytidine 3',5'-cyclic monophosphate (cyclic CMP) as one of the products resulting from the incubation of dialysed cell-free preparations from rat brain, liver and kidney with cytidine 5'-triphosphate (CTP) is described. The non-acidic precipitable products after incubation of the tissue preparations with unlabelled, with 14C-single labelled, and with 14C- and 32P-dual labelled CTP were examined by thin-layer chromatography and high-pressure liquid chromatography, isotopic ratio determination, UV absorbance spectrophotometry, selective hydrolysis with nucleotidase, phosphodiesterase and acid, and by fast atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to cyclic CMP and unchanged CTP, the products of the reaction were found to include cytidine monophosphate (CMP) and cytidine diphosphate (CDP) together with four novel cytidine compounds identified as cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate 3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine 2'-O-glutamyl-3',5'-cyclic monophosphate. The evidence presented constitutes conclusive proof of the natural occurrence of cytidylate cyclase activity; the four novel cytidine cyclic phosphates described provide a feasible explanation of the discrepancies in previous reports which have led to the controversy which exists concerning the existence of cytidylate cyclase activity.     

Cytosine to uracil conversion through hydrolytic deamination of cytidine monophosphate hydroxy‐alkylated on the amino group: a liquid chromatography – electrospray ionization – mass spectrometry investigation

A novel pathway for cytosine to uracil conversion performed in a micellar environment, leading to the generation of uridine monophosphate (UMP), was evidenced during the alkylation reaction of cytidine monophosphate (CMP) by dodecyl epoxide. Liquid chromatography‐electrospray ionization – ion trap ‐ mass spectrometry was used to separate and identify the reaction products and to follow their formation over time. The detection of hydroxy‐amino‐dodecane, concurrently with free UMP, in the reaction mixture suggested that, among the various alkyl‐derivatives formed, CMP alkylated on the amino group of cytosine could undergo tautomerization to an imine and hydrolytic deamination, generating UMP. Interestingly, no evidence for this peculiar conversion pathway was obtained when guanosine monophosphate (GMP), the complementary ribonucleotide of CMP, was also present in the reaction mixture, due to the fact that NH₂‐alkylated CMP was not formed in this case. The last finding emphasized the role played by CMP–GMP molecular interactions, mediated by a micellar environment, in hindering the alkylation reaction at the level of the cytosine amino group. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid and simple chemical method for the preparation of Ag colloids for surface-enhanced Raman spectroscopy using the Ag mirror reaction

Colloidal silver (Ag) nanoparticles (AgNP) have been widely used for surface-enhanced Raman spectroscopy (SERS) applications. We report a simple, rapid and effective method to prepare AgNP colloids for SERS using the classic organic chemistry Ag mirror reaction with Tollens’ reagent. The AgNP colloid prepared with this process was characterized using SEM, and the reaction conditions further optimized using SERS measurements. It was found that Ag mirror reaction conditions that included 20 mM AgNO₃, 5 min reaction time, and 0.5 M glucose produced AgNP colloids with an average size of 319.1 nm (s.d ± 128.1). These AgNP colloids exhibited a significant SERS response when adenine was used as the reporter molecule. The usefulness of these new AgNP colloids was demonstrated by detecting the nucleotides adenosine 5′-mono-phosphate (AMP), guanosine 5′-monophosphate (GMP), cytidine 5′-monophosphate (CMP), and uridine 5′-monophosphate (UMP). A detection limit of 500 nM for AMP was achieved with the as-prepared AgNP colloid. The bacterium Mycoplasma pneumoniae was also easily detected in laboratory culture with these SERS substrates. These findings attest to the applicability of this AgNP colloid for the sensitive and specific detection of both small biomolecules and microorganisms.     

INVESTIGATION OF THE SOLUTION STRUCTURE OF Cu(II) MIXED-LIGAND COMPLEXES OF ADENOSINE 5′-MONOPHOSPHATE AND CYTIDINE 5′-MONOPHOSPHATE AND POLYAMINES

Abstract

The coordination mode of complexes formed in the systems Cu(II)/NMP/PA; (NMP =adenosine 5′-monophosphate, cytidine 5′-monophosphate; PA = 1,4-diaminopropane (putrescine, Put), 1,7-diamino-4-azaheptane (3,3-tri) and 1,11-diamino-4,8-diazaundekane (3,3,3-tet)) was determined on the basis of the equilibrium and spectroscopic studies. The presence of the following mixed complexes was established: Cu(CMP)H(Put), Cu(AMP)H₂(3,3-tri) and Cu(CMP)H₂(3,3-tri), Cu(CMP)H₄(3,3-tri) and coordination compounds of MLL′ type-Cu(CMP)(3,3,3-tet), Cu(AMP)(3,3,3-tet). A significant influence of the polyamine length on the solution structure of the complexes was observed. In mixed-ligand complexes Cu(NMP)(3,3,3-tet) a {N4, O} chromophore is formed, and metallation involves all nitrogen atoms from 3,3,3-tet. In the analogous system with 3,3-tri, protonated complexes occur. Non-covalent intramolecular interaction between the protonated amine groups and donor atoms from the purine ring from the nucleotide results in an increase of complex stability.     

Quadruplex–Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5′- or 3′-terminus to mimic a quadruplex–duplex (Q–D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q–D hybrids with a very high affinity in the order K_a≈10⁷ m ⁻¹ irrespective of the duplex location at the quadruplex 3′- or 5′-end. NMR chemical shift perturbations identified the tetrad face of the Q–D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3′- or 5′-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3′-Q–D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5′-Q–D hybrid.     

Nucleotide coordination with 14 lanthanides studied by isothermal titration calorimetry

ITC reveals the increasingly importance of entropy for heavier lanthanides binding to nucleotides. The phosphate group forming chelating effect with purine bases but not with pyrimidines.     

A supramolecular optic receptor for selective recognition CDP in neutral aqueous solution

We designed and synthesized a Cu-coordination complex based on a seven-membered amide cycle and studied its binding ability with nucleotides (cytidine 5′-monophosphate (CMP), cytidine 5′-diphosphate (CDP), cytidine 5′-triphosphate (CTP), cytidine d-5′-monophosphate (dCMP), and thymidine d-5′-monophosphate (dTMP)) by UV-Vis spectroscopy. Results indicate that the compound shows the highest binding ability with CDP among the studied nucleotides and can selectively and strongly bind nucleotides in neutral aqueous solution. The compound can be used as optical receptor for the detection of CDP.     

A study of the binding of manganese (II) with the sodium salts of nucleosides in aqueous solutions by the13C NMR method

The binding of manganese(II) with nucleosides — adenosine (A), guanosine (G), cytidine (C), and uridine (U) — in an alkaline D₂O solution has been investigated by the¹³C NMR method. It has been established that the structure of the paramagnetic Mn(II)—nucleoside complexes differs substantially in neutral and in alkaline media. The broadening of the resonance lines (C-2′, C-3′ > C-1′, C-4′ > C-5′) shows the localization of the Mn(II) in the C-2′ and C-3′ hydroxyls of the ribose in an alkaline medium. It has been shown for the case of U that the degree of complex-formation depends on the pH of the solution. It is assumed that the nucleoside forms intramolecular complexes (I) with Mn(OH)₂.     

Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides: acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu).

As part of a series of peptides designed to have binding ability selective for each of the nucleic acid bases, five tripeptides consisting of N-acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu) were synthesized, and their abilities to form complexes with four different nucleotides were examined by the fluorescence and phase distribution methods. The association constants obtained indicated that, depending on the sort of X residue, the peptides showed a variation in their interaction with guanosine monophosphate (GMP), while no noticeable selectivity was observed for other nucleotides adenosine monophosphate (AMP), uridine monophosphate (UMP) and cytidine monophosphate (CMP). The binding mode of N-acetyl-Trp-Asp-Trp-NHCH3 for the guanine base was further investigated using the proton nuclear magnetic resonance (1H-NMR) method. The mode was suggested to involve intimate cooperation of (1) the hydrogen bond formation between the carboxyl group of the Asp side chain and the guanine C2-amino group, and (2) the stacking interaction of the base with two terminal Trp residues of the peptide. Such interaction was strengthened by the protonation of the guanine base. A tentative binding mode is proposed based on these results.     

Optimization of Electrospray Ionization by Statistical Design of Experiments and Response Surface Methodology: Protein–Ligand Equilibrium Dissociation Constant Determinations

Electrospray ionization mass spectrometry (ESI-MS) binding studies between proteins and ligands under native conditions require that instrumental ESI source conditions are optimized if relative solution-phase equilibrium concentrations between the protein–ligand complex and free protein are to be retained. Instrumental ESI source conditions that simultaneously maximize the relative ionization efficiency of the protein–ligand complex over free protein and minimize the protein–ligand complex dissociation during the ESI process and the transfer from atmospheric pressure to vacuum are generally specific for each protein–ligand system and should be established when an accurate equilibrium dissociation constant (K_D) is to be determined via titration. In this paper, a straightforward and systematic approach for ESI source optimization is presented. The method uses statistical design of experiments (DOE) in conjunction with response surface methodology (RSM) and is demonstrated for the complexes between Plasmodium vivax guanylate kinase (PvGK) and two ligands: 5′-guanosine monophosphate (GMP) and 5′-guanosine diphosphate (GDP). It was verified that even though the ligands are structurally similar, the most appropriate ESI conditions for K_D determination by titration are different for each.

Open image in new window

Graphical Abstract ?

     

Spectroscopic and thermal characterization of biologically and anticancer active novel Schiff base metal complexes

The novel Schiff base ligand 2,2′-((1Z,1′Z)-(1,3-phenylenebis(azanylylidene))-bis(phenylmethanylylidene))dibenzoic acid (H₂L) was obtained by the condensation of m-phenylenediamine with o-benzoylbenzoic acid. The molecular and electronic structure of Schiff base ligand (H₂L) was optimized theoretically, and the quantum chemical parameters are calculated. Molecular docking was used to predict the binding between Schiff base ligand (H₂L) and the receptors of breast cancer mutant 3hb5-oxidoreductase, crystal structure E. coli (3t88) and crystal structure of S. aureus (3q8u). The newly synthesized Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) complexes were characterized by elemental microanalysis, molar conductance, spectroscopic techniques (IR, ¹H NMR, ESI-mass, ESR, UV–Vis), magnetic susceptibility, thermal (TG/DTG) and powder X-ray diffraction data to explicate their structures. The data showed that the complexes had composition of MH₂L type. The IR results confirmed the bidentate binding of the ligand involving two azomethine nitrogens. ¹H NMR spectral data of the ligand (H₂L) and its Zn(II) and Cd(II) complexes agreed well with the proposed structures. On the basis of electronic spectra and the magnetic measurements, octahedral geometry of the complexes was proposed. Thermogravimetric data (TG and DTG) were also studied. The kinetic and thermodynamic parameters for thermal decomposition of the complexes were calculated using the Coats–Redfern and Horowitz–Metzger methods. In order to appraise the effect of antimicrobial activity of metal ions upon chelation, the newly synthesized ligand and its metal complexes were screened against a number of bacteria organisms as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Neisseria gonorrhoeae and against one fungus, Candida albicans, to assess their inhibiting potential by using the disc diffusion method. The results showed that in some cases the antimicrobial activity of complexes was more biologically active than the Schiff base ligand. Anticancer activity of the ligand and its metal complexes were evaluated in human cancer (MCF-7 cells viability). It was found that [Cd(H₂L)(H₂O)₂Cl₂]2H₂O complex showed lowest IC₅₀ than the others, and hence was the more active. The activity index was calculated.     

Carbamoylmethylphosphoryl Compounds: Ch-Acidity and Basicity

Abstract

Carbamoylmethylphosphoryl compounds (CMP) are used as neutral bifunctional extractants. However, little is known about acid-base properties of these compounds. We have studied CH-acidity and basicity of selected CMP, RR′P(O)CH₂C(O)NEt₂ with R,R′ = Alk, Alk₂N, Ar, AlkO, and also H-complexes of CMP with HNO₃.     

Equilibrium and spectroscopic studies of ternary cadmium(II) and mercury(II) complexes with CMP and triamines

Formation of ternary Cd(II) and Hg(II) complexes with cytidine 5′-monophosphate (CMP) and triamines has been studied. Complexes M(CMP)(H _x PA) and M(CMP)(PA) (M?=?Cd, Hg; PA?=?polyamine) were detected and overall stability constants and equilibrium constants for their formation determined. The mode of coordination in the complexes has been proposed on the basis of the equilibrium and ¹³C, ³¹P NMR and IR studies. In the Hg(II) systems, metalation involves the donor endocyclic N(3) atom, the CMP phosphate group and nitrogen donor atoms of PA. Relative to the Hg/CMP binary systems, the presence of a polyamine in ternary systems does not change the metal–nucleotide mode of coordination. In ternary systems including Hg(II) ions, the occurrence of noncovalent interactions has not been detected. Cd(II) ions form molecular complexes as well as protonated species. Introduction of a polyamine to the Cd/CMP system changes the coordination mode of the nucleotide. The phosphate group of CMP is inactive in binary complexes (metalation by the N(3) atom) but is involved in coordination in heteroligand species. In contrast to other polyamines studied, in the system including 1,7-diamino-4-azaheptane (3,3-tri), the phosphate group of CMP in Cd(CMP)(H3,3-tri) does not participate in metalation but is engaged in intramolecular noncovalent interactions that stabilize the complex.     

Synthesis and spectral studies on palladium and platinum complexes with mono- and bidentate N-donor organic ligands and their interaction with calf thymus DNA in solution

Palladium(II) and platinum(II) complexes of the types PdLX₂, PdL₂X₂, PtL₂X₂ and the Pt(IV) complexes PtLX₂Y₂, PtL₂X₂Y′₂ (where L = mono- or bidentate organic ligand containing nitrogen donor atoms; X = Cl or Br; Y = Br and Y′ = OH) have been synthesized and characterized by elemental analysis, IR and X-ray photoelectron spectral data. The Pd 3d_5/2 binding energies indicate that the 8-aminoquinoline ligand is a better electron donor to the metal than other ligands studied. The Cl 2p_3/2 binding energies in the square planar pd(II) complexes are observed in the range 198.0–199.56 eV. The ν(PdCl) vibrations (ca 340 and 320 cm^?1) corresponding to two cis-Cl ligands were observed in the IR spectra. Binding through probably N-7 of the guanine residue and the phosphate oxygen in a chelate form is implied from UV difference spectral data.     

Thermodynamic investigations of interaction of aqueous solutions of 2′,3′-dideoxyinosine and 2′,3′-dideoxyadenosine with glycyl-glycine

2′3′-dideoxyinosine (ddI) and 2′3′-dideoxyadenosine (ddA) are known to exhibit relatively selective activityvs. HIV strains in cell cultures and low toxicityin vivo; ddI has been approved for the treatment of HIV infection in humans. It is therefore interesting to determine the thermodynamic properties of aqueous solutions of these compounds. For this purpose, we determined their apparent molar volumesV _? and heat capacitiesC _P?. The preliminary measurements of interaction of these compounds with peptides were made. The volume and molar heat capacities of transfer of ddI and ddA from aqueous solutions to glycyl-glycine (Gly-Gly) ones were calculated. For both compounds the significant values ofC _P?3,tr which depended on the concentration of Gly-Gly were observed.     

He I photoelectron spectra of [3]-ferrocenophane and its 7-oxa derivative

He I photoelectron spectra of [3]-ferrocenophane and 6,8-dimethyl-7-oxa-[3]-ferrocenophane have been measured. In comparison with ferrocene the energy gap between ²E′₂(a′₁²e′₂³) and ²A′₁(a′₁¹e′₂⁴) is decreased to 0.28 and 0.22 eV due to the destabilization of the a′₁(d)-level originating in its more pronounced mixing with ligand orbitals. The increase of photoionization cross section of the a′₁(d)-level in the 6,8-dimethyl-7-oxa-[3]-ferrocenophane spectrum supports this interpretation. The band shapes and ionization potentials of ligand π-levels are strongly influenced by the hydrocarbon bridge.     

Self‐Assembly of Tricuprous Double Helicates: Thermodynamics,Kinetics, and Mechanism

We report in this paper the coordination and kinetic properties of two oligobipyridine strands, which contain three 2,2′‐bipyridine subunits separated by oxydimethylene bridges, the 4,4′‐bis(CONET₂)‐substituted L and the 4,4′‐bis(CO₂Et)‐substituted L ′. Spectrophotometric measurements allowed the characterization of thermodynamic complexes and kinetic intermediates* which are involved in the self‐assembly process of L ₂Cu₃ and L Cu₃ helicates. The reaction presents positive cooperativity for the binding of two 2,2′‐bipyridine strands to the cuprous cations. While reactive kinetic intermediates* present distorted coordination geometries around Cu^I, the final rearrangement of the tricuprous bistranded helicates allows more closely tetrahedral coordination of each cation and reduces the interactions. Differences in the bulkiness and electronic properties of the L and L ′ substituents do not affect significantly the stability of the corresponding helicates, but greatly influence binding rates in the self‐assembly process.