Separation of Cytidine 5′-triphosphate Biosynthesized from Cytidine 5′-monophosphate on Ion-exchange Resin and HPLC Analysis of Cytidine Compounds

Conditions were studied in the biosynthesis of cytidine 5′-triphosphate (CTP) from cytidine 5′-monophosphate (CMP). A 201 × 7 anion ion-exchange resin was applied for the separation of CTP from CMP. Adsorption isotherm and elution conditions (eluant, eluant concentration, flow rate, sample volume loaded) were investigated. At the same time, a new high-performance liquid chromatography on an anion ion-exchange column WAX-1 with UV detector at 260 nm was developed to measure CMP, cytidine 5′-diphosphate (CDP), and CTP. The retention time for CMP, CDP, and CTP are 0.723, 1.448, and 4.432 min, respectively. This new rapid high-performance liquid chromatography (HPLC) method for the analysis of cytidine compounds in biological sample has a wide linear range with high precision and repeatability.     

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.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.

     

Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate, inosine 5′-monophosphate and guanosine 5′-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min⁻¹. The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 °C.     

Interaction of dipropyltin(IV) with amino acids,peptides, dicarboxylic acids and DNA constituents

Interaction of dipropyltin(IV) with selected amino acids, peptides, dicarboxylic acids or DNA constituents was investigated using potentiometric techniques. Amino acids form 1?:?1 and 1?:?2 complexes and, in some cases, protonated complexes. The amino acid is bound to dipropyltin(IV) by the amino and carboxylate groups. Serine is complexed to dipropyltin(IV) with ionization of the alcoholic group. A relationship exists between the acid dissociation constant of the amino acids and the formation constants of the corresponding complexes. Dicarboxylic acids form both 1?:?1 and 1?:?2 complexes. Diacids forming five- and six-membered chelate rings are the most stable. Peptides form complexes with stoichiometric coefficients 111(MLH), 110(ML) and 11-1(MLH_?1)(tin: peptide: H⁺). The mode of coordination is discussed based on existing data and previous investigations. DNA constituents inosine, adenosine, uracil, uridine, and thymine form 1?:?1 and 1?:?2 complexes and the binding sites are assigned. Inosine 5′-monophosphate, guanosine 5′-monophosphate, adenosine 5′-monophosphate and adenine form protonated species in addition to 1?:?1 and 1?:?2 complexes. The protonation sites and tin-binding sites were elucidated. Cytosine and cytidine do not form complexes with dipropyltin(IV) due to low basicity of the donor sites. The stepwise formation constants of the complexes formed in solution were calculated using the non-linear least-square program MINIQUAD-75. The concentration distribution of the various complex species was evaluated as a function of pH.     

An Fe2+- and α-Ketoglutarate-Dependent Halogenase Acts on Nucleotide Substrates

While halogenated nucleosides are used as common anticancer and antiviral drugs, naturally occurring halogenated nucleosides are rare. Adechlorin (ade) is a 2′-chloro nucleoside natural product first identified from Actinomadura sp. ATCC 39365. However, the installation of chlorine in the ade biosynthetic pathway remains elusive. Reported herein is a Fe²⁺-α-ketoglutarate halogenase AdeV that can install a chlorine atom at the C2′ position of 2′-deoxyadenosine monophosphate to afford 2′-chloro-2′-deoxyadenosine monophosphate. Furthermore, 2′,3′-dideoxyadenosine-5′-monophosphate and 2′-deoxyinosine-5′-monophosphate can also be converted, albeit 20-fold and 2-fold, respectively, less efficiently relative to the conversion of 2′-deoxyadenosine monophosphate. AdeV represents the first example of a Fe²⁺-α-ketoglutarate-dependent halogenase that converts nucleotides into chlorinated analogues.     

Noncovalent Interaction of Uridine 5′-Monophosphate with Adenosine, Cytidine, and Thymidine, as well as Adenosine 5′-Monophosphate and Cytidine 5′-Monophosphate in Aqueous Solution

Adduct formation has been studied in the systems of uridine 5′-monophosphate (UMP) with adenosine (Ado), cytidine (Cyd), thymidine (Thd), adenosine 5′-monophosphate (AMP), and cytidine 5′-monophosphate (CMP) by the potentiometric method with computer analysis of the data and ¹³C and ³¹P NMR spectroscopic measurements. It has been established that in the complexes identified, ion–dipole and dipole–dipole interactions occur with the positive reaction centers being protonated nitrogen atoms N(3) of UMP or Thd, and at low pH values, endocyclic nitrogen atoms of the other nucleosides and nucleotides, as e.g., in (UMP)H₂(Ado). The negative reaction centers are the high-electron density atoms N(1) and N(7) from Ado or AMP and N(3) from Cyd or CMP, and the phosphate group of the nucleotides studied, which already undergo partial deprotonation at low pH values. The NMR results have established the presence of noncovalent stacking-type interactions in certain molecular complexes, e.g., (UMP)H₂(AMP). The sites of ion–dipole or dipole–dipole interactions are generated as a result of deprotonation of the nucleosides and nucleotides in the pH range of formation of molecular complexes. Analysis of the equilibrium constants of the reaction allowed a determination of the effectiveness of the phosphate groups and donor atoms of heterocyclic rings in the process of molecular complex formation.     

Mass spectrometry of protein-ligand complexes: Enhanced gas-phase stability of ribonuclease-nucleotide complexes

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.     

Separation of isomeric and nonisomeric monoribonucleotides by isocratic ion pair high performance liquid chromatography

A simple, isocratic, high performance liquid chromatographic procedure is described for the first time for the separation of nine monoribonucleotides using the ion-pairing technique. An aqueous mobile phase containing 100 mM KH₂PO₄ and 12.5 mM tetramethylammonium hydroxide as the solvophobic ion, pH 3.9, was used with a reverse phase RP-18 column. The nine monoribonucleotides studied were separated and eluted in the following order: cytidine-5′ -phosphate, uridine-5′ -phosphate, cytidine-3′ -phosphate, guanosine-5′ -phosphate, uridine-3′ -phosphate, uridine-2′ -phosphate, adenosine-5′ -phosphate, guanosine-3′ -phosphate, and adenosine-3′ -phosphate. Generally the 5′ nucleotides eluted faster than the 3′ and the order of elution within each series was: cytidine, uridine, guanosine, and adenosine. The only nucleotide where three isomers were studied was uridine, and the 2′ eluted later than the 3′. Baseline separation was attained for a mixture containing four 3′ nucleotides and uridine-2′ -phosphate. When the four 5′ nucleotides were chromatographed, baseline separation was also obtained except between cytidine-5′ -phosphate and uridine-5′ -phosphate. The coefficient of variation of the retention characteristics, which reflected day-to-day variation, averaged 6.4%.     

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.     

Determination of Bromide Production in Radiolysis of Nucleobases,Nucleosides, and Nucleotides Using HPLC

Abstract

Determination of the formation of bromide ions in intermolecular electron transfer in 5-bromouracil (BrUr) and its nucleoside and nucleotide derivatives with nucleobases, nucleosides, and nucleotides was carried out with high performance liquid chromatography (HPLC). Initial electron attachment, at high concentration of nucleobases, nucleosides, or nucleotides, is mainly on these molecules; intermolecular electron transfer then occurs between theses molecules and BrUr and the derivatives. The elimination of bromide ions from BrUr and the derivatives then follows. It is concluded that in neutral and basic solution (pH 6 to 10) there is a significant electron transfer from thymine (T), uracil (Ur), thymidine (dT), 2′-deoxyuridine (dU), or 2′-deoxyuridine-5′-monophosphate (dUMP) to BrUr and the derivatives. For example, at a concentration ratio of BrUr and T of 1 : 100, the yield of bromide ions is about 1.6, amounting to 59% of hydrated electron (e^{aq .}) yield in the radiolysis, in which the pseudo-first-order rate constants predict a bromide yield of less than 0.03.     

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.     

Complex formation reactions of two sterically hindered platinum(II) complexes with some N-bonding ligands

The kinetics of the complex formation reactions of two [(TL ^tBu)PtCl]⁺ and [Pt(tpdm)Cl]⁺ complexes (TL ^tBu = 2,6-bis[(1,3-di-tert-butylimidazolin-2-imino)methyl]pyridine and tpdm = terpyridinedimethane) with N-donor ligands, l-histidine (L-His), inosine (Ino), inosine-5′-monophosphate (5′-IMP) and guanosine-5′-monophosphate (5′-GMP), were studied. All reactions were studied under pseudo-first-order conditions as a function of nucleophile concentration and temperature in aqueous 0.1 M NaClO₄ solution in the presence of 10 mM NaCl using variable-temperature Uv–Vis spectrophotometry. The order of reactivity of the studied ligands is L-His > Ino > 5′-GMP > 5′-IMP. This order of reactivity is in relation to their electronic properties and structures. The mechanism of the substitution reactions is associative in nature as supported by the negative entropy of activation.     

Modeling hole transfer in DNA. II. Molecular basis of charge transport in the DNA chain

A theoretical multiconfigurational second-order perturbation method, CASPT2, has been employed to determine the binding energies and electronic couplings for all pairs of stacked canonical nucleobases in the standard conformation of the B-DNA. The existence and relevance of conical intersections mediating the hole transfer process has been shown in different systems in vacuo and, by using hybrid QM/MM techniques, in a more realistic biological environment, formed by a double helix of 18 oligomers of DNA surrounded by water molecules. The present results support, therefore, the cooperative micro-hopping mechanism proposed in a previous work for the migration of the hole between adjacent π-stacked 2′-deoxycytidine 5′-monophosphate (dCMP) or alternate 2′-deoxyadenosine 5′-monophosphate and dCMP oligonucleotides.     

Quantitative phosphorescence study of interactions of cytosine and cytidine and its nucleotides in frozen aqueous solution: evidence for anomalous heavy-atom effect

Phosphorescence vs. pH titration curves of cytosine, cytidine, cytidine-5'-mono-phosphate (CMP), -diphosphate (CDP) and -triphosphate (CTP) were obtained in methanol/water 10/90 v/v and in various aqueous sodium halide solutions frozen at 77 degrees K. As shown by the shape of the titration curve, molecular aggregates or "puddles" of cytosine and cytidine were shown to occur only in relatively concentrated frozen solution (10(-3)M), these aggregates being dissociated in dilute frozen solutions (相似文献   

Electron Attachment to Hydrated Oligonucleotide Dimers: Guanylyl‐3′,5′‐Cytidine and Cytidylyl‐3′,5′‐Guanosine

The dinucleoside phosphate deoxycytidylyl‐3′,5′‐deoxyguanosine (dCpdG) and deoxyguanylyl‐3′,5′‐deoxycytidine (dGpdC) systems are among the largest to be studied by reliable theoretical methods. Exploring electron attachment to these subunits of DNA single strands provides significant progress toward definitive predictions of the electron affinities of DNA single strands. The adiabatic electron affinities of the oligonucleotides are found to be sequence dependent. Deoxycytidine (dC) on the 5′ end, dCpdG, has larger adiabatic electron affinity (AEA, 0.90 eV) than dC on the 3′ end of the oligomer (dGpdC, 0.66 eV). The geometric features, molecular orbital analyses, and charge distribution studies for the radical anions of the cytidine‐containing oligonucleotides demonstrate that the excess electron in these anionic systems is dominantly located on the cytosine nucleobase moiety. The π‐stacking interaction between nucleobases G and C seems unlikely to improve the electron‐capturing ability of the oligonucleotide dimers. The influence of the neighboring base on the electron‐capturing ability of cytosine should be attributed to the intensified proton accepting–donating interaction between the bases. The present investigation demonstrates that the vertical detachment energies (VDEs) of the radical anions of the oligonucleotides dGpdC and dCpdG are significantly larger than those of the corresponding nucleotides. Consequently, reactions with low activation barriers, such as those for O? C σ bond and N‐glycosidic bond breakage, might be expected for the radical anions of the guanosine–cytosine mixed oligonucleotides.     

Photoelectron spectroscopy of the parent anions of the nucleotides, adenosine-5'-monophosphate and 2'deoxyadenosine-5'-monophosphate

The parent anions of the nucleotides, adenosine-5(')-monophosphate (AMPH) and 2(')deoxyadenosine-5(')-monophosphate (dAMPH) were generated in a novel source and their photoelectron spectra recorded with 3.49 eV photons. Vertical detachment energy (VDE) and the adiabatic electron affinity (EA(a)) values were extracted from each of the two spectra. Concurrently, Kobylecka et al. [J. Chem. Phys. 128, 044315 (2008)] conducted calculations which explored electron attachment to dAMPH. Based on the agreement between their calculated and our measured VDE and EA(a) values, we conclude that the dAMPH(-) anions studied in these experiments were formed by electron-induced, intramolecular, (barrier-free) proton-transfer as predicted by the calculations. Given the similarities between the photoelectron spectra of dAMPH(-) and AMPH(-), it is likely that AMPH(-) can be described in the same manner.     

Ligand-induced biphasic thermal denaturation of RNAase A

DSC measurements have been accomplished in aqueous solutions of bovine pancreatic ribonuclease A (RNAase A) in the presence of subsaturating amounts of 3′ cytidine monophosphate (3′ CMP) and 2′ cytidine monophosphate (2′ CMP) atpH 5.0 and 5.5. In these conditions the experimental profiles do not conform to a one-step unfolding process. It can be emphasized, as a general phenomenon, that a strong linkage between the temperature-induced protein unfolding and the ligand binding, when the ligand is less than the saturation level, causes marked distortions from a two-state transition. A purely equilibrium thermodynamic analysis gives a correct account of this behaviour and allows to simulate calorimetric curves. It is thus possible to obtain, in an indirect manner, information about the thermodynamic parameters concerning the binding process, namely the association constant and the binding enthalpy. The values ofKb and Δ_b H for 3′CMP and 2′CMP, so determined, are consistent with the literature data.     

Synthesis, characterization, antitumor activity of nickel(Ⅱ) and cobalt (Ⅱ) complexes of 2,2''''-diamino-4,4''''-bithiazole and their interaction with dGMP

New complexes,of bis(2,2'-diamino-4,4'-bithiazole)sulfate nickel(Ⅱ) and bis(2,2'-diami-no-4,4'-bithiazole)sulfate cobalt(Ⅱ),have been prepared.The complexes were characterized by infrared and UV-Vis spectroscopy,1H NMR,elemental analyses and molar conductivity.The effect of these complexes on the DNA synthesis of sarcoma 180 cells has been studied by the technique of isotopic liquid scintillation.The results indicated that complexes show ability to inhibit DNA synthesis of the tumor cells.In order to provide a molecular basis for understanding the biological effects,the probe,[trana-en2Os(η2-H2)](CF3SO3)2 (en,ethylenediamine) as a monitor was first used to explore interaction of the complexes with 2'-deoxyguanosine-5'-monophosphate (dGMP).