Large steric effect in the substitution reaction of amines with phosphoimidazolide-activated nucleosides

Aliphatic amines react with phosphoimidazolide-activated derivatives of guanosine and cytidine (ImpN) by replacing the imidazole group. The kinetics of reaction of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) with glycine ethyl ester, glycinamide, 2-methoxyethylamine, n-butylamine, morpholine, dimethylamine (Me2NH), ethylmethylamine (EtNHMe), diethylamine (Et2NH), pyrrolidine, and piperidine were determined in water at 37 degrees C. With primary amines, a plot of the logarithm of the rate constant for attack by the amine on the protonated substrate, log kSH(A), versus the pKa of the amine exhibits a good linear correlation with a Bronsted slope, beta nuc = 0.48. Most of the secondary amines tested react with slightly higher reactivity than primary amines of similar pKa. Interestingly, some secondary amines show substantially lower reactivity than might be expected: EtNHMe reacts about eight times, and Et2NH at least 100 times, more slowly than Me2NH although all three amines are of similar basicity. For comparison, the kinetics of reaction of guanosine 5'-phosphoimidazolide (ImpG) and cytidine 5'-phosphoimidazolide (ImpC) were determined with Me2NH, EtNHMe, and Et2NH, and similar results were obtained. These results establish that the increased steric hindrance observed with the successive addition of ethyl groups are not due to any special steric requirements imposed by the guanosine or the methyl on the 2-methylimidazole leaving group of 2-MeImpG. It is concluded that addition of ethyl and, perhaps, groups larger than ethyl dramatically increases the kinetic barrier for addition of aliphatic secondary amines to the P-N bond of ImpN. This study supports the observation that the primary amino groups on the natural polyamines are at least 2 orders of magnitude more reactive than the secondary amino groups in the reaction with ImpN.     

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. 2. Templates containing cytidine and guanosine residues

We have prepared hairpin oligonucleotides in which a 5'-terminal single-stranded segment contains cytidylate (C) and guanylate (G) residues. When these hairpin substrates are incubated with a mixture of cytidine 5'-phosphoro(2-methly)imidazolide (2-MeImpC) and guanosine 5'-phosphoro(2-methyl)imidazolide (2-MeImpG), the 5'-terminal segment acts as a template to facilitate sequence-specific addition of G and C residues to the 3'-terminus of the hairpin. If an isolated G residue is present at the 3'-end of the template strand, it is copied regiospecifically in the presence of 2-MeImpC and 2-MeImpG to give a product containing an isolated C residue linked to its G neighbors by 3'-5'-internucleotide bonds. However, if only 2-MeImpC is present in the reaction mixture, very little reaction occurs. Thus, the presence of 2-MeImpG catalyzes the incorporation of C. If the template strand contains a short sequence of G residues, it is copied in the presence of a mixture of 2-MeImpC and 2-MeImpG. If only 2-MeImpC is present in the reaction mixture, efficient synthesis occurs to give a final product containing one fewer C residue than the number of G residues in the template.     

Kinetic dissection of individual steps in the poly(C)-directed oligoguanylate synthesis from guanosine 5'-monophosphate 2-methylimidazolide

A kinetic study of oligoguanylate synthesis on a polycytidylate template, poly(C), as a function of the concentration of the activated monomer, guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, is reported. Reactions were run with 0.005-0.045 M 2-MeImpG in the presence of 0.05 M poly(C) at 23 degrees C. The kinetic results are consistent with a reaction scheme (eq 1) that consists of a series of consecutive steps, each step representing the addition of one molecule of 2-MeImpG to the growing oligomer. This scheme allows the calculation of second-order rate constants for every step by analyzing the time-dependent growth of each oligomer. Computer simulations of the course of reaction based on the determined rate constants and eq 1 are in excellent agreement with the product distributions seen in the HPLC profiles. In accord with an earlier study (Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahedron 1984, 40, 39), rate constants, ki, for the formation of the tetramer and longer oligomers up to the 16-mer were found to be independent of length and somewhat higher than k3 (formation of trimer), which in turn is much higher than k2 (formation of dimer). The ki (i > or = 4), k3, and k2 values are not true second-order rate constants but vary with monomer concentration. Mechanistic models for the dimerization (Scheme I) and elongation reactions (Scheme II) are proposed that are consistent with our results. These models take into account that the monomer associates with the template in a cooperative manner. Our kinetic analysis allowed the determination of rate constants for the elementary processes of covalent bond formation between two monomers (dimerization) and between an oligomer and a monomer (elongation) on the template. A major conclusion from our study is that bond formation between two monomer units or between a primer and a monomer is assisted by the presence of additional next-neighbor monomer units. This is consistent with recent findings with hairpin oligonucleotides (Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317). Our study is the first of its kind that shows the feasibility of a thorough kinetic analysis of a template-directed oligomerization and provides a detailed mechanistic model of these reactions.     

Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligation

The role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, with Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pKa values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large delta S(++) value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.     

Kinetics of template-directed pyrophosphate-linked dideoxyguanylate synthesis as a function of 2-MeImpdG and poly(C) concentration: insights into the mechanism.

Aqueous solutions of deoxyguanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpdG, yield primarily deoxyguanosine 5'-monophosphate, 5'dGMP, and pyrophosphate-linked dideoxyguanylate, dG5'ppdG, abbreviated G2p (see Chart 1). The initial rate of G2p formation, d[G2p]/dt in M h-1, determined at 23 degrees C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid chromatography (HPLC) analysis, exhibits a second-order dependence on 2-MeImpdG concentration, [G]o, indicating a bimolecular mechanism of dimerization in the range 0.02 M < or = [G]o < or = 0.09 M. In the presence of polycytidylate, poly(C), G2p synthesis is accelerated and oligodeoxyguanylate products are formed by incorporation of 2-MeImpdG molecules. The kinetics of G2p formation as a function of both monomer and polymer concentration, expressed in C equivalents, were also determined under the above conditions and exhibited a complex behavior. Specifically, at a constant [poly(C)], values of d[G2p]/dt typically increased with [G]o with a parabolic upward curvature. At a constant [G]o, values of d[G2p]/dt increase with [poly(C)], but level off at the higher poly(C) concentrations. As [G]o increases this saturation occurs at a higher poly(C) concentration, a result opposite to expectation for a simple complexation of two reacting monomers with the catalyst prior to reaction. Nevertheless, these results are shown to be quantitatively consistent with a template-directed (TD) mechanism of dimerization where poly(C) acts as the template to bind 2-MeImpdG in a cooperative manner and lead, for the first time, to the formulation of principles that govern template-directed chemistry. Analysis of the kinetic data via a proposed TD cooperative model provides association constants for the affinity between polymer and monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate synthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first system that could serve as a textbook example of a TD reaction under conditions such that the template is fully saturated by monomers and under conditions that it is not.     

Kinetic preference for the 3'-5'-linked dimer in the reaction of guanosine 5'-phosphorylmorpholinamide with deoxyguanosine 5'-phosphoryl-2-methylimidazolide as a function of poly(C) concentration.

The formation of the internucleotide bond in diguanylate synthesis was studied in aqueous solution at pH 8 and 0.2 M Mg2+ in the presence and absence of polycytidylate, poly(C). The investigation was simplified by using guanosine 5'-phosphorylmorpholinamide, mor-pG, which can act only as a nucleophile, and deoxyguanosine 5'-phosphoryl-2-methylimidazolide, 2-MeImpdG, which can act only as an electrophile. The time-dependent product distribution was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS). In the absence of poly(C) the reaction between mor-pG and 2-MeImpdG yielded small amounts of the dimer mor-pGpdG with a regioselectivity of 2'-5':3'-5' = 3.5. In the presence of poly(C) dimer yields increased and a reversal in regioselectivity occurred; both effects were in proportion to the concentration of the polymer. The results can be quantitatively explained with the proposition that poly(C), acting as the template, catalyzes the reaction between template-bound monomers by about a factor of 4-5 over the reaction in solution and yields dimers with a regioselectivity of 2'-5':3'-5' approximately 0.33. These findings illustrate the intrinsic preference of guanosine monomers to correctly self-assemble on the appropriate template.     

Nonenzymatic template-directed synthesis on oligodeoxycytidylate sequences in hairpin oligonucleotides

We have developed a novel method for studying template-directed synthesis in hairpin oligonucleotides. An unpaired segment at the 5'-terminus of the hairpin acts as an intramolecular template for the extension of the paired 3'-terminus. Products are analyzed by denaturing gel electrophoresis of [32P]-labeled hairpins. Using this system, we have studied the synthesis of oligoguanylates on an oligodeoxycytidylate template. We find that guanosine 5'-phosphoro(2-methyl)imidazolide adds efficiently to a terminal riboguanylate residue at temperatures in the range 0-37 degrees C but not at 50 degrees C. At 0 degree C, the half-time for addition of the first G residue is about 3 h, and the reaction rate is independent of pH in the range 6.5-8.0. The first addition reaction results in the formation of a predominantly 3'-5'-internucleotide bond. When the 3'-terminal riboguanylate residue is placed by a deoxyguanylate residue, the half-time for the first addition increases from about 3 to about 30 h.     

Use of phosphoimidazolide-activated guanosine to investigate the nucleophilicity of spermine and spermidine.

Guanosine 5'-phosphate 2-methylimidazolide (2-MeImpG), a labile phosphoimidazolide analog of guanosine triphosphate, was used to test the reactivity of the natural polyamines (PAs), spermine (spm) and spermidine (spd). The products are the guanosine 5'-phosphate-polyamine derivatives (PA-pG: spd-pG and spm-pG) which are quite stable in the range 4 < pH < 11. Our study is the first of which we are aware that reports on the nucleophilicity of these amines. The main findings are as follows. (i) HPLC analysis of the products indicates the formation of only two of the three possible spd products and only one of the two possible spm products. These results can be explained if only the primary amino groups of the two polyamines are reactive, while the secondary amino groups are rendered unreactive by a steric effect. The reactions of 2-MeImpG and other phosphoimidazolide derivatives of nucleosides (ImpNs) with primary and secondary monoamines support this interpretation (Kanavarioti et al. J. Org. Chem. 1995, 60, 632). (ii) The product ratio of the two spd-pG adducts derived from the primary amino groups varies between 2.40 and 0.71 in the range 6.1 < or equal to pH < or equal to 11.9. Such small variation in the product ratio can only be rationalized by the similar, but not identical, basicity of the two primary amino groups and provides strong support for a previously reported model for polyamine ionization (Onasch et. al. Biophys. Chem. 1984, 19, 245). (iii) On the basis of our kinetic determinations conditions at which the nucleophilicity of these amines is at a minimum and at which other interactions with ImpNs could be tested can be chosen.     

Catalysis of hydrolysis and nucleophilic substitution at the P-N bond of phosphoimidazolide-activated nucleotides in phosphate buffers

Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).     

Long-range solvent effects on the orbital interaction mechanism of water acidity enhancement in metal ion solutions: a comparative study of the electronic structure of aqueous Mg and Zn dications

We study the dissociation of water coordinated to a divalent metal ion center, M2+ = Mg2+, Zn2+ using density functional theory (DFT) and ab initio molecular dynamics (AIMD) simulations. First, the proton affinity of a coordinated OH- group is computed from gas-phase Mg2+(H2O)5(OH-), which yields a relative higher gas-phase acidity for a Zn2+-coordinated as compared to a Mg2+-coordinated water molecule, DeltapKa(gp) = 5.3. We explain this difference on the basis of a gain in stabilization energy of the Zn2+(H2O)5(OH-) system arising from direct orbital interaction between the coordinated OH- and the empty 4s state of the cation. Next, we compute the acidity of coordinated water molecules in solution using free-energy thermodynamic integration with constrained AIMD. This approach yields pKa Mg2+ = 11.2 and pKa Zn2+ = 8.4, which compare favorably to experimental data. Finally, we examine the factors responsible for the apparent decrease in the relative Zn2+-coordinated water acidity in going from the gas-phase (DeltapKa(gp) = 5.3) to the solvated (DeltapKa = 2.8) regime. We propose two simultaneously occurring solvation-induced processes affecting the relative stability of Zn2+(H2O)5(OH-), namely: (a) reduction of the Zn 4s character in solution states near the bottom of the conduction band; (b) hybridization between OH- orbitals and valence-band states of the solvent. Both effects contribute to hindering the OH- --> Zn2+ charge transfer, either by making it energetically unfavorable or by delocalizing the ligand charge density over several water molecules.     

A FMO-controlled reaction path in the benzil-benzilic acid rearrangement

Reaction paths for the title rearrangement along with its methyl analogue were investigated by density functional theory calculations. The reaction model is R-CO-CO-R + OH(-)(H2O)4 --> R2C(OH)-COO- + (H2O)4 (R = Me and Ph), where the water tetramer is employed both for solvation to OH- and for the proton relay along hydrogen bonds. The reaction is composed of OH- addition, C-C rotation, carbanion [1,2] migration, and proton relay toward the product anions. The rate-determining step was calculated to be the carbanion migration. Apparently, carbanion [1,2] migration is unlikely relative to the carbonium ion one. However, LUMOs of the 1,2-diketones have large and nodeless lobes at the reaction center, the C1-C2 bond. The specific LUMO character is reflected both in the [2+1]-like one-center nucleophilic addition and in the carbanion [1,2] shift. The proton relay involved in the isomerization from the oxo intermediate to the carboxylate was calculated to take place via the water tetramer.     

The elimination of a hydrogen atom in Na(H2O)n

By a systematic examination on Na(H2O)n, with n = 4-7, 9, 10, and 15, we demonstrate that a hydrogen loss reaction can be initiated by a single sodium atom with water molecules. This reaction is similar to the well-known size-dependent intracluster hydrogen loss in Mg+(H2O)n, which is isoelectronic to Na(H2O)n. However, with one less charge on Na(H2O)n than that on Mg+(H2O)n, the hydrogen loss for Na(H2O)n is characterized by a higher barrier and a more flexible solvation shell around the metal ion, although the reaction should be accessible, as the lowest barrier is around 8 kcal/mol. Interestingly, the hydroxide ion OH- produced in the process is stabilized by the solvation of H2O molecules and the formation of an ion pair Na+(H2O)4(H2O)n-l-4[OH-(H2O)l]. The activation barrier is reduced as the unpaired electron in Na(H2O)n moves to higher solvation shells with increasing cluster size, and the reaction is not switched off for larger clusters. This is in sharp contrast to the reaction for Mg+(H2O)n, in which the OH- ion is stabilized by direct coordination with Mg2+ and the reaction is switched off for n > 17, as the unpaired electron moved to higher solvation shells. Such a contrast illustrates the important link between microsolvation environment and chemical reactivity in solvation clusters.     

Direct detection of potassium cations bound to G-quadruplex structures by solid-state 39K NMR at 19.6 T

We report solid-state 39K NMR detection of the K+ ions bound to three G-quadruplex structures formed by self-assembly of 5'-tert-butyl-dimethylsilyl-2',3'-O-isopropylidene guanosine, guanosine, and guanosine 5'-monophosphate. The 39K NMR spectra clearly show different spectral signatures for K+ ions inside the G-quadruplex channel and for K+ ions bound to the phosphate groups. Solid-state 39K NMR spectra for hydrated K salts of adenosine 2'-monophosphate and adenosine 5'-diphosphate are also reported.     

Reactive sulfur species: hydrolysis of hypothiocyanite to give thiocarbamate-S-oxide

Hypothiocyanite (OSCN-) hydrolyzes under alkaline conditions to give thiocarbamate-S-oxide (H2NC(=O)SO-, the conjugate base of carbamothioperoxoic acid) via a mechanism that involves rate-limiting nucleophilic attack of OH- on OSCN-, followed by fast protonation (with no net consumption of H+/OH- at pH 11.7). Thiocarbamate-S-oxide has been characterized by 13C NMR, 15N NMR, UV spectroscopy, and ion chromatography. It has also been independently synthesized by the reaction of thiocarbamate (H2NC(=O)S-) and hypochlorite (OCl-). The properties of thiocarbamate-S-oxide that is produced by hydrolysis of OSCN- and by oxidation of H2NC(=O)S- are the same. The possible relevance of thiocarbamate-S-oxide in human peroxidase defense mechanisms remains to be explored.     

Effect of the ribose versus 2'-deoxyribose residue on the metal ion-binding properties of purine nucleotides

The interaction between metal ions and nucleotides is well characterized, as is their importance for metabolic processes, e.g. in the synthesis of nucleic acids. Hence, it is surprising to find that no detailed comparison is available of the metal ion-binding properties between nucleoside 5'-phosphates and 2'-deoxynucleoside 5'-phosphates. Therefore, we have measured here by potentiometric pH titrations the stabilities of several metal ion complexes formed with 2'-deoxyadenosine 5'-monophosphate (dAMP2-), 2'-deoxyadenosine 5'-diphosphate (dADP3-) and 2'-deoxyadenosine 5'-triphosphate (dATP4-). These results are compared with previous data measured under the same conditions and available in the literature for the adenosine 5'-phosphates, AMP(2-), ADP(3-) and ATP(4-), as well as guanosine 5'-monophosphate (GMP(2-)) and 2'-deoxyguanosine 5'-monophosphate (dGMP(2-)). Hence, in total four nucleotide pairs, GMP(2-)/dGMP(2-), AMP(2-)/dAMP(2-), ADP(3-)/dADP(3-) and ATP(4-)/dATP(4-) (= NP/dNP), could be compared for the four metal ions Mg2+, Ni2+, Cu2+ and Zn2+ (= M2+). The comparisons show that complex stability and extent of macrochelate formation between the phosphate-coordinated metal ion and N7 of the purine residue is very similar (or even identical) for the AMP(2-)/dAMP(2-) and ADP(3-)/dADP(3-) pairs. In the case of the complexes formed with ATP(4-)/dATP(4-) the 2'-deoxy complexes are somewhat more stable and show also a slightly enhanced tendency for macrochelate formation. This is different for guanine nucleotides: the stabilities of the M(dGMP) complexes are clearly higher, as are the formation degrees of their macrochelates, than is the case with the M(GMP) complexes. This enhanced complex stability and greater tendency to form macrochelates can be attributed to the enhanced basicity (DeltapKaca. 0.2) of N7 in the 2'-deoxy compound. These results allow general conclusions regarding nucleic acids to be made.     

Redox pathways in DNA oxidation: kinetic studies of guanine and sugar oxidation by para-substituted derivatives of oxoruthenium(IV)

The oxidation of nucleotides and DNA by a series of complexes based on Ru(tpy)(bpy)O2+ (1) was investigated (tpy = 2,2':6',2"-terpyridine; bpy = 2,2'-bipyridine). These complexes were substituted with electron-donating or-withdrawing substituents in the para positions of the polypyridyl ligands so that the oxidation potentials of the complexes were affected but the reaction trajectory of the oxo ligand with DNA was the same throughout the series. The prepared complexes were (with E1/2(III/II) and E1/2(IV/III) values in volts versus Ag/AgCl) Ru(4'-EtO-tpy)(bpy)O2+ (2; 0.47, 0.60), Ru(4'-Cl-tpy)(bpy)O2+ (3; 0.55, 0.63), Ru(tpy)(4,4'-Me2-bpy)O2+ (4; 0.48, 0.62), and Ru(tpy)(4,4'-Cl2-bpy)O2+ (5; 0.58, 0.63). The complexes oxidized deoxycytosine 5'-monophosphate at the sugar moiety (k = 0.24-0.47 M-1 s-1) and guanosine 5'-monophosphate at the base moiety (k = 6.1-15 M-1 s-1). The rate constants increase across these ranges in the order 3 > 1 > 4 > 2, which is the same order as the redox potentials of the complexes. The effect of the base on these reactions was also studied, and xanthine was found to react with 1 much faster than guanine while hypoxanthine was less reactive than the sugar moiety. The complexes all oxidized oligonucleotides to generate base-labile lesions at guanine and a combination of spontaneous and base-labile scission at the sugar functionalities. The selectivity of cleavage in duplex and single-stranded DNA was not a strong function of the substituents on the metal complex.     

Influence of substitution on kinetics and mechanism of ring transformation of substituted S-[1-phenylpyrrolidin-2-on-3-yl]isothiuronium salts

Twelve new substituted S-(1-phenylpyrrolidin-2-on-3-yl)isothiuronium bromides and twelve corresponding 2-imino-5-(2-phenylaminoethyl)thiazolidin-4-ones have been prepared and characterised. Kinetics and mechanism of transformation reaction of S-[1-(4-methoxyphenyl)pyrrolidin-2-on-3-yl]isothiuronium bromide and its N,N-dimethyl derivative 5a into corresponding substituted thiazolidin-4-ones 2a and 6a have been studied in aqueous solutions of amine buffers (pH 8.1-11.5) and sodium hydroxide solutions (0.005-0.5 mol l(-1)) at 25 degrees C and at I= 1 mol l(-1) under pseudo-first-order reaction conditions. The kinetics observed show that the transformation reaction is subject to general acid-base, and hydroxide ion catalyses. Acid catalysis does not operate in the transformation of 1a; the rate-limiting step of the base-catalysed transformation is the decomposition of bicyclic tetrahedral intermediate In(+/-) and the Br?nsted dependence is non-linear (pK(a) approximately 9.8). In the case of derivative 5a both base and acid catalyses make themselves felt. In the base catalysis, the rate-limiting step consists of the decomposition of bicyclic intermediate In, and the Br?nsted dependence is linear (beta = 0.9; pK(a) > 11.5). The acid-catalysed transformation of 5a also proceeds via the intermediate In, and the reaction is controlled by diffusion (alpha approximately equal to 0). With compound 5a in triethylamine and butylamine buffers, the general base catalysis changes into specific base catalysis. The effect of substitution in aromatic moiety of compounds 1a-h and 3a-h on the course of the transformation reaction has been studied in solutions of sodium hydroxide (0.005-0.5 mol l(-1)) at 25 degrees C by the stopped-flow method. The electron-acceptor substituents 4-NO(2) and 4-CN do not obey the Hammett correlation, which is due to a suppression of cross-conjugation in the ring-closure step of the transformation reaction.     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.     

Reaction monitoring of platinum(II) complex--5'-guanosine monophosphate adduct formation by ion exchange liquid chromatography/electrospray ionization mass spectrometry

Cisplatin and four structurally related platinum(II) complexes were incubated with guanosine 5'-monophosphate (5'-GMP) in water at 37 degrees C. The adduct formation reactions were monitored with cation- and anion-exchange liquid chromatography/electrospray ionization mass spectrometry. In addition to mono- and bis-adducts of guanosine 5'-monophosphate with the platinum(II) complexes, other molecular species, presumably with a binuclear structure (two platinum(II) centres), were detected in the reaction mixtures, which have not been reported previously, indicating an unexpected complexity of adduct formation. Anion-exchange chromatography revealed the presence of isomers of two complexes which presumably result from the restricted rotation at the platinum-- N-7 (5'-GMP) bonds. All reaction products were characterized in both the positive and negative ion modes. Furthermore, preliminary kinetics and half-times of complex formation were investigated for cisplatin and two other platinum(II) complexes, monitoring the relative concentrations of free 5'-GMP and of mono- and bis-GMP adducts as a function of time (250 h) using an internal standard protocol with thymidine 5'-monophosphate.     

Bisphosphonate derivatives of nucleoside antimetabolites: hydrolytic stability and hydroxyapatite adsorption of 5'-beta,gamma-methylene and 5'-beta,gamma-(1-hydroxyethylidene) triphosphates of 5-fluorouridine and ara-cytidine

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.