Influence of the nucleobase and anchimeric assistance of the carboxyl acid groups in the hydrolysis of amino acid nucleoside phosphoramidates

Nucleoside phosphoramidates (NPs) are a class of nucleotide analogues that has been developed as potential antiviral/antitumor prodrugs. Recently, we have shown that some amino acid nucleoside phosphoramidates (aaNPs) can act as substrates for viral polymerases like HIV‐1 RT. Herein, we report the synthesis and hydrolysis of a series of new aaNPs, containing either natural or modified nucleobases to define the basis for their differential reactivity. Aqueous stability, kinetics, and hydrolysis pathways were studied by NMR spectroscopy at different solution pD values (5–7) and temperatures. It was observed that the kinetics and mechanism (P? N and/or P? O bond cleavage) of the hydrolysis reaction largely depend on the nature of the nucleobase and amino acid moieties. Aspartyl NPs were found to be more reactive than Gly or β‐Ala NPs. For aspartyl NPs, the order of reactivity of the nucleobase was 1‐deazaadenine>7‐deazaadenine>adenine>thymine≥3‐deazaadenine. Notably, neutral aqueous solutions of Asp‐1‐deaza‐dAMP degraded spontaneously even at 4 °C through exclusive P? O bond hydrolysis (a 50‐fold reactivity difference for Asp‐1‐deaza‐dAMP vs. Asp‐3‐deaza‐dAMP at pD 5 and 70 °C). Conformational studies by NMR spectroscopy and molecular modeling suggest the involvement of the protonated N3 atom in adenine and 1‐ and 7‐deazaadenine in the intramolecular catalysis of the hydrolysis reaction through the rare syn conformation.     

The effect of hydrolysis time on amino acid analysis

Determining the amino acid content of a protein involves the hydrolysis of that protein, usually in acid, until the protein-bound amino acids are released and made available for detection. Both the variability in the ease of peptide bond cleavage and differences in the acid stability of certain amino acids can significantly affect determination of a protein's amino acid content. By using multiple hydrolysis intervals, a greater degree of accuracy can be obtained in amino acid analysis. Correction factors derived by linear extrapolation of serial hydrolysis data are currently used. Compartmental modeling of the simultaneous hydrolysis (yield) and degradation (decay) of amino acids by nonlinear multiple regression of serial hydrolysis data has also been validated and applied to determine the amino acid composition of various biological samples, including egg-white lysozyme, human milk protein, and hair. Implicit in the routine application of serial hydrolysis in amino acid analysis, however, is an understanding that correction factors, derived either linearly or through the more accurate nonlinear multiple regression approach, need to be determined for individual proteins rather than be applied uniformly across all protein types.     

Comparison of the conformational behavior of amino acids and N-acetylated amino acids: a theoretical and matrix-isolation FT-IR study of N-acetylglycine

Within the structure determination task for peptides, which is of large interest due to the relation between structure and functionality, infrared spectra can provide detailed information on the conformational behavior. The conformational landscape ofN-acetylgycine has been studied by a combined theoretical and matrix-isolation FT-IR study. The acetylation simulates an amino acid a peptide bond. Four stable conformations were found at the MP2/6-31++G** level of theory. Among these, only one contains an intramolecular H-bond that has a small abundance at the considered temperature. Apart from this one, three other different conformations could be detected in an Ar matrix. The experimental rotamerization constants NAG2 ? NAG1 and NAG3 ? NAG1 could be estimated. The values of the rotamerization constants as well as the mean frequency deviation of N-acetylglycine were combined with previously obtained data of other N-acetylated amino acids and they appeared to be similar to the data for nonsubstituted amino acids. This suggests that the used methodology can be in the future applied to investigate small peptides. Analysis of H-bond frequency shifts and distance demonstrates that the intramolecular H-bonds in N-acetylated amino acids are stronger compared to those in nonsubstituted amino acids.     

A simple capillary electrophoresis with electrochemical detection method for determination of the hydrolysis rate constant of chlorogenic acid

A method based on the kinetics stability study on hydrolysis of chlorogenic acid by capillary zone electrophoresis with electrochemical detection (CE-ED) has been developed in this paper. Both cyclic and hydrodynamic voltammograms of chlorogenic acid and its hydrolysis product caffeic acid have been investigated. The conditions for separation of chlorogenic acid and caffeic acid, such as the buffer pH and concentration, the separation voltage, and the injection time have been optimized. Under the optimum CE running conditions, the effects of reaction temperature and pH values of the hydrolysis solutions on the hydrolysis rate constants were further studied. The hydrolysis rate constants of chlorogenic acid were obtained from the concentration change of hydrolysis during the process of hydrolysis. Based on the fact, a simple and economical method for the determination of the hydrolysis rate constant and activation energy of hydrolysis reaction has been developed.     

Characterization of ester hydrolysis in terms of microscopic rate constants

Hydroxide-catalyzed ester hydrolysis for molecules of coexisting species is quantitated in terms of microscopic rate constants, a new, species-specific physicochemical parameter. Relationships between the overall and component reactions, as well as the macroscopic and microscopic rate constants are deduced. Experimental techniques, evaluation methods, and feasibility are discussed. Species-specific, pH-independent rate constants of four coexisting, differently hydrolyzing microspecies are determined for the first time. Protonation of an alpha-amino and beta-imidazolyl site in amino acid esters has been found to accelerate the hydroxide-catalyzed hydrolysis by factors of 120 and 7.5, respectively, whereas they jointly exert a nearly 3000-fold acceleration. A total of 20 microscopic protonation equilibrium constants, as component parameters in the rate equations, have also been determined. The species-specific rate constants have been found to correlate with the site- and species-specific basicity of the leaving group and the NMR chemical shift of an adjacent proton. Individual contributions of the various microforms to the overall hydrolysis rate are depicted in microscopic reaction fraction diagrams.     

Influence of (hydroxy)alkylamino substituents on enantioseparation ability of single-isomer amino-beta-cyclodextrin derivatives in chiral capillary electrophoresis

A family of single-isomer amino-beta-cyclodextrin (amino-beta-CD) derivatives containing an amino or (hydroxy)alkylamino group in one of the primary positions has been synthesized. The steric effect and hydrogen bond forming ability of the different substituents on enantioseparation of acidic enantiomers has been studied by capillary electrophoresis (CE). Three enantiomeric model compounds (mandelic acid, cis-permethrinic acid, and cis-deltamethrinic acid) having significantly different apparent complex stability constants with beta-CD were applied in the experiments. Dependence of separation selectivity, resolution as well as mobility difference on chiral selector concentration (0.1-20 mM, pH 6.0) was investigated. Each amino-beta-CD showed higher enantioselectivity than the native beta-CD. One hydroxyalkyl group attached to the primary amino N-atom significantly increased both the enantioselectivity and the resolution compared to the primary amino-beta-CD, while two hydroxyalkyl moieties decreased them due to the predominance of steric hindrance. The value of the apparent complex stability constants obtained suited well the mobility difference model (by Wren). On the other hand, the optimum selector concentrations calculated according to the model were slightly lower than the experienced concentrations giving the maximum enantioresolution of enantiomers.     

Complexes of vanadyl and uranyl ions with the chelating groups of humic matter

The uranyl and vanadyl complexes formed with salicylic, phthalic and 3,4-dihydroxybenzoic acids have been studied by potentiometry in order to determine the stability constants of the M(m)L(n) species formed in solution, and the constants for the hydrolysis and polymeric complexes, at 25.0 degrees , in 0.10, 0.40 and 0.70M sodium perchlorate. MINIQUAD was used to process the data to find the best models for the species in solution, and calculate the formation constants. The uranyl-salicylic acid system was also studied by spectrophotometry and the program SQUAD used to process the data obtained. The best models for these systems show that co-ordination of the uranyl ion by carboxylate groups is easier than for the vanadyl ion, whereas the vanadyl ion seems to form more stable complexes with phenolate groups. Both oxo-cations seem to tend to hydrolyse rather than form complexes when the L:M ratios are greater than unity. Although the change in the constants with ionic strength is small, the activity coefficients of the salicylate and phthalate species have been calculated at ionic strengths 0.40 and 0.70M, along with the interaction parameters with Na(+), from the stability constants found for the species ML and H(2)L, according to the Br?nsted-Guggenheim expression.     

Control of the stability of Hydrogenobacter thermophilus cytochrome C(552) through alteration of the basicity of the N-terminal amino group of the polypeptide chain

In the denatured state of Hydrogenobacter thermophilus cytochrome c(552) (HT), the N-terminal amino group of the polypeptide chain is coordinated to the heme Fe in place of the axial Met, the His-N(term) form being formed [Tai, H., Munegumi, T., Yamamoto, Y. Inorg. Chem. 2009, 48, 331-338]. Since the His-N(term) form can be considered as an ordered residual structure in the denatured protein, its stability significantly influences the energy of the denatured state. In this study, the His-N(term) forms of the wild-type HT and its mutants possessing a series of amino acid residues at the N-terminal, such as N1D, N1E, and N1G, have been characterized to elucidate the physicochemical properties of the N-terminal residue responsible for the control of the thermodynamic stability of the His-N(term) form. The study revealed that the thermodynamic stability of the His-N(term) form depends highly on the basicity of the N-terminal amino group of the polypeptide chain in such a manner that an increase in the pK(a) value of the N-terminal amino group by 1 unit results in stabilization of the bond between heme Fe and the N-terminal amino group (Fe-N(term) bond) in the His-N(term) form by ～4 kJ mol(-1). The empirical hard and soft acid and base principle could account for the observed relationship between the pK(a) value of the N-terminal amino group and the stability of the Fe-N(term) bond in the His-N(term) form. In addition, the study demonstrated that the overall stability of the protein can be manipulated through the energy of the denatured protein by changing the thermodynamic stability of the His-N(term) form. Consequently, the overall stability of the protein has been shown to be controlled through alteration of the basicity of the N-terminal amino group of the polypeptide chain. These findings provide new insights into the stabilizing interactions in the denatured protein, which are relevant as to characterization of the protein stability and folding.     

Kinetic Study on Stability of Schiff Base of Pyridoxal 5′-Phosphate and Leucine in Water Media with Cationic Surfactants

We studied the stability of the Schiff bases formed between pyridoxal 5′-phosphate (PLP) and leucine in the presence of (hexadecyl)trimethylammonium bromide (CTAB) over a wide pH range by determining the kinetic constants of formation and hydrolysis of these compounds. The results show that the stability of the Schiff bases is increased by the presence of CTAB as a result of increased rates of formation and decreased hydrolysis rate constants. The ionic head groups of CTAB favour the formation of the bases, while its hydrophobic rests protect the imine double bond from hydrolysis. This model system permits one to obtain partially hydrophobic media with no need for any non-aqueous solvents.     

Relative importance of secondary structure and solvent accessibility to the stability of protein mutants. A case study with amino acid properties and energetics on T4 and human lysozymes

Understanding the factors influencing the stability of protein mutants is an important task in molecular and computational biology. In this work, we have approached this problem by examining the relative importance of secondary structure and solvent accessibility of the mutant residue for understanding/predicting the stability of protein mutants. We have used hydrophobic, electrostatic and hydrogen bond free energy terms and nine unique physicochemical, energetic and conformational properties of amino acids in the present study and these parameters have been related with changes in thermal stability (DeltaTm) of all the single mutants of lysozymes based on single and multiple correlation coefficients. As expected the properties reflecting hydrophobicity and hydrophobic free energy play a major role to distinguish stabilizing and destabilizing mutants. The hydrophobic free energy due to carbon and nitrogen atoms distinguish the stability of coil and strand mutations to the accuracy of 100 and 90%, respectively. In agreement with previous results, the subgroup classification based on secondary structure and the information about its location in the structure yielded good relationship with the experimental DeltaTm. We revealed that the secondary structure information is equally or more important than solvent accessibility for understanding the stability of protein mutants. The comparison of amino acid properties with free-energy terms indicate that the energetic contribution explains the mutant stability better in coil region whereas the amino acid properties do better in strand region. Further, the combination of free energies with amino acid properties increased the correlation significantly. The present study demonstrates the importance of classifying the mutants based on secondary structure to the stability of proteins upon mutations.     

Recent Advances in the Metal‐Catalyzed Activation of Amide Bonds

The amide functional group is commonly found in peptides, proteins, pharmaceutical compounds, natural products, and polymers. The synthesis of amides is typically performed by using classical approaches that involve the reaction between a carboxylic acid and an amine in the presence of an activator. Amides are thought to be an inert functional group, because they are unsusceptible to nucleophile attack, owing to their low electrophilicity. The reason for this resistance is clear: the resonance stability of the amide bond. However, transition metal catalysis can circumvent this stability by selectively rupturing the N?C bond of the amide, thereby facilitating further cross‐coupling or other reactions. In this Focus Review, we discuss the recent advances in this area and present a summary of methods that have been developed for activating the amide N?C bond by using precious and non‐precious metals.     

Equilibrium investigation of complex formation reactions involving copper(II), nitrilo-tris(methyl phosphonic acid) and amino acids,peptides or DNA constitutents. The kinetics,mechanism and correlation of rates with complex stability for metal ion promoted hydrolysis of glycine methyl ester

The complex formation reactions of [Cu(NTP)(OH₂)]^4? (NTP?=?nitrilo-tris(methyl phosphonic acid)) with some selected bio-relevant ligands containing different functional groups, are investigated. Stoichiometry and stability constants for the complexes formed are reported. The results show that the ternary complexes are formed in a stepwise mechanism whereby NTP binds to copper(II), followed by coordination of amino acid, peptide or DNA. Copper(II) is found to form Cu(NTP)H _n species with n?=?0, 1, 2 or 3. The concentration distribution of the various complex species has been evaluated. The kinetics of base hydrolysis of glycine methyl ester in the presence of copper(II)-NTP complex is studied in aqueous solution at different temperatures. It is proposed that the catalysis of GlyOMe ester occurs by attack of OH^? ion on the uncoordinated carbonyl carbon atom of the ester group. Activation parameters for the base hydrolysis of the complex [Cu(NTP)NH₂CH₂CO₂Me]^4? are, ΔH^±?=?9.5?±?0.3?kJ?mol^?1 and ΔS^±?=??179.3?±?0.9?J?K^?1?mol^?1. These show that catalysis is due to a substantial lowering of ΔH^±.     

镧(Ⅲ)-氨基酸配合物的温度滴定量热法研究

稀土配合物因其特有的性质而日益受到人们的重视 [1] .对于稀土与氨基酸配合物的已有研究 [2 ,3] ,但尚未见到关于镧 ( )与不同种氨基酸配合反应的较系统的报道 .文献 [2 ,3]大多采用 p H电位法或其它非热化学方法 ,且大多只得到反应的平衡常数 ,而用滴定量热法直接测定其配合常数和反应焓变的研究尚未见报道 .用滴定热量法可同时测定配合反应的焓变和熵变 [4 ] ,而且在滴定量热过程中 ,反应体系基本上是一封闭体系 ,除了对体系的温度进行监测外 ,对反应体系没有其它任何干扰 ,因而可以减少外界因素的影响 ,从而减少测定的误差 .本文用滴…     

Hydrolytic and redox transformations of chromium(III) bis-oxalato complexes with glutaminic acid and glutamine: a kinetic,UV–Vis and EPR,study

The title complexes have been synthesized, chromatographically isolated and characterized by their ligands to metal ratio determinations and spectroscopic analyses. The kinetics of the first aquation stage, i.e., the amino acid chelate ring opening via the Cr–N bond cleavage, has been studied spectrophotometrically in acidic and alkaline media. Hydrogen peroxide oxidizes the complexes in alkaline media to CrO ₄ ^2? anion and a relatively stable Cr(V) complex. Consecutive biphasic kinetics through two first-order steps were observed for the base hydrolysis and the oxidation process, whereas the acid-catalyzed aquation obeys a simple first-order pattern. Based on the kinetic and spectroscopic data, mechanisms of the coordinated amino acid liberation and chromium(III) oxidation are discussed.     

Koordinationsverbindungen des Siliciums mit C3-substituierten Acetylacetonen

Hexacoordinated silicon?complexes with C₃?substituted acetylacetones have been prepared. The reaction rates of hydrolysis of these compounds have been compared with UV- and IR-spectra and thermogravimetric data. There are no significant correlations between reaction rate constants and spectroscopic data, but hydrolytic and thermal stability have a similar dependence from the substituents.     

The Theoretical and Experimental Studies on Oxidation of Straight Chain Amino Acids in Moderately Concentrated Sulfuric Acid Medium

The kinetics of the permanganic oxidation process of some straight chain amino acids in moderately concentrated sulfuric acid medium have been investigated using a spectrophotometric technique. Conclusive evidences have proven autocatalytic activity of Mn(II) for these reactions. It is determined that even and odd effects of the number carbon atom in a carbon chain are annihilated when it's the number of carbon atoms is increased more than of three in a noncatalytic oxidation pathway. Thus, rate constants belonging to glycine, l ‐α‐amino‐n‐butyric acid, l ‐norleucine, and l ‐α‐amino‐n‐heptanoic acid satisfy Taft's equation involving the induction factor in the noncatalytic pathway, whereas l ‐α‐amino‐n‐heptanoic acid has an odd number of carbon atom in its chain carbon. On the other hand, in the catalytic pathway, rate constants satisfy Taft' equation including inductive and steric factors, when rate constants belonging to amino acids with an even number of carbon atoms are separated from those with an odd number of carbon atoms. The oxidation process of amino acids in the noncatalytic pathway and those with the even number of carbon atoms in the carbon chain in the catalytic pathway speeds up by an increase in the length of chain that is accompanied with an increase in the carbon chain's electron‐donating characteristic. On the other hand, an increase in the length of the carbon chain is accompanied with more steric hindrance, which counteracts its electron‐donating character, thereby decreasing reaction rate in the catalytic pathway. Finally, amino acid–Mn(II) complexes were studied using a density functional theory method. Results obtained show that such a complex is less stable than reactants, namely it is formed in an endothermic reaction. The number and strength of hydrogen bonding belonging to amino acid is more than those of the amino acid–Mn(II) complex. Besides, it has been illustrated that natural bond orbital analysis and molecular orbital calculations satisfy the findings.     

Mixed-Ligand Complex Formation Equilibria of Vanadium(III) with 2,2′-Bipyridine and the Amino Acids Glycine, Proline, α-Alanine and β-Alanine Studied in 3.0 mol?dm?3 KCl at 25 °C

We studied speciation of the mixed-ligand complex formation equilibria of vanadium(III) with both 2,2??-bipyridine (Bipy) and the amino acids glycine (HGly), proline (HPro), ??-alanine (H??Ala), and ??-alanine (H??Ala) by means of electromotive forces measurements emf(H) using 3.0?mol?dm^?3 KCl as the ionic medium at 25 °C. The experimental data were analyzed by means of the computational least-squares program LETAGROP, taking into account the hydrolysis of the vanadium(III) cation, the respective stability constants of the binary complexes, and the acid/base reactions of the ligands which were kept fixed during the analysis. In all four amino acid systems studied we observed the complexes [V₂O(Bipy)(B)]³⁺, [V₂O(Bipy)₂(B)₂]²⁺, [V(OH)(Bipy)(B)₂] and [V(OH)₂(Bipy)(B)], where B represents the deprotonated form of the amino acids studied in this work. The respective stability constants were determined and the species distribution diagrams as a function of pH are briefly discussed.     

Spectroscopic study of chloranil charge-transfer complexes of amino acids and related compounds

The absorption maxima, molar absorptivities, infrared spectra, compositions, formation constants, and pH dependence of amino acid—chloranil complexes have been determined with purified chloranil The n-π charge-transfer interaction depends on the presence of an unprotonated amino group; pH 9 is optimal for complex formation, but once formed, the complex is stable in a highly acidic medium and may be quantitatively extracted by hexanol. The molar absorptivities of the chloranil complexes of glycine, iminodiacetic acid, NTA, EDTA, DTPA and TTHA were measured. There is a linear relationship between the logarithm of the molar absorptivities of their chloranil complexes and the number of carboxylic groups in the molecule. There is an inverse linear relationship between the molar absorptivities of chloranil—metal—EDTA complexes and the logarithm of the stability constants of the EDTA chelates. This leads to a new method of determining the stability constants of complexes involving a nitrogen-donating group.     

Electronic structure of the peptide linkage. I. Equilibrium geometry and electronic properties of formhydroxamic acid

The equilibrium geometry of formhydroxamic acid has been calculated within the framework of the INDO –MO formalism. Various structural factors are analyzed and discussed in terms of the calculated force constants and charge distribution. The possibility of internal rotation around the C? N bond of formhydroxamic acid has been examined. The potential energy surface for the amide-imide tautomerism is explored by calculating the geometries and characterizing saddle points on that surface. The cyclic and open dimers of formhydroxamic acid are examined and the hydrogenbond energy and length are calculated.     

Enthalpy and entropy changes associated with mixed ligand chelates of Cu(II) and Ni(II) with 4-methoxy picolinic acid N-oxide and some amino acids in aqueous medium

The stability constants of mixed complexes of Cu(II) and Ni(II) with 4-methoxy picolinic acid N-oxide, and glycine, α-alanine, proline and hydroxy-proline have been determined at various temperatures by the potentiometric method in 0·1 M ionic strength. The formation constants of the mixed complexes have been evaluated and are in good agreement with statistically expected values. The enthalpy and entropy values have been calculated from 1∶1∶1 stability constants temperature coefficient data. From the enthalpy values of the mixed complexes it may be concluded that the bond strengths are not equal to the average of the bond strengths inMA ₂ andMB ₂ type parent complexes. The entropy values have been found to be favourable for ternary complex formation.