Synthesis of a pH dependent covalent imprinted polymer able to recognize organotin species

The covalent imprinting approach has for the first time been successfully applied for the synthesis of an imprinted polymer able to recognize organotin species. The synthesis has been accomplished by co-polymerization of the complex Bu(2)SnO-m-vinylbenzoin as the imprinting template plus co-monomer sodium methacrylate, and ethylene glycol dimethacrylate as cross-linker. The imprinting effect has been evidenced within the narrow pH range 2.5< pH< 3.5. At lower pH values, the imprinting effect is prevented by the exclusive existence of non-specific interactions, whereas pH>3.5 provokes a strong rebind of the template in both imprinted and non-imprinted polymers. This pH dependency can be explained as a selective chemical modification which reduces bind diversity following a model based on enolization by protonation of the specific cavities. Characterization of the adsorption isotherms showed good agreement with the Langmuir-Freundlich (LF) model, presenting quite homogeneous binding sites for a bulk material and high capacity in the imprinting pH range. In addition, the affinity spectrum (AS) method has been represented showing the typical profiles of LF isotherm for both sub-saturation and saturation levels, being in general agreement with the encountered values for fitting coefficients. The covalent molecular imprinted polymer has been successfully evaluated in a SPE process for further OTC determination in the certified mussel tissue (CRM 477).     

Fluorescent pH probes for alkaline pH range based on perylene tetra-(alkoxycarbonyl) derivatives

The pH detection in the alkaline range is particularly important in many fields such as leather processing, waste water treatment, paper industry, and metal mining and finishing. Compared with traditional analysis methods such as colorimetric sensors and electrochemical sensors, the fluorescence and colorimetric probes for pH measurements have attracted much more attention due to their advantages of high sensitivity, excellent selectivity, noninvasiveness, low cost, fast response time, the possibility of continuously measurement, etc. However, there are few fluorescent probes fiting for alkaline pH monitoring. Acturally, the design and synthesis of them were more significant for new probes producing. In this study, the design, synthesis, and practical application of two novel fluorescent pH probes for alkaline pH assay were discussed. Both of the two probes were derived from perylene tetra-(alkoxycarbonyl). The red or blue shift of the absorption/fluorescence spectrum was caused by the introduction of electron donor amino or oxygen ring in the bay region. Due to electronic separation of the OH group from the electron-withdrawing core, the probes have high pKa values and cover the pH range from 8 to 12. They exist in either fluorescent acid form or non-fluorescent basic form. It was investigated that the amino substituent at bay region had a higher pKa value than O-heterocyclic annulated perylene, which showed that the adjustable pKa value could be achieved by the modification of electron withdrawing groups. The probes would have a wide use for testing strips measurements and monitoring pH changes in concrete.     

High-throughput evaluation of lipophilicity and acidity by new gradient HPLC methods

There is a need for fast testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity. These two parameters can conveniently be estimated by gradient reversed-phase HPLC. Appropriate conventional organic solvent gradient and the new pH gradient HPLC procedures are presented. The chromatographic parameter of lipophilicity, log kw, can be determined from two organic solvent gradient runs instead of 6-8 runs necessary in the standard isocratic (polycratic) approach. The newly introduced pH gradient reversed-phase HPLC consists in a programmed increase during the chromatographic run of the eluting power of the mobile phase with regards to ionizable analytes. The eluting strength of the mobile phase increases due to its increasing (in case of acidic analytes) or decreasing (basic analytes) pH, whereas the content of organic modifier remains constant. It has been theoretically and experimentally demonstrated that the pKa and log kw values can be evaluated based on retention data from a pH gradient run, combined with appropriate data from two organic solvent gradient runs. The gradient HPLC-derived log kw parameters correlate well with analogous parameters determined isocratically as well as with reference lipophilicity parameter log P (logarithm of n-octanol/water partition coefficient). Also, the HPLC-derived pKa parameters correlate to the literature pKa values (w(w)pKa), conventionally determined by titrations in water. The approach described allows rapid and high-throughput assessment of log kw and pKa for large series of drugs candidates, also when the analytes are available in a form of mixture, e.g. produced by combinatorial synthesis.     

Chemoselective acylation of fully deprotected hydrazino acetyl peptides. Application to the synthesis of lipopetides.

Fully deprotected N-terminal alpha-hydrazino acetyl peptides were synthesized and chemoselectively acylated on the hydrazine moiety with various fatty acid succinimidyl esters or N-(cholesterylcarbonyloxy) succinimide to give lipopeptides of high purity. The buffer and pH were adjusted in order to minimize the oxidation of the hydrazine moiety and to achieve the best conversion and selectivity. The acylation was performed in a citrate-phosphate buffer/2-methylpropan-2-ol mixture of pH 5.1. The pKa of the alpha-hydrazino acetyl group on our model peptide was found to be 6.45, i.e., about 2 units lower than the pKa of a glycyl residue. The reaction was subsequently applied to the synthesis of a 38AA peptide derivatized by a palmitoyl group.     

Retention of ionisable compounds on high-performance liquid chromatography XVI. Estimation of retention with acetonitrile/water mobile phases from aqueous buffer pH and analyte pKa

In agreement with our previous studies and those of other authors, it is shown that much better fits of retention time as a function of pH are obtained for acid-base analytes when pH is measured in the mobile phase, than when pH is measured in the aqueous buffer when buffers of different nature are used. However, in some instances it may be more practical to measure the pH in the aqueous buffer before addition of the organic modifier. Thus, an open methodology is presented that allows prediction of chromatographic retention of acid-base analytes from the pH measured in the aqueous buffer. The model presented estimates the pH of the buffer and the pKa of the analyte in a particular acetonitrile/water mobile phase from the pH and pKa values in water. The retention of the analyte can be easily estimated, at a buffer pH close to the solute pKa, from these values and from the retentions of the pure acidic and basic forms of the analyte. Since in many instances, the analyte pKa values in water are not known, the methodology has been also tested by using Internet software, at reach of many chemists, which calculates analyte pKa values from chemical structure. The approach is successfully tested for some pharmaceutical drugs.     

Influence of mobile phase acid-base equilibria on the chromatographic behaviour of protolytic compounds

A review about the influence of mobile phase acid-base equilibria on the liquid chromatography retention of protolytic analytes with acid-base properties is presented. The general equations that relate retention to mobile phase pH are derived and the different procedures to measure the pH of the mobile phase are explained. These procedures lead to different pH scales and the relationships between these scales are presented. IUPAC rules for nomenclature of the different pH are also presented. Proposed literature buffers for pH standardization in chromatographic mobile phases are reviewed too. Since relationships between analyte retention and mobile phase pH depends also on the pKa value of the analyte, the solute pKa data in water-organic solvent mixtures more commonly used as chromatographic mobile phase are also reviewed. The solvent properties that produce variation of the pKa values with solvent composition are discussed. Chromatographic examples of the results obtained with the different procedures for pH measurement are presented too. Application to the determination of aqueous pKa values from chromatographic retention data is also critically discussed.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

pH effect on cysteine and cystine behaviour at hanging mercury drop electrode

The pH effects on the electrochemical reactions of thiol and disulphide groups on mercury electrodes have been studied. These groups facilitate the oxidation of mercury from the electrode and its conversion into mercury thiolates. Under the appropriate experimental conditions, these thiolates form a compact film around the electrode. The formation of this film can be detected by the appearance of a spike current by cyclic voltammetry. Adsorption at the mercury surface of these groups is conditioned by the charge of the molecule, which in turn, is a consequence of the pH. Therefore, in cysteine solution, the compact film appears only when pH lies between the pKa1 and pKa2, and between the pKa2 and pKa3 in cystine solutions. At these pH values cysteine and cystine carry a zero net charge.     

Determination of pKa values of anthraquinone compounds by capillary electrophoresis

In this paper, the dissociation constants (pKa1, pKa2) of five anthraquinones were determined from the relation between the effective mobility at different pH values and the buffer pH value, which was derived from the basic electrophoresis theory and the dissociation equilibrium of a binary acid. In addition, the changes of pKa values of the five compounds were also investigated when organic modifiers were added to the buffer system.     

Molecular Design of Trypsin Towards High Substrate Selectivity

Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin. If the enzyme could react effectively at a pH higher than lysine's pKa but lower than that of arginine, it would react more selectively with arginine-type substrates, since in that pH range, the side chain of arginie remain protonated, while that of lysine is deprotonated. For trypsin. the change of histidine (57)'s pKa reflects the shift in reaction optimum pH. Electrostatic calculations showed that when surface positive residues were mutated into neutral or negative ones, the pKa of histidine(57) would be raised and those surface charges within a cone of 70 degree around histidine(57) have strong influence on its pKa. Several sites were suggested in rat trypsin which might serve as potential mutation locations to make trypsin active at a higher pH, thus more selective towards arginine t     

Ruthenium(II) complex of Hbopip: synthesis, characterization, pH-induced luminescence "off-on-off" switch, and avid binding to DNA

A novel ruthenium(II) complex of [Ru(bpy)2(Hbopip)](ClO4)2 (in which bpy=2,2'-bipyridine, Hbopip=2-(4-benzoxazolyl)phenylimidazo[4,5-f][1,10]phenanthroline) was synthesized and characterized. The spectrophotometric pH titrations of the complex showed that it acted as a pH-induced luminescence "off-on-off" switch: a luminescence off-on switch with a luminescence enhancement factor of IpH=3.0/IpH=1.0=20 occurring over a narrow pH range of 1.00-3.00 plus a luminescence on-off switch with a luminescence enhancement factor of 3 over a pH range of 3.20-9.40. The excited-state ionization constant of the complex derived, pKa1*=3.06, is 1.36 pKa units greater than the ground-state pKa1=1.70, and pKa2*=5.01 and pKa3*=8.22 are comparable to the ground-state pKa2=5.23 and pKa3=8.22, respectively. The complex avidly bound to calf thymus DNA with a large binding constant of (1.2+/-0.3)x10(7) M-1 in buffered 50 mM NaCl, as evidenced by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, viscosity measurements, and DNA melting experiments.     

Rapid screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis combined with short-end injection

A method applying pressure-assisted capillary electrophoresis combined with short-end injection has been developed for the rapid screening of the pKa values of pharmaceuticals. The electrophoretic separation is performed on a short capillary length with short-end injection under an applied pressure, and the effective mobility is measured in a series of 10 different buffers with constant ionic strength (I = 0.05). The application of pressure not only reduces migration times, particularly in lower pH buffers, but also improves the repeatability of effective mobility measurements. The influence of pressure on the effective mobility was investigated at various pH values. It was observed for the first time that an increase in pressure resulted in a slight decrease in the effective mobility when the pH was above the pKa for acidic analytes, whereas an increased effective mobility with increasing pressures was observed when the pH was below the pKa. However, the observed effective mobility shift by the applied pressure did not significantly affect the determined pKa values. The determined pKa values were in good agreement with published data. Furthermore, a stacking condition was applied to increase the sensitivity, and a concentration down to 2 microM could readily be detected with UV detection using a 50 microm I.D. capillary. This technique is particularly suitable for measurement of pKa values for compounds with poor aqueous solubility. The method also omits the commonly used preconditioning steps with sodium hydroxide and water. The exclusion of excessive preconditioning steps and the use of pressure reduces the total cycling analysis time, and makes it possible to determine the pKa in less than 40 min per compound without loss of accuracy.     

Resolution in capillary electrophoresis with nonaqueous methanol as solvent: theoretical prediction and experimental confirmation.

The resolution of the analytes was predicted from their known pKa* values and actual mobilities in nonaqueous methanolic solutions according to theory taking longitudinal diffusion as the only cause for peak dispersion. This leads to an equation of the resolution as a function of the pH*, as both selectivity and efficiency are dependent on the pH of the buffer. The experimentally obtained resolution values were in acceptable agreement with the predicted theoretical ones in most cases. This was demonstrated for substituted benzoic acids as analytes. The pKa* values needed for the calculation of the resolution were derived from the pH* dependence of the effective mobility. The pH* scale in methanol was based on conventional pKa* values of acetic acid and chloroacetic acid taken from the literature.     

A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.     

Evaluation of calibration data in capillary electrophoresis using artificial neural networks to increase precision of analysis

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.     

Linear-scaling molecular orbital calculations for the pKa values of ionizable residues in proteins

In this report, we present a computational methodology for the pKa prediction of proteins, based on linear-scaling molecular orbital calculations for their solution-conformations obtained from NMR measurements. The method is used to predict the pKa values of five carboxylic acids (Asp7, Glu10, Glu19, Asp27, and Glu43) in turkey ovomucoid third domain (OMTKY3), and six aspartates residues (Asp 22, Asp 44, Asp 54, Asp 75, Asp 83, and Asp 93) in barnase. For OMTKY3, all the predicted pKa values are within 1 pH units from the available experimental ones, except for the case of Glu 43. For barnase, the root-mean-square deviation from experiment is 1.46 pH units. As a result, the proposed pKa calculation method correctly reproduces the relative order of the pKa values among the carboxylic acids located in different sites of the proteins. The calculated pKa values are decomposed into the contributions of short- and long-range structural difference effects. The results indicate that in both proteins the pKa value of the given carboxylic acid is partially influenced by long-range interactions with distant charged residues, which significantly contribute to determining the relative order of the pKa values. The current methodology based on LSMO provides us useful information about the titration behavior in a protein.     

Capillary zone electrophoresis of cationic and anionic drugs in methanol

The actual mobilities and dissociation constants of acidic and basic pharmaceuticals were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the pharmaceuticals in methanol (pK(a,MeOH)) were calculated by non-linear curve fitting to the measured mobility values. It was found that the shift in pKa value (when compounds were transferred from water to methanol) increased with the acidity of the analyte. The average pKa shift for compounds exhibiting acidic properties in water was ca. 5.5 units, and the shift for basic compounds about 2 units. As was shown for a mixture of beta-blockers, the calculated actual mobilities and pKa values can be utilised in the optimisation of pH conditions for separation. The practical value of the method was illustrated by the analysis of urine samples.     

Monolayer studies of single-chain polyprenyl phosphates

The monolayer properties of some single-chain polyprenyl phosphates (phytanyl, phytyl, and geranylgeranyl phosphates), which we regard as hypothetical primitive membrane lipids, were investigated at the air-water interface by surface pressure-area (pi-A) isotherm measurements. The molecular area/ pressure at various pH conditions dependence revealed the acid dissociation constants (pKa values) of the phosphate. The pKa values thus obtained at the air-water interface (pKa1 = 7.1 and pKa2 = 9.4 for phytanyl phosphate) were significantly shifted to higher pH than those observed in the bilayer state in water (pKa1 = 2.9 and pKa2 = 7.8). The difference in pKa values leads to a stability of the phosphate as both monolayer and bilayer states in a pH range of 2-6. In addition, the presence of ions such as sodium, magnesium, calcium, and lanthanum in the subphase significantly altered the stability of the polyprenyl phosphate monolayers, as shown by the determination of monolayer collapse and compression/expansion hysteresis. Although sodium ions in the subphase showed only a weak effect on the stabilization of the monolayer, addition of magnesium ions or of a small amount of calcium ions significantly suppressed the dissolution of the monolayer into the subphase and increased its mechanical stability against collapse. In contrast, the presence of larger amounts of calcium or of lanthanum ions induced collapse of the monolayers. Based on these experimental facts, a plausible scenario for the formation of primitive cell membrane by transformation of a monolayer to vesicle structures is proposed.     

Copper(ii) complexes of a polydentate imidazole-based ligand. pH effect on magnetic coupling and catecholase activity

A potentially dodecadentate N8O4-donor ligand obtained from 2,2'-biimidazole and l-valine and its tetranuclear Cu(ii) complexes in different degrees of protonation were characterized by chemical and spectroscopic methods. The extensive solution studies performed reveal that the rise in pH media leads successively to the formation of imidazolato (pKa(1) and pKa(2) and hydroxido (pKa(3) and pKa(4)) bridges. A frozen solution EPR study shows a decrease in the signal intensity until an EPR silent spectrum is observed, upon increasing the basicity of the solution. The catalytic performance of the oxidation of 3,5-di-tert-butylcatechol to its corresponding quinone was studied using UV-Vis-NIR absorption spectroscopic methods in CH3CN-H2O and in CH3OH-H2O at pH = 7.5, 8.0 and 8.5. A marked increase in activity, consistent with the formation of the hydroxide bridged species, is observed at pH = 8.5 in both solvent mixtures, but the activity is significantly higher in CH3OH-H2O.     

The pH inside a swollen polyelectrolyte gel: poly(N-vinylimidazole)

The pH inside a dissolved polyelectrolyte coil or a swollen ionic polymer network is not accessible to direct measurement. It is here calculated through a simple model, based on Donnan equilibrium, counterion condensation (for charge density exceeding the critical value), and balance of mobile ions, without any assumption on the pKa of the ionizable groups. The data needed for the calculation with this model are polymer concentration, pH value in the initial solution, and pH value in the bath at equilibrium. All three can be determined experimentally by a batch method where the polymer is immersed in a different pot for each starting pH. The model is applied to a sample system, namely, chemically cross-linked poly(N-vinylimidazole) immersed in acidic baths of different pH values. The imidazole units are basic and become protonated by the acid, thus changing the pH of the initial bath. The model shows how the pH developed inside the swollen gel is several units higher than the pH of the bath at equilibrium, both with or without the correction for counterion condensation. Consequently, when the pKa of the polyelectrolyte is determined in the usual way (with the pH measured in the external bath), it gives an apparent value that is several units below the pKa determined from the actual pH inside the swollen gel at equilibrium. The inclusion of the counterion condensation decreases very slightly the polymer basicity. Surface effects and intramolecular association between protonated and unprotonated imidazole rings are discussed to explain the pKa behavior in the limit of low degree of ionization.