Solvent Effects on the Structure-Activity Relationship of Phenytoin-like Anticonvulsant Drugs

The absorption spectra of nine compounds structurally related to phenytoin (5,5-diphenylhydantoin) were recorded in twelve solvents over the range of 200 to 400 nm. The effects of solvent dipolarity/polarizability and solvent/solute hydrogen bonding interactions were analyzed by means of the linear solvation energy relationship (LSER) concept proposed by Kamlet and Taft. The lipophilic activity of the investigated hydantoins was estimated by calculation of their log ₁₀ P values. The calculated values of log ₁₀ P were correlated with the ratio of the contributions of specific and non-specific solute/solvent interactions. The correlation equations were combined with the corresponding ED₅₀ values to generate new equations that demonstrate exact relationship between solute/solvent interactions and the structure-activity parameters.     

Synthesis, structure, and solvatochromic properties of pharmacologically active 5-substituted 5-phenylhydantoins

Abstract  

A series of 5-substituted 5-phenylhydantoins was synthesized and their UV absorption spectra were recorded in the region 200–400 nm in selected solvents of different polarity. The effects of solvent dipolarity/polarizability and solvent–solute hydrogen-bonding interactions were analyzed by means of the linear solvation energy relationship concept proposed by Kamlet and Taft. The lipophilicities of the investigated hydantoins were estimated by calculation of their log P values. The quantitative relationship between the ratio of the contributions of specific solvent interactions and the corresponding lipophilicity parameter is discussed. The correlation equations were combined with the corresponding ED₅₀ values and different physicochemical parameters to generate new equations that demonstrate the reasonable relationships between solute–solvent interactions and the structure–activity parameters. In order to determine a spectroscopic assignment of the absorption bands in different solvents, quantum chemical calculations were done.     

Solvent Effect on Protonation Constants of 5, 10, 15, 20-Tetrakis(4-sulfonatophenyl)porphyrin in Different Aqueous Solutions of Methanol and Ethanol

The protonation constants of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin, H₂tpps⁴⁻, were determined in water–ethanol and water–methanol mixed solvents, using a combination of spectrophotometric and potentiometric methods at 20 °C and 0.1 mol⋅dm⁻³ sodium perchlorate as supporting electrolyte. Two protonation constants, K ₁ and K ₂, were characterized and were analyzed in various media in terms of the Kamlet, Abboud and Taft (KAT) parameters. Single-parameter correlations of the protonation constant K ₁ versus α (hydrogen-bond donor acidity) and π ^* (dipolarity/polarizability) are poor in all solutions, but dual-parameter (α and π ^*) correlation represents a significant improvement with regard to the single- and multi-parameter models. However, the single-parameter correlation of log ₁₀ K ₂ in terms of β (hydrogen-bond acceptor basicity) shows a better result than dual- and multi-parameter correlations. Linear correlation is observed when the experimental log ₁₀ K ₁ and log ₁₀ K ₂ values are plotted versus the calculated ones when the KAT parameters are considered. To evaluate the protonation constants of H₂tpps⁴⁻, the Yasuda-Shedlovsky extrapolation is used to obtain the log ₁₀ K ₁ and log ₁₀ K ₂ values at zero percent organic solvent. Finally, the results are discussed in terms of the effect of the solvent on protonation.     

Complexation of M–(buffer) x –(OH) y Systems Involving Divalent Ions (Cobalt or Nickel) and Zwitterionic Biological Buffers (AMPSO, DIPSO, TAPS and TAPSO) in Aqueous Solution

The complexation behavior of eight M–(buffer) _x –(OH) _y systems involving two divalent ions (cobalt and nickel) and four zwitterionic biological buffers (AMPSO, DIPSO, TAPS and TAPSO) were characterized. Complex formation was detected for all eight M–(buffer) _x –(OH) _y systems studied, but fully defined final models were obtained for only four of these systems. For systems involving cobalt or nickel with AMPSO or TAPS, a complete characterization of the systems was not possible in the studied buffer pH-range. Metal complexation was studied by glass-electrode potentiometry (GEP) and UV-Vis spectroscopy at 25.0 °C and I=0.1 mol⋅dm⁻³ KNO₃ ionic strength. For the Ni–(L) _x –(OH) _y and Co–(L) _x –(OH) _y systems, with L = TAPSO or DIPSO, the proposed final models and overall stability constants were obtained by combining results from both techniques. For the Ni–(L) _x –(OH) _y systems, the measured values of the stability constants are log ₁₀ β _NiL=3.0±0.1 and log ₁₀ β _NiL2=4.8±0.1 for L = TAPSO, and log ₁₀ β _NiL=2.7±0.1 and log ₁₀ β _NiL2=4.6±0.1 for L = DIPSO. For the Co–(L) _x –(OH) _y systems, the overall stability constants are log ₁₀ β _CoL=2.2±0.1, log ₁₀ β _CoL2=3.6±0.2 and log ₁₀ β _CoL(OH)=7.6±0.1 for L = TAPSO, and log ₁₀ β _CoL=2.0±0.1 and log ₁₀ β _CoL(OH)=7.8±0.1 for L = DIPSO. For both buffers, the CoL(OH) species is characterized by a major structural rearrangement.     

Complexation Studies of N,N′-ethylenedi-L-cysteine with Some Metal Ions

As part of a search for environmentally friendly metal chelating ligands, the stability constants of N, N′-ethylenedi-L-cysteine (EC) complexes with Ca(II), Cu(II), Mg(II) and Mn(II) were determined by potentiometry with a glass electrode in aqueous solutions containing 0.1 mol⋅L⁻¹ KCl at 25 °C. Final models are proposed. For the Ca(II)–EC system, the overall stability constants are log ₁₀ β _CaHL=14.53±0.03, log ₁₀ β _CaL=4.79±0.01 and log ₁₀ β _CaL2=8.38±0.04. For the M(II)–EC systems, where M=Cu(II) or Mg(II), the overall stability constants are log₁₀ β _CuHL=31.19±0.02 and log ₁₀ β _CuL=27.02±0.06 for Cu(II), and are log ₁₀ β _MgHL=14.84±0.02 and log ₁₀ β _MgL=6.164±0.008 for Mg(II). For the Mn(II)–EC system, the overall stability constant is log ₁₀ β _MnL=10.12±0.01. Metal–chelate speciations simulations showed that EC is an efficient chelating agent for Cd(II), Co(II), Cu(II), Ni(II), Pb(II) and Zn(II) for pH≥7.     

D<Subscript>2</Subscript>O Isotope Effects on the Ionization of <Emphasis Type="Italic">β</Emphasis>-Naphthol and Boric Acid at Temperatures from 225 to 300 °C using UV-Visible Spectroscopy

The deuterium-isotope effects on the ionization constants of β-naphthol (2-naphthol) and boric acid, Δlog ₁₀ K=[log ₁₀ K _D2O−log ₁₀ K _H2O], have been determined from measurements in light and heavy water at temperatures from 225 °C≤t≤300 °C and pressures near steam saturation. β-Naphthol is a thermally-stable colorimetric pH indicator, whose ionization constant lies close to that of H₂PO₄⁻ (aq), the only acid for which Δlog ₁₀ K is accurately known at elevated temperatures. A newly designed platinum flow cell was used to measure UV-visible spectra of β-naphthol in acid, base, and buffer solutions of H₂PO₄⁻/HPO₄²⁻ and D₂PO₄⁻/DPO₄²⁻, from which the degree of ionization at known values of pH and pD was determined. Values of the ionization constants of β-naphthol in light and heavy water were calculated from these results, and used to derive a model for and over the experimental temperature range with an estimated precision of ±0.02 in log ₁₀ K. The new values of K _H2O and K _D2O allowed us to use β-naphthol as a colorimetric indicator, to measure the equilibrium pH and pD of the buffer solutions B(OH)₃/B(OH)₄⁻ and B(OD)₃/B(OD)₄⁻ up to 300 °C, from which the ionization constants of boric acid were calculated. The magnitude of the deuterium isotope effect for H₂PO₄⁻ (aq) is known to fall from Δlog ₁₀ K=−0.62 to Δlog ₁₀ K=−0.47, on the “aquamolal” concentration scale, as the temperature rises above 125 °C, but then remains almost constant. Although the temperature range is more limited, the new results for β-naphthol and boric acid appear to show a similar trend.     

Sorption of Lead(II) on Two Chelating Resins: From the Exchange Coefficient to the Intrinsic Complexation Constant

The sorption of inorganic lead(II) on two cationic resins containing different complexing groups, the iminodiacetic Chelex 100 and the carboxylic Amberlite CG-50, was investigated. The Gibbs-Donnan model was used to describe and predict the sorption through the determination of the intrinsic complexation constants. These quantities, even though non-thermodynamic, characterize the sorption as being independent of experimental conditions. The sorption mechanism for metals on complexing resins was also studied by adding a competitive soluble ligand that shifts the sorption curves to higher pH. The ligand competes with the resin for complexation with the metal ion. Lead(II) is strongly sorbed on Chelex 100 through the formation of two complexes in the resin phase: MHL with log ₁₀ β _111i =−0.3 and ML with log ₁₀ β _111i =−3.7. The presence of the competitive ligand in solution allows for the determination of a third complex. Furthermore, on Amberlite CG-50 the sorption is rather strong and involves the formation of the complex ML, in more acidic solution, with log ₁₀ β _110i =−2.0. In the presence of the ligand PyDA, the ML(OH) complex was characterized by log ₁₀ β _11−1i =−5.6. In all the experiments the hydrolysis reactions in the aqueous phase are considered quantitatively.     

Sequestering Ability of Dicarboxylic Ligands Towards Dioxouranium(VI) in NaCl and KNO<Subscript>3</Subscript> Aqueous Solutions at <Emphasis Type="Italic">T</Emphasis>=298.15 K

The formation constants of dioxouranium(VI)-2,2′-oxydiacetic acid (diglycolic acid, ODA) and 3,6,9-trioxaundecanedioic acid (diethylenetrioxydiacetic acid, TODA) complexes were determined in NaCl (0.1≤I≤1.0 mol⋅L⁻¹) and KNO₃ (I=0.1 mol⋅L⁻¹) aqueous solutions at T=298.15 K by ISE-[H⁺] glass electrode potentiometry and visible spectrophotometry. Quite different speciation models were obtained for the systems investigated, namely: ML⁰, MLOH⁻, ML₂²⁻, M₂L₂(OH)⁻, and M₂L₂(OH)₂²⁻, for the dioxouranium(VI)–ODA system, and ML⁰, MLH⁺, and MLOH⁻ for the dioxouranium(VI)–TODA system (M=UO₂²⁺ and L = ODA or TODA), respectively. The dependence on ionic strength of the protonation constants of ODA and TODA and of both metal-ligand complexes was investigated using the SIT (Specific Ion Interaction Theory) approach. Formation constants at infinite dilution are [for the generic equilibrium pUO₂²⁺+q(L²⁻)+rH⁺ ⇌(UO₂²⁺) _p (L) _q H _r ^(2p−2q+r);β _pqr ]: log ₁₀ β ₁₁₀=6.146, log ₁₀ β ₁₁₋₁=0.196, log ₁₀ β ₁₂₀=8.360, log ₁₀ β ₂₂₋₁=8.966, log ₁₀ β ₂₂₋₂=3.529, for the dioxouranium(VI)–ODA system and log β ₁₁₀=3.636, log ₁₀ β ₁₁₁=6.650, log ₁₀ β ₁₁₋₁=−1.242 for dioxouranium(VI)–TODA system. The influence of etheric oxygen(s) on the interaction towards the metal ion was discussed, and this effect was quantified by means of a sigmoid Boltzman type equation that allows definition of a quantitative parameter (pL ₅₀) that expresses the sequestering capacity of ODA and TODA towards UO₂²⁺; a comparison with other dicarboxylates was made. A visible absorption spectrum for each complex reaching a significant percentage of formation in solution (KNO₃ medium) has been calculated to better characterize the compounds found by pH-metric refinement.     

Mixed Ligand Complex Formation Reactions and Equilibrium Studies of Cu(II) with Bidentate Heterocyclic Alcohol (N,O) and Some Bio-Relevant Ligands

The formation equilibria of copper(II) complexes and the ternary complexes Cu(HMI)L (HMI=4-Hydroxymethyl-imidazole, L=amino acid, amides or DNA constituents) have been investigated. Ternary complexes are formed by a simultaneous mechanism. The results showed the formation of Cu(HMI)L and Cu(HMI,H₋₁)(L) complexes. The stability of ternary complexes was quantitatively compared with their corresponding binary complexes in terms of the parameters Δlog ₁₀ K and log ₁₀ X. The effect of the side chains of amino acid ligands (Δ_R) on complex formation was discussed. The concentration distributions of various species formed in solution were also evaluated as a function of pH. The thermodynamic parameters ΔH° and ΔS° calculated from the temperature dependence of the equilibrium constants are investigated. The effects of dioxane as a solvent, on the protonation constant of HMI and the formation constants of Cu^II–HMI complexes, were discussed.     

The Interaction of Arsenazo III with Nd(III)—A Chemometric and Metrological Analysis

Interactions of Arsenazo III with Nd(III) in aqueous solutions (pH range 3 to 4) were studied using a spectrophotometric method. Some discrepancies are present in literature concerning the concentrations of the prevailing species and their composition. Threshold bootstrap computer-assisted target factor analysis (TB CAT) was applied to the evaluation of UV-VIS spectra of Arsenazo III in aqueous solutions containing varying amounts of neodymium(III) ions. The thus obtained experimental data are interpreted with consideration of the measurement uncertainties affecting this system. Within the limits of resolution of the experimental method, two complexes NdAazo and Nd(Aazo)₂ were indicated in the studied pH range. The values of formation constants for log ₁₀ β ₁₁ fall in the range 4.9 to 6.3 and for log ₁₀ β ₁₂ fall between 10.5 and 12.1.     

The Examination of the Activity Coefficients of Cu(II) Complexes with OH<Superscript>−</Superscript> and Cl<Superscript>−</Superscript> in NaClO<Subscript>4</Subscript> Using Pitzer Equations: Application to Other Divalent Cations

The stability constants for the hydrolysis of Cu(II) and formation of chloride complexes in NaClO₄ solution, at 25 °C, have been examined using the Pitzer equations. The calculated activity coefficients of CuOH⁺, Cu(OH)₂, Cu₂(OH)³⁺, Cu₂(OH)₂²⁺, CuCl⁺ and CuCl₂ have been used to determine the Pitzer parameter (β _i ⁽⁰⁾, β _i ⁽¹⁾, and C _i ) for these complexes. These parameters yield values for the hydrolysis constants (log ₁₀ β ₁^*, log ₁₀ β ₂^*, log ₁₀ β _2,1^* and log ₁₀ β _2,2^*) and the formation of the chloride complexes (log ₁₀ β _CuCl^* and that agree with the experimental measurements, respectively to ±0.01,±0.02,±0.03,±0.06,±0.03 and ±0.07. The stability constants for the hydrolysis and chloride complexes of Cu(II) were found to be related to those of other divalent metals over a wide range of ionic strength. This has allowed us to use the calculated Pitzer parameters for copper complexes to model the stability constants and activity coefficients of hydroxide and chloride complexes of other divalent metals. The applicability of the Pitzer Cu(II) model to the ionic strength dependence of hydrolysis of zinc and cadmium is presented. The resulting thermodynamic hydroxide and chloride constants for zinc are and . For cadmium the thermodynamic hydrolysis constants are and . The Cu(II) model allows one to determine the stability of other divalent metal complexes over a wide range of concentration when little experimental data are available. More reliable stepwise stability constants for divalent metals are needed to test the linearity found for the chloro complexes.     

Accurate quantification of freely dissolved organochlorine pesticides in water in the presence of dissolved organic matter using triolein-embedded cellulose acetate membrane

A novel method is described using triolein-embedded cellulose acetate membrane (TECAM) for accurate determination of the freely dissolved fraction of organochlorine pesticides (OCPs) in waters rich in dissolved organic matter (DOM). The performance of the method was tested with an air-bridge system for extracting OCPs from aqueous solutions with and without humic acid. In addition, the partition coefficients between humic acid and water (K _docs) for 20 OCPs were determined by TECAM with negligible depletion extraction. Results show that TECAM predominantly extracts the freely dissolved compounds and its extraction efficiency decreases significantly with an increase in concentration of humic acid in water. The proposed methodology is suitable for facile laboratory K _doc measurement for moderate to high hydrophobic compounds (log K _ow > 4). The linear relationship between log K _ow and log K _doc obtained in this study agrees well with the results reported earlier. The kinetic uptake rate constants (k _us) and TECAM–water partition coefficients (K _TECAMs) for the 20 OCPs were obtained using the controlled laboratory continuous-flow and static exposure system, respectively. These calibration parameters were used in the field experiment to estimate the freely dissolved concentrations of OCPs in the water of Taihu Lake in China. Our results show that TECAM can be used successfully to determine the freely dissolved OCPs in aquatic environments containing DOM, and the method is particularly suited for long-term water sampling. Figure Schematic diagram of water sampling with a triolein-embedded cellulose acetate membrane (TECAM)     

Spectrophotometric Determination of the Ionization Constants of Aqueous Nitrophenols at Temperatures up to 225 °C

The UV-visible spectra of aqueous o-, m-, and p-nitrophenol were measured as a function of pH at temperatures from 50 to 225 °C at a pressure of 7 MPa. These were used to determine equilibrium constants for the acid ionization reaction of each isomer. The new results were combined with literature data on the ionization of nitrophenols and used for parameter optimization in the thermodynamic model of Marshall and Franck (J. Phys. Chem. Ref. Data 10:295–304, [1981]), to describe the dependence of ionization properties on temperature and pressure. The model yields predictions of the ionization constants for o-, m-, and p-nitrophenol, log ₁₀ K _a, to at least 250 °C and 20 MPa with an estimated uncertainty in log ₁₀ K _a of less than ±0.06.     

Equilibrium Study of the Mixed Complexes of Copper(II) with Adenine and Amino Acids in Aqueous Solution

Solution equilibria of the systems Cu(II)-adenine (A)-amino acids (L) have been studied pH-metrically. The formation constants of the resulting mixed ligand complexes have been calculated at 25 °C and ionic strength 0.1 mol⋅dm⁻³ NaNO₃. Ternary complexes are formed by simultaneous reactions. The relative stability of each ternary complex was compared with that of the corresponding binary complexes in terms of Δlog ₁₀ K values. The concentration distribution of the complexes in solution was evaluated.     

Modeling of Protonation Constants of Linear Aliphatic Dicarboxylates Containing -S-Groups in Aqueous Chloride Salt Solutions,at Different Ionic Strengths,Using the SIT and Pitzer Equations and Empirical Relationships

The protonation constants of ethylenedithiodiacetic, dithiodipropionic and dithiodibutyric acids were obtained from potentiometric measurements in NaCl(aq) (I≤5 mol⋅L⁻¹) and (CH₃)₄NCl(aq) (I≤3 mol⋅L⁻¹) at t=25 °C. Their dependences on ionic strength were modeled by the SIT and Pitzer approaches. The activity coefficients of the neutral species were obtained by solubility measurements. The literature values of the protonation constants of (HOOC)-(CH₂) _n -S-(CH₂) _n -(COOH) (n=1 to 3) and (HOOC)-(CH₂)-S-(CH₂) _n -S-(CH₂)-(COOH) (n=0 to 5) in NaCl(aq) and KCl(aq) (I≤3 mol⋅L⁻¹) at 18 °C were also analyzed using the above approaches. Both the log ₁₀ K _i ^H and interaction parameter values follow simple linear trends as a function of certain structural characteristics of the ligands. Examples of modeling these trends are reported. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Studies on Complexation of Resorcinol with Some Divalent Transition Metal Ions and Aliphatic Dicarboxylic Acids in Aqueous Media

The binary and ternary complexes of Cu²⁺, Ni²⁺, Co²⁺ and Zn²⁺ metal ions with resorcinol (R) as primary ligand and some biologically important aliphatic dicarboxylic acids (adipic, succinic, malic, malonic, maleic, tartaric and oxalic acids) as secondary ligands were studied in aqueous solution at 25 °C and I=0.1 mol⋅dm⁻³ NaNO₃ using the potentiometric technique. The formation of different 1:1 and 1:2 binary complexes and 1:1:1 ternary complexes is inferred from the corresponding potentiometric pH-titration curves. The ternary complex formation was found to take place in a stepwise manner. The protonation constants of the ligands were determined and used for determining the stability constants of the different complexes formed in aqueous solutions. The lower stability of the 1:2 binary complexes compared to the corresponding 1:1 systems of all ligands studied were in accordance with statistical considerations. The order of stability of the complexes formed in solution was investigated in terms of the nature of the resorcinol, carboxylic acid, and metal ion used. The values of Δlog ₁₀ K, percentage of relative stabilization (% R.S.), and log ₁₀ X for mixed-ligand complexes studied have been evaluated and discussed. The concentration distribution of the various species formed in solution was evaluated. The mode of chelation of the ternary complexes was ascertained by conductivity measurements.     

Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method

The dissociation equilibrium constant (K _D) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K _D values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The K _D values obtained using frontal analysis were 3.36 × 10⁻⁶ M for nifedipine, 1.34 × 10⁻⁶ M for nimodipine, 6.83 × 10⁻⁷ M for nitrendipine, 1.23 × 10⁻⁷ M for nicardipine, 1.09 × 10⁻⁷ M for amlodipine, and 8.51 × 10⁻⁸ M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions.     

Study of Complex Formation of Dibenzo-18-Crown-6 with Ce3+, Y3+, $\mathrm{UO}_{2}^{2 +}$ and Sr2+ Cations in Acetonitrile–Dioxane Binary Solvent Mixtures

The complexation reactions of dibenzo-18-crown-6 (DB18C6) with Ce³⁺, Y³⁺, UO₂^{2 +}\mathrm{UO}_{2}^{2 +} and Sr²⁺ cations were studied in acetonitrile–dioxane (AN–dioxane) binary solvent solutions at different temperatures by the conductometric method. The stability constants of the resulting 1:1 complexes were determined from computer fitting of the conductance–mole ratio data. The results show that dibenzo-18-crown-6 does not exhibit selectivity for the cation whose ionic size is closest to the cavity size of this macrocyclic ligand in AN–dioxane binary solvent solutions. A nonlinear relationship was observed between the stability constants (log ₁₀ K _f) of these complexes with the composition of the AN–dioxane binary solvent. Values of thermodynamic parameters (DH_c^°, DS_c^°\Delta H_{\mathrm{c}}^{\circ}, \Delta S_{\mathrm{c}}^{\circ}) for complexation reactions were obtained from the temperature dependence of the stability constants. The results show that the values along with the sign of these parameters are influenced by the nature and composition of the mixed solvent.     

Complexation Studies of Pyridyl Sulfonamide Ligands for Sensing Zinc and Copper Ions

Ligand protonation and stepwise dissociation constants, formation constants and speciation of four pyridyl sulfonamide ligands (Congreeve et al., New J. Chem. 27:98–106, 2003) were assessed, using potentiometric and UV/Visible spectrophotometric pH titrations (in 80% MeOH − 20% H₂O). The suitability of these ligands as Cu(II) and Zn(II) sensors for physiological applications was assessed. Two ligands L¹ and L⁴ were p-toluenesulfonamide derivatives while L² and L³ were triflurosulfonamide derivatives. Additionally L³ and L⁴ were appended with α-methyl groups. The most stable complex was formed by L¹ with Cu(II) owing to the fact that this complex was square planar (log ₁₀  K ₁=12.15±0.004 and log ₁₀  β ₂=15.42±0.006). The rest of the complexes invariably formed distorted tetrahedron geometry and complexation was weaker. Speciation diagrams show the effect of ligand to metal concentration, revealing that the L² and L³ ligands are the most suitable for forming ML₂ complexes at physiological pH.