Protonation Equilibria Research Articles

DNA-interactive anticancer aza-anthrapyrazoles: biophysical and biochemical studies relevant to the mechanism of action.

The physicochemical and DNA-binding properties of anticancer 9-aza-anthrapyrazoles (9-aza-APs) were investigated and compared with the carbocyclic analogs losoxantrone (LX) and mitoxantrone (MX). Unlike their carbocyclic counterparts, the tested 9-aza-APs do not undergo self-aggregation phenomena. The pyridine nitrogen at position 9, missing in the carbocyclic derivatives, is involved in protonation equilibria at physiological pH. In addition, 9-aza-APs are electrochemically reduced at a potential intermediate between LX and MX. These data fully agree with quantum mechanical calculations. Binding to nucleic acids was examined by spectroscopic, chiroptical, and DNase I footprinting techniques as a function of ionic strength and base composition. The 9-aza-APs exhibit prominent affinity for DNA, with an important electrostatic contribution to the binding free energy. A very remarkable sequence preference pattern dramatically favors GC steps in double-helical DNA, whereas the carbocyclic reference compounds show a substantially lower selectivity for GC. A common DNA complexation geometry, considerably differing from that of MX, characterizes all anthrapyrazoles. Hence, bioisosteric substitution and ring-hydroxy deletion play an important role in defining the physicochemical properties and in modulating the affinity of anthrapyrazoles for the nucleic acid, the geometry of the intercalation complex, and the sequence specific contacts along the DNA chain. Drug stimulation of topoisomerase II-mediated DNA cleavage is remarkably attenuated in the aza-bioisosteric derivatives, suggesting that other non-enzyme-mediated cytotoxic mechanism(s), possibly connected with free radical production, are responsible for efficient cell killing. The biophysical and biochemical properties exhibited by 9-aza-APs contribute to clarifying the peculiar pharmacological profile of this family of compounds.

Solvent effects in chiral ligand exchange systems

The influence of solvent composition on equilibria in chiral ligand-exchange systems is explored by measuring protonation constants and equilibrium formation constants for all free species and complexes, respectively, in systems at 25°C and an ionic strength of 0.1 M containing one of two classic chiral selector molecules ( l-proline or l-hydroxy proline), the copper(II) ion, and an amino acid enantiomer. This set of thermodynamic constants is combined with multiple-chemical-equilibria theory to determine solution composition as a function of pH and mole-fraction methanol (MeOH) in the solvent. Results indicate that the addition of MeOH increases the stabilities of both the hetero- and the homo-chiral ternary complexes. However, it also increases, often to a greater extent, the stability of each bis binary complex. As a result, the influence of MeOH addition on equilibria in chiral ligand-exchange systems is complex and cannot be predicted by mere inspection of the equilibrium constants for the hetero- and homo-chiral ternary complexes. These results therefore identify a potential limitation of using common global stoichiometric models to predict equilibria in chiral ligand-exchange systems. A more comprehensive model, based on the complete description of the multiple chemical equilibria, is proposed. Finally, the role of solvent in stabilizing ligand-exchange complexes is explored through a series of molecular mechanics simulations in the absence or presence of solvent molecules. Results suggest that solvent stabilizes both bis binary and ternary complexes, primarily through direct participation in the complex structure in the form of occupancy of one or both distal coordination sites.

Oxonium and quinonoid intermediates in the sulfonation of dimethoxynaphthalenes (DMONs)

Monoprotonated oxonium cations (such as 2) and quinonoid dications (such as 5) of 2,3-, 2,6- and 2,7-dimethoxynaphthalenes are described. Their role as direct substrates in sulfonation reactions is discussed together with details of the sulfonation mechanism for 2,3-dimethoxynaphthalene (2,3-DMON), which is presented as a characteristic example. Steady sulfonation rate constants of 2,6- and 2,7-DMON in 80–88% H2SO4 are a result of the DMON protonation equilibria. Binding energies and heats of formation were calculated for several mono- and disulfonic acids of DMONs. Substituent effects are discussed.

Some Theoretical and Computational Aspects of the Inclusion of Proton Isomerism in the Protonation Equilibrium of Proteins

The present article discusses some aspects concerning the inclusion of proton isomerism in simulations of the global protonation equilibrium of protein molecules. In the context of continuum electrostatic methods, the usual basis for these simulations, this isomerism can be treated as a coexistence of tautomeric forms in equilibrium in a rigid structure; furthermore, it can be formally extended to nontitrable sites with proton isomerism, such as alcohol groups and water molecules. We follow the previously adopted approach of transforming the real system of tautomeric sites into a thermodynamically equivalent one of nontautomeric pseudosites, establishing a proper relation between the two systems. The necessary energetic and entropic modifications of model compound pKa values are also discussed. Additionally, we discuss the new entropy term, named tautomeric entropy, that results from the explicit inclusion of tautomerism in the simulations and how it can be computed together with the occupational entropy....

Investigating the differences in acid separation behaviour on weak base ion exchange resins

Differences in acid separation behaviour on weak base ion exchange resins have been investigated in this work. Experimental data has been obtained on sorption equilibrium using various types of weak base resins and both inorganic and organic monobasic acids. A detailed analysis has been presented using the experimental data of present work and also that reported in the literature. Apart from acid concentration and type of acid, other factors such as resin type and basicity are also discussed. It was found that, resins with high basicity are most suitable for the acid removal applications. For resins with medium and weak basicity, large variation in the sorption capacity with concentration was observed. A new theoretical approach incorporating effect of resin basicity has been proposed and examined qualitatively and quantitatively using the sorption data. The results substantiate reversibility of sorption through lower values of protonation equilibrium constants and also indicate incomplete resin salt dissociation in some cases.

Protonation of CpW(CO)2(PMe3)H: Is the Metal or the Hydride the Kinetic Site?

We have compared the rate at which a proton is transferred to the hydride ligand of CpW(CO)2(PMe3)H (3) with the rate at which one is transferred to the metal. Qualitative evidence that protonation of the hydride ligand is faster is offered by observation of fast exchange between 3 and CF3SO3D and selective broadening of the trans hydride resonance of 3 during exchange with anilinium. Rate constants were obtained by 1H NMR for H/D exchange between 3 and 4-tert-butyl-N,N-dimethylanilinium-N-d1 (5-d1); an unusual EIE of 0.19 was observed and explained by location of the vibrational normal modes of 3, 5, and their deuterated analogues via IR and Raman spectroscopy. A pKa of 5.6(1) in CH3CN was determined for CpW(CO)2(PMe3)H2+ (4a) by IR; the importance of homoconjugate pair interactions in protonation equilibria is illustrated and discussed. The exchange rate data and the rate constant for deprotonation of 4a by 4-tert-butyl-N,N-dimethylaniline (6), combined with the pKa data, provide quantitative evidence that the kinetic site of protonation of 3 is the hydride ligand.

Effect of Protonation on the Solution and Phase Behavior of Aqueous Sodium Myristate

Aqueous sodium myristate solutions have been shown to have unusually low dynamic tensions (1–10 mN/m) under pulsating area conditions. These solutions have no sharp solubility limit, evidently because they are protonated (or “hydrolyzed”) to form the much less soluble myristic acid and acid soaps. With no added electrolytes, the protonation fraction is 1% or less. The apparent protonation equilibrium “constant” increases with increasing concentration, indicating strong solution nonidealities, in addition to micellization. This protonation seems to affect the solution and phase behavior of aqueous sodium myristate strongly, as evidenced by the effect of added NaOH. Ion-selective electrodes (for Na+ and H+) and conductimetry indicate that at 25°C dissolved surfactant concentrations keep increasing well after dispersed particles are observed (2 mM). A cmc of about 4.5 mM, micelles of aggregation number n=70 and counterion binding parameter β=0.7 are inferred from these techniques. The cmc of sodium myristate increases slightly with temperature from 25 to 45°C. FTIR analysis of the filtered particles indicates that the dispersed particles are mainly acid soaps for concentrations less than 6 mM. With 10 mM NaOH, the particles observed above 2 mM consist mostly of sodium myristate. From both conductivity and IR data, the solubility of sodium myristate in water at 25°C is estimated to be about 6 mM, and as expected, it increases with increasing temperature and decreases with increasing sodium ion concentration.

PH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus.

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.

Permeation and Activation of the M2 Ion Channel of Influenza A Virus

The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).

1999 R.U. Lemieux Award Lecture Adventures with azo-, azoxy-, and hydrazoarenes: from the Wallach to the benzidine rearrangement. Molecular electronics

The author's studies with aromatic azo-, azoxy-, and hydrazo-dye molecules, comprising kinetic and equilibrium investigations, as well as synthesis of novel molecules having photogenic properties, are described under the following highlights: A. Wallach rearrangement and cognate studies with azoxyarenes — (1) Elucidation of the mechanism of the Wallach rearrangement of azoxybenzene through the kinetic observation of a two-proton process which, together with a pKa study, was interpreted on the basis of formation of a deoxygenated, dicationic, symmetrical species as a key, short-lived reaction intermediate. (2) The proposal of a general acid-catalyzed pathway in concentrated sulfuric acid (catalysis by H2SO4 and H3SO+4. (3) Elucidation of the consecutive sulfonations of reaction products of azoxybenzene in the 100% H2SO4 region, and the diprotonation equilibria for p-hydroxyazobenzene, thus shedding light on past reaction pathway and product studies. (4) The observation of a novel reaction pathway for 2,4,6,2',4',6'-hexamethy lazoxybenzene. (5) The observation of a dichotomy of reaction pathways for α- and β-2-phenylazoxynaphthalenes: reaction via the dicationic intermediate and via quinoid intermediate species; comprising two isomeric compounds reacting by different pathways to give the same product. (6) Identification and structure proof of α- and β-isomers observed for the first time in the peracid oxidation of phenylazopyridine. (7) Observation of a rate constant ratio of 22 000 in the rearrangement of these α- and β-isomers, and the proposal of differential barriers for transition states leading to a tricationic intermediate. B. Benzidine rearrangement and cognate studies — (8) Observation of the acid-catalyzed hydroxylation of phenylazopyridine to p-hydroxyphenylazopyridine and the proposal of an SNAr mechanism with formation of an intermediate hydrazo species in the reaction. (9) First study of benzidine type rearrangement-disproportionation of phenylhydrazopyridine in acid media. (10) Proposal of a A [Formula: see text] B [Formula: see text] C [Formula: see text] D type reaction profile for the consecutive hydroxylation[Formula: see text]disproportionation processes of phenylazopyridine in aq H2SO4. (11) Proposal of 10-π and 14π-electron electrocyclic processes in the benzidine type rearrangement-disproportionation of phenylhydrazopyridine. (12) Identification and structural elucidation of a dimer formed from phenylazopyridine as a minor product and proposal of a reaction mechanism. C. Facile acid-catalyzed demethylation via SNAr/A-SE2 mechanisms and studies of tautomerism — (13) Observation of an abnormally facile acid-catalyzed cleavage (demethylation) of 4-methoxyphenylazopyridine via an SNAr mechanism. (14) Observation of two reaction pathways, SNAr and A-SE2, for the consecutive demethylations of 3,4-dimethoxyphenylazopyridine, with rate constant ratio of 7 000:1 favoring the SNAr process. (15) Quantitation of the tautomeric and protonation equilibria of 4-hydroxyphenylazopyridine, produced in (13). D. A new solvent polarity scale, molecular switches, and molecular electronics — (16) Establishment of a π*azo solvent polarity scale based on solvatochromism of a series of azomerocyanine molecules ("Buncel's dye"). (17) Some glimpses are presented of current forays into molecular electronics, as emanating from the above studies: (a) spiropyran (SP) <—> merocyanine (MC) thermo- and photochromic "molecular switch" systems; (b) synthesis and characterization of azo-functionalized star-burst dendrimers with photoswitchable properties and potential applications in optical data storage systems, holographic gratings, and drug delivery systems as host molecules.Key words: Wallach rearrangement, benzidine disproportionation, azoarenes, azoxyarenes, dendrimers, hydrazoarenes, dendrimers, solvatochromism, photochromism, thermochromism, spiropyran-merocyanine molecular switch.

Signaling States of Rhodopsin: RETINAL PROVIDES A SCAFFOLD FOR ACTIVATING PROTON TRANSFER SWITCHES

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.

Proton equilibria in the manganese cluster of photosystem II control the intensities of the S(0) and S(2) state g approximately 2 electron paramagnetic resonance signals.

We have studied the pH effect on the S(0) and S(2) multiline electron paramagnetic resonance (EPR) signals from the water-oxidizing complex of photosystem II. Around pH 6, the maximum signal intensities were detected. On both the acidic and alkaline sides of pH 6, the intensities of the EPR signals decreased. Two pKs were determined for the S(0) multiline signal; pK(1) = 4.2 +/- 0.2 and pK(2) = 8.0 +/- 0.1, and for the S(2) multiline signal the pKs were pK(1) = 4.5 +/- 0.1 and pK(2) = 7.6 +/- 0.1. The intensity of the S(0)-state EPR signal was partly restored when the pH was changed from acidic or alkaline pH back to pH approximately 6. In the S(2) state we observed partial recovery of the multiline signal when going from alkaline pH back to pH approximately 6, whereas no significant recovery of the S(2) multiline signal was observed when the pH was changed from acidic pH back to pH approximately 6. Several possible explanations for the intensity changes as a function of pH are discussed. Some are ruled out, such as disintegration of the Mn cluster or decay of the S states and formal Cl(-) and Ca(2+) depletion. The altered EPR signal intensities probably reflect the protonation/deprotonation of ligands to the Mn cluster or the oxo bridges between the Mn ions. Also, the possibility of decreased multiline signal intensities at alkaline pH as an effect of changed redox potential of Y(Z) is put forward.

Proton and water transport in nano-separated polymer membranes

The microstructure of proton conducting polymers (NAFION and a sulfonated polyetherketone) has been investigated by SAXS and the results are related to coefficients of transport in thermodynamic equilibrium (water-self diffusion and proton mobility) as well as hydrodynamic transport (water permeation and electroosmotic drag) as obtained from NMR, impedance spectroscopic and permeation experiments. A model comprising (i) simple geometric effects, (ii) effects of chemical interactions and (iii) the formation of inner space charges provides a semiquantitative scheme which relates the microstructure to the transport properties of such types of polymers.

Coupled kinetic traps in cytochrome c folding: His-Heme misligation and proline isomerization

The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH < 5, the observed refolding at final pH 6 is dominated by a fast phase (τ2f = 20 ms, α2f = 90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (α2f ∼ 20 %) but the same rate (τ2f = 20 ms), and in two slower phases (τm = 6-8 seconds, αm ∼ 45 %; and τ1b = 16-20 seconds, α1b ∼ 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.

Capillary electrophoresis behavior of water-soluble anionic porphyrins in the presence of beta-cyclodextrin and its O-methylated derivatives.

The electrophoretic behavior of water-soluble anionic porphyrins, such as meso-tetrakis(4-carboxyphenyl) porphyrin (TCPP), meso-tetrakis(4-sulfonatophenyl) porphyrin (TSPP) and its zinc(II) and copper(II) complexes (ZnTSPP and CuTSPP, respectively) has been studied by capillary zone electrophoresis using fused-silica capillaries. The selectivity of the separation is strongly dependent on the type and concentration of betacyclodextrin (betaCD) or the O-methylated derivatives added to the background electrolyte. CuTSPP and TSPP can be separated using a pH 2.5 aqueous sodium phosphate buffer in the presence of 1 mM betaCD. Resolution is poorer or absent employing alkylated betaCDs, such as the heptakis (2,6-di-O-methyl)-beta-cyclodextrin or the heptakis(2,3,6-triO-methyl)-beta-cyclodextrin, as additives. On the other hand, separation of TSPP from its copper and zinc complexes has been achieved using a pH 7.0 aqueous sodium phosphate buffer, in the presence of 0.75 mM betaCD and 20% dimethyl sulfoxide (DMSO) as organic modifier. Under such conditions, the calibration curve for quantitative analysis of copper(II) was obtained. A rationale for the observed behavior will be presented and discussed on the basis of binding and protonation equilibria and a simple mathematical model.

Binding properties of octaaminocryptands.

Complexation and protonation equilibria were studied in aqueous solution for a new range of aminocryptand ligands, N(CH2CH2NHCH2RCH2NHCH2CH2)3N, (R = m-xylyl, p-xylyl, 2,5-furan, 2,6-pyridine) and demonstrate that stability constants for first transition series ions Co2+ to Zn2+ are relatively high. X-ray crystallography shows that the cryptands are reasonably well preorganized for complexation. The furan-spaced cryptand L6.H2O crystallizes in the rhomobohedral space group R3 (no. 148) with a = 14.645(1), b = 14.645(1), and c = 25.530(4) A, whereas the m-xylyl-spaced cryptand L4 crystallizes in the triclinic space group P1 (no. 2) with a = 9.517(1), b = 15.584(2), and c = 23.617(4) A. The highest formation constant (log beta21 = 33.07) is observed for the dicopper cryptate of a pyridine-spaced cryptand, suggesting involvement in complexation of donors from the spacer link. This pyridine-spaced host also shows good selectivity for copper(II) over zinc(II), making it a possible candidate for treatment of copper-excess pathology.

Comparative Studies on the Complex-Formation Equilibria of Mercury(II) with Acetylcysteine and Captopril

The complex-formation equilibria of mercury(II) with acetylcysteine and captopril have been studied by pH-metric and liquid-liquid extraction methods applying dithizone as a competitive ligand during the latter measurements. The stability constants of three differently protonated Hg(II)/ligand 1:2 complexes of extreme stability have been determined by the combination of extraction and pH-metric data.The formation of the less stabil Hg(II)-ligand 1:3 complexes with the coordination of three sulfhydrilate groups has been followed and the values of βThe differences in the Hg(II) complex formation and protonation equilibria between the two ligands and the possible binding sites in the complexes have been discussed.

Spectroscopic and magnetochemical studies on the active site copper complex in galactose oxidase

Galactose oxidase is a radical copper oxidase, an enzyme making use of a covalently modified tyrosine residue as a free radical redox cofactor in alcohol oxidation catalysis. We report here a combination of spectroscopic and magnetochemical studies developing insight into the interactions between the active site Cu(II) and two distinct tyrosine ligands in the biological complex. One of the tyrosine ligands (Y495) is coordinated to the Cu(II) metal center as a phenolate in the resting enzyme and serves as a general base to abstract a proton from the coordinated substrate, thus activating it for oxidation. The structure of the resting enzyme is temperature-dependent as a consequence of an internal proton equilibrium associated with this tyrosine that mimics this catalytic proton transfer step. The other tyrosine ligand (Y272) is covalently crosslinked to a cysteine residue forming a tyrosine–cysteine dimer free radical redox site that is required for hydrogen atom abstraction from the activated substrate alkoxide. The presence of the free radical in the oxidized active enzyme results in formation of an EPR-silent Cu(II) complex shown by multifield magnetic saturation experiments to be a diamagnetic singlet arising from antiferromagnetic exchange coupling between the metal and radical spins. A paramagnetic contribution observed at higher temperature may be associated with thermal population of the triplet state, thus permitting an estimate of the magnitude of the isotropic exchange coupling ( J>200 cm −1, JS 1· S 2) in this complex. Structural correlations and the possible mechanistic significance of metal–radical coupling in the active enzyme are discussed.

The redox mechanism of polyaniline studied by simultaneous ESR–UV–vis spectroelectrochemistry

The electrochemical redox process to charge and discharge polyaniline layers is studied by simultaneous Electron Spin Resonance (ESR) and UV–vis spectroscopic measurements. For this purpose, optically transparent electrodes were applied under potential-controlled conditions. The use of a calibrated manganese ESR standard ensures the calculation of the absolute numbers of free spins in the polymer layer during electrochemical doping. A general procedure is given to analyse the simultaneously recorded electrochemical and spectroscopic data. The procedure proposed following the time dependence of charge, ESR absorption and the absorbance demonstrates that the electrochemical charging/discharging mechanism of polyaniline can be described on the base of three oxidation states. In this way, the separated UV–vis spectra and the time/potential dependence of each redox state can be shown. Only little changes are observed in the shape of the separated spectra using the polaron/bipolaron model in comparison to the polaron/ σ-dimer model. A square ladder scheme of the redox mechanism is discussed, considering neutral, polaronic and bipolaronic states and the protonation equilibria. Furthermore, experimental evidence for the Faradaic nature of the “capacitive” current plateau is presented.

Inframolecular Studies of the Protonation of Adenophostin A: Comparison with 1-d-myo-Inositol 1,4,5-Trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the d-myo-inositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic protonation process of this compound by performing 31P NMR, 1H NMR, and potentiometric titration experiments. The logarithms of the first to the fourth stepwise protonation constants are, respectively, log K1 = 8.48, log K2 = 6.20, log K3 = 4.96, and log K4 = 3.80. The latter constant refers to the protonation equilibrium involving the N1 adenine nitrogen. From the microconstants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophostin A at near physiological pH is very similar to those of inositol 1,4,5-trisphosphate, indicating that differences in phosphate charge cannot account for the high potency of this molecule. The analysis of the 1H chemical shifts vs pH provided complementary conformational information. In particular, a slight “wrongway shift” of H1" can be related to the protonation of P2, thus indicating a short H1"–P2 distance. Our results are in line with a recently published model in which, however, a certain degree of constraint would keep the ribose 2′-phosphate moiety close to the glucose ring phosphates.