1H NMR spectroscopic identification of protonable sites in cryptolepines with C‐11 substituents containing two amino functionalities

Knowledge of protonable sites and acid dissociation constants of cryptolepine derivatives having C‐11 substituents containing two amino functionalities is of great importance to the understanding of the mechanism of their antimalarial action, which may contribute to their further development as drug candidates. In this work, we applied ¹H NMR titration to investigate the acid–base characteristics of these polyprotic compounds in the pH range 3–13. We identified three acid–base equilibria with most acid dissociation constants (pK_a*) being greater than 10.5, which prevented us from using the potentiometric method. Overall, ¹H NMR titration was sensitive and suitable for the determination of pK_a values for these drug leads. Copyright © 2012 John Wiley & Sons, Ltd.     

From proton nuclear magnetic resonance spectra to pH. Assessment of H NMR pH indicator compound set for deuterium oxide solutions

In this study, a protocol for pH determination from D₂O samples using ¹H NMR pH indicator compounds was developed and assessed by exploring the pH-dependency of 13 compounds giving pH-dependent ¹H NMR signals. The indicators cover the pH range from pH* 0 to 7.2. Equations to transform the indicator chemical shifts to pH estimates are given here for acetic acid, formic acid, chloroacetic acid, dichloroacetic acid, creatine, creatinine, glycine, histidine, 1,2,4-triazole, and TSP (2,2,3,3-tetradeutero-3-(trimethylsilyl)-propionic acid). To characterize the method in presence of typical solutes, the effects of common metabolites, albumin and ionic strength were also evaluated. For the ionic strengths, the effects were also modelled. The experiments showed that the use of pH sensitive ¹H NMR chemical shifts allows the pH determination of typical metabolite solutions with accuracy of 0.01-0.05 pH units. Also, when the ionic strength is known with accuracy better than 0.1 mol dm⁻³ and the solute concentrations are low, (the NMR estimate of pH) can be assumed to be within 0.05 pH units from potentiometrically determined pH.     

Accurate determination of low pK values by (1)H NMR titration

The NMR titration methodology to determine acid dissociation constants in aqueous solutions is extended for pK(a) values between 0 and 2, where potentiometric titrations are no longer applicable. (1)H NMR spectra are acquired for single samples of constant acid concentration (e.g. 0.02M), controlled ionic strength (I=1M with HNO(3)/NaNO(3)) and varying pH. To avoid biased pH readings due to the acid error of the glass electrode, true, concentration-based pH values are deduced by combination of the charge balance equation with information from (1)H NMR chemical shifts of the investigated acid. The method has been tested on histidine (pK(1)=1.83+/-0.02) and yielded the dissociation constant of dichloroacetic acid (pK=1.06+/-0.01) for the first time with good accuracy and precision. Dichloroacetic acid is recommended as an NMR spectroscopical "indicator molecule" for in situ monitoring the pH in strong acidic solutions of other equilibrium systems.     

Determination of pKa values of tenoxicam from H NMR chemical shifts and of oxicams from electrophoretic mobilities (CZE) with the aid of programs SQUAD and HYPNMR

In this work it is explained, by the first time, the application of programs SQUAD and HYPNMR to refine equilibrium constant values through the fit of electrophoretic mobilities determined by capillary zone electrophoresis experiments, due to the mathematical isomorphism of UV-vis absorptivity coefficients, NMR chemical shifts and electrophoretic mobilities as a function of pH. Then, the pK_a values of tenoxicam in H₂O/DMSO 1:4 (v/v) have been obtained from ¹H NMR chemical shifts, as well as of oxicams in aqueous solution from electrophoretic mobilities determined by CZE, at 25 °C. These values are in very good agreement with those reported by spectrophotometric and potentiometric measurements.     

31P nuclear magnetic resonance titrations: Simultaneous evaluation of all pH-dependent resonance signals

The simultaneous evaluation of ali pH-dependent resonance signals (or multiplets) of an NMR titration offers a substantially increased accuracy and significance. The number of linearly independent titration equilibria is determined by graphical matrix rank analysis. The chemical shifts of all pH-dependent resonance lines are plotted against each other (chemical shift or CS diagrams) indicating whether a single or more titration equilibria are NMR spectrometrically observable and how far they overlap with each other. An iterative curve-fitting program allowing the simultaneous evaluation of all (pH) curves is available, from which pK values and chemical shifts of all species can be calculated. The starting pK values for the iteration need only be estimated very approximately (accuracy ±1–2 units). The titration end-points do not have to be experimentally accessible. The different methods for the simultaneous evaluation of all pH-dependent NMR signals are exemplified in the ³¹P NMR titration of thiamine pyrophosphate. In this case either the observed resonance lines (two doublets in a broad band proton decoupled spectrum) or the calculated chemical shifts for this AB system can be evaluated. A titration of sodium pyrophosphate was performed and evaluated for comparison.     

Acid dissociation constants of uridine-5′-diphosphate compounds determined by phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing

The acid dissociation constant (pK_a) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the ³¹P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a ³¹P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pK_a values of the imide and second diphosphate of uridine-5′-diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pK_a values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pK_a values.     

Assignment of pH‐dependent NMR spectra of the scorpiand macrocycle: an application of titration profiles and spectral linewidths

Routine homo‐ and heteronuclear 2D NMR experiments (COSY, HMQC, TOCSY and ROESY) were performed for the complex macrocyclic pentaamine ligand 1‐(2′‐aminoethyl)‐1,4,8,11‐tetraazacyclotetradecane ( 1 , scorpiand), derived from 1,4,8,11‐tetraazacyclotetradecane (cyclam). However, some its ¹³C and ¹H resonances were distinguished only based on the similarity between profiles of relevant pH–chemical shift plots and/or linewidth considerations. Following this empirical method relying on the assumption that titration curves of nuclei in comparable chemical environments have comparable shapes, it was possible to assign all chemical shifts (¹³C, pH 0.25–13.4; ¹H, pH 10–11.5) for some different protonated species H_n 1 ⁿ⁺. A multiple experimental approach used for recognizing their NMR signals is offered as a general assignment procedure in the case of polyazamacrocycles. Copyright © 2002 John Wiley & Sons, Ltd.     

Determination of pK a of N-alkyl-N,N-dimethylamine-N-oxides using 1H NMR and 13C NMR spectroscopy

The pK _a values of N-alkyl-N,N-dimethylamine-N-oxides were determined from the pH dependences of chemical shifts in ¹H NMR and ¹³C NMR spectra. The dependences were measured at concentrations below and above the critical micelle concentration of the amine oxides. Above the critical micelle concentration, the plots of the peak positions vs. pH were either sigmoid (the groups close to the nitrogen) or “peak type” (farther groups). The sigmoidal behaviour was a direct result of the acid-base reaction; on the other hand, the “peak type” behaviour was probably an indirect consequence of the pH variation, mediated with the change in micelle size.     

The first automated 470.59 MHz 19F NMR‐controlled titration: dissociation constants and ion‐specific chemical shifts of 2‐amino‐4‐fluoro 2‐methylpent‐4‐enoic acid

2‐Amino‐4‐fluoro‐2‐methylpent‐4‐enoic acid, obtained as a 1 : 1 salt with trifluoro‐acetic acid, was characterized by ¹H and ¹⁹F high‐resolution NMR spectroscopy. High‐precision potentiometry led to the dissociation constants pK = 1.879 and pK = 9.054. The first automated 470.59 MHz ¹⁹F NMR‐controlled titration yielded the dynamic chemical shift 〈δ_F〉 as a function of pcH or τ and the ion‐specific chemical shifts: δ_F(H₂L⁺) = ?94.81 ppm, δ_F(HL) = ?94.21 ppm, δ_F(L^?) = ?92.45 ppm. The deprotonation gradients were found to be Δ₁ = ?0.60 ppm and Δ₂ = ?1.76 ppm. Copyright © 2002 John Wiley & Sons, Ltd.     

The determination of sulfoxide configuration in six-membered rings using NMR spectroscopy and DFT calculations

Cyclic six-membered ring sulfoxides and sulfones were prepared by a stepwise in situ oxidation of the corresponding sulfides with meta-chloroperbenzoic acid in an NMR tube. The oxidation was followed by NMR spectra and the ¹H and ¹³C NMR data were collected. The geometries of all of the compounds were optimized using the DFT B3LYP/6-31G^∗∗ method and the ¹³C and ¹H NMR chemical shifts were calculated for geometry-optimized structures with the DFT B3LYP/6-31++G^∗∗ method. The calculated ¹³C NMR chemical shifts induced by oxidation (Δδ values) are in very good agreement with the experimental data and can be used to determine the oxidation state of the sulfur atom (-S-, -SO-, -SO₂-). The characteristic differences of the induced oxidation chemical shifts of carbon atoms at the α- and β-position to sulfur were successfully used for distinguishing between the diastereoisomeric sulfoxides.     

Complexation of the [Rh(H2O)6]3+ ion with phosphoric acid as probed by 31P NMR

The kinetics of the reaction [Rh(H₂O)₆]³⁺ + H₃PO₄ ? [Rh(H₂O)₅H₂PO₄]²⁺ + H₃O⁺ has been studied by ³¹P NMR; E _a = 142 ± 12 kJ/mol, logA = 17 ± 2. An empirical dependence of the ³¹P NMR chemical shift on the equilibrium pH value has been found. The acid dissociation constant of the coordinated ion has been evaluated: pK = 1.5. The ³¹P NMR chemical shifts of individual [Rh(H₂O)₅H₂PO₄]²⁺ and [Rh(H₂O)₅HPO₄]⁺ complex ions are, respectively, 14.5 and 15.8 ppm at 323 K.     

Study of ion exchange selectivity of organic ions by using (31)P NMR spectroscopy

Ion exchange distribution of the phenylphosphoric acid has been studied on various types of chloride form strong base resins as a function of pH at 25 °C, at 0.1 M ionic strength. Equilibrium measurements were supplemented by the study of pH dependence of the ³¹P NMR spectra of the resin-phase phenylphosphate species. Equations were derived to describe the pH dependence of the overall distribution coefficient and the chemical shift of the resin-phase solute species. Experimental data were evaluated by using these model equations and the values of the individual distribution coefficients, ion exchange selectivity coefficients and the resin-phase ³¹P chemical shifts of the mono- and divalent ions have been calculated. Comparison of distribution data of the individual species corroborated the significance of the role of hydrophobic interaction in the selectivity of organic ion exchange processes. A well-defined correlation between the ion exchange selectivity and the resin-phase ³¹P NMR chemical shift data has been pointed out.     

15N NMR studies of amino acids and their reaction products with formaldehyde

Natural abundance ¹⁵N NMR spectroscopy has been used to investigate the effect of pH on the ¹⁵N chemical shifts of lysine and of ε-hydroxymethyllysine. A computer calcualtion which fits the chemical shifts of both α-and ε-nitrogen atoms versus pH has been used to predict the pK_a values. ¹⁵N chemical shifts and some ¹J(¹⁵NH) values of some other amino acids and of their reaction products with formaldehyde are also reported.     

Thermodynamic and19F NMR studies of antimony trifluoride in water

Densities, specific heat capacities per unit volume and enthalpies of dilution at 25°C and osmotic coefficients at 37°C were measured for antimony trifluoride in water as functions of concentration. From the first three properties the apparent and partial molar volumes, heat capacities and relative enthalpies were derived. As well, pH measurements in water at 25°C and¹⁹F NMR spectra in water and methanol at 33°C were also carried out. All the thermodynamic properties together with the chemical shifts abruptly change in the very dilute concentration region (<0.1m) and, then, tend to a constant value. These trends have been rationalized through a simple model based on an equilibrium of dissociation of SbF₃ into two ionic species. From the simulation of all the data it is derived that two concomitant equilibria are present in solution: the hydrolysis process of SbF₃ which explains the pH values and the ionic dissociation of SbF₃ which accounts for the¹⁹F NMR data.     

13C Pulse fourier transform NMR of menthol stereoisomers and related compounds

¹³C NMR spectra of menthol stereoisomers have been determined. The correlations of chemical shifts of these ring carbons with those of stereoisomeric 2-isopropylcyclohexanols are examined. Observed chemical shifts of 1-Me carbons are compared with those predicted from the chemical shifts of stereoisomeric 1-methyl-4-t-butylcyclohexanes. ¹³C NMR spectra of menthyl acetates, and cis and trans p -menthanes have also been examined.     

NMR determination of pKa values of indoloquinoline alkaloids

Malaria is one of the most serious global health problems. Isolating new therapeutic agents with potential antimalarial activity from natural sources or preparing such agents either semisynthetically or synthetically is one strategy for solving the problem of resistance constantly evolving to the drugs currently in use. For alkaloids, the acid–base dissociation constant, pK_a, is an important characteristic, thought to be associated with biological activity. In this contribution, pK_a values for several indoloquinoline alkaloids were determined by using ¹H NMR spectroscopy in a mixture of solvents. The data were recalculated for water solutions using the correction factors reported previously. The structural dependence of the pK_a values for cryptolepine and its isomers neocryptolepine, isocryptolepine and isoneocryptolepine as well as some substituted neocryptolepine derivatives is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Dynamic method as a simple approach for wide range pH measurements using optodes

In this paper, a flow system equipped with an optode has been suggested for wide range pH measurements. Triacetyl cellulose was used as the optode membrane in which different pH indicators were immobilized. For extending the pH range, the dynamic response rather than the steady-state response of the optode was measured. Since diffusion is the main process governing the system response, different parameters having influence on diffusion of the analyte into the membrane were optimized. Under the optimum conditions, wide range pH determination (up to 11 pH units) is simply achieved regardless of the pK_a of the pH indicator immobilized in the membrane. To validate the application of the method different indicators with different structures and pK_a values were tested and the results were all confirming the precision and accuracy of the method. The suggested method also has combined advantages of flow systems together with inherent advantages of kinetic systems.     

Elucidation of spermidine interaction with nucleotide ATP by multiple NMR techniques

Interaction of polyamines with nucleotides plays a key role in many biological processes. Here we use multiple NMR techniques to characterize interaction of spermidine with adenosine 5′‐triphosphate (ATP). Two‐dimensional ¹H‐¹⁵N spectra obtained from gs‐HMBC experiments at varied pH show significant shift of N‐1 peak around pH 2.0–7.0 range, suggesting that spermidine binds to N‐1 site of ATP base. The binding facilitates N‐1 deprotonation, shifting its pK_a from 4.3 to 3.4. By correlating ¹⁵N and ³¹P chemical shift data, it is clear that spermidine is capable of concurrently binding to ATP base and phosphate sites around pH 4.0–7.0. The self‐diffusion constants derived from ¹H PFG‐diffusion measurements provide evidence that binding of spermidine to ATP is in 1:1 ratio, and pH variations do not induce significant nucleotide self‐association in our samples. ³¹P spectral analysis suggests that at neutral pH, Mg²⁺ ion competes with spermidine and shows stronger binding to ATP phosphates. From ³¹P kinetic measurements of myosin‐catalyzed ATP hydrolysis, it is found that binding of spermidine affects the stability and reactivity of ATP. These NMR results are important for advancing the studies on nucleotide–polyamine interaction and its impact on nucleotide structures and activities under varied conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

13C NMR measurements of acid-base equilibrium constants in alkaline aqueous solutions of D-glucose anomers

The concentration dissociation constants pK _c of the α and β D-glucose anomers were measured at pH 12–14 by the ¹³C NMR method. The pK _c values for L ? H_?1L + H equilibria at 25°C and an ionic strength of 1.0 mol/l (NaCl) were pK _c ^α = 12.31 ± 0.03 and pK _c ^β = 12.03 ± 0.01. Dissociation was found to shift all the spectrum lines downfield. The most probable hydroxyl group responsible for the dissociation of β-D-glucose was determined.     

Synthesis and spectrophotometric studies of water-soluble amino[bis(ethanesulfonate)] azobenzene pH indicators

Three azobenzene pH indicators with amino[bis(ethanesulfonate)] substituents were synthesized and studied by UV–visible absorption spectroscopy in aqueous solution. The indicators exhibit brilliant and distinct colour changes with transitions between pH 1 and 4. Significant changes were observed in the UV–visible spectra on titration with acid with pK_a values ranging from 2.2 to 2.8. The indicators demonstrate individual changes in colour as a function of pH. These novel pH indicators complement the existing library of azobenzene indicator dyes and may be useful for environmental situations with high proton concentrations.