Cross-modulation of physicochemical character of aglycones in dinucleoside (3'-->5') monophosphates by the nearest neighbor interaction in the stacked state

Each nucleobase in a series of stacked dinucleoside (3'-->5') monophosphates, in both acidic and alkaline pH, shows ((1)H NMR) not only its own pK(a) but also the pK(a) of the neighboring nucleobase as a result of cross-modulation of two-coupled pi systems of neighboring aglycones. This means that the electronic character of two nearest neighbors are not like the monomeric counterparts anymore; they have simultaneously changed, almost quantitatively, to something that is a hybrid of the two due to two-way transmission of charge (i.e. 3'-->5' as well as 5'-->3'). This change is permanent due to total modulation of each others pseudoaromatic character by intramolecular stacking, which can be tuned by the nature of the medium across the whole pH range. The small difference observed in the pK(a) of the dimer compared to the monomer is a result of the change in microenvironment in the former. The charge transfer takes place between two stacked nucleobases from the negatively charged end because of the attempt to minimize the charge difference between the two neighboring pseudoaromatic aglycones. Experimental evidence points that the charge transmission in the stacked state takes place by atom-pisigma interaction between nearest neighbor nucleobases in 1-6. The net result of this cross-talk between two neighboring aglycones is a unique set of aglycones in an oligo- or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the sequence makeup, and whether they are stacked or unstacked. Thus, the physicochemical property of individual nucleobases in an oligonucleotide is determined in a tunable manner, depending upon who the nearest neighbors are, which may have considerable implication in the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability in a microenvironment, in the use of codon triplets in the protein biosynthesis or in the triplet usage by the anticodon-codon interaction.     

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constants in aqueous solutions is extended for pK(a) values between 0 and 2, where potentiometric titrations are no longer applicable.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.     

Hydrogen ion titration of alkyldimethylamine oxides by 13C and 1H NMR and conventional methods

In the hydrogen ion titration of micelles, the degree of ionization of the micelle at a given pH has to be evaluated to obtain a pKa value of micelles (Ka being the proton dissociation constant) at the pH. We compared the degree of ionization obtained from 13C and 1H NMR spectra with that obtained from the stoichiometric method. We used dodecyldimethylamine oxide (C12DMAO) and hexyldimethylamine oxide (C6DMAO) to examine the titration behavior of micelles and monomers, respectively. We determined pKa values of amine oxides both in H2O and D2O. As to the monomer (C6DMAO), the degree of ionization from NMR, alpha(NMR), coincided with that from the conventional stoichiometric method alpha. The difference of pK1 of amine oxide monomer between D2O and H2O was about 0.5: pK1(D) approximately pK1(H) + 0.5. The difference was about the same as that for carboxylic acids. As to the C12DMAO micelle, alphaNMR did not coincide with alpha over a considerable range of alpha. The NMR chemical shift might be influenced by micellar structure changes induced by the ionization, such as the sphere-to-rod transition. The intrinsic logarithmic dissociation constants of the micelle were 5.9+/-0.1 for H2O, and 6.5+/-0.1 for D2O.     

Thermal dissociation of the protein homodimer ecotin in the gas phase

The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique. Dissociation of the protonated dimer, (E₂ + nH)ⁿ⁺ E₂ⁿ⁺ where n = 14–17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175 °C. The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50–60% of the monomer product ions correspond to an asymmetric partitioning of charge. The relative abundance of the different monomer ion pairs produced from E₂¹⁴⁺, E₂¹⁵⁺, and E₂¹⁶⁺ depends on reaction time, with the more symmetric charge distribution pair dominating at longer times. The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers. For E₂¹⁷⁺, the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution. The charge distribution in the E₂¹⁵⁺ and E₂¹⁶⁺ dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity. In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E₂¹⁷⁺ dimer.     

Efficient implementation of constant pH molecular dynamics on modern graphics processors

The treatment of pH sensitive ionization states for titratable residues in proteins is often omitted from molecular dynamics (MD) simulations. While static charge models can answer many questions regarding protein conformational equilibrium and protein–ligand interactions, pH‐sensitive phenomena such as acid‐activated chaperones and amyloidogenic protein aggregation are inaccessible to such models. Constant pH molecular dynamics (CPHMD) coupled with the Generalized Born with a Simple sWitching function (GBSW) implicit solvent model provide an accurate framework for simulating pH sensitive processes in biological systems. Although this combination has demonstrated success in predicting pK_a values of protein structures, and in exploring dynamics of ionizable side‐chains, its speed has been an impediment to routine application. The recent availability of low‐cost graphics processing unit (GPU) chipsets with thousands of processing cores, together with the implementation of the accurate GBSW implicit solvent model on those chipsets (Arthur and Brooks, J. Comput. Chem. 2016, 37, 927), provide an opportunity to improve the speed of CPHMD and ionization modeling greatly. Here, we present a first implementation of GPU‐enabled CPHMD within the CHARMM‐OpenMM simulation package interface. Depending on the system size and nonbonded force cutoff parameters, we find speed increases of between one and three orders of magnitude. Additionally, the algorithm scales better with system size than the CPU‐based algorithm, thus allowing for larger systems to be modeled in a cost effective manner. We anticipate that the improved performance of this methodology will open the door for broad‐spread application of CPHMD in its modeling pH‐mediated biological processes. © 2016 Wiley Periodicals, Inc.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING II: REACTIONS INVOLVING PYRIMIDINE RADICAL ANION INTERMEDIATES

A series of photo-CIDNP (chemically induced dynamic nuclear polarization) experiments were performed on pyrimidine monomers and dimers, using the electron-donor Nα-acetyltryptophan (AcTrp) as a photosensitizer. The CIDNP spectra give evidence for the existence of both the dimer radical anion, which is formed by electron transfer from the excited AcTrp* to the dimer, and its dissociation product, the monomer radical anion. The AcTrp spectra are completely different from those obtained with an oxidizing sensitizer like anthraquinone-2-sulfonate, because of different unpaired electron spin density distributions in pyrimidine radical anion and cation. In the spectra of the anti (1,3-dimethyluracil) dimers, polarization is detected that originates from a spin-sorting process in the dimer radical pair, pointing to a relatively long lifetime of the dimer radical anions involved. Although the dimer radical anions of the 1,1′-trimethylene-bridged pyrimidines may have a relatively long lifetime as well, their protons have only very weak hyperfine interaction, which explains why no polarization originating from the dimer radical pair is detected. In the spectra of the bridged pyrimidines, polarized dimer protons are observed as a result of spin sorting in the monomer radical pair, from which it follows that the dissociation of dimer radical anion into monomer radical anion is reversible. A study of CIDNP intensities as a function of pH shows that a pH between 3 and 4 is optimal for observing monomer polarization that originates from spin-sorting in the monomer radical pair. At higher pH the geminate recombination polarization is partly cancelled by escape polarization arising in the same product.     

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

In this paper, we report nano‐electrospray ionization‐ion mobility mass spectrometry (nano‐ESI‐IM‐MS) characterization of bovine superoxide dismutase (SOD‐1) and human SOD‐1 purified from erythrocytes. SOD‐1 aggregates are characteristic of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans that could be triggered by dissociation of the native dimeric enzyme (Cu₂,Zn₂‐dimer SOD‐1). In contrast to ESI‐MS, nano‐ESI‐IM‐MS allowed an extra dimension for ion separation, yielding three‐way mass spectra (drift time, mass‐to‐charge ratio and intensity). Drift time provided valuable structural information related to ion size, which proved useful to differentiate between the dimeric and monomeric forms of SOD‐1 under non denaturing conditions. In order to obtain detailed structural information, including the most relevant post‐translational modifications, we evaluated several parameters of the IM method, such as sample composition (10 mM ammonium acetate, pH 7) and activation voltages (trap collision energy and cone voltage). Neutral pH and a careful selection of the most appropriate activation voltages were necessary to minimize dimer dissociation, although human enzyme resulted less prone to dissociation. Under optimum conditions, a comparison between monomer‐to‐dimer abundance ratios of two small sets of blood samples from healthy control and ALS patients demonstrated the presence of a higher relative abundance of Cu,Zn‐monomer SOD‐1 in patient samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemistry of the diazeniumdiolates. 2. Kinetics and mechanism of dissociation to nitric oxide in aqueous solution

Diazeniumdiolate ions of structure R(2)N[N(O)NO](-) (1) are of pharmacological interest because they spontaneously generate the natural bioregulatory species, nitric oxide (NO), when dissolved in aqueous media. Here we report the kinetic details for four representative reactivity patterns: (a) straightforward dissociation of the otherwise unfunctionalized diethylamine derivative 2 (anion 1, where R = Et) to diethylamine and NO; (b) results for the zwitterionic piperazin-1-yl analogue 4, for which the protonation state of the neighboring basic amine site is an important determinant of dissociation rate; (c) data for 5, a diazeniumdiolate derived from the polyamine spermine, whose complex rate equation can include terms for a variety of medium effects; and (d) the outcome for triamine 6 (R = CH(2)CH(2)NH(3)(+)), the most stable structure 1 ion identified to date. All of these dissociations are acid-catalyzed, with equilibrium protonation of the substrate preceding release of NO. Specific rate constants and pK(a) values for 2-6 have been determined from pH/rate profiles. Additionally, a hypsochromic shift (from approximately 250 to approximately 230 nm) was observed on acidifying these ions, allowing determination of a separate pK(a) for each substrate. For 6, the pK(a) value obtained kinetically was 2-3 pK(a) units higher than the value obtained from the spectral shift. Comparison of the ultraviolet spectra for 6 at various pH values with those for O- and N-alkylated diazeniumdiolates suggests that protonation at the R(2)N nitrogen initiates dissociation to NO at physiological pH, with a second protonation (at oxygen) accounting for both the spectral change and the enhanced dissociation rate at pH <4. Our results help to explain the previously noted variability in dissociation rate of 5, whose half-life we found to increase by an order of magnitude when its concentration was raised from near-zero to 1 mM, and provide mechanistic insight into the factors that govern dissociation rates among diazeniumdiolates of importance as pharmacologic progenitors of NO.     

Comparison of bovine and porcine beta-lactoglobulin: a mass spectrometric analysis

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is applied to comparison of bovine and porcine beta-lactoglobulin (BLG and PLG). The conformational and oligomeric properties of the two proteins under different solvent and experimental conditions are analyzed. The pH-dependence of dimerization is described for the pH range 2-11. The results indicate maximal dimer accumulation at pH 6 for BLG and pH 4 for PLG, as well as a lower stability of the PLG dimer at pH 4 compared to BLG at pH 6. Conformational stability appears to be higher for BLG at acidic pH, but higher for PLG at basic pH. The higher stability of BLG at low pH is revealed by means of either chemical or thermal denaturation. Equilibrium folding intermediates of both proteins are detected. Finally, conditions are found that promote dissociation of the BLG dimer at pH 6 into folded monomers.     

A review of: “Micelles: Theoretical and Applied Aspects.” Yoshikiyo Moroi. Plenum,New York, 1992. pp. ix + 252. 559.50. (ISBN 0-306-43996-4).

Abstract

We examine here the adsorption of weak, acidic, rigid macro‐ions onto oppositely charged surfaces using a mean field model. The analysis takes into account the effect of the nominal suspension pH on the charge distribution inside the macro‐ion layer, as well as the counter‐ion distribution in the adsorbed layer and in the solution surrounding the substrate. We find that, as expected, the adsorbed layer thickness decreases with the pH (namely, with the degree of charge dissociation) and with the solution ionic strength. The macro‐ion adsorption can, in some cases, over‐compensate for the substrate charge, thereby allowing layer‐by‐layer deposition. We find that charge inversion is obtained, for a given substrate, if the macro‐ion pK is lower than a critical value. For a given macro‐ion, charge inversion takes place if the substrate charge density exceeds a critical value that scales as the square root of the macro‐ion charge density. In both cases charge inversion is obtained only in the regime where the suspension pH is comparable to the pK.     

Imidazolinone herbicides in strongly acidic media: Speciation and electroreduction

From UV-visible measurements and potentiometric titrations it follows that the lowest pK values (pK₁) of imidazolinone herbicides correspond to the simultaneous protonation/dissociation equilibria of both the pyridinic (or quinolinic) nitrogen and the carboxyl group, the following pK (pK₂) to the imminium nitrogen and the basic pK (pK₃) to the dissociation of the imido nitrogen. Below pH 6 and down to pH c.a. 2.5, the dominant form of the herbicide is a double ion having both positive and negative charges, this being important in discussing the effect of pH in the natural dynamics of imidazolinone herbicides, especially in their soil sorption. Electrochemical studies of the reduction of the herbicides were made on mercury and carbon electrodes in strongly acidic media (0.1 to 2.7 M H₂SO₄) as well up to pH 7. The reduction signals were all attributed to the reduction of the imidazolinone ring except the second peak/wave that was found to have originated by the reduction of the pyridine/quinoline ring. A signal observed in strongly acidic media and at highly negative overpotentials was attributed to the reduction of the imidazolinone ring of the product of the previous reduction in a process consisting of two reversible electron transfers followed by a protonation reaction.     

Determination of cationic mobilities and pKa values of 22 amino acids by capillary zone electrophoresis

The effective mobilities of the cationic forms of common amino acids--mostly proteinogenic--were determined by capillary zone electrophoresis in acidic background electrolytes at pH between 2.0 and 3.2. The underivatized amino acids were detected by the double contactless conductivity detector. Experimentally measured effective mobilities were fitted with the suitable regression functions in dependence on pH of the background electrolyte. The parameters of the given regression function corresponded to the values of the actual mobilities and the mixed dissociation constants (combining activities and concentrations) of the compound related to the actual ionic strength. McInnes approximation and Onsager theory were used to obtain thermodynamic dissociation constants (pK(a)) and limiting (absolute) ionic mobilities.     

Heme-Peptide Models for Hemoproteins. 1. Solution Chemistry of N-Acetylmicroperoxidase-8

An improved method for the preparation of the heme octapeptide acetyl-MP8, obtained by proteolysis of horse heart cytochrome c, is described. AcMP8 obeys Beer's law at pH 7.0 in aqueous solution up to a concentration of 3 x 10(-)(5) M. The self-association constant measured at 25 degrees C (log K(D) = 4.04) is an order of magnitude lower than that for MP8, reflecting the role of the N-acetyl protecting group in abolishing intermolecular coordination. However, AcMP8 does form pi-stacked dimers in aqueous solution with increasing ionic strength. A more weakly packed pi-pi dimer reaches a maximum abundance at approximately 3 M ionic strength, but a more tightly packed dimer is favored at &mgr; > 3 M. An equilibrium model based on charge neutralization by specific binding of Na(+) ions gives a total molecular charge of 3- for AcMP8 at pH 7.0 and a self-association constant log K(D) = 4.20. AcMP8 exhibits six spectroscopically active pH-dependent transitions. The Glu-21 c-terminal carboxylate binds to the heme iron at low pH (pK(a) = 2.1) but is substituted by His-18 (pK(a) = 3.12) as the pH increases. The two heme propanoic acid substituents ionize with pK(a)'s of 4.95 and 6.1. This is followed by ionization of iron-bound water with a pK(a) = 9.59, DeltaH = 48 +/- 1 kJ mol(-)(1), and DeltaS = -22 +/- 3 J K(-)(1) mol(-)(1). The electronic spectra indicate that AcMP8 is predominantly in the S = (5)/(2) state at pH 7.0, while the hydroxo complex at pH 10.5 corresponds to an equilibrium mixture of S = (5)/(2) and S = (1)/(2) states at 25 degrees C. In the final transition, His-18 ionizes to form the S = (1)/(2) histidinate complex with a pK(a) of 12.71. AcMP8 is relatively stable under alkaline conditions, dimerizing slowly at high pH (k = 2.59 +/- 0.14 M(-)(1) s(-)(1)) to form a high-spin &mgr;-oxo-bridged species. The pH-dependent behavior of AcMP8 in the presence of excess 3-cyanopyridine, however, is markedly different. At low pH, AcMP8 simultaneously binds the exogenous ligand and the Glu-21 c-terminal carboxylate with a pK(a) < 2. His-18 replaces the carboxylate ligand at higher pH (pK(a) = 2.60), and both heme propanoic acid groups ionize with a mean pK(a) = 5.10. Unlike AcMP8.OH(-), the axial histidine of the 3-CNPy complex ionizes at near neutral pH (pK(a) = 7.83), prior to being replaced by OH(-) (pK(a) = 10.13). The sixth transition in the AcMP8/3-CNPy system produces the bis(hydroxo) complex (pK(a) > 13).     

Engineering an obligate domain-swapped dimer of cyanovirin-N with enhanced anti-HIV activity

The anti-HIV cyanobacterial protein cyanovirin-N can undergo domain swapping to form an intertwined dimer. The dimeric form is stable at low pH and millimolar concentrations. By deleting an amino acid from the hinge linker about which domain swapping occurs, we have constructed an obligate domain-swapped dimer of cyanovirin-N that represents a new tetravalent carbohydrate binding protein that is stable over a large range of pH values. This obligate dimer displays enhanced anti-HIV activity relative to the wild-type cyanovirin-N monomer with an observed 3.5-fold decrease in IC(50) (9nM for the dimer vs 32 nM for the monomer) for inhibition of HIV-1 envelope-mediated cell fusion and, when expressed in Escherichia coli, can be rapidly obtained in >98% purity in a single chromatographic step.     

Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis

Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.     

Amplified potentiometric determination of pK(00), pK(0), pK(l), and pK(2) of hydrogen sulfides with Ag(2)S ISE

The chemical capacitor theory has been applied to accurately determine dissociation constants of H(2)S with the Ag(2)S ion-selective electrode (ISE). The theory's principle is based on the measurement of the change in electrode charge density as a result of protonated or unprotonated sulfide adsorbed on the electrode surface. This charge density is related to the potential. Connection of each individual capacitor in series amplifies the potential according to the equation, E(total)=E(1)+E(2)+E(3)+cdots, three dots, centeredE(n). As the charges of individual capacitors are concentrated to one capacitor area, the charge density rises, and the potential increases. The pK(00), pK(0), pK(1), and pK(2) are reported as 1.8, 2.12, 7.05, and 12.0, respectively. The pK(00) and pK(0) are reported here for the first time. The pK(1) agrees well with the literature values; however, the pK(2) differs from those reported recently under extreme conditions. Reasons for disproving the unreasonably high pK(2)>17-19 values are given based on calculations. Mainly, when pK(2)>17-19, the experimental results do not fit the equilibrium equations, pH=(pK(1)+pK(2))/2, pK(1)=(pK(0)+pK(2))/2, and pH=pK(2)+log(HS(-))/(S(2-)).     

利用光交联探针研究pH值调控的蛋白-蛋白相互作用

pH值是几乎影响到所有蛋白质分子表面电荷分布和相关结构变化的关键因素,许多蛋白质分子之间的相互作用也受到pH值的调控.近年来,基于非天然氨基酸的光交联探针被广泛应用于捕捉活细胞内的蛋白-蛋白相互作用.然而,由于环境pH值的改变往往导致蛋白质分子结构、带电性质的显著变化,因此现有的非天然氨基酸光交联探针难以实现在极端pH值条件下对相互作用的蛋白质分子的捕获和研究.本文将介绍本课题组新近发展的基于烷基双吖丙啶活性基团的非天然氨基酸光交联探针-DIZPK,通过这一探针,我们成功捕获到大肠杆菌中一种重要的酸性分子伴侣HdeA在膜间质内酸性胁迫过程中的作用对象.在捕获到的HdeA底物中,我们发现了两个膜间质中重要的分子伴侣蛋白：DegP和SurA.通过实验我们证明了在酸性胁迫条件下,DegP和SurA能够被HdeA保护不形成聚集体,并进而在随后的回复中性过程中能够协助HdeA对其他底物进行重折叠.这种不依赖于ATP的分子伴侣间协作模式可能起到了帮助肠道型细菌抵抗酸性胁迫的功能.基于上述实验结果,我们提出了一个＂分子伴侣协同作用＂的模型,用以阐释细菌利用抗酸性分子伴侣提高其在酸胁迫下逃逸的机理.推而广之,在原核和真核细胞中定点引入高可适性的非天然氨基酸光交联探针可广泛适用于在活体内探测众多的由pH值调控的蛋白-蛋白相互作用.     

Aggregation and reactivity of the cesium enolate of 6-phenyl-alpha-tetralone: comparison with the lithium enolate

The cesium enolate of 6-phenyl-alpha-tetralone (CsPAT) has a lambda(max) in THF at about 387 nm, but the variation with concentration is too small for application of singular value decomposition. Proton-transfer studies with several indicators show that CsPAT forms monomer-tetramer mixtures with a tetramerization equilibrium constant, K(1,4) = 2.3 x 10(11) M(-3). The pK of the monomer is 23.39 on a scale where fluorene is assigned 22.9 (per hydrogen). For comparison, the lithium enolate, LiPAT, is also a monomer-tetramer with K(1,4) = 4.7 x 10(10) M(-3) and a monomer pK = 14.22. HMPA in large amounts promotes dissociation to monomer with both enolates. Ion-pair S(N)2 initial rates were measured for CsPAT with several alkyl halides and with methyl tosylate and compared with other rates with LiPAT. In all cases, the enolate monomers are much more reactive than the aggregates. Reaction of CsPAT with alkyl halides is generally C-alkylation but HMPA promotes increasing amounts of O-alkylation. A new indicator, 11-methyl-11H-benzo[b]fluorene, has a pK on the cesium scale of 23.39.     

Rational design of ratiometric near-infrared fluorescent pH probes with various pKa values, based on aminocyanine

Novel ratiometric, near-infrared fluorescent pH probes with various pK(a) values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pK(a) values of these probes can be predicted from the calculated pK(a) values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications.     

Effect of temperature on the chromatographic retention of ionizable compounds. III. Modeling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e. g., at least +/- 2 pH units far from the analyte pK(a)) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pK(a )and enthalpies of acid-base dissociation of both the solute and the buffer components in the hydroorganic solvent mixture. The performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity, in mobile phases containing different buffer solutions prepared in 25% w/w ACN in water and at several temperatures.