Sharp Isosbestic Points Research Articles

Conformational Stability Effect of Polymeric Iron Chelators.

SummaryThe design and synthesis of metal chelators with extraordinary metal affinities is a basic and challenging scientific problem of both fundamental and practical importance. Here, we demonstrate a “conformational stability effect” that can significantly enhance the metal affinity of ligands after conjugation to polymer chains with the ability to spontaneously adopt a specific conformation as an optimal “soft” scaffold to ensure maximum thermodynamic stability of the metal complexes. Using iron chelators as models, we show that simple conjugation of small molecule catechol ligands to a polyallylamine chain resulted in more than 8–9 orders of magnitude enhancement of the iron-binding affinity, which is comparable to that of enterobactin, the strongest iron chelator ever known. This study demonstrates that flexible polymer chelators may realize the highest possible metal affinities of the conjugated ligands owing to their ability to achieve an optimal conformation, which could advance the identification of strong metal chelators.

Multiwavelength UV-metric and pH-metric determination of the dissociation constants of the hypoxia-inducible factor prolyl hydroxylase inhibitor Roxadustat

The dissociation constant pKa of the Roxadustat with UV-VIS spectra, molar absorption coefficients and protonation equilibria are the key physicochemical parameters influencing many biopharmaceutical characteristics. Roxadustat is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase inhibitor with potential anti-anemic activity. It belongs to Active Pharmacenutical Ingredients, which have acidic/basic functionalities, their ionization state is controlled by solution pH and acid dissociation constants. Nonlinear regression of the pH-spectra with programs REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined four multiple consecutive dissociation constants with the protonation scheme. A sparingly soluble neutral molecule LH3 of Roxadustat was dissociated to the soluble anions LH2−, LH2− and L3− or protonated to the cation LH4+ in an aqueous medium. The graph of molar absorption coefficients of variously protonated species according to wavelength shows that the spectra of two anions LH2− and LH2− are nearly the same in colour. The Roxadustat spectrum exhibited five sharp isosbestic points, which were related to the LH2−/L3− equilibrium. Four consecutive thermodynamic dissociation constants were estimated using UV-metric data pKTa1 = 3.60(04), pKTa2 = 5.62(14), pKTa3 = 7.66(16), pKTa4 = 9.08(02) at 25 °C and pKTa1 = 3.60(04), pKTa2 = 5.73(10), pKTa3 = 7.52(10), pKTa4 = 8.99(02) at 37 °C and using pH-metric data pKTa1 = 4.33(09), pKTa2 = 6.57(11), pKTa3 = 8.88(05), pKTa4 = 9.03(04) at 25 °C and pKTa1 = 4.25(09), pKTa2 = 6.49(10), pKTa3 = 8.80(06), pKTa4 = 9.00(05) at 37 °C The positive values of the enthalpy ΔH0 showed that the dissociation process is endothermic and the positive values of the Gibbs free energy ΔG0 at 25 °C indicated that the dissociation process was not spontaneous, which also was confirmed by a negative value of the entropy ΔS0. Four macro-dissociation constants of Roxadustat and six protonation locations were predicted by MARVIN.

Synthesis and application of Vis/NIR dialkylaminophenylbuta-1,3-dienyl borondipyrromethene dyes

Abstract Mono- and bis-dialkylaminophenylbuta-1,3-dienyl boron-dipyrromethenes (BODIPYs) 1–12 were synthesized in 36%–42% yields by a Knoevenagel-type condensation. The absorption and emission maxima (λabs = 614–739 nm; λem = 655–776 nm in CHCl3) of 1–12 covered from the visible to the near-infrared region. Probe 1 was ratiometric Vis pH probes. Such probe was almost non-fluorescent. Upon the protonation of the tertiary amine function of 1, the strong fluorescence (Φf = 0.97) was released and the florescence intensity was dramatically increased by one thousand folds. The sharp isosbestic points were discovered at 590 nm, which was a ratiometric pH probe.

Protonation-deprotonation equilibria in tetrapyrroles Part 4: Mono- and diprotonations of deutero-, hemato-, meso-, and protoporphyrin IX dimethyl esters in methanolic hydrochloric acid

The N-protonations in the deutero, hemato, meso and protoporphyrin IX dimethyl esters (DME) were investigated by spectrophotometric titrations using HCl as the acid and methanol as the solvent. Two spectroscopically different protonated species were observed for each porphyrin DME in addition to the neutral form. These were assigned to the N-protonated monocation and dication. There were no difficulties encountered in observing the monocation formation in the HCl – MeOH system. Very sharp isosbestic points were characteristic of each protonation stage. The p K3 values for the porphyrins in the above order were 3.23, 4.70, 2.93 and 3.37; the p K4 values were 2.48, 2.62, 2.41 and 2.64, respectively. For all porphyrins studied, no further spectral changes were observed after the dication was completely formed. This was interpreted as indicating that the formation of more highly protonated species is not possible in fullydelocalized porphyrins possessing the 18 π-electron [18]diazaannulene delocalization pathway. When the titration was performed on the free dicarboxylic acid porphyrins, aggregation obscured the first protonation step and no clear monocation spectrum could be distinguished. However, also in that case the titration ended up to a UVvis spectrum typical of the dication and the effect of aggregation on the p K4 values was negligible. The UVvis spectrometric parameters are given for the neutral forms and for the protonated species of the porphyrin DMEs. The results are discussed in terms of the NH tautomerization connected to the π-electron delocalization pathway (aromaticity), which tends to hinder outofplane distortions in the porphyrin plane, and in terms of solvation and counterion stabilization of the protonated forms.

Synthesis, Structural Characterization, and Photophysical, Spectroelectrochemical, and Anion-Sensing Studies of Heteroleptic Ruthenium(II) Complexes Derived from 4′-Polyaromatic-Substituted Terpyridine Derivatives and 2,6-Bis(benzimidazol-2-yl)pyridine

Heteroleptic bis-tridentate ruthenium(II) complexes of composition [(H2pbbzim)Ru(tpy-Ar)](ClO4)2, where H2pbbzim = 2,6-bis(benzimidazol-2-yl)pyridine and tpy-Ar = 4'-substituted terpyridine ligands with Ar = phenyl (2), 2-naphthyl (3), 9-anthryl (4), and 1-pyrenyl (5) groups, have been synthesized and characterized by using standard analytical and spectroscopic techniques. The X-ray crystal structures of the complexes [(H2pbbzim)Ru(tpy-Naph)](ClO4)2 (3), [(pbbzim)Ru(tpy-Naph)]·(CH3)2CO·H2O (3a), and [(H2pbbzim)Ru(tpy-Py)](ClO4)2 (5) have been determined. The absorption, steady-state, and time-resolved luminescence spectral properties of the complexes were thoroughly investigated in dichloromethane. The compounds display strong luminescence at room temperature with lifetimes (τ2) in the range of 5.5-62 ns, depending upon the nature of the polycyclic aromatic moiety as well as the solvents. The complexes are found to undergo one reversible oxidation in the positive potential window (0 to +1.5 V) and four successive quasi-reversible reductions in the negative potential window (0 to -2.4 V). The anion-sensing properties of the receptors were thoroughly investigated in acetonitrile/dichloromethane (1/9 v/v) solutions (2 × 10(-5) M) using absorption, steady-state, and time-resolved emission spectroscopic studies. (1)H NMR titration experiments, on the other hand, were carried out in either CD3CN or DMSO-d6. The anion-sensing studies revealed that the receptors act as sensors for F(-), CN(-), AcO(-), and SO4(2-) and to some extent for HSO4(-) and H2PO4(-). It is evident that, in the presence of excess anions, deprotonation of the imidazole N-H fragments of the receptors occurs, which is signaled by the change of color from yellow-orange to violet visible with the naked eye. From the absorption and emission titration studies the binding/equilibrium constants of the receptors with the anions have also been determined. Anion-induced lifetime quenching and/or enhancement make the receptors suitable lifetime-based sensors for selective anions. Cyclic voltammetric (CV) measurements of the compounds carried out in acetonitrile have provided evidence in favor of anion-dependent electrochemical responses with F(-) and AcO(-) ions. Spectroelectrochemical studies have also been carried out for both the protonated and deprotonated forms of the complexes in the range of 300-1200 nm. With successive oxidation of the Ru(II) center, replacement of MLCT bands by LMCT bands occurs gradually with observation of sharp isosbestic points in all cases.

Structural, Spectroscopic, and Proton‐Coupled Electron‐transfer Behavior of Pyrazolyl‐3,5‐bis(benzimidazole)‐Bridged Homo‐ and Heterochiral RuIIRuII, OsIIOsII, and OsIIRuII 2,2′‐Bipyridine Complexes

AbstractThe homo‐ and heterobimetallic complexes [(bpy)2MII(H2pzbzim)M′II(bpy)2](ClO4)3·nH2O (1, 3, 5) and their corresponding deprotonated complexes [(bpy)2MII(pzbzim)M′II(bpy)2](ClO4)·nH2O (2, 4, 6) [where MII, M′II = Ru (1, 2) = Os (3, 4); MII = Os and M′II = Ru (3, 5); bpy = 2,2′‐bipyridine; H3pzbzim = pyrazole‐3,5‐bis(benzimidazole)] were synthesized, separated to their heterochiral (a, ΛΔ/ΔΛ) and homochiral (b, ΛΛ/ΔΔ) diastereoisomers, and characterized by elemental analyses, ESI‐MS, and 1H NMR spectroscopy. The X‐ray structures of 1a, 3a, and 5a show the involvement of two pyridine rings of two bpy ligands in strong intramolecular nonbonded π–π interaction. The occurrence of a C–H···π interaction between an aromatic C–H and the π‐cloud of a pyridine ring leads to strong electronic shielding of this proton (1H NMR). In all cases, the two diastereoisomers show practically no differences in their absorption spectra, redox potentials, and pK values. The large shifts in the E1/2 values to less positive potentials and substantial redshifts in the MLCT bands that occur on deprotonation of 1, 3, and 5 are energetically correlated. From the profiles of E1/2(1), (2) vs. pH over the pH range 1–12, the equilibrium constants and standard redox potentials for all the complex species in the metal oxidation states II·II, II·III, and III·III and the bridged ligand in the protonation states H2pzbzim–, Hpzbzim2–, and pzbzim3– have been evaluated. Using these values the bond dissociation free energies for the benzimidazole N–H bonds have been estimated. Spectroelectrochemical studies have been carried out for 1a, 3a, and 5a in the range 400–1100 nm. With stepwise oxidation of the metal centers replacement of MLCT bands by LMCT bands occur gradually with the observation of sharp isosbestic points. In the case of 1a, a band observed at λmax = 910 nm for the RuIIRuIII species has been ascribed to intervalence charge transfer (IVCT) transition.

Halide Coordination to Zinc Porphyrin Sensitizers Anchored to Nanocrystalline TiO2

The coordination of halide ions to 5-(3,5-dicarboxyphenyl)-10,15,20-tri- p-tolylporphinatozinc(II) anchored to mesoporous nanocrystalline (anatase) TiO 2 thin films (TiO 2/ZnP) immersed in propylene carbonate was quantified. The addition of tetrabutylammonium halide salts to the external propylene carbonate electrolyte resulted in a red shift in the absorption spectrum with the maintenance of five isosbestic points. The absorption spectra were within experimental error the same for ZnP and ZnP-X (-) compared to TiO 2/ZnP and TiO 2/ZnP-X (-): A SoretZnP = 427 nm (epsilon = 574 000 M (-1) cm (-1)), A SoretZnP-Cl (-) = 435 nm (epsilon = 905 000 +/- 12 000 M (-1) cm (-1)), A SoretZnP-Br (-) = 436 nm (epsilon = 776 000 +/- 30 000 M (-1) cm (-1)), and A SoretZnP-I (-) = 437 nm (epsilon = 620 000 +/- 56 000 M (-1) cm (-1)). Titration studies with the halides revealed sharp isosbestic points consistent with formation of a 1:1 halide/porphyrin adduct. Equilibrium constants for ZnP were found to be 1670 M (-1) for Cl (-), 96 M (-1) for Br (-), and 5.5 M (-1) for I (-), and the corresponding values for TiO 2/ZnP were significantly smaller, 780 M (-1), 70 M (-1) and 3.4 M (-1). A quasi-reversible wave was observed by cyclic voltammetry of TiO 2/ZnP, E 1/2(ZnP (+/0)) = +790 mV vs Ag/AgCl, that was shifted 160 mV after addition of excess chloride, E 1/2(ZnP-Cl (0/-)) = +630 mV. In regenerative solar cells with quinone/hydroquinone redox mediators, TiO 2/ZnP and TiO 2/ZnP-X (-), where X is Cl, Br, or I, were found to convert light into electrical power. The photocurrent action spectrum demonstrated that energy conversion was initiated by light absorption of ZnP and/or the halide adduct.

A stable monomeric nickel borohydride.

A stable discrete nickel borohydride complex (Tp*NiBH(4) or Tp*NiBD(4)) was prepared using the nitrogen-donor ligand hydrotris(3,5-dimethylpyrazolyl)borate (Tp*-). This complex represents one of the best characterized nickel(II) borohydrides to date. Tp*NiBH(4) and Tp*NiBD(4) are stable toward air, boiling water, and high temperatures (mp > 230 degrees C dec). X-ray crystallographic measurements for Tp*NiBH(4) showed a six-coordinate geometry for the complex, with the nickel(II) center facially coordinated by three bridging hydrogen atoms from borohydride and a tridentate Tp(-) ligand. For Tp*NiBH(4), the empirical formula is C(15)H(26)B(2)N(6)Ni, a = 13.469(9) A, b = 7.740(1) A, c = 18.851(2) A, beta = 107.605(9) degrees, the space group is monoclinic P2(1)/c, and Z = 4. Infrared measurements confirmed the presence of bridging hydrogen atoms; both nu(B[bond]H)(terminal) and nu(B[bond]H)(bridging) are assignable and shifted relative to nu(B-D) of Tp*NiBD(4) by amounts in agreement with theory. Despite their hydrolytic stability, Tp*NiBH(4) and Tp*NiBD(4) readily reduce halocarbon substrates, leading to the complete series of Tp*NiX complexes (X = Cl, Br, I). These reactions showed a pronounced hydrogen/deuterium rate dependence (k(H)/k(D) approximately 3) and sharp isosbestic points in progressive electronic spectra. Nickel K-edge X-ray absorption spectroscopy (XAS) measurements of a hydride-rich nickel center were obtained for Tp*NiBH(4), Tp*NiBD(4), and Tp*NiCl. X-ray absorption near-edge spectroscopy results confirmed the similar six-coordinate geometries for Tp*NiBH(4) and Tp*NiBD(4). These contrasted with XAS results for the crystallographically characterized pseudotetrahedral Tp*NiCl complex. The stability of Tp*Ni-coordinated borohydride is significant given this ion's accelerated decomposition and hydrolysis in the presence of transition metals and simple metal salts.

Palladium carbonyl clusters entrapped in NaY zeolite cages: ligand dissociation and cluster-wall interactions

Highly structured FTIR spectra, which are indicative of Pd carbonyl clusters, are obtained after adsorption of CO on Pd/NaY which was reduced at 200{degree}C, but broad IR bands are observed with samples which were reduced at higher temperatures. Pd carbonyl clusters with only CO ligands have not been reported previously, which indicates that the geometry of NaY supercages favors their formation. As the clusters lose some of their CO ligands, old IR bands erode and new IR bands with sharp isosbestic points emerge. This process is entirely reversed upon reintroduction of CO. The low activation energy of CO release and concomitant changes in the IR band characteristic of zeolite O-H vibrations indicate a chemical interaction of zeolite protons and Pd carbonyl clusters.

General occurrence of isosbestic points in the metachromatic dye complexes of sulphated glycosaminoglycans

The absorption spectra of acridine orange and the thiazine dyes, dimethylmethylene blue, azure A and toluidine blue, were recorded in the presence of several types of glycosaminoglycans. In all cases, over a range of 14 to 60°C and dye:uronic acid ratios between 0.03 and 0.70, sharp isosbestic points were seen between the dye dimer and metachromatic absorptions. Generally large positive entropic contributions to the binding free energy were observed. These results are discussed in terms of preferential binding of the dye dimer to the polyanion. This represents a much simpler model of the metachromatic interaction than has previously been considered. Our results also show that the isosbestic points are sensitive indicators of the homogeneity of a glycosaminoglycan preparation, and may be useful in distinguishing between heparin and chondroitin sulphate polysaccharides, but not heparan sulphates. As such, they may provide a useful extension of currently popular metachromatic dye-binding assays for total glycosaminoglycan concentration.

Hydride transfer from cyclohexadienyl anions: the reaction between the hydride Meisenheimer adduct of 2,4-dinitroaniline and 1,3,5-trinitrobenzene

The hydride Meisenheimer adduct of 2,4-dinitroaniline transfers hydride to 1,3,5-trinitrobenzene to form its corresponding hydride Meisenheimer adduct. The reaction is practically irreversible. In dilute solution in dimethyl sulphoxide it exhibits second-order kinetics and the observation of several sharp isosbestic points shows that it occurs without formation of any detectable intermediate species.

Light-induced spectral changes of carotenoids in chromatophores of Rhodopseudomonas sphaeroides

Carotenoid difference spectra were recorded of chromatophores of Rhodopseudomonas sphaeroides energized with low light intensities versus chromatophores under dark conditions. The amplitudes of the peaks and troughs were dependent on the light intensities and the duration of illumination, but the shape of the difference spectra remained constant. Sharp isosbestic points were found at 515, 500, 481, 465, and 452 mm. When potassium-valinomycin-induced diffusion potentials (inside negative) were imposed on the chromatophores “mirror images” of the light-induced difference spectra were recorded with the same isosbestic points and the same positions but different relative amplitudes of the peaks and troughs. The results are explained in terms of changes in the shape of the vibrational splittings of the carotenoid main electronic transition. This could be the result of changes in the fluidity of the environment of the carotenoids upon energization of the membrane. Prolonged periods of illumination with low or high light intensities resulted in irreversible changes of the difference spectra. Short periods of illumination with high light intensities resulted in reversible elevations of the baseline and red shifts of peaks, troughs, and baseline crossings.

The Reaction of Chlorite with Horseradish Peroxidase and Chloroperoxidase: ENZYMATIC CHLORINATION AND SPECTRAL INTERMEDIATES

Chloroperoxidase and horseradish peroxidase use NaClO2 as both the oxidant and the halogen donor for the peroxidative chlorination of monochlorodimedone. Previous studies have shown that both horseradish peroxidase and chloroperoxidase can catalyze iodination reactions with hydrogen peroxide as the oxidant; however, only chloroperoxidase catalyzes chlorination reactions under these conditions. The chlorite chlorination reactions obey normal Michaelis-Menten kinetics with respect to chlorite. Free chloride ion is not required for the chlorite-promoted chlorination reaction and has no effect on the Km values for chlorite or the Vmax values for either enzyme. The Vmax for the chlorination of monochlorodimedone by chloroperoxidase when chlorite is the substrate is approximately 3 times greater than when hydrogen peroxide is the oxidant, suggesting that chlorite is an exceptionally good substrate. The pH optimum for the chlorination reaction with horseradish peroxidase as catalyst is 4.1 and with chloroperoxidase a broad optimum from pH 2.25 to 3.0 is observed. Chloroperoxidase can also utilize chlorite as the oxidant for the oxidation of classical peroxidase substrates such as guaiacol, pyrogallol, and thiourea. Horseradish peroxidase reacts with chlorite to form a relatively stable product (half-life greater than 20 min) which is spectrally indistinguishable from Compound I, the first detectable intermediate formed upon the reaction of horseradish peroxidase with peroxides. The reaction yielding the horseradish peroxidase-Compound I spectrum has a precise stoichiometry. The addition of increments of chlorite to horseradish peroxidase results in the generation of a family of spectra exhibiting sharp isosbestic points at 420, 450, and 540 nm. One mole of chlorite converts 2 moles of horseradish peroxidase to the intermediate product or products having the Compound I spectrum. The addition of reducing agents such as potassium ferrocyanide to the Compound I-type intermediate results in the rapid decomposition of the intermediate and regeneration of the spectrum of the native enzyme. Incorporation studies using 36Cl indicate that the chlorine atom derived from chlorite is incorporated directly into the substrate acceptor molecule during chlorination and does not undergo exchange with free chloride ion, even in the presence of exceedingly high levels of free halide. This suggests that the reaction of chlorite with both horseradish peroxidase and chloroperoxidase results in the formation of an enzyme-bound activated halogenating intermediate. These results, in conjunction with our previous studies on the chemical nature of Compound I, suggest that the activated halogenating intermediate may be represented as an enzyme-bound halogenium ion (—X+) or hypohalite ion (—OX+) coordinated to the heme prosthetic group.

The Binding of Carbon Monoxide to α and β Chains in Tetrameric Mammalian Hemoglobin

Hemoglobins stripped of organic phosphate by gel filtration and dissolved in 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (bis-tris) buffers at pH 7, or in borate buffers at pH 9, show sharp isosbestic points when their reaction with carbon monoxide is examined by the stopped flow method. The time course of the reaction is independent of the wave length of the observing light. In phosphate buffers at pH 7, or on the addition of 2,3-diphosphoglycerate or of inositol hexaphosphate to bis-tris buffers, isosbestic points are no longer found and the time course of the reaction varies with the wave length of the observing light. This result is attributed to differences between the α and β chains. Sheep hemoglobin is exceptional among the mammalian hemoglobins examined because it shows sharp isosbestic points under all conditions tested.

Synthesis and properties of α and ε pyridoxyl lysines and their phosphorylated derivatives

The structure of the coenzyme-binding site of several pyridoxal 5'-phosphate containing enzymes has recently been determined after fixation of the cofactor by sodium borohydride reduction. In all instances, pyridoxal 5'-phosphate was found to be linked to a unique lysyl residue of the protein. This work describes the synthesis, separation and characterization of several pyridoxyl-lysine derivatives, namely N α-pyridoxyl-, N ε-pyridoxyl-, N ε-phosphopyridoxyl-, and N α, N ε-dipyridoxyl-lysine that should allow for their unambiguous identification and determination. Their fluorescence properties, quantum yields and differential responses to 2,6-dichloroquinone chloroimide and diazotized p-amino acetophenone are also described together with a number of solvent and buffer systems enabling their separation on thin layer chromatography, paper chromatography and electrophoresis and various automated methods of analysis. Absorption spectra of all four derivatives were determined as a function of pH from which ionization constants were calculated. Sharp isosbestic points were observed with N ε-pyridoxyl- and phosphopyridoxyl-lysine but not with the unphosphorylated N α-substituted derivatives, probably because of the slow ionization of their phenolic hydroxyl groups. This effect was particularly pronounced with N α-pyridoxyl-lysine which showed a marked hysteresis during titration in the alkaline range.