Porphyrin and tetrabenzoporphyrin dendrimers: tunable membrane-impermeable fluorescent pH nanosensors

The pH dependencies of the UV-vis and fluorescent spectra of new water-soluble dendritic porphyrins and tetrabenzoporphyrins were studied. Because of extended pi-conjugation and nonplanar distortion, the absorption and the emission bands of tetraaryltetrabenzoporphyrins (Ar(4)TBP) are red-shifted and do not overlap with those of regular tetraarylporphyrins (Ar(4)P). When encapsulated inside dendrimers with hydrophilic outer layers, Ar(4)Ps and Ar(4)TBPs become water soluble and can serve as pH indicators, with pK's adjustable by the peripheral charges on the dendrimers. Two new dendritic porphyrins, Gen 4 polyglutamic porphyrin dendrimer H(2)P-Glu(4)OH (1) with 64 peripheral carboxylates and Gen 1 poly(ester amide) Newkome-type tetrabenzoporphyrin dendrimer H(2)TBP-Nw(1)OH (2) with 36 peripheral carboxylates, were synthesized and characterized. The pK's of the encapsulated porphyrins (pK(H)()2(P)(-)(Glu)()4(OH) = 6.2 and pK(H)()2(TBP)(-)(Nw)()1(OH) = 6.3) were found to be strongly influenced by the dendrimers, revealing significant electrostatic shielding of the cores by the peripheral charges. The titration curves obtained by differential excitation using the mixtures of the dendrimers were shown to be identical to those determined for the dendrimers individually. Due to their peripheral carboxylates and nanometric molecular size, porphyrin dendrimers cannot penetrate through phospholipid membranes. Dendrimer 1 was captured inside phospholipid liposomes, which were suspended in a solution containing dendrimer 2. No response from 1 was detected upon pH changes in the bulk solution, while the response from 2 was predictably strong. When proton channels were created in the liposome walls, both compounds responded equally to the bulk pH changes. These results suggest that porphyrin dendrimers can be used as fluorescent pH indicators for proton gradient measurements.     

Interactions of porphyrin covalently attached to poly(methacrylic acid) with liposomal membranes

The interactions between the 5-(4-acryloyloxyphenyl)-10,15,20-tritolylporphyrin covalently attached to poly(methacrylic acid) chain (PMA-Po) and phosphatidylcholine liposomes in aqueous solution at different pH values were studied. The binding constants (K(b)) for the liposome- PMA-Po in solutions in the pH range from 6.5 to 9.2 were determined using fluorescence spectroscopy. The binding was found to be efficient. The acid-base properties of the porphyrin chromophores were also studied. Both pK values associated with imine-N protonation of the porphyrin core were found to be 6.4. The quantum yield (Phi(Delta)) of singlet oxygen production by Po in the lipid-PMA-Po system was found to be high (0.88 +/- 0.05).     

J-aggregation of a sulfonated amphiphilic porphyrin at the air-water interface as a function of pH

π-A isotherms, ellipsometric measurements, Brewster angle microscopy (BAM) and reflection spectroscopy have been utilized to characterize the films of an amphiphilic porphyrin ((OD)(3)TPPS(3)) at the air-water interface as a function of pH. This porphyrin forms stable mono-molecular layers at such interfaces, and exhibits different J-aggregation as a function of pH. The J-aggregation of (OD)(3)TPPS(3) on neutral pH subphases is notable considering that the nitrogen atoms at the central macrocycle have a pK(a)≈4.9. The type of aggregates at neutral pH is like those detected at pH<4, because the central porphyrin ring is already protonated. However at basic pH the aggregation happens without protonation of the central ring but can be instead controlled by application of the surface pressure. At the air-water interface, (OD)(3)TPPS(3) shows two bands, a red component and a blue component, which have characteristics of non-degenerate linear oscillators being perpendicularly polarized between each other. The spectral behavior observed on subphases at different pHs is qualitatively interpreted by means of exciton coupling theory, assuming that the degenerate transitions attributed to the Soret band are split. Additionally, highly oriented molecular films of these J-aggregates were deposited onto transparent quartz slides.     

Speciation, luminescence, and alkaline fluorescence quenching of 4-(2-methylbutyl)aminodipicolinic acid (H2MEBADPA)

4-(2-Methylbutyl)aminodipicolinic acid (H(2)MEBADPA) has been synthesized and fully characterized in terms of aqueous phase protonation constants (pK(a)'s) and photophysical measurements. The pK(a)'s were determined by spectrophotometric titrations, utilizing a fully sealed titration system. Photophysical measurements consisted of room temperature fluorescence and frozen solution phosphorescence as well as quantum yield determinations at various pH, which showed that only fully deprotonated MEBADPA(2-) is appreciably emissive. The fluorescence of MEBADPA(2-) has been determined to be quenched by hydroxide and methoxide anions, most likely through base-catalyzed excited-state tautomerism or proton transfer. This quenching phenomenon has been quantitatively explored through steady-state and time-resolved fluorescence measurements. Utilizing the determined pK(a)s and quenching constants, the fluorescent intensity of MEBADPA(2-) has been successfully modeled as a function of pH.     

Chemistry of covalent inhibition of the gastric (H+, K+)-ATPase by proton pump inhibitors

Proton pump inhibitors (PPIs), drugs that are widely used for treatment of acid related diseases, are either substituted pyridylmethylsulfinyl benzimidazole or imidazopyridine derivatives. They are all prodrugs that inhibit the acid-secreting gastric (H(+), K(+))-ATPase by acid activation to reactive thiophiles that form disulfide bonds with one or more cysteines accessible from the exoplasmic surface of the enzyme. This unique acid-catalysis mechanism had been ascribed to the nucleophilicity of the pyridine ring. However, the data obtained here show that their conversion to the reactive cationic thiophilic sulfenic acid or sulfenamide depends mainly not on pyridine protonation but on a second protonation of the imidazole component that increases the electrophilicity of the C-2 position on the imidazole. This protonation results in reaction of the C-2 with the unprotonated fraction of the pyridine ring to form the reactive derivatives. The relevant PPI pK(a)'s were determined by UV spectroscopy of the benzimidazole or imidazopyridine sulfinylmethyl moieties at different medium pH. Synthesis of a relatively acid stable analogue, N(1)-methyl lansoprazole, (6b), allowed direct determination of both pK(a) values of this intact PPI allowing calculation of the two pK(a) values for all the PPIs. These values predict their relative acid stability and thus the rate of reaction with cysteines of the active proton pump at the pH of the secreting parietal cell. The PPI accumulates in the secretory canaliculus of the parietal cell due to pyridine protonation then binds to the pump and is activated by the second protonation on the surface of the protein to allow disulfide formation.     

Interaction of porphyrins with a dendrimer template: self-aggregation controlled by pH

The interaction between self-aggregated porphyrins such as 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and 5,10,15,20-tetrakis(4-phosphonatophenyl)porphyrin (TPPP), and a generation 5 (G5) PAMAM dendrimer template is governed by minute differences of porphyrin acido-basic properties. While at neutral pH both monomeric TPPS and TPPP form complexes with G5, decreasing pH did not lead to porphyrin ring protonation (pK(a) approximately 5) but rather to the preferential formation of H-aggregates (probably H-dimers), most likely due to protonation of the G5. Upon further acidification of the solution, this face-to-face orientation of the porphyrin units is being converted to edge-to-edge aligned J-aggregates with a tightly defined structure. This process starts by protonation of the porphyrin ring at pH below 2.3 and 2.8 for TPPS and TPPP, respectively. The AFM imaging of porphyrin/G5 nanostructures obtained at pH 0.7 shows the formation of long nanorods of TPPS with partially aggregated G5 and small aggregates of TPPP connected to individual G5 molecules.     

Interaction of meso-tetrakis (4-sulfonatophenyl) porphyrin with cationic CTAC micelles investigated by small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR)

Small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been used to investigate the interaction of the water-soluble meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS(4)) with cationic cethyltrimethylammonium chloride (CTAC) micelles. To evaluate if the porphyrin protonation state affects its interaction with the micelle, both SAXS and EPR measurements were performed at pH 4.0 and 9.0. The best-fit SAXS curves were obtained assuming for CTAC micelle a prolate ellipsoidal shape in the absence and upon incorporation of 2-10 mM TPPS(4). SAXS results show that the presence of porphyrin impacts on micellar hydrophobic core, leading to a micellar reassembling into smaller micelles. Lineshapes of EPR spectra of 5- and 16-doxyl stearic acids (5- and 16-DSA, respectively) bound to 100 mM CTAC micelles exhibited slight changes as a function of porphyrin concentration. Spectral simulations revealed an increase of mobility restriction for both spin probes, especially at higher porphyrin concentration, where a small reduction of environment polarity was also observed for 16-DSA. The spin labels monitored only slight differences between pH 4.0 and 9.0, in agreement with the SAXS results.     

Synthesis, reactivity, and properties of N-fused porphyrin rhenium(I) tricarbonyl complexes

The thermal reactions of N-fused tetraarylporphyrins or N-confused tetraarylporphyrins with Re2(CO)10 gave the rhenium(I) tricarbonyl complexes bearing N-fused porphyrinato ligands (4) in moderate to good yields. The rhenium complexes 4 are characterized by mass, IR, 1H, and 13C NMR spectroscopy, and the structures of tetraphenylporphynato complex 4a and its nitro derivative 15 are determined by X-ray single crystal analysis. The rhenium complexes 4 show excellent stability against heat, light, acids, bases, and oxidants. The aromatic substitution reactions of 4 proceed without a loss of the center metal to give the nitro (15), formyl (16), benzoyl (17), and cyano derivatives (19), regioselectively. In the electrochemical measurements for 4, one reversible oxidation wave and two reversible reduction waves are observed. Their redox potentials imply narrow HOMO-LUMO band gaps of 4 and are consistent with their electronic absorption spectra, in which the absorption edges exceed 1000 nm. Theoretical study reveals that the HOMO and LUMO of the rhenium complexes are exclusively composed of the N-fused porphyrin skeleton. Protonation of 4 takes place at the 21-position regioselectively, reflecting the high coefficient of the C21 atom in the HOMO orbital. The skeletal rearrangement reaction from N-confused porphyrin Re(I) complex (8) to N-fused porphyrin Re(I) complex (4) is suggested from the mechanistic study as well as DFT calculations.     

DFT/electrostatic calculations of pK(a) values in cytochrome c oxidase

Using classical electrostatic calculations, earlier we examined the dependence of the protonation state of bovine cytochrome c oxidase (CcO) on its redox state. Based on these calculations, we have proposed a model of CcO proton pumping that involves His291, one of the Cu(B) histidine ligands, which was found to respond to redox changes of the enzyme Fe(a)(3)-Cu(B) catalytic center. In this work, we employ combined density functional and continuum electrostatic calculations to evaluate the pK(a)() values of His291 and Glu242, two key residues of the model. The pK(a) values are calculated for different redox states of the enzyme, and the influence of different factors on the pK(a)'s is analyzed in detail. The calculated pK(a)() values of Glu242 are between 9.4 and 12.0, depending on the redox state of the protein, which is in excellent agreement with recent experimental measurements. Assuming the reduced state of heme a(3), His291 of the oxidized Cu(B) center possesses a pK(a)() between 2.1 and 4.0, while His291 of the reduced Cu(B) center has a pK(a) above 17. The obtained results support the proposal that the His291 ligand of the Cu(B) center in CcO is a proton pump element.     

pH at the micellar interface: synthesis of pH probes derived from salicylic acid, acid-base dissociation in sodium dodecyl sulfate micelles, and Poisson-Boltzmann simulation

The study of the H+ concentration at the micellar interface is a convenient system for modeling the distribution of H+ at interfaces. We have synthesized salicylic acid derivatives to analyze the proton dissociation of both the carboxylic and phenol groups of the probes, determining spectrophotometrically the apparent pK(a)'s (pK(ap)) in sodium dodecyl sulfate, SDS, micelles with and without added salt. The synthesized probes were 2-hydroxy-5-(2-trimethylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumhexanoyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumundecanoyl)benzoate; 2-hydroxy-5-acetylbenzoic acid; and 2-hydroxy-5-dodecanoylbenzoic acid. Upon incorporation into SDS micelles the pK(ap)'s of both carboxylic and phenol groups increased by ca. 3 pH units and NaCl addition caused a decrease in the probe-incorporated pK(ap). The experimental results were fitted with a cell model Poisson-Boltzmann (P-B) equation taking in consideration the effect of salt on the aggregation number of SDS and using the distance of the dissociating group as a parameter. The conformations of the probes were analyzed theoretically using two dielectric constants, e.g., 2 and 78. Both the P-B analysis and conformation calculations can be interpreted by assuming that the acid groups dissociate very close to, or at, the interface. Our results are consistent with the assumption that the intrinsic pK(a)'s of both carboxylic and phenol groups of the salicylic acid probes used here can be taken as those in water. Using this assumption the micellar and salt effects on the pK(ap)'s of the (trialkylammonium)benzoate probes were described accurately using a cell model P-B analysis.     

A robust microporous zinc porphyrin framework solid

A robust microporous zinc(II) metalloporphyrin framework solid has been synthesized. The proposed structural model developed from X-ray single crystal data has an interpenetrated three-dimensional framework of zinc trans-biscarboxylate tetraarylporphyrins whose carboxylates coordinate the six edges of tetrahedral Zn(4)O(6+) clusters, maintaining a charge-neutral framework. This cubic framework has 74% free volume and 4 x 7 A pores. N(2) adsorption gives a type I isotherm with a surface area of 800 m(2)/g, which is greater than that of a typical zeolite. Experimental evidence indicates that the interpenetrated frameworks of the evacuated solid remain intact and retain a microporous structure. This is a versatile framework system: alteration of the metal in the porphyrin may create a catalytically active solid, and modification of the 10-, 20-substituents of the porphyrin can provide control over both the polarity and the size of the pores.     

Anion chelation-induced porphyrin protonation and its application for chloride anion sensing

The protonation of a simple meso-tetraphenylporphyrin in an organic-aqueous system was found to be induced by the counteranions. During the process of protonation, the counteranion of the proton sources binds with the porphyrin core and thus promotes the complexation of the porphyrin and protons. The interaction of porphyrin and anion was characterized by fluorescence, UV-visible, cyclic voltammetry, (1)H NMR, and IR. Moreover, it could be exploited in selective fluorescent sensing of Cl(-). The sensing mechanism was based on extraction of protons from the aqueous phase into the organic phase by free base porphyrin and simultaneous coextraction of Cl(-), which promoted porphyrin protonation, and hence resulted in significant changes of the porphyrin fluorescence spectra. Selectivity trends turned out to be dependent upon the lipophilicity of anion and the binding affinity and structure complementarity between the protonated porphyrin and anions. The fluorescence enhancement of the porphyrin band at 684 nm showed modest selectivity for Cl(-) and NO(3)(-).     

Porphyrins meso-substituted with phenanthrene and 1,10-phenanthroline

Meso-tetrakis(9-phenanthryl)porphyrin and meso-tetrakis(1,10-phenanthrolin-4-yl)porphyrin were obtained and characterized by spectroscopy methods. The porphyrin containing phenanthroline substituents was formed with much smaller yield. It showed much slower progress of metallation with Cu(I) and Zn(II) ions than the phenanthreneporphyrin. While attachment of phenanthrene did not result in any meaningful changes in the uv vis spectrum when compared to other meso-substituted tetraarylporphyrins (except that of [2.2]paracyclophanylporphyrin), the appearance of two N centers in each meso substituent substantially altered porphyrin absorption in the 530–600 nm region. Although the 1 H nmr 300 MHz spectra of both porphyrins showed the same deshielding of β-pyrrole protons, the shielding of NH protons was more advanced in phenanthrolineporphyrin.     

Tunable pK of amino acid residues at the air-water interface gives an L-zyme (langmuir enzyme)

Various amino acid-carrying amphiphiles were synthesized, and the pK values of the attached amino acid residues were investigated at the air-water interface and in aqueous vesicles using pi-A isotherm measurements, (1)H NMR titration, and IR spectroscopy in reflection-adsorption mode. The epsilon-amino group of the Lys residue embedded at the air-water interface displays a significant pK shift (4 or 5 unit) compared with that observed in bulk water, while the pK shift in aqueous vesicles was not prominent (ca. 1 unit). Moreover, pK values of the amino acids at the air-water interface can be tuned simply by control of the subphase ionic strength as well as by molecular design of the amphiphiles. A simple equation based on the dominant contribution by the electrostatic energy to the pK shift reproduces well the surface pressure difference between protonated and unprotonated species, suggesting a reduction in the apparent dielectric constant at the air-water interface. Hydrolysis of a p-nitrophenyl ester derivative was used as a model reaction to demonstrate the use of the Lys-functionalized monolayer. Efficient hydrolysis was observed, even at neutral pH, after tuning of pK for the Lys residue in the monolayer, which is a similar case to that occurring in biological catalysis.     

Nonenzymatic breakdown of the tetrahedral (alpha-carboxyketal phosphate) intermediates of MurA and AroA, two carboxyvinyl transferases. Protonation of different functional groups controls the rate and fate of breakdown

The mechanisms of nonenzymatic breakdown of the tetrahedral intermediates (THIs) of the carboxyvinyl transferases MurA and AroA were examined in order to illuminate the interplay between the inherent reactivities of the THIs and the enzymatic strategies used to promote catalysis. THI degradation was through phosphate departure, with C-O bond cleavage. It was acid catalyzed and dependent on the protonation state of the carboxyl of the alpha-carboxyketal phosphate functionality, with ionizations at pK(a) = 3.2 +/- 0.1 and 4.3 +/- 0.1 for MurA and AroA THIs, respectively. The solvent deuterium kinetic isotope effect for MurA THI at pL 2.0 was 1.3 +/- 0.4, consistent with general acid catalysis. The pK(a)'s suggested intramolecular general acid catalysis through protonation of the bridging oxygen of the phosphate, though H(3)O(+) catalysis was also possible. The product distribution varied with pH. The dominant breakdown products were pyruvate + phosphate + R-OH (R-OH = UDP-GlcNAc or shikimate 3-phosphate) at all pH's, particularly low pH. At higher pH's, increasing proportions of ketal, arising from intramolecular substitution of phosphate by the adjacent hydroxyl and the enolpyruvyl products of phosphate elimination were observed. With MurA THI, the product distribution fitted to pK(a)'s 1.6 and 6.2, corresponding to the expected pK(a)'s of a phosphate monoester. C-O bond cleavage was demonstrated by the lack of monomethyl [(33)P]phosphate formed upon degrading MurA [(33)P]THI in 50% methanol. General acid catalysis through the bridging oxygen is consistent with the location of the previously proposed general acid catalyst for THI breakdown in AroA, Lys22.     

Synthesis and characterization of a water-soluble porphyrin with a cyclic sulfone

Water-soluble sulfonated tetraarylporphyrins are studied in a wide variety of contexts including as analytical reagents and as possible agents in cancer photodynamic therapy as well as in antiviral and antidiabetic applications. Herein, we report the first synthesis of a pentasulfonated porphyrin bearing an internal cyclic sulfone ring. Treatment of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) with fuming H₂SO₄ gave a structure consistent with initial sulfonation followed by dehydration to give a sulfone bridge between an ortho-position of one of the phenyl groups and a β-pyrrole position on the porphine ring (TPPS4Sc). The structure was established by electrospray mass spectrometry and ¹H NMR. The Soret UV-visible absorption is red shifted by about 32 nm compared to that of TPPS4.     

Tunable sustained release properties of "onion-like" phospholipids multilamellar vesicles

"Onion-type" multilamellar micro-vesicles of phospholipids (spherulites) were doped with different amounts of a cationic cosurfactant ((-)N-dodecyl-N-methylephedrinium bromide) for the purpose of controlling the sustained release of anionic drugs. Three weak acid probes (methyl red, chlorophenol red, and ibuprofen) were encapsulated in the vesicles as drug models. The kinetics and rate of release were studied by absorption spectroscopy and HPLC. The effect of probe charge (pH above and below pKa of the probes), of cosurfactant concentration and of added salt was investigated. It was found that, above pKa (i.e., when the probes are anionic), the release can be almost totally inhibited by doping the vesicles with 2.4 wt% of cationic cosurfactant. The release properties can even be finely tuned by controlling the amounts of the cosurfactant. Salt and pH effects demonstrate the role of electrostatic interactions in sustaining the release.     

Structurally homologous beta- and meso-alkynyl amidinium porphyrins

Alkynylamidinium groups have been introduced at the beta and meso positions of a nickel(II) porphyrin (PNi(II)) framework. The modification permits the distance between the amidinium-amidine acid-base group and porphyrin to be increased while effectively maintaining pi conjugation between the porphyrin macrocycle and the acid-base functionality. Use of an ethynyl spacer as a linker (i) extends the amidinium functionality away from the sterically bulky mesityl groups of the porphyrin, allowing it to be nearly planar with respect to the porphyrin ring, and (ii) draws the pi-orbital character of the porphyrin out toward the amidinium functionality, thereby engendering sensitivity of the electronic properties of the porphyrin macrocycle to the protonation state of the amidinium. The barrier for rotation of the amidinium group, as calculated by time-dependent density functional theory (TDDFT), is approximately 8.5 kT (5 kcal/mol) for both porphyrins. Analysis of UV-visible absorption profiles for the beta- and meso-alkynylamidinium PNi(II) upon deprotonation enables accurate determination of the amidinium acidity constants for the ground state (pK(a)(beta) = 7.03 +/- 0.1, pK(a)(meso) = 7.74 +/- 0.1 in CH(3)CN) and excited state (pK(a)*(beta) = 6.89 +/- 0.1, pK(a)*(meso) = 8.37 +/- 0.1 in CH(3)CN) porphyrins. Whereas pK(a)* < pK(a) for the beta-alkynylamidinium porphyrin, pK(a)* > pK(a) for the meso-alkynylamidinium porphyrin, indicating that beta-alkynylamidinium PNi(II) is a photoacid and meso-alkynylamidinium PNi(II) is a photobase. These divergent behaviors are supported by analysis of the frontier molecular orbitals of the homologous pair with TDDFT.     

Association behavior of porphyrin pendants in pH-sensitive water-soluble polymer

A novel tripyridylporphyrin monomer,5-[4-[2-(acryloyloxy)ethoxy]phenyl]-10,15,20-tris(4-pyridyl)porphyrin (TrPyP),was synthesized and polymerized with acrylamide(AM) to prepare the hydrophobically associating water-soluble polymer PAM-TrPyP.The aggregation behavior of porphyrin pendants was investigated by UV-Visible and fluorescence spectra.The polymer displays a strong tendency of hydrophobic association even in dilute solutions.With increasing the concentration,the maximum absorption wavelength of Soret band changes from 416 nm to 407 nm,and the fluorescence corrected for the inner filter effect exhibits moderate concentration quenching.All the results indicate thatπ-πinteraction of porphyrin pendants plays a key role in association of PAM-TrPyP,and H-aggregates of porphyrins are mainly formed in the concentrated solution.On the other hand,dynamic light scattering(DLS) and transmission electron microscopy(TEM) were used to follow the changes in size and structure of the macromolecular assemblies with the concentration increase.The polymer aggregation conformation changes from loose "vesicle-like" morphology to solid globule accordingly.When pH value of solution decreases to 4.3,pyridine moieties on porphyrin pendants could be protonated and the H-aggregates formed in macromolecular matrix are destroyed by electrostatic repulsion interactions.     

Photophysics of a series of efficient fluorescent pH probes for dual-emission-wavelength measurements in aqueous solutions

This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission.