High pKa Research Articles

An atomic-scale understanding of the initial stage of nucleation of heavy metal cations on clay edges

Abstract For the purpose of revealing the microscopic mechanism of the early stage in the nucleation of heavy metals on clay edges, we performed first principles molecular dynamics (FPMD) simulations systematically to characterize the chemistry of the incipient clustering processes by taking Pb2+ and Ni2+ as the model cations. We found that Pb2+ and Ni2+ have different complexation mechanisms: Pb2+ cations are adsorbed onto different sites with the similar probability whereas Ni2+ cations prefer octahedral vacancy to the other sites. The reason is that Pb2+ does not enter the vacancy due to the large ionic size. pKa calculations indicate that Ni2+ adions can provide available complexing sites (i.e., OH− groups) for the subsequent cations through hydrolysis and free energy calculations show that the complexation of subsequent cations is thermodynamically favored, which can thus result in nucleation and precipitation eventually. In contrast, the first adsorbed Pb2+ cannot provide available complexing sites due to the extremely high pKa, thus making nucleation impossible. Our findings, consistent with the experiment, reveal the role of the surface in heterogeneous nucleation, which should also apply to other similar systems. The results derived in this study are directly useful for understanding the mobility and fate of heavy metals in surficial environments.

Study of Protonation Behaviour and Distribution Ratios of Hydroxamic Acids in Hydrochloric and Perchloric Acid Solutions Through Hammett Acidity Function, Bunnett-Olsen and Excess Acidity Method

The protonation parameters, dissociation constants (pKBH+) of conjugate acid, slope values (m, φ and m*) and correlation coefficients (r) of hydroxamic acids were determined by Hammett acidity function method, Bunnett- Olsen method and excess acidity method in hydrochloric and perchloric acid solutions. Effect of acid concentration on partition and percentage protonation was also studied. pKBH+ values show that hydroxamic acids do not behave as Hammett bases, but hydroxamic acids behave as weak bases in strong acidic solutions. The values of pKBH+ obtained through Bunnett-Olsen method and excess acidity method were compared with the Hammett acidity function. ChemAxon’s MarvinSketch 6.1.5 software was also used for determining pKa, pI and microspecies distribution (%) of hydroxamic acids with pH. Hydrogen donor and acceptor values and logD were also obtained. The results show that N-p-chlorophenyl-4- bromobenzohydroxamic acid has the highest pKa and lowest logD values. On the contrary, N-phenyl-3,5-dinitrobenzohydroxamic acid has lowest the pKa and highest logD values.

Factors contributing to radiation dose for patients and operators during diagnostic cardiac angiography.

IntroductionDiagnostic coronary angiography (CA) uses ionising radiation with relatively high doses, which impact on both patients and staff. This study sought to identify which patient and procedural factors impact patient and operator dose the most during CA.MethodsPatient and procedure related variables impacting on Kerma area product (PKA) and operator dose (OD) were collected for 16 months. Procedures were separated into 10 different procedure categories. PKA was used for patient dose and OD was measured with an instantly downloadable dosimeter (IDD) – downloaded at the end of each procedure. High and low radiation dose was defined by binary variables based on the 75th percentile of the continuous measures. Univariate and multivariate regression were used to identify predictors.ResultsOf 3860 patients included, the IDD was worn for 2591 (61.7%). Obesity (BMI > 30 compared to BMI < 25) was the strongest predictor for both a PKA (odds ratio (OR) = 19.1 (95% CI 13.5–26.9) P < 0.001) and OD (OR = 3.3 (2.4–4.4) P < 0.001) above the 75th percentile. Male gender, biplane imaging, the X‐ray unit used, operator experience and procedure type also predicted a high PKA. Radial access, male gender, biplane imaging and procedure type also predicted a high OD.ConclusionRadiation dose during CA is multifactorial and is dependent on patient and procedure related variables. Many factors impact on both PKA and OD but obesity is the strongest predictor for both patients and operators to receive a high radiation dose.

Grown of highly porous ZnO-nanoparticles by pulsed laser ablation in liquid technique for sensing applications

Pulsed laser ablation technique in deionized water with low laser fluency has been explored to prepare uniform dispersed porous ZnO nanoparticles for sensing applications. Surface morphology, particle size, porous structure, roughness, elemental distribution, and chemical bonding of the synthesized ZnO are analyzed by TEM, FESEM, AFM, EDX, and FTIR spectroscopy, respectively. Sensing behavior is observed by UV–Vis absorption measurements. TEM and FESEM analysis show that the prepared ZnO-coated film has homogeneous, dispersed, highly porous, and crack-free surface; the average particle size are observed ~ 24.72 ± 2.97 nm. The porous structure is responsible for appropriate sensing behavior. Low roughness value ~ 1.52 nm which is analyzed by AFM is advantageous for sensing behavior. EDX spectrum and elements mapping clearly show the uniform Zn and O distribution. XRD analysis confirms the hexagonal wurtzite structure of ZnO. FTIR reveals the Zn and O chemical bonding successfully. UV-Visible analysis exhibits that the prepared ZnO matrix has good incorporation with multi-dyes solutions at pH values 10–12 with significant changes in color behavior. The highest pKa value ~ 9.77 at a wavelength of 598.28 nm was calculated for multi-dyes immobilized ZnO matrix. So, it can be concluded that prepared ZnO nanostructures are potential candidates for sensing application.

Anion-Induced Electron Transfer.

As counterintuitive as it might seem, in aprotic media, electron transfer (ET) from strong Lewis basic anions, particularly F-, OH-, and CN-, to certain π-acids (πA) is not only spectroscopically evident from the formation of paramagnetic πA•- radical anions and πA2- dianions, but also thermodynamically justified because these anions' highest occupied molecular orbitals (HOMOs) lie above the π-acids' lowest unoccupied molecular orbitals (LUMOs) creating negative free energy changes (Δ G°ET < 0). Depending on the relative HOMO and LUMO energies of participating anions and π-acids, respectively, the anion-induced ET (AIET) events take placeeither in the ground state or upon photosensitization of the π-acids. The mild basic and charge-diffuse anions with lower HOMO levels fail to trigger ET, but they often form charge transfer (CT) and anion-π complexes. Owing to their high HOMO levels in aprotic environments, strong Lewis basic anions, such as F- enjoy much greater ET driving force (Δ G°ET) than mild and non-basic anions, such as iodide. In protic solvents, however, the former become more solvated and stabilized and lose their electron donating ability more than the latter, creating an illusion that F- is a poor electron donor due to the high electronegativity of fluorine. However, UV-vis, EPR, and NMR studies consistently show that in aprotic environments, F- reduces essentially any π-acid with LUMO levels of -3.8 eV or less, revealing that contrary to a common perception, the electron donating ability of F- anion is not dictated by the electronegativity of fluorine atom but is a true reflection of high Lewis basicity of the anion itself. Thus, the neutral fluorine atoms with zero formal charge and F- anion have little in common when it comes to their electronic properties. The F- anion can also legitimately act as a Brønsted base when the proton source has a p Ka lower than that of its conjugate acid HF (15), not the other way around, and ET from F- to a poor electron acceptor is not thermodynamically feasible. While there is no shortage of indisputable evidence and clear-cut thermodynamic justifications for ET from F- and other Lewis basic anions to various π-acids in aprotic solvents, because of the aforesaid misconception, it had been posited that F- perhaps formed diamagnetic Meisenheimer complexes via nucleophilic attack, deprotonated an aprotic solvent DMSO against an insurmountably high p Ka (35) leading to a π-acid reduction, or formed [F-/πA•+] complexes via a thermodynamically prohibited oxidation of π-acids. Unlike AIET, however, none of these hypotheses was thermodynamically viable nor supported by any experimental evidence. First, by defining the thermodynamic criteria of AIET pathways and all other alternate hypotheses and then evaluating the spectroscopic signals emanating from the interactions between different anions and π-acids and Lewis acids in the light of these criteria, this Account comes to a conclusion that AIET is the only viable mechanism that can rationalize the reduction of π-acids without violating any thermodynamic rules. The paradigm-shifting discovery of AIET not only exposed a common misconception about the electron donating ability of F- but also enabled naked-eye detection of toxic anions, electrode-free silver plating, luminescent silver nanoparticle synthesis, light-harvesting, and conductivity enhancement of conjugated polymers, with more innovative applications still to come.

Bifunctional iminophosphorane superbases: Potent organocatalysts for enantio- and diastereoselective Michael addition reactions

Bifunctional iminophosphorane (BIMP) organocatalysts catalyze the enantio- and diastereoselective Michael additions of high pKa cyclic malonamate ester pro-nucleophiles to nitroalkenes with reactivity profiles of up to 3 orders of magnitude greater than tertiary amine bifunctional Brønsted base/(thio)urea organocatalysts. The unrivalled performance of the BIMPs allows reaction times of challenging reactions to be slashed from weeks to minutes and has enabled new flow chemistry applications using polystyrene-supported versions contained within a flow reactor.

Coumarin-based, switchable fluorescent substrates for enzymatic bacterial detection

Enzymatically-switchable fluorescent substrates, such as the commercially available 4-methyl umbelliferones (4-MU) are used as standard indicators of enzymatic activity for the detection of various microorganisms and pathogens. However, a major disadvantage of 4-MU is its relatively high pKa leading to only partial dissociation of the fluorescent anion under the conditions where the enzymes are most effective (pH 6–6.5). Here we present a method for new, enzymatically-switchable, fluorescent substrates with improved photo-physico/chemical properties. The lead derivative, 4-AAU, shows excellent solubility in aqueous media (0.81 mg/mL) when compared to 4-MU (0.16 mg/mL), significantly improved quantum yield and wider dynamic range of its fluorescence properties. The corresponding bacterial substrate β-4-AAUG showed superior selectivity in the detection of clinically relevant amounts of E. coli, Enterococcus and K. pneumonia (1 CFU). The fluorescence intensity of β-4-AAUG was almost 5 times higher than that of the standard, the detection was possible in reasonably short time (∼ 2.5 h) and with excellent sensitivity.

Voltage and pH sensing by the voltage-gated proton channel, HV1.

Voltage-gated proton channels are unique ion channels, membrane proteins that allow protons but no other ions to cross cell membranes. They are found in diverse species, from unicellular marine life to humans. In all cells, their function requires that they open and conduct current only under certain conditions, typically when the electrochemical gradient for protons is outwards. Consequently, these proteins behave like rectifiers, conducting protons out of cells. Their activity has electrical consequences and also changes the pH on both sides of the membrane. Here we summarize what is known about the way these proteins sense the membrane potential and the pH inside and outside the cell. Currently, it is hypothesized that membrane potential is sensed by permanently charged arginines (with very high pKa) within the protein, which results in parts of the protein moving to produce a conduction pathway. The mechanism of pH sensing appears to involve titratable side chains of particular amino acids. For this purpose their pKa needs to be within the operational pH range. We propose a ‘counter-charge’ model for pH sensing in which electrostatic interactions within the protein are selectively disrupted by protonation of internally or externally accessible groups.

UPLC-ESI-MS/MS method for the quantitative measurement of aliphatic diamines, trimethylamine N-oxide, and β-methylamino-l-alanine in human urine

This work describes a quantitative high-throughput analytical method for the simultaneous measurement of small aliphatic nitrogenous biomarkers, i.e., 1,6-hexamethylenediamine (HDA), isophoronediamine (IPDA), β-methylamino-l-alanine (BMAA), and trimethylamine N-oxide (TMAO), in human urine. Urinary aliphatic diamines, HDA and IPDA, are potential biomarkers of environmental exposure to their corresponding diisocyanates. Urinary BMAA forms as a result of human exposure to blue-green algae contaminated food. And, TMAO is excreted in urine due to the consumption of carnitine- and choline-rich diets. These urinary biomarkers represent classes of small aliphatic nitrogen-containing compounds (N-compounds) that have a high aqueous solubility, low logP, and/or high basic pKa. Because of the highly polar characteristics, analysis of these compounds in complex sample matrices is often challenging. We report on the development of ion-pairing chemistry based ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) method for the simultaneous measurement of these biomarkers in human urine. Chromatographic separation was optimized using heptafluorobutyric acid-(HFBA-) based mobile phase and a reversed-phase C18 column. All four analytes were baseline separated within 2.6 min with an overall run time of 5 min per sample injection. Sample preparation involved 4 h of acid hydrolysis followed by automated solid phase extraction (SPE) performed using strong cation exchange sorbent bed with 7 N ammonia solution in methanol as eluent. Limits of detection ranged from 0.05 ng/mL to 1.60 ng/mL. The inter-day and intra-day accuracy were within 10%, and reproducibility within 15%. The method is accurate, fast, and well-suited for biomonitoring studies within targeted groups, as well as larger population-based studies such as the U. S. National Health and Nutrition Examination Survey (NHANES).

Agrocin 108 is a 5'-cytidine nucleotide bacteriocin containing a carbocyclic phosphoryl-ascorbate group.

Agrocin 108 is a 3'-O-β-D-xylopyranosyl-cytidine-5'-O-phosphodiester of an ascorbate-carbocyclic cyclopentenone analogue, with bacteriocin-like properties. This bacteriocin exhibits orders of magnitude greater than the inhibition zone diameter towards the indicator strain than either ampicillin or streptomycin. It has been isolated from cultures of Rhizobium rhizogenes strain K108. The structure of the agrocin 108 without detail, has been previously published. We now report a detailed structure elucidation, including the hitherto undetermined residual 5'-phospho-diester fragment by a combination of 1D and 2D NMR studies at various pH values in H2O/D2O, high resolution MS, pKa determination, and chemical degradation.

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities.

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate-Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Complexation of heavy metal cations on clay edges at elevated temperatures

Aiming at the complexation mechanism of heavy metals on clay edges at elevated temperatures, we carried out systematic first principles molecular dynamics (FPMD) simulations at 423K by taking Cd(II) and Ni(II) as model cations. The results show that stable surface complexes include monodentate complexes on ≡SiO site, bidentate complexes on ≡Al(OH)2 site, and tetradentate complexes on octahedral vacancy on (010) edges and monodentate complexes on ≡SiO site, bidentate complexes on ≡AlOH≡AlSiO site, and tetradentate complexes on vacancy on (110) edges. Constrained FPMD simulations were conducted to calculate the desorption free energies of the complexes of the two cations on (010) surface. The results show that the octahedral vacancy has significantly higher free energy than the other sites regardless of the coordination of Al(III), indicating that the vacancy is the most favorable complexing site at elevated temperatures. The same conclusion has been deduced for (110) edges. Computed pKa values show that only the complexes on vacancy sites can hydrolyze in the common pH range, whereas those on the other sites hardly dissociate due to the extremely high pKa values, that is, the former complexes can serve as complexing sites for more metal cations. Additional simulations show that multinuclear complexes of Cd(II) and Ni(II) hold stably at 423K, which may act as precursors for epitaxial growth of new mineral phases. This study forms a physical basis for understanding the behaviors of heavy metals in various environments of elevated temperatures, and provides fundamental data for further experimental and modelling studies.

Retinal Binding to Apo-Gloeobacter Rhodopsin: The Role of pH and Retinal-Carotenoid Interaction.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pKa 3.5 and another at the higher pKa 8.4, that affect the pigment's formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal-protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate.

Synthesis of optically active bromophenol blue encapsulated mesoporous silica–titania nanomatrix: structural and sensing characteristics

Mesostructured silica-titania (ST) and bromophenol blue encapsulated silica–titania (BPB-ST) nanomatrices are synthesized by sol–gel method at low temperature. FESEM analysis shows uniform crack-free porous surface of ST matrix. EDX mapping analysis shows that the BPB species are uniformly distributed/immobilized throughout the ST matrix lattices. AFM images reveal 45.68 nm thick film with low average surface roughness ~1.27 nm, after encapsulation of BPB dye in ST matrix. TEM analysis exhibits uniform interior titania core and silica shell network, with average particle size 2.4 ± 0.4 nm for ST sample which reduced to 0.8 ± 0.3 nm after BPB encapsulation. Mesoporous high surface area 488 m2/g and pore volume of 0.55 cm3/g encapsulated matrix is found to be beneficial for sensing analysis. Transparent (67%) mesoporous nanoparticles with low refractive index 1.41 is obtained for encapsulated nanomatrix. The synthesized sensing material is fast responsive <1 sec and found to be linear for pH 3–12. High pKa value of 7.57 at 580 nm for ST-BPB leads to the broader sensitivity range, repetitive, easy-to use, and color indication system. The ability to change color in response to external stimulus makes ST-BPB a potential sensing material in food.

Influence of pKa on solvent performance of MAPA promoted tertiary amines

In the present work, absorption and desorption performance of ten single tertiary amines and their blends with MAPA was studied using a screening apparatus. Absorption experiments were conducted at 40°C and up to 9.5kPa partial pressure of CO2, while desorption experiments were conducted at 80°C and down to 1.0kPa partial pressure of CO2. In addition, the pKa value of the studied amines was measured at 25°C.The results indicated that the optimal pKa of the tertiary amines, giving the highest absorption capacity and cyclic capacity, was in the range of 9.48–10.13. At a higher pKa value, the cyclic capacity was decreased. Further, addition of MAPA to the tertiary amines influenced the absorption performance by increasing the absorption capacity for tertiary amines with low pKa (7.79–9.03) and decreasing the absorption capacity for tertiary amines with high pKa (9.48–10.42). Also, the CO2 absorption rate was significant enhanced by addition of MAPA.

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal

Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the Nη1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.

Oxetane Substrates of Human Microsomal Epoxide Hydrolase.

Oxetanyl building blocks are increasingly used in drug discovery because of the improved drug-like properties they confer on drug candidates, yet little is currently known about their biotransformation. A series of oxetane-containing analogs was studied and we provide the first direct evidence of oxetane hydrolysis by human recombinant microsomal epoxide hydrolase (mEH). Incubations with human liver fractions and hepatocytes were performed with and without inhibitors of cytochrome P450 (P450), mEH and soluble epoxide hydrolase (sEH). Reaction dependence on NADPH was investigated in subcellular fractions. A full kinetic characterization of oxetane hydrolysis is presented, in both human liver microsomes and human recombinant mEH. In human liver fractions and hepatocytes, hydrolysis by mEH was the only oxetane ring-opening metabolic route, with no contribution from sEH or from cytochrome P450-catalyzed oxidation. Minimally altering the structural elements in the immediate vicinity of the oxetane can greatly modulate the efficiency of hydrolytic ring cleavage. In particular, higher pKa in the vicinity of the oxetane and an increased distance between the oxetane ring and the benzylic nitrogen improve reaction rate, which is further enhanced by the presence of methyl groups near or on the oxetane. This work defines oxetanes as the first nonepoxide class of substrates for human mEH, which was previously known to catalyze the hydrolytic ring opening of electrophilic and potentially toxic epoxide-containing drugs, drug metabolites, and exogenous organochemicals. These findings will be of value for the development of biologically active oxetanes and may be exploited for the biocatalytic generation of enantiomerically pure oxetanes and diols.

Insight into the Mechanism of Reversible Ring-Opening of 1,3-Benzoxazine with Thiols.

The reversible ring-opening addition and fragmentation reaction of p-cresol-based N-phenylbenzoxazine with aliphatic and aromatic thiols was investigated in solvent-mediated and solvent-free reactions. Independently of the used thiol, N-phenylbenzoxazine and the thiols reacted to equilibrium with comparable amounts of reactants and products in aprotic solvent, whereas in protic solvent almost full conversions were reached. In contrast, thiol reactivity was a crucial factor in solvent-free reactions yielding fast and complete conversions for a more acidic thiol and balanced equilibrium concentrations in case of thiols with high pKa values. The strong influence of thiols with low pKa values emphasizes the relevance of the protonation step in the ring-opening reactions of 1,3-benzoxazines with thiols in absence of solvents where acidity predominates nucleophilicity. The reverse reactions, namely adduct dissociation and benzoxazine recovery, were successfully conducted at elevated temperatures and reduced pressure facilitated by the removal of the formed thiols yielding up to 95% recovered 1,3-benzoxazine. These results provide deeper understanding of the reversible ring-opening reaction mechanism of 1,3-benzoxazine with thiols.

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.