Identification of reaction sites and chlorinated products of purine bases and nucleosides during chlorination: a computational study.

Hypochlorous acid (HOCl) released from activated leukocytes plays a significant role in the human immune system, but is also implicated in numerous diseases due to its inappropriate production. Chlorinated nucleobases induce genetic changes that potentially enable and stimulate carcinogenesis, and thus have attracted considerable attention. However, their multiple halogenation sites pose challenges to identify them. As a good complement to experiments, quantum chemical computation was used to uncover chlorination sites and chlorinated products in this study. The results indicate that anion salt forms of all purine compounds play significant roles in chlorination except for adenosine. The kinetic reactivity order of all reaction sites in terms of the estimated apparent rate constant kobs-est (in M-1 s-1) is heterocyclic NH/N (102-107) > exocyclic NH2 (10-2-10) > heterocyclic C8 (10-5-10-1), but the order is reversed for thermodynamics. Combining kinetics and thermodynamics, the numerical simulation results show that N9 is the most reactive site for purine bases to form the main initial chlorinated product, while for purine nucleosides N1 and exocyclic N2/N6 are the most reactive sites to produce the main products controlled by kinetics and thermodynamics, respectively, and C8 is a possible site to generate the minor product. The formation mechanisms of biomarker 8-Cl- and 8-oxo-purine derivatives were also investigated. Additionally, the structure-kinetic reactivity relationship study reveals a good correlation between lg kobs-est and APT charge in all purine compounds compared to FED2 (HOMO), which proves again that the electrostatic interaction plays a key role. The results are helpful to further understand the reactivity of various reaction sites in aromatic compounds during chlorination.

Design, synthesis, and biological application of A-D-A-type boranil fluorescent dyes.

For the first time, three acceptor-donor-acceptor (A-D-A)-type boranil fluorescent dyes, CSU-BF-R (R = H, CH3, and OCH3), featuring phenothiazine as the donor, were designed and synthesized. CSU-BF-R exhibited remarkable photophysical characteristics, including large Stokes shifts (>150 nm), high fluorescence quantum yields (up to 40%), long-wavelength emissions, and strong red solid-state fluorescence. Moreover, these CSU-BF-R fluorescent dyes were demonstrated to function as highly selective and sensitive ratiometric fluorescent probes for detecting hypochlorous acid (HClO). The preliminary biological applications of CSU-BF-OCH3 for sensing intracellular HClO in living cells and zebrafish were demonstrated. Therefore, CSU-BF-R possess the potential to further explore the physiological and pathological functions associated with HClO and provide valuable insights into the design of high-performance A-D-A-type fluorescent dyes.

A turn-on fluorescent probe for detecting and bioimaging of HOCl in inflammatory and liver disease models.

Hypochlorous acid (HOCl) is a common reactive oxygen species (ROS) associated with the development of liver, tumor, inflammatory, and other diseases. In this work, the turn-on fluorescent probe named (WZ-HOCl) with a naphthalimide structure was designed and synthesized to detect endogenous HOCl in disease models. WZ-HOCl can achieve a fast response to HOCl with good linearity in the range of 0-45 µM (LOD = 147 nM). The application of WZ-HOCl in bioimaging was investigated by constructing a series of cellular disease models, and the results showed that WZ-HOCl could sensitively detect endogenous HOCl in inflammatory and liver disease models. It can also be used to differentiate between hepatocytes and hepatoma cells. WZ-HOCl will provide new methods and ideas for fluorescent probes in detecting drug-induced liver injury, alcoholic and non-alcoholic steatohepatitis, and some inflammation-related diseases.

Heterogeneous hydrochlorination of lipids mediated by fatty acids in an indoor environment.

Fatty acids from cooking fumes and hypochlorous acid (HOCl) released from indoor cleaning adversely affect respiratory health, but the molecular-level mechanism remains unclear. Here, the effect of cooking oil fumes [palmitic acid (PA), oleic acid (OA), and linoleic acid (LA)] on lung model phospholipid (POPG) hydrochlorination mediated by HOCl at the air-water interface of the hanged droplets was investigated. Interfacial hydrochlorination of POPG was impeded by OA and LA, while that of POPG was facilitated by PA. The effect on POPG hydrochlorination increased with the decrease in oil fume concentration. A potential mechanism with respect to the chain length of these oil fumes, regardless of their saturation, was proposed. PA with a short carbon chain looses the POPG packing and leads to the exposure of the C=C double bonds of POPG, whereas OA and LA with a long carbon chain hinder HOCl from reaching the C=C bonds of POPG. These results for short chain and low concentration dependence suggest that the decay of oil fumes or the conversion of short-chain species by indoor interfacial chemistry might be adverse to lung health. These results provide insights into the relationship between indoor multicomponent pollutants and the respiratory system.

A novel dehydroabietic acid-based multifunctional fluorescent probe for the detection and bioimaging of Cu2+/Zn2+/ClO.

A multifunctional dehydroabietic acid-based fluorescent probe (CPS) was designed and synthesized by introducing the 2,6-bis(1H-benzo[d]imidazol-2-yl)phenol fluorophore. The probe CPS could selectively recognize Cu2+, Zn2+ and ClO- ions from other analytes, and it showed fluorescence quenching behavior toward Cu2+ and a ratiometric response to Zn2+ and ClO- by changing from green fluorescence to blue and cyan, respectively. The detection limits toward Cu2+, Zn2+ and ClO- ions were 3.8 nM, 0.253 µM and 0.452 µM, respectively. In addition, CPS presented many fascinating merits, such as high selectivity, a short response time (15-20 s), a wide pH range (3-10) and high photostability. The sensing mechanisms of CPS were verified by 1H-NMR, ESI-MS, FT-IR and Job's plot methods. Meanwhile, CPS exhibited satisfactory detection performance in water samples. More importantly, the probe could be applied as a promising tool for visual bioimaging of three ions in living cells and zebrafishes.

Fabrication of a paper-based facile and low-cost microfluidic device and digital imaging technique for point-of-need monitoring of hypochlorite.

Lab-on-a-paper-based devices are promising alternatives to the existing arduous techniques for point-of-need monitoring. The present work reports an instant and facile method to produce a microfluidic paper-based analytical device (µPAD). The fabricated µPAD has been used to detect hypochlorite (OCl-) by incorporating newly synthesized chromo-fluorogenic ratiometric probes 1 and 2 into the sample reception zone. The probes showed high selectivity and fast response (<10 s) toward OCl- with an excellent linear relationship in the concentration range of 0-100 µM. The concentration-dependent fluorometric change driven by the reaction of 1@µPAD with OCl- has been monitored using gel-doc imaging systems, which is unprecedented. Digitizing the intensity of the colour solution with different mathematical models of colour has developed a straightforward method for monitoring OCl- without any interference from its competitors. 1@µPAD can detect OCl- at ∼10 times lower than the WHO recommended limit. The detection limit of 1@µPAD via a digital camera-based fluorescence technique was found to be better over digital camera-based cuvette assays. Therefore, 1@µPAD has been successfully utilized to monitor OCl- in actual environmental water samples with portability, ease of use, and sensitivity. The analytical RSD was found to be ≤3% based on fluorimetric detection using 1@µPAD. The chemodosimetric reaction between OCl- and the probe was evidenced by UV-vis and fluorescence spectroscopy, 1H NMR, and ESI-MS. The rapid response time, biocompatibility, low cytotoxicity, 100% aqueous solubility, ratiometric feature, and exclusive OCl- selectivity over other competitive ROS/RNS successfully lead to the application of the probes for bioimaging of exogenous as well as endogenous OCl- in normal cells (HEK293) and cancerous cells (HeLa).

A Near-Infrared Fluorescent Probe for Recognition of Hypochlorite Anions Based on Dicyanoisophorone Skeleton.

A novel near-infrared (NIR) fluorescent probe (SWJT-9) was designed and synthesized for the detection of hypochlorite anion (ClO-) using a diaminomaleonitrile group as the recognition site. SWJT-9 had large Stokes shift (237 nm) and showed an excellent NIR fluorescence response to ClO- with the color change under the visible light. It showed a low detection limit (24.7 nM), high selectivity, and rapid detection (within 2 min) for ClO-. The new detection mechanism of SWJT-9 on ClO- was confirmed by 1H NMR, MS spectrum, and the density functional theory (DFT) calculations. In addition, the probe was successfully used to detect ClO- in HeLa cells.

Point-of-Care and Dual-Response Detection of Hydrazine/Hypochlorite-Based on a Smart Hydrogel Sensor and Applications in Information Security and Bioimaging.

A novel dual-response fluorescence probe (XBT-CN) was developed by using a fluorescence priming strategy for quantitative monitoring and visualization of hydrazine (N2H4) and hypochlorite (ClO-). With the addition of N2H4/ClO-, the cleavage reaction of C=C bond initiated by N2H4/ClO- was transformed into corresponding hydrazone and aldehyde derivatives, inducing the probe XBT-CN appeared a fluorescence "off-on" response, which was verified by DFT calculation. HRMS spectra were also conducted to confirm the sensitive mechanism of XBT-CN to N2H4 and ClO-. The probe XBT-CN had an obvious fluorescence response to N2H4 and ClO-, which caused a significant color change in unprotected eyes. In addition, the detection limits of XBT-CN for N2H4 and ClO- were 27 nM and 34 nM, respectively. Interference tests showed that other competitive analytes could hardly interfere with the detection of N2H4 and ClO- in a complex environment. In order to realize the point-of-care detection of N2H4 and ClO-, an XBT-CN@hydrogel test kit combined with a portable smartphone was developed. Furthermore, the portable test kit has been applied to the detection of N2H4 and ClO- in a real-world environment and food samples, and a series of good results have been achieved. Attractively, we demonstrated that XBT-CN@hydrogel was successfully applied as an encryption ink in the field of information security. Finally, the probe can also be used to monitor and distinguish N2H4 and ClO- in living cells, exhibiting excellent biocompatibility and low cytotoxicity.

Rational Design of a Dual-Channel Fluorescent Probe for the Simultaneous Imaging of Hypochlorous Acid and Peroxynitrite in Living Organisms.

Hypochlorous acid (HOCl) and peroxynitrite (ONOO-) are two important highly reactive oxygen/nitrogen species, which commonly coexist in biosystems and play pivotal roles in many physiological and pathological processes. To investigate their function and correlations, it is urgently needed to construct chemical tools that can track the production of HOCl and ONOO- in biological systems with distinct fluorescence signals. Here, we found that the coumarin fluorescence of coumarin-benzopyrylium (CB) hydrazides (spirocyclic form) is dim, and their fluorescence properties are controlled by their benzopyran moiety via an intramolecular photo-induced electron transfer (PET) process. Based on this mechanism, we report the development of a fluorescent probe CB2-H for the simultaneous detection of HOCl and ONOO-. ONOO- can selectively oxidize the hydrazide group of CB2-H to afford the parent dye CB2 (Absmax/Emmax = 631/669 nm). In the case of HOCl, it undergoes an electrophilic attack on the benzopyran moiety of CB2-H to give a chlorinated product CB2-H-Cl, which inhibits the PET process within the probe and thus affords a turn-on fluorescence response at the coumarin channel (Absmax/Emmax = 407/468 nm). Due to the marked differences in absorption/emission wavelengths between the HOCl and ONOO- products, CB2-H enables the concurrent detection of HOCl and ONOO- at two independent channels without spectral cross-interference. CB2-H has been applied for dual-channel fluorescence imaging of endogenously produced HOCl and ONOO- in living cells and zebrafish under different stimulants. The present probe provides a useful tool for further exploring the distribution and correlation of HOCl and ONOO- in more biosystems.

Reaction Mechanisms of Histidine and Carnosine with Hypochlorous Acid Along with Chlorination Reactivity of N-Chlorinated Intermediates: A Computational Study.

Hypochlorous acid (HOCl) released from activated leukocytes not only plays a significant role in the human immune system but is also implicated in numerous diseases including atherosclerosis and some cancers due to its inappropriate production. Histidine (His) and carnosine (Car), as a respective mediator and protective agent of HOCl damage, have attracted considerable attention; however, their detailed reaction mechanisms are still unclear. In this study, using a His residue with two peptide bond groups (HisRes) as a model, the reaction mechanisms of HisRes and Car including NεH and NδH tautomers with HOCl along with the chlorination reactivity of N-chlorinated intermediates were investigated by quantum chemical methods. The obtained results indicate that in the imidazole side chain, the pyridine-like N is the most reactive site rather than the pyrrole-like N, and the kinetic order of all of the possible reaction sites in HisRes follows pyridine-like N > imidazole Cδ ≫ imidazole Cε > pyrrole-like N, while that in Car is pyridine-like N ≫ imidazole Cδ ≫ amide N. As for N-chlorinated intermediates at imidazole, although the unprotonated form has a low chlorination reactivity as expected, it can still chlorinate tyrosine. Especially, the protonated form exhibits similar ability to HOCl, causing secondary damage in vivo. N-Chlorinated Car features higher internal chlorine migration ability than its intermolecular transchlorination, preventing further HOCl-induced damage. Additionally, a generally overlooked nucleophilic Cl- shift is also found in N-chlorinated Car/HisRes, indicating that nucleophilic sites in biomolecules also need to be considered. The outcomes of this study are expected to expand our understanding of secondary damage and protective mechanisms involved in HOCl in humans.

A benzocoumarin-based fluorescent probe for ultra-sensitive and fast detection of endogenous/exogenous hypochlorous acid and its applications.

Hypochlorous acid (HOCl) is widely used in daily production and life because of its green and strongly oxidizing properties. Additionally, as a vital reactive oxygen species (ROS), it is an innate immune system weapon and performs a critical function in many pathophysiology processes. In this paper, a novel water-soluble fluorescent probe, BMH, with excellent performance is designed and synthesized by simple condensation of benzocoumarin and 2-mercaptoethanol. BMH has specific selectivity, excellent sensitivity, ultra-fast response (<3 s), and a wide pH detection range. The fluorescence intensity of BMH has an excellent linear correlation with the concentration of HOCl in the scope of 0-10 µM, and the calculated detection limit (DL) is 2.45 nM. The intramolecular charge transfer (ICT) sensing mechanism of BL has been verified by fluorescence, UV, and MS studies as well as density functional theory (DFT) calculations. Furthermore, BMH can be incorporated into a solid-state visual sensor to detect HOCl conveniently. BMH was applied to detect HOCl-spiked actual water samples and achieved satisfying recovery rates. Also, the low-toxicity BMH can be successfully used to track changes in endogenous/exogenous HOCl in living cells. In short, BL provides a robust and reliable monitoring tool to reveal the biological functions of HOCl and ensure its safe use.

A dual-channel chemodosimetric sensor for discrimination between hypochlorite and nerve-agent mimic DCP: application on human breast cancer cells.

A styryl bridge containing a triphenylamine-thioimidazole hydrazine-based dual-analyte-responsive fluorescent sensor was designed and synthesized for the detection of the nerve gas simulant diethyl chlorophosphate (DCP) and hypochlorite (OCl-) for the first time. Hypochlorite induces oxidative intramolecular cyclization to give a triazole structure, which exhibited blue fluorescence with excellent selectivity and a low detection limit (8.05 × 10-7 M) in solution. Conversely, the probe forms a phosphorylated intermediate with diethyl chlorophosphate, which undergoes further hydrolyzation and presents green fluorescence in a ratiometric mode with a low detection limit (3.56 × 10-8 M). Additionally, the as-designed sensor was utilized to construct a portable kit for real-time monitoring of DCP in a discriminatory, simple and safe manner. Lastly, the probe was also productively employed for in situ imaging of OCl- and DCP in the living cell.

Chloride Enhances DNA Reactivity with Chlorine under Conditions Relevant to Water Treatment.

Free available chlorine (FAC) is widely used to inactivate viruses by oxidizing viral components, including genomes. It is commonly assumed that hypochlorous acid (HOCl) is the chlorinating agent responsible for virus inactivation; however, recent studies have underscored that minor constituents of FAC existing in equilibrium with HOCl, such as molecular chlorine (Cl2), can influence FAC reactivity toward select organic compounds. This study measures the FAC reaction kinetics with dsDNA and ssDNA extracted from representative bacteriophages (T3 and ϕX174) in samples augmented with chloride. Herein, chloride enhances FAC reactivity toward dsDNA and, to a lesser extent, toward ssDNA, especially at pH < 7.5. The enhanced reactivity can be attributed to the formation of Cl2. Second-order rate constants were determined for reactions of ssDNA and dsDNA with HOCl and Cl2. DNA chlorination kinetics followed the reactivity-selectivity principle, where the more-reactive nucleophilic species (ssDNA, ∼100× more reactive than dsDNA) reacted less selectively with electrophilic FAC species. The addition of chloride was also shown to enhance the inactivation of bacteriophage T3 (dsDNA genome) by FAC but did not enhance the inactivation of bacteriophage ϕX174 (ssDNA genome). Overall, the results suggest that Cl2 is an important chlorinating agent of nucleic acids and viruses.

A new ratiometric switch "two-way" detects hydrazine and hypochlorite via a "dye-release" mechanism with a PBMC bioimaging study.

A new ratiometric fluorescent probe (E)-2-(benzo[d]thiazol-2-yl)-3-(8-methoxyquinolin-2-yl)acrylonitrile (HQCN) was synthesised by the perfect blending of quinoline and a 2-benzothiazoleacetonitrile unit. In a mixed aqueous solution, HQCN reacts with hydrazine (N2H4) to give a new product 2-(hydrazonomethyl)-8-methoxyquinoline along with the liberation of the 2-benzothiazoleacetonitrile moiety. In contrast, the reaction of hypochlorite ions (OCl-) with the probe gives 8-methoxyquinoline-2-carbaldehyde. In both cases, the chemodosimetric approaches of hydrazine and hypochlorite selectively occur at the olefinic carbon but give two different products with two different outputs, as observed from the fluorescence study exhibiting signals at 455 nm and 500 nm for hydrazine and hypochlorite, respectively. A UV-vis spectroscopy study also depicts a distinct change in the spectrum of HQCN in the presence of hydrazine and hypochlorite. The hydrazinolysis of HQCN exhibits a prominent chromogenic as well as ratiometric fluorescence change with a 165 nm left-shift in the fluorescence spectrum. Similarly, the probe in hand (HQCN) can selectively detect hypochlorite in a ratiometric manner with a shift of 120 nm, as observed from the fluorescence emission spectra. HQCN can detect hydrazine and OCl- as low as 2.25 × 10-8 M and 3.46 × 10-8 M, respectively, as evaluated from the fluorescence experiments again. The excited state behaviour of the probe HQCN and the chemodosimetric products with hydrazine and hypochlorite are studied by the nanosecond time-resolved fluorescence technique. Computational studies (DFT and TDDFT) with the probe and the hydrazine and hypochlorite products were also performed. The observations made in the fluorescence imaging studies with human blood cells manifest that HQCN can be employed to monitor hydrazine and OCl- in human peripheral blood mononuclear cells (PBMCs). It is indeed a rare case that the single probe HQCN is found to be successfully able to detect hydrazine and hypochlorite in PBMCs, with two different outputs.

Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator.

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.

Critical Computational Evidence Regarding the Long-Standing Controversy over the Main Electrophilic Species in Hypochlorous Acid Solution.

Although hypochlorous acid (HOCl) solution has become a popular electrophilic reagent for industrial uses, the question of which molecule (HOCl or Cl2) undergoes electrophilic addition with olefins remains a controversial issue in some literature and textbooks, and this problem has been largely underexplored in theoretical studies. In this work, we computationally studied the electrophilic addition mechanism of olefins using three experimentally predicted effective electrophilic chlorinating agents, i.e., HOCl, Cl2, and Cl2O molecules. Our results demonstrate that Cl2 and Cl2O are the main electrophilic agents in HOCl solution, whereas the HOCl molecule cannot be the electrophile since the energy barrier when directly adding HOCl molecule to olefins is too high to overcome and the "anti-Markovnikov" regioselectivity for tri-substituted olefin is not consistent with experiments. Notably, the HOCl molecule prefers to form oxonium ion intermediate with a double bond, rather than the generally believed chlorium ion intermediate. This work could benefit mechanistic studies of critical biological and chemical processes with HOCl solution and may be used to update textbooks.

High concentrations of hypochlorous acid-based disinfectant in the environment reduced the load of SARS-CoV-2 in nucleic acid amplification testing.

During the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic, chlorine-containing disinfectants have been widely used in nucleic acid amplification testing laboratories. Whether the use of disinfectants affect the results of viral nucleic acid amplification is unknown. We examined the impact of different hypochlorous acid (HOCl) concentrations on the quantitative results of SARS-CoV-2 by real-time reverse-transcription polymerase chain reaction (RT-PCR). We also explored the mechanisms and models of action of chlorine-containing disinfectants that affected the detection of SARS-CoV-2. The results showed that different HOCl concentrations and different action times had an impact on the SARS-CoV-2 results. High concentrations of ambient HOCl have a greater impact than low concentrations, and this effect will increase with the extension of the action time and with the increase in ambient humidity. Compared with the enzymes or the extracted RNA required for RT-PCR, the impact of HOCl on the SARS-CoV-2 detection is more likely to be caused by damage to primers and probes in the PCR system. The false negative result still existed after changing the ambient disinfectant to ethanol but not peracetic acid. The use of HOCl in the environment will have an unpredictable impact on the nucleic acid test results of SARS-CoV-2. In order to reduce the possibility of false negative of SARS-CoV-2 nucleic acid test and prevent the spread of epidemic disease, environmental disinfectants should be used at the beginning and end of the experiment rather than during the experimental operation.

Mechanism of cyanocobalamin chlorination by hypochlorous acid.

Hypochlorous acid (HOCl) is a strong oxidant produced by myeloperoxidase. Previous work suggested that HOCl modifies the corrin ring of cobalamins to yield chlorinated species via mechanisms that are incompletely understood. Herein, we report a mechanistic study on the reaction between cyanocobalamin (CNCbl, vitamin B12) and HOCl. Under weakly acidic, neutral and weakly alkaline conditions, the reaction produces the c-lactone derivative of CNCbl chlorinated at the C10-position of corrin ring (C10-Cl-CNCbl-c-lactone). Formation of C10-Cl-CNCbl-c-lactone was not observed at pH ≥ 9.9. The chlorination of CNCbl by HOCl proceeds via two pathways involving one and two HOCl molecules: the reaction is initiated by the very fast formation of a complex between CNCbl and HOCl, which either undergoes slow transformation to chlorinated species, or rapidly reacts with a second HOCl molecule to produce C10-Cl-CNCbl. Subsequent reaction of C10-Cl-CNCbl with HOCl proceeds rapidly toward lactone ring formation by H-atom abstraction at position C8. This work uncovered mechanisms and products of the reaction of a biologically active and therapeutically used cobalamin, CNCbl and the endogenous oxidant HOCl. Binding and reactivity studies of C10-Cl-CNCbl and C10-Cl-CNCbl-c-lactone with relevant proteins of the cobalamin pathway and with cultured cells are necessary to elucidate the potential physiological effects of these species.

A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.

Uric Acid as a Photosensitizer in the Reaction of Deoxyribonucleosides with UV Light of Wavelength Longer than 300 nm: Identification of Products from 2'-Deoxycytidine.

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.