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We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) from 6-(benzo[ d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[ d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi-network peptide (SDYQRL) as the Golgi-apparatus-targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon-excited fluorescence at 462 and 560 nm, respectively, with effective two-photon-action cross-section values of 1860 and 1600 × 10

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca

Amyloid-β (Aβ) tracers have made a significant contribution to the treatment of Alzheimer's disease (AD) by allowing a definitive diagnosis in living patients. Unfortunately, they also detect tau and other protein aggregates that compromise test accuracy. In AD research, there has been a growing need for in vivo Aβ imaging by two-photon microscopy, which enables deep-brain-fluorescence imaging. There is no suitable neuritic Aβ probe for two-photon microscopy. Here we report PyrPeg, a novel two-photon fluorescent probe that can selectively target insoluble Aβ rather than tau and α-synuclein aggregates in the AD model brain and postmortem brain. When injected intravenously, PyrPeg detects the neuritic plaques in the brain and olfactory bulb of the AD model. PyrPeg may serve as a useful blood-brain-barrier-penetrating diagnostic tool for optical and functional monitoring of insoluble forms of Aβ aggregates in the living AD brain.

We report blue- and green-emitting two-photon probes derived from naphthalene and fluorene derivatives (as fluorophores) and an endoplasmic reticulum (ER) retrieval peptide (KDEL; as an ER-targeting moiety) that can detect the ER in a live cell by both one-photon and two-photon microscopy (TPM) and in a live tissue by TPM.

Ketene-forming elimination from C4H3(O)CH2C(O)OC6H3-2-X-4-NO2 (1) promoted by R2NH in MeCN has been studied. The reactions produced elimination products and exhibited second-order kinetics with Bronsted β = 0.51, and |βlg| = 0.47–0.53, indicating that the reaction proceeds by the E2 mechanism via an E2-central transition state. Comparison of β, |βlg|, ΔH≠, and ΔS≠ values for R2NH-promoted eliminations from ArCH2C(O)OC6H3-2-X-4-NO2 reveals that the transition-state structures for Ar = furyl and thienyl are similar and more symmetrical than that for Ar = Ph. This outcome has been attributed to the greater double bond stabilizing ability of the former than that of the latter.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10–50cm4s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/3) solution. The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the TPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger Pacid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating...

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10–50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90–130 μm in live mouse tissue by TPM.

Herein we report 22 acedan-derived, two-photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two-photon excitation with single wavelength, and to design a turn-on, two-photon fluorescence sensor for endogenous H2 O2 in Raw 264.7 macrophage and rat brain hippocampus ex vivo.

Elimination reactions of (E)-2,4,6-(NO2 ) 3C6H2CHNOC(O)C6H4X (3) promoted by R2NH/R2NH2 + in 70 mol% MeCN (aq) have been studied. The reactions produced elimination products and exhibited second-order kinetics. The β and |βlg| values remained nearly the same for all leaving groups and bases. The results can be described by the negligible pxy interaction coefficient, pxy = ∂β/∂pK lg = ∂βlg/∂pK BH ≈ 0, which provides a strong support for the (E1cb)irr mechanism. For eliminations from (E)-ArCHNOC(O)C6H4X (1, 3) and (E)-2,4,6-(NO2)3C6H2CHNOAr′ (2, 3), the change of the β-aryl group (Ar) from 2,4-dinitrophenyl (1) to 2,4,6-trinitrophenyl (3) increased the rate by 270-fold without appreciable change in the transition state structure. On the other hand, the leaving group (OAr′) variation from benzoate (3) to 4-nitrophenoxy (2) induced a change in reaction mechanism from (E1cb)irr to E2. These results have been attributed to the cyclic transition state for the nitrile-forming eliminations involving the benzoate leaving group.

Nucleophilic substitution reactions of 5-XC4H2 (S)C(O)OC6H3 -2-Y-4-NO2 (1) promoted by 4-Z-C6H4O −/4-Z-C6H4OH in 20 mol % dimethyl sulfoxide (DMSO)(aq) have been studied kinetically. The reactions exhibited second-order kinetics with βacyl = −2.52 to −2.83, ρ(x) = 2.81–3.16, βnuc = 0.88–0.04 and βlg = −0.94, respectively. The results have been interpreted with an addition–elimination mechanism in which the nucleophilic attack occurs in the rate-determining step. Comparison with existing data reveals that the rate-determining step changes from the second to the first step by the change in the nucleophile from R2NH/R2NH2 + to 4-Z-C6H4O −/4-Z-C6H4OH.

A new visible-light-induced photocatalyst based on several transition metals (iron, magnesium and manganese)-loaded TiO2 was evaluated for its anti-viral activity with influenza virus H1N1. Under a fluorescent lamp of 1000 lx, λ > 410 nm, the virus was eradicated to more than 99% within 30 min. Since this photocatalyst can be used for coating plastics, wall papers and walls, it would be desirable to use this photocatalyst to reduce viral transmission via droplets and aerosols as well as surface contact for disinfection.

A two-photon probe for near-membrane zinc ions that can be excited by 800 nm femtosecond laser pulses and emit at 500 nm is reported. The probe can be easily loaded into cells and tissue slices to visualize near-membrane zinc ions in live cells and tissues for a long period of time through the use of two-photon microscopy.

the effect of the β-aryl group on the ketene-formingelimination was assessed.Key Words : Elimination, E2 and E1cb Mechanism, β–Aryl Group effectIntroductionExtensive studies of the structure-reactivity relationshipsin the ketene-forming elimination reactions have led to thequalitative understanding of the relationship between thereactant structure and the mechanism.

Copper ions play a crucial role in living systems as cofactors of numerous metalloenzymes. To quantitatively estimate the Cu2+ concentration in human tissue, we have developed a two-photon (TP) probe with an internal reference (ACCu2) that shows significant TP action cross-section and high selectivity for Cu2+ and can quantitatively estimate the Cu2+ concentration in human colon tissues by dual-color two-photon microscopy (TPM) imaging with minimum interference from other competing metal ions or pH and minimum cytotoxicity and photostability problems. The Cu2+ concentrations in human normal colon, polyp, and colon cancer tissues were found to be 8.3 ± 0.3, 13 ± 2, and 22 ± 3 μM, respectively. This result suggests that ACCu2 may be useful for the diagnosis of human colon cancer.

Two-photon microscopy (TPM) is a new imaging tool that can detect biological targets deep inside a live tissue. To faciltate the use of TPM in biomedical research, a variety of two-photon (TP) probes for specific applications are needed. In this Forum Article, we describe the design strategy, photophysical properties, and biological imaging applications of a selection of our recent studies in the development of TP probes for metal ions. Small-molecule TP turn-on probes, organelle-targeted probes, and multicolor emissive probes for dual-color imaging are briefly reviewed.

The feed value of sesame meal, a main agricultural by-product from the northern area of Gyeongsangbuk-do province, was assessed in Hanwoo steers. A total of twelve Hanwoo steers (average BW = 412.08 kg) were allocated into a Control (fed no sesame meal) or Treatment (fed sesame meal from 14.6 months of age) group, considering BW and ages, and the animals were fed for a total of 480 days. The final BW, total gain and ADG for the Control and Treatment groups were 741.17 kg and 762.67 kg, 331.84 kg and 347.84 kg, and 0.69 kg and 0.72 kg, respectively, showing better performances in sesame meal-fed animals. Feed requirements during the whole experimental period improved by feeding sesame meal (15.31 and 14.87 for Control and Treatment group, respectively). Hanwoo steers fed sesame meal also showed a 22.83 kg heavier cold carcass weight (424.17 kg and 447.00 kg in the Control and Treatment group, respectively), and 2.41% improved dressing percentages (58.60% and 57.22% for the Control and Treatment group, respectively). The area of the M. longissimus dorsi was 91.83 cm 2 and 88.33 cm 2 in the Control and Treatment group, respectively. The final yield of A and B grade meat were 50 : 50% and 33.3 : 66.7% from the Control and Treatment group, respectively. The average marbling score was 4.83 and 5.50, and the numeric values for quality grade was 2.17 and 2.67 for the Control and Treatment group, respectively, showing better beef quality in sesame meal-fed Hanwoo steers. In fact, there were two animals with beef quality 1 ++ in the sesame meal-fed group. The melting point of lipid extracted from the M. longissimus dorsi of Hanwoo steers was significantly (p

Hydrogen sulfide (H2S) is a newly recognized transmitter, which protects various organs from oxidative stress. In this article, we report a ratiometric two-photon probe, TFCA, which can be excited by 750 nm femtosecond pulses, shows a 110-fold increase in the intensity ratio upon reaction with HS(-) and high selectivity for HS(-) and can visualize the total sulfide ([H2S] + [HS(-)]) distribution in live tissue by two-photon microscopy (TPM). We also developed a kinetic method to quantitatively estimate the total sulfide concentration ([H2S] + [HS(-)]) in live tissues. The kinetic method allowed us to measure the observed rate constants (kobs) for the sulfide-induced deazidation reaction of TFCA in live cells and tissues using TPM. The total sulfide concentration was calculated by using kobs = k2[HS(-)], with the k2 value determined in HEPES/EtOH (1/1, pH = 7.2), and [H2S]/[HS(-)] = [H(+)]/Ka. The total sulfide concentration was found to be nearly zero in HeLa cells and 4-7 μM in rat colon tissues.

value increased with a stronger electron-withdrawing 5-thienyl substituent, the Hammett plots arelinear except for X = MeO, and Yukawa-Tsuno plots are linear with ρ = 0.79-1.32 and r = 0.28-0.93,respectively. The ρ value increased and r value decreased with a stronger nucleophile, indicating an increase inthe electron density at the C=O bond and a decrease in the resonance demand. These results have beeninterpreted with enhanced N-C bond formation in the transition state with the reactivity increase. Key Words : Aminolysis, Bronsted-type plot, Hammett plot, Yukawa-Tsuno plot IntroductionExtensive studies of the structure-reactivity relationshipsin the acyl transfer reactions from XC

Hydrogen sulfide (H2S) is a multifunctional signaling molecule that exerts neuroprotective effects in oxidative stress. In this article, we report a mitochondria-localized two-photon probe, SHS-M2, that can be excited by 750 nm femtosecond pulses and employed for ratiometric detection of H2S in live astrocytes and living brain slices using two-photon microscopy (TPM). SHS-M2 shows bright two-photon-excited fluorescence and a marked change in emission color from blue to yellow in response to H2S, low cytotoxicity, easy loading, and minimum interference from other biologically relevant species including reactive sulfur, oxygen, and nitrogen species, thereby allowing quantitative analysis of H2S levels. Molecular TPM imaging with SHS-M2 in astrocytes revealed that there is a correlation between the ratiometric analysis and expression levels of cystathionine β-synthase (CBS), the major enzyme that catalyzes H2S production. In studies involving DJ-1, a Parkinson's disease (PD) gene, attenuated H2S production in comparison with wild-type controls was observed in DJ-1-knockout astrocytes and brain slices, where CBS expression was decreased. These findings demonstrate that reduced H2S levels in astrocytes may contribute to the development of PD and that SHS-M2 may be useful as a marker to detect a risk of neurodegenerative diseases, including PD.

Elimination reactions of (E)-2,4-(NO2)2C6H2CH=NOC6H3-2-X-4-NO2 (1a-e) promoted by R3N/R3NH + in 70 mol % MeCN(aq) have been studied kinetically. The reactions are second-order and exhibit Bronsted β = 0.800.84 and |βlg| = 0.39-0.42, respectively. For all leaving groups and bases employed in this study, the β and |βlg| values remained almost the same. The results can be described by a negligible pxy interaction coefficient, pxy = ∂β/pKlg = ∂βlg/pKBH ≈ 0, which describes the interaction between the base catalyst and the leaving group. The negligible pxy interaction coefficient is consistent with the (E1cb)irr mechanism. Change of the base-solvent system from R3N/MeCN to R3N/R3NH + -70 mol % MeCN(aq) changed the reaction mechanism from E2 to (E1cb)irr. Noteworthy was the relative insensitivity of the transition state structure to the reaction mechanism change.

Mitochondria provide the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species. In mitochondria, metal ions and glutathione play vital roles in maintaining their structure and the redox environment. To understand their roles in mitochondria, it is crucial to monitor each of these chemical species in the mitochondria at the cell, tissue, and organism levels. An ideal tool for such purpose is the use of two-photon microscopy (TPM). Until recently, however, there has been no report on the two-photon (TP) probes suitable for such applications. In this paper, we summarize the mitochondria-targeted TP probes for Zn2+, H2O2, and thiols, as well as their bioimaging applications.

The transition state was shifted toward theE1cb corner by an electron-withdrawing β-aryl substituentand toward the E1 intermediate and the product by astronger base in the reaction coordinate diagram. On theother hand, when the leaving group was changed from 2,4-dinitrophenoxide to picrate, the ρ value decreased but the βvalue increased. The results are in conflict with the predic-tion of the reaction coordinate diagram because both ρ and βvalues should be decreased with a better leaving group. Theunusual changes in the transition-state structure noted withthe leaving group variation have been interpreted as result-ing from a large difference in the anion-stabilizing ability ofthe leaving groups. However, the validity of this interpreta-tion remains to be verified. To determine whether a change in mechanism could berealized by introducing an even more electron-withdrawingβ-aryl substituent, we have now studied the reactions of ( E)-2,4-dinitrobenzaldehyde O-aryloxime with R

We report two-photon probes (FMg1 and FMg2) that can selectively detect intracellular free Mg(2+) ([Mg(2+)](i)) in live cells and tissues by two-photon microscopy. Combined with BCaM, a two-photon probe for near-membrane Ca(2+) ([Ca(2+)](m)), FMg2 allows dual-color imaging of Mg(2+)/Ca(2+) activities in live cells and [Mg(2+)](i) /[Ca(2+)](m) distributions in live tissues at a depth of 100-200 μm.

Two-photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two-photon (TP) probes for specific applications are needed. In this article, we report a small-molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68-fold fluorescence enhancement in response to NO, and a maximum TP-action cross-section of 170 GM (GM: 10(-50) cm(4) photon(-1)) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real-time monitor NO in living tissues at 100-180 μm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.

pH values go live! A ratiometric two-photon (TP) probe (NP1, see scheme) that has a significant TP action cross-section, high photostability, negligible toxicity, and can estimate pH values in live cells and human tissues by two-photon microscopy is described. NP1 can detect the difference in pH between live cells from the gastroesophageal junction (GEJ) and the lower esophageal sphincter of patients with and without esophagitis.