Naoya Ieda

A Reductant-Resistant and Metal-Free Fluorescent Probe for Nitroxyl Applicable to Living Cells

Nitroxyl (HNO) is a one-electron reduced and protonated derivative of nitric oxide (NO) and has characteristic biological and pharmacological effects distinct from those of NO. However, studies of its biosynthesis and activities are restricted by the lack of versatile HNO detection methods applicable to living cells. Here, we report the first metal-free and reductant-resistant HNO imaging probe available for use in living cells, P-Rhod. It consists of a rhodol derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent rhodol derivative. P-Rhod showed high selectivity for HNO in the presence of various biologically relevant reductants, such as glutathione and ascorbate, in comparison with previous HNO probes. We show that P-Rhod can detect not only HNO enzymatically generated in the horseradish peroxidase-hydroxylamine system in vitro but also intracellular HNO release from Angelis salt in living cells. These results suggest that P-Rhod is suitable for detection of HNO in living cells.

Photomanipulation of Vasodilation with a Blue-Light-Controllable Nitric Oxide Releaser

Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluorogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.

Photocontrollable Peroxynitrite Generator Based on N-Methyl-N-nitrosoaminophenol for Cellular Application

We designed and synthesized a photocontrollable peroxynitrite (ONOO(-)) generator, P-NAP, which has N-methyl-N-nitrosoaminophenol structure with four methyl groups introduced onto the benzene ring to block reaction of the photodecomposition product with ONOO(-) and to lower the semiquinoneimines redox potential. The semiquinoneimine intermediate generated by photoinduced release of nitric oxide (NO) reduces dissolved molecular oxygen to generate superoxide radical anion (O(2)(•-)), which reacts with NO to afford ONOO(-) under diffusion control (k = 6.7 × 10(9) M(-1) s(-1)). NO release from P-NAP under UV-A (330-380 nm) irradiation was confirmed by ESR spin trapping. Tyrosine nitration, characteristic of ONOO(-), was demonstrated by HPLC analysis of a photoirradiated aqueous solution of P-NAP and N-acetyl-l-tyrosine ethyl ester. ONOO(-) formation was confirmed with a ONOO(-)-specific fluorogenic probe, HKGreen-3, and compared with that from 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), which is the most widely used ONOO(-) generator at present. The photoreaction of P-NAP was influenced by superoxide dismutase, indicating that generation of O(2)(•-) occurs before ONOO(-) formation. The quantum yield for formation of duroquinone, the main P-NAP photodecomposition product, was measured as 0.86 ± 0.07 at 334 nm with a potassium ferrioxalate actinometer. Generation of ONOO(-) from P-NAP in HCT-116 cells upon photoirradiation was successfully imaged with HKGreen-3A. This is the first example of a photocontrollable ONOO(-) donor applicable to cultured cells.

Piloty's acid derivative with improved nitroxyl-releasing characteristics.

Recent studies have shown that nitroxyl (HNO) ((1)HNO/(3)NO(-)), which is the one-electron-reduced form of nitric oxide (NO), has unique biological activities, especially in the cardiovascular system, and HNO-releasing agents may have therapeutic potential. Since few HNO donors are available for use under physiological conditions, we synthesized and evaluated a series of Pilotys acid (PA) derivatives and evaluated their HNO-releasing activity under physiological conditions. N-Hydroxy-2-nitrobenzenesulfonamide (17) was the most efficient HNO donor among our synthesized PA derivatives, including the lead compound, 2-bromo-N-hydroxybenzenesulfonamide (2). The high HNO-releasing activity is suggested to be due to electronic and steric effects. Compound 17 may be a useful tool for biological experiments.

Development of photo-controllable hydrogen sulfide donor applicable in live cells

Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

Visible light-induced nitric oxide release from a novel nitrobenzene derivative cross-conjugated with a coumarin fluorophore

Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to be potentially useful. However, most of the conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylnitrobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic visible wavelength range (400-430 nm).

Fine Spatiotemporal Control of Nitric Oxide Release by Infrared Pulse-Laser Irradiation of a Photolabile Donor

Two-photon-excitation release of nitric oxide (NO) from our recently synthesized photolabile NO donor, Flu-DNB, was confirmed to allow fine spatial and temporal control of NO release at the subcellular level in vitro. We then evaluated in vivo applications. Femtosecond near-infrared pulse laser irradiation of predefined regions of interest in living mouse brain treated with Flu-DNB induced NO-release-dependent, transient vasodilation specifically at the irradiated site. Photoirradiation in the absence of Flu-DNB had no effect. Further, NO release from Flu-DNB by pulse laser irradiation was shown to cause chemoattraction of microglial processes to the irradiated area in living mouse brain. To our knowledge, this is the first demonstration of induction of biological responses in vitro and in vivo by means of precisely controlled, two-photon-mediated release of NO.

Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics

Nitric oxide (NO) is a physiological signaling molecule, whose biological production is precisely regulated at the subcellular level. Here, we describe the design, synthesis, and evaluation of novel mitochondria-targeted NO releasers, Rol-DNB-mor and Rol-DNB-pyr, that are photocontrollable not only in the UV wavelength range but also in the biologically favorable visible wavelength range (530-590 nm). These caged NO compounds consist of a hindered nitrobenzene as the NO-releasing moiety and a rhodamine chromophore. Their NO-release properties were characterized by an electron spin resonance (ESR) spin trapping method and fluorometric analysis using NO probes, and their mitochondrial localization in live cells was confirmed by costaining. Furthermore, we demonstrated visible light control of mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1) by means of precisely controlled NO delivery into mitochondria of cultured HEK293 cells, utilizing Rol-DNB-pyr.

Peroxynitrite generation from a NO-releasing nitrobenzene derivative in response to photoirradiation.

Photocontrollable ONOO(-) generation from a nitrobenzene derivative was demonstrated. The designed compound released NO in response to photoirradiation, and the resulting semiquinone reduced molecular oxygen to generate O(2)˙(-); reaction of the two generated ONOO(-), as confirmed with an ONOO(-) fluorescent probe, HKGreen-3.

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.