Effect of Two-Photon Excitation to 8-Azacoumarin Derivatives as Photolabile Protecting Groups.

An improvement of the two-photon excitation was achieved using 8-azacoumarin-type caged compounds, which showed large values of the two-photon uncaging action cross-section (δu >0.1 Goeppert-Mayer (GM)). In particular, the 7-hydroxy-6-iodo-8-azacoumarin (8-aza-Ihc)-caged compound showed an excellent uncaging action cross-section value (δu = 1.28 GM). Therefore, 8-azacoumarin-type photolabile protecting groups (PPGs) can be used as two-photon excitation sources.

Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.

The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell-mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

Synthesis of hydrophilic caged DAG-lactones for chemical biology applications.

The 6-bromo-7-hydroxy-coumarin-4-ylmethyl (Bhc) group has been used widely in cage chemistry because of its high molar absorptivity and photolytic efficiency. One of the drawbacks of coumarins however is their low aqueous solubility. Aqueous solubility is important in the behavior of caged compounds because hydrophobic caged compounds might be aggregated in physiological conditions and consequently the photocleavage would be impaired. The 8-azacoumarin-4-ylmethyl derivatives with bromine (8-aza-Bhc) or iodine (8-aza-Ihc), which were previously developed in this laboratory, have aqueous solubilities that are higher than those of related coumarins. Here, to improve the hydrophilicity and management of caged diacylglycerol lactones (DAG-lactones), 8-aza-Bhc and 8-aza-Ihc were introduced into the DAG-lactone structure. The synthesized caged compounds showed high hydrophilicity compared with the parent Bhc-caged DAG-lactone, and the 8-aza-Ihc-caged DAG-lactone (2) showed excellent photolytic efficiency, which allows rapid release of the DAG-lactone (1) by brief photoirradiation. The 8-aza-7-hydroxy-6-iodo-coumarin-4-ylmethyl group might be useful for caging of bioactive compounds, especially hydrophobic compounds.

A double bond-conjugated dimethylnitrobenzene-type photolabile nitric oxide donor with improved two-photon cross section.

Photocontrollable NO donors enable precise spatiotemporal release of NO under physiological conditions. We designed and synthesized a novel dimethylnitrobenzene-type NO donor, Flu-DNB-DB, which contains a carbon-carbon double bond in place of the amide bond of previously reported Flu-DNB. Flu-DNB-DB releases NO in response to one-photon activation in the blue wavelength region, and shows a greatly increased two-photon cross-section (δu) at 720 nm (Flu-DNB: 0.12 GM, Flu-DNB-DB: 0.98 GM). We show that Flu-DNB-DB enables precisely controlled intracellular release of NO in response to 950 nm pulse laser irradiation for as little as 1s. This near-infrared-light-controllable NO source should be a valuable tool for studies on the biological roles of NO.

Synthesis of nucleobase-caged peptide nucleic acids having improved photochemical properties.

A nucleobase-caged peptide nucleic acid (PNA) having a (6-bromo-7-methoxycoumarin)-4-ylmethoxycarbonyl (Bmcmoc) caging group was newly synthesized. The Bmcmoc-caged PNAs were photolyzed to produce parent PNAs with a high photochemical efficiency. Introduction of a single Bmcmoc group was sufficient to suppress polymerase chain reaction (PCR) clamping activity and triplex invasion complex formation. Photo-mediated restoration of the PCR clamping activity was also demonstrated.

Liquid chromatography-mass spectrometry analyses of polyprenyl phosphates in Escherichia coli cells in which genes for isoprenoid synthesis are amplified.

Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E. coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.

[Design and Synthesis of Gene-directed Caged Compounds toward Photopharmacology].

Photoresponsive molecules can be used to manipulate the physiological functions of cells with high spatiotemporal resolution. Caged compounds are photoresponsive molecules designed to temporarily mask their original biological activity through covalently bound photoremovable protecting groups. The introduction of additional properties into caged compounds without compromising photosensitivity would help expand the repertoire of caging groups. Therefore, we designed a modular caged compound consisting of three parts: a photoresponsive core, a chemical handle to introduce additional functionalities, and a molecule to be masked. We designed two modular precursors, NHS-Bhc-diazo, for caged phosphates and paBhcmoc-X, for caged alcohols and amines. NHS-Bhc-diazo was successfully applied to the synthesis of affinity-purifiable caged dsDNA with biotin tags. The modular precursor paBhcmoc-X was used to prepare a new water-soluble caged anticancer agent with improved photochemical properties. One of the barriers to the in vivo use of conventional caged compounds is that the caged compounds are not genetically encoded and cannot target the cells of interest. To overcome these limitations, we demonstrated the concept of gene-directed caged compounds that can be photoactivated with cell-type selectivity. We designed and synthesized new caged cyclic nucleotides as proof of concept. Photo-mediated release of the parent nucleotide was observed only in live mammalian cells expressing Escherichia coli ß-galactosidase. As cells or tissues can be genetically tagged by an exogenously expressed enzyme, this new method can serve as a strategy for adding targeting abilities to photocaged compounds.

Elucidation of the working principle of a gene-directed caged HDAC inhibitor with cell-type selectivity.

Histone deacetylases (HDACs) play crucial roles in the epigenetic regulation of gene expression. Here, we report CM-Bhc-SAHA, a novel caged HDAC inhibitor, genetically targeting cells of interest. Mammalian cells expressing porcine liver esterase led to the optochemical inhibition of endogenous HDAC activity when treated with CM-Bhc-SAHA and irradiated with 405 nm light.

Synthetic caged DAG-lactones for photochemically controlled activation of protein kinase C.

Switching on kinases: Synthetic caged DAG-lactones have been developed and showed decreases of two orders of magnitude, relative to the corresponding parent compounds, in their binding affinities towards PKC. The caged compounds had no effect on the translocation of PKC until after photoactivation. This approach is a potentially powerful tool for probing the PKC signaling cascade.

Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity.

We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.

Chemo-optogenetic Protein Translocation System Using a Photoactivatable Self-Localizing Ligand.

Manipulating subcellular protein localization using light is a powerful approach for controlling signaling processes with high spatiotemporal precision. The most widely used strategy for this is based on light-induced protein heterodimerization. The use of small synthetic molecules that can control the localization of target proteins in response to light without the need for a second protein has several advantages. However, such methods have not been well established. Herein, we present a chemo-optogenetic approach for controlling protein localization using a photoactivatable self-localizing ligand (paSL). We developed a paSL that can recruit tag-fused proteins of interest from the cytoplasm to the plasma membrane within seconds upon light illumination. This paSL-induced protein translocation (paSLIPT) is reversible and enables the spatiotemporal control of signaling processes in living cells, even in a local region. paSLIPT can also be used to implement simultaneous optical stimulation and multiplexed imaging of molecular processes in a single cell, offering an attractive and novel chemo-optogenetic platform for interrogating and engineering dynamic cellular functions.

Photocontrolled compound release system using caged antimicrobial peptide.

A novel photocontrolled compound release system using liposomes and a caged antimicrobial peptide was developed. The caged antimicrobial peptide was activated by UV irradiation, resulting in the formation of pores on the liposome surface to release the contained fluorophores. The compound release could be observed using fluorescence measurements and time-lapse fluorescence microscopy. UV irradiation resulted in a quick release of the inclusion compounds (within 1 min in most cases) under simulated physiological conditions. The proposed system is expected to be applicable in a wide range of fields from cell biology to clinical sciences.

Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm.

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.

Multiple bond-conjugated photoinduced nitric oxide releaser working with two-photon excitation.

Four novel nitric oxide (NO) releasers working via two-photon excitation (TPE), based on an acceptor-donor-acceptor (A-D-A) molecular design, were synthesized. Their decomposition and NO release in response to one-photon excitation, and their decomposition in response to two-photon excitation were examined. Their photoinduced decomposition characteristics are discussed.

Photoinduced nitric oxide release from a hindered nitrobenzene derivative by two-photon excitation.

Here, we demonstrated photoinduced NO generation from a 2,6-dimethylnitrobenzene-based compound (Flu-DNB) via a two-photon excitation (TPE) process. After pulse laser irradiation to a solution of Flu-DNB, oxidation products of NO were observed. This is the first account of a non-nitrosyl-chelated metal ion containing NO donor which can be controlled by the TPE technique.

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds.

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A clickable caging group as a new platform for modular caged compounds with improved photochemical properties.

A 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) caged compound having a click-modifiable chemical handle was designed and synthesized. This molecule was applied to the synthesis of modular caged paclitaxels (PTXs) in which additional functional units could be easily installed. This system was used to prepare water-soluble caged PTXs with improved photolysis efficiencies.

Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede.

A coral fluorescent protein from Trachyphyllia geoffroyi, Kaede, possesses a tripeptide of His62-Tyr63-Gly64, which forms a chromophore with green fluorescence. This chromophore's fluorescence turns red following UV light irradiation. We have previously shown that such photoconversion is achieved by a formal beta-elimination reaction, which results in a cleavage of the peptide bond found between the amide nitrogen and the alpha-carbon at His62. However, the stereochemical arrangement of the chromophore and the precise structural basis for this reaction mechanism previously remained unknown. Here, we report the crystal structures of the green and red form of Kaede at 1.4 A and 1.6 A resolutions, respectively. Our structures depict the cleaved peptide bond in the red form. The chromophore conformations both in the green and red forms are similar, except a well-defined water molecule in the proximity of the His62 imidazole ring in the green form. We propose a molecular mechanism for green-to-red photoconversion, which is assisted by the water molecule.

A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity.

A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptides and proteins with high photolytic efficiency, which have many potential applications in the study and engineering of biological systems.

Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins.

Rapid and efficient light-induced fluorescence enhancement is demonstrated on a DMNPB-"caged" coumarin derivative carrying a His-tag recognition motif.