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.

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.

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.

[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.

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.

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.

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.

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.

7-Hydroxy- N-Methylquinolinium Chromophore: A Photolabile Protecting Group for Blue-Light Uncaging.

The development of the N-methyl-7-hydroxymethylquinolinium ( N-Me-7-HQm) caging chromophore as a novel visible-light-sensitive photolabile protecting group is described. N-Me-7-HQm-caged compounds can be photoactivated by blue-light-emitting diode (LED) light (458 nm) with high photolytic efficiency, supporting applications to caging chemistry, and they also have sufficient water solubility and high resistance to spontaneous hydrolysis.

Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.

cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 µM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-µM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.

Utilization of the Heavy Atom Effect for the Development of a Photosensitive 8-Azacoumarin-Type Photolabile Protecting Group.

A remarkable improvement of the photochemical properties of coumarin-type photolabile protecting groups was achieved by iodine substitution. The newly identified 7-hydroxy-6-iodo-8-azacoumarin (8-aza-Ihc)-caged acetate showed excellent photolytic efficiency, significantly higher than that of the corresponding bromine-containing coumarin- and azacoumarin-type caging groups. The results provide a solid approach to improving the photosensitivity of photolabile protecting groups.

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.

Isostere-based design of 8-azacoumarin-type photolabile protecting groups: a hydrophilicity-increasing strategy for coumarin-4-ylmethyls.

Described is the development of 8-azacoumarin-4-ylmethyl groups as aqueous photolabile protecting groups. A key feature of the strategy is the isosteric replacement of the C7-C8 enol double bond of the Bhc derivative with an amide bond, resulting in conversion of the chromophore from coumarin to 8-azacoumarin. This strategy makes dramatically enhanced water solubility and facile photocleavage possible.

Preparation and affinity-based purification of caged linear DNA for light-controlled gene expression in mammalian cells.

A facile and useful method for preparing caged DNAs was developed. The method includes a caging reaction of a linear dsDNA having a minimal sequence of protein expression with Bio-Bhc-diazo and affinity separation of the caged DNA. Effective suppression and photo-mediated restoration of protein expression were demonstrated.

Design, synthesis, and photochemistry of modular caging groups for photoreleasable nucleotides.

A modular approach to preparing caged nucleotides having additional properties has been achieved. The modular caging agent includes three components: an amine reactive NHS ester moiety, a photoactive Bhc group, and tosylhydrazone as a precursor of the diazomethyl group. Various amines including biotin and an Arg-Gly-Asp (RGD) peptide were introduced into the key intermediate via amide linkage. The Bio-Bhc-diazo thus synthesized enables the preparation of a photoreleasable siRNA with additional properties.

Free flap blood flow evaluated using two-dimensional laser speckle flowgraphy.

Objective. We investigated the efficiency of laser speckle flowgraphy for evaluating blood flow in free flaps used for plastic surgery. Methods. We measured blood flow using a visual laser meter capable of providing two-dimensional color graphic representations of flow distribution for a given area using a dynamic laser speckle effect. Using laser speckle flowgraphy, we examined the blood flow of 20 free flaps applied following the excision of head and neck tumors. Results. After anastomosis of the feeding and draining blood vessels and sewing the flap, musculocutaneous (MC) flaps showed significantly lower blood flow than jejunal or omental flaps (P < .05). The ratio of blood flow decrease from the edge to the center was significantly greater in MC flaps than in jejunal or omental flaps (P < .001). Conclusion. Laser speckle flowgraphy is useful for the perioperative measurement of blood flow in free flaps used in plastic surgery. This method is a highly useful, practical, and reliable tool for assessing cutaneous blood flow and is expected to be applicable to several clinical fields.

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.