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Here we developed a new cell-permeative lucigenin derivative, 10,10'-dimethyl-9,9'-biacridinium bis(monomethyl terephthalate) (MMT), to detect intracellular superoxide production. Both MMT and lucigenin were specific to superoxide among reactive oxygen species tested. Although lucigenin barely penetrated into cells, MMT accumulated in mitochondria in a variety of cells such as neutrophils. By employing MMT, we found that, upon activation of neutrophils with phorbol myristate acetate, superoxide was generated extracellularly as well as intramitochondrially and that such intramitochondrial superoxide production was dependent on oxidative phosphorylation. We also found that, during apoptosis, superoxide was gradually produced in mitochondria in association with phosphatidylserine exposure and that the kinetics of superoxide production was very heterogeneous at the single-cell level. Thus this study demonstrates that MMT could serve as a specific probe for intramitochondrial superoxide in either activated or apoptotic cells.

A new AB 2 type building block for synthesis of dendritic compounds is made starting from Michael addition of 2 equiv of 3-hydroxyacetophenone to acrylate, followed by decarboxylation and deprotection of either carboxyl or hydroxyl protecting groups. [G1]–[G4] dendrons and [G2] dendrimer were synthesized by the reiterative [hydrogenolysis then DCC/DPTS coupling] sequence using a combination of convergent and divergent growth methods.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Caged compounds can be used to regulate the spatial and temporal dynamics of signaling molecules in live cells. Photochemical properties of coumarin-4-ylmethoxy carbonates (1a-d) are investigated to construct caged compounds of hydroxy-containing molecules. All the compounds possess desired properties as phototriggers for alcohols and phenols. The 6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl (Bhcmoc) group has the highest photochemical efficiency and is applied to make caged compounds of 1,2-dioctanoylglycerol (diC(8)), Tyr-OMe, and adenosine. [reaction: see text]

Photolabile derivatives of bile acids ( 8 – 10 and 13 ) were synthesized via silver (I) oxide promoted selective etherification of 3α-hydroxyls. Quantitative production of the parent cholic acid was detected from the photolytic mixture of 3-NB-CA ( 8 ) in Tris buffered solution. Interestingly, the unexpectedly stable nitroso-hemiacetal intermediate ( 14 ) was detected when the photolysis was conducted in methanol. The enzymatic analysis using 7α-HSDH showed 8 and 9 could serve as caged bile acids that might be able to regulate certain biological processes upon UV irradiation.

PAMAM and DAB dendrimers with 4–64 terminal caged compounds on their surface were synthesized. They released bioactive compound (LeuLeuOMe) upon irradiation at 350 nm. The amount of LeuLeuOMe released per molecule increased with the increase in the generation of dendrimers. Sugar ball-type caged compounds showed high solubility in PBS and cluster effect of sugars.

The synthesis of two key precursors for caging bioactive compounds, an o -nitrobenzyl p -nitrophenyl carbonate and an o -nitrobenzyl diazo compound with a steroid skeleton, is reported. The former is a suitable caging group for an amino group, while the latter is appropriate for a carboxylic acid. The resulting caged compounds were irradiated at 350 nm to afford the corresponding amine and carboxylic acid. A caged compound released bioactive compounds more efficiently when embedded in liposome than when in methanol.

The effects of solvent polarity on the fluorescence spectra and fluorescence decays of β-(1-pyrenyl)ethyl p-cyanobenzoate (P2CN) were investigated in detail using binary solvents consisting of various mixing ratios of isooctane-ethyl acetate or ethyl acetate-acetonitrile (dielectric constants (e)=1.94–36.2). Whereas both the intensity and wavelength maxima of an intramolecular exciplex emission (EX) are dependent on the solvent polarity, only the intensity of an emission from the locally excited pyrene (LE) is dependent on the solvents used. When monitored at 377 nm, the picosecond SPC (single photon counting) measurements reveal a slow decay (>150 ns) in addition to a fast decay (

Two types of immunosuppressants, cycloprodigiosin hydrochloride (cPrG) and l -leucyl- l -leucine methyl ester (LeuLeuOMe), both have the ability to selectively inhibit the lysosomal function, and a related compound to cPrG, prodigiosin 25-C, and LeuLeuOMe have been reported to selectively inhibit the T cell function in vitro. We therefore examined the cell-type specificity of cPrG and LeuLeuOMe using murine splenocytes. Concanavalin A (Con A)- and lentil lectin-induced proliferation was suppressed by cPrG more profoundly than lipopolysaccharide-induced proliferation. At the optimal concentration, Con A induced the proliferation of both CD4+ and CD8+ cells, whereas at a supra-optimal concentration Con A induced rather selective proliferation of CD8+ cells. Irrespective of the dose of Con A, CD4+ and CD8+ cells were equally affected by cPrG. In contrast, LeuLeuOMe induced the selective loss of CD8+ cells. cPrG enhanced the apoptosis of murine splenocytes and nylon fiber column-purified T cells cultured in the presence of Con A, as shown by the decrease in cell size and/or DNA fragmentation. Overall, this study revealed that the cell-type specificity of cPrG is different from that of LeuLeuOMe, and that the immunosuppression by cPrG is associated with apoptosis.

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN2 (GFCHN2), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN2 or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.

Two kinds of model compounds for a bichromophoric ester, 2-(1-pyrenyl)ethyl p-cyanobenzoate (P2CN), were synthesized: 1-substituted 2-(1-pyrenyl)ethyl p-cyanobenzoates (RP2CN, R = Me, Et, and Ph) as flexible model compounds and trans-2-(1-pyrenyl)cyclohexyl p-cyanobenzoate (cyclo-P2CN) as a rigid model compound. P2CN and all the model compounds had an intramolecular exciplex emission (φEX ≈ 0.010) and efficient quenching of fluorescence from the locally excited pyrene part (φLE ≈ 0.001). The decay lifetimes of the locally excited pyrene were 365 ps for P2CN and 66 ps for cyclo-P2CN, which were respectively identical with the risetime for the exciplex formation. The 1H NMR of both P2CN and RP2CN showed the shifts of H2″ and H3″ of the p-cyanobenzoyl to the higher and H10′ of 1-pyrenyl signals to the lower magnetic field compared with those of the reference compounds, approaching towards those of cyclo-P2CN. NOE was observed between H10′ and H2″ of cyclo-P2CN, and, although very weak, between H2′ and H3″ of...

An o-nitrobenzyl and an o-nitrophenethyl derivatized 2-deoxyglucose (caged 2-deoxyglucoses) were synthesized from 3,4,6-tri-O-acetyl-D-glucal in only two steps in moderate to good yields as isomeric mixtures, which were irradiated at 350 nm to afford a 2-deoxyglucose. Decomposition of the o-nitrophenethyl derivative upon photolysis proceeded more efficiently than that of the o-nitrobenzyl derivative.

We report the synthesis of a new type of a caged L-leucyl-L-leucine methyl ester, a sugar derivative 2, and study its photochemical and immunological properties. Compared with those of the previously reportedo-nitrobenzyl caged compound, 1, and another new 4, 5-dimethoxy-2-nitorobenzyl caged compound 3, 2 was found to be almost 30 times more soluble in PBS containing 1% DMSO, and released leucyl-leucine methyl ester upon irradiation more efficiently than 1. Efficiency of induction of apoptosis of HL60 cells by irradiation of a solution containing 2 was only slightly lower than that by leucyl-leucine methyl ester itself.

Michael addition reactions of acetoacetates and malonates with acrylates in a 5 M KOH aqueous solution without using any other catalysts are described. When they are either tert-butyl or benzyl esters, the reaction proceeded very rapidly to afford moderate to high yields of the corresponding bis-Michael adducts that are unexpectedly stable under these conditions.

In this study, we newly synthesized caged compounds of L-leucyl-L-leucine methyl ester and examined their photochemical and immunological properties. From the viewpoints of solubility in phosphate-buffered saline containing 1% dimethyl sulfoxide as well as photoreactivity, we chose two caged compounds, 1 and 2, for study. Upon irradiation, L-leucyl-L-leucine methyl ester with a trans-o-hydroxycinnamoyl group (1) releases L-leucyl-L-leucine methyl ester quite slowly, whereas L-leucyl-L-leucine methyl ester with an o-nitrobenzyl group (2) releases L-leucyl-L-leucine methyl ester quickly. Both irradiated compounds induced apoptosis of U937 cells, as evidenced by a decrease in cell size, although 1 itself caused necrosis (cell swelling) of these cells.

A highly fluorescent 1-pyrenylmethyloxycarbonyl amino acid (Pmoc-amino acid) is obtained in moderate yield by the reaction of (1-pyrenylmethyl)-4-nitrophenylcarbonate with an amino acid in the presence of sodium carbonate. The condensation of Pmoc-amino acid with an amino acid gives Pmoc-peptide in the presence of 1-ethyl-3-(3-dimethylaminepropyl)carbodiimide and 1-hydroxyben-zotriazole. The amino acid is recovered from an H2O-dioxane (2:3) solution of Pmoc-amino acid by irradiation through a Pyrex filter with a medium pressure Hg lamp or at 340 nm. Although the quantum yield of the photolysis is rather low (ca 0.01), the photolysis proceeds fast and efficiently due to the large absorption coefficient of Pmoc-amino acid at around 340 nm. Thus, the use of Pmoc-amino acid as a “caged” amino acid is promising.

The formation of molecular ions, M+., under fast atom bombardment (FAB) conditions using a liquid matrix was examined by using a new type of synthesized compounds in which preferential M+. peaks appear in their FAB spectra. The FAB spectra were compared with the corresponding mass spectra obtained by the electron impact (EI) ionization, chemical ionization (CI) and charge-exchange ionization (CEI) methods. All of the spectra showed preferential peaks of M+. ion and a characteristic intense fragment ion peak originating from a β-fission. The FAB spectra were similar in the fragment ions appearing in the EI spectra and were very similar in the fragmentation pattern to the CEI spectra using Ar+. and Xe+. as the reagent ions. Further, the FAB spectra did not show any doubly charged ion peaks, while the 70 eV EI spectra showed the peaks of doubly charged molecular and/or fragment ions. The isobutane CI spectra of the synthesized compounds suggested that the formation of M+. ions occurred through the CE reaction with isobutane ion, C4H10+., and the CI spectra showed a marked intense fragment ion peak originating from the β-fission which seemed to occur characteristically in CEI processes. The results obtained suggested that the formation of M+. ions under matrix FAB conditions occurred mainly by CE reactions between the analytes M and matrix molecular ions B+. and/or fragment ions b+..

Measurements of absorption and emission spectra, fluorescence quantum yields (Φf) and lifetimes (τf) of ω-(1-pyrenyl)alkyl para-substituted benzoates PnX (X = H, Cl, CF3, CN, NO2, with n = 1–4) were carried out in solvents of various polarity. Intramolecular interaction in the ground state is not observed in any of these compounds. PnCN (n = 2–4) shows an intramolecular exciplex emission in solvents of low to high polarity. The broad, structureless emission at longer wavelength observed in ethyl acetate solution of P1CN is ascribed to an ‘exciplex-type’ emission which does not require actual overlapping of the two chromophores. P2CF3 also shows an exciplex emission in solvents of medium to high polarity. The solvent dependence of both Φf and τf increases as the electron-withdrawing ability of the para-sustituents increases. The relationship between fluorescence quenching by electron transfer and para-substituent of PnX is discussed by means of the free energy for electron transfer, ΔGET, obtained from the oxidation and reduction potentials of pyrene and methyl para-substituted benzoates in acetonitrile, respectively. PnH, with positive ΔGET, does not show a solvent dependence of Φf, except for P1H, in which ca 40% of the fluorescence is quenched in acetonitrile. PnCl, with slightly negative ΔGET, shows more efficient quenching, but does not show exciplex emission. PnCN, PnCF3 and PnNO2 have ΔGET values between −0·36 and −0·65 eV, and their fluorescence is fairly efficiently quenched. The fluorescence of PnCN is concluded to be strongly quenched by intersystem crossing from the singlet exciplex to the locally excited pyrene, and by electron transfer from the pyrene part to the benzoate part. That the formation of a singlet exciplex is necessary for intersystem crossing in bichromophoric compounds containing pyrene is thus clearly illustrated.

New photochemically labile phosphate protecting group was developed. These phosphate esters have high molar extinction coefficient (e340 = 34500 dm3 mol−1 cm−1) and rapidly release parent phosphates upon irradiation ( >300 nm) with high quantum efficiency for disappearance (φdis = 0.22 at 340 nm).

New photochemically labile phosphate protecting group was developed. These phosphate esters have high molar extinction coefficient (e340 = 34500 dm3 mol−1 cm−1) and rapidly release parent phosphates upon irradiation ( >300 nm) with high quantum efficiency for disappearance (φdis = 0.22 at 340 nm).

Esterification of phosphates with various halides, including acid chlorides, phosphoryl trichloride and tin chloride, was achieved in 57–99% isolated yield using silver(I) oxide. The reaction was successfully applied to the preparation of phosphate triesters sensitive to acids and bases. cAMP benzyl esters were also prepared by this method.

The activation of selenoglycosides is achieved by photo-induced electron transfer between aromatic compounds and the phenylselenyl group, while cleavage of the C–Se bond of the resulting radical cation generates a glycosyl cation that reacts with various alcohols to give the O-glycosides.

A 6-O-o-nitrobenzyl methylglucoside and methylmannoside were synthesized by reacting 4,6-O-o-nitrobenzylidene acetals with triethylsilane and boron trifluoride etherate. A 2,6-di-O-o-nitrobenzyl and a 3,6-di-O-o-nitrobenzyl methylmannoside were obtained from a 2,3:4,6-di-O-o-nitrobenzylidene methylmannoside by the same method. The photolabile sugars obtained were deprotected by irradiation at 350 nm to afford methylglycosides.

[see structure]. Synthesis and photochemistry of a new photochemically removable protecting group for alcohols is described. Four carbonates of galactose derivatives (1-4) were synthesized from the corresponding arylmethanols via 4-nitrophenyl carbonate intermediates. Among them, photolysis of anthraquinon-2-ylmethoxycarbonyl (Aqmoc) galactose (1) proceeded with overall photolysis efficiency of 150 (quantum yield 0.10, and molar absorptivity 1500 M(-1) x cm(-1)) and rate constant of approximately 10(6) s(-1). To demonstrate its application to a biologically related molecule, 5'-Aqmoc-adenosine (5) was synthesized and photolyzed to yield adenosine in 91% yield.

L-Leucyl-L-leucine methyl esters (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. Glucose-carrying 2-nitrobenzyl (2-NB) and 2-nitrophenethyl (2-NPE) caged LeuLeuOMe, 1a and b, were synthesized and their photochemical and immunological properties were studied. Caged glycine methyl esters (GlyOMe), 2a,b, were also prepared to examine the cytotoxic activity of the photolytic byproducts from 1a,b. All the caged compounds were soluble in PBS containing 1% DMSO more than 400 microM, and efficiently released the substrates upon irradiation at 350 nm. While both 1a and 1b were not toxic to HL60 cells, 1b released LeuLeuOMe more quickly and induced apoptosis of HL60 cells far more efficiently than 1a. Although GlyOMe was not cytotoxic, 2a and b were slightly toxic before and after irradiation almost to the similar extent. Therefore, the photolytic products from the caging groups appear to be not toxic to the cells, and the apoptosis inducing activity of 1a and b may be for the most part due to LeuLeuOMe.

The photochemical reactions of 9-benzyloxytriptycenes in methanol were found to give 2-(9-fluorenyl)benzaldehyde acetals and benzyl 2-(9-fluorenyl)phenyl ketones. Intermediacy of norcaradienes and two kinds of singlet carbenes has been demonstrated by quantum yields, light intensity dependence, competitive reactions, and substituent effects.

β-(1-Pyrenyl)ethyl p-cyanobenzoate (P2CN) which forms an emissive intramolecular exciplex has a “folded” conformation as detected by 1H-NMR NOE. Precise decay lifetime analysis also confirms that the exciplex emission is mainly from that conformer. Exciplex emission was not observed for the p-chlorobenzoate (P2Cl) which did not show NOE.

[figure: see text] A 6-O-o-nitrobenzyl methylglucoside and methylmannoside were synthesized by reacting 4,6-O-o-nitrobenzylidene acetals with triethylsilane and boron trifluoride etherate. A 2,6-di-O-o-nitrobenzyl and a 3,6-di-O-o-nitrobenzyl methylmannoside were obtained from a 2,3:4,6-di-O-o-nitrobenzylidene methylmannoside by the same method. The photolabile sugars obtained were deprotected by irradiation at 350 nm to afford methylglycosides.

Three bromopyrrole alkaloids, ageliferin (1), bromoageliferin (2), and dibromoageliferin (3), have been isolated as potent actomyosin ATPase activators from the Okinawan marine sponge Agelas sp. and the structures elucidated on the basis of spectral data, especially two dimensional NMR spectra.

Dendrimers are synthetic, highly branched and nanometer-sized macromolecules that offer potential application to various fields. They have uniform size and molecular weight, wide internal cavity and modifiable surface functionality that made them very attractive for biological and drug delivery applications. Commercially available PAMAM (polyamidoamine) dendrimers are most frequently used for construction of delivery system by modification of surface amino groups. One of the examples is anticancer drug-PAMAM conjugate with folic acid (FA) for targeting. DNA linked FA-PAMAM and FITC-PAMAM conjugates have been recently developed. Polyester dendrimers are expected to be biodegradable and less toxic to cells. An example is shown. Lastly, construction of a delivery system using caged compounds for photochemical release of drug is described.

New aliphatic polyester-type dendrimers were synthesized using a new AB2-type building block 3, prepared from benzyl acetoacetate and 2 equiv of tert-butyl acrylate by acetoacetic acid ester synthesis. The reiterative [deprotection by HCO2H, then EDCI/DMAP coupling] sequence using divergent growth method gave [G1]-4tBu-[G5]-64tBu dendrimers. 13C NMR relaxation time (T1) measurements on the carboxy carbons show that the extended chain conformations are predominant in CDCl3. [structure: see text]

L-Leucyl-L-leucine methyl esters (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. Glucose-carrying 2-nitrobenzyl (2-NB) and 2-nitrophenethyl (2-NPE) caged LeuLeuOMe, 1a and b, were synthesized and their photochemical and immunological properties were studied. Caged glycine methyl esters (GlyOMe), 2a,b, were also prepared to examine the cytotoxic activity of the photolytic byproducts from 1a,b. All the caged compounds were soluble in PBS containing 1% DMSO more than 400 microM, and efficiently released the substrates upon irradiation at 350 nm. While both 1a and 1b were not toxic to HL60 cells, 1b released LeuLeuOMe more quickly and induced apoptosis of HL60 cells far more efficiently than 1a. Although GlyOMe was not cytotoxic, 2a and b were slightly toxic before and after irradiation almost to the similar extent. Therefore, the photolytic products from the caging groups appear to be not toxic to the cells, and the apoptosis inducing activity of 1a and b may be for the most part due to LeuLeuOMe.

[see structure]. Synthesis and photochemistry of a new photochemically removable protecting group for alcohols is described. Four carbonates of galactose derivatives (1-4) were synthesized from the corresponding arylmethanols via 4-nitrophenyl carbonate intermediates. Among them, photolysis of anthraquinon-2-ylmethoxycarbonyl (Aqmoc) galactose (1) proceeded with overall photolysis efficiency of 150 (quantum yield 0.10, and molar absorptivity 1500 M(-1) x cm(-1)) and rate constant of approximately 10(6) s(-1). To demonstrate its application to a biologically related molecule, 5'-Aqmoc-adenosine (5) was synthesized and photolyzed to yield adenosine in 91% yield.

l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.

Publisher Summary This article summarizes the methods used in laboratory for the preparation and characterization of 4-coumarinylmethyl-caged phosphate esters and their application in studies of the role of cAMP on the dispersion and aggregation of pigment granules in live cells. The coumarin-caged cAMP compounds, MCM-cAMP, 4-(7-acetoxycoumarinyl) methyladenosine (ACM)-cAMP, 4-(7-propionyloxycoumarinyl) methyladenosine (PCM)-cAMP and 4-(7-hydroxycoumarinyl)methyladenosine (HCM)-cAMP cyclic 3',5'-monophosphates that differ from each other only by the substituent at the 7-position of the coumarin ring, have been prepared from reactions of the corresponding 4- coumarinylmethyl bromide with cAMP using the silver(I) oxide promoted method. These caged cAMP compounds exhibit similar photocleavage properties and efficiently release cAMP in Ringer's solution on irradiation with near-ultraviolet light. Another significant feature of these caged cAMPs is their long half-life in the dark in physiological salt solutions compared to those of o-nitrobenzyl- or onitrophenethyl-caged cAMPs, which in the experience readily hydrolyze in aqueous solution.

Two acyloxycoumarinylmethyl-caged cAMPs (ACM- and PCM-cAMP) have been synthesized using a silver (I) oxide promoted method. Introduction of the acyloxy group to the 7-position on the coumarin ring not only enhanced the membrane permeability but diminished the photolability of the coumarin-cage. Because intracellular enzymatic hydrolysis of the 7-acyloxy group would produce the 7-hydroxy moiety which is more hydrophilic and photolabile, application of acyloxycoumarinylmethyl-caged cAMPs in biological studies would be expected to be efficient. Thus, the effect of extracellularly applied ACM- and PCM-cAMP had been investigated using the motile response of fish melanophores. After irradiation, a significant enhancement in the motility responses was observed. The observed magnitudes of the dispersions are comparable to that of Bt2cAMP/AM which is known as a membrane permeable cAMP derivative.

I,-Leucyl-L-leucine methyl ester (Leu-Leu-OMe), an apoptosis inducer in natural killer cells and macro-phages, was caged with trans-o-hydroxycinnamoyl (3ad), trans-o-mercaptocinnamoyl (4) and o-nitrobenzyl derivatives (5a, b), and the photochemical reactivity of these derivatives in phosphate-buffered saline containing 1% dimethyl sulfoxide and their immunological properties were studied. All of the derivatives exhibited absorplion at wavelengths longer than the UVB region. Although 3a–d and 4 were expected to isomerize to a cis isomer, which thgn cyclizes intramolecularly to give Leu-Leu-OMe and a coumarin derivative, cyclization efficiency was not satisfactory except for 3a. However, 3a itself caused necrosis (cell swelling) of U937 cells (a myeloid cell line). In contrast, 5a and b released Leu-Leu-OMe quickly and efficiently and did not affect U937 cells. Although irradiated 5b induced necrosis, irradiated 3a and 5a induced apoptosis in these cells, as evidenced by a decrease in cell size.

Stereodivergent synthesis of both diastereoisomeric enoates, including vinylogous acids, is achieved in the kinetic resoultion of branched aldehydes and asymmetrization of prochiral cyclic ketone by chiral phosphorus reagents derived from (–)-8-phenylmenthol.

The activation of selenoglycosides is achieved by photo-induced electron transfer between aromatic compounds and the phenylselenyl group, while cleavage of the C–Se bond of the resulting radical cation generates a glycosyl cation that reacts with various alcohols to give the O-glycosides.

Esterification of phosphates with various halides, including acid chlorides, phosphoryl trichloride and tin chloride, was achieved in 57–99% isolated yield using silver(I) oxide. The reaction was successfully applied to the preparation of phosphate triesters sensitive to acids and bases. cAMP benzyl esters were also prepared by this method.

The population ratios of the syn to the anti conformers in 9-(2-substituted phenyl)fluorenes have been determined by 1H NMR spectroscopy to range from 0.25 (for 2′;-NH2) to >20 (2′;-CHNNHC6H3(NO2)-2,4). They appear to increase, firstly, with the steric bulk of the 2′;-substituents and, secondly, with the electronic repulsion between the substituent and the π electrons on the fluorene ring. The unusually low population of the syn isomer in the 2′;-NH2 compound was found by IR absorption to be due to stabilization of the anti form by NH … π hydrogen bonding. Whereas the substituents on the fluorene ring do not affect the population of the two isomers in 9-(2-tolyl)fluorenes, the same series of substituents exhibit a positive slope in the Hammett plots of the equilibrium constants in 9-(2-methoxymethylphenyl)fluorenes. Through-space interaction between the methoxymethyl group and the π-donating fluorene ring is postulated. The free energy of activation (ΔG‡) for the interconversion between the two conformers in 9-(2-substitued phenyl)fluorenes have been obtained from the line-shape depindence on temperatures. The ΔG‡ values ranged from 56.8 (for 2′-NH2) ot 78.6kJ mol−1 when there is no fluorene ring substituent. IN three typical examples, a full line-shape analysis gave, additionally, the ΔH‡ and ΔS‡ data of the interconversion. Introduction of a methyl group at position 1 of the fluorene ring raised. ΔG‡ by c. 13kJ mol−1.