Michiko Iwamura

Ageliferins, potent actomyosin Atpase activators from the Okinawan marine sponge Agelas sp

Abstract 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.

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

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.

Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

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 (Φε) 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 (δu) of up to 2.28 GM (1 GM=10−50 cm4 s photon−1), which is the largest value among those of the reported Bhc‐caged compounds. The wavelength dependence of the δu 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 μM), 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.

Induction of apoptosis of activated murine splenic T cells by cycloprodigiosin hydrochloride, a novel immunosuppressant

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.

1-Pyrenylmethyl esters, photolabile protecting groups for carboxylic acids

Abstract 1-Diazomethylpyrenes were prepared and reacted with carboxylic acids to give 1-pyrenylmethyl esters. The fluorescent esters were photolysed at 340 nm in methanol to form the starting acids and the corresponding 1-methoxymethylpyrenes in high yield.

Direct esterification of phosphates with various halides and its application to synthesis of cAMP alkyl triesters

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.

(1-PYRENYL)METHYL CARBAMATES FOR FLUORESCENT CAGED AMINO ACIDS AND PEPTIDES

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.

Design, synthesis, photochemical properties and cytotoxic activities of water-Soluble caged l-Leucyl-l-leucine methyl esters that control apoptosis of immune cells

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.

Design, synthesis and photochemical properties of caged bile acids

Photolabile derivatives of bile acids (8-10 and 13) were synthesized via silver (I) oxide promoted selective etherification of 3alpha-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 7alpha-HSDH showed 8 and 9 could serve as caged bile acids that might be able to regulate certain biological processes upon UV irradiation.

Synthesis, Photoreactivity and Cytotoxic Activity of Caged Compounds of L‐Leucyl‐L‐Leucine Methyl Ester, an Apoptosis Inducer

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.