Specific detection of intramitochondrial superoxide produced by either cell activation or apoptosis by employing a newly developed cell-permeative lucigenin derivative, 10,10'-dimethyl-9,9'-biacridinium bis(monomethyl terephthalate).

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.

[Dendritic systems for drug delivery applications].

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.

Synthesis and characterization of polyester dendrimers from acetoacetate and acrylate.

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]

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 (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.

Coumarin-4-ylmethoxycarbonyls as phototriggers for alcohols and phenols.

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]

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.

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.