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.

Extending the Palette of Green Coumarin Photocages - Oligonucleotide Fragmentation and Superior 5'-Caps.

The possibilities of dicyanocoumarin (DCCM)-modified oligonucleotides are expanded to not just allow their release and therefore activation with green light (OFF→ON) but to also now offer a solution for their fragmentation after exposure to green light (ON→OFF). Furthermore, an answer to the decreasing uncaging quantum yields often faced when working with red-shifted photocages is given and showed that rigidified DCCM 5'-caps outperform their predecessors. Those two new 5'-caps with ATTO 390 motif or julolidine core are compatible with copper(I)-catalyzed alkyne-azide cycloadditions (CuAACs) and therefore suitable for efficient caging through cyclization or more bioconjugation reactions. Due to their planarization, they even experience an additional red-shift which is important for their use in biological applications.

Synthesis of a photocleavable bola-phosphatidylcholine.

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Template-Assisted Assembly of Hybrid DNA/RNA Nanostructures Using Branched Oligodeoxy- and Oligoribonucleotides.

A template-assisted assembly approach to a C24 fullerene-like double-stranded DNA polyhedral shell is proposed. The assembly employed a supramolecular oligonucleotide dendrimer as a 3D template that was obtained via the hybridization of siRNA strands and a single-stranded DNA oligonucleotide joined to three- or four-way branched junctions. A four-way branched oligonucleotide building block (a starlet) was designed for the assembly of the shell composed of three identical self-complementary DNA single strands and a single RNA strand for hybridization to the DNA oligonucleotides of the template. To prevent premature auto-hybridization of the self-complementary oligonucleotides in the starlet, a photolabile protecting group was introduced via the N3-substituted thymidine phosphoramidite. Cleavable linkers such as a disulfide linkage, RNase A sensitive triribonucleotides, and di- and trideoxynucleotides were incorporated into the starlet and template at specific points to guide the post-assembly disconnection of the shell from the template, and enzymatic disassembly of the template and the shell in biological media. At the same time, siRNA strands were modified with 2'-OMe ribonucleotides and phosphorothioate groups in certain positions to stabilize toward enzymatic digestion. We report herein a solid-phase synthesis of branched oligodeoxy and oligoribonucleotide building blocks for the DNA/RNA dendritic template and the branched DNA starlet for a template-assisted construction of a C24 fullerene-like DNA shell after initial molecular modeling, followed by the assembly of the shell around the DNA-coated RNA dendritic template, and visualization of the resulting nanostructure by transmission electron microscopy.

Sunscreen-Based Photocages for Topical Drugs: A Photophysical and Photochemical Study of A Diclofenac-Avobenzone Dyad.

Photosensitization by drugs is a problem of increasing importance in modern life. This phenomenon occurs when a chemical substance in the skin is exposed to sunlight. Photosensitizing drugs are reported to cause severe skin dermatitis, and indeed, it is generally advised to avoid sunbathing and to apply sunscreen. In this context, the nonsteroidal anti-inflammatory drug (NSAID) diclofenac is a photosensitive drug, especially when administered in topical form. In this work, efforts have been made to design and study an innovative pro-drug/pro-filter system containing diclofenac and the UVA filter avobenzone in order to develop a safer use of this topical drug. The design is based on the presence of a well-established photoremovable phenacyl group in the avobenzone structure. Steady-state photolysis of the dyad in hydrogen-donor solvents, monitored by UV-Vis spectrophotometry and HPLC, confirms the simultaneous photorelease of diclofenac and avobenzone. Laser flash photolysis and phosphorescence emission experiments allow us to gain insight into the photoactive triplet excited-state properties of the dyad. Finally, it is shown that avobenzone provides partial photoprotection to diclofenac from photocyclization to carbazole derivatives.

Chemical synthesis of long RNAs with terminal 5'-phosphate groups.

Long structured RNAs are useful biochemical and biological tools. They are usually prepared enzymatically, but this precludes their site-specific modification with functional groups for chemical biology studies. One solution is to perform solid-phase synthesis of multiple RNAs loaded with 5'-terminal phosphate groups, so that RNAs can be concatenated using template ligation reactions. However, there are currently no readily available reagents suitable for the incorporation of the phosphate group into long RNAs by solid-phase synthesis. Here we describe an easy-to-prepare phosphoramidite reagent suitable for the chemical introduction of 5'-terminal phosphate groups into long RNAs. The phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide by-products. The phosphate is unmasked quantitatively by brief UV irradiation. We demonstrate the value of this reagent in the preparation of a library of long structured RNAs that are site-specifically modified with functional groups.

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity.

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.

Development of photo-controllable hydrogen sulfide donor applicable in live cells.

Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

Mitochondrion-targeting and in situ photocontrolled protein delivery via photocages.

Defects in mitochondrial proteostasis contribute to many disorders, including cancer, neurodegeneration, and metabolic and genetic diseases. A strategy aimed at restoring the damaged mitochondrial proteostasis is the mitochondrion-targeting and carrier-free delivery of exogenous functional proteins that can replace the endogenous dysfunctional proteins. The modification of a protein with a photolabile protecting group (PPG, i.e., photocage group) can be activated in situ by response to illumination, leading to release of the protein from its photocage. Here, the Cys and peptide photocages with coumarin were first prepared and characterized for proof of concept. Then, we designed a pair of photocage groups PPG-RhB and PPG-TPP using coumarin and mitochondrion-targeting Rhodamine B (RhB) and triphenylphosphine (TPP), and another pair of organelle-nontarget photocage groups Br-PPG and NO2-PPG for comparison. The proteins modified with these two pairs of photocage groups undergo photolysis in solutions, and can penetrate cell membrane toward their destinations in the carrier-free fashions. The intracellular protein photocages are in situ activated by illumination at 405 nm, and the proteins are released from their photocages in mitochondria and cytoplasm, respectively. This strategy of light-responsive and carrier-free cellular delivery enables mitochondrial and cytoplasmic accumulation of exogenous proteins.

[Novel Visible Light Photoactivatable Caged Neurotransmitters Based on a N-Methyl Quinolinium Chromophore].

The development of novel photolabile protecting groups with practical levels of photolytic efficiency and hydrophilicity can provide smart photochemical tools, such as caged compounds. One of the long-standing problems of most reported photolabile protecting groups is the requirement for one-photon activation, of ultraviolet light (250-400 nm), that is harmful to living cells and has low tissue penetration power. An attractive approach to overcome this would be the use of longer-wavelength light for one-photon activation; advantages would include both lower phototoxicity and higher tissue penetration power than UV irradiation. As part of our research aimed at developing new photochemical tools, we have developed the N-methyl-7-hydroxyquinolinium (N-Me-7-HQm) caging chromophore as a novel photocage, sensitive to visible light. A key to the success of the development of the N-Me-7-HQm photocage was simple N-methylation of the 7-hydroxyquinoline chromophore. This modification allows access to visible light absorbance, facile photoactivation by blue-LED light (458 nm) with high photolytic efficiency, excellent water solubility, and high resistance to spontaneous hydrolysis. The success of the late stage upgrading of a chromophore in the synthetic sequence suggests that further functionalization of the caging chromophore will be possible, and should aid in the rapid generation of structurally diverse libraries of visible light-sensitive photocages.