Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site‐specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate‐caged siRNA and the single‐stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg²⁺. Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate‐caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate‐caged siRNA or single‐stranded caged RNA prefers to form π–π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.     

Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations.     

Synthesis and photolytic evaluation of a nitroindoline-caged glycine with a side chain of high negative charge for use in neuroscience

A photolabile precursor of the neuroinhibitory amino acid glycine has been synthesised with two phosphate groups attached to the indoline nucleus at a 4-alkoxy substituent. In common with the photochemical properties of other 1-acyl-7-nitroindolines, this releases glycine on a sub-μs time scale upon irradiation with near-UV light. The synthetic route previously developed for the preparation of the GABA analogue required some modifications because of the greater hydrolytic sensitivity of the glycine compound. The phosphorylation method used here could be beneficial to the synthesis of other nitroindoline-caged amino acids, especially the related caged GABA derivative. Glycine released by laser photolysis on spinal cord neurons generated fast-rising responses and the pharmacological properties of the reagent are such that it is useful for physiological experiments.     

A Coumarin Triflate Reagent Enables One-Step Synthesis of Photo-Caged Lipid Metabolites for Studying Cell Signaling

Photorelease of caged compounds is among the most powerful experimental approaches for studying cellular functions on fast timescales. However, its full potential has yet to be exploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, a straightforward, one-step procedure for efficiently synthesizing caged compounds was developed. An in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating various caged lipid metabolites, including a number of GPCR ligands. By combining the photo-caged ligands with the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells was developed. Ultimately, the described synthetic strategy allows rapid generation of photo-caged small molecules and thus greatly facilitates the analysis of their biological roles in live cell microscopy assays.     

Synthesis and evaluation of novel caged DNA alkylating agents bearing 3,4-epoxypiperidine structure

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.     

Design,Synthesis and Evaluation of Novel Carbazole-Derived Photocages

We report the design, synthesis and evaluation of two novel photocages, NCARB and isoNCARB, belonging to the o-nitrobenzyl chemotype and based on the carbazole ring system. The synthesis of each of these isomeric caging molecules was achieved in five steps and in 29 % overall yield, and their photochemical properties were evaluated using benzoic acid as a model for caging. In the event, upon irradiation at 400 nm for 60 min, 82 % and 42 % of benzoic acid was freed from the NCARB and isoNCARB photocages, respectively, whereas only 22 % was released from the nitrodibenzofuran (NDBF) cage. Moreover, the photochemical decaging efficiencies, ϵΦ, of the benzoates photocaged with NCARB and isoNCARB are about 150- and 20-fold better, respectively, at 400 nm than the corresponding caged benzoate derived from NDBF. The water solubility of molecules caged with nitrocarbazole analogs was improved by N-alkylation of NCARB, the better of the two new photocages, with an aminodicarboxylate group. This modified cage, NCARB-DA, was exploited in the design of a caged fluoroquinolone antibiotic, the efficacy of which was illustrated in a bacterial growth inhibition assay, and a phenol-caged tyrosine derivative.     

Kinetics study of the photocleavage of (coumarin-4-yl)methyl esters

Photolabile coumarinylmethyl esters of biomolecules (caged compounds) are new tools for studying spatial and time-dependent aspects of signal transduction in living cells. Herein we describe a fluoresence spectroscopic method for the determination of the rate constants of the photolysis steps of such caged compounds using (6.7-dimethoxycoumarin-4-yl)methyl diethyl phosphate (DMCM-DEP) and sodium (6,7-dimethoxycoumarin-4-yl)methyl sulfate (DMCM-S). DMCM-DEP and DMCM-S are caged compounds which photorelease a proton, the corresponding acid anion, and the strongly fluorescent alcohol DMCM-OH upon excitation. The results of stationary and time-resolved measurements of the photochemistry and the luminescence of both caged compounds indicate that DMCM-OH is produced already during the excitation pulse. The quantitative analysis of the data demonstrates that the first step of the reaction--heterolytic bond cleavage of the coumarinylmethyl ester leading to the ion pair of a DMCM cation and an acid anion--is very fast with a rate constant of k1 approximately 2 x 10(10) s(-1). Recombination of the ion pair occurs with a rate constant of k(rec) approximately 2.3 x 10(9) s(-1) and is about 10 times faster than the competing hydrolysis reaction of the DMCM cation yielding DMCM-OH and a proton. Thus, both caged compounds belong to the fastest phototriggers known.     

Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

In situ quantification of the conjugation efficiency of azide‐terminated synthetic polymers/imaging probes and thiol‐functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self‐sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide‐ and thiol‐functionalized precursors led to highly fluorescent bioconjugates, whereas single‐click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn‐on of C1 , whereas prominent changes in FRET ratios occurred for antibody–imaging‐probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.     

A light-activated probe of intracellular protein kinase activity

The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points.     

硅倍半氧烷改性聚酰亚胺(PI/Si)复合膜性能简

通过在聚酰亚胺(PI)中分别添加笼型八氨基苯基硅倍半氧烷(OAPS)、笼型八苯基硅倍半氧烷(OPS)、梯形聚苯基硅倍半氧烷(PPSQ)和无机纳米SiO2,制备了4种含硅聚酰亚胺(PI/Si)复合膜. 对PI/Si复合膜的相容性、力学性能、热性能和阻燃性能进行了研究. 结果表明,OAPS与PI间展现出较好的相容性,PPSQ次之,而OPS,SiO2与PI的相容性较差;但相容性与复合膜的力学和热性能无明显的对应关系. SiO2可提高PI的力学性能;PI/OAPS复合膜的Tg最高;OAPS,PPSQ或SiO2的加入使PI复合膜的热稳定性稍有提高,而少量OPS的加入大大降低PI膜的热稳定性. 这类PI/Si复合膜的显著特点是能够大幅提高PI膜的极限氧指数,含硅化合物能够增加PI燃烧后残炭量,使残炭的形貌得到显著改善. PI/Si复合膜在燃烧过程中在表面形成一层白色含硅包裹层,起到隔热隔氧及保护内层有机物不被燃烧的作用. 硅倍半氧烷对炭层形貌的改善显著,展现出比SiO2更好的阻燃性能.     

Photolabile Protecting Groups for Nucleosides: Mechanistic Studies of the 2‐(2‐Nitrophenyl)ethyl Group

The photochemistry of several 2‐(2‐nitrophenyl)ethyl‐caged compounds including caged thymidine nucleosides was studied by nanosecond laser flash photolysis and stationary illumination experiments with quantitative HPLC analysis for quantum yields and product distribution. Effects of solvent basicity and acidity were investigated by varying the H₂O content and HCl concentration, respectively, in MeCN/H₂O mixtures. For all compounds 1 – 7 investigated, intramolecular H abstraction by the nitro group from the exocyclic α‐position with respect to the aryl moiety was found to be the primary process. The protolytic dissociation equilibrium of the resulting aci‐nitro compound was kinetically characterized in the 0.1 – 10 μs time region. In general, two reaction channels compete for the aci‐nitro compound and its anion: β‐elimination of the caged compound occurs from the anion, while from the undissociated aci‐nitro compound, a nitrosobenzene derivative is formed with no release of the caged compound. The yield ratio of these two reaction channels can be controlled through shifts in the protolytic dissociation equilibrium of the aci‐nitro compound. In solutions with either low basicity (H₂O‐free MeCN) or high acidity (higher concentration of HCl in H₂O/MeCN), two as yet unidentified products are formed, each one specifically for one of the mentioned conditions.     

Design of Photocaged Puromycin for Nascent Polypeptide Release and Spatiotemporal Monitoring of Translation

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC‐puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome‐bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC‐puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2 % and the NVOC‐puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two‐photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons.     

Synthesis and evaluation of caged Garcinia xanthones

Inspired by the combination of unique structure and potent bioactivities exhibited by several family members of the caged Garcinia xanthones, we developed a synthesis of simplified analogues that maintain the overall caged motif. The caged structure of these compounds was constructed via a site-selective Claisen/Diels-Alder reaction cascade. We found that the fully substituted caged structure, in which are included the C18 and C23 geminal methyl groups, is necessary to maintain bioactivity. Analogue had comparable activity to the natural products of this family, such as gambogic acid. These compounds exhibit cytotoxicity in a variety of tumor cell lines at low micromolar concentrations and were found to induce apoptosis in HUVE cells. In addition, studies with HL-60 and HL-60/ADR cells indicate that these compounds are not affected by the mechanisms of multidrug resistance, conferred by P glycoprotein expression, typical of relapsed cancers and thus represent a new and potent pharmacophore.     

硅倍半氧烷改性聚酰亚胺(PI/Si)复合膜性能

通过在聚酰亚胺(PI)中分别添加笼型八氨基苯基硅倍半氧烷(OAPS)、 笼型八苯基硅倍半氧烷(OPS)、 梯形聚苯基硅倍半氧烷(PPSQ)和无机纳米SiO₂, 制备了4种含硅聚酰亚胺(PI/Si)复合膜. 对PI/Si复合膜的相容性、 力学性能、 热性能和阻燃性能进行了研究. 结果表明, OAPS与PI间展现出较好的相容性, PPSQ次之, 而OPS, SiO₂与PI的相容性较差; 但相容性与复合膜的力学和热性能无明显的对应关系. SiO₂可提高PI的力学性能; PI/OAPS复合膜的T_g最高; OAPS, PPSQ或SiO₂的加入使PI复合膜的热稳定性稍有提高, 而少量OPS的加入大大降低PI膜的热稳定性. 这类PI/Si复合膜的显著特点是能够大幅提高PI膜的极限氧指数, 含硅化合物能够增加PI燃烧后残炭量, 使残炭的形貌得到显著改善. PI/Si复合膜在燃烧过程中在表面形成一层白色含硅包裹层, 起到隔热隔氧及保护内层有机物不被燃烧的作用. 硅倍半氧烷对炭层形貌的改善显著, 展现出比SiO₂更好的阻燃性能.     

A Telomerase‐Responsive DNA Icosahedron for Precise Delivery of Platinum Nanodrugs to Cisplatin‐Resistant Cancer

A telomerase‐responsive DNA icosahedron was designed to precisely release caged platinum nanodrugs into cisplatin‐resistance tumor cells for effective therapy. This DNA icosahedron was constructed from two pyramidal DNA cages connected with telomerase primers and telomeric repeats, and platinum nanodrugs were then encapsulated into the DNA structure. In the presence of telomerase, the primers are extended, leading to inner‐chain substitution of the DNA icosahedron and subsequent release of the caged nanodrugs. This DNA icosahedron can precisely release caged nanodrugs in response to telomerase in tumor cells, giving enhanced anticancer efficacy in drug‐resistant carcinoma and with reduced toxicity to normal tissues. We speculate that this precisely designed, well controlled DNA cage could be generalized to diverse anticancer drugs.     

Arraying heterotypic single cells on photoactivatable cell-culturing substrates

This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH 2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH 2 substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH 2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH 2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH 2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.     

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.     

Catalytic subunit of protein kinase A caged at the activating phosphothreonine

Caged reagents are photoactivatable molecules with applications in biological research. While a great deal of work has been carried out on small caged molecules, less has been done on caged macromolecules, such as proteins. Caged proteins would be especially useful in signal transduction research. Since most proteins involved in cell signaling are regulated by phosphorylation, a means to cage phosphorylated proteins would be generally applicable. Here we show that the catalytic subunit of protein kinase A can be activated by thiophosphorylation at Thr-197. The modified protein can then be caged with 4-hydroxyphenacyl bromide to yield a derivative with a specific catalytic activity that is reduced by approximately 17-fold. Upon photolysis at near UV wavelengths, an approximately 15-fold increase in activity is observed, representing an approximately 85-90% yield of uncaged product with a quantum yield phi(P) = 0.21. Because protein kinases belong to a superfamily with structurally related catalytic domains, the protein chemistry demonstrated here should be applicable to a wide range of signaling proteins.     

Most stable conformation of the cyclopropane ring attached at a carbon atom in a 1,2-dicarba-closo-dodecaborane(12) system

We obtained two crystal structures of electronically interesting dicarba-closo-dodecaborane(12)s (hereafter, "carboranes") substituted with a cyclopropyl group at a caged carbon atom, i.e., C-cyclopropyl-o-carborane (4) and C-cyclopropyl-o-carboranylphenylmethanol (9), at 123 K. In these C-cyclopropyl-o-carboranes, the cyclopropyl group adopted a slightly twisted perpendicular conformation with respect to the electron-deficient carbon-carbon (C-C) bond axis in the o-carborane cage. In contrast, it has previously been shown that a phenyl group substituted at the caged carbon atom, i.e., C-phenyl-o-carborane (3), is almost parallel to this axis at both 150 and 199 K. In other words, the pi system of the phenyl ring adopted an almost bisected conformation in 3. The preferred conformation of the cyclopropane ring in these C-cyclopropyl-o-carboranes was compared among the solid, the solution, and the gaseous states and was retained under the present conditions. Moreover, we found that the C-C bond length in the o-carborane cage not only varied along the rotation of the cyclopropyl group in 4 but also was longer than that in 3, which bears a phenyl group at the dominant conformation. These phenomena may be related to homoconjugations between the caged o-carborane system that serves as an electron acceptor and the homoconjugative substituent that serves as an electron donor. In these C-cyclopropyl-o-carboranes, two types of homoconjugations would result in a slightly twisted perpendicular conformation and 4 would be more strongly stabilized than would the phenyl derivative 3. On the basis of these studies, we propose the existence of a third type of strongly stabilizing interactive geometry for a cyclopropane ring in an o-carborane system.     

New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4‐dinitrobenzenesulfinate (DNBS) derivative of 9‐[1‐(4‐tert‐butyl‐2‐methoxyphenyl)]‐6‐hydroxy‐3H‐xanthen‐3‐one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS‐GG by biological thiols at near‐neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence‐decay time. The new dual probe was tested in solution by using steady‐state and time‐resolved fluorescence and intracellularly by using fluorescence‐lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.