One- and Two-Photon Uncaging of Carbon Monoxide (CO) with Real-Time Monitoring: On-Demand Carbazole-Based Dual CO-Releasing Platform to Test over Single and Combinatorial Approaches for the Efficient Regression of Orthotopic Murine Melanoma In Vivo.

Herein, we report three new metal-free, photochemically active single, dual, and combinatorial CORMs (photoCORMs) based on a carbazole-fused 1,3-dioxol-2-one moiety which released one equivalent of CO, two equivalent of CO, and a combination of one equivalent of each CO and anticancer drug upon one- and two-photon excitation, respectively. The photoCORMs exhibited good cellular uptake and real-time monitoring ability of CO uncaging by a color change approach in cancerous B16F10 cells. Interestingly, the cytotoxicity assay on B16F10 cells indicated that the dual photoCORM has increased anticancer activity over the single and combinatorial photoCORMs upon irradiation. Our results also showed that CO could accelerate the effectiveness of the well-known anticancer drug (chlorambucil). Finally, the in vivo evaluation of the dual photoCORM on an established murine melanoma tumor (C57BL/6J mouse model) manifested a significant regression of tumor volume and led to significant improvement (>50%) in the overall survivability.

N,N'-bicarbazole-benzothiadiazole-based conjugated porous organic polymer for reactive oxygen species generation in live cells.

A π-conjugated porous organic polymer (BCzBz) was fabricated employing N,N'-bicarbazole and benzothiadiazole as molecular building units exhibiting broad visible light absorption. The photostable, water-dispersible, and cytocompatible BCzBz was demonstrated as an efficient probe for intracellular reactive oxygen species generation under photoirradiation.

Ionic Carbazole-Based Water-Soluble Two-Photon Photoinitiator and the Fabrication of Biocompatible 3D Hydrogel Scaffold.

Two-photon polymerization of a three-dimensional (3D) hydrogel structure has been widely applied in biological tissue engineering. For improving the biocompatibility of hydrogel structures, a new kind of ionic carbazole water-soluble photoinitiator was prepared to realize the fabrication of a 3D hydrogel structure in aqueous phase. 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC) and cucurbit[7]uril (CB7) have been employed to generate a complex with better water solubility by host-guest interactions. The binding ratio of the complex was demonstrated to be 1:1 through the characterization of isothermal titration calorimetry (ITC). The two-photon absorption (TPA) cross section of the complex increases to 2500 GM compared with the 750 GM of the BMVMC molecule. Then, an aqueous-phase photoresist was obtained using the CB7/BMVMC complex as the photoinitiator and poly(ethylene glycol) diacrylate (PEGda) as the hydrogel monomer. Two-photon fabrication capability in aqueous phase has been studied using the as-prepared photoresist. A low laser threshold of 3.7 mW as well as a high resolution of 180 nm are achieved. Benefiting from the fluorescence properties of the photoinitiator, we can achieve the confocal fluorescence images without any assistance of fluorescent probes. Subsequently, a 3D engineered hydrogel scaffold microstructure was fabricated by the two-photon polymerization technology, whose biocompatibility was demonstrated by culturing the structure with living cells of L929. The BMVMC-CB7 complex and the as-prepared photoresist are demonstrated to have good biocompatibility, which is prospective for further application in tissue engineering.

Reversible photo-cross-linking of the GCN4 peptide containing 3-cyanovinylcarbazole amino acid to double-stranded DNA.

Interaction analysis in vivo greatly promotes the analyses and understanding of biological functions. The interaction between DNA and peptides or proteins is very important in terms of readout and amplifying information from genomic DNA. In this study, we designed and synthesized a photo-cross-linkable amino acid, l-3-cyanovinlycarbazole amino acid (l-CNVA), to double-stranded DNA. Reversible photo-cross-linking between DNA and peptides containing CNVA, having 3-cyanovinylcarbazole moieties capable of photo-cross-linking to nucleic acids, was demonstrated. As a result, it was shown that the GCN4 peptide, containing CNVA, can be photo-cross-linked to DNA, and its adduct was photo-split into the original peptide and DNA with 312 nm-irradiation. This is the first report that reversibly manipulates photo-crosslinking between double stranded DNA and peptides. In addition, this reversible photo-cross-linking, using l-CNVA, is faster and with higher yield than that using diazirine and psoralen.

Sensitive Discrimination of Nerve Agent and Sulfur Mustard Simulants Using Fluorescent Coassembled Nanofibers with Förster Resonance Energy Transfer-Enhanced Photostability and Emission.

In this work, highly sensitive discrimination of nerve agent and sulfur mustard simulants is achieved by using photostable and fluorescent coassembled nanofibers from molecules 1 and 2. We demonstrate that the introduction of 2 as a Förster resonance energy transfer (FRET) acceptor not only enhances the photostability and emission efficiency compared to individual 1 nanofibers but also induces different binding interactions between analytes and 1-2 coassembled nanofibers and thereby distinct fluorescence quenching behaviors used for the discrimination of nerve agent and sulfur mustard simulants. Our findings represent an important advance toward sensitive detection and discrimination of chemical warfare agents (CWAs).

Implications of tryptophan photoproduct FICZ in oxidative stress and terminal differentiation of keratinocytes.

Ultraviolet B (UVB) irradiation activates aryl hydrocarbon receptor (AHR), generates reactive oxygen species (ROS) and mediates photocarcinogenesis and photoaging. 6-Formylindolo[3,2-b]carbazole (FICZ) is a tryptophan photoproduct generated by UVB exposure. FICZ exhibits similar biological effects to UVB, including AHR ligation and ROS production. FICZ also acts as a potent photosensitizer for UVA and the production of ROS is synergistically augmented in the simultaneous presence of FICZ and UVA. In contrast, FICZ upregulates the expression of terminal differentiation molecules such as filaggrin and loricrin via AHR. In parallel with this, the administration of FICZ inhibits skin inflammation in a murine psoriasis and dermatitis model. In this article, we summarize the harmful and beneficial aspects of FICZ in skin pathology.

NIR two-photon fluorescent probe for biothiol detection and imaging of living cells in vivo.

A fluorescence probe, Cz-BDP-NBD, for detecting biothiols with two photon excited fluorescence has been designed and used under irradiation from sapphire pulsed lasers at 800 nm. Upon addition of biothiols, NBD was removed through a SNAr reaction and the fluorophore Carbazyl BODIPY (Cz-BDP) was released simultaneously. A two photon excited fluorescence was turned on and accompanied by a strong red emission at 660 nm, the two-photon absorption (TPA) cross section of the Cz-BDP reached a value of 198.5 GM at 800 nm. Moreover, Hcy/Cys and GSH could be distinguished between by a two emission channel, which resulted from the different mechanisms. For Hcy and Cys, N-NBD-Hcy and N-NBD-Cys were formed through a 5/6-membered cyclic transition, they displayed distinct fluorescent turn-on signal changes at 540 nm (Φ = 0.28, Φ = 0.24) and 670 nm, while GSH only has a single emission channel at 670 nm. Therefore, the Cz-BDP-NBD can distinguish GSH, Hcy and Cys with a fast response time (within one minute), high selectivity and sensitivity. In addition, it has been successfully applied for the detection of Cys/Hcy and GSH in living cells and ratio imaging. The Cz-BDP-NBD probe could be used for discriminative sensing of intracellular Cys, Hcy and GSH, and has the potential for use in biological applications.

An endogenous tryptophan photo-product, FICZ, is potentially involved in photo-aging by reducing TGF-ß-regulated collagen homeostasis.

BACKGROUND: Persistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-ß transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-ß signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. OBJECTIVE: To evaluate the effects of FICZ on the TGF-ß-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-ß. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-ß-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. RESULTS: We found that FICZ significantly inhibited the TGF-ß-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-ß. The inhibitory actions of FICZ on the TGF-ß-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-ß-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. CONCLUSIONS: These findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging.

A Versatile Probe for Caffeine Detection in Real-Life Samples via Excitation-Triggered Alteration in the Sensing Behavior of Fluorescent Organic Nanoaggregates.

Excitation triggered alteration in the sensing behavior of fluorescent nanoaggregates was explored in water, considering caffeine as the "target analyte". Merely by changing the excitation wavelength, we could specifically excite either the monomeric species or the fluorescent nanoaggregates. The monomer showed highly sensitive interaction with caffeine despite poor selectivity, while the "strongly associated" fluorescent nanoaggregates displayed relatively high selectivity with low sensitivity. In addition, the extent of self-aggregation was also found to be influenced by the micropolarity of the local surroundings and the electronics of the core carbazole unit. Furthermore, the present protocol was utilized for the estimation of caffeine in different beverages and biological fluids with reasonably high accuracy. Inexpensive, portable paper strips were designed for a rapid, on-site detection of caffeine without involving sophisticated instruments or trained technicians.

Photodegradative fate and potential phototoxic products of bromocarbazoles and chlorocarbazoles in water.

Bromocarbazoles and chlorocarbazoles are emerging environmental contaminants that have been reported to be persistent and possessing dioxin-like toxicity; however, their photodegradative fate in water is unknown. The photodegradation of 3-bromocarbazole, 3-chlorocarbazole, and 3,6-dichlorocarbazole was determined in ultrapure water. They proceeded by direct photolysis and followed first-order kinetics. The rate constants (k) were 0.4838, 0.3454, and 0.4422 h-1 corresponding to half-lives (t 1/2) 1.81, 2.01, and 1.62, while the quantum yields (Ф) were 0.232, 0.180, and 0.295 respectively. The maximum wavelengths of absorption (λ max) were in the near ultraviolet region (295, 296, 299, and 301 nm) implying these compounds are likely to degrade slowly under sunlight in natural aquatic environment. The molar extinction coefficients (ε) determined in acetonitrile were 18,573, 17,028, 13,385, and 14,010 L mol-1 cm-1, respectively, the latter being 3,6-dibromocarbazole. A bathochromic shift was observed with halogen addition on their respective mono-substituted congeners. Bromocarbazoles were observed to degrade faster in water than chlorocarbazoles. In addition, photodegradation was estimated to proceed faster in summer than in winter, in natural water system at 50° N latitude. In the absence of light, hydrolytic degradation occurred but proceeded very slowly. Hexahydroxybenzene and trihydroxycarbazole were positively identified as the likely photoproducts with the former being a known toxic compound. Dehalogenation, oxidative cleavage, hydroxylation, and hydrolysis are suggested as the major photodegradation mechanisms in water, yielding phototoxic products that may be of enhanced toxicity than the parent compounds.

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct.

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80-100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

Cyclobutane pyrimidine dimers are photosensitised by carprofen plus UVA in human HaCaT cells.

Every year in the UK about 75,000 cases of non-melanoma skin cancer (NMSC) are registered, and about 9500 people are diagnosed with cutaneous melanoma (CM). The main risk factor for these cancers is exposure to sunlight. The effects of light on skin are wavelength dependent, with wavelengths in the UVB waveband (280-315 nm) being the most carcinogenic. UVB is directly absorbed by DNA, producing dimeric pyrimidine photoproducts including cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimodone photoproducts (6-4PP). However UVA (315-400 nm) can also produce CPD, induce skin tumours in mice, and has been shown to be mutagenic in cell culture. Although the precise role of UVA in human skin cancer remains to be elucidated, it comprises the major portion of solar UV radiation, transmits through window glass and can be delivered in high doses from tanning lamps. Non-steroidal anti-inflammatory drugs (NSAIDs), in particular the 2-aryl propionic acid derivatives, are a well-documented group of photosensitising chemicals producing clinical phototoxic and photoallergic reactions. We have used carprofen, a model compound from this group to see if it could amplify the effects of UVA and contribute to the formation of CPD by UVA. Preliminary work has shown that carprofen combined with low doses of UVA (lambda(max): 365 nm; 5 J/cm(2)) can produce both strand breaks (SB) and CPD in human skin or blood cells. CPD were detected indirectly by both an immunofluorescence method and as T4 endonuclease V sensitive sites in the comet assay. These findings show that compounds other than fluoroquinolones and psoralen derivatives may contribute to CPD formation in skin cells in combination with UVA.

Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation.

We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( (CNV)K). Oligodeoxynucleotide (ODN) containing (CNV)K can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.

Stereodifferentiating drug-biomolecule interactions in the triplet excited state: studies on supramolecular carprofen/protein systems and on carprofen-tryptophan model dyads.

Stereoselective interaction between a chiral nonsteroidal antiinflammatory drug, namely carprofen (CP), and human serum albumin (HSA) was studied, and the results were compared with those obtained with model dyads. In the presence of albumin the same triplet-triplet transition was detected for both CP stereoisomers; however, time-resolved measurements revealed a remarkable stereodifferentiation in the CP/HSA interaction. For each stereoisomer, the decay dynamics evidenced the presence of two components with different lifetimes that can be correlated with complexation of CP to the two possible albumin binding sites (site I and site II). This assignment was confirmed by using ibuprofen, a site II displacer. Thus, the shorter lived components, for which stereodifferentiation was more important (tauR/tauS ca. 4), were ascribed to the CP triplet state in site I; the lifetime shortening can be attributed to electron-transfer quenching by the only tryptophan (Trp) of the protein. Laser flash photolysis of model dyads containing covalently linked CP and Trp revealed formation of the expected Trp radical cation, providing support for such a mechanism. Moreover, significant stereodifferentiation was observed between the (R)- and (S)-CP-Trp dyads. In the case of CP/HSA complexes, as well as in the model compounds, the stereodifferentiation detected in the decays is in good agreement with that observed in the formation of the only CP photoproduct, resulting from a photodehalogenation process. Moreover, stereodifferentiation was also found to occur for the photobinding of CP to the protein.

Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity.

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.

Photophysical properties of a carbazolyl mesogen of 8PCzC as revealed by absorption, fluorescence, and transient absorption measurements.

Fluorescence spectra in three phases [crystalline, smectic A liquid crystal (SmA), and isotropic] of a mesogen including one carbazolyl (Cz) chromophore, 4'-n-octylphenyl carbazole-2-carboxylate (8PCzC), were measured at various temperatures and compared with fluorescence spectra in solvents with various dielectric constants. Further, for the first time, picosecond transient absorption spectroscopy was applied to elucidating photoinduced electron-transfer dynamics (charge separation, charge recombination, and hole transfer reactions) in the three phases of 8PCzC doped with a small amount of an electron acceptor. The fluorescence spectra showed clearly the presence of two crystalline phases, which was supported by DSC measurement. On the basis of a comparison of the fluorescence spectra and those in solution, the 8PCzC molecule in the first crystal structure was suggested to be in polar environment compared to that in the second one. The fluorescence yields of 8PCzC in both SmA and isotropic phases were remarkably small compared to that in crystalline one. This was ascribed to the formation of hydrogen bonds between 8PCzC molecules in both SmA and isotropic phases. As a first approximation, the picosecond transient absorption spectra in the three phases of 8PCzC doped with 2,4,7-trinitrofluorenone were analyzed by the simple model of one-dimensional hole migration. The rate constants obtained for the hole transfer from the cation state of Cz chromophore to neighboring Czs decreased in the order of SmA (approximately 10(9) s(-1)) > isotropic (approximately 10(8) s(-1)) > crystalline (approximately 10(7) s(-1)) phases, although these measurement temperatures were different.

Microwave-assisted synthesis of novel thiazolocarbazoles and evaluation as potential anticancer agents. Part III.

Novel 6-substituted thiazolocarbazole derivatives have been synthesized under microwave irradiation via the corresponding imino-1,2,3-dithiazoles. In vitro antitumor potential of these polyheterocyclic compounds was evaluated. Among all the tested thiazolocarbazoles, compound 10 is the most effective in inhibiting cell growth.

Molecular mechanism of drug photosensitization. 4. Photohemolysis sensitized by carprofen.

Red blood cell lysis photosensitized by carprofen (CPF) was investigated. The photohemolysis process was observed in both aerobic and anaerobic conditions. Irradiation (310-390 nm) of buffered CPF solutions at pH 7.4 in deaerated conditions leads to a dehalogenation process via intermediate radicals with formation of the compound 2-(2-carbazolyl)propanoic acid (I) in the presence of hydrogen donors. Irradiation of I produces decarboxylation via free radicals and the formation of a stable decarboxylated compound, 2-ethylcarbazole (II). The photodegradation products I and II do not show lytic activity. The dechlorinated product I shows photosensitizing ability which was studied in the presence of the red blood cells in both aerated and deaerated solution. When carried out in the presence of additives, the observed photohemolysis suggests the involvement of free radicals and singlet oxygen in the membrane damage induced by both CPF and photoproduct I irradiation, whereas there is no evidence of any role for hydroxyl radicals. Superoxide anion is involved only in the photosensitization process induced by photoproduct I.