A Photolabile Carboxyl Protecting Group for Solid Phase Peptide Synthesis.

A new kind of photolabile protecting group (PLPG) for carboxyl moieties was designed and synthesized as the linker between resin and peptide. This group can be used for the protection of amino acid carboxyl groups. The peptide was synthesized on Nph (2-hydroxy-3-(2-nitrophenyl)-heptanoic acid)-derivatized resins and could be cleaved under UV exposure, thus avoiding the necessity for harsh acid-mediated resin cleavage. The PLPG has been successfully used for solid-phase synthesis of peptides.

Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell's response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome in vivo analysis (TIVA). Successful application of the TIVA technique hinges upon its oligonucleotide probe remaining completely inert (or "caged") to mRNA unless photoactivated. To improve the reliability of TIVA probe caging in diverse and challenging biological conditions, we applied a rational design process involving iterative modifications to the oligonucleotide construct. In this work, we discuss these design motivations and present an optimized probe with minimal background binding to mRNA prior to photolysis. We assess its caging performance through multiple in vitro assays including FRET analysis, native gel comigration, and pull down with model mRNA transcripts. Finally, we demonstrate that this improved probe can also isolate mRNA from single living neurons in brain tissue slices with excellent caging control.

Photolabile Linkers: Exploiting Labile Bond Chemistry to Control Mode and Rate of Hydrogel Degradation and Protein Release.

Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl (NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.

Synthesis of metallic copper modified g-C3N4 by molecular self-assembly structure and its combined catalytic performance with activated sludge.

Copper modified carbon nitride (CuCN) was prepared by a hydrothermal self-assembly reaction and following high temperature thermal polymerization process. Finally, the sample exhibits uniform one-dimensional tubular structure. Interestingly, the separation efficiency of electron-hole pair is improved, and more catalytic active sites are exposed due to the special hollow structure. Meanwhile, the presence of copper element narrows its band gap, leading to the enhancement of photocatalytic degradation performance under simulated sunlight. In addition, the effect of CuCN on dehydrogenase activity of activated sludge was determined by TTC reduction method. After adding CuCN-2, the activity of activated sludge reached 0.134 µmol g-1 min-1, which indicated that the prepared CuCN-2 had good biocompatibility. It is suitable for both photocatalytic process and activated sludge treatment process. Therefore, the combination of photocatalytic technology and activated sludge process can further completely degrade organic pollutants. We found that CuCN could protect the survival and growth of microorganisms in activated sludge, so that the degradation efficiency of CuCN to nitrobenzene could reach 94.4 %. Therefore, CuCN has broad application prospects in photocatalytic-activated sludge combined treatment.

A NIR light-triggered drug delivery system using core-shell gold nanostars-mesoporous silica nanoparticles based on multiphoton absorption photo-dissociation of 2-nitrobenzyl PEG.

Gold nanostars coated with a mesoporous silica shell and functionalized with poly(ethylene glycol) containing photolabile 2-nitrobenzyl moieties are able to release doxorubicin after NIR light irradiation at low power irradiance via a multiphoton absorption photo-dissociation process.

Traceless Photolabile Linker Expedites the Chemical Synthesis of Complex Oligosaccharides by Automated Glycan Assembly.

Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the demand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the solid support play a pivotal role in the synthesis strategy as they determine all chemical conditions used during the synthesis and the form of the glycan obtained at the end of it. Here, we describe a traceless photolabile linker used to prepare carbohydrates with a free reducing end. Modification of the o-nitrobenzyl scaffold of the linker is key to high yields and compatibility with the AGA workflow. The assembly of an asymmetrical biantennary N-glycan from oligosaccharide fragments prepared by AGA and linear as well as branched ß-oligoglucans is described to illustrate the power of the method. These substrates will serve as standards and biomarkers to examine the unique specificity of glycosyl hydrolases.

Dual activity of amphiphilic Zn(II) nitroporphyrin derivatives as HIV-1 entry inhibitors and in cancer photodynamic therapy.

Two Zn(II) nitro porphyrin derivatives bearing combinations of meso-4-nitrophenyl and meso-4-methylpyridinium moieties and their free-base precursors were synthesized through one-pot microwave process, purified and characterized. The biological activity of these nitroporphyrins was assessed under both photodynamic and non-photodynamic conditions to correlate their structure-activity relationship (SAR). Unlike, the free-base precursors, Zn(II) complexes of these nitroporphyrins displayed nearly complete inhibition in the entry of lentiviruses such as HIV-1 and SIVmac under non-photodynamic conditions. In addition, the Zn(II) complexes also exhibited a higher in vitro photodynamic activity towards human lung cancer cell-line A549 than their free-base precursors. Our results strongly suggest that incorporation of Zn(II) has improved the antiviral and anticancer properties of the nitroporphyrins. To the best of our knowledge, this is the first report demonstrating the dual activity of nitroporphyrin-zinc complexes as antiviral and anti-cancer, which will aid in their development as therapeutics in clinics.

Sequential Photoactivation of Self-Assembled Monolayers to Direct Cell Adhesion and Migration.

Dynamic substrates for cell culture control the spatial and temporal presentation of extracellular matrix ligands that interact with adherent cells. This paper reports a photoactive surface chemistry that can repeatedly activate regions of the substrate for cell adhesion, spreading, and migration. The approach uses self-assembled monolayers presenting the integrin ligand RGD that is caged with a nitrophenyl-based photoprotecting group. The group is also modified with a maltoheptaose oligosaccharide to prevent nonspecific protein adsorption and cell attachment. The peptide is uncaged when irradiated with a laser source at 405 nm on a microscope to reveal micron-size regions for single cell attachment. This method is applied to studies of gap junction-mediated communication between two neighboring cells and requires the patterning of an initial receiver cell population and then the patterning of a second sender population to give a culture wherein each pair of cells are separated by 30 µm. Finally, activation of the region between the cells permits cell-cell contact and gap junction assembly between the sender and receiver cells. This example demonstrates the broad relevance of this method to studying complex phenotypes in cell culture.

Photoactivatable Prodrug of Doxazolidine Targeting Exosomes.

Natural lipid nanocarriers, exosomes, carry cell-signaling materials such as DNA and RNA for intercellular communications. Exosomes derived from cancer cells contribute to the progression and metastasis of cancer cells by transferring oncogenic signaling molecules to neighboring and remote premetastatic sites. Therefore, applying the unique properties of exosomes for cancer therapy has been expected in science, medicine, and drug discovery fields. Herein, we report that an exosome-targeting prodrug system, designated MARCKS-ED-photodoxaz, could spatiotemporally control the activation of an exquisitely cytotoxic agent, doxazolidine (doxaz), with UV light. The MARCKS-ED peptide enters a cell by forming a complex with the exosomes in situ at its plasma membrane and in the media. MARCKS-ED-photodoxaz releases doxaz under near-UV irradiation to inhibit cell growth with low nanomolar IC50 values. The MARCKS-ED-photodoxaz system targeting exosomes and utilizing photochemistry will potentially provide a new approach for the treatment of cancer, especially for highly progressive and invasive metastatic cancers.

Light-control of the ultra-fast Gp41-1 split intein with preserved stability of a genetically encoded photo-caged amino acid in bacterial cells.

Inteins change the structure and function of their host protein in a unique way and the Gp41-1 split intein is the fastest protein trans-splicing intein known to date. To design a photo-activatable variant, we have incorporated ortho-nitrobenzyl-tyrosine (ONBY) at the position of a structurally conserved phenylalanine in the Gp41-1-N fragment. Using irradiation at 365 nm, the splicing reaction was triggered with virtually unchanged rates. The partial cellular reduction of the nitro group in ONBY, previously observed during bacterial protein expression for several photo-caged amino acids, was overcome by periplasmatic expression and by using an E. coli K12(DE3) strain instead of BL21(DE3). Together, our findings provide new tools for the artificial photo-control of proteins.

Novel Light-Responsive Hydrogels with Antimicrobial and Antifouling Capabilities.

Smart materials with both bactericidal and bacteria-resistant functions are promising for combating the infection concern of medical devices. Current work mostly utilizes hydrolysis to switch materials from antimicrobial to antifouling forms by incubating materials in aqueous solutions for hours to days. In this work, a new photoresponsive poly[2-((4,5-dimethoxy-2-nitrobenzyl)oxy)- N-(2-(methacryloyloxy)ethyl)- N, N-dimethyl-2-oxoethan-1-aminium] (polyCBNA) hydrogel was developed, incorporating the photolabile 4,5-dimethoxy-2-nitrobenzyl and cationic quaternary ammonium groups. The photolabile groups were readily cleaved from the hydrogel shortly upon UV irradiation at 365 nm (a long wavelength widely used for biomedical applications), leading to polymer surface charge switching from cationic to zwitterionic form. Protein adsorbed significantly on polyCBNA but easily desorbed from surfaces after UV irradiation. The cationic hydrogel as a precursor was shown to effectively kill the attached bacteria, and then quickly switched to zwitterionic antifouling form via photolysis, which released the attached bacteria from surfaces and prevented further bacterial attachment. Moreover, the adhered endothelial cells were easily detached from polyCBNA surfaces triggered by light, providing a facile and less destructive nonenzymatic approach to harvest cells. This smart photoresponsive polyCBNA polymer, with integrated antimicrobial and antifouling properties, holds great potential in biomedical applications such as self-sterilizing and self-cleaning coatings for implants, cell harvesting, and cell patterning.

An Application of Photoactivatable Substrate for the Evaluation of Epithelial-mesenchymal Transition Inhibitors.

Epithelial-mesenchymal transition (EMT), phenotypic changes in cell adhesion and migration, is involved in cancer invasion and metastasis, hence becoming a target for anti-cancer drugs. In this study, we report a method for the evaluation of EMT inhibitors by using a photoactivatable gold substrate, which changes from non-cell-adhesive to cell-adhesive in response to light. The method is based on the geometrical confinement of cell clusters and the subsequent migration induction by controlled photoirradiation of the substrate. As a proof-of-concept experiment, a known EMT inhibitor was successfully evaluated in terms of the changes in cluster area or leader cell appearance, in response to biochemically and mechanically induced EMT. Furthermore, an application of the present method for microbial secondary metabolites identified nanaomycin H as an EMT inhibitor, potentially killing EMTed cells in disseminated conditions. These results demonstrate the potential of the present method for screening new EMT inhibitors.

Tailored photocleavable peptides: fragmentation and neutralization pathways in high vacuum.

Photocleavable tags (PCTs) have the potential for excellent spatio-temporal control over the release of subunits of complex molecules. Here, we show that electrosprayed oligopeptides, functionalized by a tailored ortho-nitroarylether can undergo site-specific photo-activated cleavage under UV irradiation (266 nm) in high vacuum. The comparison of UV photodissociation (UVPD) and collision-induced dissociation (CID) points to the thermal nature of the cleavage mechanism, a picture corroborated by the temperature dependence of the process. Two competing photodissociation pathways can be identified. In one case a phenolate anion is separated from a neutral zwitterion. In the other case a neutral phenol derivative leaves a negatively charged peptide behind. To understand the factors favoring one channel over the other, we investigate the influence of the peptide length, the nature of the phenolic group and the position of the nitro-group (ortho vs. para). The observed gas phase cleavage of a para-nitro benzylic ether markedly differs from the established behavior in solution.

Modeling the photochemical transformation of nitrobenzene under conditions relevant to sunlit surface waters: Reaction pathways and formation of intermediates.

Nitrobenzene (NB) would undergo photodegradation in sunlit surface waters, mainly by direct photolysis and triplet-sensitized oxidation, with a secondary role of the *OH reaction. Its photochemical half-life time would range from a few days to a couple of months under fair-weather summertime irradiation, depending on water chemistry and depth. NB phototransformation gives phenol and the three nitrophenol isomers, in different yields depending on the considered pathway. The minor *OH role in degradation would make NB unsuitable as *OH probe in irradiated natural water samples, but the selectivity towards *OH could be increased by monitoring the formation of phenol from NB+*OH. The relevant reaction would proceed through ipso-addition of *OH on the carbon atom bearing the nitro-group, forming a pre-reactive complex that would evolve into a transition state (and then into a radical addition intermediate) with very low activation energy barrier.

Fluorescent Pyrene Assisted Photodeprotection of 2-(2-nitrophenyl)Propyloxycarbonyl Groups on Self-Assembled Monolayers.

Accelerating the photodeprotection rate of photolabile protecting group is conducive to a light-directed chemical reaction, especially for the in situ synthesis of a biochip. Herein, a photosensitizer pyrene was applied to the photodeprotection of 2-(2-nitrophenyl)propyloxycarbony (NPPOC) groups on self-assembled monolayers (SAMs). It was found that the addition of pyrene could largely improve photodeprotection rate, and effectively prevent molecule damage that are often encountered by the photosensitizer 2-isopropyl thioxanthone (ITX). The most likely explanation for this result is that the whole photodeprotection process involves three joint actions, including ultraviolet light irradiation, triplet energy transfer by pyrene, and singlet fluorescence irradiation from pyrene. The joint actions enable the transfer of over-absorbed energy from pyrene to protecting groups in terms of fluorescence rather than free radicals produced by ITX that are detrimental to the molecules modified on glass substrates. Pyrene dissolved in an optimized combination of mixed solvent of dimethylacetamide (DMAC), ethanol, and dioxane with a volume ratio of 1:1:1 was tested to produce a complete photodeprotection of NPPOC groups within 6 min under 365 nm ultraviolet with an intensity of 10.8 mW/cm2. Meanwhile, tens to hundreds of cycles of photodeprotection could be conducted at a high efficiency. This research will shed light on the deprotection of photolabile groups with weak ultraviolet using a fluorescent sensitizer.

Intimately coupling of photolysis accelerates nitrobenzene biodegradation, but sequential coupling slows biodegradation.

Photo(cata)lysis coupled with biodegradation is superior to photo(cata)lysis or biodegradation alone for removal of recalcitrant organic compounds. The two steps can be carried out sequentially or simultaneously via intimate coupling. We studied nitrobenzene (NB) removal and mineralization to evaluate why intimate coupling of photolysis with biodegradation was superior to sequential coupling. Employing an internal circulation baffled biofilm reactor, we compared direct biodegradation (B), biodegradation after photolysis (P+B), simultaneous photolysis and biodegradation (P&B), and biodegradation with nitrophenol (NP) and oxalic acid (OA) added individually and simultaneously (B+NP, B+OA, and B+NP+OA); NP and OA were NB's main UV-photolysis products. Compared with B, the biodegradation rate P+B was lower by 13-29%, but intimately coupling (P&B) had a removal rate that was 10-13% higher; mineralization showed similar trends. B+OA gave results similar to P&B, B+NP gave results similar to P+B, and B+OA+NP gave results between P+B and P&B, depending on the amount of OA and NP added. The photolysis product OA accelerated NB biodegradation through a co-substrate effect, but NP was inhibitory. Although decreasing the UV photolysis time could minimize the inhibition impact of NP in P+B, P&B gave the fastest removal of NB by accentuating the co-substrate effect of OA.

The roles of reactive species in micropollutant degradation in the UV/free chlorine system.

The UV/free chlorine process forms reactive species such as hydroxyl radicals (HO(•)), chlorine atoms (Cl(•)), Cl2(•-), and O(•-). The specific roles of these reactive species in aqueous micropollutant degradation in the UV/chlorine process under different conditions were investigated using a steady-state kinetic model. Benzoic acid (BA) was chosen as the model micropollutant. The steady-state kinetic model developed fitted the experimental data well. The results showed that HO(•) and Cl(•) contributed substantially to BA degradation, while the roles of the other reactive species such as Cl2(•-) and O(•-) were negligible. The overall degradation rate of BA decreased as the pH increased from 6 to 9. In particular, the relative contributions of HO(•) and Cl(•) to the degradation changed from 34.7% and 65.3% respectively at pH 6 to 37.9% and 62% respectively at pH 9 under the conditions evaluated. Their relative contributions also changed slightly with variations in chlorine dosage, BA concentration and chloride concentration. The scavenging effect of natural organic matter (NOM) on Cl(•) was relatively small compared to that on HO(•), while bicarbonate preferentially reduced the contribution of Cl(•). This study is the first to demonstrate the contributions of different reactive species to the micropollutant degradation in the UV/chlorine system under environmentally relevant conditions.

Degradation kinetics and mechanism of trace nitrobenzene by granular activated carbon enhanced microwave/hydrogen peroxide system.

The kinetics of the degradation of trace nitrobenzene (NB) by a granular activated carbon (GAC) enhanced microwave (MW)/hydrogen peroxide (H202) system was studied. Effects of pH, NB initial concentration and tert-butyl alcohol on the removal efficiency were examined. It was found that the reaction rate fits well to first-order reaction kinetics in the MW/GAC/H202 process. Moreover, GAC greatly enhanced the degradation rate of NB in water. Under a given condition (MW power 300 W, H202 dosage 10 mg/L, pH 6.85 and temperature (60 +/- 5)degrees C), the degradation rate of NB was 0.05214 min-1when 4 g/L GAC was added. In general, alkaline pH was better for NB degradation; however, the optimum pH was 8.0 in the tested pH value range of 4.0-12.0. At H202 dosage of 10 mg/L and GAC dosage of 4 g/L, the removal of NB was decreased with increasing initial concentrations of NB, indicating that a low initial concentration was beneficial for the degradation of NB. These results indicated that the MW/GAC/H202 process was effective for trace NB degradation in water. Gas chromatography-mass spectrometry analysis indicated that a hydroxyl radical addition reaction and dehydrogenation reaction enhanced NB degradation.

Synthesis of a photocaged tamoxifen for light-dependent activation of Cre-ER recombinase-driven gene modification.

We report the design of a water-soluble, quaternized tamoxifen photoprobe and demonstrate its application in light-controlled induction of green fluorescent protein expression via a Cre-ER recombinase system.

Femtosecond spectroscopy on the photochemistry of ortho-nitrotoluene.

The photo tautomerisations of ortho-nitrotoluene (oNT) and its methylated derivative ortho-ethylnitrobenzene (oENB) have been studied by means of femtosecond spectroscopy and (TD)-DFT computations. In UV/Vis transient absorption spectroscopy a band peaking at 400 nm is seen to rise in a bi-modal manner with time constants of 1-10 ps and 1500 ps. Femtosecond stimulated Raman experiments clearly identify aci-nitro forms as the spectroscopic carriers of the 400 nm band. The assignment of the Raman spectra is based on TD-DFT computations. The quantum yields of the aci-nitro forms after 3 ns are 0.08 (oNT) and 0.3 (oENB). The aci-nitro forms are formed via a singlet channel (1-10 ps) and a triplet channel (1500 ps). There are indications that the triplet channel involves a bi-radical intermediate. In between 3 ns and 1 ms the spectrum of the aci-nitro form shifts from 400 to 390 nm. This could indicate a tautomerisation from Z-aci-nitro to an E form.