Photocaged activity-based probes for improved monitoring of protein S-sulfenylation in living cells

Protein S-sulfenylation(protein sulfenic acid),as one of the most significant oxidative post-translational modifications(OxiPTMs),plays a vital role in regulating protein function.A variety of activity-based probes have been developed to profile sulfenic acid in living cells.However,due to the transient presence and low content of sulfenic acid in living cell,high doses of probes are needed to achieve efficient labeling.More importantly,current probes have no temporal control over sulfenic acid labeling.To overcome these limitations,two caged cysteine sulfenic acid probes DYn-2-ONB and DYn-2-Cou with either an o-nitrobenzyl or coumarin protecting group were developed in this study.Both probes can be efficiently uncaged via irradiation to produce the active C-nucleophile probe DYn-2.Labeling assay in living cells demonstrated DYn-2-ONB exhibited better labeling capacity compared with DYn-2,providing it as a powerful tool for improved monitoring of protein S-sulfenylation in living cells.

Genetic Encoding of a Photocaged Glutamate for Optical Control of Protein Functions

Genetic encoding of photocaged noncanonical amino acids provides a powerful tool to study protein functions through optical control but is not yet available for acidic amino acids.Herein,we report the first site-specific genetic encoding of a photocaged glutamate,4-methoxy-7-nitroindolinyl caged glutamate(MNI-Glu),into recombinant proteins via an expanded genetic code through evolved EcLeuRS/tRNA pair.Using two enzymes as examples,we demonstrate that substituting the conserved-active-site glutamate of a secreted alkaline phosphatase and a protease HRV3C to MNI-Glu allows photoregulatory control of their enzymatic activities.Our approach is an important addition to the photocaged noncanonical amino-acid toolbox and provides a general method to photocontrol protein activity based on caging a critical glutamate.

A thioxanthone-based photocaged superbase for highly effective free radical photopolymerization

Thioxanthone-based N-phthalimidoamino acid ammonium salt（thioxanthen-DBU） as a photocaged base was synthesized and characterized. The photochemical properties and initiation mechanism were analyzed. It was found that the compound absorbs over the UV and visible region with relatively high absorption coefficients. Furthermore, the covalent binding of N-phthalimidoamino acid and type II chromophores（thioxanthone, TX） remarkably improved the photoreactivity. Specifically, in combination with a benzoyl peroxide initiator, thioxanthen-DBU was able to initiate the amine-mediated redox photopolymerization of trimethylol propane triacrylate（TMPTA）, and an excellent photopolymerization profile was obtained.

Benzoylformamides as new photocaged bases for photo-latent anion polymerization

Benzoylfonnamide（BFA） derivatives are proposed as new photocagcd bases with good solubility in epoxy resin.Initially their structures were confirmed by ~1H NMR,^（13）C NMR,and elemental analysis.Next,we detail their thermal stability,solubility behavior,and photolysis products.Furthermore,the model photo-latent anion polymerization（AP） of epoxide system in the presence of BFA-dBA（N,N-dibenzyl-2-oxo-2-phenylacetamide） as a photocaged base has been investigated,and excellent photopolymerization profile is obtained.

Controlling antifungal activity with light:Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors

Invasive fungal infections(IFIs)have been associated with high mortality,highlighting the urgent need for developing novel antifungal strategies.Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14a-demethylase(CYP51)inhibitors.The photocaged triazoles completely shielded the CYP51inhibition.The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4%and 83.7%,respectively.Importantly,the shielded antifungal activity(MIC80≥64μg/m L)could be efficiently recovered(MIC80=0.5—8μg/m L)by light irradiation.The new chemical tools enable optical control of fungal growth arrest,morphological conversion and biofilm formation.The ability for highprecision antifungal treatment was validated by in vivo models.The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model.Overall,this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

Peptide photocaging:A brief account of the chemistry and biological applications

Light is arguably the most convement non-invasive stimulant to perturb or control specinc cnemical reactions in the biological systems. Upon light illumination, photosensitive molecules incur conformational changes or formation/breaking of chemical bonds. Consequently, these molecules can be used to transfer signals from one location to another in the cell, from outside of the cell to the inside, or from a light bulb to the interior of animal tissues. The development of the photochemical reactions of organic compounds has paved the road towards their use in peptides and peptide-based biological applications. In this mini-review, we summarized the state-of-the-art development of photo-protecting groups for peptide photocaging including the un-caging mechanism of different PPGs, the synthesis of photo-caged peptides, and the recent applications of peptide photocaging in chemical biology.

A Genetically Encoded Toolkit for Probing the Post Translational Modifications of Lysine in Mammalian Cells

Post translational modifications (PTMs) of lysine play a crucial role on modulating the activity and stability of essential proteins in eukaryotic cells including the tumor

White-light-driven fluorescence switch for super-resolution imaging guided photodynamic and photoacid therapy

Photocaged fluorophores with photoactivatable characteristics presented important applications in imaging the biological structures and processes.Taking advantage of their super-resolution imaging merits to manipulate and visualize anti-cancer treatment is always a goal of modern clinical medicine.Traditional photodynamic therapy(PDT) is a noninvasive treatment but limited in intracellular oxygen content.Type I PDT and photoacid therapy(PAT) are two effective supplements of traditional PDT especially in hypoxic condition.Herein,a novel white-light-driven fluorescence switch(7H-dibenzo[c,g]carbazol-7-yl)(2-iodophenyl)methanone(2IB) was designed and synthesized as an unprecedent “all in one” platform for stochastic optical reconstruction microscopy(STORM) imaging guided Type Ⅰ/Ⅱ PDT and PAT.The experimental and theoretical studies revealed that the working mechanism is based on two competing paths under excitation:photosensitization and photocyclization reaction.Efficient intersystem crossing(ISC) ensured the generation of reactive oxygen species(ROS) for PDT,while low energy barrier facilitated the photocyclization reaction that simultaneously yielded emissive fluorophores(2IBC) and H^(+) for super-resolution imaging and photoacid,respectively.Impressively,the fluorescent intensity of mitochondria-targeted 2IBC was positively correlated with treatment efficacy,which is beneficial to spatiotemporally visualized therapeutic process and outcome.As a result,superior anti-tumor performance was achieved in vitro and in vivo.This contribution provided a multifunctional nanodrug paradigm for multimode cancer diagnosis and treatment.

Regulable DNA–Protein Interactions in Vitro and Vivo at Epigenetic DNA Marks

5-Formyluracil(5fU)is a vital DNA marker that is widely distributed in the cells of organisms.A unique feature of 5fU is the possession of a potentially reactive aldehyde group in its structure that could realize addition and condensation reactions.However,the biological functional details of 5fU remain mostly elusive,especially,regarding its relatedness with proteins.In this current study,we show that 5fU bases have a strong affinity toward nucleosome core particles,and that could yield regulable DNA–protein conjugates(DPCs)via chemical interactions between amino and aldehyde groups,and reductants could be applied to stabilize or dissociate the interactions.Besides,we developed a photocaged method to exploit the relationship between 5fU and nucleosomes.Finally,by applying a combination of the existence of 5fU–histone interactions in vivo by ChIP analysis of histone H4 with liquid chromatography–mass spectrometry(LC–MS),we probed further,the DPCs’influence on nucleosome and enzyme.Collectively,our results showed that the 5fU–protein interactions increase the occupancy and stability of nucleosomes,affect enzyme recognition,and block DNA replication.These might imply that,in vivo,the DPCs between 5fU and nucleosome core particles might play a key role in 5fU-associated pathways such as DNA repair,transcriptional regulation,or development.

Light-triggered release of insecticidally active spirotetramat-enol

Spirotetramat metabolizes to its active enol form in the plant. We described here a photocaged pesticide delivery system that can release insecticidal spirotetramat enol form upon light irradiation. Covalently linking spirotetramat-enol with photoresponsive coumarin generated the caged insecticide. The photophysical and photochemical properties, deprotection photolysis and insecticidal activities of the caged spirotetramat enol were studied. This light-triggered system can undergo cleavage to release free spirotetramat enol form at the presence of blue light （420 nm） or sunlight, Bioassays indicated that the triggered molecule has no obvious insecticidal activity against Aphis craccivora Koch at dark and could be activated by light to release the insecticidal ingredients, which provides precise control over insecticide delivery.

Systematic investigation of bioorthogonal cellular DNA metabolic labeling in a photo-controlled manner

Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.