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The first bench-stable triple-diazonium reagent (TDA-1) was rationally designed and synthesized for coupling and crosslinking. The three reactive sites of TDA-1 can react with phenol-containing molecules as well as plant viruses in aqueous buffers efficiently. In addition, a new-type azo-linked cage was constructed by the direct reaction of TDA-1 with a triple-phenol molecule and was characterized by X-ray crystallography.

Gene therapy offers an alternative and promising avenue to lung cancer treatment. Here, we used dibenzocyclooctyne (DBCO)-branched primers to construct a PTEN gene nanovector (NP-PTEN) through branch-PCR. NP-PTEN showed the nanoscale structure with biocompatible size (84.7±11.2 nm) and retained the improved serum stability compared to linear DNA. When transfected into NCI-H1299 cancer cells, NP-PTEN could overexpress PTEN protein to restore PTEN functions through the deactivation of PI3K-AKT-mTOR signaling pathway to inhibit cell proliferation and induce cell apoptosis. The apoptosis rate of NCI-H1299 cancer cells could reach up to 54.5 %±4.6 % when the transfection concentration of NP-PTEN was 4.0 μg/mL. In mice bearing NCI-H1299 tumor xenograft intratumorally administrated with NP-PTEN, the average tumor volume and tumor weight was separately reduced by 61.7 % and 63.9 %, respectively, compared with the PBS group on the 18

In order to evaluate 7-sulfonamide benzoxadiazole (SBD) derivatives for the development of fluorescent probes, herein we investigated the thiolysis reactivity and selectivity of a series of SBD compounds with different atoms (N/O/S/Se) at the 4-position. Both SBD-amine and SBD-ether are stable toward biothiols in buffer (pH 7.4), while SBD-selenoether can react efficiently with biothiols GSH/Hcy, Cys, and H2S to produce SBD-SG/S-Hcy, SBD-NH-Cys, and SBD-SH, respectively, with three different sets of spectral signals. Therefore, the SBD-selenoether compounds should be useful platforms for the differentiation of these biothiols. Though SBD-alkylthioether shows much lower reactivity than SBD-selenoether, SBD-arylthioether is a tunable motif and structural modifications at the aryl moiety enable the rate of thiol-mediated thiolysis to be modified. To this end, an ER-targeted GSH-selective fluorescent probe 7 was rationally designed via thiolysis of SBD-arylthioether. Compared with control probe SBD-Cl, probe 7 exhibits improved GSH selectivity and better biocompatibility. In total, this study highlights that the modification at the 4-position of SBD is an efficient strategy for the development of new fluorescent probes with tunable reactivity and selectivity.

The development of advanced bioorthogonal reactions for detection and labeling of biomolecules is significant in chemical biology. Recently, researchers have found that multifluorinated aryl azides hold great potential for the development of improved bioorthogonal reactions. The fluorine atom can be a perfect substituent group because of its properties of excellent electronegativity and small steric hindrance. In this Minireview, we discuss recent developments of improved hydrogen sulfide (H2 S) fluorescence probes, fast strain-promoted azide-alkyne cycloaddition (SPAAC) and nonhydrolysis Staudinger reactions based on the use of multifluorinated aryl azides. Additionally, kinetic studies and biological applications of these reactions are also presented.

The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100-200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.

Cancer immunotherapy is a powerful weapon in the fight against cancers. Cyclic dinucleotides (CDNs) have demonstrated the great potential by evoking the immune system to fight cancers. There are still a lot of unmet needs for highly active CDNs in clinical applications due to low cell permeation and serum stability. Here we reported S-acylthioalkyl ester (SATE)-based prodrugs of deoxyribose cyclic dinucleotides (dCDNs) with three different types of internucleotide linkages (3',3':11a; 2',3':11b; 2',2':11c). The parent dCDNs could be efficiently released from SATE-dCDNs by cellular esterases. Compared to 2',3'-cGAMP and ADU-S100, 11a exhibited much higher potency of activating STING pathway and higher serum stability. In a CT26-Luc tumor-bearing animal model, 11a showed the efficient antitumor activity in eliminating the established tumor and induced significant increase of mRNA expression of IFN-β and other related inflammatory cytokines. Hence, SATE-dCDN prodrugs demonstrated their benefits in promoting cell penetration, improving serum stability, and thus enhancing bioactivity, suggesting their potential application as immunotherapy in a variety of malignancies.

The fast pyrolysis mechanism of xylose, xylobiose and xylan was investigated with combined fast pyrolysis experiments and quantum chemistry calculations. The results indicate that acyclic d -xylose is an important intermediate for the formation of the major pyrolytic products in xylose pyrolysis, i.e., 1,4-anhydro- d -xylopyranose (ADX), furfural (FF) and hydroxyacetaldehyde (HAA). ADX derives from the successive cyclization of d -xylose which is the only source for ADX formation. 3-Deoxy-xylose is the most important intermediate for FF formation. HAA results from the C C bond scission by retro-aldol and cyclic Grob fragment. In addition, the five-membered intermediate derived from the ring-contraction reaction can transform into FF with high selectivity. The dehydrated xylose is also another source for HAA formation. The fast pyrolysis mechanism of xylobiose is similar to that of xylose, and thus similar pyrolytic product distribution can be obtained from them. However, the pyrolytic products of xylan differ significantly from those of xylose and xylobiose, which is ascribed to its high degree of polymerization and the branches in it.

To selectively produce monocyclic aromatic hydrocarbons (MAHs) with low amount of polycyclic aromatic hydrocarbons (PAHs), modified HZSM-5 catalysts (Mo2N/HZSM-5, Ti(SO4)2/HZSM-5 and Ti(SO4)2-Mo2N/HZSM-5) were prepared and employed for ex-situ catalytic fast pyrolysis (CFP) of pine wood. Both analytical pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and lab-scale pyrolysis experiments were conducted to investigate the effects of catalyst, pyrolysis temperature and catalyst-to-pine ratio on the distribution of aromatic hydrocarbon products. Py-GC/MS results showed that the maximal MAHs yields from the HZSM-5, Mo2N/HZSM-5, Ti(SO4)2/HZSM-5 and Ti(SO4)2-Mo2N/HZSM-5 catalysts were 7.57 wt%, 8.11 wt%, 8.71 wt% and 12.53 wt% at 650 °C and catalyst-to-pine ratio of 7. Corresponding PAHs yields from the four catalysts were 2.55 wt%, 0.97 wt%, 1.47 wt% and 1.10 wt%, which clearly indicated that the Ti(SO4)2-Mo2N/HZSM-5 significantly increased MAHs formation and decreased PAHs formation as compared with HZSM-5. Furthermore, lab-scale experiments were conducted to quantitatively determine the pyrolytic product distribution. Results showed that Ti(SO4)2-Mo2N/HZSM-5 catalyst increased the yield of liquid and gas products, and decreased solid products as compared with HZSM-5. Under optimal conditions of 650 °C and catalyst-to-pine ratio of 4/3, the actual yields of MAHs and PAHs from Ti(SO4)2-Mo2N/HZSM-5 catalyst were 9.71 wt% and 1.45 wt%, respectively. In addition, stability tests confirmed that the Ti(SO4)2-Mo2N/HZSM-5 catalyst possessed better effects on MAHs production as well as anti-deactivation than the HZSM-5.

The delivery of siRNAs to selectively target cells poses a great challenge in RNAi-based cancer therapy. The lack of suitable cell-targeting methods seriously restricts the advance in delivering siRNAs to extrahepatic tissues. Based on prostate-specific membrane antigen (PSMA)-targeting ligands, we have synthesized a series of lysine-urea-glutamate (KUE)-siRNA conjugates and verified their effective cell uptake and gene silencing properties in prostate cancers. The results indicated that the KUE-siRNA conjugates could selectively enter PSMA+ LNCaP cells, eventually down-regulating STAT3 expression. Based on post-synthesis modification and receptor-mediated endocytosis, this strategy of constructing ligand-siRNA conjugates might provide a general method of siRNA delivery for cell-targeted gene silencing.

Understanding the role of H2 S in host defense mechanisms against RNA viruses may provide opportunities for the development of antivirals to combat viral infections. Here, we have developed a green-emitting fluorogenic probe, which exhibits a large fluorescence response at 520 nm (>560-fold) when treated with 100 μM H2 S for 1 h. It is highly selective for H2 S over biothiols (>400-fold F/F0 ) and has a detection limit of 12.9 nM. We demonstrate the application of the probe for endogenous H2 S detection in vivo for the understanding of its roles in antiviral host defense. Such virus-induced H2 S inhibits viral replication by reducing gene expression of RNA-dependent RNA polymerase (RdRp) and coat protein (CP). Additionally, a H2 S donor GYY4137 showed significantly antiviral activity as ribavirin, a broad-spectrum drug against RNA viruses. Furtherly, we propose a possible molecular mechanism for the TMV-induced H2 S biogenesis. This work provides a proof-of-principle in support of further studies identifying endogenous H2 S and its donors as potential antivirals toward RNA viruses.

Chemical labelling can significantly extend the structures and functions of proteins for advanced applications. Herein, a highly efficient bench-stable reagent diazo-azide was designed and synthesized for the incorporation of tetrafluorinated aromatic azides into proteins via the diazonium coupling. The diazo-azide-labelled proteins could be further functionalized via the nonhydrolysis Staudinger reaction to achieve fluorescence labelling, PEGylation and biotinylation. The whole protein labelling processes were catalysis-free and could be finished within several hours under the mild conditions. To this end, we have prepared thickened viral nanoparticles with controllable diameters.

The combined usage of two bioorthogonal reactions can provide hetero-bifunctional molecules under physiological conditions for various applications. Based on the Nonhydrolysis Staudinger Reaction (NSR), we design and develop a bisazido linker 1 for chemoselective dual-functionalization without the need of protection using catalyst-free and one-pot procedures. The NSR is much faster with tetrafluorinated aromatic azide than that the Staudinger-Bertozzi or SPAAC ligation with alkyl azide, as revealed by HPLC analysis and fluorescence kinetics. Based on the tandem NSR and Staudinger-Bertozzi ligation, we prepare a molecular beacon 7 from 1 in one-pot synthesis with a recovery yield of 32%. When a faster SPAAC ligation is used instead of the Staudinger-Bertozzi ligation, compound 8 is prepared in the tandem NSR and SPAAC reactions with a recovery yield of 59%. As a proof-of-concept study, the tandem NSR and SPAAC ligation is further used to produce a FRET-based dyad in living cells, as revealed by dual-color bioimaging. This work shows that NSR can be combined with other bioorthogonal reactions without the need of protection in one-pot.

Herein, we report the synthesis of 4'-C-trifluoromethyl (4'-CF3) thymidine (T4'-CF3) and its incorporation into oligodeoxynucleotides (ODNs) through solid-supported DNA synthesis. The 4'-CF3 modification leads to a marginal effect on the deoxyribose conformation and a local helical structure perturbation for ODN/RNA duplexes. This type of modification slightly decreases the thermal stability of ODN/RNA duplexes (-1 °C/modification) and leads to improved nuclease resistance. Like the well-known phosphorothioate (PS) modification, heavy 4'-CF3 modifications enable direct cellular uptake of the modified ODNs without any delivery reagents. This work highlights that 4'-CF3 modified ODNs are promising candidates for antisense-based therapeutics, which will, in turn, inspire us to develop more potent modifications for antisense ODNs and siRNAs.

Biothiols are implicated in different physiological and pathological processes, while abnormal levels of biothiols are related to numerous diseases. It is essential to discriminatively detect these molecules and explore their inherent transformation in living biological samples. Herein, dual-reactive and dual-quenching fluorescent probes based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) thioether/ether/amine for separate detection of H2S and Cys/Hcy were rationally designed. Probe 2 was able to sense H2S (λex/em = 405/455 nm) and Cys/Hcy (λex/em = 470/550 nm) in the presence of GSH in aqueous buffer. Moreover, the probe not only could be applied for visualization of exogenous H2S and Cys in living cells but also successfully showcased the potential to verify the biosynthesis of endogenous H2S from Cys.

Sulfuric acid (H2SO4) is widely used as a strong acid catalyst in biomass pyrolysis process, and exhibits prominent catalytic effects on the pyrolytic reactions and product distribution. In this study, the fundamental catalytic mechanism of H2SO4 on cellulose pyrolysis process was investigated via combined experimental and computational methods. Both thermographic analysis (TGA) and pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) experiments were performed to reveal the pyrolytic characteristics and product distribution in the H2SO4-catalyzed pyrolysis of cellulose. In addition, quantum chemistry methods were employed to build the reaction models and investigate the major H2SO4-assisted reactions in initial cellulose pyrolysis process, i.e., depolymerization of the cellulose chain, bridged dehydration, ring-opening, ring-contraction and dehydration of the free hydroxyl groups. The results indicate that the H2SO4-assisted depolymerization via C1-O1 bond scission and bridged dehydration reactions take place in a two-step mechanism involving a sulfate ester intermediate. While other reactions occur via a one-step mechanism involving only hydroxyl group or both hydroxyl and sulfonic groups of H2SO4. The activation energies of above reactions are all decreased by adding H2SO4, and thus lowering the degradation temperature of cellulose. Among all initial pyrolytic reactions, dehydration reactions which are inconspicuous in the non-catalytic process, become very important in the catalytic process, because their activation energies are decreased dramatically by adding H2SO4 (from 322.7 to 372.1 to 152.2–237.7 kJ/mol). The promoting effect on the dehydration reactions plays a vital role in the increased char yield and significant change of organic volatile product distribution. Through the promoted dehydration reactions, unsaturated structures of cellulose are formed, which is favorable for the formation of certain dehydrated products at the expense of depolymerized and ring scission products.

A series of Mo2N/HZSM-5 and transition metal modified Mo2N/HZSM-5 catalysts were prepared for the catalytic upgrading of pine wood-derived pyrolytic vapors for the selective production of monocyclic aromatic hydrocarbons (MAHs), while restraining the formation of polycyclic aromatic hydrocarbons (PAHs). Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) experiments were performed to determine the effects of several factors on selective MAHs production, including Mo2N loading on HZSM-5, transition metal (Fe, Ce, La, Cu, Cr) modification of Mo2N/HZSM-5, pyrolysis temperature, and catalyst-to-biomass ratio. In addition, quantitative experiments were conducted to determine the actual yields of major aromatic hydrocarbons and the source of aromatic hydrocarbons from basic biomass components. Results indicated that among the various catalysts, the Ce-10%Mo2N/HZSM-5 exhibited the best performance on promoting the formation of MAHs and restraining the generation of PAHs. Under the optimal conditions, the actual yields of MAHs and PAHs from Ce-10%Mo2N/HZSM-5 catalytic process were 99.8mg/g and 7.5mg/g, while those from HZSM catalyst were only 77.2mg/g and 23.7mg/g respectively. Furthermore, the possible catalytic mechanism of the Ce-Mo2N/HZSM-5 catalyst was proposed based on the catalyst characterization.

A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

Based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) thioether, we designed and synthesized fast-response fluorescent probes for discriminative detection of biothiols and Cys/Hcy. Probes treated with GSH/H2S could only release one emission; while reactions with Cys/Hcy generated two. Compared with probe 1, probe 2 exhibited good stability and high sensitivity toward Cys/Hcy in PBS buffer. Moreover, 2 could eliminate the effect of probe consumption by GSH/H2S when used to selectively detect Cys/Hcy and was applied for selective bioimaging of Cys in living mammalian cells successfully.

5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly, and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

Discovering new protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors is a promising direction for agrochemical research. Herein, we reported the discovery and in silico structure-guided optimization of N-phenyltetrahydroquinazolinones 1 and 2 as new PPO inhibitors. Most of the obtained compounds 1 and 2 exhibited significantly enhanced Nicotiana tabacum PPO (NtPPO) inhibitory potency than that of flumioxazin. Promisingly, 1-(tert-butoxy)-1-oxopropan-2-yl 2-chloro-4-fluoro-5-(4-oxo-5,6,7,8-tetrahydroquinazolin-3(4H)-yl)benzoate, 2o, with a Ki value of 4 nM, showed ten folds more enhanced NtPPO-inhibiting potency than flumioxazin. Additionally, compounds 2b and 2i showed a broad spectrum of broadleaf weeds control at 37.5–150 g ai/ha, and selective for wheat at 150 g ai/ha in the post-emergent application. The molecular simulation studies revealed the vital basis between N-phenyltetrahydroquinazolinones and NtPPO. The present work indicated that the N-phenyltetrahydroquinazolinone motif might be a potential scaffold for herbicide discovery.

Cyclic dinucleotides (CDNs) have been recognized as second messengers in both prokaryotes and eukaryotes. Until now, four CDNs were identified in the natural organism. In mammalian cells, CDNs could activate stimulator of interferon genes (STING), leading to the induction of type I interferon pro-inflammatory cytokines. It indicated that CDNs could be used for the treatment of tumor and infectious diseases in immune-therapy. Here, we will review the latest progress of the CDNs analogs and their bioactivities. What’s more, the preparation of CDNs through biosynthetic and chemical synthetic methods was summarized as well. It was hoped that this review would provide a comprehensive understanding of CDNs based STING agonists on the chemical perspective for readers who are interested in this area.

The development of efficient bioorthogonal reactions for sensing of endogenous biomolecules and for bioconjugation should be of paramount importance in the field of chemical biology. In this work, the o,o′-difluorinated aromatic azide was firstly employed to develop a new fast-response fluorescent probe 1 for H2S detection and for bioorthogonal reactions. Compared with non- and mono-fluorinated probes, 1 showed faster reaction toward H2S, the third gasotransmitter, in buffer (pH 7.4), implying that the reaction rate could be enhanced by the dual-fluorine groups. Furthermore, such enhanced reaction rates of 1 were also observed in the Staudinger reaction and strain-promoted azide–alkyne cycloaddition (SPAAC) based on the comparison studies of the non-fluorinated probe. Our results firstly highlight that the o,o′-difluorinated aromatic azide group should be useful for fast bioorthogonal reactions and H2S detection.

Hydrogen sulfide (H2S) is an important endogenous signaling molecule with multiple biological functions. The development of colorimetric and fluorescent probes for the visualization of H2S is useful for studying H2S biology. This review details up-to-date research on H2S probes based on thiolysis reactions of NBD (7-nitro-1,2,3-benzoxadiazole) amines or ethers (thioethers). The reactivity and selectivity of NBD-based dyes are discussed. The design principles of NBD-based probes are highlighted. The advantages and disadvantages of NBD-based probes are compared, and their biological applications are discussed.

RNA interference (RNAi) is a cellular process for gene silencing. Because of poor serum stability, transferring dsRNA directly into the target cells is a challenge. We report a facile and universal strategy to construct short hairpin RNA (shRNA) transcription nanoparticles with multiple shRNA transcription templates by PCR with flexible branched primers (branch-PCR). Compared with conventional linear shRNA transcription templates, these shRNA transcription nanoparticles show excellent stability against digestion by exonuclease III. Importantly, we found that our highly stable shRNA transcription nanoparticles can also be transcribed and thus induce efficient and long-lasting RNAi with picomolar activity in living mammalian cells. These chemically well-defined branch-PCR-generated stable shRNA transcription nanoparticles might facilitate RNAi delivery with a long-lasting RNAi effects.

Hydrogen sulfide (H2S) is an important endogenous signaling molecule with a variety of biological functions. To detect H2S in living biological systems, herein we developed a new fluorescent probe for highly sensitive and selective sensing of H2S in cells. The probe is based on coumarin–triazole as the fluorophore and thiolysis of the NBD (7-nitro-1,2,3-benzoxadiazole) amine as the receptor. Bioimaging experiments indicated that this probe could be used to monitor H2O2-induced H2S biosynthesis in yeast cells. Our results show that such thiolysis of the NBD amine can be used for the development of fluorescent H2S probes.

Cyclic dinucleotides (CDNs) could activate stimulator of interferon genes (STING) protein to produce type I interferon and other pro-inflammation cytokines in mammalian cells. To explore new types of potentially efficient STING activators targeting all five major hSTING variants (WT, R232H, HAQ, AQ and R293Q), we here reported the synthesis of a total of 19 inosine-containing CDNs based on the combinations of hypoxanthine with four natural bases (A, G, C and U) and three phosphodiester linkage backbones (3'-3', 2'-3', 2'-2'). The IFN-β induction results showed that all of the 2'-3' and 2'-2' CDNs linked by inosine and purine nucleosides favored the stacking interaction with Y167 and R238 residues of hSTING protein, and several CDNs constructed by hypoxanthine and pyrimidine like c[I(2',5')U(2',5')] could also activate all five hSTING variants. The molecular dynamic simulation and the isothermal titration calorimetric (ITC) assay further demonstrated the potential of cAIMP isomers with 2'-5' phosphate to form the hydrogen binding with R232 and R238 residues of hSTING in an entropically driven manner compared to cGAMP isomers. It would be promising to exploit novel inosine-mixed CDNs as activators of hSTING variants in immune therapy.

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is a target of several types of potent herbicides and antimicrobials. AHAS contains the catalytic subunit (CS) and the regulatory subunit (RS). The AHAS RS is usually composed of ACT domains and C-terminal domains. Herein, it is reported that the ACT domain of AHAS RS from different species could efficiently activate its respective CS. Moreover, the universal cross-activation between the CSs and the ACT domains of RSs across species has been discovered. Based on these biochemical and structural analyses, a molecular basis for the universal ACT-triggered CS activation is proposed, which would help to design broad-spectrum herbicides by targeting the interaction interface between CS and ACT from different species.

To achieve multisite-targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA-array-guided FnCpf1 nuclease showed target-activated, nonspecific DNase activity on both target DNA and nontarget DNA at random sites. Through optimization of the ratio of nuclease and CRIPSR RNAs, CRISPRay should further enhance gene interference in cells. This work presents a simple approach through which to improve multisite-directed gene disruption by fusing four guide RNAs (sgRNAs or crRNAs) into a CRISPR RNA string.

The development of highly efficient bioorthogonal reactions is of paramount importance for the research fields of biomaterials and chemical biology. We found that the o,o'-difluorinated aromatic azide was able to react with triphenylphosphine to produce water-stable phosphanimine. To further improve the efficiency of this kind of nonhydrolysis Staudinger reaction, a tetrafluorinated aromatic azide was employed to develop a faster nonhydrolysis Staudinger reaction with a rate of up to 51 m-1 s-1 , as revealed by high-performance liquid chromatography (HPLC) analysis and fluorescence kinetics. As a proof-of-concept study, the highly efficient Staudinger reaction was successfully used for chemoselective fluorescence labeling of proteins and nucleic acids (DNA and RNA) as well as for protein polyethyleneglycol (PEG)ylation. We believe that this bioorthogonal reaction can provide a broadly useful tool for various bioconjugations.

In order to evaluate the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines for development of H2S probes, herein we investigated the reactivity and selectivity of a series of NBD amines for the first time. The piperazinyl- and piperidyl-based NBD probes could react efficiently with micromolar H2S in buffer (pH 7.4), while such NBD(S) (nitrobenzothiadiazole) derivatives showed much slow thiolysis even in the presence of millimolar H2S. Low reactivity was also observed for thiolysis of these ethylamino-, ethanolamino- and anilino-based NBD probes. Therefore, almost all NBD amines used in bioimaging should be stable, in consideration of the presence of only micromolar endogenous H2S in vivo. Moreover, the piperazinyl-NBD derivatives could be efficient in the development of fluorescent H2S probes and for directly visualizing H2S by paper-based detection.

Small-molecule thiols are essential for maintaining the cellular redox homeostasis and play important roles in regulating various cellular functions. New reaction-based fluorescent turn-on probes based on fast thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole)ether were rationally designed for sensing of biothiols in physiological buffer and in cells. The blue-light-excited probes were based on a new developed excited-state intramolecular proton transfer (ESIPT) fluorophore. The combination use of ESIPT fluorophore and electron-withdrawing effect in the probe design led to fast-response fluorescent probes toward cysteine (Cys) with reaction rate (k2) up to 616.7 M−1 s−1. The probes were highly selective toward biothiols over other amino acids and could be used for bioimaging in cells.

The two signaling molecules H2 S and H2 O2 play key roles in maintaining intracellular redox homeostasis. The biological relationship between H2 O2 and H2 S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single- and dual-response fluorescent probes for detecting both H2 O2 and H2 S in living cells. The dual-response probe was shown to be capable of mono- and dual-detection of H2 O2 and H2 S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H2 O2 could induce H2 S biogenesis in living cells. By using gene-knockdown techniques with bioimaging, the H2 S biogenesis was found to be majorly cystathionine β-synthase (CBS)-dependent. Our finding shows the first direct evidence on the biological communication between H2 O2 (ROS) and H2 S (RSS) in vivo.

Biothiols including cysteine (Cys), reduced glutathione (GSH), and hydrogen sulfide (H2S) play critical roles in living organisms. New reaction-based fluorescent turn-on probes based on fast thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) ether were explored for sensing of biothiols in aqueous buffer. The probes showed fast response toward biothiols with more than 100-fold off–on signal enhancement. Moreover, the probes were highly selective toward thiols over other amino acids and could be used for bioimaging in mammalian cells.

Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H2S in real time, new coumarin- and boron-dipyrromethene-based fluorescent turn-on probes were developed for fast sensing of H2S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H2S. On the basis of o-fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H2S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H2S and D-cysteine-dependent endogenously produced H2S in living cells, which makes them promising tools for potential applications in H2S biology.

Hydrogen sulfide (H2S) is an important endogenous signalling molecule and also an important environmental target for detection. New reaction-based colorimetric and fluorescent turn-on probes based on selective thiolyling of NBD (7-nitro-1,2,3-benzoxadiazole) ether were explored for sensing of H2S in aqueous buffer. The syntheses of both probes are simple and quite straightforward. The probes are highly sensitive and selective toward H2S over other biologically relevant species. Probe 1 can be used to directly visualize H2S by the naked eye and shows more than 1000-fold fluorescence increase upon reaction with H2S. Probe 2 is a near-infrared fluorescent sensor for H2S at physiological pH.

Thiol-Michael-cross-linked oligodeoxynucleotides were synthesized through the thiol–maleamic acid methyl ester addition reaction. As a metal-free ‘click’ reaction, the cross-linked method was fast responsive and highly sensitive. The newly synthesized oligodeoxynucleotides were thermally stable and their global structures retained those of non-cross-linked oligodeoxynucleotides and showed responsive reaction to different DNA restriction enzymes.

A method of masking 3′-H-phosphonate group for the solution-phase synthesis of ribonucleotide by H-phosphonate approach was described. The phosphonic acid group was masked by bis-(2-cyanoethyl) boranophosphate during the reactions. After a successive demasking treatment by triethylamine, trityl cation, and triethylamine, the triethylammonium 3′-H-phosphonate nucleotide can be obtained efficiently ready for the coupling cycle in synthesis of oligonucleotide.

During the formation of RNA-induced silencing complex (RISC), the passenger and guide strand of an siRNA duplex separate from each other to generate an active RISC complex. Accumulating evidence shows that an siRNA passenger strand can also assemble into a RISC complex and mediate RNA interference, thereby causing undesired off-target effects. To reduce this effect, the so-called "universal base" 5-nitroindole nucleotides were incorporated into an siRNA passenger strand. Melting temperature and circular dichroism spectrum measurements showed no significant changes compared to the unmodified duplex, thus indicating the formation of normal A-form conformation. Using a dual luciferase reporter assay, we have further shown that 5-nitroindole modification at position 15 of the siRNA passenger strand drastically decreased the RNAi (RNA interfering) potency of this strand, whereas the potency of the RNA guide strand was not much affected. These results could provide a practical approach for reducing off-target effects mediated by the siRNA passenger strand.

A protecting group AZMB was introduced to ribonucleosides 3′-hydroxyl group to facilitate solution phase synthesis of siRNA. The protection and cleavage reaction were carried out in mild conditions, that is protection by acyl chloride and cleavage by triphenylphosphine. The synthesized siRNA showed good biological activity to suppress targeted superoxide dismutase gene expression.

Protoporphyrinogen oxidase (Protox, EC 1.3.3.4) has attracted great interest during the last decades due to its unique biochemical characteristics and biomedical significance. As a continuation of our research work on the development of new PPO inhibitors, 23 new 1,3,4-thiadiazol-2(3H)-ones bearing benzothiazole substructure were designed and synthesized. The in vitro assay indicated that the newly synthesized compounds 1a–w displayed good inhibition activity against human PPO (hPPO) with Ki values ranging from 0.04 μM to 245 μM. To the knowledge, compound 1a, O-ethyl S-(5-(5-(tert-butyl)-2-oxo-1,3,4-thiadiazol-3(2H)-yl)-6-fluorobenzothiazol-2-yl)carbonothioate, with the Ki value of 40 nM, is so far known as the most potent inhibitor against hPPO. Based on the molecular docking and modified molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) calculations, the quantitative structure–activity relationships of 1,3,4-thiadiazol-2(3H)-ones and 1,3,4-oxadiazol-2(3H)-one derivatives were established with excellent correlation relationships (r2 = 0.81) between the calculated and experimental binding free energies. Some important insights were also concluded for guiding the future rational design of new hPPO inhibitors with improved potency.

The characteristics of low application rates, good crop selectivity, low residue and environmental safety exhibited by Protoporphyrinogen oxidase (PPO, EC 1.3.3.4)-inhibiting herbicides have attracted a world-wide research interests. As continuation of our research work on the development of new PPO inhibitors, a series of mono-carbonyl analogues of cyclic imides, N-phenyl pyrrolidin-2-ones and N-phenyl-1H-pyrrol-2-ones, were designed and synthesized based on previously established DFT-QSAR results. The PPO inhibition activities of 29 newly synthesized compounds were tested and a predictive comparative molecular field analysis (CoMFA) model was established with the conventional correlation coefficient r(2)=0.980 and the cross-validated coefficient q(2)=0.518. According to the CoMFA model, the substituent effects on the PPO inhibition activity were explained reasonably. Further greenhouse assay showed that 2-(4-chloro-2-fluoro-5-propoxy-phenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(6), k(i)=0.095μM) and 2-(5-allyloxy-4-chloro-2-fluorophenyl)-2,3,4,5,6,7-hexahydro-isoindol-1-one (C(7), k(i)=0.12μM) displayed excellent post-emergency herbicidal activity at the concentration of 150g.ai/ha against seven tested weeds. Due to their high PPO inhibition effect and broad spectrum herbicidal activity, these two compounds have the potential for further study on crop selectivity and field trial. These results confirmed once again that only one of the carbonyl groups of cyclic imides is essential to the PPO inhibition activity.