A highly selective and sensitive endoplasmic reticulum-targeted probe reveals HOCl- and cisplatin-induced H2S biogenesis in live cells.

Reactive oxygen species (ROS) and reactive sulfur species (RSS) are involved in many physiological processes and act as collaborators with crosstalk. As an important member of gasotransmitters and RSS, hydrogen sulfide (H2S) carries out signaling functions at submicromolar levels because of its high reactivity. Mechanisms of dynamic regulation of ROS and H2S production are poorly understood, and the development of a highly selective and organelle-targeted chemical tool will advance the further understanding of H2S chemical biology and ROS/RSS crosstalk. Herein, we report a highly selective and sensitive, endoplasmic reticulum (ER)-targeted fluorescent probe (ER-BODIPY-NBD) for revealing cisplatin-induced H2S biogenesis for the first time. The probe demonstrates a 152-fold fluorescence enhancement at 520 nm after reaction with H2S to release a bright BODIPY product (quantum yield 0.36). The probe is highly selective toward H2S over biothiols, ER-targeted, and biocompatible. In addition, the probe was successfully employed to track H2S biogenesis in live cells via stimulation from exogenous hypochlorous acid and the drug cisplatin.

Multiple-Gene Regulation for Enhanced Antitumor Efficacy with Branch-PCR-Assembled TP53 and MYC Gene Nanovector.

Multiple proteins are involved in network regulation through the crosstalk of different signaling pathways in cancers. Here, we propose a novel strategy of genome therapy with branch-PCR-assembled gene nanovectors to perform network-based gene regulation at multiple levels for cancer therapy. To validate network-based multiplex-gene regulation for genome therapy, we chose to simultaneously target one tumor suppressor gene (TP53) and one oncogene (MYC) in two different signaling pathways. The results showed that, compared to gene nanovectors targeting single genes (NP-TP53 and NP-shMYC), branch-PCR-assembled gene nanovectors simultaneously expressing p53 proteins and MYC shRNA arrays (NP-TP53-shMYC) showed enhanced antitumor efficacy in both MDA-MB-231 cancer cells and an MDA-MB-231-tumor-bearing mouse model. These findings indicate the feasibility and effectiveness of genome therapy in cancer therapy.

The Potential Application of Branch-PCR Assembled PTEN Gene Nanovector in Lung Cancer Gene Therapy.

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 18th  day of administration. The anticancer efficacy of NP-PTEN in NCI-H1299 tumor xenograft suggests the promising therapeutic potential of branch-PCR assembled PTEN gene nanovectors in lung cancer gene therapy and also provided more opportunities to introduce two or more tumor suppressor genes as an all-in-one gene nanovector for multiple gene-based cancer gene therapy.

Rational design of a dual-reactive probe for imaging the biogenesis of both H2S and GSH from l-Cys rather than d-Cys in live cells.

Biothiols and their interconversion are involved in cellular redox homeostasis as well as many physiological processes. Here, a dual-reactive dual-quenching fluorescent probe was rationally developed based on thiolysis reactions of 7-nitrobenzoxadiazole (NBD) tertiary amine and 7-cyanobenzoxadiazole (CBD) arylether for imaging of the biothiol interconversion. We demonstrate that the NBD-CBD probe exhibits very weak background fluorescence due to the dual-quenching effects, and can be dual-activated by H2S and GSH with an over 500-fold fluorescence increase at 525 nm. In addition, the probe shows high sensitivity, excellent selectivity, and good biocompatibility, all of which promote the simultaneous detection of both H2S and GSH in live cells. Importantly, probe 1 was successfully employed to reveal the biogenesis of both H2S and GSH from l-Cys rather than from d-Cys, and therefore, d-Cys would be solely converted into H2S, which may help understand the more H2S generation from d-Cys than from l-Cys in live cells.

An NBD tertiary amine is a fluorescent quencher and/or a weak green-light fluorophore in H2S-specific probes.

The thiolysis of NBD piperazinyl amine (NBD-PZ) is highly selective for H2S over GSH and has been widely used for the development of many H2S fluorescent probes. Whether the NBD amine in H2S-specific probes could be a fluorescent quencher should be further clarified, because NBD amines have been used as environment-sensitive fluorophores for many years. Here, we compared the properties of NBD-based secondary and tertiary amines under the same conditions. For example, the emission of NBD-N(Et)2 is much smaller in water and less responsive to changes in polarity than that of NBD-NHEt. The emission of NBD-PZ-TPP is also smaller than that of NBD-NH-TPP both in aqueous buffer and in live cells. In addition, confocal bioimaging signals of NBD-PZ-TPP with excitation at 405 nm and 454 nm are much weaker than that at 488 nm. Based on these results as well as the previous work on NBD-based probes, we discuss and summarize the design strategies and sensing mechanisms for different NBD-based H2S probes. Moreover, NBD-PZ-TPP may be a useful tool for reaction with and imaging of mitochondrial H2S in live cells. This work should be useful for clarification of the roles of NBD in H2S-specific fluorescent probes as well as for facilitating the development of future NBD-based probes.

Cysteine-Activated Small-Molecule H2Se Donors Inspired by Synthetic H2S Donors.

The importance of selenium (Se) in biology and health has become increasingly clear. Hydrogen selenide (H2Se), the biologically available and active form of Se, is suggested to be an emerging nitric oxide (NO)-like signaling molecule. Nevertheless, the research on H2Se chemical biology has technique difficulties due to the lack of well-characterized and controllable H2Se donors under physiological conditions, as well as a robust assay for direct H2Se quantification. Motivated by these needs, here, we demonstrate that selenocyclopropenones and selenoamides are tunable donor motifs that release H2Se upon reaction with cysteine (Cys) at pH 7.4 and that structural modifications enable the rate of Cys-mediated H2Se release to be tuned. We monitored the reaction pathways for the H2Se release and confirmed H2Se generation qualitatively using different methods. We further developed a quantitative assay for direct H2Se trapping and quantitation in an aqueous solution, which should also be operative for investigating future H2Se donor motifs. In addition, we demonstrate that arylselenoamide has the capability of Cys-mediated H2Se release in cellular environments. Importantly, mechanistic investigations and density functional theory (DFT) calculations illustrate the plausible pathways of Cys-activated H2Se release from arylselenoamides in detail, which may help understand the mechanistic issues of the H2S release from pharmacologically important arylthioamides. We anticipate that the well-defined chemistries of Cys-activated H2Se donor motifs will be useful for studying Se biology and for development of new H2Se donors and bioconjugate techniques.

Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing.

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.

The one-pot nonhydrolysis Staudinger reaction and Staudinger or SPAAC ligation.

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.

Branch-PCR constructed TP53 gene nanovector for potential cancer therapy.

A novel and efficient branch PCR strategy has been used to construct a TP53 gene nanovector based on a pair of trimers as primers, which showed unique advantages compared to other existing systems for gene delivery and effective potential cancer therapy.

Tetrafluorination of Aromatic Azide Yields a Highly Efficient Staudinger Reaction: Kinetics and Biolabeling.

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.

A Redox-Nucleophilic Dual-Reactable Probe for Highly Selective and Sensitive Detection of H2S: Synthesis, Spectra and Bioimaging.

Hydrogen sulfide (H2S) is an important signalling molecule with multiple biological functions. The reported H2S fluorescent probes are majorly based on redox or nucleophilic reactions. The combination usage of both redox and nucleophilic reactions could improve the probe's selectivity, sensitivity and stability. Herein we report a new dual-reactable probe with yellow turn-on fluorescence for H2S detection. The sensing mechanism of the dual-reactable probe was based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amine (a nucleophilic reaction) and reduction of azide to amine (a redox reaction). Compared with its corresponding single-reactable probes, the dual-reactable probe has higher selectivity and fluorescence turn-on fold with magnitude of multiplication from that of each single-reactable probe. The highly selective and sensitive properties enabled the dual-reactable probe as a useful tool for efficiently sensing H2S in aqueous buffer and in living cells.

o-Fluorination of aromatic azides yields improved azido-based fluorescent probes for hydrogen sulfide: synthesis, spectra, and bioimaging.

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.

2-[4-(1H-1,2,4-Triazol-1-yl)phen-yl]-1H-benzimidazole.

In the title compound, C(15)H(11)N(5), the benzimidazole ring system is nearly planar [maximum deviation = 0.039 (2) Å], and is oriented at a dihedral angle of 28.85 (10)° with respect to the benzene ring; the dihedral angle between the triazole and benzene rings is 17.30 (15)°. In the crystal N-H⋯N hydrogen bonds link the mol-ecules into chains. Weak C-H⋯N inter-actions are also present.