Ligand-Directed H2 S Probe and Scavenger for Specific Tumor Imaging.

An expanding body of evidence suggests that specifically targeting hydrogen sulfide (H2 S) might potentially benefit both tumor diagnosis and treatment, but there is still a lack of cancer-targeted molecular tools for in vivo applications. Here, we report the first ligand-directed H2 S-specific near-infrared fluorescent sensor PSMA-Cy7-NBD and scavenger PSMA-Py-NBD that target the prostate-specific membrane antigen (PSMA). PSMA-Cy7-NBD displays a 53-fold off-on fluorescence response to H2 S at 803 nm with high specificity. PSMA-Py-NBD can scavenge H2 S fast (k2 =30.8 M-1 s-1 at 25 °C) without interference from biothiols. Both tools are highly water-soluble and can be transported selectively into PSMA-expressing prostate cancer cells. Endogenous H2 S levels in murine 22Rv1 tumor models can be imaged and downregulated by intravenous injection of PSMA-Cy7-NBD and PSMA-Py-NBD, respectively. These tools could potentially help to investigate H2 S cancer biology and with related therapies.

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.

Insights into self-degradation of cysteine esters and amides under physiological conditions yield new cleavable chemistry.

An unprecedented H2S release from cysteine esters and amides (CysO/NHR) under physiological conditions was discovered and the plausible mechanism was proposed. Alkylation of the amino moiety of cysteine esters enables the H2S release to be tuned and further provides support to the mechanistic insights. This discovery not only provides new insights into several fundamental science issues including non-enzymatic H2S-produced pathways, but also inspires new tunable cleavable motifs for sustained release of arylthiols and even for prodrug design.

A unique reaction of diphenylcyclopropenone and 1,2-aminothiol with the release of thiol for multiple bioconjugation.

Selective reaction of diphenylcyclopropenone (DPCP) and 1,2-aminothiol in water at pH 7.4 produces an amide conjugate with the release of thiol. In addition, structural modifications of DPCP enable the coupling rate to be tuned with a reaction constant of +3.68. Based on this chemistry, triple labelling was demonstrated by treating an N-terminal cysteine peptide with DPCP-Cl followed by thiol-maleimide and tyrosine-diazonium couplings in one pot. We anticipate that the DPCP motif will be a useful toolkit for multiple bioconjugation.

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.

Cell-Trappable BODIPY-NBD Dyad for Imaging of Basal and Stress-Induced H2S in Live Biosystems.

H2S is a gaseous signaling molecule that is involved in many physiological and pathological processes. In general, the level of intracellular H2S (<1 µM) is much lower than that of GSH (∼1-10 mM), leading to the remaining challenge of selective detection and differentiation of endogenous H2S in live biosystems. To this end, we quantitatively demonstrate that the thiolysis of NBD amine has much higher selectivity for H2S over GSH than that of the reduction of aryl azide. Subsequently, we developed the first NBD-based cell-trappable probe 1 (AM-BODIPY-NBD) for highly selective and ultrasensitive imaging of intracellular H2S. Probe 1 demonstrates a 207-fold fluorescence enhancement at 520 nm after reaction with H2S/esterase to produce a bright BODIPY (quantum yield 0.42) and a detection limit of 15.7 nM. Probe 1 is water-soluble, cell-trappable, and not cytotoxic. Based on this excellent chemical tool, relative levels of basal H2S in different cell lines were measured to reveal a positive correlation between endogenous H2S and the metastatic potential of colon and breast cancer cells. In addition, H2S biogenesis in vivo was also validated by probe 1 both in tobacco leaves under viral infection and in zebrafish after tail amputation. It is anticipated that probe 1 will have widespread applications in imaging and for investigating different H2S-related biological processes and diseases.

Thiolysis of CBD arylethers for development of highly GSH-selective fluorescent probes.

Thiolysis of 7-cyanobenzoxadiazole (CBD) arylether was investigated for development of GSH-selective fluorescent probes for the first time. The results demonstrate that CBD-based probes have tunable reactivities and appropriate dissociation constants for GSH, and are highly GSH-selective and suitable for bioimaging.

Investigation of thiolysis of 4-substituted SBD derivatives and rational design of a GSH-selective fluorescent probe.

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.

A H2 S-Specific Ultrasensitive Fluorogenic Probe Reveals TMV-Induced H2 S Production to Limit Virus Replication.

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.

Highly efficient H2S scavengers via thiolysis of positively-charged NBD amines.

H2S is a well-known toxic gas and also a gaseous signaling molecule involved in many biological processes. Advanced chemical tools that can regulate H2S levels in vivo are useful for understanding H2S biology as well as its potential therapeutic effects. To this end, we have developed a series of 7-nitro-1,2,3-benzoxadiazole (NBD) amines as potential H2S scavengers. The kinetic studies of thiolysis reactions revealed that incorporation of positively-charged groups onto the NBD amines greatly increased the rate of the H2S-specific thiolysis reaction. We demonstrate that these reactions proceed effectively, with second order rate constants (k 2) of >116 M-1 s-1 at 37 °C for NBD-S8. Additionally, we demonstrate that NBD-S8 can effectively scavenge enzymatically-produced and endogenous H2S in live cells. Furthering the biological significance, we demonstrate NBD-S8 mediates scavenging of H2S in mice.