Chung-Min Park

Reaction Based Fluorescent Probes for Hydrogen Sulfide

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H(2)S) was developed. This strategy was based on a H(2)S-specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.

pH-Controlled Hydrogen Sulfide Release for Myocardial Ischemia-Reperfusion Injury

Hydrogen sulfide (H2S) is a critical signaling molecule that regulates many physiological and/or pathological processes. Modulation of H2S levels could have potential therapeutic value. In this work, we report the rational design, synthesis, and biological evaluation of a class of phosphonamidothioate-based H2S-releasing agents (i.e., H2S donors). A novel pH-dependent intramolecular cyclization was employed to promote H2S release from the donors. These water-soluble compounds showed slow, controllable, and pH-sensitive production of H2S in aqueous solutions. The donors also showed significant cytoprotective effects in cellular models of oxidative damage. Most importantly, the donors were found to exhibit potent cardioprotective effects in an in vivo murine model of myocardial ischemia-reperfusion (MI/R) injury through a H2S-related mechanism.

Thiol-Activated gem-Dithiols: A New Class of Controllable Hydrogen Sulfide Donors

A class of novel thiol-activated H2S donors has been developed on the basis of the gem-dithiol template. These donors release H2S in the presence of cysteine or GSH in aqueous solutions as well as in cellular environments.

Use of metabolomics for the chemotaxonomy of legume-associated Ascochyta and allied genera.

Chemotaxonomy and the comparative analysis of metabolic features of fungi have the potential to provide valuable information relating to ecology and evolution, but have not been fully explored in fungal biology. Here, we investigated the chemical diversity of legume-associated Ascochyta and Phoma species and the possible use of a metabolomics approach using liquid chromatography-mass spectrometry for their classification. The metabolic features of 45 strains including 11 known species isolated from various legumes were extracted, and the datasets were analyzed using chemometrics methods such as principal component and hierarchical clustering analyses. We found a high degree of intra-species consistency in metabolic profiles, but inter-species diversity was high. Molecular phylogenies of the legume-associated Ascochyta/Phoma species were estimated using sequence data from three protein-coding genes and the five major chemical groups that were detected in the hierarchical clustering analysis were mapped to the phylogeny. Clusters based on similarity of metabolic features were largely congruent with the species phylogeny. These results indicated that evolutionarily distinct fungal lineages have diversified their metabolic capacities as they have evolved independently. This whole metabolomics approach may be an effective tool for chemotaxonomy of fungal taxa lacking information on their metabolic content.

Functional Analyses of the Diels-Alderase Gene sol5 of Ascochyta rabiei and Alternaria solani Indicate that the Solanapyrone Phytotoxins Are Not Required for Pathogenicity

Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.

Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-β-lactamase Superfamily.

Background: Persulfide dioxygenases (PDOs), which belong to the metallo-β-lactamase (MBL) enzyme superfamily, oxidize glutathione persulfide (GSSH). Results: Crystal structures and ITC data provide information on ligand binding by PDOs. Conclusion: MBLs share conserved amino acid residues, but the functions of these residues vary by class. Significance: These results provide criteria for distinguishing PDOs from other MBL superfamily members. Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-β-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and β-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-β-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.

Discovery of Heteroaromatic Sulfones As a New Class of Biologically Compatible Thiol-Selective Reagents

The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.

9-Fluorenylmethyl (Fm) Disulfides: Biomimetic Precursors for Persulfides

The development of a functional disulfide, FmSSPy-A (Fm = 9-fluorenylmethyl; Py = pyridinyl), is reported. It can effectively convert small molecule and protein thiols (−SH) to form −S-SFm adducts under mild conditions. This method allows for a H2S-free and biomimetic protocol to generate highly reactive persulfides (in their anionic forms). The high nucleophilicity of persulfides toward a number of thiol-blocking reagents is also demonstrated. The method holds promise for further understanding the chemical biology of persulfides and S-sulfhydration.

Use of Phosphorodithioate-Based Compounds as Hydrogen Sulfide Donors

Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays were used to determine H2S release from the donors in both aqueous buffers and in cultured cells. These donors were found to be slow-release donors, much like the well-known GYY4137. These donors also showed some protective effects against hydrogen peroxide (H2O2)-induced oxidative damage in myocytes.

Proline-based phosphoramidite reagents for the reductive ligation of S-nitrosothiols

S-Nitrosothiols (RSNOs) have many biological implications but are rarely used in organic synthesis. In this work we report the development of proline-based phosphoramidite substrates that can effectively convert RSNOs to proline-based sulfenamides through a reductive ligation process. A unique property of this method is that the phosphine oxide moiety on the ligation products can be readily removed under acidic conditions. In conjugation with the facile preparation of RSNOs from the corresponding thiols (RSHs), this method provides a new way to prepare proline-based sulfenamides from simple thiol starting materials.