2-λ5-Phosphaquinolin-2-ones as Non-cytotoxic, Targetable, and pH-Stable Fluorophores.

Several phosphaquinolinone derivatives have been synthesized and characterized to explore their usefulness in the realm of cell imaging. Solution-state photophysical properties in both aqueous and organic solutions were collected for these derivatives. Additionally, CCK-8 cell viability assays and fluorescent imaging in HeLa cells incubated with the new heterocyclic derivatives show evidence of favorable cell permeability, cell viability, and moderate intracellular localization when appended with the well-known morpholine targeting motif.

Hydrolysis-Based Small-Molecule Hydrogen Selenide (H2Se) Donors for Intracellular H2Se Delivery.

Hydrogen selenide (H2Se) is a central metabolite in the biological processing of selenium for incorporation into selenoproteins, which play crucial antioxidant roles in biological systems. Despite being integral to proper physiological function, this reactive selenium species (RSeS) has received limited attention. We recently reported an early example of a H2Se donor (TDN1042) that exhibited slow, sustained release through hydrolysis. Here we expand that technology based on the P═Se motif to develop cyclic-PSe compounds with increased rates of hydrolysis and function through well-defined mechanisms as monitored by 31P and 77Se NMR spectroscopy. In addition, we report a colorimetric method based on the reaction of H2Se with NBD-Cl to generate NBD-SeH (λmax = 551 nm), which can be used to detect free H2Se. Furthermore, we use TOF-SIMS (time of flight secondary ion mass spectroscopy) to demonstrate that these H2Se donors are cell permeable and use this technique for spatial mapping of the intracellular Se content after H2Se delivery. Moreover, these H2Se donors reduce endogenous intracellular reactive oxygen species (ROS) levels. Taken together, this work expands the toolbox of H2Se donor technology and sets the stage for future work focused on the biological activity and beneficial applications of H2Se and related bioinorganic RSeS.

Efficient inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by sulfuration with solubilized elemental sulfur.

Hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO) have garnered increasing scientific interest in recent decades due to their classifications as members of the gasotransmitter family of signaling molecules. Due to the versatility of sulfur redox chemistry in biological systems, H2S specifically is being studied for its ability to modulate cellular redox environments, particularly through the downstream production of oxidized sulfur species. A major mechanism of this regulation is through a posttranslational modification known as persulfidation, where oxidized sulfur atoms are appended to free cysteine in proteins. Currently, it is difficult to discern the activity of H2S itself versus these oxidized sulfur species, particularly sulfane sulfur (S0). We have previously developed a method of solvating S8, a source of pure S0, to more accurately study persulfidation and sulfuration in general. Here, we apply this pure S0 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has previously been shown to be inhibited by S0-containing polysulfides via persulfidation. Using solvated S0, we demonstrate that native, reduced GAPDH can be completely inhibited by sulfuration with S0. Further, oxidized GAPDH activity cannot be rescued using S0, demonstrating that it is the oxidation of reduced GAPDH by S0 that curtails its activity. We also compare inhibition of GAPDH by pure S0 to different polysulfides and demonstrate the modulating effects that pendant alkyl groups have on GAPDH inhibition. These results highlight the promise of this novel, simplified system for the study of S0.

Subcellular Targeted Nanohoop for One- and Two-Photon Live Cell Imaging.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 µM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

Modified cyclodextrins solubilize elemental sulfur in water and enable biological sulfane sulfur delivery.

An important form of biological sulfur is sulfane sulfur, or S0, which is found in polysulfide and persulfide compounds as well as in elemental sulfur. Sulfane sulfur, often in the form of S8, functions as a key energy source in the metabolic processes of thermophilic Archaean organisms found in sulfur-rich environments and can be metabolized both aerobically and anaerobically by different archaeons. Despite this importance, S8 has a low solubility in water (∼19 nM), raising questions of how it can be made chemically accessible in complex environments. Motivated by prior crystallographic data showing S8 binding to hydrophobic motifs in filamentous glycoproteins from the sulfur reducing Staphylothermus marinus anaerobe, we demonstrate that simple macrocyclic hydrophobic motifs, such as 2-hydroxypropyl ß-cyclodextrin (2HPß), are sufficient to solubilize S8 at concentrations up to 2.0 ± 0.2 mM in aqueous solution. We demonstrate that the solubilized S8 can be reduced with the common reductant tris(2-carboxyethyl)phosphine (TCEP) and reacts with thiols to generate H2S. The thiol-mediated conversion of 2HPß/S8 to H2S ranges from 80% to quantitative efficiency for Cys and glutathione (GSH). Moreover, we demonstrate that 2HPß can catalyze the Cys-mediated reduction of S8 to H2S in water. Adding to the biological relevance of the developed systems, we demonstrate that treatment of Raw 264.7 macrophage cells with the 2HPß/S8 complex prior to LPS stimulation decreases NO2 - levels, which is consistent with known activities of bioavailable H2S and sulfane sulfur. Taken together, these investigations provide a new strategy for delivering H2S and sulfane sulfur in complex systems and more importantly provide new insights into the chemical accessibility and storage of S0 and S8 in biological environments.

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.

Effects of sulfane sulfur content in benzyl polysulfides on thiol-triggered H2S release and cell proliferation.

Investigations into hydrogen sulfide (H2S) signaling pathways have demonstrated both the generation and importance of persulfides, which are reactive sulfur species that contain both reduced and oxidized sulfur. These observations have led researchers to suggest that oxidized sulfur species, including sulfane sulfur (S0), are responsible for many of the physiological phenomena initially attributed to H2S. A common method of introducing S0 to biological systems is the administration of organic polysulfides, such as diallyl trisulfide (DATS). However, prior reports have demonstrated that commercially-available DATS often contains a mixture of polysulfides, and furthermore a lack of structure-activity relationships for organic polysulfides has limited our overall understanding of different polysulfides and their function in biological systems. Advancing our interests in the chemical biology of reactive sulfur species including H2S and S0, we report here our investigations into the rates and quantities of H2S release from a series of synthetic, pure benzyl polysulfides, ranging from monosulfide to tetrasulfide. We demonstrate that H2S is only released from the trisulfide and tetrasulfide, and that this release requires thiol-mediated reduction in the presence of cysteine or reduced glutathione. Additionally, we demonstrate the different effects of trisulfides and tetrasulfides on cell proliferation in murine epithelial bEnd.3 cells.

Light-Activated COS/H2S Donation from Photocaged Thiocarbamates.

Hydrogen sulfide (H2S) is an important biomolecule, and responsive chemical tools for its delivery are needed. Here, we utilize the photocleavable o-nitrobenzyl group to unmask caged thiocarbamates and to access photoactivated H2S releasing molecules. These donors function by the initial release of carbonyl sulfide (COS), which is quickly hydrolyzed to H2S by carbonic anhydrase (CA). Our investigations demonstrate that o-nitrobenzyl-caged thiocarbamates can serve as a donor platform for the bio-orthogonal stimulated release of COS/H2S.