Hydrogen Sulfide in Mammalian Cells: Comparison
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Hydrogen sulfide (H2S) was recognized as a gaseous signaling molecule, similar to nitric oxide (-NO) and carbon monoxide (CO). The aim of this review is to provide an overview of the formation of hydrogen sulfide (H2S) in the human body. H2S is synthesized by enzymatic processes involving cysteine and several enzymes, including cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (3MST) and D-amino acid oxidase (DAO). The physiological and pathological effects of hydrogen sulfide (H2S) on various systems in the human body have led to extensive research efforts to develop appropriate methods to deliver H2S under conditions that mimic physiological settings and respond to various stimuli. These functions span a wide spectrum, ranging from effects on the endocrine system and cellular lifespan to protection of liver and kidney function. The exact physiological and hazardous thresholds of hydrogen sulfide (H2S) in the human body are currently not well understood and need to be researched in depth. 

  • hydrogen sulfide
  • chemistry
  • gasotransmitter
  • physiology

1. Biological Functions of H2S

Endogenous H2S refers to the natural presence of this compound in the body because of regular metabolic processes in humans, animals and other organisms. Research conducted from the early 2000s to the present has revealed that endogenous H2S plays a critical role in regulating specific systems and processes in living organisms [82][1]. H2S has a high affinity for lipids, which makes it extremely lipophilic and facilitates its penetration of cell membranes, allowing it to enter various cell types. H2S plays a central role in the regulation of various physiological and pathological processes [83,84][2][3]. The potential consequences of decreasing H2S levels in the human body include the development and progression of various health conditions, including hypertension, atherosclerosis, gastrointestinal ulcers, cirrhosis of the liver, diabetes, inflammation, Alzheimer’s disease, cancer and other diseases [85][4]. The accompanying diagram provides a comprehensive representation of the biological mechanisms in human physiology that are controlled by endogenous H2S or show a response to pharmacological intervention with H2S or its derivatives (Figure 18).
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Figure 18.
Examples of diseases related to low levels of H
2
S.
Among the variety of effects attributed to H2S, it can be mention that its participation in cardiovascular processes (in vasodilation, one of the first effects described), the central nervous system, the gastrointestinal system, the endocrine system, cytoprotection, etc. [86,87,88,89][5][6][7][8] (Figure 192).
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Figure 192. Physiological roles of H2S.

1.1. Cardiovascular System

H2S plays a central role in modulating and regulating various signaling pathways involved in metabolism, cardiac function and cellular viability in mammalian organisms [90][9], and it has a significant effect on the cardiovascular system, blood vessels and blood components. Both mitochondrial activity and cellular metabolism are affected [21][10].
H2S has been found to be associated with hypertension, atherosclerosis and myocardial damage in the cardiovascular system. The potential efficacy of this compound in the treatment of hypertension may be due to its ability to induce vasodilation. Relaxation of the rat thoracic aorta, portal vein and mesenteric artery by H2S has been demonstrated, suggesting that hydrogen sulfide plays an important role in regulating contractility and blood pressure. In contrast, another study suggests that H2S exhibits vasoconstrictor properties at low concentrations, possibly through a mechanism that inhibits the activity of nitric oxide (-NO), a molecule also involved in contractility [91,92][11][12]. H2S may help patients recover from myocardial injury, particularly ischemia–reperfusion injury. Numerous cardiovascular diseases have been associated with H2S, suggesting a potentially broad applicability of H2S in the context of heart disease [90][9].
H2S exerts several effects on the cardiovascular system. These include attenuating ischemia–reperfusion injury to cardiac tissue, facilitating angiogenesis, relaxing smooth muscle cells and regulating blood pressure [93][13].

1.2. Gastrointestinal System

H2S is known to have a significant effect on reducing gastric mucosal damage and has the potential to act as a crucial mediator of gastrointestinal motility.
Insulin secretion and diabetes mellitus may be affected by H2S because the pancreas is among the targets of H2S [94,95,96][14][15][16]. In the pancreas, CSE is the main enzyme that converts cysteine to H2S. H2S concentration is elevated in response to the presence of pancreatitis, which is attributed to its proinflammatory effect. H2S administration contributes to chloride secretion, which aggravates certain types of gastritis [97][17]. H2S concentration increases when abdominal sepsis or endotoxemia occurs [98][18].
H2S has a protective anti-inflammatory effect on the gastrointestinal system in some types of gastritis and colitis [99,100,101][19][20][21]. H2S elicits both proinflammatory and anti-inflammatory responses in various models of inflammation [102][22]. The synthesis of H2S is markedly increased in colon ulcers, resulting in accelerated restoration of epithelial barrier integrity and healing of injured tissues [103][23].

1.3. Respiratory System

A study that investigated the relationship between H2S and pulmonary hypertension represents a pioneering achievement in the field of pathophysiology and H2S on a global scale [104][24]. In recent years, numerous studies have been conducted to investigate the involvement of H2S in the development of pulmonary hypertension. These studies have primarily focused on the administration of H2S to animal models suffering from chronic hypoxia.
H2S is implicated in the pathogenesis and therapeutic interventions of chronic obstructive pulmonary disease, as endogenous H2S is involved in the regulation of physiological functions of the respiratory system and pathophysiological alterations, such as chronic obstructive pulmonary disease, asthma, pulmonary fibrosis and hypoxia-induced pulmonary hypertension [105][25].

1.4. Nervous System

In addition, H2S exerts its action on important functions of the central nervous system and provides neuroprotective protection against oxidative stress. There is a belief that it has potentially protective properties against neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease [106][26]; spinocerebellar ataxia; and traumatic brain injury. It has been published that H2S levels are reduced in the brains of patients with Alzheimer’s disease compared to healthy individuals [107][27]. In the central nervous system, both sides of H2S are observed: it ameliorates ischemic lesions, but it leads to the aggravation of stroke.

1.5. Endocrine System

The variation in H2S concentration is related to a variety of endocrine disorders [108][28]. Understanding the effect of H2S concentration on the endocrine system is useful for the treatment of hypertension, diabetes and other diseases. H2S can affect the secretion of many hormones and participate in the onset and development of endocrine diseases. H2S can regulate hormone secretion through antioxidant stress and regulation of ion channels and protect endocrine organs. H2S can regulate glucose and fat metabolism through the pancreas, liver, adipose tissue and skeletal muscle [109][29].

1.6. Visual System

H2S protects retinal photoreceptor cells from light-induced degeneration. Deficiency of H2S or its substrates was found to be associated with ectopialentis, myopia, cataracts, optic atrophy and retinal detachment [110,111,112][30][31][32].

1.7. Age-Related Diseases

H2S is a reducing agent and can be metabolized by various oxidants in the human body. It counteracts oxidative species, such as reactive oxygen species (ROS and reactive nitrogen species (RNS), in the human body. The activation of antioxidant enzymes serves to limit the reactions of free radicals and thus protects against the harmful effects of aging [113][33].
H2S has several cytoprotective and physiological functions related to age-related diseases. Most notably, it serves as a potent antioxidant and gasotransmitter. Oxidative stress plays an important role in the development and progression of many age-related diseases [114][34].

1.8. H2S in Cancer

It is documented that several types of cancer such as colon, breast, ovarian and prostate cancers have higher levels of CBS, CSE or 3MST enzymes or synthesize greater amounts of H2S compared to adjacent non-tumor tissue [115,116,117,118][35][36][37][38].
The outcome of treating cancer cells with H2S donors depends on the concentration/dose, time, cell type and drug used. The effects of both natural and synthetic donors vary from potent cancer suppressors to promoters [40,119][39][40]. The administration of H2S donors to various cancer cell lines has been shown to produce cell death, with this effect being dependent on increasing concentrations of H2S. This indicates that H2S donors could represent a therapeutic potential as anticancer drugs. The combination of non-steroidal anti-inflammatory drugs with slow-release H2S donors has been shown to effectively inhibit the growth of human colon, mammary, pancreatic, prostate, lung and bone marrow cancer cells by promoting cell apoptosis via the activation of p38 MAPK [120][41].

1.9. H2S and Antimicrobial Resistance

The prevalence of antimicrobial resistance (AMR) is increasing and represents a major public health challenge [121][42]. Similar to mammalian organisms, bacterial cells also have three enzymes involved in the synthesis of H2S [122][43]. These enzymes are known as cystathionine-γ-lyase (CSE), cystathionine-β-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3MST) [123][44]. Bacteria have been shown to produce H2S as a cytoprotective agent in response to host-induced stress, such as oxidative stress and antibiotics [124][45]. Endogenously produced H2S stimulates ROS-scavenging enzymes and interferes with the Fenton reaction, reducing the amount of ROS produced by cells and promoting antibiotic tolerance [125][46]. Antibiotics such as quinolones, beta-lactams and aminoglycosides are more effective against bacterial pathogens in vitro and in mouse models when combined with small compounds that block a bacterial enzyme involved in the formation of hydrogen sulfide [126][47]. Therefore, it has been hypothesized that this messenger is a fundamental anti-antibiotic defense mechanism in bacteria [126,127][47][48]. In biofilms, persisting cells had significantly higher H2S content than active cells, supporting the notion that H2S is a critical component in bacterial biofilm formation [128][49]. However, not all significant pathogenic bacteria encode the H2S biosynthetic pathway. Pathogenic Acinetobacter baumannii bacteria do not produce endogenous H2S. By manipulating the sulfide content of A. baumannii with a H2S-releasing chemical, researchers showed that exogenous-H2S-sensitized A. baumannii was able to reverse acquired resistance to gentamicin [129][50]. It appears that the presence of exogenous H2S triggered a disruption of redox and energy balance that ultimately led to increased susceptibility to the lethal effects of antibiotics [126][47]. Therefore, it was hypothesized that H2S can be used as an antibiotic-potentiating and resistance-converting agent in bacteria that do not produce it themselves [129][50]. The recognition of H2S biogenesis is a promising focus for the development of antibacterial adjuvants to combat tolerance and resistance [130][51]. Influencing hydrogen sulfide-based defenses is a largely unexplored alternative to conventional antibiotic discovery.

2. Mechanisms of Action of H2S and Molecular Targets

The mechanisms through which H2S exerts its effects are not yet fully understood. However, there is sufficient consensus regarding four mechanisms of action or molecular targets (Figure 203).
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Figure 203. H2S signaling mechanisms.
H2S is of great importance as a secondary messenger that binds to specific target proteins to facilitate signal transduction, especially in the field of mammalian physiology. It is now known that the signal transduction function of H2S occurs through at least three main mechanisms: (i) interactions with metal centers [131][52], (ii) scavenging of ROS and RNS [132][53], (iii) S-persulfidation [6][54] and (iv) effects on ion channels.

2.1. Effect of H2S on Ion Channels

There is abundant literature on the effect of H2S on ion channels. For example, H2S opens ATP-dependent potassium channels [133,134][55][56] and modulates various types of calcium [135,136,137][57][58][59] and chloride channels [138][60].
Ion channels are proteins that form pores in the membranes of cells and organelles, having the function of regulating the flow of ions through them. H2S can act directly or indirectly on the channels. ATP-dependent potassium channels (K+ATP) are the most studied in terms of their interaction with H2S.
K+ATP is a hetero-octamer consisting of four pore-forming subunits, Kir6.x, and four regulatory subunits, SURx, and their activity is inhibited by binding to ATP [139][61] (Figure 214).
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Figure 214. KATP channel subunits.
Cysteine 43 of the Kir6.1 subunit is the target of H2S persulfuration. The interaction of the gas with the protein was accompanied by decreased ATP binding and increased binding of phosphatidyl inositol diphosphate, suggesting that the effect of the interaction with H2S decreases the affinity of the protein for ATP, thus activating the channel. Other authors showed with targeted mutagenesis experiments that the effect of H2S was only observed when the two subunits, Kir6.1 and rvSUR1, were present, but not with Kir6.1 alone [139][61]. They also identified cysteine residues in the regulatory subunit, cysteine 6 and cysteine 26, necessary for the effect.
H2S is unable to regulate the function of K+ATP channels directly [140][62]. It has been observed that the cardioprotective effects of H2S partially disappear when K+ATP channels are chemically blocked [141][63].

2.2. Direct Reaction of H2S with ROS and RNS

There is conflicting evidence for the influence of H2S on various organisms, mainly due to its toxic nature and its ability to scavenge reactive oxygen species (ROS) and thus mitigate oxidative stress [7,20][64][65]. Hydrogen peroxide, peroxynitrite, hypochlorite and the superoxide radical anion are examples of ROS and reactive nitrogen species (RNS) that can react with H2S. This means that it can inhibit the harmful effects of ROS and/or RNS on organic substances.
Recently, there has been increased interest in the study of H2S due to its toxic nature and its ability to protect bacteria from the harmful effects of oxidative stress induced by antibiotic therapy [142][66]. The process has the ability to render the redox centers of metalloenzymes inactive [143[67][68][69][70],144,145,146], causing DNA damage [147][71] and protein denaturation by disrupting disulfide bonds [148][72].

2.2.1. Non-Radical Species (Two-Electron Oxidation)

H2S reacts with two-electron oxidants such as hydrogen peroxide, peroxonitrous acid and hypochlorous acid and chloramines to transform into sulfenic acid (HSOH) which is an unstable intermediate (Figure 225).
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Figure 225. Oxidation of H2S by H2O2, ONOOH and HOCl. Formation of sulfenic acid (HSOH).
The primary outcome of the chemical reaction between H2S and hydrogen peroxide (H2O2) is the formation of hydroxylthiol (HSOH). The final product consists largely of polysulfides, elemental sulfur and, in the case of excess oxidant, sulfate, and it depends on the initial ratios of hydrogen peroxide and H2S.
The direct reaction between peroxynitrous acid and HS− involves a nucleophilic substitution of HS−, leading to the formation of HSOH and NO2− as starting products. In the presence of an excess amount of H2S, HSOH further reacts with a second HS−, leading to the formation of HSS−/HSSH and other compounds.
It is likely that the interaction between hypochlorous acid and HS− occurs through the formation of HSCl, which is subsequently subjected to rapid hydrolysis to produce HSOH. Chloramines, particularly RHNCl and R2NCl, exhibit lower reactivity but higher selectivity as oxidants compared to hypochlorous acid.
HSOH is the main product. Polysulfides, elemental sulfur and sulfate can all be produced by this reaction, but the exact composition of the final product depends on the ratio of hydrogen peroxide to hydrogen sulfide. It is noteworthy that the system exhibits the typical properties of a chemical oscillator. The oxidation of H2S can lead to the formation of a very reactive reductant: sulfoxylic acid. In many respects, H2S exhibits a reactivity profile similar to cysteine. It is a powerful nucleophile and, like thiols, can react with electrophiles as well as oxidants.
The oxidation process of H2S can lead to the formation of many compounds in which the sulfur atom can have oxidation states from −2 to +6. The oxidation products include several chemical compounds such as sulfate (SO42−), sulfite (SO32−), thiosulfate (S2O32−), persulfides (RSS−), organic and inorganic polysulfides and elemental sulfur (Sn).
The oxidation pathways of thiols lead to the formation of sulfenic acids as transient intermediates. The main mechanism by which they are degraded is the formation of disulfide bonds in response to the presence of another thiol, and they are widely recognized for their instability [153,154][73][74].

2.2.2. Radical Species (One-Electron Oxidation)

H2S can be oxidized to HS−/S•− by a limited number of strong one-electron oxidizing agents, namely the hydroxyl radical (HO−), the carbonate radical (CO3•−), nitrogen dioxide (NO2−) and the peroxidase compounds oxoferryl I and II.
The initial product of the one-electron oxidation of H2S is the sulfyl radical (HS−/S−) (Figure 236).
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Figure 236. Oxidation of H2S and formation of the sulfiyl radical (HS•/S•−).
This particular radical exhibits oxidizing properties and is capable of undergoing reactions with a number of electron donors or hydrogen atoms. The compound is capable of undergoing a reaction with a secondary radical HS−/S•−, forming HSSH/HSS− [155][75], or it can react with HS− to form HSS•2−, which is a reducing radical that has the ability to undergo a reaction with oxygen that leads to the formation of the superoxide radical. HS−/S•− can [155][75] also react with oxygen to form SO2•−, which again can react with oxygen to form a superoxide radical [156][76]. The one-electron oxidation of H2S has the ability to trigger oxygen-dependent free radical chain reactions that can lead to an amplification of the original oxidation species (Figure 247).
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Figure 247. Chain reactions produced by the sulfiyl radical (HS•/S•−), which is an oxidant.
In addition to ROS and RNS, it is also necessary to consider reactive sulfur species, including hydropersulfides, polysulfides and H2S. The concept of RSS was postulated in 2001 [157][77], RSS that are produced under oxidative stress include RS−, sulfenic acids (RSOH), disulfides (RSSR), thiosulfinate (RS(O)SR), thiosulfonate (RS(O)2SR) and S-nitrosothiols (SNTs), the products of cysteine transformations, H2S and sulfane sulfur-containing compounds. SSRs that are produced under physiological conditions (without oxidative stress) are referred to in the scientific literature as “the first class of SSRs”. The “second class of SSRs”, on the other hand, “refers to species that are formed by the initial action of oxidative stress” [158][78].
H2S plays an important role in combating oxidative species such as ROS and RNS in the body. Filipovic, in 2012 [159][79], studied the reaction of H2S with peroxynitrite in vitro and in different cell models. The results showed that H2S can remove peroxynitrite with a second-order rate constant and that the reaction does not proceed through radicals. In this reaction, a new product is formed, which was characterized by spectral and computational studies as HSNO2 (thionitrate), mostly as sulfinyl nitrite HS(O)NO.
The ability of HS(O)NO to function as a nitric oxide (NO•) donor in response to pH and its ability to release NO• in cellular environments have been successfully demonstrated. Therefore, H2S removal plays a role in modulating the chemical and biological effects of peroxynitrite. This process effectively suppresses the pro-apoptotic, oxidative and nitratative properties associated with peroxynitrite (Figure 258).
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Figure 258. Reaction of H2S with peroxynitrite with formation of sulfinyl nitrite.

3. Persulfidation or S-Sulfhydration of Protein Thiols

Sulfhydration of cysteine residues and nitration of tyrosine are H2S-induced post-translational modifications induced by H2S and RNS, respectively [160][80]. H2S is an important biological messenger molecule that transmits signals through the formation of persulfide bonds (SSH) in proteins or low-molecular-weight thiols [1,161,162][81][82][83]. S-Persulfidation is the process in which a thiol (R-SH) is converted into a perthiol (R-SSH). The modification of thiols to form persulfides is one of the mechanisms by which sulfide exerts signaling functions. Protein modification by persulfurization of cysteine residues is able to modulate the activity of different proteins. The formation of persulfides is associated with the body’s sulfur reserve [1][81]. The direct reaction of H2S with protein cysteines does not take place; for this reaction to occur, the presence of an oxidant is required. The proposed mechanism for the formation of persulfides is the reaction of sulfur with oxidized cysteines such as sulfenic acid (RSOH) or disulfide (RSSR) [163][84]. Persulfides can also be formed via radicals, through the reaction of the sulfhydryl radical (HS•−) with the RS−, although the low concentration of these species means that this reaction is of little biological relevance [148,164][72][85]. HS•− can also react with a non-radical thiol to generate the radical anion RSSH•−, which gives up its unpaired electron to molecular oxygen to give the persulfide and superoxide radical anion [165][86] (Figure 269).
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Figure 269. Mechanisms of protein persulfidation.
Persulfides are unstable and have an electrophilic character. They also retain the nucleophilic character of the original thiol, or even enhance it due to the presence of an adjacent sulfur containing unshared electron pairs, i.e., the α-effect [166,167,168][87][88][89]. It has been proposed that these compounds are responsible for the biological effects initially assigned to H2S [169][90].
One of the ways in which H2S functions as a messenger molecule is by sulfhydration of reactive cysteine residues of target proteins in a manner analogous to protein nitrosylation [1,28][81][91].
Due to a decrease in pKa and an increase in nucleophilicity of perthiols compared to thiols, S-persulfidation can affect the biological activity of proteins [170][92]. For example, the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is primarily involved in glycolysis and gluconeogenesis, undergoes an activity shift after persulfidation to prevent cell death [170][92]. Persulfidation at KATP channels is a factor contributing to vasodilation caused by H2S [171][93].
The reaction of persulfides with cyanide gives thiols and thiocyanate (Figure 2710).
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Figure 2710. Hydropersulfide cyanolysis reaction.
The cyanolysis reaction is a common reaction involving hydrosulfides and other sulfur compounds. This reaction serves as a reliable method to confirm the presence of the -SSH group on a protein. In addition, this reaction has characteristic properties of hydrosulfides and can be used for the detection of persulfides. The reaction between thiocyanate and ferric ions leads to the formation of a red complex that exhibits absorption at a wavelength of 460 nm. This complex can be accurately measured and quantified by spectrophotometric methods [172][94] (Figure 2811).
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Figure 2811. Persulfide detection reaction.

3. Detection of H2S

The two deprotonated H2S species (HS− and S2−) absorb in UV at 230 nm with molar extinction coefficients of 8 × 103 and 4.6 × 103 M−1 cm−1, respectively, at 25 °C, so the concentration of the predominant species (HS−) could be measured by absorbance [173][95]. In practice, their oxidation products generate interferences.
Several research groups have focused their efforts on the development of H2S detection probes. Classical instrumental methods for H2S detection include the following: (i) colorimetric and electrochemical assays, (ii) gas chromatography and (iii) sulfide precipitation [38,75,140,174,175][62][96][97][98][99]. These techniques often require complex sample processing. The results of these methods may differ due to the high reactivity of H2S [63,163,176][84][100][101]. The high sensitivity of fluorescence-based assays suggests that they could be potentially valuable in this area. However, the limited number of fluorescence techniques available today for the detection of H2S poses a challenge when it comes to monitoring this gas in biological samples in real time [75,177][97][102]. A major challenge is to develop molecular probes that can detect aqueous sulfides (H2S and HS− at neuronal pH) in the presence of other thiols found inside most cells.
Numerous H2S detection techniques have been described, including spectrophotometric methods, where sulfide can be monitored by the formation of lead sulfide or methylene blue at 390 or 670 nm, respectively; fluorimetric methods, using fluorescein mercuric acetate; and polarographic methods with sulfide-specific electrodes, as well as by liquid or gas chromatography [178,179][103][104].
Another methodology used is the trapping of sulfide on ZnS particles by a reaction with zinc acetate. This technique is used in conjunction with other methods [14][105].
Classical iodometric titrations are used to prepare a standard solution. H2S is first immobilized in zinc acetate to reduce its dispersion and then reacts with an excess of iodine in an acidic environment. The remaining iodine is subjected to titration with sodium thiosulfate, with starch added as an indicator (Figure 129).
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Figure 129. Classical iodometric titration.
However, this method leads to errors due to the presence of other reductants. Numerous research groups have focused primarily on the development of probes for the detection of H2S. The approaches currently described for the detection of sulfur are usually based on its nucleophilicity or its reductive capacity, both of which are common to other thiols (glutathione and protein thiols) in biological studies and can easily mask the signal corresponding to sulfur. Figure 130 shows the most commonly used methods for H2S detection.
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Figure 130. Methods for H2S measurement.

3.1. Lead Acetate

The enzymatic synthesis of hydrogen sulfide can be traced back to a simple approach involving the use of lead acetate and the determination of the formation of lead sulfide (Figure 314), which is insoluble and can be detected by increasing turbidity at 390 nm.
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Figure 314. Hydrogen sulfide reacts with lead acetate to form a brown solid of lead sulfide (PbS).
When using this approach to calculate H2S concentrations, it is required to compare the results with a calibration curve created with already-known lead sulfide concentrations [180][106]. This approach provides semi-quantitative data and has relatively low sensitivity.

3.2. Methylene Blue Method

Methylene blue is formed when an oxidizing agent, usually ferric iron, reacts with H2Saq and N,N-dimethyl-p-phenylenediamine (N,N-dimethylbenzene-1,4-diamine) in an acidic environment (Figure 3215).
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Figure 3215. Reaction in the methylene blue method for sulfide detection.
The methylene blue formation reaction can be used to determine the H2S concentration. The concentration of methylene blue is determined at a wavelength of 670 nm and then compared with calibration curves generated with samples of known H2S concentrations subjected to comparable processing procedures [179,181][104][107]. This method also has a number of disadvantages.

3.3. Monobromobimane Derivatization

H2S undergoes a nucleophilic substitution reaction with monobromobimane, resulting in the formation of a bimane-substituted thiol compound. This biman-substituted thiol can further react with a second monobromobiman molecule, leading to the formation of dibiman sulfide. The fluorescence of dibiman sulfide can be observed upon its separation by high-performance liquid chromatography (HPLC) or mass spectrometry. This method has found wide use recently; however, the reaction rate is relatively slow (K ≈ 10 M−1 s−1 at pH 8) [182][108] (Figure 3316).
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Figure 3316. Bromobrimane reaction to obtain dibiman sulfide.

3.4. Methods Based on the Reducing Capacity of H2S

The sensitivity of fluorescent probes can be quite high. Certain probes, which have been described in detail, use nitro or azide derivatives of rhodamine, dansyl, coumarins or naphthylamides, which can be reduced by H2S to produce fluorescent amines (Figure 3417).
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Figure 3417. Fluorescent probes for the detection of H2S using the reduction of azide (a–c) or nitro (d) groups.
In chemical synthesis, H2S is frequently used for the reduction of azido groups (N3) [183][109] and aromatic nitro groups [184][110] to aniline derivatives due to its strong reducing agent properties.
By attaching an N3 group to an SF1 rhodamine core, the research team led by Chang was able to produce a selective probe for the detection of H2S. Such probes exhibit strong selectivity for H2S over oxygen and nitrogen [185][111], two other reactive sulfur species that are physiologically important, and the fluorescent signal is released upon reduction to the amine. Using a similar strategy, Wang’s lab has developed a sulfonyl azidedansyl derivative, whose electrical and fluorescent properties are due to the different electronegativity of the azide and amine groups. H2S could be captured with a fluorescent probe to detect and visualize hydrogen sulfide. However, its slow kinetics are a disadvantage.
To track changes in mitochondrial H2S content in living organisms, Mike Murphy’s team synthesized and characterized MitoA [186][112]. MitoA consists of an aryl azide coupled with a lipophilic triphenylphosphonium cation (TPP). In living organisms, the TPP cation causes MitoA to accumulate in the mitochondrial structures of the cell. The arylazide group forms MitoN, an arylamine species, when it interacts with H2S (Figure 3518).
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Figure 3518. Reaction of MitoA with H2S to form MitoN.
Therefore, the extent to which MitoA is converted to MitoN serves as an indicator of the amount of H2S in the mitochondria of a living organism. Detection and quantification of these chemicals in tissues can be performed with high sensitivity by liquid chromatography–tandem mass spectrometry (LC-MS/MS), using deuterated internal standards for accurate measurement.
Certain electrochemical techniques utilize the inherent reducing capability of sulfide as a fundamental principle. A polarographic technique based on the oxidation reaction between sulfide and ferricyanide can be used [175][99].

3.5. Methods Based on Nucleophilicity

H2S is considered a nucleophile that usually occurs as HS− under physiological pH conditions. Consequently, it exhibits stronger nucleophilic activity than various other thiols in the cellular environment, which are predominantly present in their protonated form (RSH). The observed difference indicates specific advantages in terms of the nucleophilicity of sulfur compared to thiols. For the selective detection of H2S, it is crucial to distinguish H2S from other nucleophilic compounds present in biological systems, especially thiols such as cysteine and glutathione. In a theoretical context, it is plausible to classify H2S as an unmodified thiol that has the potential to perform two nucleophilic attacks. In contrast, other thiols, such as cysteine, are capable of performing only a single nucleophilic attack. In view of these considerations, probes containing electrophilic functions capable of transforming in the presence of H2S have been described.
HS− is known for its strong nucleophilic properties, which allow it to readily bind to electrophilic sites in luminescent compounds such as cyanine dyes. This approach has been used in the development of radiometric H2S sensors, where fluorescence emission is altered by disrupting an extended pi system [187,188,189][113][114][115].
This review introduces a novel radiometric fluorescence probe called CouMC, which is described in a recently published research article. The functionality of this probe is based on the process of selective nucleophilic addition of HS− to a merocyanine derivative in a near-neutral pH environment. In addition to its ability to rapidly and specifically detect H2S, this probe also shows potential for the selective visualization of H2S in the mitochondria of living cells (Figure 3619).
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Figure 3619.
Structures of ratiometric H
2
S probes that function by disrupting the conjugated p-system within a fluorophore. Also shown is the process by which these probes react with H
2
S.
To construct the CouMC probe, an ethylene group was used to link a coumarin fluorophore to an indole block. By aligning with the electrically positive benzopyrilium moiety of the fluorescent probe, H2S can be distinguished from biothiols and other biological products. This allows the probe to detect H2S based on a flavilium derivative.
The sensitivity of fluorescent probes can be of a considerable order of magnitude. The Xian research group postulated that the introduction of a probe with two electrophilic cores could potentially lead to selectivity for H2S, prompting them to investigate this particular property. The synthesis of a fluorescein ester of thiosalicylic acid was achieved by introducing a thiopyridyl disulfide functionality into the thiol group. This result was achieved by a synthesis process. Active disulfides can undergo disulfide exchange reactions with thiols and H2S. It is important to note that further rearrangement can only take place with the disulfide intermediate generated from H2S. The rearrangement of the ester occurs because of an intramolecular nucleophilic attack on the carboxyl carbon atom, resulting in the release of benzodithiolone and a fluorophore [75][97] (Figure 3720).
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Figure 3720. Fluorescent probe for the detection of hydrogen sulfide on the basis of H2S-mediated benzodithiolone formation.
Aldehydes and acrylates are two examples of electrophilic centers that have been used in the development of probes. Hemithioacetal is formed by the addition of hydrogen ions to the aldehyde compound. Subsequently, in a neutral pH aqueous solution, the nucleophilic behavior of S is observed; S undergoes an intramolecular Michael addition, specifically adding to the beta position of the α,β-unsaturated ester. The process described above leads to a cyclic thioacetal compound. The results of this research represent a significant advance for Chuan’s research team, as they have effectively visualized the enzymatic production of H2S in living cell systems [190][116] (Figure 3821).
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Figure 3821. Fluorescent probes and reaction of methyl (E)-3-(5-(3-(3,5-difluorophenyl)-1-phenyl-1H-pyrazol-5-yl)-2-formylphenyl)acrylate with H2S.

3.6. Methods Based on the Ability to Bind Metal Cations

Another property that can be exploited for the detection of H2S is its remarkable affinity for metals. Novel probes consisting of a fluorophore coupled with Cu2+ have been developed. The precipitation of CuS and subsequent increase in fluorescence are the result of H2S binding to the copper ion [191][117] (Figure 3922).
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Figure 3922. Use of azamacrocyclic copper(II) ion complex chemistry to modulate fluorescence in the fluorescent probe for the detection of H2S, known as HSip-1.

4. H2S Donors

There is an urgent need for the development of novel chemical tools that facilitate the controlled release of H2S to study its biological activities for research purposes and potential therapeutic applications. This need arises from the frequently observed low concentrations of endogenous H2S, which pose a challenge in studying its biological effects. Different categories of hydrogen sulfide donors can release hydrogen sulfide at different rates. Since a synthetic source of H2S is needed, chemists have developed chemical tools to analyze H2S in biological contexts.
Given the problems associated with the direct administration of H2S gas in various biological contexts, inorganic sulfide salts such as sodium (NaSH) and sodium sulfide (Na2S) are often used as alternative sulfide sources. Potential problems associated with these salts include their susceptibility to oxidation, their volatility and/or shortened effective residence time, and the rapid release of hydrogen sulfide upon dissolution, which can be challenging in some contexts [192][118]. To address this problem, researchers have developed and studied small H2S donors that release H2S at a controlled rate, like enzymatic H2S synthesis [193][119]. Currently, there is a wide range of chemicals that are commonly used as donors of H2S. These compounds include aryl isothiocyanates [194][120], phosphinodithioates [195][121], thioacids [196][122]/amides [197][123], dithiolethiones [198][124], thiolysis of protected trisulfides [199][125] and persulfides [200][126]. In addition, the emission of carbonyl sulfide (COS) from thiocarbamates, followed by its conversion to H2S by the action of carbonate anhydrase [201][127], is also considered to be a component of these donors.

4.1. Sulfur Salts

Inorganic salts, such as sodium acid sulfide (NaHS) and sodium sulfide (Na2S), were historically the first compounds used and are still the most widely used to generate H2S. These salts are very soluble in water and upon dissolution release a large amount of H2S quickly, and for this reason, they are called fast-releasing donors (Figure 4023).
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Figure 4023. Sodium acid sulfide and sodium sulfide spontaneously release H2S.
More recently, slow-releasing donors that gradually release small amounts of H2S have been developed. These are usually organic compounds, and their effect more closely resembles a physiological situation, where the gas is released at a low but constant rate [202][128]. One of the most widely used slow releasers is GYYY4137 [195][121]. In addition, peptide-based releasers capable of releasing H2S in a more controlled manner are being explored [203][129].

4.2. Natural Donors

The human body is able to convert sulfur compounds contained in some foods such as garlic, onions, mushrooms and selected edible legumes and fruits into hydrogen sulfide through chemical or enzymatic processes. The compounds isolated from natural products are polysulfides substituted with allyl residues. The garlic derivatives diallyl sulfide, diallyl disulfide and diallyl trisulfide (DAS, DADS and DATS) are capable of releasing H2S in the presence of GSH.
There has long been speculation about the possible health benefits associated with the consumption of garlic.
The major bioactive compound in freshly pressed garlic and certain other Allium plants is allicin [204][130]. Allicin is a naturally occurring compound that is chemically synthesized during the mechanical crushing of a garlic clove [205][131]. The compound exhibits inherent instability and is converted into many secondary metabolites that possess bioactive properties. Allicin and its secondary metabolites are a class of organosulfur compounds (OSCs) that exhibit various physiological functions, such as anticancer properties, activity against pathogenic organisms, modulation of the gut microbiota and antioxidant and anti-inflammatory effects. Their effects on pathogenic organisms include antibacterial, antifungal, antiviral and antiparasitic activities [206][132] (Figure 241). Numerous studies have demonstrated the following beneficial effects of garlic on cardiovascular health: (i) reducing blood pressure, (ii) reducing blood cholesterol levels and aggregation of platelets and (iii) reducing oxidative stress. S-allyl-l-cysteine (SAC) is proposed to be responsible for the cardioprotective effects of garlic.
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Figure 241. The biological functions of allicin and its secondary metabolites.
When garlic cloves are crushed or chopped, the enzyme allinase, which is stored in the vacuoles, is released. When it comes into contact with the cytosolic allicin, it converts it into a series of thiosulfinates, of which allicin is the best known (Figure 425).
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Figure 425. Enzymatic conversion of allin to allicin. Two molecules of 2-propenesulfenic acid interact to form allicin and remove water.
The chemical compound allicin exhibits a high degree of instability, as it is susceptible to decomposition and subsequent rearrangement, which leads to the formation of various organosulfur compounds (Figure 4326).
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Figure 4326. Organosulfur compounds derived from allicin.
Ajoene is a colorless liquid containing a sulfoxide group and a disulfide group and is found as a mixture of up to four stereoisomers and two geometric isomers (E-Z) due to the chirality of sulfoxide (R-S). This chemical that gives garlic its distinctive aroma and flavor is released when the cloves are mechanically crushed or cut. To synthesize ajoene, allicin is dissolved in various solvents, such as edible oils. It has antioxidant properties and thus prevents the formation of superoxides. In addition, it shows antithrombotic and antiviral effects, especially against vaccinia, herpes simplex, rhinovirus, human parainfluenza virus and vesicular stomatitis virus. In addition, ajoene has been observed to have an inhibitory effect on integrin-mediated viral mechanisms in the context of HIV infections. In addition, this molecule has shown antibacterial and antifungal properties, particularly against Candida albicans and Tinea pedis infections.
The chemical composition of the garlic juice produced by the mechanical crushing of the garlic cloves differs significantly from that of the intact garlic clove, particularly with regard to the content of organosulfur components. Unprocessed garlic consists mainly of sulfur derivatives (Figure 4427).
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Figure 4427. The main compounds found in intact garlic cloves.
The proposed mechanism of H2S release from DADS by GSH attack of sulfur to form an allyl perthiol is shown in Figure 4528. Similarly, red blood cells rapidly released H2S from diallyl disulfide (DADS) under oxygen deprivation and in the presence of glutathione [199][125].
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Figure 4528.
Proposed mechanism of H
2
S release from trisulfide in the presence of GSH by initial GSH attack on sulfur and a second GSH attack on α-carbon with the formation of S-allyi glutathione.
The identification of DATS signifies the early recognition of a garlic-derived chemical that possesses vasoactive properties as well as a marked ability to selectively inhibit the proliferation of cancer cells [207][133]. Garlic-derived chemical compounds are commonly known as precursors of H2S during the absorption and metabolization process in the circulatory system.
The antiviral potential of garlic and its CSOs has been demonstrated in both in vivo and in vitro studies against a variety of viruses, including those belonging to the families Adenoviridae, Arteriviridae, Coronaviridae, Flaviviridae, Flaviviridae, Herpesviridae, Orthomyxoviridae, Picornaviridae, Paramyxoviridae, Poxviridae, Rhabdoviridae and Retroviridae. Blocking viral entry and fusion into host cells; inhibiting viral RNA polymerase, reverse transcriptase and viral replication; and enhancing the host immune response are the primary mechanisms by which garlic and its CSOs exert their antiviral activity. Garlic and its CSOs were also responsible for enhancing the host immune response. Further research is needed to better understand the properties of garlic and its active compounds, known as CSOs, in relation to their role in antiviral therapy. This means that additional research needs to be conducted focusing on the pharmacokinetics and clinical aspects of the therapeutic effect of garlic.
Other natural sulfur compounds extracted from other plants are isothiocyanates (or sulforaphane), present in cabbages such as broccoli and cauliflower, and erucin, present in seeds and leaves of rocket [101[21][134],208], (Figure 4629).
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Figure 4629. Natural sulfur compounds present in plants that have been associated with the formation of hydrogen sulfide.

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