Isolation, Determination and Analysis of Bioactive Natural Sulfur Compounds

Sulfur-containing products contribute significantly to natural chemical diversity and allow fundamental biological functions that no other compounds allow. Natural sulfur compounds are utilized by all living beings and are distributed across the different kingdoms depending on their role. Marine organisms are one of the most important sources of sulfur-containing natural products, since most of the inorganic sulfur is metabolized in ocean environments where this element is abundant. Terrestrial and marine organisms, such as plants and microorganisms, are also able to incorporate sulfur into organic molecules to produce primary metabolites (e.g., methionine, cysteine) and more complex unique chemical structures with diverse biological roles (e.g., glucosinolates, ergothioneine, ovothiols). Animals are not able to fix inorganic sulfur into biomolecules and are completely dependent on preformed organic sulfurous compounds to satisfy their sulfur needs. However, some higher species, such as humans, are able to build new sulfur-containing chemical entities, starting, particularly, from the organosulfur precursors of plants. The aim of this Special Issue is to provide fundamental and new research works on sulfur compound separations at the preparative and analytical scale, as well as new findings in bioactive natural sulfur products.

This Special Issue includes five articles that covered various topics, including analysis of natural sulfur compounds, extraction and chromatography of chiral sulfur-containing products, development of spectrofluorometric methods, isolation of novel bioactive molecules and synthesis of unusual peculiar sulfur-containing compounds. The papers published in this Special Issue are briefly described below.

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In one of the papers presented in this Special Issue, Francioso and co-workers set up an optimized fluorometric method for the direct determination of total glutathione (GSH and GSSG) in red blood cells [1]. GSH is a tripeptide (γ-L-glutamyl-L-cysteinyl-glycine) characterized by a non-canonical peptide bond, with an amide moiety linking the nitrogen of cysteine to the carboxyl in the γ-position of the glutamic acid, and it is found ubiquitously in nature, animals, plants and microorganisms. GSH in the red blood cells is present in the millimolar (mM) concentration range and plays a focal role in the thiol-based cell redox signaling and defense. The determination of total glutathione (reduced and oxidized) content in red blood cells comprises very important data, which indicate the amount of synthetized and transported GSH by the erythrocytes, being indicative of their functionality and of particular oxidative stress conditions, such as diabetes and other oxidative metabolic pathologies. In this work, an optimized rapid and simple fluorometric method for total GSH determination in red blood cells was developed, avoiding the use of chromatographic separation and allowing the simultaneous analysis of a high number of samples with minor costs, shorter analysis time and no extensive sample processing.

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Another interesting original article by Manurung et al. reported the presence of unusual sulfated constituents in two Indonesian mangroves, and their anti-infective properties [2]. Medicinally active compounds from mangroves are not always produced by the plant itself, but often by associated microorganisms such as endophytic fungi. For example, the extracts of endophytic fungi isolated from leaves of ten mangrove species from Thailand, including L. littorea, showed some cytotoxic activity against cancer cell lines. In line with the agenda of discovering new anti-infective and neuroactive constituents while at the same time promoting the protection and sustainable development of mangrove ecosystems, the work focused on two mangrove species, namely Lumnitzera littorea and L. racemosa. The researchers aimed to investigate the diversity of natural products present in the roots of L. littorea and L. racemosa from Indonesia, evaluate selected biological effects, and investigate the phylogenetic relationships of the two species, as well as the chemophenetic patterns of their natural products across Indonesia. Therefore, they combined a molecular phylogeny of Lumnitzera based on the internal transcribed spacer (ITS) region with phytochemical analyses using hyphenated chromatographic and tandem mass spectrometric techniques. In this study, a series of unusual sulfated constituents was characterized in root samples from the mangrove species Lumnitzera littorea and L. racemosa (Combretaceae). Thus, most of the methylated ellagic acid derivatives isolated from both species possess a sulfate moiety. However, L. racemosa samples from North Sumatra (LR1), Aceh (LR2, LR3), East Kalimantan (LR4), and Maluku (LR8) completely lack sulfated and non-sulfated trimethylellagic acid, being instead dominated by dimethylellagic acid and its sulfate. This phytochemical pattern was corroborated with phylogenetic data, where these specific samples form a well-supported clade in the ITS tree. Interestingly, the occurrence of antimicrobial activity and sulfated ellagic acid derivatives are not connected. Although the ellagic acid derivatives were present within all samples, the antibacterial effects were limited to samples from particular locations in Indonesia, suggesting that other compounds from the plant or root-associated microorganisms might be responsible. In summary, the authors suggest that Lumnitzera roots represent a potentially promising source for sulfated ellagic acid derivatives and further sulfur-containing plant metabolites.

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Stereochemistry and, in particular, chirality are strongly connected to the biological and environmental world. This is the case for particular sulfur-containing compounds used as pesticides in agriculture. Overall, the asymmetric center of a chiral compound is a carbon atom attached to four different groups, although the chirality is also possible due to the presence of an asymmetric nitrogen, phosphorus, or sulfur atom. Sulfur has a lone pair of electrons that can act as a fourth “group”, which results in a chiral center when combined with three other functional groups that are different from each other. In a review article published in this Special Issue by Lopez Cabeza et al., the authors described and summarized the extraction and analytical methods associated with these particular chiral pesticides with asymmetric sulfur atoms [3]. In general, the analysis of the enantiomers of a chiral compound represents a significant analytical challenge since, as mentioned above, the physicochemical properties of the enantiomers are identical, which makes their individual determination considerably difficult. In this review, the most frequently used techniques for the extraction and determination of pesticide enantiomers from environmental samples are described, emphasizing the analysis of chiral pesticides with an asymmetrical sulfur atom in their structure. As for the rest of the chiral pesticides, the separate determination of the stereoisomers has become crucial in the environmental risk assessment of these pesticides. Therefore, the development of suitable extraction and clean-up methods, as well as efficient stereoselective analytical techniques for stereoisomers determination in environmental samples, is essential. The authors summarized the extraction and clean-up methods along with the analytical methodology used for the chiral pesticides with a chiral sulfur described in this review. Overall, QuEChERS is the most widely method used for the extraction of these chiral pesticides from environmental samples. In the case of analytical techniques, liquid chromatography has proven to be the most frequently used for the resolution of all the pesticides reviewed. Absorbance detectors (UV, UV/Vis, and DAD) are widely used coupled to an HPLC system. However, the most recent studies on the stereoselective determination of new sulfur pesticides preferred the use of MS detection due to the high sensitivity and selectivity of this technique, which is essential in complex environmental samples with a high matrix effect.

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The last century has been very important from the point of view of research and investigation in the fields of the chemistry and biochemistry of sulfur-containing natural products. One of the most important contributions to the discovery and study of human sulfur-containing metabolites was performed by the research group of Professor Doriano Cavallini at Sapienza University of Rome, during the last 80 years. His research brought to light the discovery of unusual sulfur metabolites that were chemically synthesized and determined in different biological specimens. Francioso and co-workers, in another contribution to this Special Issue, described most of Prof. Cavallini’s synthetical strategies that were performed in aqueous conditions, and which, nowadays, can be considered to be completely in line with the recent concepts of green chemistry [4]. The review starts with a memorandum of Prof. Mario Fontana, the last disciple of the prestigious school founded by Prof Cavallini:

“I joined Prof. Cavallini’s research group in 1984, as medical student at Sapienza University of Rome. I had followed the one-year course in biochemistry held by Prof. Carlo De Marco, one of his collaborators, and decided to carry out the experimental thesis at the Department of Biochemical Sciences. I was thus introduced to Doriano Cavallini, who invited me to follow him to his laboratories, located in an L-shaped corridor, whose entrance was at the back of the department building. An entire wall of the long corridor was occupied by a wooden cupboard with glass doors; I was struck by the hundreds of chemical compounds and reagents and the most disparate glassware they contained. He introduced me to his young collaborators, who assigned me the task of developing, under their guidance, a method for the determination of mercaptolactate in human urine [1]. Since then, I have been working with Cavallini and his group for more than 20 years [2]. The purification of the mammalian liver enzyme involved in the formation of cystathionine ketimine was my first article published with the Cavallini research school [3]. Years later, I received the Cavallini black notebook, as a gift, where he had noted over the years the several chemical syntheses developed or carried out in his laboratory”.

In this paper dedicated to Prof. Cavallini, the authors describe and summarize both synthetic procedures, and purification and analytical methods from the Cavallini school, with the purpose of providing efficient and green methodologies for the preparation and obtainment of peculiar unique sulfur-containing metabolites. All the reported chemical syntheses were taken from the “Cavallini black notebook”. Some syntheses were originally developed by Cavallini and his early pupils, while others were transcribed by Cavallini from original papers. These last have been retranslated into English, taking into account the original works and the observations that Cavallini himself reported in the notebook. Many of the reported peculiar sulfur metabolites are not yet commercially available. Moreover, the preparations of these sulfur-containing compounds are currently difficult to find and often not available in digital form. The aim of this work is to share these preparations with researchers interested in sulfur chemistry, biochemistry and metabolism. The study of sulfur-containing compounds was the leitmotif of Doriano Cavallini’s scientific life at Sapienza University of Rome (Rome, Italy). His research brought to light the discovery of unusual sulfur metabolites and bioactive derivatives. Noteworthily, the synthetical strategies designed and set up in Cavallini’s laboratories were performed in aqueous hydrophilic environments and with aqueous chromatographic conditions, avoiding the use of large amounts of organic solvents and conforming to the eco-friendly and biocompatible practice of synthetic chemistry. Nowadays, this approach used by Prof. Cavallini can be considered to be completely in line with the concepts of green chemistry and sustainable science.

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In the last contribution of this Special Issue dedicated to sulfur-containing natural products, we find the work of Di Risola et. al, in which the synthesis, the chromatographic behavior and the reactivity of of S-Nitrosopantetheine is described. S-nitrosothiols (RSNOs) are a group of physiological sulfur-containing compounds biologically involved in nitric oxide (NO) release and signaling. RSNOs have been shown to have the same physiological role as nitric oxide itself. Levels of RSNOs in respiratory airways are affected by diseases such as asthma and pneumonia, and it is believed that RSNOs act as bronchodilators and relax bronchial smooth muscle, thus potentially playing a role in the treatment of these diseases. S-nitroso-glutathione (GSNO) is already being administered at lower concentrations than those required for vasodilation in coronary angioplasty clinical trials, where it can be used to reduce blood clotting. GSNO has also been successful in treating the hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome in pregnant women [5]. RSNOs, together with NO˙, have also been implicated in the suppression of HIV-1 replication through the S-nitrosylation of the cysteine residues in the HIV-1 protease. All of these and other pharmacological features of RSNOs make them ideal candidates for the development of novel drugs in different therapeutic fields. This notwithstanding, the most relevant pharmaceutical problem related to RSNOs’ use as drugs is their poor stability in aqueous environments, which limits their potential as effective therapeutic agents. Considering that pantetheine is a biological thiol with a crucial role in the metabolism, both as the intermediate of the biosynthesis of Coenzyme A and as the most important form of storage of vitamin B5, its S-nitrosylation in vivo could play an interesting role in various metabolic signaling pathways. Chemically, RSNOs can be easily obtained by treating thiols with nitrite in aqueous acidic media. In this paper, the authors used this strategy to synthesize S-nitrosopantetheine (SNOPANT) for the first time, starting from potassium nitrite and pantetheine. The chemical structure of the product was elucidated using HPLC, UV-visible spectroscopy and high resolution and tandem mass spectrometry (HR-MS/MS). The effect of copper and ascorbic acid/ascorbate has been explored to assess the stability of this novel nitrosothiol. Interestingly, the authors found that SNOPANT can be obtained in high yields and in a relatively stable form through the mutual and opposite effect of the copper ion and ascorbic acid, avoiding the use of strongly acidic conditions.