As previously stated, the important part of the GSH molecule in terms of its chemical activity is the nucleophilic sulfhydryl (thiol, -SH) group of the cysteine residue. Generally, as illustrated in Figure 4, conjugation with GSH involves attachment of the xenobiotic molecule or its phase I metabolite (assigned as R-X) with this group to form the GSH S-conjugate (assigned as R-SG). In fact, the reactive nucleophilic species is the thiolate anion of GSH (GS⁻), whose concentrations at physiological pH are approximately 1% of GSH concentration (pK_a of GSH = 9.2) [38]. Due to the low electronegativity and high polarizability of the sulfur atom, GSH acts as a ‘soft’ nucleophile and, as such, can be used to sense the reactivity of ‘soft’ electrophiles (i.e., compounds that contain an acceptor atom with high polarizability, low electronegativity, and often unshared p or d valence electrons) [51]. Xenobiotics that are conjugated with GSH are either very electrophilic right away or are metabolized to electrophilic compounds. Some reactions of the tripeptide GSH with cellular electrophiles have spontaneous rates which vary considerably depending on the reactivity of the electrophile, and frequently, but not always, are catalyzed by various GST isoenzymes [34]. However, non-enzymatic reactions are usually much slower than those catalyzed by GSTs.

Substitution reactions, which involve the reaction of a nucleophile with an electrophile, are prevalent in physiological and metabolic processes, in the action of some drugs, and in the chemical synthesis of nearly all drugs [57]. Thus, nucleophilic substitution is also the basic and the most widespread mechanism of GSH conjugation of electrophilic compounds. It is observed in several families of anticancer drugs, including alkyl, allylic, benzylic, and aryl halides, nitrogen mustard derivatives, or platinum complexes [33]. Electrophiles are positively charged or have a polarized bond with a partial positive character. Electrophiles capable of undergoing substitution reactions have a leaving group—a species that can accept and stabilize the pair of electrons that make up the bond being broken [57].

A common type of nucleophilic substitution reaction is the bimolecular nucleophilic substitution reaction, or S_N2 reaction for short, where one atom or functional group is replaced with an electronegative GSH molecule (the frame in Figure 5). In this case, bond formation and bond breaking occur simultaneously and the leaving group tends to be a weaker base than the nucleophile. Halide ions, such as I⁻, Br⁻, and Cl⁻, are very good leaving groups and thus give fast reactions [58].

The S_N2 reaction mechanism of alkyl halide with GSH can be illustrated by the conjugation of 3-bromopyruvic acid (3-BrPA) with GSH (Figure 5A). 3-BrPA is the brominated derivative of pyruvic acid with proven anticancer activity against many different cancers in children and adults [59,60]. Like other α-bromoketones, it is widely known as a strong alkylating agent with a high affinity for protein binding as well as an antimetabolite [17]. Moreover, being an inhibitor of key glycolysis enzymes, including hexokinase II [61] and glyceraldehyde 3-phosphate dehydrogenase [62], 3-BrPA inhibits the growth of neoplastic cells that perform aerobic glycolysis known as the ‘Warburg effect’ [63]. Additionally, it also selectively blocks mitochondrial oxidative phosphorylation, angiogenesis, and energy production in cancer cells [63,64].

Based on a chemical view, the thiolate anion of GSH (GS⁻) easily attacks the carbon atom to which the bromine atom is attached. This reaction occurs because of the imbalance of the electron density between the carbon and halogen since it is a polar covalent bond. The more electronegative bromine atom pulls the electron density, thus making the carbon partially positively charged (an electrophilic center) and susceptible to a nucleophilic GS⁻ attack [66]. A bromine atom built into the structure of the 3-BrPA molecule is a good leaving group because the negatively charged bromine atom (bromide) is stable enough to exist on its own when it leaves the molecule. Hence, the conjugation to GSH does not require any prior metabolic activation of the parent compound. 3-BrPA was reported to form GSH S-conjugate both under GST catalysis and also in an enzyme-free system. Further, it is eliminated through the mercapturic acid synthesis pathway where it is excreted from the cell by ATP-binding cassette transporter proteins [17,67].

Another example of a similar S_N2 reaction is the GSH conjugation of chlorambucil (CBL; the brand name: Leukeran) (Figure 5B). CBL is an alkylating agent approved for use in various malignant and non-malignant neoplasms, such as chronic lymphocytic leukemia [68], lymphosarcoma [69], and giant follicular lymphoma [70]. In the presence of GSH, CBL behaves as an efficient substrate for GSTA1-1 and GSTP1-1 isoenzymes [65,71]. As with the 3-BrPA, the formation of the corresponding GSH S-conjugate undergoes without prior CBL activation as the negatively charged chlorine atom (chloride) is a good enough leaving group. Moreover, kinetic data suggest that the rate-limiting of the catalytic reaction between CBL and GSH is the reaction product release [65].

The next type of GSH conjugation mechanism is the attachment of the thiolate anion (GS^–) to the epoxide, four-membered lactone, or three-membered aziridine resulting in ring-opening (Figure 6). Such reactions take place quite easily because the rings are composed of three or four carbon atoms which are highly tensed and their energy is quite high. Additionally, such a process will be easier, if the tensed system includes a heteroatom, because it will inductively decrease the electron density on adjacent carbon atoms [72,73]. Under basic conditions, ring-opening occurs by an S_N2 mechanism, and the less substituted carbon is the site of GS^– nucleophilic attack. The described transformation can take place stereoselectively, depending on the GST isoenzyme that catalyzes a reaction.

Formation of GSH S-conjugate, accompanied by the aziridine ring-opening, takes place, e.g., in the case of cyclophosphamide and thiotepa. Cyclophosphamide (CP; the brand name: Cytoxan) belongs to the alkylating agent and nitrogen mustard family of medications [74,75]. Its mechanism of action, quite similar to that of chlorambucil, relies on interfering with the duplication of DNA and RNA synthesis [76]. It is a chemotherapeutic approved to treat malignant lymphomas, neuroblastoma, multiple myeloma, leukemias, ovarian, breast, and many other cancers [77]. Such a wide spectrum of clinical uses makes it an essential component of numerous combination chemotherapeutic regimens. Moreover, CP is also used to suppress the immune system [78].

A prerequisite for conjugation with GSH is the presence of a reactive electrophilic metabolite(s). Unlike 3-BrPA and CBL, CP must undergo previous metabolic activation by hepatic microsomal mixed-function oxidases (i.e., P450s, mainly 2B6, 2C8, and 2C9) (Figure 7) [79,80]. The generally accepted mechanism for the generation of active metabolites of CP involves oxidation to the primary 4-hydroxycyclophosphamide (4-OH-CP) metabolite, which stays in tautomeric equilibrium with the ring-opened aldophosphamide (APA). Then, non-enzymatic cleavage of APA results in the formation of two toxic species—phosphoramide mustard (PAM) and acrolein. PAM is believed to unfold cell toxicity by DNA alkylation [81] while acrolein is held to be responsible for some aspects of host toxicity, such as hemorrhagic cystitis [82]. Therefore, GSH conjugation can result in the formation of three types of GSH S-conjugates, i.e., 4-monoglutathionyl CP (4-GSCP) (1), mono- (2) and diglutathionyl PAMs (3) [13,83]. The formation of 4-GSCP was found to be reversible, and by APA hydrolysis, PAM was formed. Thus, 4-GSCP can be considered a stable reservoir for the generation of PAM that subsequently undergoes two consecutive GSH conjugations. The reaction with nucleophilic GSH was established to proceed through the positively charged and highly polarized aziridinium ion (aziridinium intermediate) that can be opened in both the enzyme-catalyzed and the chemical reactions. On the other hand, the second GSH conjugation reaction was shown to occur through the direct displacement of chloride [13]. The 4-GSCP formation reaction can be catalyzed by various GSTs isoenzymes, whereas GST1A-1 has the greatest effect on the rate of monoglutathionyl PAM formation. Melphalan, mechlorethamine, ifosfamide, carmustine, lomustine, and nimustine are examples of other anticancer-active nitrogen mustard derivatives that react with GSH in a manner similar to that described for CP [13,80].

Thiotepa (N,N′,N″-triethylenethiophosphoramide; the brand name: Tepadina) also acts as an alkylating agent [84]. Being active against a wide variety of cancers, it is commonly used in combination with other chemotherapeutic agents to treat ovarian cancer, bladder cancer, and breast cancer [85,86]. Chemically, it is an organophosphorus compound containing a four-coordinated phosphorus atom and three aziridine moieties (Figure 8) through which the drug probably induces crosslinks with DNA, interfering with DNA replication and cell division [87]. The major metabolite formed from thiotepa during P450-catalyzed transformation (i.e., desulfuration reaction) is N,N′,N″-triethylenephosphoramide (tepa) [88]. In turn, when reacting with GSH, the tensioned ring of thiotepa containing a nitrogen atom as a heteroatom is opened, leading to the formation of mono- (1) and diglutathionyl thiotepa (2). In the metabolic pathway of thiotepa, 2-aminoethyl GSH (3), which is the product of the direct GSH conjugation of aziridine, was also characterized. The results confirmed that only thiotepa but not its monoglutathionyl S-conjugate is a substrate for GSTs (mainly A1-1 and P1-1 isoenzymes). Moreover, the non-enzymatic reaction of the aziridinium moieties of thiotepa with GSH is strongly dependent on the pH, and the yield of the reaction is greatest at low pH [89].

3.2. Nucleophilic Addition (Michael Addition)