Definition and importance of ligand protonation constants

The protonation constant, also known as the acid constant, is the equilibrium constant for the reaction of a ligand L (or any chemical species) with a proton (H⁺) to form the corresponding protonated species. The equilibrium reaction is written as:

$$ \mathrm{L}+{\mathrm{H}}^{+}\hskip0.6em \rightleftarrows \hskip0.6em \mathrm{L}{\mathrm{H}}^{+}, $$

where L is the unprotonated form of the ligand, H⁺ is the proton and LH⁺ is the protonated form of the ligand. The protonation constant (K) is defined as the ratio of the concentrations of the protonated and unprotonated forms at equilibrium:

$$ K=\frac{\left[\mathrm{L}{\mathrm{H}}^{+}\right]}{\left[\mathrm{L}\right]\left[{\mathrm{H}}^{+}\right]}. $$

Take the negative logarithm of K to obtain the pK value:

$$ \mathrm{p}K=-\log K. $$

The pK is a measure of the acidity of the ligand: lower pK values indicate greater acidity, meaning the ligand is more likely to donate the proton (Zhou et al., Reference Zhou, Jhamb, Liang, Sundmacher and Gani2018).

Reasons for the determination of ligand protonation constants

Protonation constants are the equilibrium constants for the interaction of the proton with charged or uncharged ligands depending on the availability of hydrogen ions in aqueous and mixed solutions. These parameters are used to predict the ionization state of the molecule as a function of pH. The main purposes of determining the protonation constants of ligands in solution are to:

1. a) determine the pH values for the different forms of the compound under investigation,

2. b) perform a quantitative spectrophotometric analysis by selecting a suitable pH on the basis of the different UV spectra,

3. c) isolate the compound with maximum yield by identifying the pH range at which the minimum ionization of the compound occurs,

4. d) provide additional information on the structure of the newly synthesized ligand (agreement between theoretically calculated and experimental protonation constants indicates the potential correctness of the proposed structure),

5. e) prepare buffer solutions at different pH values (Martell and Calvin, Reference Martell and Calvin1952; Liptay et al., Reference Liptay, Rossotti and Rossotti1962; Rossotti, Reference Rossotti1978),

6. f) calculate the stability constants of complex formation between biologically active ligands and metal ions in coordination chemistry,

7. g) elucidate the intricate mechanisms of metalloenzymes and their catalytic activities (Sigel and Martin, Reference Sigel and Martin1982),

8. h) study the mobility and bioavailability of metal ions in natural waters (metal speciation and contaminant fate in environmental chemistry) (Flaschka, Reference Flaschka1974).

The side chain groups of amino acids are involved in a variety of functions, including metal binding, weak interactions, and cofactor activities. In chemical systems, these groups are useful in the construction of artificial functionalized complexes, such as the model active site complexes of metalloenzymes and in the imidazolate – bridged multinuclear complexes that form molecular assemblies (Shimazaki et al., Reference Shimazaki, Takani and Yamauchi2009).

Thanks to the protonation constants of amino acids and their derivatives, we are still discovering new metal ion binding sites in proteins. The amino acids form stable five-membered chelates with various metal ions via the amine and carboxylate moieties (N, O – chelation). Several amino acids possess an additional metal binding site in the side chain and thus form metal complexes with a variety of structures. Groups such as the imidazole ring of histidine, the phenol ring of tyrosine, and the thiol group of cysteine are important metal binding sites in proteins. Similarly, the carboxylate groups of aspartate and glutamate and the thioether moiety of methionine are often involved in metal binding. These and other amino acid side chain groups form a microenvironment around the metal center which is necessary for substrate recognition and fixation and for the catalytic exercise of enzyme functions through non-covalent or weak interactions (hydrogen bonding, π-π stacking interactions, and hydrophobic interactions) (Berthon, Reference Berthon1995).

Methods for the determination of ligand protonation constants

There are several techniques such as potentiometry, conductometry, and spectrophotometry that can be used to determine protonation constants. This paper presents data and results from the use of the potentiometric method as it has the widest range of applicability and reliability (Woermann, Reference Woermann1973).

Potentiometric titrations are widely used for their precision in determining the acid–base equilibria of ligands. In this method, a ligand solution is titrated with a strong base while monitoring changes in pH. The most commonly used programs for solution equilibrium constants are PKPOT (Barbosa et al., Reference Barbosa, Barron, Beltrán and Sanz-Nebot1995), PKAS (Martell and Motekaitis, Reference Martell and Motekaitis1992), BEST (Motekaitis and Martell, Reference Motekaitis and Martell1982), MINIQUAD (Sabatini et al., Reference Sabatini, Vacca and Gans1974), MINIQUAD75 (Gans et al., Reference Gans, Sabatini and Vacca1976), SUPERQUAD (Gans et al., Reference Gans, Sabatini and Vacca1985), PSEQUAD (Vacca and Sabatini, Reference Vacca and Sabatini1985), and HYPERQUAD (Gans et al., Reference Gans, Sabatini and Vacca1996). All of these programs use least squares refinement to reduce the differences between calculated and experimental data to obtain the best model that gives the best fit. The sum of the squares of the residuals between experimental and calculated values is usually very small, typically between 10⁻⁶ and 10⁻⁹.

Protonation equilibria in aqueous solution

Amino acid molecules possess at least one acidic group and one basic group. This allows an intramolecular acid–base equilibrium reaction to take place, resulting in the formation of a dipolar tautomeric ion known as a zwitterion or internal salt. From the above we know that an amino acid has at least two dissociation constants, the first corresponding to the case where the COOH group is deprotonated and the second corresponding to the case where the NH₃⁺ group is deprotonated in an aqueous solution (Jhamb et al., Reference Jhamb, Liang, Gani and Hukkerikar2018). Thus, for all amino acids, the proton association constants can be expressed as stepwise protonation constants:

$$ {\mathrm{H}}^{+}+{\mathrm{L}}^{-}\hskip0.6em \rightleftarrows \hskip0.6em \mathrm{HL}\hskip0.4em \left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{NH}}^2\hskip0.5em \mathrm{group}\right) $$

$$ {\mathrm{H}}^{+}+\mathrm{HL}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}}_2{\mathrm{L}}^{+}\left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{COO}}^{-}\hskip0.6em \mathrm{group}\right). $$

Amino acids that are positively charged at pH 7, such as lysine, arginine, and histidine, undergo protonation equilibrium reactions:

$$ {\mathrm{H}}^{+}+{\mathrm{L}}^{-}\hskip0.6em \rightleftarrows \hskip0.6em \mathrm{HL}\;\left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{NH}}^2\hskip0.4em \mathrm{group}\right) $$

$$ {\mathrm{H}}^{+}+\mathrm{HL}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}}_2{\mathrm{L}}^{+}\left(\mathrm{protonation}\ \mathrm{of}\ \mathrm{side}\ \mathrm{chain}\right) $$

$$ {\mathrm{H}}^{+}+{\mathrm{H}}_2{\mathrm{L}}^{+}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}}_3{\mathrm{L}}^{2+}\left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{COO}}^{-}\hskip0.4em \mathrm{group}\right). $$

The representative equilibria for amino acids that are negatively charged at pH 8, including glutamic acid, aspartic acid, and cysteine:

$$ {\mathrm{H}}^{+}+{\mathrm{L}}^{2-}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}\mathrm{L}}^{-}\left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{NH}}^2\hskip0.4em \mathrm{group}\right) $$

$$ {\mathrm{H}}^{+}+{\mathrm{H}\mathrm{L}}^{-}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}}_2\mathrm{L}\;\left(\mathrm{protonation}\ \mathrm{of}\ \mathrm{side}\ \mathrm{chain}\right) $$

$ {\mathrm{H}}^{+}+{\mathrm{H}}_2\mathrm{L}\hskip0.6em \rightleftarrows \hskip0.6em {\mathrm{H}}_3{\mathrm{L}}^{+}\left(\mathrm{protonation}\ \mathrm{of}\hbox{--} {\mathrm{COO}}^{-}\hskip0.4em \mathrm{group}\right) $ .

In aqueous solutions, solvent molecules act as proton acceptors and therefore play a crucial role in chemical and biochemical reactions (Murphy et al., Reference Murphy, Powell and Brumaghim2020). The study of glycine, alanine, glutamic acid, histidine, tryptophan, and leucine derivatives and their complexes is of enormous biological interest because such complexes display interesting antibacterial, antifungal, anticancer, and antiviral properties (Quyoom, Reference Quyoom2014). Leucine, isoleucine, and valine are metabolically active. In peripheral tissues, they can be oxidized to produce energy or act as anti-catabolic factors (especially leucine) by stimulating the synthesis and reducing the rate of breakdown of muscle protein (Soomro et al., Reference Soomro, Devrajani, Ghori, Lohana and Qureshi2008). L-lysine oxidase (LysOx) isolated from the extracellular growth medium of Trichoderma cf. aureoviride was reported by Pokrovsky et al. (Reference Pokrovsky, Treshalina, Lukasheva, Sedakova, Medentzev, Arinbasarova and Berezov2013) to exhibit significant cytotoxicity and antitumor activity in vitro against a panel of murine and human tumor cell lines and in vivo on murine tumors and on animals with human tumor xenografts (breast cancer SKBR3, melanoma Bro, colon cancer HCT116 and ovarian adenocarcinoma SCOV3).

Amino acids are involved in pathways that feed cancer cells and provide building blocks for cancer cell growth. The citric acid cycle (the TCA cycle) is an important mechanistic example of the involvement of amino acids in cancer. The branched-chain amino acids and threonine fuel the TCA cycle intermediates, resulting in the release of ATP and providing the energy required for oncogenic activities. Cancer cells also use valine, leucine, and isoleucine as ‘alternative fuels’ to compete with other cells in the tumor stroma for energy and to optimize nutrient use during tumor development (Green et al., Reference Green, Wallace, Divakaruni, Phillips, Murphy, Ciaraldi and Metallo2016). The recently discovered V-9302, a selective inhibitor of a glutamine transporter ASCT2, showed anti-tumor activity in vivo, demonstrating the utility of a pharmacological agent in oncology (Schulte et al., Reference Schulte, Fu, Zhao, Li, Geng, Smith and Manning2018). Cysteine uptake also plays an important role in breast cancer by maintaining redox balance and numerous studies have shown the efficacy of xCT inhibition on tumor growth (Timmerman et al., Reference Timmerman, Holton, Yuneva, Louie, Padró, Daemen and Gray2013). Amino acids also form derivatives that contribute to tumor growth and metastasis. Arginine-derived polyamines alter gene expression by modulating global chromatin structure and cancer cell proliferation (Pegg, Reference Pegg2009). Kynurenine, derived from tryptophan, induces immunosuppression by binding to and activating the aryl hydrocarbon receptor (AhR) transcription factor. This impairs the ability of immune-tolerant dendritic cells (DCs) and regulatory T cells to target and eliminate cancer cells (Fallarino et al., Reference Fallarino, Grohmann, Hwang, Orabona, Vacca, Bianchi, Belladonna, Fioretti, Alegre and Puccetti2003).

In addition to the direct involvement of amino acids and their derivatives in metabolic reprogramming processes, amino acids are also fundamental in mediating epigenetic regulation and post-transcriptional modification. For example, DNA and histone methylation are regulated by balanced metabolite levels in the methionine cycle, which is influenced by methionine, serine, and glycine (Maddocks et al., Reference Maddocks, Athineos, Cheung, Lee, Zhang, Van Den Broek and Vousden2017).

The protonation constants of the aqueous amino acids are required to determine the stability constants of metal-amino acid complexes. Aiyelabola et al. prepared coordination complexes of aspartic acid in both basic and acidic media, for Mn²⁺: L in a stoichiometric ratio of 1: 2. The antimicrobial activity of the compound [Mn²⁺(asp)₂] was antibacterial and antifungal (Aiyelabola et al., Reference Aiyelabola, Isabirye, Akinkunmi, Ogunkunle and Ojo2016). Jasmin et al. synthesized a ligand Cu²⁺ complex by condensation reaction of isatin for cysteine, glycine, leucine, and alanine. The ligand Cu²⁺ complex was tested in antimicrobial studies using the disk diffusion method. All synthesized complexes exhibited strong antibacterial activity (Shampa et al., Reference Shampa, Islam, Hossain, Rahman, Zakaria and Zahan2017).

Al Naimi et al. synthesized complexes of Mn²⁺, Co²⁺, Zn²⁺, Cd²⁺, and Hg²⁺ with mixed ligands of 5-chlorosalicylic acid and L-valine. The results obtained in antibacterial studies of models through agar well diffusion bioassay evidenced the biological efficacy of ligands as well complexes (Al Naimi et al., Reference Al Naimi, Kindeel, Jarad and Al-Noor2016). Mixed iron(III) and zinc(II) complexes with isonitrosoacetophenone (HINAP) and histidine, proline, and phenylalanine were formed and characterized. The antimicrobial activity was evaluated against bacteria (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli), and Candida albicans fungi. The Fe²⁺ and Zn²⁺complexes were more active against Gram+ bacteria than Gram- bacteria. They also showed a significant inhibitory effect on the fungi tested. In vitro antitumor activity assayed against HEP2-type cancer cell lines (laryngeal cancer cells) showed significant toxicity of the ligands and their mixed complexes (Rahmouni et al., Reference Rahmouni, el Houda Bensiradj, Megatli, Djebbar and Baitich2019).

A potentiometric titration technique was used to determine the protonation constants of glycine, leucine, methionine, phenylalanine, tryptophan, asparagine, glutamine (T = 25 °C, I = 0.1 M KCl) (İnci and Aydın, Reference İnci and Aydın2021); cysteine, tyrosine, histidine, lysine (T = 25 °C, I = 0.1 M NaCl) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014); alanine, serine, isoleucine, threonine, valine (T = 25 °C, I = 0.1 M NaClO₄) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014); methylcystein, methylselenocysteine, selenomethionine (T = 25 °C, I = 0.1 M NaClO₄) (Murphy et al., Reference Murphy, Gaertner, Williams, McMillen, Powell and Brumaghim2019); proline, arginine, glutamic acid, aspartic acid, 3,4-dihydroxyphenylalanine, hydroxyproline (T = 25 °C) (Burger, Reference Burger1990); substituted salicylaldehyde amino acid (Sal–alanine, SalCl–alanine, SalBr–alanine, Sal–glycine, SalCl–glycine, SalBr–glycine, Sal–serine, Sal–tyrosine, Sal–phenylalanine) (T = 25 °C, I = 0.1 M KCl) (El-Sherif and Aljahdali, Reference El-Sherif and Aljahdali2013) in aqueous solution (Table 1).

Table 1. Protonation constants of L-amino acids in water, ethanol– water mixtures (E), dioxane–water mixtures (D), and dimethyl sulfoxide–water media (DMSO)

Sal-amino acid, substituted salicylaldehyde amino acid. The % is expressed as volume.

Protonation equilibria of amino acids in a mixed medium (ethanol–water, dioxane–water, dimethyl sulfoxide–water)

Water–organic solvent mixtures have attracted much interest due to their frequent use and a wide range of applications (reaction media for a variety of organic and analytical processes such as synthesis, titrations, or liquid chromatographic separations). Therefore, studies in media other than water provide insight into the chemistry of bioligands in living organisms (Diaz-Cruz et al., Reference Diaz-Cruz, Diaz-Cruz, Mendieta, Tauler and Esteban2000).

Ethanol is the closest organic solvent to water in terms of structure and properties and therefore the behavior of dissociation or protonation in ethanol is similar to that in aqueous solution. The water–ethanol mixture is of very interesting binary character. One reason for this is that ethanol dissolves most organic acids and bases better than water. Solvents such as water–ethanol mixtures are thus a better model for in vivo reactions (Crosby et al., Reference Crosby, Stone and Lienhard1970; Hughes et al., Reference Hughes, Bergan and Grabowski1986) because the mixtures possess both a low polar character and partially aqueous fractions, as in all biological systems. Their mixtures are macroscopically homogeneous, but it has been reported that the water and organic solvent molecules are not microscopically homogeneously dispersed due to the formation of hydrogen bonding networks and hydrophobic interactions. Consequently, the molecular composition of the solvation layer around a solute molecule is not the same as the bulk mixing ratio of water and organic solvent (Canel et al., Reference Canel, Gültepe, Doğan and Kilıç2006).

Mixed solvents, such as mixtures of water and dioxane, provide an even better model for in vivo reactions because they are not only less polar than pure water, but also partially aqueous, as in all biological systems (Fiol et al., Reference Fiol, Brandariz and Vicente1995). These mixtures are a favorite mixed solvent system in which to study the association and mobility of ions because the dielectric constant can be varied over a wide range. Changes in protonation constants upon the addition of 1,4-dioxane to aqueous solutions are due to increased ion–ion interactions and changes in solvent–ion and solvent–solvent interactions (Roy et al., Reference Roy, Sinha, Dey and Sinha2005).

The use of a water–DMSO mixture has several advantages: (a) the DMSO–water 50%: 50% mixture has a low hygroscopic character (pure DMSO is very hygroscopic and it is difficult to control its water content); (b) compatibility with the standard glass electrode, so that pH measurements can be carried out in a similar way to a purely aqueous solution; (c) a wide acidity range (pk _w = 15.50), which allows the study of deprotonation equilibria of weak acids that are difficult to study in water (Hernández-Molina et al., Reference Hernández-Molina, Mederos, Gili, Domínguez, Núñez, Germain and Debaerdemaeker1997).

The autoprotolysis constant (K _ap) of a solvent is an important parameter in understanding acid–base equilibria in mixed solvents. It determines the extreme limits of acidity and basicity in a given solvent medium. Kilic and Aslan derived a convenient and rapid potentiometric technique using a combined glass pH electrode for the determination of autoprotolysis constants in a variety of aqueous-organic mixed solvents (Kiliç and Aslan, Reference Kiliç and Aslan2005).

Ciolan et al. synthesized the binuclear complex [M₂L(AcO)₂(H₂O)₄] (M = Cu²⁺, Ni²⁺, Co²⁺, Mn²⁺ and L = (C₃₉H₃₄N₄O₆)²⁻) from 1,3-bis(2′-formylphenyl)-1,3-dioxapropane, L-tryptophan and metal acetate in methanolic medium. The complex [Cu₂L(AcO)₂(H₂O)₄] was the most effective in terms of antimicrobial efficacy and microbial spectra (Ciolan et al., Reference Ciolan, Patron, Marutescu and Chifiriuc2015). Mabrouk et al. synthesized and characterized Co²⁺ complexes of the Schiff base with salicylaldehyde and three amino acids (valine, leucine, isoleucine) in an ethyl alcohol solution. The metal: ligand ratio was 1: 2. Schiff bases and complexes were tested against different strains of microbes to determine their biological effects. Co(II) complexes showed greater bacterial activity against most bacterial species and the fungus C. albicans (Salama et al., Reference Salama, Ahmed and Hassan2017). Obaid et al. prepared novel mixed metal–ligand complexes for metal(II) chloride (Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺) (L = glycine: alanine: valine) using 50% ethanolic solution as well as 50% distilled water in a molar ratio of 1:1:1:1. The Cd⁺² complex had a higher antibacterial activity than the other complexes (Obaid et al., Reference Obaid, Al Naemi and Al-Hamdani2018). Bougherra et al. prepared Cu²⁺ complexes for dimethylglyoxime as well as the tryptophan, glutamate, proline, arginine, and valine. The antimicrobial activity of ligand complexes was tested using the agar diffusion technique with DMSO as solvent against pathogenic bacteria and fungi. The complexes were more antimicrobial than the free ligand. Metal chelation significantly affected the antimicrobial activity of the ligands (Bougherra et al., Reference Bougherra, Berradj, Adkhis and Amrouche2018).

The protonation constants of cysteine, tyrosine, histidine, lysine, tryptophan (T = 25 °C, I = 0.1 M NaCl) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014); glycine, alanine, valine, leucine, isoleucine, phenylalanine, serine, methionine (T = 25 °C, I = 0.1 M NaClO₄) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014) in various ethanol–water mixtures; leucine, isoleucine, serine, threonine (T = 25 °C, I = 0.1 M NaClO₄) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014) in dioxane–water mixtures; alanine, valine, leucine, threonine, phenylalanine, serine, methionine (T = 25 °C, I = 0.1 M NaNO₃) (El-Sherif et al., Reference El-Sherif, Shoukry, Abd Elkarim and Barakat2014) in DMSO–water media are given in Table 1.