Carboxyl Group as a Radical Scavenging Moiety : Thermodynamics of 2 H + / 2 e – Processes of Phloretic Acid

Phloretic acid is one of the most abundant colon catabolites of various classes of polyphenols (e.g., polymeric proanthocyanidins, tea catechins, and ellagitannins). In this paper thermodynamics of 2H+/2e– radical scavenging mechanisms of phloretic acid was studied. For the first time the involvement of carboxyl group in double hydrogen atom transfer (dHAT), double electron transfer-proton transfer (dET-PT) and sequential double proton loss double electron transfer (SdPLdET) processes was investigated. Obtained results indicate that phloretic acid possesses potential for inactivating radicals of different characteristics (HO•, HOO•, CH3O•, CH3OO•, CH2=CH–O–O•, PhO•, Cl3COO• etc.) via dHAT and SdPLdET mechanisms. Because phloretic acid is usually better absorbed than its precursor molecules, it may contribute to health benefits associated with regular intake of polyphenol-rich diet by direct scavenging of radicals.


INTRODUCTION
HLORETIC acid (3-(4-hydroxyphenyl)propanoic acid; dihydro-p-coumaric acid) can be found in very small quantities in plant kingdom [1] where it is involved as a precursor in the biosynthesis of cyclic diarylheptanoids, a class of natural products. [2]Some processed fruits, such as black brined olives and raisins, contain phloretic acid as a product of bacterial activity on phenolic acids. [3,4]In man and animal, among numerous products formed during colonic microbial catabolism of various (poly)phenols (e.g., polymeric proanthocyanidins, acylated flavonoid glycosides, tea catechins, gallotannins, ellagitannins and hydroxycinnamates), [5][6][7] phloretic acid is one of the most abundantly produced. [8][11] Concentrations of intact (poly)phenols in systemic circulation are mainly in nM to very low M range, [10] which is insufficient to exert in vivo direct radical scavenging (antioxidant) activity. [12]However, colonic catabolites produced in high M concentrations [8] have potential to promote human health via several possible in vivo mechanisms. [13]hloretic acid is a weak acid (pKa1 (-COOH) = 4.76; [14] pKa2 (-OH) = 10.1). [15]It is slightly soluble in water and at the physiological pH of 7.4 (pH of colon is in the range of 6.6 to 7.5) [16] the majority of phloretic acid is in the ionized form, i.e. as a carboxylate anion.Its radical scavenging activity has been investigated by inhibition of chemiluminescence in human granulocytes. [17]Phloretic acid concentration in colon may reach values of 200 M, [8] which could be sufficient to exert biological activity and effective direct radical inactivation.

P
Radicals are continuously produced in all cells as a part of normal cellular function.They are essential for human health because of involvement in specific metabolic processes (such as energy production), intercellular signalling and destroying of pathogenic microbes. [18]Body defence mechanisms ensure delicate balance between the production and the removal of radicals, maintaining their optimal concentrations. [19,20]If this homeostasis is interrupted, excess of radicals initiate a chain of oxidative reactions, giving rise to protein, lipid and DNA damage.(Poly)phenolic colon catabolites produced in high M concentrations could be important endogenous antioxidants capable to scavenge excess of radicals and suppress their deleterious effects.
[23] They can be grouped into two types of processes: H-atom abstraction and radical adduct formation (RAF).H-atom abstraction processes may occur via at least three different mechanisms: hydrogen atom transfer (HAT), electron transfer followed by proton transfer (ET-PT) and sequential proton loss electron transfer (SPLET).These and some other possible mechanisms (such as proton-coupled electron transfer (PCET) and sequential proton-loss hydrogen-atom transfer (SPLHAT)) have been recently reviewed. [24]Already published studies dealing with radical scavenging mechanisms of natural (poly)phenols have mostly been based on single, 1H + /1e -processes. [25][28][29] Recently, we have performed a DFT investigation of radical scavenging potency of phloretic acid by considering single 1H + /1e - mechanisms related to homolytic and heterolytic O-H bond cleavage in phenolic OH group. [30]In this study, we have computationally investigated energetics of double, i.e., 2H + /2e -mechanisms (two sequential 1H + /1e -processes) by including carboxyl group of phloretic acid in consideration.To the best of our knowledge, involvement of carboxyl group as a radical scavenging moiety in such processes has not been studied before.

COMPUTATIONAL DETAILS
Phloretic acid (HO-Phl-COOH) could be able to scavenge two radicals via double HAT (dHAT) mechanism.The first step in this mechanism is the homolytic cleavage of phenolic O-H bond which results in formation of phenoxyl radical ( • O-Phl-COOH) (Eq.1).In the second step the homolytic cleavage of O-H bond of carboxyl group occurs producing dienone lactone ( • O-Phl-COO • ), Eq. (2).
The HAT mechanism can be characterized by the bond dissociation enthalpy (BDE).The first and the second BDE are calculated according to Eqs. ( 3) and (4), respectively. where ) and H(H • ) are enthalpies of phloretic acid, phenoxyl radical, dienone lactone and H-atom, respectively.The double ET-PT mechanism (dET-PT) proceeds in four steps.The first step is transfer of an electron from HO-Phl-COOH by which the radical cation (HO-Phl-COOH •+ ) is formed, Eq. ( 5).The second step is deprotonation of HO-Phl-COOH •+ , which results in formation of phenoxyl radical, Eq. ( 6).Electron transfer from phenoxyl radical (the third step, Eq. ( 7)) gives cationic intermediate ([O-Phl-COOH] + ) which produces dienone lactone by the proton transfer in the final step, Eq. ( 8).
  The reaction enthalpies related to these processes can be calculated by the following equations: In the double SPLET mechanism deprotonation of carboxyl group occurs first followed by the heterolytic cleavage of phenolic O-H bond in the second step.Obtained dianion ( -O-Phl-COO -) by two-step process of electron donation produces dienone lactone: Accordingly, this mechanism can be abbreviated as SdPLdET because sequential double proton loss followed by double electron transfer occurs.Related reaction enthalpies are as follows: PA1 (PA2) and ETE1 (ETE2) are proton affinity and electron transfer enthalpy related to the corresponding steps.
are enthalpies of carboxylate anion, dianion and radical anion, respectively.The net result of all three mechanisms is the same, that is, dienone lactone production.Inactivation of radicals is a very complex process influenced by many factors. [31]Among them, chemical nature of scavenged radicals particularly influences scavenging processes. [24,32]To investigate radical scavenging potential of phloretic acid we used the same set of radicals as in our recent papers. [29,30]It embraces oxygen-derived radicals with very different characteristics: (superoxide anion).The • OH radical is the main source of biological damage in living organisms because it is the most reactive and electrophilic of the oxygen-centered radicals.It may withdraw an electron or H-atom from almost any compound in its vicinity. [33] O-O • is very electronegative, highly reactive peroxyl radical.O2 •-is an important radical with rather low reactivity that can occur during in vivo metabolism.Phenoxyl type radicals (PhO • ) are involved in biological redox processes and in the biosynthesis of natural products. [34]o take into account electronic properties of selected radicals on reaction with phloretic acid, calculations of Gibbs free energy of reactants and products involved in studied mechanisms have been performed.In general, Gibbs free energy of a reaction represents the criterion for definition of thermodynamically preferred process.The reaction of phloretic acid with particular radical (RO • ) is thermodynamically favourable if it is exergonic: rG o BDE1 and rG o BDE2 represent the free energies of double HAT mechanism (Eqs. 1 and 2) and are given by Eqs.22 and 23: Lower rG o BDE1 and rG o BDE2 values are assumed to correspond to a higher reactivity of phloretic acid with a radical considered.
Croat.Chem.Acta 2016, 89( 4) In the case of the SdPLdET mechanism (Eqs.13-16) All three radical scavenging mechanisms have the same reactants and the same products, and hence they have the same net thermodynamic balance, Eq. ( 32): Accordingly, they may proceed in parallel and could be competitive.All electronic calculations were performed using the Gaussian 09 program package. [35]The density functional theory (DFT)-based methods are able to accurately predict antioxidant potencies and reaction mechanisms involved in radical scavenging reactions of (poly)phenolic compounds. [24]Geometry optimizations and frequency calculations for phloretic acid and its radical cation, radicals, anions, radical anions, as well as for ten selected radicals and their species involved in radical scavenging pathways were carried out using the M06-2X functional [36] and the 6-311++G(d,p) basis set.Among the suite of density functionals, M05-2X, M06-2X and M-11L Minnesota functionals are particularly suitable for systems where main-group thermochemistry, kinetics, and noncovalent interactions are important. [37]The M06-2X has been chosen because it is among the best performing functionals for modelling reaction energies involving radicals. [38]The influence of water and pentyl ethanoate as solvents was calculated with an implicit continuum solvation model -SMD, [39] which takes into account the full solute electron density in estimation of energy of solvation.SMD is a universal solvation model and in conjunction with the M06-2X density functional has been successfully used for study of thermodynamics and kinetics of radical scavenging mechanisms. [40]Spin unrestricted calculations were used for radical species.Since spin contamination can affect the accuracy of enthalpies of open-shell systems, the spin operator S 2  values for all the open-shell species have been checked.If there is no spin contamination, this should equal s(s+1), where s equals ½ times the number of unpaired electrons.In all cases, the deviations from the ideal value (S 2  = 0.75) were lower than 4.87 % and 0.11 % before and after annihilation of the first spin contaminant.Because spin contamination can be considered negligible if the value of S 2  differs from s(s+1) by less than 10 %, [41] the obtained enthalpy values of radical species studied in this work are reliable.4][45]

RESULTS AND DISCUSSION
The most stable structures of phloretic acid in water and pentyl ethanoate obtained by the conformational analysis are presented in Figure 1.Conformational analysis indicates that the difference in energy between them is small which indicates that structures interconvert easily at room temperature.All obtained results and related discussion presented in this paper are based on syn conformation in both solvents.It should be noted that more than a half century ago Iwasaki et al. indicated that syn conformation of phloretic acid converts into its dienone lactone via 2H + /2e -oxidation. [46]As mentioned in Introductory section, recently we investigated single, i.e., 1H + /1e -radical scavenging mechanisms of anti conformation of phloretic acid [30] by considering solely phenolic O-H group as antiradical moiety.Here presented results additionally embrace contribution of carboxyl moiety in radical inactivation by syn conformation of phloretic acid via 2H + /2e -mechanisms.
The major problem in evaluation of correctness of theoretical results related to reaction enthalpies of radical scavenging mechanisms of (poly)phenolic compounds is lack of corresponding experimental results.Only limited number of published data for simple phenolic compounds can be found in literature.To ascertain reliability of applied approach experimentally determined BDE for phenol and 3,5-di-tert-butylcatechol may be used.For these compounds BDE values of 88.30 kcal/mol and 79.30 kcal/mol have been obtained in benzene as a solvent, respectively. [47,48]We recently showed that chosen level of theory (SMD/M06-2X/6-311++G(d,p)) may be considered as appropriate because obtained calculated values of 89.40 kcal/mol and 77.29 kcal/mol in the same solvent are in good agreement with experimental results. [30]eaction enthalpies related to dHAT, dET-PT and SdPLdET mechanisms of phloretic acid calculated at the SMD/M06-2X/6-311++G(d,p) level of theory are listed in Table 1.Calculations were performed in water pentyl ethanoate, representing biological liquids and membrane lipids, respectively, i.e., the two kinds of natural environments of radical inactivation.
To conclude which radical scavenging mechanism represents the most probable reaction pathway from the thermodynamic point of view, BDE, IP, PA and ETE should be considered.Their values may indicate thermodynamically preferred mechanism and point out the active site for initial radical inactivation. [23,43]Data presented in Table 1 indicate that in both solvents dET-PT mechanism (Figure 2) is thermodynamically unfavourable pathway because IP values (for the first and second 1H + /1e -process) are much higher than the corresponding BDE and PA (ETE) values.
In water solution the SdPLdET mechanism is more probable than the dHAT mechanism because PA values are much lower than the corresponding BDE values.This mechanism starts with deprotonation of carboxyl group, which is more acidic than the phenolic OH group (PA1 values 15.79 kcal/mol vs 26.58 kcal/mol, respectively).Obtained carboxylate anion easily deprotonates (abstracts phenolic proton) rather than undergoes electron transfer due to the much lower PA2 value (15.73 kcal/mol) than  ETE1 value (77.48 kcal/mol).Then, resulting dianion yields dienone lactone by the double electron transfer (Figure 3).Regarding the dHAT mechanism, data presented in Table 1 indicate that it is a favourable mechanism in nonpolar medium (pentyl ethanoate).The first H-atom abstraction occurs from phenolic O-H group due to the lower BDE1 value (87.15 kcal/mol vs 112.73 kcal/mol for -OH and -COOH group, respectively).The second H-atom abstraction from -COOH group, which is less energy demanding than the first one by 25.59 kcal/mol (61.56 kcal/mol vs 87.15 kcal/mol), gives dienone lactone (Figure 4).Lower energetic costs of second processes, which indicate 2H + /2e -processes as plausible, have been recognized earlier in the case of guaiacyl moiety, [49] catechol moiety [27,29] and uric acid. [28]cause the scavenging processes are highly influenced by the properties of the scavenged radical species, we also calculated the free energy of reactions (rG o ) of phloretic acid with each of ten selected radicals ( • OH, • OOH, • OCH3, • OC(CH3)3, PhO • , O2 •-, CH3-OO • , CH2=CH-OO • , CH2=CH-CH2-OO • and Cl3C-OO • ) for dHAT, dET-PT and SdPLdET mechanisms.Obtained thermodynamic results for these mechanisms in water and pentyl ethanoate are summarized in Table 2 and Table 3, respectively.The second and seventh columns in Table 2 and Table 3 are related to free energy change of reactions of inactivation of radicals by phloretic acid via the first HAT (rG o BDE1) and second HAT (rG o BDE2) mechanisms, respectively.The sum of these numerical values is the total energy requirement for dHAT (ΔrG o HAT1 + ΔrG o HAT2).It is equal to the total energy    toward radicals decreases as follows: hydroxyl >> alkoxyls > phenoxyl ≈ peroxyls >> superoxide.Because phloretic acid is produced in colon in high M concentrations and is usually better absorbed than its precursor molecules, it has potential to contribute to health benefits associated with regular intake of polyphenol-rich diet via direct radical quenching.

Figure 2 .
Figure 2. The dET-PT mechanism in water.

Figure 5 .
Figure 5. Overall energy requirements for scavenging of selected radicals via dHAT, dET-PT and SdPLdET by phloretic acid.
The double ET-PT mechanism (Eqs.5-8) is characterized by rG o IP1, rG o PDE1, rG o IP2 and rG o PDE2 which are calculated by Eqs.24-27, respectively: , rG o PA1, rG o PA2, rG o ETE1 and rG o ETE2 are related to involved processes, and are calculated by Eqs.28-31, respectively:

Table 2 .
Standard Gibbs energies of reaction (rG o in kcal/mol) for dHAT, dET-PT and SdPLdET mechanisms of radical scavenging by phloretic acid in aqueous medium