lopachin2012

Published: November 04, 2011

r 2011 American Chemical Society 239 dx.doi.org/10.1021/tx2003257 | Chem. Res. Toxicol. 2012, 25, 239–251

PERSPECTIVE

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Application of the Hard and Soft, Acids and Bases (HSAB) Theoryto Toxicant�Target InteractionsRichard M. LoPachin,*,† Terrence Gavin,*,‡ Anthony DeCaprio,§ and David S. Barber||

†Department of Anesthesiology, Montefiore Medical Center, 111 East 210th Street, Bronx, New York 10467, United States‡Department of Chemistry, Iona College, New Rochelle, New York 10804, United States§Department of Chemistry and Biochemistry, Florida International University, 11200 S.W. Eighth Street, Miami, Florida 33199,United States

)Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611, United States

’CONTENTS

Introduction 240HSAB Definitions: Soft and Hard, Electrophiles and

Nucleophiles 240Physiochemical Principles Determining the Selective

Interactions of Electrophiles and Nucleophiles 240

HSAB Descriptors of Electrophile�Nucleophile AdductFormation 241

Toxicological Application of HSAB Principles 242α,β-Unsaturated Carbonyl Derivatives:

Toxicological Consequences of Exposure 242

α,β-Unsaturated Carbonyl Derivatives: Softnessand Electrophilicity 242

α,β-Unsaturated Carbonyl Electrophilicity: InterveningVariables That Influence Interpretation 243

α,β-Unsaturated Carbonyl Derivatives: NucleophilicTargets and Molecular Mechanisms of Toxicity 244

α,β-Unsaturated Carbonyl Derivatives: Relevance ofHSAB Calculations to in Vivo Toxicokinetics andToxicodynamics 246

Cytoprotective Properties of 1,3-Dicarbonyl Enols:Mechanistic Insights Gained by HSAB QuantumMechanical Calculations 247Cellular Oxidative Stress: Phytopolyphenol

Cytoprotection 247

1,3-Dicarbonyl Enols: ANewClass of Cytoprotectants 2471,3-Dicarbonyl Enols: HSAB Parameters 247

Summary 248Author Information 248Abbreviations 248References 248

Received: August 5, 2011

ABSTRACT: Many chemical toxicants and/or their active metabolites are electrophiles thatcause cell injury by forming covalent bonds with nucleophilic targets on biological macro-molecules. Covalent reactions between nucleophilic and electrophilic reagents are, however,discriminatory since there is a significant degree of selectivity associated with these interactions.Over the course of the past few decades, the theory of Hard and Soft, Acids and Bases (HSAB)has proven to be a useful tool in predicting the outcome of such reactions. This concept utilizesthe inherent electronic characteristic of polarizability to define, for example, reacting electro-philes and nucleophiles as either hard or soft. These HSAB definitions have been successfullyapplied to chemical-induced toxicity in biological systems. Thus, according to this principle, atoxic electrophile reacts preferentially with biological targets of similar hardness or softness. Thesoft/hard classification of a xenobiotic electrophile has obvious utility in discerning plausiblebiological targets and molecular mechanisms of toxicity. The purpose of this perspective is todiscuss the HSAB theory of electrophiles and nucleophiles within a toxicological framework. Inprinciple, covalent bond formation can be described by using the properties of their outermost or frontier orbitals. Because theseorbital energies for most chemicals can be calculated using quantummechanical models, it is possible to quantify the relative softness(σ) or hardness (η) of electrophiles or nucleophiles and to subsequently convert this information into useful indices of reactivity.This atomic level information can provide insight into the design of corroborative laboratory research and thereby help investigatorsdiscern corresponding molecular sites and mechanisms of toxicant action. The use of HSAB parameters has also been instrumentalin the development and identification of potential nucleophilic cytoprotectants that can scavenge toxic electrophiles. Clearly, thedifficult task of delineating molecular sites andmechanisms of toxicant action can be facilitated by the application of this quantitativeapproach.

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240 dx.doi.org/10.1021/tx2003257 |Chem. Res. Toxicol. 2012, 25, 239–251

Chemical Research in Toxicology PERSPECTIVE

’ INTRODUCTION

Unlike the formation of drug�receptor complexes that primarilyinvolve short distance forces such as hydrogen-bonding, hydro-phobic, or van der Waals interactions, many chemical xenobioticsand/or their active metabolites are electrophiles (electron deficient)that form covalent bonds (e.g., SN2, Schiff base formation, Michaeladdition) with cognate nucleophilic (electron rich) sites on bio-logical targets.1�10 Cytotoxicity occurs because the formation ofcovalently bonded adducts can impair the functions of enzymes,DNA, cytoskeletal proteins, and other macromolecules, therebyleading to defective cell processes. Thus, it is now recognized thatelectrophilic α,β-unsaturated carbonyl derivatives of the type-2alkene chemical class (e.g., acrolein, acrylamide, or methylvinylketone) cause broad organ system toxicity by forming covalentMichael-type adducts with nucleophilic sulfhydryl groups on func-tionally critical proteins.11�20 However, electrophiles do not reactindiscriminately with nucleophiles, and instead, such interactionsoccur along a continuumof relative reactivity. The significant degreeof selectivity that occurs in electrophile�nucleophile interactions ispredicted by Pearson's Hard and Soft, Acids and Bases (HSAB)theory.5,17,21 On the basis of this concept, reactive molecules areassessed on the basis of their respective polarizability such thatelectrophiles and nucleophiles are classified as either soft (relativelypolarizable) or hard (relatively nonpolarizable). A useful theoremstemming from this principle is that toxic electrophiles reactpreferentially with biological targets of similar hardness or softness.Therefore, the α,β-unsaturated carbonyls can be viewed as softelectrophiles that cause cytotoxicity by selectively forming adductswith soft nucleophilic anionic thiolates on functionally critical pro-teins.9,14�16,18Whereas the HSAB theory initially described hard-ness and softness in terms of the experimental ionization potentialand electron affinity of the reactingmolecules, these parameters alsocan be related (e.g., by Koopmans theorem) to the respectiveenergies of the outermost or frontier molecular orbitals (FMOs).Since small molecule FMO energies can be determined computa-tionally using various quantum mechanical models, HSAB param-eters such as the softness (σ) or hardness (η) of an electrophileand its nucleophilic target can be readily derived. Correspondingly,the values for σ and η can be used in a more recently developedalgorithm to calculate the electrophilic index (ω) of a toxicant, thevalue of which reflects the propensity of the electrophile to formadducts with a given biological nucleophile. Although more com-plex, the respective σ and η values for the nucleophile can also beused to derive an index of nucleophilicity (ω�, see ahead). On apragmatic basis, these parameters can be used to predict the toxicpotential of electrophilic xenobiotic chemicals and to facilitate theidentification of corresponding nucleophilic molecular sites ofaction.5,8,17 Indeed, our research has shown that, excluding stericand other physiochemical issues (see ahead), the calculated values ofthese descriptors (σ, ω, and ω�) directly corresponded to thesecond order rate constants for α,β-unsaturated carbonyl adduc-tion of cysteine sulfhydryl groups. For individual members of aα,β-unsaturated carbonyl series, the kinetic and HSAB parameterswere closely correlated to the respective magnitude of in vitrotoxicity, i.e., disruption of synaptosomal function.14�16,18 This abilityof HSAB parameters (σ, η,ω, andω�) to predict the potency of agiven chemical toxicant has significant implications for risk asses-sment.19,20 In addition, these parameters are a useful tool in thesearch for macromolecular targets and subsequent elucidation ofmolecular mechanisms of toxicity. Therefore, in this perspective, wewill discuss the HSAB theory and define the physiochemical

characteristics of hard and soft, and electrophiles and nucleo-philes. As we will point out, establishing structure�activityrelationships (SAR) based on kinetic and HSAB properties canreveal the substructure (e.g., α,β-unsaturated aldehyde) ormoiety (e.g., carbonyl group) responsible for the toxicity of theelectrophile. This forms a rational basis for categorizing achemical series and for understanding the corresponding toxicmechanism.19,22,23 Finally, in this perspective we will presentexamples from the literature as a framework for demonstratinghow quantum chemical calculations can be effectively appliedtoward understanding molecular toxicodynamic issues.

’HSAB DEFINITIONS: SOFT AND HARD, ELECTRO-PHILES AND NUCLEOPHILES

Physiochemical Principles Determining the Selective In-teractions of Electrophiles and Nucleophiles. The HSABtheory classifies reacting species as either relatively “hard” or“soft”, based on polarizability, i.e., the ease with which electrondensity can be displaced or delocalized to form new covalentbonds. Polarizability is an inherent characteristic of electrondistribution in atoms or molecules. Thus, electrons that are farfrom the influence of the nucleus or that occupy a large volume(electron “cloud”) are more likely to be displaced, i.e., redis-tributed into a new bonding pattern. For example, the conjugatedα,β-unsaturated carbonyl structure of many type-2 alkenesis considered to be a soft electrophile (Table 1) because the

Table 1. Hardness/Softness Values for Selected Electrophilea

aGround state equilibrium geometries were calculated for each structurewith DF B3LYP-6-31G* in water from 6-31G* initial geometries. LUMOand HOMO energies were generated exclusively from corresponding s-cisconformations and were used to calculate η and σ (see text).

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delocalized π electrons are mobile. This mobility can be consid-ered the extension of an electron cloud over four nuclear centers(CdC�CdO) based on interactions between the orbitals ofthe electron-withdrawing carbonyl group and those of thealkene moiety. The extended cloud is easily distorted, hence soft(polarizable). In contrast, hard electrophilic toxicants, e.g., chloro-ethylene oxide or vinyl chloride (Table 1), have highly localized,nonextended charge densitites at specific electrophilic centers, e.g.,the respective C1 carbon atoms. Consequently, these chemicalsare characterized by low electron polarizability. The softness orhardness of a nucleophile is also determined by the polarizability ofcorresponding electrons. Sulfur has a large atomic radius so that itsvalence electrons are relatively far from the nucleus and, as such,are highly polarizable. Upon ionization of the thiol (i.e., SHf S�),the consequent expansion of the anionic cloud yields the relativelysoft (most easily polarizable) thiolate nucleophile, e.g., comparethe respective thiol- and thiolate-states of the amino acid residue,cysteine (Table 2). In contrast, nitrogen and oxygen nucleophileshave relatively small atomic radii and, accordingly, their electronclouds are less readily distorted. Consequently, such compoundsare much harder nucleophiles, e.g., the methoxide ion and theprimary nitrogen of the amino acid lysine (Table 2). Expansion ofthe electron cloud via nearby conjugation with unsaturated sitesyields a less hard nucleophile, e.g., the secondary amine ofhistidine, the phenolate ion, or the enolate of 2-acetylcyclopenta-none (Table 2).According to the selectivity principle of the HSAB model, soft

electrophiles preferentially form adducts with nucleophiles of

comparable softness, whereas hard electrophiles form adductswith hard nucleophiles (see ahead). The data presented inTable 2 indicate that, among the biological nucleophiles, thesulfhydryl thiolate state of cysteine residues is the softest.5,8,17

Therefore, the preferred nucleophilic cellular targets of the softelectrophilic α,β-unsaturated carbonyls are cysteine sulfhydrylthiolate groups. In contrast, the neurotoxic n-hexane metabolite,2, 5-hexanedione is a hard electrophile (Table 1) that preferen-tially forms adducts with harder nucleophiles such as nitrogenatoms on the ε-amino group of lysine residues. Additional hardnucleophilic targets include the secondary imidazole nitrogenof histidine and the carbon, nitrogen, and oxygen groups ofDNA/RNA.HSAB Descriptors of Electrophile�Nucleophile Adduct

Formation. As indicated above, covalent bond formation be-tween reacting molecules can be described by using the proper-ties of their outermost orbitals. The two most important of theseare the highest energy orbital that contains electrons (known bythe acronym HOMO = Highest Occupied Molecular Orbital)and the lowest energy orbital that is vacant (LUMO = LowestUnoccupied Molecular Orbital). Covalent bonding such asadduct formation occurs when electron density is transferredbetween the orbitals of the reacting species (nucleophile toelectrophile) to form the new bonds of the product (the adduct).FMO energies (EHOMO and ELUMO) for a wide variety ofchemical structures can be calculated from quantum mechanicalmodels, and the corresponding hardness (η) and softness (σ)can then be derived using eqs 1 and 2.

η ¼ ½ELUMO � EHOMO�=2 ð1Þ

σ ¼ 1=η ð2ÞIn essence, softness measures the ease with which electron

redistribution takes place during covalent bonding, i.e., nucleo-phile donation of electrons and acceptance by the electrophile.Therefore, with respect to electrophilic species, it is often the casethat softness (higher σ value) is correlated with ease of adductformation (expressed, for example, as a reaction rate). Theelectrophilic index (ω) is a parameter that combines softnessand chemical potential (μ; eq 3).24 The latter parameter, μ, is thepropensity of a species to undergo chemical change. Thus, ω iscalculated according to eq 4 and represents a more comprehen-sive measure of electrophilic reactivity.

μ ¼ ½ELUMO þ EHOMO�=2Þ ð3Þ

ω ¼ μ2=2η ð4ÞWith respect to the nucleophile, the corresponding molecularorbital energies can also be used to calculate nucleophilic softnessand chemical potential as shown above in eqs 2 and 3. In addi-tion, the likelihood that a given nucleophile (A) will form anadduct with a given electrophile (B) can be predicted bycalculating a nucleophilicity index (ω�) using eq 5.25

ω� ¼ ηAðμA � μBÞ2=2ðηA þ ηBÞ2� ð5ÞThis parameter considers the hardness (η) and chemical poten-tial (μ) of both the electrophilic and nucleophilic reactants. Theelectrophilic (ω) and nucleophilic (ω�) indices have been demon-strated to be reliable descriptors for a variety of electrophile�nucleophile interactions.8,18,27,28

Table 2. Hardness/Softness Values for SelectedNucleophilesa

aGround state equilibrium geometries were calculated for each structurewith DF B3LYP-6-31G* in water from 6-31G* initial geometries. Valuesobtained were used to calculate σ and η (see text).




To perform the quantum mechanical computations, numerouscomputer software packages such as Gaussian (www.gaussian.com),HyperChem (www.hyper.com), Q-Chem (www.q-chem.com),Spartan (www.wavefun.com) are available commercially. In addi-tion, there are many open source programs like GAMESS (www.msg.chem.iastate.edu), deMon (www.demon-software.com) andAbinit (www.abinit.org) among others that can be used to makethese types of calculations. While various models exist, densityfunctional theory (DFT) provides the most consistently em-ployed approach (the B3LYP model) for the purposes describedby this perspective. However, in one case (see ahead) we used aHartree�Fock (HF) model with good results. Although in mostof the work described here molecular geometries (or energies)are calculated in water, we have also reported an applicationusing gas phase (in vacuum) calculations. The use of eitherstate can be rationalized, but in our applications, no differe-nces in the results were observed as long as the method chosenwas consistently employed. Throughout this study, only global(i.e., whole molecule) parameters are considered, but the use oflocalized functions at sites of reactivity within a molecule has beensuccessfully applied by others in the development of computa-tional models.23,24

’TOXICOLOGICAL APPLICATION OF HSABPRINCIPLES

Whereas exceptions exist, most toxic chemicals are electro-philes that produce toxicity by interacting with biological nucleo-philes,5,6,8,10,29 and therefore, the HSAB concepts should havebroad applicability to the field of molecular toxicology. In thefollowing sections, we will demonstrate how the application ofHSAB principles can help identify the sites and mechanisms oftoxicant action. Thus, we will consider the application of HSABprinciples to recent structure�toxicity studies involving acrolein,4-hydroxy-2-nonenal, and other α,β-unsaturated carbonyl andaldehyde derivatives. In addition, we will discuss the role ofHSAB principles in the development of nucleophilic 1,3-dicar-bonyl compounds that might be useful in treating diseaseprocesses involving endogenous α,β-unsaturated carbonyl com-pounds. Although the HSAB calculations are broadly applicableand straightforward, our discussions of these studies will alsoserve to identify certain physiochemical toxicant characteristicsand intervening biological factors that can influence toxic expres-sion and interpretation of the quantum mechanical data.

α,β-Unsaturated Carbonyl Derivatives: Toxicological Con-sequences of Exposure.Acrylamide (ACR), acrolein, 4-hydroxy-2-nonenal (HNE), and other structurally related derivatives(Table 1) are characterized by a conjugated α,β-unsaturatedcarbonyl substructure that is formed when an electron-withdrawing group (e.g., a carbonyl group) is linked to an alkene.These are therefore soft electrophiles of the type-2 alkenechemical class. Members of this class are used extensively inthe manufacturing, agricultural, and polymer industries, andconsequently, occupational human exposure is significant. Inaddition, these chemicals are recognized as environmental pol-lutants and dietary contaminants, e.g., acrolein from the combus-tion of petrochemical fuels, methyl vinyl ketone (MVK) inautomobile exhaust, and ACR in French fries. α,β-Unsaturatedcarbonyl derivatives are also prevalent components of cigarettesmoke, e.g., acrolein, ACR, and acrylonitrile. Acute or chronicexposure to these type-2 alkenes has been linked to major organsystem toxicity and to possible carcinogenicity in humansand laboratory animals.5,30�34 In addition to environmental orexogenous intoxication, acrolein, HNE, and certain other α,β-unsaturated aldehydes are highly toxic byproducts of membranelipid peroxidation associated with cellular oxidative stress. Grow-ing evidence indicates that these endogenous type-2 alkenes playa pathogenic role in many diseases that have oxidative stress asa common molecular etiology, e.g., atherosclerosis, Alzheimer’sdisease, and diabetes.5,13,17,35,36 Clearly, the type-2 alkenes are animportant class of chemicals with respect to environmentallyacquired toxicity and endogenous disease pathogenesis. Asdescribed in the following subsection, deciphering the mecha-nism of type-2 alkene cytotoxicity can be aided significantly byapplication ofHSAB concepts and quantummechanical descriptors.α,β-UnsaturatedCarbonylDerivatives: Softness andElectro-

philicity. As discussed above, the relative softness and electro-philicity of a α,β-unsaturated carbonyl derivative can be calcu-lated using LUMO andHOMO energies determined via quantummechanical models. As the data in Table 3 illustrate, acroleinand HNE are the strongest electrophiles among the type-2alkenes tested, i.e., the respective gas phase electrophilicityindex (ω) is numerically larger than other constituents in thechemical series. In contrast, acrylamide (ACR) and methylacrylate (MA) are substantially weaker electrophiles, whereasMVK exhibits intermediate electrophilic reactivity. Overall, thetype-2 alkene electrophilic indices suggest the following rank

Table 3. Calculated and Experimental Parameters for Conjugated Type-2 Alkenes and Non-Conjugated Analogues

type-2 alkene ELUMOa (ev) EHOMO (ev) σ (�10�3 ev�1) ω (ev) log k2

b (pH 7.4) uptake inhibitionc (log IC50)

acrolein �1.70 �6.98 379 3.57 2.596 �4.28

MVK �1.46 �6.69 382 3.18 2.048 �3.48

HNE �1.84 �6.93 393 3.78 0.938 �3.40

MA �1.01 �7.36 315 2.76 �1.893 �0.34

ACR �1.00 �6.78 346 2.62 �1.804 �0.36

Nonconjugated Analogues

propanald �0.65 �6.84 323 2.26

allyl alcohold +0.18 �7.06 276 1.63a ELUMO and EHOMO were obtained after calculating ground state equilibrium geometries for each compound with DF B3LYP-6-31G* in vacuum from6-31G* initial geometries. These values were generated exclusively from corresponding s-cis conformations and were used to calculate softness (σ) andthe electrophilic index (ω) of each electrophile as described in LoPachin et al.15 b Second-order reaction rates (k2) were determined for type-2 alkenereactions with L-cysteine at pH 7.4 (n = 4�6 experiments). c Inhibition of synaptosomal membrane 3H-DA uptake was determined in striatalsynaptosomes exposed to type-2 alkenes (n = 4�6 experiments).14 dDoes not undergo the Michael Reaction.



order of electrophilicity: acrolein ≈ HNE > MVK . MA gACR. In this type-2 alkene series, the relative differences inelectrophilicity are determined by substituent functional groupsand their respective contributions to the electron density of theα,β-unsaturated carbonyl structure. Allyl alcohol and propanaldo not contain extended π electron systems and therefore havesubstantially lower values of electrophilicity compared to that ofacrolein and related conjugated analogues (Table 3). Since allylalcohol and propanal lack a conjugated structure, neither of thesesubstances can undergo Michael-type reactions, i.e., conjugateaddition of a nucleophile. Subsequent experimental researchshowed that softness and electrophilicity were determinants ofthe chemical reactions that mediate type-2 alkene toxicity.14�16

Excluding HNE (see below), the rank order of electrophilicitywas closely correlated (r2 > 0.90) to the corresponding secondorder rate constants for the adduct reaction with cysteine (log k2)and to the respective potencies (log IC50) for in vitro nerveterminal (synaptosomes) toxicity (Table 3). While this corre-lated data set consists of only four compounds, it should berecognized that each structure represents a different functionalgroup (i.e., aldehyde, ketone, ester, and amide) and thus con-stitutes a significant range of potential variability. Within theconfines of a particular functional group (e.g., esters), electro-philicity is consistently expressed (see Table 5). Molecularsoftness was also correlated to these experimental parametersbut to a slightly lesser extent (r2≈ 0.80). These data suggest thatelectrophilicity governs the cysteine adduction rate and toxicityof a chemical and that softness also is an important factor.α,β-Unsaturated Carbonyl Electrophilicity: Intervening

Variables That Influence Interpretation. The preceding dataindicate that, although values for HNE electrophilicity andsoftness were higher than those of acrolein and MVK,37 the rankorder of the respective adduct rate constant (log k2) and toxicpotency (log IC50) were substantially lower than those predictedby the HSAB parameters. This discrepancy would seem to indi-cate a limited role for electrophilicity in determining toxic expres-sion. However, the extended alkane tail of HNE (Tables 1 and 4)imposes significant steric hindrance that can impede access to theactive site of many enzymes and thereby slow the correspondingkinetics of adduct formation and reduce toxic potency.37 Suchdisagreement between direct chemical measurements and calcu-lated HSAB parameters should be expected since the respectivealgorithms for σ and ω do not consider steric factors. As anadditional illustration of steric influences, Seiner et al.38 demon-strated that acrolein and a series of structurally related aldehydesinhibited protein tyrosine phosphatase 1B (PTP1B) activity viacovalent modification of a specific cysteine residue (Cys215).The order of potency was acrolein. crotonaldehyde > 3-methyl-2-butanal ≈ propanal. When the corresponding aqueousphase electrophilic indices (ω) were calculated retrospectively,the greater ability of acrolein to inactivate PTP1B was clearlyrelated to correspondingly higher electrophilicity (ω = 3.82 ev),whereas the reduced potency of crotonaldehyde was a function oflower electrophilicity (ω = 3.27 ev). However, the ω value for3-methyl-2-butanal (ω = 3.31) was significantly higher than thatof propanal (ω = 2.32) and equivalent to that of crotonaldehyde(ω = 3.27). Similar to the previous HNE study (Table 4), thisorder of electrophilicity is not consistent with the rank order ofpotency. Nonetheless, as the authors indicate, it can be rationa-lized by steric hindrance imposed through the bulky tertiaryalkene terminus of 3-methyl-2-butanal. It is noteworthy that, dueto its significant ω value, this compound would likely inactivate

PTP1B at higher concentrations and/or longer exposure timesthat overcome steric hindrance. However, this is not the case forpropanal since it is neither structurally nor electronically capableof irreversible sulfhydryl adduction.As part of a structure�toxicity analysis of lipid electrophiles

generated in vivo during phospholipid peroxidation, McGrathet al.39 determined the corresponding half-lives (t1/2) and secondorder rate constants (k2) for the reactions of HNE, 4-oxonenal(ONE) and analogue metabolites (Table 4) with N-acetyl-cysteine (NAC). As indicated above, these lipid derivatives areα,β-unsaturated carbonyl compounds that mediate cytotoxicitythrough irreversible modification of macromolecules and disrup-tion of cellular response pathways.17,40,41 When the correspond-ing electrophilicity values (ω) for the HNE analogues werecalculated, the kinetic parameters (t1/2 and k2) were onlyqualitatively correlated to ω unless the acidity of certain meta-bolites (RCOOH f RCOO�) was considered. Specifically,when structures of the dominant anionic species (RCOO�) atpH 7.4 were used in a HF quantum mechanical model, correla-tion of both k2 and t1/2 withω improved from r2 = 0.553 to 0.906and r2 = 0.752 to 0.911, respectively. In addition, the lack ofreactivity exhibited by HNEA in this study can be related to theextremely low value of ω calculated for its conjugate base(Table 4). It can also be seen from Table 4 that although the

Table 4. Reaction of HNE/ONE Analogues withN-Acetylcysteine

aData are from McGrath et al.39, and shaded entries indicate dominantspecies (see corresponding text). bHalf-lives and rate constants fromreaction with N-acetylcysteine at pH 7.4, 37 �C. cω = μ2/2η (see text).Orbital energies were generated exclusively from corresponding s-cisconformations and using ground state equilibrium geometries computedusing HF 6-31G* in water starting from 3-21G geometry. dOrbitalenergies were generated exclusively from corresponding s-cis conforma-tions and were obtained from ground state equilibrium geometriescomputed using DF B3LYP 6-31G* in water starting from 6-31G*geometry. e Failed to converge.

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DF model yielded numerically different values for ω, the sameresult was obtained, i.e., rate data were well correlated to electro-philicity when the relevant anionic species were considered.Esters of acrylic acid and methacrylic acid are used extensively

in the production of polymers for textiles, latex paints, andmedical/dental cements. The acrylates (CH2dCHCO2R) andmethacrylates (CH2dCCH3CO2R) are conjugated α,β-unsatu-rated ester derivatives. Several lines of evidence suggest thatthese compounds produce toxicity (e.g., carcinogenicity andskin sensitization) through adduction of protein sulfhydrylgroups.20,42�44 Early studies by Tanii and Hashimoto42 deter-mined the lethal oral doses (LD50) in mice for a broad spectrumof acrylates/methacrylates. Results indicated that the LD50 wasrelated to both solubility (as a partition coefficient, log P) and thereaction rate (log k) of these esters with glutathione in a bufferedphysiological solution. When the electrophilic index (ω) wascalculated for the 14 compounds (Table 5), we found that thecorresponding rate constants (log k) were highly correlated (r2 =0.97) to the ω parameter. In contrast, both log k (r2 = 0.65) andω (r2 = 0.60) were only modestly correlated with the in vivoLD50 values. This discrepancy can, however, be explained by thevariable solubility (log P; Table 5) of the acrylate and methacry-late compounds18 and by the fact that toxicant solubility is aphysiochemical determinant of tissue distribution and, therefore,target accessibility. This is evidenced by comparing the respectivedata for the water-soluble 2-hydroxyethyl acrylate (log P =�0.21) to the less soluble ethyl acrylate (P = 1.33), i.e., bothhave virtually identical ω values (∼3.200 ev) but significantlydifferent reaction rates (log k = 1.92 vs 1.43, respectively) andtoxic potencies (LD50 = 5.2 mmol/kg vs 18 mmol/kg,respectively). Clearly, the chemical factors that establish solubi-lity do not necessarily produce commensurate changes inelectronic characteristics such as electrophilicity. This is notsurprising since, although the hydroxyl group improves solubility(i.e., CH2dCHCO2CH2CH2OH vs CH2dCHCO2CH2CH3),this substituent is distant from the electrophilic site of the

molecule (i.e., carbon�carbon double bond) and might havelittle effect on electrophilic reactivity. Thus, it should be evidentfrom this example that, when individual differences in solubilityare considered, the relative electrophilicity (ω) of the acrylates/methacrylates determines both the corresponding in vitro rate(log k) of cysteine adduct formation and the expression of in vivotoxicity.18,29,44

The preceding discussion indicates that a variety of chemicaltoxicants can be classified according to the quantitative HSABdescriptors of hardness (η) and softness (σ), as determined byquantum mechanical calculations. Furthermore, the degree ofelectrophilic reactivity (ω) for these compounds can also bequantitatively determined. This latter parameter is criticallyimportant since it is directly related the second order rateconstant (k2) of the target adduct reaction and, therefore, reflectstoxicological potential. Accordingly, ω has been suggested tobe an in silico surrogate parameter for experimentally derivedrate constants (k2) and, as such, might be a useful tool forpredicting the toxicity of industrial chemicals.10,19,20,22,44 Thepreceding discussion also pointed out that the HSAB algorithmsdo not account for possible intervening physiochemical variables,e.g., solubility, acid�base equilibrium and steric hindrance, thatcould independently modify the toxicological outcome and leadto discrepancies in predicted vs observed results. These issueshave been addressed to some extent, and in at least one case,electrophilic and steric parameters were combined in a computa-tional algorithm.24

The α,β-unsaturated ester and carbonyl derivatives discussedhere form adducts with cysteine and other amino acid targets onproteins via second order (covalent) reactions. The rates of thesereactions are not only dependent upon the toxicant ω but alsothe nucleophilic status of the target residues. Therefore, in thenext section, we will discuss the application of HSAB parametersto the identification of amino acid sites of toxicant action.α,β-Unsaturated Carbonyl Derivatives: Nucleophilic Targets

and Molecular Mechanisms of Toxicity. According to theselectivity principle of theHSAB theory, soft electrophiles shouldpreferentially react with soft nucleophiles. There are severalnucleophilic amino acid side chains that might be relevantprotein targets for the α,β-unsaturated carbonyl compounds,i.e., sulfhydryl groups on cysteine (Cys) residues, the primarynitrogen atoms of the lysine (Lys) ε-amino group, and thesecondary nitrogen atoms of the imidazole group of histidine(His). Although the type-2 alkenes can form adducts with lysineand histidine residues,40,45�52 the weight of kinetic and proteo-mic data indicate that cysteine sulfhydryl groups are the prefer-ential targets for the type-2 alkenes.11,12,14,16,37,40,41,49,52�58 On aquantitative basis, this cysteine preference is several orders ofmagnitude greater than that for the other biological nucleophiles.This differential is consistent with the fact that the side chainamino nitrogen groups of lysine and histidine are harder nucleo-philes (Table 2) and have inherently lower reactivity for softelectrophiles such as the type-2 alkenes. The likelihood that agiven nucleophile will form a covalent adduct with a type-2alkene can be predicted by calculating an index of nucleophilicity(ω�, see above). This comprehensive parameter considers thehardness (η) and chemical potential (μ) of both the electrophilic(type-2 alkene) and nucleophilic (cysteine, histidine, or lysine)reactants.25 As suggested by the respective ω� values (Table 6),the type-2 alkenes preferentially form adducts with cysteinethiolate sites. However, based on a pKa of 8.3, the cysteinesulfhydryl side chain is mostly protonated at physiological

Table 5. Acrylate and Methacrylate Oral Lethality in Mice

ester

LD50a

(mmol/kg) log Pa log kaω

(ev)b

methyl acrylate 9.6 0.8 1.602 3.2245

ethyl acrylate 17.97 1.33 1.428 3.1981

n-butyl acrylate 58.98 2.36 1.401 3.196067

isobutyl acrylate 47.63 2.22 1.431 3.196067

2-hydroxyethyl acrylate 5.177 �0.21 1.919 3.20026

2-hydroxypropyl acrylate 8.112 0.35 1.758 3.214

methyl methacrylate 51.97 1.38 �0.675 2.9967

ethyl methacrylate 68.64 1.94 �0.403 2.9729

isopropyl methacrylate 67.84 2.25 �0.876 2.9708

n-butyl methacrylate 142.7 2.88 �0.655 2.95895

isobutyl methacrylate 83.14 2.66 �0.769 2.9708

2-hydroxyethyl methacrylate 45.24 0.47 �0.273 3.0108

2-hydroxypropyl methacrylate 55.24 0.97 �0.273 2.9848

tert-butyl methacrylate 60.12 2.54 �0.632 2.9708aData from Tanii and Hashimoto.42 bω = μ2/2η (see text). Orbitalenergies were generated exclusively from corresponding s-cis conforma-tions and were obtained from ground state equilibrium geometriescomputed using DF B3LYP 6-31G* in water starting from 6-31G*geometry.



pH (7.4) and, therefore, exists primarily as the neutral (0) thiol.Also at this pH, the imidazole secondary amine of histidine (pKa

of 6.0) ismostly deprotonated (0), and the primary ε-amino groupamine of lysine (pKa = 10.5) is completely protonated (+1). Asthe corresponding HSAB parameters indicate (Table 2), therelative nucleophilicity values for the thiol and nitrogen sidechain groups are low, and hence, these side chain states are notparticularly reactive. Instead, a large body of evidence indicatesthat the α,β-unsaturated carbonyls selectively form adducts withthe highly nucleophilic thiolate of cysteine sulfhydryl groups.This anionic state can be found within specialized pKa-loweringmicroenvironments such as catalytic triads,5,17 and as the respec-tive HSAB descriptors (Tables 2 and 6) demonstrate, cysteinethiolates are a much softer and significantly more reactivenucleophile than the Lys, His, or thiol sulfhydryl groups.To provide experimental evidence for the thiolate-state pre-

ference of the α,β-unsaturated carbonyls, we determined theeffects of pH on the corresponding rates of adduct formation.The thiolate concentration in solution will vary as a function ofpH, i.e., given a cysteine pKa of 8.3, at physiological pH (7.4) themajority (∼90%) of sulfhydryl groups will be in the nonreactivethiol-state, whereas at higher pH, the thiolate/thiol ratio willbecome increasingly larger. Thus, we found that the reactions ofL-cysteine (pKa = 8.3) with selected type-2 alkenes were 10�15times faster when the pH was increased from 7.4 to 8.8.14�16

The observed changes in second-order rate constant (k2) reflectthe increased thiolate concentration at the higher pH value. Theexperimentally determined k2 can be used to derive an anionicrate constant (kRS�) as a quantifiable measure of the inherent

(concentration independent) nucleophilic strength.58 Using theexpression log (kRS� � k2) = log k2 + pKa � pH (where kRS�represents the anionic rate constant), a representative series ofthiolate rate constants were calculated. For each α,β-unsaturatedcarbonyl derivative, the corresponding thiolate rate constants(kRS�) were highly correlated to ω� (r2 = 0.91; Table 6).Furthermore, the fact that ω� and kRS� were closely correlatedto the neurotoxic potencies (IC50’s; Table 3) provided evidencethat thiolate targeting by acrolein and HNE has toxicologicalrelevance.15,16 Similarly, previously reported rate data (k2, kRS�)for the reactions of unhindered cysteine related thiolate nucleo-philes58 with acrylonitrile were well correlated (r2 > 0.97) to ourcorresponding calculations of ω� values (Table 7).As a more relevant experimental approach to defining the

interactions of type-2 alkenes with cysteine sulfhydryl groups, wedetermined the effects of acrolein, MVK, and ACR on erythro-cyte glyceraldehyde-3-phosphate dehydrogenase (GAPDH)activity.18 Consistent with the concepts of the previous section,the softness (σ) and electrophilicity (ω) values of the selectedtype-2 alkenes were related to the corresponding second orderrate constants (log k2) and potencies (log KI) for GAPDHinhibition (Table 8). Whereas earlier proteomic studies demon-strated the cysteine preference of type-2 alkene adduct forma-tion,40,41,56,59,60 it is unlikely that these adducts all have toxico-logical relevance, i.e., the corresponding cysteine residues all playa vital role in protein function. Indeed, other studies indicatedthat the type-2 alkenes impaired protein function by reactingwith specific cysteine residues on important cellular proteins, e.g.,N-ethylmaleimide inhibits vesicle (H+)-ATPase activity byreacting with Cys254;61 and HNE adduct formation at Cys280inhibits mitochondrial SIRT3 acitivity.62 However, it was unclearwhy these residues were selectively targeted. Therefore, we usedtandem mass spectrometry to quantify the adduct formationassociated with graded concentrations of a weak electrophile,ACR.18 Results indicated that lower in vitro concentrations ofACR inhibited GAPDH activity by selectively forming adductswith Cys 152 in the active site of this enzyme, whereas at higherconcentrations, ACR also reacted with Cys 156 and Cys247.Calculations using the PROPKA program revealed a pKa of6.03 for Cys152, whereas the pKa values for Cys156 and Cys247were higher. Furthermore, we found that GAPDH inhibition bythe selected type-2 alkenes was pH-dependent indicating thiolatemediation. These data suggest that Cys152 is contained within alow pKa microenvironment63,64 and that ACR reacted preferen-tially with the resulting sulfhydryl thiolate.Considered together, our data indicate that α,β-unsaturated

carbonyl chemicals produce cytotoxicity by forming Michael-type adductswith sulfhydryl groups on specific cysteine residues.5,17,59

The elucidation of this mechanism was guided by HSAB-basedcalculations of softness and hardness, which indicated that thetype-2 alkenes were electrophiles of varying softness and that, assuch, these chemicals could form covalent bonds (adducts) withnucleophiles of comparable softness. Calculation of ω� sug-gested nucleophilic anionic thiolates as potential molecularsites of electrophile action. Cysteine thiolates are present in theactive sites of many proteins where they play critical roles inmodulating protein activities and, therefore, post translationalmodification via adduct formation would cause cytotoxicity byinhibiting the function of important proteins, e.g.,N-ethylmaleimidesensitive factor (NSF), GAPDH, ubiquitin carboxyl terminalhydrolase-L1 (UCH-L1), clathrin, and vesicular-ATPase.11�14

These predictions were corroborated by direct evidence from a

Table 6. Calculated Nucleophilic Indices (ω�) for Type-2Alkene Reactions with Cysteine Sulfhydryl Groups

electrophile ω� Cysa (�1) ω� Cys (0) log kRS�b log IC50

c

acrolein 2.03 0.103 3.417 �4.28

HNE 1.93 0.083 1.759 �3.40

MVK 1.83 0.064 2.953 �3.48

MA 1.59 0.069 1.011 �0.34

ACR 1.50 0.036 0.767 �0.36aThe nucleophilicity index (ω�) was calculated asω�=ηA (μA�μB)

2/2(ηA� ηB)

2, whereη = (ELUMO� EHOMO)/2,μ= (ELUMO +EHOMO)/2,A = reacting nucleophile, and B = reacting electrophile.15 FMO energies(ELUMO and EHOMO) were generated exclusively from correspondings-cis conformations andwere derived from equilibrium geometries of theelectrophiles at the ground state calculated using DF B3LYP-6-31G* inwater starting from 6-31G* geometries. For the sulfhydryl thiolate andthiol states, the respective ionization-states are presented in parentheses.The nucleophilicity index considers the respective hardness and chemi-cal potential of the electrophilic (type-2 alkene) and nucleophilic(cysteine, histidine, or lysine) reactants and is, therefore, a measure ofthe likelihood of subsequent adduct formation. As suggested by therespectiveω� values, the type-2 alkenes preferentially form adducts withcysteine thiolate sites as opposed to the thiol state. bThe k2 values at pH7.4 were corrected for the corresponding cysteine thiolate concentration(kRS�) according to the algorithm: log (kRS� � k2) = log k2 + pKa� pH.Inhibition of membrane 3H-dopamine transport was determined in ratstriatal synaptosomes exposed in vitro to graded concentrations of eachtype-2 alkene. cThe concentration�response data for transport werefitted by nonlinear regression analysis and the respective IC50’s werecalculated by the Cheng�Prusoff equation.14,15 Abbreviations: HNE =4-hydroxy-2-nonenal, MVK = methyl vinyl ketone, MA = methylacrylate, and ACR = acrylamide.



variety of proteomic and kinetic experimental approaches in bothin vitro and in vivo models of type-2 alkene toxicity.5,17,31,59

Because these thiolate targets are acceptors for nitric oxide (NO)and other modulatory electrophiles, we have proposed that toxi-city induced by α,β-unsaturated carbonyl exposure is mediatedby the disruption of cellular redox signaling pathways.5,17,31,65 Aswill be discussed, a similar approach was used to decipher themechanism of protection against cellular oxidative stress pro-vided by 1,3-dicarbonyl enols.α,β-Unsaturated Carbonyl Derivatives: Relevance of HSAB

Calculations to in Vivo Toxicokinetics and Toxicodynamics.The evidence reviewed above indicates that the type-2 alkenescause cytotoxicity through a common molecular mechanism.

This common mode of action suggests that members of thischemical family should, depending upon route of exposure,produce a similar pattern of organ system toxicity. However,ACR intoxication in laboratory animals or humans (e.g., occupa-tional exposure or poisoning) produces selective neurotoxicity,whereas similar intoxication with acrolein, MVK, or other type-2alkenes causes peripheral organ toxicity.5,31 This seeminglyinconsistent diversity in toxicological responses is not due tovariations in mechanism but to relative differences in electro-philicity that correspondingly influences tissue distribution andtoxicokinetics. As we have discussed, acrolein is a highly reactivesoft electrophile (Table 1) that rapidly forms adducts with softnucleophilic thiolate sites on cysteine residues of proteins(Table 6). Following systemic intoxication with a reactive type-2 alkene, the rapid formation of protein adducts essentially limitstissue distribution. Consequently, the resulting toxic manifesta-tions are determined by the site of absorption, e.g., acroleininhalation (cigarette smoke and pollution) produces pulmonarytoxicity, whereas systemic acrolein intoxication is associated withhepatic and/or vascular toxicity.66,67 In contrast to more reac-tive type-2 alkenes, ACR is a weak water-soluble electrophile(Table 1) that slowly forms adducts with protein thiolate sites(Table 6). Correspondingly, ACR is less influenced by systemic“adduct buffering” and therefore has a relatively large volume ofdistribution that includes the central nervous system.68,69 Inaccordance with these concepts, oral acrolein intoxication of rats(0.05�0.5 mg/kg) produced centrolobular liver necrosis andvascular hypertension,70 whereas oral ACR (10�50 mg/kg)caused selective neurotoxicity in this species.71 However, theproduction of neurotoxicity is not simply a function of brain

Table 7. Reaction of Thiolate Nucleophiles with Acrylonitrile at pH 8.1 and 30 �C

aData from Friedman et al.58 bCalculated as log (k � k2) = log k2 + pKa � pH. cOrbital energies obtained from ground state equilibrium geometriescomputed using DF B3LYP 6-31G* in water starting from 6-31G* geometry.

Table 8. HSAB and Kinetic Parameters for Type-2 AlkeneInteractions with GAPDH

electrophilea σ (� 10�3 ev�1) ω (ev) log k2 log KI

acroleinb 371 3.82 4.250 �4.419

MVK 363 3.38 3.885 �4.220

ACR 315 2.61 0.502 �0.607aHSAB (σ, ω) and kinetic parameters (k2, KI) were calculated asdescribed in Martyniuk et al.18 bOn the basis of the HSAB parameters,acrolein andMVK are significantly softer andmore reactive electrophilesthan ACR, i.e., larger values of σ andω, respectively. The rank orders ofrespective σ and ω values for each type-2 alkene were closely correlatedto the corresponding rate constants (k2; r2 = 0.9996 and 0.9359,respectively) and relative potencies (KI; r2 = 0.9926 and 0.9004,respectively) for the inhibition of GAPDH activity.




accessibility since ACR has an equal propensity to form thiolateadducts in both nervous and non-nervous organ systems. Instead,our studies indicate that ACR neurotoxicity is mediated byselective nerve terminal damage and that several unique anato-mical and functional characteristics predispose this neuronalregion to electrophile-induced toxicity.5,17 Specifically, as indi-cated above, we suspect that ACR (and other type-2 alkenes)causes cytotoxicity by reacting with thiolate sites that act as NOacceptors. Neurotransmission is a complex multistep processthat is highly regulated by NO signaling,59 and therefore,disruption of this pathway is likely to have a significant impacton presynaptic activity. Furthermore, the nerve terminal isanatomically separated from the cell body and is, as a result,devoid of transcriptional and translational abilities. Therefore,unlike the cell body, the nerve terminal is vulnerable to electro-phile attack since it cannot mount transcription-based cytopro-tective mechanisms such as the Nrf2-Keap1 response.72 Finally,relative to proteins in other nerve regions or cell types, theturnover of nerve terminal proteins is exceptionally slow.73 Thus,when these proteins are rendered dysfunctional through cysteineadduct formation, they are replaced slowly. This leads to theaccumulation of nerve terminal adducts,11,12 progressive impair-ment of presynaptic processes,13,57 and cumulative neurotoxi-city.71 Thus, although the type-2 alkenes have a common mech-anism of cytotoxicity, differences in electrophilic strength amongthese chemicals can influence respective tissue distributions andtherefore their toxicological manifestations.

’CYTOPROTECTIVE PROPERTIES OF 1,3-DICARBO-NYL ENOLS:MECHANISTIC INSIGHTSGAINEDBYHSABQUANTUM MECHANICAL CALCULATIONS

Cellular Oxidative Stress: Phytopolyphenol Cytoprotec-tion. There is general agreement that cellular oxidative stressis a common molecular etiology in many chronic disease states(e.g., Alzheimer’s disease, atherosclerosis) and acute injuries(e.g., stroke and traumatic spinal cord injury). A confluence ofepidemiological data and clinical trials,74,75 corroborated bylaboratory evidence,76�78 indicate that the course of these con-ditions can be significantly improved by the administration ofplant-derived polyphenolic compounds such as curcumin (curryspice), resveratrol (red grapes), or phloretin (apple skins). It hasbeen largely assumed that the protective effects of these phyto-polyphenols were related to their abilities as antioxidants totrap reactive oxygen species. However, recent evidence indicatesthat polyphenolic cytoprotection involves scavenging of otherelectron-deficient species that also mediate oxidative stress, i.e.,divalent metal ions (e.g., Fe2+ and Cu2+) and toxic unsaturatedaldehydes (e.g., acrolein and HNE).79�81 Structure�activitystudies have indicated that the molecular determinant of theseprotective actions is resident enol moieties.82�84 For example,theα-carbon hydrogen atoms on the central β-diketone bridge ofcurcumin are acidic, and upon ionization, in physiological buffers,a carbanionic enolate is formed as the corresponding conjugatebase. Moreover, the characteristic phenols of flavonoids such asphloretin are enols since they consist of a hydroxyl groupattached to a carbon�carbon double bond. These enols will alsoform nucleophilic enolate ions. Despite therapeutic potential,however, the clinical utility of the phytopolyphenols couldbe limited by poor bioavailability, toxicity, and chemicalinstability.36,85,86

1,3-Dicarbonyl Enols: A New Class of Cytoprotectants.The search for safe, water-soluble phytopolyphenol analoguesled to the discovery that 1,3-dicarbonyl enols such as 2-acetyl-cyclopentanone (2-ACP) and acetylacetone (AcAc; Table 2)might be cytoprotective.87 The structure of these simple solubleenols resembles the central diketone�bridge of curcumin. Likethe phytopolyphenols, the α-carbon hydrogen atoms of thesesimple enols can also readily ionize to form highly nucleophilicsoft enolates. Indeed, our studies87 showed that the 1,3-dicarbo-nyl compounds provided levels of cytoprotection in several invitro models of oxidative stress that exceeded those of thephytopolyphenols. The evidence suggested at least two mecha-nisms of 1,3-dicarbonyl cytoprotection in oxidative stress-inducedcell injury. First, metal ion chelation mediated by bidentatecoordination through the β-diketone structure. Presumably,the resulting inhibition or modification of the metal-catalyzedFenton reaction reduces the cellular free radical burden. Second,the nucleophilic β-dicarbonyl enolates act as soft surrogatetargets for free acrolein and other electrophilic α,β-unsaturatedaldehydes. The reduction in toxic aldehyde content preventssecondary protein dysfunction in exposed cells. In studies thatspecifically examined the latter cytoprotective component, weidentified the following rank order of 1,3-dicarbonyl analogueswith respect to protection in acrolein-exposed neuronal cellculture systems: 2-ACP >AcAc, 1,1,1-trifluoro-2,4-pentanedione(TFPD). diethylmalonate (DEM). This sequence reflects thedifferences in second order rate constants (k2) for the reaction ofthese 1,3-dicarbonyls with acrolein. The differential reactivity ofthe 1,3-dicarbonyls can be explained by the acidity (pKa) of theparent compound and by the inherent nucleophilicity of thecorresponding enolate. The pKa values indicate the relativeextent of ionization (enolate formation) and hence reflect nucleo-philic concentration as a factor contributing to reaction rate.1,3-Dicarbonyl Enols: HSAB Parameters. As discussed pre-

viously, the nucleophilic strength of an enolate carbanion can bequantified by ω�.5,17 The ω� values computed for reactionsbetween acrolein and various nucleophiles are presented inTable 9. A nucleophile of significant reactivity (high ω� value)will increase the reaction rate (k2) with a given electrophile bylowering the energy of the transition state. Therefore, ω� valuesshould correlate with the relative rate at which a given concen-tration of enolate reacts with a specific type-2 alkene (Table 9).Inherent differences in the respective ω� and known pKa values

Table 9. HSAB and Kinetic Parameters for Reactions of 1,3-Dicarbonyl Compounds with Acrolein

compd ω� (�10�3 ev)a pKa k2 rate constantb

2-ACP 401 7.8 0

AcAc 160 8.9 �24.9

TFPD 108 4.7 �52.1

DEM 228 12.9 �78.7

phloretin 139 7.3 �1.9

NAC (�2) 316 9.5 ndaThe nucleophilic index (ω�) was calculated based on the reaction ofacrolein with the selected 1,3-dicarbonyl and was calculated according tothe algorithm provided in the text. FMO energies were generatedexclusively from corresponding s-cis conformations and were calculatedas equilibrium geometries at the ground state using DF B3LYP-6-31G*in water starting from 6-31G* geometries. b Second order rate con-stants (k2) for the reactions of 1,3-dicarbonyls with acrolein are derivedfrom LoPachin et al.87 and are expressed as nM sulfhydryl loss s�1.



provide a molecular explanation of the corresponding adductreaction rates and, hence, cytoprotective potency. Thus, 2-ACP isreasonably acidic (30�40% ionized at pH 7.4; pKa = 7.8) andforms an enolate that is a powerful nucleophile (relative ω� =401). Correspondingly, it exhibited superior protective capacityin all toxicity models. In contrast, AcAc is less reactive towardelectrophiles, not only because it ionizes to a lesser extent (2% atpH 7.4; pKa = 8.9) but also because it is a weaker nucleophile(ω� = 160). Although TFPD is highly acidic (pKa = 4.7), theresulting higher concentration of enolate at physiological pH isoffset by its weakness as a nucleophile (ω� = 108). Conversely,the enolate form of DEM is a significant nucleophile (ω� = 228).However, the low acidity (pKa = 12.9) of this chemical precludesany potential capability as a protectant since less than 0.001%would be in the enolate state at physiological pH. NAC (pKa =9.5) ionizes poorly in biological solutions, but is protectiveagainst acrolein-induced thiol loss and toxicity because thenucleophilicity of the thiolate anion is relatively high (ω� =316). Phloretin has several enolizable sites, and although thenucleophilicity of these sites is modest (e.g., the relative ω� ofthe C-3 enol on the A ring = 139), the corresponding pKa value of7.3 indicates significant (>50%) ionization. Therefore, at phy-siological pH the high concentration of enolate and multiple sitepotential enable phloretin to provide significant thiol preserva-tion through acrolein adduction. Together, these results indicatethat the ability of 1,3-dicarbonyl compounds to act as acroleintargets is a predictable function of enolate formation and theinherent nucleophilicity of these carbanions. We are currentlyusing the ability to quantify nucleophilicity (ω�), in conjunctionwith a high level of chemical understanding (i.e., conformationalflexibility, pKa, and nucleophilicity requirements) derived fromextensive experimentation, to design and identify efficaciouscytoprotective 1,3-dicarbonyl analogues.

’SUMMARY

In the field of toxicology, the irreversible covalent interactionof a toxic electrophile and its cognate nucleophilic target isrecognized as a basic reaction mediating chemical-induced cellinjury. It is now understood that electrophile�nucleophilereactions exhibit a significant degree of selectivity, in that a givenelectrophile preferentially reacts with specific nucleophiles ofcomparable softness or hardness. This selectivity is based onelectronic and structural characteristics that constitute the softand hard classifications of the HSAB theory. In this perspective,we have shown that the soft�soft interactions of electrophilicα,β-unsaturated carbonyl and aldehyde derivatives with theirnucleophilic sulfhydryl thiolate targets can be understood andpredicted through the calculation of HSAB parameters. Hard�hard interactions can also be quantitatively described and used toclarify toxicodynamics. Accordingly, we have employed HSABcalculations to describe the interactions of 2,5-hexanedione, thehard electrophilic metabolite of the parent compound, n-hexane,with possible hard nucleophilic targets in nervous tissue, e.g.,ε-amino groups on lysine residues of axon cytoskeletal proteins.88

Similar to the n-hexane/2,5-hexanedione relationship, manytoxicants are metabolic derivatives of a less reactive parent orprotoxicant, e.g., desulfuration of the relatively soft electrophilicchlorpyrifos parent to the highly reactive hard electrophilic oxonmetabolite (chlorpyrifos oxon). Therefore, on a theoretical basis,HSAB calculations could also be used to determine the electro-philic reactivity and potential toxicity of metabolites derived from

less reactive protoxicants. However, despite these obvious benefi-cial applications of the HSAB calculations, in vivo and in vitromodel systems and their presumed macromolecular targets arehighly complex, and accordingly, accurate formulation of kineticor mechanistic predictions is dependent upon the knowledge ofadditional chemical features, such as steric hindrance, that mightindependently influence adduct reaction rates. Furthermore, itshould be recognized that the relative electrophilicity of atoxicant will influence corresponding in vivo toxicokinetics andthe organ systems affected, as evidenced in our study of type-2alkene toxicity. Regardless, HSAB calculations provide molecularlevel information that can offer guidance for corroborativelaboratory research and thereby help investigators discern corre-sponding electrophile molecular mechanisms and sites of tox-icant action. The use of quantum chemical calculations hasalso been instrumental in the development and identifica-tion of potential nucleophilic antagonists, which can scavengeelectron-deficient species that mediate many diseases with acommon etiology of cellular oxidative stress. Clearly, the difficulttask of delineating molecular sites and mechanisms of toxicantaction can be advanced by the application of quantummechanicalapproaches.

’AUTHOR INFORMATION

Corresponding Author*(R.M.L.)Tel: (718) 920-5054. Fax: (718) 515-4903. E-mail:[email protected]. (T.G.) Department of Chemistry,Iona College, 402 North Ave., New Rochelle, NY 10804. Tel:(914) 633-2301. E-mail: [email protected].

Funding SourcesThis research was supported by NIH grants from the NationalInstitutes of Environmental Health Sciences to R.M.L. (NIEHSRO1 ES03830-24; NIEHS RO1 07912-13).

’ABBREVIATIONS

HSAB, hard and soft, acids and bases; FMOs, frontier molecularorbitals; SAR, stucture�activity relationship; LUMO, lowestunoccupied molecular orbital;HOMO, highest occupied molecularorbital; DFT, density-functional theory; HTT, Hartree�Fock theory; ACR, acrylamide; HNE, 4-hydroxy-2-nonenal;MVK, methylvinyl ketone;MA, methyl acrylate; PTP1B, proteintyrosine phosphate 1B; NAC, N-acetylcysteine; LD50, lethaloral dose for 50% of the population; GAPDH, glyceraldehydes3-phosphate dehydrogenase; SIRT3, mitochondrial sirtuin3;NSF,N-ethylmaleimide sensitive factor;UCH-L1, ubiquitin carboxylterminal hydrolase-L1; NO, nitric oxide; Nrf2/Keap1, nuclearfactor erythroid 2-related factor 2/kelch-like erythroid cell-derived protein with CNS homology-associated protein 1; 2-ACP, 2-acetylcyclopentanone; AcAc, acetylacetone; TFPD,1,1,1-trifluoro-2,4-pentanedione; DEM, diethylmalonate

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