International Journal of Molecular Sciences

Review Reactivities of Quinone Methides versus o-Quinones in Catecholamine Metabolism and Eumelanin Biosynthesis

Manickam Sugumaran

Department of Biology, University of Massachusetts Boston, Boston, MA 02125, USA; [email protected]; Tel.: +1-617-287-6598

Academic Editor: David Arráez-Román Received: 18 August 2016; Accepted: 12 September 2016; Published: 20 September 2016

Abstract: Melanin is an important biopolymeric pigment produced in a vast majority of organisms. Tyrosine and its hydroxylated product, dopa, form the starting material for melanin biosynthesis. Earlier studies by Raper and Mason resulted in the identification of dopachrome and dihydroxyindoles as important intermediates and paved way for the establishment of well-known Raper–Mason pathway for the biogenesis of brown to black eumelanins. Tyrosinase catalyzes the oxidation of tyrosine as well as dopa to dopaquinone. Dopaquinone thus formed, undergoes intramolecular cyclization to form leucochrome, which is further oxidized to dopachrome. Dopachrome is either converted into 5,6-dihydroxyindole by decarboxylative aromatization or isomerized into 5,6-dihydroxyindole-2-carboxylic acid. Oxidative polymerization of these two dihydroxyindoles eventually produces eumelanin pigments via melanochrome. While the role of quinones in the biosynthetic pathway is very well acknowledged, that of isomeric quinone methides, however, remained marginalized. This review article summarizes the key role of quinone methides during the oxidative transformation of a vast array of catecholamine derivatives and brings out the importance of these transient reactive species during the melanogenic process. In addition, possible reactions of quinone methides at various stages of melanogenesis are discussed.

Keywords: catecholamine metabolism; quinone methides; quinone isomerization; eumelanin biosynthesis; dihydroxyindole polymers; quinone reactivity

1. Introduction Tyrosine and its hydroxylated product dopa constitute major precursors for cellular catecholamines in practically all organisms. They serve as the starting point for the biosynthesis of crucial hormones, neurotransmitters, alkaloids, coenzymes, and a plethora of pigments. Melanin is an important phenolic pigment formed from the amino acid tyrosine and its derivatives in nature. It is widely distributed in practically all organisms and is extensively used by animals for pigmentation, camouflage, mimicry, UV protection and thermoregulation [1–5]. There are two kinds of melanins biosynthesized in animals. The yellow to red pheomelanin arises by the oxidative polymerization of cysteinyldopa derivatives formed by the condensation of the amino acid, cysteine and dopaquinone (or its derivatives). The brown to black eumelanin is generated by the oxidative polymerization of 5,6-dihydroxyindoles, which in turn arise by intramolecular cyclization and further transformation of dopaquinone [1–5]. The focus of this review is on eumelanin. Raper–Mason pathway for the biosynthesis of eumelanin was established in early 1930s–1940s by the pioneering work of Raper and Mason [6–10]. They studied the enzymatic oxidation of tyrosine and dopa systematically and delineated a pathway for the biosynthesis of eumelanin, which is depicted in Figure1. According to this pathway, tyrosinase converts tyrosine to dopaquinone via dopa.

Int. J. Mol. Sci. 2016, 17, 1576; doi:10.3390/ijms17091576 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1576 2 of 23

Int. J. Mol. Sci. 2016, 17, 1576 2 of 23 However, dopa is not directly formed but indirectly produced by the reduction of dopaquinone to dopa. However, dopa is not directly formed but indirectly produced by the reduction of dopa.dopaquinone Dopaquinone to dopa. exhibits Dopaquinon a rapid nonenzymatice exhibits a intramolecularrapid nonenzymatic cyclization intramolecular producing leucochrome,cyclization whichproducing reduces leucochrome, dopaquinone which to dopa reduces and gets dopaquinone oxidized toto dopachrome dopa and gets (Figure oxidized1). After to dopachrome the formation of(Figure dopaquinone, 1). After most the formation of the reaction of dopaquinone, leading to most melanin of the biosynthesis reaction leading can to occur melanin with biosynthesis out the need forcan any occur enzyme. with Thus,out the the need common for any beliefenzyme. was Thus, that the tyrosinase common is belief the sole was enzyme that tyrosinase responsible is the forsole the productionenzyme responsible of eumelanin for the pigment production in nature. of eumelanin pigment in nature.

FigureFigure 1. 1.Raper–Mason Raper–Mason PathwayPathway for for the the biosynthesis biosynthesis ofof eumelanin. eumelanin. Tyrosinase Tyrosinase converts converts tyrosine tyrosine to dopaquinone via dopa However, dopa is not formed directly from tyrosine but indirectly by the to dopaquinone via dopa However, dopa is not formed directly from tyrosine but indirectly by the reduction of dopaquinone. Dopaquinone undergoes intrmolecular cyclization producing reduction of dopaquinone. Dopaquinone undergoes intrmolecular cyclization producing leucochrome leucochrome nonenzymatically, which is further oxidized to dopachrome by dopaquinone. nonenzymatically, which is further oxidized to dopachrome by dopaquinone. Dopachrome is converted Dopachrome is converted to 5,6-dihydroxyindole (DHI) as the major product and to 5,6-dihydroxyindole (DHI) as the major product and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as the minor product. Further oxidation of DHI to as the minor product. Further oxidation of DHI to melanochrome and its eventual polymerization melanochrome and its eventual polymerization leads to melanin pigment. leads to melanin pigment. Dopachrome was initially believed to undergo transformation to produce 5,6-dihydroxyindole (DHI),Dopachrome as the major was initiallyproduct believedwith small to undergoamounts transformationof DHICA. The todihydroxyindoles produce 5,6-dihydroxyindole are further (DHI),oxidized as the to major melanochrome product with and smalleventually amounts produces of DHICA. dark Thecolored dihydroxyindoles melanin polymer. are However, further oxidized this tosimple melanochrome scheme was and modified eventually with produces the discovery dark of colored two new melanin enzymes polymer. associated However, with melanogenic this simple schemepathway. was An modified enzyme with converting the discovery dopachrome of two newto 5,6-dihydroxyindole-2-carboxylic enzymes associated with melanogenic acid (DHICA) pathway. Anwas enzyme first discovered converting in dopachromemammalian systems to 5,6-dihydroxyindole-2-carboxylic [11–16]. Subsequently a sister enzyme acid (DHICA) catalyzing was the first discoveredconversion in of mammalian dopachrome systems to DHI [ 11was–16 characterized]. Subsequently from a insects sister enzyme [17–19]. catalyzingAlthough the the mammalian conversion of dopachromeenzyme was to initially DHI was named characterized as dopachrome from isomerase, insects [17 the–19 current]. Although accepted the mammalianname for this enzymeenzyme is was initiallydopachrome named tautomerase as dopachrome (DCT) isomerase, [13,14]. The the sister current enzyme accepted in insects name forconverts this enzyme dopachrome is dopachrome to DHI and hence should be called dopachrome decarboxylase. However, it also converts dopachrome tautomerase (DCT) [13,14]. The sister enzyme in insects converts dopachrome to DHI and hence methyl ester to DHICA methyl ester thus catalyzing a perfect tautomerization reaction [19]. should be called dopachrome decarboxylase. However, it also converts dopachrome methyl ester to Therefore, this enzyme will be referred to as dopachrome decarboxylase/tautomerase (DCDT). DHICA methyl ester thus catalyzing a perfect tautomerization reaction [19]. Therefore, this enzyme Another enzyme named DHICA oxidase that catalyzed the oxidation of DHICA to its corresponding will be referred to as dopachrome decarboxylase/tautomerase (DCDT). Another enzyme named quinone was discovered in mouse [20,21]. With the help of these enzymes, DHI melanin, DHICA DHICAmelanin oxidase and mixed that catalyzed DHI/DHICA the oxidation eumelanins of DHICAare formed to its in correspondingvarious animal quinone systems. was Thus, discovered melanin in mousebiosynthesis [20,21]. Within animals the help seems of these to occur enzymes, with the DHI he melanin,lp of three DHICA (or more) melanin enzymes and as mixed opposed DHI/DHICA to one. eumelaninsAs shown arein Figure formed 1, inquinone various reactivity animal systems.plays a central Thus, role melanin in eumelanin biosynthesis biosynthesis. in animals However, seems to occurless withknown the and help more of three reactive (or more) quinone enzymes methides, as opposed which toare one. isomers As shown of 4-alkylquinones, in Figure1, quinone also reactivityparticipates plays in a key central conversions role in eumelanin and contributes biosynthesis. to the However,reactivity lessof different known andcatecholamines more reactive quinoneassociated methides, with eumelanogenic which are isomers pathway. of 4-alkylquinones, This review summarizes also participates the importance in key conversions of quinone and contributesmethides during to the reactivitythe oxidative of differenttransformation catecholamines of a variety associated of catecholamines with eumelanogenic related to eumelanin pathway. Thisand review serves summarizes to illustrate their the importance importance. of quinone methides during the oxidative transformation of a variety of catecholamines related to eumelanin and serves to illustrate their importance. Int. J. Mol. Sci. 2016, 17, 1576 3 of 23

Int. J. Mol. Sci. 2016, 17, 1576 3 of 23 2. Reactions of Quinones 2. Reactions of Quinones Catecholamine derivatives are prone to easy oxidation. Their two-electron oxidation leads to the mostCatecholamine common product, derivatives 4-alkyl are quinones. prone to Theeasy reactivitiesoxidation. Their of quinones two-electron are extremely oxidation vital leads for to the biologicalthe most role common of many product, catechols. 4-alkyl Therefore, quinones. the The reactions reactivities of quinones of quinones will beare considered extremely vital first. for Only the the reactionsbiological of roleo-quinones of many arecatechols. given Therefore, in this review the reactions and the of reactions quinones of willp-quinones be considered are notfirst. covered. Only Thethe quinonoid reactions of nucleus o-quinones is very arereactive given in andthis unstable.review and Hence, the reactions it exhibits of p-quinones rapid Michael-1,4-addition are not covered. reactionsThe quinonoid with a nucleus variety is of very nucleophiles reactive and to generateunstable. Hence, substituted it exhibits catechols. rapid Michael-1,4-addition The reaction leads to 1,4-adductreactions ofwith nucleophiles a variety onof thenucleophiles ring and endsto genera up withte substituted the re-aromatization catechols. The of the reaction quinone leads nucleus. to Quinones1,4-adduct can of react nucleophiles with water on forming the ring 1,4-addition and ends up product. with the The re-aromatization resultant trihydroxy of the phenols quinone are nucleus. Quinones can react with water forming 1,4-addition product. The resultant trihydroxy not very stable and undergo rapid nonenzymatic aerial oxidation (Figure2). Reaction with molecular phenols are not very stable and undergo rapid nonenzymatic aerial oxidation (Figure 2). Reaction oxygen leads to the production of superoxide anion and semiquinone radicals, which will exhibit with molecular oxygen leads to the production of superoxide anion and semiquinone radicals, radical coupling to produce dimers and other oligomers. Moreover, trihydroxyphenol can also be which will exhibit radical coupling to produce dimers and other oligomers. Moreover, oxidized to its two-electron oxidation product, hydroxy-p-quinone. The importance of the above trihydroxyphenol can also be oxidized to its two-electron oxidation product, hydroxy-p-quinone. reactionThe importance becomes evident of the whenabove onereaction looks becomes at the biosynthesis evident when of topaquinone one looks at cofactor the biosynthesis found in amine of oxidasestopaquinone [22,23 ].cofactor A special found tyrosine in amine group oxidases in the [22,23]. precursor A special protein tyrosine is oxidized group to in dopa the precursor and further convertedprotein is to oxidized 6-hydroxydopa. to dopa Aerialand further oxidation converted of this trihydroxyto 6-hydroxyd compoundopa. Aerial generates oxidation the of required this topaquinonetrihydroxy cofactorcompound in generates these enzymes the required [22,23]. topaquinone cofactor in these enzymes [22,23].

FigureFigure 2. 2.Formation Formation of of hydroxylated hydroxylated catecholscatechols and their further reactions. reactions. o-Quinoneo-Quinone formed formed from from catecholcatechol by by two-electron two-electron oxidationoxidation cancan undergoundergo addition with with water water forming forming hydroxyl hydroxyl catechol, catechol, whichwhich will will readily readily undergo undergo oxidation oxidation toto hydroxy-hydroxy-p-quinone as as well well as as semiquinone semiquinone by by reaction reaction with with molecular oxygen. Eventually, these quinonoid compounds will exhibit polymerization. molecular oxygen. Eventually, these quinonoid compounds will exhibit polymerization.

Similar to water reaction, alcohol group also reacts with the quinones forming the Michael-1,4-additionSimilar to water products. reaction, The alcohol resultant group subs alsotituted reacts ether withdoes not the exhibit quinones any formingsignificant the Michael-1,4-additionreactivity with molecular products. oxygen The but resultant is capable substituted of further ether oxidation does not to exhibit typical any o-quinones. significant reactivityInterestingly with molecularquinones oxygencan also but react is capable with the of further carboxyl oxidation groups toforming typical oesters.-quinones. However, Interestingly this quinonesreaction canoccurs also most react likely with if thethe carboxylcarboxyl group groups is formingtied up to esters. the quinonoid However, nucleus this reaction in a suitable occurs mostposition likely such if the as carboxyl those found group in is4-carboxymethyl- tied up to the quinonoido-quinone and nucleus 4-carboxyethyl- in a suitableo-quinone position [24,25]. such as thoseOxidation found inof 4-carboxymethyl- both 3,4-dihydroxyo-quinone phenylacetic and 4-carboxyethyl- acid and 3,4-dihydroxyo-quinone [24 ,hydrocinnamic25]. Oxidation ofacid both 3,4-dihydroxyleads to the phenylacetic corresponding acid andquinones 3,4-dihydroxy (Figure hydrocinnamic3). The quinones acid leadsthus to formed, the corresponding exhibit quinonesinstantaneous (Figure 3).intramolecular The quinones thuscyclization formed, exhibitgenerating instantaneous 2,5,6-trihydroxybenzofuran intramolecular cyclization and generating6,7-dihydroxyhydrocoumarin, 2,5,6-trihydroxybenzofuran respectively. and 6,7-dihydroxyhydrocoumarin, The quinones of N-acetyl dehydro respectively. dopa also The quinonesexhibit of intramolecularN-acetyl dehydro cyclization dopa also producing exhibit intramolecular coumarin derivatives cyclization [26]. producing coumarin derivatives [26]. Importantly,Importantly, amines—both amines—both primary primary andand secondarysecondary amines—react amines—react under under facile facile conditions conditions with with quinones producing amino substituted catechols, which suffer rapid oxidation yielding quinones producing amino substituted catechols, which suffer rapid oxidation yielding quinoneimines. quinoneimines. As witnessed in the case of carboxyl group, internal amino group will react As witnessed in the case of carboxyl group, internal amino group will react extremely fast with the extremely fast with the quinone forming cyclized product. It is this reaction that in fact forms the quinone forming cyclized product. It is this reaction that in fact forms the central theme in melanin central theme in melanin biosynthesis. As shown in Figure 1, dopaquinone undergoes rapid biosynthesis. As shown in Figure1, dopaquinone undergoes rapid intramolecular cyclization with intramolecular cyclization with the internal amino group generating the leucochrome, which is the internal amino group generating the leucochrome, which is rapidly oxidized by the dopaquinone rapidly oxidized by the dopaquinone to dopachrome. Dopamine quinone also exhibits the same toreaction dopachrome. (Figure Dopamine 4) [27]. Furthermore, quinone also 6-hydroxydopamine, exhibits the same the reaction hydroxylated (Figure product4)[ 27]. of Furthermore, dopamine Int. J. Mol. Sci. 2016, 17, 1576 4 of 23 Int. J. Mol. Sci. 2016, 17, 1576 4 of 23 Int. J. Mol. Sci. 2016, 17, 1576 4 of 23 6-hydroxydopamine,quinone, upon oxidation the hydroxylated produces the product same ofdopa dopamineminechrome quinone, as that upon produced oxidation by produces dopamine the quinone, upon oxidation produces the same dopaminechrome as that produced by dopamine sameFigure dopaminechrome 4 [28]. as that produced by dopamine Figure4[28]. Figure 4 [28].

Figure 3. Nucleophilic addition of internal carboxyl group on quinone. Both Figure 3. Nucleophilic3. Nucleophilic addition addition of internal of carboxyl internal group oncarboxyl quinone. group Bothcarboxymethyl- on quinone.o-quinone Both carboxymethyl-o-quinone and carboxyethyl-o-quinone exhibit rapid intramolecular cyclization due andcarboxymethyl- carboxyethyl-o-quinoneo-quinone and exhibit carboxyethyl- rapid intramolecularo-quinone exhibit cyclization rapid intr dueamolecular to the presence cyclization of suitably due to the presence of suitably substituted internal carboxyl group yielding cyclic products. substitutedto the presence internal of suitably carboxyl substituted group yielding intern cyclical carboxyl products. group yielding cyclic products.

Figure 4. Reactions of quinone with internal amines. Dopamine quinone formed by the two-electron Figure 4. Reactions of quinone with internal amines. Dopamine quinone formed by the two-electron Figureoxidation 4. Reactions of dopamine of quinone reacts with rapidly internal with amines. the internal Dopamine amino quinone group formed forming by the leucochrome, two-electron oxidation of dopamine reacts rapidly with the internal amino group forming leucochrome, oxidationwhich is ofconverted dopamine readily reacts to rapidly dopaminechrome with the internal similar amino to groupthe dopachrome forming leucochrome, conversion whichreaction is which is converted readily to dopaminechrome similar to the dopachrome conversion reaction convertedshown in readilyFigure to 1. dopaminechrome Interestingly dopaminechrome similar to the dopachrome is also conversiongenerated reactionduring shownthe oxidation in Figure of1. shown in Figure 1. Interestingly dopaminechrome is also generated during the oxidation of Interestingly6-hydroxydopamine dopaminechrome as a condensation is also of generated product of during 6-hydroxydopaminequinone. the oxidation of 6-hydroxydopamine as a6-hydroxydopamine condensation of product as a condensation of 6-hydroxydopaminequinone. of product of 6-hydroxydopaminequinone. The above reactions can be translated to external nucleophilic addition of amines also. Figure 5 The above reactions can be translated to external nucleophilic addition of amines also. Figure 5 summarizesThe above the reactions reactions can of simple be translated quinone to with external external nucleophilic amine. Note addition the remarkable of amines also. resemblance Figure5 summarizes the reactions of simple quinone with external amine. Note the remarkable resemblance summarizesbetween the theinternal reactions and ofexternal simple reactions quinone withof amines. external In amine. the case Note of thesecondary remarkable amines, resemblance the best between the internal and external reactions of amines. In the case of secondary amines, the best betweencharacterized the internal reaction and being external the one reactions that takes of pl amines.ace between In the the case imidazole of secondary group amines, of histidine the bestand characterized reaction being the one that takes place between the imidazole group of histidine and characterizedquinone [29]. The reaction addition being leads the oneto 4-substituted that takes place catechols between that the do imidazole not readily group oxidize of histidine to quinones. and quinone [29]. The addition leads to 4-substituted catechols that do not readily oxidize to quinones. quinoneThis reaction [29]. plays The addition a vital role leads in toinsect 4-substituted cuticular catecholssclerotization that doreaction, not readily which oxidize has been to quinones.shown to This reaction plays a vital role in insect cuticular sclerotization reaction, which has been shown to Thisremarkably reaction parallel plays amelani vital rolen biosynthesis in insect cuticular [30–32]. sclerotization reaction, which has been shown to remarkably parallel melanin biosynthesis [30–32]. remarkablyThiols react parallel with melanin quinones biosynthesis more rapidly [30–32 than]. any other nucleophiles. Initial studies assumed Thiols react with quinones more rapidly than any other nucleophiles. Initial studies assumed that Thiolsthey also react follow with Michael-1,4-addition quinones more rapidly reactions. than any Thus, other the nucleophiles. reaction of enzymatically Initial studies generated assumed that they also follow Michael-1,4-addition reactions. Thus, the reaction of enzymatically generated thatdopaquinone they also with follow cysteine Michael-1,4-addition was assumed to reactions. produce 6-cysteinyldopa. Thus, the reaction However, of enzymatically careful examination generated dopaquinone with cysteine was assumed to produce 6-cysteinyldopa. However, careful examination dopaquinoneof the structure with of cysteinethe product was assumedas well as to quantita producetive 6-cysteinyldopa. product analysis However, revealed careful that examinationthe majority of the structure of the product as well as quantitative product analysis revealed that the majority of(about the structure3/4th) of ofthe the product product is as5-cysteinyldopa, well as quantitative which productis 1,6-addition analysis product. revealed Another that the isomer majority of (about 3/4th) of the product is 5-cysteinyldopa, which is 1,6-addition product. Another isomer of (about1,6-adduct, 3/4th) namely of the 2-cysteinyldopa product is 5-cysteinyldopa, is produced whichat about is 1,6-addition14% level and product. the Michael-1,4-adduct Another isomer 1,6-adduct, namely 2-cysteinyldopa is produced at about 14% level and the Michael-1,4-adduct of(6-cysteinyldopa) 1,6-adduct, namely is produced 2-cysteinyldopa only at is1% produced level [1,33,34]. at about The 14% reaction level and of the N-acetylcysteine Michael-1,4-adduct with (6-cysteinyldopa) is produced only at 1% level [1,33,34]. The reaction of N-acetylcysteine with (6-cysteinyldopa)N-acetyldopamine is quinone produced alsoonly gives at similar 1% level products [1,33, 34[35].]. The The reason reaction for ofthisN abnormal-acetylcysteine addition with is N-acetyldopamine quinone also gives similar products [35]. The reason for this abnormal addition is Nnot-acetyldopamine clear although various quinone authors also gives have similar proposed products different [35]. hypothesis The reason to for account this abnormal for this deviation. addition not clear although various authors have proposed different hypothesis to account for this deviation. Thiol addition plays a crucial role in pheomelanin biosynthesis as cysteinyldopa and Thiol addition plays a crucial role in pheomelanin biosynthesis as cysteinyldopa and Int. J. Mol. Sci. 2016, 17, 1576 5 of 23 is not clear although various authors have proposed different hypothesis to account for this Int. J. Mol. Sci. 2016, 17, 1576 5 of 23 deviation.Int. J. Mol. Sci. Thiol 2016, addition17, 1576 plays a crucial role in pheomelanin biosynthesis as cysteinyldopa5 of 23 and cysteinyldopamine formed by the condensation of cysteine with dopaquinone and dopaminequinone cysteinyldopamine formed by the condensation of cysteine with dopaquinone and formcysteinyldopamine the important precursors formed forby pheomelanin the condensa biosynthesistion of incysteine all animals. with Even dopaquinone the thioether and group dopaminequinone form the important precursors for pheomelanin biosynthesis in all animals. Even ofthe methionine thioether isgroup known of tomethionine exhibit addition is known reaction to exhibit with addition quinones reaction that may with parallel quinones the that reaction may of thiolsthe [thioether36–38]. Thus, group practically of methionine all-available is known biological to exhibit nucleophiles addition reaction can react with with quinones quinones that producing may parallel the reaction of thiols [36–38]. Thus, practically all-available biological nucleophiles can react various adducts. Apart from the reactions with these nucleophiles, certain if not all quinones are with quinones producing various adducts. Apart from the reactions with these nucleophiles, certain capable of undergoing isomerization to more reactive quinone methides. if not all quinones are capable of undergoing isomerization to more reactive quinone methides.

FigureFigure 5. 5.Reactions Reactions of of quinone quinone with withexternal external amines.amines. Quinones Quinones formed formed by by the the oxidation oxidation of ofcatechols catechols reactreact with with external external amino amino group group forming forming 4-substituted4-substituted catechols. catechols. These These catechols catechols are are unstable unstable and and form further adducts via substituted quinones. formform further further adducts adducts via via substituted substituted quinones.quinones.

3. Quinone Methides—General Consideration 3.3. Quinone Quinone Methides—General Methides—General Consideration Quinone methides are analogs of quinones with one of the carbonyl oxygen replaced with a QuinoneQuinone methides methides areare analogsanalogs of quinones quinones with with one one ofof the the carbonyl carbonyl oxygen oxygen replaced replaced with with a benzylic methylene group. There are two types of quinone methides: o-quinone methide and a benzylic methylene group. There are two types of quinone methides: o-quinone methide and p-quinone methide. Since only p-quinone methides are typically associated with catecholamines, the p-quinone methide. Since only p-quinone methides are typically associated with catecholamines, rest of the article will deal with the reactions of these novel intermediates and no aspect of o-quinone the rest of the article will deal with the reactions of these novel intermediates and no aspect of methide chemistry will be discussed. o-quinone methide chemistry will be discussed. Quinone methides are extremely unstable and reactive. The alignment of dipoles in the functionalQuinone groups methides of arep-quinone extremely methide unstable lie andin the reactive. same Thedirection, alignment which of dipolescauses the in theoxygen functional to groupsabstract of pa-quinone proton rapidly methide and lie inforce the aromatization same direction, of whichthe quinonoid causes the nucleus. oxygen This to abstract results ain proton the rapidlyaddition and of force even aromatization weaker nucleophiles of the quinonoidat 6-position nucleus. (Figure This6). Thus, results the in simplest the addition p-quinone of even methide weaker nucleophilesdue to its atextreme 6-position reactivity (Figure 6has). Thus, not thebeen simplest isolatedp-quinone but has methide been characterized due to its extreme only reactivityas its hasMichael-1,6-adduct not been isolated [39]. but has been characterized only as its Michael-1,6-adduct [39].

Figure 6. Rapid reaction of p-quinone methide. The simplest p-quinone methide arises from the FigureFigure 6. 6.Rapid Rapid reaction reactionof ofp -quinonep-quinone methide.methide. The The simplest simplest pp-quinone-quinone methide methide arises arises from from the the two-electron oxidation of p-cresol. It is extremely unstable and reacts instantaneously with water two-electrontwo-electron oxidation oxidation of ofp -cresol.p-cresol. ItIt isis extremelyextremely unstable unstable and and reacts reacts instantaneously instantaneously with with water water forming the Michael-1,6-adduct, 4-hydroxybenzyl alcohol. forming the Michael-1,6-adduct, 4-hydroxybenzyl alcohol.

In the case of catecholamine derivatives, as early as 1958, Witkop and others proposed that a similarIn the route case involving of catecholamine oxidation of derivatives, dopamine to as its early p-quinone as 1958, methide Witkop and andsubsequent others proposedhydration of that a similarthe quinone route methide involving to oxidationform the norepinephrine of dopamine to de itsrivativesp-quinone [40,41]. methide However, and subsequent this novel hydrationroute was of thedisproved quinone for methide the biosynthesis to form theof norepinephrine norepinephrine and derivatives a direct enzymatic [40,41]. hydroxylation However, this of dopamine novel route wasside disproved chain to forgive the stereospecific biosynthesis norepinephri of norepinephrinene was andsoon aestablished direct enzymatic [42]. Thus, hydroxylation dopamine of dopamineβ-hydroxylase side chain catalyzes to give the stereospecific stereospecific norepinephrine incorporation of was one soon atom established of molecular [42 ].oxygen Thus, into dopamine the side chain of dopamine with the use of two reducing equivalents (Figure 7). Following this Int. J. Mol. Sci. 2016, 17, 1576 6 of 23

β-hydroxylase catalyzes the stereospecific incorporation of one atom of molecular oxygen into the sideInt. chain J. Mol. ofSci.dopamine 2016, 17, 1576 with the use of two reducing equivalents (Figure7). Following this discovery,6 of 23 Int. J. Mol. Sci. 2016, 17, 1576 6 of 23 quinone methide involvement in catecholamine metabolism was completely abandoned until our discovery, quinone methide involvement in catecholamine metabolism was completely abandoned group first reported a peculiar reaction of catecholamine derivatives with cuticular enzymes isolated discovery,until our groupquinone first methide reported involvement a peculiar in catereacticholamineon of catecholamine metabolism wasderi completelyvatives with abandoned cuticular Sarcophaga bullata fromuntilenzymes our isolatedgroup first from [reported43 Sarcophaga]. Incubation a peculiar bullata of intactreacti[43]. on cuticleIncubation of catecholamine isolated of intact from deri thecuticlevatives wandering isolated with stagefromcuticular ofthe the Sarcophagaenzymeswandering bullataisolated stagelarva, of from the containing Sarcophaga Sarcophaga phenol bullata bullata oxidizinglarva, [43]. containing Incubation enzymes phenol withof intact oxidizing various cuticle catecholamines enzymes isolated with from various produced the sidewanderingcatecholamines chain hydroxylated stage produced of the catecholsSarcophaga side chain and bullata covalenthydroxylated larva, bindingcontaining catechols of phenol catechols and oxidizing covalent through enzymesbinding their side withof chaincatechols various to the cuticlecatecholaminesthrough [43]. their The reactionside produced chain was toside accounted the chain cuticle byhydroxylated [43]. the formation The reaction catechols and subsequentwas and accounted covalent reactions by binding the of quinoneformation of catechols methide and (Figurethroughsubsequent8). their reactions side chain of qu toinone the methidecuticle [43].(Figure The 8). reaction was accounted by the formation and subsequent reactions of quinone methide (Figure 8).

FigureFigure 7. 7.Mechanism Mechanism ofof dopaminedopamine hydroxylation reaction. reaction. Initially, Initially, oxidation oxidation of dopamine of dopamine to a to Figure 7. Mechanism of dopamine hydroxylation reaction. Initially, oxidation of dopamine to a a quinonequinone methide and and hydration hydration of of the the resultant resultant quinone quinone methide methide was wasproposed proposed to be tothe be route the routefor quinone methide and hydration of the resultant quinone methide was proposed to be the route for forthe the biosynthesis biosynthesis of of norepinephrine. norepinephrine. However, However, studies studies with with labeled labeled oxygen oxygen revealed revealed that that the the thehydroxylation biosynthesis is accompaniedof norepinephrine. by the incorporationHowever, studies of one withatom oflabeled molecular oxygen oxygen revealed into dopamine that the hydroxylation is accompanied by the incorporation of one atom of molecular oxygen into dopamine by hydroxylationby a specific dopamine- is accompaniedβ-hydroxylase by the incorporation reaction. of one atom of molecular oxygen into dopamine a specificby a specific dopamine- dopamine-β-hydroxylaseβ-hydroxylase reaction. reaction.

Figure 8. Initial observation on the quinone methide formation in insect cuticle. Incubation of FigureFigure4-alkylcatechols 8. 8.Initial Initial observation withobservation intact cuticle onon thethe resulted quinonequinone in methidethmethidee covalent formationformation binding in inof insect insectcatechols cuticle. cuticle. through Incubation Incubation their side of of 4-alkylcatechols4-alkylcatecholschain to the cuticle with with and intact intact generation cuticle cuticle ofresultedresulted side chain in theth hydroxylatede covalent binding binding products. of of catechols catechols through through their their side side chain to the cuticle and generation of side chain hydroxylated products. chain to the cuticle and generation of side chain hydroxylated products. Evidence that such an oxidation of 4-alkylphenols to quinone methide can occur easily comes fromEvidence studies withthat suchlaccase. an oxidationLaccase form of 4-alkylphe a heterogeneousnols to quinone group methideof phenoloxidases. can occur easily They comes often Evidence that such an oxidation of 4-alkylphenols to quinone methide can occur easily comes fromcatalyze studies the oxidation with laccase. of p-diphenols Laccase form to p -quinones.a heterogeneous However, group they of also phenoloxidases. catalyze the oxidationThey often of from studies with laccase. Laccase form a heterogeneous group of phenoloxidases. They often catalyzeo-diphenols the tooxidation o-quinones. of p Most-diphenols laccases to arep-quinones. associated However, with plants they and also fungi catalyze and are the not oxidation present ofin catalyze the oxidation of p-diphenols to p-quinones. However, they also catalyze the oxidation of ohigher-diphenols animals. to o -quinones.However, insectsMost laccases possess are a largeassociated group with of laccases plants andfor bothfungi cuticular and are notpigmentation present in o-diphenolshigherreaction animals. and to o -quinones.hardening However, Most reactions.insects laccases possess The are ainsect large associated grouplaccases withof laccasesspecifically plants for and bothuse fungi cuticularo-diphenolic and are pigmentation not substrate present in higherreactionsuch animals.as dopamine.and However,hardening Laccases insectsreactions. will possess oxidize The a insecttheir large su grouplaccasesbstrates of specifically laccasesto semiquinones for use both o -diphenolic cuticularthrough one pigmentation substrate electron reactionsuchoxidation as and dopamine. [44]. hardening However, Laccases reactions. the willsemiquinones, The oxidize insect their laccases bein sugbstrates unstable, specifically to semiquinonesreact use witho-diphenolic one through another substrate oneundergoing electron such as dopamine.oxidationdisproportionation, Laccases [44]. However, will which oxidize leadsthe semiquinones, their to the substrates formation bein to of semiquinonesg the unstable, two-electron react through oxidationwith one product, electronanother quinone oxidationundergoing and [44 ]. However,disproportionation,regeneration the semiquinones, of the parent which compound,leads being to unstable,the catecholformation react (Figure of with the 9).two-electron one Intere anotherstingly oxidation undergoing some but product, not disproportionation, all quinonelaccases willand whichregenerationalso leadsoxidize to theofcertain the formation parent phenols compound, of thesuch two-electron as catechol syringalda (Figure oxidationzine 9). and Intere product, 2,6-dimethoxy-4-allylstingly quinone some but and not regenerationphenol all laccases to their will of the parentalsocorresponding compound,oxidize certain quinone catechol phenols methides (Figure such (Figure9). as Interestingly syringalda 10) [45]. Agaizine somen andthe but reaction not2,6-dimethoxy-4-allyl all laccasesoccurs by will disproportionation also phenol oxidize to certaintheir of phenolscorrespondingthe semiquinone such as quinone syringaldazine to quinone methides andmethide (Figure 2,6-dimethoxy-4-allyl 10)and [45]. parent Again phenol.the reaction phenol However, occurs to their bytill corresponding disproportionation now no one quinone has of thedemonstrated semiquinone an enzyme to quinone that will methide oxidize and4-alkylcatechols parent phenol. to hydroxyquinone However, till methides. now no one has demonstrated an enzyme that will oxidize 4-alkylcatechols to hydroxyquinone methides. Int. J. Mol. Sci. 2016, 17, 1576 7 of 23 methides (Figure 10)[45]. Again the reaction occurs by disproportionation of the semiquinone to quinone methide and parent phenol. However, till now no one has demonstrated an enzyme that will oxidizeInt. J. Mol. 4-alkylcatechols Sci. 2016, 17, 1576 to hydroxyquinone methides. 7 of 23 Int. J. Mol. Sci. 2016, 17, 1576 7 of 23 Int. J. Mol. Sci. 2016, 17, 1576 7 of 23

Figure 9. Laccase catalyzed oxidation of catechol. Laccase oxidizes catechols to the semiquinone. FigureFigure 9. 9.Laccase Laccase catalyzed catalyzedoxidation oxidation ofof catechol.catechol. Lacca Laccasese oxidizes oxidizes catechols catechols to tothe the semiquinone. semiquinone. Two molecules of semiquinone undergo rapid disproportionation generating the starting material TwoTwoFigure molecules molecules 9. Laccase of semiquinoneof catalyzedsemiquinone oxidation undergo undergo rapidof rapidcatechol. disproportionation dispro Laccaportionationse oxidizes generating generating catechols the theto starting thestarting semiquinone. material material and and quinone product. quinoneandTwo quinone molecules product. product. of semiquinone undergo rapid disproportionation generating the starting material and quinone product.

Figure 10. Laccase catalyzed quinone methide production from 4-alkyl phenols. Laccase produces Figure 10. Laccase catalyzed quinone methide production from 4-alkyl phenols. Laccase produces Figurequinone 10. Laccasemethide catalyzedas the final quinone product methide in some production cases. For from example, 4-alkyl it phenols.oxidizes certain Laccase phenolic produces quinoneFigure 10. methide Laccase as catalyzed the final quinone product methide in some production cases. For from example, 4-alkyl it phenols.oxidizes Laccase certain producesphenolic quinonesubstrates methide such as theas finalsyringald productazine in someand cases.2,6-dimethoxy-4-allylp For example, it oxidizeshenol to certain their phenolic corresponding substrates substratesquinone methide such as thesyringald final productazine and in some2,6-dimethoxy-4-allylp cases. For example,henol it oxidizesto their certain corresponding phenolic suchsemiquinones as syringaldazine that undergo and 2,6-dimethoxy-4-allylphenol disproportionation to quinone tometh theirides corresponding and starting compounds. semiquinones that undergosemiquinonessubstrates disproportionation such that asundergo syringaldto disp quinoneazineroportionation methidesand 2,6-dimethoxy-4-allylp to andquinone starting meth compounds.ideshenol and starting to their compounds. corresponding Allsemiquinones our efforts that to undergo identify disp theroportionation enzyme that to quinonecould generatemethides andquinone starting methides compounds. directly from All our efforts to identify the enzyme that could generate quinone methides directly from 4-alkylcatechols by two electron oxidation ended up in vain. However, we were fortunate to identify 4-alkylcatecholsAllAll our our efforts efforts by totwo to identify identifyelectron the oxidationthe enzymeenzyme ended thatthat up couldcould in vain. generate However, quinone quinone we were methides methides fortunate directly directly to identify from from a two-step route for this reaction [46,47]. Rather than direct generation of quinone methide from 4-alkylcatecholsa4-alkylcatechols two-step route by by for two two this electron electron reaction oxidation oxidation [46,47]. endedendedRather up than in vain. vain.direct However, However, generation we we ofwere were quinone fortunate fortunate methide to toidentify identifyfrom 4-alkylcatechols, we discovered a route involving initial oxidation of catechol to 4-alkylquinone by a two-step4-alkylcatechols,a two-step route route for wefor this discoveredthis reaction reaction a [ route46[46,47].,47 ].involving RatherRather thaninitial direct direct oxidation generation generation of catechol of of quinone quinoneto 4-alkylquinone methide methide from fromby phenoloxidase and subsequent enzyme catalyzed isomerization of quinone to quinone methide by a 4-alkylcatechols,phenoloxidase4-alkylcatechols, and we we discoveredsubsequent discovered enzy aa routerouteme involvingcatalyzedinvolving isomerization initial oxidation oxidation of quinoneof of catechol catechol to quinone to to 4-alkylquinone 4-alkylquinone methide by by a by quinone isomerase (Figure 11). Thus, our extensive efforts to search for the enzymes generating phenoloxidasequinonephenoloxidase isomerase and and subsequent subsequent(Figure 11). enzymeenzy Thus,me our catalyzed extensive isomerization isomerization efforts to ofsearch ofquinone quinone for theto quinone to enzymes quinone methide generating methide by a by quinone methides resulted in the discovery of a new enzyme that isomerizes 4-alkyl quinones to a quinonequinone isomerasemethidesisomerase resulted (Figure in11).11 the). Thus, Thus, discovery our our extensive extensiveof a new efforts effortsenzyme to to thatsearch search isomerizes for for the the enzymes4-alkyl enzymes quinones generating generating to hydroxyl quinone methides [47]. quinonehydroxylquinone methides methides quinone resulted methidesresulted in in[47]. the the discoverydiscovery ofof aa newnew enzyme that that isomerizes isomerizes 4-alkyl 4-alkyl quinones quinones to to hydroxylhydroxyl quinone quinone methides methides [47 [47].].

Figure 11. Mechanism of quinone methide production in insect cuticle. Quinone methide production Figure 11. Mechanism of quinone methide production in insect cuticle. Quinone methide production from 4-alkylcatechols is accomplished in insect cuticle by a two-enzyme system consisting of fromFigure 4-alkylcatechols 11. Mechanism ofis quinoneaccomplished methide in production insect cuticle in insect by a cuticle. two-enzyme Quinone system methide consisting production of Figurephenoloxidase, 11. Mechanism which of oxidizes quinone the methide catechols production to o-quinones in insect and cuticle. quinone Quinone isomerase methide that productionconverts phenoloxidase,from 4-alkylcatechols which isoxidizes accomplished the catechols in insect to o -quinonescuticle by anda two-enzyme quinone isomerase system thatconsisting converts of fromphenoloxidase 4-alkylcatechols generated is accomplished 4-alkylquinones in to insect p-quinone cuticle methides by a (R two-enzyme = alkyl substitution). system consisting of phenoloxidase,phenoloxidasephenoloxidase, whichgenerated which oxidizes oxidizes 4-alkylquinones thethe catecholscatechols to p-quinone toto o-quinones methides and and (R quinone quinone= alkyl substitution).isomerase isomerase that that converts converts 4. Predominancephenoloxidasephenoloxidase generated of generated Quinone 4-alkylquinones 4-alkylquinones to Quinone toMethidetop p-quinone-quinone Tautomerism methides (R (R =in = alkyl alkylSimple substitution). substitution). Catechols 4. Predominance of Quinone to Quinone Methide Tautomerism in Simple Catechols 4. PredominanceQuinone isomerase of Quinone is certainly to Quinone needed Methide for the Tautomerism conversion inof SimpleN-acetyldopamine Catechols quinone to Quinone isomerase is certainly needed for the conversion of N-acetyldopamine quinone to quinone methide [47] but a number of 4-substituted quinones exhibit spontaneous nonenzymatic quinoneQuinone methide isomerase [47] but is a certainlynumber ofneeded 4-substituted for the conversionquinones ex ofhibit N-acetyldopamine spontaneous nonenzymatic quinone to isomerization reaction to quinone methides. Since these reactions are highly pertinent to isomerizationquinone methide reaction [47] but to a quinonenumber ofmethides. 4-substituted Since quinones these reactions exhibit spontaneous are highly nonenzymaticpertinent to isomerization reaction to quinone methides. Since these reactions are highly pertinent to Int. J. Mol. Sci. 2016, 17, 1576 8 of 23

4. Predominance of Quinone to Quinone Methide Tautomerism in Simple Catechols Quinone isomerase is certainly needed for the conversion of N-acetyldopamine quinone to quinone methide [47] but a number of 4-substituted quinones exhibit spontaneous nonenzymatic isomerizationInt. J. Mol. Sci. reaction2016, 17, 1576 to quinone methides. Since these reactions are highly pertinent8 of 23 to eumelanogenesis, they are discussed in this section. Let us examine these reactions starting from C –Ceumelanogenesis,compounds. Oxidationthey are discussed of 3,4-Dihydroxybenzylamine in this section. Let us examine either these chemically reactions orstarting enzymatically from 6 Int.1 J. Mol. Sci. 2016, 17, 1576 8 of 23 producesC6–C1 tocompounds. its corresponding Oxidation quinone,of 3,4-Dihydroxybenzy which is verylamine unstable either andchemically isomerizes or enzymatically rapidly to the quinoneeumelanogenesis,produces methide. to its Michael-1,4-addition corresponding they are discussed quinone, in of this which water sectio toisn. very quinoneLet usunstable examine methide and these isomerizes generates reactions therapidly starting carbinolamine to from the quinone methide. Michael-1,4-addition of water to quinone methide generates the carbinolamine intermediateC6–C1 compounds. (Figure 12 Oxidation). Substitution of 3,4-Dihydroxybenzy of hydroxyl grouplamine and either amino chemically group or on enzymatically the same carbon intermediate (Figure 12). Substitution of hydroxyl group and amino group on the same carbon atomproduces results into theits corresponding loss of ammonia quinone, and which generation is very of unstable 3,4-dihydroxybenzaldehyde and isomerizes rapidly asto thethe final atom results in the loss of ammonia and generation of 3,4-dihydroxybenzaldehyde as the final product.quinone [48]. methide. 3,4-Dihydroxybenzyl Michael-1,4-addi alcoholtion of water also veryto quinone much methide behaves generates the same the way carbinolamine and produces intermediateproduct. [48]. (Figure 3,4-Dihydroxybenzyl 12). Substitution alcohol of hydrox also veryyl group much and behaves amino the group same on way the andsame produces carbon 3,4-dihydroxybenzaldehyde as the final product, as shown in Figure 12 via another transient quinone atom3,4-dihydroxybenzaldehyde results in the loss of ammoniaas the final and product, generation as shownof 3,4-dihydroxybenzaldehyde in Figure 12 via another as transientthe final methide [49]. product.quinone methide[48]. 3,4-Dihydroxybenzyl [49]. alcohol also very much behaves the same way and produces 3,4-dihydroxybenzaldehyde as the final product, as shown in Figure 12 via another transient quinone methide [49].

Figure 12. Oxidation chemistry of 3,4-dihydroxybenzylamine and 3,4-dihydroxybenzyl alcohol. Figure 12. Oxidation chemistry of 3,4-dihydroxybenzylamine and 3,4-dihydroxybenzyl alcohol. 3,4-Dihydroxybenzylamine is easily oxidized to its quinone, which undergoes rapid nonenzymatic 3,4-Dihydroxybenzylamine is easily oxidized to its quinone, which undergoes rapid nonenzymatic isomerization to quinone methide. Quinone methide reacts with water to form carbinolamine and Figure 12. Oxidation chemistry of 3,4-dihydroxybenzylamine and 3,4-dihydroxybenzyl alcohol. isomerizationthen looses to ammonia quinone generating methide. 3,4-dihydroxybenzaldehyde Quinone methide reacts withas the water final toproduct. form carbinolamineA similar 3,4-Dihydroxybenzylamine is easily oxidized to its quinone, which undergoes rapid nonenzymatic and thenconversion looses of ammonia 3,4-dihydroxybenzyl generating alcohol 3,4-dihydroxybenzaldehyde to 3,4-dihydroxybenzaldehyde as the also final occurs product. through A similara isomerization to quinone methide. Quinone methide reacts with water to form carbinolamine and conversionquinone ofmethide 3,4-dihydroxybenzyl intermediate. alcohol to 3,4-dihydroxybenzaldehyde also occurs through then looses ammonia generating 3,4-dihydroxybenzaldehyde as the final product. A similar a quinone methide intermediate. conversion of 3,4-dihydroxybenzyl alcohol to 3,4-dihydroxybenzaldehyde also occurs through a The next group of compounds that can exhibit quinone to quinone methide tautomerism is the quinone methide intermediate. C6–C2 series. 3,4-Dihydroxybenzyl cyanide upon oxidation readily produces the corresponding The next group of compounds that can exhibit quinone to quinone methide tautomerism is the o-quinone. The benzylic carbon in this o-quinone is acidic due to the presence of electron C –C series.The next 3,4-Dihydroxybenzyl group of compounds cyanidethat can exhibit upon quinone oxidation to quinone readily methide produces tautomerism the corresponding is the 6 withdrawing2 quinone nucleus and the cyanide group (Figure 13). As a result, it rapidly isomerizes to o-quinone.C6–C2 series. The 3,4-Dihydroxybenzyl benzylic carbon in cyanide this o -quinoneupon oxidation is acidic readily due produces to the the presence corresponding of electron oquinone-quinone. methide. The benzylic The quinone carbon methide in this formed o-quinone seems is to acidicundergo due dimerization to the presence and perhaps of electron other withdrawing quinone nucleus and the cyanide group (Figure 13). As a result, it rapidly isomerizes to withdrawingoligomerization quinone reactions nucleus [50]. and the cyanide group (Figure 13). As a result, it rapidly isomerizes to quinonequinone methide. methide. The The quinone quinone methide methide formedformed seemsseems to to undergo undergo dimerization dimerization and and perhaps perhaps other other oligomerizationoligomerization reactions reactions [50 [50].].

Figure 13. Oxidative transformation of 3,4-dihydroxybenzyl cyanide. 3,4-Dihydroxybenzyl cyanide upon oxidation to its quinone undergoes rapid isomerization to quinone methide, which readily polymerizes to uncharacterized products. Figure 13. Oxidative transformation of 3,4-dihydroxybenzyl cyanide. 3,4-Dihydroxybenzyl cyanide Figure 13. Oxidative transformation of 3,4-dihydroxybenzyl cyanide. 3,4-Dihydroxybenzyl cyanide upon oxidation to its quinone undergoes rapid isomerization to quinone methide, which readily upon3,4-dihydroxyphenylglycine oxidation to its quinone undergoes is the lower rapid homolo isomerizationgue of dopa. to quinone We synthesized methide, this which compound readily polymerizes to uncharacterized products. polymerizesand studied its to uncharacterizedmetabolic fate by products. oxidation reactions [51]. Tyrosinase readily oxidizes this compound to its3,4-dihydroxyphenylglycine quinone, which undergoes rapid is the decarboxylation lower homologue to produce of dopa. the We same synthesized quinone thismethide compound as that and studied its metabolic fate by oxidation reactions [51]. Tyrosinase readily oxidizes this compound to its quinone, which undergoes rapid decarboxylation to produce the same quinone methide as that Int. J. Mol. Sci. 2016, 17, 1576 9 of 23

3,4-dihydroxyphenylglycine is the lower homologue of dopa. We synthesized this compound and studied its metabolic fate by oxidation reactions [51]. Tyrosinase readily oxidizes this compound to its quinone, which undergoes rapid decarboxylation to produce the same quinone methide as that producedInt. J. Mol. by Sci. 3,4-dihydroxybenzylamine 2016, 17, 1576 (shown in Figure 12). Thus, its oxidation also culminates9 of 23 in the same product viz., 3,4-dihydroxybenzaldehyde. 3,4-Dihydroxyphenylaceticproduced by 3,4-dihydroxybenzylamine acid however, (shown exhibits in Figure a 12). more Thus, complex its oxidation pattern also of culminates reactions in which are summarizedthe same product in Figure viz., 3,4-dihydroxybenzaldehyde. 14. A number of quinone to quinone methide tautomerism has been 3,4-Dihydroxyphenylacetic acid however, exhibits a more complex pattern of reactions which identified in this case [24,52–54]. Oxidation of 3,4-dihydroxyphenylacetic acid results in the formation are summarized in Figure 14. A number of quinone to quinone methide tautomerism has been of carboxymethyl-o-benzoquinone, which has three different fates. First it can undergo decarboxylation identified in this case [24,52–54]. Oxidation of 3,4-dihydroxyphenylacetic acid results in the generatingformation the of simple carboxymethyl- hydroxylo-benzoquinone,p-quinone methide which has [53 three,54]. diffe Thisrent quinonefates. First methide it can undergo undergoes rapiddecarboxylation Michael-1,6-addition generating reaction the simple with hydroxyl water producingp-quinone methide 3,4-dihydroxybenzyl [53,54]. This quinone alcohol methide (reaction 2 → 12undergoes→ 13; Figure rapid Michael-1,6-addition14). The second reaction reaction of wi carboxymethyl-th water producingo-benzoquinone 3,4-dihydroxybenzyl is the alcohol conversion to isomeric(reaction quinone 2 → 12 methide→ 13; Figure and 14). rapid The addition second reaction of water of producingcarboxymethyl- 3,4-dihydroxymandelico-benzoquinone is the acid (reactionconversion2 → 7 →to 8;isomeric Figure 14 )[quinone24,54]. Themethide mandelic and acidrapid derivative addition thus of formedwater alsoproducing undergoes further3,4-dihydroxymandelic oxidation by tyrosinase. acid (reaction Initially 2 we → proposed7 → 8; Figure a direct 14) [24,54]. oxidative The decarboxylationmandelic acid derivative route to the formationthus formed of the also quinone undergoes methide further (compound oxidation 8bygoing tyrosinase. directly Initially to 10; we Figure proposed 14)[ a55 direct]. However, oxidative it was decarboxylation route to the formation of the quinone methide (compound 8 going directly to 10; subsequently demonstrated that mandeloquinone 9 is a compulsory intermediate, which undergoes Figure 14) [55]. However, it was subsequently demonstrated that mandeloquinone 9 is a compulsory β γ rapidintermediate, decarboxylation which generatingundergoes rapid quinone decarboxylation methide as generating it is also aquinone, -unsaturated methide as it acid is also [54 ,56a ,57]. The dihydroxymandelateβ,γ-unsaturated acid [54,56,57]. derivative The in dihydroxymandelate which the α-hydrogen derivative is replaced in which with the a methylα-hydrogen group is also exhibitsreplaced a similar with reactiona methyl [group58]. also exhibits a similar reaction [58].

Figure 14. Multiple routes for the oxidative transformation of 3,4-dihydroxyphenylacetic acid. 3,4 - Figure 14. Multiple routes for the oxidative transformation of 3,4-dihydroxyphenylacetic acid. Dihydroxyphenylacetic acid (1) is readily oxidized by tyrosinase to its quinone (2). This quinone 3,4-Dihydroxyphenylacetic acid (1) is readily oxidized by tyrosinase to its quinone (2). This quinone exhibits three different reactions. It can undergo decarboxylation to the quinone methide exhibits(12), three which different reacts with reactions. water producing It can undergo 3,4-dihydroxybenylalcohol decarboxylation to (13 the). 3,4-dihydroxybenylalcohol quinone methide (12), which reactsis with also water oxidized producing by tyrosinase 3,4-dihydroxybenylalcohol to its quinone ((1314).) 3,4-dihydroxybenylalcoholand nonenzymatically converted is also oxidizedto by tyrosinase3,4-dihydroxybenzaldehyde to its quinone ( 14) and(11) nonenzymaticallythrough quinone converted methide to 3,4-dihydroxybenzaldehyde intermediate (10). (11) throughCarboxymethyl- quinoneo-quinone methide (2) intermediate also isomerizes (10). to Carboxymethyl- quinone methideo-quinone (7), which (2) also generates isomerizes to quinone3,4-dihydroxymandelic methide (7), which acid generates(8) by 1,6-addtion 3,4-dihydroxymandelic of water. Tyrosinase acid (8catalyzed) by 1,6-addtion oxidation of of water. Tyrosinase3,4-dihydroxy catalyzed mandelic oxidation acid of 3,4-dihydroxy produces the mandelic mandeloquinone acid produces (9), thethat mandeloquinone is nonenzymatically (9), that is nonenzymaticallydecarboxylated decarboxylated to the same quinone to the methide same quinone (10) produced methide by ( 103,4-dihydroxybenzyl) produced by 3,4-dihydroxybenzyl alcohol, thus alcohol,yielding thus the yielding same final the sameproduct, final 3,4-dihydroxy product, 3,4-dihydroxy benzaldehyde benzaldehyde (11). Finally, quinone (11). Finally, (2) undergoes quinone (2) rapid intramolecular cyclization to form 2,5,6-trihydroxy benzofuran (4) via furanone (3). This furan undergoes rapid intramolecular cyclization to form 2,5,6-trihydroxy benzofuran (4) via furanone (3). is initially believed to form the quinone (5) before getting converted to quinone methide (6), and This furan is initially believed to form the quinone (5) before getting converted to quinone methide (6), eventual polymerization. and eventual polymerization. Finally, carboxymethyl-o-quinone exhibits a unique intramolecular cyclization reaction [24] that Finally,is similar carboxymethyl- to the intramolecularo-quinone reaction exhibits observ aed unique with dopaquinone intramolecular and cyclization dopaminequinone. reaction This [24] that is similarreaction to theproduces intramolecular 5,6-dihydroxybenzofuran-2-one reaction observed (compound with dopaquinone 3; Figure 14). and However, dopaminequinone. the furanone This undergoes rapid oxidation producing a red colored compound, which was identified as the corresponding two-electron oxidation product—quinone methide (reactions 3 → 4 → 5 → 6; Int. J. Mol. Sci. 2016, 17, 1576 10 of 23 reaction produces 5,6-dihydroxybenzofuran-2-one (compound 3; Figure 14). However, the furanone undergoes rapid oxidation producing a red colored compound, which was identified as the corresponding two-electron oxidation product—quinone methide (reactions 3 → 4 → 5 → 6; Figure 14). At physiologicalInt. J. Mol. Sci. 2016 pH,, 17, 1576 the formation of red colored quinone methide 6 could not be10 decisively of 23 identified [24]. However, with the use of fluorine substitution at the aromatic ring, we were able to showFigure that the 14). fluoroquinone At physiological looses pH, fluoridethe formation ion during of red cyclizationcolored quinone and producesmethide 6 thecould furanolactone, not be whichdecisively is further identified oxidized [24]. to red However, colored with quinone the use methide of fluorine (6). The substitution quinone (at5) the shown aromatic in Figure ring, 14we is not were able to show that the fluoroquinone looses fluoride ion during cyclization and produces the formed at physiological pH, but the more stable yet highly reactive quinone methide (6) is generated furanolactone, which is further oxidized to red colored quinone methide (6). The quinone (5) shown duringin theFigure oxidation 14 is not [52 formed]. at physiological pH, but the more stable yet highly reactive quinone Apartmethide from (6) is these generated important during compounds, the oxidation two[52]. additional compounds—3,4-dihydroxyphenethyl alcohol asApart well from as itsthese side important chain hydroxylatedcompounds, two product, additional 3,4-dihydroxyphenyl compounds—3,4-dihydroxyphenethyl glycol also exhibit quinonealcohol to quinoneas well as methide its side tautomerismchain hydroxylated as shown product, in Figure 3,4-dihydroxyphenyl 15. The quinone glycol isomerization also exhibit could be enzymequinone catalyzed to quinone [59methide] as well tautomerism as nonenzymatic as shown in reaction Figure 15. [54 The]. quinone Thus, o -quinoneisomerization formed could from 3,4-dihydoxyphenethylbe enzyme catalyzed alcohol [59] as is well converted as nonenzymatic by quinone reaction isomerase [54]. to Thus, quinone o-quinone methide, formed which from rapidly reacts3,4-dihydoxyphenethyl with water producing alcohol 3,4-dihydroxyphenyl is converted by quinone glycol. isomerase Glycol undergoesto quinone furthermethide, oxidation which to rapidly reacts with water producing 3,4-dihydroxyphenyl glycol. Glycol undergoes further quinone and isomerization to quinone methide. This quinone methide product of 3,4-dihydroxyphenyl oxidation to quinone and isomerization to quinone methide. This quinone methide product of glycol readily transforms by isomerization reaction to 2-hydroxy-3,4-dihydroxyacetophenone similar 3,4-dihydroxyphenyl glycol readily transforms by isomerization reaction to to the2-hydroxy-3,4-dihydroxyacetophenone transformation of 3,4-dihydroxybenzyl similar alcohol to the quinonetransformation methide of 3,4-dihydroxybenzyl to 3,4-dihydroxybenzaldehyde alcohol as shownquinone in Figuremethide 12 to[54 3,4-dihydroxybenzaldehyde,59]. as shown in Figure 12 [54,59].

Figure 15. Oxidation of transformations of 3,4-dihydroxyphenethyl alcohol. Tyrosinase (A) catalyzes Figure 15. Oxidation of transformations of 3,4-dihydroxyphenethyl alcohol. Tyrosinase (A) catalyzes the oxidation of 3,4-dihydroxyphenethyl alcohol to its quinone, which can undergo either enzyme the oxidation of 3,4-dihydroxyphenethyl alcohol to its quinone, which can undergo either enzyme catalyzed isomerization or nonenzymatic isomerization (B) to its quinone methide. Water addition to catalyzed isomerization or nonenzymatic isomerization (B) to its quinone methide. Water addition to this quinone methide and reoxidation of 3,4-dihydroxyphenyl glycol and yet another isomerization this quinoneproduces methidea transient and quinone reoxidation methide of that 3,4-dihydroxyphenyl readily yields 2-hydro glycolxy-3,4-dihydroxy and yet another acetophenone isomerization as producesthe final a transient product. quinone methide that readily yields 2-hydroxy-3,4-dihydroxy acetophenone as the final product. Quinone to quinone methide tautomerism is much more prevalent in C6–C3 series, which is pertinent to melanin biosynthesis as dopa falls in this category. Even though dopamine and its Quinone to quinone methide tautomerism is much more prevalent in C6–C3 series, which is derivatives belong to C6–C2 series, due to their participation in melanogenic process and their pertinent to melanin biosynthesis as dopa falls in this category. Even though dopamine and its relation to dopa, their chemistry is considered in this section. Our group first showed that the derivativesquinones belong of dihydrocaffeic to C6–C2 series, acid derivatives due to their (both participation methylamide in and melanogenic methyl ester process derivatives) and their exhibit relation to dopa,facile their tautomerization chemistry isto consideredquinone methide in this [25,60] section.. Oxidation Our group of dihydrocaffeic first showed acid that derivatives the quinones of dihydrocaffeicreadily produced acid the derivatives unstable quinones, (both methylamide which rapidly and isomerized methyl to ester quinone derivatives) methide and exhibit then facile tautomerizationproduced caffeic to quinone acid derivatives methide [as25 ,60the]. final Oxidation products of dihydrocaffeicthrough yet another acid derivativesnonenzymatic readily producedisomerization the unstable reaction quinones, as shown which in rapidlyFigure 16. isomerized Subsequently, to quinone N-acetyldopa methide ethyl and thenester producedand caffeicN-acetyldopa acid derivatives methyl as ester, the final possessing products the through same skeleton, yet another were nonenzymaticalso shown to exhibit isomerization similar side reaction as shownchain desaturation in Figure 16 reac. Subsequently,tions producing dehydro-N-acetyldopaN-acetyldopa ethyl esteresters and[61,62].N -acetyldopa methyl ester, possessing the same skeleton, were also shown to exhibit similar side chain desaturation reactions producing dehydro-N-acetyldopa esters [61,62]. Int. J. Mol. Sci. 2016, 17, 1576 11 of 23 Int. J. Mol. Sci. 2016, 17, 1576 11 of 23

FigureFigure 16. Oxidative16. Oxidative transformation transformation of of dihydrocaffeate dihydrocaffeate derivatives. Dihydrocaffeate Dihydrocaffeate derivatives derivatives (R” = NHCH3 or OCH3) upon oxidation produce the unstable quinones that exhibit rapid (R” = NHCH or OCH ) upon oxidation produce the unstable quinones that exhibit rapid tautomerization3 to quinone3 methides. These quinone methides suffer another nonenzymatic tautomerization to quinone methides. These quinone methides suffer another nonenzymatic isomerization producing more stable caffeic acid derivatives. This reaction also occurs with isomerization producing more stable caffeic acid derivatives. This reaction also occurs with N-acetyldopa esters (R = NHCOCH3; R” = OCH3 or OCH2CH3). N-acetyldopa esters (R = NHCOCH3; R” = OCH3 or OCH2CH3). Extensive kinetic studies carried out by Bolton and her associates [63,64] indicate that the rate of Extensivequinone isomerization kinetic studies to quinone carried methide out by heavily Bolton depends and her on associates the stability [63, 64of ]the indicate quinone that methide. the rate of quinoneExtended isomerization π conjugation to quinone on the side methide chain carbon heavily atom depends increased on the the stability stability of ofthe the quinone quinone methide methide. Thus, for example quinone methide formation from quinones of 4-propyl catechol, hydroxychavicol, Extended π conjugation on the side chain carbon atom increased the stability of the quinone methide and 4-cinnamoyl catechol increased dramatically with increased conjugation [63]. Bolton et al. [64] Thus,also for examined example quinonethe mechanism methide of isomerization formation from of 4-propyl- quinoneso-quinone of 4-propyl to its catechol, quinone hydroxychavicol,methide and and 4-cinnamoyldeduced that catechol the isomerization increased reaction dramatically is a base with catalyzed increased reaction conjugation with [the63]. abstraction Bolton et al.of [the64] also examinedproton the at mechanismthe quinone ofmethide isomerization carbon by of 4-propyl-the base aso-quinone the rate determining to its quinone step methide of the andreaction. deduced that theConsistent isomerization with this reaction finding they is a also base observed catalyzed a la reactionrge kinetic with isotope the effect abstraction with the of substitution the proton of at the quinonebenzylic methide methylene carbon protons by the with base deuterium as the rate [64]. determining From the stepforegoing of the discussion, reaction. it Consistent is evident withthat this findingquinone they alsoto quinone observed methide a large tautomerism kinetic isotope plays a effectdominant with role the in substitution the metabolic of transformation benzylic methylene of protonsnumerous with deuterium catechols. The [64 importance]. From the of foregoingquinone me discussion,thide chemistry it is for evident melanin that biosynthesis quinone will to quinone be methidebrought tautomerism to light in the plays rest a of dominant this review. role in the metabolic transformation of numerous catechols. The importance5. Dopachrome of quinone Conversion methide and Quinone chemistry Me forthide melanin Formation biosynthesis in Melanin will Biochemistry be brought to light in the rest of this review. As early as 1980, Musson et al. [65] oxidized α-methyl dopa methyl ester by potassium 5. Dopachromeferricyanide and Conversion isolated a and yellow Quinone colored Methide solid whose Formation structure in was Melanin determined Biochemistry to be a quinone methide. However, its relation to melanin biosynthesis was not clarified until 1990 when we first Asreported early asthe 1980, production Musson of etthis al. quinone [65] oxidized methideα-methyl by isomerization dopa methyl of the ester dopachrome by potassium derived ferricyanide from and isolatedα-methyl adopa yellow methyl colored ester (Figure solid whose 17) [66]. structure Following was our determined work, Prota toand be his a associates quinone methide.also However,published its relation evidence to to melaninsupport quinone biosynthesis methide was from not dopachrome clarified untilderivatives 1990 when[67,68]. we Based first on reported the the productionfacile isomerization of this quinone of substituted methide dopachrome by isomerization to a stable ofquinone the dopachrome methide [66], derived we postulated from α that-methyl dopaa methylsimilar esterquinone (Figure methide 17)[ should66]. Following be formed our du work,ring melanin Prota andbiosynthes his associatesis as well. also For published the evidenceconversion to support of dopachrome quinone methideto 5,6-dihydroxyindol from dopachromee, initially derivatives a route involving [67,68]. Basedindolidine on thewas facile proposed [69]. However, this route invokes electron donating properties for the C=N bond. isomerization of substituted dopachrome to a stable quinone methide [66], we postulated that a similar However, the C=N bond is electron withdrawing in the C → N direction. As a consequence, it is quinoneeasier methide to protonate should the be nitrogen. formed This during will melanin trigger electron biosynthesis flow around as well. the For ring the causing conversion the of dopachromeproduction to 5,6-dihydroxyindole,of a transient quinone initially methide a routerather involving than the indolidineindolidine. was Thus, proposed quinone [69 methide]. However, this routeproduction invokes is a electron more likely donating possibility. properties Once forformed, the C=N the quinone bond. However, methide can the loose C=N the bond carboxyl is electron withdrawinggroup as incarbon the C dioxide→ N direction.or the α-proton As a consequence,to generate the it 5,6-dihydroxyindole is easier to protonate derivatives. the nitrogen. Note Thisthat will triggerthe electron quinone flow methide around is thea β ring,γ-unsaturated causing the acid. production Therefore, of ait transient can spontaneously quinone methide undergo rather thandecarboxylation the indolidine. generating Thus, quinone DHI. In methidedolidine however, production is doubtful is a more to exhibit likely this possibility. reaction. OnceAlthough formed, the quinoneboth indolidine methide and can quinone loose themethide carboxyl can exhibit group isomerization as carbon dioxide to DHICA. or the It shouldα-proton be pointed to generate out the 5,6-dihydroxyindolehere that various derivatives. metal ions favor Note the that producti the quinoneon of methideDHI under is anonenzymaticβ,γ-unsaturated conditions acid. Therefore,[69]. Dopachrome generated at pH 6.5 also undergoes spontaneous decarboxylation to DHI while the it can spontaneously undergo decarboxylation generating DHI. Indolidine however, is doubtful to same at pH 13 exhibits tautomerization to DHICA [70]. exhibit this reaction. Although both indolidine and quinone methide can exhibit isomerization to DHICA. It should be pointed out here that various metal ions favor the production of DHI under nonenzymatic conditions [69]. Dopachrome generated at pH 6.5 also undergoes spontaneous decarboxylation to DHI while the same at pH 13 exhibits tautomerization to DHICA [70]. Int. J. Mol. Sci. 2016, 17, 1576 12 of 23 Int. J. Mol. Sci. 2016, 17, 1576 12 of 23

Int. J. Mol. Sci. 2016, 17, 1576 12 of 23

Figure 17. First demonstration of quinone methide production in a dopachrome derivative. α-Methyl Figure 17. First demonstration of quinone methide production in a dopachrome derivative. α-Methyl dopa methyl ester upon oxidation produces its dopachrome derivative, which rapidly isomerizes to

dopaquinone methyl methide ester upon under oxidation physiological produces conditions. its dopachrome derivative, which rapidly isomerizes to quinoneFigure methide 17. First under demonstration physiological of quinone conditions. methide production in a dopachrome derivative. α-Methyl dopaOur groupmethyl establishedester upon oxidation concrete produces evidence its fordopachrome the participation derivative, of which quinone rapidly methide isomerizes in melanin to Ourbiosynthesisquinone group establishedmethidein 1990 under with concretephysiological the use evidenceof conditions.insect DCDT for the [19]. participation Insect enzyme of quinone exhibits methide wide substrate in melanin biosynthesisspecificity in and 1990 catalyzes with the the conversion use of insect of a number DCDT of [ L19-dopachrome]. Insect enzyme derivatives exhibits but does wide not touch substrate their OurD-form group [19]. established It converts concrete L-dopachrome evidence to for DHI. the Similarlyparticipation it converts of quinone α-methyldopachrome methide in melanin to specificity and catalyzes the conversion of a number of L-dopachrome derivatives but does not touch biosynthesis2-methyl-DHI, in by 1990 decarboxylative with the use rearrangement. of insect DCDT However, [19]. Insect with enzymedopachrome exhibits methyl wide ester, substrate which their D-form [19]. It converts L-dopachrome to DHI. Similarly it converts α-methyldopachrome specificitycannot undergo and catalyzes decarboxylation, the conversion it produces of a number the ofisomeric L-dopachrome product, derivatives 2-carboxymethyl but does DHInot touch [19]. to 2-methyl-DHI,theirFinally D-form it even [19]. acts by It decarboxylativeon converts α-methyldopachrome L-dopachrome rearrangement. methylto DHI. ester, Similarly However,which it cannot converts with undergo α dopachrome-methyldopachrome decarboxylation methyl orto ester, which2-methyl-DHI,deprotonation cannot undergo byat decarboxylativethe decarboxylation, α-carbon producing rearrangement. it produces the stable However, the quinone isomeric with methide product,dopachrome [19]. 2-carboxymethyl methylThus, dopachromeester, which DHI [19]. Finallycannotisomerase it even undergo does acts onnotdecarboxylation,α touch-methyldopachrome the α-proton it produces or methyldecarboxylate the isomeric ester, whichthe prod carboxyluct, cannot 2-carboxymethyl group, undergo but removes decarboxylation DHI [19]. the or deprotonationFinallyβ-proton it evenas the atacts initial the on α step-methyldopachrome-carbon leading producing to quinone methyl the methide stable ester, quinoneproductionwhich cannot methide (Figure undergo 18). [19 decarboxylation]. Quinone Thus, dopachromemethide or deprotonation at the α-carbon producing the stable quinone methide [19]. Thus, dopachrome isomeraseundergoes does either not touch rapid the decarboxylationα-proton or decarboxylateto DHI with the theinsect carboxyl enzyme group, and isomerization but removes to theDHICAβ-proton isomerase does not touch the α-proton or decarboxylate the carboxyl group, but removes the as thewith initial the stepmammalian leading enzyme to quinone (Figure methide 18). production (Figure 18). Quinone methide undergoes β-proton as the initial step leading to quinone methide production (Figure 18). Quinone methide eitherundergoes rapid decarboxylation either rapid decarboxylation to DHI with to the DHI insect with the enzyme insect enzyme and isomerization and isomerization to DHICA to DHICA with the mammalianwith the enzymemammalian (Figure enzyme 18). (Figure 18).

Figure 18. Reaction catalyzed by dopachrome converting enzymes. A base on the enzyme abstracts the β-proton on dopachrome causing isomerization of dopachrome to quinone methide. Quinone

methide can readily exhibit another isomerization generating DHICA in the case of mammalian FigureFigureDCT. 18. SinceReaction 18. Reaction quinone catalyzed catalyzed methide by isby dopachrome a dopachrome β,γ-unsaturated convertingconverting acid, it enzymes. can enzymes. also readilyA Abase base onexhibit onthe the enzymedecarboxylation. enzyme abstracts abstracts the β-proton on dopachrome causing isomerization of dopachrome to quinone methide. Quinone the βWith-proton insect on DCDT, dopachrome the reaction causing accompanies isomerization decarboxylation-coupled of dopachrome aromatization to quinone methide.resulting in Quinone the methide can readily exhibit another isomerization generating DHICA in the case of mammalian methideformation can readily of DHI. exhibit another isomerization generating DHICA in the case of mammalian DCT. DCT. Since quinone methide is a β,γ-unsaturated acid, it can also readily exhibit decarboxylation. Since quinone methide is a β,γ-unsaturated acid, it can also readily exhibit decarboxylation. With insect 6. QuinoneWith insect Methide DCDT, Intermediate the reaction accompanies in Other Catecholamine decarboxylation-coupled Reactions aromatization resulting in the DCDT,formation the reaction of DHI. accompanies decarboxylation-coupled aromatization resulting in the formation of DHI.Much like the same way dopa is converted to DHICA, dopamine is transformed into DHI by 6.oxidative Quinone transformation Methide Intermediate [27]. Thus, in Otherdopaminequinone Catecholamine formed Reactions by the two-electron oxidation of dopamine undergoes rapid nonenzymatic intramolecular cyclization forming leucochrome, which is 6. Quinone Methide Intermediate in Other Catecholamine Reactions quicklyMuch oxidized like the bysame dopamine way dopa quinone is converted to dopa to DHICA,minechrome dopamine (Figure is transformed19). The conversion into DHI byof Muchoxidativedopaminechrome like transformation the same to DHI way [27].parallels dopa Thus, is the converteddopaminequinone conversion to of DHICA, dopachrome formed dopamine by to the DHICA, two-electron is transformed thus obligating oxidation into the DHIof by dopamine undergoes rapid nonenzymatic intramolecular cyclization forming leucochrome, which is oxidativeformation transformation of quinone methide [27].Thus, intermediate. dopaminequinone In this case, concrete formed evidence by the for two-electron the quinone oxidationmethide of quickly oxidized by dopamine quinone to dopaminechrome (Figure 19). The conversion of dopamineintermediate undergoes comes rapid from nonenzymatic the product analysis intramolecular studies. cyclizationA quinone formingmethide leucochrome,adduct of DHI which at is dopaminechrome2-postion was identified to DHI in parallels the reaction the mixture,conversion the offormation dopachrome of which to DHICA, is shown thus in Figure obligating 19 [27]. the quicklyformation oxidized of quinone by dopamine methide intermediate. quinone to In dopaminechrome this case, concrete evidence (Figure for 19 the). quinone The conversion methide of dopaminechromeintermediate comes to DHI from parallels the product the conversion analysis studies. of dopachrome A quinone tomethide DHICA, adduct thus of obligating DHI at the formation2-postion of quinone was identified methide in the intermediate. reaction mixture, In this the case,formation concrete of which evidence is shown for in the Figure quinone 19 [27]. methide Int. J. Mol. Sci. 2016, 17, 1576 13 of 23 intermediate comes from the product analysis studies. A quinone methide adduct of DHI at 2-postion was identifiedInt. J. Mol. Sci. in 2016 the, 17 reaction, 1576 mixture, the formation of which is shown in Figure 19[27]. 13 of 23

Int. J. Mol. Sci. 2016, 17, 1576 13 of 23

FigureFigure 19. Formation19. Formation of quinoneof quinone methide methideadducts adducts of dopamine during during its its oxidation. oxidation. Dopamine Dopamine quinone formed by the oxidation of dopamine undergoes intramolecular cyclization producing the quinone formed by the oxidation of dopamine undergoes intramolecular cyclization producing the Figureleucochrome 19. Formation and then ofdopaminechrome. quinone methide Isomerizat adducts ionof dopamineof dopaminechrome during its to oxidation. its quinone Dopamine methide leucochrome and then dopaminechrome. Isomerization of dopaminechrome to its quinone methide quinoneand aromatization formed by ofthe the oxidation catecholic of dopaminering produces undergoes DHI. A intramolecular quinone methide cyclization adduct producing of DHI in the its and aromatizationleucochromeoxidative stage and has of then thebeen dopaminechrome. catecholic characterized ring fromproduces Isomerizat the reaction DHI.ion ofmixture. Adopaminechrome quinone methide to itsadduct quinone of methide DHI in its oxidativeand aromatization stage has been of characterizedthe catecholic ring from produces the reaction DHI. mixture. A quinone methide adduct of DHI in its oxidativeOxidations stage of hasnorepinephrine been characteri zedas well from asthe epinephrine reaction mixture. exhibit the quinone → leucochrome → Oxidationsiminochrome of → norepinephrine indole transformations as well (Figure as epinephrine 20). The presence exhibit of the hydroxyl quinone group→ leucochromeat 3-position → → → iminochromewill allowOxidations→ theindole quinone of norepinephrine transformations methide intermediate as (Figurewell as to 20epinephrine rearrange). The presence and exhibit form of the the hydroxyl quinone adrenolutin/noradrenolutin group leucochrome at 3-position will iminochrome → indole transformations (Figure 20). The presence of hydroxyl group at 3-position allow[71,72]. the quinone Norepinephrine methide intermediate quinone has tobeen rearrange shown andto isomerize form the to adrenolutin/noradrenolutin quinone methide and produce[71 ,72]. will2-hydroxy-3 allow the′,4 quinone′-dihydroxyacetophenone methide intermediate and ato number rearrange of sideand chainform theoxidized adrenolutin/noradrenolutin products [72]. Norepinephrine[71,72]. Norepinephrine quinone quinon has beene has shownbeen shown to isomerizeto isomerize to to quinonequinone methide methide and and produce produce 0 0 2-hydroxy-32-hydroxy-3,4 -dihydroxyacetophenone′,4′-dihydroxyacetophenone and and aa numbernumber of of side side chain chain oxidized oxidized products products [72]. [ 72].

Figure 20. Oxidation of epinephrine and norepinephrine. Oxidation of epinephrine (R = methyl group) and norepinephrine (R = H) results in their corresponding quinones. These quinones also FigureFigurecyclize 20. Oxidation 20.and Oxidation get oxidized of epinephrine of epinephrineto form andiminochromes. and norepinephrine. norepinephri The iminochromesne. Oxidation Oxidation of upon epinephrineof epinephrine conversion (R (R =to methyl= quinone methyl group) group)methide and easily norepinephrine isomerize and (R produce= H) results adrenolutin/ in their noradrenolutin,corresponding quinoneswhich w. illThese also quinonesform melanin also and norepinephrine (R = H) results in their corresponding quinones. These quinones also cyclize and cyclizepigment and eventually. get oxidized to form iminochromes. The iminochromes upon conversion to quinone get oxidized to form iminochromes. The iminochromes upon conversion to quinone methide easily methide easily isomerize and produce adrenolutin/noradrenolutin, which will also form melanin isomerize7. Reactivitypigment and eventually.of produce Dihydroxyindoles adrenolutin/noradrenolutin, which will also form melanin pigment eventually. A major difference between simple catechols and dihydroxyindoles is the presence of amino 7. Reactivity7. Reactivity of Dihydroxyindolesof Dihydroxyindoles group attached to the catecholic ring in the latter compounds. This introduces extreme reactivity to Adihydroxyindoles. majorA major difference difference People between between working simple simple with catecholsDHI catechols for exam an anddple dihydroxyindoles dihydroxyindoles are well aware of is its the is extreme thepresence presence sensitivity of amino of to amino groupgroupoxygen. attached attached DHI to like the to its the catecholic leucochrome catecholic ring ring precursor in in the the latter latterwill also compounds.compounds. undergo rapid This This introducesoxidation introduces and extreme extremeproduce reactivity the reactivity more to to dihydroxyindoles.dihydroxyindoles.stable quinone imine People People form, working working which withwithwill beDHI in forequilib for exam exampleriumple arewith are well the well aware isomeric aware of its quinone ofextreme its extreme as sensitivity well sensitivityas the to oxygen. DHI like its leucochrome precursor will also undergo rapid oxidation and produce the more to oxygen.quinone DHI methide. like its The leucochrome indole quinone precursor is less favored will also structure undergo over rapid the indolechrome oxidation and or producequinone the stablemethide, quinone as both imine of these form, will which give will some be instability equilib duerium to with internal the isomerichydrogen quinone bonding as (Figurewell as 21).the more stable quinone imine form, which will be in equilibrium with the isomeric quinone as well as quinoneThus, the methide. two-electron The indoleoxidation quinone of dihydroxyind is less favoredole produce structure quinone, over the quinone indolechrome imine and or quinone the quinonemethide, methide. as both of The these indole will quinonegive some is stability less favored due to structure internal overhydrogen the indolechrome bonding (Figure or quinone21). methide,Thus, as the both two-electron of these oxidation will give of somedihydroxyind stabilityole due produce to internal quinone, hydrogen quinone imine bonding and quinone (Figure 21). Int. J. Mol. Sci. 2016, 17, 1576 14 of 23

Thus, the two-electron oxidation of dihydroxyindole produce quinone, quinone imine and quinone methide isomers each one of which are capable of a plethora of reactions. Among the two electron Int. J. Mol. Sci. 2016, 17, 1576 14 of 23 oxidation products, the iminochrome form will be less reactive compared to the quinone methide form. A goodmethide example isomers for this each is one the of reactivity which are of capable dopachrome of a plethora and its of quinone reactions. methide Among the isomer. two electron The quinone methideoxidation is very products, reactive the compared iminochrome to dopachrome form will be in less its reactive iminochrome compared form. to the Thus, quinone quinone methide methide form ofform. two A electrongood example oxidation for this product is the reactivity of DHI of willdopachrome have a and dominant its quinone effect methide on the isomer. course The of the reactionquinone because methide quinone is very methide reactive can compared undergo to easydopachrome Michael-1,6-addition in its iminochrome reaction. form. Thus, Molecular quinone oxygen methide form of two electron oxidation product of DHI will have a dominant effect on the course of as well as other oxidants such as dopachrome and dopaquinone can also cause the oxidation of DHI. the reaction because quinone methide can undergo easy Michael-1,6-addition reaction. Molecular Oxidasesoxygen including as well as peroxidase other oxidants and such tyrosinase as dopach willrome also and dopaquinone catalyze the can oxidation. also cause Boththe oxidation one electron oxidationof DHI. and Oxidases two-electron including oxidation peroxidase are possibleand tyrosinase for DHI will (Figurealso catalyze 21). Aminothe oxidation. catechols Both asone well as trihydroxyphenolselectron oxidation such and as two-electron 6-hydroxydopa oxidation derivatives are possible exhibit for DHI easy (Figure one electron 21). Amino oxidation catechols to produceas semiquinonewell as trihydroxyphenols radicals both by such peroxidase as 6-hydroxydopa catalyzed de reactionrivatives exhibit and oxidation easy one electron with molecular oxidation to oxygen. Furtherproduce coupling semiquinone of free radicals radicals will both produce by peroxidase a number catalyzed of oligomeric reaction and products. oxidation At with present molecular a separate enzymeoxygen. catalyzing Further the coupling oxidation of free ofradicals DHI seemswill produce to be a doubtful,number of oligomeric as there are products. a number At present of reactants a that canseparate easily enzyme oxidize catalyzing this compound. the oxidation However, of DHI the seems same to cannotbe doubtful, be said as there for DHICA. are a number The presence of reactants that can easily oxidize this compound. However, the same cannot be said for DHICA. The of carboxyl group in DHICA stabilizes the amino group by hydrogen bonding and prevents easy presence of carboxyl group in DHICA stabilizes the amino group by hydrogen bonding and oxidationprevents of this easy compound. oxidation of Thus, this compound DHICA. is Thus, more DHICA stable is that more DHI. stable that DHI.

Figure 21. Possible reactive intermediates generated from DHI. DHI can undergo easily oxidation Figure 21. Possible reactive intermediates generated from DHI. DHI can undergo easily oxidation with with molecular oxygen producing reactive semiquinones. In addition it is also susceptible to molecular oxygen producing reactive semiquinones. In addition it is also susceptible to two-electron two-electron oxidation generating quinone, quinone methide and iminochrome. oxidation generating quinone, quinone methide and iminochrome. The reactive species formed from dihydroxyindoles also react with each other undergoing Theself-polymerization reactive species thereby formed produc froming dihydroxyindolesmelanin pigment. Of alsothe seven react possible with eachpositions, other hydroxyl undergoing self-polymerizationgroups occupy two thereby positions producing and one melanin position pigment.is used up Of by the NH seven group possible in the ring. positions, This leaves hydroxyl position 2 and 3 on the indole ring and position 4 and 7 on the catechol ring for polymerization groups occupy two positions and one position is used up by NH group in the ring. This leaves reactions. Quinones typically undergo Michael-1,4-addition reactions; but these two positions are positionnot 2available and 3 onin the quinone indole ringstructure. and There position is some 4 and possibility 7 on the of catecholreaction occurring ring for ortho polymerization to the reactions.quinone Quinones carbonyl typically group. In undergocomparison, Michael-1,4-addition the quinone methide carbon reactions; should but be these extremely two positionsreactive are not availablefor addition in thereactions. quinone This structure. will produce There 3-substituted is some indoles possibility and regeneration of reaction of occurringthe aromaticortho ring.to the quinoneEvidence carbonyl for group.the quinone In comparison, methide reactivity the quinone comes from methide mass carbon spectral should studies be of extremelythe oxidative reactive for additiontransformation reactions. of dopamine. This will Kroesche produce and 3-substituted Peter [73] observed indoles that and during regeneration the oligomerization of the aromatic ring. Evidencereaction of for DHI the originating quinone from methide dopamine, reactivity there comeswas an fromincorporation mass spectral of one atom studies of oxygen of the oxidativeinto the indole units. Such incorporation can be accounted by the reaction of quinone methide with water transformation of dopamine. Kroesche and Peter [73] observed that during the oligomerization to generate 3-hydroxy 5,6-dihydroxyindole units. Al-Kazwini et al. [74,75] also witnessed the reactionquinone of DHI methide originating formation from during dopamine, both two-electron there was anand incorporation one-electron oxidation of one atom of DHI. of oxygenHowever, into the indolethe units. majority Such of incorporation the dimeric products can be accountedidentified during by the the reaction oxidative of coupling quinone of methide DHI seem with to waterbe to generatecouple 3-hydroxy at the 2-position 5,6-dihydroxyindole [76–80]. Figure units. 22, fo Al-Kazwinir example, shows et al. the [74 ,structure75] also witnessedof the three the major quinone methideproducts formation characterized during both from two-electronthe DHI reaction and mixture. one-electron oxidation of DHI. However, the majority of the dimeric products identified during the oxidative coupling of DHI seem to be couple at the 2-position [76–80]. Figure 22, for example, shows the structure of the three major products characterized from the DHI reaction mixture. Int. J. Mol. Sci. 2016, 17, 1576 15 of 23 Int. J. Mol. Sci. 2016, 17, 1576 15 of 23

Int. J. Mol. Sci. 2016, 17, 1576 15 of 23 Int. J. Mol. Sci. 2016, 17, 1576 15 of 23

Figure 22. Structure of DHI dimers. Oxidative polymerization of DHI yields 2,2′, 2,4′ and 2,7′ coupled Figure 22. Structure of DHI dimers. Oxidative polymerization of DHI yields 2,20, 2,40 and 2,70 coupled DHI dimers as the major products. DHI dimers as the major products.

Interestingly,Figure 22. Structure the ofoxidation DHI dime rs.products Oxidative of polymerizat some of ionthese of DHI dimers yields 2,2employ′, 2,4′ and quinone 2,7′ coupled methide Interestingly,reactivityFigureDHI dimers [79–81]. 22. Structure theas theA oxidation2,3 major of′ DHIcoupled products. dime products tetramerrs. Oxidative was of polymerizat someidentified ofion during these of DHI dimersthe yields oxidative 2,2 employ′, 2,4′ dimerizationand 2,7 quinone′ coupled of methidethe reactivity2,4′ dimerDHI [79– dimers81 [80].]. A Twoas 2,3 the 0different coupledmajor products. dimeric tetramer products was identified of quinone during methide the coupled oxidative products dimerization (2,3′ and of 3,3 the′) 2,40 dimerhave [80Interestingly, ].been Two characterized different the dimericoxidation during productsthe products oxidative of of quinonedimerization some methideof these of 2,7 ′ coupleddimers dimer [80].employ products quinone (2,30 and methide 3,30) have Interestingly, the oxidation products of some of these dimers employ quinone methide beenreactivity characterizedTwo-electron [79–81]. during Aoxidation 2,3 the′ coupled oxidative of DHICA tetramer dimerization can was produce identified ofthree 2,7 during 0differentdimer the [ 80 isomericoxidative]. quinonoid dimerization products. of the reactivityThese are quinone[79–81]. Aimine, 2,3′ coupledquinone tetramermethide andwas iminochromeidentified during forms the which oxidative are depicted dimerization in Figure of the23. Two-electron2,4′ dimer [80]. oxidationTwo different of dimeric DHICA products can produce of quinone three methide different coupled isomeric products quinonoid (2,3′ and products.3,3′) 2,4haveAll′ ofdimer been which [80].characterized are Two capable different duringof having dimeric the hydrogen-bondingoxidative products dimerization of quinone stabilization ofmethide 2,7′ dimer ascoupled shown [80]. products in the same (2,3 figure.′ and 3,3 Of′) These are quinone imine, quinone methide and iminochrome forms which are depicted in Figure 23. havewhich Two-electronbeen iminochrome characterized oxidation form during ofshould theDHICA oxidative be cantheoretica produce dimerizationlly three more of different 2,7stable′ dimer isomericthan [80]. the quinonoid other two products. forms. All ofIminochromeThese whichTwo-electron are arequinone capablegets imine,oxidationits stabilization of quinone having of DHICA methide hydrogen-bondingby thecan and presproduce iminochromeence threeof opposing stabilization different forms dipolewhich isomeric as aremovements shown depictedquinonoid in theinby Figureproducts. the same para 23. figure. Of whichTheseAllpositioned of arewhich iminochrome quinone C=O are andcapable imine, C=N form ofquinone bonds. having should On methide hydrogen-bonding the be other and theoretically hand,iminochrome both stabilization quinone more forms stable andaswhich shown quinone thanare indepicted themethide same other in Figureshouldfigure. two 23.Ofbe forms. IminochromeAllwhichmore of reactive whichiminochrome gets are as its capabletheir stabilization quinonoidform of havingshould by group the hydrogen-bonding be presence is theoreticapoised of re opposingadilylly stabilizationmore for addition dipolestable as movementsreactions. thanshown the in There theother by same the are twopara onlyfigure. forms.positioned three Of C=OwhichIminochromepossible and C=N iminochrome positions bonds. gets Ontoits exhibitform thestabilization other should reactivity hand, bybe thebothintheoretica thespres quinoneeence moleculeslly of andmore opposing quinone thatstable can dipole methide thancause movementsthe re-aromatization. shouldother betwoby morethe forms. paraThe reactive as theirIminochromefavorablepositioned quinonoid 2-postion C=O gets group and itsthat C=N is stabilizationpoised is bonds. free in readily OnDHI by the is forthe otheroccupi additionpres hand,edence in reactions. bothofDHICA opposing quinone by There a andcarboxyldipole are quinone onlymovements group three methide thus possible by preventing should the positionspara be to exhibitpositionedanymore coupling reactivityreactive C=O reactionas and intheir these C=N quinonoidat this moleculesbonds. position. groupOn the thatThus, is other poised can only hand, cause re3-,adily 4-, both re-aromatization. and for quinone 7-positionsaddition and reactions. arequinone The available favorable There methide for are coupling only 2-postionshould three bein that more reactive as their quinonoid group is poised readily for addition reactions. There are only three is freethispossible in molecule. DHI positions is occupied Again, to exhibitthe in DHICAreactive reactivity position by a in carboxyl thesshoulde molecules groupbe theoretically thus that preventing can the cause 3-position, re-aromatization. any coupling but so far reaction onlyThe at possibleproductsfavorable positions coupled2-postion atto that 4-exhibit isand free 7-positionreactivity in DHI is in haveoccupi thes beeed enmolecules in identified DHICA that by as a can majorcarboxyl cause products groupre-aromatization. thusand preventing3 coupled The this position. Thus, only 3-, 4-, and 7-positions are available for coupling in this molecule. Again, favorableproductsany coupling have2-postion reaction been identifiedthat at isthis free position. as in minor DHI Thus, isproducts occupi onlyed (Figure3-, in 4-, DHICA and 24) 7-positions[82–84]. by a carboxyl are available group thus for couplingpreventing in the reactive position should be theoretically the 3-position, but so far only products coupled at 4- and anythis couplingmolecule. reaction Again, atthe this reactive position. position Thus, shouldonly 3-, be4-, theoreticallyand 7-positions the are 3-position, available butfor couplingso far only in 7-positionthisproducts molecule. have coupled been Again, identifiedat 4-the and reactive as7-position major position productshave should been and beidentified theoretically 3 coupled as major productsthe 3-position, products have butand been so3 identifiedfarcoupled only as minorproducts products havecoupled (Figure been at identified 24 4-)[ and82– 84 7-positionas]. minor products have be (Figureen identified 24) [82–84]. as major products and 3 coupled products have been identified as minor products (Figure 24) [82–84].

Figure 23. Two-electron oxidation products of DHICA. Two electron oxidation of DHICA will produce imino quinone, quinone methide and iminochrome products.

FigureFigure 23. Two-electron 23. Two-electron oxidation oxidation products products of DHICA. of DHIC TwoA. Two electron electron oxidation oxidation of DHICA of DHICA will will produce Figureproduce 23. imino Two-electron quinone, quinone oxidation me thideproducts and iminochromeof DHICA. Two products. electron oxidation of DHICA will iminoproduce quinone, imino quinone quinone, methide quinone and me iminochromethide and iminochrome products. products.

Figure 24. Structure of DHICA dimers. Oxidative polymerization of DHIDA yields 4,4′, 4,7′ and 7,7′ coupled DHICA dimers as the major products and 3,4′ and 3,7′ coupled dimers as the minor products.

Figure 24. Structure of DHICA dimers. Oxidative polymerization of DHIDA yields 4,4′, 4,7′ and FigureFigure7,7 24.′ coupledStructure 24. Structure DHICA of DHICA of dimers DHICA dimers. as dimers. the Oxidativemajor Oxidative products polymerization polymerization and 3,4′ and ofof DHIDA3,7DHIDA′ coupled yieldsyields dimers 4,4 ′0, ,4,7 4,7as′ 0 andandthe 7,70 7,7′ coupled DHICA dimers as the major products and 3,4′ and 3,7′ coupled dimers as the coupledminor DHICA products. dimers as the major products and 3,40 and 3,70 coupled dimers as the minor products. minor products. Int. J. Mol. Sci. 2016, 17, 1576 16 of 23

8. Comparative Oxidation Chemistry of 1,2-Dehydro-N-acyldopamines and Dihydroxyindoles Int. J. Mol. Sci. 2016, 17, 1576 16 of 23 In order to understand the significance of quinone methide reactivity for eumelanogenesis, it is important8. Comparative to consider Oxidation the reactivities Chemistry ofof 1,2-Dehydro- a sister groupN-acyldopamines of compounds and called Dihydroxyindoles 1,2-dehydro- N-acyl dopamineIn derivatives.order to understand The simplest the significance compound of quinone in this methide group reactivity is 1,2-dehydro- for eumelanogenesis,N-acetyldopamine it is (dehydroimportant NADA). to consider It is biosynthesizedthe reactivities of ina sister a vast group majority of compounds of insects called for 1,2-dehydro- cuticularN hardening-acyl processdopamine also known derivatives. as sclerotizationThe simplest compound [3,30–32]. in Thethis reactivegroup is intermediates1,2-dehydro-N-acetyldopamine generated during sclerotization(dehydro NADA). form adducts It is biosynthesized with cuticular in a proteins vast majority and chitinof insects backbone for cuticular that hardening results in process a hardened cuticle.also Thisknown process as sclerotization is very similar [3,30–32]. to melaninThe reactive biosynthesis intermediates with generated the exception during sclerotization that the reactive intermediatesform adducts generated with cuticular in cuticle proteins are betterand chitin suited backbone to exhibit that externalresults in reactionsa hardened while cuticle. the This reactive intermediatesprocess is ofvery melanogenic similar to melanin pathway biosynthesis are poised wi forth internalthe exception polymerization that the reactive reaction. intermediates The reactions generated in cuticle are better suited to exhibit external reactions while the reactive intermediates of associated with both sclerotization and melanization exhibit remarkable similarity between each melanogenic pathway are poised for internal polymerization reaction. The reactions associated with other [32]. The catecholamine derivatives N-acetyldopamine and N-β-alanyldopamine are precursor both sclerotization and melanization exhibit remarkable similarity between each other [32]. The for sclerotizationcatecholamine whilederivatives dopa andN-acetyldopamine dopamine are precursorand N-β-alanyldopamine for melanization, are the precursor major difference for betweensclerotization them being while the protectiondopa and dopamine of amino groupare precursor by acylation, for melanization, which prevents the major internal difference reactivity in the casebetween of sclerotizing them being precursors.the protection Tyrosinase of amino group initiates by acylation, melanogenesis which prevents by oxidizing internal the reactivity catechols to quinones,in the whilecase of the sclerotizing related enzyme, precursors. phenoloxidase Tyrosinase initiates initiates melanogenesis sclerotization by oxidizing doing the the same catechols oxidation. N-acyldopamineto quinones, quinoneswhile the exhibit related external enzyme, reactivity, phenoloxidase while initiates dopa/dopamine sclerotization quinones doing exhibit the same internal reactivity.oxidation.N-acyldopamine N-acyldopamine quinones quinones areexhibit converted external toreactivity, quinone while methide dopa/dopamine derivatives quinones by quinone isomeraseexhibit [ 46internal,47]. Dopachromereactivity. N-acyldopamine tautomerases—DCT quinones are and converted DCDT—convert to quinone dopachrome methide derivatives to DHICA by quinone isomerase [46,47]. Dopachrome tautomerases—DCT and DCDT—convert dopachrome and DHI respectively in melanogenesis [12–19]. A separate quinone methide isomerase causes to DHICA and DHI respectively in melanogenesis [12–19]. A separate quinone methide isomerase the isomerization of N-acyldopamine quinone methides to 1,2-dehydro-N-acyldopamines [85–87]. causes the isomerization of N-acyldopamine quinone methides to 1,2-dehydro-N-acyldopamines Thus,[85–87]. the double Thus, bondthe double is introduced bond is introduced in the side in the chain sideof chain dopa of derivativesdopa derivatives byextremely by extremely similar enzymaticsimilar reactions enzymatic in reactions both the in pathways. both the pathways. ExtensiveExtensive studies studies carried carried out out on on the the oxidationoxidation chemistry of of 1,2-dehydro- 1,2-dehydro-N-acetyldopamineN-acetyldopamine (dehydro(dehydro NADA) NADA) paved paved way way to to understand understand thethe ke keyy role role played played by by quinone quinone methides methides in the in the sclerotizationsclerotization process process [88 [88–91].–91]. Dehydro Dehydro NADANADA upon two two electron electron oxidation oxidation produces produces a transient a transient quinonequinone methide methide and and not not the the conventional conventional quinonequinone product. This This quinone quinone methide methide forms forms adduct adduct with proteinswith proteins and chitinand chitin generating generating adducts adducts and crosslinks and crosslinks necessary necessary for strengthening for strengthening and hardening and hardening the cuticle. In addition, it also forms adduct with the parent catechols generating the cuticle. In addition, it also forms adduct with the parent catechols generating benzodioxan type benzodioxan type compounds as shown in Figure 25. Moreover, the quinone methide also reacts compoundswith the ascatecholic shown group in Figure of dimeric 25. Moreover, products gene the quinonerating trimers, methide then alsotetramers, reacts and with a number the catecholic of groupoligomers of dimeric [91]. products generating trimers, then tetramers, and a number of oligomers [91].

Figure 25. Oxidative transformations of dehydro NADA. Dehydro NADA upon oxidation produces Figure 25. Oxidative transformations of dehydro NADA. Dehydro NADA upon oxidation produces directly a highly reactive quinone methide, which crosslinks proteins and chitin (not shown in directly a highly reactive quinone methide, which crosslinks proteins and chitin (not shown in figure). figure). It also reacts with the starting compound forming a benzodioxan dimer. The accumulated It also reacts with the starting compound forming a benzodioxan dimer. The accumulated dimer dimer reacts with another molecule of quinone methide forming trimer. Oligomers up to hexamer reactshave with been another characterized. molecule One-electron of quinone oxidatio methiden of forming dehydro trimer. NADA Oligomers generates the up tosemiquinone, hexamer have beenwhich characterized. is in equilibrium One-electron with the oxidation quinone methide of dehydro radical. NADA Radical generates coupling the in this semiquinone, case produces which the is in equilibriumsame dimer with but the not quinone the oligomers. methide radical. Radical coupling in this case produces the same dimer but not the oligomers. Int. J. Mol. Sci. 2016, 17, 1576 17 of 23 Int. J. Mol. Sci. 2016, 17, 1576 17 of 23

Interestingly even the the one-electron one-electron oxidation oxidation product product forms forms the the same same dimeric dimeric adduct adduct through through a aradical radical coupling coupling reaction. reaction. Since Since radical radical coupling coupling results results in in the the precipitous precipitous loss of semiquinonesemiquinone radicals, in this case, only dimers are formedformed asas shownshown inin FigureFigure 2525[ [91].91]. ThisThis kindkind ofof dimerizationdimerization reaction isis manifested byby a numbernumber of dehydro dopyl derivatives [[2266,88–94].,88–94]. The quinone to quinone methide tautomerism is very much in favor of quinone methides in the case of dehydro NADA [[90]90] as wellwell asas 1,2-dehydro-1,2-dehydro-NN-acetyldopa-acetyldopa [[26].26]. However, with 1,2-dehydro-N-acetyldopa-acetyldopa methyl methyl ester, ester, quinone product is slightly more stable than the quinone methide [94]. [94]. However, even in this case, the quinonequinone exhibitsexhibits anan ionicionic Diels–AlderDiels–Alder typetype additionaddition formingforming thethe samesame benzodioxanbenzodioxan dimericdimeric products (Figure(Figure 2626))[ [94].94]. Thus, in dehydro dopa derivativesderivatives both the side chain positions seem to be extremely reactive.reactive. Oxidized DHI andand DHICADHICA shouldshould alsoalso exhibitexhibit thethe samesame kindkind ofof additions.additions.

Figure 26.26. OxidativeOxidative dimerization of 1,2-dehydro-1,2-dehydro-NN-acetyldopa-acetyldopa methylmethyl ester.ester. Oxidation ofof 1,2-Dehydro-1,2-Dehydro-N-acetyldopa methyl ester by tyrosinase resultsresults in thethe productionproduction ofof conventionalconventional quinone product and and not not the the quinon quinonee methide methide analog. analog. This This quinone quinone also also dimerizes dimerizes and and produces produces the thesame same benzodioxan benzodioxan type type adduct adduct as asthat that produced produced by by dehydro dehydro NADA NADA but but by by an an ionic Diels–Alder type reaction.reaction.

9. Oligomerization Oligomerization of of DHI DHI and and DHICA DHICA and and the the Final Final Eumelanogenic Eumelanogenic Pathway Pathway Several authors have demonstrated the facile oligomerization of both DHI and DHICA duringduring eumelanogenesis. As As stated stated earlier, earlier, a number of dimeric structures of DHI and DHICA have beenbeen characterized [73[73–78].–78]. A A mixed mixed dimer dimer of DHIof DHI and and DHICA DHICA has alsohas beenalso identifiedbeen identified [95]. Trimeric [95]. Trimeric [77,83] as[77,83] well as tetramericwell as tetrameric products products [79–81] of [79–81] DHI and of DHICADHI and have DHICA also been have characterized. also been characterized. Using mass spectralUsing mass analysis, spectral Kroesche analysis, and Kroesche Peter [73 and] identified Peter [73] oligomers identified of oligomers up to eleven of unitsup to ofeleven DHI. units Studies of withDHI. matrix-assistedStudies with matrix-assisted laser desorption/ionization laser desorption mass/ionization spectrometry mass havespectrometry revealed have the production revealed the of production of dimeric to heptomeric linear oligomers of DHICA coupled at 4,7-positions [96]. dimeric to heptomeric linear oligomers of DHICA coupled at 4,7-positions [96]. During a study of During a study of tyrosinase catalyzed oxidation of tyrosine, Bertazzo et al. [97] witnessed the tyrosinase catalyzed oxidation of tyrosine, Bertazzo et al. [97] witnessed the production of not only production of not only DHI oligomers as well as DHICA oligomers, but also mixed DHI-DHICA DHI oligomers as well as DHICA oligomers, but also mixed DHI-DHICA oligomers. Furthermore, they oligomers. Furthermore, they were able to observe the production of DHI oligomers up to nanomer. were able to observe the production of DHI oligomers up to nanomer. Structural analyses of several of Structural analyses of several of these compounds are extremely difficult due to nonavailability of these compounds are extremely difficult due to nonavailability of sufficient materials and the extreme sufficient materials and the extreme instability of oligomeric products. As pointed out in Section 7 on instability of oligomeric products. As pointed out in Section7 on the reactivities of dihydroxyindoles, the reactivities of dihydroxyindoles, a number of these adduct formation involves quinone methide a number of these adduct formation involves quinone methide participation. Thus, eumelanogenic participation. Thus, eumelanogenic pathway has emerged to be a more complex biochemical pathway has emerged to be a more complex biochemical pathway than what is described in Figure1. pathway than what is described in Figure 1. Based on the foregoing discussion and taking into consideration of the key roles played by Based on the foregoing discussion and taking into consideration of the key roles played by quinone methide intermediates during oxidative transformation of many catecholamine derivatives, quinone methide intermediates during oxidative transformation of many catecholamine derivatives, one can formulate a modified Raper–Mason pathway for eumelanin biosynthesis as shown in one can formulate a modified Raper–Mason pathway for eumelanin biosynthesis as shown in Figure 27. Tyrosinase has been shown to convert tyrosine to dopaquinone directly and not through Figure 27. Tyrosinase has been shown to convert tyrosine to dopaquinone directly and not through the intermediacy of dopa [98]. Tyrosinase can also oxidize dopa to dopaquinone. The intramolecular the intermediacy of dopa [98]. Tyrosinase can also oxidize dopa to dopaquinone. The intramolecular cyclization of dopaquinone to leucochrome and its further oxidation by dopaquinone produces cyclization of dopaquinone to leucochrome and its further oxidation by dopaquinone produces dopachrome and dopa. Thus, dopa is generated only nonenzymatically by an indirect route. While the dopachrome and dopa. Thus, dopa is generated only nonenzymatically by an indirect route. While above reactions corroborate the importance of quinones, the subsequent reactions attest the key role the above reactions corroborate the importance of quinones, the subsequent reactions attest the key role of quinone methides in eumelanogenic pathway. Two quinone methide intermediates—one at Int. J. Mol. Sci. 2016, 17, 1576 18 of 23 Int. J. Mol. Sci. 2016, 17, 1576 18 of 23 ofthe quinone stage of methides dopachrome in eumelanogenic conversion and pathway. the othe Twor at the quinone stage methideof dihydroxyindole intermediates—one oxidation—are at the stageshown. of dopachromeThe monomeric conversion units of and indole the otherquinone, at the iminochrome stage of dihydroxyindole and quinone oxidation—aremethide thus formed shown. Theundergo monomeric oligomerization units of indole and eventual quinone, polymeriza iminochrometion. and The quinone dimers, methide trimers, thus tetramers formed and undergo other oligomerizationoligomers upon andoxidation eventual will polymerization. form reactive Thequinone dimers, methide trimers, derivatives tetramers andand otherexhibit oligomers further uponpolymerization oxidation willas demonstrated form reactive quinoneby the pioneering methide derivatives work of the and Italian exhibit group further [76–80,95,96]. polymerization Thus, as demonstratedquinone methides by the seem pioneering to play worka dominant of the Italianrole not group only [in76 –the80 ,middle95,96]. Thus,part but quinone also in methides the later seem part toof playeumelanin a dominant biosynthesis. role not only As inindicated the middle in partSection but also 2, inthe the reactions later part of of quinones eumelanin are biosynthesis. typically Asmanifested indicated through in Section the2, ring the reactions additions, of quinoneswhere as arethe typicallyreactions manifested of quinone through methides the are ring centered additions, on wherethe side as chain the reactions additions. of quinone A combination methides of are all centered these reactivities on the side makes chain the additions. eumelanin A combination biosynthesis of allvery these intriguing reactivities and makescomplex. the eumelanin biosynthesis very intriguing and complex.

Figure 27.27. ModifiedModified Raper–Mason Pathway for eumelanineumelanin biosynthesis.biosynthesis. Tyrosinase (A) convertsconverts tyrosine to dopaquinone and and oxidizes oxidizes dopa dopa to to dopaquinone. dopaquinone. External External addition addition of ofcysteine cysteine to todopaquinone dopaquinone and and the the oxidative oxidative polymerization polymerization of of cysteinyldopa cysteinyldopa result result in in the the production of pheomelanin pigments (not shown in Figure).Figure). IntramolecularIntramolecular cyclization of dopaquinonedopaquinone produces leucochrome, whichwhich isis furtherfurther oxidized oxidized to to dopachrome dopachrome by by nonenzymatic nonenzymatic reactions reactions (B ().B Dopachrome). Dopachrome is isomerizedis isomerized to transientto transient quinone quin methideone methide (1) that (1) isthat converted is converted to either to DHICAeither DHICA by mammalian by mammalian DCT (C) orDCT DHI (C by) or insect DHI by DCDT insect (C DCDT0). Oxidation (C′). Oxidation of these two of these indoles two by indoles tyrosinase by tyrosinase or nonenzymatic or nonenzymatic reactions orreactions by DHICA or by oxidase DHICA (D oxidase) produces (D) produces among other among reactive other species, reactive quinone species, methides, quinone methides, which will which have awill major have role a major in eventual role in polymerization eventual polymerization of these monomeric of these monomeric compounds compounds (R = H for DHI(R = derivative;H for DHI andderivative; COOH and for DHICACOOH for derivative). DHICA derivative).

10. Conclusions Conclusions In spite spite of of many many decades decades of of intensive intensive research, research, several several aspects aspects of structure, of structure, reaction reaction course course and andbiochemical biochemical mechanism mechanism of eumelanin of eumelanin pigment pigment still still remains remains un unresolved.resolved. One One such such aspect aspect is is the production andand use use of reactiveof reactive quinone quinone methide methid intermediates.e intermediates. Ever since Ever our groupsince firstour demonstrated group first thedemonstrated involvement the of involvement quinone methide of quinone in the methide oxidation in the chemistry oxidation of αchemistry-methyl dopaof α-methyl methyl dopa ester, amethyl number ester, of quinone a number methide of derivativesquinone methide have been derivatives shown to behave reactive been intermediatesshown to be during reactive the metabolicintermediates transformation during the ofmetabolic a plethora transformation of catecholamine of a compoundsplethora of catecholamine as summarized compounds in this review. as Quinonesummarized methides in this playreview. crucial Quinone role methides not only play in melanin crucial biosynthesisrole not only butin melanin also in biosynthesis insect cuticular but sclerotizationalso in insect processcuticular that sclerotization is very much process related that to melanin is very biosynthesis.much related To to date, melanin four enzymesbiosynthesis. that areTo solelydate, four involved enzymes in quinone that are methide solely biochemistryinvolved in quinone have been methide identified: biochemistry mammalian have DCT been that identified: produces DHICAmammalian from DCT dopachrome; that produces insect DCDTDHICA that from produces dopach DHIrome; from insect dopachrome; DCDT that quinone produces isomerase DHI from that convertsdopachrome;N-acyldopamine quinone isomerase to their correspondingthat converts quinoneN-acyldopamine methides; to and their quinone corresponding methide isomerase quinone thatmethides; isomerizes and Nquinone-acyldopamine methide quinone isomerase methide that toisomerizes dehydro- NN-acyldopamine.-acyldopamine Duequinone to high methide reactivity to dehydro-N-acyldopamine. Due to high reactivity and extreme instability quinone methide formation and utilization in several biological processes including melanogenesis remains still to be Int. J. Mol. Sci. 2016, 17, 1576 19 of 23 and extreme instability quinone methide formation and utilization in several biological processes including melanogenesis remains still to be under appreciated. Only future studies can shed more light on the many aspects of quinone methide reactivity in melanogenesis process.

Acknowledgments: I thank Hanine Barek for critically examining this manuscript and offering many suggestions to improve it. Conflicts of Interest: The author declares no conflict of interest.

Abbreviations

DHI 5,6-dihydroxyindole DHICA 5,6-dihydroxyindole-2-carboxylic acid Dehydro NADA 1,2-dehydro-N-acetyldopamine DCT Dopachrome tautomerase DCDT Dopachrome decarboxylase/tautomerase

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