International Journal of Molecular Sciences

Review Chemical Reactivities of ortho- Produced in Living Organisms: Fate of Quinonoid Products Formed by and Phenoloxidase Action on and

Shosuke Ito 1,* , Manickam Sugumaran 2 and Kazumasa Wakamatsu 1,*

1 Department of Chemistry, Fujita Health University School of Medical Sciences, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan 2 Department of , University of Massachusetts, Boston, MA 02125, USA; [email protected] * Correspondence: [email protected] (S.I.); [email protected] (K.W.); Tel.: +81-562-93-9849 (S.I. & K.W.); Fax: +81-562-93-4595 (S.I. & K.W.)  Received: 30 July 2020; Accepted: 20 August 2020; Published: 24 August 2020 

Abstract: Tyrosinase catalyzes the oxidation of phenols and catechols (o-diphenols) to o-quinones. The reactivities of o-quinones thus generated are responsible for oxidative browning of products, sclerotization of insect cuticle, defense reaction in arthropods, tunichrome in tunicates, production of mussel glue, and most importantly biosynthesis in all organisms. These reactions also form a set of major reactions that are of nonenzymatic origin in nature. In this review, we summarized the chemical fates of o-quinones. Many of the reactions of o-quinones proceed extremely fast with a half-life of less than a second. As a result, the corresponding production can only be detected through rapid scanning spectrophotometry. Michael-1,6-addition with thiols, intramolecular cyclization reaction with side chain amino groups, and the redox regeneration to original represent some of the fast reactions exhibited by o-quinones, while, nucleophilic addition of carboxyl group, alcoholic group, and water are mostly slow reactions. A variety of catecholamines also exhibit side chain desaturation through tautomeric formation. Therefore, quinone methide tautomers also play a pivotal role in the fate of numerous o-quinones. Armed with such wide and dangerous reactivity, o-quinones are capable of modifying the structure of important cellular components especially proteins and DNA and causing severe cytotoxicity and carcinogenic effects. The reactivities of different o-quinones involved in these processes along with special emphasis on mechanism of melanogenesis are discussed.

Keywords: o-quinones; quinone methides; phenols; catechols; tyrosinase; thiols; amines; dopaquinone; sclerotization; melanization

1. Introduction ortho-Quinones are ubiquitous in nature. especially produce a wide variety of o-quinones as secondary metabolites. It is not possible to cover all naturally occurring quinones and their reactions in a short review. Therefore, only quinones arising from catecholamine derivatives and few other related molecules will be considered in this review. Unlike the related p-quinones that are reasonably stable towards isolation and characterization, several of the o-quinones are extremely reactive and difficult to isolate. Often, they are present in the transient form in a reaction thus even avoiding detection. A most important and prominent example of the o-quinones is dopaquinone that plays pivotal roles in melanogenesis, the process of production of eumelanin and pheomelanin [1]. Due to its

Int. J. Mol. Sci. 2020, 21, 6080; doi:10.3390/ijms21176080 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 35

Int. J. Mol. Sci. 2020, 21, 6080 2 of 36 plays pivotal roles in melanogenesis, the process of production of eumelanin and pheomelanin [1]. Due to its inherent high reactivity dopaquinone can easily undergo both Michael 1,6- and 1,4- additionsinherent highwith reactivity nucleophiles dopaquinone such as thiols can easily and amines. undergo These both Michaelreactions 1,6- lead and to 1,4-additionsthe production with of melaninnucleophiles such found as thiols in andthe amines.skin, hair, These and reactions eyes of leadall animals. to the production The reactivity of melanin of the pigment related dopaminequinonefound in the skin, hair, produces and eyes neuromelanin of all animals. in the The brain reactivity [2,3]. of Protein the related dopaquinones dopaminequinone formed in produces mussel proteinsneuromelanin are responsible in the brain for [2 ,3gluing]. Protein of mussels dopaquinones to rocks formed and other in mussel hard proteins surfaces are [4]. responsible The defense for reactiongluing of exhibited mussels toby rocks tunichromes and other found hard surfacesin the blood [4]. The of defensetunicates reaction are due exhibited to the reactivities by tunichromes of o- quinonesfound in the[5]. bloodIn insects, of tunicates catecholamine are due derivates to the reactivities such as N of-acetyldopamineo-quinones [5]. and In insects, N-β-alanyldopamine catecholamine formderivates key components such as N-acetyldopamine of hardened exoskeleton and N-β -alanyldopaminethat protects the soft form bodied key componentsanimals [6]. In of plants hardened and fungi,exoskeleton oxidative that browning protects the reactions soft bodied that animalsare part [of6]. their In plants defense and mechanism fungi, oxidative against browning invading reactions insects, isthat caused are part by the of theirreactivities defense of mechanismenzymatically against generated invading o-quinones. insects, While is caused some by of the the reactivities reactions are of beneficialenzymatically to the generated organisms,o-quinones. several While of the some reactions of the reactions of o-quinones are beneficial especially to the binding organisms, to macromoleculesseveral of the reactions such as of proteinso-quinones and especially DNA through binding the to sulfhydryl macromolecules and/or such amino as proteinsgroups, andresults DNA in deterioratingthrough the sulfhydryl biological and consequences/or amino groups, such resultsas cytotoxicity in deteriorating and carcinogenesis. biological consequences Therefore, such it asis importantcytotoxicity to and understand carcinogenesis. the biological Therefore, activities it is important of these to transient understand compounds. the biological o-Quinones activities are of mostlythese transient produced compounds. in biologicalo-Quinones systems by are th mostlye oxidation produced of the in corresponding biological systems catechols by the (o oxidation-diphenols) of bythe two corresponding electron removal. catechols However, (o-diphenols) tyrosinase by two and electron related removal. phenoloxidases However, tyrosinasepresent in andmammals, related plants,phenoloxidases fungi, and present insects in are mammals, capable plants,of oxidizing fungi, andsimple insects phenols are capable to the corresponding of oxidizing simple quinones phenols [7] (Figureto the corresponding 1). This review quinones summarizes [7] (Figure briefly1). the This function review summarizes of tyrosinase briefly and the the chemical function ofreactivity tyrosinase of oand-quinones the chemical with various reactivity nucleophiles of o-quinones. Special with emphasis various is nucleophiles. given to the mechanisms Special emphasis of melanogenesis. is given to Atthe this mechanisms point, we want of melanogenesis. to point out that At practically this point, weall biological want to point reactions out that are practicallyenzyme-catalyzed, all biological with thereactions exception are enzyme-catalyzed, of ribozymes, which with theare exceptionRNA catalysts. of ribozymes, Without which enzymatic are RNA intervention, catalysts. Without some reactionsenzymatic can intervention, proceed, but some for reactions them to canbe useful proceed, for butthe forbiological them to system, be useful enzymatic for the biological intervention system, is absolutelyenzymatic interventionnecessary. An is example absolutely is necessary.hydration Anof carbon example dioxide is hydration to carbonic of carbon acid which dioxide can to carbonicproceed quiteacid whichrapidly can without proceed the quiteneed rapidlyfor an enzyme, without but the in need biological for an systems, enzyme, ca butrbonic in biological anhydrase systems, makes thiscarbonic hydration anhydrase go faster. makes However, this hydration a very go small faster. set However, of reactions, a very form small a group set of of reactions, nonenzymatic form a reactionsgroup of that nonenzymatic often occur reactions in biological that systems. often occur Glycation in biological of hemoglobin systems. is Glycationone such example. of hemoglobin While itis is one a useful such example. index to Whiledetermine it is athe useful seriousness index to of determine diabetic conditions, the seriousness the glycation of diabetic is conditions,entirely of nonenzymaticthe glycation is origin. entirely In of nonenzymaticthe case of o-quinone origin. In chemistry, the case of oonce-quinone these chemistry, reactive onceintermediates these reactive are generatedintermediates by enzyme are generated assisted by reactions, enzyme assistedthe rest of reactions, the reactions the rest seem of theto proceed reactions nonenzymatically seem to proceed withoutnonenzymatically the need for without any enzymatic the need assistance. for any enzymatic These nonenzymatic assistance. These reactions nonenzymatic are absolutely reactions essential are forabsolutely parts of essential melanin for biosynthesis, parts of melanin insect biosynthesis, cuticular sclerotization, insect cuticular innate sclerotization, immunity innate in invertebrate immunity animalsin invertebrate where encapsulation animals where and encapsulation melanization and of melanizationforeign objects of occur, foreign mussel objects glue occur, formation mussel (1,4– glue 6).formation Therefore, (1,4–6). it is important Therefore, to it isinvestigate important the to investigatefate of o-quinones the fate in of biologicalo-quinones systems, in biological which systems, forms thewhich main forms goal the of this main review. goal of this review.

Figure 1. Tyrosinase-catalyzed oxidation of 4-substituted phenols and the corresponding catechols producing o-quinones. Note Note that that catechols catechols are not produced directly from phenols [[7].7].

2. oo-Quinone-Quinone Formation Formation from from Phenols Phenols and and Catechols Catechols by by Tyrosinase Tyrosinase Tyrosinase (EC 1.14.18.1) is a rate-limiting enzyme for the biosynthesis of melanin and isis present widespread in nature,nature, rangingranging fromfrom microorganisms,microorganisms, plants,plants, insectsinsects toto mammals.mammals. The primary function of tyrosinase is the production of o-quinones from both phenolsphenols and catechols. This

Int. J. Mol. Sci. 2020, 21, 6080 3 of 36 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 35 enzyme exhibitsexhibits two two diff erentdifferent activities. activities. Often describedOften described in literature in as monophenolliterature as monooxygenase monophenol (ormonooxygenase monophenolase) (or activitymonophenolase) is assumed activity to convert is assumed monophenols to convert to o-diphenols, monophenols although to o-diphenols, in reality thealthough diphenol in reality is produced the diphenol by the reductionis produced of o by-quinone the reduction primary of product. o-quinone Thus, primary this activity product. converts Thus, monophenolsthis activity converts to o-quinones monophenols first. The to second o-quinones activity first. is theThe oxidation second activity of o-diphenols is the oxidation to o-quinones of o- anddiphenols is referred to o-quinones to as catechol and oxidase is referred or diphenolase to as catechol activity oxidase (Figure or 1diphenolase). Oxidases suchactivity as peroxidase, (Figure 1). laccase,Oxidases and such polyphenol as peroxidase, oxidase laccase, are alsoand polyphenol able to catalyze oxidase the are oxidation also able of to catechols catalyze to theo-quinones. oxidation Typically,of catechols many to o-quinones. biological oxidantsTypically, produce many biologicalo-quinones oxidants from catechols produce but o-quinones not from phenols.from catechols Thus, onlybut not tyrosinase from phenols. is capable Thus, of exhibitingonly tyrosinase both phenolis capable monooxygenase of exhibiting activityboth and diphenoloxidasemonooxygenase activity.activity and Therefore, diphenoloxidase the function activity. of tyrosinase Therefore, is briefly the function summarized. of tyrosinase is briefly summarized. cancan be be classified classified into into type-3 type-3 copper copper proteins. proteins. This classThis of class functional of functional proteins includesproteins tyrosinases,includes tyrosinases, catechol oxidases, catechol hemocyanins oxidases, hemocyan (the oxygen-carryingins (the oxygen-carrying proteins of arthropods proteins andof arthropods mollusks), and arthropodmollusks), phenol and arthropod oxidases. phenol The active oxidases. site of The these acti proteinsve site of consists these proteins of two copper consists atoms of two (identified copper asatoms CuA (identified and CuB), as each CuA coordinated and CuB), each by three coordinated histidine by residues three histidine (Figure2 ).residues Several (Figure tyrosinases 2). Several have beentyrosinases isolated have and been their isolated crystalline and structurestheir crystalline elucidated. structures For elucid example,ated. the For tyrosinase example, fromthe tyrosinaseAgaricus bisporusfrom Agaricusis a heterotetramer bisporus is a heterotetramer composed of two composed heavy chains of two of heavy about chains 48 kDa of and about two 48 light kDa chains and two of 14light kDa chains [8]. The of 14 first kDa 3D structure[8]. The first study 3D of structure this protein study indicates of this that protein three indicates histidine that residues three each histidine in the heavyresidues chain each coordinate in the heavy totwo chain copper coordinate atoms to (CuA two andcopper CuB) atoms [8]. (CuA and CuB) [8].

Figure 2. Relationship among the four oxidativeoxidative states of tyrosinase [[7].7]. Tyrosinase possesses four distinctly different oxidation states (deoxy-, oxy-, met-, and deact-) and Tyrosinase possesses four distinctly different oxidation states (deoxy-, oxy-, met-, and deact-) and understanding their roles and inter-conversions is essential for understanding catalytic activities of understanding their roles and inter-conversions is essential for understanding catalytic activities of tyrosinase [7,9]. Tyrosinase is usually present mostly as a stable met-tyrosinase [Cu(II)2], in which a tyrosinase [7,9]. Tyrosinase is usually present mostly as a stable met-tyrosinase [Cu(II)2], in which a hydroxide ion is bound to two Cu(II) atoms. This form of the enzyme does not react with phenols but hydroxide ion is bound to two Cu(II) atoms. This form of the enzyme does not react with phenols but is able to oxidize catechols to o-quinones. During this oxidation process, met-tyrosinase [Cu(II)2] is is able to oxidize catechols to o-quinones. During this oxidation process, met-tyrosinase [Cu(II)2] is reduced to deoxy-tyrosinase [Cu(I)2]. The latter rapidly reacts with molecular oxygen to form the bound reduced to deoxy-tyrosinase [Cu(I)2]. The latter rapidly reacts with molecular oxygen to form the peroxide ion in oxy-tyrosinase [Cu(II)2-O2]. oxy-Tyrosinase is the most important oxidizing form of this bound peroxide ion in oxy-tyrosinase [Cu(II)2-O2]. oxy-Tyrosinase is the most important oxidizing enzyme and oxidizes phenols to o-quinones while it returns reversibly to deoxy-tyrosinase. The latter form of this enzyme and oxidizes phenols to o-quinones while it returns reversibly to deoxy- binds to molecular oxygen to regenerate oxy-tyrosinase and the oxidation of phenols continues until tyrosinase. The latter binds to molecular oxygen to regenerate oxy-tyrosinase and the oxidation of the substrates phenol and molecular oxygen are depleted. This is the phenol-oxidase cycle. phenols continues until the substrates phenol and molecular oxygen are depleted. This is the phenol- oxy-Tyrosinase is able to oxidize catechols to o-quinones and itself is reduced to met-tyrosinase. oxidase cycle. The latter oxidizes the second molecule of catechol to o-quinone through the catechol oxidase oxy-Tyrosinase is able to oxidize catechols to o-quinones and itself is reduced to met-tyrosinase. The latter oxidizes the second molecule of catechol to o-quinone through the catechol oxidase

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 35 Int. J. Mol. Sci. 2020, 21, 6080 4 of 36 mechanism and itself is reduced to deoxy-tyrosinase (mentioned above). This is the catechol-oxidase mechanismcycle. The hydroxyl and itself group is reduced of phenols to deoxy binds-tyrosinase to the (mentioned active site above).through This the isCuA, the catechol-oxidasewhile catechols initially bind to the CuB [10]. Therefore, phenols and catechols act competitively against oxy- cycle. The hydroxyl group of phenols binds to the active site through the CuA, while catechols tyrosinase. Usually oxy-tyrosinase exhibits a higher specificity for catechol oxidation than for the initially bind to the CuB [10]. Therefore, phenols and catechols act competitively against oxy-tyrosinase. corresponding phenol oxygenation reaction. Usually oxy-tyrosinase exhibits a higher specificity for catechol oxidation than for the corresponding As pointed out earlier, it is still generally claimed that tyrosinase first hydroxylates phenols to phenol oxygenation reaction. catechols that subsequently undergo oxidation to o-quinones. However, it should be emphasized that As pointed out earlier, it is still generally claimed that tyrosinase first hydroxylates phenols to in the monooxygenase mechanism (Figure 1), the o-quinone is formed directly from the phenol and catechols that subsequently undergo oxidation to o-quinones. However, it should be emphasized that catechol formation is not an intermediate step [7]. A possible reason for this misunderstanding is the in the monooxygenase mechanism (Figure1), the o-quinone is formed directly from the phenol and fact that the quinone is reduced by a nonenzymatic redox exchange mechanism thus producing the catechol formation is not an intermediate step [7]. A possible reason for this misunderstanding is the catechol [11] (see Section 6). fact that the quinone is reduced by a nonenzymatic redox exchange mechanism thus producing the It has been known for years that tyrosinase is slowly inactivated during the oxidation of catechol [11] (see Section6). catechols. The reason for such suicidal inactivation remained unidentified till Land et al. [12,13] It has been known for years that tyrosinase is slowly inactivated during the oxidation of catechols. pointed out that catechol substrates may sometimes (once in 2000 turnover) be oxidized by oxy- The reason for such suicidal inactivation remained unidentified till Land et al. [12,13] pointed out that tyrosinase through the monooxygenase mechanism and oxy-tyrosinase is irreversibly reduced to catechol substrates may sometimes (once in 2000 turnover) be oxidized by oxy-tyrosinase through the deact-tyrosinase (Figure 2). During this process, one of the two copper atom is reduced to the Cu(0) monooxygenase mechanism and oxy-tyrosinase is irreversibly reduced to deact-tyrosinase (Figure2). state and is released from the active center. This proposal is consistent with the observation the During this process, one of the two copper atom is reduced to the Cu(0) state and is released from the inactivation of tyrosinase activity is accompanied by a loss of half the active-center copper atoms [7]. active center. This proposal is consistent with the observation the inactivation of tyrosinase activity is Recently, a similar mechanism was proposed for the inactivation of tyrosinase by resorcinol [14]. accompanied by a loss of half the active-center copper atoms [7]. Recently, a similar mechanism was The catecholic substrate that is formed through redox exchange is able to activate the native form proposed for the inactivation of tyrosinase by resorcinol [14]. of tyrosinase, met-tyrosinase, to the active form, oxy-tyrosinase. Evidence in support of this proposal The catecholic substrate that is formed through redox exchange is able to activate the native form came from the study by Cooksey et al. [11]. N,N-Dimethyltyramine is oxidized to the corresponding of tyrosinase, met-tyrosinase, to the active form, oxy-tyrosinase. Evidence in support of this proposal o-quinone and undergoes cyclization. The cyclized product, indolium salt, is unable to act as a came from the study by Cooksey et al. [11]. N,N-Dimethyltyramine is oxidized to the corresponding catecholic (diphenolic) substrate for met-tyrosinase due to the presence of the electron-withdrawing o-quinone and undergoes cyclization. The cyclized product, indolium salt, is unable to act as a Me2N+ group keeping one of the hydroxyl groups as oxide anion [9] (Figure 3). With this background catecholic (diphenolic) substrate for met-tyrosinase due to the presence of the electron-withdrawing in mind,+ let us examine the reactivities of quinonoid products that are formed in the biological Me2N group keeping one of the hydroxyl groups as oxide anion [9] (Figure3). With this background in processes. mind, let us examine the reactivities of quinonoid products that are formed in the biological processes.

Figure 3. Tyrosinase-catalyzed oxidation of N,N-dimethyltyramine [11]. This reaction requires a catalyticFigure 3. amount Tyrosinase-catalyzed of L-dopa. The oxidationo-quinone of rapidly N,N-dimethyltyramine gives the indolium [11]. salt inThis a spontaneous reaction requires reaction. a Thiscatalytic salt doesamount not of have L-dopa. an activity The o to-quinone activate rapidlymet-tyrosinase gives the and, indolium thus, the salt oxidation in a spontaneous stops at this reaction. stage. This salt does not have an activity to activate met-tyrosinase and, thus, the oxidation stops at this 3. Reactivitystage. of o-Quinones with Small Molecules Leading to Adducts Formation

3.1.3. Reactivity Chemical Fateof o-Quinones of o-Quinones with Small Molecules Leading to Adducts Formation In this section, chemical fates of o-quinones are summarized. Readers are advised to refer also to excellent3.1. Chemical reviews Fate of on o-Quinones related topics with different points of view [6,9,15–22]. oIn-Quinones this section, are chemical extremely fates reactive of o-quinones electrophiles are summarized. that undergo Readers Michael-1,4-addition are advised to refer reactions also withto excellent available reviews nucleophilic on related molecules. topics wi However,th different thiols points exhibit of view a di[6,9,15–22].fferent behavior. The reactions of thiolso-Quinones with quinones are extremely are extremely reactive fast–faster electrophile thans anythat otherundergo nucleophiles. Michael-1,4-addition But they also reactions deviate fromwith available other nucleophiles nucleophilic in theirmolecules. regiospecificity However, to th theiols addition exhibit a site. different They addbehavior. on to theThe carbon reactions atom of nextthiols to with the quinoneso-quinone are moiety extremely (O3 andfast–faster O4) at than C2- any and other C5-positions nucleophiles. (1,6-Michael But they addition) also deviate and from not toother the nucleophiles remote C6-position in their (1,4-Michaelregiospecificity addition), to the addi thetion site preferredsite. They byadd the on most to the other carbon nucleophiles. atom next Thisto the regiospecificity o-quinone moiety of thiol (O3 additionand O4) remainsat C2- and enigma C5-positions in the chemistry (1,6-Michael of o addition)-quinones. and Kishida not to and the Kasairemote [23 C6-position] investigated (1,4-Michael this process addition), through density the site functional preferred theory-based by the most calculations, other nucleophiles. showing This that theregiospecificity binding of Cys–S of thiol- to dopaquinoneaddition remains proceeds enigma with in recruiting the chemistry of Cys–S of o--quinones.to C3–C4 bridge, Kishida migration and Kasai of - + Cys–S[23] investigatedto C5, proton this rearrangementprocess through from density cysteinyl-NH functional3 theory-basedto O4, and from calculations, C5 to O3. Butshowing more that studies the arebinding needed of Cys–S to get a- to clear dopaquinone picture. With proceeds this exception with recruiting in mind, of the Cys–S chemical- to C3–C4 reactivity bridge, of o-quinones migration can of Cys–S- to C5, proton rearrangement from cysteinyl-NH3+ to O4, and from C5 to O3. But more studies

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 35 Int. J. Mol. Sci. 2020, 21, 6080 5 of 36 are needed to get a clear picture. With this exception in mind, the chemical reactivity of o-quinones can be classified into four categories based on the appropriate speed of reactions (Figure 4): (1) be classified into four categories based on the appropriate speed of reactions (Figure4): (1) addition of addition of thiols (through sulfhydryl group) by 1,6-Michael reaction, (2) reduction to the parent thiols (through sulfhydryl group) by 1,6-Michael reaction, (2) reduction to the parent catechols through catechols through redox exchange, (3) addition of amines (through amino group) by 1,4-Michael redox exchange, (3) addition of amines (through amino group) by 1,4-Michael reaction or Schiff base reaction or Schiff base formation, and (4) addition of other nucleophiles such as carboxylic acids formation, and (4) addition of other nucleophiles such as carboxylic acids (through carboxylate anion), (through carboxylate anion), phenols, alcohols, and water (in this order of reactivity). The rate of phenols, alcohols, and water (in this order of reactivity). The rate of these four sets of the reactions are: these four sets of the reactions are: Reaction 1 (fast) > Reaction 2 (next fast) >> Reaction 3 (slow) > Reaction 1 (fast) > Reaction 2 (next fast) >> Reaction 3 (slow) > Reaction 4 (very slow). In Figure4, Reaction 4 (very slow). In Figure 4, the thickness of the arrows corresponds to the fastness of these the thickness of the arrows corresponds to the fastness of these reactions. In this review, the “fast” reactions. In this review, the “fast” reactions complete within about a second, while the “slow” reactions complete within about a second, while the “slow” reactions require minutes to hours to reactions require minutes to hours to complete. The type 4 reactions often do not proceed unless the complete. The type 4 reactions often do not proceed unless the functional group is present within the functional group is present within the same molecule of o-quinone. In this last category one can also same molecule of o-quinone. In this last category one can also include slow dimerization and other include slow dimerization and other oligomerization reactions. However, such reactions are so oligomerization reactions. However, such reactions are so complex that general conclusions cannot be complex that general conclusions cannot be drawn and thus are not discussed in this review. drawn and thus are not discussed in this review.

Figure 4. Summary of intrinsic chemical reactivity of o-quinones. The relative reactivity is shown in the Figure 4. Summary of intrinsic chemical reactivity of o-quinones. The relative reactivity is shown in thickness of the arrows. Note that the reactions with carboxylic acids and alcohol (water) are possible the thickness of the arrows. Note that the reactions with carboxylic acids and alcohol (water) are only when the functional groups are present in the side chain of o-quinones. For the sake of simplicity, possible only when the functional groups are present in the side chain of o-quinones. For the sake of the numbering on the o-quinone ring is made so that R- is attached to the C1 position. The addition simplicity, the numbering on the o-quinone ring is made so that R- is attached to the C1 position. The of thiols proceeds mostly at the C5 (major) and C2 (minor) positions (1,6-Michael addition) while the addition of thiols proceeds mostly at the C5 (major) and C2 (minor) positions (1,6-Michael addition) addition of other nucleophiles proceeds mostly at the C6 position (1,4-Michael addition). while the addition of other nucleophiles proceeds mostly at the C6 position (1,4-Michael addition). 3.2. Reaction of o-Quinones with Thiols 3.2. Reaction of o-Quinones with Thiols The reactivity of thiols with o-quinone is astonishingly very fast. For example, one would expect that aThe nucleophile reactivity whichof thiols is with present o-quinone internally is astonishingly on the quinone very at fa ast. suitable For example, position one would would have expect the thatgreatest a nucleophile advantage which of using is present proximity internally effect to on exhibit the quinone extremely at a fastsuitable intramolecular position would cyclization have the as greatestopposed advantage to a nucleophile of using presented proximity externally effect to toex thehibit quinone. extremely However, fast intram thiololecular addition cyclization defies such as opposedlogical explanation to a nucleophile and proceeds presented remarkably externally faster to the than quinone. even the However, intramolecular thiol addition cyclization defies reactions. such logicalAn early explanation study, by Tse and et al. proceeds [24], examined remarkably nucleophilic faster addition than even rate constantsthe intramolecular of nucleophiles cyclization against reactions.electrochemically An early generated study, dopaminequinone.by Tse et al. [24], At examined pH 7.4, cysteine nucleophilic and addition wererate foundconstants to react of nucleophilesfaster than the against intramolecular electrochemically cyclization bygenerated 2100 and 1400dopaminequinone. times, respectively. At LysinepH 7.4, was cysteine less reactive and glutathionewith a relative were rate found of 0.47 to compared react faster to thethan cyclization. the intramolecular Ascorbic acidcyclization reduced by dopaminequinone 2100 and 1400 times, back respectively.to Lysine through was redox less exchangereactive with much a relative faster than rate theof 0.47 cyclization, compared but to surprisingly, the cyclization. the Ascorbic addition acidof glutathione reduced dopaminequinone was found to proceed back a to little dopamine faster than through the reduction redox exchange reaction. much The detailedfaster than kinetic the cyclization,study conducted but surprisingly, by Jameson the et addition al. [25] also of glutathione pointed out was the found extremely to proceed fast addition a little faster of cysteine than the to reductiondopaminequinone. reaction. The At adetailed concentration kinetic ofstudy 50 µ conducM (physiologicalted by Jameson concentration et al. [25] in also the pointed brain) cysteine out the extremelyreacted 2000-times fast addition faster of than cysteine the intramolecular to dopaminequin cyclization.one. At a concentration of 50 µM (physiological concentrationThe high in reactivity the brain) of sulfhydrylcysteine reacted group 2000-ti in thiolsmes is illustratedfaster than bythe the intramolecular addition reaction cyclization. of cysteine to dopaquinoneThe high reactivity generated of sulfhydryl in situ by group tyrosinase-catalyzed in thiols is illustrated oxidation by the addition of dopa reaction [26]. The of cysteine reaction toaff ordeddopaquinone cysteine adductsgenerated in ain quantitative situ by tyrosinase-catalyzed yield: 5-S-cysteinyldopa oxidation (5SCD), of 2-dopaS-cysteinyldopa [26]. The reaction (2SCD), afforded6-S-cysteinyldopa cysteine (6SCD),adducts andin a 2,5- quantitativeS, S-dicysteinyldopa yield: 5-S-cysteinyldopa (2,5DiCD) in yields (5SCD), of 74%,2-S-cysteinyldopa 14%, 1%, and

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 35 Int. J. Mol. Sci. 2020, 21, 6080 6 of 36 (2SCD), 6-S-cysteinyldopa (6SCD), and 2,5-S, S-dicysteinyldopa (2,5DiCD) in yields of 74%, 14%, 1%, and 5%, respectively (Figure 5). As shown in Section 6.1, these cysteinyldopa isomers are further 5%, respectively (Figure5). As shown in Section 6.1, these cysteinyldopa isomers are further oxidized to give rise to pheomelanin. The facile production of 5-S-cysteinyldopa also makes it an oxidized to give rise to pheomelanin. The facile production of 5-S-cysteinyldopa also makes it an excellent biochemical marker for melanoma progression [27,28]. Interestingly, 2,5-S,S- excellent biochemical marker for melanoma progression [27,28]. Interestingly, 2,5-S,S-dicysteinyldopa dicysteinyldopa was found as a reflecting material in the tapetum lucidum (a reflecting later lying was found as a reflecting material in the tapetum lucidum (a reflecting later lying behind the behind the retina) of the eyes of the alligator gar Lepisosteus [29]. Dopaminequinone reacted similarly retina) of the eyes of the alligator gar Lepisosteus [29]. Dopaminequinone reacted similarly with with cysteine to afford cysteinyldopamine isomers in comparable yields [30] and dopaquinone cysteine to afford cysteinyldopamine isomers in comparable yields [30] and dopaquinone reacted reacted similarly with glutathione [31]. Cysteinyldopamine isomers are important precursors of the similarly with glutathione [31]. Cysteinyldopamine isomers are important precursors of the brain brain melanin, neuromelanin [32,33]. Regarding the reactivity of various other o-quinones with thiols, melanin, neuromelanin [32,33]. Regarding the reactivity of various other o-quinones with thiols, Cooksey et al. [34] obtained rate constants for reactions of cysteine and glutathione with 17 different Cooksey et al. [34] obtained rate constants for reactions of cysteine and glutathione with 17 different o-quinones and concluded that the rate constants increase with the electron withdrawing capacities o-quinones and concluded that the rate constants increase with the electron withdrawing capacities of of the substituent groups. This is principally due to the resonance effect, with a smaller but significant the substituent groups. This is principally due to the resonance effect, with a smaller but significant contribution attributable to the field effect. contribution attributable to the field effect.

Figure 5. Reaction of dopaquinone with cysteine. 5-S-Cysteinyldopa (5SCD) is the major isomer [26] Figure 5. Reaction of dopaquinone with cysteine. 5-S-Cysteinyldopa (5SCD) is the major isomer [26] and serves as a biochemical marker of malignant melanoma [28]. and serves as a biochemical marker of malignant melanoma [28]. As shown for the reaction of dopaquinone or dopaminequinone with thiols (Figure5), di-adduct As shown for the reaction of dopaquinone or dopaminequinone with thiols (Figure 5), di-adduct formation is usually a minor pathway. A unique exception is the reactivity of resveratrol quinone formation is usually a minor pathway. A unique exception is the reactivity of resveratrol quinone (Figure6). Tyrosinase-catalyzed oxidation of resveratrol, 3,5,4 -trihydroxy-trans-stilbene, in the (Figure 6). Tyrosinase-catalyzed oxidation of resveratrol, 3,5,40′-trihydroxy-trans-stilbene, in the presence of four equivalent N- (NAC) afforded triNAC-resveratrol-catechol and presence of four equivalent N-acetylcysteine (NAC) afforded triNAC-resveratrol-catechol and diNAC-resveratrol-catechol in 24% and 22% yields, respectively [35]. Surprisingly, no mono-adduct diNAC-resveratrol-catechol in 24% and 22% yields, respectively [35]. Surprisingly, no mono-adduct was isolated. The production of triGSH adduct of resveratrol was also reported by De Lucia et al. [36]. was isolated. The production of triGSH adduct of resveratrol was also reported by De Lucia et al. Similarly, chlorogenic acid, a catecholic compound with a conjugated double bond, produced the [36]. Similarly, chlorogenic acid, a catecholic compound with a conjugated double bond, produced triGSH adduct along with diGSH and monoGSH adducts showing that the C2 position is most the triGSH adduct along with diGSH and monoGSH adducts showing that the C2 position is most reactive [37] (Figure6). The reactions of N-acetylcysteine with the quinones produced from catechol, reactive [37] (Figure 6). The reactions of N-acetylcysteine with the quinones produced from catechol, 4-methylcatechol and N-acetyldopamine, have also been reported [38]. The quinone formed from 4-methylcatechol and N-acetyldopamine, have also been reported [38]. The quinone formed from 1,2- 1,2-dehydro-N-acetyldopamine also trapped N-acetylcysteine in a complex reaction yielding multiple dehydro-N-acetyldopamine also trapped N-acetylcysteine in a complex reaction yielding multiple productsInt. J. Mol. Sci. [5]. 2020, 21, x FOR PEER REVIEW 7 of 35 products [5]. A unique example of di-thiol adducts of dopa was found in adenochrome, an iron (III)-binding peptide from Octopus vulgaris [39]. Acid hydrolysis of desferri-adenochrome afforded a mixture of adenochromine isomers and glycine in a molar ratio of 1:2. Structure elucidation of adenochromines indicated that they consisted of the three possible isomers of di-adducts of 5-thiolhistidine to dopa (Figure 7). Biosynthetic pathway to adenochromines is not well clarified as tyrosinase-catalyzed oxidation of dopa in the presence of 5-thiolhistidine afforded 85% yield of the three possible mono- adducts with only 3% yield of the di-adducts [40].

Figure 6. Reaction of resveratrol quinone and chlorogenic acid quinone with thiols [35,37]. Note that Figure 6. Reaction of resveratrol quinone and chlorogenic acid quinone with thiols [35,37]. Note that the C2 position is most reactive. the C2 position is most reactive.

Figure 7. Three structural isomers of di-adducts of 5-thiohistidine to dopa isolated from Octopus vulgaris [39,40]. Note that adenochromine A, B, and C correspond to the 2,5-, 5,6-, and 2,6-adducts, respectively.

Methionine which possesses a thioether group also reacts with o-quinones but at much less rate compared to the free thiol group of cysteine and related compounds. Vithayathil and his group [41– 45] reported that reaction of simple o-benzoquinone with N-acetylmethionine in acetic acid resulted in the formation of N-acetylmethionylcatechol (Figure 8). Subsequently, they were able to show that this reaction is of general occurrence and a number of o-quinones could react with methionyl group. They showed that even methionine residues present in ribonuclease were able to react with o- benzoquinone through the thioether group [41]. The physiological relevance of this reaction was later demonstrated by Sugumaran and Nelson [46], who reported the production of N-acetylmethionine adducts during the tyrosinase-catalyzed oxidation of catechol, 4-methylcatechol, and N- acetyldopamine. These authors also proposed that such reactions could be of biological significance during sclerotization insect cuticle, although it was acknowledged by them that insect cuticular proteins have low concentration of sulfur containing amino acids. Moreover, these addition reactions also required large mole excess of N-acetylmethionine. Therefore, occurrence of such reactions although theoretically possible, nevertheless is unlikely to witness in insect systems that biological significance of this type of conjugation with proteins is questionable.

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Int. J. Mol. Sci. 2020, 21, 6080 7 of 36

A unique example of di-thiol adducts of dopa was found in adenochrome, an iron (III)-binding peptide from Octopus vulgaris [39]. Acid hydrolysis of desferri-adenochrome afforded a mixture of adenochromine isomers and glycine in a molar ratio of 1:2. Structure elucidation of adenochromines indicated that they consisted of the three possible isomers of di-adducts of 5-thiolhistidine to dopa (Figure7). Biosynthetic pathway to adenochromines is not well clarified as tyrosinase-catalyzed oxidationFigure of 6. dopaReaction in of the resveratrol presence quinone of 5-thiolhistidine and chlorogenic a acidfforded quinone 85% with yield thiols of [35,37]. the three Note possiblethat mono-adductsthe C2 position with is only most 3% reactive. yield of the di-adducts [40].

FigureFigure 7.7. ThreeThree structural structural isomers isomers of di-adducts of di-adducts of 5-thiohistidine of 5-thiohistidine to dopa to isolated dopa isolated from Octopus from Octopusvulgaris vulgaris[39,40]. Note[39,40 that]. Note adenochromine that adenochromine A, B, and A,C correspond B, and C correspond to the 2,5-, to5,6-, the and 2,5-, 2,6-adducts, 5,6-, and 2,6-adducts,respectively. respectively.

MethionineMethionine which possesses a a thio thioetherether group group also also reacts reacts with with o-quinoneso-quinones but but at much at much less lessrate ratecompared compared to the to free the thiol free group thiol groupof cysteine of cysteine and related and compounds. related compounds. Vithayathil Vithayathil and his group and [41– his group45] reported [41–45 ]that reported reaction that of reactionsimple o-benzoquinone of simple o-benzoquinone with N-acetylmethionine with N-acetylmethionine in acetic acid in resulted acetic acidin the resulted formation in theof N formation-acetylmethionylcatechol of N-acetylmethionylcatechol (Figure 8). Subsequently, (Figure8). they Subsequently, were able to they show were that ablethis toreaction show thatis of thisgeneral reaction occurrence is of general and a occurrencenumber of o and-quinones a number could of oreact-quinones with methionyl could react group. with methionylThey showed group. that They even showed methionine that evenresidues methionine present residuesin ribonuclease present were in ribonuclease able to react were with able o- tobenzoquinone react with o-benzoquinone through the thioether through group the thioether [41]. The groupphysiological [41]. The relevance physiological of this reaction relevance was of later this reactiondemonstrated was later by Sugumaran demonstrated and by Nelson Sugumaran [46], who and reported Nelson [the46], production who reported of N the-acetylmethionine production of Nadducts-acetylmethionine during the adducts tyrosinase-catalyzed during the tyrosinase-catalyzed oxidation of oxidationcatechol, of4-methylcatechol, catechol, 4-methylcatechol, and N- andacetyldopamine.N-acetyldopamine. These authors These also authors proposed also proposed that such thatreactions such could reactions be of could biological be of significance biological significanceduring sclerotization during sclerotization insect cuticle, insect although cuticle, it although was acknowledged it was acknowledged by them that by them insect that cuticular insect cuticularproteins proteinshave low have concentration low concentration of sulfur ofcontaining sulfur containing amino acids. amino Moreover, acids. Moreover, these addition these additionreactions reactionsalso required also required large mole large moleexcess excess of N of-acetylmethionine.N-acetylmethionine. Therefore, Therefore, occurrence occurrence of of such such reactionsreactions althoughalthough theoreticallytheoretically possible,possible, neverthelessnevertheless isis unlikelyunlikely toto witnesswitness inin insectinsect systemssystems thatthat biologicalbiological Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 35 significancesignificance ofof this this type type of of conjugation conjugation with with proteins proteins is is questionable. questionable.

Figure 8. ReactionReaction of of oo-quinones-quinones with with methionine methionine and and N-acetylmethionine-acetylmethionine [[46].46]. Note Note that that the the position position of attachment of thio-ether group has not been confirmed.confirmed. 3.3. Reaction of o-Quinones with Amines 3.3. Reaction of o-Quinones with Amines Next to the sulfhydryl group, the amino group reacts with o-quinones at rates that are biologically Next to the sulfhydryl group, the amino group reacts with o-quinones at rates that are relevant especially when it is present within the same molecule of o-quinone. Three types of reactions biologically relevant especially when it is present within the same molecule of o-quinone. Three types have been characterized so far for amines: (1) intramolecular cyclization if the amino group is of reactions have been characterized so far for amines: (1) intramolecular cyclization if the amino group is situated within the molecule at a suitable position, (2) intermolecular nucleophilic addition, and (3) Schiff base formation. The intramolecular cyclization of side chain amino group in dopaquinone and dopaminequinone results in the production of cyclodopa and cyclodopamine. These two reactions form critical steps in eumelanogenesis and neuromelanogenesis (Figure 9) and will be discussed in detail in Section 6. Such unique reactivity is not specific for a particular o-quinone alone, but is of general occurrence is demonstrated by the pioneer work by Hawley et al. [47]. Electrochemically generated o-quinones from epinephrine and were found to cyclize 140- and 10- times faster than dopaminequinone. Interestingly, the high tendency of epinephrinequinone and norepinephrinequinone to cyclize to form stable aminochromes is related to the lower cytotoxicity of epinephrine and norepinephrine [48]. Reactivity of dopaminequinone was compared with dopaquinone by Li and Christensen [49]. Cyclization of dopaquinone proceeded ca. 10 times faster than dopaminequinone. The faster cyclization of dopaquinone is related to the lower cytotoxicity of dopa as compared to dopamine [48]. Another example of cyclization is presented during the oxidation of 4-S-cysteaminylphenol. Mascagna et al. [50] showed that a violet pigment, dihydro-1,4-benzothiazine-6,7-dione (BQ), was produced upon tyrosinase oxidation of 4-S- cysteaminylphenol (4SCAP; Figure 9). A seven-membered homologue of BQ can also be produced from 4-S-homocysteaminylphenol [51]. 4SCAP is highly cytotoxic to melanoma cells [52], which can be attributed to the tyrosinase-dependent production of BQ [53].

Figure 9. Intramolecular addition of amino group to o-quinone group to form aminochromes. A unique example of the intramolecular addition of amino group to form a six-membered ring is presented for 4SCAP quinone [50].

Nucleophilic addition of amines to o-quinones has not been extensively examined as compared to thiols. This may be ascribed to the lower reactivity of amines and the instability of the amine adducts produced. Li et al. [54] compared reactivity of small thiols and amines with o-quinones. They found the reaction of 4-methyl-o-benzoquinone with the amino group of amino acids (Gly, Nα-acetyl- L-Lys, Nε-acetyl-L-Lys, and L-Lys) and the guanidine group of Nα-acetyl-L-Arg was at least 5 × 105 slower compared to the reaction with the low molecular mass thiols such as cysteine. In addition, they also observed formation of transient reduced amine-quinone adducts followed by reoxidation to the more stable, oxidized form having absorbance near 500 nm (Figure 10). More recently, Li et al., [55] compared the reactivity of different o-quinones with lysine and cysteine using cyclic voltammetry. The study indicated the order of reactivity of various quinones as: protocatechuic acid quinone = o-benzoquinone > 4-methyl-o-benzoquinone = caffeic acid quinone > chlorogenic acid

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Figure 8. Reaction of o-quinones with methionine and N-acetylmethionine [46]. Note that the position of attachment of thio-ether group has not been confirmed.

3.3. Reaction of o-Quinones with Amines Next to the sulfhydryl group, the amino group reacts with o-quinones at rates that are Int. J. Mol. Sci. 2020 21 biologically relevant, , 6080 especially when it is present within the same molecule of o-quinone. Three 8types of 36 of reactions have been characterized so far for amines: (1) intramolecular cyclization if the amino situatedgroup is withinsituated the within molecule the molecule at a suitable at a position,suitable position, (2) intermolecular (2) intermolecular nucleophilic nucleophilic addition, addition, and (3) Schiandff (3)base Schiff formation. base formation. The intramolecular The intramolecular cyclization ofcyclization side chain of amino side chain group amino in dopaquinone group in anddopaquinone dopaminequinone and dopaminequinone results in the results production in the of production cyclodopa of and cyclodopa cyclodopamine. and cyclodopamine. These two reactionsThese two form reactions critical form steps critical in eumelanogenesis steps in eumelanogenesis and neuromelanogenesis and neuromelanogenesis (Figure9) (Figure and will 9) beand discussedwill be discussed in detail in indetail Section in Section6. Such 6. Such unique unique reactivity reactivity is not is not specific specific for for a particulara particularo o-quinone-quinone alone,alone, butbut isis ofof generalgeneral occurrenceoccurrence isis demonstrateddemonstrated byby thethe pioneerpioneer workwork byby HawleyHawley et al. [[47].47]. ElectrochemicallyElectrochemically generated generated o-quinoneso-quinones from from epinephrine epinephrine and and norepinephrine norepinephrine were werefound found to cyclize to cyclize140- and 140- 10- and times 10- times faster faster than than dopaminequinone. dopaminequinone. Interestingly, Interestingly, the the high high tendency ofof epinephrinequinoneepinephrinequinone andand norepinephrinequinonenorepinephrinequinone toto cyclizecyclize toto formform stablestable aminochromesaminochromes isis relatedrelated toto thethe lower cytotoxicity cytotoxicity of of epinephrine epinephrine and and norepi norepinephrinenephrine [48]. [48 Reactivity]. Reactivity of dopaminequinone of dopaminequinone was wascompared compared with with dopaquinone dopaquinone by Li by and Li and Christense Christensenn [49]. [49 Cyclizat]. Cyclizationion of dopaquinone of dopaquinone proceeded proceeded ca. ca.10 times 10 times faster faster than thandopaminequinone. dopaminequinone. The faster The cy fasterclization cyclization of dopaquinone of dopaquinone is related is to related the lower to thecytotoxicity lower cytotoxicity of dopa as of compared dopa as compared to dopamine to dopamine [48]. Another [48]. example Another of example cyclization of cyclization is presented is presentedduring the during oxidation the oxidationof 4-S-cysteaminylphenol. of 4-S-cysteaminylphenol. Mascagna Mascagna et al. [50] etshowed al. [50 ]that showed a violet that pigment, a violet pigment,dihydro-1,4-benzothiazine-6,7-dione dihydro-1,4-benzothiazine-6,7-dione (BQ), wa (BQ),s produced was produced upon tyrosinase upon tyrosinase oxidation oxidation of 4- ofS- 4-cysteaminylphenolS-cysteaminylphenol (4SCAP; (4SCAP; Figure Figure 9).9). A A seven-me seven-memberedmbered homologue homologue of BQ cancan alsoalso bebe produced produced fromfrom 4-4-SS-homocysteaminylphenol-homocysteaminylphenol [[51].51]. 4SCAP4SCAP isis highlyhighly cytotoxiccytotoxic toto melanomamelanoma cellscells [[52],52], whichwhich cancan bebe attributedattributed toto thethe tyrosinase-dependenttyrosinase-dependent production production of of BQ BQ [ 53[53].].

FigureFigure 9.9.Intramolecular Intramolecular addition addition of aminoof amino group group to o-quinone to o-quinone group group to form to aminochromes. form aminochromes. A unique A exampleunique example of the intramolecular of the intramolecular addition of addition amino group of amino to form group a six-membered to form a six-membered ring is presented ring for is 4SCAPpresented quinone for 4SCAP [50]. quinone [50].

NucleophilicNucleophilic addition addition of of amines amines to too -quinoneso-quinones has has not not been been extensively extensively examined examined as comparedas compared to thiols.to thiols. This This may may be ascribed be ascribed to the to lower the lower reactivity reacti ofvity amines of amines and the and instability the instability of the amine of the adducts amine produced.adducts produced. Li et al. [ 54Li ]et compared al. [54] compared reactivity reactivity of small of thiols small and thiols amines and withamineso-quinones. with o-quinones. They found They α thefound reaction the reaction of 4-methyl- of 4-methyl-o-benzoquinoneo-benzoquinone with the with amino the amino group ofgroup amino of amino acids (Gly, acidsN (Gly,-acetyl-L-Lys, Nα-acetyl- Nε-acetyl-L-Lys, and L-Lys) and the guanidine group of Nα-acetyl-L-Arg was at least 5 105 slower L-Lys, Nε-acetyl-L-Lys, and L-Lys) and the guanidine group of Nα-acetyl-L-Arg was at× least 5 × 105 comparedslower compared to the reaction to the reaction with the with low molecularthe low mole masscular thiols mass such thiols as cysteine. such as Incysteine. addition, In theyaddition, also observedthey also formationobserved offormation transient of reduced transient amine-quinone reduced amine-quinone adducts followed adducts by followed reoxidation by toreoxidation the more stable,to the oxidizedmore stable, form oxidized having absorbanceform having near absorbance 500 nm (Figure near 500 10 ).nm More (Figure recently, 10). More Li et al., recently, [55] compared Li et al., the[55] reactivity compared of dithefferent reacotivity-quinones of different with lysine o-quinones and cysteine with using lysine cyclic and voltammetry. cysteine using The studycyclic indicatedvoltammetry. the order The ofstudy reactivity indicated of various the order quinones of reacti as:vity protocatechuic of various quinones acid quinone as: protocatechuic= o-benzoquinone acid >quinone4-methyl- = o-benzoquinone >= 4-methyl-caffeic acido-benzoquinone quinone > chlorogenic = caffeic acid acid quinone quinone. > Thechlorogenic reactivity acid of quinones appear to decrease with steric hinderance of substituents on quinone ring and weakened by enhancing electron cloud density on the quinone ring. Adducts generated by the interaction of 4-methyl-o-benzoquinone with amines were identified as amine-quinone adducts connected at the C6 position (Figure4). The reactions of primary amine with o-quinone is very complex and the products suffer further reactions and hence a clear picture is very difficult to obtain. However, the reactions of secondary amine type—especially imidazole with o-quinone lead to clean products that have been fully characterized. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 35 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 35 quinone. The reactivity of quinones appear to decrease with steric hinderance of substituents on quinone.quinone Thering reactivityand weakened of quinon by enhancinges appear toelectron decrease cloud with density steric hinderanceon the quinone of substituents ring. Adducts on quinonegenerated ring by and the weakenedinteraction byof enhancing4-methyl-o -benzoquinoneelectron cloud densitywith amines on the were quinone identified ring. asAdducts amine- generatedquinone adductsby the interactionconnected atof the4-methyl- C6 positiono-benzoquinone (Figure 4). withThe reactionsamines were of primary identified amine as amine-with o- quinonequinone adducts is very complexconnected and at thethe productsC6 position suffer (Figure further 4). reactions The reactions and hence of primary a clear aminepicture with is very o- quinonedifficult is to very obtain. complex However, and the the products reactions suffer of secondary further reactions amine type—especially and hence a clear imidazole picture iswith very o - difficultquinone to lead obtain. to clean However, products the that reactions have been of secondary fully characterized. amine type—especially imidazole with o- Int. J. Mol. Sci. 2020 21 quinone lead to, clean, 6080 products that have been fully characterized. 9 of 36

Figure 10. Reaction of o-quinones with amines. Note that the oxidized form of amine adducts are Figure 10. Reaction of o-quinones with amines. Note that the oxidized form of amine adducts are more Figuremore stable10. Reaction than the of reduced o-quinones form with [54]. amines. Note that the oxidized form of amine adducts are stable than the reduced form [54]. more stable than the reduced form [54]. In 1982, Sugumaran and Lipke [56] pointed out the involvement of both side chains of lysine In 1982, Sugumaran and Lipke [56] pointed out the involvement of both side chains of lysine andIn histidine 1982, Sugumaran in cross-linking and Lipke reactions [56] pointed with outinsect the involvementcuticular quinonoid of both side molecules chains ofthrough lysine and histidine in cross-linking reactions with insect cuticular quinonoid molecules through radioactive andradioactive histidine experiments. in cross-linking Following reactions this study, with Xuinsect et al. cuticular [57] reported quinonoid using moleculesmodel sclerotization through experiments. Following this study, Xu et al. [57] reported using model sclerotization studies with radioactivestudies with experiments. N-acetyldopaminequinone Following this study, and NXu-acetylhistidine et al. [57] reported the formation using model of 6-sclerotizationN- and 2-N - N-acetyldopaminequinone and N-acetylhistidine the formation of 6-N- and 2-N-imidazole adducts studiesimidazole with adducts N-acetyldopaminequinone in a ratio of 5:1 (Figure and 11). NThe-acetylhistidine position of amine the formationsubstitution of was 6-N confirmed- and 2-N by- in a ratio of 5:1 (Figure 11). The position of amine substitution was confirmed by NMR studies. imidazoleNMR studies. adducts Cyclic in avoltammetry ratio of 5:1 (Figure studies 11). with The these position adducts of amineand parent substitution catechol was revealed confirmed that they by Cyclic voltammetry studies with these adducts and parent catechol revealed that they are much more NMRare much studies. more Cyclic resistant voltammetry to oxidation studies in withcomparison these adducts with N and-acetyldopamine, parent catechol which revealed explains that they why resistant to oxidation in comparison with N-acetyldopamine, which explains why these compounds arethese much compounds more resistant are produced to oxidation as stable in comparison products in with contract N-acetyldopamine, to the reaction which products explains of primary why are produced as stable products in contract to the reaction products of primary amines with quinone, theseamines compounds with quinone, are produced which are as extrstableemely products unstable. in contract The production to the reaction of 6-N products-imidazole of adductprimary is which are extremely unstable. The production of 6-N-imidazole adduct is consistent with the fact aminesconsistent with with quinone, the fact which that aminesare extr obeyemely predominantly unstable. The typicalproduction Michael-1,4-nucleophilic of 6-N-imidazole adduct addition is that amines obey predominantly typical Michael-1,4-nucleophilic addition reaction, contrary to the consistentreaction, contrarywith the tofact the that thiol amines additions. obey However,predominantly the formation typical Michael-1,4-nucleophilic of 2-N-imidazole adduct addition and the thiol additions. However, the formation of 2-N-imidazole adduct and the absence of 5-N-imidazole reaction,absence contraryof 5-N-imidazole to the thiol adduct additions. is intriguing However, as sterically the formation 5-N-adduct of 2-N is-imidazole a more preferred adduct andproduct the adduct is intriguing as sterically 5-N-adduct is a more preferred product than the 2-N-adduct yet, absencethan the of 2- 5-NN-adduct-imidazole yet, itadduct is not reportedlyis intriguing formed as sterically in the reaction 5-N-adduct mixture. is a moreMore preferredstudies are product needed it is not reportedly formed in the reaction mixture. More studies are needed to shed light on this thanto shed the 2-lightN-adduct on this yet, inconsistent it is not reportedly observation. formed in the reaction mixture. More studies are needed inconsistent observation. to shed light on this inconsistent observation.

Figure 11. Reaction of o-quinones with N-acetylhistidine. Note that the position of attachment of the Figure 11. Reaction of o-quinones with N-acetylhistidine. Note that the position of attachment of the imidazole group was confirmed [57]. Figureimidazole 11. Reaction group was of oconfirmed-quinones [57].with N-acetylhistidine. Note that the position of attachment of the Reactionsimidazole group of o-quinones was confirmed with [57]. amino group in deoxyribonucleosides have also been reported. CavalieriReactions et al. [ 58of] examinedo-quinones the with reaction amino of groupo-quinones in deoxyribonucleosides generated by chemical have oxidation also been of catechol,reported. CavalieriReactions et al. of [58] o-quinones examined with the reaction amino groupof o-quinones in deoxyribonucleosides generated by chemical have oxidationalso been of reported. catechol, dopamine, and N-acetyldopamine with Ag2O or NaIO4, and witnessed their addition reaction with adenineCavalieridopamine, (Ade) et al.and and[58] N -acetyldopamine deoxyguanosineexamined the reaction (dG)with (FigureAgof o2O-quinones or 12 NaIO). The generated4, positionand witnessed by of aminechemical their addition additionoxidation was reaction of confirmed catechol, with todopamine,adenine be C6 by (Ade) and NMR, andN-acetyldopamine again deoxyguanosine in accordance with (dG) with Ag (Figure2O the or Michael-1,4-addtion. NaIO 12). The4, and position witnessed of amine These their adductsadditionaddition also wasreaction servedconfirmed with as standardsadenineto be C6 (Ade) forby evaluatingNMR, and deoxyguanosine again the in binding accordance of (dG) DNA with(Figure to othe-quinones 12). Michael-1,4-addtion. The (see position Section of 5.2 amine ).These Interestingly, addition adducts was thesealso confirmed served adducts as weretostandards be producedC6 by forNMR, atevaluating the again reduced, in the accordance catecholicbinding ofwith state. DNA the Under toMichael-1,4-addtion. o-quinones the same conditions, (see Section These however, 5.2).adducts Interestingly, deoxyadenosine, also served these as deoxycytidine,standardsInt.adducts J. Mol. Sci. werefor 2020 evaluating and, produced21, x thymidine FOR PEER theat REVIEWthebinding did reduced, not exhibitof DNA catecho any to reactionolic-quinones state. with Under (seeo-benzoquinone. Section the same 5.2). conditions, Interestingly, however, 10 these of 35 adductsdeoxyadenosine, were produced deoxycytidine, at the reduced,and thymid catechoine didlic notstate. exhibit Under any the reaction same with conditions, o-benzoquinone. however, deoxyadenosine, deoxycytidine, and thymidine did not exhibit any reaction with o-benzoquinone.

FigureFigure 12.12.Reaction Reaction of ofo -quinoneso-quinones with with DNA DNA bases bases [58 ].[58]. These These adducts adducts are isolated are isolated in the in reduced the reduced form. form.

The third type of reaction of o-quinone with amine is the Schiff’s base formation. The of quinone and the primary amino groups can easily condense and loose water to form Schiff bases (Figure 4). The quinone imine formation is vital for the biosynthesis of pheomelanin pigment. Cysteinyldopa quinone exhibits intramolecular cyclization and forms a bicyclic quinone imine which is the precursor for all pheomelanin production. More about this will be discussed in Section 6.2. But the reaction does not have to occur only intramolecularly. Even intermolecular addition of amines to o-quinones have been witnessed. Such reactions occur during the oxidative transformation of insect cuticular sclerotizing precursor, N-β-alanyldopamine. It accounts for the brown coloration of insect cuticle. Mass spectral studies have tentatively identified the production of different colored quinone imine adducts [59].

3.4. Reaction of o-Quinones with Carboxylic Acids, Phenols, and Alcohols (Water) The reaction of quinones with carboxyl groups are sluggish. Thus, the reactions of o-quinones with carboxylic acids (carboxylates) would not proceed unless the carboxylate group is present in the side chain of the o-quinone (Figure 4). Sugumaran et al. [60] showed that carboxyethyl-o- benzoquinone undergoes cyclization to form the six-membered lactone derivative, dihydroesculetin (Figure 13). Interestingly, the lower homologue, carboxymethyl-o-benzoquinone (DOPAC quinone), also afforded the five membered lactone ring that tautomerizes to form 2,5,6-trihydroxybenzofuran (Figure 13) along with 3,4-dihydroxymandelic acid (DOMA) and 3,4-dihydroxybenzaldehyde (DHIBAld) formed via quinone methide intermediate (see the next section; [61]). Another example is that 1,2-dehydro-N-acetyldopa upon oxidation rapidly undergoes intramolecular cyclization [62]. Even though quinone may be the initial product of the reaction, it rapidly and instantaneously isomerized to a tautomeric quinone methide imine amide that exhibited rapid intramolecular cyclization generating dihydroxy coumarin derivative (see the next section; Figure 13).

Figure 13. Intramolecular addition of carboxylate group to o-quinone group to form lactones [60–62].

The 2,5,6-trihydroxybenzofuran formed from the cyclization of carboxymethyl-o-benzoquinone suffered further oxidation but characterization of the product turned out to be tough [61]. To overcome this problem, fluorine substituted 3,4-dihydroxyphenylacetic acid was synthesized and its oxidation examined. Fluorine positioned at the cyclization site left during cyclization of the quinonoid product, due to its high electronegativity, thus directly producing a quinonoid product but not catechol. This compound was identified to be quinone methide tautomer of furanoquinone [63].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 35

Figure 12. Reaction of o-quinones with DNA bases [58]. These adducts are isolated in the reduced Int. J. Mol. Sci. 2020, 21, 6080 10 of 36 form.

The third type of reaction of oo-quinone-quinone withwith amineamine isis thethe SchiSchiff’sff’s basebase formation.formation. The carbonyl group of quinone and the primary amino groups can easily condense and loose water to form Schiff Schiff bases (Figure 44).). TheThe quinonequinone imineimine formationformation isis vitalvital forfor thethe biosynthesisbiosynthesis ofof pheomelaninpheomelanin pigment.pigment. Cysteinyldopa quinone exhibits intramolecular cyclization and forms a bicyclic quinone imine which is the the precursor precursor for for all all pheomelanin pheomelanin production. production. More More about about this this will will be discussed be discussed in Section in Section 6.2. But6.2. Butthe reaction the reaction does does not nothave have to occur to occur only only intramolec intramolecularly.ularly. Even Even intermolecular intermolecular addition addition of amines of amines to too-quinoneso-quinones have have been been witnessed. witnessed. Such Such reactions reactions occu occurr during during the the oxidative oxidative transformation transformation of of insect cuticular sclerotizing precursor, NN--β-alanyldopamine. It It accounts accounts for for the brown coloration of insect cuticle. Mass Mass spectral spectral studies have tentatively i identifieddentified the production of different different colored quinone imine adducts [[59].59].

3.4. Reaction of o-Quinones with Carboxy Carboxyliclic Acids, Phenols, and Alcohols (Water) The reaction of quinones with carboxyl groupsgroups are sluggish.sluggish. Thus, the reactions of oo-quinones-quinones with carboxylic acids (carboxylates) would not proceedproceed unless the carboxylate group is present in the side chainchain of of the theo-quinone o-quinone (Figure (Figure4). Sugumaran 4). Sugumaran et al. [ 60 ]et showed al. [60] that showed carboxyethyl- that ocarboxyethyl--benzoquinoneo- undergoesbenzoquinone cyclization undergoes to formcyclization the six-membered to form the six-membered lactone derivative, lactone dihydroesculetin derivative, dihydroesculetin (Figure 13). Interestingly,(Figure 13). Interestingly, the lower the homologue, lower homologue, carboxymethyl- carboxymethyl-o-benzoquinoneo-benzoquinone (DOPAC (DOPAC quinone), quinone), also aalsofforded afforded the fivethe five membered membered lactone lactone ring ring that that tautomerizes tautomerizes to toform form 2,5,6-trihydroxybenzofuran2,5,6-trihydroxybenzofuran (Figure 1313)) alongalong withwith 3,4-dihydroxymandelic3,4-dihydroxymandelic acidacid (DOMA)(DOMA) andand 3,4-dihydroxybenzaldehyde3,4-dihydroxybenzaldehyde (DHIBAld) formed viavia quinonequinone methide methide intermediate intermediate (see (see the the ne nextxt section; section; [61]). [61]). Another Another example example is isthat that 1,2-dehydro- 1,2-dehydro-N-acetyldopaN-acetyldopa upon upon oxidation oxidation rapidly rapidly under undergoesgoes intramolecular intramolecular cyclization cyclization [62]. [62]. Even though quinone may be the initial product of the reaction, it rapidly and instantaneously isomerized toto a tautomerica tautomeric quinone quinone methide methide imine im amideine thatamide exhibited that exhibited rapid intramolecular rapid intramolecular cyclization generatingcyclization dihydroxygenerating coumarindihydroxy derivative coumarin (see derivative the next (see section; the next Figure section; 13). Figure 13).

FigureFigure 13. 13.Intramolecular Intramolecular additionaddition ofof carboxylate group to to oo-quinone-quinone group group to to form form lactones lactones [60–62]. [60–62].

The 2,5,6-trihydroxybenzofuran formed from the cyclization of carboxymethyl-o-benzoquinone susufferedffered furtherfurther oxidation oxidation but but characterization characterization of the of productthe product turned turned out to beout tough to be [61 tough]. To overcome [61]. To thisovercome problem, this fluorineproblem, substituted fluorine substituted 3,4-dihydroxyphenylacetic 3,4-dihydroxyphenylacetic acid was synthesized acid was synthesized and its oxidation and its examined.oxidation examined. Fluorine positioned Fluorine atpositioned the cyclization at the site cyclization left during cyclizationsite left during of the quinonoidcyclization product, of the duequinonoid to its highproduct, electronegativity, due to its high thus electronegativ directly producingity, thus directly a quinonoid producing product a quinonoid but not catechol.product Thisbut not compound catechol. was This identified compound to bewas quinone identified methide to betautomer quinone ofmethide furanoquinone tautomer [63of ].furanoquinone [63]. Reaction of o-quinones with hydroxyl group in phenols (and catechols) may proceed smoothly because of the acidity of the phenolic hydroxyl group (pKa ca. 10). Some o-quinones undergo 1,4-Michael reaction with the parent catechols to gives rise to dimeric products with a dibenzodioxin skeleton. An example was seen in the tyrosinase-catalyzed oxidation of hydroxytyrosol [(2-(3,4-dihydroxyphenylethanol)) [64] (Figure 14). However, this type of self-coupling reactions may not be a dominant pathway for the fate of o-quinones, because the reaction requires high concentrations of the substrate catechol and the product o-quinone for an efficient formation of the dimer through a bimolecular reaction. A unique method of preparing the dibenzodioxins was reported by Stratford et al. [65] who witnessed Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 35 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 35

ReactionReaction of of o o-quinones-quinones with with hydroxyl hydroxyl group group in in phen phenolsols (and (and catechols) catechols) may may proceed proceed smoothly smoothly becausebecause of of the the acidity acidity of of the the phenolic phenolic hydroxyl hydroxyl group group (p (pKKaa ca. ca. 10). 10). Some Some o o-quinones-quinones undergo undergo 1,4- 1,4- MichaelMichael reaction reaction with with the the parent parent catechols catechols to to gives gives rise rise to to dimeric dimeric products products with with a a dibenzodioxin dibenzodioxin skeleton.skeleton. AnAn exampleexample waswas seenseen inin thethe tyrosinasetyrosinase-catalyzed-catalyzed oxidationoxidation ofof hydroxytyrosolhydroxytyrosol [(2-(3,4-[(2-(3,4- dihydroxyphenylethanol))dihydroxyphenylethanol)) [64] [64] (Figure (Figure 14). 14). However, However, this this type type of of self-coupling self-coupling reactions reactions may may not not be be aa dominant dominant pathway pathway for for the the fate fate of of o o-quinones,-quinones, because because the the reaction reaction re requiresquires high high concentrations concentrations of of Int. J. Mol. Sci. 2020, 21, 6080 11 of 36 thethe substrate substrate catechol catechol and and the the product product o o-quinone-quinone for for an an efficient efficient formation formation of of the the dimer dimer through through a a bimolecularbimolecular reaction. reaction. A A unique unique method method of of prepar preparinging the the dibenzodioxins dibenzodioxins was was reported reported by by Stratford Stratford et al. [65] who witnessed the tyrosinase-catalyzed oxidation of 4-halogenated catechols giving rise to theet al. tyrosinase-catalyzed [65] who witnessed the oxidation tyrosinase-catalyzed of 4-halogenated oxidation catechols of 4-halogenated giving rise catechols to the dibenzodioxin giving rise to the dibenzodioxin derivatives efficiently. derivativesthe dibenzodioxin efficiently. derivatives efficiently.

Figure 14. Self-coupling of tyrosol quinone with hydroxytyrosol (HT) to form a dibenzodioxin [64]. FigureFigure 14.14.Self-coupling Self-coupling ofof tyrosoltyrosol quinonequinone withwith hydroxytyrosolhydroxytyrosol (HT)(HT) toto formform aa dibenzodioxindibenzodioxin [[64].64]. An isomer differing in the position of the substituent (R-) was also isolated. An efficient formation of AnAn isomer isomer di differingffering in in the the position position of of the the substituent substituent (R-) (R-) was was also also isolated. isolated.An Ane efficientfficient formation formation of of dibenzodioxins was reported for 4-halogenated quinones [65]. dibenzodioxinsdibenzodioxins was was reported reported for for 4-halogenated 4-halogenated quinones quinones [ 65[65].].

ReactionReaction of ofof o o-quinones-quinones-quinones with withwith hydroxyl hydroxylhydroxyl group groupgroup in inin water waterwater or oror alcohols alcoholsalcohols on onon the thethe other other hand handhand would would be bebe difficultdidifficultfficult to toto occur occuroccur (Figure (Figure(Figure4). 4). Addition4). AdditionAddition of water ofof waterwater molecules moleculesmolecules to o-quinones toto oo-quinones-quinones will produce willwill produceproduce trihydroxy trihydroxytrihydroxy product productthatproduct will bethat that rapidly will will be be oxidized rapidly rapidly with oxidized oxidized the generation with with the the ofgeneration generation the p-quinone of of the the product, p p-quinone-quinone 2-hydroxy-1,4-benzoquinone product, product, 2-hydroxy-1,4- 2-hydroxy-1,4- benzoquinone(Figurebenzoquinone 15). This (Figure (Figure reaction 15). 15). seemsThis This reaction reaction possible seems seems for some possible possibleo-quinones. for for some some However, o o-quinones.-quinones. in reality,However, However, it is in in di reality, ffireality,cult toit it isisolateis difficult difficult a single to to isolate isolate product a a single single and product product show the and and occurrence show show th thee occurrence ofoccurrence such a reaction. of of such such a a Withreaction. reaction. tyrosinase With With tyrosinase tyrosinase for instance, for for instance,theinstance, nucleophilic the the nucleophilic nucleophilic addition additionof addition water toof of owater -quinonewater to to o- o- productsquinonequinone products formedproducts during formed formed the during during reaction the the hasreaction reaction not been has has notreported.not been been reported. reported. However, However, However, formation formation formation of 6-hydroxydopa of of 6-hydroxydopa 6-hydroxydopa in peptidyl in in peptidyl dopapeptidyl moiety dopa dopa of moiety moiety topaquinone of of topaquinone topaquinone cofactor cofactorcontainedcofactor contained contained in amine in in oxidasesamine amine oxidases oxidases appear appear appear to proceed to to proceed proceed by a by nucleophilicby a a nucleophilic nucleophilic addition addition addition reaction reaction reaction of waterof of water water to todopaquinoneto dopaquinone dopaquinone followed followed followed by by by re-oxidation re-oxidation re-oxidation [66 [66]. [66].]. Whether Whether Whether suchsuch such aa a hydroxylationhydroxylation hydroxylation reactionreaction reaction is is of of enzyme enzyme catalyzedcatalyzed or oror of ofof nonenzymatic nonenzymaticnonenzymatic origin originorigin is is yet yet to to be be resolved. resolved. Hydroxylation Hydroxylation Hydroxylation of of of quinones quinones quinones can can can occur occur occur by by by a a differenta different route route in insome some cases. cases. For Forexample, example, in peroxidase/H in peroxidase2O/H2 systemO system hydroperoxide hydroperoxide anion anioncan serve can different route in some cases. For example, in peroxidase/H2O2 2system2 hydroperoxide anion can serve asserve a nucleophile as a nucleophile (pKa of (p HK2aO of2 is H 11.6)O isand 11.6) cause and this cause reaction. this reaction. Thus, two Thus, group two of group workers of workers have shown have as a nucleophile (pKa of H2O2 is 211.6)2 and cause this reaction. Thus, two group of workers have shown thatshown the that peroxidase/H the peroxidase2O2 /oxidationH O oxidation of N-acetyldopamine of N-acetyldopamine and andhydroxytyrosol hydroxytyrosol afforded afforded the the2- that the peroxidase/H2O2 oxidation2 2 of N-acetyldopamine and hydroxytyrosol afforded the 2- hydroxy-1,4-benzoquinone2-hydroxy-1,4-benzoquinonehydroxy-1,4-benzoquinone derivatives derivatives derivatives in in ingood good good yields yields yields [67,68] [67,68] [67,68 (Figure ](Figure (Figure 15). 15). 15 ).

Figure 15. 15. ReactionReaction of o-quinones of o-quinones with with hydrogenperoxide lead peroxideing the production leading the of production2-hydroxy-1,4- of Figure 15. Reaction of o-quinones with leading the production of 2-hydroxy-1,4- quinones2-hydroxy-1,4-quinones [67,68]. Note that [67 the,68 ].direct Note addition that the of water direct molecule addition to of o water-quinone molecule is unlikely to o -quinoneto occur. is quinones [67,68]. Note that the direct addition of water molecule to o-quinone is unlikely to occur. unlikely to occur.

Rhododendrol, 4-(4-hydroxyphenyl)-2-butanol, is a phenol that was widely used as a skin lightening compound until 2013 in Japan. Continuous application of this compound has caused severe leucoderma in many consumers [69]. Although rhododendrol inhibits mushroom tyrosinase [70], it also serves as a good substrate not only for mushroom tyrosinase [71,72] but also for human tyrosinase [73]. In fact, rhododendrol is a better substrate for human tyrosinase than even the natural substrate, L-, [73]. There is a possibility that o-quinone from rhododendrol might Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 35

Rhododendrol, 4-(4-hydroxyphenyl)-2-butanol, is a phenol that was widely used as a skin lightening compound until 2013 in Japan. Continuous application of this compound has caused severe leucoderma in many consumers [69]. Although rhododendrol inhibits mushroom tyrosinase [70], it also serves as a good substrate not only for mushroom tyrosinase [71,72] but also for human Int. J. Mol. Sci. 2020, 21, 6080 12 of 36 tyrosinase [73]. In fact, rhododendrol is a better substrate for human tyrosinase than even the natural substrate, L-tyrosine, [73]. There is a possibility that o-quinone from rhododendrol might form the formsix-membered the six-membered ring cyclic ring ether cyclic (RD-cyclic ether (RD-cycliccatechol) through catechol) addition through of additionthe hydroxyl of the group hydroxyl to o- groupquinone to moietyo-quinone within moiety the same within molecule the same (Figure molecule 16). However, (Figure 16 the). However,feasibility of the this feasibility intramolecular of this intramolecularaddition reaction addition has been reaction questioned has been by questionedKishida et al. by [74] Kishida using et density al. [74] usingfunctional density theory-based functional theory-basedfirst principles first calculations. principles Nevertheless, calculations. Nevertheless,the production the of productionrhododendrol-cyclic of rhododendrol-cyclic quinone was quinoneunambiguously was unambiguously confirmed because confirmed rhododendrol-cyclic because rhododendrol-cyclic catechol was isolated catechol after was reduction isolated afterwith reductionNaBH4 [71]. with Interestingly, NaBH4 [71 ].rhododendrol-cyclic Interestingly, rhododendrol-cyclic quinone is gradually quinone conver is graduallyted to rhododendrol- converted to rhododendrol-hydroxy-1,4-quinonehydroxy-1,4-quinone by the addition byof wa theter addition molecule of and water decays molecule to oligomeric and decays products. to oligomeric Thus, the products.mechanism Thus, of production the mechanism of RD-cyclic of production catechol of still RD-cyclic remains catechol to be elucidated. still remains to be elucidated.

FigureFigure 16.16. Reaction of rhododendrolrhododendrol (RD)(RD) quinonequinone toto formform RD-cyclicRD-cyclic catecholcatechol [[71].71]. NoteNote thatthat thisthis cyclizationcyclization reactionreaction isis lessless likelylikely toto proceedproceed [[72,74].72,74]. 3.5. Redox Exchange of o-Quinones with Reducing Agents 3.5. Redox Exchange of o-Quinones with Reducing Agents The next important reaction of o-quinones that occurs very fast is its redox exchange with The next important reaction of o-quinones that occurs very fast is its redox exchange with reducing agents. These reductions proceed so fast to be of significant importance to biological systems. reducing agents. These reductions proceed so fast to be of significant importance to biological Ascorbic acid is the most important biological reducing agent because of its high reactivity and abundant systems. Ascorbic acid is the most important biological reducing agent because of its high reactivity levels in various tissues. o-Quinones produced in biological systems are often reduced back to catechols and abundant levels in various tissues. o-Quinones produced in biological systems are often reduced by ascorbic acid instantaneously. Such reduction prevents the drastic effects caused by highly reactive back to catechols by ascorbic acid instantaneously. Such reduction prevents the drastic effects caused quinones. But the disadvantage of this process is ascorbic acid being consumed over and over until it by highly reactive quinones. But the disadvantage of this process is ascorbic acid being consumed is completely depleted as the reduced catechol can be easily re-oxidized by the endogenous oxidizing over and over until it is completely depleted as the reduced catechol can be easily re-oxidized by the enzymes. Thus, oxidative browning of plant products, mushrooms etc., which often accompanies endogenous oxidizing enzymes. Thus, oxidative browning of plant products, mushrooms etc., which wounding will deplete cellular reduction potential and cause deleterious effects. Again, cellular thiols often accompanies wounding will deplete cellular reduction potential and cause deleterious effects. and other nucleophiles will also undergo addition reaction with quinones thereby adding more damage Again, cellular thiols and other nucleophiles will also undergo addition reaction with quinones to the systems. NADH is also able to reduce o-quinones to catechols [75]. For synthetic purposes, thereby adding more damage to the systems. NADH is also able to reduce o-quinones to catechols ascorbate reduction of quinones formed by the tyrosinase-catalyzed oxidation of monophenols proved [75]. For synthetic purposes, ascorbate reduction of quinones formed by the tyrosinase-catalyzed to be a very convenient and efficient way to synthesize catecholic compounds [75,76]. Some quinones oxidation of monophenols proved to be a very convenient and efficient way to synthesize catecholic can also be reduced by catechols. In this process, catechol providing the reducing equivalent gets compounds [75,76]. Some quinones can also be reduced by catechols. In this process, catechol oxidized to form quinone and the quinone substrate is reduced to catecholic product. This process providing the reducing equivalent gets oxidized to form quinone and the quinone substrate is known as redox exchange reaction or double decomposition is biologically very important as it is reduced to catecholic product. This process known as redox exchange reaction or double heavily involved in both eumelanin biosynthesis and pheomelanin biosynthesis. Significance of redox decomposition is biologically very important as it is heavily involved in both eumelanin biosynthesis exchange reactions in melanogenesis will be discussed in Section6. and pheomelanin biosynthesis. Significance of redox exchange reactions in melanogenesis will be 3.6.discussed Reaction in ofSection o-Quinone 6. from

3.6. ReactionHydroquinone of o-Quinone is a from unique Hydroquinone phenol in that it belongs to the para-diphenols but at the same time to the 4-substituted phenols. In fact, hydroquinone is oxidized to p-benzoquinone when oxidizedHydroquinone by tyrosinase is a in unique the presence phenol of in dopa that [it77 belongs]. This reactionto the para proceeds-diphenols through but at redox the same exchange time betweento the 4-substituted hydroquinone phenols. anddopaquinone. In fact, hydroquinone Hydroquinone is oxidized is cytotoxic to p-benzoquinone to melanocytes when and oxidized is widely by usedtyrosinase as a topical in the hypopigmentingpresence of dopa agent [77]. [This78]. reaction Hydroquinone proceeds appears through to exertredox its exchange effects mainly between in melanocyteshydroquinone with and activedopaquinone. tyrosinase Hydroquinone activity [79]. is Palumbocytotoxic etto al.melanocytes [77] showed and that is widely hydroquinone, used as a astopical a phenol, hypopigmenting acts as a substrate agent [78]. of tyrosinase Hydroquinone and generated appears to the exert hydroxylated its effects mainly product, in melanocytes which was characterizedwith active tyrosinase as a red coloredactivity chromophore[79]. Palumbo [ 80et ]al. (Figure [77] showed 17). The that red hydroquinone, chromophore wasas a identifiedphenol, acts as 2-hydroxy-1,4-benzoquinoneas a substrate of tyrosinase and [77]. generated The immediate the hydroxylated oxidation product product, from which hydroquinone was characterized by tyrosinase as a is most likely 4-hydroxy-1,2-benzoquinone, but it is immediately tautomerized to the more stable

2-hydroxy-1,4-benzoquinone [81]. The production of 2-hydroxy-1,4-benzoquinone precludes the generation of catecholic products that may activate tyrosinase (see Section2), which is why the Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 35

red colored chromophore [80] (Figure 17). The red chromophore was identified as 2-hydroxy-1,4- benzoquinone [77]. The immediate oxidation product from hydroquinone by tyrosinase is most likely 4-hydroxy-1,2-benzoquinone, but it is immediately tautomerized to the more stable 2-hydroxy-1,4- Int.benzoquinone J. Mol. Sci. 2020 ,[81].21, 6080 The production of 2-hydroxy-1,4-benzoquinone precludes the generation13 of 36of catecholic products that may activate tyrosinase (see Section 2), which is why the oxidation of oxidationhydroquinone of hydroquinone by tyrosinase by terminates tyrosinase at terminates an initial phase at an initial when phase a catecholic when acofactor catecholic is not cofactor present is not[81]. present [81].

Figure 17. Figure 17. Reaction of hydroquinonehydroquinone inin thethe tyrosinase-catalyzedtyrosinase-catalyzed oxidationoxidation [[77,80,81].77,80,81]. 4. Reactivity of o-Quinones through Quinone Methide Tautomers 4. Reactivity of o-Quinones through Quinone Methide Tautomers 4.1. Addition of Water to Quinone Methide Tautomers 4.1. Addition of Water to Quinone Methide Tautomers With 4-alkyl substituted quinones a novel isomerization reaction occurs. The occurrence of this reactionWith depends 4-alkyl onsubstituted the substituents quinones present a novel on isomerization the alkyl group. reaction The simplestoccurs. The 4-alkyl occurrence quinone of isthis 4- methyl-reactiono -benzoquinone.depends on the substituents Isomerization present of this on compound the alkyl group. would The generate simplest the 4-alkylp-quinone quinone methide, is 4- althoughmethyl-o-benzoquinone. with this simple Isomerization compound the of chance this compound of isomerization would seemsgenerate to bethe negligible. p-quinone However, methide, aalthough number with of side this chainsimple alkyl compound substituted the chance compounds of isomerization can easily seems isomerize to be tonegligible.p-quinone However, methide a derivatives.number of sidep-Quinone chain alkyl methides substitute are tautomersd compounds of o -quinonescan easily with isomerize one of to the p-quinone carbonyl methide oxygen replacedderivatives. with p-Quinone a benzylic methides are grouptautomers (Figure of 18o-quinones). The weak with acidity one of of the benzylic carbonyl hydrogen oxygen atomreplaced renders with a the benzylic conversion methylene to quinone group (Figure methide 18). tautomers The weak possible. acidity of Thebenzylic deprotonation hydrogen atom step duringrenders thethe quinoneconversion methide to quinone formation methide is provedtautomers to bepossible. base-catalyzed The deprotonation using deuterium-labeled step during the 4-propionylcatecholquinone methide formation [82]. Quinone is proved methides to arebe muchbase-catalyzed more reactive using than deuterium-labeled the corresponding 4- opropionylcatechol-quinones [6,19,83 ].[82].p-Quinone Quinone methides methides rapidly are addmuch on more to any reactive nucleophiles than presentthe corresponding in the medium. o- Ifquinones there are [6,19,83]. no other p-Quinone nucleophiles methides present rapidly in the add medium, on to any they nucleophiles form dimers present and otherin the potentialmedium. oligomers.If there are They no other are even nucleophiles capable of present adding onin th toe water medium, molecules they form forming dimers side chainand other hydroxylated potential products.oligomers. BothThey 3,4-dihydroxybenzylalcoholare even capable of adding on [84 to] water and 3,4-dihydroxybenzylamine molecules forming side chain [85 hydroxylated] are rapidly oxidizedproducts. to Both form 3,4-dihydroxybenzy their correspondinglalcohol quinone [84] derivatives. and 3,4-dihydroxybenzylamine These quinones rapidly [85] isomerize are rapidly to unstableoxidized quinoneto form methidestheir corresponding which add quinone on to water derivatives. molecules These forming quinones side rapidly chain hydroxylated isomerize to products.unstable quinone The product methides formed which with add 3,4-dihydroxybenzylalcohol on to water molecules losesforming a water side moleculechain hydroxylated in the side chainproducts. and producesThe product 3,4-dihydroxybenzaldehyde formed with 3,4-dihydroxyben as thezylalcohol end product loses [84 ].a water Loss of molecule ammonia in from the side the productchain and of 3,4-dihydroxybenzylamineproduces 3,4-dihydroxybenzaldehyde also produces as 3,4-dihydroxybenzaldehydethe end product [84]. Loss of as ammonia final product from [ 85the]. 3,4-dihydroxyphenylglycineproduct of 3,4-dihydroxybenzylamine also exhibits also a similarproduces reaction 3,4-dihydroxybenzaldehyde producing 3,4-dihydroxybenzaldehyde as final product as[85]. the end3,4-dihydroxyphenylglycine product [86]. Another side chainalso hydroxylationexhibits a wassimilar witnessed reaction during producing the oxidation 3,4- of 3,4-dihydroxyphenylaceticdihydroxybenzaldehyde as acid the [end61]. Theproduct quinone [86]. generatedAnother side form chain this compoundhydroxylation rapidly was isomerized witnessed andduring reacted the withoxidation water of producing 3,4-dihydroxyphenylaceti 3,4-dihydroxymandelicc acid acid,[61].along The withquinone other generated products. form Recently, this Brookscompound et al., rapidly [87] isomerized has demonstrated and reacted the with addition water water producing to the 3,4-dihydroxymandelic side chain with 4-ethylphenol. acid, along Tyrosinase-catalyzedwith other products. oxidationRecently, Brook of thes 4-ethylphenolet al., [87] has produceddemonstrated the correspondingthe addition water quinone to the which side afterchain isomerization with 4-ethylphenol. and water Tyro additionsinase-catalyzed generated 1-(3,4-dihydroxyphenyl). oxidation of the 4-ethylphenol produced the corresponding quinone which after isomerization and water addition generated 1-(3,4- dihydroxyphenyl)ethanol.

Int. J. Mol. Sci. 2020, 21, 6080 14 of 36 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 35

Figure 18.18. QuinoneQuinone methide methide formation formation from fromo-quinones o-quinones and theand subsequent the subsequent addition addition of water of molecule water moleculeto form alcohol to form derivatives alcohol derivatives [84]. [84]. 4.2. Reaction of Quinone Methides from Catecholamine Metabolites 4.2. Reaction of Quinone Methides from Catecholamine Metabolites The significance of quinone methide intermediates in biosynthesis of catecholamines was first The significance of quinone methide intermediates in biosynthesis of catecholamines was first proposed by Senoh and Witkop as early as 1959 [88]. They suggested that norepinephrine is produced proposed by Senoh and Witkop as early as 1959 [88]. They suggested that norepinephrine is produced by the hydration of p-quinone methide formed from dopaminequinone. However, this novel concept by the hydration of p-quinone methide formed from dopaminequinone. However, this novel concept was subsequently disproved with discovery of dopamine β-hydroxylase, which was shown to catalyze was subsequently disproved with discovery of dopamine β-hydroxylase, which was shown to the direct hydroxylation of the β-carbon atom of dopamine by a stereospecific reaction [89]. Much later, catalyze the direct hydroxylation of the β-carbon atom of dopamine by a stereospecific reaction [89]. Sugumaran and Lipke [90] rediscovered the significance of quinone methides in catecholamine Much later, Sugumaran and Lipke [90] rediscovered the significance of quinone methides in by demonstrating the possible occurrence of the side chain hydroxylation reaction during catecholamine metabolism by demonstrating the possible occurrence of the side chain hydroxylation cuticular enzyme catalyzed oxidation of a number of catecholic compounds. Although initially reaction during cuticular enzyme catalyzed oxidation of a number of catecholic compounds. they proposed a direct 1,6-oxidation of 4-alkyl catechols to p-quinone methides, careful fraction of Although initially they proposed a direct 1,6-oxidation of 4-alkyl catechols to p-quinone methides, insect enzyme system revealed that this reaction proceeds with the intermediary formation of 4-alkyl careful fraction of insect enzyme system revealed that this reaction proceeds with the intermediary quinone by a tyrosinase-catalyzed oxidation reaction and then the isomerization of 4-alkyl quinones to formation of 4-alkyl quinone by a tyrosinase-catalyzed oxidation reaction and then the isomerization quinone methides catalyzed by a specific quinone isomerase [91] (Figure 18). Their study revealed of 4-alkyl quinones to quinone methides catalyzed by a specific quinone isomerase [91] (Figure 18). that not all 4-alkyl quinone exhibit spontaneous isomerization to p-quinone methides. Some such as Their study revealed that not all 4-alkyl quinone exhibit spontaneous isomerization to p-quinone N-acetyldopamine quinone required specific enzyme to catalyze the reaction. Electron withdrawing methides. Some such as N-acetyldopamine quinone required specific enzyme to catalyze the reaction. substituents such as carbonyl and carboxyl groups facilitated the nonenzymatic tautomerization and Electron withdrawing substituents such as carbonyl and carboxyl groups facilitated the fully reduced side chains such as -CH2-CH2-X (X not being an electron withdrawing group) strictly nonenzymatic tautomerization and fully reduced side chains such as -CH2-CH2-X (X not being an required enzymatic assistance. electron withdrawing group) strictly required enzymatic assistance. Reactivity of o-quinones with 4-substituted side chain having hydroxyl or carboxyl group Reactivity of o-quinones with 4-substituted side chain having hydroxyl or carboxyl group has has been extensively studied because they are likely intermediates in sclerotization in insect been extensively studied because they are likely intermediates in sclerotization in insect cuticle cuticle [6,19,83] or they are incorporated into neuromelanin in brain [2,92]. Sclerotization is a [6,19,83] or they are incorporated into neuromelanin in brain [2,92]. Sclerotization is a process in process in which the cuticle of an arthropod is hardened by cross-linking of the cuticle proteins. which the cuticle of an arthropod is hardened by cross-linking of the cuticle proteins. 3,4- 3,4-Dihydroxyphenylethanol (DOPE) and 3,4-dihydroxyphenylethyleneglycol (DOPEG) are alcohol Dihydroxyphenylethanol (DOPE) and 3,4-dihydroxyphenylethyleneglycol (DOPEG) are alcohol metabolites, while 3,4-dihydroxyphenylacetic acid (DOPAC), and DOMA are carboxylic acid metabolites, while 3,4-dihydroxyphenylacetic acid (DOPAC), and DOMA are carboxylic acid metabolites of catecholamines dopamine and norepinephrine, respectively. Reactivity of DOPE metabolites of catecholamines dopamine and norepinephrine, respectively. Reactivity of DOPE quinone was first reported by Sugumaran et al. [93] using a preparation of cuticular enzyme(s) quinone was first reported by Sugumaran et al. [93] using a preparation of cuticular enzyme(s) from from Sarcophaga bullata. They showed a rapid conversion to DOPEG through the quinone Sarcophaga bullata. They showed a rapid conversion to DOPEG through the quinone methide methide intermediate (Figure 19). DOPEG was then gradually converted to 2-oxo-DOPE through intermediate (Figure 19). DOPEG was then gradually converted to 2-oxo-DOPE through another another quinone methide intermediate. DOMA quinone, generated either enzymatically [94] quinone methide intermediate. DOMA quinone, generated either enzymatically [94] or or electrochemically [95] was converted to 3,4-dihydroxybenzaldehyde (DHBAld) through the electrochemically [95] was converted to 3,4-dihydroxybenzaldehyde (DHBAld) through the decarboxylative rearrangement giving a quinone methide intermediate. DOPAC quinone also rapidly decarboxylative rearrangement giving a quinone methide intermediate. DOPAC quinone also rearranged to give 3,4-dihydroxybenzylalcohol (DHBAlc) through a quinone methide intermediate, rapidly rearranged to give 3,4-dihydroxybenzylalcohol (DHBAlc) through a quinone methide which was then gradually converted to DHBAld through the same quinone methide as from DOMA intermediate, which was then gradually converted to DHBAld through the same quinone methide as quinone [84,96]. Ito et al. [97] compared chemical reactivity of o-quinones from DOPE, DOPEG, from DOMA quinone [84,96]. Ito et al. [97] compared chemical reactivity of o-quinones from DOPE, DOPAC, and DOMA to be: DOMA (a half-life, t1/2, of 3.0 min at pH 6.8) > DOPAC (9.1 min) > DOPE DOPEG, DOPAC, and DOMA to be: DOMA (a half-life, t1/2, of 3.0 min at pH 6.8) > DOPAC (9.1 min) (14.6 min) > DOPEG (30.9 min). It appears that the presence of carboxyl group next to the benzylic > DOPE (14.6 min) > DOPEG (30.9 min). It appears that the presence of carboxyl group next to the benzylic carbon in DOPAC and DOMA renders the o-quinone intermediates more reactive than in

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 35 Int. J. Mol. Sci. 2020, 21, 6080 15 of 36 Int.the J. alcohols Mol. Sci. 2020 DOPE, 21, xand FOR DOPEG. PEER REVIEW On the other hand, the presence of hydroxyl groups on the benzylic 15 of 35 carbon showed ambiguous effect, either suppressive (DOMA > DOPAC) or accelerating (DOPE > carbonthe alcohols in DOPAC DOPE and and DOMA DOPEG. renders On the the othero-quinone hand, th intermediatese presence of more hydroxyl reactive groups than on in the benzylic alcohols DOPEG). Those o-quinones show a common metabolite fate: conversion to quinone methide DOPEcarbon andshowed DOPEG. ambiguous On the effect, other hand,either thesuppressive presence (DOMA of hydroxyl > DOPAC) groups or on accelerating the benzylic (DOPE carbon > tautomers followed by secondary reactions such as addition of water molecule or tautomerization to showedDOPEG). ambiguous Those o-quinones effect, either show suppressive a common (DOMA metabolite> DOPAC) fate: orconversion accelerating to (DOPEquinone> DOPEG).methide carbonyl group (Figure 19). α-(3,4-Dihydroxyphenyl)lactic acid which is a homolog of DOMA also Thosetautomerso-quinones followed show by asecondary common metabolitereactions such fate: as conversion addition of to water quinone molecule methide or tautomers tautomerization followed to exhibits the same oxidative decarboxylation reaction and produces 3,4-dihydroxyacetophenone as bycarbonyl secondary group reactions (Figure such19). α as-(3,4-Dihydroxyphenyl)lactic addition of water molecule acid or tautomerizationwhich is a homolog to carbonyl of DOMA group also the end product [98]. (Figureexhibits 19 the). αsame-(3,4-Dihydroxyphenyl)lactic oxidative decarboxylation acid reac whichtion isand a homolog produces of 3,4-dihydroxyacetophenone DOMA also exhibits the same as oxidativethe end product decarboxylation [98]. reaction and produces 3,4-dihydroxyacetophenone as the end product [98].

Figure 19. Reaction of o-quinone products from catecholamine metabolites 3,4- FigureDihydroxyphenylethanol 19. o (DOPE), 3,4-dihydroxyphenylethyleneglycol (DOPEG), 3,4- Figure 19.Reaction Reaction of -quinone of productso-quinone from products catecholamine from metabolites catecholamine 3,4-Dihydroxyphenylethanol metabolites 3,4- (DOPE),dihydroxyphenylacetic 3,4-dihydroxyphenylethyleneglycol acid (DOPAC), and (DOPEG),3,4-dihydroxymandelic 3,4-dihydroxyphenylacetic acid (DOMA) acid [94]. (DOPAC), Tyrosinase- and Dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethyleneglycol (DOPEG), 3,4- 3,4-dihydroxymandeliccatalyzed oxidation of the acid catechols (DOMA) was [94 terminated]. Tyrosinase-catalyzed by the addition oxidation of ascorbic of acid. the catechols Note that was the dihydroxyphenylacetic acid (DOPAC), and 3,4-dihydroxymandelic acid (DOMA) [94]. Tyrosinase- terminatedformation of by quinone the addition methides of ascorbic proceeds acid. slowly, Note wh thatich the is quickly formation followed of quinone by the methides addition proceeds of water catalyzed oxidation of the catechols was terminated by the addition of ascorbic acid. Note that the slowly,molecule which or the is quicklyformation followed of carbonyl by the group. addition of water molecule or the formation of carbonyl group. formation of quinone methides proceeds slowly, which is quickly followed by the addition of water InmoleculeIn papilionidpapilionid or the butterflies, butterflies,formation of thethe carbonyl wingwing colorgroup.color is is producedproduced byby thethe interaction interaction of ofN N-β-β-alanyldopamine-alanyldopamine withwith kynurenine.kynurenine. Of the several pigments formedformed by this interaction, one pigment compound— In papilionid butterflies, the wing color is produced by the interaction of N-β-alanyldopamine papilliochromepapilliochrome II, was identified identified as as diastereoi diastereoisomericsomeric mixtures mixtures of ofkynurenine kynurenine adduct adduct of ofN- Nβ- with kynurenine. Of the several pigments formed by this interaction, one pigment compound— βalanyldopamine-alanyldopamine [99,100], [99,100 the], the formation formation of which of which was biochemically was biochemically examined examined by one by of oneour groups of our papilliochrome II, was identified as diastereoisomeric mixtures of kynurenine adduct of N- β- groups[101]. Nonenzymatic [101]. Nonenzymatic addition addition of the amino of the group amino of group kynurenine of kynurenine to the N- toβ-alanyldopamine the N-β-alanyldopamine quinone alanyldopamine [99,100], the formation of which was biochemically examined by one of our groups quinonemethide enzymatically methide enzymatically generated by generated tyrosinase by and tyrosinase quinone isomerase and quinone gives isomerase rise to diastereoisomers gives rise to [101]. Nonenzymatic addition of the amino group of kynurenine to the N-β-alanyldopamine quinone diastereoisomersof this compound of(Figure this compound20). During (Figurethis reaction, 20). water During addition this reaction, to form N water-β-alanylnorepinephine addition to form methide enzymatically generated by tyrosinase and quinone isomerase gives rise to diastereoisomers Nas- βa-alanylnorepinephine side product is also witnessed as a side product[101]. is also witnessed [101]. of this compound (Figure 20). During this reaction, water addition to form N-β-alanylnorepinephine as a side product is also witnessed [101].

Figure 20. Reaction of N-β-alanyldopamine quinone with kynurenine to form papilliochrome Figure 20. Reaction of N-β-alanyldopamine quinone with kynurenine to form papilliochrome II [99– II101]. [99– Note101]. that Note quinone that quinone isomerase isomerase was required was requiredfor the production for the production of papilliochrome of papilliochrome II and that N II- Figure 20. Reaction of N-β-alanyldopamine quinone with kynurenine to form papilliochrome II [99– andβ-alanylnorepinephrine that N-β-alanylnorepinephrine was also prod wasuced also through produced addition through of additionwater. of water. 101]. Note that quinone isomerase was required for the production of papilliochrome II and that N- β-alanylnorepinephrine was also produced through addition of water.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 35

Int.4.3. J. Side Mol. Chain Sci. 2020 Desaturation, 21, 6080 (Dehydrogenation) Reactions 16 of 36 Another interesting reaction of 4-alkyl quinone is their tendency to undergo side chain 4.3.desaturation. Side Chain The Desaturation side chain (Dehydrogenation) desaturation reactions Reactions play pivotal role in key biological processes such as melaninAnother biosynthesis interesting reaction and sclerotization of 4-alkyl quinone of inse is theirct cuticle tendency [6,19,83,102,103]. to undergo side Therefore, chain desaturation. we will Theexamine side chainthe side desaturation chain reactivity reactions of play 4-alkyl pivotal quinones role in in key this biological section. processes Side chain such desaturation as melanin biosynthesis(dehydrogenation) and sclerotization can proceed of when insect a cuticlecarbonyl [6, 19group,83,102 (ketone,103]. or Therefore, ) is wepresent will examineat the γ-position the side chainof the reactivityside chain of to 4-alkyl make an quinones extension in thisof conjug section.ation Side system chain possible desaturation through (dehydrogenation) the quinone methide can proceedintermediate. when The a carbonyl first example group(ketone of side chain or ester) desaturation is present at was the witnessedγ-position with of the dihydrocaffeic side chain to make acid [3- an extension(3,4-dihydroxyphenyl)propionic of conjugation system possible acid] derivatives through the (Figure quinone 21). methide Oxidation intermediate. of 3,4-dihydrocaffeic The first example acid ofester side produced chain desaturation its quinone was readily, witnessed which with rapidly dihydroca isomerizedffeic acid [3-(3,4-dihydroxyphenyl)propionicto quinone methide and further acid]exhibited derivatives another (Figure isomerization 21). Oxidation to yield of side 3,4-dihydroca chain desaturatedffeic acid compound, ester produced caffeic its acid quinone ester readily,[60]. A whichsimilar rapidly behavior isomerized was also to exhibited quinone by methide the methyl and further amide exhibited derivative another [104]. isomerizationEven N-acetyldopa to yield side chainexhibited desaturated this side compound, chain desatura caffeiction acid reactions ester [60]. producing A similar behaviordehydro- wasN-acetyldopa also exhibited esters by the [105,106]. methyl amideDehydrodopa derivative is a [constituent104]. Even ofN -acetyldopatunichromes esters found exhibited in blood cells this sideof tunicates, chain desaturation which are implicated reactions producingin metal sequestering dehydro-N-acetyldopa and other esters functions [105,106 [107].]. Dehydrodopa The need for is aanother constituent enzyme of tunichromes associated found with inquinone blood cellsmethide of tunicates, metabolism which was are implicateddiscovered in when metal the sequestering reactions of and N-acetyldopamine other functions [107 quinone]. The need and forisomeric another dihydrocaffeiyl enzyme associated methyl with amide quinone quinone methide were metabolism compared was [104]. discovered While dihydrocaffeiyl when the reactions methyl of Namide-acetyldopamine quinone exhibited quinone rapid and isomericisomerizat dihydrocaion to sideffeiyl chain methyl desaturated amide quinone product, were N-acetyldopamine compared [104]. Whilequinone dihydroca requiredffeiyl the methylassistance amide of quinonean enzyme. exhibited This enzyme rapid isomerization was subsequently to side chaindiscovered desaturated as N- product,acetyldopamineN-acetyldopamine quinone methide/1,2-dehydro- quinone requiredN the-acetyldopamine assistance of tautomeras an enzyme.e [108,109]. This enzyme Therefore, was subsequentlyleft unassisted discovered by this isomerase, as N-acetyldopamine N-acetyldopamine quinone quinone methide methide/1,2-dehydro- simply givesN-acetyldopamine rise to water tautomeraseadduct N-acetylnorepinephrine [108,109]. Therefore, [88,101]. left unassisted Such a mark by thised difference isomerase, inN the-acetyldopamine reactivity between quinone N- methideacetyldopa simply esters gives and rise N-acetyldopamine to water adduct Ncan-acetylnorepinephrine be ascribed to the presence [88,101]. Suchof electron-withdrawing a marked difference incarboxyl the reactivity group in between the former.N-acetyldopa Dopachrome esters isomeras and N-acetyldopaminee reaction represents can be the ascribed second toimportant the presence side ofchain electron-withdrawing desaturation reaction. carboxyl Dopach grouprome inundergoes the former. slow Dopachrome isomerization isomerase to its quinone reaction methide represents and thethe secondquinone important methide then side exhibits chain desaturation side chain desaturation reaction. Dopachrome producing 5,6-dihydroxyindole undergoes slow isomerization (DHI) and to5.6-dihydroxyindole-2-carboxylic its quinone methide and the quinone acid (DHICA) methide [102,103]. then exhibits More about side chain this reaction desaturation will be producing discussed 5,6-dihydroxyindoleunder melanin biosynthesis (DHI) and (Section 5.6-dihydroxyindole-2-carboxylic 6.2). acid (DHICA) [102,103]. More about this reactionRaspberry will ketone, be discussed 4-(4-hydroxyph under melaninenyl)-2-butanone, biosynthesis is (Section a ketone 6.2 analogue). of rhododendrol and knownRaspberry to have ketone,caused 4-(4-hydroxyphenyl)-2-butanone,chemical leukoderma in 1998 [110]. is aRaspberry ketone analogue ketone quinone of rhododendrol undergoes and a knownside chain to havedesaturation caused chemicalgiving 3,4-dihydroxybenza leukoderma in 1998lacetone [110]. Raspberry(DBL) quinone ketone due quinone to the presence undergoes of aa sidecarbonyl chain group desaturation at the γ giving-position 3,4-dihydroxybenzalacetone of the side chain [111]. Interestingly, (DBL) quinone the C2 due position to the presenceof o-quinone of a carbonylmoiety in group DBL quinone at the γ -positionbecomes ofmore the reactive side chain than [111 the]. C5 Interestingly, position in theaddition C2 position reaction of witho-quinone thiols moietythrough in the DBL electron-withdrawing quinone becomes more effect reactive of 3-oxo-1-butenyl than the C5 position group (Figure in addition 21). A reaction similar with activation thiols throughby a conjugated the electron-withdrawing double bond was eff observedect of 3-oxo-1-butenyl in resveratrol group quinone (Figure and 21 ).chlorogenic A similar activation acid quinone by a conjugated(Figure 6). double bond was observed in resveratrol quinone and chlorogenic acid quinone (Figure6).

Figure 21. Side chain chain desaturation desaturation from quinone methides from dihydrocaffeic dihydrocaffeic acidacid quinonesquinones [[60]60] andand raspberryraspberry ketone quinone [[111].111]. Note that dihydrocadihydrocaffeicffeic acid (caffffeiceic acid) with thethe NN-acetyl-acetyl groupgroup can bebe consideredconsidered asas dopadopa (dehydrodopa)(dehydrodopa) derivative.derivative. The position of thiol addition didiffersffers betweenbetween raspberryraspberry ketoneketone quinonequinone andand DBLDBL quinone.quinone.

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 35 4.4. Reactivity of Side Chain Desaturated Quinones 4.4. Reactivity of Side Chain Desaturated Quinones Earlier we examined the side chain desaturation reactions of different catecholamine derivatives.Earlier we The examined side chain the side desaturated chain desaturation compounds reactions play of very different crucial catecholamine role in a derivatives. number of biologicalThe side chain process. desaturated Three compounds uncyclized play unsaturated very crucia dopal role derivativesin a number thatof biological are central process. to Three other biologicaluncyclized processunsaturated are—1,2-dehydro- dopa derivativesN-acyldopamines that are central which to areother absolutely biological essential process forare—1,2- insect sclerotizationdehydro-N-acyldopamines [6,83,112,113]; which 1,2-dehydro- are absolutelyN-acyldopa essential esters for whichinsect sclerotization is partly responsible [6,83,112,113]; for gluing 1,2- propertiesdehydro-N-acyldopa observed withesters dopyl which proteins is partly [ 114responsible], and 1,2-dehydrodopa for gluing properties which observed forms a with basic dopyl unit inproteins a number [114], ofand tunichrome 1,2-dehydrodopa derivatives which that forms could a basic act unit as in a number compounds of tunichrome and derivatives cementing materialthat could [5, 62act,107 as,115 antibiotic]. 1,2-Dehydro- compoundsN-aceyldopamines and cementing upon material tyrosinase [5,62,107,115]. oxidation may 1,2-Dehydro- generate theN- quinoneaceyldopamines as the immediate upon tyrosinase product oxidation but characterizing may generate the the quinone quinone even as the transiently immediate was product practically but impossiblecharacterizing due the to thequinone fact that even it instantaneously transiently was isomerized practically to impossible tautomeric quinonedue to the methide fact that imine it amideinstantaneously [19,112,113 isomerized,116,117] (Figure to tautomeric 22). These quinone compounds methide exhibitedimine amide rapid [19,112,113,116,117] reaction with the (Figure parent catechol22). These generating compounds benzodioxan exhibited dimersrapid reaction and other with oligomers. the parent 1,2-Dehydro- catechol generatingN-acyldopa benzodioxan was even moredimers reactive and other in generatingoligomers. the1,2-Dehydro- quinone methideN-acyldopa product was even [62]. more As stated reactive earlier, in generating oxidation the of 1,2-dehydro-quinone methideN-acetyldopa product resulted[62]. As instated rapid earlier, quinone oxidation formation of followed1,2-dehydro- by intramolecularN-acetyldopa cyclization.resulted in Therapid dihydroxy quinone coumarinformation thusfollowed formed by furtherintramolecular suffered morecyclization. oxidation The in dihydroxy the reaction coumarin mixture thus and generatedformed further a number suffered of polymericmore oxidation derivatives in the reactivia anotheron mixture quinone and generated methide imine a number amide of derivative.polymeric Surprisingly,derivatives via 1,2-dehydro- another quinoneN-acyldopa methide ester producedimine amide the corresponding derivative. Surprisi quinonengly, and not1,2-dehydro- the quinoneN- methideacyldopa tautomer ester produced as the immediate the corresponding product [114quinon]. Irrespectivee and not ofthe the quinone nature methide of the quinonoid tautomer species, as the allimmediate of these compoundsproduct [114]. exhibited Irrespecti oxidativeve of the polymerization nature of the quinonoid to form di ffspecies,erent benzodioxan all of these compounds derivatives. Theseexhibited transformations oxidative polymerization attest the versatile to form reactivity different benzodioxan of side chain derivative substituteds. These quinones transformations (see [19] for moreattest details).the versatile reactivity of side chain substituted quinones (see [19] for more details).

Figure 22.22. ReactionReaction ofof side side chain chain desaturated desaturated quinones quinon producinges producing benzodioxan benzodioxan dimers dimers [19,62 [19,62,113–,113–117]. 4.5. Reaction117]. of Estradiol Quinones

4.5. ReactionMetabolic of Estradiol fate of 17- Quinonesβ-estradiol, a phenolic hormone, was examined by Pezzella et al. [118] showing the involvement of quinone methide intermediates. Tyrosinase-catalyzed oxidation of Metabolic fate of 17-β-estradiol, a phenolic steroid hormone, was examined by Pezzella et al. 17-β-estradiol afforded catechol estrogens, 2-hydroxyestradiol and 4-hydroxyestradiol at the low, but [118] showing the involvement of quinone methide intermediates. Tyrosinase-catalyzed oxidation of physiologically relevant substrate concentrations of 1–10 nM [118]. In addition, they isolated metabolites, 17-β-estradiol afforded catechol estrogens, 2-hydroxyestradiol and 4-hydroxyestradiol at the low, but 6-oxo-2-hydroxyestradiol, 9,11-dehydro-2-hydroxyestradiol, 6,7-dehydro-2-hydroxyestradiol, and physiologically relevant substrate concentrations of 1–10 nM [118]. In addition, they isolated 9,11-dehydro-4-hydroxyestradiol (Figure 23). At higher estradiol concentrations, e.g., 1 µM, additional metabolites, 6-oxo-2-hydroxyestradiol, 9,11-dehydro-2-hydroxyestradiol, 6,7-dehydro-2- products including 6,7,8,9-dehydro-2-hydroxyestradiol and dimers were also detected. These products hydroxyestradiol, and 9,11-dehydro-4-hydroxyestradiol (Figure 23). At higher estradiol arise from quinone methide intermediates. Reactivity of quinone (or quinone methide) from estradiol concentrations, e.g., 1 µM, additional products including 6,7,8,9-dehydro-2-hydroxyestradiol and with DNA is discussed in the following section. dimers were also detected. These products arise from quinone methide intermediates. Reactivity of quinone (or quinone methide) from estradiol with DNA is discussed in the following section.

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Figure 23. Reaction of β-estradiol quinones [118]. Note that those quinones undergo tautomerization Figure 23. Reaction of β-estradiol quinones [[118].118]. Note Note that that those quinones undergo tautomerization to quinone methides which give rise to the secondary products. to quinonequinone methidesmethides whichwhich givegive riserise toto thethe secondarysecondary products.products. 5. Reactivity of o-Quinones with Macromolecules 5. Reactivity of o-Quinones with Macromolecules 5.1. Covalent Binding with Proteins 5.1. Covalent Binding with Proteins The high high reactivity reactivity of of thiol thiol group group renders renders proteins proteins to react to react with witho-quinones.o-quinones. This type This of typeprotein of The high reactivity of thiol group renders proteins to react with o-quinones. This type of protein proteinmodifications modifications is biologically is biologically important important because this because may lead this to may denaturation lead to denaturation of thiol proteins of thiol and modifications is biologically important because this may lead to denaturation of thiol proteins and proteinsinhibition and of thiol inhibition enzymes, of thiol leading enzymes, to cytotoxicity leading [48,53]. to cytotoxicity Kato et al. [48 [119],53]. found Kato etthat al. dopaquinone [119] found inhibition of thiol enzymes, leading to cytotoxicity [48,53]. Kato et al. [119] found that dopaquinone thatreacts dopaquinone with thiol reactsgroup with in thiolcysteine group residue in cysteine of thiol residue proteins, of thiol bovine proteins, serum bovine albumin, serum albumin, alcohol reacts with thiol group in cysteine residue of thiol proteins, bovine serum albumin, alcohol alcoholdehydrogenase, dehydrogenase, and isocitrate and isocitrate dehydrogenase dehydrogenase in yields of in 5.4, yields 44, ofand 5.4, 33%, 44, respectively. and 33%, respectively. The yields dehydrogenase, and isocitrate dehydrogenase in yields of 5.4, 44, and 33%, respectively. The yields Thecan yieldsbe determined can be determined by acid hydrolysis by acid hydrolysis to release to cy releasesteinyldopas cysteinyldopas (or other (or cy othersteinylcatecho cysteinylcatechols;ls; Figure Figurecan24). beThis determined24 ).hydrolysis This hydrolysis by method acid hydrolysis methodis generally isto generally release applicable cy applicablesteinyldopas to evaluate to (or evaluate binding other cy bindingofsteinylcatecho thiol ofproteins thiolls; proteins with Figure o- 24). This hydrolysis method is generally applicable to evaluate binding of thiol proteins with o- withquinones,o-quinones, e.g., rhododendrol e.g., rhododendrol quinone quinone [120] and [120 o-quinone] and o-quinone from N-propionyl-4SCAP from N-propionyl-4SCAP [121] (Figure [121] quinones, e.g., rhododendrol quinone [120] and o-quinone from N-propionyl-4SCAP [121] (Figure (Figure24). During 24). Duringacid hydrolysis, acid hydrolysis, some functional some functional groups groupsundergo undergo additional additional modifications modifications such as such the 24). During acid hydrolysis, some functional groups undergo additional modifications such as the assubstitution the substitution of a chlorine of a chlorinegroup in groupRD and in th RDe hydrolytic and the hydrolyticcleavage of cleavagean amide ofgroup an amide in N-propionyl- group in substitution of a chlorine group in RD and the hydrolytic cleavage of an amide group in N-propionyl- N4SCAP-propionyl-4SCAP (Figure 24). (Figure 24). 4SCAP (Figure 24).

Figure 24.24. Reaction of o-quinones with protein-SH and acid hydrolysishydrolysis of the adducts to liberateliberate Figure 24. Reaction of o-quinones with protein-SH and acid hydrolysis of the adducts to liberate cysteinyl derivativesderivatives [[30,120–122].30,120–122]. cysteinyl derivatives [30,120–122]. Reactivity of of biologically biologically relevant relevant o-quinoneso-quinones with with bovine bovine serum serum albumin albumin was compared was compared by Ito Reactivity of biologically relevant o-quinones with bovine serum albumin was compared by Ito byet Itoal. et[123] al. [showing123] showing the thereactivity reactivity of ofo-quinoneso-quinones from from catecholamines catecholamines to to be be dopamine dopamine >> et al. [123] showing the reactivity of o-quinones from catecholamines to be dopamine >

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Int.norepinephrine J. Mol. Sci. 2020, 21> ,dopa 6080 > epinephrine; this reactivity is inversely correlated to the rate of cyclization19 of 36 of the side chain amino group (see above). norepinephrineThe covalent> dopa binding> epinephrine; of o-quinones this with reactivity thiol proteins is inversely has correlated been proposed to the as rate the of mechanism cyclization ofof thecytotoxic side chain action amino of various group (see4-substituted above). phenols in melanocytes and production of neo-antigens. It shouldThe be covalent stressed binding that the of initialo-quinones step in withthis cy thioltotoxic proteins and immunologic has been proposed action asof thephenols mechanism is their ofconversion cytotoxic actionto o-quinones of various through 4-substituted tyrosinase-catalyzed phenols in melanocytes oxidation and[124]. production The covalent of neo-antigens. binding of Itrhododendrol should be stressed quinone that to proteins the initial through step in cysteine this cytotoxic residues and was immunologic found to be 20- action to 30-fold of phenols greater is theirthan conversiondopaquinone to oin-quinones B16F1 mouse through melanoma tyrosinase-catalyzed cells [120] (Figure oxidation 24). The [124 addition]. The covalent of thiol bindingproteins ofto rhododendrolrhododendrol quinonequinone towas proteins also confirmed through in cysteine vivo in residues a mousewas model found mimicking to be 20- Japanese to 30-fold skin greater [122]. thanMonobenzone dopaquinone (hydroquinone in B16F1 mouse benzyl melanoma ether) is cells a 4-substitued [120] (Figure phenol 24). The known addition for inducing of thiol proteins whitening to rhododendrolof the skin due quinone to loss of was melanocytes. also confirmed It servesin vivo as ain good a mouse substrate model for mimicking tyrosinase Japanese[124,125] skinand [the122 o].- Monobenzonequinone produced (hydroquinone reacts not only benzyl with ether) small is athiols 4-substitued but also phenolthe sulfhydryl known forgroup inducing in bovine whitening serum ofalbumin the skin [125]. due toIt losswas ofproposed melanocytes. that monobenzone It serves as a goodquinone, substrate acting for as tyrosinase a hapten, [124reacts,125 with] and thiol the oproteins-quinone to produced form a neo-antigen reacts not that only triggers with small immune thiols response but also leading the sulfhydryl to a specific group loss in of bovine melanocytes serum albumin[125–127] [125 One]. It waspossible proposed mechanis that monobenzonem of rhododendrol-induced quinone, acting as aleukoderma hapten, reacts is with the thiol binding proteins of torhododendrol form a neo-antigen quinone that with triggers thiol immune proteins response that leadingleads to aimmune specific loss response of melanocytes through [125 antigen–127] Onepresentation possible mechanismor ER stress of through rhododendrol-induced inactivation/den leukodermaaturation of is thethiol binding enzymes/proteins of rhododendrol [72]. quinone4SCAP, withN-propionyl-4SCAP, thiol proteins that and leads related to immune phenolic response thio-ether throughs were antigen examined presentation for inhibiting or ER stressthe growth through of inactivationmalignant melanoma/denaturation in ofvitro thiol and enzymes in vivo,/proteins in the [72 hope]. 4SCAP, for developingN-propionyl-4SCAP, antimelanona and relatedagents phenolic[53,121,128]. thio-ethers were examined for inhibiting the growth of malignant melanoma in vitro and in vivo,Li inet theal. hope[54] examined for developing reactivity antimelanona of 4-methyl- agentso-benzoquinone [53,121,128]. with bovine serum albumin, humanLi et serum al. [54 ]albumin, examined and reactivity α-lactalbumin of 4-methyl- (no ocysteine-benzoquinone residue) with showing bovine their serum second albumin, order human rate 4 −1 −1 3 −1 −1 2 −1 −1 serumconstants albumin, of 3.1 × and 10 αM-lactalbumin s , 4.8 × 10 (no M cysteine s , and residue)4.0 × 10 showingM s , respectively. their second When order we rate consider constants the oflarge 3.1 excess104 M of amine/guanidine1 s 1, 4.8 103 M groups1 s 1, andin proteins 4.0 10 (832 M vs.1 ones 1, cysteine respectively. residue When in serum we consider albumin), the it × − − × − − × − − largeis the excess cysteine of amineresidues/guanidine that are kinetically groups in proteinsthe most (83 important vs. one cysteinetargets in residue proteins. in serumThe reactivity albumin), of itdopaminequinone is the cysteine residues with proteins that are kineticallywas recently the re-visited most important by Wakamatsu targets in et proteins. al. [129] showing The reactivity that the of dopaminequinoneefficacy of binding with of proteinsproteins was through recently cysteine re-visited residue by Wakamatsu depends eton al. its [129 reactivity] showing (or that steric the eavailability),fficacy of binding e.g., of70% proteins in bovine through serum cysteine albumin residue and depends20% in β on-lactoglobulin, its reactivity (orboth steric containing availability), one e.g.,cysteine 70% residue. in bovine serum albumin and 20% in β-lactoglobulin, both containing one cysteine residue. 3,4-Dihydroxybenzaldehyde3,4-Dihydroxybenzaldehyde (DOPAL) isis formedformed fromfrom enzymaticenzymatic oxidationoxidation ofof dopaminedopamine byby monoaminemonoamine oxidase.oxidase. DOPAL is toxic to cellscells andand animalsanimals suchsuch thatthat DOPALDOPAL accumulationaccumulation mightmight contributecontribute toto neuronalneuronal malfunctionsmalfunctions andand eventualeventual lossloss ofof dopaminergicdopaminergic neuronneuron [[130].130]. DOPALDOPAL itselfitself reactsreacts withwith NN--αα-acetyllysine-acetyllysine toto formform thethe reversiblereversible SchiSchiffff basebase butbut notnot withwith NN-acetylcysteine-acetylcysteine [[131]131] (Figure(Figure 25 25).). Jinsmaa Jinsmaa et al.et [al.132 [132]] compared compared reactivity reactivity of dopaminequinone of dopaminequinone and DOPAL and quinoneDOPAL showingquinone thatshowing DOPAL that quinone DOPAL is more quinone efficient is more than dopaminequinoneefficient than dopaminequinone in modifications ofinα -synuclein,modifications a protein of α- whosesynuclein, oligomerization a protein whose plays oligomer a role inization pathogenesis plays a of role Parkinson in pathogenesis disease (Figureof Parkinson 25). DOPAL disease quinone (Figure appears25). DOPAL highly quinone reactive appears due to highly the presence reactive of due both to othe-quinone presence and of aldehyde both o-quinone groups and [131 aldehyde], but its reactivitygroups [131], toward but variousits reactivity nucleophiles toward various remains nucleophiles to be studied. remains to be studied.

FigureFigure 25. ReactionReaction of of 3,4-Dihydroxybenzaldehyde 3,4-Dihydroxybenzaldehyde (DOPAL (DOPAL)) and and its quinone its quinone with withamines amines and thiols and thiols[131]. [Note131]. that Note the that reaction the reaction with thiols with thiolsrequires requires oxidation oxidation to the to quinone the quinone form. form.

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5.2. Covalent Binding with DNA 5.2. Covalent Binding with DNA DNA is also susceptible to modification by o-quinones. Cavalieri et al. [58] showed that dopaminequinone,DNA is also susceptibleformed with to tyrosinase, modification horser by oadish-quinones. peroxidase, Cavalieri or liver et al.microsomes, [58] showed reacted that dopaminequinone,with DNA to form formed the depurinating with tyrosinase, dopamine horseradish adducts, peroxidase, dopamine-6-N3Ade or liver microsomes, and dopamine-6- reacted with DNAN7Gua, to formalbeit the at depurinatinglow yields (Figure dopamine 12). adducts,This type dopamine-6-N3Ade of DNA modification and would dopamine-6-N7Gua, lead to mutation albeit of atDNA low initiating yields (Figure cancer 12 and). This other type diseases of DNA such modification as Parkinson’s would disease lead in to the mutation case of dopaminequinone of DNA initiating cancer[133]. o and-Quinones other diseases from estradiol, such as Parkinson’s estradiol-3,4-quinone disease in the and case estradiol-2,3-quinone, of dopaminequinone [133 were]. o -Quinonesshown to fromreact estradiol,with DNA estradiol-3,4-quinone to form the depurinating and estradiol-2,3-quinone, adducts [134]. Estradiol-3,4-quin were shownone to react afforded with DNAthe 1-N3Ade to form theand depurinating 1-N7Gua adducts, adducts whereas [134]. Estradiol-3,4-quinone estradiol-2,3-quinone a ffaffordedorded the the 1-N3Ade 6-N3Ade and adduct 1-N7Gua (Figure adducts, 26). whereasEstradiol-3,4-quinone estradiol-2,3-quinone was found aff toorded be much the 6-N3Ade more reactive adduct than (Figure estradiol-2,3-quinone. 26). Estradiol-3,4-quinone Interestingly, was foundthe former to be reacted much more through reactive the o than-quinone estradiol-2,3-quinone. while the latter through Interestingly, the quinone the former methide reacted tautomer. through It theis possibleo-quinone that while this DNA the lattermodification through by the estradiol quinone quinones methide might tautomer. lead to It breast, is possible prostate, that thisand DNAother modificationhuman cancers by [134]. estradiol quinones might lead to breast, prostate, and other human cancers [134].

Figure 26. Reaction of β-estradiol-estradiol quinones with DNA to pr produceoduce depurinating products [[134].134]. 6. Melanogenesis in Relation to o-Quinone Chemistry 6. Melanogenesis in Relation to o-Quinone Chemistry 6.1. Early Stages of Mixed Melanogenesis 6.1. Early Stages of Mixed Melanogenesis Melanogenesis is a complex pathway leading to the production of the dark brown to black eumelaninMelanogenesis and the yellow is a complex to reddish-brown pathway pheomelaninleading to the [1 production,135–141]. The of earlythe dark stages brown of eumelanin to black productioneumelanin startingand the fromyellow tyrosine to reddish-brown were established pheomela by thenin pioneer [1,135–141]. works ofThe Raper early [142 stages] and of Mason eumelanin [143], andproduction are thus starting called “Raper-Mason from tyrosine pathway”,were establis whilehed those by the of pioneer pheomelanin works production of Raper [142] were and elucidated Mason by[143], Prota, and Nicolaus, are thus and called collaborators “Raper-Mason in the latepathway” 1960s [,135 while–137 those,144]. of However, pheomelanin most of production natural melanin were pigmentselucidated are by actually Prota, Nicolaus, co-polymers and or collaborators mixtures of eumelaninin the late and1960s pheomelanin [135–137,144]. in varying However, ratios most [145 of] andnatural thus melanin melanin pigments production are actually can be consideredco-polymers as or “mixedmixtures melanogenesis” of eumelanin and [1,146 pheomelanin]. Therefore, in avarying unified ratios scheme [145] for mixedand thus melanogenesis melanin producti was proposedon can be to accountconsidered for howas “mixed this complex melanogenesis” process is chemically[1,146]. Therefore, regulated. a unified scheme for mixed melanogenesis was proposed to account for how this complexThe earlyprocess stages is chemically of mixed melanogenesisregulated. are now well elucidated using the technique called pulse radiolysisThe early [17,147 stages]. Pulse of mixed radiolysis melanogenesis is a powerful are toolnow to well study elucidated the chemical using fate the of technique highly reactive called pulse radiolysis [17,147]. Pulse radiolysis is a powerful tool to study the chemical fate of highly melanin precursors. Pulse radiolysis of N2O-saturated KBr solution produces dibromine radical reactive melanin precursors. Pulse radiolysis of N2O-saturated KBr solution produces dibromine anions Br2− within 1 nanosecond. Addition of dopa to this system produces dopasemiquinone which − thenradical disproportionates anions Br2 within to 1 generate nanosecond. dopaquinone Addition andof dopa dopa to within this system around produces a millisecond dopasemiquinone (Figure 27). Thenwhich the then fate disproportionates of dopaquinone can to begenerate followed dopa by spectrophotometryquinone and dopa inwithin the presence around ora absencemillisecond of a target(Figure molecule 27). Then such the asfate cysteine. of dopaquinone can be followed by spectrophotometry in the presence or absence of a target molecule such as cysteine.

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Figure 27. Kinetics of early stages of mixed melanogenesis. Note that the rate constants r1–r4 are Figure 27. Kinetics of early stages of mixed melanogenesis. Note that the rate constants r1–r4 are controlled by the intrinsic chemical reactivity of dopaquinone (DQ) [17,148–150]. controlled by the intrinsic chemical reactivity of dopaquinone (DQ) [17,148–150]. 1 When cysteine is not present, the first step (r1 = 3.8 s− ) in eumelanogenesis proceeds through the −1 intramolecular,When cysteine 1,4-Michael is not additionpresent, ofthe the first amino step group (r1 = to3.8 produce s ) in eumelanogenesis cyclodopa (leucodopachrome) proceeds through [148]. the intramolecular, 1,4-Michael addition of the amino group to produce1 cyclodopa This cyclization is a base-catalyzed reaction as shown by the rate constants of 0.91 s− at pH 6.6 and (leucodopachrome)1 [148]. This cyclization is a base-catalyzed reaction as shown by the rate constants 7.6 s− at pH 7.6, because the amino group needs to be a non-protonated –NH2 [149]. As cyclodopa is −1 −1 formed,of 0.91 s it isat rapidlypH 6.6 oxidizedand 7.6 s by at dopaquinone pH 7.6, because to form the dopachromeamino group through needs to a redoxbe a non-protonated exchange giving – NH2 [149]. As cyclodopa6 1 is1 formed, it is rapidly oxidized by dopaquinone to form dopachrome dopa (r2 = 5.3 10 M− s− )[150]. When cysteine is present, the first step in pheomelanogenesis through a redox× exchange giving dopa (r2 = 5.3 × 106 M−1 s−1) [150]. When cysteine is present, the7 first1 proceeds very quickly through the intermolecular, 1,6-Michael addition of cysteine (r3 = 3 10 M− step1 in pheomelanogenesis proceeds very quickly through the intermolecular, 1,6-Michael× addition s− )[149]. The second step in pheomelanogenesis is the redox exchange with dopaquinone to generate of cysteine (r3 = 3 × 107 M−1 s−1) [149]. The second step in pheomelanogenesis5 1 is1 the redox exchange cysteinyldopa quinone, which proceeds at a slower rate (r4 = 8.8 10 M− s− )[148]. It should be × 5 emphasizedwith dopaquinone that the to chemicalgenerate cysteinyldopa reactivity of dopaquinone quinone, which controls proceeds these at foura slower reactions rate (r4 through = 8.8 × 10 an −1 −1 intramolecularM s ) [148]. It addition should be of emphasized amino group, that an intermolecularthe chemical reactivity addition of of dopaquinone sulfhydryl group, controls and twothese redox four exchangereactions reactions.through an From intramolecular these kinetic data,addition an “Index of amino of Divergence” group, an (D) interm betweenolecular eumelanogenesis addition of andsulfhydryl pheomelanogenesis group, and cantwo nowredox be derived.exchange By reacti takingons. dopachrome From these and kinetic cysteinyldopa data, an quinone“Index asof representativesDivergence” (D) of between those two eumelanogenesis pathways, Land and et al. pheo [150melanogenesis] proposed the can formula: now be derived. By taking dopachrome and cysteinyldopa quinone as representatives of those two pathways, Land et al. [150] proposed the formula: D = r3 r4 [cysteine]/r1 r2. × × D = r3 × r4 [cysteine]/r1 × r2. This leads to a “crossover value” (i.e., for D = 1) for switching between eumelanogenesis to pheomelanogenesisThis leads to a when“crossover cysteine value” concentration (i.e., for D at the= 1) site for ofswitching melanogenesis between reaches eumelanogenesis 0.8 µM. to pheomelanogenesis when cysteine concentration at the site of melanogenesis reaches 0.8 µM. 6.2. Late Stages of Mixed Melanogenesis 6.2. Late Stages of Mixed Melanogenesis Dopachrome accumulated during early stages of eumelanogenesis. The orange-red pigment dopachromeDopachrome is fairly accumulated stable having during a half-life early stages of about of eumelanogenesis. 30 min (first-order The rate orange-red constant pigment of 4.0 4 1 × 10dopachrome− s− ). It spontaneously is fairly stable decomposes having a half-life to give of mostly about 5,6-dihydroxyindole 30 min (first-order (DHI)rate constant by decarboxylation of 4.0 × 10−4 ats−1 neutral). It spontaneously pH values (Figure decomposes 28). The to ratiogive ofmostly DHI to5,6-dihydroxyindole DHICA produced in (DHI) the absence by decarboxylation of dopachrome at tautomeraseneutral pH values (DCT) or(Figure metal 28). cations The is ratio 70:1 of [151 DHI]. However, to DHICA in theproduced presence in ofthe DCT absence (Tyrp2), of dopachrome undergoestautomerase tautomerization (DCT) or metal to preferentiallycations is 70:1 produce [151]. However, DHICA [ 152in ,153the]. presence Chemically, of DCT dopachrome (Tyrp2), solutiondopachrome prepared undergoes by ferricyanide tautomerization oxidation to of prefer DL-dopaentially afforded produce DHI DHICA at pH 6.5 [152,153]. and DHICA Chemically, at pH 13, whichdopachrome is a basis solution of preparation prepared of thoseby ferricyanide dihydroxyindoles oxidation at sub-gramof DL-dopa scales afforded in ca. 70%DHI yields at pH [138 6.5, 154and]. DHICA at pH 13, which is a basis of preparation of those dihydroxyindoles at sub-gram scales in ca. 70% yields [138,154].

Int. J. Mol. Sci. 2020, 21, 6080 22 of 36 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 22 of 35

Figure 28. Reaction of dopachrome to produce 5,6-dihydroxyindole (DHI) and Figure 28. Reaction of dopachrome to produce 5,6-dihydroxyindole (DHI) and 5.6-dihydroxyindole- 5.6-dihydroxyindole-2-carboxylic acid (DHICA) and roles of dopachrome tautomerase (DCT) and Cu2+ 2-carboxylic acid (DHICA) and roles of dopachrome tautomerase (DCT) and Cu2+ ions [102,151– ions [102,151–153,155]. 153,155]. Ito et al. [155] examined in detail the effects of pH and Cu2+ ions on the conversion of dopachrome Ito et al. [155] examined in detail the effects of pH and Cu2+ ions on the conversion of to DHI and DHICA and their subsequent oxidation to form eumelanin. The results indicate that acidic dopachrome to DHI and DHICA and their subsequent oxidation to form eumelanin. The results pHs suppress these late stages of eumelanogenesis and Cu2+ ions greatly accelerate the conversion of indicate that acidic pHs suppress these late stages of eumelanogenesis and Cu2+ ions greatly dopachrome to DHICA (and hence increase the ratio of DHICA to DHI) and its subsequent oxidation. accelerate the conversion of dopachrome to DHICA (and hence increase the ratio of DHICA to DHI) The conversion of dopachrome to DHI or DHICA was first suggested to involve the quinone methide and its subsequent oxidation. The conversion of dopachrome to DHI or DHICA was first suggested intermediate by Sugumaran’s group [103,141]. Sugumaran and Semensi [102] examined the enzymatic to involve the quinone methide intermediate by Sugumaran’s group [103,141]. Sugumaran and conversion of dopachrome, dopachrome methyl ester, and α-methyldopachrome methyl ester. In the Semensi [102] examined the enzymatic conversion of dopachrome, dopachrome methyl ester, and α- presence of a dopachrome-converting factor isolated from the hemolymph of insect, Manduca sexta, methyldopachrome methyl ester. In the presence of a dopachrome-converting factor isolated from dopachrome was converted to DHI while dopachrome methyl ester was tautomerized to DHICA the hemolymph of insect, Manduca sexta, dopachrome was converted to DHI while dopachrome methyl ester. At a weakly alkaline pH of 8.0 without any enzyme, a-methyldopachrome methyl methyl ester was tautomerized to DHICA methyl ester. At a weakly alkaline pH of 8.0 without any ester produced a stable quinone methide tautomer [100]. However, at the physiological pH value, enzyme, a-methyldopachrome methyl ester produced a stable quinone methide tautomer [100]. the enzyme catalyzed this reaction [102]. These results conclusively proved the transient production However, at the physiological pH value, the enzyme catalyzed this reaction [102]. These results of quinone methide tautomer during conversion of dopachrome to DHI (Figure 28). Formation of conclusively proved the transient production of quinone methide tautomer during conversion of the same quinone methide intermediate during the conversion of dopachrome to DHICA is similarly dopachrome to DHI (Figure 28). Formation of the same quinone methide intermediate during the likely, but it has not yet been demonstrated. conversion of dopachrome to DHICA is similarly likely, but it has not yet been demonstrated. Then, how can DHI and DHICA be oxidized finally to give rise to eumelanin? The redox Then, how can DHI and DHICA be oxidized finally to give rise to eumelanin? The redox exchange between DHI and dopaquinone was found to proceed but not quite to completion with a exchange between DHI and 6dopaquinone1 1 was found to proceed but not quite to completion with a rate constant of r5 = 1.4 10 M− s− [156] (Figure 29). This is in the same rate as the rate constants rate constant of r5 = 1.4 ×× 106 M−1 s−1 [156] (Figure 29). This is in the same rate as the rate constants with cyclodopa (r2) and with 5SCD (r4). The reaction with DHICA is much slower (r6 = 1.6 105 5 −1 with1 cyclodopa1 (r2) and with 5SCD (r4). The reaction with DHICA is much slower (r6 = 1.6 × 10× M M− s− ) and does not go to completion. It thus appears that during eumelanogenesis, DHI oxidation s−1) and does not go to completion. It thus appears that during eumelanogenesis, DHI oxidation proceeds by redox exchange with dopaquinone but DHICA oxidation may require its oxidation to the proceeds by redox exchange with dopaquinone but DHICA oxidation may require its oxidation to quinone form catalyzed by tyrosinase [157] or tyrosinase-related protein 1 [158,159]. DHI quinone and the quinone form catalyzed by tyrosinase [157] or tyrosinase-related protein 1 [158,159]. DHI quinone DHICA quinone can readily dimerize with the parent DHI and/or DHICA to give homodimers and and DHICA quinone can readily dimerize with the parent DHI and/or DHICA to give homodimers heterodimers (for a heterodimer, see [160]). Various oligomers up to the stage of tetramers have been and heterodimers (for a heterodimer, see [160]). Various oligomers up to the stage of tetramers have isolated and characterized in the form of acetylated derivatives for DHI or non-derivatized form for been isolated and characterized in the form of acetylated derivatives for DHI or non-derivatized form DHICA [161–163]. for DHICA [161–163]. The late stages of pheomelanogenesis beyond cysteinyldopa (CD) quinones are summarized The late stages of pheomelanogenesis beyond cysteinyldopa (CD) quinones are summarized in in Figure 30. Conversion of cysteinyldopa to its dehydrated form, the dihydro-1,4-benzothiazine-3- Figure 30. Conversion of cysteinyldopa to its dehydrated form, the dihydro-1,4-benzothiazine-3- carboxylic acid (DHBTCA), takes place under biomimetic conditions [164,165]. Interestingly, carboxylic acid (DHBTCA), takes place under biomimetic conditions [164,165]. Interestingly, during during tyrosinase oxidation of dopa in the presence of equimolar cysteine, cysteinyldopa isomers are tyrosinase oxidation of dopa in the presence of equimolar cysteine, cysteinyldopa isomers are the the first major products that are then dehydrated to give DHBTCA isomers [166]. DHBTCA (isomers) first major products that are then dehydrated to give DHBTCA isomers [166]. DHBTCA (isomers) accumulates transiently and then decay rapidly in the course of pheomelanogenesis. accumulates transiently and then decay rapidly in the course of pheomelanogenesis.

Int. J. Mol. Sci. 2020, 21, 6080 23 of 36 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 35

Figure 29. Kinetics of late stages of eumelanogenesis from DHI and DHICA. Note that dopaquinone Int. J. Mol.Figure Sci. 29.2020 Kinetics, 21, x FOR of PEERlate stages REVIEW of eumelanogenesis from DHI and DHICA. Note that dopaquinone 24 of 35 (DQ) acts again here as a redox exchanger [156]. (DQ) acts again here as a redox exchanger [156].

Then how is DHBTCA produced and then decayed? Kinetic experiments using the pulse radiolysis technique [148,149,167] showed that: (1) cysteinyldopa accumulates in the early stage of pheomelanogenesis because the addition reaction of cysteine to dopaquinone producing cysteinyldopa (r3) proceeds much faster than the redox exchange of cysteinyldopa with dopaquinone giving cysteinyldopa quinone (r4), (2) cysteinyldopa is gradually oxidized to cysteinyldopa quinone through the redox exchange with dopaquinone only after cysteine concentration becomes low, (3) the intramolecular cyclization of cysteinyldopa quinone produces the o-quinone imine through Schiff base formation (r7 = 10 s−1) [168], and (4) the quinone imine then rapidly rearranges with/without decarboxylation to afford the 1,4-benzothiazine (BT) and the 1,4-benzothiazine-3-carboxylic acid (BTCA)(r8 = 6.0 s−1)[168]. When the rate of production of cysteinyldopa quinone (and hence the quinone imine) is low as in the case of tyrosinase-catalyzed oxidation, the quinone imine is reduced Figure 30. Kinetics of late stages of pheomelanogenesis from 5-S-cysteinyldopa (5SCD) quinone [166]. to DHBTCAFigure 30. by Kinetics the excess of late cysteinyldopa, stages of pheomelanogenesis which in turn from is oxidized 5-S-cysteinyldopa back to cysteinyldopa (5SCD) quinone quinone. [166]. It Note that dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) is produced via redox exchange shouldNote be that stressed dihydro-1,4-benzothiazine-3-c that the redox exchangearboxylic of dopaquinone acid (DHBTCA) with isDHBTCA produced to via produce redox exchange the quinone while benzothiazine intermediates are generated by rearrangement of quinone imine intermediate iminewhile (and benzothiazine dopa) is an additionalintermediates role are of generated dopaquinon by rearrangemente [166]. This reactionof quinone acts imine as a intermediate driving force in (with/without decarboxylation). Rate constants are from Napolitano et al. [167]. Note that dopaquinone this stage(with/without of pheomelanogenesis. decarboxylation). Rate constants are from Napolitano et al. [167]. Note that chemicallydopaquinone controls chemically pheomelanogenesis controls pheomelanogenesi at a critical stages at a of critical DHBTCA stage oxidation. of DHBTCA oxidation. The last stage of pheomelanogenesis is the oxidative polymerization of BT and BTCA, which eventually leads to the production of pheomelanin (Figure 30). This process appears to be very 6.3. MetabolicThen how Fates is DHBTCAof Catecholamine produced Quinones and then decayed? Kinetic experiments using the pulse radiolysiscomplex [169] technique but involves [148,149 ,167secondary] showed modification that: (1) cysteinyldopas of the benzothiazine accumulates moiety in the leading early stage to the of pheomelanogenesisformationNeuromelanin of the 3-oxo-3,4-dihydro-1,4-benzothiazi becauseis a brown the addition to black-brown reaction of cysteineinsone luble(ODHBT) to melanin-like dopaquinone and the benzothiazole producingpigment present cysteinyldopa (BZ) in [170]. the (r3)midbrainThese proceeds intermediates, of humans much faster andBT (andother than BTCA), theprimates redox ODHBT, and exchange is knownand of BZ, cysteinyldopa to areaccumulate gradually with during incorporated dopaquinone aging [2,21,176]. into giving the cysteinyldopaDopaminepheomelanin and polymer quinonenorepinephrine [166]. (r4), (2)Using are cysteinyldopa considered chemical degradativeto isbe graduallythe major approach precursors oxidized [166,171,172], toof neuromelanin cysteinyldopa we were present quinone able into throughtheshow substantia that the the redox nigra benzothiazine exchange and locus with coeruleus moiety dopaquinone graduallyof the brain, only degrades respectively after cysteineto form [2,176 concentrationa benzothiazole,177]. However, becomes moiety biosynthesis low, in the (3) thelast of intramolecularthesestage ofneuromelanin pheomelanogenesis. cyclization appears of cysteinyldopaThisa complex is consistent process quinone with and the produces out observation of the the scopeo-quinone by Napolitanoof this imine review. throughet al. Here,[170] Schi thatweff summarize briefly what1 are known about the metabolic fates of dopaminequinone and basethe chromophore formation (r7 =of10 pheomelanin s− )[168], and (λmax (4) thearound quinone 305 iminenm) is then better rapidly characterized rearranges by with a prevalent/without decarboxylationnorepinephrinequinone.presence of the benzothiazole to afford the moiety 1,4-benzothiazine (λmax at 303 nm) (BT) than and the the dihydrobenzothiazine 1,4-benzothiazine-3-carboxylic (BT and BTCA) acid It has been a matter1 of debates whether tyrosinase plays a significant role in the production of (BTCA)(r8moiety (λmax= 6.0above s− )[340168 nm).]. When Recent the studies rate of have production shown that of cysteinyldopa melanogenesis quinone is suppressed (and hence at acidic the quinoneneuromelaninpH in melanosomes, imine) [178]. is low However, the as in organelles the caseit is ofvery specialized tyrosinase-catalyzed likely that in melaninthe first oxidation,synthesisstep of the thein neuromelanin melanocytes quinone imine [173,174].production is reduced The is tothecatalytic DHBTCA oxidation activities by of thecatecholamines of excess human cysteinyldopa, tyrosinase to o-quinone are which progre derivatives inssively turn isthrough suppressed oxidized interaction back by lowering to cysteinyldopawith redoxthe pH active [174], quinone. metal with Itionsa shouldshift such to be aas stressedmore Fe3+ andpheomelanic that Cu2+ the [2,3,129,176]. redox phenotype exchange Reactivity [173]. of dopaquinone Re ofcently, dopaminequinone we with compared DHBTCA with the to effectsmall produce thiolsof pH the andon quinone mixedthiol- imineproteinsmelanogenesis (and has dopa) been in isextensivelytyrosinase-catalyzed an additional studied role [3,129]. of oxidation dopaquinone Bec auseof dopa dopaminequinone [166 or]. Thistyrosine reaction in thecyclizes acts absence as amore driving or slowlypresence force than inof thisdopaquinone,cysteine stage [175]. of pheomelanogenesis. itThe binds results to proteins showed throughthat pheomelanin the cysteinyl production residues wasmore greatest efficiently at pH [123,129]. values of On 5.8 the to other6.3, whileThe hand, last eumelanin stagein the of pheomelanogenesisproductionabsence of wasthiols, suppressed isdopaminequinone the oxidative at pH 5.8 polymerization compared cyclizes toand pH of BTis6.3–7.3. oxidized and BTCA,This tosuggests whichform eventuallydopaminechromethat mixed leadsmelanogenesis to in the nearly production is quantitative chemically of pheomelanin shiftedyield [129,179] to more (Figure pheomelanic(Figure 30 ).31). This Jimenez states process at et weakly al. appears [179] acidic showed to pH. be very Thisthat complextyrosinasefits well with [169 oxidation] the but kinetic involves of dopamine data secondary for the proceeds cyclization modifications in a of si 5SCDmilar of quinonemanner the benzothiazine to thethat quinone of dopa, moiety imine i.e., leading cyclization(QI), which to the of is formationdopaminequinonepromoted ofby theacid 3-oxo-3,4-dihydro-1,4-benzothiazine (r7followed = 15 s−1 at pHby 5.6redox and 3.3exchange s−1 at pH (ODHBT) 7.7;of [149]).the and resulting the benzothiazole cyclodopamine (BZ) [170with]. Thesedopaminequinone intermediates, to BT form (and dopaminechrome. BTCA), ODHBT, and Dopamine BZ, are graduallychrome is incorporated then slowly intopolymerized the pheomelanin to give polymerrise to dopamine [166]. Using melanin chemical via DHI degradative [67]. In addition approach to the [166 cyclization,171,172], wepathway, were ablein the to presence show that of H2O2, tyrosinase-catalyzed oxidation of dopamine gave rise to 6-hydroxydopamine in several percent yield [67]. In a model experiment, peroxidase/H2O2 oxidation of N-acetyldopamine gave up to 55% yield of the 2-hydroxy-1,4-benzoquinone derivative (Figure 31). The slow rate of cyclization of dopaminequinone as compared to norepinephrinequinone has been shown by spectrophotometry [179,180] and by cyclic voltammetry [47]. This may possibly lead to the generation of the more reactive quinone methide tautomer of dopamine, which should give rise to secondary products including norepinephrine through the addition of water molecule [67]. However, this aspect of dopaminequinone chemistry has not been examined in detail. In this relation, when dopamine is oxidized by tyrosinase, the yield of dopamine melanin is not high, less than 50% [33], whereas the yield of dopa melanin is nearly quantitative [138]. It is possible that dopaminequinone and/or dopaminechrome may give rise to various secondary, soluble metabolites, similar to those from NE (see below). Certainly, more studies appear necessary to clarify these aspects of dopamine oxidation. In this regard, it should be mentioned that in recent years polydopamine formed by oxidation of dopamine at pH 8.5 has drawn great interests from material scientists because of its unique properties

Int. J. Mol. Sci. 2020, 21, 6080 24 of 36 the benzothiazine moiety gradually degrades to form a benzothiazole moiety in the last stage of pheomelanogenesis. This is consistent with the observation by Napolitano et al. [170] that the chromophore of pheomelanin (λmax around 305 nm) is better characterized by a prevalent presence of the benzothiazole moiety (λmax at 303 nm) than the dihydrobenzothiazine (BT and BTCA) moiety (λmax above 340 nm). Recent studies have shown that melanogenesis is suppressed at acidic pH in melanosomes, the organelles specialized in melanin synthesis in melanocytes [173,174]. The catalytic activities of human tyrosinase are progressively suppressed by lowering the pH [174], with a shift to a more pheomelanic phenotype [173]. Recently, we compared the effect of pH on mixed melanogenesis in tyrosinase-catalyzed oxidation of dopa or tyrosine in the absence or presence of cysteine [175]. The results showed that pheomelanin production was greatest at pH values of 5.8 to 6.3, while eumelanin production was suppressed at pH 5.8 compared to pH 6.3–7.3. This suggests that mixed melanogenesis is chemically shifted to more pheomelanic states at weakly acidic pH. This fits well with the kinetic data 1 for the cyclization of 5SCD quinone to the quinone imine (QI), which is promoted by acid (r7 = 15 s− 1 at pH 5.6 and 3.3 s− at pH 7.7; [149]).

6.3. Metabolic Fates of Catecholamine Quinones Neuromelanin is a brown to black-brown insoluble melanin-like pigment present in the midbrain of humans and other primates and is known to accumulate during aging [2,21,176]. Dopamine and norepinephrine are considered to be the major precursors of neuromelanin present in the substantia nigra and locus coeruleus of the brain, respectively [2,176,177]. However, biosynthesis of these neuromelanin appears a complex process and out of the scope of this review. Here, we summarize briefly what are known about the metabolic fates of dopaminequinone and norepinephrinequinone. It has been a matter of debates whether tyrosinase plays a significant role in the production of neuromelanin [178]. However, it is very likely that the first step of the neuromelanin production is the oxidation of catecholamines to o-quinone derivatives through interaction with redox active metal ions such as Fe3+ and Cu2+ [2,3,129,176]. Reactivity of dopaminequinone with small thiols and thiol-proteins has been extensively studied [3,129]. Because dopaminequinone cyclizes more slowly than dopaquinone, it binds to proteins through the cysteinyl residues more efficiently [123,129]. On the other hand, in the absence of thiols, dopaminequinone cyclizes and is oxidized to form dopaminechrome in nearly quantitative yield [129,179] (Figure 31). Jimenez et al. [179] showed that tyrosinase oxidation of dopamine proceeds in a similar manner to that of dopa, i.e., cyclization of dopaminequinone followed by redox exchange of the resulting cyclodopamine with dopaminequinone to form dopaminechrome. Dopaminechrome is then slowly polymerized to give rise to dopamine melanin via DHI [67]. In addition to the cyclization pathway, in the presence of H2O2, tyrosinase-catalyzed oxidation of dopamine gave rise to 6-hydroxydopamine in several percent yield [67]. In a model experiment, peroxidase/H2O2 oxidation of N-acetyldopamine gave up to 55% yield of the 2-hydroxy-1,4-benzoquinone derivative (Figure 31). The slow rate of cyclization of dopaminequinone as compared to norepinephrinequinone has been shown by spectrophotometry [179,180] and by cyclic voltammetry [47]. This may possibly lead to the generation of the more reactive quinone methide tautomer of dopamine, which should give rise to secondary products including norepinephrine through the addition of water molecule [67]. However, this aspect of dopaminequinone chemistry has not been examined in detail. In this relation, when dopamine is oxidized by tyrosinase, the yield of dopamine melanin is not high, less than 50% [33], whereas the yield of dopa melanin is nearly quantitative [138]. It is possible that dopaminequinone and/or dopaminechrome may give rise to various secondary, soluble metabolites, similar to those from NE (see below). Certainly, more studies appear necessary to clarify these aspects of dopamine oxidation. In this regard, it should be mentioned that in recent years polydopamine formed by oxidation of dopamine at pH 8.5 has drawn great interests from material scientists because of its unique properties to adhere to all type of surfaces [181,182]. However, structural basis of polydopamine has not been fully clarified as it is so for dopamine melanin [183,184]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 25 of 35 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 25 of 35 to adhere to all type of surfaces [181,182]. However, structural basis of polydopamine has not been to adhere to all type of surfaces [181,182]. However, structural basis of polydopamine has not been Int.fully J. Mol.clarified Sci. 2020 as, 21it ,is 6080 so for dopamine melanin [183,184]. 25 of 36 fully clarified as it is so for dopamine melanin [183,184].

Figure 31. 31. ReactionReaction of dopaminequinone of dopaminequinone leading leading to the to production the production of dopamine of dopamine melanin melaninthrough Figure 31. Reaction of dopaminequinone leading to the production of dopamine melanin through dopaminechrome [129,179]. Note that oxidation of dopamine with peroxidase/H2O2 gives 6- through dopaminechrome [129,179]. Note that oxidation of dopamine with peroxidase/H2O2 gives dopaminechrome [129,179]. Note that oxidation of dopamine with peroxidase/H2O2 gives 6- 6-hydroxydopaminehydroxydopamine after after reduction reduction [67]. [67 ]. hydroxydopamine after reduction [67]. The reactivity of norepinephrinequinone remain remainss less studied than that of dopaquinone and The reactivity of norepinephrinequinone remains less studied than that of dopaquinone and dopaminequinone. JimenezJimenez etet al.al. [[180]180] showedshowed that that tyrosinase tyrosinase oxidation oxidation of of norepinephrine norepinephrine proceeds proceeds in dopaminequinone. Jimenez et al. [180] showed that tyrosinase oxidation of norepinephrine proceeds ain similar a similar manner manner to thatto that of dopa of dopa and and dopamine dopamine but withbut with a faster a faster rate ofrate cyclization. of cyclization. Terland Terland et al. [et185 al.] in a similar manner to that of dopa and dopamine but with a faster rate of cyclization. Terland et al. compared[185] compared reactivity reactivity of one-electron of one-electron oxidation oxidation products ofproducts catecholamines of catecholamines produced by produced ferricyanide by [185] compared reactivity of one-electron oxidation products of catecholamines produced by oxidation.ferricyanide The oxidation. rate of formation The rate of of formation the corresponding of the corresponding aminochromes aminochr was foundomes to was be dopaminefound to be< ferricyanide oxidation. The rate of formation of the corresponding aminochromes was found to be norepinephrinedopamine < norepinephrine< epinephrine. < Manini epinephrine. et al. [186 Manini] examined et al. the [186] fate examined of o-quinone the produced fate of o by-quinone Fenton dopamine <2+ norepinephrine < epinephrine.2+ Manini et al. [186] examined the fate of o-quinone reagentproduced (Fe by -EDTAFenton/ Hreagent2O2) and (Fe horseradish-EDTA/H2O peroxidase2) and horseradish/H2O2. The peroxidase/H cyclization2O pathway2. The cyclization proceeds, produced by Fenton reagent (Fe2+-EDTA/H2O2) and horseradish peroxidase/H2O2. The cyclization givingpathway norepinephrinechrome proceeds, giving norepinephrinechrome and ultimately norepinephrine and ultimately melanin. norepinephrine In addition, they melanin. were able In pathway proceeds, giving norepinephrinechrome and ultimately norepinephrine melanin. In toaddition, detect 3,4-dihydroxymandelicthey were able to detect acid, 3,4-dihydr 3,4-dihydroxybenzaldehyde,oxymandelic acid, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, and3,4- addition, they were able to detect 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, 3,4- 3,4-dihydroxyphenylglyoxylicdihydroxybenzoic acid, and acid3,4-dihydroxyphenylglyoxylic in about 50% combined yields acid (Figure in about 32). It50% would combined be interesting yields dihydroxybenzoic acid, and 3,4-dihydroxyphenylglyoxylic acid in about 50% combined yields to(Figure examine 32). theIt would metabolic be interesting fate of norepinephrinequinone to examine the metabolic produced fate of norepinephrinequinone by tyrosinase to exclude produced the effect (Figure 32). It would be interesting to examine the metabolic fate of norepinephrinequinone produced ofby Htyrosinase2O2. to exclude the effect of H2O2. by tyrosinase to exclude the effect of H2O2.

Figure 32. ReactionReaction norepinephrinequinone norepinephrinequinone [186]. [186 ].The Thequinone quinone methide methide pathway pathway leads leads to the to theFigureproduction production 32. Reactionof 3,4-dihydroxymandelic of 3,4-dihydroxymandelic norepinephrinequinone acid acid(D [186].OMA), (DOMA), Th 3,4-dihydroxybenzaldehydee quinone 3,4-dihydroxybenzaldehyde methide pathway (DHBAld), leads (DHBAld), to 3,4-the 3,4-dihydroxybenzoicproductiondihydroxybenzoic of 3,4-dihydroxymandelic acid acid (DHBA), (DHBA), and and 3,4-dihydroxyphenylglyoxylic acid 3,4-dihydroxyphenylglyoxylic (DOMA), 3,4-dihydroxybenzaldehyde acid. acid. DHBAld DHBAld and and(DHBAld), DHBA DHBA arise 3,4- fromdihydroxybenzoic DOMA (see FigureFigureacid (DHBA), 1717).). Another and 3,4-dihydroxyphenylglyoxylic pathway leads to the productionproduction acid. ofof DHBAld norepinephrinenorepinephrine and DHBA melanin arise throughfrom DOMA norepinephrinechrome. (see Figure 17). Another pathway leads to the production of norepinephrine melanin through norepinephrinechrome. Oxidation of epinephrine () affords a red pigment, called adrenochrome (Figure 33). Oxidation of epinephrine (adrenaline) affords a red pigment, called adrenochrome (Figure 33). There were considerable interests in the detection of this aminochrome in various tissues in the ThereOxidation were considerable of epinephrine interests (adrenaline) in the detection affords of a this red aminochrome pigment, called in variousadrenochrome tissues in(Figure the 1960s 33). 1960s [187]. Adrenochrome seems to be the most stable among various aminochromes at neutral There[187]. wereAdrenochrome considerable seems interests to be in the detectionmost stab ofle this among aminochrome various aminochromes in various tissues at inneutral the 1960s pH pH [188,189] but at alkaline pH it gradually rearranges to give a highly fluorescent adrenolutin [187].[188,189] Adrenochrome but at alkaline seems pH itto gradually be the most rearranges stable toamong give avarious highly fluoreaminochromesscent adrenolutin at neutral (3,5,6- pH (3,5,6-trihydroxy-N-methylindole) [190]. The subsequent metabolic fate remains little known. [188,189]trihydroxy- butN -methylindole)at alkaline pH [190].it gradually The subsequent rearranges metabolic to give afate highly remains fluore littlescent known. adrenolutin Because (3,5,6- of its Becausetrihydroxy- of itsN-methylindole) stability, adrenochrome [190]. The subsequent can be quantified metabolic in biological fate remains samples little known. by various Because methods of its including HPLC-photodiode array [189].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 26 of 35 stability,Int. J. Mol. Sci.adrenochrome2020, 21, 6080 can be quantified in biological samples by various methods including HPLC-26 of 36 photodiode array [189].

Figure 33. Reaction of epinephrinequinone [190]. [190]. The The pr productoduct adrenochrome gradually rearrange to adrenolutin (3,5,6-trihydroxy- N-methylindole). 7. Conclusions 7. Conclusions Tyrosinase is able to catalyze oxidation of phenols and catechols (o-diphenols) to o-quinones. Tyrosinase is able to catalyze oxidation of phenols and catechols (o-diphenols) to o-quinones. In In this review, we summarized chemical fates of o-quinones. Some of the reactions of o-quinones this review, we summarized chemical fates of o-quinones. Some of the reactions of o-quinones proceed extremely fast with a half-life of less than one second. Some reactions proceed only after proceed extremely fast with a half-life of less than one second. Some reactions proceed only after tautomerization to the quinone methide form. Because of their high reactivity, o-quinones are able tautomerization to the quinone methide form. Because of their high reactivity, o-quinones are able to to modify the structure of proteins and DNA, leading possibly to cytotoxicity and carcinogenesis. modify the structure of proteins and DNA, leading possibly to cytotoxicity and carcinogenesis. Dopaquinone plays pivotal roles in melanogenesis; it acts chemically in several key steps including the Dopaquinone plays pivotal roles in melanogenesis; it acts chemically in several key steps including redox exchange with cyclodopa to form dopachrome in the early stages of eumelanin production and the redox exchange with cyclodopa to form dopachrome in the early stages of eumelanin production the addition of cysteine to initiate pheomelanin production. and the addition of cysteine to initiate pheomelanin production. During the preparation of this review, the authors have noticed some discrepancies in metabolic During the preparation of this review, the authors have noticed some discrepancies in metabolic fates of o-quinones from one study to another. Although most of those discrepancies may arise from fates of o-quinones from one study to another. Although most of those discrepancies may arise from differences in the substrate concentration and the pH of the medium, more studies certainly remain to differences in the substrate concentration and the pH of the medium, more studies certainly remain be performed with rigorous identification of the products. to be performed with rigorous identification of the products. Author Contributions: S.I. and K.W. established the concept of this review and wrote it together. M.S. critically Authorexamined Contributions: it and added S.I. some and essential K.W. established comments. the All concept authors of have this readreview and and agreed wrote to it the together. published M.S. version critically of examinedthe manuscript. it and added some essential comments. All authors have read and agreed to the published version of theFunding: manuscript.This research received no external funding. Funding:Conflicts ofThis Interest: researchThe received authors no declare external no funding. conflictof interest. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations

AbbreviationsBT 1,4-Benzothiazine BTCA 1,4-Benzothiazine-3-carboxylic acid BT 1,4-Benzothiazine BQ Dihydro-1,4-benzothiazine-6,7-dione BTCA 1,4-Benzothiazine-3-carboxylic acid BZ Benzothiazole BQ Dihydro-1,4-benzothiazine-6,7-dione CD quinone Cysteinyldopaquinone BZ Benzothiazole DBL 3,4-Dihydroxybenzalacetone CD quinone Cysteinyldopaquinone DCT Dopachrome tautomerase, Tyrp2 DBL 3,4-Dihydroxybenzalacetone DHBA 3,4-Dihydroxybenzoic acid DCT Dopachrome tautomerase, Tyrp2 DHBAlc 3,4-Dihydroxybenzylalcohol DHBA 3,4-Dihydroxybenzoic acid DHBAldDHBAlc 3,4-dihydroxybenzaldehyde3,4-Dihydroxybenzylalcohol DHBTCADHBAld 3,4-dihydroxybenzaldehyde Dihydro-1,4-benzothiazine-3-carboxylic acid DHIDHBTCA 5,6-DihydroxyindoleDihydro-1,4-benzothiazine-3-carboxylic acid DHICADHI 5,6-Dihydroxyindole-2-carboxylic5,6-Dihydroxyindole acid DihydrocaDHICA ffeic acid 3-(3,4-Dihydroxyphenyl)propionic5,6-Dihydroxyindole-2-carboxylic acid acid DOMADihydrocaffeic acid 3,4-Dihydromandelic3-(3,4-Dihydroxyphenyl)propionic acid acid DOPACDOMA 3,4-Dihydr 3,4-Dihydroxyphenylaceticomandelic acid acid DOPALDOPAC 3,4-Dihydroxyphenylacetic 3,4-Dihydroxybenzaldehyde acid DOPEDOPAL 3,4-Dihydroxybenzaldehyde 3,4-Dihydroxyphenylethanol DOPEGDOPE 3,4-Dihydroxyphenylethanol 3,4-Dihydroxyphenylethyleneglycol DOPEG 3,4-Dihydroxyphenylethyleneglycol DQ Dopaquinone

Int. J. Mol. Sci. 2020, 21, 6080 27 of 36

DQ Dopaquinone NAC N-Acetylcysteine ODHBT 3-Oxo-3,4-dihydro-1,4-benzothiazine 4SCAP 4-S-Cysteaminylphenol 2SCD 2-S-cysteinyldopa 5SCD 5-S-cysteinyldopa

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