P-quinone Methide Research Articles

Oxidation of 4-alkylphenols and catechols by tyrosinase: ortho-substituents alter the mechanism of quinoid formation

Numerous phenols and catechols are known to be substrates for tyrosinase. While the catalytic mechanism of phenol oxidation by tyrosinase has been well studied, little work has been done to determine the influence of substituents on the reaction. In the present investigation, we explored the effects of changing substituents at the 2 and 6 position on the mechanism of tyrosinase-catalyzed oxidation of 4-allyl and 4-propylphenols and catechols. We have previously demonstrated that tyrosinase initially oxidizes hydroxychavicol (4-allyl-catechol) to an o-quinone (3,5-cyclohexadien-1,2-dione) which because of the relatively acidic protons in the benzyl position, readily isomerizes to the tautomeric p-quinone methide (4-allylidene-2,5-cyclohexadien-1-one, QM) (Bolton et al., 1994). We have confirmed through GSH trapping studies that oxidation of 4-allylphenol by tyrosinase yields the same o-quinone GSH conjugates as hydroxychavicol. In contrast, the presence of additional ortho substituents dramatically alters the mechanism of tyrosinase-catalyzed oxidation of 4-alkylphenols. For example, eugenol (4-allyl-2-methoxyphenol), which possesses 1 ortho-methoxy substituents, is not oxidized to a o-quinone or a QM. However, when both ortho positions are substituted with methoxy groups, direct tyrosinase-catalyzed QM formation is observed. The identity of the 4-allyl-2,6-dimethoxyphenol-QM was confirmed by CDCl 3 extraction of the post incubate and characterization of the QM by 1H-NMR. Finally, the oxidation rates were determined by following the appearance of either the o-quinone or the QM spectrophotometrically or by monitoring increases in the GSH trapped conjugates by HPLC. The data showed that the tyrosinase-catalyzed rates of oxidation decreased in the following order; hydroxychavicol > 4-allylphenol > 4-allyl-6-methoxycatechol > 4-allyl-2,6-dimethoxyphenol ⪢ eugenol (no quinoids detected). These results show that changes in the substituents ortho to the phenolic hydroxy group not only lead to an alteration in the mechanism and type of quinoid formed, there is also a dramatic substituent effect on the rate of tyrosinase oxidation.

Skin sensitization to eugenol and isoeugenol in mice: possible metabolic pathways involving ortho-quinone and quinone methide intermediates.

With the aim of providing further mechanistic insights into the mode of action of eugenol (4-allyl-2-methoxyphenol) and isoeugenol (4-propenyl-2-methoxyphenol), we have synthesized two series of modified compounds which were tested in the mouse local lymph node assay for their skin sensitizing potential. The replacement of the methoxy group by an isopropoxy group led to a complete loss of sensitization for the eugenol derivative 6a, while no significant effect was observed for the isoeugenol derivative 6b. In the eugenol series, when methyl groups were present in the 3-, 5-, or 6-position a significant reduction in sensitization potential was observed while in the isoeugenol series only methyl substitution in the 3- and 5-position had a discernable effect. Introduction of three methyl groups on the aromatic ring of eugenol (3,5,6-trimethyl-4-allyl-2-methoxyphenol, 7) and of a tert-butyl substituent at the gamma-position of the alkyl chain of isoeugenol (4-[3',3',3'-trimethylpropenyl]-2-methoxyphenol, 8) led to a strong decrease of the sensitizing capacity. Our findings indicate that, at least in the mouse, eugenol could sensitize via a demethylation pathway followed by oxidation to the o-quinone which could act directly as a hapten even if we cannot exclude a reaction via its tautomeric p-quinone methide. Isoeugenol, on the other hand, could act via a mechanism not involving demethylation and for which the evidence is consistent with a direct oxidation to the p-quinone methide.

P-quinone methides as geometric analogues of quinolone carboxylate antibacterials

In an attempt to examine the role of the N(1)-alkyl or -aryl moiety of the quinolone antibacterials, several quinone methides such as perinaphthindenone carboxylates ( 6 ) were synthesized as 1-carba(sp 2) analogues of the quinolone structure, and found to have no antibacterial activity. These results together with literature information are interpreted as the necessity of at least one nitrogen atom at the position of 1, 3, 4a, 6 or 8 in the quinolone-like skeleton for the antibacterial activities.

1995 Merck Frosst Award Lecture Quinone methides: relevant intermediates in organic chemistry

ortho and para-Quinone methides (2-methylene-3,5-cyclohexadien-1-one and 4-methylene-2,5-cyclohexadien-1-one, respectively) are intermediates in a variety of important chemical systems. In particular, o-quinone methides are useful in synthesis for the construction of chroman ring systems. A brief account of the relevance of quinone methide chemistry will be provided. This is followed by a review of recent studies from our laboratory on efficient methods for the photogeneration of quinone methides, concentrating on the use of hydroxy-substituted benzyl alcohols in aqueous media. It is shown that this method is general since it provides access to o-, m-, and p-quinone methide isomers. When appropriately substituted, all of these quinone methide isomers have been spectroscopically characterized by laser flash photolysis, making this technique the one of choice for studying the dynamics of these reactive intermediates. The mechanism of photochemical generation from hydroxybenzyl alcohols and extensions of the reaction to photogeneration of fluorenyl and biphenyl quinone methides will also be presented. Key words: quinone methide, biphenyl quinone methide, carbocation, photosolvolysis, photodehydroxylation, hetero-Diels–Alder reaction.

Mechanism of isomerization of 4-propyl-o-quinone to its tautomeric p-quinone methide.

In previous work, we showed that o-quinones (3,5-cyclohexadiene-1,2-diones) can isomerize to p-quinone methides (4-alkyl-2,5-cyclohexadien-1-one) at rates which depend on the type of substituent at the para position [Iverson, S. L., Hu, L. Q., Vukomanovic, V., and Bolton, J. L. (1995) Chem. Res. Toxicol. 8, 537-544]. In the present investigation, we explored the mechanism of this isomerization reaction using 4-propyl-3,5-cyclohexadiene-1,2-dione (PQ) and its benzyl dideuterio analog 4-(1',1'-dideuteriopropyl)-3,5-cyclohexadiene-1, 2-dione (DPQ). The results show that the isomerization reaction is general base-catalyzed, which suggests that amino acids on proteins with basic side chains could catalyze the reaction in vivo. The Bronsted beta value was determined to be 0.23 +/- 0.02, consistent with the transfer of a proton in the rate-determining step. The rate/pH profile generated from the buffer dilution plots showed dependence on hydroxide ion concentration from pH 7.8 to 9, indicative of base catalysis. From pH 6 to 7.8, the reaction was independent of pH, suggesting that other processes compete at low buffer concentration in this pH region. Substitution of the benzyl CH2 group with CD2 dramatically slows the isomerization reaction. The kinetic deuterium isotope effect on quinone methide formation was determined by measuring the amount of quinone methide trapped as GSH conjugates from PQ compared with DPQ. The isotope effect on product formation was 5.5 +/- 0.6, 37 degrees C. These data provide further evidence that formation of these electrophilic quinone methides from o-quinones could be catalyzed by basic residues in vivo and that the reaction could be inhibited by deuterium substitution at the benzyl methylene group.

P-Quinone methides are the major decomposition products of catechol estrogen o-quinones.

The mechanism of catechol estrogen-induced carcinogenesis could involve alkylation of critical cellular macromolecules by electrophilic quinoids. The o-quinones formed from peroxidase/P450-catalyzed oxidation of catechol estrogens have previously been implicated as the ultimate carcinogens. In the present study, we have shown that additional reactive intermediates can be produced from isomerization of the catechol estrogen o-quinones to highly electrophilic p-quinone methides (QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. The GSH adducts were isolated and characterized by 1H-NMR, UV, and electrospray mass spectrometry. The o-quinone of 2-hydroxyestrone isomerized to two QMs; a QM stabilized by one alkyl substituent in the B ring, 2-OHE-QM1 (3-hydroxy-1-(10),3(4),5(6)-oestratrien-2,17-dione) and one having two alkyl substituents on the methylene group in the C ring, 2-OHE-QM2 (2-hydroxy-1(2),4(5),9(10)-oestratrien-3,17-dione). Only one QM was observed from the o-quinone of 4-hydroxyestrone, 4-OHE-QM2 (4-hydroxy-1(2),4(5),9(10)- oestratrien-3,17-dione) which is analogous to the C ring analog (2-OHE-QM2) from the o-quinone of 2-hydroxyestrone. The GSH adduct of 4-OHE-QM2 decomposed at pH 7.4 to give 9(11)-dehydro-4-hydroxyestrone as the major product. Finally, the disappearance of the estrogen o-quinone GSH adducts correlated with the formation of the GSH conjugates of the QMs. These data suggest that in cells with low levels of GSH, the formation of these potent electrophiles represents the major reaction pathway for estrogen o-quinones. The implications of the o-quinone/QM pathway for the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Influence of glutathione on the oxidation chemistry of the catecholaminergic neurotransmitter dopamine

In this study it is demonstrated that dopamine- o-quinone (DA- o-quinone), generated by the electrochemically driven oxidation of the catecholaminergic neurotransmitter dopamine (DA) at physicological pH, is rapidly scavenged by glutathione (GSH) to give, initially, 5- S-glutathionyldopamine (5- S-Glu-DA). The latter conjugate is more easily oxidized than DA to an o-quinone that reacts further with free GSH to give 2,5-bi- S-glutathionyldopamine ( 6), an even more easily oxidized compound. The proximate oxidation product of 6, o-quinone ( 7), is the precursor of 2,5,6-tri- S-glutathionyldopamine ( 8) and 4,7-bi- S-glutathionyl-5,6,dihydroxyindole ( 16). A p-quinone methide tautomer of o-quinone 7 is the precursor of glutathionyl conjugates of DA which contain a glutathionyl residue substituted in the ethylamino side-chain of the neurotransmitter. In the presence of equimolar or greater concentrations of GSH the normal oxidation pathway of DA to insoluble black indolic melanin polymer is blocked. The potential relevance of the in-vitro oxidation of DA in the presence of free GSH to an understanding of the degeneration of nigrostriatal dopaminergic neurons in idiopathic Parkinson's disease is discussed.

STABLE QUINONE METHIDES II: ADDITION OF THIOLS TO QUINONE METHIDES, SYNTHESIS OF BISALKYLTHIOSUBSTITUTED QUINONE METHIDES AND TRITHIO-ORTHOBENZOATES

Abstract Regioselective oxidation of the p-alkylthiomethylphenols 1 with K3[Fe(CN)6] to the monosubstituted p-quinone methides 2 followed by addition of thiols R3SH gives rise to the dithioacetals 3.1 This reaction sequence can be extended to introduce a third RS substituent by repeating the above mentioned reactions. Spectroscopic data, especially 1H-NMR and 13C-NMR data of the quinone methides 4 and the new trithioorthoesters 5 are discussed. The same regioselectivity as for the oxidation of la 1 is observed when compounds with two different activated benzylic positions e.g. 3a and 3b are oxidized with K1[Fe(CN)6]. The sequence 1 → 2 → 3 → 4 → 5 constitutes a new approach to trithio-orthoesters.

Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols.

The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycols and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred. Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the C gamma atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation. The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa < 6, 25 degrees C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcohol-oxidase-catalyzed conversion of 4-allylphenols.

Direct Evidence for Quinone-Quinone Methide Tautomerism during Tyrosinase Catalyzed Oxidation of 4-Allylcatechol

The hapatotoxicity of safrole and related compounds has been attributed to the electrophilic compounds generated from the oxidation of these chemicals. In this paper, for the first time using quasi pre-steady state conditions, we present direct evidence for the rapid production of electrophilic allyl o-quinone and its tautomerization to more stable and reactive vinyl p-quinone methide during both the enzyme catalyzed and chemical mediated oxidation of 4-allylcatechol, an in vivo product of safrole metabolism.

The influence of the p-alkyl substituent on the isomerization of o-quinones to p-quinone methides: potential bioactivation mechanism for catechols.

Previously, we have shown that an additional bioactivation pathway for the hepatocarcinogen safrole (1-allyl-3,4-(methylenedioxy)benzene) exists which may contribute to its toxic effects: initial O-dealkylation of the methylenedioxy ring, forming the catechol, hydroxychavicol (HC, 1-allyl-3,4-dihydroxybenzene), 2-electron oxidation to the o-quinone (4-allyl-3,5-cyclohexadien-1,2-dione), and isomerization, forming the more electrophilic p-quinone methide (2-hydroxy-4-allylidene-2,5-cyclohexadien-1-one) [Bolton, J. L., Acay, N. M., & Vukomanovic, V. (1994) Chem. Res. Toxicol. 7, 443-450]. In the present investigation, we explored the effects of changing pi-conjugation at the 4-position on both the rate of isomerization of the initially formed o-quinones to the QMs and the reactivity of the quinoids formed from 4-propylcatechol (1), 2,3-dihydroxy-5,6,7,8-tetrahydronaphthalene (2), and 4-cinnamylcatechol (3). We selectively oxidized the catechols to the corresponding o-quinones or p-quinone methides and trapped these reactive electrophiles with glutathione (GSH). The GSH adducts were fully characterized by UV, NMR, and mass spectrometry. Microsomal incubations with the parent catechols in the presence of glutathione produced only o-quinone glutathione conjugates. However, if the trapping agent (GSH) was added after an initial incubation time, both o-quinone and p-quinone methide GSH conjugates were observed. The results indicate that extended pi-conjugation at the para position enhances the rate of isomerization of the o-quinone to the quinone methide. Thus the half-life of the o-quinones decreased in the following order: the o-quinone of 1 > 2 > HC > 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective benzylic deprotonation in the enzymatic rearrangement of N-acetyldopamine derived o-quinone to the p-quinone methide

( R)- N-acetyl-[ β- 2H 1]-dopamine (R)- 1 undergoes an enzyme catalyzed oxidative tautomerization to quinone methide 3 with loss of the benzylic H Si , as proven by isolation of deuteriated rac. N-acetylnoradrenaline ( 4), while (S)-[β- 2 H 1]- 1 loses the deuterium label. Thus, the o-quinone- p-quinone methide isomerization is a stereoselective enzymatic reaction.

Stable Quinome Methides: Regioselective Para-Oxidation of a 2,4-Bisalkylthiomethenol and Addition Reaction Reaction to Quinonemethides

Abstract The 2.4-bisfunctionalized phenol 1, a commercial antioxidant, is dehydrogenated regioselectively with potassium ferricyanide. affording the corresponding p-quinone methide 2. 1,6-Addition of nucleophiles e.g. thiols to 2 gives rise to the corresponding addition products e.g. the dithioacetals 4 of the corresponding substituted benzaldehyde. On the other hand, treatment of 2 with αα′-azoisobutyronitrile at 90°C leads to compounds 5a-b and 6, addition products of the cyanopropyl radical to the quinone methide 2. The structures of all products are confirmed mainly by 1H-NMR-and 13C-NMR-spectroscopy and the mode of their formation is discussed.

Characterisation of electron beam generated transformation products of irganox 1010 by particle beam liquid chromatography-mass spectrometry with on-line diode array detection

Transformation products of Irganox 1010 [pentaerythritol tetrakis-3-(3,5-di- tert.-butyl-4-hydroxyphenyl) propionate], in food contact polymers subjected to electron beam irradiation have been analysed by particle beam LC-MS with on-line UV diode array detection. It has been shown that the principal transformation products arise via loss of sub-units of the parent molecule, and the subsequent transformation of these sub-units. A total of 56 transformation products has been detected, for some of which structures are proposed. These results are compared to previous work on Irganox 1330 [1,3,5-trimethyl-2,4,6-tris(3′,5′-di- tert.-butyl-4-hydroxybenzyl)benzene], where transformations to yield p-quinone methide type structures were shown to occur.

Functionalized depsipeptides, substrates and inhibitors of β-lactamases and DD-peptidases

A series of derivatives of phenyl phenylacetylglycinates (aryl phenaceturates) with a carboxylate substituent meta to the oxygen of the phenoxide leaving group and a functionalized methylene group in the ortho- or para-position have been synthesized. These molecules possess a latent o- or p-quinone methide electrophile which could be unmasked during enzymic turnover and could react with an active site nucleophile. This chemistry does seem to occur in solution where a common hydrolysis product, independent of the benzylic leaving group, presumably o- or p-hydroxymethylphenol, was observed. These depsipeptides are substrates of class A and C β-lactamases, particularly of the latter, comparable with the parent m-carboxyphenyl phenaceturate. They also have modest inhibitory activity against these enzymes and against the serine DD-peptidase of Streptomyces R61. The inhibition of a class C β-lactamase was turnover dependent, as expected of mechanism-based inhibitor, but the small leaving group dependence of the inhibition suggested that the quinone methide, if it was in fact responsible for the inhibition, was generated in solution subsequent to release of the product phenol from the active site.

Mechanism of p-quinone methide initiated cyclization reactions terminated by alkenes: 1,2- vs. 1,3-hydrogen migration

Intramolecular cyclization of benzylic cations derived from p-quinone methides 5 and 6, afforded the corresponding six- or seven-membered ring products 7 or 9 depending on the substitution of the alkene. Deuterium labeling experiments indicated that formation of 9 occurred via a 1,3-hydrogen shift, whereas formation of 7 proceeded via two sequential 1,2-hydride transfers.

Evidence that 4-allyl-o-quinones spontaneously rearrange to their more electrophilic quinone methides: potential bioactivation mechanism for the hepatocarcinogen safrole.

Several naturally occurring aromatic ethers, of which safrole [1-allyl-3,4-(methylenedioxy)-benzene] is one example, are hepatocarcinogens. One bioactivation pathway previously proposed for safrole involves hydroxylation of the benzyl carbon, conjugation with sulfate, and then alkylation of DNA with displacement of the sulfate group [Miller, J.A., and Miller, E.C. (1983) Br. J. Cancer 48, 1-15]. The fact that safrole is O-dealkylated to the corresponding catechol (hydroxychavicol, 1-allyl-3,4-dihydroxybenzene) indicates that quinoid formation is also possible and may contribute to the genotoxic and/or cytotoxic activity of this compound. In the present investigation we selectively oxidized hydroxychavicol to the corresponding o-quinone (HC-quinone, 4-allyl-3,5-cyclohexadiene-1,2-dione) or p-quinone methide (HC-QM, 2-hydroxy-4-allylidene-2,5-cyclohexadien-1-one) and trapped these reactive electrophiles with glutathione (GSH). The GSH adducts were fully characterized by UV, NMR, and mass spectrometry. Microsomal incubations with safrole or hydroxychavicol in the presence of glutathione produced only o-quinone glutathione conjugates. However, if the trapping agent (GSH) was added after an initial incubation of 10 min, both o-quinone and p-quinone methide GSH conjugates were observed. The first-order rate constant of isomerization was estimated from the decrease in HC-quinone GSH adducts to be 1.9 x 10(-3) s-1 (t1/2 = 9 min). Kinetic studies showed that while HC-QM reacts rapidly with water, the model o-quinone (4-tert-butyl-3,5-cyclohexadiene-1,2-dione), which cannot isomerize to a quinone methide, was remarkably resistant to hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzymic formation and chemical reactivity of quinone methides correlate with alkylphenol-induced toxicity in rat hepatocytes

The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM's; 4-methylene-2,5-cyclohexadien-1-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM's were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxy-tert-butyl)], BDMP (2-tert-butyl-6-methyl), BMP (2-tert-butyl), TMP (2,6-dimethyl), and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P450.min) in rat liver microsomes and 16-62 pmol/(10(6) cells.min) in isolated rat hepatocytes. QM's derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and TMP (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM's, strongly support the proposal that QM's mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.

On regioselectivity of the reaction of p-quinone methides with p-hydroxybenzyl alcohols

1H NMR analysis of the reaction of lignin model p-quinone methides with aliphatic alcohols, phenols and p-hydroxybenzyl alcohols in chloroform solution has demonstrated a pronounced predominance of non-cyclic benzyl alkyl ethers over benzyl aryl ethers in the mixture of products, as a result of faster addition of alcohols than phenols. In the case of the simplest p-quinone methides, the only competing reaction seems to be a stereoselective isomerization into E-p-hydroxystyrenes.

Cuticle-catalyzed coupling between N-acetylhistidine and N-acetyldopamine

Several types of insect cuticle contain enzymes catalyzing the formation ofof adducts between N-acetyldopamine (NADA) and N-acetylhistidine (NAH). Two such adducts, NAH-NADA-I and NAH NADA-II, have been isolated and their structures determined. In one of the adducts the link connecting the two residues occurs between the I-position (ß-position) in the NADA side chain and the 1-N atom (τ-N) in the imidazole ring of histidine. Diphenoloxidase activity alone is not sufficient for formation of this adduct, whereas extracts containing both diphenoloxidase and o-quinone- p-quinone methide isomerase activities catalyze the coupling reaction. The adduct consists of a mixture of two diastereomers and they are presumably formed by spontaneous reaction between enzymatically produced NADA- p-quinone methide and N-acetylhistidine. The other adduct has been identified as a ring addition product of N-acetylhistidine and NADA. In contrast to the former adduct it can be formed by incubation of the two substrates with mushroom tyrosinase alone. An adduct between N-acetylhistidine and the benzodioxan-type NADA-dimer is produced in vitro, when the N-acetylhistidine-NADA adduct is incubated with NADA and locust cuticle containing a 1,2-dehydro-NADA generating enzyme system. Trimeric NADA-polymerization products of the substituted benzodioxan-type have been obtained from in vivo sclerotized locust cuticle, confirming the ability of cuticle to produce NADA-oligomers. The results indicate that some insect cuticles contain enzymes promoting linkage of oxidized NADA to histidine residues. It is suggested that histidine residues in the cuticular proteins can serve as acceptors for oxidized NADA and that further addition of NADA-residues to the phenolic groups of bound NADA can occur, resulting in formation of protein-linked NADA-oligomers. The coupling reactions identified may be an important step in natural cuticular sclerotization.