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Chemical Modifications of Biopolymers by Quinones and Quinone Methides.

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Chemical Modifications of Biopolymers by Quinones
and Quinone Methides
By Martin G. Peter*
Many quinones and their precursors, which are transformed oxidatively into quinones and/or
quinone methides, are important natural products. As secondary metabolites, they frequently
possess antibiotic and cytotoxic activities, in addition to acting sometimes as pathogens.
Several plants and animals, especially insects, use quinonoid substances for defense, often with
spectacular results. On the macromolecular level, quinone methides have a key role in the plant
kingdom in lignin biosynthesis; the biosynthesis of melanoproteins exemplifies the reactions
of o-quinones in the animal kingdom. In insects, cross-linking of structural proteins through
quinones and quinone methides results in the construction of the sclerotized exoskeleton. For
mankind, the reactivity of quinones in biological systems has far-reaching consequences of
pharmaceutical, toxicological, and technical relevance. The examples in this review show that
a common principle underlies these reactions, namely, the chemical modification of biopolymers. As demonstrated particularly well in a more detailed discussion of the chemical principles of insect cuticle sclerotization, several major and important new results have emerged
from the development and applications of modern methods of sample separation and from
solid-state NMR spectroscopy.
1. Introduction
Quinones, quinone methides, and often their phenolic precursors are widely distributed in nature. A large number of
phenolic and quinonoid compounds occur as secondary
metabolites,[’’ and numerous examples are given in reviews
funon natural products of plants,[2. 31 marine
gi,‘’’ and insects.[61This review covers the irreversible reactions of quinonoids in biochemical environments. Thus,
quinonoids that participate in reversible primary metabolic
processes, such as electron transport, are not described here.
The term “quinonoids” is meant to include 0- and p quinones in addition to any reactive intermediates that can
form upon oxidation of a diphenol.
Quinonoids often show pronounced biological activities,
such as antibiotic, cytotoxic, or allergenic actions, and they
may serve the organism producing them as a weapon for
defense. The biological activity of many quinonoids consists
ultimately in chemical modification and denaturation of biopolymers, thereby damaging the exposed organism. When
we look, however, at the organism that produces phenols
together with the appropriate oxidative enzymes, interaction
of quinones or quinone methides with macromolecules is
usually beneficial, as exemplified by the biosynthesis of
melanoproteins in animals, lignopolysaccharides in plants,
and sclerotins in insects. One example of a useful technical
application of protein-quinone interactions is the tanning of
animal hides in the production of leather.
However, undesired and even deleterious results of protein-quinone interactions are exemplified by the toxicity of
certain xenobiotic phenolic substances, which can eventually
cause allergies, liver damage, or cancer. Aberrant metabolism of phenolic compounds may also have pathological
consequences. Another unfavorable effect of quinonoid sub[*] Prof. Dr. M. G. Peter
Institut fur Organische Chemie und Biochemie der Universitlt
Gerhard-Domagk-Strasse 1, D-5300 Bonn 1 (FRG)
Angew.
C%rm. h i . Ed. Engl. 28 (1989) 5SS-570
8 VCH
stances is the decrease in the nutritional value of vegetable
and fruit foodstuffs in the presence of plant phenols or tannins.
An understanding of the chemical principles of quinone
reactions in biological systems is of great importance, not
only for finding new technical applications or developing
methods for interfering to our benefit with undesired and
deleterious effects of quinones, but also for gaining insight
into fundamental processes of life.
Our views on the reactions of quinonoids in biological
systems are mostly derived from the results of studies in
preparative or mechanistic organic chemistry. A large variety of diphenols and oxidants as well as quinones and nucleophiles have been employed in organic synthesis for the construction of quinone derivatives, and the great preparative
value of the various reactions is amply documented in several
excellent re~iews.1~1‘
In contrast, studies on the reactions of quinonoid compounds in biological systems have rarely led to an unambiguous structural characterization of the reaction products.
One of the reasons is immediately obvious to the preparative
organic chemist: the primary reaction products are unstable
themselves. They readily undergo secondary reactions which
are difficult, if not impossible, to control in an aqueous environment. Some ideas on the pathways of secondary reactions
can be derived from the structures of new, unexpected products that again have been discovered during synthetic or
mechanistic studies, and several examples will be discussed in
this review. Besides this more classical approach, many significant discoveries have emerged from the availability of
advanced methods of product separation and modern spectroscopic techniques including ESR as well as high-field and
solid-state NMR spectroscopy.
An evaluation of our present knowledge on the reactions
of quinones and quinone methides in biological systems will
be useful for a critical assessment of the molecular aspects of
Verlagsge.wkhaJi mhH, D-6940 Wrinheim, 19x9
0570-0833~X9j0SOS-OS3S
$02.5010
555
their biochemical behavior and may also help to clarify some
of the remaining ambiguities. The first part of this review
provides a general description of the reactions of quinones,
including a few selected examples of obvious biological relevance. A brief review on polyphenol-biopolymer complexes
is then followed by a critical evaluation of the structural and
biological aspects of chemical modification of proteins,
polysaccharides, and nucleic acids. Radical reactions are not
treated separately. They will be mentioned when there is
evidence for the participation of radical intermediates in biopolymer modifications. Finally, the mechanisms of insect
cuticle sclerotization will be discussed as a particularly puzzling example of the fascinating roles of quinone-biopolymer interactions in nature.
2. General Principles
2.1. Biological Oxidation of Phenols
The enzyme-catalyzed oxidations that lead to quinones
and quinone methides from 4-alkyl 1,2-diphenols and 4-alkyl monophenols are summarized in Scheme 1 .I1'.
2-AIkyl 1,4-diphenols react analogously.
,
R = O H
@
R ' = H, -C-
.
H
H
R'
conjugation with the phenoxyl radical, a p-quinone methide
radical is one of the possible resonance structures. When the
side chain is saturated, radical abstraction is required for
quinone methide formation (Scheme 1; pathway @
Per-I).
oxidase/H,O, oxidation of diphenols leads to semiquinone
radicals, and the corresponding radical anions of o-diphenols have been observed by ESR spectroscopy, again as complexes with divalent metal ions." 3s Semiquinone radicals
can also form through one-electron reductions of quinones
by several flavoenzymes, such as NADPH-cytochrome P450 reductase. Reduction of oxygen by semiquinone radicals
yields superoxide anion radicals."
A quinone-mediated
shuttle of electrons from superoxide to hydrogen peroxide
then results in the formation of hydroxyl radicals.['*' A description of the mechanisms of protein modification by the
highly cytotoxic and mutagenic oxygen radicals [ I 9 ] is beyond the scope of this review.
Finally, quinone methides may form through oxidative
introduction of a leaving group into the side chain and subsequent elimination of H L (Scheme 1 ; pathway @). This
reaction sequence is important, for example, in the
metabolism of certain monophenolic xenobiotics. It starts
usually with a hydroxylation mediated by cytochrome P-450.
Quinones can form under physiological conditions also in
nonenzymatic reactions. Autooxidation of a phenolic compound leads to a phenoxyl or to a semiquinone radical that
can add molecular oxygen. The quinol hydroperoxide then
decays to the corresponding quinone.120T*'I
Quinones and quinone methides are electrophilic species
that will react with a variety of nucleophiles by vinylogous
addition. Phenoxyl and semiquinone radicals, besides being
intermediates in quinone methide formation, are likely to
react further by phenolic coupling. Both nucleophilic addition and oxidative phenolic coupling are also fundamental
processes in the polymerization of phenolic compounds.
peroxidase
2.2. Nucleophilic Addition
The vinylogous, Michael-type addition of a nucleophile to
the a, p-unsaturated carbonyl system of a quinone is a wellknown reaction (Scheme 2).
The enzymes involved in the primary oxidation step are of
three principal types. Tyrosinase (monophenol monooxygenase; EC 1.14.18.1) hydroxylates a monophenol in the o-position. This enzyme also has diphenoloxidase activity, transforming an o-diphenol into an o-quinone by two-electron
transfer. Semiquinone radical anions are detectable by ESR
spectroscopy in aqueous solution when the oxidation is performed in the presence of diamagnetic metal ions." 3 , 14'
These radicals are generated by comproportionation of an
o-quinone and excess o-diphenol.1'3. 15] Both 0-and p-diphenols are also oxidized by diphenoloxidases of the laccase
type, which catalyze one-electron transfers. Tautomerization
of the alkylquinones leads to the corresponding quinone methides (Scheme 1, pathway @).
Peroxidase/H,O,-catalyzed oxidation of monophenols
yields phenoxyl radicals in a one-electron transfer. When the
side chain of a 4-alkyl monophenol is unsaturated and in
556
Scheme 2 . R
= H,alkyl
The primary addition product is a substituted o-diphenol
which, in the presence of an oxidant (e.g., excess quinone)
will yield a substituted o-quinone. Depending on the reaction
conditions and the type of nucleophile, further reactions may
follow; if R = H, for example, a second Michael addition
followed by an oxidation may lead to a bis-substituted
quinone. According to the terminology that has been created
by Wan~lick,'~'
such reaction sequences are classified as
A-D-A-D, where A and D mean addition and dehydrogenation, respectively. The overall pathway applies to p-quinones
and to quinone methides in analogous ways.
Angew. Chem. I n t . Ed. Engl. 28 (f989)555-570
The addition of nucleophiles to quinones and quinone
methides occurs in biological systems usually as a spontaneous, nonenzymatic reaction. The nucleophiles, available
from functional groups of polypeptides, polysaccharides,
and nucleic acids, are thiols, thioethers, primary and secondary amines, and hydroxyl groups. The biological relevance of Scheme 2 is illustrated in the following with a few
examples of reactions which have led to structurally defined
products.
Oxidation of Dopa by means of tyrosinase in the presence
ofcysteine yields a mixture of the 1,4- and 1,6-thiol-addition
products 1-4,fZ2]which are important intermediates in
phaeomelanin biosynthesis (see Section 3). The main component (74 O h ) is the 1,6-addition product 5-cysteinyl-Dopa 1. However, Kalyanamaran et al.[231observed by ESR
spectroscopy only the 1,4-addition product of cysteine to
4-methyl-o-benzoquinone, generated by periodate oxidation
of 4-methylcatechol at pH 3.0 in the presence of ZnZe ions.
Vinylogous thiol addition to 2-methyl-I ,4-naphthoquinone
(menadione) leads to semiquinone radical anions even in the
absence of spin-stabilizing metal ions.[241
'2H
1-1
5
R 2 = H. R3 = SCH,CH(NH?)COOe
R 3 = H, R2 = SCH,CH(NHf')COOe
= SCH,CH(NHy)COOe, R 2 = R3 = H
4: R' = R 3 = SCH,CH(NHy)COOe, R2 = H
1: R'
2: R'
3: R'
=
=
Biogenetically related to bis(cysteiny1)-Dopa 4 is the naturally occurring adenochromine 5, which presumably is a precursor of adenochromes, the iron-binding pigments of Octopus vulgaris. Degradation of adenochrome revealed one of
the components to be the unusual amino acid 5-mercaptohistidine linked to a Dopa
ThioIs may also add to quinone methides, as has been
demonstrated conclusively through isolation of the adduct 6,
formed by addition of cysteine to the quinone methide generated from 2,6-di-tert-butyl-4-methylphenol
(Scheme 3; cf.
Scheme 1, pathway @).I261
6
Scheme 3
Primary and secondary amines yield, in general, addition
products according to Scheme 2 (for reviews, see Refs. [7, 9,
101). Owing to the basicity of the amino nitrogen, the
reaction is strongly pH dependent. Reaction of aniline
(pK, = 4.6) with o-benzoquinone in glacial acetic acid to
form the bis-addition product was first observed in 1913.f271
The same product is also obtained in aqueous buffer at physAngew. Chem. Int. Ed. Engl. 28 (1989) 555--570
iological pH when catechol is oxidized in situ by means of
tyrosinase in the presence of aniline.[28."1 Semiquinone radical anions are generated in the presence of Zn2@during
addition of aniline to 4-methyl-o-benzoq~inone.~~~~
The addition of an aromatic amine to an o-quinone methide in tautomeric equilibrium with the corresponding
p-quinone is exemplified in Scheme 4 (cf. Scheme I , pathway @). The reaction of 3- or 4-hydroxyaminophenol with
menadione in methanol/acetic acid leads to the classical
Michael-type addition product 7 and to the o-quinone methide adduct tLr301
OH
OH
OH
H
8
7
Scheme 4.
Under aprotic conditions, aliphatic amines (pK, = 9-1 1)
will also lead to A-D-A-D products in appreciable yields. A
classical example is the preparation of 4,5-bis(dimethylamino)-I ,2-benzoquinone in dry acetone.13 Some related
1,4-addition products from the tyrosinase-catalyzed oxidation of 4-methylcatechol in the presence of p-alanine methyl
ester have been isolated, along with Schiff bases, in rather
low yields and characterized by spectroscopic methods.[321
The o-quinone formed in the oxidation of dopamine can
cyclize to 2,3-dihydroindole-5,6-diol.Determination of the
relative rates of intramolecular and intermolecular additions
of amines and thiols to electrochemically generated dopamine-quinone in aqueous buffer at pH 7.4[331has revealed
that the SH group of cysteine reacts ca. two times faster than
that of glutathione and ca. 200 times faster than aniline, the
addition of which is ca. ten times faster than the intramolecular cyclization of dopamine. This again is faster than the
addition of a-amino groups of amino acids by a factor of
about two. The preference for intramolecular cyclization as
compared to intermolecular addition has also been demonstrated by conducting the oxidation of Dopa in the presence
of p - b e n z o q ~ i n o n e . [ ~ ~ ]
A fundamentally different reaction of amines is the nucleophilic 1,2 addition to a quinonoid carbonyl group. A recent
example is the formation of the tetrols 9 from several biogenic amines and 2,5-dihydroxy-p-benzoquinonein ethanolic solution.[351Under the same conditions, histamine adds
with the primary amino rather than with the imidazole nitrogen. Withp-benzoquinone, the usual 2,Sdisubstituted products are generated through the A-D-A-D sequence.
HO
9
R
=
PhCH,CH2, p-HO-C,H,CH,CH,,
4-imidazolyl-CH2CH,
557
The 1,2-carbonyl addition is usually followed by dehydration to the corresponding Schiff base. Thus, the A-D-A-D
condensation product from aniline and a-benzoquinone has
long been k n ~ w n . [ ~ ’ - ~ ’The
] corresponding products 10
have been obtained from o-quinones and aliphatic amines in
methanol[361o r glacial acetic acid,[321although the reaction
mixtures are extremely complex and the yields are sometimes
exceedingly low.
5-methyl-4-pyrrolidino-I,2-benzoquinone in aqueous metha n 0 1 . [ ~In
~ ] this case, the nitrogen atom is most likely derived
from the a-amino acid, which loses its side chain in a Strecker-type degradation sequence.
C02Me
I
B
12
11
10
R = Me, Et, nBu [36], CH,CH,COOCH, [32]
Another way of generating Schiff bases starts with vinylogous 1,4 addition to, for example, an o-quinone, followed
by reoxidation and tautomerization to the 2-hydroxy-pquinone imine. Schiff base formation with aliphatic amines,
particularly a-amino acids, may be followed by Strecker degradation, that is, rearrangement and ultimately deamination.
The rather unusual indole derivative 13 is formed in small
amounts during oxidation of 4-methylcatechol in the presence of p-alanine methyl ester in acetic
Apparently,
Nenitzescu-type addition of the enamine that forms by a
dehydrogenation of j3-alanine methyl ester and nitrogen
addition of another amino ester to the o-quinone in the usual
1,4 fashion is followed by cyclization and deamination.
C02Me
2.3. Formation of Heterocyclic Compounds
13
A considerable variety of heterocyclic compounds are
known to arise from secondary transformations of the initial
quinone and quinone methide addition products. The pertinent literature from the fields of biology and biochemistry
documents that, with the exception of the intermediates in
melanin biosynthesis (see Section 3), little attention has usually been paid to the idea that biopolymer modification may
eventually also occur in such secondary reactions. Since we
cannot a priori exclude the possibility that secondary reactions are biologically important, it is appropriate to present
a few examples of the formation of heterocyclic derivatives
from quinonoid compounds in reactions of possible biological relevance (for a review, see Ref. [37]).
Reaction of p-benzoquinone with o-aminophenol in
glacial acetic acid yields 2-aminophenoxazin-3-one, in addition to higher-condensed heterocyclic ring systems.[3s1 2Aminophenoxazin-3-one is also generated upon oxidation of
o-aminophenol mediated by oxyhemoglobin in human eryt h r o c y t e ~ . [The
~ ~ ’ phenoxazine ring system occurs in nature
as a structural element in the actinomycin antibiotics and in
certain insect pigments, the ommochromes. Their biomimetic preparation has been studied along with the enzymatic
reactions involved in their biosynthesis (for reviews, see
Refs. [7,40,41I). 2-Aminothiophenols yield the corresponding phenothiazinones in an analogous
Phenoxazinones may also form during the reaction of an
o-quinone with an aliphatic amine. Compound I 1 was isolated in low yield after oxidation of catechol in the presence
of p-alanine methyl ester in glacial acetic acid.r321An intermediate in this reaction is obviously the corresponding N alkyl-o-aminophenol, which adds in an A-D-A-D sequence
to a second molecule of o-benzoquinone. Similarly, the phenoxazine salt 12 is prepared from various a-amino acids and
558
1L
More highly substituted o-quinones, such as 3,5-di-tertbutyl-o-benzoquinone, react with primary amines of the type
R-CH,-NH, to yield stable addition/condensation prodIn this case, Schiff base
ucts, such as the benzoxazole 14.f461
formation is followed by rearrangement and intramolecular
oxidative cyclization. Likewise, the reaction of 3,Sdi-tertbutyl-o-benzoquinone with ol-amino acid esters leads to the
corresponding b e n ~ o x a z i n o n e s . ~Addition-condensation
~’~
of glycine ethyl ester with 4-methyl-o-quinone yields a product that has been described as either 15 o r 16.[481
RyJoro
’
H3C
R
15
R
=
16
NHCH,COOEt
Dimerization of 2-alkylamino-p-benzoquinonesleads to
the carbazole derivatives 17, which have been found after
reactions of primary a l k y l a m i n e ~ [or
~ ~glycine
’
ethyl ester[481
with p-benzoquinone.
OH
RHN
0
1
R
17
R
=
alkyl. CH,COOEI
Anxew. Chrm. Inr. Ed. EngI. 28 (1989) 555-570
3. On the Structures of Polyphenol-Biopolymer
Complexes
Polyphenolic biopolymers are in general highly complex
materials and our knowledge of their structures is rather
fragmentary. A tremendous amount of work involving model reactions, degradation, and biosynthetic studies has led to
recognition of partial structures from which overall constitutions have been derived. Though it is not the intention of this
article to review the chemistry of polyphenolic polymers, it
may be useful to recall first some basic facts before considering the interactions of these polymers with other biopolymers. An overview on the principal classes of polyphenolic
polymers is given in Table 1.
the presence of pyrrolecarboxylic acid units that arise from
degradation of indolic entities. ESR spectra of both synthetic
and natural eumelanins reveal the presence of o-semiquinone
radicals in these polymer^.^^^.^^] Quinone methides are not
considered as significant intermediates in eumelanin biosynthesis.
H02C
0-0
Table 1. Classes o f polyphenolic biopolymers
Polymer
Phenolic monomer
eumelanin
Dopa. dopamine, or noradrenaline
Dopa and cysteine
phaeomelanin
allomelanina
lignin
fungal lignin
brown algae tannins
condensed tannins
hydrolyzable tannins
hurntc acids
Ref.
catechols containing no nitrogen
methoxy(propy1)phenoIs
styrylpyrones
phloroglucinol
flavanols
gallic acid and glucose
various
The binding interactions between polyphenolic polymerization products of quinonoids and other biopolymers-i.e.,
proteins, polysaccharides, and nucleic acids-are caused in
principle by the following processes:
1. Covalent trapping of reactive intermediates, such as
quinones, quinone methides, or radicals, during any stage
of the polymerization process. In this case, the complex of
the polyphenol and biopolymer is formed either by continuation of the polyphenolic polymerization or by addition of a functional group of the biopolymer to a reactive
center of a polyphenol.
2. Noncovalent association between the biopolymer and the
polyphenol. In this case, stabilization is achieved predominantly by a multiplicity of hydrogen bonds, in addition to
electrostatic and hydrophobic interactions.
3. Cross-linking of biopolymers by a combination of covalent and noncovalent forces.
Tyrosinase-catalyzed oxidation of tyrosine or Dopa leads
to a highly inhomogeneous black polymer, eumelanin (see
Refs. [50,511 for reviews and definition of various melanins
and Ref. [63] for discussions of the mechanisms of melanin
formation). An intermediate in the polymerization process is
2,3-dihydro-5.6-dihydroxyindole-2-carboxylic acid which
arises from Dopa-quinone by an intramolecular 1,4addition
of the cc-amino group. A generally accepted scheme for the
structure of Dopa-melanin is depicted in Scheme 5.I5O3 5 1 1
The polymer contains C-N, C-C, and C-0 bonds which are
formed through addition of the corresponding nucleophiles
to intermediate o-quinonoid species or by oxidative phenolic
coupling reactions. The structure is further complicated by
Angrw. Chmi. In!. Ed. Engl. 28 (1989) 555-570
0
Scheme 5
Low-molecular-weight tyrosine-containing peptides also
polymerize upon oxidation with tyrosinase.[661Depending
on the position of the amino acid in the peptide sequence,
different types of intermediates are distinguishable by UV
spectroscopy: N-terminal tyrosine yields a Dopa-quinone
residue, whereas tyrosine in the peptide chain or at the C-terminus leads to N-acyl Dopa-quinone units. In contrast to the
eumelanins, which form by oxidative polymerization of free
tyrosine, the polymers of peptidic tyrosine are soluble in
water.
Natural eumelanin is always associated tightly with
protein. When Dopa is oxidized by means of tyrosinase in
the presence of various proteins, cysteinyl-Dopa residues are
isolated after acid hydrolysis of the reaction product.f671Recently, It0 et al. showed that peptide-bound 5-cysteinylDopa residues are formed when proteins are subjected to
oxidation with tyrosinase.[681The mechanism of the reaction
is understood as an enzyme-catalyzed hydroxylation of tyrosine residues followed by further oxidation of the resulting
Dopa residues and nucleophilic addition of peptidic cysteinyl SH groups (Scheme 6).
These results demonstrate conclusively that melanoprotein biosynthesis involves Michael-type 1,6additions of
cysteinyl SH groups to Dopa-quinone, generated by hydroxylation of either free or peptidic tyrosine and subsequent
oxidation of the resulting Dopa residues.
559
HO
45
40-40
S-CyS
co’
HO
1
20
-
Scheme 6.
It is assumed that the structure of dopamine melanin is
similar to that of Dopa melanin. However, application of
CP/MAS I3C N M R spectroscopy has recently revealed that
the polymer of dopamine contains high amounts of aliphatic
carbon atoms.[69]Therefore, in contrast to the formation of
Dopa melanin, the polymerization must occur largely via
dihydroindole and dopamine units.
Oxidation of Dopa in the presence of cysteine leads to the
brown phaeomelanins. The principal intermediates are the
cysteinyl-Dopa derivatives 1-4 and the benzothiazines 18
and 19, which are formed through intramolecular cyclization
via the corresponding o-quinone imine.15” Apparently,
Schiff base formation and reduction by excess diphenol is
favored over intramolecular 1,4 Michael addition of nitrogen in this case. Recent investigations by means of
3C N M R spectroscopy indicate that the polymerization of
5-cysteinyl-Dopa occurs also with partial degradation of the
side chains of the Dopa and the cysteinyl rnoietie~.[”~
Enzymatic oxidation of catechols that d o not contain nucleophilic nitrogen leads to dark polymers, the allomelanins
or catecholmelanins. Chemical investigations on their structure (reviewed in Ref. [SO]) have only revealed that C-C and
C-0 bonds are formed. The latter may arise both by
Michael-type addition of H,O to an o-quinone and by addition of phenolic hydroxyl groups of unoxidized substrate. It
is not always clear whether nucleophilic additions or radical
reactions are involved. One of the reaction products in the
enzymatic oxidation of catechol is the dibenzodioxinquinone
22.[751More recently, 22 was synthesized on a preparative
scale from c a t e c h ~ l . [ The
~ ~ ] dibenzodioxin ring system is
found in nature in the e c k l o n o q u i n o n e ~ . [ ~ ~ ]
Nitrogen has been found in various allomelanins (see
Ref. [50] for a review). It is reasonable to assume that the
nitrogen is introduced into these polymers in the form of
amino acid residues, since the biosynthesis starts from nitrogen-free catechol precursors. The presence of nitrogen is not
necessarily indicative of an intact amino acid-polyphenol
linkage, since Strecker degradation may have occurred (cf.
formula 12).
\ /
-
J Q
MeO
‘
-
MeO&
y o
R
’$
OH
22
21
101
/
OH
OMe
R
H Cl O$i
18.19
18: R I = H, ~2 = CH,CH(NH?)COOG
19: R’ = CH,CH(NH?)COO*, R 2 = H
The distinction between eumelanins and phaeomelanins
seems to be rather arbitrary. Though 5-cysteinyl-Dopa is a
poor substrate of tyrosinase, its enzymatic oxidation is accelerated by Dopa. Thus, Dopa and 5-cysteinyl-Dopa will copolymerize under oxidative conditions, and the relative
amounts of the two diphenols in the polymers are reflected
by the sulfur contents.[711Such mixed eumelanins/phaeomelanins can be characterized by ESR spectroscopy.[721
Tyrosinase-catalyzed oxidation of 5,6-dihydroxyindole
leads in the presence of glutathione to adduct 20.1731
The
tetrahydrothiazinoindole 21 was isolated from a reaction of
dopachrome methyl ester with cysteine ethyl ester or glutat h i ~ n e . ’ ~The
~ ] structures of these compounds are most
interesting with respect to melanoprotein biosynthesis. They
suggest that covalent linkages can form by SH addition not
only to Dopa-quinone but also to reactive indolic intermediates.
560
OMe
OH
J y
HO
dMe
Scheme 7. R
=
H, OMe.
Angen. Chem. In!. Ed. Engl. 28 (1989) 555-570
Freudenberg's classical work[531revealed that lignins are
formed by polymerization of p-hydroxy-m-methoxyphenylpropyl units. The initial one-electron oxidation is performed
by a peroxidase, and intermediates are free radicals and p quinone methides (cf. Scheme 1, pathway @). Some principal structural elements of the polymer are depicted in
Scheme 7.[s41Lignins do not contain detectable amounts of
nitrogen.
The tannins of brown algae contain C-0 and C-C linkages between adjacent phloroglucinol units.[57.581 Their
biosynthesis most likely follows oxidative coupling pathways
between phenoxyl and aryl radicals. Covalent binding with
amino acid residues of proteins could result from similar
processes involving tyrosine. Indeed, nitrogen has been
found in varying amounts in high-molecular-weight algal
tannins.[561Sulfur can be present in the form of sulfate.158]
Oligomeric phloroglucinols occur also in several fern spec i e ~ , ~but
~ ' ]their structure differs principally from that of the
algal polymers by a linkage of the aromatic units via methylene bridges.
The vegetable tannins are grouped into two classes, the
hydrolyzable and the condensed tannins. The hydrolyzable
tannins are composed of glucose and oIigomers of gallic acid
which are combined by oxidative phenolic coupling.[611The
condensed tannins are generated from 3- and 4-hydroxyflavanols. The elegant studies of Roux and F e r t ~ i r a Ion
~ ~the
]
biomimetic synthesis of condensed tannins suggest that intermediates are either carbocations or quinone methides.
Polymerization occurs via nucleophilic carbon addition
(Scheme 8).
HO
R
R
binding is observed at pH values in the range 3-5. This has
been taken as evidence for ionic interactions between positively charged Iysine and arginine residues with acidic groups
of the ~annins.1'~~
Strong binding occurs also with synthetic
polymers that do not contain basic functionalities, such as
polyamides, polyvinylpyrrolidones, or urea-formaldehyde
resins. In this case the polyphenol-polyamide complex must
be stabilized by hydrogen bonds between phenolic hydroxyl
and amide carbonyl groups. The degree of polymerization in
condensed tannins must be 2 6 in order to effect denaturation of protein.[801Quantitative analyses of noncovalent
protein-polypheno1 interactions have been published recently"8"
Chaotropic reagents have varying effects on the stability
of tannin-protein complexes. Thus, 8 M urea effectively
strips hydrolyzable, but not condensed tannins, from collagen. Apparently, there are also covalent linkages in the aggregates of proteins and condensed tannins. For example,
quinone methide 23, formed from polymeric proanthocyanidin at pH 4.1 according to the hypothetical mechanism in Scheme 9, could react with nucleophilic functional
groups of the protein.1821Evidence for the formation of a
quinone methide from 4,7-flavandiol in acidic solution has
been obtained by UV spectroscopy in the presence of thiop h e n 0 1 . I ~Alternatively,
~~
the corresponding carbenium ion
may be the reactive intermediate. Trapping of such intermediates by nitrogen or oxygen nucleophiles has so far not been
reported.
H20
OH
R
/
R
o+p::
\
R
H
23
\
Ho p
O
+a
o
H
OH
R
Scheme 8. R
=
Scheme 9. R = H, OH.
H. OH
Owing to the technical applications in the tanning of
leather and the undesired effects on vegetable foodstuffs,
there is considerable interest in tannin-protein interactions.
A comprehensive, though somewhat outdated, review on
this subject is available in the classical book by G~sfavson.['~]
According to traditional views, both groups of tannins interact with collagen by noncovalent forces. A maximum of
Angen. Chem. Int. Ed. Engi. 28 (1989) 555-570
,
Probably the most complex natural polymers are the humic acids, and an admirable compilation of their chemistry
and biochemistry has been published.16*]It is well established that they arise inter alia from polyphenolic polymers
and contain heteroelements of proteinaceous origin. They
are sometimes considered as the end products of the decomposition of various biomaterials in the soil.
561
4. Reactions of Functional Groups of Biopolymers
with Quinones and Quinone Methides
Replacement of the hydroxyl group of 24 by an ethoxy
group leads to another analgetic, phenacetin 26. In contrast
to 24, phenacetin is weakly carcinogenic. This difference has
been ascribed to the oxidative formation of the nitrenium ion
27 rather than a p-quinone imine from 26Is9](Scheme 11).
In Section 3, we have considered biologically important
macromolecular polyphenol-biopolymer complexes with respect to the structural principles governing the association of
the two components. We shall now discuss correlations of
observed biological activities of quinonoids with structural
modifications of the various cellular components.
HN Ac
4.1. Reactions of Thiols and Sulfides
In the aqueous intracellular environment, the strongest
nucleophiles are the cysteinyl SH groups. UV-spectroscopic
evidence for the reaction between cysteine and various
quinones was reported first by Mason and Peter~on,"~]
who
studied models for melanoprotein biosynthesis, and later by
Pierpoint[8s1
in an investigation of the enzymatic oxidation
of chlorogenic and caffeic acids in the presence of amino
acids. Actually, the first proof for the structure of the addition products of cysteine to an o-quinone was presented by
It0 and Prota,[221who isolated the cysteinyl-Dopa isomers
1-4.
Besides the reactions occurring in melanoprotein biosynthesis, addition of the thiol group of cysteine-containing peptides is amply documented by isolation of glutathionyl-SH
adducts from in vitro and in vivo transformations of several
phenols and quinonoids.
Estradiol is converted by tyrosinase into a mixture of
N Ac
HO
0
c y t . P-L50
HO
25
2L
Scheme 10.
562
qJ&y
H ~ C O 0 HO
26
27
z"::
____)
6
WH
I
H3C
I
Dau
28
29
f RSH
The analgetic drug 4-hydroxyacetanilide (paracetamol) 24
may cause liver necrosis when administered to mammals in
high doses. The cytotoxic effects result from metabolic conversion of 24 to N-acetyl-p-benzoquinone imine 25 and hydrolysis of 25 to p-benzoquinone (Scheme 10). Oxidation of
24 is catalyzed by a cytochrome P-450-dependent monooxyg e n a ~ e [ *or
~ ]by prostaglandin H synthetase.[881Formation
of 3-(gIutathion-S-yl)-4-hydroxyacetanilide has been observed in ~ i t r o . ' * ~ After
, ~ * ~ the cells are depleted of
glutathione, liver damage results most likely from covalent
binding of 25 to proteins. Besides SH addition, 25 undergoes
extensive polymerization under physiological conditions.
5 -$=- 0
OEt
The antitumor activity of daunomycin 28 and related anthraquinone antibiotics has been associated with intercalation into DNA, cross-linking of double strands, and induction of single-strand breaking, either by interactions with
oxygen radicals o r by nucleophilic addition (for a review, see
Ref. [90]). Biopolymer modification in the latter reaction is
suggested by the isolation of the products 30 from model
reactions in which the quinone methide 29 was generated by
elimination of the sugar moiety from the enzymatically reduced anthraquinone (Scheme 12).L9']
gens produce semiquinone radicals also upon autooxidation
in alkaline solution or under the action of peroxidase/H,O, .
The 3,4-catechol is presumably more cytotoxic than the 2,3c a t e ~ h o l . [ ' Addition
~]
of glutathionyl SH to the o-quinone
that results from oxidation of 17-ethynyl-3,4,17-estratriol
has been discussed in the context of 17-ethynyl-2,17-estradi01 binding to rat liver microsomes.[861
.
0Et
Scheme 11.
semiquinone
2,3,17and radicals
3,4,17-estratriol;
are observed."
in the31 presence
The catechol
of Zn2@,
estro-
HO
N Ac
- - * e e
H3C
HO
543
&$
/
H3CO t%
/
/
HO
SR
30
Scheme 12. Dau = 1-~-3-amino-2.3.6-trideoxy-~-lyxohexosyl;
R =CH,CH(NHCOCH,)COOH, p-D.glucosy~.
x 6
0
Some cysteinyl-Dopa compounds and cysteaminyl phenols show selective cytotoxic activity and inhibit growth of
certain tumors.[921It is thought that this biological activity
rests on the quinonoid oxidation product (e.g., of I) which
again may act as a scavenger for SH groups of enzymes
involved in D N A biosynthesis.
The glutaminyl derivative 31 is a natural product from the
mushroom Agraricus b i s p ~ r u s . ' Enzymatic
~~'
oxidation of 31
leads to the corresponding benzoquinone 32 (Scheme 13),
which is a powerful inhibitor of sulfhydryl-dependent respiratory enzymes[931and shows antineoplastic activity in
murine melanoma C ~ I I S . [ ~ ~ I
.
Anxew. Chem. Inl. Ed. EngI. 28 (1989)
535-570
OH
Vinylogous sulfide addition of peptidic methionine
residues to o-benzoquinone at varying pH values was suggested as a tool for investigations on the structure and conformation of
Indirect evidence for the 1,4
addition of N-acetylmethionine to o-benz~quinone['~]or
Dopa-quinone" Ool was obtained from UV-spectroscopic investigations, which suggested the structures 34 and 35, respectively. At physiological pH, however, failure to detect a
sulfide-quinone addition product was reported.[231
0
32
31
Scheme 13.
The unusual phenylhydrazine 33 occurs together with 4diazocyclohexadienone in the related fungus A . xanthoderma, extracts of which possess strong antibiotic and cytotoxic activity.[951
kH3
3 4 35
Q
,;Jcoy
N
34: R'
35: R'
H
33
Oxidation of 4-(dimethylamino)phenol by oxyhemoglobin leads to loss of a cysteine and a histidine
residue.['61 It has been postulated that cysteine-93p of
hemoglobin adds to the p-quinone iminium ion, and autooxidation of the adduct is followed by hydrolysis and addition
of histidine-146 p to the substituted p-quinone (Scheme 14).
Though the structure of the adduct has not been proven in
detail, supporting evidence for this pathway was provided by
tryptic digestion of the final product and subsequent amino
acid analysis.
\;/
\ I
0
N
Ac, R 2 = H
H, R* = C H , C H ( N H , ~ ) C O O H ~
Likewise, reaction of the rare amino acid selenomethionine (see Ref. [loll for a review) with o-benzoquinone in 1 N
HC1 has been studied."021The structure of the product was
proposed to be analogous to 34.
4.2. Reactions of Amino Groups
Incorporation of an aromatic amine into a synthetic lignin
has been observed upon peroxidase/H,O,-catalyzed copolymerization of 36 with coniferyl alcohol at pH 7.5"03'
(Scheme 15). Addition of 36 to the quinone methide 37 was
evident from 'H and 13C NMR spectra of the product mixture.
N''
CH20H
r",
I
His-lL6p-Hb-Cz-93p
0
HO
=
=
HO
/
CYS
His-1 46p-Hb
c,
HO
37
Scheme 15.
/
His-146p-Hb
cYS
Scheme 14
Aberrant metabolism of brain catecholamines may lead to
severe neurological disorders. It has been suggested that the
abnormal oxidation of certain neurotransmitters results in
covalent modification of proteins in catecholaminergic neur o n ~ . Supporting
~~~]
evidence was obtained by isolation of
6-(2-aminoethyl)-2,3,5-trihydroxyphenyl-glutathionefrom
an in vitro oxidation of the highly neurotoxic 6-hydroxydopamine in the presence of glutathione as well as from an in
vivo
A n g w Chem. Int. Ed. Engl. 28 (1989) 555-570
Heteroaromatic amines are very abundant in nucleic
acids, and additions of base residues to quinones or quinone
methides are conceivable. However, the corresponding addition products have apparently not been isolated. Though not
related directly to the reactivity of a quinone according to
Scheme 2, it is interesting to note that the antitumor agent
mitomycin C cross-links DNA via addition of guanine
residues, as depicted in Scheme 16.[1041The reaction proceeds probably via an iminium ion rather than the formerly
postulated quinone methide.
Another group of biologically active quinonoid natural
products should be mentioned here, though their mode of
action has not yet been elucidated. The sesquiterpenoid
monoaminoquinones 38 and 39 were isolated from the
sponge Dysidea avara.[1051Most likely, they arise as artifacts
563
/
e"
Scheme 16.
from the parent p-diphenol avarol 40 during the extraction
procedure. Avarol40 and the aminoquinones 38 and 39 show
pronounced inhibition of sea-urchin egg development. Related compounds from the sponge Smenospongia sp. possess
cytotoxic and antimicrobial activity.['061
OH
9338.39
3:R'
39: R'
=
=
H; R 2 = NHCH,
NHCH,, R2 = H
Aliphatic amines (pK, = 9 - 11) occur in biological systems in great variety. Primary amines include the catecholamines, the &-amino groups of either free or peptidic
lysine, and the a-amino groups of amino acids and N-terminal residues in polypeptides. Proline and hydroxyproline are
secondary amines, and the imidazole nitrogen of histidine
(pK, = 6.05) occupies an intermediate position between aromatic and aliphatic amines.
Though leading usually to intractable complex mixtures,
reactions of amino acids and proteins with enzymatically
generated quinones have been studied in great detail by specand, more recently,
troscopic methods, such as UV[84*851
ESR s p e c t r o s ~ o p yIsolation
. ~ ~ ~ ~ of a structurally defined reaction product was not attempted in these investigations.
Thus, Mason and Peterson[841described the absence of a
reaction of 3-substituted indole and 4-substituted imidazole
as well as of the serine hydroxyl group and amido, ureido,
and guanidino nitrogen, with a variety of o-quinones generated from the parent diphenols with o-diphenol oxidase in
aqueous buffer. The UV spectra indicated, however, some
reactions with primary and secondary aliphatic amines and
thiols. Similar results were obtained by Pierpoint,'851 who
interpreted UV spectra of mixtures of phenoloxidase, amino
acids, and chlorogenic or caffeic acid as an indication for the
addition of primary a-amino groups and the &-aminogroup
of lysine to the o-quinones.
Absence of the reaction of a primary amine with the oquinone of N-acetyldopamine at physiological pH has been
reported from ESR studies conducted under conditions of
564
spin stabilization with Zn2@.['41
Kalyaiiamaran et
using a similar experimental protocol, have drawn the following conclusions: a-amino groups of amino acids form
I,.?-addition products at pH 2 8; with lysine and histidine,
addition occurs with the a-amino rather than with the &-amino and imidazole nitrogen, respectively; the secondary amino group of hydroxyproline adds even at physiological pH;
the N-terminal amino groups of peptides also react at lower
pH values, owing to the decreasing basicity seen with increasing numbers of amino acids. The addition of proline
nitrogen to 4-methyl-o-benzoquinone was first suggested by
Jackson and Kend~l["'~some 40 years ago in a study on the
violet pigment formed upon exposure of crude extracts of
potatoes or mushrooms to air. However, according to
Weaver et al.,'931this color is caused by the presence of y-glutaminyl-3,4-benzoquinone 32.
Poison ivy (Toxicodeizdron radicans, Anacardiaceae) contains urushiol, which consists of a mixture of the 3-hexadec(en)yl and 3-pentadec(en)yl catechols 41. The urushiols
act as potent allergens, causing severe skin irritation upon
repeated contact with the plant. It is thought that the toxicity
of the diphenolic compounds results from their oxidation to
o-quinones, in addition to the presence of the lipophilic side
chains. Their mechanism of action has been described as an
oxidative addition to serum protein through mercapto or
terminal amino groups according to Scheme 2. This results
in chemically modified polypeptides which are allergenic.
This view has been supported indirectly by isolation of corresponding derivatives in model experiments['o81 as well as in
studies on the binding of the oxidation products (presumably
o-quinones) of various alkyl-substituted diphenols to
proteins.[' 091 When y-globulin was washed through a column cotaining quinone-coated Celite, binding of the protein
was most extensive when the quinone was derived from 3pentadecylcatechol, whereas 4-methyl-, 5-methyl-, or 6methyl-3-pentadecyl-o-benzoquinoneresulted in diminished
binding of protein. The presence of more than one methyl
group, such as in 4,Sdimethyl- or 4,5,6-trimethyl-3-pentadecyl-o-benzoquinone abolished protein binding nearly completely.[' 091
More recently, cross-reactivity of urushiol analogues in
urushiol-sensitized Guinea pigs has been demonstrated with
compounds 42 and 43.["O1 Blocking of the amino group in
42 or 43 in the form of the methanesulfonamide resulted in
loss of cross-reactivity in sensitized animals. Resorcinol
derivatives did not cross-react with urushiol. It should be
noted that the p-quinone primin 44 also shows pronounced
allergenic activity (for a review, see Ref. [3]).
0
R'
H3c0Q-
0
11-13
11
41: R 1 = R 2 = OH,
R'
= n-C,,H,,,
n-C,,H,,, n-C,,H,,,
n-C,,H,,
42: R ' = OH, R2 = NH,, R3 = C,H,,,, (n = 13, 15, 17)
43: R' = NH,, R 2 = O H , R 3 = C,H,,+, (n = 13, 15, 17)
Plant materials often undergo extensive browning when
wounded tissue is exposed to air. This browning reaction
leads to a decrease in the nutritional value of vegetable foodAngew. Chem. In(. Ed. EngI. 28 (1989) 555-570
stuffs, and it is understood as an oxidative modification of
lysine residues in proteins by plant phenolics. A novel approach towards the identification of quinonoid-lysine addition products is offered by high-resolution mass spectroscopy of degradation products of hydrogenated plant proteins.[' ", "'] The results obtained upon analysis of the
extremely complex product mixtures provided some, though
not fully convincing, evidence for the presence of an addition
product between lysine and caffeic acid.
A similar reaction between chlorogenic acid and the Eamino group of lysine has been suggested in order to explain
the partial inactivation of a potato virus upon exposure to
chlorogenic acid and phenol oxidase." ' 3 , 141 Cross-linking
of protein subunits was observed, and digestion with trypsin
(which cleaves peptide bonds adjacent to lysine residues) was
less extensive with the cross-linked than with monomeric
diphenol-protein reaction product. It was suggested that the
mechanism of cross-linking involves a D-l,4-A-D-1,6-A sequence, resulting in a 3,5-diamino-4-alkyldiphenol.
Alternatively, coupling of two catechol units via an aryl ether linkage was considered.[ll31 The stoichiometry of binding of
chlorogenoquinone was dependent on the pH of the reaction
mixture: at pH 7.0, there was a 1:l ratio between chlorogenic acid and protein subunit, whereas a ratio of 2: 1 was
observed at pH 7.8.
A highly interesting example of the occurrence of 3,4-dihydroxyphenylalanine residues in a protein has been found in
the periostracum["5' and in the phenol
of the
mussel Mytiius edulis. The byssus of the same molluscs also
contains a catechol oxidase,['"I and it is believed that the
enzyme oxidizes peptidic Dopa units to the corresponding
o-quinones, which then add &-amino groups of lysine
residues. Though cross-linking via cysteinyl SH groups is
also a possibility (cf. Scheme 6), the protein contains no cysteine and only traces of methionine.1"61 Thus, a unique type
of natural glue is formed that enables the mussel to adhere
tightly to the surface of a support. It is noteworthy in this
context that the natural product primin 44 and some homologues possess molluscicidal activity." "1
Sequential addition of two &-amino groups of lysine
residues to quinones must not necessarily represent the only
pathway of protein cross-linking in biological systems that
do not contain sulfur nucleophiles. When the addition reaction is followed by rearrangement and hydrolysis of the
azomethine (Scheme 17), an aldehyde is generated in a
Strecker-type reaction. The aldehyde is itself reactive as both
an electrophilic amino-group acceptor and a component in
aldol condensations. Further rearrangements and reactions
can follow. They represent one of the principally possible
structural modifications in collagen and related proteins (for
a review. see Ref. [I 191.
Scheme 17
A n p a , . Chem. Int. Ed. Engl. 28 (1989) 555-570
a-Aminoadipic acid could be isolated after catechol/peroxidase/H,O,-mediated protein modification, followed by
performic acid oxidation and hydrolysis.[' 201 Protein crosslinking by various catechols and peroxidase/H,O, was also
described"". l Z z l but structures of the cross-links have not
yet been determined. Treatment of proteins with peroxidase/
H,O, in the absence of catechols leads to oxidative coupling
of tyrosine residues." 2 3 1
Mechanism-based inactivation of tyrosinase during oxidation of catechol has long been known (for a review, see
Ref. [124]). An earlier report, describing covalent attachment of radiolabeled catechol, presumably through nucleophilic groups of the enzyme,['251is obviously not confirmed
by the results of L e r ~ h , [ ''261
~ ~ .who showed that, though a
histidine residue at the active site was destroyed during catalysis, only insignificant amounts of substrate were bound covalently to the protein.
Addition products of amines to an intermediate o-quinone
methide, which was derived from pyridoxol under rather
harsh conditions, have been investigated in order to provide
models for the neurotoxic action of certain vitamin B,
derivatives.r127JThough possible in principle, an addition of
amino acids or proteins to quinone methides in a biochemical environment has so far not been confirmed experimentally.
4.3. Reactions of Hydroxyl Groups
The most abundant oxygen nucleophile in biological systems is, of course, water. Other candidates for nucleophilic
addition include carbohydrates and the hydroxyl groups of
serine, threonine, and hydroxyproline residues. Furthermore, the phenolic hydroxyl groups of the substrates themselves can act as nucleophiles, and many examples for this
type of C-0 bond formation are found in the field of natural
products chemistry. Reactions of alcohols with p-quinones
are of considerable value in preparative organic chemistry.["] The addition of carboxylates should be considered in
this context also.
With respect to protein modification, no example of the
addition of hydroxyl functions of serine, threonine, or hydroxyproline to a quinone has been reported so far. However, p-quinone has been used to immobilize enzymes on hydroxyalkyl methacrylate polymers" "I
and on polysac~harides.~''~~The
nature of the reactions involved has not
been determined in detail. Since the polymers are first activated byp-benzoquinones, formation of C-0 bonds through
nucleophilic addition is conceivable. After reoxidation, the
protein is attached presumably via mercapto or amino group
addition. Thus, the sequence is A(hydroxy1 group to benzoquinone)-D-A(protein to quinone-modified polymer).
The addition of hydroxyl groups of polypeptides to intermediates in lignin biosynthesis has not yet been observed.
However, lignin does contain C-0 bonds from carbohydrates, as was first suggested by F r e ~ d e n b e r g . ~The
' ~ ] structures of the linkages have been indirectly identified after
digestion with cellulase followed by methylation analysis
of a lignin-carbohydrate complex from Pinus densiflora
wood.11301
A representative example is given in formula 45.
Other types of C-0 bonds, namely, ether linkages to ferulic
565
acid and ester linkages to p-coumaric acid, exist in wheat
straw.“ 311
5. The Mechanisms of Insect Cuticle Sclerotization
In the foregoing sections, we have seen that reactions of
quinonoids with biopolymers result in a variety of types of
covalent and noncovalent bonding, in addition to secondary
modifications such as deamination o r reactions with oxygen
radicals. We shall now turn to a fascinating process in nature,
namely, the hardening and darkening of the exoskeleton of
insects. This subject has been studied quite vigorously in
recent years and we shall ask ourselves to what extent the
results of chemical investigations of other biological systems
may also be valid for this process.
Arthropods in general require a stiff exoskeleton for maintaining such essential life functions as muscle attachment,
body structure, and protection against chemical or mechanical threats from the environment. In insects, the process of
hardening of the cuticle is termed “sclerotization”, and it is
often accompanied by darkening of the cuticle. The most
prominent examples can be seen in the more o r less dark
brown cuticles of many insect pupae. In the larval stage,
stiffening of the mandibles is necessary to provide the functionally intact organs for food intake. From a more practical
viewpoint, the study of sclerotization might suggest new approaches in insect control, since inhibition of this process
would be lethal to insects. One of the prerequisites for the
development of new insecticides is, of course, some idea
about their possible mode of action.
The molecular mechanisms of insect cuticle sclerotization
have long attracted the attention of many researchers. However, the numerous attempts to elucidate the chemistry of
this process have met with rather limited success. In fact,
several hypotheses still exist concerning the nature of the
interactions of polyphenols with structural proteins and chitin, and at least some experimental evidence has been obtained for nearly all of them.[’32]It is generally assumed that
structural proteins are covalently modified and cross-linked
by o-quinones and p-quinone methides that are generated by
enzymatic oxidation of N-acyl-3,4-dihydroxyphenethylamines. According to other hypotheses, sclerotization is effected by noncovalent polyphenol-protein interactions and
by a decrease of water in the cuticle. Several reviews have
appeared in the last few years,’132-’351 and only the most
conclusive results shall be included here together with some
important historical observations.
The earliest investigation on the mechanism of sclerotization goes back to Pryor, who, in 1940, described the hardening of cockroach egg cases“ 361 and of insect cuticle” 3 7 1 as a
result of covalent cross-linking of proteins by o-quinones. He
also suggested the name “sclerotin” for the brown proteinquinone complex. Reactions of peptidic amino groups was
566
assumed after the observation that pretreatment of the
proteins with formaldehyde prevented sclerotin formation.
Very important is also Pryor’s assumption that the stiffness
of the cuticle may increase owing to quinone polymerization
in the “meshes of the protein network”.[’361
The principal sclerotization substrate was identified in the
classical work of Karlson’s group some 25 years ago; they
isolated N-acetyldopamine 46 from larvae of the blowfly
Calliphora erythrocephala[‘381and, subsequently, from other
insect species.[’391Another principal sclerotization agent is
N-P-alanyldopamine 47/1401which, in contrast to 46, contains a free primary amino group.
Ho&NHco+
HO
L6-L0
46: R’ = H, R2 = CH,
47: R’ = H. R2 = CH,CH,NH,
48: R’ = OH, RZ = CH,
The fundamental role of the dopamine derivatives in sclerotization is now well established. Inhibition of Dopa decarboxylase by a-methyl-Dopa o r carbidopa (deamino-a-hydrazino-a-methyl-Dopa) interferes with the biosynthesis of
the sclerotization agents.[’41’ 1421 Pupae of the fly Lucilia
cuprina that were treated with such compounds during immature stages display a light color and die from dehydration.
The idea that enzymatic oxidation of the diphenols yields
o-quinones, which cannot cyclize to indole derivatives and
therefore react according to Scheme 2, has been presented
many times in the literature. According to these hypotheses,
cross-linking occurs between E-amino groups of lysine
residues through either a D-l,4-A-D-I ,6-A o r a D-1,4-A-D
condensation sequence, followed by Schiff base formation.
Since mercapto groups are present in cuticle proteins, if at
participation of E-Iysine amino
all, only to a small
groups is likely.
Inhibition of the phenoloxidase in mosquitoes with 2,6-ditert-butyl-4-(a,a-dimethylbenzyl)phenolprevents sclerotization of the pupal cuticle and the pupae die rapidly after the
molt.[’441The cross-linking of tyrosine-rich proteins, which
can be achieved with tyrosinase in the presence of 46 o r
47,[145b]
is inhibited by the resorcinol analogue N-acetyl-2hydroxytyramine by an as yet unknown mechanism.[’461
The isolation of a bis-lysine adduct from hardened insect
material has been reported only once.[1471The original data
have not been published so far, and therefore this most important result is not accessible to evaluation. Other indications for reactions of amino groups during sclerotization are
derived indirectly from differences in the amounts of lysine
in hydrolysates of sclerotized and soft cuticles.[’3331439 14*]
The participation of histidine residues in protein modification has been demonstrated by application of CP/MAS
N M R spectroscopy to cuticles of insect pupae that had been
fed [1,3-’5N,J histidine and [phenyl-’3C]dopamine.1’491
The
results revealed clearly the presence of ”N-’ 3C linkages, but
it is not possible to decide whether attachment of a dopamine
metabolite to protein results in cross-linking. Participation
Angen,. Chem. Inr. Ed. Engl. 28 (1989) 555-570
of histidine in sclerotization is also evident from comparative
amino acid analysis.[' 501
Several lines of evidence indicate that, besides cross-linking of insect cuticle proteins via basic amino acid residues
and 0-quinones, other mechanisms are involved in sclerotization. Racemic N-acetylnoradrenaline 48 was isolated from
reaction mixtures containing 46 and powdered insect cuticle.
Likewise, oxidation of 4-methylcatechol with cuticle from
flies leads to 3,4-dihydroxybenzyl alcohol and to 3,4-dihydroxybenzaldehyde." 'I These results indicate a side-chain
hydroxylation mechanism which is not consistent with a n
enzyme-controlled reaction.[152,l S 3 ] S'ince dopamine-P-hydroxylase is known to produce (R)-noradrenaline from dopamine, the occurrence of racemic 48 must be explained by
a rearrangement of the o-quinone of N-acetyldopamine to
the correspondingp-quinone methide and nonstereoselective
addition of water to this electrophilic species. In analogy to
the biosynthesis of lignin, the formation of racemic 48 is
most likely a side reaction in the polymerization of 46 in
insect cuticle (cf. Scheme 1, pathways @ and
The enzyme that catalyzes quinone methide formation has not yet
been isolated. It is interesting to note that tyrosinase catalyzes the oxidative decarboxylation of 3,4-dihydroxymandelate to protocatechuic aldehyde, presumably via the pquinone methide." 541 An impressive number of side-chainoxidized derivatives and benzodioxin dimers of 46 and 47
have been isolated from cuticles of various insects (for reviews, see Refs. [I 32, 133]), but their functional role in sclerotization has not been elucidated in molecular terms.
The tan color of most sclerotized insect cuticles indicates
extensive polymerization of the phenolic precursors. Dark
cuticles contain eumelanin in varying amounts[40*
,'41
1551
besides polyphenolic polymers that arise from the oxidation
of the sclerotization substrates via reactive o-quinones and
p-quinone methides. The presence of melanin in sclerotized
cuticle has also been demonstrated by ESR spectroscopy." 4. 641 Copolymerization of peptidic tyrosine residues
with sclerotization substrates has been observed in a few
instances.['". 15'1 In vestigation of insoluble materials by
CP/MAS I3C N M R spectroscopy provides a nondestructive
method for the observation of polyphenolic polymers, and
its application has revealed the presence of 1,2-diphenoxy
carbon atoms in sclerotized insect cuticle.[' "1
Melanization and sclerotization of insect cuticles are separate processes, as was first recognized by Karison et al. in
1962,[1591who also discovered the presence of the nonproteinogenic amino acid (3-alanine in sclerotized cuticle of
the blowfly Calliphora erythrocephala.['601 It appears now
that this amino acid is present in all sclerotized cuticles and
that it plays an essential role in the regulation of the balance
between melanin and sclerotin formation.[' 551 Mutants of
the fruit fly Drosophila melanogaster that are unable to
biosynthesize p-alanine or to transport the amino acid into
the cuticle incorporate Dopa o r dopamine into black eumelanin.['611The stiffness of the cuticle is decreased in these
cases."621 Likewise, ethyl hydrazinoacetate, an inhibitor of
p-alanine biosynthesis o r transport, causes a black coloration of the pupae of Manduca sexta, which normally are
Probably, P-alanine is necessary for acylation of
dopamine, thus preventing intramolecular oxidative cyclization and facilitating protein cross-finking by intermolecular
0).
A n g m . Cliern Jnt. Ed. Engl. 28 (1989) 855-570
nucleophilic addition. Since N-P-alanyldopamine 47 is, in
fact, a component of the sclerotization process, this hypothesis appears quite attractive. However, the question
whether cross-linking occurs on the monomeric or on the
polymeric level remains unanswered. According to another
theory, binding of p-alanine to chitin is required for the
correct packing of the macromolecular network in the cuticle.[' 6 2 1 The P-alanine-mediated browning of chitin [ ' 621 results most likely from Maillard-type reactions." 1 9 , '641
Chitin is not a necessary component for sclerotin formation, since o-quinones and protein are sufficient to harden
and darken the egg cases of cockroaches which are devoid of
However, when chitin deposition is blocked by
diflubenzuron, the cuticles may blacken" 651 and sclerotization can be affected (for a review, see Ref. [166]). Noncovalent binding of N-acetyldopamine-derived polymer to chitin
has been
Chitin appears to be tightly associated with protein, but
the types of bonds are unknown. Again, covalent polysaccharide-protein linkages are in principle possible, either
through acyl linkages between amino or hydroxyl groups or
through glycosidic coupling of polypeptides with the reducing sugar unit..Attempts to isolate conjugates of amino sugar
and amino acids have so far not been successful. Therefore,
the question concerning the molecular architecture of chitinprotein-polyphenol complexes remains unanswered. If glycosidic bonds are formed between the reducing terminus of
chitin and E-amino groups of lysine, rearrangement and subsequent hydrolysis could eventually lead to allysine residues
(cf. Scheme 17). This mechanism is analogous to that of the
glucose-mediated covalent cross-linking of collagen.['681
According to other theories, the importance of covalent
interaction is generaIly overemphasized and sclerotization
results from the reduction of the water content in the cuticle
and from noncovalent binding of proteins to polymers of the
N-acylcatecholamines.[152.
'691 T h ose polymers d o not contain appreciable amounts of indole units, since intramolecular cyclization of the quinones of N-acylcatecholamines is
very slow. Therefore, despite their origin from dopamine
derivatives, they are to be classified as allomelanins (see Section 3). In view of the possibility of quinone methide formation, one expects the properties of these polymers to be similar to those of condensed tannins and Iignin. Their
interaction with proteins would thus lead to noncovalent
interactions as well as random covalent binding.
6. Summary and Prospect
Reactions of quinones and quinone methides have many
important and often spectacular results in nature. In this
article, we have considered defensive systems, toxic effects,
and diminishing of the value of vegetable foodstuffs. We
have also seen examples of the construction of macromolecular, highly complex polymers as essential prerequisites for
the sustaining of life in various organisms. On the molecular
level, the common principle is always a chemical modification of biopolymers, either covalent o r noncovalent. Quite
often, reactions of sulfur nucleophiles with quinones and
quinone methides prevail. When sulfur is absent, modifications occur via nitrogen nucleophiles in proteins and via
567
oxygen nucleophiles in polysaccharides. Finally, biopolymer
modifications can be induced indirectly, for example,
through deamination o r quinone-mediated electron-transfer
processes which result in the formation of oxygen radicals.
Some of the past theories of covalent quinone-protein
binding, which were derived from model systems, have to be
revised. It appears that the role of E-amino groups of lysine
residues is less important for covalent binding than has been
assumed previously. Certainly, there are decreases in the
number of lysine residues after the reaction of a protein with
quinones. However, lysine may also suffer oxidative deamination. Blocking of lysine with aldehydes also decreases
binding of quinones. This does not prove covalent binding,
since noncovalent interactions will also be impaired when
the aldehyde reaction leads to deamination o r to loss of
positive charges. It appears that the imidazole nitrogens of
histidine residues are much more important in nucleophilic
addition than the E-amino groups of lysine.
The most significant and spectacular advances in the direct observation of insoluble biopolymers have been
achieved by applications of solid-state NMR spectroscop ~ . [ ~149,
' * l S s l However, not all speculations are resolved
now and nature presents many variations, including noncovalent binding and C-C bond formation by oxidative coupling. A thorough experimental investigation is mandatory
in any case if questions concerning molecular structure are
raised.
Several issues may be raised at this point for future discussions: The study of protein-quinone interactions, such as
those involved in natural defense mechanisms and sclerotization processes, provides a basis for seeking new pharmaceutical applications (cf. daunomycin) o r avoiding toxic effects
(cf. paracetamol). The knowledge of the biosynthesis and
structural principles of macromolecular networks may help
in the design of new compounds for medical use, such as
those interfering with melanogenesis in melanoma cells, or
for agricultural applications, such as those impairing the
construction of a sclerotized exoskeleton in insect pests.
Finally, new materials can be envisaged that share such
properties as hardness with lignocellulose, o r stiffness and
low density with the exoskeleton of insects, o r strength with
the byssal threads of the common fouling mussel. The construction of new materials from chemically modified
proteins has been suggested recently"
and, with advancing techniques in protein design and engineering, the time
may have arrived to look more closely into this matter. Thus,
an understanding of the principles of the reactions of
quinones in biological systems% important from a practical
viewpoint. However, our desire to analyze the unknown in
order to discover causal relationships and to unravel nature's
molecular construction plans is motivated above all by sheer
curiosity.
Much of the work citedjrom this laboratory was performed by
a number of skilled students whose names appear in the references. I am indebted to themfor their encouraging engagement
and endurance. Quite a few of the ideas discussed in this article
were born in stimulating discussions with many colleagues, and
I am grateful especially to Prof: P. Karlson (Marburg), Prof.
N Steglich (Bonn), and Dr. G. I! Weirich (Beltsville) . Several projects were only successful through the cooperativity
of colleagues in zoology: Prof: A . Egelhaaf (Cologne), the
568
late Pro$ H . Emmerich (Darmstadt), Prof: K. Scheller
(Wiirzburg),and Prof: R . Keller (Bonn). Most of the experimental work mentioned was funded generously by the
Deutsche Forschungsgemeinschaft to whom I am particularly
indebted for a Heisenberg Award. Further support from the
Fonds der Chemischen Industrie is grateful1.v acknowledged.
Received: May 5, 1988 [A 715 IE]
German version: Angew. Chem. 10/ (1989) 572
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