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Mechanism of activation of 1 2-dehydro-N-acetyldopamine for cuticular sclerotization.

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Archives of Insect Biochemistry and Physiology 14:93-109 (1990)
Mechanism of Activation of 1,2-Dehydro-NAcetyldopamine for Cuticular Sclerotization
Manickam Sugumaran, Karin Schinkmann, and Hemalata Dali
Department of Biology, University of Massachusetts at Boston, Harbor Campus, Boston,
Massachusetts
The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro
NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in p-sclerotization. While
Andersen has indicated that dehydro NADA quinone is the p-sclerotizingagent
[Andersen, 19891, we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 19881. Since dehydro NADA
quinone has not been isolated or identified till to date, we studied the
enzymatic oxidation of dehydro NADA in the presence of quinone traps to
characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trappped the transiently formed dehydro NADA quinone
as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred.
The structure of the adducts i s in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with
the fact that 4-hydroxyl or amino-substituted quinones instantly transformed
into p-quinonoid structure, indicates that dehydro NADA quinone is only a
transient intermediate and that it i s the dehydro NADA quinone methide that
is the thermodynamically stable product. However, since this compound is
chemically more reactive due to the presence of both quinone methide and
acylimine structure on it, the two side chain carbon atoms are "activated."
Based on these considerations, it is suggested that the quinone methide derived
from dehydro NADA i s the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during p-sclerotization.
Key words: phenoloxidase, quinones, quinone methides, p-sclerotization, tanning, quinone
methide sclerotiration
Acknowledgments: This work was supported in part by grant no. R01-Al-14753from the National
Institutes of Health and by grants from the University of Massachusetts (Healey, BRSG, Ed.
Needs and Fac. Dev. Grants).
Received June5,1989; accepted March 21,1990.
Address reprint requests to Dr. M. Sugumaran, Department of Biology, University of Massachusetts at Boston, Boston, MA02125.
0 1990 Wiley-Liss, Inc.
94
Sugumaran et al.
TANNING
QUINONE
O T E T
H
HO
OJQ
-J
+
-,
0
NADA
DEHYDRO NADA QUINONE
" T N T p-
SCLEROTIZATION
HO
N A D A QUINONE
H
DEHYDRO NADA
E
QUINONE METHIDE
SCLEROTIZATION
NADA QUINONE METHIDE
E
HO
0
DEHYDRO NADA
QUINONE METHIDE
Fig. 1. Mechanisms for cuticular tanning. A: Phenoloxidase. 6: NADA desaturase. C: NADA
quinone isomerase. D: NADA quinone methide : dehydro NADA tautomerase. E: Nonenzymatic transformations.
INTRODUCTION
The cuticle, or exoskeleton, which protects the soft body parts of most, if
not all, insects, is hardened by a process known as sclerotization [l].During
sclerotization, cuticular macromolecules are rendered insoluble and inextractable from cuticle by crosslinking reaction with reactive species (called sclerotizing agents) generated from sclerotizing precursors such as NADA" and
N-P-alanyldopamine by the action of cuticular enzymes [2-51. Based on the
sclerotizing agents generated, two different types of sclerotizing modes, viz.
quinone tanning and p-sclerotization, have been identified so far (Fig. 1).While
quinone tanning identifies the quinonoid nucleus of sclerotizing agents as the
site of attachment of macromolecules, p-sclerotization calls for the aliphatic
side chain of the sclerotizing agent to be the loci for binding [5 and the references cited therein].Andersen, who first discovered p-sclerotization, isolated and
characterized dehydro NADA from the sclerotized cuticle of Locustu rnigvuforiu
and proposed that the quinone of this compound is the p-sclerotizingagent [6,7].
Independently, we discovered a new mechanism for sclerotization in which
quinone methides are generated as reactive species and termed this process
quinone methide sclerotization, analogous to Pryor's quinone tanning [2-5,8,9].
By reinterpreting Andersen's results and based on our own trapping experiments, we also concluded that quinone methides are the reactive intermediates for p-sclerotization [3-5,9-111. In addition, we proposed that dehydroNADA
is not a direct product of NADA oxidation, but is formed by tautomerization
*Abbreviations used: dehydro NADA = 1,2-dehydro-N-acetyIdopamine; mp = melting point;
NADA = N = acetyldopamine.
Oxidation of Dehydro-N-Acetyldopamine
95
of enzymatically generated NADA quinone methide in the cuticle [3-S,9-11]
(Fig. 1).In support of our proposal, recently we have reported the successful
solubilization of enzymes involved in the biosynthesis of dehydro NADA from
the larval cuticle of Surcophugu bullata and demonstrated that the conversion of
NADA to dehydro NADA is caused by the combined action of phenoloxidase,
NADA quinone : quinone methide isomerase and NADA quinone methide :
dehydro NADA tautomerase [ll-131 and not by a specific NADA desaturase
as claimed by Andersen [6,7,14,15].
Andersen has proposed that the dehydro NADA quinone is the reactive species for p-sclerotization [6,7,14,15]. But, in spite of its putative key role in
P-sclerotization, it is rather surprising that Andersen has neither isolated nor
characterized this compound to substantiate his claim. Secondly, he believes
that this quinone exhibits reactivity through its side chain and not through
the quinone ring. In his opinion, dehydro NADA quinone is supposed to react
with catechols to produce the dihydrobenzodioxine-typedimers and to add
onto cuticular nucleophiles to generate the p-crosslinks [6,7,14,15]. But, he
has neither provided evidence to support this contention nor explained why
this quinone should be unusual and exhibit side chain reactivity rather than
conventional ring reactivity characteristic of quinones. Quinones with conjugated double bonds are known to be formed in nature but they do not exhibit
side chain reactivity. Thus, both chlorogenoquinone and caffeic acid quinone
do not form dimers as the dehydro NADA oxidation product, but simply exhibit
well-known Michael 1,Caddition with nucleophiles [16,17]. For example,
chlorogenoquinone readily adds onto benzene sulfinic acid through its quinonoid ring to form the expected Michael 1,Cadduct [16,17]. Therefore, we
argued that it is the quinone methide tautomer (or its radical), and not the
dehydro NADA quinone, that is the reactive species formed from dehydro
NADA [4,5,10,11,18] (Fig. 1).
In order to resolve these discrepancies, we examined the oxidation chemistry
of dehydro NADA and present evidence in this paper that dehydro NADA quinone is brmed, but only as a transient intermediate of dehydro NADA oxidation.
In addition, we will demonstrate, based on both trapping experiments and chemical considerations, that it is the quinone methide tautomer (or its radical)
which is the p-sclerotizing agent and not the dehydro NADA quinone.
MATERIALS AND METHODS
Synthesis of Catechol-DehydroNADA Adduct
A solution of catechol(10mmol) and 1,2-dehydro-N-acetyldopamine
(3 mmol)
in dimethyl sulfoxide (10 ml) containing 20% aqueous potassium carbonate
(20 ml) was stirred and aerated constantly at room temperature for 3 hr. At
the end of this time, the reaction mixture was diluted with water, saturated
with sodium chloride and extracted with ethyl acetate. The organic layer was
separated and dried over anhydrous magnesium sulfate. After removing the
ether on a rotary evaporator, the residue was chromatographed on a Biogel
P-2 column using 0.2 M acetic acid as a solvent. Fractions containing the desired
adduct were pooled and lyophilized (0.33g; 36%yield) m.p. 123-125°C Anal.
calculated for Cl6HISNO5 H20: C, 60.19; H, 5.32; N, 4.38%;found: C, 60.15;
96
Sugumaran et al.
H, 5.23; N, 4.11%. UV (0.2 M acetic acid):
, ,X
= 282 nm. IR (nujol):V = 3250
(NH, OH), 1670 (C=O), 1380 (COCH3), 1270 (C-O-C), 1045 cm-' (C-O-C).
' H - N M R (DMSO-d6): 6 = 1.80 (singlet,SH,CH3), 4.73 (doublet, J = 7 Hz,
lH,CH), 5.60 (quadruplet - changed to doublet on D20 exchange, J = 7 Hz,
1H,CH), 6.40-7.10 (multiplet,7H,ArH), 8.00-9.60 ppm (broad,3H,NH + OH exchanged by D20).
Synthesis of Dehydro NADA-NADA Adduct
A similar reaction containing NADA (2.5 mmol) and dehydro NADA (1mmol)
gave the desired product as a white solid (0.05 g; 12% yield). m.p. 128-130°C
Anal. calculated for C20H22N206
" H20 : C, 56.87; H, 6.16; N, 6.64%; found:
C, 56.36; H, 6.34; N, 6.64%.UV (0.2 M acetic acid): A,,
= 282 nm. IR (nujol):
P = 3150 (OH,NH), 1670(C = 0),1380(COCH3), 1270cm-l (C-0-C). 'H-NMR
(DMSO-d6)-S = 1.80(singlet,6H,CH3),2.40-2.80 (rnultiplet,2H,CH2),2.95-3.50
(multiplet,2H,CH2),4.73 (doublet, J = 7 Hz, lH,CH), 5.50 (quadruplet-changed
to doublet on D20 exchange, J = 7 Hz, lH,CH), 6.30-6.95 (multiplet,6H,ArH),
7.80 (broad,lH,NH), 8.40-9.60 ppm (broad,3H,NH,OH). NH and OH protons were exchanged by D20.
Synthesis of Dehydro NADA Dimer
A solution of dehydro NADA (1 mmol) in dimethyl sulfoxide (2 ml) was
mixed with 20% aqueous potassium carbonate (5 ml). The contents were stirred
with constant aeration. After 1 hr at room temperature, the reaction mixture
was diluted with water, saturated with sodium chloride, and extracted with
ethylacetate. The organic solution was dried over anhydrous magnesium sulfate and concentrated on a rotary evaporator. The residue was taken up in 0.2
M acetic acid and chromatographed on Biogel P-2 column using the same solvent to get pure product; yield -90%. white crystals, mp. 165-167°C. Anal.
Calcd. for C20H20N206"HZO:
C, 59.70; H, 5.47; N, 7.00%; found: C, 59.31; H,
5.35; N, 6.81%. UV (CH30H):A,,
= 282,292,305 nm. IR (nujol): V = 3250-3150
(OH,NH), 1670 (C = 0),1380 (COCH3),1270 (C-0-C), 1040 (C-OX), 950 cm-'
(CH = CH). The NMR spectrum of the synthetic dimer corresponded well with
that of enzymatic product (10,18). In addition the synthetic compound exhibited the same chromatographic properties as the biological isolate (18).
Dehydro NADA was synthesized by the published procedure [19]. All other
chemicals were obtained from Sigma Chemical Co., St. Louis, MO.
HPLC studies were carried out as outlined in earlier publications [9,20]. Two
HPLC systems were used to study the oxidation of dehydro NADA. System I
consisted of Waters model 510 pumps, model 440 automated gradient controller, and model 740 absorbance detector. HPLC system I1 included Altex
model 100 A pumps, Hitachi model 100-30 spectrophotometer, Altex model
C-R1A integrator. Separations were achieved on Beckman Ultrasphere ODS
column (5 p m , 4.6 rnm x 150 mm) using jsocratic elution with 50 mM acetic
acid containing 0.2 mM sodium octyl sulfonate in 30% methanol at a flow rate
of 1 mumin (solvent I). Alternately, two gradient programs were also employed.
In Program A, the above solvent was used for the first 5 min and then the flow
rate and methanol concentrationwere increased to 1.5mumin and 40%, respectively, After 12 min, methanol concentration was brought back to 30% and
Oxidation of Dehydro-N-Acetyldopamine
97
after 15 min the flow rate was reduced back to 1 mumin. In Program B, again,
the same solvent was used with the following modifications. After 5 min,
methanol concentration and flow rate were increased to 50% and 1.5 ml/min
respectively. At 16 min, the amount of methanol was reduced to 30% and at
20 min the flow rate was reduced to 1ml/min.
Enzymatic oxidation of dehydro NADA was performed as outlined in earlier
publications [10,18]. Any modifications made are given under the legends to
figures. Catechols were detected with the nitrite molybdate reagent of Arnow’s
[21,22]. Ultraviolet and visible spectra were recorded in a Gilford model 2600
spectrophotometer.
RESULTS
Tyrosinase-CatalyzedOxidation of Dehydro NADA
Visible spectral studies. In accordance with its reactivity, mushroom tyrosinase readily generated quinones from a variety of o-diphenolic compounds.
Thus, during the oxidation of catechol, 3- and 4-methylcatechols,3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic
acid, caffeic acid, NADA, N-acetylnorepinephrine, N-P-alanyldopamine, NP-alanylnorepinephrine, 3,4-dihydroxyphenylethylalcohol and 3,4-dihydroxyphenylglycol, formation of their corresponding quinone derivatives could be
easily witnessed by the typical visible spectral characteristics. However, in the
case of dehydro NADA, we could not identify the quinone [10,18]. Earlier, we
reported a similar failure to observe the quinone of 3,4-dihydroxymandelic
acid and proposed a direct oxidative decarboxylation of this compound by
tyrosinase to a quinone methide, which ultimately tautomerized to yield stable 3,4-dihydroxybenzaldehydeas the final product [23]. However, Ortiz et al.
1241, who re-examined this reaction by electrochemical studies, did identify
the quinone as a transient intermediate. A similar situation may occur with
dehydro NADA as well and the quinone may be extremely short-lived. In such
cases, one routinely uses trapping experiments to characterize the quinone;
hence, we conducted the following experiments.
Oxidation in the presence of cysteamine. If dehydro NADA quinone (or its
semiquinone) is generated as the initial product of enzymatic oxidation, one
can successfully trap it with thiol compounds [25].Therefore, we examined
the enzymatic oxidation of dehydro NADA in the presence of cysteamine. If
cysteamine traps the dehydro NADA quinone (or semiquinone), we should
get a thiolated catechol as the product (Fig. 2, structure A). On the other hand,
if it traps the dehydro NADA quinone methide (or its radical), we should find
a catechol substituted thiazine derivative (Fig. 2, structure B). HPLC analysis
of dehydro NADA oxidation products formed in the presence of cysteamine
indicated the formation of a single product. (Fig. 2).
The UV spectrum (Fig. 2, inset, spectrum 11) had an absorption maxima at
287 nm with a shoulder at 312 nm, which are attributable to the presence of
olefin conjugated to the catecholic ring. It exhibited an absorption maximum
at 254 nm, corresponding to the sulfur addition on to the catecholic ring. On
treatment with borate, the spectrum exhibited a bathochromic shift consistent with the presence of o-dihydroxy phenolic function (spectrum 111). Based
98
Sugumaran et al.
220
290
3
WAVELENGTH (nm)
I
A !
II
A - A
6.6
13.3
20.0
ELUTION TIME (rnin)
26 6
Fig. 2. HPLC analysis of dehydro NADA-cysteamine-tyrosinase reaction mixture. A reaction
mixture containing 1 pmol of dehydro NADA, 10 prnol of cysteamine, 10 kgof mushroom tyrosinase in 1 ml of 50 mM sodium phosphate buffer, pH 6.0 was incubated at room temperature
for I0 min and a 10 pI aliquot was subjected to HPLC analysis on System I using isocratic elution. The peak at 3.87 min is due to dehydro NADA and that at 17.12 min is due to the adduct.
Inset: Ultraviolet spectrum of dehydro NADA in 0.2 M acetic acid (trace I ) and dehydro NADAcysteamine adduct (trace II, in 0.2 M acetic acid; trace Ill, in sodium borate, pH 8.0). Note the
resemblance of these two compounds (Iand II) with the exception of 254-nm peak, which is
due to sulfur addition to catecholic ring. Structure A is dehydro NADA quinone-cysteamine
adduct. Structure B i s dehydro NADA quinone methide cystearnine adduct.
on these studies, this compound was identified to be thiolated catechol (Fig.
2, structure A) and not catechol-substituted thiazine (Fig. 2, structure B).
Oxidation in the presence of o-phenylenediamine. Although the above experiments indicate the trapping of dehydro NADA quinone by cysteamine, it was
essential to confirm the quinone formation by an entirely different experimental route. To this end, we chose o-phenylenediamine as an alternate trapping
agent because it is a highly selective and sensitive reagent for the detection of
o-benzoquinone.Its two amino groups condense with the two carbonyl groups
of o-quinones with the elimination of water molecules to form phenazine derivatives. It has been successfully used to detect several quinones [26]. If the
dehydro NADA quinone is formed, it will be trapped by phenylenediamine to
colored phenazine derivatives which will also lack the o-diphenolic function
(Fig. 3, structure A). If dehydro NADA quinone methide is formed, its trapping will generate a colorless catecholic dihydrobenzopyrazine (Fig. 3, structure B). Hence we examined the oxidation products of dehydro NADA in the
presence of o-phenylenediamine. HPLC analysis of the reaction mixture (Fig.
3) showed the formation of several minor and one major product, which was
dependent on the presence of o-phenylenediamine.The major product (marked
A in Fig. 3) was found to be a very labile phenazine derivative, based on its
UV and visible spectra (Fig. 4). The adduct neither exhibited a bathochromic
shift in borate buffer nor reacted with Arnaw’s reagent specific for o-diphenols
(Data not shown), thereby indicating the absence of o-diphenolic group. The
visible absorption maximum at 468 nm was consistent with the quinonoid phenazine structure. Based on the spectra and known reactivity of the phenyl-
Oxidation of Dehydro-N-Acetyldopamine
99
A
B
1
0
10.0
20.0
ELUTION TIME (min)
Fig. 3. HPLC analysis of dehydro NADA-o-phenylenediamine-tyrosinasereaction mixture. A
reaction mixture containing 1 pmol of dehydro NADA, 2 pmol of o-phenylenediamine, 200 pg
of mushroom tyrosinase in 1 ml of SO mM sodium phosphate buffer, pH 6.0 was incubated at
room temperature for 60 min and 10 11.1 of the reaction mixture was subjected to HPLC analysis
on System Ii using gradient program A. The peaks at 3.88,5.83, 7.71, 10.25, 11.66 min are due
to dehydro NADA, o-phenylene diamine, dehydro NADA dimer, adduct and an oxidation product of o-phenylene diamine, respectively. The rest of the peaks are unknown.
0.5
w
0
2
U
m
a:
0
v)
m
a
0
2
WAVELENGTH (nm)
Fig. 4. (A) Ultraviolet and (B) visible spectra of o-phenyienediamine-dehydro NADA adduct
(solid line). For comparison, the ultraviolet spectrum of o-phenylenediamine (dotted line) is
also shown. Spectra were recorded in 0.2 M acetic acid.
enediamine with o-quinone, this compound was identified to be the quinonoid
phenazine derivative (Fig. 3, structure A) and not the dihydrobenzopyrazine
derivative (Structure B). However, unlike other phenazine derivatives, this
adduct was not stable and rapidly decomposed even on drying. Hence further characterization could not be carried out on this compound.
Oxidation in the presence of o-aminophenol. Replacement of one of the amino
100
Sugurnaran et al.
‘OH
E
/ A
C
a
m
cu
IU
W
0
z
C
m
U
0
v)
m
U
1
i.
0
10.0
20.0
ELUTION TIME ( m i n )
Fig. 5. HPLC analysis of dehydro NADA, o-aminophenol, tyrosinase reaction mixture. A reaction mixture containing 1 Fmol of dehydro NADA, 10 pmol of o-arninophenol, 20 pg of mushroom tyrosinase in 1 rnl of 50 mM sodium phosphate buffer, pH 6.0 was incubated at room
temperature for 40 min and 10 ~1 of the reaction mixture was subjected to HPLC analysis on
System I using gradient program B. The peaks at 3.86,8.96 and 14.22 are due to dehydro NADA,
adduct and phenoxazinone, respectively.
groups in o-phenylene diamine with a hydroxyl group gives o-aminophenol.
This modification accompanies the loss of quinone trapping ability. Hence it
was of interest to examine dehydro NADA oxidation in the presence of oaminophenol. HPLC analysis of dehydro NADA-tyrosinase-o-aminophenol
reaction mixture indicated the formation of two new products (Fig. 5). The
compound eluting at 14.22 min was formed even in the absence of dehydro
NADA from o-aminophenol-tyrosinase reaction and was tentatively identified to
be the known dimerization product of o-aminophenol,viz., phenoxazinone derivative [27l (Fig. 5, structure B). Hence it was not subjected to further characterization. The UV spectrum of the major product (Fig. 6, spectrum A) exhibited a
simple absorption at 286 nm with the lack of shoulder to characterize the presence of side chain double bond, indicating that the addition has occurred at the
side chain and not on the ring. Accordingly, the presence of o-diphenolic group
could be confirmed both by the bathochromic spectral shift in borate (Fig. 6,
spectrum B) and by a positive response to the Arnow’s reagent (Fig. 6, inset).
Based on these studies, it was tentatively identified to be a dihydrobenzoxazine-type compound (Fig. 5, structure A). Like the phenylene diamine adduct,
Oxidation of Dehydro-N-Acetyldopamine
101
W
0
Z
a
m
U
0
cn
m
220
290
360
WAVELENGTH (nm)
Fig. 6. Ultraviolet absorption spectra of o-aminophenol-dehydro NADA adduct (A) in 0.2 M
acetic acid (-)
and (B) in sodium borate, pH 8.0 (-), Note the bathochromic shift in borate.
Also note the difference between the ultraviolet spectrum of the adduct and that of o-phenylenediamine-dehydroNADA adduct shown in Figure 4. Inset: Visible spectrum of nitrite-molybdate complex of o-aminophenol-dehydro NADA adduct.
this adduct was also found to be unstable and hence a detailed characterization could not be performed.
Oxidation in the presence of catechol. We then took catechol, which is not
only an analog of o-aminophenol, but is also an o-diphenol, which is expected
to form a dihydrobenzodioxine-typeadduct with dehydro NADA side chain.
HPLC analysis of the reaction mixture containing dehydro NADA-catecholtyrosinase indeed revealed the generation of two products which are dependent on the presence of catechol (Fig. 7). The UV spectrum (Fig. 7, inset,
spectrum A) of the major compound lacked the absorbance due to the double
bond (present on the parent compound) suggesting that the addition had
occurred on this side chain. In addition, it shaved bathochromic shift in borate
and a positive response to Arnow’s reagent, confirming the presence of at
least one o-diphenolicfunction (Fig. 7, inset, spectrum B). To characterize this
compound in detail, a large-scale chemical synthesis was carried out as outlined in Materials and Methods and the adduct was purified. The purified
adduct was indistinguishable from the biological isolate in its UV spectral characteristics, and response to Arnow’s reagent [21,22]. On HPLC, both the compounds exhibited the same retention time under different conditions and
co-chromatographed as a single symmetrical peak, confirming that they are
one and the same. The NMR spectrum of the synthetic adduct confirmed the
assigned structure (see under Materials and Methods).
Oxidation in the presence of NADA. Finally, it was of interest to see whether
dehydro NADA can form mixed dimer with NADA as it does in cuticular reac-
102
Sugumaran et al.
E
C
0
m
N
+
a
Lu
0
Z
4
220
m
[r
0
tn
m
270
321
WAVELENGTH ( n m )
4
r
0
L
,
.
10.0
20.0
ELUTION TIME ( m i n )
Fig. 7. HPLC analysis of catechol-dehydro NADA-tyrosinase reaction mixture. A reaction mixture containing1 prnol of dehydro NADA, 10 kmol of catechol, 5 pg of mushroom tyrosinase in
1 ml of 50 rnM of sodium phosphate buffer pH 6.0was incubatedat room temperature for50 min
and 10 pI of the reaction mixture was subjected to HPLC analysis on system II using gradient
program A. The peak at 3.87 min i s due to catechol and dehydro NADA; 8.51 min peak is due
to catechol-dehydroNADA adduct and the peaks at 9.83 and 13.68 may be due to NADA dimers
or unidentified products. Inset: Ultraviolet spectrum of catechol-dehydro NADA adduct A) in
0.2 M acetic acid (-)
and B) in sodium borate buffer, pH 8.0 (---I. Note the bathochromic
shift in borate and the similarity of spectral characteristic with that of o-aminophenol-dehydro
NADA adduct (Fig. 6).
tion. Incubation of NADA, dehydro NADA and tyrosinase indeed readily generated dimers characteristic of NADA-dehydro NADA mixed adduct. These
adducts could also be chemically synthesized as outlined under Materials and
Methods. The NMR spectrum of synthetic adduct confirmed that it is indeed
a NADA-dehydro NADA adduct and corresponded well with that of published
spectrum by Andersen et al. [28].The adducts were resolved on HPLC as three
peaks [ll]and the ultraviolet spectra of these isomers are given in Figure 8.1
We are investigating the detailed structure of these isomers currently.
Oxidation of dehydro NADA alone. We have already reported both nonenzymatic as well as tyrosinase-catalyzed dimerization of dehydro NADA [10,18].
As outlined under Materials and Methods, the dimer could also be synthesized by chemical oxidation. The isomers of dehydro NADA dimer can be
resolved on HPLC [ll]and Figure 8.11 shows the UV spectra of the resolved
isomers. Recently Andersen 1141 has also reported a similar separation of
dimers. We are currently working on elucidating the structure of these isomers.
DISCUSSION
For sclerotization reactions involving the side chain of sclerotizing precursors, Andersen [6,7,14,15], mainly working with locust cuticle, proposed
Oxidation of Dehydro-N-Acetyldopamine
103
0.5
W
0
z
4
m
U
0
O
v)
m
4
0.4
0
220
290
360
WAVELENGTH ( n m )
Fig. 8. Ultraviolet spectra of I . isomers of dehydro NADA-NADA mixed dirners A, B and C and
II. isomers of dehydro NADA dimers A, 5, and C in 0.2 M acetic acid. Note the absence of
absorption due to the free double bond in the mixed adduct.
dehydro-NADA as the key intermediate and claimed that it is the quinone of
this compound which is the p-sclerotizing agent (see Fig. 1).However, he neither provided any evidence for the formation of quinone from dehydro NADA,
nor explained how the quinone interacts with cuticular components through
its side chain while related quinones react only through their quinonoid
nucleus. Thus, for example, chlorogenoquinone readily reacts with benzenesulfinic acid by conventional Michael-l,4-addition reaction to give the ring
adduct rather than side chain adduct [16,17]. In this respect, it is interesting
to recall that Andersen’s group has identified about eight different dimers from
sclerotized locust cuticle but has not accounted for their formation mechanistically with their dehydro NADA quinone [29]. Therefore, not only does
dehydro NADA quinone formation need to be demonstrated, but also its
104
Sugumaran et al.
unusual side chain reactivity has to be confirmed before this compound can
be considered as the p-sclerotizing agent.
The quinone methide hypothesis was originally proposed to account for the
several anomalous observations made by several groups of workers [2]. Only
in subsequent years have we provided evidence for quinone methide production in insect cuticle through a series of studies involving trapping experiments,
tritium release from side chain labelled NADA and mechanistic considerations
with substrate analogs, and confirmed the widespread occurrence of quinone
methide sclerotization [3-5,8,9,20,30-321. In support of quinone methide formation, we have also recently characterized the enzyme responsible for its
synthesis as the o-quinone - p-quinone methide isomerase - in the cuticle of S.
bullata, Manduca sexta, Periplaneta americana, Drosoyhila melanogaster, and Tenebrio
molitor [20,30-321. We have also reported the purification of o-quinone isomerase from the hemolymph of s. bullata and invoked it even in insect immunity
as well 1331. According to our model of p-sclerotization (see Fig. l), some
sclerotizing precursors are converted to quinone methides which are the reactive species for p-sclerotization. As a specific case, NADA is oxidized by cuticular enzyme(s) to NADA quinone and subsequently isomerized by quinone
isomerase to generate NADA quinone methide as the p-sclerotizing agent
[20,30].
Andersen isolated dehydro NADA a s a naturally formed metabolite in cuticle in small amounts [6,14,15] and invoked a desaturase for its synthesis, while
we insisted in the absence of any other supporting data (e.g., characterizing
the enzyme) this could be due to nonenzymatic tautomerization of transiently
formed NADA quinone methide in cuticle although a separate isomerase carrying out this reaction is also possible [3-5,111. Recently, we have successfully
solubilized the enzymes responsible for the biosynthesis of dehydro NADA
from the larval cuticle of S. bullata and our results indicate that dehydro NADA
is synthesized from NADA not directly by the action of a NADA desaturase
but indirectly through the combined action of three enzymes viz., phenoloxidase, quinone isomerase and quinone methide isomerase [ll-131.
During enzymatic oxidation of dehydro NADA, we could not identify the
corresponding quinone by visible spectral studies [3,4,10,18]. This could be
due to either the lack of quinone formation or formation below the detection
levels. In the latter case, further transformation of quinone might make it
difficult to identify this transient intermediate. In such cases one routinely
uses trapping experiments to characterize these compounds. For example,
transiently-formed dopaquinone and dopamine quinone, which defy isolation and characterization, have been successfully trapped as their thiol adducts
[25]. We have used this approach to characterize the transiently generated quinones during cuticular enzyme-mediated oxidation of NADA and related compounds [20,30,31]. Application of this technique also aided us in confirming
the transient formation of dehydro NADA quinone (Figs. 2,9). In addition,
using the well-known ability of o-phenylenediamine to trap the o-quinones
[XI, we further confirmed the generation of dehydro NADA quinone (Figs. 3,
4, 9). Trapping of dehydro NADA quinone as thiol adduct resulted in prevention of its further transformation, such as dimer formation (Fig. 9). Since dehydro
NADA quinone is formed only as a transient metabolite and theoretically can-
Oxidation of Dehydro-N-Acetyldopamine
+
j
J
f
105
- [,r%]
"%-r';, ]
J O "H O
DEHYDRO N A D A
DEHYDRO N A D A QUINONE
THIOLATED DEHYDRO NADA
DEHYDRO N A D A
QUINONE METHIDE
D I HYDROBENZODI OX I N E
Fig. 9. Summary of the observed reactions. Dehydro NADA is oxidized to its corresponding
quinone which can be trapped either by cystearnine to form the cysteamine-dehydro NADA
adduct or by o-phenylenediamine to form orange-colored phenazine derivative. In the presence of cystearnine, dehydro NADA does not form dimers. Also, if high concentration of
o-phenylene diamine is present, dehydro NADA does not form dimers. However, if catechols
(R= H) or o-aminophenol was used to trap the reactive species, they trap the quinone methide
tautomer to form dihydrobenzodioxine-type adduct and dihydrobenzoxazine-type adduct,
respectively. If no additive is present, dehydro NADA (R i s CH =CH-NHCOCH3) can add on to
the quinone methide to form the unsaturated dihydrobenzodioxine dimer.
not be the p-sclerotizing agent, some other product(s) must be responsible for
the attributed reactions. The following chemical considerations indicate that
this alternative reactive species is indeed the quinone methide tautomer.
The stable product of hydroxyquinol oxidation is not the o-quinone, 4-hydroxy-o-benzoquinone, but the p-quinone, 2-hydroxy-p-benzoquinone,
because
the p-hydroxy group destabilizes the o-quinonoid structure of the former by
electron donation (Fig. 10). p-Quinones are more stable than their corrcsponding o-quinones and in the case of hydroxy quinone, hydrogen bonding further stabilizes the p-quinonoid structure. Hence, the more stable p-quinone
rather than the less stable o-quinone is the isolatable product of the oxidation
of hydroxyquinol. If the 4-hydroxyl group in this compound is replaced with
an amino group, the resultant amino catechol is easily susceptible to oxidation and in this case the product is p-quinoneimine and not amino-o-quinone
[34]. Such stabilization is very common, a well-known example being dopachrome formation during melanin biosynthesis [35].The stable structure for
Sugumaran et al.
106
HO
-
H YD ROX Y QUI N OL
HYDROXYQUINONE
HoDNH2
HO
A MI N OCATECH OL
li
LEUCOCHROME
Ho)-f$-+NHR
D OPACH ROME
QUlNONE
-
H
J
----H
f[
52
]
^,04,
--F
{
o
r
N
R
“o
HO
DEHYDRO NADA
(R-COCHJ)
DEHYDRO NADA QUINONE
DEHYDRO NADA
QUlNONE METHIDE
Fig. 10. Stability of substituted quinones. Hydroxyquinol, aminocatechol, leucochrome, and
dehydro NADA all form their corresponding o-quinones as the oxidation products. But these
quinones are thermodynamically unstable and readily transform to the comparatively more
stable p-quinonoid structure as shown.
dopachrome is the iminoquinone form rather than the o-quinone form (Fig.
10). In the case of dehydro NADA, the corresponding oxidized form encounters a similar situation due to the positioning of the amide nitrogen to the ring
with the interface of a conjugated double bond. The quinone in this case is
also less stable than the “vinylogous p-quinoneimine amide” or the ”quinone
methide imine amide” (Fig. 10). Hence, the thermodynamically more stable
quinone methide derivative is formed. The quinone methide derivative thus
formed, although thermodynamically more stable than the quinone, is chemically unstable as it has two of its side chain carbon atoms ”activated.” While
the quinone methide carbon undergoes a Michael-l,6,-addition reaction, the
imine carbon participates in a simple addition reaction with available nucleophiles. Therefore, both the side chain carbon atoms are highly susceptible to
nucleophilic attack by bases. Because of this, when this reactive intermediate
is generated, it reacts rapidly with cuticular nucleophiles forming adducts at
both the a- and p-positions with the regeneration of o-diphenolic function
[4,5,10,11,18]. In the presence of catechols it simply adds onto the catechols
Oxidation of Dehydro-N-Acetyldopamine
L
DEHYDRO NADA
I
DEHYDRO NADA
)/
DEHYDRO NADA
QUINONE
QUINONE METHIDE
I
I
-
I
A
yfjJ-JNHy
-
" r/ N H
K
0
.o
0
SEMIQUINONE
107
\
OUlNONE METHIDE ADdUCT
OUlNONE METHIDE
RADICAL
QUINONE METHIDE
SCLEROTIZATION
( j3
- SCLEROTIZATION)
OEHYDRO N A D A
DIMER
Fig. 11. Proposed mechanism for the fate of dehydro NADA in cuticle. Cuticular enzymes
oxidize dehydro NADA to its corresponding quinone derivative, which i s extremely short-lived.
It readily tautomerizes to more stable quinone methide derivative, which takes part in quinone methide sclerotization. Either through reverse dismutation of dehydro NADA and its oxidation product or by direct one electron oxidation, serniquinones may also be formed. The
radical coupling of semiquinone and quinone methide radical yields quinone methide adduct,
which upon rapid ring closure generates dehydro NADA dimers. The dirners can also be formed
by the addition of dehydro NADA quinone methide to catechols (see Fig. 9). Since quinone
methide i s adding to cuticular nucleophiles with the regeneration of o-diphenolic function
( = 0-sclerotization), this process should therefore correctly be called quinone methide sclerotization, analogous to quinone tanning in which quinones are the reactive species of
crosslinking.
to form dihydrobenzodioxine-typecompounds [lo, 181. Thus its reaction with
catechol and NADA yields the catechol-dehydro NADA adduct and NADAdehydro NADA adduct, respectively (Figs. 7, 8). Interestingly, even o-aminophenol forms an analogous adduct (Figs. 5,6).
As indicated in Figures 1,9, and 11, both dimerization and side chain addition to different catechols and/or nucleophiles is possible only for the quinone
methide tautomer (or the corresponding free radical) and is not possible for
dehydro NADA quinone as such. Therefore unless dehydro NADA quinone is
converted to its quinone methide tautomer such addition does not take place.
Accordingly, when the quinone was trapped by cysteamine, the characteristic
dihydrobenzodioxine dimer formation was totally inhibited. Hence, dehydro
NADA quinone can only be called as the sclerotizing precursor and not the
sclerotizing agent. It is indeed the dehydro NADA quinone methide (or its
radical) which is responsible for dimerization and adduct formation characteristic of @-sclerotization(Fig. 11). At this point, the unusual biochemistry of
dehydro NADA oxidation needs special consideration. Unlike NADA conver-
108
Sugumaran et al.
sion to NADA-quinone methide [20,30,31], dehydro NADA conversion to its
quinone methide does not require the action of two enzymes, viz. phenoloxidase and quinone methide tau tomerase, but requires only phenoloxidase,
eliminating the need for the tautomerase.
In light of the results presented in this paper, the course of sclerotization
starting from NADA can be accounted as follows: the sclerotizing precursor,
NADA, is converted to NADA quinone by cuticular phenoloxidase (Fig. 1).
NADA quinone thus formed participates in quinone tanning by non-enzymatic
reactions or is isomerized to NADA quinone methide by quinone isomerase
(Fig. 1). NADA quinone methide is one of the two reactive species responsible for quinone methide sclerotization ( = p-sclerotization) (Fig. 1). Initially
we proposed a nonenzymatic route for the conversion of NADA quinone
methide to dehydro NADA [3-51. However, recent studies from our laboratory
reveal that this conversion is indeed catalyzed by the enzyme, NADA quinone methide : dehydro NADA tautomerase [ll-131. The dehydro NADA
formed is further acted upon by cuticular phenoloxidase to produce either
the semiquinone or the quinone (Fig. 11).The dehydro NADA quinone will
rapidly tautomerize to generate dehydro NADA quinone methide, which is
the other reactive species of quinone methide sclerotization. The quinone
methide radical formed by isomerization of semiquinone can also be a sclerotking agent but again contributingonly to quinone methide sclerotization.Dimer
formation can occur either by radical coupling route or by direct addition of
dehydro NADA quinone methide to catechols accounting for all observed
reactions.
LITERATURE CITED
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Org Prep
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31. Sugumaran M, Semensi V, Saul SJ: On the oxidation of 3,4-dihydroxyphenethyl alcohol
and 3,4-dihydroxyphenyl glycol by cuticular enzyme(s) from Sarcophagu bullata. Arch Insect
Biochem Physiol10, 13 (1989).
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135 (1989).
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