1.5 Biosynthesis of Pheomelanin—Reaction Process After Binding to Cysteine
19
1.5 Biosynthesis of Pheomelanin—Reaction Process After
Binding to Cysteine
Dopaquinone can react with thiols (R-SH) such as intracellular cysteine (Fig. 1.4).
The binding of thiols takes place by nucleophilic addition with cysteine thiolate
ion at 5-carbon and 2-carbon of dopaquinone, resulting in the formation of 5S-cysteinyldopa and 2-S-cysteinyldopa, respectively [93]. (Both substituted dicysteinyldopa is also formed.) When these cysteinyldopas were oxidized by pulse radiolysis, the absorption maximum at around 310 nm of cysteinyldopas (note that the
peak slightly shifts depending on the sulfur binding site) was immediately diminished, and then a new peak near 380 nm appeared instead. This new peak was further
spontaneously replaced by two peaks around 330 and 540 nm [94]. Since the absorption maximum near 380 nm is a characteristic property of o-quinones, the oxidation of cysteinyldopas was likely to produce the corresponding o-quinones, namely
cysteinyldopaquinones, as the initial products. The consumption rate of cysteinyldopas was proportional to the concentration of cysteinyldopas and of the molecules
with the absorption maximum at 380 nm (considered to be cysteinyldopaquinones)
[94]. In addition, the transient species with the absorption maximum at 380 nm were
consumed at a rate proportional to their own concentrations, thereby producing a
molecule having absorption maxima near 330 and 540 nm [94]. Here, the peak near
330 nm was stable for several tens of seconds, but the peak near 540 nm was unstable,
which decayed by the first-order kinetics.
From the above spectrophotometric observation, a reaction scheme as shown in
Fig. 1.9 was proposed. First, cysteinyldopa is oxidatively transformed to cysteinyldopaquinone, and the amino N in cysteine forms a bond with the carbonyl C in
o-quinone to form a ring structure. (The carbonyl O is eliminated as H 2 O together
with the amino H.) The quinoneimine body thus formed is likely to be responsible
for the absorption maximum at 540 nm. Through decarboxylation or tautomerization, this quinoneimine is converted to 1,4-benzothiazine (absorption maximum at
330 nm), which is a pheomelanin monomer.
The conversion to the quinoneimine was later demonstrated in a more direct
manner [95]. 3,4-Dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA), which
is a reduced form of the quinoneimine, was subjected to pulse radiolysis. As a result,
the radiolytic oxidation product showed an absorption peak at 540 nm, which is the
same observed in the oxidation of cysteinyldopa, confirming the production of the
quinoneimine intermediate [95].
The second-order kinetics of cysteinyldopa consumption can be explained by
considering (partial) reduction of the quinoneimine by the unreacted cysteinyldopa,
which produces DHBTCA (and cysteinyldopaquinone). This was proposed based
on an HPLC analysis that showed the formation of DHBTCA [96]. This indicates
that the quinoneimine and DHBTCA are in a chemical equilibrium. The formation
of 1,4-benzothiazine was later directly confirmed by HPLC analysis of the NaBH 4
(or NaBD 4 ) reduction products [97]. Note that two types of 1,4-benzothiazine (i.e.
3-decarboxylated and carboxyl-retained cases) were found. Although HPLC cannot
19
1.5 Biosynthesis of Pheomelanin—Reaction Process After
Binding to Cysteine
Dopaquinone can react with thiols (R-SH) such as intracellular cysteine (Fig. 1.4).
The binding of thiols takes place by nucleophilic addition with cysteine thiolate
ion at 5-carbon and 2-carbon of dopaquinone, resulting in the formation of 5S-cysteinyldopa and 2-S-cysteinyldopa, respectively [93]. (Both substituted dicysteinyldopa is also formed.) When these cysteinyldopas were oxidized by pulse radiolysis, the absorption maximum at around 310 nm of cysteinyldopas (note that the
peak slightly shifts depending on the sulfur binding site) was immediately diminished, and then a new peak near 380 nm appeared instead. This new peak was further
spontaneously replaced by two peaks around 330 and 540 nm [94]. Since the absorption maximum near 380 nm is a characteristic property of o-quinones, the oxidation of cysteinyldopas was likely to produce the corresponding o-quinones, namely
cysteinyldopaquinones, as the initial products. The consumption rate of cysteinyldopas was proportional to the concentration of cysteinyldopas and of the molecules
with the absorption maximum at 380 nm (considered to be cysteinyldopaquinones)
[94]. In addition, the transient species with the absorption maximum at 380 nm were
consumed at a rate proportional to their own concentrations, thereby producing a
molecule having absorption maxima near 330 and 540 nm [94]. Here, the peak near
330 nm was stable for several tens of seconds, but the peak near 540 nm was unstable,
which decayed by the first-order kinetics.
From the above spectrophotometric observation, a reaction scheme as shown in
Fig. 1.9 was proposed. First, cysteinyldopa is oxidatively transformed to cysteinyldopaquinone, and the amino N in cysteine forms a bond with the carbonyl C in
o-quinone to form a ring structure. (The carbonyl O is eliminated as H 2 O together
with the amino H.) The quinoneimine body thus formed is likely to be responsible
for the absorption maximum at 540 nm. Through decarboxylation or tautomerization, this quinoneimine is converted to 1,4-benzothiazine (absorption maximum at
330 nm), which is a pheomelanin monomer.
The conversion to the quinoneimine was later demonstrated in a more direct
manner [95]. 3,4-Dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA), which
is a reduced form of the quinoneimine, was subjected to pulse radiolysis. As a result,
the radiolytic oxidation product showed an absorption peak at 540 nm, which is the
same observed in the oxidation of cysteinyldopa, confirming the production of the
quinoneimine intermediate [95].
The second-order kinetics of cysteinyldopa consumption can be explained by
considering (partial) reduction of the quinoneimine by the unreacted cysteinyldopa,
which produces DHBTCA (and cysteinyldopaquinone). This was proposed based
on an HPLC analysis that showed the formation of DHBTCA [96]. This indicates
that the quinoneimine and DHBTCA are in a chemical equilibrium. The formation
of 1,4-benzothiazine was later directly confirmed by HPLC analysis of the NaBH 4
(or NaBD 4 ) reduction products [97]. Note that two types of 1,4-benzothiazine (i.e.
3-decarboxylated and carboxyl-retained cases) were found. Although HPLC cannot