1.3 Biosynthesis of Eumelanin—Formation of Dopaquinone and Dopachrome
9
1.3.2 Exploration of Raper-Mason Pathway
In parallel with the above-mentioned studies on tyrosinase, there was research
progress on intermediates involved in melanogenesis. The oxidation of tyrosine to
form an intermediate dopa is followed by the formation of another intermediate
presenting red to orange color, which further spontaneously changes to colorless,
and then converts into the final product black eumelanin. The colorless and the red
to orange compounds were identified from a solution by Raper and Mason. Raper
showed that the colorless compounds possess nitrogen atom(s) in the non-amino form
[32]. The non-amino nitrogen was thought to be involved in a ring structure resulting
from intramolecular cyclization (ring-closure reaction via bonding between amino N
and benzene ring C). As tyrosine and dopa are not cyclizable, the oxidized substance
of them, namely dopaquinone (a kind of o-quinones), must be responsible for the
cyclization [33]. Raper successfully isolated the (O-methyl-substituted) colorless
compound in crystalline form by employing several conditions to avoid autooxidation of the compounds, including careful control of atmosphere [33]. The CHN
analyses and measurement of melting points were conducted with mixed synthetic
compounds. As a result, DHI was identified as the major product, whereas DHICA
was principally formed when the decolorization was conducted with sulfurous acid
(an acidic reducing agent) [33]. Currently, these products were also identified by
the HPLC retention time, and simplified preparation methods for DHI, DHICA,
and O-methyl derivatives were established [34]. Raper also indicated that the redto orange-colored pigment is probably 2,3-dihydroindole-5,6-quinone-2-carboxylic
acid, namely dopachrome, based on the identified structure of the decolorization
product DHI.
In 1948, Mason spectrophotometrically followed the tyrosinase-catalyzed oxidation of dopa to investigate the oxidation mechanism [35]. The oxidation of dopa at
neutral pH gave rise to a sequential change in UV-visible spectra, in which at the first
conversion the absorption maxima appeared at 305, 475 nm (red to orange), and next
at 275, 298 nm (colorless), followed by 300, 540 nm (purple) of wavelength, and
then finally the spectrum showed a broad absorption profile (black), corresponding
to eumelanin. The absorption maxima at 275, 298 nm confirms the formation of
DHI since its O-methyl derivative gives an identical absorption profile. At strongly
acidic conditions (pH 1.3 or 2.0), instead, an absorption maximum at 310 nm was
observed, which is identical to that of O-methylated DHICA. Although the purple
pigment (called melanochrome) with absorption maxima at 300, 540 nm was thought
as 5,6-indolequinone (IQ), subsequent reports denied this possibility and identified
this pigment as a newly identified dimers [36]. The color of IQ was later found
to be yellow [37]. The remaining absorption maxima at 305, 475 nm of the red to
orange pigment were almost identical to adrenochrome and rubreserine (a kind of
aminochromes), indicating that structurally related dopachrome is the corresponding
compound.
The spectrophotometric findings given by the Mason’s experiments provided
a foundation for the development of melanin chemistry. Thus, the intermediates
9
1.3.2 Exploration of Raper-Mason Pathway
In parallel with the above-mentioned studies on tyrosinase, there was research
progress on intermediates involved in melanogenesis. The oxidation of tyrosine to
form an intermediate dopa is followed by the formation of another intermediate
presenting red to orange color, which further spontaneously changes to colorless,
and then converts into the final product black eumelanin. The colorless and the red
to orange compounds were identified from a solution by Raper and Mason. Raper
showed that the colorless compounds possess nitrogen atom(s) in the non-amino form
[32]. The non-amino nitrogen was thought to be involved in a ring structure resulting
from intramolecular cyclization (ring-closure reaction via bonding between amino N
and benzene ring C). As tyrosine and dopa are not cyclizable, the oxidized substance
of them, namely dopaquinone (a kind of o-quinones), must be responsible for the
cyclization [33]. Raper successfully isolated the (O-methyl-substituted) colorless
compound in crystalline form by employing several conditions to avoid autooxidation of the compounds, including careful control of atmosphere [33]. The CHN
analyses and measurement of melting points were conducted with mixed synthetic
compounds. As a result, DHI was identified as the major product, whereas DHICA
was principally formed when the decolorization was conducted with sulfurous acid
(an acidic reducing agent) [33]. Currently, these products were also identified by
the HPLC retention time, and simplified preparation methods for DHI, DHICA,
and O-methyl derivatives were established [34]. Raper also indicated that the redto orange-colored pigment is probably 2,3-dihydroindole-5,6-quinone-2-carboxylic
acid, namely dopachrome, based on the identified structure of the decolorization
product DHI.
In 1948, Mason spectrophotometrically followed the tyrosinase-catalyzed oxidation of dopa to investigate the oxidation mechanism [35]. The oxidation of dopa at
neutral pH gave rise to a sequential change in UV-visible spectra, in which at the first
conversion the absorption maxima appeared at 305, 475 nm (red to orange), and next
at 275, 298 nm (colorless), followed by 300, 540 nm (purple) of wavelength, and
then finally the spectrum showed a broad absorption profile (black), corresponding
to eumelanin. The absorption maxima at 275, 298 nm confirms the formation of
DHI since its O-methyl derivative gives an identical absorption profile. At strongly
acidic conditions (pH 1.3 or 2.0), instead, an absorption maximum at 310 nm was
observed, which is identical to that of O-methylated DHICA. Although the purple
pigment (called melanochrome) with absorption maxima at 300, 540 nm was thought
as 5,6-indolequinone (IQ), subsequent reports denied this possibility and identified
this pigment as a newly identified dimers [36]. The color of IQ was later found
to be yellow [37]. The remaining absorption maxima at 305, 475 nm of the red to
orange pigment were almost identical to adrenochrome and rubreserine (a kind of
aminochromes), indicating that structurally related dopachrome is the corresponding
compound.
The spectrophotometric findings given by the Mason’s experiments provided
a foundation for the development of melanin chemistry. Thus, the intermediates