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The structure of melanins and melanogenesis—II
Tetrahedron, I%Z. Vol. 18,
pp,941to 949.
Petgmon
THE STRUCTURE
Press Ltd. Printed In Northern Ireland
OF MELANINS GENESIS-II*
SEPIOMELANIN
AND
SYNTHETIC
AND MELANOPIGMENTS
and R. A. NICOLAUS University of Naples, Italy
M. PIATTELLI,E. FATTORUSSO, S. MAGNO Institute of Organic Chemistry,
(Received 4 Junuizry 1962) Abe&act-A comparative study on sepiomeianin and three synthetic pigments, namely dopa melanin, S,&dihydroxyindole melanin and pyrroie black, has been carried out. Sepiomelanin is structurally related to 5,6_dihydroxyindoie and dopa meianins. The only observed differences are the absence of carboxyi groups in position 2 of the indoie nuclei in the synthetic pigments (this result, obvious for 5,6dihydroxyindoie melanin, is rather surprising for dopa melanin) and the presence of a-amino acid chains in dopa melanin. Some carboxyi groups found in dopa and S,&dihydroxyindole meianins are located on pyrroie units. Considerable differences arise when comparing pyrroie black with sepiomeianin.
IN the study of sepiomelanin previous work, lp2 by assuming
the analytical data can be accounted for, as shown in that units of the type I and 11 are present in the pigment.
C N 111
H”:;JIJ.Lc N
Ip
Units 1 and II can occur in an oxidation state varying among the fully reduced, semiquinoid and quinoid types. During melanogenesis, oxidative breakdown of the benzenoid portion of indolequinone units may result in the formation of nuclei of types III and IV. In the polymer molecule, pyrrole nuclei probably occur, in low amounts, as suggested by the value of the C:N ratio which is approximately 8. A comparison of the chemical and physical properties of sepiomelanin with those of dopa and 5,6-dihydroxyindole melanins was undertaken in order to elucidate these problems and at the same time the anaIogy between natural melanins and pyrrole black was investigated. l This investigation was supported by the National and Cl-5220, Public Health Service, U.S.A.
Cancer Institute,
research Grant C-5220
I M. Piattelii and R. A. Nicolaus, Tetrahedron in press. z M. Piattelli, E. Fattorusso, S. Magno and R. A.Nicoiaus, Rend. AC. Sci. js. e mat. (1961).
XXVIII, 165
942
M.
PIAITXLLI,
E. FATTORUSSO, S. MAGNO and R. A. NUXLAUS
The only chemical data available in the literature on synthetic melanins are elemental analyse&j and the identification of pyrrole-2,3-dicarboxylic and pyrrole-2,3,5tricarboxylic acids during oxidative degradation of 5,6-dihydroxyindole and dopa melanins.” Further information may be deduced from Raper’s work7 on melanogenesis ; according to this author, 5,6-dihydroxyindole shoufd be considered as an intermediate in the melanin formation from tyrosine. He succeeded by means of a complex procedure in isolating 5,6-dimethoxyindole from the enzymatic oxidation mixture of tyrosine. Since 5,6_dihydroxyindole has bcen isolated during enzymatic oxidation of dopa at pH 648, and identified by chromatography,8 indole nuclei seem to be actually present in tyrosine melanin and dopa melanin in Iarge amounts. PyrroIe black has been compared with melanins- because AngeP related it with tyrosine melanin in accordance with the following scheme:
CH2 NH
NH
=
u
f
-
NH
melanin
The chemica1 investigations carried out on pyrrole black reveal that small amounts of maleimide, succinic acid and succinimide are obtained by oxidationrO and more recently, pyrrole-2,3-dicarboxylic and pyrrole-2,3,5-tricarboxylic acids have also been identified? The insufficiency of data on these pigments induced further investigation. Dopa melanin was prepared from_3,&dihydroxyphenylalanine by oxidation with mushroom tyrosinase either in the absence of or in the presence of catalase at a buffer pH of 6-8, since dopachrome undergoes rapid decarboxylative rearrangement at even slightly alkaline pH? The oxidative polymerization of 5,6dihydroxyindole at pH 8 was performed under one of three experimental conditions, namely: autoxidation, oxidation with oxygen in the presence of tyrosinase, oxidation with oxygen in the presence of tyrosinase and a B. Bloch and F. Schaaf, Biochem. Z. 162, 181 (1926); H. Burton, Chem. & Ind. 23 (1947); H. S. Mason and A. Lada, Fed. Proc. 11, 254 (1952). 4 G. A. Swan and D. Wright, J. Chem. Sot. 381 (1954). s R. J. S. Beer, T. Broadhurst and A. Robertson, J. Chem. Sot. 1947 (1954). * F. Binns and G. A. Swan, Chem. & Ind. 396 (1957). 7 H. S. Raper, Biochem. J. 20, 735 (1926); H. S. Raper, Ibid. 21, 89 (1927); W. L. Duliere and H. S. Raper, Ibid. 24, 239 (1930). a M. Piattelli, E. Fattorusso and S. Magno, Rend. AC. Sci. fis. e mat. XXVIII, 168 (1961). 9 A. Angeli, Arti. Accad. Lincei 6 (VI) 2nd sem., 87 (1927); 2. E. Jolles, Chem. & M. 845 (1953). lo A. Quilico, Ipigmenti net-i animali e uegetali. Tip. Fusi Pavia (1937). I* R. A. Nicolaus and L. Mangoni, Gazz. Chim. ital. 85, 1397 (1955). l2 H. S. Mason, J. Biol. Chem. 180, 235 (1949).
943
The structure of melanins and melanogenesis-II
Higher yields of dopa and 5,Gdihydroxyindole melanins were obtained when the oxidation was carried out in the presence of catalase. Pyrrole black was prepared by prolonged oxidation of pyrrole with hydrogen peroxide in acetic acid. These pigments, as well as sepiomelanin, are insoluble in organic solvents and are very hygroscopic. Table 1 gives the nitrogen content and the percentage of carbon dioxide evolved during the heating of the pigments at 19Q-200”. TABLE 1.
NITROGEN
CONTENT EVOLVED
OF
DURING
PIGMENTS HEATING
AND AT
THE
PERCENTAGE
OF
co,
190-200
Sample
%N
%CO,
Sepiomelanin
8.5
9-l
Dopa melanin (tyrosinase)
7.29
7-5
Dopa melanin (tyrosinase + catalase)
6.29
5-2
5,GDihydroxyindole
melanin (autoxidation)
7.64
9-3
5,GDihydroxyindole
melanin (tyrosinase)
8.0
9.6
5,6_Dihydroxyindole
melanin (tyrosinase + catalase) _--
8.24
Pyrrole black
13.82
~-
4.7 4.9
Pyrrole black differs from the other pigments in hating a much higher nitrogen content. Similar amounts of carbon dioxide are obtained by heating sepiomelanin, dopa melanin prepared in the presence of tyrosinase and S&dihydroxyindole melanins prepared by autoxidation or by tyrosinase-catalysed oxidation. Pyrrole black and melanins prepared from dopa and 5,6-dihydroxyindole in the presence of tyrosinase and catalase, evolve lower values of carbon dioxide. Surprisingly, the heating of melanins from 5,Gdihydroxyindole also result in the production of large amounts of carbon dioxide, arising no doubt from carboxyl groups the presence of which may only be explained by assuming a partial breakdown of some indole units, which probably subsequently lead to the presence of pyrrole nuclei in these pigments. Since the pigments prepared in the presence of catalase yield, when heated, about one half the normal quantity of carbon dioxide, it may be presumed that the breakdown is caused by hydrogen peroxide formed during melanogenesis? These results agree with those of Swan and Wright,4 who have shown that during formation of melanin from 5,6_dihydroxyindole, the addition of catalase almost halves the amount of carbon dioxide evolved. Similarly, in the case of dopa melanin, the amount of carbon dioxide evolved on heating is approximately halved if catalase was added during preparation of the pigment, i.e. the pigment prepared in the presence of catalase contains about one half of the normal number of free carboxyl groups. These results indicate that for both dopa melanin and 5,Gdihydroxyindole melanin, a fission due to hydrogen peroxide is likely to take place; but in the case of dopa
944
M.
PIAITELLI,E. FATTORIJSO,S. MAGNOand K A. NIC~LAUS
melanin the presence of carboxyl groups derived from those already present in dopa cannot be excluded. All these meIanins and pyrrole black were methylated with diazomethane. The methylated pigments are brown in colour, amorphous, infusible and insoluble. TABLE 2. TEST
PERCENTAGE OF
FOR
N~OGEN
METHYLENEDIOXY
AK-D
GROUPS
IN
GRO~-F~AND
METHOXYL DIFFERENT
Sample
METHYLATED
QUALITATIVE
PIGMENTS
%N
%--0cNl
--O-cH~-o--
Sepiomelanin
7.4
18.8
positive
Dopa melanin (tyrosinase)
7.43
20.6
positive
Dopa melanin (tyrosinase +- catalase)
5.81
19.6
positive
5,dDihydroxyindole (autoxidation)
7.55
19.1
positive
5,GDihydroxyindole (tyrosinase)
7.72
21.6
positive
5,6_Dihydroxyindole (tyrosinase - catalase)
7.73
21.5
positive
14-06
8.6
negative
Pyrrole black
The methylated pigments with the exception of pyrrole black, a11 show similar percentages of nitrogen and methoxyl groups. It should be noted that melanins prepared in the presence of catalase, i.e. under conditions designed to minimize the breakdown of indole nuclei, show percentages of methoxyl groups no lower than those of the other pigments. An explanation of this
fact is afforded by the action of hydrogen peroxide on the hydroxylated benzenoid portion of some indole nuclei with the formation following scheme :
of carboxyl
groups according
to the
t 0
omc 4 5
3 I
05 m
I
2
N
=
HO0
D
NH
Such decomposition has little effect on the percentages of nitrogen and methoxyl groups in methylated melanins, since the difference between the molecular weights of V and VI is smal1, and also because the number of VI units present in the polymer is not large. According to this scheme, during the melanogenesis from the rupture of one indole unit, two molecules of carbon dioxide (from carbon atoms 5 and 6) and one carboxyl group should be formed. Therefore, the amount of carbon dioxide evolved during the heating of melanins should be half that obtained during melanogenesis. This consideration is supported by comparison of qur experimenta results with those obtained by Swan and Wright!
The structure of melanins and melanogenesis--II
945
Methylated pyrrole black differs from the other pigments because of its lower methoxyl content and its negative test for methylenedioxy groups. 3. F%RROLICACIDS
TABLE
OBTAINED
u_
Sample
BY OXIDATION
wrm
KMnO,*
l----J _l----J_ J---J--
_I_Q
Sepiomelanin
-
7
-
++++
+
Dopa melanin (tyrosinase)
-
+
-
+++
I
Dopa melanin (tyrosinase + cat&se)
-
+
-
-!-++
+
5,GDihydroxyindole (autoxidation)
melanin -
-I- t
-
t++
+
5,6_Dihydroxyindole (tyrosinase)
melanin -
++
-
+++
+ --
5,dDihydroxyindole melanin (tyrosinase + catalase)
-
++
-
+++
+
Pyrrole black
+
+
+
-
l
+ = present;
+++A
- = absent
It is noteworthy that although the relative amounts are different, all melanins on oxidation yieId the same pyrrolic acids, Only traces of pyrrole-2,3-dicarboxylic acid are detected among the oxidation products of sepiomelanin and small amounts are present among those of dopa melanin, while relatively large quantities are formed during oxidation of 5,Cdihydroxyindole meIanin. Pyrrole-2,Sdicarboxylic acid is the main product during oxidation of pyrrole black, although pyrrole-Zcarboxylic, pyrrole-2,3-dicarboxylic and pyrrole-2,3,StricarboxyIic acids are also produced. TABLE
4.
P~RROLIC
ACIDS OBTAINED
KMnOo Decarboxylated
BY OXIDATION
AFTER DECARBOXYLATION
AT
OF
THE PIGMENT
wm
190200”
pigments
-Sepiomelanin
-
-i--t
+
+
+++
+
Dopa melanin (tyrosinase)
-
7
+
+
+++
+
+
+++
+
Dopa melanin + cat&se) 5,GDihydroxyindole (autoxidation)
melanin
5,6Dihydroxyindole (tyrosinase)
melanin
-
++
+
t-
+ + t-
+
-
+t
+
+
+ + i-
+
5,dDihydroxyindole melanin (tyrosinase + catalase)
-
-t +
+
+
+ -I- +
.L
Pyrrole bIack
+
+
++++
-
+
-
5
946
M. PIATIELLI,E. FA~ORUSSO, S. MAGNO and R. A. NICOLAUS
Chromatographic analyses of the oxidation products of the pigments after decarboxylation are given in Table 4: The presence of pyrrole-2,4dicarboxylic and pyrrole-2,5-dicarboxylic acids among the oxidation products of the decarboxylated melanins is proof of the oxidation cleavage in the benzenoid portion of some indole units during melanogenesis. The identification of these acids supports the assumption that pyrrole units are present in melanins? Higher yields of pyrrole-2,3-dicarboxylic acid and lower yields of pyrtole-2,3,5tricarboxylic acid are produced by oxidation of sepiomelanin after decarboxylation than by oxidation of the undecarboxylated pigment .l This evidence proves the presence of carboxyl groups in position 2 of some indole nuclei. In the case of dopa melanin and 5,Cdihydroxyindole melanin, no significant variation in the yields of the two pyrrolic acids could be observed; therefore, dopa melanin cannot have carboxyl groups in position 2 of the indole nuclei. Furthermore, since pyrrole-2,3,5-tricarboxylic acid is not found among the hydrolysed oxidation products of methylated dopa melanin, this pigment, unlike sepiomelaninl has no carboxyl groups in position 2 of the indole nuclei. The retention of carboxyl groups of some dopa molecules in dopa melanin, proved by use of carboxyl-labelled dopa,13 is explained by assuming the incorporation of carboxylated intermediate molecules which may either be cyclized or uncyclized. The hypothesis of incorporation of uncyclized intermediate molecules is supported by the occurrence of trimethylamine among the oxidation products of methylated dopa melanin. Therefore, it seems that dopa melanin differs from all other pigments in having a-amino acid chains in its molecule, i.e. dopa quinone (Raper’s scheme) participates in the building up of dopa melanin. In spite of differences, all the other data being in agreement, tyrosine (or dopa) very probably is the precursor of sepiomelanin. In connection with the Raper’s scheme, the carboxyl group in position 2 of some indole nuclei in sepiomelanin originates from participation of a carboxylated intermediate (probably dopachrome) in the building up of the pigment. Such a participation does not occur in vitro to any extent, probably owing to the fact that conditions of pigment formation in do are not reproducible. Analytical data and behaviour on oxidation suggests that pyrrole black is very On the other hand, the occurrence in nature of pigments different from sepiomelanin. similar to pyrrole black cannot apriori be excluded. EXPERIMENTAL
The different C, l-I, N, percentages in various analyses of the same sampleareascribed to the difficult combustion of melanins. Greater differences are observed when samples from different runs of the same preparation are analysed. This can be explained on the basis that melanogenesis takes place with oxidation reactions, (e.g. breakdown of benzenoid parts) decarboxylation and participation of intermediates different from 5,bindolequinone (e.g. dopaquinone and dopachrome) resulting in molecules of high complexity and irregularity. These reactions even occur when several preparations of the same pigment are carried out under apparently identical experimental conditions. The N percentages given in Tables I and 2 are mean values. Sepiomelanin. Pigment was purified according to the cold purification method reported in the ftrst paper of this series.’ I8 G, R. CIemo, F. K. Duxbury and G. A. Swan, J. Chem. Sot. 3464 (1952).
The structure of melanins and melanogenesis-IT
947
Dopa melanin. Mushroom tyrosinase (loo0 U/mg; 38 mg) was added to a solution of L-B(3,4-dihydroxyphcnyl) alanin (1.5 g) in M/20 phosphate buffer at pH 6.8 (2 I.) and a rapid stream of oxygen was passed for 6 hr through the solution, kept at 35”. The black precipitate+ collected by centrifugation, was kept in cone HCl at room temp for 7 days. After centrifugation, the melanin was thoroughly washed with 1% HCl, distilled water and finally acetone. After drying over phosphoric oxide, 550 mg of melanin were obtained. (Found: (a) C, 60.64; H, 4.23; N, 8*77-C, 60-84; H, 4.10; N, 8~23; (b) C, 59~70; H, 3.55; N, 5032-C 59.93; H, 3.65; N, 6.74; (c) C, 59~33; H, 2.87; N, 7937%). A similar preparation carried out with addition of catalase (ex beef liver; 240 Kiel units) resulted in a larger yield of pigment (800 mg). (Found: C, 56.92; H, 346; N, 6.48-C 56-97; H, 3-39; N, 6~17%). These melanins were amorphous, infusible compounds, insoluble in any solvent and highly hygroscopic. 5,bDihydroxyindole melanin. 5,6_Dihydroxyindole was prepared from 2-nitro4,5-dihydroxybenzaldehyde according to Beer ez al. ;I4 a method more recently described by Harley-MasorP resulted in poor and erratic yields in the last step of the synthesis. A rapid stream of oxygen was passed for 12 hr through a solution of 5,bdihydroxyindole (2 g) in M/20 phosphate buffer (660 ml) at pH 8. The solution turned purple in colour with the formation of a brown precipitate. The latter was collected by centrifugation (the mother liquors were colourless) and kept in cone HCl for 7 days. After centrifugation the melanin was repeatedly washed with 1% HCI, distilled water and finally acetone. After drying over phosphoric oxide, 1.75 g of melanin were obtained. [Found: (a) C, 59.06; H, 3.45; N, 9*44-C, 63.24; H, 2.70; N, 10.74; (b) C, 59.31; H, 3.43; N, 9.21-C 58-86; H, 3.31; N, 9.35 YJ. Another sample of S,&dihydroxyindole melanin was similarly prepared with addition of mushroom tyrosinase (50 mg). In this case the oxidation was more rapid, requiring only 6 hr and 1.75 g of melanin were obtained from 2 g of 5,6_dihydroxyindole. [Found: (a) C, 59.24; H, 3.24; N, 8.63C, 59.38; H, 3.02; N, 744; (b) C, 59~89; H, 2.53; N, 7.96-C 59.27; H, 2-54; N, 7*99x]. A third preparation was carried out with tyrosinase (50 mg) and catalase (300 Kiel units), and 2 g of melanin from 2 g of S$dihydroxyindole were obtained [Found: (a) C, 57-61; H, 4.0; N, 8.09; (b) C, 59~77; H, 2.86; N, 8*10--C, 58.74; H, 2~71; N, 8.66x]. The pigments were black, amorphous, infusible compounds, insoluble in all solvents and highly hygroscopic. Pyrrole black. Pyrrole (10 g) was oxidised with hydrogen peroxide in acetic acid according to Angeli.16 After dilution with water (2: I), the pigment was coflected by ccntrifugation and washed with 1 o/0HCI, distilled water and acetone. The dried pigment was powdered, placed in a glass column and washed first with acetone and subsequently with ethanol. After drying, a black, amorphous pigment (2.7 8) was obtained. [Found: C, 57.75; H, 5.03; N, 14.7X,56.79; H, 5.28; N, 12.91 %J About 500 mg of each pigment, previously dried in vacua Thermic decurboxylation of ~hepigments. over phosphoric oxide at 80’ for 12 hr were suspended in vaseline oil and heated at 190--200° (bath temp) in a current of nitrogen for 10 hr. The amount of CO, evolved was measured by absorbing the gas in a saturated solution of barium hydroxide and weighing the barium carbonate formed. The following results were obtained : Sample
Pigment in mg
ElaC.0, in mg
540.8 316 408 475.2 365 426 428
214.6 106 48.7 194 157-5 90 93
Sepiomelanin Dopa melanin (tyrosinase) Dopa melanin (tyrosinase + 5,dDihydroxyindole melanin 5,6-Dihydroxyindole melanin S&Dihydroxyindole melanin Pyrrole black
cat&se) (autoxidation) (tyrosinase) (tyrosinase + catalase)
I4 R. J. S. Beer, K. Clarke, H. G. Khorana and A Robertson, J. Chem. Sot. 223 (1948). l5 J. Harley-Mason, 3. Chem. Sm. 200 (1953). 1@A. Angeh and L. Alessandri, Gorr. Chim. ital. 46,283 (1916).
’
948
M. PIAITELLI,E. FATTORUSSO,S. MAGNO and R. A. NICOLAUS
Reaction uf the pigments with diozomethane. About 500 mg of each pigment were treated with a solution of diazomethane (500 mg) in ether (50 ml) and allowed to stand at room temp for 24 hr. Unreacted diazomethane was removed with acetic acid. The pigment, collected by centrifugation, was successively washed with methanol, ethanol and ether. After determination of methoxyl groups, the pigment was again treated with diazomethane until the percentage of methoxyl groups was constant. All the methylated pigments were brown, amorphous compounds. [Found : sepiomeliznin: C, 64-l; H, 5.5; N, 7.4; OCH*, 18.8; dopa melanin (tyrosinase): C, 62.80; H, 5.17; N, 7.42; OCHS, 20.&C, 62.92; H, 5.14; N, 7.45; dopu melunin (tyrosinase + catalase) (a) C, 62.14; H, 5.97; N, 6.17; OCH*, 19-w, 62.23; H, 5.63; N, 7.95; (b) C, 62.31; H, 4.95; N, 5.01-C, 6267; H, 5.18; N, 4.12; 5,6_dihydroxyindole melanin (autoxidation): C, 61.53; H, 5.83; N, 7.55; OCHa, 19.1-C, 61.63; H, 5-58; N, 760; 5,GdihydroxyindoZe mefunin (tyrosinase): C, 62.31; H, 5.19; N, 7.83; OCHS, 21*5-C, 63.39; H, 5.08; N, 7.62; 5$-dihydroxyindole melanin (tyrosinase + catalase) (a) C, 63.66; H, 5-30; N, 7.51; OCHI, 21.5-C, 6362; H, 5.16; N, 7.32; (b) C, 61.60; H, 5.67; N, 8*59-C, 62.48; H, 5.49; N, 7.61; pyrrole black: C, 60.72; H, 5-24; N, 14-05; OCH*, 8.6; C, 60-56; H, 5.52; N, 14.08%]. The methylenedioxy groups test was positive for all the pigments except pyrrole black, which dissolves in the reagent, the solution turning brown in colour. Oxidation of the pigmertts. 50 mg of each pigment, suspended in 0.6 ml 2N KICOs, were oxidized with 3 % KMnO, until the colour persisted for 15 min. The following quantities of 3 *AKMnOl were employed : sepiomelanin: 6-3 ml; dopa melanin (tyrosinase): 5 ml; dopu melanin (tyrosinase + catalase) : 7 ml ; 5,6_dihydroxyindole melanin (autoxidation) : 6.2 ml ; 5,6_dihydroxyin&le melanin (tyrosinase): 6-4 ml; 5&dihydroxyindo/e melanin (tyrosinase + catalase): 6.6 ml; pyrrole black: 6 ml. The solution was boiled for a few minutes, filtered and acidified with 2N HCl to pH 4-5. Oxalic acid was precipitated with 20% CaCit and the solution filtered, strongly acidified with cone HCI and extracted with ether (50 ml in 5 portions). After separation of the solvent, the residue was dissolved in distilled water (0.2 ml) and the filtered solution used for chromatographic analysis; O-03 ml of solution were spotted on Whatman No. 1 paper and compared by descending chromatography with reference aliquots of pyrrolic acids [propanol : 33 %NHI : water (60: 30 : 10) and ethanol : 33 %NH, : water (80 :4: 16) as solvents]. The chromatograms were sprayed with the diazonium salt of sulphanilic acid and N NaOH. From the intensity and size of the developed red spots the following conclusions could be drawn: dopu melanin yielded pyrrole-2,3,5-tricarboxylic acid and a smaller amount of pyrrote-2,3_dicarboxylic acid, while pyrrole-2,3,4,5-tetracarboxylic acid was present only in traces; 5,ddihydroxyindole melanin yielded relatively large amounts of pyrrole-2,3,5-tricarboxylic and pyrrole 2,3dicarboxylic acids and a smaller quantity of pyrrole-2,3,4,5_tetracarboxylic acid. The chief oxidation product of pyrrole black was pyrrole-2,5dicarboxylic acid although pyrrole-2,3,5-tricarboxylic, pyrrole-2,3-dicarboxylic and pyrrole-2-carboxylic acids were also found in smaller amounts. Paper chromatography of the mixture obtained by oxidation of pyrrole black did not lead to the identification of pyrrole-2-carboxylic acid but its presence was shown by thin layer chromatography [silica gel; chloroform : formic acid (80 :20) and benzene : acetic acid (50 : 50) as solvents.] Oxi&tiun of the methyiated pigments. Methylated pigments (100 mg) were oxidized as described above and the typical smell of aliphatic amines was perceived during the oxidation. The following quantities of 3% KMnO, were employed: sepiomelanin: 14-l ml; dopa melanin (tyrosinase): 10.8 ml; dupa melanin (tyrosinase + catalase): 14-5 ml ; 5,ddihydruxyinMe melanin 14.6 ml; 5,GdihydroxyindoZe autoxidation) : 13.8 ml; 5,6-dihydroxyindole melanin (tyrosinase): melanin (tyrosinase + catalase): 15 ml; pyrofe black : 11 ml. In order to avoid the hydrolysis of pyrrolic esters which might have been present, MnOs was removed, without previous heating, by centrifugation after adding some sodium sulphite. A portion of the ether extract, chromatographically examined as described above, showed no presence of pyrrolic acids. Only pyrrole black yielded pyrrole-2,5-dicarboxylic and pyrrole-2,3,5tricarboxylic acids, (pyrrole-2,3dicarboxylic and pyrrole-2-carboxylic acids wereonlydetectable when spotting larger amounts of the solution on the paper.) The remaining portion of the ether extract was evaporated to dryness and the residue hydrolysed with 30% NaOH (0.2 ml) at 100” for 40 min. The solution, after addition of water and acidification with cone HCI, was extracted with ether (25 ml in 5 portions). After solvent evaporation, the residue was dissolved in water (O-2 ml) and the filtered solution used for paper chromatography. Pyrrole-2,3,Qricarboxylic acid could again be identified after hydrolysis only in the case of sepiomelanin, whereas this did not occur in the other pigments.
.
The structure of melanins and melanogenesis--11
949
Since the presence of p-methyl esters of pyrrolic acids among the degradation products could not be excluded (on account of their higher resistance to hydrolysis than a-esters), hydrolysis was also performed under more drastic conditions (10% KOH in ethanol for 12 hr) but this did not modify the results. Oxidation of the decarboxyluted pigments. Decarboxytated pigments (50 mgJ were oxidized as usual with 3 % KMnO,. The following quantities of 3 % KMnO, were employed: sepiomehin: 6.4 ml; dopu melanin (tyrosinase): 7.5 ml; dopa melonin (tyrosinase t catalase): 7.2 ml; 5,6-dil hydroxyindok melanin (autoxidation) : 6-9 ml; 5,6_dihydroxyindofe melanin (tyrosinase) : 5.1 ml ; 5$-dihydruxyindole melanin (tyrosinase t. catalase) : 7.3 ml; pyrrole black: 4.8 ml. The oxidation products were analysed by paper chromatography (ethanol:33 % NHg:water (8014: 16) and propanol:33% NHs:water (60:30: 10) as solvents) and by thin layer chromatography [chloroform:formic acid (50: 50) as solvent]. Decarboxylated sepiomelanin when compared with undecarboxylated pigment yielded lower amount of pyrrole-2,3,5-tricarboxylic acid and larger quantities of pyrrole-2,3dicarboxylic acid ; the yields of pyrrole-2,3,4,5-tetracarboxylic acid were the same and small amounts of pyrrole-2,4_dicarboxylic and pyrrole-2,Sdicarboxylic acids were also obtained, Dopa melanin and 5,6dihydroxyindole melanin, after decarboxylation, gave the same results as the undecarboxylated pigments ; the only difference being the appearance of small amounts of pyrrole-2,4-dicarboxylic and of pyrrole-2,5dicarboxylic acids. Permanganic oxidation of decarboxylated pyrrole black gave results similar to those of undecarboxylated pigment . Volatile bases from oxidation of methylated dopu melanin. Methylated dopa melanin (tyrosinasecatalysed oxidation ; 200 mg) was oxidised with 3 % KMnOl. The liquid was distilled and collected in 2N HCI. The solution containing the volatile bases as hydrochlorides was evaporated. From the residue, free bases were obtained by alkali addition. Gas chromatography [C. Erba’s Fractovap; 20”; column (1 m length of Celite (85x), triethanolamine (7.5x), Vaseline oil (7*5x))] showed the presence of ammonia and trimethylamine. The same results were obtained from the methylation product of dopa melanin prepared in the presence of tyrosinase and catalase. Acknowledgements-We wish to record our thanks to G. Ferracane for technical assistance and to E. Thommen, Dept. Org. Chem., University of Base], Switzerland, for elementary analyses.
pp,941to 949.
Petgmon
THE STRUCTURE
Press Ltd. Printed In Northern Ireland
OF MELANINS GENESIS-II*
SEPIOMELANIN
AND
SYNTHETIC
AND MELANOPIGMENTS
and R. A. NICOLAUS University of Naples, Italy
M. PIATTELLI,E. FATTORUSSO, S. MAGNO Institute of Organic Chemistry,
(Received 4 Junuizry 1962) Abe&act-A comparative study on sepiomeianin and three synthetic pigments, namely dopa melanin, S,&dihydroxyindole melanin and pyrroie black, has been carried out. Sepiomelanin is structurally related to 5,6_dihydroxyindoie and dopa meianins. The only observed differences are the absence of carboxyi groups in position 2 of the indoie nuclei in the synthetic pigments (this result, obvious for 5,6dihydroxyindoie melanin, is rather surprising for dopa melanin) and the presence of a-amino acid chains in dopa melanin. Some carboxyi groups found in dopa and S,&dihydroxyindole meianins are located on pyrroie units. Considerable differences arise when comparing pyrroie black with sepiomeianin.
IN the study of sepiomelanin previous work, lp2 by assuming
the analytical data can be accounted for, as shown in that units of the type I and 11 are present in the pigment.
C N 111
H”:;JIJ.Lc N
Ip
Units 1 and II can occur in an oxidation state varying among the fully reduced, semiquinoid and quinoid types. During melanogenesis, oxidative breakdown of the benzenoid portion of indolequinone units may result in the formation of nuclei of types III and IV. In the polymer molecule, pyrrole nuclei probably occur, in low amounts, as suggested by the value of the C:N ratio which is approximately 8. A comparison of the chemical and physical properties of sepiomelanin with those of dopa and 5,6-dihydroxyindole melanins was undertaken in order to elucidate these problems and at the same time the anaIogy between natural melanins and pyrrole black was investigated. l This investigation was supported by the National and Cl-5220, Public Health Service, U.S.A.
Cancer Institute,
research Grant C-5220
I M. Piattelii and R. A. Nicolaus, Tetrahedron in press. z M. Piattelli, E. Fattorusso, S. Magno and R. A.Nicoiaus, Rend. AC. Sci. js. e mat. (1961).
XXVIII, 165
942
M.
PIAITXLLI,
E. FATTORUSSO, S. MAGNO and R. A. NUXLAUS
The only chemical data available in the literature on synthetic melanins are elemental analyse&j and the identification of pyrrole-2,3-dicarboxylic and pyrrole-2,3,5tricarboxylic acids during oxidative degradation of 5,6-dihydroxyindole and dopa melanins.” Further information may be deduced from Raper’s work7 on melanogenesis ; according to this author, 5,6-dihydroxyindole shoufd be considered as an intermediate in the melanin formation from tyrosine. He succeeded by means of a complex procedure in isolating 5,6-dimethoxyindole from the enzymatic oxidation mixture of tyrosine. Since 5,6_dihydroxyindole has bcen isolated during enzymatic oxidation of dopa at pH 648, and identified by chromatography,8 indole nuclei seem to be actually present in tyrosine melanin and dopa melanin in Iarge amounts. PyrroIe black has been compared with melanins- because AngeP related it with tyrosine melanin in accordance with the following scheme:
CH2 NH
NH
=
u
f
-
NH
melanin
The chemica1 investigations carried out on pyrrole black reveal that small amounts of maleimide, succinic acid and succinimide are obtained by oxidationrO and more recently, pyrrole-2,3-dicarboxylic and pyrrole-2,3,5-tricarboxylic acids have also been identified? The insufficiency of data on these pigments induced further investigation. Dopa melanin was prepared from_3,&dihydroxyphenylalanine by oxidation with mushroom tyrosinase either in the absence of or in the presence of catalase at a buffer pH of 6-8, since dopachrome undergoes rapid decarboxylative rearrangement at even slightly alkaline pH? The oxidative polymerization of 5,6dihydroxyindole at pH 8 was performed under one of three experimental conditions, namely: autoxidation, oxidation with oxygen in the presence of tyrosinase, oxidation with oxygen in the presence of tyrosinase and a B. Bloch and F. Schaaf, Biochem. Z. 162, 181 (1926); H. Burton, Chem. & Ind. 23 (1947); H. S. Mason and A. Lada, Fed. Proc. 11, 254 (1952). 4 G. A. Swan and D. Wright, J. Chem. Sot. 381 (1954). s R. J. S. Beer, T. Broadhurst and A. Robertson, J. Chem. Sot. 1947 (1954). * F. Binns and G. A. Swan, Chem. & Ind. 396 (1957). 7 H. S. Raper, Biochem. J. 20, 735 (1926); H. S. Raper, Ibid. 21, 89 (1927); W. L. Duliere and H. S. Raper, Ibid. 24, 239 (1930). a M. Piattelli, E. Fattorusso and S. Magno, Rend. AC. Sci. fis. e mat. XXVIII, 168 (1961). 9 A. Angeli, Arti. Accad. Lincei 6 (VI) 2nd sem., 87 (1927); 2. E. Jolles, Chem. & M. 845 (1953). lo A. Quilico, Ipigmenti net-i animali e uegetali. Tip. Fusi Pavia (1937). I* R. A. Nicolaus and L. Mangoni, Gazz. Chim. ital. 85, 1397 (1955). l2 H. S. Mason, J. Biol. Chem. 180, 235 (1949).
943
The structure of melanins and melanogenesis-II
Higher yields of dopa and 5,Gdihydroxyindole melanins were obtained when the oxidation was carried out in the presence of catalase. Pyrrole black was prepared by prolonged oxidation of pyrrole with hydrogen peroxide in acetic acid. These pigments, as well as sepiomelanin, are insoluble in organic solvents and are very hygroscopic. Table 1 gives the nitrogen content and the percentage of carbon dioxide evolved during the heating of the pigments at 19Q-200”. TABLE 1.
NITROGEN
CONTENT EVOLVED
OF
DURING
PIGMENTS HEATING
AND AT
THE
PERCENTAGE
OF
co,
190-200
Sample
%N
%CO,
Sepiomelanin
8.5
9-l
Dopa melanin (tyrosinase)
7.29
7-5
Dopa melanin (tyrosinase + catalase)
6.29
5-2
5,GDihydroxyindole
melanin (autoxidation)
7.64
9-3
5,GDihydroxyindole
melanin (tyrosinase)
8.0
9.6
5,6_Dihydroxyindole
melanin (tyrosinase + catalase) _--
8.24
Pyrrole black
13.82
~-
4.7 4.9
Pyrrole black differs from the other pigments in hating a much higher nitrogen content. Similar amounts of carbon dioxide are obtained by heating sepiomelanin, dopa melanin prepared in the presence of tyrosinase and S&dihydroxyindole melanins prepared by autoxidation or by tyrosinase-catalysed oxidation. Pyrrole black and melanins prepared from dopa and 5,6-dihydroxyindole in the presence of tyrosinase and catalase, evolve lower values of carbon dioxide. Surprisingly, the heating of melanins from 5,Gdihydroxyindole also result in the production of large amounts of carbon dioxide, arising no doubt from carboxyl groups the presence of which may only be explained by assuming a partial breakdown of some indole units, which probably subsequently lead to the presence of pyrrole nuclei in these pigments. Since the pigments prepared in the presence of catalase yield, when heated, about one half the normal quantity of carbon dioxide, it may be presumed that the breakdown is caused by hydrogen peroxide formed during melanogenesis? These results agree with those of Swan and Wright,4 who have shown that during formation of melanin from 5,6_dihydroxyindole, the addition of catalase almost halves the amount of carbon dioxide evolved. Similarly, in the case of dopa melanin, the amount of carbon dioxide evolved on heating is approximately halved if catalase was added during preparation of the pigment, i.e. the pigment prepared in the presence of catalase contains about one half of the normal number of free carboxyl groups. These results indicate that for both dopa melanin and 5,Gdihydroxyindole melanin, a fission due to hydrogen peroxide is likely to take place; but in the case of dopa
944
M.
PIAITELLI,E. FATTORIJSO,S. MAGNOand K A. NIC~LAUS
melanin the presence of carboxyl groups derived from those already present in dopa cannot be excluded. All these meIanins and pyrrole black were methylated with diazomethane. The methylated pigments are brown in colour, amorphous, infusible and insoluble. TABLE 2. TEST
PERCENTAGE OF
FOR
N~OGEN
METHYLENEDIOXY
AK-D
GROUPS
IN
GRO~-F~AND
METHOXYL DIFFERENT
Sample
METHYLATED
QUALITATIVE
PIGMENTS
%N
%--0cNl
--O-cH~-o--
Sepiomelanin
7.4
18.8
positive
Dopa melanin (tyrosinase)
7.43
20.6
positive
Dopa melanin (tyrosinase +- catalase)
5.81
19.6
positive
5,dDihydroxyindole (autoxidation)
7.55
19.1
positive
5,GDihydroxyindole (tyrosinase)
7.72
21.6
positive
5,6_Dihydroxyindole (tyrosinase - catalase)
7.73
21.5
positive
14-06
8.6
negative
Pyrrole black
The methylated pigments with the exception of pyrrole black, a11 show similar percentages of nitrogen and methoxyl groups. It should be noted that melanins prepared in the presence of catalase, i.e. under conditions designed to minimize the breakdown of indole nuclei, show percentages of methoxyl groups no lower than those of the other pigments. An explanation of this
fact is afforded by the action of hydrogen peroxide on the hydroxylated benzenoid portion of some indole nuclei with the formation following scheme :
of carboxyl
groups according
to the
t 0
omc 4 5
3 I
05 m
I
2
N
=
HO0
D
NH
Such decomposition has little effect on the percentages of nitrogen and methoxyl groups in methylated melanins, since the difference between the molecular weights of V and VI is smal1, and also because the number of VI units present in the polymer is not large. According to this scheme, during the melanogenesis from the rupture of one indole unit, two molecules of carbon dioxide (from carbon atoms 5 and 6) and one carboxyl group should be formed. Therefore, the amount of carbon dioxide evolved during the heating of melanins should be half that obtained during melanogenesis. This consideration is supported by comparison of qur experimenta results with those obtained by Swan and Wright!
The structure of melanins and melanogenesis--II
945
Methylated pyrrole black differs from the other pigments because of its lower methoxyl content and its negative test for methylenedioxy groups. 3. F%RROLICACIDS
TABLE
OBTAINED
u_
Sample
BY OXIDATION
wrm
KMnO,*
l----J _l----J_ J---J--
_I_Q
Sepiomelanin
-
7
-
++++
+
Dopa melanin (tyrosinase)
-
+
-
+++
I
Dopa melanin (tyrosinase + cat&se)
-
+
-
-!-++
+
5,GDihydroxyindole (autoxidation)
melanin -
-I- t
-
t++
+
5,6_Dihydroxyindole (tyrosinase)
melanin -
++
-
+++
+ --
5,dDihydroxyindole melanin (tyrosinase + catalase)
-
++
-
+++
+
Pyrrole black
+
+
+
-
l
+ = present;
+++A
- = absent
It is noteworthy that although the relative amounts are different, all melanins on oxidation yieId the same pyrrolic acids, Only traces of pyrrole-2,3-dicarboxylic acid are detected among the oxidation products of sepiomelanin and small amounts are present among those of dopa melanin, while relatively large quantities are formed during oxidation of 5,Cdihydroxyindole meIanin. Pyrrole-2,Sdicarboxylic acid is the main product during oxidation of pyrrole black, although pyrrole-Zcarboxylic, pyrrole-2,3-dicarboxylic and pyrrole-2,3,StricarboxyIic acids are also produced. TABLE
4.
P~RROLIC
ACIDS OBTAINED
KMnOo Decarboxylated
BY OXIDATION
AFTER DECARBOXYLATION
AT
OF
THE PIGMENT
wm
190200”
pigments
-Sepiomelanin
-
-i--t
+
+
+++
+
Dopa melanin (tyrosinase)
-
7
+
+
+++
+
+
+++
+
Dopa melanin + cat&se) 5,GDihydroxyindole (autoxidation)
melanin
5,6Dihydroxyindole (tyrosinase)
melanin
-
++
+
t-
+ + t-
+
-
+t
+
+
+ + i-
+
5,dDihydroxyindole melanin (tyrosinase + catalase)
-
-t +
+
+
+ -I- +
.L
Pyrrole bIack
+
+
++++
-
+
-
5
946
M. PIATIELLI,E. FA~ORUSSO, S. MAGNO and R. A. NICOLAUS
Chromatographic analyses of the oxidation products of the pigments after decarboxylation are given in Table 4: The presence of pyrrole-2,4dicarboxylic and pyrrole-2,5-dicarboxylic acids among the oxidation products of the decarboxylated melanins is proof of the oxidation cleavage in the benzenoid portion of some indole units during melanogenesis. The identification of these acids supports the assumption that pyrrole units are present in melanins? Higher yields of pyrrole-2,3-dicarboxylic acid and lower yields of pyrtole-2,3,5tricarboxylic acid are produced by oxidation of sepiomelanin after decarboxylation than by oxidation of the undecarboxylated pigment .l This evidence proves the presence of carboxyl groups in position 2 of some indole nuclei. In the case of dopa melanin and 5,Cdihydroxyindole melanin, no significant variation in the yields of the two pyrrolic acids could be observed; therefore, dopa melanin cannot have carboxyl groups in position 2 of the indole nuclei. Furthermore, since pyrrole-2,3,5-tricarboxylic acid is not found among the hydrolysed oxidation products of methylated dopa melanin, this pigment, unlike sepiomelaninl has no carboxyl groups in position 2 of the indole nuclei. The retention of carboxyl groups of some dopa molecules in dopa melanin, proved by use of carboxyl-labelled dopa,13 is explained by assuming the incorporation of carboxylated intermediate molecules which may either be cyclized or uncyclized. The hypothesis of incorporation of uncyclized intermediate molecules is supported by the occurrence of trimethylamine among the oxidation products of methylated dopa melanin. Therefore, it seems that dopa melanin differs from all other pigments in having a-amino acid chains in its molecule, i.e. dopa quinone (Raper’s scheme) participates in the building up of dopa melanin. In spite of differences, all the other data being in agreement, tyrosine (or dopa) very probably is the precursor of sepiomelanin. In connection with the Raper’s scheme, the carboxyl group in position 2 of some indole nuclei in sepiomelanin originates from participation of a carboxylated intermediate (probably dopachrome) in the building up of the pigment. Such a participation does not occur in vitro to any extent, probably owing to the fact that conditions of pigment formation in do are not reproducible. Analytical data and behaviour on oxidation suggests that pyrrole black is very On the other hand, the occurrence in nature of pigments different from sepiomelanin. similar to pyrrole black cannot apriori be excluded. EXPERIMENTAL
The different C, l-I, N, percentages in various analyses of the same sampleareascribed to the difficult combustion of melanins. Greater differences are observed when samples from different runs of the same preparation are analysed. This can be explained on the basis that melanogenesis takes place with oxidation reactions, (e.g. breakdown of benzenoid parts) decarboxylation and participation of intermediates different from 5,bindolequinone (e.g. dopaquinone and dopachrome) resulting in molecules of high complexity and irregularity. These reactions even occur when several preparations of the same pigment are carried out under apparently identical experimental conditions. The N percentages given in Tables I and 2 are mean values. Sepiomelanin. Pigment was purified according to the cold purification method reported in the ftrst paper of this series.’ I8 G, R. CIemo, F. K. Duxbury and G. A. Swan, J. Chem. Sot. 3464 (1952).
The structure of melanins and melanogenesis-IT
947
Dopa melanin. Mushroom tyrosinase (loo0 U/mg; 38 mg) was added to a solution of L-B(3,4-dihydroxyphcnyl) alanin (1.5 g) in M/20 phosphate buffer at pH 6.8 (2 I.) and a rapid stream of oxygen was passed for 6 hr through the solution, kept at 35”. The black precipitate+ collected by centrifugation, was kept in cone HCl at room temp for 7 days. After centrifugation, the melanin was thoroughly washed with 1% HCl, distilled water and finally acetone. After drying over phosphoric oxide, 550 mg of melanin were obtained. (Found: (a) C, 60.64; H, 4.23; N, 8*77-C, 60-84; H, 4.10; N, 8~23; (b) C, 59~70; H, 3.55; N, 5032-C 59.93; H, 3.65; N, 6.74; (c) C, 59~33; H, 2.87; N, 7937%). A similar preparation carried out with addition of catalase (ex beef liver; 240 Kiel units) resulted in a larger yield of pigment (800 mg). (Found: C, 56.92; H, 346; N, 6.48-C 56-97; H, 3-39; N, 6~17%). These melanins were amorphous, infusible compounds, insoluble in any solvent and highly hygroscopic. 5,bDihydroxyindole melanin. 5,6_Dihydroxyindole was prepared from 2-nitro4,5-dihydroxybenzaldehyde according to Beer ez al. ;I4 a method more recently described by Harley-MasorP resulted in poor and erratic yields in the last step of the synthesis. A rapid stream of oxygen was passed for 12 hr through a solution of 5,bdihydroxyindole (2 g) in M/20 phosphate buffer (660 ml) at pH 8. The solution turned purple in colour with the formation of a brown precipitate. The latter was collected by centrifugation (the mother liquors were colourless) and kept in cone HCl for 7 days. After centrifugation the melanin was repeatedly washed with 1% HCI, distilled water and finally acetone. After drying over phosphoric oxide, 1.75 g of melanin were obtained. [Found: (a) C, 59.06; H, 3.45; N, 9*44-C, 63.24; H, 2.70; N, 10.74; (b) C, 59.31; H, 3.43; N, 9.21-C 58-86; H, 3.31; N, 9.35 YJ. Another sample of S,&dihydroxyindole melanin was similarly prepared with addition of mushroom tyrosinase (50 mg). In this case the oxidation was more rapid, requiring only 6 hr and 1.75 g of melanin were obtained from 2 g of 5,6_dihydroxyindole. [Found: (a) C, 59.24; H, 3.24; N, 8.63C, 59.38; H, 3.02; N, 744; (b) C, 59~89; H, 2.53; N, 7.96-C 59.27; H, 2-54; N, 7*99x]. A third preparation was carried out with tyrosinase (50 mg) and catalase (300 Kiel units), and 2 g of melanin from 2 g of S$dihydroxyindole were obtained [Found: (a) C, 57-61; H, 4.0; N, 8.09; (b) C, 59~77; H, 2.86; N, 8*10--C, 58.74; H, 2~71; N, 8.66x]. The pigments were black, amorphous, infusible compounds, insoluble in all solvents and highly hygroscopic. Pyrrole black. Pyrrole (10 g) was oxidised with hydrogen peroxide in acetic acid according to Angeli.16 After dilution with water (2: I), the pigment was coflected by ccntrifugation and washed with 1 o/0HCI, distilled water and acetone. The dried pigment was powdered, placed in a glass column and washed first with acetone and subsequently with ethanol. After drying, a black, amorphous pigment (2.7 8) was obtained. [Found: C, 57.75; H, 5.03; N, 14.7X,56.79; H, 5.28; N, 12.91 %J About 500 mg of each pigment, previously dried in vacua Thermic decurboxylation of ~hepigments. over phosphoric oxide at 80’ for 12 hr were suspended in vaseline oil and heated at 190--200° (bath temp) in a current of nitrogen for 10 hr. The amount of CO, evolved was measured by absorbing the gas in a saturated solution of barium hydroxide and weighing the barium carbonate formed. The following results were obtained : Sample
Pigment in mg
ElaC.0, in mg
540.8 316 408 475.2 365 426 428
214.6 106 48.7 194 157-5 90 93
Sepiomelanin Dopa melanin (tyrosinase) Dopa melanin (tyrosinase + 5,dDihydroxyindole melanin 5,6-Dihydroxyindole melanin S&Dihydroxyindole melanin Pyrrole black
cat&se) (autoxidation) (tyrosinase) (tyrosinase + catalase)
I4 R. J. S. Beer, K. Clarke, H. G. Khorana and A Robertson, J. Chem. Sot. 223 (1948). l5 J. Harley-Mason, 3. Chem. Sm. 200 (1953). 1@A. Angeh and L. Alessandri, Gorr. Chim. ital. 46,283 (1916).
’
948
M. PIAITELLI,E. FATTORUSSO,S. MAGNO and R. A. NICOLAUS
Reaction uf the pigments with diozomethane. About 500 mg of each pigment were treated with a solution of diazomethane (500 mg) in ether (50 ml) and allowed to stand at room temp for 24 hr. Unreacted diazomethane was removed with acetic acid. The pigment, collected by centrifugation, was successively washed with methanol, ethanol and ether. After determination of methoxyl groups, the pigment was again treated with diazomethane until the percentage of methoxyl groups was constant. All the methylated pigments were brown, amorphous compounds. [Found : sepiomeliznin: C, 64-l; H, 5.5; N, 7.4; OCH*, 18.8; dopa melanin (tyrosinase): C, 62.80; H, 5.17; N, 7.42; OCHS, 20.&C, 62.92; H, 5.14; N, 7.45; dopu melunin (tyrosinase + catalase) (a) C, 62.14; H, 5.97; N, 6.17; OCH*, 19-w, 62.23; H, 5.63; N, 7.95; (b) C, 62.31; H, 4.95; N, 5.01-C, 6267; H, 5.18; N, 4.12; 5,6_dihydroxyindole melanin (autoxidation): C, 61.53; H, 5.83; N, 7.55; OCHa, 19.1-C, 61.63; H, 5-58; N, 760; 5,GdihydroxyindoZe mefunin (tyrosinase): C, 62.31; H, 5.19; N, 7.83; OCHS, 21*5-C, 63.39; H, 5.08; N, 7.62; 5$-dihydroxyindole melanin (tyrosinase + catalase) (a) C, 63.66; H, 5-30; N, 7.51; OCHI, 21.5-C, 6362; H, 5.16; N, 7.32; (b) C, 61.60; H, 5.67; N, 8*59-C, 62.48; H, 5.49; N, 7.61; pyrrole black: C, 60.72; H, 5-24; N, 14-05; OCH*, 8.6; C, 60-56; H, 5.52; N, 14.08%]. The methylenedioxy groups test was positive for all the pigments except pyrrole black, which dissolves in the reagent, the solution turning brown in colour. Oxidation of the pigmertts. 50 mg of each pigment, suspended in 0.6 ml 2N KICOs, were oxidized with 3 % KMnO, until the colour persisted for 15 min. The following quantities of 3 *AKMnOl were employed : sepiomelanin: 6-3 ml; dopa melanin (tyrosinase): 5 ml; dopu melanin (tyrosinase + catalase) : 7 ml ; 5,6_dihydroxyindole melanin (autoxidation) : 6.2 ml ; 5,6_dihydroxyin&le melanin (tyrosinase): 6-4 ml; 5&dihydroxyindo/e melanin (tyrosinase + catalase): 6.6 ml; pyrrole black: 6 ml. The solution was boiled for a few minutes, filtered and acidified with 2N HCl to pH 4-5. Oxalic acid was precipitated with 20% CaCit and the solution filtered, strongly acidified with cone HCI and extracted with ether (50 ml in 5 portions). After separation of the solvent, the residue was dissolved in distilled water (0.2 ml) and the filtered solution used for chromatographic analysis; O-03 ml of solution were spotted on Whatman No. 1 paper and compared by descending chromatography with reference aliquots of pyrrolic acids [propanol : 33 %NHI : water (60: 30 : 10) and ethanol : 33 %NH, : water (80 :4: 16) as solvents]. The chromatograms were sprayed with the diazonium salt of sulphanilic acid and N NaOH. From the intensity and size of the developed red spots the following conclusions could be drawn: dopu melanin yielded pyrrole-2,3,5-tricarboxylic acid and a smaller amount of pyrrote-2,3_dicarboxylic acid, while pyrrole-2,3,4,5-tetracarboxylic acid was present only in traces; 5,ddihydroxyindole melanin yielded relatively large amounts of pyrrole-2,3,5-tricarboxylic and pyrrole 2,3dicarboxylic acids and a smaller quantity of pyrrole-2,3,4,5_tetracarboxylic acid. The chief oxidation product of pyrrole black was pyrrole-2,5dicarboxylic acid although pyrrole-2,3,5-tricarboxylic, pyrrole-2,3-dicarboxylic and pyrrole-2-carboxylic acids were also found in smaller amounts. Paper chromatography of the mixture obtained by oxidation of pyrrole black did not lead to the identification of pyrrole-2-carboxylic acid but its presence was shown by thin layer chromatography [silica gel; chloroform : formic acid (80 :20) and benzene : acetic acid (50 : 50) as solvents.] Oxi&tiun of the methyiated pigments. Methylated pigments (100 mg) were oxidized as described above and the typical smell of aliphatic amines was perceived during the oxidation. The following quantities of 3% KMnO, were employed: sepiomelanin: 14-l ml; dopa melanin (tyrosinase): 10.8 ml; dupa melanin (tyrosinase + catalase): 14-5 ml ; 5,ddihydruxyinMe melanin 14.6 ml; 5,GdihydroxyindoZe autoxidation) : 13.8 ml; 5,6-dihydroxyindole melanin (tyrosinase): melanin (tyrosinase + catalase): 15 ml; pyrofe black : 11 ml. In order to avoid the hydrolysis of pyrrolic esters which might have been present, MnOs was removed, without previous heating, by centrifugation after adding some sodium sulphite. A portion of the ether extract, chromatographically examined as described above, showed no presence of pyrrolic acids. Only pyrrole black yielded pyrrole-2,5-dicarboxylic and pyrrole-2,3,5tricarboxylic acids, (pyrrole-2,3dicarboxylic and pyrrole-2-carboxylic acids wereonlydetectable when spotting larger amounts of the solution on the paper.) The remaining portion of the ether extract was evaporated to dryness and the residue hydrolysed with 30% NaOH (0.2 ml) at 100” for 40 min. The solution, after addition of water and acidification with cone HCI, was extracted with ether (25 ml in 5 portions). After solvent evaporation, the residue was dissolved in water (O-2 ml) and the filtered solution used for paper chromatography. Pyrrole-2,3,Qricarboxylic acid could again be identified after hydrolysis only in the case of sepiomelanin, whereas this did not occur in the other pigments.
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The structure of melanins and melanogenesis--11
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Since the presence of p-methyl esters of pyrrolic acids among the degradation products could not be excluded (on account of their higher resistance to hydrolysis than a-esters), hydrolysis was also performed under more drastic conditions (10% KOH in ethanol for 12 hr) but this did not modify the results. Oxidation of the decarboxyluted pigments. Decarboxytated pigments (50 mgJ were oxidized as usual with 3 % KMnO,. The following quantities of 3 % KMnO, were employed: sepiomehin: 6.4 ml; dopu melanin (tyrosinase): 7.5 ml; dopa melonin (tyrosinase t catalase): 7.2 ml; 5,6-dil hydroxyindok melanin (autoxidation) : 6-9 ml; 5,6_dihydroxyindofe melanin (tyrosinase) : 5.1 ml ; 5$-dihydruxyindole melanin (tyrosinase t. catalase) : 7.3 ml; pyrrole black: 4.8 ml. The oxidation products were analysed by paper chromatography (ethanol:33 % NHg:water (8014: 16) and propanol:33% NHs:water (60:30: 10) as solvents) and by thin layer chromatography [chloroform:formic acid (50: 50) as solvent]. Decarboxylated sepiomelanin when compared with undecarboxylated pigment yielded lower amount of pyrrole-2,3,5-tricarboxylic acid and larger quantities of pyrrole-2,3dicarboxylic acid ; the yields of pyrrole-2,3,4,5-tetracarboxylic acid were the same and small amounts of pyrrole-2,4_dicarboxylic and pyrrole-2,Sdicarboxylic acids were also obtained, Dopa melanin and 5,6dihydroxyindole melanin, after decarboxylation, gave the same results as the undecarboxylated pigments ; the only difference being the appearance of small amounts of pyrrole-2,4-dicarboxylic and of pyrrole-2,5dicarboxylic acids. Permanganic oxidation of decarboxylated pyrrole black gave results similar to those of undecarboxylated pigment . Volatile bases from oxidation of methylated dopu melanin. Methylated dopa melanin (tyrosinasecatalysed oxidation ; 200 mg) was oxidised with 3 % KMnOl. The liquid was distilled and collected in 2N HCI. The solution containing the volatile bases as hydrochlorides was evaporated. From the residue, free bases were obtained by alkali addition. Gas chromatography [C. Erba’s Fractovap; 20”; column (1 m length of Celite (85x), triethanolamine (7.5x), Vaseline oil (7*5x))] showed the presence of ammonia and trimethylamine. The same results were obtained from the methylation product of dopa melanin prepared in the presence of tyrosinase and catalase. Acknowledgements-We wish to record our thanks to G. Ferracane for technical assistance and to E. Thommen, Dept. Org. Chem., University of Base], Switzerland, for elementary analyses.