STRUCTURE AND BIOSYNTHESIS OF MELANIN - PDF document

1 STRUCTURE AND BIOSYNTHESIS OF MELANIN o
STRUCTURE AND BIOSYNTHESIS OF MELANIN of the UNIVERSITY OF LONDON' Imperi

2 al College, ABSTRACT II ACKNOWLEDGEIO
al College, ABSTRACT II ACKNOWLEDGEIONTS I wish to record my sincere gr

3 atitude to CONTENTS REVIEW #41.411
atitude to CONTENTS REVIEW #41.411 19 € 51 1 INTRODUCTION Defini

4 tion of Melanin 1 3 Thomas 4 BIOGENE
tion of Melanin 1 3 Thomas 4 BIOGENETIC STUDIES 47 has shown the Uinto

5 the melanin of the 5 skin and hair. I
the melanin of the 5 skin and hair. It is now firmly established that the

6 COOH 1st stare HO 2 OOH 9 000H
COOH 1st stare HO 2 OOH 9 000H Scheme 1 COOH 2nd stage + 00 10

7 7 3rd stage: 9 and 10 and by silver o
7 3rd stage: 9 and 10 and by silver oxide to the same end€products. More

8 recently17 5,6-dihydroxyindole has been
recently17 5,6-dihydroxyindole has been isolated and 8 ButLoOk.9., and Ma

9 son to be general for dihydroindole-5,6
son to be general for dihydroindole-5,6-quinones and has been 11 12 10 c

10 hromophore of which was deducedyi in wat
hromophore of which was deducedyi in water) 22 H fit Me H 14 when the

11 catechol was oxidised with silver oxide
catechol was oxidised with silver oxide in ) formed from the oxidation Olig

12 omers 16 12 13 experiments with dopa
omers 16 12 13 experiments with dopamine, 7iitkop and his coworkers HO

13 0 000H How
0 000H How Me€ 0 20 21 m (E: 10,800; 15 2

14 /ijHowever, by using (‚) [14c0 2 n carb
/ijHowever, by using (‚) [14c0 2 n carbon dioxide evolved, during the enzym

15 atic oxidation of the former E.,- benzen
atic oxidation of the former E.,- benzene nucleus and only 1.9% and 2.3% res

16 pectively from the labelled positions (2
pectively from the labelled positions (22). These authors, using 16 4anucl

17 ear labelled dopamines, later showed tha
ear labelled dopamines, later showed that 3 2 destroys hydrogen peroxide

18 inhibits the evolution of 17 and 5,6-d
inhibits the evolution of 17 and 5,6-dihydroxyindole HOOC, HOOC 18 HOW

19 I HOOC COON STRUCTURAL STUDIES 19 2
I HOOC COON STRUCTURAL STUDIES 19 20 21 22 Commoner and his coworker

20 s HO 23 26 melanin biosynthesis is ac
s HO 23 26 melanin biosynthesis is accepted, it still remains to 5 (28)

21 Scheme 3 27 1R2 + 2 24 quinonimin
Scheme 3 27 1R2 + 2 24 quinonimines (29) are formed, or side-chain su

22 bsti- R H etc. 0 OH R 0 2
bsti- R H etc. 0 OH R 0 2 Similarly 0 2xArNH 25 0 OH 28 A

23 lthough the nitrogen atom in heterocycli
lthough the nitrogen atom in heterocyclic substances 3-indolyl-quinones (30

24 ), (31), (3Scheme 4) 26 011 35 etc.
), (31), (3Scheme 4) 26 011 35 etc. 34 If the 2-position of indole is

25 free, then a 2,3- 28 3-7 linked structu
free, then a 2,3- 28 3-7 linked structure were valid, then 5-substituted 3

26 8 0, 40 37 37(a) 30 isopropyl-,
8 0, 40 37 37(a) 30 isopropyl-, 3-methyl-4-isopropyl-, and 3-methyl-

27 7- 31 43- reported an average consump
7- 31 43- reported an average consumption of 4.6 atoms of 3 inter-unit l

28 inkages A Nff , 0 eV A 43 33 ,ti
inkages A Nff , 0 eV A 43 33 ,tible to oxidation than 5,6-dihydroxyi

29 ndole itself and, 34
ndole itself and, 34

30 44 46 35 The
44 46 35 The predominance of 4-7 linkage has bee

31 n strongly by autoxidation: of .dopa 36
n strongly by autoxidation: of .dopa 36 deuterium retention obtained above

32 suggested to Swan . 48 37 and his co
suggested to Swan . 48 37 and his coworkers do raise the very serious qu

33 estion as to whether or not 5,6-dihydrox
estion as to whether or not 5,6-dihydroxyindole is in fact 38 information

34 on the probable nature of the nuclei HO
on the probable nature of the nuclei HOOC H OOH 50 OPM 39 Nicolaus an

35 d his collaborators" later identified H
d his collaborators" later identified HOOC '/It% H 49 000H HOOC The mos

36 t significant was the failure to detect
t significant was the failure to detect 3-7 backbone structure is correct. S

37 ince this acid was 3 of the indole- 40
ince this acid was 3 of the indole- 40 41 degradations of melanins is that

38 the same pyrrole HOOC 24 H00"3 25 54
the same pyrrole HOOC 24 H00"3 25 54 CH II OOH COON 53 \t/ COOlfe

39 58 COON 1 Me0 59 43 the following w
58 COON 1 Me0 59 43 the following way. Sepia melanin was methylated 44

40 CCOH coworkers favoured the idea of u
CCOH coworkers favoured the idea of uncyclised amino acid 45 after deca

41 rboxylation. Furthermore, since pyrrole-
rboxylation. Furthermore, since pyrrole-melonha (see:blicrie)', is unlikely

42 to have carboxyl groups in position 2 of
to have carboxyl groups in position 2 of 46 A, (ed. K. Paech and M.V. Trac

43 ey) Springer, Berlin, 47 12. Chem. So
ey) Springer, Berlin, 47 12. Chem. Soc., 1954, 1947. 48 49 37. 689.

44 39. 1951, 703. 44. 50 51 OOH I S
39. 1951, 703. 44. 50 51 OOH I Scheme 1 (Contd.) 52 Quinonoid inte

45 rmediates H Melanin V n The problem
rmediates H Melanin V n The problem of melanin structure therefore has

46 been to 53 were invariably detected in
been to 53 were invariably detected in very low yields (for 54 for compa

47 ring the structures ,of melanins produce
ring the structures ,of melanins produced of specifically labelled dopa will

48 be next. Finally the use of such precur
be next. Finally the use of such precursors 3 55 _Isotope Dilution Study

49 of Melanin Degradation II 7 OOH OH
of Melanin Degradation II 7 OOH OH VI obtained from the oxidative degr

50 adation of melanin ..]as follows47.
adation of melanin ..]as follows47. 0 ) H

51 n Melanin
n Melanin 56 A sample of this material,

52 of known total activity, 57 An intens
of known total activity, 57 An intensive study of melanin oxidation with

53 7 and graph C). The stability of alkali
7 and graph C). The stability of alkaline permanga- 58 59 Table 5 (10 m

54 l. of N-permanganate plus 0.6 ml. 2N-pot
l. of N-permanganate plus 0.6 ml. 2N-potassium *N/40 13.50 Table 6 (Mel

55 anin, 22.2 mg.; N-permanganate, 10 ml.;
anin, 22.2 mg.; N-permanganate, 10 ml.; 2N-potassium, N/40 11.20 *N/40 T

56 able 7 (melanin, 18.2 mg.; diacid, 20.
able 7 (melanin, 18.2 mg.; diacid, 20.8 mg.; N- 10.35 .. 8.30 € Graph

57 A Stability of alkaline permanganate
A Stability of alkaline permanganate (see Table 5) 16 12 lo 10 30 Tab

58 le 7 (contd.) 61 Vol. of titrant Oxid
le 7 (contd.) 61 Vol. of titrant Oxidation of melanin 12 14 16. 12 (

59 ml. Graph C 12 62 9, Recovery of Py
ml. Graph C 12 62 9, Recovery of Pyrrole-2,3-dicarboxylic acid from the

60 oxi- 63 of the unreacted diacid had b
oxi- 63 of the unreacted diacid had been established, mela-: 64 Exchang

61 e Reactions in Phenols 65 a convenien
e Reactions in Phenols 65 a convenient and inexpensive procedure for the

62 prepa- 66 Base-catalysed Exchange React
prepa- 66 Base-catalysed Exchange Reactions in Phenols valent of deuterox

63 ide or a molar equivalent of amine. 311
ide or a molar equivalent of amine. 311:1Tyrosine was obtained in a similar

64 67 [cf. (Ix)] was unaffected. 68 0.
67 [cf. (Ix)] was unaffected. 68 0.5 60 Graph D Deuteration of l

65 ee-oresƒ Deuteration Time (hrs.) H 0
ee-oresƒ Deuteration Time (hrs.) H 0 MeO 69 Iz KIT Vanillin (

66 X; R=Me) likewise gave only the 5- 70 m
X; R=Me) likewise gave only the 5- 70 monodeutero-derivative in the usual w

67 ay. Benzylation, 6) ortho and para 5 wh
ay. Benzylation, 6) ortho and para 5 which is meta to the 71 72 deactiva

68 ting effect of the aldehyde group no lon
ting effect of the aldehyde group no longer Indeed specific ortho and para

69 labelling of phenolic morphine (XII; R=1
labelling of phenolic morphine (XII; R=11) when heated alone in dimethylform

70 amide containing deuterium oxide was con
amide containing deuterium oxide was converted in good yield into the aryl p

71 rotons in undeuterated material gave 3.4
rotons in undeuterated material gave 3.47Z (T=8 cps) in the 3.47t was assign

72 ed to produced broadening and 73 Table
ed to produced broadening and 73 Table (%) in * * Incomplete deuteration

73 arose from introduction of atmospheric
arose from introduction of atmospheric water during repeated sampling. Deu

74 teration10 8o 60 20 (see Table 4)
teration10 8o 60 20 (see Table 4) A 4 20 40 120 Time (mins.) Graph F

75 1 2 as expected, in addition to the o
1 2 as expected, in addition to the ortho and Para hydrogens 76 The fol

76 lowing general observations were made.
lowing general observations were made. 77 Synthesis of Labelled 3,4-Dihydr

77 oxyphenylalanine 78 r Bromoproto- 7
oxyphenylalanine 78 r Bromoproto- 79 6-Bromo-1-3g isovanillin was most

78 conveniently 80 RO wr 2 V dope H
conveniently 80 RO wr 2 V dope H Ro GOGH H RO B RO r xii 8

79 2 83 deuterium oxide. The resulting di
2 83 deuterium oxide. The resulting dideuterated-derivative 0 of the orig

80 inal activity. With the theo(-or the /3-
inal activity. With the theo(-or the /3-position. 84 85 Study of Melanin

81 Structure with Labelled EG-dopa and the
Structure with Labelled EG-dopa and the mixture recrystallised to give effe

82 ctively 86 written for convenience as
ctively 86 written for convenience as a polymer of the indole€ HO

83 1 (XXIII) 314 Previous trac
1 (XXIII) 314 Previous tracer work by Swan and 7right 87 of ca

84 rbon from any other part of its molecule
rbon from any other part of its molecule during 88 (xv) H XIVn tl The

85 first set "Cfa) of the 89 Table 10 3
first set "Cfa) of the 89 Table 10 3H] dopa retained ti 90 %C 3v/ n

86 tr3y5 HO 91 manner.E(- 92 Table 12
tr3y5 HO 91 manner.E(- 92 Table 12 (Melanins were precipitated with a

87 mmonium nitrate; 0 u14 E5- 93 Table
mmonium nitrate; 0 u14 E5- 93 Table 13 3H retained 0,11 * In the 1st

88 set of results, no correction was nece-
set of results, no correction was nece- 94 1 that 33 -precursor especial

89 ly when it is realised that The result
ly when it is realised that The result with @-melanin from this precursor,

90 as expected, differed 95 from all the
as expected, differed 95 from all the others in that it lost most of the

91 52 (13) was retained. Clemo and his cow
52 (13) was retained. Clemo and his coworkers had ajdopa was precipitated w

92 ith ammo- 96 adsorption of unreacted do
ith ammo- 96 adsorption of unreacted dopa might contribute the absence of a

93 n acid. are a bit more difficult to int
n acid. are a bit more difficult to interpret. pH Dopa should give the dopa

94 chrome (XVI) labelled as 97 H N.N H
chrome (XVI) labelled as 97 H N.N H .VII N'` C 00H * 3 XVIII This en

95 tity might aromatise before coupling by
tity might aromatise before coupling by the gromatise the ring before coupl

96 ing. If there is a large mass (46f.)will
ing. If there is a large mass (46f.)will be due 98 almost entirely to coup

97 ling. If on the other hand, melanin for
ling. If on the other hand, melanin formed in an intact organism is fundame

98 ntally different from that 99 "meetrat
ntally different from that 99 "meetratrimmermlelo 1) 100 0.50 mg.) in d

99 imethylformamide (0.1 ml.) was in the f
imethylformamide (0.1 ml.) was in the form of its hydrochloride. (%) in 1.

100 2 ml. Vol. of DMF 0.10 101 BaE ge0 E
2 ml. Vol. of DMF 0.10 101 BaE ge0 Edjperspex (60% carbon of known activ

101 ity) which 102 as usual. Counting at t
ity) which 102 as usual. Counting at this low [14a-dopa was to dissolve c

102 ompletely and the solution evaporated 1
ompletely and the solution evaporated 103 their more labile protons, for e

103 xample, protons 104 .-cresol (100 mg.)
xample, protons 104 .-cresol (100 mg.) in 105 3.2e'C which diminished in

104 106 p,5-tyrosine rapidly by centrifug
106 p,5-tyrosine rapidly by centrifugation 9 cps) corresponding to the pr

105 otons disappeared while the meta-proton
otons disappeared while the meta-proton doublet 2.7815 107 (J = 9 cps) ha

106 d been replaced by a broad singlet. spec
d been replaced by a broad singlet. spect7um consisting of an approxi-proton

107 s (an ABC pattern) remained with a solu
s (an ABC pattern) remained with a solution of thionyl chloride in glacial

108 (J = 2 cps) 108 50 of this value. 109
(J = 2 cps) 108 50 of this value. 109 determined by appropriate dilution

109 (c/f tyrosine), 5lityrosine.11-Tyrosin
(c/f tyrosine), 5lityrosine.11-Tyrosine treated 110 hydroxyl group re-ex

110 changed for hydrogens (n.m.r. 2.951r (J
changed for hydrogens (n.m.r. 2.951r (J = 8 cps) 111 52o protocatechuic a

111 ldehyde. D-Atproto-catechuic aldehyde m.
ldehyde. D-Atproto-catechuic aldehyde m.p. 151-153 112 (195 mg.) suspended

112 in ethanol (30 ml.) containing as prisms
in ethanol (30 ml.) containing as prisms m.p. 62-64. Its n.m.r. spectrum run

113 in 665. Bromination of a 113 3-0Benzy
in 665. Bromination of a 113 3-0Benzy1-5-bromoprotocatechuic aldehyde. A

114 solu- CP 54.7;'H, 3.7. 03.60). 00-Dibenz
solu- CP 54.7;'H, 3.7. 03.60). 00-Dibenzyl-@- manner, was chromatographed on

115 alumina (Grade V) to 114 2H0 isovanil
alumina (Grade V) to 114 2H0 isovanillin (60ro yield) m.p. 113-115ƒ6 The

116 n.m.r. spectrum of isovanillin (in dimet
n.m.r. spectrum of isovanillin (in dimethylsul-J = 8 and 2 cps), 2.691(H-C(1

117 1 34 ƒ for 1 hr. and steam 115 116 6
1 34 ƒ for 1 hr. and steam 115 116 6-Bromoisovanillin, even when obtaine

118 d as well formed 2-Bromo-1 [ 117 (18.1
d as well formed 2-Bromo-1 [ 117 (18.1 and 28. D- 118 L. With the undeute

119 rated material, both In a similar way,
rated material, both In a similar way, 6-bromo-12- 119 pyridine (1.5 ml.)

120 containing anhydrous aluminium 120 121
containing anhydrous aluminium 120 121 anhydride (9 ml.) were refluxed (7

121 0 min.) in presence 122 H9 5.5). H, 5
0 min.) in presence 122 H9 5.5). H, 5.7. 0 123 Hydrolysis of the benzylh

122 ydantoin with ƒ V-1 (Found: 0, 68.2; H
ydantoin with ƒ V-1 (Found: 0, 68.2; H, 6.1. Crequires 0, 68.6; H, 5.75%)…

123 4-5 by ƒ. ƒ (2 hrs.) was collected, w
4-5 by ƒ. ƒ (2 hrs.) was collected, washed ƒ. The on paper chromatograph

124 developed in n-butanol: acetic acid:wate
developed in n-butanol: acetic acid:water = 3:1:1 and sprayed with 0.1% nihy

125 drin in 124. Hydrolysis of 5-(3,4-Dibe
drin in 124. Hydrolysis of 5-(3,4-Dibenzyloxybenzy1)-- 125 2,5,6- (?- A

126 solution 2-bromoisovanillin (95 mg.) in
solution 2-bromoisovanillin (95 mg.) in dimethylforma- 126 alcohol (242 mg.

127 ) which was characterised as its 127 a
) which was characterised as its 127 alumina (Grade 3). Elution with benze

128 ne gave 42-43). 128 3) MELANIN DEGRADA
ne gave 42-43). 128 3) MELANIN DEGRADATION 129 powder and dried at room

129 temperature in vacuo over ammonium nitr
temperature in vacuo over ammonium nitrate (5.1 gm.) (sodium chloride (3.9

130 gm.) has also been used) and well stirre
gm.) has also been used) and well stirred to then acetone (2 x 25 ml. porti

131 ons) and dried as above to give 66-78 mg
ons) and dried as above to give 66-78 mg. of product. Dopa (100 mgs.) in M

132 /15 phosphate acids and bases and in al
/15 phosphate acids and bases and in all common organic their infrared spec

133 tra 130 in Nujol mull revealed no dist
tra 130 in Nujol mull revealed no distinguishing features. following are t

134 ypical analytical results: Tyrosine Mel
ypical analytical results: Tyrosine Melanins f H 131 (Found: C, 52.6; H

135 , 4.85. Calculated for C C9 46.2; H, 3.2
, 4.85. Calculated for C C9 46.2; H, 3.25. Calculated e 8,900 and 11,800. L

136 it; E 2,900 xylic acid was treated brie
it; E 2,900 xylic acid was treated briefly at room temperature H, 4.2. Cal

137 culated 9 72-73). 132 Rate of Oxidati
culated 9 72-73). 132 Rate of Oxidation of Melanin in the Absence and the

138 13.3 (b) N/4-ferrous 134 0.10 ml.
13.3 (b) N/4-ferrous 134 0.10 ml. At the end of oxidations in (a) and

139 (b) 135 EtOHLmg.) which crystallised
(b) 135 EtOHLmg.) which crystallised from benzene-petroleum ether as Tab

140 le 8 [14 136 Melanin Degradation
le 8 [14 136 Melanin Degradation Oxidation Oxidation

141 I Chromatographic dimethyl ester (of
I Chromatographic dimethyl ester (of 137 Collection and assay of carbon

142 dioxide evolved during 9 E, tyrosine
dioxide evolved during 9 E, tyrosine The solution in the absorption tube

143 remained clear 138 The test solutions
remained clear 138 The test solutions remained clear throughout the 139

144 acid (0.5 ml.) for 32 hrs. The solution
acid (0.5 ml.) for 32 hrs. The solution of desorbed 140 References Obschc

145 h. Khim. 1958, 28, 2644; G.E. Hall, E.M.
h. Khim. 1958, 28, 2644; G.E. Hall, E.M. 141 D.H.R. Barton, G.W. Kirby and

146 H.P. Tiwari, 142 143 28. a, 423. 33
H.P. Tiwari, 142 143 28. a, 423. 33. 22 1843. Personal Communications.

147 See (1958) purchased from Koch-Light
See (1958) purchased from Koch-Light 36. 37. and Haarman, Ber., 1874, 7,

148 615. 38. Nicolaus, Gazetta, 1953, 83,
615. 38. Nicolaus, Gazetta, 1953, 83, 239. 41. 1948, 172, 83. Fattorus

149 so, E. Magno and R.A. Rend. Ac. Sci. fis
so, E. Magno and R.A. Rend. Ac. Sci. fis. e Mat., 1961, 28 144 43. 12, 5