U.S. patent application number 09/802189 was filed with the patent office on 2002-01-17 for dyeing substrates.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Desler, Torben, Kirk, Ole, Lolck, Rikke, Sorensen, Niels Henrik.
Application Number | 20020004958 09/802189 |
Document ID | / |
Family ID | 8101047 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020004958 |
Kind Code |
A1 |
Sorensen, Niels Henrik ; et
al. |
January 17, 2002 |
Dyeing substrates
Abstract
The invention relates to substrates, which develops colour on
oxidation of the general formula 1 1 The invention also relates to
the use of said substrates for dyeing e.g. keratinous fibers, in
particular hair, dyeing textiles, a dyeing composition and a method
for dyeing keratinous fibers.
Inventors: |
Sorensen, Niels Henrik;
(Skaevinge, DK) ; Kirk, Ole; (Virum, DK) ;
Lolck, Rikke; (Smorum, DK) ; Desler, Torben;
(Copenhagen, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
C/O NOVO NORDISK OF NORTH AMERICA, INC.
405 LEXINGTON AVENUE, SUITE 6400
NEW YORK
NY
10174
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
8101047 |
Appl. No.: |
09/802189 |
Filed: |
March 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09802189 |
Mar 8, 2001 |
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09271540 |
Mar 18, 1999 |
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6231621 |
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09271540 |
Mar 18, 1999 |
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PCT/DK97/00435 |
Oct 8, 1997 |
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Current U.S.
Class: |
8/405 ; 8/406;
8/407; 8/411; 8/412; 8/421 |
Current CPC
Class: |
G01N 33/535 20130101;
A61K 8/445 20130101; A61Q 5/10 20130101; D06P 1/32 20130101; C09B
17/02 20130101 |
Class at
Publication: |
8/405 ; 8/406;
8/407; 8/411; 8/412; 8/421 |
International
Class: |
A61K 007/13 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 1996 |
DK |
1104/96 |
Claims
1. A substrate, which develops colour on oxidation characterized by
the inclusion of an amino benzoic acid compound with the general
formula 1 7wherein R is an amino, mono- or a distributed amino or
OR', where R is H, alkyl, alkenyl, alkynyl, halogenalkyl, nitro,
benzyl, phenyl or substituted phenyl, X, Y and Z may each
independently be hydrogen alkyl, alkenyl, alkynyl, halogenalkyl,
nitro, benzyl, phenyl, substituted phenyl, amino, hydroxy or
mercapto with the proviso that at least one of the groups X, Y and
Z is an amino group or an salt thereof.
2. The substrate according to claim 1, wherein R' is a methyl,
ethyl or isopropyl ester.
3. The use of amino benzoic acid compounds with the general formula
1 as well as the salt thereof as a colour producing substrate in
analysis based on the use of peroxidase.
4. The use of amino benzoic acid compounds with the general formula
1 as well as the salt thereof as a colour producing substrate in
hair dyeing compositions as well as for compositions for dying of
natural and synthetic fibres including textiles, thread and
yarns.
5. Use according to claims 3 or 4 wherein the compound is selected
from the group of methyl, ethyl or isopropyl esters of amino
benzoic acid.
6. Method for quantitative and/or qualitative analysis of a
material of biological interest, wherein a peroxidase enzyme
together with a marker is bound to the compound of interest,
hydrogen peroxide is then converted with a colour producing
substrate in the presence of the peroxidase, characterized by the
use of a substrate selected from the group made up of amino benzoic
acid compounds with the general formula 1 as well as its salts.
7. The method according to claim 8 wherein the compound is selected
from the group of methyl, ethyl or isopropyl esters of amino
benzoic acid as well as its salts.
8. The method according to claim 6 wherein the peroxidase marker is
capable of forming an immunological link to the materiel of
biological interest.
9. The method according to claim 6 wherein the materiel of
biological interest is an antigen and the marker is an antibody to
the antigen.
10. Method for producing a colored product wherein the substrate
according to claim 1 or 2 is converted by means of an oxidation
system.
11. The method according to claim 10, wherein the oxidation system
comprises an inorganic compound, preferably one of the compound
hypochlorite, hypobromite, permanganate, dichromate, or iron as
Fe3+).
12. The method according to claim 10, wherein the oxidation system
includes an enzyme and an electron acceptor.
13. The method according to claim 12, wherein the enzyme is a
peroxidase and the electron acceptor is hydrogen peroxide or that
the enzyme is oxidase and the electron acceptor is oxygen.
14. The method according to claim 13, wherein the oxidase is chosen
from the group catechol oxidase, laccase and related enzymes
o-amino phenoloxidase.
15. A composition adapted for dyeing of keratinous fibres,
comprising 1) at least one oxidation enzyme, 2) at least one
substrate according to any of claims 1 or 2, and optionally 3) at
least one modifier.
16. The composition according to claim 15, wherein the oxidation
enzyme is an oxidoreductase selected from laccases and related
enzymes, oxidases or peroxidases.
17. The composition of claim 16, wherein the oxidation enzyme is a
laccase, in particular a laccase derived from a strain of Polyporus
sp., in particular a strain of P. pinsitus or P. versicolor, a
strain of Myceliophthora sp., in particular M. thermophila, a
strain of Rhizoctonia sp., in particular Rh. praticola or Rh.
solani, a strain of a Rhus sp., in particular Rhus vernicifera, or
a strain of Scytalidium, in particular S. thermophilium, a strain
of Pyricularia sp., in particular P. oryzae.
18. A method for dyeing keratinous fibres, comprising contacting
the fibers with a composition comprising 1) at least one oxidation
enzyme, 2) at least one substrate according to claims 1 or 2, and
optionally 3) at least one modifier, for a period of time and under
conditions sufficient to permit oxidation of the substrate to a
colored compound.
19. The method of claim 18, wherein the dyeing is carried out at a
pH in the range from 3.0 to 9.0, preferably 4.0 to 8.5, especially
6.0 to 8.0.
20. The method of claims 18 or 19, wherein the composition is as
defined in any of claims 15 to 17.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns the use of aminobenzoic acid
(DABA) as a substitute for e.g. o-phenylendiamine (OPD) in analyses
based on peroxidases as well as a dyeing substrate (i.e. a dye
precursor) in dyeing compositions, and as an substrate for dyeing
natural and synthetic fibres including textiles, thread and yarns.
The invention also relates to a composition adapted for dyeing
keratinous fibres, e.g. hair, wool, fur and hides, and a method for
dyeing such keratinous fibres.
BACKGROUND OF THE INVENTION
[0002] Immuno-chemical Assays
[0003] Several different substrates are known in the part to be
used for enzyme systems in connection with peroxidase-based immuno-
hemical assays, for example ELISA. These substrates are often
toxic, mutagenic or carcinogenic.
[0004] ELISA (Enzyme Linked Immuno Sorbent Assay) is a method used
to assess the amount of antibody in serum. The main principle is
that in a much diluted solution many antigens will become bound to
the surface of plastic. Thus, if a much diluted solution of
antigens is incubated for a time in plastic trays it is possible to
wash the cavities in a buffer solution and still retain the film of
antigens on the surface of the plastic. If it is required to
determine the amount of antibodies in, for instance, serum then the
trays with their deposits of antigens are incubated with the serum.
The antibodies attach themselves to the antigens and, after
thorough washing the trays are again incubated this time with a
marker, for example anti-immune globulin serum to which there is
attached a suitable enzyme by covalent bonding. In this particular
case peroxidase is used.
[0005] The peroxidase-marker complex will attach itself to those
locations where there is already a deposit of antibodies. After
thorough washing to remove all un-combined material the enzyme
activity is measured, normally by the use of a suitable colour
indicator. Enzyme activity may be determined by the addition of a
cromogenetic substrate (i.e. colour producing compound) and
hydrogen peroxide. The enzyme catalyses the reduction of the
substrate to a coloured compound and the resultant degree of
absorbency provides a measure of the enzyme activity. If the serum
contains no antibodies there will be no enzyme activity, on the
other hand if there is much antibody present there will be very
considerable enzyme activity. A standard curve can be drawn showing
enzyme activity as a function of the concentration of antibodies.
This may be used to estimate the content of antibodies in unknown
serum samples by interpolation.
[0006] Many of the peroxidase substrates are aromatic amines and
include diaminobenziden (DAB), 3,3'-diamino benzid tetra
hydrochloride, 3,3',5,5'-tetra methyl benzidin (TMD). Another
peroxidase substrate, which does not belong to the group of
aromatic amines, is 2,2-azino-di (3-ethyl-benzo
thiazolin-6-sulphonic acid) (ABTS), this has been used as a
standard for the establishment of the activity of peroxidase
preparations. According to Voogd, Van der Stel and Jacobs (1980)
this material is also a mutagent.
[0007] o-phenylendiamine (OPD) is another peroxidase substrate
which is widely used in hospital and development laboratories. OPD
is known to be both mutagenic and carcinogenic.
[0008] The staff of laboratories in which analyses involving the
use of toxic, mutagenic or carcinogenic materials are carried out
are exposed to a significant degree of risk of coming into direct
contact with these materials. In order to provide a safe working
environment considerable efforts are now made to substitute these
dangerous materials with less dangerous ones.
[0009] Hair Dyeing Composition
[0010] In addition to being used as a substrate in immuno-chemical
assays OPD is used to dye hair. In this connection too it is
desirable to substitute a dangerous material with one less
dangerous so that the user is not exposed to danger by coming into
contact with it. To protect the hands against the dangerous
material it is normal for gloves to be used while the hair dye is
being applied. Gloves cannot, of course, protect the scalp of the
person to whom the dye is applied.
[0011] In general hair dyeing compositions on the market today can
be divided into three main groups:
[0012] temporary hair dyes,
[0013] semi-permanent hair dyes, and
[0014] permanent oxidative hair dyes.
[0015] The temporary hair dyes are only intended to change the
natural hair colour for a short period of time and usually
functions by depositing dyes on the surface of the hair. Such hair
dyes are easy to remove with normal shampooing.
[0016] When using semi-permanent hair dyes the colour of the dyed
hair can survive for five or more shampooings. This is achieved by
using dyes having a high affinity for hair keratin and which is
able penetrate into the interior of the hair shaft.
[0017] Permanent hair dyes are very durable to sunlight, shampooing
and other hair treatments and need only to be refreshed once a
month as new hair grows out. With these dyeing systems the dyes are
created directly in and on the hair. Small aromatic colourless dye
precursors (e.g. p-phenylene-diamine, o-aminophenol,
o-phenylendiamine (OPD)) penetrate deep into the hair where said
dye precursors are oxidised by an oxidising agent into coloured
polymeric compounds. These coloured compounds are larger than the
dye precursors and can not be washed out of the hair.
[0018] By including compounds referred to as modifiers (or
couplers) in the hair dyeing composition a number of hair colour
tints can be obtained. Cathecol and Resorcinol are examples of such
modifiers.
[0019] Some of the today most widely used dye precursors such as
OPD are known to be both mutagenic and carcinogenic.
[0020] Further, traditionally H.sub.2O.sub.2 is used as the
oxidizing agent (colour builder), but also as a bleaching agent.
Dyeing compositions comprising H.sub.2O.sub.2 are often referred to
as "lightening dyes" due to this lightening effect of
H.sub.2O.sub.2.
[0021] The use of H.sub.2O.sub.2 in dyeing compositions have some
disadvantages as H.sub.2O.sub.2 damages the hair. Further,
oxidative dyeing often demands high pH (normally around pH 9-10),
which also inflicts damage on the hair and on the skin.
Consequently, if using dye compositions comprising H.sub.2O.sub.2
it is not recommendable to dye the hair often.
[0022] To overcome the disadvantages of using H.sub.2O.sub.2 it has
been suggested to use oxidation enzymes to replace
H.sub.2O.sub.2.
[0023] U.S. Pat. No. 3,251,742 (Revlon) describes a method for
dyeing human hair by dye formation in situ (i.e. on the hair). An
oxidation enzyme is used for the colour formation reactions at a
substantially neutral pH (7-8.5). Laccases, tyrosinases,
polyphenolases and catacolases are mentioned as suitable oxidation
enzymes. The hair colour pigment is formed by controlled oxidation
of various quinone-forming compounds and mono or poly aromatic
amines having the amino groups on the aromatic rings to form
natural appearing pigments. Specifically mentioned dye precursors
are 2-amino-4-nitrophenol, p-phenylene diamine, m-phenylene
diamine, o-phenylene diamine, 2-amino-1,4-naphthoquenone,
m-aminophenol, p-aminophenol, o-aminophenol, 2-amino resorcinol,
1,2,4-benzene triamine, nitro-p-phenylene diamine,
2-amino-5-diethyl amino toluene.
[0024] EP patent no. 504.005 (Perma S.A.) concerns dyeing
compositions for keratinous fibres, in particular hair, which do
not require the presence of H.sub.2O.sub.2 (hydrogen peroxide). The
composition comprises an enzyme capable of catalysing the formation
of the polymeric dyes and also dye precursors, such as bases and
couplers, in a buffer solution wherein the pH of said composition
is between 6.5 and 8 and said enzyme has an optimal activity in the
pH range between 6.5 and 8. Rhizoctonia praticola laccase and Rhus
vernicifera laccase are exemplified as the oxidation enzyme to
oxidize the dye precursor(s). The following dye precursors are
specifically mentioned: p-phenylene diamine, o-aminophenol,
p-methylaminophenol, p-aminophenol, p-toluylenediamine and
N-phenyl-p-phenylene diamine.
[0025] The aim of the present invention is to use the present
findings to make available a substrate which to all intents and
purposes is non-toxic, non-mutagenic and/or non-carcinogenic and
which may be used in immuno-chemical assays, for the dying of
keratinous fibres, in particular hair and for dying both natural
and synthetic fibres, e.g. textiles. This aim is achieved by
utilising the discovery of a substrate which includes the group
with the general formulae shown in 1. 2
[0026] wherein
[0027] R is an amino, mono- or a distributed amino or OR', where R
is H, alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl, phenyl
or substituted phenyl. X, Y and Z may each be any one of the
following: alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl,
phenyl, substituted phenyl, amino, hydroxy or mercapto with the
proviso that at least one of the groups X, Y and Z is an amino
group or an amino salt.
[0028] In a special embodiment of the invention a substrate is made
available which includes a connection with formula 1 where R' is a
methyl, ethyl or isopropyl group.
[0029] In an preferred embodiment the substrate is a benzoic acid
ester, in particular 3,4-diaminobenzoic acid methyl ester
(DABA-Me), 3,4-diaminibenzoic acid ethyl ester and 3,4-diamino
benzoic acid isopropyl ester.
[0030] Comparison of the very toxic aniline with the carboxyl acid
derivative of aniline, p-amino benzoic acid (PABA) shows that the
toxicity of the molecule is radically altered by the addition of
the carboxyl group. PABA is generally considered to be non-toxic
and, among other applications, is used as an ultra violet filter in
sun lotions. If the substrate OPD is thought of along the same
lines it can be seen that a possible analogue is 3,4-diamino
benzoic acid (3,4-DABA). This material is comparatively cheap and
readily obtainable. 3
[0031] Investigation using enzymes showed that 3,4-DABA is a
considerably poorer substrate for peroxidase than is OPD. In other
words 3,4-DABA has a higher K.sub.m-value at the same V.sub.max
when compared with OPD. This is apparently due to the carboxyl
group's inductive (deactivating) effect upon the aromatic ring.
This effect can be countered by modifying the carboxyl group, for
example by esterification with an alcohol. The preferred alcohols
are methanol, ethanol and isopropanol. Methyl, ethyl and isopropyl
esters were examined in connection with enzymes and the materials
were shown to have significantly improved properties than 3,4-DABA.
Especially the ethyl ester was found to have a very high V.sub.max,
i.e. at the same concentration it gives a very much higher reaction
speed than OPD.
[0032] The reaction mechanism for the oxidation of OPD with, for
instance, hydrogen peroxide, both with and without an enzyme, is
described by the following equations: 4
[0033] It may be seen from this that the product of oxidation is
2,3-diamino phenazin.
[0034] When one of the compounds described here is employed the
product of oxidation is also an amino phenazine. In the case of
3,4-DABA the oxidation product is 4,7-dicarboxy-1,2-diamino
phenazine as shown in the following equation. 5
[0035] The substrate should have two amino groups at the
3,4-location for the reaction to take place. However, substrates
which have the amino groups at either the 2,3-location or the
2,3,4-location may be used but only if the carboxyl group does not
hinder the reaction.
[0036] Substrates which have the amino group at the para location
may also be used to produce a coloured product. An example of this
is 3,6-DABA.
[0037] In order to counteract the carboxyl groups inductive effect
(i.e. deactivation of the aromatic ring caused by the groups
attractive effect upon electrons) esterification of the carboxyl
group was investigated using electron donating groups to see if
deactivation could be counteracted while at the same time retaining
non-mutagenic attributes. To investigate the effect of different
alkyl groups on the material's enzymatic properties as well as
possible mutagenic properties the methyl, ethyl and isopropyl
esters of 3,4-DABA were synthesised.
[0038] Compounds with the general formulae 1 are preferably
dissolved in DMF (Dimethyl formamide) but other organic solvents
may be used for this purpose. If a compound with the general
formulae 1 is in the form of a salt it can be dissolved in water
and this is preferred when using organic solvents.
[0039] In another aspect the invention relates to a method for
quantitative and/or qualitative analysis of a material of
biological interest. In this case a peroxidase enzyme together with
a marker is bound to the compound in question. Hydrogen peroxide is
then converted with a cromogenetic substrate (i.e. colour forming
compound) in the presence of the peroxidase, the substrate includes
a bond with the general formulae 1.
[0040] In a preferred embodiment of the method of the invention one
of the following substrates are used: the methyl, propyl or
isopropyl ester of amino benzoic acid.
[0041] In those cases where the material of biological interest is
an antigen the associated antibody is used. In this connection
other combinations will suggest themselves to the skilled
person.
[0042] The coloured product produced by the method of the invention
is especially suited to the dying of textiles, thread, yarn, wool,
hides and skins and human hair. Other natural fibres such as cotton
and silk may also be dyed with the product as may synthetic fibres
such as polyamides, polyurethane and polyester.
[0043] The coloured product may either be made immediately before
it is to be used for dying or it may be synthesised in the
immediate vicinity of the substance to be dyed. For example this
may be done by mixing the substrate and the oxidation system in a
person's hair.
[0044] The dying process may be carried out rinsing the person's
hair with a mixture of the substrate of the invention and hydrogen
peroxide or an oxidation enzyme. A peroxidase is then added and
distributed in the hair. When the desired degree of colouring has
been obtained the hair is rinsed with water.
[0045] The substrate may be mixed with the oxidation system before
it is applied to the hair. As stated above the substrate may be
oxidised with hydrogen peroxide or an oxidation enzyme generating
hydrogen peroxidase in the presence of a peroxidase.
[0046] Peroxidases belongs to the group of enzymes which is known
as the oxidoreductases. The group also includes the classes of
enzymes dehydrogenase, oxygenase, oxidase, laccase and related
enzymes. These enzymes may also be used for as an oxidation
system/agent for e.g. dyeing keratinous fibres, such as hair, wool,
fur and hides and the like. Dyeing composition and preferred
oxidation enzymes will be described further below.
[0047] In oxidation reactions which are catalysed by the enzyme
peroxidase the oxygen donor is hydrogen peroxide which is used as
an electron acceptor. Oxidases employ oxygen as an electron
acceptor.
[0048] Examples of suitable oxidases include catecholoxidase,
laccase and o-amino phenoloxidase.
[0049] Oxidation systems which may be used for the oxidation of the
substrate in this connection therefore include peroxidase and
hydrogen peroxide as well as oxidases, laccases and related enzymes
and oxygen. When the system consists of only an oxidase and oxygen
it is only necessary to add the oxidase to the substrate as the
oxygen in the air is used as an oxidant.
[0050] Dyeing Composition
[0051] In an aspect the invention relates to a composition in
particular adapted for dyeing keratinous fibres comprises, e.g.
hair, fur, hide or wool. Comprising 1) at least one oxidation
enzyme 2) at least one substrate as defined by the formulae 1 and
optionally 3) at least one modifier.
[0052] A preferred use of the composition is as a permanent dye for
the dyeing of human hair.
[0053] The oxidation enzyme is as also indicated above an
oxidoreductase, i.e. an enzyme classified under the Enzyme
Classification number E.C. 1 (Oxidoreductases) in accordance with
the Recommendations (1992) of the International Union of
Biochemistry and Molecular Biology (IUBMB)) which catalyses
oxidoreduction reactions.
[0054] Within the class of oxidoreductase enzymes are preferred
enzymes which catalyse the oxidation of a substrate (an electron or
hydrogen donor) by acting on oxygen (O.sub.2) and/or a peroxide as
the acceptor. Such enzymes include enzymes classified within the
enzyme classes comprising oxidases, including E.C. 1.1.3. E.C.
1.2.3, E.C. 1.3.3, E.C. 1.4.3, E.C. 1.5.3, E.C. 1.7.3, E.C. 1.8.3
and E.C. 1.9.3, laccases and related enzymes in E.C. 1.10.3, and
peroxidases in E.C. 1.11.
[0055] According to the invention three types of oxidoreductases
are specifically contemplated:
[0056] a) Laccases or related enzymes, which act on molecular
oxygen and yield water (H.sub.2O) without any need for peroxide
(e.g. H.sub.2O.sub.2),
[0057] b) Oxidases, which act on molecular oxygen (O.sub.2) and
yield peroxide (H.sub.2O.sub.2), and
[0058] c) Peroxidases, which act on peroxide (e.g. H.sub.2O.sub.2)
and yield water (H.sub.2O).
[0059] Also, enzyme systems which comprise a combination of more
than one enzyme from a single class or from different classes among
the three types of enzymes are contemplated. In the present
specification, although reference will often be made to a single
enzyme for the sake of simplicity, it is to be understood that the
description is generally applicable to such combinations of more
than one enzyme. Further, although the invention is generally
described in terms of the preferred aspect relating to the dyeing
of hair, it is to be understood that the description is generally
applicable to compositions according to the invention adapted for
dyeing of other types of keratinous fibres.
[0060] Particularly preferred enzymes are laccases and related
enzymes, the term "laccases and related enzymes" including enzymes
comprised by the enzyme classification E.C. 1.10.3.2 (laccases) and
catechol oxidase enzymes comprised by E.C. 1.10.3.1, bilirubin
oxidase enzymes comprised by the enzyme classification E.C. 1.3.3.5
and mono-phenol mono-oxygenase enzymes comprised by the enzyme
classification E.C. 1.14.99.1. Laccases are multi-copper containing
enzymes that catalyze the oxidation of phenols and aromatic amines.
Laccase-mediated oxidation results in the production of
aryloxy-radical intermediates from suitable phenolic substrates;
the ultimate coupling of the intermediates so produced provides a
combination of dimeric, oligomeric, and polymeric reaction
products. Certain reaction products can be used to form dyes
suitable for dyeing hair.
[0061] Preferably, the laccase employed may be derived from a
strain of Polyporus sp., in particular a strain of P. pinsitus or
P. versicolor, a strain of Myceliophthora sp., e.g. M. thermophila,
a strain of Rhizoctonia sp., in particular a strain of Rh.
praticola or Rh. solani, a strain of a Rhus sp., in particular Rhus
vernicifera, a strain of Pyricularia sp, in particular P. oryzae,
or a strain of Scytalidium, such as S. thermophilium.
[0062] In specific embodiments of the invention the oxidoreductase
is a laccase such as a Polyporus sp. laccase, especially the
Polyporus pinisitus laccase (also called Trametes villosa laccase)
described in WO 96/00290 (from Novo Nordisk Biotec Inc.) or a
Myceliophthora sp. laccase, especially the Myceliophthora
thermophila laccase described in WO 95/33836 (from Novo Nordisk
Biotech Inc.).
[0063] Further, the laccase may be a Scytalidium sp. laccase such
as the S. thermophilium laccase described in WO 95/33837 and WO
97/19998 (from Novo Nordisk Biotech Inc.), the contents of which is
incorporated herein by reference, or a Pyricularia sp. laccase,
such as the Pyricularia oryzae laccase which can be purchased from
SIGMA under the trade name SIGMA No. L5510, or a Coprinus sp.
laccase, such as a C. cinereus laccase, especially a C. cinereus
IFO 30116 laccase, or a Rhizoctonia sp. laccase, such as a Rh.
solani laccase, especially the neutral Rh. solani laccase described
WO 95/07988 (from Novo Nordisk A/S) having a pH optimum in the
range from 6.0 to 8.5.
[0064] The laccase may also be derived from a fungus such as
Collybia, Fomes, Lentinus, Pleurotus, Aspergillus, Neurospora,
Podospora, Phlebia, e.g. P. radiata (WO 92/01046), Coriolus sp.,
e.g. C. hirsitus (JP 2-238885), or Botrytis.
[0065] Bilirubin oxidase may preferably be derived from a strain of
Myrothecium sp., such as M. verrucaria.
[0066] The substrates (i.e. dye precursors) may according to the
dyeing composition of the invention be any of the above within the
definition of the general formulae 1.
[0067] Preferred dye precursors (i.e. substrates) are benzoic acid
esters, especially diamino benzoic acid esters, in particular
3,4-diamino benzoic acid methyl ester (DABA-Me), 3,4-diamino
benzoic acid ethyl ester and 3,4-diamino benzoic acid isopropyl
ester.
[0068] Other Oxidation Agents
[0069] The substrate may also be oxidised by a number of inorganic
compounds, among these are compounds which include hypochlorite
(ClO.sup.-), hypobromite (BrO.sup.-), permanganate
(MnO.sub.4.sup.-) dicromate (Cr.sub.2O.sub.7.sup.2-) and the iron
ion (Fe.sup.3+).
[0070] Oxidation systems are taken to be either an oxidant per se
or a combination of an enzyme and an oxidant.
[0071] Modifiers
[0072] Modifiers typically incorporation in a dye compositions
include m-aromatic diamines, m-aminophenols, polyphenols, amino
naphthalines or naphthols. The modifier (coupler) reacts with the
dye precursor in the presence of the oxidative enzyme or the like,
converting it into a coloured compound. Examples of specific
modifiers (couplers) include m-phenylene-diamine,
2,4-diaminoanisole, 1-hydroxynaphthalene (.alpha.-naphthol),
1,4-dihydroxybenzene(hydroquinone), 1,5-dihydroxynapthalene,
1,2-dihydroxybenzene(pyrocatechol), 1,3-dihydroxybenzene
(resorcinol), 1,3-dihydroxy-2-methylbenzene,
1,3-dihydroxy-4-chlorobenzene(4-chlororesorcinol),
1,2,3,trihydroxybenzene, 1,2,4-trihydroxybenzene,
1,2,4-trihydroxy-5-meth- ylbenzene, and
1,2,4-trihydroxytoluene.
[0073] Method of Dyeing Keratinous Fibres
[0074] In a further aspect the invention relates to a method for
dyeing keratinous fibres, in particular hair, fur, hide and wool,
using a composition as described above. The dyeing method can be
conducted with one or more dye precursors (i.e. substrates of the
invention) and optionally in combination with one or more
modifiers. The amount of dye precursor(s) and other ingredients
used in the composition of the invention for this purpose are in
accordance with usual commercial amounts and therefore known for
the skilled person. Hair dyeing is typically carried out at or near
room temperature, preferably around the optimum temperature of the
enzyme being used, and at a pH in the range of from 3.0 to 9.0,
preferably 4.0 to 8.5, especially 6.0 to 8.0. Dye precursors (i.e.
substrates of the invention) and optional modifiers are described
above.
[0075] The invention is further illustrated in the following
non-limiting example.
EXAMPLE 1
[0076] Synthesis of 3,4-diamino Benzoic Acid Esters
[0077] The esters were made starting from 4-amino-3-nitrotoluene,
followed by the esterification and reduction of
4-amino-3-nitrobenzoicacid esters to 3,4-diamino benzoic acidester.
The reactions are shown below. 6
[0078] The amino group in 4-amino-3-nitrotoluene is protected by
boiling in anhydrous acetic acid in acetic acid. The methyl group
is oxidized with permanganate in an aqueous solution containing
magnesium sulphate. The acetyl group is removed by boiling with 0.1
hydrochloric acid. After isolation and drying
4-amino-3-nitrobenzoicacid is dissolved in absolute alcohol and
concentrated sulphuric acid is added. On boiling for two to five
hours the acid groups are esterified. The exact boiling time
depends on the type of alcohol. The last stage is the reduction of
the isolated product with activated iron and water in boiling
benzine for about five hours.
[0079] When the reaction was complete the iron particles were
filtered out and the residue dried for between 24 to 48 hours over
anhydrous sodium sulphate. After filtration and evaporation the
ester was recrystallised in a mixture of n-butanol and benzine in
the ratio of 1 to 10.
[0080] The above procedure was used to produce 3,4-DABA esters for
measurement of enzymes and for mutagenetic testing. If larger
amounts were to be required at a reasonable price then direct
esterification of 3,4-DABA would be preferred. Thus the methyl
ester may be prepared by bubbling hydrochloric acid gas through a
solution of 3,4-DABA containing methanol. This last method finds
only limited use in the production of the ethyl ester and cannot be
used to produce the isopropyl ester.
[0081] Characterization of 3,4-DABA Esters
[0082] Thin Layer Chromatography
[0083] The DABA esters that were prepared were analysed and
compared with the help of thin layer chromatography (TLC) using
silica gel plates of the type MERC 60 F.sub.254 the stock number of
the zone of concentration was 5583. A mixture of chloroform,
methanol and acetic acid in the ratio of 90:5:5 on a volumetric
basis was used as a solvent For the purpose of comparison OPD and
3,4-DABA were analyzed on the same plate. The Rf values obtained
are shown in Table 1.
1 TABLE 1 Chemical bond Rf-value 3,4-DABA 0.15 OPD 0.19 Methyl
ester 0.29 Ethyl ester 0.31 Isopropyl ester 0.33
[0084] It can be seen from table 1 that the bigger alkyl groups
give a measurably greater displacement in the solvent which
contains chloroform. The least displacement is seen, as might be
expected, with 3,4-DABA which contains a free carboxyl group. All
the combinations tested moved in the form of patches which is an
indication of their purity.
[0085] The fact that the 3,4-DABA esters are lipeds gives no
problems with regard to solubility in water when making up
solutions of substrates. A standard solution of dimethyl formamide
diluted in an aqueous buffer with a pH of 5.0. At high
concentrations of substrate oxidation products formed by the action
of enzymes may cause slight turbidity in the solution. By reducing
the pH to about 1 with 1M sulphuric acid a completely clear
solution is produced. This is due to the addition of protons to the
amino groups.
[0086] Determination of Melting Points
[0087] To verify that the materials synthesised were identical with
those described in the literature their melting points were
determined and compared with values given in a table on page 1532
of Chapman and Hall's Dictionary of Organic Compounds, Fifth
Edition, Volume 2. Melting points were determined by the use of the
capillary tube method using a silicone oil bath.
[0088] Melting points are given in Table 2.
2TABLE 2 Material Measured melting point Value from literature
methyl ester 108-109.degree. C. 108-109.degree. C. ethyl ester
112-113.degree. C. 112-113.degree. C. isopropyl ester 73-74.degree.
C. --
[0089] It can be seen from the above table that there is close
agreement between the melting points determined by experiment and
the melting points as given in the literature. It may, therefore,
reasonably be assumed that the synthesised material are identical
with those described in the literature. It was not possible to find
a value for the isopropyl ester in the literature.
[0090] Ultraviolet Spectroscopy
[0091] UV spectra were taken of OPD as well as the methyl, ethyl
and isopropyl esters.
[0092] Scanning was done from 360 nm to 210 nm using a solution of
the compounds in methanol. The concentration was 0.1 /1. Table 3
shows the absorption maxima and the extinction coefficients for the
compounds investigated.
3 TABLE 3 Material Absorption max. Extinction coefficient OPD 290.0
nm 2000 M.sup.-1 231.3 nm 3400 M.sup.-1 methyl ester 310.0 nm 6000
M.sup.-1 277.5 nm 6000 M.sup.-1 232.5 nm 8400 M.sup.-1 ethyl ester
310.0 nm 6200 M.sup.-1 277.5 nm 6000 M.sup.-1 232.5 nm 8600
M.sup.-1 isopropyl 310.0 nm 6500 M.sup.-1 ester 277.5 nm 6200
M.sup.-1 232.5 nm 8700 M.sup.-1
[0093] As can be seen from the above table all three esters absorb
at the same wavelength. At 237.5 nm there is a slight increase in
extinction with increasing molecular weight of the alkyl group.
3,4-DABA-esters show a typical maximum at 277.5 nm. This maximum is
not found in OPD because of the ester carbonyl group.
[0094] In order to investigate the properties of 3,4-DABA and the
three esters with respect to oxidation catalyzed by peroxidase a
series of measurements were carried out on the enzymatic reactions
initial velocity with increasing concentration of the substrate.
Measurements were carried out on OPD,3,4-DABA, 3,4-DABA-methyl
ester, 3,4-DABA-ethyl ester, and 3,4-DABA isopropyl ester.
[0095] Absorbancy at 492 nm was used as a measure of the course of
the reaction. The initial velocity was taken to be the absorbancy
at 492 nm two minutes after the addition of the peroxidase to a
mixture of the substrate and hydrogen peroxide in a buffer with a
pH of 5.0. The wavelength of 492 nm was chosen because it is
employed in the standard assay procedures for OPD. None of the
substrates has a specially high absorption at this wavelength.
[0096] By varying the concentration of the substrate and at the
same time measuring the initial velocity of the reaction it is
posible to apply the Michaelis/Menten equation to the system.
[0097] Under ideal conditions the values determined experimentally
will approach the value of the expression: 1 V init = V max 1 + K m
[ S ]
[0098] where [S] is the concentration of the substrate, V.sub.max
is the maximum initial concentration which is reached in the
particular assay. K.sub.m is defined as the concentration of the
substrate at V.sub.max/2. A small K.sub.m value will therefore be
characteristic for an enzyme system where a low concentration of
the substrates produces saturation of the enzyme.
[0099] The expression implies that the initial velocity will
increase with increasing concentration of the substrate, but the
curve for the velocity will flatten out and approach V.sub.max for
very high concentrations of the substrate. In actual fact the
parameter K.sub.kat be calculated as V.sub.max/[E] where [E] is the
molar concentration of the enzyme in the reaction. K.sub.kat is the
same as min.sup.-1 and reflects the activity of the enzyme in a
saturated solution upon the substrate.
[0100] The peroxidase system has an extremely complicated energy
balance (Arnoa et al. (1990)) for short periods of less than a
minute the system may nevertheless be described in terms of the
above given formulae.
EXAMPLE 2
[0101] Enzymatic Determination
[0102] The following solutions were prepared for use in the enzyme
assay:
[0103] a) Assay buffer
[0104] A 50 mM phosphate/citrate buffer with a pH of 5.0 was made
by mixing a 50 mm Na.sub.2HPO.sub.4 solution and a 50 mm solution
of citric acid. The pH was measured while mixing was in
progress.
[0105] b) DMF diluted 1:10
[0106] By means of pipette 10 ml were placed in a graduated flask
which was then topped up to 100 ml with assay buffer.
[0107] c) Solution of hydrogen peroxide 0.018%
[0108] 15 .mu.l of a 30% solution of hydrogen peroxide (PERHYDROL,
Merck) was thinned down with 25 ml of the assay buffer.
[0109] d) Stabilizing buffer for peroxidase
[0110] The stabilizing buffer for peroxidase, i.e. a buffer which
stabilizes the enzyme, was prepared according to the method of
Olsen and Little (1983). A 0.1M Na-acetate buffer, which was 0.5M
in terms of CaCl.sub.2 was adjusted to pH 5.6.
[0111] 37.5 mg N-acetyl-trimethyl-ammonium-bromide was dissolved in
75 ml of the buffer. To this solution 25 ml of glycerol was added.
The enzyme activity is maintained in this buffer because the
molecules of the enzyme are prevented from aggregating.
[0112] e) Standard solution of peroxidase
[0113] 10 mg of horseradish peroxidase type VI-A (Sigma no. 6782)
were dissolved in 10 ml of the stabilizing buffer. This was stored
at -15.degree. C. This will keep for several months (Olsen and
Little (1983).
[0114] f) Bench solution of peroxidase
[0115] The standard peroxidase solution was diluted to 1:1000 with
the 10 ml assay-buffer solution in 10 .mu.l standard solution.
[0116] g) Standard solutions of the substrates
[0117] 0.5 mmol of each substrate in 5 ml DMF. These solutions will
keep for several weeks at -15.degree. C.
[0118] h) Bench solutions of the substrates
[0119] The standard solutions were diluted by 1:10 with the assay
buffer. Before being used the substrate solutions were diluted by
1:10. 0.5 ml of the standard solution was thinned down with 4.5 ml
of the assay buffer.
[0120] Measurement of the Velocity of Reaction as a Function of the
Concentration of the Substrates
[0121] For each of the compounds OPD, 3,4-DABA-, and the methyl,
ethyl, and isopropyl esters of 3,4-DABA 15 measurements were
carried out and absorbency was measured twice in each case at 492
nm one minute after the addition of 100 .mu.l dilute peroxidase
solution (bench solution). The concentration of the peroxidase was
held constant at 50 ng/ml during the whole investigation. By using
the volume of substrate and the volumes of 10 vol-% DMF solution as
given in table 4 it was possible to employ a constant reaction
volume and a constant concentration of DMF. For all measurements
there was used 1550 .mu.l buffer and 50 .mu.l bench solution of
peroxidase. The total volume is then as follows: 300 .mu.I DMF and
substrate solution, 1550 .mu.l buffer, 100 .mu.l bench solution of
peroxidase and 50 .mu.l hydrogen peroxide solution. That is 2000
.mu.l in total.
4TABLE 4 Substrate .sub..mu.l substrate .sub..mu.l 10% DMF-
concentration solution solution (.sub..mu.mol/l) 0 300 0 5 295 25
10 290 50 15 285 75 20 280 100 25 275 125 30 270 150 35 265 175 40
260 200 50 250 250 60 240 300 80 220 400 100 200 500 150 150 750
200 100 1000 300 0 1500
[0122] Table 5 contains information about the values measured for
K.sub.m, V.sub.max and K.sub.kat for the five compounds.
5 TABLE 5 K.sub.m V.sub.max K.sub.kat Substrate
(.sub..mu.mol/.sub..mu.l) (min.sup.-1) (1*mol.sup.-1*min.sup.-1- )
OPD 47.35 0.16 1.6 * 10.sup.8 3,4-DABA 251.22 0.20 2.0 * 10.sup.8
methyl 125.97 0.22 2.2 * 10.sup.8 ester ethyl 212.39 0.27 2.7 *
10.sup.8 ester isopropyl 118.46 0.22 2.2 * 10.sup.8 ester
[0123] The results of the measurements of initial velocities are
stated in units of absorbency and not in molar units. In order to
be able to measure the "true" velocity of reaction it is necessary
to isolate the oxidation product for each substrate and determine
the molar extinction coefficient. The value of Kkat is worked out
from 1 mole of peroxidase of 50,000 g/mol.
[0124] FIG. 1 +L-5 is a graphic representation of the result of the
measurement of initial velocity on OPD, 3,4-DABA, 3,4-DABA-methyl
ester, 3,4-DABA-ethyl ester, 3,4-DABA-isopropyl ester.
[0125] As is shown by Table 5 and FIGS. 1 to 6 the carboxyl esters
of 3,4-DABA are effective substrates in a peroxidase/hydrogen
peroxide system. It is possible to obtain much higher initial
velocities with these compounds than with OPD. K.sub.m per se is a
poor indicator of the effectiveness in enzyme assays of the
substrates in question as it actually only shows the sensitivity of
the system at low concentrations of substrate. In practice
substrate concentrations would be chosen to allow maximum and
linear colour development with different concentrations of enzymes.
In other words V.sub.max and K.sub.kat are more relevant parameters
for the comparison of different substrates. It was found that, for
all the esters investigated, the values of V.sub.max and K.sub.kat
were considerably larger than the comparable values for OPD.
[0126] All reaction velocities are expressed as absorption units,
this is partly because most practical enzyme essays are based on
the measurement of absorption and partly because the products of
reaction are not isolated from the reaction mixture.
EXAMPLE 3
[0127] Determination of the Mutagenicity of the Compounds
[0128] The Ames test (Maron and Ames (1983)) was used to determine
the mutagenic properties of the compounds. Nutrient media were
prepared in the manner described by Venitt and Parry (1984). To
3.times.2 ml melted agar at 45.degree. C. were added 100 .mu.l of
50, 100 and 200 mM of solutions of the compounds dissolved in DMSO.
By means of a pipette 100 .mu.l of a well-grown culture of
Salmonella tphimurium TA 98 (BIO-TEST gl. skolevej 47, 6731
Tiaereborg) were added to the same test-tube. The bacteria contain
a frameshift mutation on the histinol-dehydrogenase gene and
require histidin in order to grow. Mutagenic aromatic amines can
cause the bacteria to mutate to His+ and they can then grow on the
nutrient medium. The number of colonies after incubation therefore
give a quantitative measure of the ability of the added compound to
cause mutation. In all trials spontaneous mutations take place in
the absence of a mutagen. These provide a measure of "background"
mutation.
[0129] OPD is not a direct mutagen, it must first be activated by
the liver enzyme system P450. All trials were therefore carried out
both with and without the addition of "S9-mix" from the livers of
rats, this contains the P450 system. 0.5 ml of "59-mix" was added
to each test-tube. The criteria for mutation are that increasing
concentrations of the compound under investigation give a marked
increase in the number of His+ mutants. The results of these trial
are given in FIG. 7 +L-11. It may be seen from this figure that
only OPD has mutagenic properties.
EXAMPLE 4
[0130] Dyeing Effect of Dye Precursors of the Invention
[0131] The permanent oxidative dyeing effect of different dye
precursors using 0.05 mg active enzyme protein Myceliophthora
thermophila laccase (available from Novo Nordisk and described in
WO 95/33836) per ml reaction mixture were tested.
[0132] The dye precursors tested were
[0133] 0.1% w/w 3,4 diamino benzoic acid (DABA) in 0.1M
K-phosphatebuffer, pH 7.0.
[0134] 0.1% w/w 3,4-diaminobenzoic acid methyl ester (DABA-Me) in
0.1M K-phosphatebuffer, pH 7.0.
[0135] Modifier used
[0136] 0.1% w/w m-phenylenediamine (MPD) in 0.1M K-phosphatebuffer,
pH 7.0.
[0137] Dye precursor solutions were prepared by mixing the
indicated modifier so that the final concentration in the dyeing
solution was 0.1% w/w with respect to dye precursor (i.e. substrate
of the invention) and 0.1% w/w with respect to modifier.
[0138] Hair Dyeing
[0139] 1 gram 6" De Meo Virgin natural white hair tresses (De Meo
Brothers Inc. USA) were used.
[0140] 4 ml dye precursor solution (including modifier) was mixed
with 1 ml laccase on a Whirley mixer, applied to the hair tresses
and incubated at 30.degree. C. for 30 minutes.
[0141] The hair tresses were then rinsed with running water, washed
with shampoo, rinsed with water, combed, and air dried.
[0142] a*, b* and L* were determined on the Chroma Meter and
.DELTA.E* was then calculated as described below.
[0143] Hair tress samples treated without enzyme were used as a
blind.
[0144] The result of the test is shown in Table 6.
6TABLE 6 DABA and DABA-Me 0.1% w/w dye with/without MPD
precursor/modifier .DELTA..sup.L .DELTA..sup.a .DELTA..sup.b
.DELTA..sup.E Assessment DABA -4.27 -0.63 -2.55 5.01 no colour DABA
+ MPD -23 -2.21 -18.29 29.47 grayish DABA-Me -7.58 6.53 -2.14 10.23
light orange DABA-Me + MPD -32.31 2.35 -25.07 40.96 grayish
violet
[0145] Assessment of the Hair Colour
[0146] The quantitative colour of the hair tresses was determined
on a Minolta CR200 Chroma Meter by the use the parameters L*
("0"=black and "100"=white), a* ("-"=green and "+"=red) and b* ("-"
blue and "+" yellow).
[0147] .DELTA.L*, .DELTA.a* and .DELTA.b* are the delta values of
L*, a* and b* respectively compared to L*, a* and b* of untreated
hair (e.g. .DELTA.L*=L*.sub.sample-L*.sub.untreated hair).
[0148] .DELTA.E* was calculated as .DELTA.E*=square
root(.DELTA.L*.sup.2+.DELTA.a*.sup.2+.DELTA.b*.sup.2) and is an
expression for the total quantitative colour change.
EXAMPLE 5
[0149] Dyeing Effect of DAB-Me and Various Modifiers
[0150] Using the procedure described in Example 4 the permanent
dyeing effect of the dye precursor (i.e. substrates) DABA-Me with
various modifiers were tested, except that 0.2% w/w dye precursor
and 0.2% modifier were used.
7TABLE 2 0.2% DABA-Me and 0.2% w/w modifier .DELTA..sup.L*
.DELTA..sup.a* .DELTA..sup.b* .DELTA..sup.E* Assessment
4-chlor-resorcinol -22.36 1.19 -6.28 23.26 Gray-green
5-amino-o-cresol -14.1 5.69 -2.1 15.35 light orange
m-phenylene-diamine -33.25 2.17 -23.71 40.9 Grey (Bluish)
pyrogallol -29.47 5.74 -7.69 30.99 Brown 4-methoxy-1,3-phenyl-
-39.24 2.33 -19.73 43.98 Brown-gray/ enediamine black
[0151] As can be seen the keratinous fibers can be dyed using
DABA-Me and a modifier.
* * * * *