Dyeing substrates

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.

EXAMPLE 1 Synthesis of 3,4-diamino Benzoic Acid Esters 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 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. 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. 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. Characterization of 3,4-DABA Esters Thin Layer Chromatography 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 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 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. 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. Determination of Melting Points 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. Melting points are given in Table 2. 2 TABLE 2 Material Measured melting point Value from literature methyl ester 108-109° C. 108-109° C. ethyl ester 112-113° C. 112-113° C. isopropyl ester 73-74° C. — 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. Ultraviolet Spectroscopy UV spectra were taken of OPD as well as the methyl, ethyl and isopropyl esters. 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 −1 231.3 nm 3400 M −1 methyl ester 310.0 nm 6000 M −1 277.5 nm 6000 M −1 232.5 nm 8400 M −1 ethyl ester 310.0 nm 6200 M −1 277.5 nm 6000 M −1 232.5 nm 8600 M −1 isopropyl 310.0 nm 6500 M −1 ester 277.5 nm 6200 M −1 232.5 nm 8700 M −1 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. 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. 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. 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. Under ideal conditions the values determined experimentally will approach the value of the expression: 1 V init = V max 1 + K m [ S ] where &lsqb;S&rsqb; is the concentration of the substrate, V max is the maximum initial concentration which is reached in the particular assay. K m is defined as the concentration of the substrate at V max /2. A small K m value will therefore be characteristic for an enzyme system where a low concentration of the substrates produces saturation of the enzyme. 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 max for very high concentrations of the substrate. In actual fact the parameter K kat be calculated as V max /&lsqb;E&rsqb; where &lsqb;E&rsqb; is the molar concentration of the enzyme in the reaction. K kat is the same as min −1 and reflects the activity of the enzyme in a saturated solution upon the substrate. 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 Enzymatic Determination The following solutions were prepared for use in the enzyme assay: a) Assay buffer A 50 mM phosphate/citrate buffer with a pH of 5.0 was made by mixing a 50 mm Na 2 HPO 4 solution and a 50 mm solution of citric acid. The pH was measured while mixing was in progress. b) DMF diluted 1:10 By means of pipette 10 ml were placed in a graduated flask which was then topped up to 100 ml with assay buffer. c) Solution of hydrogen peroxide 0.018% 15 &mgr;l of a 30% solution of hydrogen peroxide (PERHYDROL, Merck) was thinned down with 25 ml of the assay buffer. d) Stabilizing buffer for peroxidase 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 2 was adjusted to pH 5.6. 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. e) Standard solution of peroxidase 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° C. This will keep for several months (Olsen and Little (1983). f) Bench solution of peroxidase The standard peroxidase solution was diluted to 1:1000 with the 10 ml assay-buffer solution in 10 &mgr;l standard solution. g) Standard solutions of the substrates 0.5 mmol of each substrate in 5 ml DMF. These solutions will keep for several weeks at −15° C. h) Bench solutions of the substrates 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. Measurement of the Velocity of Reaction as a Function of the Concentration of the Substrates 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 &mgr;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 &mgr;l buffer and 50 &mgr;l bench solution of peroxidase. The total volume is then as follows: 300 &mgr;I DMF and substrate solution, 1550 &mgr;l buffer, 100 &mgr;l bench solution of peroxidase and 50 &mgr;l hydrogen peroxide solution. That is 2000 &mgr;l in total. 4 TABLE 4 Substrate &mgr; l substrate &mgr; l 10% DMF- concentration solution solution ( &mgr; 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 Table 5 contains information about the values measured for K m , V max and K kat for the five compounds. 5 TABLE 5 K m V max K kat Substrate ( &mgr; mol/ &mgr; l) (min −1 ) (1*mol −1 *min −1 ) OPD 47.35 0.16 1.6 * 10 8 3,4-DABA 251.22 0.20 2.0 * 10 8 methyl 125.97 0.22 2.2 * 10 8 ester ethyl 212.39 0.27 2.7 * 10 8 ester isopropyl 118.46 0.22 2.2 * 10 8 ester 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. 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. 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 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 max and K kat are more relevant parameters for the comparison of different substrates. It was found that, for all the esters investigated, the values of V max and K kat were considerably larger than the comparable values for OPD. 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 Determination of the Mutagenicity of the Compounds 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 3×2 ml melted agar at 45° C. were added 100 &mgr;l of 50, 100 and 200 mM of solutions of the compounds dissolved in DMSO. By means of a pipette 100 &mgr;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&plus; 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. 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&plus; 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 Dyeing Effect of Dye Precursors of the Invention 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. The dye precursors tested were 0.1% w/w 3,4 diamino benzoic acid (DABA) in 0.1M K-phosphatebuffer, pH 7.0. 0 . 1 % w/w 3,4-diaminobenzoic acid methyl ester (DABA-Me) in 0.1M K-phosphatebuffer, pH 7.0. Modifier used 0.1% w/w m-phenylenediamine (MPD) in 0.1M K-phosphatebuffer, pH 7.0. 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. Hair Dyeing 1 gram 6″ De Meo Virgin natural white hair tresses (De Meo Brothers Inc. USA) were used. 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° C. for 30 minutes. The hair tresses were then rinsed with running water, washed with shampoo, rinsed with water, combed, and air dried. a*, b* and L* were determined on the Chroma Meter and &Dgr;E* was then calculated as described below. Hair tress samples treated without enzyme were used as a blind. The result of the test is shown in Table 6. 6 TABLE 6 DABA and DABA-Me 0.1% w/w dye with/without MPD precursor/modifier &Dgr; L &Dgr; a &Dgr; b &Dgr; E Assessment DABA −4.27 −0.63 −2.55 5.01 no colour DABA &plus; MPD −23 −2.21 −18.29 29.47 grayish DABA-Me −7.58 6.53 −2.14 10.23 light orange DABA-Me &plus; MPD −32.31 2.35 −25.07 40.96 grayish violet Assessment of the Hair Colour The quantitative colour of the hair tresses was determined on a Minolta CR200 Chroma Meter by the use the parameters L* (“0”&equals;black and “100”&equals;white), a* (“−”&equals;green and “&plus;”&equals;red) and b* (“−” blue and “&plus;” yellow). &Dgr;L*, &Dgr;a* and &Dgr;b* are the delta values of L*, a* and b* respectively compared to L*, a* and b* of untreated hair (e.g. &Dgr;L*&equals;L* sample −L* untreated hair ). &Dgr;E* was calculated as &Dgr;E*&equals;square root(&Dgr;L* 2 &plus;&Dgr;a* 2 &plus;&Dgr;b* 2 ) and is an expression for the total quantitative colour change. 
 EXAMPLE 5 Dyeing Effect of DAB-Me and Various Modifiers 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. 7 TABLE 2 0.2% DABA-Me and 0.2% w/w modifier &Dgr; L* &Dgr; a* &Dgr; b* &Dgr; 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 As can be seen the keratinous fibers can be dyed using DABA-Me and a modifier.