Substrates For Mushroom Tyrosinase Research Articles

Synthesis of dialkylaminoaryl phosphonate bioisosteres of tyrosine and tyramine: a novel application of allene phosphonate chemistry for the synthesis of false substrates of tyrosinase

Tyrosinase is the rate-limiting oxidase in the synthesis of melanin, making it an obvious target for the treatment of melanotic melanomas. Tyrosine and tyramine are its natural substrates, but many of their derivatives are inhibitors or false substrates, and are therefore prime candidates for melanoma chemotherapy. A series of dialkylphosphonate derivatives of tyramine have now been synthesized in order to extend the chemical diversity of tyrosinase substrates. The known reactivity between alkenephosphonates and nucleophiles was exploited by the addition of 4-(2-aminoethyl)phenol (tyramine) across the 2,3-double bond of dialkyl 1,2-alkadiene phosphonates, to obtain the desired bisphosphonate derivatives. These reactions were highly chemoselective and regioselective but not stereoselective. Five of the reported novel dialkylphosphonate aminophenols were substrates for mushroom tyrosinase in vitro: dimethyl 2-[2(4-hydroxyphenyl)ethylamino]-3-methyl-1-butene phosphonate (3); diethyl 2-[2(4-hydroxyphenyl)ethylamino]-3-methyl-1-butene phosphonate (4); dimethyl 2-[2-(4-hydroxyphenyl)ethylamino]-2-cyclohexyl-1-ethene phosphonate (5); diethyl 2-[2-(4-hydroxyphenyl)ethylamino]-2-cyclohexyl-1-ethene phosphonate (6); diethyl 2-[2-(4-hydroxyphenyl)ethylamino]ethane phosphonate (7). Compound 3 blocked the pigmentation of anagen hair in vivo in a murine animal model, a further demonstration that these compounds are able to enter and disrupt the melanogenic pathway.

Mushroom tyrosinase inhibitors from mung bean (Vigna radiatae L.) extracts

A seventy percent ethanol from mung bean (Vigna radiatae L.) was extracted further with CH2Cl2, EtOAc and n-BuOH to afford four fractions: CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. When using l-3,4-dihydroxyphenylalanine as the substrate for mushroom tyrosinase, the EtOAc-soluble fractions showed the highest inhibitory activity. Two pure flavonoid compounds, vitexin and isovitexin, were isolated (using the enzyme assay-guided fractionation method) from the EtOAc-soluble fractions. Vitexin and isovitexin showed high inhibitory activities, with IC50 values of 6.3 and 5.6 mg/ml, respectively. This is the first study on the active compositions of azuki beans against mushroom tyrosinase.

Inhibitory effects of the water extracts of Lavendula sp. on mushroom tyrosinase activity

This study aimed to evaluate the inhibition properties of six lavender species, including Lavendula angustifolia, Lavandula angustifolia “Vera”, Lavendula X allardii, Lavendula stoechas, Lavendula viridis and Lavendula X heterophylla, toward the activity of mushroom tyrosinase. When using l-3,4-dihydroxyphenylalanine ( l-Dopa) as the substrate for mushroom tyrosinase, the water extracts of leaves and stems from L. stoechas and L. angustifolia “Vera” showed strong inhibitory effects against the activity of mushroom tyrosinase (70% and 66.4% inhibition, respectively). Oven-drying the leaves and stems or free-drying the water extracts significantly decreased the inhibitory abilities of the water extracts from all lavender species. The water extract from L. stoechas decreased the V max values when using l-Dopa, catechol and 3,4-dihydroxyphenylacetic acid (DHPAA) as the substrates. It increased the value of K m when l-Dopa and catechol were the substrates but it decreased the K m when DHPAA was used. It behaved as a mixed-type inhibitor toward mushroom tyrosinase.

Synthesis and Antimelanoma Activity of Sterically Congested Tertiary Amide Analogues of N-Acetyl-4-S-cysteaminylphenol

Interference with the biosynthetic pathway to melanin may be a useful means for developing new chemotherapeutic drugs to combat malignant melanoma. N-Acetyl-4-S-cysteaminylphenol (1) is an analogue of tyrosine that is involved in the pathway to melanin. It is probably oxidized selectively in melanocytes to an o-quinone that can alkylate thiol groups on important cellular enzymes, resulting in interference with cell growth and proliferation. We previously synthesized a range of more lipophilic analogues of 1 by independently varying the acyl portion and introducing substitution alpha to the nitrogen. Most of the new compounds displayed greater cytotoxicity than the original lead compound 1. We also made a series of tertiary amides that again showed higher cytotoxicity than 1. In this work three new acetamides and two new cyclohexanecarboxamides containing 4-S-cysteaminylphenol were prepared incorporating both substitution alpha to the nitrogen and different substituents on the nitrogen of the amide in each compound to increase lipophilicity and to reduce further the possibility of hydrolysis of the amides. Most of the new tertiary amides showed greater cytotoxicity towards five representative melanoma cell lines than the parent secondary amide. The highest cytotoxicity against these five cell lines with IC50 values of 1-15 nicroM, comparable to cisplatin, was observed for N-[2[(4-hydroxyphenyl)thio]-1,1-dimethylethyl]-N-methylcyclohexanecarboxamide (8c). The IC50 values of 14.5 and 5.4 microM for this compound against SK-Mel-24 (not containing tyrosinase) and an ovarian cell line, respectively, suggest that interference with the melanin pathway may not be the only mode of action of this new compound. The cyclohexanecarboxamides were better substrates for mushroom tyrosinase (EC 1.14.18.1) than the acetamides.

Amperometric quantification of Alternaria mycotoxins with a mushroom tyrosinase modified carbon paste electrode

The affinity of mushroom tyrosinase for alternariol (AOH) and alternariol monomethyl ether (AME), mycotoxins of the Alternaria alternata genus, is verified both spectroscopically and electrochemically. Commercial enzyme and fresh mushroom tissue were employed for electrochemical measurements. The results presented here demonstrate, for the first time, that both AME and AOH are substrates for mushroom tyrosinase. The kinetic parameters were evaluated from Eadie–Hofstee plots. The analytical performance was also evaluated in the detection limits 2.4×10 −5 and 1.9×10 −5 M for AME and AOH, respectively, with a linear range up to 1.8×10 −4 and 2.0×10 −4 M for the same analytes. It is concluded that the enzyme has relatively good affinities for both substrates, which is a promising result for developing an enzymatic biosensor.

N-acetyl-L-tyrosine (NAT) as a substrate for mushroom tyrosinase.

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (lambda max = 390 +/- 10 nm). H2O2 and NH2OH each shortened the lag period of NAT hydroxylation by the enzyme. H2O2 had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H2O2 converts N-acetyl dopaquinone to a pink-violet product(s) (lambda max = 490 nm), whereas such a product(s) was not formed in the absence of H2O2. A pink-violet product(s) (lambda max 490 +/- 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH2OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH2OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H2O2. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490-530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320 +/- 10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol (3-hydroxy-2-methyl-4H-pyran-4-one), a gamma-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H2O2.

2, 4, 5-TRIHYDROXYBUTYROPHENONE (2, 4, 5-THBP)aAS A SUBSTRATE FOR MUSHROOM TYROSINASE

2, 4, 5-THBP (2, 4, 5-trihydroxybutyrophenone) is a substrate for mushroom tyrosinase with a Km value of 1.00 mM. The final product(s) formed is red (λ.max 480–490 nm) and it is rather stable with time. An intermediate red product(s), having a maximum at λ 505–515 nm, was detected and the time course of its conversion to the final red product(s) was studied. The data suggest that the intermediate red product(s) is 2, 4, 5-THBP-quinone which is converted to colorless polymerized 2, 4, 5-THBP-OH, which is then oxidized to a red polymerized 2, 4, 5-THBP-quinone(s), which is the final red product(s).

2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AS A SUBSTRATE FOR MUSHROOM TYROSINASE

2,3,4-Trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase with a Km value of 1.2 mM. The product(s) formed is yellow, characterized by a peak at 420–430 nm. A lag period in 2,3,4-THAP oxidation to the yellow product(s) in the presence of ascorbate indicates that the initial product(s) is an o-quinone of 2,3,4-THAP. An oxime, characterized by a broad peak at 510–650 nm, is the likely product formed between o-quinone of 2,3,4-THAP and NH2OH. The ɛm of the o-quinone of 2,3,4-THAP was estimated to be 1.6 × 104 M−1 cm−1 at 425 nm. During relatively long incubation periods, the peak of the yellow product(s) shifts from 420–425 nm to 430–440 nm; the solution remains yellow and transparent for at least a week and no precipitate is formed. The final yellow product(s) is probably a low molecular weight polymer of THAP-o-quinone (dimer, tetramer, etc).

Mechanism of selective toxicity of 4-S-cysteinylphenol and 4-S-cysteaminylphenol to melanocytes

Our previous studies showed that 4-S-cysteinylphenol (4-S-CP) and 4-S-cysteaminylphenol (4-S-CAP) inhibit the growth of malignant melanoma and cause depigmentation of black skin. In this study we examined kinetic constants of CP and CAP as substrates for tyrosinases and their properties as sulphydryl scavengers. 4-S-CP and 4-S-CAP were found to be much better substrates for mushroom tyrosinase than l-tyrosine while their 2-S isomers were not the substrates. 4-S-CP and 4-S-CAP were also good substrates for mammalian tyrosinase. Upon tyrosinase oxidation the two phenols conjugated with cysteine to form the cysteinyl derivatives of the corresponding catechols via o-quinone forms. The tyrosinase oxidation product of 4-S-CP had a poor ability to conjugate with alcohol dehydrogenase, a sulphydryl enzyme, while that of 4-S-CAP had a much higher ability. These results suggest that in melanocytes these phenols are oxidised by tyrosinase to the corresponding o-quinone forms, some of which conjugate with sulphydryl enzymes through cysteine residues, thus exerting cytotoxic effects.

Phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) influence mushroom tyrosinase activity.

A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate.

Tiron as a substrate for mushroom tyrosinase

Tiron (4,5-dihydroxy-1,3-benzene disulphonic acid) can serve as a substrate for mushroom tyrosinase with a K m value of 34 mM. The product(s) formed is yellow (λ max 435 nm) and is stable with time. An intermediate product, having significant absorbance at 3685 nm (probably tiron- o-quinone), was detected and the kinetics of its conversion to the final yellow product(s) was studied. Hydrogen peroxide (at 0.03–1.3 mM) accelerated the conversion of the intermediate compound to the yellow product(s). Tiron-semiquinone was detected by EPR spectroscopy during the initial phase of Tiron oxidation by mushroom tyrosinase. Maximum EPR signal intensity due to Tiron-semiquinone and the time required to reach maximum intensity were dependent on the amount of mushroom tyrosinase but not on the presence or absence of hydrogen peroxide (3.3 mM). In view of separate studies showing that the yellow product(s) is mainly a low M, polymerized Tiron-quinone, suggestions as to possible pathways by which such a product(s) is formed are discussed.

The synthesis of a DOPA analogue, 1-amino-2-(3,4-dihydroxyphenyl)-ethylphosphonic acid and distribution studies in mice bearing the Harding-Passey melanoma

The aminophosphonic acid analogue of DOPA, DL-1-amino-2-(3,4-dihydroxy-phenyl) ethylphosphonic acid (ADEP) has been synthesised. The compound was of low toxicity; a single dose of 2 g/kg given to mice subcutaneously was not lethal. [ 3H]ADEP was injected subcutaneously into mice carrying the established Harding-Passey melanoma, and the distribution of the tritium determined. The highest initial concentration of radioactivity was in the kidneys, adrenal glands and eyes. Isotope content fell to low values in all tissues within 8 days or less but the tumour retained radioactivity for a longer period than did the other tissues examined. ADEP served as a substrate for mushroom tyrosinase.