【作者】 宋康康；

【导师】 陈清西；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 酪氨酸酶（EC.1.14.18.1）是一种含铜的氧化还原酶,广泛存在于生物体中,是生物体合成黑色素的关键酶,通过调控其活性可以调控黑色素的生成量。本论文首先以蘑菇酪氨酸酶为研究对象,筛选酶抑制剂并探讨其对单酚酶和二酚酶活性的抑制作用机理。在总结抑制剂构效关系的基础上,设计合成了两大类含硫化合物。并且研究了合成抑制剂对单酚酶和二酚酶的抑制作用机理。再以粗提的小鼠B16细胞的酪氨酸酶为研究对象,筛选该酶的抑制剂。并通过可见紫外光谱和DPPH法分别研究了巯基化合物的抑酶机理,以及效应物的抗氧化活性。最后以小鼠B16黑色素瘤细胞为研究对象,研究了合成的缩氨基硫脲类化合物对细胞增殖率、酪氨酸酶活性、黑色素含量以及酪氨酸酶、TRP-1、TRP-2蛋白的mRNA和蛋白表达水平的影响。相关研究内容及结果如下:（1）分别比较研究了烷基苯甲醛、羟基苯甲醛、卤代苯甲醛、4-乙烯苯甲醛和苯甲酸、烷氧基苯甲酸对蘑菇酪氨酸酶活力的抑制作用。它们均对酪氨酸酶具有抑制作用,但苯甲醛类化合物对酪氨酸酶的抑制活性明显比苯甲酸类化合物强。通过研究烷基苯甲醛和烷氧基苯甲酸对酶的抑制机理表明,苯环的4-位的取代基团可以提高化合物的抑酶活性,并且当4-取代基团碳链长度达到一定长度时,促进化合物的抑酶活性增强。（2）分别研究半胱氨酸及其衍生物、硫脲类化合物对酪氨酸酶的抑制机理。研究表明,半胱氨酸、N-乙酰半胱氨酸、半胱氨酸乙酯均是酪氨酸酶高效的抑制剂,并均是该酶的不可逆抑制剂。而硫脲类化合物对酪氨酸酶的抑制机理均表现为可逆的抑制,并表现出不同的抑制类型:硫脲和氨基硫脲均是非竞争型,而烯丙基硫脲和苯基硫脲均是混合型抑制剂。并且,通过底物反应动力学,测定了苯基硫脲对酪氨酸酶反应抑制动力学的相关参数。（3）设计合成两类含硫的酪氨酸酶抑制剂:半胱氨酸席夫碱类化合物和缩氨基硫脲类化合物。（4）研究合成化合物对酪氨酸酶活性的抑制机理。研究表明,合成的化合物均可以高效的抑制酶的活性,均为酶的可逆抑制剂。但随着化合物苯环取代基团的不同,表现出不同的抑制类型。（5）采用可见紫外光谱研究巯基化合物对酪氨酸酶的抑制机理。半胱氨酸和四个合成的半胱氨酸席夫碱类化合物均可以不同程度的与L-DOPA的氧化产物形成无色复合物,导致产物特征峰吸收值的下降。（6）通过DPPH方法研究了化合物的清除氧自由基的活性。研究表明,苯甲醛类化合物除了3,4-二羟基苯甲醛外,清除氧自由基的活性均很弱。而硫脲类化合物在浓度较高时,才有清除氧自由基的活性。但合成的缩氨基硫脲类化合物则表现出不同的清除氧自由基的能力。（7）粗提得到小鼠B16黑色素瘤细胞的酪氨酸酶,以此为研究对象,筛选酶抑制剂。熊果甙、维生素类化合物（维生素B₁、C、K₁）、苯甲酸类化合物、苯甲醛类化合物、硫脲类化合物、合成的缩氨基硫脲类化合物均对酪氨酸酶有不同程度的抑制作用。其中烯丙基硫脲、苯基硫脲、缩氨基硫脲类化合物表现出高效的抑制酪氨酸酶的活性。（8）以曲酸为阳性对照,研究合成的缩氨基硫脲类化合物对B16细胞增殖率、细胞内酪氨酸酶活性、黑色素含量的影响。根据这三方面因素,4-二甲氨基苯甲醛缩氨基硫脲具有最佳的降低黑色素合成的活性。其对细胞中酪氨酸酶、TRP-1、TRP-2蛋白的mRNA表达水平影响不大,但可以显著降低三种蛋白的蛋白表达水平。总之,通过本课题的研究,设计合成了新型的高效酪氨酸酶抑制剂,并总结了抑制剂的构效关系。研究了酶抑制剂在小鼠B16黑色素瘤细胞中的抑制黑色素形成的效果,为其应用于美白化妆品奠定了理论基础。更多还原

【Abstract】 Tyrosinase （EC 1.14.18.1） is a kind of redoxidase containing copper, widely distributed in nature. It is a key enzyme involved in the melanin biosynthesis. Its inhibitors are usuallyused in whitening cosmetic.Firstly, screening the tyrosinase inhibitors and studying their inhibitory mechanism have been conducted. In view of the relationship between structure and activity （SAR） of tyrosinase inhibitors, two series of compounds containing sulfur were designed and synthesized. The inhibitory effects and mechanism of these synthetical compounds had been elucidated. Then, some tyrosinase inhibitors were screened out through studying their inhibitory capacities on prepared cell-free tyrosinase from B16. Furthermore, through UV-Vis spectrum and DPPH method, the inhibitory mechanism of thiols on mushroom tyrosinase and the anti-oxidant activity of compounds were investigated, respectively. In the end, the B16 cell viability, tyrosinase activity, melanin content and mRNA and protein levels of tyrosinase, TRP-1, TRP-2 were assayed in presence of different concentrations of thiosemicarbazones. The contents and results were as follows:The effects of alky-substituted benzaldehydes, hydroxy-substituted benzaldehydes, halide-substituted benzaldehydes, 4-vinylbenzaldehyde, 4-vinylbenzoic acid and alkoxy-substituted benzoic acids on mushroom tyrosinase were studied. They can all inhibit the tyrosinase activity. And it is concluded that the inhibitory ability of benzaldehydes is apparently stronger than that of benzoic acids. According to the inhibitory mechanism of alky-substituted benzaldehydes and alkoxy-substituted benzoic acid, it is proved that the substituted group on 4-position and the certain length of carbon chain are benefit for the inhibitory capacity of inhibitors.The inhibitory effects and mechanism of cysteine, cysteine derivatives and thioureas on mushroom tyrosinase were investigated. Cysteine and its derivatives are all irreversible inhibitors, while thioureas were all reversible inhibitors on mushroom tyrosinase. Moreover, thiourea and N-aminothiourea are noncompetitive inhibitor, and allythiourea and phenylthiourea both are Mixed-I type inhibitor. Then, according to the kinetics of substrate, the inhibitory kinetics constants of phenylthiourea on mushroom tyrosinase were determined.The design and synthesis of two kinds of tyrosinase inhibitors containing sulfur were done. They were cysteine schiff bases and thiosemicarbazones. The inhibitory mechanisms of the synthetical compounds on mushroom tyrosinase were elucidated, too. It is studied that they are all potent reversible inhibitors of tyrosinase. With the changing of substituted group on benzene ring in compounds, they appear different inhibitory type.The inhibitory mechanisms of thiols on mushroom tyrosinase were investigated through UV-Vis spectrum. It is indicated that cysteine and four kinds of cysteine schiff bases can react with the oxidant product of L-DOPA by tyrosinase or NaIO₄ forming colorless compounds in order to reduce the quantity of the oxidant product.The anti-oxidant activity of compounds were assayed though DPPH method. Except for 3,4-dihydroxybenzaldehyde, other benzaldehydes can’t scavenge free radical. They are of free radical scavenging activity in presence of high concetration of thioureas. And, different thiosemicarbazones appear different ability of scavenging free radicalArbutin, vitamins （vitamin B₁, C, K₁）, benzoic acids, benzaldehydes, thioureas and thiosemicarbazones all can inhibit the activity of cell-free tyrosinase in B16. Among them, allythiourea, phenylthiourea and thiosemicarbazones are all potent this enzyme inhibitors.Kojic acid as positive control, the cell viability, tyrosinase activity, melanin content and the mRNA level of tyrosinase in B16 by thiosemicarbazones were studied. According that, 4-（dimethylamino）-benzaldehyde can more effectively inhibit melaningenesis than others. But, these four compounds can’t apparently affect the mRNA levels of tyrosinase, TRP-1, TRP-2. While, 4-（dimethylamino）-benzaldehyde can dramatically reduce the protein levels of tyrosinase, TRP-1, TRP-2.In a word, the synthesis and inhibitory mechanism of new potent tyrosinase inhibitors were done in this study. Furthermore, the inhibition of melaningenesis by these compounds were investigated, which provided a foundation to use for whiten cosmetic.更多还原