WO2015018176A1 - 葡萄糖醛酸转移酶ugt1a1的特异性荧光探针及其应用 - Google Patents

葡萄糖醛酸转移酶ugt1a1的特异性荧光探针及其应用 Download PDF

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WO2015018176A1
WO2015018176A1 PCT/CN2014/000333 CN2014000333W WO2015018176A1 WO 2015018176 A1 WO2015018176 A1 WO 2015018176A1 CN 2014000333 W CN2014000333 W CN 2014000333W WO 2015018176 A1 WO2015018176 A1 WO 2015018176A1
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ugt1a1
substrate
probe substrate
fluorescent probe
specific fluorescent
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杨凌
崔京南
葛广波
吕侠
冯磊
刘兆明
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Dalian Institute of Chemical Physics of CAS
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    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
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    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a specific fluorescent probe substrate of glucuronyltransferase UGT1A1 and application thereof.
  • Uridine diphosphate-glucuronosyltransferase (UGT) superfamily is the most important phase II drug metabolizing enzyme in the body, catalyzing the lipophilic compound and the cofactor uridine diphosphate glucuronic acid (UDPGA) by the S N 2 reaction mechanism.
  • UDPGA cofactor uridine diphosphate glucuronic acid
  • Glucuronic acid (GA) binds, thereby increasing the hydrophilicity of the substrate, allowing it to be excreted more efficiently from urine or bile.
  • UGT enzyme-mediated glucuronic acid binding is an important detoxification process in the body.
  • UGTs Many endogenous compounds, mutagens, drugs, and their metabolites are substrates for UGTs, such as endogenous substances such as bilirubin and estradiol, and exogenous substances SN-38 and nitrosamines. Detoxification of amine compounds is achieved by the glucuronylation pathway.
  • UGT1 and UGT2 family members play an important role in the metabolism of endogenous and exogenous substances.
  • UGT1 and UGT2 family members play an important role in the metabolism of endogenous and exogenous substances.
  • UGT1 and UGT2 family members play an important role in the metabolism of endogenous and exogenous substances.
  • UGT subtypes identified, including 9 of UGT1 A (UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10) and 7 subtypes of UGT2B. (UGT2B4, 2B7, 2B10, 2B15 and 2B17).
  • UGT1A1, 1A4, 1A6, 1A9 have moderate expression in human liver, but UGT1A3 is extremely low in human liver, and UGT1A7, UGT1A8 and UGT1A10 are only expressed in the human gut.
  • UGTIAI is an enzyme that catalyzes the endogenous substance bilirubin glucuronidation. It mediates the glucuronidation reaction, which is a necessary step in the elimination of bilirubin, and is most closely related to human health. Numerous studies at home and abroad have confirmed that the mutation of UGT1A1 gene causes the loss of bilirubin glucuronidation in whole or in part, which in turn affects the metabolism of bilirubin, leading to severe hyperbilirubinemia such as Crigler-Najjar syndrome and Gilbert's Syndrome (Annu. Rev. Pharmacol. Toxicol. 2000. 40:581-616).
  • UGTIAI is also a major metabolic enzyme of various clinical drugs, such as Etoposide, SN-38 (active metabolite of the anticancer drug irinotecan), and the loss of UGT1A1 activity has been confirmed to be associated with the toxicity of irinotecan (J.Clin .Oncol.2006. 24:4534-8). Therefore, the study of individual differences in UGT1A1 enzyme activity is important for clinical personalized safety medications.
  • UGT1A1 some clinical drugs will inhibit UGT1A1, thereby reducing the body's ability to clear bilirubin, causing an increase in blood bilirubin, which in turn leads to hyperbilirubinemia or exacerbation of patients with Crigler-Najjar syndrome and Gilbert's syndrome.
  • domestic and foreign pharmaceutical giants need to evaluate the ability of each candidate new drug to inhibit UGT1A1 in vitro during drug development. Therefore, the efficient and sensitive specific UGT1A1 probe substrate is essential for the efficient screening of UGT1A1 inhibitors and for the quantitative determination of UGT1A1 activity in biological systems.
  • each subtype of the UGT1A subfamily has a similar amino acid sequence, its substrates usually overlap each other, and each subtype of enzyme has a few specific substrates.
  • bilirubin has a high single-enzyme selectivity, it has poor chemical stability and low detection sensitivity.
  • estradiol and The single-enzyme selectivity of etoposide is not high and quantitative analysis of the product can be achieved by means of expensive analytical instruments such as LC-MS MS. Therefore, the development of a highly selective UGT1A1 specific fluorescent probe substrate and its associated high-throughput detection method have important practical value.
  • the 1,8-naphthylimide compound has a single metabolite (only one monoglucuronidation product is produced), a highly selective metabolic enzyme (mainly metabolized by UGT1A1), a substrate and a metabolite which are easy to detect and sensitive. Higher characteristics.
  • the object of the present invention is to provide a specific fluorescent probe substrate of glucuronyltransferase UGT1A1 and an application thereof, wherein the fluorescent emission wavelength of the specific fluorescent probe substrate and the glucuronidation product are significantly different, and the product is The fluorescence quantum yield is higher and easier to detect.
  • This probe reaction allows quantitative assessment of the distribution and function of UGT1A1 in a variety of biological systems.
  • the present invention provides a specific fluorescent probe substrate of glucuronyltransferase UGT1A1, which can be specifically catalyzed by UGT1A1 to form a corresponding 0-glucuronidation product, and its structural formula is as follows (1)
  • the substrate has a 1,8-naphthylimide-based structure, wherein R is one of an organic acidic group such as -COOH, benzoic acid or -S0 3 H, and n is 2 to 10.
  • the invention also provides the application of the specific fluorescent probe substrate of the glucuronyltransferase UGT1A1, and the compound of the above formula (1) is used as a specific substrate of the UGT1A1 sub-enzyme, and the glucuronidation binding reaction is carried out by quantification. Detection of the substrate elimination rate per unit time or the production rate of glucuronidation products to quantitatively determine UGT1A1 in different biological systems (including recombinant expression of UGT1A1 single enzyme, human or animal tissue preparation, various tissue cells, etc.) Activity; specific determination method is:
  • K m concentration of the substrate is preferably a single-point measurement; substrate concentration to select l / 10 ⁇ 10 K m.
  • the reaction temperature is between 20 ° C and 60 ° C, preferably 37 ° C is the optimal reaction time;
  • the incubation system pH is between 5.5 and 10.5, preferably pH 7.4 is the most Excellent reaction pH;
  • the reaction time is 0 to 140 minutes, and the reaction is terminated when the corresponding glucuronidation product of the above substrate reaches the limit of quantitation and the substrate conversion rate does not exceed 20%;
  • the probe substrate and the glucuronidation product thereof have fluorescence properties, and the two have different optical properties, and the fluorescence detection can be adopted. Simultaneously, the substrate and the product are detected rapidly and sensitively.
  • the fluorescence detection conditions of the glucuronidation product and the substrate are as follows: excitation wavelength 362, 450 nm, maximum emission wavelength is 450, 564 nm (as shown in Fig. 4).
  • the specific probe substrate is a ratiometric fluorescent probe, which is not easily interfered by biological system matrix and impurities during the UGT1A1 activity detection process, and can be used for various recombinant UGT1A1, human and animal tissue preparation liquids and UGT1A1 in various tissue cells. Quantitative determination of enzyme activity; also as a probe substrate for UGT1A1 in vivo and in vivo, to assess individual and species differences in the bilirubin metabolism enzyme UGT1A1.
  • the probe substrate and the fluorescence detection method of glucuronidation metabolites can also be used for rapid screening of UGT1A1 inhibitors and quantitative evaluation of inhibitory ability.
  • the recombinant glucuronide UTG1A1 single enzyme was used to investigate the liver microsome incubation system. By correlation analysis, specific inhibition experiments, recombinant single enzyme metabolic reactions, and evidence of enzyme reaction kinetics, it was proved that 1, 8-naphthoyl group
  • the imine compound can be specifically metabolized by glucuronic acid UTG1A1 (as shown in Figure 9) to form a glucuronidation product of a hydroxyl group.
  • glucuronic acid UTG1A1 as shown in Figure 9
  • this compound can be used to detect the activity of UTG1A1, especially for UTG1A1 produced by bacterial, insect cell, mammalian cell and yeast clone expression systems. Enzyme Live assays, as well as the activity calibration of UTG1A1 in preparations of microsomes, S-9, etc. from a variety of mammalian tissues and organs.
  • 1, 8-naphthylimide compound can be highly specifically metabolized by a glucuronic acid UTG1A1 single enzyme into a metabolite, that is, a glucuronidation product of a hydroxyl group.
  • 1, 8-naphthalimide compounds can be obtained by chemical synthesis, the synthesis process is simple and easy, and the detection method by fluorescence method is low in cost.
  • FIG. 1 the structural formula of 8-naphthylimide compounds
  • Figure 2. ifi-NMR spectrum of N-(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide; Figure 3. ⁇ (3-carboxypropyl)-4-hydroxy-1, 13 C-NMR spectrum of 8-naphthalimide; Figure 4. Ultraviolet absorption spectrum of N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthalimide and its glucuronidation metabolite Figure (maximum absorption at 362 nm and 450 nm, respectively); Figure 5.14 Metabolite map of HLM versus N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthalimide; Figure 6.
  • Figure 8 Lower limit of detection of N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthylimide UGT1A1 single enzyme;
  • Figure 9 N-(3-carboxypropyl)-4-hydroxy-1, a metabolic pathway in which 8-naphthylimide is acidified by UGT1A1 gluconic acid;
  • Example 3 In vitro inhibition assay for determination of IC S () of human liver microsomes and UGT1A1 (1) Prepare 190 ⁇ human liver microsomes and UGTIAI metabolic reaction system, including Tris-Hcl buffer (50 mM) at pH 7.4, human liver microsomes (0.2.5 mg/ml).
  • UGTIAI (0.06 mg/ml) , N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthalimide has a final concentration of 10 ⁇ , and different concentrations of protopanaxatriol are pre-incubated for 3 minutes at 37 °C;
  • HMM human liver microsomes
  • the assay was performed on a microplate reader using a 96-well plate, N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthalimide 10 ⁇ , uridine diphosphate glucuronic acid 2 mM, twelve Alkyl polyglycol ether 1 mg/ml, MgCl 2 5 mM, UGTIAI monozyme 20 ng/m ⁇ 500 ng'ml, pH 7.4 Tris-Hcl buffer 50 mM, total volume 100 L, 37 °C After incubation for 5 h, the average value of each group was compared with the control group without UGT1A1. The results are shown in Fig. 8. The UGT1A1 of 50 and 100 ng was statistically significant (P ⁇ 0.05), and the UGT1A1 was determined. The lower limit of detection is 50 ng.
  • the experiment was performed on a microplate reader using a % well plate, N-(3-carboxypropyl)-4-hydroxy-1, 8-naphthalimide 10 ⁇ , uridine diphosphate glucuronic acid 2 mM, twelve Alkyl polyglycol ether 1 mg/ml, MgCl 2 5 mM, UGTIAI monozyme 0.01 mg/ml ⁇ 0.09 mg/ml, pH 7.4 Tris-Hcl buffer 50 mM, total volume 100 ⁇ , 37 °C After incubating for 140 min, the plate was analyzed every 10 minutes. The ratio of the fluorescence intensity of the product to the fluorescence intensity of the substrate was compared with the incubation time. The R 2 ⁇ 0.99 of each standard curve indicates that the linear range of the standard curve is broad. 7, can accurately quantify the content of UGT1A1.

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Abstract

一种式（1）所示化合物用于葡萄糖醛酸转移酶UGT1A1的特异性荧光探针的应用。

Description

说 明 书 葡萄糖醛酸转移酶 UGT1A1的特异性荧光探针及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种葡萄糖醛酸转移酶 UGT1A1的特异性荧光探针底物及其应用。
背景技术
葡萄糖醛酸转移酶 ( Uridine diphosphate-glucuronosyltransferase, UGT)超家族是机体内最重要的 II相药物代谢酶， 以 SN2反应机制催 化亲脂性化合物与辅因子尿苷二磷酸葡糖醛酸（UDPGA) 的葡萄糖 醛酸 （GA) 结合， 从而增加底物的亲水性， 使其能更有效地从尿或 胆汁中排出体外。 通常 UGT酶介导的葡萄糖醛酸结合反应是机体的 一个重要的解毒过程。许多内源性化合物， 诱变剂， 药物， 以及它们 的代谢产物都是 UGTs的底物， 如内源性物质， 如胆红素和雌二醇， 以及外源性物质 SN-38和亚硝胺类化合物的脱毒都通过葡萄糖醛酸 化途径来实现。
人体的 UGT酶可以分为 4个家族: UGT1 , UGT2, UGT3和 UGT8。 其中 UGT1和 UGT2家族各成员在内源性及外源性物质代谢中发挥了 重要作用。目前，已被鉴定的人源 UGT亚型有 18个，其中包括 UGT1 A 中的 9个（UGT1A1 , 1A3， 1A4, 1A5, 1A6, 1A7, 1A8， 1A9, 1A10) 以及 UGT2B中的 7个亚型 （UGT2B4, 2B7, 2B10, 2B15和 2B17 )。 值得注意的是， UGT1A1 , 1A4, 1A6, 1A9在人肝脏中都有中等程 度的表达， 但 UGT1A3 在人肝脏中表达量极低， 同时 UGT1A7, UGT1A8及 UGT1A10仅在人肠道中表达。
UGTIAI是催化毒性内源性物质胆红素葡萄糖醛酸化的酶，它介 导的葡萄糖醛酸化反应是胆红素排除体外的必须步骤，和人体健康的 关系最为密切。 已有国内外大量研究证实 UGT1A1 的基因突变使得 胆红素葡萄糖醛酸化的能力全部或部分的缺失进而影响胆红素的代 谢，从而导致严重的高胆红素血症如 Crigler-Najjar综合症和 Gilbert's 综合症 （Annu. Rev. Pharmacol. Toxicol.2000. 40:581-616) 。 同时 UGTIAI也是多种临床药物， 如 Etoposide、 SN-38 (抗癌药物伊立替 康的活性代谢产物） 的主要代谢酶， 且 UGT1A1 的活性缺失已被证 实和伊立替康的毒性相关（J.Clin.Oncol.2006. 24:4534-8) 。 因此， 开 展 UGT1A1酶活的个体差异研宄对于临床个性化安全用药有着重要 意义。此外， 部分临床药物会抑制 UGT1A1 , 从而减少机体对胆红素 的代谢清除能力， 引起血液中胆红素的上升，进而导致高胆红素血症 或加剧 Crigler-Najjar综合症和 Gilbert's综合症患者的病情。目前国内 外制药巨头在药物开发过程中， 需要在体外评估各候选新药抑制 UGT1A1的能力。 因此， 幵发高效、 灵敏的特异性 UGT1A1探针底 物对于高效筛选 UGT1A1抑制剂， 及定量测定生物体系内 UGT1A1 的活性至关重要。
由于 UGT1A亚家族中的各亚型具有相似的氨基酸序列， 其底物 通常相互交叠，各亚型酶鲜有特异性的底物。目前，已报道的 UGT1A1 的探针底物有 3个， 分别是胆红素， 雌二醇和依托泊甙。 虽然胆红素 的单酶选择性较高， 但其化学稳定性差、检测灵敏度低。而雌二醇和 依托泊甙的单酶选择性并不高且需借助 LC-MS MS等昂贵分析仪器 才能实现产物的定量分析。 因此， 开发选择性高的 UGT1A1 的特异 性荧光探针底物及其配套的高通量检测方法具有重要的实用价值。
本发明中 1, 8-萘酰亚胺类化合物具有代谢产物单一（仅生成一个 单葡萄糖醛酸化产物）、 代谢酶高选择性（主要由 UGT1A1 代谢）、 底物及代谢产物易于捡测且灵敏度高等特点。
发明内容
本发明的目的在于提供一种葡萄糖醛酸转移酶 UGT1A1 的特异 性荧光探针底物及其应用，该特异性荧光探针底物和葡萄糖醛酸化产 物的荧光发射波长具有明显差异，且产物的荧光量子产率更高更易检 测。 利用该探针反应可对多种生物体系中 UGT1A1 的分布和功能进 行定量评价。
本发明提供了一种葡萄糖醛酸转移酶 UGT1A1 的特异性荧光探 针底物， 该探针底物可被 UGT1A1特异性催化生成相应的 0-葡萄糖 醛酸化产物， 其结构通式如式 （1 )所示， 该底物具有 1， 8-萘酰亚胺 类结构， 其中 R为 -COOH、 苯甲酸、 -S03H等有机酸性基团中的一 种， n为 2〜10。
Figure imgf000006_0001
式 （1 )
本发明还提供了所述葡萄糖醛酸转移酶 UGT1A1 的特异性荧光 探针底物的应用， 采用上述式（1 )化合物作为 UGT1A1亚酶的特异 性底物，进行葡萄糖醛酸化结合反应，通过定量检测单位时间内的底 物消除率或其葡萄糖醛酸化产物的生成率来定量测定不同生物体系 (包括重组表达 UGT1A1 单酶、 人或动物组织制备液、 各类组织细 胞等生物体系） 中 UGT1A1的活性； 具体测定方法为：
——体系中以 1，8-萘酰亚胺类化合物作为特异性探针底物；底物 浓度选择 l/10~10 Km; 单点测定时底物浓度优选 Km。
——在 Tris-Hcl缓冲液中， 反应温度为 20 °C至 60 °C之间， 优选 37°C为最优反应时间； 孵育体系 pH介于 5.5〜10.5之间， 优选 pH7.4 为最优反应 pH值；
——反应时间为 0〜140分钟，确保以上底物相应的葡萄糖醛酸化 产物达到定量限且底物转化率不超过 20%时终止反应；
——测定单位时间内底物减少量或葡萄糖醛酸化产物生成量作 为 UTG1A1活性的评价指标。 本发明提供的葡萄糖醛酸转移酶 UGT1A1 的特异性荧光探针底 物的应用，该探针底物及其葡萄糖醛酸化产物均具有荧光属性，且两 者具有不同的光学属性，可采用荧光检测器同时实现底物及产物的快 速、灵敏检测； 葡萄糖醛酸化产物及底物荧光检测条件分别为： 激发 波长 362， 450 nm, 最大发射波长为 450， 564 nm (如图 4所示）。
该特异性探针底物为比率型荧光探针， 其在 UGT1A1 活性检测 过程不易受生物体系基质及杂质的干扰， 可用于各种重组 UGT1A1、 人及动物组织制备液及各类组织细胞中 UGT1A1 酶活的定量测定； 同时也可作为在体及动物整体 UGT1A1 的探针底物， 评估胆红素代 谢酶 UGT1A1 的个体及种属差异。 该探针底物及葡萄糖醛酸化代谢 产物的荧光检测方法还可用于 UGT1A1 抑制剂的快速筛选及抑制能 力的定量评价。
采用重组葡萄糖醛酸 UTG1A1 单酶， 肝微粒体孵育体系进行考 察， 通过相关性分析， 特异性抑制实验， 重组单酶代谢反应， 以及酶 反应动力学几方面的证据，证明 1, 8-萘酰亚胺类化合物可特异性的经 葡萄糖醛酸 UTG1A1代谢（如图 9所示)， 生成羟基的葡萄糖醛酸化 产物。进一步采用各种哺乳动物的新鲜提取的肝细胞、 原代培养肝细 胞、肝切片、 肝灌流等代谢评价体系迸行考察，发现该代谢反应具有 非常良好的特异性。
作为高特异性的葡萄糖醛酸 UTG1A1 单酶的荧光探针底物， 该 化合物可以用来检测 UTG1A1 的活性， 尤其适合用于对细菌、 昆虫 细胞、 哺乳动物细胞以及酵母菌克隆表达体系生产的 UTG1A1 的酶 活测定， 以及多种哺乳动物组织器官来源的微粒体、 S-9等制备物中 UTG1A1的活性标定。
选用本发明所述葡萄糖酸酸 UTG1A1 单酶的特异性探针底物检 葡萄糖醛酸 UTG1A1单酶体外活性具有以下突出优势：
(1)高特异性： 1， 8-萘酰亚胺类化合物可被葡萄糖醛酸 UTG1A1 单酶高特异性地代谢成一个代谢产物， 即羟基的葡萄糖醛酸化产物。
(2) 廉价易得: 1， 8-萘酰亚胺类化合物可经化学合成获得， 合成 工艺简单易行， 荧光方法检测成本低。
(3)高灵敏度： 具有 1, 8-萘酰亚胺母核结构的化合物均具有良好 的荧光发射光谱特性（450〜700nm)， 且该底物及其葡萄糖醛酸化代 谢产物具有不同的荧光发射光谱特征，能较好的进行区分检测， 同时 可通过比率型标准曲线的建立进行定量测定， UGT1A1单酶的检测下 限为 50 ng。
附图说明
图 1. 1, 8-萘酰亚胺类化合物的结构通式；
图 2. N-(3-羧丙基 )-4-羟基 -1， 8-萘酰亚胺的 ifi-NMR谱图； 图 3.^(3-羧丙基)-4-羟基-1， 8-萘酰亚胺的 13C-NMR谱图； 图 4.N-(3-羧丙基 )-4-羟基 -1， 8-萘酰亚胺及其葡萄糖醛酸化代谢 产物的紫外吸收光谱图 （分别在 362 nm和 450 nm有最大吸收） ； 图 5.14例 HLM对 N-(3-羧丙基 )-4-羟基 -1, 8-萘酰亚胺的代谢图； 图 6. N-(3-羧丙基 )-4-羟基 -1, 8-萘酰亚胺的人 UGT重组单酶筛选 试验结果； 图 7.葡萄糖醛酸化代谢产物的生成量随孵育时间变化的线性拟 合.
图 8. N-(3-羧丙基 )-4-羟基 -1， 8-萘酰亚胺 UGT1A1单酶检测下限； 图 9. N-(3-羧丙基 )-4-羟基 -1， 8-萘酰亚胺被 UGT1A1葡萄糖酸酸 化的代谢通路；
图 10. N-(3-羧烷基 4-羟基 -1， 8-萘酰亚胺的合成路线。
具体实施方式
下面的实施例将对本发明予以迸一步的说明，但并不因此而限制 本发明。
实施例 1. N-P-羧烷基 )-4-羟基 -1， 8-萘酰亚胺的合成路线
( 1 )化合物 1的合成
将 4.2 mmol 4-氨基丁酸加入到含有 1 g(3.61 mmol) 4-溴 -1， 8萘酐 的 50 ml乙醇溶液中， 70-80 °C反应过夜后， 加入 200 ml水， 析出大 量固体， 过滤， 真空干燥得到米黄色固体 N-(3-羧丙基 )-4-溴 -1， 8-萘 酰亚胺， 产率 80-90%。
(2)化合物 2的合成
将 800 mg化合物 1与 2.54 g碳酸钾置于 100 ml单口瓶中， 加入 30 ml甲醇， 60-70°C反应过夜后， 冷却， 用 1 M的盐酸将 pH调至酸 性， 析出大量黄色固体， 过滤， 大量水洗， 真空干燥得到黄色固体 N-(3-羧丙基 )-4-甲氧基 -1， 8-萘酰亚胺， 产率 80-90%。
(3 )化合物 3的合成
将 300 mg化合物 2置于 25 ml两口瓶中，氩气保护下加入 10 ml 55-58%氢碘酸水溶液， 120-130°C搅拌过夜后， 加大量水稀释， 过滤， 用水洗涤至滤液为中性，真空干燥得到黄色固体，产率 60-70%。 N-(3- 羧烷基 )-4-羟基 -1, 8-萘酰亚胺（化合物 3)， 该化合物的氢谱碳谱见图 2和图 3， 氢碳化学位移如下：
lH NMR (400 MHz, DMSO) δ = 11.87 (s， 1Η), 8.54 (dd( J=8.3， 1.1, 1H), 8.48 (dd, J=7.3, 1.1, 1H), 8.37 (d， J=8.2, 1H)， 7.84-7.70 (m， 1H)， 7.17 (d, J-8.2, 1H), 4,07 (t, J=6.9, 2H), 2.29 (t, J=7.4， 2H), 2.01-1.71 (m， 2H). 13C NMR (100 MHz, DMSO) 23.59, 31.82, 39.33, 110.32, 113.05: 122.22, 122.75, 125.91, 129.21, 129.61, 131.46, 133.89, 160.61, 162.52, 164.20， 174.42. HRMS [M+H] + 300.0866, found 330.0864.
注： 化合物 1、 2、 3的结构如图 10所示。
实施例 2.体外测定人重组 VGT单酶的选择性
( 1 )预先准备 95 μΐ UGT代谢反应体系，包括 ρΗ 7.4的 Tris-Hcl 缓冲液（50 mM)、重组人 UGT各单酶（0.06 mg/ml), N-(3..羧丙基 )-4- 羟基 -1， 8-萘酰亚胺终浓度为 10 μΜ,于 37°C条件下震荡预孵 3分钟；
(2) 向反应体系中加入 5 μΐ浓度为 40 mM (终浓度 2 mM) 的 UDPGA起始反应；
(3 ) 30分钟后， 加入 100 μΐ冰乙腈， 剧烈震荡后， 终止反应；
(4)用高速冷冻离心机在 4 C， 20,000xg的条件下， 高速离心 20分钟后， 取上清， 进行荧光检测 （Ex-362 nm， Em-450 nm); 重 组人 UGT1A1酶的选择性最高约是其它单酶的 25倍左右 （图 6)。 实施例 3.体外抑制试验测定人肝微粒体和 UGT1A1的 ICS() ( 1 )预先准备 190 μΐ人肝微粒体和 UGTIAI代谢反应体系， 包 括 pH 7.4的 Tris-Hcl缓冲液（50 mM)、 人肝微粒体（0.2.5 mg/ml). UGTIAI (0.06 mg/ml), N-(3-羧丙基 )-4-羟基 -1， 8-萘酰亚胺终浓度为 10 μΜ， 不同浓度的原人参三醇于 37°C条件下震荡预孵 3分钟；
(2) 向反应体系中加入 10 μΐ浓度为 40 mM的 UDPGA起始反 应；
(3 ) 30分钟后， 加入 200 μΐ冰乙腈， 剧烈震荡后， 终止反应；
(4)用高速冷冻离心机在 4t， 20,000xg的条件下:， 高速离心 20分钟后， 取上清， 进行荧光检测（Ex=362 nm， Em=450 nm); 计 算其对人肝微粒体和 UGT1A1的 IC5Q值分别为 6.5 μΜ和 2.9 μΜ。 实施例 4.不同个体来源肝微粒体中 UGT1A1的活性定量评估
( 1 )选取 14例人肝微粒体 （HLM) 稀释至 10 mg/ml， 准备 UGT1A1代谢反应体系， 包括 pH 7.4的 Tris-Hcl缓冲液 （50 mM)、 人肝微粒体 (0.25 mg/ml) UGTIAI (0.06 mg/ml), N-(3-羧丙基 )-4-羟 基 -1, 8-萘酰亚胺终浓度为 20μΜ， 于 37°C条件下震荡预孵 3分钟；
(2) 向反应体系中加入 10 μΐ浓度为 40 mM的 UDPGA起始反 应；
(3 ) 30分钟后， 加入 200 μΐ冰乙腈， 剧烈震荡后， 终止反应；
(4) 用高速冷冻离心机在 4°C， 20,000xg的条件下， 高速离心 20分钟后， 取上清， 进行荧光检测 （Ex=362 nm， Em=4:50 nm), 将 所获荧光强度代入标准曲线后得到 14例人肝微粒体（HLM)对 N-(3- 羧丙基 )-4-羟基 -1， 8-萘酰亚胺的代谢速率 （图 5)。 实施例 5. UGTIAI捡测下限测定
实验在酶标仪上使用 96孔板进行测定， N-(3-羧丙基 )-4-羟基 -1, 8- 萘酰亚胺 10 μΜ， 尿苷二磷酸葡萄糖醛酸 2 mM, 十二烷基聚乙二醇 醚 1 mg/ml， MgCl2 5 mM, UGTIAI单酶 20 ng/m〜 500 ng'ml, pH 7.4 的 Tris-Hcl缓冲液 50 mM， 总体积为 100 L， 37°C下孵育 5 h后通过 酶标仪分析， 每组的平均值与不加 UGT1A1 的对照组比较， 结果如 图 8， 表明 50和 lOO ng的 UGT1A1有统计学意义 （P<0.05)， 确定 UGT1A1的检测下限为 50 ng。
实施例 6. UGT1A1时间标准曲线测定
实验在酶标仪上使用％孔板进行测定， N-(3-羧丙基 )-4-羟基 -1， 8- 萘酰亚胺 10 μΜ， 尿苷二磷酸葡萄糖醛酸 2 mM， 十二烷基聚乙二醇 醚 1 mg/ml， MgCl2 5 mM, UGTIAI单酶 0.01 mg/ml~0.09 mg/ml, pH 7.4的 Tris-Hcl缓冲液 50 mM,总体积为 100 μ , 37°C下孵育 140min， 每隔 10分钟酶标仪分析， 产物的荧光强度比底物的荧光强度的比值 与孵育时间做标准曲线，每条标准曲线的 R2〉0.99,表明标准曲线线 性范围宽广如图 7， 可准确定量 UGT1A1的含量。

Claims

权 利 要 求 书
1、 一种葡萄糖醛酸转移酶 UGT】 A】的特异性荧光探针底物， 其 特征在于： 该探针底物可被 UGT1A1特异性催化生成相应的 0-葡萄 糖醛酸化产物， 其结构通式如式（1 ) 所示， 该底物具有 1， 8-萘酰亚 胺类结构， 其中 R为- COOH、 苯甲酸、 -S03H中的一种， n为 2〜10。
Figure imgf000013_0001
式 （1 )
2、一种权利要求 1所述葡萄糖醛酸转移酶 UGT1A1的特异性荧 光探针底物的应用，其特征在于:采用上述式（ 1 )化合物作为 UGT1 A1 亚酶的特异性底物，进行水解反应，通过定量检测单位时间内的底物 消除率或其葡萄糖醛酸化产物的生成率来定量测定不同生物体系中 UGT1A1的活性。
3、按照权利要求 2所述葡萄糖醛酸转移酶 UGT1A1的特异性荧 光探针底物的应用， 其特征在于： 所述体外孵育反应条件为： 底物浓 度介于 l/10~10 Km之间；孵育体系 pH介于 5.5〜10.5之间； 反应温度 介于 20~60°C之间。
4、按照权利要求 2所述葡萄糖醛酸转移酶 UGT1A]的特异性荧 光探针底物的应用， 其特征在于： 所述的生物体系为重组表达 UGT1A1单酶、 人或动物组织制备液、 各类组织细胞。
5、按照权利要求 2所述葡萄糖醛酸转移酶 UGT1A1的特异性荧 光探针底物的应用，其特征在于：所述底物消除率或产物的生成率低 于 20%。
6、按照权利要求 2所述葡萄糖酸酸转移酶 UGT1A]的特异性荧 光探针底物的应用，其特征在于： 该探针底物及其葡萄糖醛酸化产物 均具有荧光属性，且两者具有不同的光学属性，可采用荧光检测器同 时实现底物及产物的快速、灵敏检测；葡萄糖醛酸化产物及底物荧光 检测条件分别为： 激发波长 362， 450 nm, 最大发射波长为 450， 564 nm。
7、权利要求 2所述葡萄糖醛酸转移酶 UGT1A1的特异性荧光探 针底物的应用， 其特征在于： 该探针底物还可用于 UGT1A1抑制剂 的快速筛选及抑制能力的定量评价。
8、权利要求 1所述葡萄糖醛酸转移酶 UGT1A1的特异性荧光探 针底物的应用， 其特征在于： 该探针底物也可作为在体及动物整体 UGT1A1的探针底物， 评估胆红素代谢酶 UGT1A1的个体及种属差 异。
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CN113640263A (zh) * 2021-08-09 2021-11-12 桂林电子科技大学 葡萄糖醛酸功能化金纳米簇lb膜及其制备方法和应用
CN113640263B (zh) * 2021-08-09 2022-11-25 桂林电子科技大学 葡萄糖醛酸功能化金纳米簇lb膜及其制备方法和应用
CN114703159A (zh) * 2022-03-15 2022-07-05 华南理工大学 一种葡糖基转移酶突变体及其应用
CN114703159B (zh) * 2022-03-15 2023-05-26 林影 一种葡糖基转移酶突变体及其应用

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