fluorescein mono(β-D-galactopyranoside)

• 分子结构分类: 有机化合物 - 有机氧化合物
• 英文名称: fluorescein mono(β-D-galactopyranoside)
• 英文别名: fluorescein mono-β-D-galactopyranoside；2-[3-oxo-6-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyxanthen-9-yl]benzoic acid
• 化学式: C₂₆H₂₂O₁₀
• 分子量: 494.455
• InChiKey: QSWRFZUITMFLIC-WLPYWIPGSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  0.6
• 重原子数：
  36
• 可旋转键数：
  5
• 环数：
  5.0
• sp3杂化的碳原子比例：
  0.23
• 拓扑面积：
  163
• 氢给体数：
  5
• 氢受体数：
  10

反应信息

• 作为反应物：
  描述：

  fluorescein mono(β-D-galactopyranoside) 在 biotinylated β-galactosidase 、 2-巯基乙醇 、 magnesium chloride 作用下， 以 aq. phosphate buffer 为溶剂， 生成 fluorescein free acid
  参考文献：
  名称：

  Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient

  摘要：

  Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated beta-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K-m) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K-m value for biotinylated beta-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5 x 10(-10) m(2) s(-1). Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent. (C) 2012 Elsevier Ltd. All rights reserved.

  DOI：

  10.1016/j.carres.2012.05.017
• 作为产物：
  描述：

  fluorescein methyl ester mono(β-D-galactopyranoside) 在 sodium hydroxide 作用下， 以 水 为溶剂， 反应 1.0h， 以53%的产率得到fluorescein mono(β-D-galactopyranoside)
  参考文献：
  名称：

  Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient

  摘要：

  Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated beta-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K-m) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K-m value for biotinylated beta-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5 x 10(-10) m(2) s(-1). Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent. (C) 2012 Elsevier Ltd. All rights reserved.

  DOI：

  10.1016/j.carres.2012.05.017

文献信息

• Stopped-Flow Enzyme Assays on a Chip Using a Microfabricated Mixer
  作者：Brian J. Burke、Fred E. Regnier

  DOI：10.1021/ac026173j

  日期：2003.4.1

  This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in <1s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. β-Galactosidase (β-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-β-d-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 ± 13 μM and 44 ± 3 s-1 were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 ± 6 μM and kcat was 47 ± 4 s-1. Enzyme inhibition assays with phenylethyl-β-d-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.

  本文介绍了一种微加工酶测定系统，包括一个可用于进行停流反应的微搅拌器。样品和试剂通过电渗流（EOF）进入系统。试剂流在小于 1 秒的时间内合并并通过 100 升的微搅拌器。这项工作的目的是利用一个容积约为 6 nL 的系统，在停流模式下进行基于动力学的酶测定。β-半乳糖苷酶（β-Gal）被选为这些研究的模型酶，用于将底物荧光素单-β-d-吡喃半乳糖苷（FMG）转化为荧光素。使用微型搅拌器的微加工系统所获得的结果与使用外部 T 型混合装置所获得的结果相比毫不逊色。相比之下，在微加工装置中通过合并两股液流并允许横向扩散进行混合而进行的化验结果并不理想。使用微制造混合器测定的 Km 和 kcat 值分别为 75 ± 13 μM 和 44 ± 3 s-1。这些数值与使用传统止流装置测定出的 Km 为 60 ± 6 μM 和 kcat 为 47 ± 4 s-1 的数值相比，相差无几。用苯基乙基-β-d-硫代半乳糖苷（PETG）进行的酶抑制测定也具有可比性。结论是，如果使用所述装置辅助混合，则可在 60 秒或更短的时间内使用约 6 nL 液体容积的微型系统进行基于动力学的停流酶测定。