Synthesis of the Adenosine A2A Receptor Fluorescent Agonist MRS5424
腺苷 A2A 受体荧光激动剂 MRS5424的合成
发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1069 浏览次数: 8264
评审: Cheng Zhang
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参见作者原研究论文
The authors used this protocol in:
Nov 2012
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Abstract
MRS5424 is a functional fluorescent agonist for the adenosine A2A receptor (A2AR) in which the fluorescent dye Alexa Fluor 532 is covalently attached to the A2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethyl-carboxamidoadenosine (APEC). This easy-to-synthesize new A2AR fluorescent ligand was shown to be extremely useful for determining the binding kinetic constants of A2AR in a real-time mode (Fernandez-Duenas et al., 2012). In addition, this fluorescent A2AR ligand is compatible with ligand-receptor interaction studies using fluorescent plate readers. Finally, it is important to mention that even though the sensitivity of this A2AR fluorescent ligand may not be as high as that observed for the marketed A2AR radioactive compounds, the use of such fluorescent derivative may have some advantages over radioactive probes, for example its safe delivery, manipulation and disposal, the short signal acquisition times, the feasibility to automate and to miniaturize, and finally its cost.
Keywords: Fluorescent ligand (荧光配体)
Adenosine receptor (Adenosine受体)
APEC (亚太经合组织)
MRS5424 (mrs5424)
A2AR (A2aR)
Materials and Reagents
- Alexa Fluor 532 carboxylic acid, N-succinimidyl ester (Life Technologies, InvitrogenTM)
- Anhydrous dimethylformamide (DMF; HPLC grade) (Alfa Aesar)
- Sodium tetraborate labeling buffer (0.1 M, pH 8.5)
- 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethyl-carboxamidoadenosine (APEC) (NIMH Chemical Synthesis and Drug Supply Program, http://nimh-repository.rti.org/)
- Triethylammonium acetate (TEAA)-CH3CN (BioUltra grade) (Sigma-Aldrich)
- Tetrabutylammonium dihydrogenphosphate-CH3CN (TBAP) [puriss. ≥99.0% (T)] (Sigma-Aldrich)
Equipment
- RP-C18(2) semipreparative column (250 x 10.0 mm) (Phenomenex)
- Hewlett-Packard 1100 HPLC equipped with a Luna 5 µm RP-C18(2) semipreparative column (250 x 10.0 mm) (Figure 1B) (Phenomenex) or a Zorbax SB-Aq 5 µm analytical column (50 x 4.6 mm) (Agilent) (Figure 1A)
Figure 1. Picture of the Luna 5 µm RP-C18(2) semipreparative column (250 x 10.0 mm) (B) and the Zorbax SB-Aq 5 µm analytical column (50 x 4.6 mm) (A)
- Diode array detector
- POLARstar Optima plate-reader (BMG LABTECH)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jacobson, K. A. and Ciruela, F. (2014). Synthesis of the Adenosine A2A Receptor Fluorescent Agonist MRS5424 . Bio-protocol 4(6): e1069. DOI: 10.21769/BioProtoc.1069.
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分类
生物化学 > 蛋白质 > 相互作用 > 蛋白质-配体相互作用
生物化学 > 蛋白质 > 荧光
细胞生物学 > 细胞成像 > 荧光
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