腺苷 A2A 受体荧光激动剂 MRS5424的合成

Kenneth A. Jacobson; Francisco Ciruela

doi:10.21769/BioProtoc.1069

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Peer-reviewed

Synthesis of the Adenosine A_2A Receptor Fluorescent Agonist MRS5424

腺苷 A2A 受体荧光激动剂 MRS5424的合成

Kenneth A. Jacobson

Francisco Ciruela email

发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1069 浏览次数: 7966

评审: Cheng Zhang

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Abstract

MRS5424 is a functional fluorescent agonist for the adenosine A_2A receptor (A_2AR) in which the fluorescent dye Alexa Fluor 532 is covalently attached to the A_2AR agonist 2-[[2-[4-[2-(2-aminoethyl)-aminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethyl-carboxamidoadenosine (APEC). This easy-to-synthesize new A_2AR fluorescent ligand was shown to be extremely useful for determining the binding kinetic constants of A_2AR in a real-time mode (Fernandez-Duenas et al., 2012). In addition, this fluorescent A_2AR 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 A_2AR fluorescent ligand may not be as high as that observed for the marketed A_2AR 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, Invitrogen^TM)
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

English

中文翻译

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如何引用

Jacobson, K. A. and Ciruela, F. (2014). Synthesis of the Adenosine A_2A Receptor Fluorescent Agonist MRS5424 . Bio-protocol 4(6): e1069. DOI: 10.21769/BioProtoc.1069.