Synthesis of the Adenosine A2A Receptor Fluorescent Agonist MRS5424
腺苷 A2A 受体荧光激动剂 MRS5424的合成   

Cheng Zhang Cheng Zhang
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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

  1. Alexa Fluor 532 carboxylic acid, N-succinimidyl ester (Life Technologies, InvitrogenTM)
  2. Anhydrous dimethylformamide (DMF; HPLC grade) (Alfa Aesar)
  3. Sodium tetraborate labeling buffer (0.1 M, pH 8.5)
  4. 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/)
  5. Triethylammonium acetate (TEAA)-CH3CN (BioUltra grade) (Sigma-Aldrich)
  6. Tetrabutylammonium dihydrogenphosphate-CH3CN (TBAP) [puriss. ≥99.0% (T)] (Sigma-Aldrich)


  1. RP-C18(2) semipreparative column (250 x 10.0 mm) (Phenomenex)
  2. 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)

  3. Diode array detector
  4. POLARstar Optima plate-reader (BMG LABTECH)


Briefly, MRS5424 (Fernandez-Duenas et al., 2012) was synthesized as follows.

  1. Firstly, Alexa Fluor 532 carboxylic acid, N-succinimidyl ester (1.0 mg, 1.38 µmol) was dissolved in anhydrous DMF (200 µl).
  2. Next, make a 0.1 M sodium tetraborate buffer by dissolving 0.038 g of sodium tetraborate decahydrate for every ml of water. Adjust pH with HCl to 8.5. The labeling buffer should be made just before using it (i.e. fresh) since air exposure of this solution will result in carbon dioxide absorption, which will change its pH.
  3. Then, 200 µl of freshly prepared sodium tetraborate labeling buffer (0.1 M, 1 ml, pH 8.5) containing APEC (1.12 mg, 2.07 µmol) - initially dissolved in anhydrous DMF - was added to the Alexa Fluor 532 solution.
  4. The reaction mixture was protected from light and after stirring for 18 h at 4 °C, the mixture was diluted with H2O (600 µl) and purification was performed by HPLC with a Luna 5 µm RP-C18(2) semipreparative column under the following conditions: flow rate of 2 ml/min; 10 mM triethylammonium acetate (TEAA)-CH3CN from 100:0 (v/v) to 70:30 (v/v) in 30 min.
  5. An homogeneous product corresponding to the MRS5424 was isolated in the triethylammonium salt form with an HPLC retention time of 13.5 min.
  6. Analytical purity of this conjugate was checked using a Hewlett-Packard 1100 HPLC equipped with a Zorbax SB-Aq 5 µm analytical column. Mobile phase: linear gradient solvent system: 5 mM TBAP from 80:20 to 40:60 in 13 min; the flow rate was 0.5 ml/min (retention time 9.08 min).
  7. Peaks were detected by UV absorption with a diode array detector at 254, 275, and 280 nm, and the yield of MRS5424 was 0.67 mg (31%). ESI-HRMS m/z 1150.4142 [M + H]+, C55H63N11O13S2.H+: Calcd. 1150.4127.
  8. Finally, in order to check the fluorescence features of the MRS5424 the excitation/emission spectrum was assessed by means of a POLARstar Optima plate-reader.


This work was supported by grants SAF2011-24779, Consolider-Ingenio CSD2008-00005 and PCIN-2013-019-C03-03 from Ministerio de Economía y Competitividad and ICREA Academia-2010 from the Catalan Institution for Research and Advanced Studies (to FC), by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Intramural Research Program (to KAJ). FC belong to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). We thank E. Castaño and B. Torrejón from the Scientific and Technical Services (SCT) group at the Bellvitge Campus of the University of Barcelona for their technical assistance.


  1. Fernandez-Duenas, V., Gomez-Soler, M., Jacobson, K. A., Kumar, S. T., Fuxe, K., Borroto-Escuela, D. O. and Ciruela, F. (2012). Molecular determinants of A2AR-D2R allosterism: role of the intracellular loop 3 of the D2R. J Neurochem 123(3): 373-384.


MRS5424是腺苷A 2A受体(A 2A 2A R)的功能性荧光激动剂,其中荧光染料Alexa Fluor 532共价连接到Aβ2A受体2 - [[2- [4- [2-(2-氨基乙基) - 氨基羰基]乙基]苯基]乙基氨基] -5'-N-乙基 - 甲酰胺基腺苷(APEC)。这种容易合成的新A 2A 2A荧光配体显示对于在实时模式中确定A 2A 2A R的结合动力学常数是非常有用的(Fernandez -Duenas等人,2012)。此外,这种荧光A 2A 2A配体与使用荧光读板器的配体 - 受体相互作用研究相容。最后,重要的是提到即使该A 2A 2A R荧光配体的灵敏度可能不如市售的A 2A 2B放射性化合物所观察到的那么高,使用这种荧光衍生物可能具有超过放射性探针的一些优点,例如其安全递送,操作和处置,短信号获取时间,自动化和小型化的可行性,以及最终其成本。

关键字:荧光配体, Adenosine受体, 亚太经合组织, mrs5424, A2aR


  1. Alexa Fluor 532羧酸,N' - 琥珀酰亚胺酯(Life Technologies,Invitrogen TM
  2. 无水二甲基甲酰胺(DMF; HPLC级)(Alfa Aesar)
  3. 四硼酸钠标记缓冲液(0.1M,pH8.5)
  4. 2 - [[2- [4- [2-(2-氨基乙基) - 氨基羰基]乙基]苯基]乙基氨基] -5'-N-乙基 - 甲酰胺基腺苷(APEC)(NIMH Chemical Synthesis and Drug Supply Program, ="_ blank"href ="http://nimh-repository.rti.org/"> http://nimh-repository.rti.org/)
  5. 三乙基乙酸铵(TEAA)-CH 3 CN(BioUltra级)(Sigma-Aldrich)
  6. 四丁基铵二氢磷酸盐-CH 3 CN(TBAP)[puriss。 ≥99.0%(T)](Sigma-Aldrich)


  1. RP-C18(2)半制备柱(250×10.0mm)(Phenomenex)
  2. 装备有Luna5μmRP-C18(2)半制备柱(250×10.0mm)(图1B)(Phenomenex)或Zorbax SB-Aq5μm分析柱(50×4.6mm)(Agilent)的Hewlett-Packard 1100HPLC )(图1A)

    图1. Luna5μmRP-C18(2)半制备柱(250×10.0mm)(B)和Zorbax SB-Aq5μm分析柱(50×4.6mm)(A)的图片。 强>

  3. 二极管阵列检测器
  4. POLARstar Optima读板器(BMG LABTECH)



  1. 首先,将Alexa Fluor 532羧酸Nuc-琥珀酰亚胺酯(1.0mg,1.38μmol)溶于无水DMF(200μl)中。
  2. 接下来,通过对于每ml水溶解0.038g十水合四硼酸钠来制备0.1M四硼酸钠缓冲液。 用HCl调节pH至8.5。 标记缓冲液应在使用前进行(即 新鲜),因为该溶液的空气暴露将导致二氧化碳吸收,这将改变其pH。
  3. 然后,将含有最初溶解于无水DMF中的APEC(1.12mg,2.07μmol)的200μl新鲜制备的四硼酸钠标记缓冲液(0.1M,1ml,pH8.5)加入到Alexa Fluor 532溶液中。
  4. 将反应混合物避光,在4℃下搅拌18小时后,将混合物用H 2 O(600μl)稀释,通过HPLC进行纯化,使用Luna5μmRP- C18(2)半制备柱在以下条件下:流速为2ml/min;在30分钟内从100:0(v/v)至70:30(v/v)的10mM三乙基乙酸铵(TEAA)-CH 3 CN。
  5. 以三乙基铵盐形式分离对应于MRS5424的均相产物,HPLC保留时间为13.5分钟。
  6. 使用配备有Zorbax SB-Aq5μm分析柱的Hewlett-Packard 1100 HPLC检查该缀合物的分析纯度。流动相:线性梯度溶剂系统:5mM TBAP,在13分钟内从80:20至40:60;流速为0.5ml/min(保留时间9.08min)。
  7. 通过用二极管阵列检测器在254,275和280nm的UV吸收检测峰,MRS5424的产率为0.67mg(31%)。 ESI-HRMS m/z 1150.4142 [M + H] +,C 55 H 63 N 11 O 11。 sub> 13 S 2 H + :Calcd。 1150.4127。
  8. 最后,为了检查MRS5424的荧光特征,通过POLARstar Optima读板器评估激发/发射光谱。


这项工作得到了来自加拿大研究和高等研究机构(FC)的经济部长奖学金SAF2011-24779,Consolider-Ingenio CSD2008-00005和PCIN-2013-019-C03-03以及加泰罗尼亚研究与高等研究学院ICREA学术成员2010 ,由国家糖尿病和消化和肾脏疾病研究所(NIDDK)校内研究计划(KAJ)。 FC属于"神经药理学和疼痛"认证研究组(Generalitat de Catalunya,2014 SGR 1251)。我们感谢巴塞罗那大学Bellvitge校区科学技术服务(SCT)小组的E.Castaño和B.Torrejón的技术援助。


  1. Fernandez-Duenas,V.,Gomez-Soler,M.,Jacobson,K.A.,Kumar,S.T.,Fuxe,K.,Borroto-Escuela,D.O.和Ciruela,F。(2012)。 分子决定因素 > A 2A亚型R 2亚基的变体:D 2 R 3的细胞内环3的作用。神经化学 123(3):373-384。
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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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