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

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

Firstly, Alexa Fluor 532 carboxylic acid, N-succinimidyl ester (1.0 mg, 1.38 µmol) was dissolved in anhydrous DMF (200 µl).
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.
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.
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.
An homogeneous product corresponding to the MRS5424 was isolated in the triethylammonium salt form with an HPLC retention time of 13.5 min.
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).
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.
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.

Acknowledgments

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.

References

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 A_2AR-D₂R allosterism: role of the intracellular loop 3 of the D₂R. J Neurochem 123(3): 373-384.