Analytical Sciences

Chemical Synthesis and Application of C-Terminally 5-Carboxyfluorescein-labelled Thymopentin as a Fluorescent Probe for Thymopoietin Receptor

Satomi ONOUE,*^1,*² Baosheng LIU,*³ Yoshitaka NEMOTO,*³ Mariko HIROSE,*⁴ and Takehiko YAJIMA*¹

*1 Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
*2 Analytical Research and Development, Pfizer Global Research and Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
*3 American Peptide Company, 777 East Evelyn Ave., Sunnyvale, CA, 94086, USA
*4 Pharmaceutical Division, Ito Life Sciences Inc., 1-2-1 Kubogaoka, Moriya, Ibaraki 302-0104, Japan

Thymopentin (TP5) is a synthetic pentapeptide fragment, which corresponds to position 32 - 36 of thymic polypeptide thymopoietin. Thymopoietin and TP5 display a variety of biological functions, including phenotypic differentiation of T cells and the regulation of immune systems. Previous chemical modification experiments suggested that there was an absolute requirement for N-terminal amino acids to maintain the biological activity of TP5. On the basis of this structure-activity relationship, we designed and synthesized the C-terminally 5-carboxyfluorescein-coupled TP5 (TP5-FAM) as a fluorescent probe for thymopoietin receptor. TP5-FAM could bind to the membrane of human lymphoid cell lines, MOLT-4 cells, in which the thymopoietin receptor is expressed. The binding is specific and saturable (K_d = 33 µM). TP5 and human splenopentin are nearly equipotent inhibitors of TP5-FAM binding to the thymopoietin receptor, but porcine secretin did not show any significant inhibition of TP5-FAM binding to MOLT-4 cells. Thus, TP5-FAM is suggested to be a potent and biologically active ligand that would be useful for studying the binding and functional characteristics of the human thymopoietin receptor.

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