DOI: 10.1002/cbic.200700761 Synthesis and Application of Fluorescein- and Biotin- Labeled Molecular Probes for the Chemokine Receptor CXCR4 Shinya Oishi,* [a] Ryo Masuda, [a] Barry Evans, [b] Satoshi Ueda, [a] Yukiko Goto, [a] Hiroaki Ohno, [a] Akira Hirasawa, [a] Gozoh Tsujimoto, [a] Zixuan Wang, [b] Stephen C. Peiper, [b] Takeshi Naito, [c] Eiichi Kodama, [c] Masao Matsuoka, [c] and Nobutaka Fujii* [a] Introduction The CXC chemokine receptor 4 (CXCR4) is a G-protein-coupled cell-surface receptor that was identified previously as a core- ceptor for infection by the T-cell-line-tropic (X4) human immu- nodeficiency virus type 1 (HIV-1). [1,2] Stromal cell-derived factor 1 (SDF-1)/CXCL12 is a homeostatic chemokine that regu- lates a number of physiological and pathologic processes through its interaction with and activation of CXCR4. SDF-1 se- creted in bone-marrow stromal cells supports the retention of hematopoietic stem cells (HSCs), progenitor cells, and B-cell precursors in the hematopoietic microenvironment. [3] SDF-1 ex- pression is implicated in the survival, growth, and develop- ment of CXCR4-expressing cells, including normal and malig- nant B lymphocytes, hematopoietic progenitors, and carcino- ma cells. [3,4] It has also been demonstrated that concentration gradients of SDF-1 promote the homing of HSCs to bone marrow, the recruitment of progenitor cells to sites of ischemic tissue damage, and the metastasis of CXCR4-expressing neo- plastic cells to target organs. [4,5] Recently, CXCR7 (RDC1, CCX-CKR2) was reported to be ACHTUNGTRENNUNGanother receptor for SDF-1. [6,7] CXCR7 promotes cell survival, growth, and adhesion in vitro and in vivo. [7,8] Furthermore, the expression pattern of CXCR7 is complementary to that of CXCR4 in the migrating primordium. [9,10] Therefore, the SDF-1– CXCR7 axis, like SDF-1–CXCR4, is relevant to the control pro- cesses of cell growth, migration, and recruitment. To investi- gate the distribution and localization of two binding partners of SDF-1, CXCR4 and CXCR7, both in vitro and in vivo, it would be useful to have access to selective and specific fluorescence- and otherwise-labeled ligands for these receptors. To date, several CXCR4-receptor probes have been prepared and applied both in vitro [11–14] and in vivo. [15] Fluorescein- labeled SDF-1 was utilized to detect the CXCR4-dependent in- ternalization of SDF-1 by stromal bone-marrow cells. [11] This ACHTUNGTRENNUNGlabeled agonist was useful for evaluating the mechanism of re- ceptor activation. We developed a potential radiopharmaceuti- cal agent based on the polyphemusin II derived CXCR4 antago- nist T140 (Scheme 1). Thus, [ 111 In]–diethyleneACHTUNGTRENNUNGtriACHTUNGTRENNUNGamineACHTUNGTRENNUNGpenACHTUNGTRENNUNGta- [a] Dr. S. Oishi, R. Masuda, Dr. S. Ueda, Y. Goto, Dr. H. Ohno, Dr. A. Hirasawa, Prof. Dr. G. Tsujimoto, Prof. Dr. N. Fujii Graduate School of Pharmaceutical Sciences, Kyoto University Sakyo-ku, Kyoto 606-8501 (Japan) Fax: (+ 81)75-753-4570 E-mail: soishi@pharm.kyoto-u.ac.jp nfujii@pharm.kyoto-u.ac.jp [b] B. Evans, Dr. Z. Wang, Prof. Dr. S. C. Peiper Department of Pathology, Medical College of Georgia Georgia 30912 (USA) [c] T. Naito, Dr. E. Kodama, Prof. Dr. M. Matsuoka Institute for Virus Research, Kyoto University Sakyo-ku, Kyoto 606-8507 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. The design, synthesis, and bioevaluation of fluorescence- and biotin-labeled CXCR4 antagonists are described. The modification of d-Lys8 at an e-amino group in the peptide antagonist Ac- TZ14011 derived from polyphemusin II had no significant influ- ence on the potent binding of the peptide to the CXCR4 receptor. The application of the labeled peptides in flow cytometry and confocal microscopy studies demonstrated the selectivity of their binding to the CXCR4 receptor, but not to CXCR7, which was ACHTUNGTRENNUNGrecently reported to be another receptor for stromal cell-derived factor 1 (SDF-1)/CXCL12. Scheme 1. Structure of the selective CXCR4 antagonists T140, which was used to design probe Ac-TZ14011 (1). Bold type indicates the pharmaco- phore residues. 1154 www.chembiochem.org # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2008, 9, 1154 – 1158