Exploration of labeling by near infrared dyes of the polyproline linker for bivalent-type CXCR4 ligands

doi:10.1016/j.bmc.2015.09.040

Bioorganic & Medicinal Chemistry

Volume 23, Issue 21, 1 November 2015, Pages 6967-6973

https://doi.org/10.1016/j.bmc.2015.09.040 Get rights and content

Abstract

We have previously used poly-l-proline linkers for the development of bivalent-type ligands for the chemokine receptor, CXCR4. The bivalent ligands with optimum linkers showed specific binding to CXCR4, suggesting the existence of CXCR4 possibly as a dimer on the cell membrane, and enabled definition of the amount of CXCR4 expressed. This paper reports the synthesis by a copper-catalyzed azide–alkyne cycloaddition reaction as the key reaction, of bivalent CXCR4 ligands with near infrared (NIR) dyes at the terminus or the center of the poly-l-proline linker. Some of the NIR-labeled ligands, which would be valuable probes useful in studies of the behavior of cells expressing CXCR4, have been obtained. The information concerning the effects of the labeling positions of NIR dyes on their binding properties is useful for the design of modified bivalent-type CXCR4 ligands.

Graphical abstract

Introduction

Bi- or multi-modality involving some functional groups in a molecule such as a ligand moiety for specific binding, a fluorophore for imaging or spectroscopic analyses, or a photo-reactive moiety for the search for target molecules, are valuable in research in chemical biology. Integrated peptide templates are useful because they enable the design of such molecules.1, 2 Among these templates, poly-l-proline linkers have been used in several studies as ‘molecular rulers’ to obtain information of biological activity or to adjust the structures and thus the activity of the target molecule.3, 4, 5 The most important property of poly-l-proline is the rigidity of its stable helical structure, even in vivo.6, 7 Two functional moieties connected by a poly-l-proline linker could be placed at the predetermined distance from each other at opposite ends of the linker. The distances between the moieties can be precisely controlled by changing the number of prolines that separate them. One possible application of the poly-l-proline linker is the design of bivalent ligands, several of which have been reported to target membrane proteins.8, 9 An advantage of the bivalency is that the binding of each ligand could work in a concerted fashion, resulting in a synergistic increase of the binding affinity and specificity. Although flexible linkers such as PEG, alkyl groups or random amino acids could be utilized for bivalent ligands, it is difficult to determine the optimal linker length associated with the highest affinity.¹⁰ In our laboratory, poly-l-proline linkers were successfully used for the construction of bivalent-type ligands for a G protein-coupled chemokine receptor, CXCR4.¹¹ The ligand with the optimal linker 1, a poly-l-proline 18-mer, showed specific binding to CXCR4 and suggested a possible dimeric state of CXCR4 on the cell membrane.8, 9 Furthermore, the bivalent ligand distinguished the expressed amount of CXCR4 on the cell surface.

CXCR4 is a drug target important in the therapy of AIDS, cancer metastasis and rheumatoid arthritis.12, 13, 14, 15, 16, 17 The X-ray crystallographic structure of CXCR4 has been reported,¹⁸ but it is possible that CXCR4 exists as a dimer or multimer on the cell surface, especially when the receptor is overexpressed as seen in cancer cells. Specific recognition by bivalent ligands would be useful for the detection of cells that have overexpressed CXCR4 in vitro or in vivo. In case of detection in vivo, fluorescent probes analyzed in a near infrared (NIR) region are required. To conjugate fluorescent dyes with the ligand, it must be shown that the attachment of dyes does not interfere with the binding of the ligand. Most of NIR dyes such as cyanine dyes have relatively large molecular weights and structures. A variety of bivalent ligands with a poly-l-proline linker could be applied. Thus, information concerning the effect of dye labeling will be useful in studies of the in vivo behavior of cells expressing target receptors.

In this study, the effect of the NIR dye labeling on poly-l-proline linkers was addressed by taking advantage of our bivalent-type CXCR4 ligand. Bivalent-type ligands with poly-l-proline linkers having the NIR-dye at the center or end of the linkers were prepared, and their fluorescent and binding properties were analyzed.

Section snippets

Results and discussion

Studies on the dimeric state of CXCR4 on the cell surface have been conducted with bivalent-type CXCR4 ligands having two FC131 derivatives19, 20, 21, 22 (cFC131, Fig. 1) connected via poly-l-proline linkers. When the ligand is labeled with a fluorescent moiety, the labeling positions could be important for maintenance of the ligand activity. The middle and the end of the linkers were selected as labeling positions. Replacement of any of the proline residues in poly-l-proline by a different

Acknowledgements

This work was supported in part by KAKENHI, Grant-in-Aid for Scientific Research (B) (24390024 to H.T.); JSPS Core-to-Core Program, A. Advanced Research Networks; and the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT, Japan. The authors are grateful to Dr. Tomohiro Tanaka and Dr. Tetsuo Narumi for their assistance in setting up the research and for helpful discussions, and to Prof. Takaki Koide and Dr. Ryo Masuda, Department of Chemistry and Biochemistry, Waseda

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Exploration of labeling by near infrared dyes of the polyproline linker for bivalent-type CXCR4 ligands

Abstract

Graphical abstract

Introduction

Section snippets

Results and discussion

Acknowledgements

Curr. Opin. Immunol.

Biochem. Biophys. Res. Commun.

Tetrahedron Lett.

ChemBioChem

J. Med. Chem.

J. Am. Chem. Soc.

Proc. Natl. Acad. Sci. U.S.A.

J. Am. Chem. Soc.

J. Am. Chem. Soc.

J. Am. Chem. Soc.

Bioconjugate Chem.

J. Med. Chem.

ChemMedChem

J. Am. Chem. Soc.

Nature