Convenient and efficient synthesis of a lanthanide³⁺‐coordinated, diethylene triamine pentaacetic acid labeled biopolymer as an assay for the cholecystokinin B receptor

Abstract

To develop an assay for the cholecystokinin B receptor with an Eu 3+ -labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide 3+ -coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study.

Solid-Phase Synthesis of Europium-Labeled Human INSL3 as a Novel Probe for the Study of Ligand−Receptor Interactions

“…The peptide purity was shown by analytical RP-HPLC to be greater than 95%, and to have a peptide content of 64% as assessed by amino acid analysis. A monoisotopic m / z of 6821 corresponding to the singly Eu-coordinated DTPA-labeled INSL3 was obtained using MALDI-TOF MS, which contrasts to previous studies on synthetic lanthanide complexes of synthetic peptides, in which multiple isomers were obtained . The overall yield of synthesis was 7.7%, which compares favorably to that obtained for native human INSL3 (10%) obtained using a similar synthesis protocol .…”

Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor

“…25 The overall yield of 3 was 26%, significantly higher than the 16% yield reported for the synthesis of probe 1 . 16 …”

“…During the past decade several europium-diethylenetriaminepentaacetic acid (Eu-DTPA) chelates linked to peptide recognition elements have been reported. 10,13–16 These probes were used to evaluate multivalent molecules targeted to melanocortin or cholecystokinin receptors via dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)-based ligand binding assays. 17 The functional readout of such assays comes from the time-resolved fluorescence (TRF) generated by the europium ions.…”

“…17 The functional readout of such assays comes from the time-resolved fluorescence (TRF) generated by the europium ions. While TRF probes 1 and 2 based on a CCK8 recognition element 16 have previously been used to characterize ligand binding to CCK2R in competitive binding assays, 18–20 a redesign of these probes could simplify and improve probe preparation and purification, possibly enhance probe solubility and receptor binding, and provide an additional tool for use in bioassays.…”

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

“…In order to circumvent these problems, site‐specific incorporation of the chelate into solid phase‐assembled and bound biomolecules can be successfully undertaken. Solid‐phase synthesis has been reported for labelling oligonucleotides for use in DNA studies13 as well as for the preparation of small, labelled peptides14, 15. Recently, we have reported a highly efficient solid‐phase synthesis methodology for site‐specifically incorporating a single lanthanide chelate into two‐chain peptides having multiple disulfide bonds16.…”