Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

Abstract

Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination, as well as the success of metal ion removal upon attempted purification. We compared the use of Empore™ chelating disks and Chelex® 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore™ chelating disks were found to give much higher recoveries of the probes under the conditions utilized. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.

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

“…Fortunately, passage of 3 through an Empore chelating disk removed the unchelated europium ions. 25 The overall yield of 3 was 26%, significantly higher than the 16% yield reported for the synthesis of probe 1 . 16 …”

“…Compound 5 was characterized by ESI-ICR MS. Following complexation of Eu 3+ by 5 , purification and partial desalting by reversed-phase chromatography, final desalting by passage through an Empore chelating disk, 25 and recovery by lyophilization afforded Eu-DTPA-PEGO-CCK4 ( 3 ) in 73% yield. Compound 3 was characterized by analytical HPLC and ESI-ICR MS.…”

“…The absence of unchelated Eu 3+ ion (within a 10 μM limit of detection) was confirmed by means of a xylenol orange spectrophotometric assay. 25 …”

“…Treatment of 5 with excess EuCl 3 in a pH 7-8 buffer gave the chelate 3 , but passage through a Sep-Pak ® C 18 column left unchelated europium ions as a contaminant as determined by a xylenol orange assay. 25 Complete removal of unchelated europium ions is essential to obtaining reliable bioassay readings. Fortunately, passage of 3 through an Empore chelating disk removed the unchelated europium ions.…”

“…The removal of unchelated Eu ions was confirmed by a xylenol orange assay. 25 Recovery was 14.6 mg (97%); HRMS (ESI-ICR) m/z calculated for C 58 H 84 EuN 11 O 21 [M+2H] 2+ 711.7527, observed 711.7527; analytical HPLC, t R = 13.82 min.…”