Solid Phase Synthesis of Short Peptide-Based Multimetal Tags for Biomolecule Labeling
Abstract
We describe an unprecedented solid phase peptide synthesis (SPPS) of short peptide-based multimetal tags designated as elemental tags for the quantification of biomolecules via inductively coupled plasma mass spectrometry (ICP-MS). The macrocyclic chelator 1,4,7,10-tetraazacyclododecane N,N′,N″,N‴-tetra acetic acid (DOTA) was attached to the side chain of N-α-(9-fluorenylmethoxycarbonyl)-l-lysine (Fmoc-Lys-OH) and metalated with a lanthanide to provide a building block for Fmoc-based SPPS. Thereby, in contrast to existing strategies for the synthesis of DOTA–peptide conjugates, an already metalated DOTA-amino acid was used as a building block for SPPS. The DOTA-lanthanide complex was stable throughout the whole SPPS, even during the final cleavage in concentrated trifluoroacetic acid. This indicates that the strategy to first metalate the Fmoc-Lys(DOTA)-OH and to utilize the metal coordination to protect the carboxyl groups of DOTA offers an alternative to conventional synthetic routes using tert-butyl protected DOTA. Several small peptides containing up to four metal ions were synthesized, among them peptides carrying defined metal sequences consisting of two different lanthanides. The peptides were N-terminally maleimide-functionalized, thus introducing a moiety for conjugation to thiol-bearing biomolecules. The final objective of this work was the signal enhancement in ICP-MS-based DNA quantification assays. To evaluate the performance of the multimetal peptide tags in assay, they were applied to label thiol-modified 15mer DNA oligonucleotide probes. These served as reporter probes in a model sandwich-type hybridization assay. Thereby, we found that the ICP-MS signal increased linearly with the number of lanthanide ions attached to the reporter probe.
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