Solid phase synthesis of short peptide-based multimetal tags for biomolecule labeling.
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.
Department of Chemistry, Humboldt-Universitaet zu Berlin , Brook-Taylor-Strasse 2, 12489 Berlin, Germany.
Kathrin
K
Brückner
Robert
R
Zitterbart
Oliver
O
Seitz
Sebastian
S
Beck
Michael W
MW
Linscheid
D004247:DNA / Q000032:analysis*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D013058:Mass Spectrometry; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D060327:Solid-Phase Synthesis Techniques
D006573:Heterocyclic Compounds, 1-Ring; D028581:Lanthanoid Series Elements; D009942:Organometallic Compounds; D010455:Peptides; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D004247:DNA