Tritium Labeling of Neuromedin S by Conjugation with [3H] N-Succinimidyl Propionate
- PMID: 36687043
- PMCID: PMC9851027
- DOI: 10.1021/acsomega.2c06758
Tritium Labeling of Neuromedin S by Conjugation with [3H] N-Succinimidyl Propionate
Abstract
The human neuropeptide neuromedin S (NMS) consists of 33 amino acids. The introduction of tritium atoms into NMS has not been described so far. This represents a gap for using [3H]NMS in radioreceptor binding assays or in tracking and monitoring their metabolic pathway. Two approaches for the incorporation of tritium into NMS were explored in this study: (1) halogenation at the His-18 residue followed by catalyzed iodine-127/tritium exchange and (2) conjugation of tritiated N-succinimidyl-[2,3-3H3]propionate ([3H]NSP) to at least one of the three available primary amines of amino acids Ile-1, Lys-15, and Lys-16 in the peptide sequence. Although iodination of histidine was achieved, subsequent iodine-127/deuterium exchange was unsuccessful. Derivatization at the three possible amino positions in the peptide using nonradioactive NSP resulted in a mixture of unconjugated NSM and 1- to 3-conjugations at different amino acids in the peptide sequence. Each labeling position in the mixture was assigned following detailed LC-MS/MS analysis. After separating the mixture, it was shown in an in vitro fluorometric imaging plate reader (FLIPR) and in a competitive binding assay that the propionyl-modified NMS derivatives were comparable to the unlabeled NMS, regardless of the degree of labeling and the labeling position(s). A molecular simulation with NMS in the binding pocket of the protein neuromedin U receptor 2 (NMUR2) confirmed that the possible labeling positions are located outside the binding region of NMUR2. Tritium labeling was achieved at the N-terminal Ile-1 using [3H]NSP in 7% yield with a radiochemical purity of >95% and a molar activity of 90 Ci/mmol. This approach provides access to tritiated NMS and enables new investigations to characterize NMS or corresponding NMS ligands.
© 2023 The Authors. Published by American Chemical Society.
Conflict of interest statement
The authors declare the following competing financial interest(s): All authors are in paid employment by the company F. Hoffmann-La Roche AG during the completion of the study.
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