Tritium Labelling of Novel Endomorphin Analogues Containing Unnatural alpha-and beta-Amino Acids

摘要

New endomorphin-2 analogues containing 2',6'-dimethyl-tyrosine (Dmt) or alicyclic-beta-amino acids ((1S,2R)-aminocyclopentanecarboxylic acid (Acpc) and (1S,2R) 2-aminocyclohexanecarboxylic acid (Achc)) were designed and synthesized to obtain more active and more selective mu-and 5-opioid receptor ligands with higher stability against proteolytic enzymes. The most promising analogues (Dmt-Pro-Phe-Phe-NH_2, Tyr-(1S,2R)Acpc-Phe-Phe-NH_2 and Tyr(1S,2R)Achc-Phe-Phe-NH_2) were labelled with tritium. First, we synthesized the precursor peptides (3',5'I_2Dmt-Pro-Phe-Phe-NH_2, Dmt-DPro-Phe-Phe-NH_2, Tyr-A(1S,2R)Acpc-Phe-Phe-NH_2 and Tyr-A(1S,2R)Achc-Phe-Phe-NH_2 by SPPS method. The dehydro-beta-amino acids and their Boc-protected derivatives were synthesized in racemic forms. The diastereomeric peptides were separated by HPLC. The configuration of the dehydro-beta-amino acids in the peptide isomer was determined by chiral TLC using enantiomeric standard. The appropriate precursor peptide isomer was used for the tritium labelling. The tritium labels were introduced in the different amino acids with saturation of the double bond in the peptides containing dehydro-proline or dehydro-beta-amino acids or dehalogenation of the peptide containing diiodo-dimethyl-tyrosine using tritium gas and PdO/BaSO_4 catalyst. The crude triti-ated peptides were purified by HPLC with radioactive detector. The specific radioactivities of the final products were 1.4-2.8 TBq/mmol. The novel labelled peptides have become useful tools for the opioid research in our laboratory.