Fluorescent pseudo-peptide linear vasopressin antagonists: Design, synthesis, and applications

Fluoresceinyl and rhodamyl groups have been coupled by an amide link to side-chain amino groups at positions 1, 6, and 8 of pseudo-peptide linear vasopressin antagonists (Manning et al. Int. J. Pept. Protein Res. 1992, 40, 261-267) through different positions on the fluorophore, to give tetraethylrhodamyl-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (2), 4- HOPh(CH2)2-CO-DTyr(Me)-Phe-Gln-Asn-Lys(5-carboxyfluoresceinyl)-Pro-Arg- NH2 (4), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5- or 6- carboxytetramethylrhodamyl)-Pro-Arg-NH2 (5, 6), 4-HOPh-(CH2)2CO-DTyr(Me)- Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxyfluoresceinyl)-NH2 (8, 9), and 4- HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxytetramethylrhodamyl)-NH2 (10, 11). The closer to the C-terminus the fluorophore, the higher the affinities of the fluorescent derivatives for the human vasopressin V(1a) receptor transfected in CHO cells. The compound 10 has a K(i) of 70 pM, as determined by competition experiments with [125I]- 4-HOPh-CH2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2. It showed a good selectivity for human V(1a) receptor versus human OT (K(i) = 1.2 nM), human vasopressin V(1b) (K(i) approximately 27 nM), and human vasopressin V2 (K(i) > 5000 nM) receptor subtypes. All fluorescent analogues were antagonists as shown by the inhibition of vasopressin induced inositol phosphate accumulation. These fluorescent ligands are efficient for labeling cells expressing the human V(1a) receptor subtype, as shown by flow cytofluorometric experiments or fluorescence microscopy. They are also appropriate tools for structural analysis of the vasopressin receptors by fluorescence.