Synthesis and bioactivity of monobiotinylated DALDA: a mu-specific opioid peptide designed for targeted brain delivery.

Delivery through the blood-brain barrier of opioid peptide-based therapeutic agents may be achieved with the use of conjugation of avidin and blood-brain barrier transport vectors. However, this drug delivery strategy requires that 1) the peptide is monobiotinylated and 2) the peptide is biologically active after cleavage of a disulfide linker and peptide release from the avidin-vector conjugate. Whether these criteria may be successfully fulfilled was examined in the present studies. The highly mu receptor-specific dermorphin analog, Tyr-D-Arg-Phe-Lys-NH2 (DALDA), was selectively monobiotinylated at the epsilon-NH2 group of Lys4 with the cleavable biotin linker, sulfosuccinimidyl-2-(biotinamidoethyl) 1,3'-dithioproprionate to obtain biotinylated DALDA (bio-DALDA). The N-terminal alpha-NH2 group of the peptide was protected during biotinylation with the N-9-fluorenylmethoxycarbonyl group. Cleavage of the disulfide bridge yielded the desbiotinylated derivative, desbio-DALDA. The identity of these peptides was verified by secondary ion mass spectrometry. In receptor binding assays with 3H-Tyr-D-Ala-Gly-Phe-(N-Me)-Gly-ol, the Kis of DALDA, bio-DALDA and desbio-DALDA for mu opioid receptors were determined to be 2.3 +/- 0.4, 6.5 +/- 1.1 and 4.0 +/- 0.9 nM, respectively. Binding of bio-DALDA to avidin resulted in a Ki of 14.5 +/- 2.4 nM. The i.c.v. administration of DALDA and desbio-DALDA induced potent and long-lasting analgesia in the rat tail-flick assay. It was found that 1 microgram of DALDA was equipotent to 3 micrograms of desbio-DALDA and 20 micrograms of morphine. The analgesic effect could be blocked by naloxone pretreatment. In conclusion, these studies 1) described methods for the preparation of a biologically active monobiotinylated mu opioid receptor-specific ligand and 2) demonstrated the advantages of using cleavable biotinylation of opioid peptides because the affinity of desbio-DALDA for the receptor approximated the affinity of DALDA and had a 3- to 4-fold higher affinity than did the bio-DALDA-avidin complex.