BIOTINYLATION OF AN ENKEPHALIN-CONTAINING HEPTAPEPTIDE VIA VARIOUS SPACER ARMS - SYNTHESIS, COMPARATIVE BINDING-STUDIES TOWARD AVIDIN, AND APPLICATION AS SUBSTRATES IN ENZYMATIC-REACTIONS

The preparation of an enkephalin-containing heptapeptide of the sequence Tyr-Gly-Gly-Phe-Leu-Arg-Arg-OH with a biotinyl moiety linked to the carboxy terminus is described. A series of biotinylated derivatives, each containing a different linker (LC) moiety between the biotin function and the carboxy-terminal Arg residue, were synthesized by solution-phase chemistry following the coupling of the side chain protected peptide with previously prepared appropriate biotinylamine derivative. Both linear and flexible spacer arms of variable chain lengths [LC = (CH2)x, X = 2, 4, or 6] as well as semirigid cyclohexyl spacers (racemic 1,2-cyclohexane, cis or trans) were incorporated. The relative binding aptitudes of these molecules toward the glycoprotein, avidin, either in immobilized form or in solution were compared using both I-125-labeled and unlabeled peptide derivatives and were found to be in the following order, trans greater-than-or-equal-to 6C > 4C > 2C > cis. The potential application of these materials as substrates for enzymatic analysis is illustrated for one of the derivatives, namely the LC-2C analogue.