P-(4-HYDROXYBENZOYL)PHENYLALANINE - A PHOTOREACTIVE AMINO-ACID ANALOGAMENABLE TO RADIOIODINATION FOR ELUCIDATION OF PEPTIDE-PROTEIN INTERACTION APPLICATION TO SUBSTANCE-P RECEPTOR

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with t heir receptors. This probe, however, lacks versatility because it is n ot detectable by Edman sequencing and because it cannot be labeled wit h radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radiois otope and photoaffinity labels along the primary sequence limits ident ification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesize d p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving rea ction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphor ic acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)- 4'-hydroxybenzophenone followed by reaction of the benzophenone deriva tive with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolab ile amino acid was incorporated into substance P (replacing Phe(8) or Lys(3)) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substa nce P analogs resulted in the incorporation of I-125 at the photoreact ive amino acid residue, yielding probes of high (similar to 2000 Ci/mm ol) specific activity. Subsequent photolysis of the radiolabeled pepti des in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approx imate to 30%); photolabeling was abolished in the presence of excess u nlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenyl alanine's high insertion yield and low reactivity with water, but in c ontrast allows placement of radioiodine and the photoactive moieties w ithin the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman se quencing. This novel amino acid may be useful in the elucidation of th e interaction of a variety of peptides with their receptors.