p-(4-Hydroxybenzoyl)phenylalanine: A Photoreactive Amino Acid Analog Amenable to Radioiodination for Elucidation of Peptide-Protein Interaction Application to Substance P Receptor
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- 作者：Carol J. Wilson ; S. Shaukat Husain ; Evelyn R. Stimson ; Lawrence J. Dangott ; Keith W. Miller ; and John E. Maggio
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：April 15, 1997
- 年：1997
- 卷：36
- 期：15
- 页码：4542 - 4551
- 全文大小：192K
- 年卷期：v.36,no.15(April 15, 1997)
- ISSN：1520-4995
文摘
Benzoylphenylalanine, a photoreactive phenylalanine analog thatcan be incorporated into apeptide during solid-phase synthesis, is a useful probe forinvestigating the interactions of bioactive peptideswith their receptors. This probe, however, lacks versatilitybecause it is not detectable by Edman sequencingand because it cannot be labeled with radioiodine, requiringradiolabeling of the peptide ligand at a sitedistal to the photoreactive amino acid. The separation of theradioisotope and photoaffinity labels alongthe primary sequence limits identification of the photoinsertion siteto a peptide fragment rather than aspecific amino acid of the receptor protein. We have nowsynthesized p-(4-hydroxybenzoyl)phenylalanineby a synthetic route involving reaction of 4-(chloromethyl)benzoicanhydride with phenol in polyphosphoricacid to give the 4-(chloromethyl)benzoyl ester of4-(chloromethyl)-4'-hydroxybenzophenone followed byreaction of the benzophenone derivative with ethylacetamidocyanoacetate and subsequent hydrolysis ofthe product to give p-(4-hydroxybenzoyl)phenylalanine.The novel photolabile amino acid was incorporatedinto substance P (replacing Phe8 or Lys3) togive 11-mer peptides that bind with high (nM) affinity andspecificity to the substance P receptor. Radioiodination of thesubstance P analogs resulted in theincorporation of 125I at the photoreactive amino acidresidue, yielding probes of high (~2000 Ci/mmol)specific activity. Subsequent photolysis of the radiolabeledpeptides in the presence of substance P receptorcaused covalent attachment of the peptide to the receptor with highphotoinsertion yield (
30%);photolabeling was abolished in the presence of excess unlabeled SP.p-(4-Hydroxybenzoyl)phenylalanineretains p-benzoylphenylalanine's high insertion yield andlow reactivity with water, but in contrast allowsplacement of radioiodine and the photoactive moieties within the sameresidue, providing the ability toidentify the specific site(s) of interaction, and identification of theresidue by Edman sequencing. Thisnovel amino acid may be useful in the elucidation of the interaction ofa variety of peptides with theirreceptors.
30%);photolabeling was abolished in the presence of excess unlabeled SP.p-(4-Hydroxybenzoyl)phenylalanineretains p-benzoylphenylalanine's high insertion yield andlow reactivity with water, but in contrast allowsplacement of radioiodine and the photoactive moieties within the sameresidue, providing the ability toidentify the specific site(s) of interaction, and identification of theresidue by Edman sequencing. Thisnovel amino acid may be useful in the elucidation of the interaction ofa variety of peptides with theirreceptors.