Peptides photoreactions

Peptidic photoprobes can be based on the photoreactive amino acid p-benzoyl-L-phenylalanine inserted into a peptide in place of a natural aromatic residue by peptide synthesis [65] or by manipulation of the genetic code [66]. The use of p-benzoyl-L-phenylalanine for this purpose is not new, but the nature of peptide probes naturally offers opportunities for the location of linkage sites by proteomic analysis [67]. 

In the chemical communication many peptide-protein (e.g., peptide hormone agonist) or protein-protein signaling interaction take place. Various photoreactive amino acids, e.g., azido-phenyalanines (Apa, TFApa) [40, 41], benzoyl-phenyalanines (Bpa,p-OH-Bpa) [41,42],trifluorometil-diazirine-phenylalanine (TMDPhe) [43], were developed (Fig. 3), which can be incorporated into any places in peptide sequences by standard solid-phase synthetic techniques. 

A systematic replacement of any amino acid in the sequence for photoreac-tive analogues allows a photoaffinity scanning of the binding interface. Since solid-phase synthesis is limited in the length of the peptide, Schultz et al. developed a sophisticated method which makes it possible to incorporate unnatural amino acids into large peptide sequences. The photoreactive amino acid was linked to transfer RNA, which inserted the amino acid into the required position by in vivo translation [44]. 

Parathyroid hormone (PTH) regulates calcium levels in blood and bone remodeling. The activation domain of that 84-amino acid polypeptide locates around the N-terminal (1-34 amino acids). Parathyroid hormone receptor is a typical G-protein coupled receptor, which is coupled to both adenyl cyclase/cAMP and PLCy/IP3/cytosolic Ca2+ intracellular signaling pathways. In order to identify the structural elements involved in the peptide hormone binding and signal initiation, Chorev et al. employed a photoaffinity scanning approach. The N-terminal amino acids were successively deleted or modified and the new N-terminus was replaced for photoreactive Bpa. The most active peptide ana-... 

Stony Brook synthesized a photoreactive benzophenone containing inhibitor (39, Fig. 14) which efficiently labeled the active site of the enzyme. The photoinhibition was prevented by adding native Ras to the reaction mixture. That competition indicated that the labeling was specific at the active site. Peptide mapping of the labeled enzyme by HPLC, Edman sequencing and MALDI-MS allowed the identification of key amino acids in the substrate binding, as Asp-110 and Asp-112 in the a-subunit [126]. 

There are two principal modes for introducing the photophore into a peptide (1) Coupling a photoreactive N-protected amino acid during the stepwise assembly of the peptide e.g. in Scheme 3, the amino acid 4-benzoylphenylalanine (3) is incorporated into position 8 of substance P (9). (2) Post-synthetic modification of a fully assembled peptide, at either a free N-terminal a-amino group or a side-chain functionality, by the photophore in a site-specific or nonspecific manner e.g. in Scheme 3, treatment of cyclic R-G-D-containing peptide 10 with 4-benzoylbenzoic anhydride (11) gives the modified peptide 12. 

Table 1 Photoreactive Peptides Modified with 4-Azido-Based Photophores 16-18-2234-283133-91 ...

Table 2 Photoreactive Peptides Modified with 4-Nitroaryl-Based Photophores116 25-27 34 4348 84100-1081...

Table 3 Photoreactive Peptides Modified with Benzenediazonium-Based Photophores 25-27-34115 ...

Table 4 Photoreactive Peptides Modified with 4-[3-(Trifluoromethyl)-3//-diazirin-3-yl]aryl-Based...

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