1. New chromogenic substrates of human neutrophil cathepsin G containing non-natural aromatic amino acid residues in position P(1) selected by combinatorial chemistry methods
Magdalena Wysocka, Anna Legowska, Elzbieta Bulak, Anna Jaśkiewicz, Hanna Miecznikowska, Adam Lesner, Krzysztf Rolka Mol Divers. 2007 May;11(2):93-9. doi: 10.1007/s11030-007-9063-7. Epub 2007 Jul 25.
Specificity of human cathepsin G was explored using combinatorial chemistry methods. Deconvolution of a tetrapeptide library, where 5-amino-2-nitrobenzoic acid served as a chromophore attached at the C-terminus, yielded the active sequence Phe-Val-Thr-Tyr-Anb(5,2)-NH(2). This sequence was used for a second-generation library with the general formula Ac-Phe-Val-Thr-X-Anb(5,2)-NH(2), where position X was replaced with several amino acids: L-pyridyl- alanine (Pal), 4-nitro-L-phenylalanine (Nif), 4-amino-L- phenylalanine (Amf), 4-carboxy-L-phenylalanine (Cbf), 4-guanidine-L-phenylalanine (Gnf), 4-methyloxycarbonyl- L-phenylalanine (Mcf), 4-cyano-L-phenylalanine (Cyf), Phe, Tyr, Arg and Lys. Specificity ligand parameters, k(cat) and K(M), with human cathepsin G were determined for all chromogenic substrates synthesized. The highest value of the specificity constant (k(cat)/K(M)) was obtained for a substrate with the Gnf residue in position P(1). This peptide was 10 times more active than the second most active substrate which contained the Amf residue. The following order of potency was established: Gnf > > Amf > Tyr = Phe > Arg= Lys > Cyf. Substrate specificity for cathepsin G is greatly enhanced when an aromatic side chain and a strong positive charge are incorporated in residue P(1).
2. The design, synthesis and activity of pentapeptide pp60c-src inhibitors containing L-phosphotyrosine mimics
J H Lai, T H Marsilje, S Choi, S A Nair, D G Hangauer J Pept Res. 1998 Apr;51(4):271-81. doi: 10.1111/j.1399-3011.1998.tb00424.x.
Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine and 4-carboxy-L-phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH2 (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH2. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH2 with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-L-phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-L-phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60c-src than the corresponding pentapeptide wherein X = L-phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.
