NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. II has been shown that this tripeptide Is a neutrophil chemoattractant. Exi sting data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In o rder to design inhibitors of this tripeptide chemoattractant that would blo ck neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptid e to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution a s a mixture of four isomers with different cis and trans conformations abou t the two X-proline amide bonds. The isomer with two trans conformations (t rans-trans) was the most dominant (41%) in aqueous solution. This was follo wed by the isomers with mixed cis and trans conformations (trans-cis, 26% a nd cis-trans. 20%). The isomer with two cis conformations (cis-cis) was the least Savored (13%). The populations of these these isomers were investiga ted in DMSO and they were similar to those reported in aqueous solutions ex cept that the ordering of the trans-cis and cis-trans isomers were reversed . NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measu rements as well as CD spectroscopy were used to demonstrate that the four i somers exist primarily in an extended conformation with little hydrogen bon ding. The available (NOE) information M as used with molecular dynamics cal culations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of pe ptide and nonpeptide inhibitors of the chemoattractant. (C) 2001 John Wiley & Sons, Inc.