Toward new designed proteins derived from bovine pancreatic trypsin inhibitor (BPTI): Covalent cross-linking of two 'core modules' by oxime-forming ligation

A 25-residue disulfide-cross-linked peptide, termed 'oxidized core module' (OxCM), that includes essentially all of the secondary structural elements of bovine pancreatic trypsin inhibitor (BPTI) most refractory to hydrogen e xchange, was shown previously to favor nativelike P-sheet structure [Carull a, N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization o f a P-Hairpin Peptide That Represents a 'Core Module' of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 7927-7937]. The present work pre pares to explore the hypothesis that the energies of nativelike conformatio ns, relative to other possible conformations, could be decreased further by covalent linkage of two OxCMs. Optimized syntheses of six similar to 50-re sidue OxCM dimers are reported herein, featuring appropriate monomer modifi cations followed by oxime-forming ligation chemistry to create covalent cro ss-links at various positions and with differing lengths. Several side reac tions were recognized through this work, and modified procedures to lessen or mitigate their occurrence were developed. Particularly noteworthy, guani dine or area denaturants that were included as peptide-solubilizing compone nts for some reaction mixtures were proven to form adducts with glyoxylyl m oieties, thus affecting rates and outcomes. All six synthetic OxCM dimers w ere characterized by 1D H-1 NMR; three of them showed considerable chemical shift dispersion suggestive of self-association and mutual stabilization b etween the monomer units.