| The effect of secondary structure on the rate of asparagine (Asn) deamidation in the solid state was investigated using model beta-turn cyclic peptides. Two pairs of model peptides, each containing a cyclic beta-turn peptide and a linear analog, were used as models of Asn residues in structured and unstructured domains, and incorporated into poly (vinyl pyrrolidone) (PVP) based lyophilized solids. Structural characterization was performed in solution and the solid state using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy and solid-state NMR (SSNMR) spectroscopy. The model beta-turn cyclic peptides were found to be type II beta-turns and the linear analogs were determined to be predominantly unstructured. Quantitatively, the cyclic models were determined to be approximately 80% beta-turn and the linear analogs were found to be approximately 30% beta-turn. Accelerated stability studies were performed in the solid state and the effect of matrix mobility was investigated by changing the moisture content through variation of relative humidity or the addition of a plasticizer. The observed deamidation rate constants for the cyclic peptides were slower (1.2--8 times) than the linear analogs at all conditions studied, showing the importance of secondary structure. However, the effects of matrix mobility, as measured by the glass transition temperature (Tg), in controlling the reaction rate were greater and decreased the observed rate constant by 1 to 4 orders of magnitude. An unexpected result during accelerated stability studies was that, while both cyclic peptides were equivalent in type and amount of beta-turn, the observed deamidation rates differed by a factor of 2--3. Molecular dynamics (MD) simulations were performed for solutions of varying viscosity and the dihedral angles near the Asn residue and the C-N reaction distance were determined to assess peptide structure. The simulations showed a difference in turn type for the two cyclic peptides (type II vs. type II' ) that was not detected in the previous extensive spectroscopic studies, supporting the importance of subtle differences in secondary structure in determining deamidation rates. |
