Solution stability of linear vs. cyclic RGD peptides

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological act ivity. Our hypothesis is that cyclization of RGD peptides via disulphide bo nd linkage can induce structural rigidity, thereby preventing degradation m ediated by the aspartic acid residue. In this paper, we compared the soluti on stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic pepti de (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2, 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in bu ffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation Fates and pathways of both pe ptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ioni zation mass spectrometry (ESI-MS), matrix-assisted laser desorption/ ioniza tion-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-ma ss spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic reso nance spectroscopy (NMR) were used to characterize peptides 1 and 2 and the ir degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-firs t-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the asp artic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides a lso exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, es pecially at neutral pH, may be due to decreased structural flexibility impo sed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.