Synthesis of cyclic tetrapeptides via ligation of peptide hydrazides

Abstract

Naturally occurring or man-made cyclic tetrapeptides have unique rigid skeletons and therefore, represent an interesting class of candidate bioactive molecules in drug discovery. However, efficient chemical synthesis of cyclic tetrapeptides often presents a difficult problem due to the large strain involved in this category of target compounds. To overcome this problem we describe in the present study the use of peptide hydrazides for the preparation of highly strained 12-membered all-L cyclic tetrapeptides. The new synthetic route starts with the easy Fmoc solid phase synthesis of a linear N-Cys-tetrapeptide hydrazide precursor. Upon quick activation by NaNO2 at pH 3 and -10°C for 20 min followed by treatment with a pH 7 buffer containing an external thiol (4-mercaptophenylacetic acid, 40 equiv.) at room temperature, the N-Cys-tetrapeptide hydrazide precursor is converted in situ to an N-Cys-tetrapeptide thioester. This thioester undergoes a fast intramolecular thioester exchange reaction to generate a 13-membered thiolactone intermediate. Then an S-to-N acyl shift is expected to take place to create an amide bond, which affords the desired cyclic tetrapeptide in a modest overall yield (ca. 40%). Finally, desulfurization of the cyclic peptide product can be carried out to produce the target cyclic tetrapeptide that does not contain any Cys residue. Through detailed 1H, 13C, and TOSCY NMR and HPLC analyses, it is found that the new method for tetrapeptide synthesis can be carried out at relatively high substrate concentrations (0.8-1.0 mmol/L) without causing the formation of much cyclic octapeptide byproduct. Our test also showed that the reaction can generate the desired cyclic tetrapeptide in an epimerization free manner. By using the new method we have successfully prepared several cyclic tetrapeptides including cyclo(Ala-Leu-Ala-Leu), cyclo(Ala-His-Gly-Trp), and cyclo(Ala-Val-Gly-Ile) in 18%-25% overall yields. We expect that our method of cyclic tetrapeptide synthesis may find applications in the development of cyclic peptide libraries for bioactivity screening. © 2012 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.