Bidirectional tandem pseudoproline ligations of proline-rich helical peptides

We have developed a bidirectional ligation strategy for preparing proline-rich peptides that couples three unprotected segments in tandem to form two pseudoproline bonds (thia- or oxaproline) without the need for a protection scheme. Ligation in the C-->N direction exploits the regioselectivity of an amino terminal (NT)-Cys in forming a thiaproline bond over an NT-Ser or NT-Thr peptide in forming an oxaproline bond with a peptide that bears a carboxyl terminal (CT)-glycoaldehyde ester. Thus, successive ligations of three unprotected segments in a predetermined order formed a thiaproline and then an oxaproline bond. However, ligation through the N-->C direction is flexible. An NT-Cys, NT-Ser, or NT-Thr segment bearing a CT-glycerol ester as a masked CT-glycoaldehyde was used to form a pseudoproline bond with another CT-glycoaldehyde ester segment. Oxidative activation of the glycerol ester product to a CT-glycoaldehyde ester effected another round of pseudoproline ligation with an NT-Ser, NT-Thr, or NT-Cys segment. This sequential process could be extended for ligating three or more segments. Optimized conditions for this bidirectional strategy were applied successfully to syntheses of five analogues of a proline-rich helical antimicrobial peptide, the 59-residue bactenecin 7 (Bac 7), using three segments containing 24, 14, and 21 amino acids, respectively. CD spectra showed that Pac 7 and its analogues displayed typical polyproline II helical structures in phosphate buffers. Furthermore, the psi Pro-containing analogues exhibited antibacterial activity similar to Bac 7.