Bidirectional tandem pseudoproline ligations of proline-rich helical peptides

We have developed a bidirectional ligation strategy for preparing proline-r ich 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 a n amino terminal (NT)-Cys in forming a thiaproline bond over an NT-Ser or N T-Thr peptide in forming an oxaproline bond with a peptide that bears a car boxyl terminal (CT)-glycoaldehyde ester. Thus, successive ligations of thre e unprotected segments in a predetermined order formed a thiaproline and th en an oxaproline bond. However, ligation through the N-->C direction is fle xible. 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 anothe r CT-glycoaldehyde ester segment. Oxidative activation of the glycerol este r product to a CT-glycoaldehyde ester effected another round of pseudoproli ne ligation with an NT-Ser, NT-Thr, or NT-Cys segment. This sequential proc ess could be extended for ligating three or more segments. Optimized condit ions 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 2 1 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 activ ity similar to Bac 7.