Peptide Synthesis

The synthesis of cyclic peptides 2605-4 and 2612-8.1 was performed following the strategy described by Bionda and Cudic (2011). The 2605-4 peptide (Figure 1A) macrocycle is composed of Fmoc-D-aspartic acid α-allyl ester, Fmoc-D-homophenylalanine, Fmoc-L-homophenylalanine, Fmoc-D-homophenylalanine, N^α –Fmoc-N^β -4-methyltrityl-L-2,3-diaminopropionic acid and N^α -Fmoc-N^ω -(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-D-arginine (Chemimpex), respectively. The Fmoc-D-aspartic acid α-allyl ester, Fmoc-D-homophenylalanine, Fmoc-L-homophenylalanine and Fmoc-D-homophenylalanine, N^α –Fmoc-N^β -4-methyltrityl -L-2,3-diaminopropionic acid were added sequentially onto the solid support Tentagel XV RAM resin (RAPP Polymere) followed by addition of the Fmoc-12-aminododecanoic acid (Chemimpex) using standard Fmoc chemistry. The Fmoc-12-amino dodecanoic acid tail was then coupled with 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine with 4 equivalents of triethylamine (TEA) in dichloromethane (DCM) to cap the lipid tail. Following the addition of the guanidine cap, the 4-methyltrityl (Mtt) protecting group from the diaminopropionic acid residue was cleaved using 1% trifluoroacetic acid (TFA)/3% triisopropylsilane (TIS)/96% DCM and the last residue (N^α -Fmoc-N^ω -(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-D-arg ini ne) is coupled. Once the last residue was coupled onto the solid support, the allyl ester protecting group was cleaved by using 3 equivalents of palladium-tetrakis(triphenylphosphine) in a solution of DCM: acetic acid: N-methylmorpholine (20:2:1) volumetric ratio. Post removal of the allyl protecting group, the Fmoc protecting group from the D-arginine residue was removed using a solution of 20% piperidine/DMF for 10 min (repeated twice) and the cyclization of the lipopeptide was completed on solid support using (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIEA) in dimethylformamide (DMF). Following overnight cyclization, the bags were rinsed with anhydrous DMF and DCM and dried under vacuum. The final cyclic lipopeptide (CLP) was cleaved from the resin using 88% TFA/6% water/6% TIS. The cleaved oligomers were then transferred to scintillation vials, frozen and lyophilized. Compounds were then reconstituted in 50% acetonitrile and water, frozen and lyophilized three more times.

Structures of cyclic lipopeptides: (A) 2605-4 (B) 2612-8.1.

The 2612-8.1 macrocycle is composed of Fmoc-D-aspartic acid α-allyl ester, Fmoc-D-tryptophan, Fmoc-L-tryptophan, Fmoc-L-leucine, N^α –Fmoc-N^β -4-methyltrityl-L-2,3-diaminopropionic acid and N^α -Fmoc-N^ω -(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-D-argin ine (Chemimpex), respectively. The synthesis of 2612-8.1 (Figure 1B) involved sequential insertion of two additional exocyclic residues comprising of N^α -Fmoc-N^γ -Boc-L-2,4-diaminobutyric acid (linker), between the macrocycle and the Fmoc-12-amino dodecanoic acid using standard Fmoc chemistry. Post addition of the Fmoc-12-amino dodecanoic acid similar methodology (mentioned above) was used to complete the capping of the C-12 tail with guanidine functional group, followed by Mtt removal, addition of N^α -Fmoc-N^ω -(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-D-arginine, allyl deprotection, Fmoc deprotection and lastly cyclization. The CLP was then cleaved from the solid support following identical strategy as above and subjected to 3 rounds of lyophilization. Purity and identity of the peptides were also verified by HPLC, LC-MS and MALDI-TOF (Supplementary Data).