Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6

Abstract

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared “classical” fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the “classical” method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the “classical” SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a “working day” (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.

Dual FGFR-targeting and pH-activatable ruthenium–peptide conjugates for targeted therapy of breast cancer

“…The peptides were prepared on a rink amide resin by ultrasound-assisted solid-phase peptide synthesis (US-SPPS), according to our previously reported methodology. 50 Peptide P1 was N -functionalized with a ketone group by treating the resin with levulinic acid. Analogously, N -derivatization of P2 with the hydrazide group was performed in two steps, first by treating the resin with succinic anhydride, and then by reaction of the product with Fmoc-hydrazine, followed by final Fmoc deprotection.…”

“…This sequence is described to bind with high affinity and specificity to the extracellular domain of FGFR and is not toxic to the tumoral cells, being used in this approach only as a carrier. 49,50…”