Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment.

The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition 'click' chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the 'staples' as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere.

Improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative.

The lipophilic amino acid, (S)-2-aminoundecanoic acid, was synthesized and incorporated at a number of specific positions within the peptide sequence of anoplin. These lipophilic anoplin analogs showed to be more active against Escherichia coli and Staphylococcus aureus compared to native anoplin, while the EC50-value of hemolysis was at least one order of magnitude lower than the MIC values. This was in sharp contrast to the N-acylated anoplin derivative, where a gain in activity also led to a complete loss of selectivity. Thus, the incorporation of a lipophilic amino acid residue into anoplin enhanced the antimicrobial activity, while selectivity towards microbial membranes was retained.

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 µM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Efficient synthesis of protein mimics by sequential native chemical ligation.

Synthetic mimics of protein surfaces have the potential to become inhibitors of protein-protein interactions or even synthetic vaccines. However, the synthesis of these complicated molecular constructs is still difficult. Here we describe an efficient and versatile synthesis of protein mimics containing up to three different cyclic peptides. Using a sequential native chemical ligation strategy, peptide loops containing a thioester handle were introduced onto a triazacyclophane scaffold bearing orthogonal protected cysteine residues.

Convenient Preparation of Bactericidal Hydrogels by Covalent Attachment of Stabilized Antimicrobial Peptides Using Thiol-ene Click Chemistry.

This report describes the design and synthesis of a bactericidal poly(ethylene glycol)-based (PEG) hydrogel coating with covalently attached antimicrobial peptides (AMP) stabilized against proteolytic degradation. As such, mimics of the highly active AMP HHC10 (H-KRWWKWIRW-NH2) were designed for optimal stability in human serum while retaining strong antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis, the major causative agents of biomaterial associated infection. In order to investigate the selectivity of the AMPs, their hemolytic activity was determined. A N-terminal cysteine facilitated thiol-ene chemistry for a fast, single-step immobilization/photopolymerization strategy. The antimicrobial activity of the resulting thin layer hydrogel coating on a PET surface was established using the Japanese Industrial Standard (JIS) Z2801 assay, showing complete killing (>99.9%) of inocula of S. aureus ATCC 49230, S. epidermidis ATCC 35984, and E. coli ATCC 8739.