title: Design, synthesis and structural determination of antimicrobial peptides and peptidomimetics
Creators: name: Personne, Hippolyte
Publisher: name: Universität Bern
Year: 2024
Subjects: subject: 540 Chemistry
subject: 570 Life sciences; biology

description: This thesis was guided by two major objectives. The first one, consisting in one global project, was the optimization and the structural determination of mixed-chirality α-helical antimicrobial peptides. To start with, I synthesized the fucosylated analogs of two antimicrobial peptides ln65 (homochiral L-) and ln69 (mixed-chirality) discovered in our group. The addition of the sugar moiety aimed to obtain their X-ray structures by co-crystallization with lectin LecB. Therefore, I resolved the X-ray structure of both fucosylated ln65 and dln69, the enantiomer of ln69, both showing a α-helical structure. This represents the first α-helix composed only of L- and D- natural amino acids. As ln69 showed reduced hemolysis but similar antimicrobial activity compared to ln65, I explored the diastereomeric chemical space of ln65 sequence by synthesizing 31 diastereomers. Through the biological assays and structural determination by circular dichroism, I highlighted a correlation between antimicrobial activity, hemolysis and α-helical structure. This study led to new potent and non-toxic mixed-chirality α-helical AMPs. The structural behaviour of these diastereomers was supported by X-ray crystallography and Molecular Dynamics. As a last investigation, I applied the ln69 chirality pattern to reported α-helical peptides. The stereochemical modifications led to a disappearance of membrane-disruptive ability, underlining the sequence dependence of stereochemical optimization.
The second objective was to contribute to improving solid-phase synthesis methodology for peptides and peptidomimetics. This objective contained two subprojects. In the first one, I addressed the question of substituting piperidine, which is toxic, expensive, and regulated due to its use in illegal drug manufacturing, by a more benign reagent for 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. We screened a broad range of bases and identified dipropylamine (DPA) as a potential new Fmoc deprotection agent to replace piperidine in the context of high-temperature Solid-Phase Peptide Synthesis. The use of DPA on aspartimide-prone sequences showed significant decrease of aspartimide and related byproducts formation. In addition, DPA gave high yields for the syntheses of challenging therapeutical peptides and peptide dendrimers. This new Fmoc removal agent is cheaper, less malodorous than piperidine and non-regulated.
In the second project, I investigated how to improve the synthesis of inverse polyamidoamine (i-PAMAM) dendrimers, a new type of dendritic molecules developed in our group. The synthesis of i-PAMAMs employed iterative peptide coupling and deprotection of the very expensive N,N-bis(3-fluorenylmethoxycarbonyl-aminopropyl)glycine building block. I designed a new method, inspired from the submonomer approach of peptoid synthesis in which dendrimer extension occurs by on-bead acylation of a free N-terminus with bromoacetic acid, followed by nucleophilic substitution of bromide by N,N-bis(3-trifluoroacetyl-aminopropyl)amine and finally trifluoroacetyl deprotection with aqueous piperidine. This method is less expensive and time-consuming compared to the previous i-PAMAMs synthesis. The effectiveness of this innovative synthesis was showcased through the successful obtention of i-PAMAMs of different sizes.