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Cyclative Release Strategy to Obtain Pure Cyclic Peptides Directly from the Solid Phase

Sevan Mleh Habeshian, Christian Heinis, Manuel Leonardo Merz, Ganesh Ashokrao Sable

The synthesis of large numbers of cyclic peptides-required, for example, in screens for drug development-is currently limited by the need of chromatographic purification of individual peptides. Herein, we have developed a strategy in which cyclic peptides are released from the solid phase in the pure form and do not need purification. Peptides with an N-terminal thiol group are synthesized on the solid phase via a C-terminal disulfide linker, their sidechain-protecting groups are removed while the peptides remain on the solid phase, and the peptides are finally released via a cyclative mechanism by the addition of a base that deprotonates the N-terminal thiol group and triggers an intramolecular disulfide-exchange reaction. The method yields disulfide-cyclized peptides, a format on which many important peptide drugs such as o based. We demonstrate that the method is applicable for facile synthesis in 96-well plates and allows for synthesis and screening of hundreds of cyclic peptides.

AMER CHEMICAL SOC2022

De novo developed small cyclic peptides that are orally available

Manuel Leonardo Merz

Cyclic peptides are ring-shaped molecules that emerged as a promising class of therapeutics. While it is often difficult to find small molecule binders for challenging disease targets, cyclic peptides can bind to featureless surfaces or inhibit protein-protein interactions. However, due to their typically large size and polar surface area, most peptides have a limited membrane permeability which restricts them to inhibiting extracellular targets and prevents an oral administration. The few peptides that are orally bioavailable generally have a molecular weight being below 1 kDa, five or less hydrogen-bond donors, and a polar surface area smaller than 200 Ã2, which indicates the requirements for cyclic peptides to be orally available. The de novo development of cyclic peptides that bind to new disease targets and that fulfill those requirements is challenging due to the lack of sufficiently large libraries that could be screened. A limiting factor in accessing such libraries is the low synthetic throughput associated with challenging cyclization reactions and time-consuming chromatographic purifications.The overall goal of my thesis was to establish a procedure that enables de novo development of biologically active cyclic peptides that can be administrated orally. Towards this aim, I build onto the nanomole-scale cyclic peptide synthesis approach established in our group and implemented a dithiol cyclization with bis-electrophilic crosslinkers. Using a two-step combinatorial synthesis, I cyclized 384 peptide sequences with 2 crosslinkers and diversified a peripheral amino group via N-acylation with 11 carboxylic acids to generate a library of 8,448 cyclic peptides. This approach implements acoustic droplet ejection technology (ADE) for combinatorial dispending of nanoliter droplet of the reactants. Screening of the 8,448-member cyclic peptide library against human ï¡-thrombin identified a double digit nanomolar inhibitor with a molecular weight of 700 Da. I then applied the approach to improve the membrane permeability and metabolic stability in iterative synthesis and screening cycles. This resulted in the de novo development of a cyclic peptide that inhibits human ï¡-thrombin with nanomolar affinity (Ki = 65 nM) and has an oral bioavailability of 18% in rats.The automated nanoscale-synthesis on the acoustic dispenser can rapidly generate large and diverse libraries that may even develop ligands for challenging targets such as protein-protein interactions. The established method is general and may be applied for generating orally bioavailable peptide ligands for many other targets.

EPFL2022

Solid-phase peptide synthesis on disulfide-linker resin followed by reductive release affords pure thiol-functionalized peptides

Zsolt Bognár, Christian Heinis, Manuel Leonardo Merz, Ganesh Kumar Mothukuri, Alexander Lund Nielsen, Peter Matei Frisius Pânzar

Thiol groups are suitable handles for site-selectively modifying, immobilizing or cyclizing individual peptides or entire peptide libraries. A limiting step in producing the thiol-functionalized peptides is the chromatographic purification, which is particularly laborious and costly if many peptides or even large libraries are to be produced. Herein, we present a strategy in which thiol-functionalized peptides are obtained in >90% purity and free of reducing agent, without a single chromatographic purification step. In brief, peptides are synthesized on a solid support linked via a disulfide bridge, the side-chain protecting groups are eliminated and washed away while the peptides remain on resin, and rather pure peptides are released from the solid support by reductive cleavage of the disulfide linker. Application of a volatile reducing agent, 1,4-butanedithiol (BDT), enabled removal of the agent by evaporation. We demonstrate that the approach is suited for the parallel synthesis of many peptides and that peptides containing a second thiol group can directly be cyclized by bis-electrophilic alkylating reagents for producing libraries of cyclic peptides.

ROYAL SOC CHEMISTRY2022

De novo developed small cyclic peptides that are orally available

Manuel Leonardo Merz

EPFL2022