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

Abstract

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 oxytocin, vasopressin, and octreotide are 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.

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale

“…We next assessed whether the procedure for picomole-scale synthesis was suitable for generating a large macrocycle library. We synthesized 384 randomly selected cyclic peptide scaffolds that contained a peripheral amino group, using a recently developed approach for the production of small cyclic peptides in 96-well plates 31 . In brief, we synthesized short peptides on solid phase via a disulfide linker, removed the protecting groups, and released the peptide via a cyclative reaction, which yielded disulfide-cyclized peptides with 90% or greater purity (Fig.…”

“…The cyclic peptide model scaffold 1 was synthesized using the cyclative disulfide release strategy (CDR) described in the paper Habeshian, S. et al 2022 31 . The linear peptide precursor was synthesized on a 25 μmol scale in a 5 mL polypropylene synthesis column (MultiSyntech GmbH, V051PE076) using Rapp Polymere HA40004.0 Polystyrene A SH resin (200-400 mesh), 0.95 mmol/gram loading resin and following the procedure described in Habeshian, S. et al 2022 31 . The peptide was released as follows.…”

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

“…7 Our laboratory has recently developed a strategy for efficiently accessing disulfide-cyclized peptides by synthesizing peptides via a disulfide linker on solid phase and releasing them via a cyclative disulfide exchange reaction. 8…”

“…In order to omit peptide purification after SPPS, strategies were developed in which thiol-functionalized peptides are deprotected while still linked to the solid phase, allowing washing away the protecting groups and releasing peptides in a rather pure form. [6][7][8] Tegge and co-workers synthesized cyclic peptides on a solid support via a disulfide linker and released them by reducing the disulfide bond by dithiothreitol (DTT). 6 Gless and Olsen produced thiol-functionalized cyclic peptides applying Dawson's 3-amino-4-(methylamino)benzoic acid (MeDbz) linker 9 which yielded unprotected thioester peptides that were cyclized by native chemical ligation.…”

“…7 Our laboratory has recently developed a strategy for efficiently accessing disulfidecyclized peptides by synthesizing peptides via a disulfide linker on solid phase and releasing them via a cyclative disulfide exchange reaction. 8 In peptides produced with all the described strategies, the thiol groups tend to oxidize during the production or storage, or are fully oxidized such as in the cyclative release approach, and thus require to be treated with reducing agents prior to conjugation reactions. The reducing agents applied for breaking the disulfide bridges, however, require to be removed afterwards as they would interfere with the chemical reactions, and this involves a cumbersome purification step.…”