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

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.

Methods for the generation of large combinatorial macrocycle libraries

Sevan Mleh Habeshian

Macrocycles are an attractive class of molecules due to their good binding properties and yet rather small size that allows, in many cases, crossing membranes to reach intracellular targets. In comparison to classical small molecule drugs, macrocycles are generally better at binding to challenging targets such as proteins having flat, featureless surfaces or protein-protein interactions. However, the development of macrocycle-based ligands has been challenging due to the lack of sufficiently large libraries of macrocyclic compounds for screening. The preparation of large numbers of macrocycles is limited due to the chromatographic purification that is typically required following low-yielding cyclization reactions. As a result, the full potential of macrocycles cannot be exploited in drug discovery efforts.The goal of my thesis was to develop new methods for the efficient generation of large macrocycle libraries. Towards this end, I envisioned to establish a new macrocycle library synthesis principle in which a large panel of m small cyclic peptides are combinatorially ligated in microwell plates with a large number of n chemical fragments, and the resulting m x n products screened without purification. For accessing large libraries, I planned to perform the reactions and screens at a nanomole scale and using contact-less liquid transfer.In a first project, I developed a method for obtaining readily pure macrocyclic peptides directly from solid phase. Peptide synthesis took place on a disulfide linker to solid phase, which allowed for side chain deprotection with acid without simultaneous peptide cleavage. When a thiol group was included at the N-terminus, treatment with base in DMSO resulted in an intramolecular disulfide exchange to release peptides cyclized by a disulfide bond. The method was tolerant of diverse peptide sequences, and released all tested compounds in high purity. With this method, I was able to synthesize cyclic peptides in 96-well plates, and thus hundreds of peptides in parallel with a moderate effort. I screened a test library and identified a weak inhibitor of thrombin (Ki = 13 ± 1 µM), which validated the method. In the second project, I intended to combinatorially diversify the above macrocyclic peptides with chemical fragments in nanoliter volumes, as described above. For chemical ligation, I chose to acylate amines built into the macrocyclic peptides with carboxylic acid building blocks. Following proof-of-concept experiments, I diversified a 384-member library with 10 carboxylic acids in nanoliter volumes to give 3,840 macrocyclic compounds, which I screened against thrombin. I identified a potent thrombin inhibitor (Ki = 44 ± 1 nM), and a co-crystal structure was obtained which confirmed binding to the active site of the protease. In a final application, I generated and screened a library of 19,968 macrocycles against MDM2, an important oncology target forming a protein-protein interaction with p53. The screen identified a high nanomolar binder (KD = 394 ± 41 nM) that I optimized in two rounds of iterative library synthesis and screening (KD = 31 ± 6 nM).The methods developed in the two projects are currently applied by our laboratory, and for the development of macrocycle-based ligands to diverse disease targets.

EPFL2021

Synthesis of DNA‐encoded disulfide‐ and thioether‐cyclized peptides

Christian Heinis, Van Manh Pham

DNA-encoded chemical library technologies enable the screening of large combinatorial libraries of chemically and structurally diverse molecules, including short cyclic peptides. A challenge in the combinatorial synthesis of cyclic peptides is the final step, the cyclization of linear peptides that typically suffers from incomplete reactions and large variability between substrates. Several efficient peptide cyclization strategies rely on the modification of thiol groups, such as the formation of disulfide or thioether bonds between cysteines. In this work, we established a strategy and reaction conditions for the efficient chemical synthesis of cyclic peptide-DNA conjugates based on linking the side chains of cysteines. We tested two different thiol-protecting groups and found that tert-butylthio (S-tBu) works best for incorporating a pair of cysteines, and we show that the DNA-linked peptides can be efficiently cyclized through disulfide and thioether bond formation. In combination with established procedures for DNA encoding, the strategy for incorporation of cysteines may be readily applied for the generation and screening of disulfide- and thioether-cyclized peptide libraries.

2019

Structurally Diverse Cyclisation Linkers Impose Different Backbone Conformations in Bicyclic Peptides

Alessandro Angelini, Shiyu Chen, Christian Heinis, Julia Morales Sanfrutos

Combinatorial libraries of structurally diverse peptide macrocycles offer a rich source for the development of high-affinity ligands to targets of interest. In this work we have developed linkers for the generation of genetically encoded bicyclic peptides and tested whether the peptides cyclised by them have significant variations in their backbone conformations. Two new cyclisation reagents, each containing three thiol-reactive groups, efficiently and selectively cyclised linear peptides containing three cysteine moieties. When the mesitylene linker of the bicyclic peptide PK15, a potent inhibitor of plasma kallikrein (Ki=2 nM), was replaced by the new linkers, its inhibitory activity dropped by a factor of more than 1000, suggesting that the linkers impose different conformations on the peptide. Indeed, structural analysis by solution-state NMR revealed different NOE constraints in the three bicyclic peptides, indicating that these relatively small linkers at the centres of bicyclic peptide structures significantly influence the conformations of the peptides. These results demonstrate the prominent structural role of linkers in peptide macrocycles and suggest that application of different cyclisation linkers in a combinatorial fashion could be an attractive means to generate topologically diverse macrocycle libraries.

2012

Methods for the generation of large combinatorial macrocycle libraries

Sevan Mleh Habeshian

EPFL2021