Traceless parallel peptide purification by a first-in-class reductively cleavable linker system featuring a safety-release

Abstract

Hundreds of peptides can be synthesized by automated parallel synthesizers in a single run. In contrast, the most widely used peptide purification method - high-pressure liquid chromatography (HPLC) - only allows one-by-one processing of each sample. The chromatographic purification of many peptides, therefore, remains a time-consuming and costly effort. Catch-and-release methods can be processed in parallel and potentially provide a remedy. However, no such system has yet provided a true alternative to HPLC. Herein we present the development of a side-reaction free, reductively cleavable linker. The linker is added to the target peptide as the last building block during peptide synthesis. After acidic cleavage from synthetic resin, the linker-tagged full-length peptide is caught onto an aldehyde-modified solid support by rapid oxime ligation, allowing removal of all impurities lacking the linker by washing. Reducing the aryl azide to an aniline sensitizes the linker for cleavage. However, scission does not occur at non-acidic pH enabling wash out of reducing agent. Final acidic treatment safely liberates the peptide by an acid-catalysed 1,6-elimination. We showcase this first-in-class reductively cleavable linker system in the parallel purification of a personalized neoantigen cocktail, containing 20 peptides for cancer immunotherapy within six hours.

Scheme 1. (a) General scheme of oxime-based c&r purification of peptides. (b) Molecular structure of previously reported base-labile linker molecules 1a, 1b and (c) of the novel reductively cleavable linker units 2a, 2b and 2c described in this paper. aa: amino acid; PG: protecting group; MA: modified agarose beads; L_CU: cleavable linker unit; LG: leaving group.

Scheme 2. (a) Application of reductively cleavable linker 2a, b, c in c&r purification of peptides with PPh₃ as reductive stimulus; MA: modified agarose beads, GdmCl: Guanidinium chloride. (b) Usage of dithiothreitol (DTT) as reductive stimulus, *: linker-modified peptide in solution.

Fig. 1. PEC-purification of base-sensitive peptides P1–3 and Histone H3 (1–15) peptide P4. (i) Trial cleavage of peptide before linker coupling; (ii) full cleavage after linker coupling of 1a or 2a, (iii) after PEC-process using 1a or 2a. *Assigned masses of co-eluting truncated peptides in P4 trial cleavage; red traces using base-labile 1a and black traces using reductively cleavable linker 2a.

Fig. 2. (a) Generation of propylamine carbamates 9a–c from 2a–c. (b) TFA-stability analysis of carbamates 9a–c relative to the sum of UV-UPLC integrals of TFA-cleavage products 11. reagent K: TFA/H₂O/PhOH/PhSMe/ethanedithiol 82.5 : 5 : 5 : 5 : 2.4.

Fig. 3. Amount of released peptide P5 in DTT supernatant (undesired) and after DTT wash-out in 5% aq. TFA supernatant (desired) calculated by the UV(210 nm) integral of product peak relative to the sum of DTT and TFA released peptide from 6a.

Fig. 4. (a) Box-diagram of crude vs. final purities of the 20 neoantigen peptides. IQR: interquartile range. (b) UPLC chromatograms before (i) and after the PEC process (ii) of peptide P14 from a personalized peptide set of 20 peptides purified in parallel.