High-Density Immobilization of TCEP on Silica Beads for Efficient Disulfide Reduction and Thiol Alkylation in Peptides

Chembiochem. 2023 Dec 4:e202300592. doi: 10.1002/cbic.202300592. Online ahead of print.ABSTRACTTris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15 μmol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129±16 μmol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.PMID:38047532 | DOI:10.1002/cbic.202300592

Source: Chembiochem - December 4, 2023 Category: Biochemistry Authors: Mischa Sch üttel Christian Heinis Source Type: research