A two-component 'double-click' approach to peptide stapling

Abstract

Peptide cyclization is a useful strategy for the stabilization of short flexible peptides into well-defined bioactive conformations, thereby enhancing their ability to interact with proteins and other important biomolecules. We present an optimized procedure for the stabilization of linear diazido peptides in an α-helical conformation upon reaction with dialkynyl linkers under Cu(I) catalysis. As this procedure generates side chain-cyclized peptides bearing a bis-triazole linkage, it is referred to as 'double-click' stapling. Double-click stapling can enhance the binding affinity, proteolytic stability and cellular activity of a peptide inhibitor. A distinguishing feature of double-click stapling is the efficiency with which peptides bearing different staple linkages can be synthesized, thus allowing for modular control over peptide bioactivity. This protocol describes the double-click reaction between a 1,3-dialkynylbenzene linker and peptides that contain azidoornithine. Subsequent peptide purification and confirmation steps are also described. The entire double-click stapling protocol can be completed in ∼48 h, including two overnight lyophilization steps.

Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

“…Of particular note are modular two-component strategies, where the staple linkage and peptide are separate moieties prior to cyclisation (Figure 1). [14] We recently developed a two-component stapling technique that makes use of double Cu-catalyzed azide–alkyne cycloaddition (CuAAC),[7, 17, 18] following related work by Bong[19] and other click systems. [20–22] Combining a two-component approach with click chemistry enables easy access to peptides bearing different functional staples.…”

Bridged Analogues for p53-Dependent Cancer Therapy Obtained by S-Alkylation

“…Remaining residue was purified on RP-HPLC. Obtained ArB-alkyne derivative was then “clicked” with AZT using published protocol (Lau et al, 2015b) using CuSO 4 /sodium ascorbate catalyst in 50% tert-buthanol in water. Subsequently, reaction mixture was freeze-dried and purified using RP-HPLC.…”

“…These include hydrazone bridge (Cabezas and Satterthwait, 1999), oxime bridge (Haney et al, 2011), 1,4-disubstituted-[1,2,3]-triazole linkage (Holland-Nell and Meldal, 2011; Ingale and Dawson, 2011; Kawamoto et al, 2012; Lau et al, 2014c; Lau et al, 2014b; Lau et al, 2014a; Lau et al, 2015b; Scrima et al, 2010), metal chelation (Ghadiri and Choi, 1990; Ruan et al, 1990), disulfide bond formation (Almeida et al, 2012; Jackson et al, 1991; Leduc et al, 2003), lactam ring formation (Fujimoto et al, 2008; Geistlinger and Guy, 2001; Geistlinger and Guy, 2003; Houston, Jr. et al, 1995; Osapay and Taylor, 1992; Phelan et al, 1997) and S-alkylation based staples employing either α-haloacetamide alkylation of single cysteine (Brunel and Dawson, 2005; Cardoso et al, 2007; Galande et al, 2004; Woolley, 2005) or bridging two cysteines with bis-S-alkylating linker(s) (de Araujo et al, 2014; Jo et al, 2012; Muppidi et al, 2011b; Muppidi et al, 2011a; Muppidi et al, 2012; Spokoyny et al, 2013; Szewczuk et al, 1992; Timmerman et al, 2005; Wilkinson et al, 2007; Zhang et al, 2007; Zhang et al, 2008). Among these, the last seems to be most flexible approach as a wide range of inexpensive bis-thiol-reactive linkers is commercially available, including rigid aromatic derivatives (Chua et al, 2015; Jo et al, 2012; Muppidi et al, 2011b; Muppidi et al, 2011a; Muppidi et al, 2012; Timmerman et al, 2005; Zhang et al, 2007) and aliphatic counterparts (Byrne and Stites, 1995; Chua et al, 2015; Lindman et al, 2001; Wilkinson et al, 2007).…”

Macrocyclized Extended Peptides: Inhibiting the Substrate-Recognition Domain of Tankyrase

“…83 The aliphatic linkers were purchased from Sigma-Aldrich, and all others were synthesized according to protocols previously described. 56−58 All solvents for the reaction were degassed with nitrogen for 1 h before use.…”