Conformational Restriction of Peptides Using Dithiol Bis-Alkylation

Abstract

Macrocyclic peptides are highly promising as inhibitors of protein–protein interactions. While many bond-forming reactions can be used to make cyclic peptides, most have limitations that make this chemical space challenging to access. Recently, a variety of cysteine alkylation reactions have been used in rational design and library approaches for cyclic peptide discovery and development. We and others have found that this chemistry is versatile and robust enough to produce a large variety of conformationally constrained cyclic peptides. In this chapter, we describe applications, methods, mechanistic insights, and troubleshooting for dithiol bis-alkylation reactions for the production of cyclic peptides. This method for efficient solution-phase macrocyclization is highly useful for the rapid production and screening of loop-based inhibitors of protein–protein interactions.

Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy

“…48−52 For all the stapled peptides described herein, we observed nearly quantitative yields and little formation of dimer or other side products. 52 The location of the staple was varied and included locations that are proximal to each other in the crystal structure of the ECD of Beclin 1 (Figure 1b). For each staple position, all permutations of l - and d -cysteines were tested, along with a variety of different linkers.…”

“…Similar chemistry has been applied to phage display libraries and to the design of protein–protein interaction inhibitors and can incorporate further diversity using alternative linkers or artificial thiol-containing amino acids. 51,52,67,68 Here we used this approach to convert an 10-mer peptide with minimal activity into a cell-penetrant stapled peptide with potent autophagy-inducing activity in vitro and in vivo. Previous work had shown that bis-alkylation of l -cysteines at ( i , i+ 4) positions using the m -xylene linker can stabilize α-helical structure.…”

“…After the identity of the major product was confirmed by MALDI-TOF mass spectrometry, peptides were subjected to bis-alkylation conditions as described. 52 All peptides were purified by reverse-phase HPLC on a C 8 preparative column. Purity of the final product was confirmed on a C 18 analytical column and observed masses of final products are given in SI Table 1.…”

Cell Penetration Profiling Using the Chloroalkane Penetration Assay

“…Peptide ct-DD5o was cyclized using thiol bisalkylation chemistry as previously described in detail. 45 The linear peptide DP1 (FΦRRRREKW) was synthesized as described above with Fmoc-Glu-OAll and Fmoc-Lys(Mtt)-OH. To cyclize the peptide, first the Glu was deprotected using 0.1 eq.…”

A Solid‐Phase Approach to Accessing Bisthioether‐Stapled Peptides Resulting in a Potent Inhibitor of PRC2 Catalytic Activity

“…[3] Alternatively, a-helical cyclopeptides can also be efficiently prepared through lactamization reactions, [4a] via metal-catalyzed arylations, [4b] disulfide bridges, [4c] through oxime or hydrazones linkers, [2] nitrogen arylations, [4d] and by Cu I -catalyzed cycloaddition between azide and alkyne functionalized unnatural amino acids. [5,6] In contrast, cysteine bis-alkylation with more flexible spacers such as dibromo or diiodoalkanes have been poorly explored, which is mainly due to the low reactivity of such electrophiles. [5,6] In contrast, cysteine bis-alkylation with more flexible spacers such as dibromo or diiodoalkanes have been poorly explored, which is mainly due to the low reactivity of such electrophiles.…”