REVIEW Effects of chiral center on an all-hydrocarbon tethered peptide Xiaodong Shi | Kuan Hu | Hao Geng | Zhihong Liu | Feng Yin | Zigang Li State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China Correspondence Feng Yin and Zigang Li, State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. Emails: yinfeng@pkusz.edu.cn; lizg@pkusz. edu.cn Funding information National Natural Science Foundation of China, Grant/Award Numbers: 21778009, 81701818, 81572198; Shenzhen Science and Technology Innovation Committee, Grant/Award Numbers: JCYJ20170412150609690, KQJSCX20170728101942700, JCYJ20170807144449135 Abstract Recently, our group reported that a precisely positioned chiral center on a thioether tether could dominate the backbone peptides' secondary structures and modulate the peptides' biophysical properties. Helical peptides constructed with this chirality induced helicity (CIH) method were named as CIH peptide. In this work, we examined the effects of substituting the thioether tether with an all hydrocarbon tether for the biophysical property differences. Two peptide epimers were prepared and showed distinct secondary structures and the R epimer is helical. Comparing with its thioether counterpart, the all-hydrocarbon R epimer showed slightly higher helical con- tent, modest improved binding affinity with mammal double minute 2 (MDM2), while similar cell permeability and slightly higher membrane toxicity. KEYWORDS cell permeability, chirality induced helicity, membrance toxicity, P53, peptide 1 | INTRODUCTION Protein-protein interactions (PPIs) have been demonstrated as important therapeutic targets in many biological process. Many PPIs involve multi-protein complex formation via large, shallow, and/or discontinued interacting surfaces. [1–3] In this regard, peptide ligands mimicing the interacting motif with the conserved key interacting residues show potentials to target these intracellular targets. Peptides constraint with suitable methods were shown to have improved biophysical properties, including enhanced protease resistance, cell-penetration, and/or target binding affinity. [4,5] Many chemical methods have been developed to constrain peptides into helical conformation by attaching different tethers to the peptide side chains, including disulfide/thioether bond formation, [6,7] ring-closing metathesis, [8] lactam ring formation, [9] “click” chemistry, [10] addition of perfluoroarenes, [11,12] vinyl-sulfide formation, [13] and so on. Among which, all-hydrocarbon stapled peptides containing two α,α-disubstituted unnatural amino acids which were then cross-linked by olefin methasis have been broadly utilized in many bio- logical studies, including Bcl2, Notch complex, p53/Mdm2, and HIV-1 capsid assembly. [14,15] Recently, our group developed a chirality induced helicity (CIH) strategy by which a precisely positioned chiral center on the tether could dominate the backbone peptides’helicity, with improved cell permeability and binding affinity towards mammal double minute 2 (MDM2) and estrogen receptor alpha (ERα). [16] This strategy could also be extended to sulfilimide chiral center and sulfoxide chiral center, which provide a valuable modifiable site on the tether. [17,18,36,37] Meanwhile, Moore et al. also reported that the N-terminal γ-methylation on a stapled pep- tide's tether showed significant influences on the peptides’ helicity and binding affinity with ERα. [19] Based on previous study of others and our own, the hydrophobicity of the additional tether shows tremendous impact on the peptides’cellular uptakes. [13,20,21] We are interested in how the biophysical properties of an all-hydrocarbon tethered CIH pep- tide different from its thioether counterpart (Figure 1). In addition, simu- lation of an all-hydrocarbon tethered CIH peptide using the recently developed force field RSFF2 [22] predicted a more ordered helical confor- mation (Figure 2). Thus, in this report, we successful translated the CIH concept into all-hydrocarbon tethered peptides. Peptide epimers carry- ing a on-tether carbon chiral center showed distinct secondary struc- tures and biophysical/biochemical properties. Comparing with their thioether tethered counterparts, the all-hydrocarbon CIH peptides showed higher helical contents, slightly improved binding affinity with MDM2, similar cell permeability and slightly higher membrane toxicity. 2 | MATERIALS AND METHODS 2.1 | Peptide synthesis and characterization All peptides were synthesized on Rink amide MBHA resin by standard Fmoc (9-fluorenylmethyloxycarbonyl)-based solid-phase peptide Received: 4 August 2018 Revised: 23 November 2018 Accepted: 5 December 2018 DOI: 10.1002/pep2.24110 Peptide Science. 2018;e224110. wileyonlinelibrary.com/peptidesci © 2018 Wiley Periodicals, Inc. 1 of 7 https://doi.org/10.1002/pep2.24110