An amide to thioamide substitution improves the permeability and bioavailability of macrocyclic peptides.

Solvent shielding of the amide hydrogen bond donor (NH groups) through chemical modification or conformational control has been successfully utilized to impart membrane permeability to macrocyclic peptides. We demonstrate that passive membrane permeability can also be conferred by masking the amide hydrogen bond acceptor (>C = O) through a thioamide substitution (>C = S). The membrane permeability is a consequence of the lower desolvation penalty of the macrocycle resulting from a concerted effect of conformational restriction, local desolvation of the thioamide bond, and solvent shielding of the amide NH groups.

Structural impact of thioamide incorporation into a β-hairpin.

The thioamide is a naturally-occurring single atom substitution of the canonical amide bond. The exchange of oxygen to sulfur alters the amide's physical and chemical characteristics, thereby expanding its functionality. Incorporation of thioamides in prevalent secondary structures has demonstrated that they can either have stabilizing, destabilizing, or neutral effects.

A dimeric proteomimetic prevents SARS-CoV-2 infection by dimerizing the spike protein.

Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin.

Graphene oxide as a dual template for induced helicity of peptides.

Artificial template-mediated fabrication of secondary structures within peptides always attracts great interest in biological systems due to several biomimetic interactions. In all earlier studies, a uniform template containing molecules/nanomaterials was used to target only one type of peptide at a time, which extensively limits the diversity in the generation of artificial protein surface/binding sites. This limitation can be overcome by the incorporation of more than one binding template (heterogeneity) in a single system, for example, Janus nanomaterials, which are challenging and difficult to synthesize.

Desolvation of Peptide Bond by O to S Substitution Impacts Protein Stability.

Amino acid side chains are key to fine-tuning the microenvironment polarity in proteins composed of polar amide bonds. Here, we report that substituting an oxygen atom of the backbone amide bond with sulfur atom desolvates the thioamide bond, thereby increasing its lipophilicity. The impact of such local desolvation by O to S substitution in proteins was tested by synthesizing thioamidated variants of Pin1 WW domain.

Opportunities and challenges in the synthesis of thioamidated peptides.

Chemical modifications of peptides hold great promise for modulating their pharmacological properties. In the last few decades amide to thioamide substitution has been widely explored to modulate the conformation, non-covalent interactions, and proteolytic stability of peptides. Despite widespread utilization, there are some potential limitations including epimerization and degradation under basic and acidic conditions, respectively.

Increasing protein stability by engineering the n → π* interaction at the β-turn.

Abundant n → π* interactions between adjacent backbone carbonyl groups, identified by statistical analysis of protein structures, are predicted to play an important role in dictating the structure of proteins. However, experimentally testing the prediction in proteins has been challenging due to the weak nature of this interaction. By amplifying the strength of the n → π* interaction amino acid substitution and thioamide incorporation at a solvent exposed β-turn within the proteins and Pin 1 WW domain, we demonstrate that an n → π* interaction increases the structural stability of proteins by restricting the torsion angle.

Convenient synthesis of thioamidated peptides and proteins.

The unique physicochemical properties of a thioamide bond, which is an ideal isostere of an amide bond, have not been fully exploited because of the tedious synthesis of thionated amino acid building blocks. Here, we report a purification-free and highly efficient synthesis of thiobenzotriazolides of Fmoc-protected and orthogonally protected 20 naturally occurring amino acids including asparagine, glutamine, and histidine. The near-quantitative conversion to the respective thioamidated peptides on solid support demonstrates the robustness of the synthetic route.

Strategies to Enhance Metabolic Stabilities.

Macrocyclic peptides are a unique class of molecules that display a relatively constrained peptidic backbone as compared to their linear counterparts leading to the defined 3-D orientation of the constituent amino acids (pharmacophore). Although they are attractive candidates for lead discovery owing to the unique conformational features, their peptidic backbone is susceptible to proteolytic cleavage in various biological fluids that compromise their efficacy. In this chapter we review the various classical and contemporary chemical and biological approaches that have been utilized to combat the metabolic instability of macrocyclic peptides.

Increasing the bioactive space of peptide macrocycles by thioamide substitution.

We show that substituting a single atom, O to S (amide to thioamide), in a peptide bond results in global restriction of the conformational flexibility in peptide macrocycles with minimal perturbation of the parent conformation. The van der Waals interactions between the C[double bond, length as m-dash]S group and the surrounding atoms are the major driving force in inducing the conformational restriction, resulting in well-defined structures of these cyclic peptides with static 3-D presentation of the pharmacophores. Utilizing this property of thioamides, we report the development of a superactive antagonist of pro-angiogenic αvβ3, αvβ5 and α5β1 integrins, which are responsible for cancer cell proliferation and survival.