A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 μM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 μM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications

Di- and Triphenylacetates of Lanthanum and Neodymium. Synthesis, Structural Diversity, and Application in Diene Polymerization

A new family of lanthanide complexes with di- and triphenylacetate ligands was prepared from LnCl₃. Dimeric diphenylacetates were the only isolated products of the reaction between LnCl₃ (Ln = La, Nd) and Ph₂CHCOONa. The exchange reaction between LnCl₃ and Ph₃CCOONa led to the formation of three structurally different types of neodymium and lanthanum complexes under different reaction conditions. Various coordination modes of the carboxylate ligands were revealed, including κ²- and κ¹-terminal coordination, μ₂-κ¹:κ¹-bridging, and μ₂-κ¹:κ²-semibridging. The treatment of La and Nd tris-triphenylacetates with excess AlEt₃ produces the dimeric tetraethylaluminates [Ln₂((μ₂-κ¹:κ¹-Ph₃CCOO)₄(AlEt₄)₂]. The structures of the key La and Nd triphenylacetates that were obtained from these reactions were verified by X-ray diffraction, and the complex [(THF)₄Nd₂(Ph₂CHCOO)₂(μ₂-κ¹:κ²-Ph₂CHCOO)₂(μ₂- κ¹:κ¹-Ph₂CHCOO)₂] was studied by high-resolution X-ray diffraction. Neodymium di- and triphenylacetate complexes can serve as precursors of catalysts for butadiene and isoprene stereospecific 1,4-cis polymerization

d‑Amino Acid Scan of Two Small Proteins

A “D-scan” of two small proteins, the disulfide-rich <i>Ecballium elaterium</i> trypsin inhibitor II (EETI-II) and a minimized Z domain of protein A (Z33), is reported. For each protein, the stereochemistry of one amino acid at a time was inverted to generate a series of diastereomers. In much the same way an alanine scan determines necessary residues for protein function, the D-scan elucidated the critical stereocenters of the 30-residue EETI-II and the 33-residue Z33. The folding properties and activity of each variant were investigated. A total of 24 out of 30 EETI-II D-scan analogues folded to give a three-disulfide product. Of the 24 variants that folded, half were high-affinity trypsin inhibitors, and three were as active as the wild type (WT). Of these 12 active variants, most were substantially less stable to reduction than WT EETI-II (WT first reduction potential −270.0 ± 1.5 mV, WT second reduction potential −307.2 ± 1.1 mV). Similarly, ten Z33 analogues retained high binding affinity to IgG (<i>K</i>_D < 250 nM, WT: 24 ± 1 nM) and 12 additional analogues had reduced but appreciable IgG binding affinity (<i>K</i>_D between 250 nM and 2.5 μM). As with EETI-II, most Z33 analogues were substantially less stable than the WT (Δ<i>G</i>(H₂O, 263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show that the D-scan is powerful new strategy for studying how the stereochemistry of amino acids affects the structure and function of proteins

Polyphenylcyclopentadienyl Ligands as an Effective Light-Harvesting π‑Bonded Antenna for Lanthanide +3 Ions

A new approach to design “antenna-ligands” to enhance the photoluminescence of lanthanide coordination compounds has been developed based on a π-type ligandthe polyphenyl-substituted cyclopentadienyl. The complexes of di-, tri-, and tetraphenyl cyclopentadienyl ligands with Tb and Gd have been synthesized and all the possible structural types from mononuclear to di- and tetranuclear complexes, as well as a coordination polymer were obtained. All types of the complexes have been studied by single-crystal X-ray diffraction and optical spectroscopy. All terbium complexes are luminescent at ambient temperature and two of them have relatively high quantum yields (50 and 60%). Analysis of energy transfer process has been performed and supported by quantum chemical calculations. The role of a low-lying intraligand charge transfer state formed by extra coordination with K⁺ in the Tb³⁺ ion luminescence sensitization is discussed. New aspects for design of lanthanide complexes containing π-type ligands with desired luminescence properties have been proposed