Application and development of solid-phase peptide synthesis techniques and the unique conformational effects on 4S-substituted proline derivatives
Date
2019
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Native chemical ligation (NCL) is a method used to combine two unprotected peptide fragments together to form one longer peptide. Standard NCL requires an N-terminal cysteine and a C-terminal thioester. Native chemical ligation surrogates have been previously developed; however, many surrogates see limited use due to long synthetic sequences in the production of the cysteine-substitute. 2-Thiolphenylalanine was developed and synthesized to replace cysteine in the NCL reaction. The NCL surrogate was synthesized from a commercially available 2-iodophenylalanine and copper-mediated cross-coupling chemistry developed in the Zondlo lab. 2- Thiolphenylalanine was synthesized separately via solid-phase and solution-phase. ☐ 2-Thiolphenylalanine efficiently participates in NCL with a C-terminal thioester. A masked-thioester strategy was also implemented to avoid hydrolysis of the C-terminal thioester in larger peptides. The product of the native chemical ligation reaction was also desulfurized in model peptides to yield the native peptide product. Nickel boride and Pd/Al2O3-mediated desulfurization strategies were compared to determine the optimal method to obtain the native phenylalanine from 2- thiolphenylalanine. ☐ Phosphorylation is one of the most prevalent protein post-translational modifications and is known to impact the local and global structure of proteins and peptides. Understanding the conformational effects can lead to better computational modeling, drug design, and protein design. A library of phosphodipeptides was synthesized in collaboration with the University of Delaware CHEM334 laboratory. Students enrolled in CHEM334 synthesized phosphodipeptides via solid-phase peptide synthesis, obtained 1-D and 2-D NMR spectral information, and analyzed their own data. Phosphodipeptides were also enzymatically dephosphorylated to obtain NMR spectral data on the non-phosphorylated dipeptides. ☐ The Ф torsion is one of the main backbone torsion angles in proteins. Ф can be determined through the 3JαN coupling constant and the Karplus relationship. The 3JαN coupling constant was obtained with 1H NMR and chemical shifts were assigned based on TOCSY spectra. The lab was designed to engage students in discovery-oriented research and identify protein features that impact structure. NMR data was obtained for phosphoserine (pS)- and phosphothreonine (pT)-containing dipeptides at different pH values. At pH = 4.0, the monoanionic phosphate effects were observed. The dianionic phosphate effects were observed in the pH range of 6.8-7.2. In the monoanionic phosphate-containing peptides, the pS and pT Ф values become slightly more compact, while the dianionic phosphate induces a significantly more compact Ф for pS and pT. ☐ The n→π* interaction is an important interaction which stabilizes proteins. Previous work from the Zondlo lab demonstrated that the n→π* interaction can be modulated in exo ring pucker (2S,4R)-4-nitrobenzoyl-hydroxyproline methyl ester derivatives. The identity of the N-capping group was changed, and the work demonstrated that electronic effects could tune the strength of the n→π* interaction. ☐ To understand the effects of an inverted stereocenter, 4S-proline derivatives were examined. Through a Mitsunobu reaction, 4S-proline derivatives were synthesized. (2S,4S)-4-Nitrobenzoyl-hydroxyproline methyl ester derivatives were synthesized with pivaloyl, iso-butyryl, acetyl, formyl, Boc, and trifluoroacetyl Ncapping groups. The 4S-derivatives were analyzed through NMR and X-ray crystallography. Through 1H NMR, the ratio of cis and trans isomers was quantified in solution. The proportion of trans isomers was higher than cis isomers in all cases except for the Boc and formyl N-caps. The pivaloyl group was particularly interesting because only the trans isomer was detected within the limits of NMR spectroscopy. ☐ X-ray crystallography demonstrated that all 4S-hydroxyproline derivatives exhibited an endo ring pucker. When crystallized, the pivaloyl and formyl N-capping derivatives were the only derivatives that exhibited a trans amide bond. Only minor electronic effects due to an n→π* interaction were observed. The steric bulk of the Ncapping derivative was the major determinant for a trans or cis conformation. The previously generated 4R-hydroxyproline derivatives exhibited an exo ring pucker that orients the N-capping group for an n→π* interaction. The ability to control conformation has applications in medicinal chemistry, catalysis, and protein design. ☐