Synthesis of conformationally diverse peptides to control peptide structure and function and investigation of unique serine/threonine phosphorylation effects on peptide conformation
Date
2016
Authors
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Publisher
University of Delaware
Abstract
The amino acid proline is unique and finds special identity in proteins, occupying critical positions in turns, loops, bends, and in secondary structures. It performs functional as well as structural role in proteins and is an integral part of polyproline helices and is also found to be closely involved in biomolecular recognitions. Proline is exceptional in its propensity to induce cis amide bonds. Proline analogues have gained significance for applications in medicinal chemistry, protein engineering, structural biology, molecular probes, and in material science. Despite their significance, practical access to these analogues is limited due to tedious solution synthetic operations. We devised a general and practical approach called proline editing to access stereospecifically defined proline analogues, which uses commercially available and inexpensive Fmoc-4R-hydroxyproline (Fmoc-4R-Hyp), introduced into a model peptide via standard solid phase synthesis. The hydroxyl group is protected via a trityl ether (OTrt) or other orthogonal protecting groups in a fully automated fashion and the rest of the amino acids are coupled to complete the peptide synthesis. Selective deprotection of the trityl group under mild acidic conditions yielded a fully protected peptide on solid phase with a reactive hydroxyl functional group amenable for solid phase chemical modifications. In a model Ac-TYXN-NH2 peptide context, the hydroxyl group on Hyp was stereospecifically modified to produce 122 different peptides, with 4R- and 4S-configuration, by conducting reactions like Mitsunobu inversion, acylation, oxidation, reduction, and substitution reactions. 4-Substituted proline derivatives synthesized via proline editing include incorporated structured amino acid mimetics, recognition motifs, electron-withdrawing groups to induce stereoelectronic effects, handles for heteronuclear NMR and other spectroscopies (fluorescence and IR), leaving groups, and other reactive handles. Proline editing provides access to these diverse proline derivatives with no solution phase synthesis, and is thus broadly accessible and highly practical. In addition, proline editing provides the ability to readily modify a single peptide on solid phase to generate multiple peptides with diverse substitution. All proline derivatives were analyzed by NMR in the model tetrapeptide context for their structural effects, via their Ktrans/cis and 3JαN. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, which allowed three parallel bioorthogonal reactions (tetrazine-trans -cyclooctene, oxime, and Diels-Alder) to be conducted in a single solution, demonstrating the potential applications of structured and functionalized proline derivatives. We have also used aromatic-proline interactions to tune polyproline helix conformation and cis-trans isomerism in proline-rich sequences. Using a wide range of electron-rich and electron-poor aromatic amino acids in proline-rich sequences we demonstrated that electron-rich aromatic residues strongly disfavor PPII while the electron-poor aromatic residues exhibit smaller populations of cis amide bonds and favor the PPII conformation. In addition the peptides with block proline-aromatic (PPX)n sequences, where X = 4-aminophenylalanine, exhibited electronically switchable pH-dependent structures. We demonstrated that these electron-poor aromatic amino acid residues can potentially be introduced in polyproline helices and can provide basis for electronically switchable architectures. Phosphorylations and dephosphorylations are key intracellular post-translational modifications, which regulates signaling processes in cells. OGlcNAcylation of Ser/Thr is a competing post-translational modification to phosphorylation. We investigated the conformational effects of Ser/Thr phosphorylation and OGlcNAcylation in peptides derived from the longest isoform of human tau (441 amino acids). CD and NMR (1-D and 2-D) studies revealed that phosphorylation of serine and/or threonine residues in tau-derived peptides led to an induction of PPII while OGlcNAcylation mostly opposed it. To identify the differences between serine and threonine phosphorylation, the structural effects of phoshorylation were examined in model peptides, tau-derived peptides, proline-rich model peptides, and peptides derived from proteins and/or protein substrates. In every case, phosphorylation of serine/threonine led to induction of PPII as identified by CD. Examination by NMR (1H, 1H- 13C HSQC, 1H-15N HSQC, 1H- 13C HMBC, and 31P) revealed that the conformational change, in peptides, due to dianionic phosphorylated threonine was larger than dianionic phosphorylated serine. The analysis of the PDB and ab initio calculations supported the observed data from analysis of peptides in aqueous solution. Upon phosphorylation, threonine exhibits a particularly strong disorder to order transition, preferentially adopting a cyclic conformation with restricted &phis; (mean &phis; = –57°) stabilized by three noncovalent interactions, a strong intraresidue phosphate-amide hydrogen bond, an n–π* interaction between consecutive carbonyls, and a novel stabilizing n–σ* interaction between the phosphate O? and the antibonding orbital of C–Hβ.