Proline Editing: A General and Practical Approach to the Synthesis of Functionally and Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides

Abstract

Functionalized proline residues have diverse applications. Herein we describe a practical approach, proline editing, for the synthesis of peptides with stereospecifically modified proline residues. Peptides are synthesized by standard solid-phase-peptide-synthesis to incorporate Fmoc-Hydroxyproline (4R-Hyp). In an automated manner, the Hyp hydroxyl is protected and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In a model tetrapeptide (Ac-TYPN-NH2), 4R-Hyp was stereospecifically converted to 122 different 4-substituted prolyl amino acids, with 4R or 4S stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted prolines synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, nitrobenzoate), handles for heteronuclear NMR (19F:fluoro; pentafluorophenyl or perfluoro-tert-butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence, IR: cyanophenyl ether), leaving groups (sulfonate, halide, NHS, bromoacetate), and other reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, 1,2-aminothiol). Proline editing provides access to these proline derivatives with no solution phase synthesis. All peptides were analyzed by NMR to identify stereoelectronic and steric effects on conformation. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including azide-alkyne, tetrazinetrans-cyclooctene, oxime, reductive amination, native chemical ligation, Suzuki, Sonogashira, cross-metathesis, and Diels-Alder reactions. These proline derivatives allowed three parallel bioorthogonal reactions to be conducted in one solution.

Replacing a single atom accelerates the folding of a protein and increases its thermostability

“…26–28 The ensuing gauche effect alters the pyrrolidine ring pucker, which in turn changes the distance between main-chain carbonyl groups. 21,22,29 Specifically, a 4 S fluoro group increases the C=O···C=O distance, which weakens the n → π * interaction and disfavors the trans isomer (Figure 1C).…”

(2S,4R)- and (2S,4S)-Perfluoro-tert-butyl 4-Hydroxyproline: Two Conformationally Distinct Proline Amino Acids for Sensitive Application in 19F NMR

“…42 Hyp = 4 R -substituted ( trans relative stereochemistry) hydroxyproline, indicated by use of capitalized three-letter code and red color; hyp = 4 S -substituted ( cis relative stereochemistry) hydroxyproline, indicated by lower-case three-letter code and blue color. Proline exhibits a mixture of exo and endo ring puckers.…”

“…12,13,42,47 Notably, 4 S -perfluoro- tert -butyl hydroxyproline exhibited one of the largest conformational preferences in these model peptides that were designed to promote cis prolyl amide bonds, with similar populations of cis and trans amide bonds ( K trans/cis = 1.2, compared to K trans/cis = 2.7 for the peptide containing proline, ΔΔ G trans/cis = +0.48 kcal mol –1 ) observed by 1 H and 19 F NMR, indicating the potential of the perfluoro- tert -butyl group, appropriately installed, to broadly modulate protein structure. In peptides and proteins, the structural effects of 4-substituted prolines are a balance of both the sterics and the electron-withdrawing nature of the proline 4-substituent, which determines the preference for proline ring pucker conformation ( exo versus endo ), which is coupled to the protein main chain conformation (compact versus extended, respectively) (Figure 1).…”

(2S,4R)-4-Hydroxyproline(4-nitrobenzoate): Strong Induction of Stereoelectronic Effects via a Readily Synthesized Proline Derivative. Crystallographic Observation of a Correlation between Torsion Angle and Bond Length in a Hyperconjugative Interaction

“…6−8,11,18 Right: nomenclature of proline H β and H δ hydrogens, with hydrogens capable of engaging in hyperconjugative interactions (as the σ of C β –H β3 or C δ –H δ2 bonds), via overlap with the σ* of the C γ -nitrobenzoate, indicated in bold.…”