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.

[reaction: see text] Strong conformational biases in peptides and proteins can be achieved with 4-substituted proline residues (cis-, trans-, or disubstituted fluoroproline or hydroxyproline). The practical, divergent synthesis of peptides containing these residues, via postsynthetic modification of a peptide containing an internal trans-hydroxyproline residue, is described. Significant differences in the conformations of the peptides Ac-TYXN-NH2 were observed, including K(trans/cis) values, which varied from 1.5 (X = cis-fluoroproline) to 7.0 (X = trans-fluoroproline).

The cis-trans isomerization of prolyl amide bonds results in large structural and functional changes in proteins and is a rate-determining step in protein folding. We describe a novel electronic strategy to control cis-trans isomerization, based on the demonstration that interactions between aromatic residues and proline are tunable by aromatic electronics. A series of peptides of sequence TXPN, X = Trp, pyridylalanine, pentafluorophenylalanine, or 4-Z-phenylalanine derivatives (Z = electron-donating, electron-withdrawing, or electron-neutral substituents), was synthesized and Ktrans/cis analyzed by NMR. Electron-rich aromatic residues stabilized cis amide bond formation, while electron-poor aromatics relatively favored trans amide bond formation. A Hammett correlation between aromatic electronics and cis-trans isomerization was observed. These results indicate that the interaction between aromatic residues and proline, which is observed to stabilize cis amide bonds and is also a general stabilizing interaction ubiquitous in proteins and protein-protein complexes, is not stabilized exclusively by a classical hydrophobic effect. To a large extent, the aromatic-prolyl interaction is driven and controllable by an electronic effect between the aromatic ring pi-electrons and the proline ring, consistent with a C-H-pi interaction as the key stabilizing force. The aromatic-prolyl interaction is electronically tunable by 0.9 kcal/mol and is enthalpic in nature. In addition, by combining aromatic ring electronics and stereoelectronic effects using 4-fluoroprolines, we demonstrate broad tuning (2.0 kcal/mol) of cis-trans isomerism in tetrapeptides. We demonstrate a simple tetrapeptide, TWflpN, that exhibits 60% cis amide bond and adopts a type VIa1 beta-turn conformation.

Proline residues are critical structural elements in proteins, defining turns, loops, secondary structure boundaries, and polyproline helices. Control of proline conformation therefore may be used to define protein structure and stability. 4-Substituted proline derivatives may be used to control proline ring pucker, which correlates with protein main chain conformation. To examine the use of proline conformational restriction to tune globular protein stability, a series of peptides derived from the trp cage miniprotein was synthesized. Proline at residue 12 of the trp cage miniprotein, which adopts a Cgamma-exo ring pucker in the NMR structure, was replaced with 4-substituted proline derivatives, including 4R derivatives favoring a Cgamma-exo ring pucker and 4S derivatives favoring a Cgamma-endo ring pucker. Eight trp cage peptides were synthesized, five of which included residues that are not commercially available, without requiring any solution phase chemistry. Analysis of the trp cage peptides by circular dichroism and NMR indicated that the structure and stability of the trp cage miniprotein was controllable based on the conformational bias of the proline derivative. Replacement of Pro12 with 4S-substituted proline derivatives that favor the Cgamma-endo ring pucker destabilized the trp cage, while replacement of Pro12 with 4R-substituted proline derivatives that favor a Cgamma-exo ring pucker resulted in increased alpha-helicity and thermal stability of the trp cage. The most stable trp cage derivatives contained benzoates of 4R-hydroxyproline, which also exhibited the most pronounced stereoelectronic effects in TYProxN model peptides. Overall, the stability of the trp cage was tunable by over 50 degrees C depending on the identity of the proline side chain at residue 12.

Hepatitis C virus afflicts approximately 180 million people worldwide, and the development of direct acting antivirals may offer substantial benefit compared to the current standard of care. Accordingly, prodrugs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate analogues were prepared and evaluated for their anti-HCV efficacy and tolerability. These prodrugs demonstrated >1000 fold greater potency than the parent nucleoside in a cell-based replicon assay as a result of higher intracellular triphosphate levels. Further optimization led to the discovery of the clinical candidate PSI-353661, which has demonstrated strong in vitro inhibition against HCV without cytotoxicity and equipotent activity against both the wild type and the known S282T nucleoside/tide resistant replicon. PSI-353661 is currently in preclinical development for the treatment of HCV.