4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation.

Native chemical ligation (NCL) at proline has been limited by cost and synthetic access. In addition, prior examples of NCL using mercaptoproline have exhibited stalling of the reaction after thioester exchange, due to inefficient S → N acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing N-terminal 4R-mercaptoproline and 4R-selenoproline. The synthesis proceeds via proline editing on the N-terminus of fully synthesized peptides on the solid phase, converting an N-terminal Boc-4R-hydroxyproline to the 4S-bromoproline, followed by an SN2 reaction with potassium thioacetate or selenobenzoic acid. After cleavage from the resin and deprotection, peptides with functionalized N-terminal proline amino acids were obtained. NCL reactions with mercaptoproline proceeded slowly under standard NCL conditions, with the S-acyl transthioesterification intermediate observed as a major species. Computational investigations indicated that the bicyclic intermediates and transition states for S → N acyl transfer are sufficiently low in energy (10-15 kcal mol-1 above starting material) that ring strain cannot explain the slow S → N acyl transfer. Instead, the bicyclic zwitterionic tetrahedral intermediate has a low barrier for reversion to the S-acyl intermediate, causing reversion to the thioester (reverse reaction) to occur preferentially over elimination to generate the amide (forward reaction). We hypothesized that a buffer capable of general acid and/or general base catalysis could promote S → N acyl transfer and thus achieve greater efficiency in proline NCL. In the presence of 2 M imidazole at pH 6.8, NCL with mercaptoproline proceeded efficiently to generate the peptide with a native amide bond. NCL with selenoproline also proceeded efficiently to generate the desired products when a thiophenol thioester was employed as a ligation partner. After desulfurization or deselenization, the products obtained were identical to those synthesized directly, confirming that the solid-phase proline editing reactions proceeded stereospecifically and without epimerization.