Bimodal Use of Chiral α-Trifluoromethylalanine in Aib Foldamers: Study of the Position Impact Towards the Helical Screw-Sense Preference.

Oligomers of the achiral α-aminoisobutyric acid (Aib) adopt a 310 helical conformation in which the screw-sense preference can be controlled by a single chiral residue. The use of the fluorinated residue α-Trifluoromethylalanine (α-TfmAla) revealed a unique way to both induce and measure the screw-sense preference of such oligomers acting as 19F NMR probe. This work proposes a systematic study of the effect of this fluorinated chiral inducer on the helical screw-sense preference of poly-Aib oligomers. The impact of the position of the fluorinated residue into pentamers (N-terminal, central or C-terminal) as well as the nature of the C-terminal capping of the peptides was thoroughly studied in light of complete structural analysis. A deeper understanding of the fluorine effect was achieved confirming the unique ability of α-TfmAla as a helical screw-sense controller.

Fluorinated Peptide Hydrogels Result in Longer In Vivo Residence Time after Subcutaneous Administration.

Peptide-based hydrogels are of interest to biomedical applications. Herein, we have explored the introduction of fluorinated amino acids in hydrogelator H-FQFQFK-NH2 (P1) to design a series of fluorinated peptide hydrogels and evaluate the in vitro and in vivo properties of the most promising analogues. The impact of fluorinated groups on peptide gelation, secondary structure, and self-assembly processes was assessed. We show that fluorine can significantly improve hydrogel stiffness, compared to the nonfluorinated reference P1. For P15 (H-FQFQF(o-CF3)K-NH2), P18 (H-FQFQF(F5)K-NH2), and P19 (H-FQFQM(CF3)K-NH2), microscopy studies scrutinized fiber morphologies and alignment in the network. In vitro release studies of hydrogels loaded with an opioid cargo suggested improved hydrogel stability for P15 and P18. This improved stability was further validated in vivo, notably for P15, giving the most significant increased gel residence time, with more than 20% of hydrogel still present 9 days post-injection, as monitored by nuclear SPECT-CT imaging.

Asymmetric α-Fluoroalkyl-α-Amino Acids: Recent Advances in Their Synthesis and Applications.

Due to the specific properties provided by fluorine atoms to biomolecules, amino acids with fluorinated side chains are of great interest for medicinal chemistry and chemical biology. Among them, α-fluoroalkyl-α-amino acids constitute a unique class of compounds. In this review, we outline the strategies adopted for their syntheses in enantiopure or enantioenriched forms and their incorporation into peptides. We then describe the consequences of the introduction of fluorine atoms in these compounds for the modulation of their hydrophobicity and the control of their conformation. Emerging applications are presented in the areas of enzyme inhibition, medicinal chemistry, hydrolytic stability of peptides, antimicrobial peptides, PET, and 19F NMR probes.

Inviting C5-Trifluoromethylated Pseudoprolines into Collagen Mimetic Peptides.

Numerous collagen mimetic peptides (CMPs) have been engineered using proline derivatives substituted at their C(3) and/or C(4) position in order to stabilize or functionalize collagen triple-helix mimics. However, no example has been reported so far with C(5) substitutions. Here, we introduce a fluorinated CMP incorporating trifluoromethyl groups at the C(5) position of pseudoproline residues. In tripeptide models, our CD, NMR, and molecular dynamics (MD) studies have shown that, when properly arranged, these residues meet the structural requirements for a triple-helix assembly. Two host-guest CMPs were synthesized and analyzed by CD spectroscopy. The NMR analysis in solution of the most stable confirmed the presence of structured homotrimers that we interpret as triple helices. MD calculations showed that the triple-helix model remained stable throughout the simulation with all six trifluoromethyl groups pointing outward from the triple helix. Pseudoprolines substituted at the C(5) positions appeared as valuable tools for the design of new fluorinated collagen mimetic peptides.

Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides.

The incorporation of fluorinated groups into peptides significantly affects their biophysical properties. We report herein the synthesis of Fmoc-protected trifluoromethylthiolated tyrosine (CF3S-Tyr) and tryptophan (CF3S-Trp) analogues on a gram scale (77-93% yield) and demonstrate their use as highly hydrophobic fluorinated building blocks for peptide chemistry. The developed methodology was successfully applied to the late-stage regioselective trifluoromethylthiolation of Trp residues in short peptides (66-80% yield) and the synthesis of various CF3S-analogues of biologically active monoamines. To prove the concept, Fmoc-(CF3S)Tyr and -Trp were incorporated into the endomorphin-1 chain (EM-1) and into model tripeptides by solid-phase peptide synthesis. A remarkable enhancement of the local hydrophobicity of the trifluoromethylthiolated peptides was quantified by the chromatographic hydrophobicity index determination method, demonstrating the high potential of CF3S-containing amino acids for the rational design of bioactive peptides.

Introducing the Chiral Constrained α-Trifluoromethylalanine in Aib Foldamers to Control, Quantify and Assign the Helical Screw-Sense.

Oligomers of α-aminoisobutyric acid (Aib) are achiral peptides that adopt 310 helical structures with equal population of left- and right-handed conformers. The screw-sense preference of the helical chain may be controlled by a single chiral residue located at one terminus. 1 H and 19 F NMR, X-ray crystallography and circular dichroism studies on new Aib oligomers show that the incorporation of a chiral quaternary α-trifluoromethylalanine at their N-terminus induces a reversal of the screw-sense preference of the 310 -helix compared to that of a non-fluorinated analogue having an l-α-methyl valine residue. This work demonstrates that, among the many particular properties of introducing a trifluoromethyl group into foldamers, its stereo-electronic properties are of major interest to control the helical screw sense. Its use as an easy-to-handle 19 F NMR probe to reliably determine both the magnitude of the screw-sense preference and its sign assignment is also of remarkable interest.

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.

Orthogonal 19 F-Labeling for Solid-State NMR Spectroscopy Reveals the Conformation and Orientation of Short Peptaibols in Membranes.

Peptaibols are promising drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structure is needed to understand their activity and should be, in principle, accessible by solid-state NMR spectroscopy. However, their unusual amino acid composition and noncanonical conformations make it very challenging to find suitable labels for NMR spectroscopy. Particularly in the case of short sequences, new strategies are required to maximize the structural information that can be obtained from each label. Herein, l-3-(trifluoromethyl)bicyclopent[1.1.1]-1-ylglycine, (R)- and (S)-trifluoromethylalanine, and 15 N-backbone labels, each probing a different direction in the molecule, have been combined to elucidate the conformation and membrane alignment of harzianin HK-VI. For the short sequence of 11 amino acids, 12 orientational constraints have been obtained by using 19 F and 15 N NMR spectroscopy. This strategy revealed a ß-bend ribbon structure, which becomes realigned in the membrane from a surface-parallel state towards a membrane-spanning state, with increasing positive spontaneous curvature of the lipids.

Structural Behavior of the Peptaibol Harzianin HK VI in a DMPC Bilayer: Insights from MD Simulations.

Microsecond molecular dynamics simulations of harzianin HK VI (HZ) interacting with a dimyristoylphosphatidylcholine bilayer were performed at the condition of low peptide-to-lipid ratio. Two orientations of HZ molecule in the bilayer were found and characterized. In the orientation perpendicular to the bilayer surface, HZ induces a local thinning of the bilayer. When inserted into the bilayer parallel to its surface, HZ is located nearly completely within the hydrophobic region of the bilayer. A combination of solid-state NMR and circular dichroism experiments found the latter orientation to be dominant. An extended sampling simulation provided qualitative results and showed the same orientation to be a global minimum of free energy. The secondary structure of HZ was characterized, and it was found to be located in the 310-helical family. The specific challenges of computer simulation of nonpolar peptides are discussed briefly.

Homochiral versus Heterochiral Trifluoromethylated Pseudoproline Containing Dipeptides: A Powerful Tool to Switch the Prolyl-Amide Bond Conformation.

The design of constrained peptides is of prime importance in the development of bioactive compounds and for applications in supramolecular chemistry. Due to its nature, the peptide bond undergoes a spontaneous cis-trans isomerism, and the cis isomers are much more difficult to stabilize than the trans forms. By using oxazolidine-based pseudoprolines (ΨPro) substituted by a trifluoromethyl group, we show that the cis peptide bond can be readily switched from 0% to 100% in Xaa-ΨPro dipeptides. Our results prove that changing the configuration of the Cα in Xaa or in ΨPro is sufficient to invert the cis:trans populations while changing the nature of the Xaa side chain finely tuned the conformers ratio. Moreover, a strong correlation is found between the puckering of the oxazolidine ring and the peptide bond conformation. This finding highlights the role of the trifluoromethyl group in the stabilization of the peptide bond geometry. We anticipate that such templates will be very useful to constrain the backbone geometry of longer peptides.

Incorporation of Trifluoromethylated Proline and Surrogates into Peptides: Application to the Synthesis of Fluorinated Analogues of the Neuroprotective Glycine-Proline-Glutamate (GPE) Tripeptide.

The incorporation into a peptide chain of highly hindered and weakly nucleophilic trifluoromethylated prolines, pseudoprolines and oxazolidines has been achieved. As an application, the synthesis of a new class of fluorinated analogues of the neuroprotective tripeptide glycine-proline-glutamate (GPE) is reported. These analogues have been elaborated from a panel of five-membered ring trifluoromethylated amino acids (Tfm-AAs) through the coupling reaction with a glutamate residue at the C-terminus and a glycine at the N-terminus. Although the peptide coupling reaction at the C-terminal position of the fluorinated amino acid was conveniently performed under standard conditions, the very challenging coupling reaction at the highly deactivated N-terminal position proved to be much more problematic. A methodological study was needed to identify suitable reaction conditions for this difficult peptide coupling.

Synthesis of enantiopure trans-2,5-disubstituted trifluoromethylpyrrolidines and (2S,5R)-5-trifluoromethylproline.

Enantiopure trans-2,5-disubstituted trifluoromethylpyrrolidines were prepared on a several gram scale starting from a readily available chiral fluorinated oxazolidine (Fox). A pure oxazolopyrrolidine intermediate could be obtained after an efficient separation by selective diastereomer destruction. The addition of various Grignard reagents on this oxazolopyrrolidine provided disubstituted pyrrolidines with moderate to complete trans diastereoselectivity. The highly valuable compound (2S,5R)-5-trifluoromethylproline could be synthesized from the same oxazolopyrrolidine intermediate via a Strecker-type reaction.

Incorporation of CF3-pseudoprolines into polyproline type II foldamers confers promising biophysical features.

The development and the use of fluorinated polyproline-type II (PPII) foldamers are still underexplored. Herein, trifluoromethyl pseudoprolines have been incorporated into polyproline backbones without affecting their PPII helicity. The ability of the trifluoromethyl groups to increase hydrophobicity and to act as 19F NMR probes is demonstrated. Moreover, the enzymatic stability and the non-cytotoxicity of these fluorinated foldamers make them valuable templates for use in medicinal chemistry.

Incorporation of CF3-pseudoprolines into peptides: a methodological study.

The peptide coupling reactions allowing the incorporation of trifluoromethyl substituted oxazolidine-type pseudoprolines (CF3-ΨPro) into peptide chains have been studied. While standard protocols can be used for the peptide coupling reaction at the C-terminal position of the CF3-ΨPro, acid chloride activation has to be used for the peptide coupling reaction at the N-terminal position to overcome the decrease of nucleophilicity of the CF3-ΨPro. We demonstrate that the N-amidification of a diastereomeric mixture of CF3-ΨPro using Fmoc-protected amino acid chloride without base gave the corresponding dipeptides as a single diastereomer (6 examples). The ratio of the cis and trans amide bond conformers was determined by NMR study, highlighting the role of the Xaa side chains in the control of the peptide backbone conformation. Finally a tripeptide bearing a central CF3-ΨPro has been successfully synthesized.

Enantiopure 5-CF3-Proline: Synthesis, Incorporation in Peptides, and Tuning of the Peptide Bond Geometry.

The straightforward synthesis of enantiopure 5-(R)-and 5-(S)-trifluoromethylproline is reported. The key steps are a Ruppert-Prakash reagent addition on l-pyroglutamic esters followed by an elimination reaction and a selective reduction. The solution-phase and solid-phase incorporation of this unprotected enantiopure fluorinated amino acid in a short peptide chain was demonstrated. Compared to proline, the CF3 group provides a decrease of the trans to cis amide bond isomerization energy and an increase of the cis conformer population.

Synthesis of 2-trifluoromethyl-1,3-oxazolidines as hydrolytically stable pseudoprolines.

Trifluoromethyl group containing oxazolidines (Fox) are conveniently synthesized by condensation of serine esters with trifluoroacetaldehyde hemiacetal or trifluoroacetone. These oxazolidines can undergo N-acylation and amidification reactions and are completely configurationally and hydrolytically stable. Therefore, they can be considered as highly valuable proline surrogates (Tfm-pseudoprolines).

Trifluoromethylated proline analogues as efficient tools to enhance the hydrophobicity and to promote passive diffusion transport of the l-prolyl-l-leucyl glycinamide (PLG) tripeptide.

The synthesis of four CF3-proline analogues of the PLG peptide is reported. Our results show that the incorporation of trifluoromethylated amino acids (Tfm-AAs) at the N-terminal position of a peptide significantly increases its hydrophobicity. In addition, depending on the relative configuration and the position of the CF3 group, Tfm-AAs can also promote passive diffusion transport.

Straightforward synthesis of (S)- and (R)-alpha-trifluoromethyl proline from chiral oxazolidines derived from ethyl trifluoropyruvate.

[Structure: see text] A concise synthesis of both enantiomers of alpha-Tfm-proline and (S)-alpha-Tfm-prolinol from ethyl trifluoropyruvate is reported. The key step is a diastereoselective allylation reaction of ethyl trifluoropyruvate and (R)-phenylglycinol-based oxazolidines or imine. The lactone obtained by cyclization of the resulting hydroxy ester proved to be a valuable intermediate for the synthesis of (S)-alpha-Tfm-allylglycine and (S)-alpha-Tfm-norvaline in enantiopure form.

Synthesis of an MIF-1 analogue containing enantiopure (S)-α-trifluoromethyl-proline and biological evaluation on nociception.

The synthesis and the effect of a novel MIF-1 analogue on nociception during acute pain in rat model are reported. The synthesis of this enantiopure trifluoromethyl group containing tripeptide was performed through a peptide coupling reaction between the HCl. Leu-Gly-NH2 and the (S)-α-Tfm-proline. The analgesic effect of the CF3-(MIF-1) 2 has been evaluated in vivo on rat model by paw pressure (PP) and hot plate (HP) tests and compared to the native peptide MIF-1. Highest analgesic effect was observed with CF3-(MIF-1) 2 only in PP test. In order to study the mechanisms of nociception induced by the studied peptides, the involvement of the opioid and the nitric oxideergic systems was investigated. The results are in favor of a participation of both system since pretreatment, 20 min before injection of the CF3-(MIF-1) 2, with the non-competitive antagonist of opiate receptors naloxone, the nitric oxide synthase (NOS) inhibitor l-N(G)-nitroarginine ester (l-NAME) or the nitric oxide (NO) donor l-arginine (l-Arg) significantly decreased the pain perception in PP and HP tests.

Local control of the cis-trans isomerization and backbone dihedral angles in peptides using trifluoromethylated pseudoprolines.

NMR studies and theoretical calculations have been performed on model peptides Ac-Ser(ΨPro)-NHMe, (S,S)Ac-Ser(Ψ(H,CF3)Pro)-NHMe, and (R,S)Ac-Ser(Ψ(CF3,H)Pro)-NHMe. Their thermodynamic and kinetic features have been analyzed in chloroform, DMSO, and water, allowing a precise description of their conformational properties. We found that trifluoromethyl C(δ)-substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. In CHCl(3), the configuration of the CF(3)-C(δ) entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn-like or PPI/PPII-like backbone conformations. Moreover, in water and DMSO, this C(δ)-configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. Theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the NMR observations.