Stabilising Peptoid Helices Using Non‐Chiral Fluoroalkyl Monomers

Abstract Stability towards protease degradation combined with modular synthesis has made peptoids of considerable interest in the fields of chemical biology, medicine, and biomaterials. Given their tertiary amide backbone, peptoids lack the capacity to hydrogen‐bond, and as such, controlling secondary structure can be challenging. The incorporation of bulky, charged, or chiral aromatic monomers can be used to control conformation but such building blocks limit applications in many areas. Through NMR and X‐ray analysis we demonstrate that non‐chiral neutral fluoroalkyl monomers can be used to influence the Kcis/trans equilibria of peptoid amide bonds in model systems. The cis‐isomer preference displayed is highly unprecedented given that neither chirality nor charge is used to control the peptoid amide conformation. The application of our fluoroalkyl monomers in the design of a series of linear peptoid oligomers that exhibit stable helical structures is also reported.


Stabilising Peptoid Helices Using Non-ChiralF luoroalkyl Monomers
Diana Gimenez, Juan A. Aguilar,Elizabeth H. C. Bromley,a nd Steven L. Cobb* Abstract: Stability towards protease degradation combined with modular synthesis has made peptoids of considerable interest in the fields of chemical biology,m edicine,a nd biomaterials.G iven their tertiary amide backbone,p eptoids lackt he capacity to hydrogen-bond, and as such,c ontrolling secondary structure can be challenging. The incorporation of bulky,c harged, or chiral aromatic monomers can be used to control conformation but such building blocks limit applications in many areas.T hrough NMR and X-ray analysis we demonstrate that non-chiral neutral fluoroalkyl monomers can be used to influence the K cis/trans equilibria of peptoid amide bonds in model systems.The cis-isomer preference displayed is highly unprecedented given that neither chirality nor charge is used to control the peptoid amide conformation. The application of our fluoroalkyl monomers in the design of as eries of linear peptoid oligomers that exhibit stable helical structures is also reported.
Peptoids ( Figure 1) are ac lass of foldamers that are being developed as potential therapeutics, [1] biomaterials, [2] chemical sensors, [3] and organocatalysts. [4] They represent an attractive platform for biological and pharmaceutical applications as they are highly resistant to protease degradation. [5] However,given their tertiary amide backbone,peptoids lack the capacity to form hydrogen bonds so that their secondary structures are dominated by relatively weak interactions. Considerable efforts have been devoted to try and understand the relationships between apeptoid primary sequence and its folded structure. [6][7][8][9][10] The cis/trans isomerization of the tertiary amide bond is the major cause of conformational heterogeneity in peptoid oligomers.D espite this,t he groups of Zuckermann and Barron have demonstrated that a-chiral aromatic monomers,s uch as NSpe (1), can stabilize the cis configuration of the peptoid amide bond largely through steric effects (Figure 1b,c). [6,7] Peptoid oligomers of NSpe (1) fold into stable all cis-amide helices,s tructurally similar to that of ap eptide PPI helix. [6,7] Gorske and Blackwell found that the synergistic application of steric and non-covalent n! p*interactions (NCIs) in aromatic systems could also be used to design stable cis-amide peptoid monomers (e.g., Ns1npe, 2). [8] However,i ti sn ot possible to use the aforementioned NCIs to stabilize the cis-amide conformation of alkyl peptoid monomers,a nd thus the design of stable peptoid helices remains dominated by the use of chiral aromatic residues (e.g., 1 and 2). [9] Recently,Faure,Taillefumier,and co-workers exploited steric effects in the design of anon-chiral tBu alkyl monomer that has ac lear cis-amide preference (NtBu, 3). [10] Whereas 3 offers ar oute to control peptoid structure that avoids the use of aromatic building blocks,the design of nonchiral but stable cis-amide alkyl monomers is an area that is still highly underdeveloped.
It is in this context that we sought to explore the potential application of fluorine incorporation as atool to modulate the conformational preferences of alkyl peptoid monomers. Fluorine is arelatively small atom, close in size to hydrogen, but aHto Fs wap can give rise to significant changes in the electronic and structural properties of am olecule. [11,12] For example,f luorine may engage in stereoelectronic hyperconjugative interactions with neighbouring CÀHb onds (s(CH)!s*(CF)). This ability of fluorine to enforce the preorganization of its local environment is most keenly observed when fluorine atoms are located b to electronwithdrawing groups.I ns uch an arrangement, the fluorine gauche effect is seen (Figure 1d). [12,13] Notably,t he fluorine gauche effect is more pronounced in b-fluoroamides than in other related systems. [12,13] However, in a-fluoroamides,C F/ C=Od ipolar interactions dominate,a nd the fluorine atom adopts a trans-periplanar arrangement (Figure 1e). [14] The peptoid amide bond cis/trans equilibrium in our model systems ( Figure 2; 10-14)w as analysed by ar ange of established NMR methods (see the Supporting Information for the synthesis of 10-14). [8b-d] Thenon-fluorinated dipeptoid 10 exhibits a cis/trans equilibrium that highly favours the trans isomer (CD 3 CN; DG cis/trans = 0.28, K cis/trans = 0.66;F igure 3a). Relative to this,a ll of the fluorinated dipeptoids (11)(12)(13) showed an enhanced preference for the cis-amide conformation ( Figure 3). Initial NMR analysis (in CD 3 CN) revealed that even the introduction of as ingle fluorine atom b to the amide bond enhanced the cis-amide preference by 0.37 kcal mol À1 when compared to 10.I ncorporation of as econd fluorine atom further increased the cis-amide preference. Indeed, unlike 10 and 11,the difluorinated dipeptoid 12 shows ah ighly predominant cis-amide conformation in solution, with DG cis/trans = À0.42 kcal mol À1 and K cis/trans = 2.05 (Figure 3a). We were surprised to note that the K cis/trans value exhibited by 12 is comparable to those seen when cis-inducing chiral aromatic monomers are used (e.g., for 14, K cis/trans = 2.08 in CD 3 CN). Initial NMR analysis revealed alinear correlation between the DG cis/trans values observed and the electronwithdrawing character of the C a carbon substituent when one or two fluorine atoms were incorporated (e.g., 10 to 12; Figure 3b,c). This correlation indicated ac lear relationship between the inductive properties of the fluorinated groups and the cis/trans ratios produced (s I ; Figure 3c,e). [15] An even greater cis-isomer preference was observed when the N3fEtcontaining dipeptoid 13 was analysed (CD 3 CN; K cis/trans = 2.24).
To determine the nature of the interactions present within 11-13,wenext examined how the solvent polarity influenced the cis/trans ratios.W hen using protic MeOD,t he K cis/trans values observed were collectively lower than those found in CD 3 CN (Figure 3a,b). However, the general increases in the cis-isomer preference produced upon fluorine incorporation were still clearly maintained. This outcome indicates that hydrogen bonding is not involved in the cis-isomer stabilization observed in 11-13 (Figure 3a-c). Theu se of CDCl 3 also reduced the K cis/trans values recorded, and this general trend is in good agreement with previous observations reported for other model peptoid systems. [8b-d] Despite this general decrease,t he cis-amide preferences of 11 and 12 in CDCl 3 were still significantly greater than that of the control 10. Upon moving from no fluorine atoms (10)toeither one (11) or two (12), relative changes in the free energy of À0.81 kcal mol À1 and À1.13 kcal mol À1 ,r espectively,were seen.
These relative DG cis/trans changes are in fact larger in CDCl 3 (non-polar) than in CD 3 CN (polar), and this finding supports the hypothesis that an electronic cis-stabilizing effect is occurring.R emarkably,i nC DCl 3 ,t he N2fEt monomer (7) actually has ag reater ability to stabilise a cis-amide preference than the chiral aromatic NRpe monomer (9; K cis/trans = 1.28 vs.0 .94;F igure 3a). TheN 3fEt-containing dipeptoid 13 was found to be more affected in CDCl 3 ,a nd it produced as trong out-of-trend shift to the trans isomer (Figure 3a,b). Given this observation, we hypothesised that the energetic penalty that 13 experiences in the cis conformation may arise from an increased solvation barrier as non-polar solvents are well-known to disfavour structures where large dipoles are  [a] From each replica, DG = ÀRT ln(K cis/trans )at258 8C. Averages and standard deviation values are given for n = 6o rn = 4( b ). b) Average K cis/trans values vs. the number of fluorine atoms present (n f ). c) Correlation between DG cis/trans and C a -substituent field/inductiveconstants (s I ). d) Schematic representation of the proposed peptoid cis-amide isomer stabilization by inductive factors. e) Inductive constantso fC a groups. [15] present. As depicted in Figure 3d,the overall dipolar moment within trans-13 is likely to be lower than that within the corresponding cis isomer as the carbonyl and side-chain dipoles are opposed. This solvation effect should be less pronounced in 11 and 12 as they have weaker dipoles (Figure 3e).
Next, we explored the role that fluorine/amide gauche interactions could play in enhancing the cis-isomer preferences.T he vicinal (three-bond coupling) 3 J HF coupling constants were thus analysed (Figure 4a). [17,18] In 11,a 3 J HF,cal value of 20.0 Hz was calculated for an ideal fluorine/amide gauche conformation of the side chain (g). As ignificantly lower value of 8.0 Hz was obtained for the alternative fluorine/amide anti configuration (a). Thee xperimental value found within the predominant cis-11 isomer was 3 J HF,obs = 25.7 Hz (CD 3 CN). This result strongly suggested an overall fluorine/amide gauche orientation within the side chain. Tw os taggered conformations for 12 were also examined, and the experimental value of 3 J HF,obs = 14.9 Hz was in perfect agreement with an anti/gauche conformation (Figure 4b). This finding indicates that only one Fatom may be actually located gauche to the peptoid amide group,and this is contrary to the more intuitive (+ g/Àg)c onfiguration that would be expected. No significant variations in the experimental 3 J HF,obs values were seen within each cis/trans pair in any of the solvents tested, indicating that the fluorine/amide relative arrangement is retained between conformers.T he NMR results suggest that fluorine gauche effects are not solely responsible for the cis-isomer preferences observed in 11 and 12.In13,fast rotation of the CF 3 group was inferred as the experimental 3 J HF,obs value greatly deviated from the calculated value,w hich assumes as tatic fluorine/amide arrangement (Figure 4c).
We were able to crystallize dipeptoids 12 and 13 from their EtOAc saturated solutions. [19] Thesolid-state structures for 12 and 13 and the conformations suggested by NMR analysis were in perfect agreement (Figure 4d,e). It is worth noting that from the crystal structures of 12 and 13,i tw ould appear that neither fluorine-oxygen repulsive interactions nor unfavourable steric clashes contribute substantially to the cis/trans conformation preferences observed in these systems. As shown in Figure 4d,e,the fluorinated groups in 12 and 13 display aw ell-defined orthogonal orientation relative to the amide bond planes.T his orientation minimizes the potential steric clashes and/or electronic repulsion imposed by the CHF 2 /CF 3 groups.O verall, our findings support the hypothesis that the enhanced cis-amide preferences observed in 11-13 arise from the inductive effects imposed by the fluorine atom(s). As the polarization at C a increases,t he peptoid cisamide preference also increases.W ep ropose that this is due to the fact that the d+ + on C a can form a syn-periplanar stabilising dipolar interaction with the amide C=O ( Figure 3d).
Encouraged by the cis/trans ratios achieved in the model systems (11)(12)(13), we then moved to see if the non-chiral fluoroalkyl monomers could be exploited to design stable peptoid helices.T ot his end, we designed ac ontrol 15-mer peptoid, Pep.1, using non-chiral alkyl ethylamine monomers ( Figure 5). As ingle NSpe residue was introduced as ac hiral reporter for circular dichroism (CD) spectroscopy.T he Pep.1 sequence was then altered by substituting in the various fluorinated monomers (6-8)i np lace of some,b ut not all, of the NEt residues (group 1, Pep.2-4). In as econd group of fluorinated peptoids,a ll of the NEt residues present were replaced (group 2, Pep.5-7). We were pleased to see that structural analysis of the peptoid oligomers Pep.2-Pep.7 by CD spectroscopy revealed the presence of stable peptoid helices (Figure 5b,c). In all of the peptoids studied, substitution of the NEt residues by any of the fluorinated monomers clearly enhanced the CD minima at 218 nm (M q,218 ), which is characteristic of an increase in helicity.W henf ive substitutions (NEt for af luoroalkyl monomer) were made in nonconsecutive positions (group 1, Pep.2-4, n f = 5), the overall increases in molar ellipticity were found to correlate with the number of fluorine atoms within the side chain. Forexample, upon going from the non-fluorinated peptoid (Pep.1) to the N1fEt-based analogue (Pep.2), achange in molar ellipticity of DM q,218 = 6660 deg cm 2 dmol À1 was observed. Similarly,i ncorporation of N2fEt (7)and N3fEt (8)produced approximately two-and threefold higher increases in M q,218 (Pep.3, DM q,218 = 12 640;Pep.4, DM q,218 = 17 000 deg cm 2 dmol À1 ).
When the more heavily substituted peptoids from group 2 were analysed, higher values of M q,218 were found, indicating that the secondary structure enhancement induced by the incorporation of fluorinated side chains has an overall accumulative behaviour (Pep.5-7;F igure 5d,e). In fact, the  [16] average increases in M q,218 produced by each N1fEt (6)a nd N2fEt (7)m onomer introduced in these sequences were higher than those observed when only five replacements were made (DM q,218 /n f ;P ep.2 vs.P ep.5 and Pep.3 vs.P ep.6; Figure 5c-e). These results revealed ab roadly cooperative effect between neighbouring fluorinated side chains.Interestingly,t his synergy between consecutive monomers did not occur when N3fEt monomers were used (Pep.4 vs.P ep.7). Based on our crystal structure data, we could evaluate that the volumes of the CHF 2 and CF 3 groups are 29.63 and 40.47 3 respectively.Based on this,wehypothesise that the behaviour seen for Pep.7 may be related to unfavourable steric and/or repulsive interactions between the CF 3 groups of adjacent N3fEt monomers.O verall, the results from the CD studies ( Figure 5) are highly unprecedented owing to the fact that none of the fluorinated monomers investigated are either chiral, aromatic,o rc harged, and yet they can support the formation of stable peptoid helices.
In summary,w eh ave shown that the selective and strategic incorporation of fluorine atom(s) offers an ew route to control the amide bond isomerism in peptoids containing alkyl side chains.T hrough NMR and X-ray analysis we demonstrated that simple non-chiral fluoroalkyl monomers can be used to influence the key K cis/trans equilibria of ap eptoid amide bond and induce ar emarkable degree of cis-amide preference.T he cis-isomer preference is highly unprecedented given that neither chirality nor charge was being used to control the peptoid amide conformation. The data gathered support the hypothesis that inductive effects imparted by the fluorine atom(s) and not fluorine gauche effects underpin the cis-isomer stabilization observed. The novel fluoroalkyl monomers were also used to prepare as eries of peptoid oligomers that exhibited stable helical structures despite only having one chiral aromatic residue. Thea pplication of fluorine in the design of alkyl monomers offers an ew approach to control amide bond isomerism in peptoid sequences,o vercoming the current need for high levels of chiral side chains.G iven the lack of alternatives available,the N1fEt, N2fEt, and N3fEt alkyl monomers offer exciting new tools to design structurally stable peptoid systems with applications in ar ange of areas,i ncluding medicine and biomaterials.