José Laxio Arenas

Dr Jacopo Lesma

Tap Ha-Duong

Dr Bikash Sahoo

Ayyalusamy Ramamoorthy

Dr Nicolo Tonali

Jean-Louis Soulier

Dr Frédéric Halgand

Francois Giraud

Dr Benoit Crousse

Dr Julia Kaffy
email: julia.kaffy@universite-paris-saclay.fr

Sandrine Ongeri
email: sandrine.ongeri@universite-paris-saclay.fr

Dr J Laxio-Arenas

Prof T Ha-Duong

Dr J.-L Soulier

Prof. S Ongeri

Prof A Rammamoorthy

Relation between conformation and activity on islet amyloid polypeptide aggregation of fluorinated foldamers based on Ndifluoromethyl-1,4-triazole amino acids

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyldifluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units reduces it, and this more efficiently that the natural peptide parent. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded β-sheets have no effect. This work demonstrates that a good balance between structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Introduction

Misfolded proteins into β-sheet rich amyloid fibrils are associated with more than 20 human diseases which include Alzheimer's (AD) and Parkinson's (PD) diseases, and type II diabetes (T2D). 1- 3 T2D is a major public health issue, affecting around 400 million people worldwide. This number is expected to exceed half a billion by 2045, while no causal treatment for this disease is available. 4,5 Human islet amyloid polypeptide (hIAPP, also known as amylin) aggregation is involved in the death of pancreatic βcells of T2D patients, by a very partially understood mechanism. hIAPP undergoes a conformational change inducing an aggregation pathway providing toxic soluble oligomers and fibers. [START_REF] Akter | [END_REF][7][8] The aggregated species adopt β-sheet rich conformations and amyloid deposits are found in the pancreas of more than 95% of diabetic patients. [START_REF] Akter | [END_REF][7][8] Preventing both the presence of soluble transient oligomers and late aggregated forms of hIAPP is crucial to prevent the cell toxicity that is caused by membrane disruption and also by other less understood phenomena induced by hIAPP aggregation. [9][10][11] This strategy has to be explored to consider a viable therapy against T2D. Some small molecules have been reported as inhibitors of hIAPP aggregation, such as polycyclic and polyphenols. [12][13][14][15] They often present the disadvantage of being not selective and thus inducing side effects. Few rationally designed peptide derivatives have been reported. [16][17][18][19] Foldamer science [20][21][22] has recently provided original solutions to address some of the peptide issues. 23,24 Their ability to mimic the secondary structure of proteins combined with an increase of selectivity and membrane permeability afford remarkable new opportunities. Nevertheless, this field is still in its infancy for application as modulators of amyloid protein aggregation. The group of Miranker has reported that helical oligopyridine and oligoquinoline foldamers interfere with the early folding steps of both Aβ1-42 (involved in AD) 25,26 and hIAPP 27,28 peptides. Some of us demonstrated that a foldamer composed by six units of 4-amino(methyl)-1,3-thiazole-5-carboxylic acid (ATC) acts as dual inhibitors of hIAPP and Aβ1-42 aggregation and preserves monomeric species. 29 Some of us also recently reported the use of diaza-peptide foldamers to decrease Aβ1-42 aggregation. 30 We currently aim at exploring the interest of fluorinated foldamers. The design of this new class of foldamers has been still poorly addressed by chemists, [31][32][33][34][35][36] and they are completely unknown as potential modulators of amyloid aggregation or even more widely in the vast field of medicinal chemistry. They might be all the more promising as fluorinated molecules constitute almost 30% of all new approved drugs 37,38 and as the introduction of fluorinated amino-acids into peptides and proteins has been shown to increase their biological activity, their chemical and enzymatic stability and their secondary structure propensity. 23,[39][40][41][42] In the present work we study the conformational preferences and activity on amyloid protein aggregation of novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units. We previously described the insertion in pseudotripeptides of this 1,4-Tz-CF2 constrained scaffold that can be considered as an analogue of the gly-difluoroGly dipeptide. 43 The stabilizing electrostatic interaction between the fluorine atoms and the neighbouring triazole and the NH of the successive amino acid induces extended conformation. We then performed the selfassembly of this architecture and carried out conformational studies of these new foldamers (Figure 1, compounds 1, 2). 44 Oligomers of 1,4-Tz, called triazolamers, have been rarely reported in literature, especially in medicinal chemistry. Triazoles act as trans-amide mimetics with higher stability to hydrolysis. They are easily available thanks to copper(I) catalysed azide-alkyne cycloaddition (CuAAC), which was described independently by the groups of Meldal and Sharpless in 2002. 45,46 Conformational influence of 1,4-Tz has been rarely described until recently. Small bivalent peptidomimetics based on 1,4-Tz adopt rotation-like conformations that replace dipeptide in an α-helical secondary structure. 47,48 In addition, in tri and pentapeptide, the tendency of Tz amino acid to induce β-turn formation has been reported. 49,50 Extended [51][52][53] or folded 51,54 conformation of peptidotriazolamers have been reported. Our fluorinated homotriazolamers based on the assembly of tri and four 1,4-Tz-CF2 adopt elongated β-strand-like structures. 44 Herein we present the rational design, the synthesis and conformational studies of new fluorinated foldamers based on 1,4-Tz-CF2 mixed with natural amino acids, that we call heterotriazolamers (Fig 1 , compounds 3-7). Their conformation preferences and ability to modulate the hIAPP aggregation process have been compared with our previous homotriazolamers (Figure 1, compounds 1-2). We have also introduced the 1,4-Tz-CF2 unit in flexible β-hairpin like structure based on a piperidine-pyrrolidine β-turn inducer to obtain fluorinated analogues of our recently reported inhibitors of hIAPP aggregation (Fig 1 , compounds 8-9). 55 To assess their influence on hIAPP aggregation and conformation, we have combined Thioflavin-T (Tht) kinetic assays, native and ion mobility mass spectrometry (MS), as well as NMR and Circular dichroism (CD) experiments.

Results and Discussion

Design and synthesis

Our hypothesis is based on the design of extended fluorinated foldamers using 1,4-Tz-CF2 as a constrained dipeptide mimic. These foldamers could act as β-sheet breakers with respect to βsheet rich oligomers and fibers of hIAPP, by interacting with hIAPP sequences and forming locally β-sheet structures with these sequences, and consequently disrupting the intra and intermolecular interactions involved in the misfolding and the aggregation processes. As mentioned above, we have shown the preferential adoption of extended conformation of homotriazolamers of 1,4-Tz-CF2 unit. 44 To go further, now we have based the design of new fluorinated foldamers on the hIAPP amyloidogenic sequence N21NFGAIL27. Experimental and in silico models of monomers and aggregates have shown the critical role in fibril formation of this region [56][57][58][59] Peptides S20NNFGAILSS29, F23GAIL27 and N21NFGAIL27 based on this sequence have been described to readily form amyloid fibrils. 60,61 The latter has been characterized with X-ray crystallography. 62 Furthermore, this sequence has been described as a promoter of hIAPP fibrils formation 63 or as an inhibitor when introduced in a macrocycle peptide derivative. [START_REF] Buchanan | Proc. Natl. Acad. Sci[END_REF] More recently, hIAPP-derived 21-residues peptides, based on FGAIL sequence, was reported by Kapurniotu et al. as inhibitors of hIAPP and Aβ1-40 aggregation. [START_REF] Tatarek-Nossol | [END_REF][66][67] In our hands, FGAIL and AIL were inserted in dynamic β-hairpin like structures built on a piperidine-pyrrolidine β-turn inducer to efficiently reduce hIAPP self-assembly. 55 Therefore, we chose to insert some amino acids from this sequence in heterotriazolamers, to further selectively interact with the amyloidogenic sequence of hIAPP, thanks to the specific amino acid lateral chains, while the 1,4-Tz-CF2 units will act as β-sheet breaker elements. For that purpose, in our first foldamers 3-5, we inserted two difluorotriazole units in the F23GAIL27 sequence to obtain three F-(1,4-Tz-CF2)-A-(1,4-Tz-CF2)-L derivatives, differing only by their Nterminus N-Boc (compound 3), NH2 (compound 4) or AcNH (compound 5). In order to potentially increase the interactions with hIAPP, we increased the size of our foldamers N-(1,4-Tz-CF2)-F-(1,4-Tz-CF2)-A-(1,4-Tz-CF2)-L 6 and 7, by inserting three 1,4-Tz-CF2 in the sequence N21NFGAIL27 and having a N-terminal NH2 (compound 6) or AcNH (compound 7). Finally, the 1,4-Tz-CF2 unit was introduced in flexible β-hairpin like structures based on a piperidine-pyrrolidine β-turn inducer. We designed the direct fluorinated analogues 8 and 9 of our recently reported inhibitors of hIAPP aggregation, bearing AIL et FLV peptide arms linked to the β-turn mimic. 53 We kept the AIL peptide at the N-terminus and replaced FLV by F-(1,4-Tz-CF2)-V and F-(1,4-Tz-CF2) at the Cterminus (Figure 1, compounds 8 and 9 respectively).

The synthesis of our fluorinated heterotriazolamers was based, first on the preparation of 1,4-Tz-CF2 building blocks bearing one or two amino acids, and secondly on their coupling. For the first foldamers, we started by coupling the Boc-protected Lphenylalanine and L-arginine with propargylamine in order to obtain Boc-protected amino acids propargylamide 10 and 11. All difluorinated azides bearing amino acid residues (Alanine, Phenylalanine and Leucine for 12, 13 and 14 respectively) were obtained following the method described in our previous work, from the direct amidification of ethyl 2-azido-2,2-difluoroacetate with the corresponding amino acids (see SI). 41 Propargylamides and difluoro-azides were put under click chemistry conditions using CuSO4.5H2O as a copper source, sodium ascorbate as a reducer of Cu(II) species, and TBTA as a ligand to stabilize Cu(I), in a mixture of MeOH/H2O. The N-difluoromethyl-triazole building blocks bearing one (17, 18) or two (15, 16) amino acids were obtained in good yields (60-81%). Building blocks 15 was submitted to hydrogenolysis (following McMurray methodology using triethylsilane and palladium on carbon in MeOH to generate hydrogen in situ 68 ) and 17 was treated by a chlorohydric acid solution in dioxane, to obtain respectively the free carboxylic acid and amine intermediates, that were coupled to give compound 3. The coupling conditions using EDC.HCl, HOBt in the presence of DIPEA in DMF for 48 hours allowed the obtention of 3 in 55% yield (Table 1, entry 1). However, two sets of peaks were clearly visible on the 19 F NMR spectrum indicating the presence of two diastereoisomers and thus the epimerization of at least one C. By decreasing the reaction time to 5 hours, no epimerization was visible and 3 was obtained in satisfactory 48% yield (Table 1, entry 2). We also tried to use a weaker base such as collidine and no epimerization was observed (Table 1, entry 3). In order to try to increase the yield, we screened other coupling conditions such as COMU/OXYMA in the presence of DIPEA or collidine (table 1, entries 4 and 5), DIC/OXYMA, HATU/HOAt, HCTU (Table 1, entries 6-8). 67 Unfortunately, none of these conditions gave better yields than the initial conditions. The Boc cleavage of 3 was conducted to afford compound 4 (97% yield) that was then acetylated to give compound 5 in satisfactory yield (88%). Similar to the procedure followed to obtain 3, compounds 16 and 19 were submitted to hydrogenolysis and acidic treatment respectively to obtain the free carboxylic acid and amine intermediates, that were coupled to give compound 20 in 42% yield. Its Boc cleavage using the cleavage cocktail TFA/H2O/TIPS/thioanisole (scavengers were necessary to avoid trityl addition on the triazole rings) gave 6 (61% yield) which was acetylated to give 7 in 50% yield. ), HOBt (1.1 eq.), EDC.HCl (1.1 eq.), DIPEA (2.5 equiv.), CH2Cl2, rt, 5h; (b) ethyl 2-azido-2,2difluoroacetate (1 eq.), Et3N (1 eq.), 4h, ))); (c) CuSO4.5H2O (5%), Asc.Na (20%), TBTA (5%), MeOH/H2O, rt, 2h; (d) HCl 4N in dioxane (10 eq.), CH2Cl2; or CH3OH, rt, 1h (e) Pd/C (30%), Et3SiH (2 eq.), MeOH, rt, 30 mn; (f) EDC.HCl (1.2 eq.), HOBt (1.2 eq.), collidine (5 eq.), DMF, rt, 3-5h; (g) Ac2O (1.5 eq.), Et3N (3 eq.), rt, 5 h. (h) TFA/H2O/TIPS/thioanisole (90/5/2.5/2.5, v/v/v/v), rt, 2h.

Scheme 2. Synthesis of compounds 8 and 9. (a) Boc2O (2.5 eq.), DMAP (2.5 eq.), DIPEA 2.5 eq., DMF, rt, 15h; (b) Pd/C 10% (1.0 eq.), H2, 30°C, 24h; (c) Fmoc-NH-Leu-OH or Fmoc-NH-Ile-OH or Fmoc-NH-Ala-OH (1.2 eq.), EDC (1.2 eq.), HOBt (1.2 eq.), collidine (2.5 eq.), DCM, rt, 15h; (d) Piperidine 20% DMF, rt, 4h; (e) aq. NaOH 2M (5.0 eq.), MeOH, 40°C, 3h then aq. KHSO4; (f) COMU (1.0 eq), Oxyma (1.0 eq), collidine (2.5 eq.), DMF, rt, 15h; (g) HCl 4N in dioxane (20.0 eq.), DCM, rt, 6h. To prepare compounds 8 and 9, we followed a slightly modified procedure reported by us. 55 The synthesis, outlined in Scheme 2, started from scaffold 21 that was prepared in accordance with our published procedure 70,71 Contrary to what we reported for the coupling of a simple tripeptide or a di-aza-tripeptide on the carboxylic group of our scaffold moiety, 55,72 here we had to prepare the doubly (Boc and Tosyl) protected scaffold 22 because the acidic proton of the NH-Tosyl led to an undesired and predominant intracyclisation during the coupling reaction between the peptidomimetic arms and the carboxylic acid. Thus, a Boc protection of the NH-Tosyl using Boc2O in the presence of DIPEA and DMAP allowed the double protected intermediate 22 (93% yield). The benzyl group on the piperidine ring was cleaved by hydrogenolysis to quantitatively give 23. The three successive coupling reactions using the amino acids N-Fmoc-L-Leu-OH, N-Fmoc-L-Ile-OH, and N-Boc-L-Ala-OH, followed by the basic hydrolysis of the ester group of

Structural characterization

Conformational analysis by NMR

The conformational studies of our oligomers 4-9 were performed by NMR in CD3OH, a polar solvent avoiding the problems of solubility in water. The 1 H, 13 C and 19 F chemical shift assignments at low temperature (278K for 6-9 or 283K for 4-5) are given in Tables 1S-16S (See SI). In compound 5, the 3 JNH-Hα coupling constant values of 7.9 and 8.0 Hz of Phe-1

and Leu-3 (see Table 3S, SI) correspond to the dihedral angles around 120° associated mostly with peptide segments in extended conformation, 73 as expected for compounds bearing the N-difluoromethyltriazole scaffold. 43 However. The 3 JNH-Hα of Ala-2 was more difficult to reliably calculate but appeared to be below 7Hz, indicating a more folded structure in this position. The amide bond NH protons temperature coefficient (NH/T) were then calculated to analyze the backbone conformational propensities. Small negative temperature coefficients (> -4.5 ppb.K -1 ) correspond to amide protons involved in hydrogen bonds (intra or intermolecular), whereas higher negative values (< -6 ppb.K -1 ) indicate solvent interactions. In 5, NH/T were calculated between 283 and 313 K (see Table 4S). These values were between -8 and -9 for amide of natural amino acid residue and around -7 for amide bearing 1,4-Tz-CF2 scaffold indicating that amide bond NH protons were not engaged in stable hydrogen bonds. Very similar 3 JNH-Hα and NH/T values were obtained for compound 4 (see tables 1S and 2S in SI). Long-range 1 H-1 H ROE correlations were observed indicating a folded conformation for heterotriazolamers 4 and 5. In both 4 and 5, ROEs were visible between the CH3 of Ala-2 and the Hc of the two triazoles, TZL-1 and TZL-2. In 5, correlations between CH3 of Ala-2 and H of Phe-1, and between H of Phe-1 and Hc of TZL-1 were also observed (Figure 2). The two amino acids on either side of the TZL-1 unit could be brought together thanks to the flexibility of the C of TZL units. In fact, this part of the N-difluoromethyltriazole scaffold has a higher flexibility than the part bearing the two fluorine atoms which is responsible for the constraining effect of the scaffold thanks to H-F interactions (Figure 2). 43 In the case of the NH2-free analogue 4, one ROE was visible between H of Phe-1 and Hc of TZL-1.

The direct rapprochement between Phe 1 and Ala-2 was not visible. Very similar NMR data were obtained for the longer heterotriazolamers 6 and 7. The amide bond NH protons temperature coefficient (NH/T) were all lower than -6 ppb.K -1 indicating that there are not involved in H-bonds (see Table 6S and 8S, SI). Although, some 3 JNH-Hα of 6 and 7 were difficult to determine, the two 3 JNH-Hα values for Tzl-1 and -2 of 6 and 7 were below 7 Hz, indicating the folding around the C of TZL units. Many long-range 1 H-1 H ROE correlations were observed further confirming folded conformations for 6 and 7 (Figure 2). In particular between CHβ of Asn-1 and Hc of TZL-1, CHβ of Phe-2 and Hc of TZL-1 and TZL-2 (only TZL-2 for 6), CH3 of Ala-2 and Hc of TZL-2 and TZL-3, and CHδ of Leu-4 and Hc of TZL-3. A very long-range 1 H-1 H ROE correlation was also observed between CHδ of Leu-4 and Hc of TZL-1 in both 6 and 7, as well as between CHβ of Asn-1 and Hc of TZL-3 in the case of 6, indicating a close proximity between the Nand C-termini of heterotriazolamers 6 and 7.

In the 2D 19 F-1 H HOESY spectra of 4 -7, only the short-range correlations involving in each 1,4-Tz-CF2 unit the two fluorine atoms with the proton of the triazole and the NH of the successive unit, were visible (Figures 2 and9-10S, 17-18S, 24-25S and 31-32S in SI). This result suggests that the two fluorine atoms of each 1,4-Tz-CF2 unit maintained the extended constraint at its C-terminal part but are not involved in the overall folding of the foldamers. The conformational behaviour of 8 and 9 was similar to that of our previously reported acyclic β-hairpin like structures bearing the tripeptide arms and the di-aza-tripeptide. 55 They displayed the presence of two major conformers in dynamic equilibrium. The complete assignment of the 1 H and 13 C signals was performed for the two conformers of compounds 8 and 9. Working at 278K allowed the observation of two different conformers in dynamic equilibrium in ratio 3:1 and 1:1 for 8 and 9 respectively. However, in contrast with 9, despite the low temperature, some protons and carbons displayed the same chemical shift in both conformers of 8. The 1 H-NMR and 13 C-NMR chemical shifts are reported in tables 9S and 10S (SI) respectively for conformers A and B of 8. In both conformations (Table 11S, SI) only the 3 JNH-Hα coupling constant for Ile-2 and Val-8 values fell within the range 8-12 Hz that correspond to the dihedral angles associated with peptide segments in extended conformation as expected for βhairpin arms. 73 These results suggested that compound 8 was not fully structured. In both conformers NH-CSD generally showed positive values as expected for the extended arms of a β-hairpin (Tables 12S and13SS, SI). Noteworthy the high values of Val-8 (+1.0 and +1.3 respectively for conformers A and B) might be ascribed to the presence of the vicinal CF2 group. Indeed, the CF2 could enhance the CSD by either de-shielding the amide (down fielded) thanks to its electron withdrawing effect or by stabilizing the extended conformation because of its electronic properties. The borderline H α -CSD values for almost all the amino acids fitted with the hypothesis of a non-fully structured β-hairpin. However, the general C-CSDs up-fielding (Table 12S and 13S, SI) of both conformers confirmed the presence of extended conformations of the arms. While the positive C α -CSD of Pro-5 indicated the presence of a β-turn inducer in both conformations. In conformer A, NH/T calculated between 278 and 313 K were smaller than -4.5 suggesting no stable H-bond (see Table 14S, SI). In both conformers, the presence of sequential CHαi/NHi+1 ROEs for the tripeptide FLV and for the segment from Pro-5 to Tzl-7 confirmed the extended conformation of the arms (Figure 4, blue arrow). Concerning the ROEs network usually observed in the β-turn region for similar compounds 55 we observed only the ROE between H α -Pro-5 and proton in position 3 of the piperidine ring (Figure 3, green arrow). The two different ROEs displayed by the H α -Leu-3 with the piperidine ring were more interesting. Indeed, in conformer A we observed a ROE signal of H α -Leu-3 with proton in position 2 while in conformer B the ROE was visible between H α -Leu-3 and proton in position 6. As we recently demonstrated, 55 these signals are diagnostic of two different architectures: one more closed (conformer A) and one more open (conformer B). Furthermore, we observed spatial proximities between the lateral chains of Leu-3 and Phe-6, reflecting the interaction between the two arms, only in the case of conformer A as expected for the more closed patterning. However, the ROE between the CH2 of Phe-6 with the CH3 of Val-8 suggested a folding of the peptidomimetic arm around the CF2-Tz. This is in accordance with the folding observed in the triazolamer 5 indicating a rapprochement between the two amino acids on each side of the TZL-1 unit.

Figure 3

In green the common ROEs, in blue the common sequential ROEs and in red the differences between conformers A and B of compound 8

Figure 4 Structure of compound 9 showing the ROE networks. In green the common ROEs, in blue the common sequential ROEs and in red the differences between conformers A and B.

The complete attribution of two conformers A and B of 9 was performed (Tables 15S and16S respectively for conformers A and B, SI). By analyzing the CSDs for each natural amino acid present in the structures (Tables 17S and18S, SI), we observed, in both conformers, that the residues Ile-2, Leu-3 and Phe-6 showed a general down-fielding for α hydrogens and amides compatible with an extended conformation, while

Ala-1 and Pro-5 displayed negative values indicating flexibility at the N-terminal and a β-turn conformation for Pro-5. The negative C-CSDs further confirmed possible extended conformation of the arms, while the very small Cα and carbonyl CSD Pro-5 values confirmed its involvement in a β-turn. The measurable 3 JNH-Hα displayed large values systematically higher than 8.9 Hz, reflecting extended arms (Tables 19S,SI). In conformer A, NH/T calculated between 278 and 313 K were smaller than -4.5 in the case of Phe-6 and Tzl-7 suggesting their involvement in H-bonds (Table 20S,

Conformational analysis by molecular dynamics

To gain better insight into their preferential structures, compounds 4 and 5 were each submitted to a 500 ns all-atom molecular dynamics simulation in explicit methanol solvent. The theoretical 3 JNH-Hα coupling constants (computed over the whole trajectories) are fairly close to the experimental measurements ( Overall, MD simulations of compounds 4 and 5 generated conformational ensembles in which the two rather rigid extended segments Phe1-TZL1-Ala2 and Ala2-TZL2-Leu3 can rotate around Ala2 residue which act as a hinge, leading to folded hairpin-like or extended linear structures (Figure 6). and Ala-TZL fragments are rather rigid, but can rotate around the 2 residues Phe and Ala which act as hinges. Thus we could observe transient hairpin-like structures or zig-zag conformations bent at these two positions which sometimes fold into ladder-like structures similar to short multi-stranded β-sheets. It could be noted that the population of these transient folded structures could be underestimated in MD simulations compared to experiments. Indeed, as shown in Figure 8, the interproton distances between non-adjacent "residues" (Asn1-TZL3, TZL1-Leu4, Phe2-TZL3, and TZL2-Leu4) are most often larger than 0.5 nm in simulations, whereas corresponding long-range 1 H-1 H ROEs are observed in NMR experiments. This indicates that NMR conformations of compounds 6 and 7 are probably more frequently folded (or structured) than those generated by simulations.

Activity on hIAPP fibrillization by ThT-fluorescence experiments

The ability of foldamers 1-9 to interact with hIAPP fibril formation was first investigated using the in vitro Thioflavin-T (ThT) fluorescence assay (representative curves are shown in Figures 9 and47S, SI). Heterotriazolamers 3-5 and 6-7 were compared to the natural peptides FGAIL-NH2 and NNFGAIL-NH2 respectively and to the homotriazolamers 1 and 2.

Flexible -hairpin like structure 8 and 9 were compared to their peptide analogues bearing AIL and FLV arms and reported as inhibitors of hIAPP aggregation. 55 ThT is the standard dye used to monitor the kinetics of aggregation of amyloid proteins because it fluoresces upon binding to βsheet rich structures. 74,75 The fluorescence curves for hIAPP controls displayed the typical sigmoidal pattern, starting with an initial lag phase lasting around 1 h, followed by a rapid polymerization phase ending with a plateau reached after 5 h. Two parameters were gathered from the fluorescence curves analysis: (1) t1/2, defined as the time at which fluorescence has reached half of its maximal value and gives insight on the kinetics of the aggregation process and (2) F, the fluorescence intensity at the plateau, which is, in a first approximation, related to the amount of fibrillar material formed. Each compound was tested at two different foldamer/amyloid peptide ratios, 10/1 and 1/1 and variations of t1/2 and F are reported in table 3. Parameters are expressed as mean ± SE, n=3. [a] Compounds were dissolved in DMSO; the final concentration kept constant at 3 % (v). The concentration of hIAPP was 5µM.

[b] See supporting information for the calculation of the t1/2 extension/reduction.

[c] See supporting information for the calculation of the F extension/reduction.

[d] compound 2, self-aggregate at 50µM, [e] ne = no effect. The aggregation curves of hIAPP in the presence of homotriazolamer 1 at 1/hIAPP ratio 10/1 (Figure 9) showed a shorter t1/2 and a higher fluorescence plateau than for the control experiment indicating that compound 1 is capable of accelerating but also increasing the aggregation of hIAPP. These observations were not found at the lower ratio 1/1. This higher fluorescence plateau could be due to the slight selfaggregation of 1 (seen in Figure 9, orange curve) which can seed and thus trigger the aggregation process. The longer homotriazolamer 2 displayed no effect on the aggregation process of hIAPP while its slight self-aggregation was clearly observed at 50 M (Figure 47S). Concerning the heterotriazolamers built on the FGAIL sequence, surprisingly, the Boc-protected analogue 3 displayed a slight acceleration of the fibrillization kinetics associated to a decreased of the plateau at 10/1 ratio (Δt1/2 =-42 %, ΔF=-49 %); with only the effect on the fluorescence plateau being maintained at the lower ratio 1/1. However, the N-terminal free amine analogue, compound 4, was found to be inactive regardless of the ratio tested. In this series, compound 5, acetylated at the N-terminal part, showed the most interesting effect, stronger than the peptide analogue FGAIL, with a pronounced activity at 5/hIAPP ratio 10/1 (Figure 9), both on the fibrillization kinetics (Δ t1/2=+112% for 5 vs no effect for FGAIL-NH2) and on the fluorescence plateau (ΔF=-80% for 5 vs =-36% for FGAIL-NH2). The activity was not retained at the lower ratio 1/1. The longer heterotriazolamers 6 and 7 designed on the NNFGAIL sequence, displayed weaker activity compared to the FGAIL built analogues The N-terminal free amine 6 slightly delayed the aggregation process at a 10-fold excess (Δ t1/2=+42%) and showed no effect on the fluorescence plateau, while at 1/1 ratio, it displayed no effect on the kinetics and a modest decrease of the fibrillization plateau (ΔF = -33 %). Unlike in the FGAIL-based series, the acetyl analogue 7 based on the NNFGAIL series was inactive at all ratios tested, which seemed consistent since its peptide parent NNFGAIL-NH2 is reported in the literature as the shortest hIAPP sequence forming fibers of identical morphology as the 37 isoform peptide. 62 The flexible β-hairpin like 8 bearing an AIL peptide at the Nterminus and a F-(1,4-Tz-CF2)-V arm at the C-terminus significantly delayed the fibrillization kinetics (Δ t1/2 = +106%) and decreased the fibrillization process (ΔF=-64 %) at ratio 10/1 as seen in figure 9 and table 3. At the lower ratio 1/1, the activity was weaker with only a modest reduction of the fluorescence plateau (ΔF=-28 %) and no effect on the kinetics.

In comparison, the shorter analogue 9 bearing a F-(1,4-Tz-CF2) arm at the C-terminus retained the activity but to a lesser extent, being half active as the longer analogue 8 (Δ t1/2 = +46% and ΔF = -37 % at 9/hIAPP 10/1 ratio). No noticeable effects were observed at the lower ratio. Overall, this activity was observed only at 10:1 ratio while the peptide analogue was still active at the lower ratios 1:1 and 0.1:1. 55 Thus, among the nine evaluated compounds, homotriazolamer 1 and heterotriazolamer 5 showed the higher ability to interact with hIAPP at compound/hIAPP 10/1 ratio. However, while 1 adopting an extended conformation dramatically increased the fibrillization process, on the contrary the folded heterotriazolamer 5 decreased it. Furthermore 5 is more active than its natural parent FGAIL-NH2.

Effect on hIAPP oligomerization by native and ion mobility Mass Spectrometry experiments

Mass spectrometry (MS) has been used as a valuable technique to identify oligomeric species formed during the early oligomerization process of amyloid peptides and proteins. 76 It represents a complementary method to the Thtfluorescence assay which is more focused on the subsequent formation of fibrils. Moreover, it is a powerful tool to study the mechanism of hIAPP aggregation modulators. [77][78][79][80][81][82] In order to have information on the effect of these series of foldamers on the oligomerization process of hIAPP, native mass spectrometry (MS) experiments, using our reported method, 77,29,55 were carried out at different incubation times (from T0h to T2h, corresponding to the period when monomers and small oligomers are visible and allowing to follow the effect of compounds). Three foldamers, displaying different Tht-fluorescence behaviours, were selected and tested: the homotriazolamer 1, accelerating hIAPP fibrillization, the FGAIL-built heterotriazolamer 5, demonstrating a delaying effect and the longer NNFGAILbased heterotriazolamer 7, being inactive on hIAPP fibrillization. Compounds were tested at 3 different foldamers/hIAPP ratios: 10/1, 5/1 and 1/1 and as their effects were similar at the 2 higher ratios and they showed no effect at the lowest ratio, we will only discuss the 5/1 ratio. As already reported, the MS spectrum of hIAPP alone at T0 showed a common set of multicharged monomeric and oligomeric species; from dimers to hexamers that decreased progressively and at T2h, only monomers to trimers were still visible (Figure 10a). In the presence of homotriazolamer 1 at T0h, only hIAPP monomeric, dimeric and trimeric species were detected (M, 2M and 3M), the larger oligomers being undetectable (Figure 10b). The spectrum was similar at T2h concerning these species. Noticeably, non-covalent complexes of 1 with hIAPP monomers and dimers ([M+1], [M+2.1], [M+3.1], [2M+1] and [2M+2.1]) were seen at T0 but become indiscernible at T2h. Some clusters of 1 were also detected, from [2.1] to [4.1], indicating self-aggregation of 1 or aspecific associations maybe due to its relatively high concentration (Figure 10b). In the presence of heterotriazolamer 5 (Figure 10c), at T0h, only hIAPP monomers and (M) dimer (2M) were detected while larger oligomers, trimers to hexamers, were invisible. Moreover, 14 non-covalent complexes of 5 with almost exclusively hIAPP monomers were seen (from [M+5] to [M+10.5]) at T0 and some were stable at T2h, indicating a strong interaction between heterotriazolamer 5 and hIAPP monomer. Like for 1, some clusters of 5, from [2.5] to [9.5], were also detected only at the intial time of the kinetics. In the presence of the longer heterotriazolamer 7, at T0h only monomeric species of hIAPP were detectable (M) with an intensity significantly weaker than for the control experiment (Figure 10d). Non-covalent complexes of 7 with hIAPP monomeric forms were seen ([M+7] to [M+5.7]) at T0h and some of them were stable at T2h ([M+7] and [2M+7]). Then, we performed some ion mobility experiments that allowed the separation of conformers according to their shape, charge and collisional cross section, in order to both describe the qualitative and quantitative conformational profile of specific ions belonging to hIAPP and foldamer complexes between T0 and T2h. 77 In contrast some ions belonging to hIAPP and 5 complexes like M+5 and M+2.5 exhibited changes in conformer abundances with the stabilization of more compact conformers between T0 and T2h (Figure 48S b), SI). Ion mobility data could not be compared for 1 since all hIAPP + 1 complexes disappeared at T2h. Finally, for 7, that display no effect on fibrilization, no change in conformer abundances between T0 and T2h were observed. Overall, these MS experiments revealed differences between the three foldamers 1, 5 and 7 in particular regarding the nature, number and shape of non-covalent complexes of foldamers with hIAPP monomers/oligomers formed. Foldamer 1, a fiber formation accelerator in the ThT experiments, interacts slightly with the monomer while these non-covalent complexes as well as hIAPP oligomers disappear very quickly. Compound 7, being inactive in the fibrillization process, also interacts with monomeric species to form non-covalent complexes which also disappear fairly quickly, and does not affect the shape and abundance of conformers. On the contrary, foldamer 5, which significantly delays the fibrillization process in ThT assays, forms numerous noncovalent complexes almost exclusively with hIAPP monomer, which are more compact and stable over time. Thus, it can be hypothesized that foldamer 5 interacts more strongly with hIAPP monomer, producing its sequestration and favoring a compact conformer that could explain its fibrillization delaying effect.

Interaction with hIAPP by NMR and CD

Proton fluorine-19 NMR experiments reveal compounds 1 and 5 interaction with hIAPP

The effect of compounds 1 and 5 on hIAPP was first studied using proton NMR experiments and the spectra are shown in Figure 10a. To evaluate the aggregation of hIAPP in the absence and in the presence of compounds 1 and 5, resonances from amide (N-H) and aromatic protons were analyzed. It should be noted that the amide proton resonances are, in particular, sensitive to the size of the aggregates and often not visible for large-aggregates and fibers. At 0.5 hour, 50 µM of hIAPP (black curve) presented a high-resolution proton NMR spectrum with peaks from amide and aromatic protons well resolved. NMR spectra of 250 µM of 1 and 5 compounds in the absence of hIAPP showed a few strong peaks that overlap with amide-N-1 H peaks from hIAPP amide protons. Therefore, for a comparative analysis of the effect of compounds 1 and 5 on hIAPP, a few nonoverlapping amide and aromatic peaks in the region between the dotted (vertical) lines were considered (Figure 11a). At 0.5 hour, a line-broadening was observed in the presence of 1 or 5 compounds, which could be due to compounds 1 or 5 interacting with monomeric hIAPP, hIAPP aggregation, or to the influence of compounds 1/5 binding on hIAPP aggregation (Figure 49S, SI). The interaction of compounds 1/5 with hIAPP was next probed using 19 F NMR experiments. The 19 F NMR spectra of the compounds with and without hIAPP are shown in Figure 11b. Two additional 19 F NMR peaks (~-75.4 and -75.6 ppm) appeared when 1 was mixed with 50 µM hIAPP, as shown in green, when compared to the spectrum of the compound alone (Figure 11b). The total intensities of these new peaks are ~12% and ~60% relative to the intensities of peaks observed at ~88 ppm in the absence of hIAPP (Figure 50S, SI). 5 that showed a single peak at -75.6 ppm in the absence of hIAPP presented two peaks ~-75.4 and -75.6 ppm (Figure 11b, blue) in the presence of hIAPP. Similar to 1, a 19 F signal intensity loss of ~40% was observed for the peaks in the ~-86-88 ppm region relative to 5 in the absence of hIAPP (Figure 50S, SI). Taken together, the appearance of new 19 F NMR peaks indicates the interaction of compounds 1 and 5 with hIAPP. The measured differences in signal intensities may be attributed to the presence of two different compound populations, hIAPP-bound and hIAPP-unbound, in the mixture containing 5 molar excess of 1 or 5.

DOSY NMR experiments reveal an increase in the species size

The size of species present in the samples of compounds 1 and 5 incubated for ~1 hour with and without hIAPP was next determined by measuring the diffusion rate constants using diffusion ordered NMR spectroscopy (DOSY). As shown in Figure 51Sb (SI), the diffusion rate constants measured for 1 in the absence of hIAPP are ~3.9×10 -10 m 2 /s for peaks near ~8.45 ppm. On the other hand, incubation of 1 with hIAPP showed a slightly slow diffusion rate constants ~4.2×10 -10 m 2 /s for the peaks at ~8.45 (green peaks), respectively, indicating an increase in particle size (Figure 51Sa). Similarly, 5 in the presence of hIAPP (blue peaks) showed a slow diffusion rate constant suggesting its complexation with hIAPP. The same diffusion rates (~4.5×10 -10 m 2 /s) measured for the two proton peaks ~7.0 to 7.1 ppm region in the absence of hIAPP, were found to be different in the presence of hIAPP. Further, the time-evolved NMR measurements showed a significant loss of amide proton signals after ~24 hours of sample incubation at room temperature (Figure 51Sa, SI) in the absence of compounds as reported elsewhere 83 and also in the presence of compound 1, which correlate to our ThT fluorescence observation.

2D SOFAST-HMQC experiments reveal hIAPP aggregation promoted by compound 1 and interaction of 5 with the Nterminal and central parts of hIAPP

To determine the compounds 1/5 binding sites in hIAPP, we probed the interaction between 1/5 with hIAPP using 2D heteronuclear 1 H/ 15 N SOFAST-HMQC NMR spectroscopy. A decrease in signal intensity and chemical shift perturbation was observed in the presence of compounds 1/5 as seen in the spectra (Figure 12a) and as summarized in plots (b and c). 70 µM of uniformly 15 N-labelled hIAPP in the presence of 5 molar excess of 1 or 5 presented a substantial loss in signals for most of the residues in hIAPP: ~40% loss for 1 (Figure 11b, green) and ~20% loss for 5 (Figure 11b, blue). The loss of NMR signal could be attributed to hIAPP aggregation, which correlates to our fluorescence observation that showed faster aggregation kinetics for hIAPP in the presence of 1. Chemical shift perturbation analysis presented a relatively higher perturbation in the presence of 5 as compared to 1 (Figure 12c). The N-terminal (10 to 17) and central (25 to 28) residues presented CSP above the average CSP in the presence of 5; on the other hand, in the presence of 1, above the average CSP value was observed only for Asn21 located in the central region of hIAPP (Figure 12c). Next, the effect of compounds 1 and 5 on hIAPP conformation was measured using far-UV circular dichroism as shown in Figure 52S (SI). 50 µM of hIAPP dissolved in 10 mM Tris, 100 mM NaCl, pH 7.4 containing 95% H2O and 5% methanol presented minima ~200 nm suggesting a disordered conformation. We note that 1 did not show a characteristic CD spectrum (black); essentially, because of the lack of chirality whereas 5 showed a minimum centered ~200 nm (Figure 52S, SI, purple). Titration of 5 molar excess of 1 or 5 to hIAPP did not significantly alter the secondary structure of hIAPP. Although hIAPP in the presence of 1 and 5 showed CD spectra indicating mainly a random-coil rich conformation, a slight increase in ~222 nm peak minimum (both green for 1 and blue for 5, Figure 52S, SI) possibly suggests an induction of short α-helical structure. This observation correlates to the NMR results that showed NMR observable peaks during the ~1-hour measurement indicating no quick hIAPP fibrillation in the presence of compounds 1 and 5.

Conclusions

We described the conformational preferences and activity on hIAPP amyloid protein aggregation of novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units. We demonstrated by NMR experiments and MD simulations that while homotriazolamers of 1,4-Tz-CF2 unit (1 and 2) preferentially adopt an extended conformation, heterotriazolamers (4-7) showed a preferential tendency to adopt folded hairpin-like conformation and sometimes ladder-like structures similar to short multi-stranded βsheets in the case of the longer compounds 6 and 7. The electrostatic interaction between the two fluorine atoms and the neighbouring triazole and the NH of the successive amino acid unit maintains the extended constraint at the C-terminal part of each 1,4-Tz-CF2 unit but the fluorine atoms are not involved in the overall folding of heterotriazolamers 4-7. The folding of these foldamers is due to a kink at the natural amino acid between two triazole units. We hypothesize that π-stacking and/or dipole-dipole interactions between triazole rings can trigger intermolecular interactions between the less flexible and extended homotriazolamers inducing their aggregation. On the contrary, these triazole-triazole interactions appear to play a critical role in intramolecular interaction and folding of the more flexible heterotriazolamers, to induce hairpin-like conformation when two triazoles are present (4 and 5) and ladder-like structures when three triazoles units are present (compounds 6 and 7). Very interestingly, these three different conformational behaviours are linked to the ability of the foldamers to interact with hIAPP and influence their activity on hIAPP aggregation. While extended homotriazolamer 1 accelerates and enhances the aggregation of hIAPP, flexible heterotriazolamer 5 decreases it and the more folded heterotriazolamer 7 has not effect, as shown in thioflavin-T fluorescence assays. The activity of 5 was dramatically increased compared to those of the natural peptide parent FGAIL. N-terminal acetylation of 5 is favorable for the inhibitory activity on hIAPP aggregation, as the free amine 4 or the N-Boc protected intermediate 3 did not show any interesting activity. 1 H and 19 F NMR studies confirmed interactions of 1 and 5 with hIAPP. In particular for 5, interactions with sequences directly involved in hIAPP aggregation process (10-17 and 21-27) were observed. 56 Mass spectrometry experiments demonstrated that foldamer 5 interacts with hIAPP monomer to trap it to favor a compact conformer delaying its aggregation. This study highlights the potential of fluorinated peptidomimetic foldamers which were previously unknown as potential modulators of amyloid aggregation or even more widely in the vast field of medicinal chemistry. This work also confirms the interest of fluorine "labeling" for studying by 19 F-NMR spectroscopy biological events such as ligandbiomolecule interactions structure and open the perspective to use fluorinated foldamers for 19 F MRI medical imaging. 84,85 Experimental Section

Synthesis of foldamers

The protocols of synthesis of the intermediates 10, 12-14, 21-31 are given in Supporting Information.

General procedure A : procedure for the synthesis of Boc-protected aminoacid propargylamide

To a solution of N-(tert-Butoxycarbonyl)-L-aminoacid (1.0 eq.) in CH2Cl2 (0.38 M) was added propargylamine (1.1 eq.), HOBt (1.1 eq.) and EDC (1.1 eq.). DIPEA (2.5 eq.) was added at 0 °C and then the mixture was let to stir at room temperature for 5 hours. After completion, the solution was diluted with water and extracted with EtOAc. The combined organic layers were successively washed with 10% aqueous solution of citric acid, 10% aqueous solution of K2CO3, H2O and brine, dried over Na2SO4, filtered and concentrated under vacuum. The crude product was used in next step without further purification.

General procedure B : procedure for difluorinated azidesbearing amino acid residues

To a solution of bromodifluoroethylacetate (1.0 eq.) in DMF (0.5 M) was added NaN3 (1.15 eq.) and the mixture was stirred at 35 °C overnight. After completion (monitored by 19 F NMR) triethylamine (1eq.) and suitable aminoacid (1 eq.) were immediately added and then the mixture was heated in an ultrasound bath for 4 h. The solution was diluted with water (10 mL) and extracted with Et2O (2 x 10 mL). The organic layers were successively washed with H2O (10 mL) and brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography using the appropriate solvents.

General procedure C : procedure for the Huisgen click reaction for 1,4-Tz-CF2 based oligomers

To a solution of corresponding azidodifluoroacetylated aminoacid (1.0 eq.) and Boc-protected propargylamide or propargylamine (1.0 eq.) in MeOH (0.124 M) were successively added CuSO4•5H2O (5 %), Asc.Na (20 %), and TBTA (5 %). The mixture was stirred for 2 to 4 hours at room temperature. After completion, monitored by TLC, the solution was diluted in water and extracted with EtOAc. The combined organic layers were successively washed with water and brine, dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography using the appropriate solvents.

General procedure D : General procedure for the coupling step First, benzyl esters (15, 16, 18) were deprotected using triethylsilane (2 eq.) and palladium on carbon 30% (0.3 eq.) in MeOH (0.1 M) for 30 minutes at room temperature. The mixture was filtered on a short pad of celite, concentrated under vacuum to provide the corresponding free acids. Secondly, Boc-protected N-difluoromethyltriazoles (17, 18) were deprotected using a solution of HCl in dioxane (4.0 M, 10 eq.) in dry CH2Cl2 (0.1 M) for 1 hour at room temperature. The mixture was concentrated under vacuum to provide respectively the free amines from 17 and 18 which were used in next step for the coupling reaction with the free acid from 15, 16 and 18 previously obtained. So, to a solution of the corresponding acid (1 eq.) in dry DMF (0.38 M) were successively added HOBt (1.2 eq.) and EDC (1.2 eq.). Then, collidine (5 eq.) and the corresponding amine were added at 0 °C. The mixture was let to stir at room temperature for 3 to 5 hours. After completion, the mixture was diluted with water and extracted with EtOAc. The combined organic layers were successively washed with 10% aqueous solution of citric acid, 10% aqueous solution of K2CO3, H2O and brine, dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by flash chromatography using the appropriate eluant.

BocNH-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Heptapeptide (3)

The product was obtained following the general procedure D from benzyl ester 15 (240 mg, 0.40 mmol) and 17 (162 mg, 0.40 mmol) which were both deprotected. The crude product obtained was purified by flash chromatography (CH2Cl2/MeOH: 99/1 to 90/10) to obtain 3 (154 mg, 0.19 mmol, 48 %) as a white solid.; Rf = 0.

NH2-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Heptapeptide (4)

To a solution of 3 (100 mg, 0.126 mmol,1 eq.) in dry CH2Cl2 was added drop by drop a solution of HCl in dioxane (4.0 M, 10 eq.) . After 2 hours, the mixture was concentrated under vacuumand the crude hydrochloride salt 4 obtained (90 mg, 0.122 mol, 97%) was purified to get a higher purity by preparative HPLC eluting with H2O + 0.2% formic acid/CH3CN (gradient 5 to 80% in 20 min) column Pursuit 5 C18 250*10.0 mm Rf = 0.1 (EtOAc/MeOH : 8/2); IR: 3256, 3104, 3016, 2904, 1675, 1586, 1454, 1224, 1192 cm -1 ; Melting point: 170-172°C; 697.2950, found 697.2922. HPLC purity: Sunfire C18 3.5 μm; H2O + 0.1 % formic acid/ACN, gradient 5-100 % in 30 min; Rt = 9.43 min, 94 %. 1 H and 13 C NMR data are given in table 1S, SI. 1 H, 13 C and 19 F spectra are respectively given in figures 4S, 5S and 6S, SI.

AcNH-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Heptapeptide (5)

To a solution of unprotected heptapeptide 4 (50 mg, 0.07 mmol, 1.0 eq.) in dry methanol (0.1 M) was added acetic anhydride (1.5 equiv.) and triethylamine (10 mg, 0.1 mmol.,1.5 eq.). After completion, monitored by TLC, the mixture was concentrated under vacuum and purified by silica column chromatography using CH2Cl2/MeOH (9/1) as eluant to provide 5 (44 mg, 0.06 mmol, 88 %) as a white solid; Rf = 0.2 (CH2Cl2/MeOH: 9/1); IR: 3268, 3123, 3100, 2976, 1611, 1546, 1521, 1412, 1402, 1318, 1211, 1132 cm -1 ; Melting point: 138-140°C; 761.2958, found 761.2961. HPLC purity: XSelect C18 3.5 μm; H2O + 0.1 % formic acid/ACN, gradient 5-100 % in 30 min; Rt = 12.17 min, 97 %. 1 H and 13 C NMR data are given in table 3S, SI. 1 H and 19 F spectra are respectively given in figures 11S and 12S, SI.

NH2-Asn-[TzlCF2]-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Decapeptide (6) 2 mL of a cleavage cocktail composed of TFA/H2O/TIPS/thioanisole (90/5/2.5/2.5, v/v/v/v) was added to compound 20 (10 mg, 0.009 mmol, 1 eq.) After 2 hours of agitation at room temperature, the mixture was precipitated in Et2O at 0°C and purified by preparative HPLC (H2O + 0.2% formic acid/CH3CN 5 to 80% in 20 min, column Pursuit 5 C18 250*10.0 mm) and the compound 6 was obtained (5.5 mg, 0.0056 mol, 61%) as a white foam after lyophilization.

AcNH-Asn-[TzlCF2]-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Decapeptide (7)

To a solution of compound 6 (50 mg, 0.051 mmol, 1eq.) in dry methanol (0.1 M) was added acetic anhydride (7.8 mg, 0.077 mmol, 1.5 eq.) and triethylamine (3 eq.). After completion, monitored by TLC, the mixture was concentrated under vacuum and purified by preparative HPLC (H2O + 0.2% formic acid/CH3CN 5 to 80% in 20 min, column Pursuit 5 C18 250*10.0 mm) and the compound 7 was obtained (25.9 mg, 0.025 mmol, 50 %) as a white solid.

BocNH-Asn(Trt)-[TzlCF2]-Phe-OBn Tetrapeptide (16)

The product was obtained following the general procedure C from 11 (600 mg, 1.2 mmol) and 13 (358 mg, 1.2 mmol). The crude product obtained was purified by column chromatography (CH2Cl2/MeOH: 99/1) to yield 16 (638 mg, 0.72 mmol, 60 %) as a white solid.; Rf = 0.4 (CH2Cl2/MeOH: 99/1); IR: 3252, 3111, 2910, 2904, 2873, 1598, 1512, 1443, 1212, 1102, 996 cm -1 ; Melting point: 82-84°C; 1 H NMR (300 MHz, CDCl3): δ 7.87 (s, 1H), 7.38-7.17 (m, 25H), 7.04-7.00 (m, 3H), 6.00 (br s, 1H), 5.24 (d, 2 J = 11.8 Hz, 1H), 5.17 (d, 2 J = 12.1 Hz, 1H), 4.97 (q, 3 J = 6.5 Hz, 1H), 4.58-4.53 (m, 2H), 4.49-4.45 (m, 1H), 3.21 (d, 3 J = 5.3 Hz, 2H), 3.12 (dd, 2 J = 15.9 Hz, 3 J = 5.4 Hz, 1H), 2.61 (dd, 2 J = 15.9 Hz, 3 J = 5.4 Hz, 1H), 1.42 (s, 9H);

BocNH-[TzlCF2]-Leu-NH2 Tripeptide (17)

The product was obtained following the general procedure C from Boc protected propargylamine (388 mg, 2.5 mmol) and 14 (623 mg, 2.5 mmol). The crude product was purified by column chromatography (c-Hex/EtOAc: 4/6) to provide 17 (727 mg, 1.8 mmol, 72 %) as a white solid.; Rf = 0.

BocNH-[TzlCF2]-Ala-OBn Tripeptide (18)

The product was obtained following the general procedure C from Boc protected propargylamine (465 mg, 3.0 mmol) and 12 (894 mg, 3.0 mmol). The crude product obtained was purified by column chromatography (c-Hex/EtOAc: 7/3) to yield 18 (1.0 mg, 2.3 mmol, 76 %) as a white solid.; Rf = 0.

BocNH-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Hexapeptide (19)

The product was obtained following the general procedure D from 18 (313 mg, 0.69 mmol) and 17 (279 mg, 0.69 mmol) which were both deprotected. The crude product obtained was purified by flash chromatography (CH2Cl2/MeOH: 99/1 to 90/10) to yield 19 (220 mg, 0.34 mmol, 49 %) as a white solid.Rf = 0.

NMR Conformational studies

Proton NMR spectra were recorded on Bruker spectrometers operating at 600 MHz, equipped with z-gradient TCI and 19 F-QCI cryprobes (ICSN, Institut de Chimie des Substances Naturelles, Gifsur-Yvette). 1 H and 13 C resonances were completely assigned using 1D 1 H WATERGATE, 2D 1 H-1 H TOCSY (mixing time set to 30, 60 or 100 ms), 2D 1 H-1 H ROESY (200 ms mixing time), 2D 1 H-13 C HSQC and 2D 1 H-13 C HMBC spectra, recorded at 278 K and 298 K. 1 H and 13 C chemical shifts were calibrated using the solvent residual peak (H2O/D2O, δ 1 H 4.70 ppm). The chemical shifts deviations were calculated as the differences between observed chemical shifts and random coil values reported in water reported by Wishart et al. ( 1 H, 13 C and 15 N random coil NMR chemical shifts of the common amino acids). Vicinal coupling constants were extracted from 1D 1 H spectrum and from 2D 1 H-1 H COSY at 283 K and 313 K. Temperature coefficient (ΔδHN/ΔT) of the amide protons were calculated between 283-313K.

Computational methods 4/5 and 6/7 three-dimensional structures were generated using the program MarvinSketch 6.2.1 from ChemAxon (https://www.chemaxon.com/). Each compound was placed in a cubic simulation box so that the minimum distance between the solute and the cube sides was 1.4 nm. Afterward, both simulation boxes were filled with explicit methanol molecules. Molecular dynamics simulations were performed by using the GROMACS 2019.1 package (Abraham et al., 2015) with the Generalized AMBER Force Field (GAFF) (Wang et al., 2004) for both solute and methanol solvent. The length of the covalent bonds involving hydrogens was kept constant using the LINCS procedure (Hess, 2008). Lennard-Jones potentials were cut-off at 1.2 nm and electrostatic interactions were treated using the smooth PME method (Essmann, 1995). After two short simulations of 1 ns each to bring the system temperature and pressure around T = 300 K and P = 1 bar, each system was simulated during 200 ns in the isothermal-isobaric ensemble using the Nose-Hoover (Nosé, 1984; Hoover, 1985) and Parrinello-Rahman (Parrinello and Rahman, 1981) coupling methods. Molecular coordinates were saved every 20 ps for subsequent analyses by GROMACS tools, including gmx angle for extracting torsion angle values.

Fluorescence-Detected ThT Binding Assay (hIAPP). hIAPP, purchased from Bachem, was dissolved in pure hexafluoroisopropanol (HFIP) at a concentration of 1 mM and incubated for 1 hour at room temperature to dissolve any preformed aggregates. Next, HFIP was evaporated with dry nitrogen gas followed by vacuum desiccation for 3 hours. The resulting peptide film was then dissolved in DMSO to obtain stock solutions of hIAPP (0.2 mM). Stock solutions of foldamers were dissolved in DMSO or in water (10, 1 and 0.1 mM). The concentration of DMSO was kept constant at 3% (v/v) in the final volume of 200 µL. Thioflavin-T binding assays were used to measure the formation of fibrils over time. A plate reader (Fluostar Optima, BmgLabtech) and standard 96-wells flat-bottom black microtiter plates in combination with a 440 nm excitation and 480 nm emission filters were used. The ThT assay was started by adding 5 µL of a 0.2 mM hIAPP stock solution to a mixture of 10 µM ThT and 10 mM Tris/HCl, 100 mM NaCl at pH 7.4 containing 1 µL of stock solutions of foldamers. The concentration of IAPP was held constant at 5 µM and foldamers were added to yield foldamer/IAPP ratios of 10/1, 1/1 and 0.1/1. The ThT assays were performed in triplicate and between 2 and 4 times on different days, with the same batch of peptide. The ability of foldamers to inhibit hIAPP aggregation was assessed considering the time of the half-aggregation (t1/2) and the intensity of the experimental fluorescence plateau (F), both values were obtained by fitting the obtained kinetic data to a Boltzmann sigmoidal curve using GraphPad Prism 5. The relative extension/reduction of t1/2 is defined as the experimental t1/2 in the presence of the tested compound relative to the one obtained without the compound and is evaluated as the following percentage: [t1/2 (hIAPP + compound) -t1/2 (hIAPP)] / t1/2 (hIAPP) × 100. The relative extension/reduction of the experimental plateau is defined as the intensity of experimental fluorescence plateau observed with the tested compound relative to the value obtained without the compound and is evaluated as the following percentage: (F hIAPP + compound -F hIAPP) / F hIAPP × 100. Curves of the tested compounds are fitted to a Boltzmann sigmoidal model, normalized to the control experiment and represented in supporting information for 1-9, FGAILNH2, and NNFGAILNH2 (Fig. 47S, SI).

ESI-MS experiments

were carried out with a Synapt G2-Si Q-TOF instrument (Waters, Manchester, UK) equipped with an ESI interface. Positive ion mode was used. Samples of hIAPP (pretreated with HFIP as previously described for the fluorescence-detected ThT binding assay) was reconstituted in 50 mM ammonium acetate buffer pH 3.7 at 100 µM. Compounds 1, 5 and 7 were dissolved in DMSO and added to the peptide solution to reach final concentrations of 1 mM, 500 and 10 µM, corresponding respectively to ratios 10/1, 5/1 and 1/1 of compounds/hIAPP; the final concentration of DMSO in the ammonium acetate buffer being of 5% (v/v). Samples were injected by direct infusion through a syringe at 5 µL/min. Main MS parameters are detailed the following: the capillary voltage was set at 2.4 kV, with the sampling cone and source offset at 70V. Source and desolvation temperatures were respectively 40 °C and 75°C. Data were processed with MassLynx.

Ion mobility experiments were performed using a Travelling Wave Ion Mobility device, equipped on the G2-Si Q-TOF. Briefly, ion mobility parameters were: Trap gas flow = 7 ml/min; Helium gas flow120, IMS gas flow = 45, IMS wave velocity = 110 m/sec, wave height 40 V. Ion mobility data were similarly processed using MassLynx™ software.

NMR experiments

Synthetic human-IAPP peptide was purchased from AnaSpec at > 95% purity. The peptide was used without any further purification. It was dissolved in hexafluoroisopropanol (HFIP) and kept on ice for 30 minutes. HFIP treated peptide aliquots each containing 0.1 mg of human-IAPP were next subjected to lyophilization for 24 hours. The lyophilized hIAPP peptide was dissolved in d11-10mM TRIS, 100 mM NaCl, pH 7.4 (or d3-30 mM sodium acetate buffer, pH 5.5) as required for biophysical measurements. Uniformly-15 N-labelled hIAPP was biologically expressed and purified. 15 N-hIAPP peptide samples were prepared in the same way as the unlabeled peptides. Proton and 19 F NMR spectra were recorded on a 500 MHz Bruker NMR spectrometer equipped with a z-axis gradient triple-resonance probe ( 1 H/ 19 F/ 13 C). 2D SOFAST-HMQC spectra of uniformly 15N labelled hIAPP peptide were recorded on a 800 MHz Bruker spectrometer equipped with a cryoprobe. Proton NMR spectra of 50 µM of unlabeled hIAPP was recorded in the absence and presence of 5 molar excess of JAL235/JLA369. The NMR sample was prepared in 10 mM TRIS, 100 mM NaCl, pH 7.4 buffer containing 87.5% H2O, 10% D2O, and 2.5% DMSO-d6. 2D SOFAST-HMQC spectra were recorded for 70 µM of uniformly-15N-labelled hIAPP in the absence and presence of 5 molar excess of 1 or 5. The NMR sample for SOFAST-HMQC measurement was prepared in 30 mM d3-sodium acetate buffer, pH 5.5 containing 87.5% H2O, 10% D2O, and 2.5% DMSO-d6. 2D SOFAST-HMQC spectra were acquired at 25 °C with 32 scans and 128 t1 increments. 1 H, 19 F and 2D SOFAST-HMQC NMR spectra were processed using TopSpin 3.5 (Bruker) and the 2D SOFAST-HMQC spectra were analyzed using Sparky. The 19 F signal intensities were calculated using the Mestrelab MNova program.

CD experiments

Compounds 1 and 5 were dissolved in 100% deuterated-DMSO (DMSO-d6) at a concentration of 20 mM. 50 L of compounds dissolved in 100% deuterated-DMSO were lyophilized and resuspended in methanol for circular dichroism (CD) measurements. Far-UV CD spectra of 50 µM hIAPP in the absence and presence of 5 molar excess of compounds (1 or 5) were recorded using a JASCO (J820) spectropolarimeter. CD measurements were carried out with 16 scans at 25 °C. The average CD spectra were plotted after subtracting the buffer signal (10 mM TRIS, 100 mM NaCl, pH 7.4) containing 95% H2O and 5% methanol. To observe the interference of compounds in the Far-UV CD spectrum of hIAPP, CD spectra of 250 µM JLA235 or JLA369 were also recorded with an identical experimental setup as described above in the absence of hIAPP.

Figure 1 . 9 Scheme 1 .

 191 Figure 1. Synthesized and studied fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2): homotriazolamers 1 and 2, heterotriazolamers 3-7 and β-hairpin like compounds 8 and 9

Figure 2 .

 2 Figure 2. NMR conformational analysis of heterotriazolamers 4, 5, 6 and 7: long-range 1 H-1 H ROE (red) and 1 H-19 F ROE (green) correlations

  SI). The arms of both conformers showed the characteristic pattern of sequential CHαi/NHi+1 ROEs typical of extended peptides (Figure4, blue arrow). This sequence was interrupted only for the terminal Ala-1, further confirming the more flexible Nterminus. Both conformers had the typical ROE pattern of a βturn inducer (Figure4, green arrows55 ). Here again, conformer A presented numerous spatial proximity signals between the upper and the lower arms, unlike conformer B, (Figure4, red arrows). H α Leu-3 had ROE signal with protons in positions 2 and 6 respectively for conformers A and B of the piperidine ring. Both observations confirmed the presence of a more closed (conformer A) and one more open (conformer B) architectures.

4 and 5

 5 measured by NMR or computed by MD simulations in methanol at 300 K Regarding interproton distances within the two molecules, the NMR observations are partially retrieved in MD simulations. As shown in Figure 5, distances between Phe1 Hα or Hβ protons and Hc of TZL1, as well as distances between Ala2 Hβ protons and Hc of both neighbouring TZL1 and TZL2, reached values below 0.5 nm in significant number of conformations, which is consistent with detected NOE. Nevertheless, contrary to NMR experiments of compound 5, but consistently with those of compound 4, we did not observe close proximity of Phe1 Hα (or Hβ) with Ala2 Hβ in MD simulations. This indicates that the Phe1-TZL1-Ala2 segment is more extended in compound 5 simulation than in vitro. A similar extended conformation of the second half Ala2-TZL2-Leu3 segment in both compounds is supported by the non detection of long range NOE between Ala2 and Leu3 protons.

Figure 5 .

 5 Figure 5. Distribution of interproton distances computed from MD trajectories of compound 4 (black lines) and compound 5 (red lines). Vertical green lines indicate the distance below which NOE can be detected.

Figure 6 .

 6 Figure 6. Representative structures of the five most populated clusters of compounds 4 (left column) and 5 (right column). The RMSD threshold for clustering is 0.3 nm. Since MD simulations of molecules 4 and 5 could generate conformational ensembles in fair agreement with NMR experiments, we used the same method to propose structural models of compounds 6 and 7. MD trajectory visualizations show that both molecules 6 and 7 are highly flexible and dynamically adopt different conformations, the most representative of which are displayed in Figure 7. Like in compounds 4 and 5, we noticed that the 3 Asn-TZL, Phe-TZL,

Figure 7 .

 7 Figure 7. Representative structures of the five most populated clusters of compounds 6 (left column) and 7 (right column). The RMSD threshold for clustering is 0.4 nm.

Figure 8 .

 8 Figure 8. Distribution of interproton distances computed from MD trajectories of compound 6 (black lines) and compound 7 (red lines). Vertical green lines indicate the distance below which NOE can be detected.

Figure 9 .

 9 Figure 9. Representative curves of ThT fluorescence assays over time showing hIAPP control aggregation (5 µM) in the absence (red curve) and in the presence of compounds 1, 5, 8 and 9 at compound/hIAPP ratios of 10/1 (purple curves) and 1/1 (green curves). The control curves are represented in orange lines and buffer in grey.

Figure 10 . 1 )

 101 Figure 10. 1) Mass spectra recorded in the the 500-5000 m/z range of hIAPP peptide control at T0h (hIAPP monomer is labeled in bold black as M and hIAPP oligomers as 2M, 3M … for respectively dimer, trimer…) (a) and at T2h (b) ; 2), 3) and 4) : Mass spectra recorded in the the 500-5000 m/z range of hIAPP in the presence of triazolamers 1, 5 and 7 at T0h (a) and T2h (b). The different species are labeled with the number of hIAPP monomer and with the addition of n molecules of dedicated triazolamer corresponding to this complex (e.g. 2M+2.1 in bold red) and the clusters of triazolamers are labeled in bold green. Peaks labeled with an asterisk belong to contaminants. For clarity reasons, the charge states of ions are not mentioned.

  hIAPP control charges states monomer did not exhibit any change in both qualitative and quantitative conformational landscape from T0 to T2h (Figure 48S a), SI).

Figure 10 .

 10 Figure 10. Proton (a) and fluorine-19 (b) NMR spectra of 50 µM hIAPP in the presence and absence of 5 molar excess compounds (1 and 5) as indicated, and the reference spectra of 250 µM compounds (1 and 5). The proton NMR spectra of hIAPP mixed with compounds shown in green and blue were recorded after ~1 hour of sample preparation.

Figure 12 a

 12 Figure 12 a. 2D SOFAST-HMQC spectra of 70 µM uniformly-15 N-labelled hIAPP in the absence (red) and presence of 5 molar excess of 1 (green) and 5 (blue). The resonance assignment was adopted from a previous study. 12 b. Relative change in intensities measured from 2D SOFAST-HMQC spectra (a); where I0 and I are the signal intensities for hIAPP and hIAPP+triazolamers, respectively. c. Observed chemical shift perturbation (CSP) for hIAPP residues in the presence of 1 (green) and 5 (blue). The average (NH) CSP was calculated using the formula CSP = √{(ωHNΔ 1 HN) 2 + (ωNΔ 15 N) 2 }, where ωH = 1 and ωN = 0.154. The asterisks in (b) and (c) represent unassigned residues of hIAPP.

2 ( 2 J

 22 CH2Cl2/MeOH: 95/5); IR: 3276, 3164, 3113, 2932, 2897, 1611, 1546, 1300, 1234 cm -1 ; Melting point: 122-124°C; 1 H NMR (400 MHz, CD3OD):δ 8.40 (s, 1H), 8.23 (s, 1H), 7.26-7.20 (m, 5H), 4.59-4.58 (m, 6H), 4.30 (t, 3 J = 7.3 Hz, 1H), 3.01 (dd, 3 J = 13.6 Hz, 2 J = 8.1Hz, 1H), 2.99 (dd, 3 J = 13.6 Hz, 2 J = 8.1 Hz, 1H), 1.81-1.69 (m, 3H), 1.50 (d, 3 J = 7.2 Hz, 3H), 1.39 (s, 9H), 0.99 (d, 3 J = 6.2 Hz, 3H), 0.96 (d, 3 J = 6.2 Hz, 3H); 13 C NMR (400 MHz, CD3OD):δ 174.6, 173.1, 172.3, 158.6 (t, (C,F) = 30.8 Hz), 158.4 (t, 2 J(C,F) = 30.8 Hz), 146.1, 145.8, 137.0, 129.0, 128.0, 126.4, 121.3, 121.1, 110.5 (t, 1 J(F,F) = 267.1 Hz), 110.3 (t, 1 J(F,F) = 267.0 Hz), 79.3, 56.2, 52.3, 50.1, 40.0, 37.7, 34.3, 34.0, 27.3, 24.6, 22.1, 20.2; 19 F NMR (400 MHz, CD3OD): δ -87.4 (d, 2 J(F,F) = 205.7 Hz), -88.0 (d, 2 J(F,F) = 205.7 Hz), -88.6 (d, 2 J(F,F) = 205.7 Hz), -89.2 (d, 2 J(F,F) = 205.7 Hz); HRMS (ESI-TOF) m/z C33H44N12O7NaF4 [M+NH4] + cal. 819.3290, found 819.3292.

19 F 2 J

 192 NMR (CD3OD, 564 MHz): δ -84.8 (d, 2 J(F,F) = 204.4 Hz), -84.9 (d, (F,F) = 204.4 Hz), -85.4 (d, 2 J(F,F) = 204.4 Hz), -85.7 (d, 2 J(F,F) = 204.4 Hz) ; HRMS (ESI-TOF) m/z C26H38N12O5NaF4 [M+Na] + cal.

19 F

 19 NMR (CD3OH, 564 MHz):δ -87.9 (d, 2 J(F,F) = 205.5 Hz), -88.1 (d, 2 J(F,F) = 205.5Hz), -89.0 (d, 2 J(F,F) = 205.5 Hz), -89.3 (d, 2 J(F,F) = 205.5 Hz); HRMS (ESI-TOF) m/z C30H38N12O6NaF4 [M+Na] + cal.

19 F 19 F 2 J 1 J 2 J

 1919212 NMR (564 MHz, CD3OH): δ -87.62 (d, 2 J(F,F) = 205.6 Hz), -87.70 (d, 2 J(F,F) = 204.0 Hz), -87.71 (d, 2 J(F,F) = 205.6 Hz), -88.59 (d, 2 J(F,F) = 205.6 Hz), -89.00 (d, 2 J(F,F) = 204.0 Hz), -89.30 (d, 2 J(F,F) = 205.6 Hz); HRMS (ESI-TOF, ion polarity positive): m/z C39H49N18O9F6 [M+H] + cal.1027.3834, found 1027.3828. HPLC purity: XBridge C18 3.5 μm; H2O + 0:2 % formic acid/ACN, gradient 5-100 % in 20 min; Rt = 6.81 min, 100 %.1 H and13 C NMR data are given in table 7S, SI. 1 H,13 C and spectra are respectively given in figures 26S, 27S and 28S, SI.Boc-Asn(Trt)-NHCH2CCH (11)The product was obtained following the general procedure A from L-Boc-Asn(Trt)-OH (1.0 g, 2.1 mmol) and propargylamine (126 mg, 2.3 mmol) to obtain 11 (665 mg, 1.3 mmol, 62 %) as a colorless solid. and was used without further purification. mp = 76-78 ºC; Rf = 0.3 (C6H12/EtOAc: 7/3); IR: 3153, 2825, 1546, 1473, 1242, 1221, 1121, 1031, 970 cm -1 ; Melting point: 76-78 ºC; 1 H NMR (300 MHz, DMSO): δ 8.49 (s, 1H), 8.17 (t, 3 J = 5.6 Hz, 1H), 7.29-7.16 (m, 15H), 6.95 (t, 3 J = 8.4 Hz, 1H), 4.22 (bs, 1H), 3.84 (dd, 3 J = 5.6 Hz, 4 J = 2.5 Hz, 2H), 3.05 (t, 4 J = 2.5 Hz, 1H), 2.63 (dd, 2 J = 14.5 Hz, 3 J = 9.8 Hz, 1H), 1.39 (s, 9H); 13 C NMR (75 MHz, DMSO): δ 171.2, 168.9, 155.0, 144.7, 128.5, 127.4, 126.3, 81.0, 78.1, 72.8, 69.4, 51.6, 38.7, 28.2, 28.1. Benzyl (2-azido-2,2-difluoroacetyl)-L-alaninate (12). 12 was obtained following the general procedure B from L-Alanine Benzyl ester hydrochloride (1.2 g, 5.6 mmol). The crude product obtained was purified by column chromatography (c-Hex/EtOAc: 92/8) to provide 12 (1.1 g, 3.8 mmol, 67 %) as a white solid. mp = 40-42 ºC; Rf = 0.3 (c-Hex/EtOAc : 9/1); 1 H NMR (300 MHz, CDCl3): δ 7.36 (s), 7.00 (s, 1H), 5.21 (s, 2H), 4.66-4.57, (m, 1H), 1.49 (d, 3 J = 7.1 Hz, 3H); 13 C NMR (75 MHz, CDCl3): δ 171.4, 159.2 (t, 2 J(C,F) = 34.6 Hz), 134.9, 128.7, 128.3, 113.4 (t, 1 J(C,F) = 269.3 Hz), 67.8, 48.7, 17.8 ; 19 F NMR (188 MHz, CDCl3): δ -82.98 (d, 2 J(F,F) = 194.5 Hz, 1F), -84.09 (d, 2 J(F,F) = 194.5 Hz, 1F); IR (neat): ʋmax 3083, 2834, 1546, 1413, 1321, 1111, 1081, 930 cm -1 ; HRMS (ESI-TOF, ion polarity positive): m/z C12H16N5O3F2 [M+NH4] + cal. 316.1221, found 316.1121. BocNH-Phe-[TzlCF2]-Ala-OBn Tetrapeptide (15) The product was obtained following the general procedure C from 10 (500 mg, 1.7 mmol) and 12 (493 mg, 1.7 mmol).The crude product was purified by column chromatography (c-Hex/EtOAc: 65/35) to provide 15 (826 mg, 1.4 mmol, 81 %) as a white solid; Rf = 0.3 (c-Hex/EtOAc: 65/35); IR: 3213, 3001, 2954, 1593, 1501, 1252, 1211, 1141, 1013, 989 cm -1 ; Melting point: 100-102 °C; 1 H NMR (300 MHz, CDCl3): δ 7.87 (s, 1H), 7.37 (s, 5H), 7.17-7.13 (m, 5H), 6.50-6.39 (m, 1H), 5.23 (s, 2H), 4.99 (br s, 1H), 4.70 (t, 3 J = 7.2 Hz, 1H), 4.51 (d, 3 J = 5.9 Hz, 2H), 4.38-4.38 (m, 1H) 3.06 (d, 2H), 1.55 (d, 3 J = 7.2 Hz, 3H), 1.40 (s, 9H); 13 C NMR (75 MHz, CDCl3): δ 172.5, 170.3, 157.9 (t, (C,F) = 31.3 Hz), 156.2, 145.5, 134.9, 128.7, 128.5, 120.9, 110.3 (t, (C,F) = 268.9 Hz), 80.3, 67.7, 55.9, 49.2, 39.9, 34.6, 28.3, 17.5; 19 F NMR (188 MHZ, CDCl3): δ -86.8 (d, 2 J(F,F) = 209.9Hz, 1F), -89.2 (d, (F,F) = 209.9Hz, 1F); HRMS (ESI-TOF) m/z C29H38N7O6F2 [M+NH4] + cal. 618.2852, found 618.2855.

  2 (c-Hex/EtOAc: 4/6); IR: 3144, 2914, 1614, 1554, 1413, 1240, 1100, 988 cm -1 ; Melting point: 94-96°C; 1 H NMR (200 MHz, CDCl3): δ 7.94 (s, 1H), 7.72 (d, 1H), 6.54 (s, 1H), 5.75 (s, 1H), 5.23 (s, 1H) 4.58-4.47 (m, 1H,), 4.36 (d, 3 J = 5.7 Hz, 2H), 1.78 (m, 3H, 14), 1.37 (s, 9H), 0.91 (d, 3 J = 2.3 Hz, 3H), 0.88 (d, 3 J = 2.3 Hz, 3H); 13 C NMR (75 MHz, CDCl3): δ 173.2, 158.1 (t, 2 J(C,F) = 31.2 Hz), 155.9, 146.6, 120.5, 110.3 (t, 1 J(C,F) = 268.4 Hz), 80.1, 52.4, 40.6, 35.8, 28.3, 24.8, 22.9, 21.6; 19 F NMR (188 MHz, CDCl3): δ -86.0 (d, 2 J(F,F) = 205.5 Hz, 1F), -89.3 (d, 2 J(F,F) = 205.5 Hz, 1F); HRMS (ESI-TOF) m/z C16H27N6O4F2 [M+Na] + cal. 405.2062, found 405.2068.

- 3 J

 3 87.5 (d, 2 J(F,F) = 205.5 Hz), -87.7 (d, 2 J(F,F) = 205.5 Hz), -89.1 (d, 2 J(F,F) = 205.5 Hz), -89.2 (d, 2 J(F,F) = 205.5Hz); HRMS (ESI-TOF) m/z C24H36N11O6F4 [M+H] + cal. 650.2801, found 650.2795. BocNH-Asn(Trt)-[TzlCF2]-Phe-[TzlCF2]-Ala-[TzlCF2]-Leu-NH2 Decapeptide (20) 20 was obtained following the general procedure D from 16 (284 mg, 0.32 mmol) and 19 (208 mg, 0.32 mmol) which were both deprotected. The crude product obtained was purified by recrystallisation in MeOH to yield 20 (180 mg, 0.14 mmol, 42 %) as a white solid. mp = 112-114 °C; Rf = 0.2 (CH2Cl2/MeOH 93/7); 1 H NMR (400 MHz, DMSO-d6): δ 9.74 (brs, 1H), 9.69 (brs, 1H), 9.56 (d, 3 J = 8.2 Hz, 1H), 8.80 (t, = 5.3 Hz, 1H), 8.66 (t, 3 J = 5.6 Hz, 1H), 8.53 (bs, 1H), 8.51 (s, 1H), 8.43 (s, 1H), 8.41 (t, 3 J = 5.8 Hz, 1H), 8.13 (s, 1H), 7.48 (bs, 1H), 7.28-7.14 (m, 20H), 7.05 (d, 3 J = 7.9 Hz, 1H), 4.69-4.63 (m, 1H), 4.51-4.31 (m, 8H), 4.25-4.20 (m, 1H), 3.14 (dd, 2 J = 13.8 Hz 3 J = 4.8 Hz, 1H), 2.98 (dd, 2 J = 13.8 Hz 3 J = 4.8 Hz, 1H) 2.64 (dd, 2 J = 14.2 Hz 3 J = 9.5 Hz, 1H), 2.53 (dd, 2 J = 14.2 Hz 3 J = 9.5 Hz, 1H), 1.70-1.56 (m, 3H), 1.39 (s, 9H), 1.36 (d, 3 J = 7.2 Hz, 6H), 0.89 (d, 3 J = 6.3 Hz, 6H), 0.86 (d, 3 J = 6.3 Hz, 6H); 13 C NMR (400 MHz, DMSO-d6):δ 172.5, 171.

Table 1 .

 1 Coupling conditions assays to obtain heterotriazolamer 3

	Entry	Coupling reagents	Base	Time (h)	Yield	de(%)
		EDC.HCl (1.2 eq.)	DIPEA (2.5 eq.)	48 h	55%	80
		HOBt (1.2 eq.)
		EDC.HCl (1.2 eq.)	DIPEA (2.5 eq.)	5 h	48%	100
		HOBt (1.2 eq.)
		EDC.HCl (1.2 eq.)	Collidine (5 eq.)	5 h	48%	100
		HOBt (1.2 eq.)
		OXYMA (1.2 eq.)	DIPEA (2.5 eq.)	5 h	27%	100
		COMU (1.2 eq.)
		OXYMA (1.2 eq.)	Collidine (5 eq.)	5 h	16%	100
		COMU (1.2 eq.)
		OXYMA (1.2 eq.)	DIPEA (2.5 eq.)	5 h	20%	100
		DIC (1.2 eq.)
		HATU (1.2 eq.)	DIPEA (2.5 eq.)	5 h	18%	100
		HOAt (1.2 eq.)
		HCTU (1.2 eq.)	DIPEA (5 eq.)	5 h	28%	100

Table 2

 2

	Residues	Compound 4	Compound 5
		NMR	MD	NMR	MD
	Phe 1	-	-	7.4	7.6 ± 1.6
	TZL 1	6.3	4.9 ± 2.6	5.9	4.4 ± 2.6
	Ala 2	6.8	7.5 ± 1.7	7.3	7.5 ± 1.7
	TZL 2	5.6	4.8 ± 2.7	5.8	4.9 ± 2.7
	Leu 3	8.8	8.0 ± 1.5	8.0	8.0 ± 1.4

) indicating that the conformational ensembles generated by MD simulations are consistent with those revealed by NMR.

Table 2 .

 2 Comparison of

3

JNH-Hα coupling constants (Hz) in compounds

Table 3 .

 3 Table

	Compound	Compound/ hIAPP ratio [a]	t 1/2 extension/ reduction (%)	[b]	F extension/ reduction (%) [c]
	1	(10/1)	-30±8%		+94±37%
		(1/1)	ne		ne
	2	(10/1) [d]	ne [e]		-24±15%
		(1/1)	ne		ne
	3	(10/1)	-42±12%		-49±4%
		(1/1)	+15±9%		-41±9%
	4	(10/1)	ne		ne
		(1/1)	ne		ne
	5	(10/1)	+112±42%		-80±8%
		(1/1) 5	+15±7%		ne
	6	(10/1)	+42±25%		ne
		(1/1)	ne		-33±15%
	7	(10/1)	ne		ne
		(1/1)	ne		ne
	8	(10/1)	+106±32%		-64±5%
		(1/1)	ne		-28±13%
	9	(10/1)	+46±18%		-37±9%
		(1/1)	ne		ne
	FGAIL-NH2	(10/1)	ne		-36±3% -46±10%	53
		(1/1)	ne		ne
	NNFGAIL-NH2 (10/1)	+14±8%		-14±10%
		(1/1)	ne		-24±13%

summarizing the effects of compounds 1-9, FGAIL-NH2 and NNFGAIL-NH2 on hIAPP fibrillization assessed by ThT-fluorescence spectroscopy at 10/1 and 1/1 ratios of compound/hIAPP.

  H and13 C NMR data are given in table 5S, SI. 1 H,13 C and 19 F spectra are respectively given in figures 19S, 20S and 21S, SI.

	19 F NMR (564 MHz,
	CD3OH): δ -87.65 (d, 2 J(F,F) = 205.8 Hz), -87.67 (d, 2 J(F,F) = 204.7
	Hz), -87.85 (d, 2 J(F,F) = 207.7 Hz), -88.67 (d, 2 J(F,F) = 205.8 Hz), -
	88.90 (d, 2 J(F,F) = 204.7 Hz), -89.49 (d, 2 J(F,F) = 205.8 Hz) ; HRMS
	(ESI-TOF, ion polarity positive): m/z C37H47N18O8F6 [M+H] + cal.
	985.3728, found 985.3747. HPLC purity: XSelect C18 3.5 μm; H2O +
	0.1 % formic acid/ACN, gradient 5-100 % in 20 min; Rt = 5.80 min,
	100 %.

1

13 C NMR (75 MHz, CDCl3):δ 171

  .6, 170.3, 169.7,157.5 (t, 2 J(C,F) = 31.1 Hz), 145.6, 144.2, 134.6, 134.5, 129.4, 128.8, 128.7, 128.6, 128.0, 127.4, 127.1, 120.8, 110.1 (t, 1 J(C,F) = 269.5 Hz), 80.5, 70.9, 67.9, 53.9, 51.5, 38.3, 37.3, 34.9, 28.3;

19 F NMR (188, MHz, CDCl3): δ

  . -86.8 (d, 2 J(F,F) = 208.9 Hz, 1F) -88.7 (d, 2 J(F,F) = 208.9 Hz, 1F); HRMS(ESI-TOF) m/z C49H50N7O7F2 [M+H] + cal. 886.3863, found 886.3869.

19 F NMR (188 MHz, CD3OD):δ

  = 267.3 Hz), 111.7 (t, 1 J(F,F) = 267.9 Hz), 53.8, 51.5, 41.4, 36.5, 35.7, 28.7, 26.0, 23.4, 21.6, 17.3;

	2 (CH2Cl2/MeOH: 95/5); IR:
	3276, 3164, 3113, 2932, 2897, 1611, 1546, 1300, 1234 cm -1 ; Melting
	point: 122-124°C; 1 H NMR (200 MHz, CD3OD):δ 8.39 (s, 1H), 8.34 (s,
	1H), 4.57 (s, 2H), 4.53-4.50 (m, 2H), 4.37 (s, 1H), 1.74-1.72 (m, 3H),
	1.49 (s, 3H), 1.44 (s, 9H); 13 C NMR (400 MHz, CD3OD):δ 176.0,
	173.7, 148.1 (t, 2 J(C,F) = 30.7 Hz), 122.6, 122.2, 126.4, 111.9 (t,

1

J(F,F)

Acknowledgements

The Ministère de l'Enseignement Supérieur et de la Recherche (MESR) is thanked for financial support for José Laxio Arenas and Jacopo Lesma.

Conflicts of interests

No conflict to declare.

Keywords: amyloid • aggregation • foldamer • peptidomimetic • beta-hairpin • triazole • fluorine