Binhao Teng

Pradeep K Mandal

Lars Allmendinger

Céline Douat

Yann Ferrand

Ivan Huc
email: ivan.huc@cup.lmu.de

Controlling aromatic helix dimerization in water by tuning charge repulsions

Several helically folded aromatic oligoamide foldamer sequences were designed and synthesized. The sequences were all water soluble thanks to the charged side chains borne by the monomers. Replacing a few, sometimes only two, charged side chains by neutral methoxy groups was shown to trigger various types of aggregation processes leading to the formation of head-to-head stacked dimers of single helices, double helical duplexes and a quadruplex, none of which would form in organic solvent with organic soluble analogues. The nature and structure of the aggregates were supported by concentration and solvent dependent NMR studies, DOSY experiments, mass spectrometry, and X-ray crystallography or energy-minimized models. The hydrophobic effect appears to be the main driving force for aggregation but it can be finely modulated by the presence or absence of a small number of charges to an extent that had no precedent in aromatic foldamer architectures. These results will serve as a benchmark for future foldamer designs in water.

Introduction

Molecular aggregation in water often rests on a balance between hydrophobic effects that act as a driving force, and hydrophilic interactions mediated by polar groups without which molecules may not be water soluble. When hydrophilic groups consist of ions, it is generally beneficial that a net total charge exists. Switterions may cancel each other's hydrophilic interactions and tend to be poorly water soluble. The hydrophobic-hydrophilic balance finds illustrations in objects as diverse as a simple charged surfactant and a polyanion such as DNA. In both cases, one may say that aggregation -micelle formation for the former and double helix association for the latter -occurs despite electrostatic repulsions between charges. Tuning these repulsions may in turn result in changes in aggregation properties. For instance, upon screening electrostatic interactions, spherical micelles may transit to cyclindrical micelles or bilayer phases. 1 Conversely, DNA forms more stable duplexes with its neutral analogue PNA than with itself. 2 Here, we show that minor changes in the hydrophobic-hydrophilic balance of helical aromatic foldamers result in great variations of their propensity to form different types of discrete aggregates in water.

Aromatic helices are an important class of foldamers with aryl groups in their main chain. They have attracted attention for their ability to serve as scaffolds for molecular recognition either in their interior [3][4][5][6][7][8][9] or at their surface, [10][11][12] in ion [13][14][15] and water [16][17][18] transport through membranes, in molecular machinery, 19,20 and for their charge transport 21,22 and chiroptical properties. 23,24 Aromatic helix folding may be driven by solvophobic effects, [25][26][27][28][START_REF] Zhao | Foldamers[END_REF][START_REF] Hua | [END_REF][31] the presence of neutral or ionic guests, [32][33][34][35][36] steric congestion combined with aryl-aryl interactions, 37,38 or local conformational preferences. [39][40][41][42][43] Aromatic helices may undergo different types of aggregation: stacking into continuous 44 or discrete 45 tubular objects, side-by-side bundling, 46 or intertwining in double, 9,[47][48][49][50][51][52][53] triple, [54][55][56] or quadruple 57,58 stranded objects. Helix aggregation may contribute to the properties mentioned above. For example, double helices may also act as molecular containers. 9,19,59,60 Double helix formation has been shown to depend on solvent 51,53,61 and on the presence of guests. 62,63 A few reports have also highlighted the role of interactions between side chains. 50,52 However, aggregation has been investigated mostly in organic solvents. Reports on aggregation in water are few 45,51,53 and have not made use of the hydrophobic-hydrophilic charge balance. We now report that removing as few as two or four out of fourteen positive charges from a helical oligoamides can drive its association into discrete stacked dimers or double helical dimers, respectively. In a shorter sequence, removing four out of seven charges allows for the formation of a tetramer.

Results and discussion

Design and synthesis

Q R and Qf R monomers bearing various R solubilizing groups in positon 4 have been have been widely investigated as building blocks of helically folded oligoamide sequences (Fig. 1). 4,39,45,46,53,58,64 In these sequences, folding is driven by local electrostatic attractions and repulsions between the amide NH or CO and neighboring quinoline endocyclic nitrogen atoms or exocyclic fluorine atoms that create a conformational bias at aryl-amide rotatable bonds, resulting in a well-defined curvature of the aromatic strand and eventually promoting a helical shape. 41,42 These helices are stable in all kinds of solvents and are particularly stable in water due to hydrophobic effects associated with aromatic stacking. 65 We recently introduced Qh Dap as an analogue of Qf Dap lacking the conformational control associated with the fluorine atom of Qf, which gives rise to a conformational equilibrium at the 7carbonylamino-quinoline bond. 66 Using sequence 1, we showed that this additional conformational degree of freedom hampers folding in DMSO but that hydrophobic effects still promote helix folding in water, thus favoring the s-trans conformation of each Qh unit. Sequence 1 comprises a central (Qh Dap )8 segment flanked with (Q Dap )3 segments. Qh, like Qf, was expected to generate a wider helix diameter possibly conducive of dimerization into multistranded helices through intertwining of the strands. 53,58 In contrast, Q oligomers possess a narrower diameter and do not form multiple helices. Also, they have been show to prevent double helix formation of wider helical segments placed between them. 4,67 Indeed, such a double helix would have two Qn segments at each end (one at each end of both strands), which would normally create steric hindrance and perturb folding. 67 As expected, sequence 1 was shown to be a single helix in water. 66 It may possess a cavity potentially capable of encapsulating guest molecules, as in designs that we and others have presented previously, 4,7,[START_REF] Hua | [END_REF] and its molecular recognition properties will be described elsewhere. The structure of 1 in solution was elucidated using 1 H NMR spectroscopy, but attempts to produce crystals suitable for x-ray diffraction were unsuccessful. We hypothesized that the high charge density at the surface of single helical 1 was, at least in part, responsible for the difficulty to produce crystals. In the context of this work, new sequences 2-5 were thus synthesized as analogues of 1 in which two, four or seven charged side chains were replaced by neutral methoxy groups in the hope to obtain crystals. In the case of 5, the seven remaining charged side chains were all made anionic. As shown in the following, these variations gave rise to various types of unanticipated aggregation behavior (and one crystal structure).

In the meantime, sequences 6-9 were prepared to purposely investigate possible double helix formation. Sequence 6 possesses Qf monomers that encode helicity regardless of the solvent. Analogues of 6 have been shown to form double helical dimers, including in water. 53 Indeed, since this sequence contains only one Q3 segment. Therefore, it may accommodate two of them in a head-to-tail double helical dimer. Because of their additional degree of conformation freedom, it was unknown whether Qh monomers would also undergo double helix formation. Sequence 7, was thus also prepared, that amounts to the seven C-terminal residues of 1. Sequences 8 and 9 are analogues of 7, bearing anionic residues and three or four methoxy side chains.

The preparation of these new sequences entailed the synthesis of two new monomers ready for oligoamide synthesis on solid phase, Fmoc-Qh Ala -OH and Fmoc-Qh Sul -OH. Details of their preparation is reported in the supporting information. All sequences were prepared on solid phase using either manual or automated synthesis using low loading Wang or Protide resin and established protocols. 68,69

Head-to-head dimerization of single helices

In order to reduce water solubility and promote crystal growth in water, oligomer 2 was produced as an analogue of 1 in which two charged aminomethyl side chains were replaced with neutral and isosteric methoxy groups near the ends. Crystals suitable for x-ray diffraction analysis could not be obtained. In contrast with 1, the 1 H NMR spectrum of 2 in water showed two sets of sharp signals, the proportion of which varies with concentration (Fig. 2a), demonstrating a process of reversible aggregation in slow exchange on the NMR timescale. The different size of the two species was also highlighted by a DOSY experiment clearly showing different hydrodynamic radii (Fig. 2c). The number of resonances indicated that the aggregate is symmetrical, i.e., its helical subcomponents are in the same environment. The signals of the aggregate are upfieldshifted, suggesting that aggregation involved aromatic stacking. The dimeric species [2M+4H] 4+ and [2M+5H] 5+ were detected in the gas phase by ESI-MS (Fig. S1 †) indicating that the aggregation is probably a dimerization. Upon integrating the NMR signals of the two species, a dimerization constant of 970 M -1 was calculated. ‡ The above observations are in principle compatible with either the formation of an unexpected anti-parallel symmetrical double helix, 52,53,70 or with head-to-head stacking of two helices as reported for Qn oligomers bearing anionic residues. 45 Evidence in favour of the latter came from the behaviour of 3, an analogue of 2 bearing a Cterminal AiB. The 1 H NMR spectrum of 3, shows a single set of signals at chemical shift values very similar to those of the monomer of 2 (Fig. S2) suggesting that the AiB has suppressed dimerization. This Cterminal extension would not be expected to much alter double helix formation but it is not compatible with the stacking of the C-terminal cross-sections of two helices which normally requires direct hydrogen bonding between a carboxylate and a carboxylic acid of the two strands involved (Fig. S3). 45 Furthermore, the ratio of monomer and dimer of 2 was shown to reach equilibrium within minutes after a dilution (Fig. S4), whereas kinetics would be expected to be much slower -of the order of hours or days -for a long double helix. 42 Based on these experiments, a plausible energy-minimized model of a stacked dimer of 2 was constructed (Fig. 2b). It is quite remarkable that the proximity between two charges at these positions is sufficient to completely suppress dimerization. These finds thus extent the formation of stacked Qn helix dimers to cationic sequences.

Dimerization was enhanced by increasing buffer concentration, which we interpreted as the result of an enhanced polarity of the solvent that promotes aggregation via a stronger hydrophobic effects and better screening of charge repulsions. In an attempt to decrease charge repulsion by neutralizing some cationic residues, the pH was to 5.6 (sodium acetate buffer, 45 mM), but this immediate precipitation and no NMR signals could be observed.

Double helices may accommodate four Q3 segments

Using a C-terminal Aib extension to avoid head-to-head stacking of single helices, sequence 4 was designed to further reduce its water solubility and favour crystal growth. Thus, two additional positively charged side chains were replaced by two neutral methoxy groups. Unexpectedly, this change led to another kind of aggregation. Despite the Aib termination, the 1 H NMR spectrum of compound 4 in water again revealed two sets of sharp signals whose proportions vary with concentration (Fig. 3a). DOSY NMR confirmed that the aggregate -the species more abundant at high concentration -has a larger hydrodynamic radius (Fig. 3b). Furthermore, dimeric species [2M+4H] 4+ and [2M+5H] 5+ were detected in the gas phase by ESI-MS (Fig. S5) hinting at a dimerization process again. Integration of the signals ‡ allowed for the calculation of a dimerization constant of 32500 M -1 at pH 4 in 13.5 mM sodium acetate buffer. The dimerization of 4 was enhanced upon increasing buffer concentration more significantly than the dimerization of 2, (4)2 being formed quantitatively at 0.5 mM in 45 mM sodium acetate while it represents only 76% at the same concentration in 13.5 mM sodium acetate (Fig. 3a).

The exchange between monomeric and dimeric 4 was found to be much slower than between monomeric and dimeric 2. After a dilution, several hours of incubation were required to reach equilibrium, i.e. until proportions stop evolving (Fig. S6). This indicates a larger kinetic barrier between the two species, that is, a change in molecular structure more significant than just stacking two single helices. We tried to obtain crystals of 4 to elucidate its structure and aggregation mode but without success, as for all cationic oligomers in our hands so far. We therefore turned to anionic derivatives that have crystallized more readily in other systems 68 and prepared oligomer 5. This sequence was not conceived as a direct analogue of 4, as it contains more and differently located neutral methoxy side chains. The solution behavior of 5 is also more complex and its NMR spectrum shows broader peaks (Fig. S7). Nevertheless, single crystals of 5 that diffracted at atomic resolution could be obtained, revealing a parallel double helix that forms despite the presence of Q3 segments at each end of the two strands (Fig. 3c, Fig. S8), and despite the greater flexibility of Qh-containing sequences.

The double helix of (5)2 only involves the central Qh8 segments and spans two full turns. The two strands are offset by half a turn and engage in extensive intermolecular aromatic stacking. The four Q3 segments are all single helical and the steric crowd that they create is somehow accommodated. For that, two outer Q3 segments are slightly tilted away from the duplex and overlap with two inner Q3 segments. This arrangement suggests that segments having more than three Q units may not be accommodated as easily, but this hypothesis was not tested. The structure also shows that terminal Aib units play no role in aggregation. A molecular model of the single helical monomer of 5 was built (Fig. S9) and its solvent accessible surface was calculated to be 1790 Å 2 , compared to 2690 Å 2 for the duplex solid state structure. Thus, the duplex surface amounts to about 75% of the surface of two monomers, giving a measure of how much is hidden from water upon dimerization. Of note, the double helix (5)2 is parallel and lacks any symmetry. Its two strands are inequivalent. In contrast, the number of 1 H NMR resonances of (4)2 suggests a symmetrical structure (Fig. 3a). Dimer (4)2 may instead be a symmetrical anti-parallel dimer, or perhaps its NMR spectrum represents an average between two degenerate parallel conformations. In any case, we think it reasonable to propose that the crystal structure of (5)2 informs about the aggregation mode of 4. We also note that the structure of (5)2 shares a key design feature with 1-4 and with other helical capsules: 4 it possesses a sizeable cavity surrounded by the helix backbone (350 Å 3 in this case, see Fig. S8). The solid state structure of (5)2 represents the first example of a water-soluble helical capsule crystal structure.

To conclude this section, it is again remarkable that differences as small as those that exist between 3 and 4 -minus two charged residues -can trigger a such a drastic change of aggregation behavior. We also infer that the monomeric nature of 1 and 3 is not just the result of the bulky terminal Q3 segments, but also of charge repulsions within double helical dimers.

Hemi-capsule dimerization

To explore further the modes of dimerization of helically folded aromatic oligoamides in water, several hemicapsule sequences were synthesized. A sequence differing from 6 only by the length of the cationic side chains has already been shown to form a double helical dimer in water. 72 The behaviour of 6 was consistent. Upon adding water to a DMSO-d6 solution of 6, a species assigned to a single helical monomer in DMSO is replaced by another species assigned to a double helical dimer (Fig. 4a). In the gas phase, MS showed a substantial population of [2M+2H] 2+ and [2M+3H] 3+ dimeric species (Fig. S10). The dimerization constant of 6 was too high to observe monomeric species by 1 H NMR upon dilution a solution in water (Fig. S11). Also, given the multiplicity of NMR signals, one can infer that (6)2 has a symmetrical structure and propose a molecular model as shown in Fig. 5a.

Similar experiments were carried out using analogous sequence 7 in which Qf units were replaced by Qh units that should provide more degrees of conformational freedom. A similar transition as for 6 was observed upon adding water to a DMSO-d6 solution of 7 (Fig. 4b). That the two species correspond to an aggregation phenomenon was ascertained by DOSY NMR and from the concentration dependence of the proportion of the two species (Fig. 4c,d) in DMSO-d6/H2O (40:60 vol/vol). A dimerization constant of 10 4 M -1 in this solvent could be calculated. ESI-MS confirmed the presence of [2M+2H] 2+ and [2M+3H] 3+ dimeric species in the gas phase (Fig. S12). Again no dissociation was observed upon diluting a solution of 7 in water (Fig. S13). Nevertheless, less DMSO is required to disrupt (7)2 than to disrupt (6)2 (Fig. 4a,b). In short, Qh units also promote double helix formation, albeit slightly less efficiently than Qf monomers, and the model shown in Fig. 5b can be proposed for (7)2. The dimeric segment involves eight Qh units, allowing some cooperative solvophobic folding to occur. In contrast, the four units of monomeric 7 cannot in principle undergo cooperative folding because they do not span a full helix turn. Estimations of the solventaccessible surface of (7)2 and of different conformations of monomeric 7 suggest that the surface of a dimer amounts to 61 to 71% of the surface of two monomers (Fig. S14), shedding light on the significant hydrophobic effects observed. Interestingly when mixing 6 and 7 in water, only homodimers (6)2 and (7)2 were observed (Fig. S15,S16), indicating a sort of narcissistic self-sorting that contrasts with heterodimerization observed in other systems. 73,74

Double helices may also dimerize

In view of the success at getting crystals of anionic sequences, oligomer 8 was introduced as an analogue of 7 having both methoxy and sulfonic acid side chains. At this stage, the neutral methoxy groups did not alter aggregation properties and 8 behaved like 7 in pure water (Fig. S17) and in water/DMSO mixtures (Fig. S18). Also, [2M-3H] 3-and [2M-2H] 2-dimeric species could be observed by ESI-MS in the gas phase (Fig. S19). However, sequence 9 in which one more sulfonic acid has been replaced by a methoxy group exhibited a new behavior. § Unlike 6, 7 and 8, the 1 H NMR spectrum of 9 in water showed two sets of signals (Fig. 6a). Upon adding water to a DMSO-d6 solution of 9, a first transition occurred near 30 % of water, which we assigned to the expected monomer-to-double-helical-dimer transition, and a second transition occurred when reaching 100 % water (Fig. 6a). All three species are in slow exchange on the NMR times scale. The proportion of the two species present in water was shown to depend on concentration: the relative intensity of the signals assigned to the double helical dimer decreases when concentration increases (Fig. 6b), indicating that the new species is an aggregate of higher molecularity than a dimer. The conversion is quantitative at 5 mM. DOSY NMR confirmed that the higher aggregate has a larger hydrodynamic radius (Fig. 6c). Native ESI-MS analysis could only be performed at concentrations too low for the higher aggregate to be present in significant amount and only the [2M-2H] 2-dimeric species was detectable in the gas phase (Fig. S20). However, the molecularity of the higher aggregate may be inferred from its number of 1 H NMR resonances. This number is double the number of resonances of (9)2, meaning the aggregate possesses two inequivalent strands in equal proportions, ruling out a possible trimer and making a tetramer the most likely possibility. The time required to form (9)4 after dissolving 9 in water was found to be of the order of hours (Fig. S21), indicating that significant structural changes are required. A plausible molecular model for (9)4 was proposed based on the structure of ( 5)2 (Fig. 7). Taking the C-terminal half of (5)2 (bottom part of Fig. 3c), a parallel duplex (9)2 can be proposed distinct from the anti-parallel duplex that prevails at lower concentration. Next, two of these parallel duplexes can be intertwined in an antiparallel fashion through their N-terminal Qh4 segments to produce the C2symmetric quadruplex shown in Fig. S7 having two pairs of inequivalent strands. This model is of course speculative. It nevertheless allows to point to some factors that could play a role: its solvent-accessible surface is smaller than that of two anti-parallel dimers (Fig. S22); removing one charge from a monomer means removing four charges from the tetramer; two MeO of 9 that replaced sulfonic acids of 8 are found to be buried between two hydrophobic aromatic surfaces pointing to the destabilization of such a structure in the case of 8. Besides electrostatic effects between charges, the fact that an electron withdrawing group in 8 has been replaced by a donor in 9 might also influence interactions between aryl rings but this is hard to estimate. Single crystals of 9 have been obtained that diffracted up to 1.5 Å but no structure could be solved so far.

Conclusions

We have shown that the formation of at least three different types of discrete aggregates of helical aromatic foldamers can be triggered in water by minor changes in side chain properties. These changes always consisted in replacing a small number of charged residues, sometimes only one, by a neutral methoxy group. Specifically, a head-to-tail stacked dimer of two single helices, a double helical duplex with four terminal Q3 segments, and a quadruplex were shown to form whereas related sequences containing two or a few more charged residues did not. The last two aggregates are new. All aggregates are kinetically stable. They are in slow exchange with their subcomponents on the NMR timescale. In the case of the double helix and quadruplex, kinetic stability is so high that equilibrium may take hours to be reached. Hydrophobic effects appear to be a major driving force for aggregation, but these effects are counterbalanced by the side chain charges. Earlier observations in organic solvents had shown that Q3 segments prevent the formation of some types of multistranded helices. We now have shown that this effect can be overcome in water unless sufficient charge repulsions are also at play. Overall, these results provide a foundation for future aromatic foldamer structure design in water. In addition, the monomeric and multimeric species presented here possess cavities in which guest molecules may be recognized, and secluded from the surrounding aqueous environment. These cavities, in particular those generated by more than one strand, may undergo rearrangements triggered by external stimuli and exhibit controlled release or guest swapping properties. Progress in this direction is being made and will be reported in due course.

Footnotes and References ‡ Care should be taken during integration as the intensity of the 1 H NMR signals of exchangeable NH protons may be affected by the water suppression. This effect is more pronounced at lower concentration and results in lower-than-expected intensities for protons that exchange most. Typically, this results in different intensities for signals belonging to the same species that would be expected to have the same integration. The NH resonances belonging to Q units found at highest chemical shit values typically exchange least and may be preferred for accurate integration. § Compound 9 was not produced as an analogue of 8 but as another candidate for crystal growth, hence the other differences between the two. Crystal structures, including that of (5)2, suggest that Cterminal Aib units, as in 9, do not influence aggregation. There is also no ground to invoke a role of Q Asp as the seventh residue in 9 instead of Q Sul in 8. We thus assign the different aggregation behavior of these compounds to the Q Sul 6Q Ala mutation.

Fig. 1

 1 Fig. 1 Structure of the building blocks Q R , Qh R and Qh R bearing R side chains in position 4 and composition of sequences 1-9 investigated in this study. Different quinoline monomers and side chains are highlighted with different colors. Monomers: Q: black, Qh: brown, Qf: purple; side chains: Dap: blue, Ala: red, Sul: gold, Asp: green).

Fig. 2

 2 Fig. 2 a) Excerpts of the 1 H NMR (500 MHz) spectra of compound 2 at different concentrations in 45 mM sodium acetate aqueous buffer pH 4.0. Selected amide proton signals belonging to either the monomeric or dimeric form are highlighted in red and blue, respectively. b) Energy minimized model (Maestro, MMFFs force field, water as implicit solvent) 71 of a dimer of 2 formed by stacking two C-terminl cross-sections. The two distinct molecules engaged in binding are colored in green and grey, respectively. The carboxylic acid and carboxylate involved in intermolecular hydrogen bonding at the C-termini are circled in red. c) Amide-region of the DOSY NMR (500 MHz) spectrum of compound 2 (0.49mM) in D2O/H2O (1:9 vol/vol). The two different hydrodynamic radii related to different levels of the signals in the DOSY spectrum are indicated by dashed lines. Signals corresponding to the monomeric or dimeric form are highlighted in red and blue, respectively.

Fig. 3

 3 Fig. 3 a) Excerpts of the 1 H NMR (500 MHz) spectra of compound 4 at different concentrations in 13.5 mM (* means in 45mM) sodium acetate aqueous buffer pH 4.0. Selected amide proton signals belonging to either the monomeric or dimeric form are highlighted in red and blue, respectively. b) Amide-region of the DOSY NMR (500 MHz) spectrum of compound 4 (0.5 mM) in 13.5 mM sodium acetate aqueous buffer pH 4.0. The two different levels of signals related to different molecularities in the DOSY spectrum are indicated by dashed lines. Signals corresponding to the monomeric or dimeric form are highlighted in red and blue. Solid-state structure of (5)2: Side view shown in c) CPK and d) stick representation for different strands. In both representations, two distinct strands are colored in green or cyan, respectively. Side chains have been omitted for clarity.

Fig. 4

 4 Fig. 4 Excerpts of the 1 H NMR (500 MHz) spectra at 298 K of 6 (a) and 7 (b) in DMSO-d6/H2O mixtures. The percentage of DMSO is from top to bottom: a) 0, 50, 66, 70, 76 and 100; and b) 0, 33, 40, 50, 57 and 100. Note: 0% DMSO means the spectrum is recorded in H2O/D2O (90:10 vol/vol). Red and blue amide proton signals stand for either the monomeric or dimeric form, respectively. c) Excerpts of the 1 H NMR spectra of 7 at different concentrations in DMSO-d6/H2O (40:60 vol/vol). Selected amide proton signals belonging to either the monomeric or dimeric form are highlighted in red and blue, respectively. d) Amide NH region of the DOSY NMR (500 MHz) spectrum of 7 (0.32 mM) in DMSO-d6/H2O (40:60 vol/vol). The two different levels of signals related to different hydrodynamic radii are indicated by dashed lines. Signals corresponding to the monomeric or dimeric form are highlighted in red and blue.

Fig. 5

 5 Fig. 5 Energy minimized models (Maestro, MMFFs force field, water as implicit solvent) 71 of (6)2 (a,b) and (7)2 (c,d). In a) and c), one strand is shown in space filling representation and the other in stick representation. In b) and d), both strands are shown in stick representation but the fluorine atoms in position 8 of Qf units and the hydrogen atoms in position 8 of Qh units are shown in space filling representations and colored in cyan and white, respectively. In all representations two distinct strands are colored in green or grey, respectively. Side chains and most hydrogen atoms have been omitted for clarity.

Fig. 6

 6 Fig. 6 Excerpts of the 1 H NMR (500 MHz) titration at 298 K of 8 (0.72 mM) in DMSO-d6/buffer mixtures in the following DMSO percentage (top to bottom): (a) 0, 16.7, 50, 66.6, 72.5, 76.9, 83.3 and 100. Note: buffer means 13 mM ammonium acetate aqueous buffer pH 8.5. 0% DMSO means 1 H NMR (500 MHz) spectrum of 8 in buffer (buffer/D2O 90:10 vol/vol). Red, blue and yellow amide proton signals stand for the monomeric, dimeric and tetrameric form, respectively. b) Excerpts of the 1 H NMR (500 MHz) spectra of compound 8 at different concentrations in 11.7 mM (* means in 47.7 mM) ammonium acetate aqueous buffer pH 8.5. Selected amide proton signals belonging to either the dimeric or tetrameric form are highlighted in blue and yellow, respectively. d) Amideregion of the DOSY NMR (500 MHz) spectrum of compound 8 (1 mM) in 11.7 mM ammonium acetate aqueous buffer pH 8.5. The two different levels of signals related to different molecularities in the DOSY spectrum are indicated by dashed lines. Signals corresponding to the dimeric or tetrameric form are highlighted in blue and yellow.

Fig. 7

 7 Fig. 7 Energy minimized model (Maestro, MMFFs force field, water as implicit solvent) 71 of (9)4: a) Side view with two strands in space-filling representation and two strands in stick representation. Side chains and hydrogen atoms have been omitted for clarity. b)Same side view including the side chains with all four strands in stick representation. In all representations, four distinct strands are colored in green, grey, cyan and pink, respectively. The symmetry of the structure is such that gray and cyan strands on one hand and green and pink strands on the other hand, are equivalent.

Acknowledgements

This work was supported by the DFG (project HU 1766/6-1). B. Teng gratefully acknowledges financial support from the China Scholarship Council. We thank D. Gill and E. Merlet for contributing some synthetic precursors and G. Leonard (ID30B, ESRF) for assistance during data collection at the Synchrotron beamline.

Conflicts of interest

There are no conflicts to declare.


This case study is part of a series that has been produced within the study on "Risks and Trust in pursuit of a well-functioning PID infrastructure for research" commissioned by the Knowledge Exchange in July 2021. The main outcome of this study will be a report examining the current PID landscape with an emphasis on its risks and trust-related issues. The report; "Building the Plane as We Fly It": the Promise of Persistent Identifiers (DOI https://

Rationale

This case study aims to explore the key role research funders are expected to play in the gradual adoption of an ever wider range of PIDs across European countries.

The involvement of national-level funders in the awareness-raising exercise around the role of PIDs will contribute to the achievement of the various use cases for PID implementation -many of which, such as their use in internal workflows for project proposal submission and review, cannot be realised without a firm support from funders. A particularly critical role will be played by the European Commission via the efforts for the consolidation of the European Open Science Cloud (EOSC). The fact that the PID Graph underpinning the vision for a widespread PID implementation lies at the core of the plans the EOSC has laid out to support a wide range of research data management-related PIDs may provide a much-needed harmonisation in the approach to multiple PIDs, some of which may not be that relevant for national funders at an early stage.

The case study also explores the possible mechanisms and forums for coordination across funders so that best practices in PID adoption by a number of them can gain traction on a wider scope. The ORCID consortia already available in many European countries -in which specific funders are already represented -may also play a relevant role by gradually expanding the scope of the PIDs whose implementation they support. Same as in other case studies in this series, the final section of this case study on the role of funders will be devoted to perceived issues around risks and trust from a research funder perspective. These include the risk of fragmentation that has been highlighted in other case studies -meaning that funders might take diverging approaches towards the implementation of specific PIDs or be left behind altogether due to the lack of awareness of the appropriate mechanisms to follow. The lack of effective forums to discuss and agree on a coordinated cross-funder approach is also seen as a potential risk, closely connected with a possible lack of trust in the real usefulness of the implementation of specific PIDs such as grant IDs.

Why should a research funder like the Wellcome Trust, the NWO or the DFG with its own internal and functional ID adopt a new, global ID?

Even if a specific funder has its own well-maintained identification system for its grants, there are three main factors that justify adopting DOI-based, Crossref-issued grant IDs: I. With so many funders out there there is always a risk of duplicate grant numbers across funders. Even if the combination of funder name + full grant number will typically be unique, this is potentially problematic and easy to fix via persistent grant IDs II. Authors often make mistakes when including the full grant numbers for their projects in their manuscript acknowledgements.

The more complex the grant number structure, the more frequent such typos are III. Self-managed grant numbers issued by funders do not allow the full power of the PID Graph to be exploited. Once DOI-based grant IDs start regularly featuring in published papers that can be shared in the article metadata by publishers, it will be simple to link publications and datasets to a funded project in an automated, machine-readable way. This is already theoretically possible on the basis of self-managed project grant numbers issued by funders, but it's much more difficult because there are hardly any links between internal grant numbers and the landing pages containing the project information.

Research funders and their coordination: a complex landscape Europe, and through the early mandate issued for its funded researchers 5 it has effectively driven the highest national-level adoption for ORCID in any country worldwide, see Figure 1 (below);

`Funders come in many sizes and often lack the technical resources to devise a policy for PID adoption or simply do not see this area as a priority;

`While the best practices mentioned above are publicly available online and the case for PID adoption by funders has been extensively made, these initiatives are relatively recent and there are not that many forums for funder coordination that may allow a specific discussion to take place in the domain. This inevitably leads to a certain degree of fragmentation in the landscape, where some countries are frontrunners while other ones are lagging. The effort to adopt persistent grant IDs will be examined below as an area that needs to be specifically driven by research funders, quite a few of whom are already engaging in the process. However, a previous step is required before even considering the implementation of PIDs for grants, namely for funders to collect and make available the information on their funded projects in a standardised and comprehensive fashion. This is only a relatively recent development and it's far from being complete due to the strong variations in research funders' nature (public vs private research funders), size, budget and technical resources.

The fact that there are not too many coordination forums available to bridge the gaps across a fragmented research funding landscape is another factor that makes it difficult to have a widespread funder movement towards sharing project data on the basis of a harmonised metadata set. The OpenAIRE project was one of the first international initiatives to push for this kind of housekeeping effort by research funders by using the European Commission-funded research framework programmes as a benchmark. Having an all-European (and beyond) set of National Open Access Desks (NOADs ii ) for stakeholder engagement purposes, OpenAIRE was able to use this network as a series of contact points and to make the case for a layer of services towards research funders based on the comprehensive collection of funding information and the linking of funded project data to publications and datasets 8 . This reference to OpenAIRE funder services is only dated 2017 though, and even if the list of funders already engaging in this international monitoring effort provided in the slides includes research funders from many different countries both in and outside Europe, the list of funders that are not on the list and may not even be aware of these services is far longer. The first attempts to make the case and discuss the technical requirements for issuing persistent grant IDs were made by Crossref. The Crossref Funder advisory group was aimed at promoting cross-funder collaboration in areas like assigning PIDs to funders as organisations and later on in exploring the technical requirements to issue grant IDs for their funded projects. Same as for PIDs in other areas, one of the first tasks was for this group to define the use cases for persistent identification of grants [START_REF]Funder advisory group[END_REF] .

One additional use case for grant IDs not specifically mentioned in the list above is to ensure the correct identification of specific grants in the references to them that researchers include in the funding acknowledgements section of their manuscripts. Funders use very different internal identification formats for their funded projects and there are frequent mistakes when typing them in these manuscripts, so an additional use case would be to improve the quality of PID graphs so that research outputs can easily be linked to their funding sources [START_REF] Cousijn | Connected Research: The Potential of the PID Graph[END_REF] .

The list of stakeholders involved in this Crossref advisory group shown in reference [START_REF]Funder advisory group[END_REF] Critically, the DOI for the first of these two persistently identified Wellcome-funded grants, https://doi. org/10.35802/207522, a snapshot of whose landing page in Europe PMC is still shown in [15] at the time of writing, does not resolve due to a minor mistake in the URL following an update in the grant record in Europe PMC (grant holder name Prof MC English was updated to Prof M English after the hyperlink has been set). This is particularly revealing in a case study looking into the risks associated with PIDs and highlights the need to be extremely careful when updating any metadata element in a third-party hosted grant ID page at which a PID is pointing. Teething issues aside, the fact that the persistently identified funding acknowledgements have already been included in a published research paper shows the way forward in terms of enhancing the PID Graph via the collaboration of multiple stakeholders in the scholarly communications community.

Moreover, it is now already possible to search Europe PMC for at the time of writing, just Wellcome-funded grants in order to identify their DOIs via the Europe PMC grant finder feature at https://europepmc.org/grantfinder/, see Figure 3 below. This will allow any interested user to check if their Europe PMC member funder of choice is also already issuing grant IDs for their funded projects.

Figure 3. EuropePMC grant finder

Grant finder

Funding iii. This is already starting to happen, see for instance June 2022 tweet showing the grant ID for the H2020 FiberEUse project, https://twitter.com/pcastromartin/status/1537350780545540100 in the acknowledgements section of a manuscript Both examples provided above for the issuing of grant IDs by the Wellcome Trust and the European Commission are following the Crossref workflow and schema. In fact, entries 13 and 15 in the references provided for this case study highlight the need for research funders to become Crossref members in order to be able to follow the procedure described above to issue grant IDs. This may pose a potential risk of lack of uptake by smaller research funders who may either be unaware of this trend or may find the workflow technically unaffordable.

The emergence of an alternative, handle-based mechanism to mint project IDs may offer an alternative way forward in these cases. This potentially alternative route is currently provided by the RAiD initiative for Research Activity identification [START_REF]RAiD: A persistent identifier for research projects[END_REF] . Originally designed by the Australian Research Data Commons (ARDC), this is a free service for minting project IDs that is being considered for its implementation (beyond Australia) in Europe, the US and the UK via independent albeit connected initiatives. In Europe, the EOSC Association is to promote a 'European RAiD' under its FAIRCORE4EOSC project which kickedoff in June 2022, https://faircore4eosc.eu/.

The RAiD Metadata Manifest potentially includes additional PIDs for researchers, research outputs (publications and datasets), organisations and instruments or facilities within the same record, see an example of a RAiD record structure in Figure 5 below [START_REF] Burton | BoF: Identifying, connecting and citing research with persistent identifiers (PIDs)[END_REF] . This PID initiative -which is currently undergoing registration as an official ISO standard 18 -has so far been adopted only in Australia. As per the FREYA project report dated Nov 2020 [START_REF] Ferguson | FREYA Project Deliverable D4.6 Emerging and New PID Graph Resource Types in Disciplinary Contexts[END_REF] , RAiDs were being used by seven Australian organisations at the time this was written, with an additional 24 having access to the RAiD-minting system but with no active integrations yet as of then. 5,336 RAiDs were said to have already been issued by then.

While RAiDs are to be issued by institutions and will not be able to include a DOI-based grant ID within their 'metadata envelope' unless this has previously been minted by the appropriate research funder, RAiD implementation could offer an alternative way forward for the persistent identification of projects in cases where their funders haven't been able to issue grant IDs for them. These grant IDs could be added at a later stage, and in the meantime RAiDs would include ORCIDs for the researchers involved in a project, OrgIDs for their institutions, Crossref DOIs for the publications, DataCite DOIs for the datasets and so on. Research funders are ideally placed to promote the use of PIDs by researchers they fund. In order to become key drivers for PID adoption, funders need to watch the progress around the PID landscape, avoid fragmentation and identify appropriate collaboration forums.

ORCID is the most consolidated PID among those whose implementation has been community-driven. Portugal's outlier position in Figure 1 showing ORCID uptake (2015-2019) provides evidence of the key role that research funders may be able to play in the adoption of PIDs (widespread ORCID uptake in Portugal was a direct outcome of the emphasis made by the Portuguese Foundation for Science and Technology in requiring ORCID registration from their funded researchers). However, all other countries shown on this graph display rather limited rates of ORCID adoption and use, even when ORCID consortia have been operating for quite some time in many of these countries.

The current PID landscape is rather complex and it could be fair to expect varying degrees of adoption for different PIDs depending on the emphasis that research funders (among others) are willing to make on each of these. The NWO PID strategy for instance -perhaps the best example available at the time of writing for a coherent funder-driven approach to PID adoptionspecifically focuses on ORCIDs, Crossref grant IDs and OrgIDs as the most urgent priorities for PID adoption from a funder's perspective. This section provides a broad analysis of the issues around risk and trust perceived to be relevant with regard to funder engagement in PID adoption.

Funder involvement in harmonised strategies for PID adoption

The lack of funder coordination initiatives has already been mentioned above as a significant risk that could prevent best practices to be widely shared across funders. Some examples have also been provided for the sort of collaborative initiatives that could help bridge this potential gap, especially the Crossref funder advisory group and Europe PMC network.

Recommendation number 5 in the NWO PID strategy suggests [NWO] "to collaborate with other funders in the international PID landscape, for instance by participating in such a coordination effort within the context of Science Europe". Issues remain nevertheless in areas like the following ones:

` Not all funders have the required technical expertise that would allow them to engage in PID-issuing initiatives;

`Most cross-funder collaborative networks are designed to deal with policy-making issues rather than to address technical ones;

`Funders, especially small ones, may not be willing (or even have the workflows in place) to pay a membership fee to an organisation for the purpose of minting their own PIDs;

`Big national-level funders like the DFG, the NWO and the UKRI and Wellcome have successfully advocated for ORCID adoption by their funded researchers, but it may be harder for them to promote more complex PIDs whose benefits may not be so evident yet;

`The collaborative workflows and endeavours between funders and institutions are often there in many countries, but are largely missing in many other ones.

The Knowledge Exchange partnership encompasses six member countries where the situation around these issues is generally an advanced one. Not only do some funders in KE countries already have PID strategies, but the collaborative networks within the wider scholarly communications community are well established. A recommendation is then for KE to consider the possibility of acting as a frontrunner in the area of funder involvement in PID adoption, with the appropriate international coordination mechanisms in place that would complement -and overlap with -the efforts that other key stakeholders in this area are already conducting such as Crossref, DataCite, ORCID iv or the Research Data Alliance (RDA) V .

Potential divergences in the technical workflows for PID adoption

Partially as a result of the lack of coordination across national-level research funders, there is a risk for their adopting different technical solutions when promoting PID adoption in their countries. Some examples of these could be the (current) ROR vs Ringgold dichotomy as a technical basis to mint OrgIDs, or using Crossref grant IDs vs allowing institutions to start implementing RAiDs as project identifiers. Transparent communication practices by research funders in terms of disseminating their support for and their progress around implementing various PIDs could tackle this risk, but some forum for this sort of exchange to take place should be available. The discontinuation of the PIDapalooza series of events may pose an issue in this regard, but other venues could rise up to the challenge.

The EU-funded FREYA project managed to bring together all relevant stakeholders in the PID landscape for a joint initiative aimed at "iteratively extending a robust environment for PIDs into a core component of European and global research e-infrastructures". In a similar way, EOSC-funded projects such as those stemming from the HORIZON-INFRA-2021-EOSC-01 call to enable an operational, open and FAIR EOSC ecosystem (2021) could also play a key role in terms of the international PID community management both from a technical and a social perspective.

Funders ideally placed to gain researchers' trust

A distinction between 'technical' and 'admin-oriented' PIDs has been made in another case study in this series devoted to PIDs for instruments and facilities. 'Technical PIDs',are those identifiers, such PIDs for geo samples or for research instruments and facilities, whose implementation has so far mainly been driven by researchers with little involvement from other stakeholders like institutions or research libraries (or indeed research funders). This is in contrast to the more 'admin-oriented PIDs' (such as ORCID, OrgIDs, grantIDs and RAiDs) whose use cases much more clearly serve the objectives of the wider scholarly communications community. Awareness of these admin-oriented PIDs among researchers is typically much lower. These are not clear-cut categories, but from the perspective of the risks associated to PID implementation this is a useful classification to bear in mind.

iv ORCID operates its own Funder Working Group, which only partially overlaps with other associations of research funders mentioned above, https://info.orcid.org/funder-working-group/. While the presence of European and specifically KE member country funders in the group is relatively small, this forum has a strong representation from relevant areas like Oceania and Latin America.

v

The RDA National PID Strategies Working Group brings together various national agencies and initiatives with the purpose of mapping common activities and reporting on the specific PIDs adopted in the context of national PID strategies, https://www.rd-alliance.org/groups/national-pid-strategies-wg. Several core KE agencies (Jisc, SURF and CSC) currently represent their respective countries (and their key funders) in the group.

While research institutions may well be able to deal with the latter group of 'admin-oriented' PIDs on their own, the research funders' involvement is seen as critical for ensuring a widespread adoption of the more technical PIDs such as identifiers for instruments or geo samples. This is because these technical PIDs are likely to see a bottom-up implementation in which researchers take the lead.

Research funders are ideally placed to identify researchers' best practices with regard to these technical PIDs and to further promote them in specific disciplines. This role would also involve discussing such best practices with institutions, which may often not be aware of them. The NWO PID strategy proposes the creation of a very timely Dutch PID advisory board in which the national research funder's involvement is strongly recommended. This would ensure a solid coordination across key stakeholders in the country and beyond. This is in fact a recommendation that other countries -and specifically Knowledge Exchange member countries -could also bear in mind.

This case study has mainly been written by Pablo de Castro (University of Strathclyde and euroCRIS, ORCID https:// orcid.org/0000-0001-6300-1033) within a team of consultants including Ulrich Herb (Saarland University, ORCID https:// orcid.org/0000-0002-3500-3119), Laura Rothfritz (Humboldt University Berlin, ORCID https://orcid.org/0000-0001-7525-0635) and Joachim Schöpfel (University of Lille and euroCRIS, ORCID https://orcid.org/0000-0002-4000-807X) under the umbrella of scidecode science consulting (ROR https://ror.org/02c0bjd31). The work has been overseen by the Knowledge Exchange Task & Finish Group whose composition is listed at https://www.knowledge-exchange.info/event/ pids-risk-and-trust.

Figure 1 .

 1 Figure 1. ORCID adoption vs completeness by country worldwide (2015-2019) 6

Figure 2 .

 2 Figure 2. Swedish research funders (public and private) involved in SweCRIS

Figure 4 .

 4 Figure 4. Example DOI-based grant ID for a EC-funded H2020 project

Figure 5 .

 5 Figure 5. RAiD record structure for funded projects

Table 1 .

 1 A snapshot of the key research funders in KE member countries

	Country	Research funder	Comments
	Netherlands	`NWO/ZonMW	`Lead 'NWO PID Strategy' 2
			`EuropePMC member
	Germany	`DFG	`Leads ORCID DE project
		`Volkswagen Foundation
	United Kingdom `UKRI	`UKRI is member of Crossref Funder advisory group
		`Wellcome Trust	`Wellcome is EuropePMC member
	Denmark	`Danish National Research	`No 'hegemonic' research funder as in other KE
		Foundation (DNRF)	countries, but a mix of public and private funders
		`Danish Ministry of Science,
		Technology & Innovation
		`Independent Research Fund
		Denmark (DFF)
		`Innovation Fund Denmark
		`Novo Nordisk
		`Carlsberg Foundation
	Finland	`Academy of Finland	`Academy of Finland is a member of "cOAlition S"
		`Business Finland
		`Kone Foundation
		`Svenska litteratursällskapet
	France	`Agence nationale de la	`French ORCID Consortium
		recherche (ANR)	`INIST is a member of DataCite and delivers DOIs to
		`Ministère de l'enseignement	research outputs
		supérieur et de la recherche	`National Program for Open Science https://www.
		`Centre national de la	ouvrirlascience.fr/second-national-plan-for-open-
		recherche scientifique (CNRS)	science-npos/

  At the time of writing the OpenAIRE funder dashboard lists 24 funders engaging with the OpenAIRE monitoring services, among them the European Commission, the French National Research Agency (ANR), the Academy of Finland, the Austrian FWF, the Portuguese FCT, the Dutch NWO, Science Foundation Ireland and the Swiss National Science Foundation (SNSF)

	360Giving is a UK non-profit launched in 2015 aiming
	to collect comprehensive funding information in an
	open and standardised way across a range of UK
	research funders including charities and Government
	departments. The data collection workflows are based
	on the 360Giving Data Standard. At the time of writing
	the list of UK funders providing their data to 360Giving
	includes over 220 funders contributing over £170bn of
	grant funding 10 . The fact that most charities that were part of the former Charity Open Access Fund (COAF) partnership -Arthritis Research UK, Breast Cancer Now, the British Heart Foundation, Cancer Research UK, Bloodwise and Parkinson's UK -are currently not listed among these over 220 data providers shows the	SweCRIS is an initiative presented at the CRIS2018 Conference to gather funding information from public and private research funders in Sweden in a harmonised way 9 . The SweCRIS database includes research projects funded by 11 different Swedish research funders, https://www.vr.se/english/swecris.html#/. This

Other international initiatives worth mentioning aimed at collecting aggregated funding information and at bringing together research funders include Europe PubMed Central (Europe PMC). National-level efforts include the SweCRIS project in Sweden and the 360Giving initiative in the UK.

Europe PMC lists 36 European Life Sciences research funders across Europe at the time of writing, https:// europepmc.org/Funders/, including the Wellcome Trust, the European Research Council (ERC), NWO and ZonMW in the Netherlands, the Austrian Research Fund FWF, Telethon Italy and the Chief Scientist Office (CSO) in Scotland among others. This funder network is strongly supporting the implementation of Open Science by all the funders involved in it, which includes making openly available their project funding information and linking it to the research outputs stemming from these funded projects. The implementation of PIDs for grants is also an area where some cross-funder technical collaboration is already taking place within this group, making it the most suitable forum at present for a harmonised push towards PID implementation from funders. example for a multiple-funder CRIS (Current Research Information System) highlights the value of such ii. OpenAIRE NOADs for KE member countries are Syddansk Universitet (DK), Couperin (FR), Universität Konstanz (DE), Helsingin yliopisto (FI), DANS (NL) and Jisc (UK), https://www.openaire.eu/contact-noads systems for collecting this kind of research information from funders. The fact that the standards for information exchange between the funders' systems and the SweCRIS national portal are directly discussed and agreed with funders means they are brought into a national-level research information management community that can also be a very useful forum when discussing PID implementation. difficulty of bringing all research funders under a single umbrella. However, the largest former COAF partner and also largest independent research funder in the UK, the Wellcome Trust is indeed included in 360Giving since April 2018. Both the remarkable growth in the number of 360Giving data providers and the fact that this initiative includes a Data Champions programme to "bring funders together to collaborate and learn how to grow a data culture in their organisations" could make it a suitable candidate to promote the gradual implementation of PIDs across their funder partners.

There are of course other international collaboration networks among research funders -Science Europe and cOAlition S being the two most prominent European examples at present. The emphasis on the three examples shown above is due to the fact that the joint effort across funders for the adoption of PIDs requires a degree of technical collaboration that the above-mentioned initiatives are already conducting. While more policy-making-oriented research funder forums may also provide a valuable contribution in this domain, it is the joint technical work that is expected to truly make a difference.

  includes research funders like the European Research Council, UK Research and Innovation, the Wellcome Trust, Science Foundation Ireland and the Austrian Research Fund FWF (plus a good number of non-European members too). Crossref is already the big one [cross-funder coordination forum] but I have had more one-toones with other funders during last year, some of them in America and one in Australia. So I think the awareness is there. And people definitely do reach out -the party was able to find me and contact me to have that chat. So I think there's definitely an awareness, and I guess there's also a demand to know more. We have just discussed, okay, here's the people, here's what we have to do. It isn't just a nice easy case of "Oh, you could just provide the data and off you go". There is always going to be work at some technical level, particularly addressing technical barriers, to produce PIDs. Grants and persistent IDs for researchers, the ORCIDs, I think, are the easiest ones, because you are already collecting a lot of this information anyway".

	provide landing pages for the DOIs assigned to their grants -thus opening the door for other Europe PMC member funders to follow the same procedure. A June 2020 post on the Europe PMC blog 15 announced the first grant IDs had been made available for Wellcome-	2. Government vs. private funding relationships e.g. which private funders work with which governments to support what kind of research?
	funded grants in Sep 2019 and provided the technical details (DOIs and metadata sets) for these two first examples that had already been included in the funding acknowledgements for a paper published Oct 2019 in	3. Co-funding e.g. which other funders do my grantees tend to receive support from as well as us?
	PLoS ONE at https://doi.org/10.1371/journal. pone.0222922.	4. Portfolio analysis e.g. a funder invests in centers and individual scientists; which effort
		generates more products?
	Wellcome Trust representative
		As the Crossref initiative for funder registration via
		FundRef (later renamed the Open Funder Registry)
		consolidated, the case for issuing persistent grant IDs
		for registered funders became more achievable. The
		conversation subsequently moved onto the technical
		requirements for this purpose and whether DOIs would
		be an appropriate standard to use 13 .
	Some use cases for the grants project
	1. Multi-country funding e.g. the Australian
	Research Council wants to know which other
	countries their awardees get additional
	funding from.

The Wellcome Trust adopted a frontrunner role in piloting the initiative to issue the first grant IDs. A Feb 2018 guest post on the Crossref blog by Wellcome representatives announced the forthcoming issuing of their grant IDs and the funder's rationale for implementing these PIDs

[START_REF] Kiley | Wellcome explains the benefits of developing an open and global grant identifier[END_REF]

. The Wellcome Trust being a founding member of Europe PMC, they used this platform to "

:

  MM, GK, JM, MZ and OT were supported by funds through a grant from the Economic and

	Because there is a pattern in the way (for instance)	been issued for all H2020 projects, the plan is to mint
	Wellcome-funded grant ID 202924/Z/16/Z is assigned	Development Planning and Coordinating Agency additional ones for Horizon Europe projects while at the
	DOI 10.35802/202924, adding this information into	(NEPAD Agency) with funding from the Wellcome same time progressing 'backwards' with grant ID
	institutional CRIS and/or project management systems	Trust (#107769, https://doi.org/10.35802/107769) minting for projects funded under previous research
	should be straightforward, allowing grant IDs to be	and the UK government. framework programmes down to FP1.
	increasingly used by researchers in their manuscript
	acknowledgements as soon as they are made available.	As shown in Figure 4 below, DOIs are already
		displayed on a number of H2020 project webpages in
		the CORDIS database. Discussions with publishers
		are also underway to gradually promote among
		EC-funded researchers the inclusion of these new
		grant IDs in their funding acknowledgement
		statement in their manuscripts iii .
	Social Research Council ESRCS ES/P004938/1)
	awarded to ME. A Senior Research Fellowship
	awarded to ME by The Wellcome Trust (#207522,
	https://doi.org/10.35802/207522) supported PM.
	MM received additional support from a grant to the
	Initiative to Develop African Research Leaders

(IDeAL) through the DELTAS Africa Initiative (DEL-15-003), an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Further work around the implementation of grant IDs has recently been undertaken by the European Commission. The EC's Directorate-General for Research and Innovation (DG RTD) in collaboration with the EU Publications Office is currently minting grant IDs (DOIs) for all Horizon 2020 projects. The current effort, which aims to reach 35,000 minted grant IDs for H2020 projects by the end of 2022, is following both the Crossref workflow and schema. Once grant IDs have

Rationale

Endorsement of PIDs by research funders and opportunities for cross-funder collaboration

Issues around risks and trust regarding the adoption of PIDs by research funders

Authorship


Vincent Laffilé

Kevin Moreno

Eric Merlet

Nathan Mcclenaghan

Yann Ferrand
email: yann.ferrand@u-bordeaux.fr

Céline Olivier
email: celine.olivier@u-bordeaux.fr

CPL-Active Water-Soluble Aromatic Oligoamide Foldamers

A series of enantiopure water-soluble quinoline-based foldamers were prepared and their optical and chiroptical properties in water were investigated. The new hexameric sequences incorporated either cationic or anionic water-solubilizing chains, and one of the oligomers was additionally functionalized by an electron donating moiety to further modulate the optoelectronic properties. A systematic study revealed strong electronic circular dichroism and circularly-polarized luminescence properties in water, with dissymetry factors up to 2 x 10 -2 in absorption and 5 x 10 -3 in emission, regardless of the nature of the solubilizing chains and functions. This study therefore highlights new opportunities for the development of water-soluble and chiroptically-active artificial systems towards chirality-associated applications in aqueous or biological media.

Introduction

Chiral organic fluorophores that emit circularly polarized luminescence (CPL) have attracted considerable attention due to their potential application in the fields of 3D imaging, advanced security inks or device applications. 1 Furthermore, the circular polarization of emitted light may be used to study dynamic processes such as molecular chiral recognition. [START_REF] Meskers | Circular Polarization of Luminescence as a Tool to Study Molecular Dynamical Processes[END_REF] However reports on effective CPL emission from chiral small organic molecules in water remain scarce, [START_REF] Pascal | Zwitterionic [4]helicene: a watersoluble and reversible pH-triggered ECD/CPL chiroptical switch in the UV and red spectral regions[END_REF][START_REF] Kitatobe | Circularly polarized luminescence from open-and closed-style axially chiral amphipathic binaphthyl fluorophores in water[END_REF][START_REF] He | Water-soluble chiral tetrazine derivatives: towards the application of circularly polarized luminescence from upperexcited states to photodynamic therapy[END_REF][START_REF] Mimura | Sign inversion of excimer circularly polarized luminescence in water-soluble bipyrenyl oligopeptides through an odd-even effect[END_REF][START_REF] Olivier | A -extended phenanthrene-fused aza[7]helicenium as novel chiroptically-active architecture in organic and aqueous media[END_REF] limiting their potential application for biological purposes such as chiral analyte sensing or chiral bio-imaging. [START_REF] Staszak | Lanthanides Complexes -Chiral Sensing of Biomolecules[END_REF] Asymmetry in organic compounds is primarily expressed as point chirality (i.e. asymmetric carbon), axial chirality (i.e. atropoisomerism) or helicity. The latter, although being the prominent expression of chirality at the macromolecular scale, involves intramolecular electronic interactions that may be sensitive to protic solvents. For this reason, water-soluble artificial helical structures represent challenging targets. In this regard, applying a bio-inspired strategy, Huc and co-workers developed several water-soluble helical architectures based on aromatic oligoamide [START_REF] Gillies | Development and Biological Assessment of Fully Water-Soluble Helical Aromatic Amide Foldamers[END_REF][START_REF] Iriondo | Cellular Internalization of Water-Soluble Helical Aromatic Amide Foldamers[END_REF][START_REF] Baptiste | Solid Phase Synthesis of Aromatic Oligoamides: Application to Helical Water-Soluble Foldamers[END_REF][START_REF] Dawson | Controlling Helix Handedness in Water-Soluble Quinoline Oligoamide Foldamers[END_REF][START_REF] Ziach | Single Helically Folded Aromatic Oligoamides that Mimic the Charge Surface of Double-Stranded B-DNA[END_REF] backbones. In addition to high helical stability in water, these oligomers have been shown to be highly tuneable. Unlike natural peptidic -helices whose folding relies strongly on their side chains, aromatic oligoamide foldamers can be functionalized by various types of functional groups without any major impact on their helical stability. These modular molecular platforms were first used for their cellpenetrating capabilities and, as their nontoxicity were established, they were considered for biological applications. 9- 10 Later, Huc et al. demonstrated that these artificial oligomers could serve as efficient DNA mimics paving the way to the design of novel inhibitors of protein-DNA interactions. [START_REF] Ziach | Single Helically Folded Aromatic Oligoamides that Mimic the Charge Surface of Double-Stranded B-DNA[END_REF] In parallel, we and others have reported on the CPL properties of related classes of quinoline oligomers in organic media. [START_REF] Merlet | Aromatic Oligoamide Foldamers as Versatile Scaffolds for Induced Circularly Polarized Luminescence at Adjustable Wavelengths[END_REF][START_REF] Zheng | Significant Enhancement of Circularly Polarized Luminescence Dissymmetry Factors in Quinoline Oligoamide Foldamers with Absolute Helicity Org[END_REF][START_REF] Zheng | Absolute Control of Helicity at the C-termini in Quinoline Oligoamide Foldamers by Chiral Oxazolylaniline Moieties[END_REF][START_REF] Moreno | Influence of Positional Isomerism on the Chiroptical Properties of Functional Aromatic Oligoamide Foldamers[END_REF][START_REF] Zheng | Red Circularly Polarized Luminescence from Intramolecular Excimers Restricted by Chiral Aromatic Foldamers[END_REF] Notably, functional oligoamide foldamers appended with different fluorophores were shown to display strong CPL emission at adjustable wavelengths. [START_REF] Merlet | Aromatic Oligoamide Foldamers as Versatile Scaffolds for Induced Circularly Polarized Luminescence at Adjustable Wavelengths[END_REF] Herein, we report on the preparation of a series of enantiomerically pure water-soluble quinoline oligomers and the evaluation of their optical and chiroptical properties in water. For all sequences, quantitative helix handedness bias was achieved by introducing a camphanic acid moiety at the N terminus. [START_REF] Kendhale | Absolute Control of Helical Handedness in Quinoline Oligoamides[END_REF] A systematic study was carried out on quinoline sequences bearing water-solubilizing chains including either cationic (e.g. ammonium or guanidinium) or anionic (phosphonate) functional groups. Cationic side-chains are known to assist cell-membrane permeation [START_REF] Iriondo | Cellular Internalization of Water-Soluble Helical Aromatic Amide Foldamers[END_REF] whereas precisely positioned anionic side-chains can mimic DNA charge-surface. [START_REF] Ziach | Single Helically Folded Aromatic Oligoamides that Mimic the Charge Surface of Double-Stranded B-DNA[END_REF] Additionally, the substitution of a cationic side chain by an electron donating morpholine moiety was implemented to tune the emission wavelength of foldamers in polar solvents.

Results and discussion

Design and synthesis

First, it is worthy to note that unlike the preparation of functionalized organic solvent soluble aromatic oligoamides prepared by conventional multi-step synthesis requiring lengthy synthesis routes and purification, the preparation of watersoluble analogues can be achieved via straightforward solidphase supported methods followed by HPLC purification. For this purpose, two new monomers Fmoc-Q Gua -OH and Fmoc-Q Mor -OH were prepared specifically. Fmoc-Q Gua -OH was obtained from NO2-Q Orn -OMe using N,N'-bis(tertbutoxycarbonyl)-1H-pyrazole-1-carboxamidine to form the Boc protected guanidine side chain (Scheme S1). Fmoc-Q Mor -OH was prepared via the nucleophilic aromatic substitution of the bromine of NO2-Q Br -OMe by a morpholine residue (Scheme S2). In both cases, after reduction of the nitro group and saponification of the methyl ester, the Fmoc group was introduced using 9-fluorenylmethyl chloroformate. Solid phase synthesis of sequences 1-4 (Fig. 1) were performed on low loading LL ProTide resin (0.17 mmol.g -1 ) using a Fmoc strategy (Scheme S4-S7). [START_REF] Baptiste | Solid Phase Synthesis of Aromatic Oligoamides: Application to Helical Water-Soluble Foldamers[END_REF] The first Fmoc-acid monomer was grafted using CsI and DIEA as a base in dry DMF. Fmoc-acid building blocks were activated in situ by generating the respective acid chlorides using an Appel reaction (PPh3/trichloroacetonitrile) prior to coupling. One should note that in situ activation of Fmoc-Q Gua -OH failed and was replaced by the pre-activation of the acid using Ghosez reagent prior to coupling (see the supporting information). Then, S-or R-camphanic chloride was reacted with the terminal amino group of sequences 1-4 to induce a P or M handedness, respectively. Finally, the oligomers were cleaved from the resin and the side chains deprotected under acidic conditions. After purification using semipreparative reverse phase HPLC and salt exchange when necessary, sequence 1, 3 and 4 were obtained in good overall purified yields (57-80%). Sequence 2 was obtained with a lower overall yield (7%) yet was sufficient for the study.

The foldamers prepared in this study are all hexameric quinoline-derived oligomers whose sequences are represented in Fig. 1. Sequence 1 is exclusively composed of Q Orn monomers whose water-solubilizing chain is a propyloxyammonium salt and as a result sequence 1 is a polycationic species. In the same way, sequence 2 is composed of Q Gua monomers only, in which the water-solubilizing chain is a propyloxyguanidinium salt, making it polycationic like 1. On the contrary, 3 is a polyanionic sequence, composed of Q Pho monomers, bearing a methyloxyphosphonate chain. Finally, 4 is a hetero-oligomeric sequence composed of five Q Orn and one Q Mor monomers. The functionalized quinoline Q Mor was introduced at position 2 of the sequence, based on previous studies related to the positional isomerism of such functionalized oligoquinoline foldamers. [START_REF] Moreno | Influence of Positional Isomerism on the Chiroptical Properties of Functional Aromatic Oligoamide Foldamers[END_REF] In addition to the four water-soluble hexameric sequences, Fig. 1 represents previously reported sequence 5, [START_REF] Moreno | Influence of Positional Isomerism on the Chiroptical Properties of Functional Aromatic Oligoamide Foldamers[END_REF] composed of Q Leu monomers, i.e. bearing isobutoxy chains that make it highly soluble in chlorinated solvents.

Optical Properties in Water

The optical properties of the water-soluble sequences 1-4 were evaluated in pure water. The normalized UV-visible absorption and emission spectra are shown in Fig. 2 and the corresponding values are regrouped in Table 1. The data corresponding to sequence 5 recorded in chloroform are indicated for comparison. All hexamers show comparable absorption profiles in solution. The spectra present two intense absorption bands around 325 nm and 345 nm, characteristic of the oligoquinoline backbone. These absorption bands correspond to n  π* and π  π* transitions within the aromatic part. The main differences in absorption lie in the corresponding molar absorption coefficients that significantly vary depending on the nature of the solubilizing chains. Hexamer 3, bearing negatively charged end-groups, exhibits the highest molar absorption coefficients of the series, max = 38 500 and 35 500 M -1 .cm -1 . These values are in the same range of those measured in chloroform for sequence 5 equipped with isobutoxy chains,  = 42 000 M -1 .cm -1 and 38 200 M -1 .cm -1 . However, the hexamers bearing positively charged end-groups show a slightly lower absorption coefficient. Hexamer 1 bearing propyloxyammonium chains shows  = 33 000 and 30 500 M - 1 .cm -1 , and upon replacing the ammonium groups by guanidinium end-groups as in sequence 2, a net decrease of the molar absorption coefficient is observed,  = 25 800 and 23 700 M -1 .cm -1 . The morpholine-functionalized analogue 4 shows the  abs /nm [a]  /M -1 .cm -1  em. /nm [b]  lum /% [c]  [d] gabs [e] glum [ (P) 1.1 x 10 -2 (400), 0.6 x 10 -2 (390) (M) -1.0 x 10 -2 (400), -0.5 x 10 -2 (390) 450) [a] Maximum absorption wavelength in H2O (C = 3 x 10 -5 M). [b] Maximum emission wavelength in H2O (exc = 365 nm). [c] Fluorescence quantum yield. [d] Molar ellipticity. [e] Absorption dissymmetry factor and corresponding wavelength. [f] Luminescence dissymmetry factor and corresponding wavelength. [g] In CHCl3 (data from ref. 17).

(P) 1.3 × 10 -2 (450) (M) -1.1 × 10 -2 (
lowest absorption intensity of the series with  = 20 400 and 19 300 M -1 .cm-1 . Overall, the molar absorption properties are thus generally preserved from an organic medium to an aqueous medium.

The emission properties of the water-soluble sequences were evaluated in pure dilute water solutions (∼10 -6 M) at room temperature. Overall, the fluorescence spectra show a broad unstructured band in the visible with red-shifted maximum emission compared to that of hexamer 5 recorded in chloroform (em = 452 nm). Sequences 1 and 2 show maximum emission at em = 502 nm and 519 nm, respectively, which represent significant red-shifts of 50 nm and 67 nm compared to 5 in CHCl3. Hexamer 3, bearing negatively charged endgroups, exhibits a smaller red-shift of 32 nm corresponding to an emission maximum at em = 484 nm. In general, the photoluminescence of discrete molecules in solution originates from the relaxation of the system from its photoexcited state to the ground-state, and this phenomenon is related to the HOMO-LUMO energy gap of the molecule. The red-shifting of the emission maxima observed in water compared to organic solvent therefore corresponds to a reduction of the HOMO-LUMO gap presumably due to interactions of the molecules with the highly polar water solvent and which also allows rapid non-radiative decay of the excited state by vibrational relaxation due to the presence of vicinal O-H oscillators.

As expected, hexamer 4 bearing ammonium end-groups plus an additional morpholine functional group, presents further increased red-shifting of the emission, with em = 568 nm, and consequently a large Stokes shift of 221 nm (Δν = 11213 cm -1 ). This appealing property of 4 might be advantageous in the context of fluorescence imaging applications, since the wide gap between the excitation and emission maxima diminishes self-absorption phenomena.

The fluorescence quantum yields evaluated for the nonfunctionalized hydrophilic hexameric sequences 1-3 in water range from 0.01% to 0.12%. These values are lower than that of sequence 5 recorded in chloroform (lum = 6%) as one could expect from the enhanced non-radiative deactivation processes occurring in polar protic solvents such as water. [START_REF] Maillard | Universal Quenching of Common Fluorescent Probes by Water and Alcohols[END_REF] Nonetheless, thanks to the presence of the morpholine group acting as electron donating unit, sequence 4 yields slightly higher fluorescence quantum yield of lum = 0.42%, thus showing that modulation of the emission properties of quinoline-based oligomers is also possible in water.

Chiroptical Properties in Water

The chiroptical properties of the two P and M enantiomers of each water-soluble sequence were evaluated in pure water using electronic circular dichroism (ECD) and circularly polarized luminescence (CPL). The spectra are shown in Fig. 3 and the corresponding values are regrouped in Table 1. Regardless of the nature of the solubilizing chains, intense ECD signals are typically observed for the different water-soluble oligoamides confirming the efficient folding of these oligomers as supramolecular helices even in aqueous medium. Similar to what was observed with sequence 5 in chloroform, the ECD spectra of the water-soluble hexamers show multiple Cotton effects corresponding to their UV-visible absorption region.

Notably, two intense absorption bands are observed around 410 and 390 nm that are characteristic of quinoline oligomers. As in organic solvents, the P enantiomers yield a positive Cotton effect at low energy, while M enantiomers yield a negative Cotton band. Two bands of weaker intensities and with sign inversions are observed between 280 and 370 nm. Finally, an intense band is systematically observed at 260 nm, which is positive for the P enantiomers and negative for M enantiomers.

Hexamers 1 and 3, bearing positively-charged propyloxyammonium chains and negatively-charged methyloxyphosphonate chains, respectively, present quasisuperimposable ECD spectra. This tends to indicate that the nature of the solubilizing end-groups (positively-or negativelycharged) has no (or little) impact on the chirality of the groundstate electronic transitions. In terms of intensity of the ECD response, those of sequences 2 and 4 are slightly weaker than that of sequences 1 and 3. The maximum molar circular dichroism values range from Δεmax = 108 M -1 . cm -1 for P-1 to 84 M -1 . cm -1 and 57 M -1 . cm -1 , for P-2 and P-4, respectively. This, in the case of 4, suggests an electronic perturbation occurring within the functionalized oligomer in its ground state, similar to previously reported functionalized quinoline hexamers. [START_REF] Moreno | Influence of Positional Isomerism on the Chiroptical Properties of Functional Aromatic Oligoamide Foldamers[END_REF] To quantitatively assess the magnitude of the ECD, the dimensionless Kuhn's anisotropy factor in the ground state, i.e. absorption dissymmetry factor, defined as gabs =  /  , was considered. Thus, the water-soluble hexameric sequences 2 and 3 show gabs up to 2 x 10 -2 , which is slightly higher than sequence 5 in chloroform. Such gabs values are one order of magnitude higher in comparison to the C2-symmetric water-soluble binaphthyl fluorophores reported by Imai, [START_REF] Kitatobe | Circularly polarized luminescence from open-and closed-style axially chiral amphipathic binaphthyl fluorophores in water[END_REF] and it is also higher than that recently reported for a chromophoric π-extended aza [7]helicenium in an aqueous medium. [START_REF] Olivier | A -extended phenanthrene-fused aza[7]helicenium as novel chiroptically-active architecture in organic and aqueous media[END_REF] The chiral emission properties of the new hexamer series were further assessed in water. Irrespective of the nature of the solubilizing chains, and similar to the chiral absorption properties, a CPL response was systematically observed for the different oligomers in aqueous solution. The sign of the CPL signals was the same as the low-energy ECD signals, i.e. positive for the P enantiomers and negative for the M enantiomers. Hence the local chirality of the corresponding electronic transitions is conserved in the ground-and excited-states. The circularly polarized emission range of the water-soluble oligomers corresponds to that of the total unpolarised photoluminescence (PL) in water. The Kuhn's anisotropy factors in the photoexcited state, i.e. luminescence dissymmetry factors glum, were therefore calculated considering the CPL intensity at maximum PL. Overall, the water-soluble sequences exhibit significant CPL activity, with glum values ranging from 1.3 x 10 -3 (M-4) to 5.3 x 10 -3 (P-2) at maximum PL, which represents remarkable chiral emission for small organic molecules. [START_REF] Sanchez-Carnerero | Circularly Polarized Luminescence from Simple Organic Molecules[END_REF][START_REF] Tanaka | Circularly Polarized Luminescence and Circular Dichroisms in Small Organic Molecules: Correlation between Excitation and Emission Dissymmetry Factors[END_REF] Sequences 1, 2 and 3 display comparable CPL intensities with glum = 3.4 x 10 -3 to 5.3 x 10 -3 for the P enantiomers, indicating that the nature of the water-solubilizing chains has only poor influence on the chiral emission properties. The three watersoluble hexameric sequences devoid of additional functional group therefore show slightly lower dissymmetry in emission than sequence 5 in chloroform (glum = 1.1 x 10 -2 for P-5). Nonetheless, the glum values reported herein are in the same range of previously reported aza [7]helicenium in H2O/MeOH solvent mixture (glum = 6 x 10 -3 ) 7 and they are significantly higher than that of functionalized binaphthyl fluorophores in water and in MeOH (glum = 4 x 10 -4 ). [START_REF] Kitatobe | Circularly polarized luminescence from open-and closed-style axially chiral amphipathic binaphthyl fluorophores in water[END_REF] As for the fluorescence emission, the electronically enriched sequence 4 exhibits red-shifted CPL signal compared to the non-functionalized water-soluble sequences, yielding CPL emission at  = 558 nm with an associated glum = 2.4 x 10 -3 for P-4. The slightly lower CPL intensity observed for the functionalized sequence 4 compared to the hexamers without additional substituents is consistent with previous observations in organic solvent and stems from the perturbation of the excited states related to the presence of the achiral fluorophore on the chiral oligomeric helices. [START_REF] Merlet | Aromatic Oligoamide Foldamers as Versatile Scaffolds for Induced Circularly Polarized Luminescence at Adjustable Wavelengths[END_REF][START_REF] Moreno | Influence of Positional Isomerism on the Chiroptical Properties of Functional Aromatic Oligoamide Foldamers[END_REF] A correlation between the absorption and emission dissymmetry factors (gabs and glum) was established by Mori et al. considering several categories of chiral small organic molecules. [START_REF] Tanaka | Circularly Polarized Luminescence and Circular Dichroisms in Small Organic Molecules: Correlation between Excitation and Emission Dissymmetry Factors[END_REF] Although the supramolecular helices reported in the present study do not fall into the categories described in this review article, their characteristics are in line with other molecules of helical chirality (i.e. helicenes and helicenoids). The general trend is glum < gabs, to a greater or lesser extent related to the conformational flexibility of the molecules in their excited state. In particular the authors consider the glum/gabs ratio to quantify the difference in asymmetry between the chiroptical properties of absorption and emission. In the present study, this ratio falls in the range of 0.25 (sequence 1) to 0.5 (sequence 3) which, as expected from these dynamic supramolecular architectures, further testifies to the importance of processes of vibrational relaxation on the excited-state through both conformational flexibility and interaction with the polar solvent.

Conclusions

In summary, a series of eight water-soluble quinoline-based oligoamide foldamers was readily prepared through solid-phase supported synthesis for assessment of their optical and chiroptical properties in water. The hexameric sequences were equipped with different types of solubilizing chains, either cationic or anionic, and derivatized with a camphanyl chiral inducer for complete handedness bias induction, leading to Pand M-helices selectively. In addition, one of the new sequences incorporated a morpholine functional group to study its influence on the optoelectronic properties of the foldamer in water. Whatever the nature of the polar end-group present on the solubilizing chains (i.e. ammonium, guanidinium or phosphonate), the helical folding of the oligoamide architectures was confirmed by spectroscopic characterizations. The systematic study carried out in pure water revealed strong chiroptical properties, as proved by ECD and CPL spectroscopies, thus validating our approach of using oligoquinoline foldamers to generate circularly polarized emission in water.

Through this study we therefore demonstrate that oligoquinoline foldamers represent a highly modular molecular platform to observe chiroptical activity, both in absorption and emission, in a variety of solvents including pure water. This represents a new step forward for (supra)molecular chiral emitters towards bio-related applications such as chiroptical bioimaging.

Experimental Section

General procedures for oligomer SPS Resin grafting. On LL ProTide resin (0.17 mmol.g -1 ), the first Fmoc(Q R )CO2H monomer (3 equiv.) was grafted using CsI (5 equiv.) and DIEA (6 equiv.) in dry DMF. The reaction mixture was shaken vigorously overnight. After reaction, the resin was filtered and washed three times with DMF and three times with dichloromethane. Fmoc deprotection. Grafted resin was washed twice with DMF, suspended in a 20% piperidine in DMF solution (4 mL) and slowly stirred for 3 minutes. The resin was then filtered, washed twice with DMF and resuspended in a 20% piperidine in DMF solution and stirred for 7 minutes. The resin was then filtered and washed three times with DMF and three times with dry THF. In situ coupling procedure (Q orn , Q Pho , Q Mor ). The resin was suspended in dry THF and collidine (9 equiv.) was added. A solution of Fmoc(Q X )CO2H monomer (3 equiv.), PPh3 (8 equiv.) and TCAN (9 equiv.) in dry CHCl3 was added to the resin. The reaction was assisted by microwave irradiation (25W, 50°C) for 15 minutes and repeated once. After reaction, the resin was filtered and washed with dry THF and DMF. Acid chloride coupling procedure (Q Gua ). The resin was suspended in dry THF and DIEA (6 equiv.) was added. Fmoc(Q Gua )COCl monomer (3 equiv., scheme S3) was dissolved in dry THF and added to the resin. The reaction was assisted by microwave irradiation (50W, 50°C) for 15 minutes and repeated once. The resin was then filtered and rinsed with dry THF and DMF. Resin cleavage. The resin was washed three times with DMF and three times with dichloromethane and was suspended in a solution of TFA/TIPS/H2O 95:2.5:2.5 (v/v/v). The mixture was vigorously stirred for 4 hours. The resin was filtered and the filtrate was evaporated under reduced pressure. The residual solid was suspended in Et2O and centrifuged at 4°C for 5 minutes. Et2O was removed and the yellow solid was dried under vacuum and then freeze-dried. Preparative HPLC purification. For sequences containing Q Orn and Q Gua monomers. Crude compounds were purified using solvents A and B. The following gradient was used: (0 min): 95 % A, 5% B then (2 min): 95 % A, 5 % B then (22 min): 0 % A, 100 % B then (27 min): 0 % A, 100 % B. Collected fractions were analyzed by analytic HPLC and the relevant ones were combined and freeze-dried twice to remove the excess of TFA. For sequences containing Q Pho monomers. Crude compounds were purified using solvents A' and B'. The following gradient was used: (0 min): 95 % A, 5 % B then (2 min): 95 % A, 5 % B then (22 min): 0 % A, 100 % B then (27 min): 0 % A, 100 % B. Collected fractions were analyzed by analytic HPLC and the relevant ones were combined and freeze-dried twice to remove the excess of triethylammonium acetate.

Fig. 1 .

 1 Fig. 1. a) Structure of Q Xxx (left) and the various side chains that were introduced in position 4 of the quinolone ring (right). The counter ions are trifluoroacetate and ammonium for cationic and anionic side chains, respectively. b) Oligoamide sequences investigated in this study. c) Formulae of the R-and S-camphanyl chiral inducers. d) Energy minimized model (MMFFs) of the P helix of a canonical Q hexamer; the helix is shown in white stick representation, the purple spheres denote the position of the side chains, non-polar hydrogens are omitted for clarity.

Fig. 2 .

 2 Fig. 2. Electronic absorption and normalised fluorescence emission spectra of the quinoline-derived hexamers 1 (blue), 2 (green), 3 (purple), 4 (orange) recorded in H2O and 5 (grey) recorded in CHCl3 (Cabs. = 30 µM ; exc = 365 nm). * Raman scattered peak of water. 21

Fig. 3 .

 3 Fig. 3. ECD (top) and CPL (bottom) spectra of the quinoline-derived hexamers P-1 (blue, solid), M-1 (blue, dashed), P-2 (green, solid), M-2 (green, dashed), P-3 (purple, solid), M-3 (purple, dashed), P-4 (orange, solid), M-4 (orange, dashed) recorded in pure water and P-5 (grey, solid), M-5 (grey, dashed) recorded in CHCl3 (C = 30 µM ; exc = 365 nm).

Table 1 .

 1 Optical and chiroptical properties of sequences 1, 2, 3 and 4. Data corresponding to sequence 5 are reported for comparison according to ref.17.

Acknowledgements

The University of Bordeaux and IdEx Bordeaux are greatly acknowledged for financial support (PhD grant to K.M). V.L thanks ANR for PhD grant (ANR POLYnESI grant nr. ANR-18-CE29-0013-01).

Conflicts of interest

There are no conflicts to declare.