Conformationally Programmable Chiral Foldamers with Compact and Extended Domains Controlled by Monomer Structure

Abstract Foldamers are an important class of abiotic macromolecules, with potential therapeutic applications in the disruption of protein–protein interactions. The majority adopt a single conformational motif such as a helix. A class of foldamer is now introduced where the choice of heterocycle within each monomer, coupled with a strong conformation‐determining dipole repulsion effect, allows both helical and extended conformations to be selected. Combining these monomers into hetero‐oligomers enables highly controlled exploration of conformational space and projection of side‐chains along multiple vectors. The foldamers were rapidly constructed via an iterative deprotection‐cross‐coupling sequence, and their solid‐ and solution‐phase conformations were analysed by X‐ray crystallography and NMR and CD spectroscopy. These molecules may find applications in protein surface recognition where the interface does not involve canonical peptide secondary structures.


General Experimental Naming and Numbering of Compounds
Systematic compound names are those generated by ChemBioDraw TM Ultra version 15.1.0.144 (Perkin Elmer) following IUPAC nomenclature.

Solvents and Reagents
Reactions were carried out under a nitrogen atmosphere in oven-dried glassware unless otherwise stated. Standard inert atmosphere techniques were used in handling all air-and moisture-sensitive reagents. Where necessary, toluene and N,N-DMF (from commercial sources) were de-gassed prior to use by sparging with argon or nitrogen for at least 15 min. Other solvents and reagents were used directly as received from commercial suppliers.

Chromatography
Flash column chromatography was carried out using Fluorochem 60 40-63 micron silica gel. Thin-layer chromatography was carried out using Merck Kieselgel 60 F254 (230-400 mesh) fluorescent treated silica, visualized under UV light (254 nm) or by staining with ninhydrin, ceric ammonium molybdate or aqueous potassium permanganate solutions. 1 H and 13 C NMR spectra were recorded using a Bruker 600, 400 or 300 MHz spectrometer running TopSpin TM software and are quoted in parts per million (ppm) for measurement against tetramethylsilane. Where no tetramethylsilane was present, spectra are referenced relative to the residual non-deuterated solvent peaks. Unless otherwise stated spectra were acquired at 298 K. Topspin TM was used for processing and viewing NMR data. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hertz (Hz). The 1 H NMR spectra are reported as follows: δ / ppm (number of protons, multiplicity, coupling constant J / Hz (where appropriate), assignment). Multiplicity is abbreviated as follows: s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet. Where peaks are not fully resolved but 2D experiments unambiguously identify the regions of multiplet corresponding to particular hydrogens, this is indicated with a subscript denoting the portion of the multiplet. For example, H15RHS would indicate that the right-hand side of the multiplet corresponds to H15. The numbering scheme used for NMR assignment is arbitrary and does not follow any particular convention. The 13 C NMR spectra are reported in δ / ppm. Where necessary or appropriate, two-dimensional (COSY, HSQC, HMBC, NOESY or ROESY) NMR experiments were used to assist the assignment of signals in the 1 H and 13 C NMR spectra. In some cases, complete assignment of spectra was not possible (in particular, where aromatic CHs corresponding to multiple phenyl groups overlapped significantly); in these cases only a partial assignment is reported.

Spectroscopy
Infra-red (IR) spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer equipped with a Perkin-Elmer Universal ATR Sampling Accessory, or an Agilent Cary 630 spectrometer equipped with a DialPath accessory. Samples were deposited on the ATR and DialPath accessories as a thin film. Only selected maximum absorbances (νmax) of the most intense peaks are reported (cm -1 ).
High resolution mass spectra (HRMS) were recorded by Analytical Services and Environmental Projects (ASEP) at Queen's University Belfast on a Waters LCT Premier ToF mass spectrometer using the electrospray ionisation (ESI) technique.
Optical rotations were recorded at the sodium D-line (589 nm) using a Perkin Elmer 341 polarimeter at a temperature of 20 °C and are reported in degrees using concentrations (c) in g . 100 mL -1 . Reported values are the average of eight readings. Circular dichroism (CD) spectra were acquired at 293 K on a Jasco J-815 CD Spectrometer. Samples were placed in a quartz cuvette with a path length of 1 mm.

Crystallography
Low temperature [1] single crystal X-ray diffraction studies were carried out using CuKα radiation on an Agilent Supernova diffractometer equipped with an area detector and graphite monochromator. Raw frame data were reduced using CrysAlisPRO [2] solved using Superflip. [3] Full-matrix least-squares refinement of the structures were carried out using CRYSTALS. [4,5] Full refinement details are given in the supplementary material (CIF). CCDC 1824641 (3), 1824637 (4), 1824639 (5), 1824642 (10), 1824638 (11), 1825640 (12) contain the supplementary crystallographic data for this paper. These data are provided free of charge by The Cambridge Crystallographic Data Centre and copies can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

Melting Points
Melting points were determined for compounds where a preparative recrystallization was carried out. These were acquired on a Stuart SMP10 digital melting point apparatus. Values are given in °C and are uncorrected.

Experimental Procedures and Characterization Data
Compounds 1, 6 and 8 were prepared according to previously reported procedures. [6]

General Procedure A (N-Nosyl Deprotection)
To a stirred, room-temperature suspension of N-nosyl urea (1.0 eq.) and K2CO3 (3.0 eq.) in N,N-DMF (ca. 0.1 M) was added thiophenol (1.5 eq.). The rapid development of a deep orange colour was invariably observed upon the addition of thiophenol; the reaction mixture also turned cloudy over time in some cases. After complete consumption of the N-nosyl-protected starting material by TLC analysis, the reaction mixture was concentrated in vacuo at 60 °C, and the crude residue was taken up in dichloromethane (ca. 20 mL/mmol urea). The solution was washed with NaHCO3 (sat. aq., ca. 10 mL/mmol urea), and the aqueous layer was extracted with dichloromethane (ca. 2 x 10 mL/mmol urea). The combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude product was then purified by flash column chromatography.

General Procedure B (Palladium-Catalysed Coupling of Deprotected Ureas with Aryl Halides)
To a Schlenk tube equipped with a magnetic stir bar was added deprotected urea (1.0 eq.), aryl halide (1.1-5.0 eq.), Pd2(dba)3 (5-10 mol%), and Xantphos (15-30 mol%). Toluene (ca. 0.1 M) was added to the flask, and the resulting suspension was de-gassed by sparging with nitrogen gas for 15-30 min. Cs2CO3 (2.5 eq.) was then added in one portion to the flask, and the reaction mixture was heated to reflux under a nitrogen atmosphere. After complete consumption of the urea starting material by TLC analysis, the reaction was cooled to room temperature, and diluted with dichloromethane (ca. 20 mL/mmol deprotected urea). The solution was washed with water (ca. 10 mL/mmol deprotected urea), and the aqueous layer was extracted with dichloromethane (ca. 2 x 10 mL/mmol deprotected urea). The combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude product was then purified by flash column chromatography.

(S)-4-Benzyl-1-(tert-butyl)imidazolidin-2-one (S11)
This reaction was carried out by analogy to the published procedure. [6] A stirred, room-temperature suspension of N-nosylurea S10 (314 mg, 0.752 mmol) and K2CO3 (311 mg, 2.25 mmol) in N,N-DMF (8 mL) was de-gassed by sparging with nitrogen for 15 min. Thiophenol (115 μL, 1.12 mmol) was added, with the immediate development of a rich yellow colour. After 1 h the reaction mixture was concentrated in vacuo and taken up in dichloromethane (50 mL). Sodium bicarbonate solution (sat. aq., 25 mL) was added, the layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 25 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (silica gel, dichloromethane:methanol, 100:1) to afford S11 (142 mg, 81%) as a beige crystalline solid. The 1 H data obtained agree with those obtained for the racemic compound. [7]

Solution-Phase Conformational Analysis by NMR
All NOESY and ROESY data were acquired on a Bruker 600 MHz spectrometer with a mixing time (tmix) of 200 ms. Unless otherwise stated, spectra were acquired in CDCl3 at room temperature. Full NOESY/ROESY spectra are given, followed by a zoomed view of the cross-peak region of greatest interest. Integrations were carried out manually. Where coloured bands are placed over spectra and with alternating red/blue colouration, this is done to aid visualization and the colours are arbitrary (unless otherwise stated). Since many of the signals examined are very weak, bands of noise are frequently present at similar levels of intensity. Where these can be identified they are marked on spectra with a green coloured band. The strong nOe between H3 and H6' was used as an internal standard for integration of peaks. Relative to this and H3↔H6, the H12↔[H15+H15'] resonance was the next strongest peak, with an intensity ~8% of H3↔H6'. Due to H15 and H15' being isochronous, the true integral per hydrogen atom is likely closer to 0.04. The presence of cross-peaks along the 'outer edge' of the molecule (H7↔H11, H11↔H15) provide evidence that the conformation adopted is as depicted, since these hydrogens would be distant in alternative conformations where the pyrimidine and urea dipoles are aligned. As noted in the main text, the combined intensities of the H3↔H6 and H3↔H6' resonances would be expected to have an intensity ratio with the combined H12↔H15 and H12↔H15' resonances of 2:1 in an unbiased system. In CDCl3, this ratio is 25:1, indicating the presence of a strong conformational biasing effect.
Comparison of this data to the data obtained by X-ray crystallography is given in the main text.
In addition to the above studies in CDCl3 (dielectric constant ε 4.8), the conformation of 10 was examined in the much more polar d6-DMSO (ε 46.7), and at elevated temperature, to determine whether the dipole repulsion-mediated conformational control would be retained.
ROESY, d6-DMSO, 600 MHz, tmix = 0.2 s. As with the CDCl3 data, the RT d6-DMSO are largely consistent with the dipole-opposed conformation about the N-Cpyrimidine bonds, in particular due to the H8↔H11, H3↔H12 and H11↔H15 resonances, all of which would not be expected to occur in the alternative, dipole-aligned conformers. The absence of H7↔H11 and H7↔H12 resonances is unexpected, but inspection of the data shows that there are no signals to H7 across the entire F2 spectral range, suggesting the reason for this is experimental, rather than conformational. When the temperature was increased to 355 K, all observable signals were strengthened relative to H3↔H6, consistent with an overall thermal increase in conformational flexibility. However, the fact that H3↔H12 is now the third most intense signal in this region of the spectrum strongly suggests that the molecule remains biased towards the dipole-opposed conformation at this temperature. As noted in the main text, the combined intensities of the H3↔H6 and H3↔H6 ' resonances would be expected to have an intensity ratio with the combined H12↔H15 and H12↔H15 resonances of 2:1 in an unbiased system. In RT DMSO, this ratio is 41:1, indicating the presence of a strong conformational biasing effect. At 355 K this value dropped to 11:1, indicating increased rotation about the N-C13 bond, but the retention of some of the dipolar bias even at this temperature. The strong nOe between H3 and H6 was used as an internal standard for integration of peaks. In addition to H3↔H6', the only other relevant cross-peaks observed were H8↔H14 and H15↔[H18+H18'], both of which were observed at ~7% the intensity of H3↔H6. The weakness of these resonances, and the fact that H7↔H14 is not observed at all, is in agreement with the proposed, dipole-opposed conformation. If the dipole mediated conformational biasing were entirely absent, it would be expected that the ratio of H15↔[H18+H18'] to (H3↔H6 + H3↔H6') would be 1:2the observed value is 1:29.

K 355 K
are close in value; as a result, weak nOes are generally observed between the imidazolidin-2-one and arene CH's with similar intensity (e.g. H14↔H18' = 0.037; H15↔H18' = 0.026). If the alternative conformation were adopted (with orthoarene nitrogens syn with the adjacent carbonyl) we would expect e.g. H15↔H18' >> H14↔H18'. The fact that H7↔H14 is stronger than H7↔H15 does not fully agree with the corresponding distances from the X-ray structure (4.6 Å and 4.1 Å respectively), suggesting there may be a small degree of rotation around the N-C13 bond placing H7 in occasional close proximity to H14. However, the overall weakness of H7↔H14 compared to H3↔H6 is still consistent with the depicted, dipole opposed conformation being predominant in solution. If the dipole mediated conformational biasing were absent, it would be expected that the ratio of (H15↔H18 + H15↔H18') to (H3↔H6 + H3↔H6') would be 1:2the observed value is 1:40. This is in good agreement with the ratios obtained for the pyrimidine (1:25) and pyridazine (1:29) dimers, suggesting the conformational biasing is of similar strength across all three heterocycles. The strong nOe between H3 and H6 was used as an internal standard for integration of peaks. Relative to this and H3↔H6', the H21↔[H24+H24'] and [H15+H24+H24']↔[H11+H20] cross-peaks were the next most intense. This is likely largely due to the isochronous nature of the peaks involved. H17↔H20 and H8↔H11 are both ranked relatively highly, and their presence strongly indicates that the dipole opposed conformation is adopted about the N-C10 and N-C19 bonds. The presence of an H11↔H15' cross-peak also suggests the dipole opposed conformation about the C13-N bond, since in the alternative dipole-aligned conformation these would be too far apart to experience an nOe. The analogous H20↔[H24+H24'] is likely also present, but due to overlap with H11↔H15 cannot be unambiguously interpreted.

Pyrimidine Trimer 13
More generally, the weakness of all imidazolidine-2-one cross peaks with adjacent ortho-aryl CHs is consistent with the proposed conformation. For example, the nOe intensity ratio between H12↔H15' and H3↔H6 is 1:20, where if there were no conformational bias around C13-N this ratio would be ~1:2.
The fully expanded spectrum is presented at two different levels of intensity scaling.
distinguished from background noise. These observations are consistent with the dipole-opposed conformation depicted.
Due to the absence of HMBC cross-peaks corresponding to either H7↔C13 or H18↔C16, it was not possible to unambiguously assign protons H14 or H15 on the 1 H NMR spectrum of pyrazine trimer 15. H26 is also isochronous with one of these two proton signals, which causes several conformationally relevant cross-peaks to overlap on the ROESY spectrum. Despite this, several clear conclusions regarding the conformational preferences of the molecule can be drawn from its ROESY spectrum.
The presence of weak cross-peaks corresponding to H26↔H30 and H26↔H32 indicates that the depicted conformation is preferred around the C28-N bond. Similarly, the presence of H19↔H27 and H20↔H27 peaks indicate the depicted conformation about the N-C25 bond, since these hydrogens would be too distant for an observable nOe in the alternative, dipoles-syn conformation. The weakness of the H19↔H26 and H27↔H30 peaks also supports the proposed conformation around the N-C25 and N-C28 bonds, since these would be expected to have ~1/2 the intensity of the H3↔H6 resonances if freely-rotating between syn-and anti-conformers.
As before, the strong nOe between H3 and H6' was used as an internal standard for the integration of peaks. Relative to this and H3 and H6, the next strongest conformationally relevant peak is ~16% the intensity of H3↔H6, which is relatively high when compared to the pyrazine dimer 12; however, the fact that this cross-peak likely corresponds to three overlapping resonances (H14/15↔H18, H14/15↔H18' and H26↔H30') accounts for the observed intensity being slightly higher than expected. By analogy to the spectral features implicating the dipole-opposed conformation about the N-C25 and N-C28 bonds, the weakness of all other cross-peaks involving H14 and H15 (not indicated on structure due to the ambiguous NMR assignment of these protons) is consistent with the depicted conformation around the N-C13 and C16-N bonds.

Pyrimidine Tetramer 16
ROESY, CDCl3, 600 MHz, tmix = 0.2 s. appear as a multiplet between 7.70-7.85 ppm, whereas in tetramer 16 the former appears at 8.41 ppm, and the latter are resolved and appear between 7.69 and 7.40 ppm. Figure S1. 1 H NMR spectra of tetramer 16 (bottom, maroon) and all other N-Ns foldamers prepared in this study (blue), highlighting the appearance of the nosyl protecting group. The CH ortho-to the nitro group appears in purple, and the remaining three peaks appear in green. The strong nOe between H3 and H6' was used as an internal standard for integration of peaks. Relative to this and H3↔H6, the H18↔H14 resonance was the next strongest peak, with an intensity ~9% of H3↔H6'. Due to H18 and H18' being isochronous, the true integral per hydrogen atom is likely closer to 0.045. However, the presence of this peak, and of H8↔H15 (the next most intense), indicate the depicted conformation, since if the N-Cpyrazine bonds were rotated 180°, the H15↔H18 and H8↔H14 nOe would be much stronger than these. Similarly, the H3↔H14 nOe strongly indicated the conformation given about the N-C13 bond. A peak of relative intensity 0.049 was observed between H20 and H26, however this peak corresponds with a strong band of T1 noise (see blue vertical band on spectrum) so no major conclusions can be drawn from its presence. A weak H27↔H30 nOe and the absence of an H19↔H26 nOe indicate the predicted, dipole-opposed conformation is adopted about the N-Cpyridazine bonds.
The strong nOe between H3 and H6' was used as an internal standard for integration of peaks. Relative to this, the next strongest conformationally relevant peak was H9'↔H13. The presence of this, and H7↔H13, indicate that the depicted conformation is preferred around the N-C10 bond. Similarly, the presence of H13↔H15 and H13↔H16 peaks indicate the depicted conformation about the N-C14 bond, since these hydrogens would be too distant for an observable nOe in the alternative, dipoles-syn conformation. The absence of H16↔H20 and H21↔H24 peaks supports the proposed conformation around the N-Cpyrazine bonds, since these would be expected to have ~1/2 the intensity of H3↔H6 resonances if freely-rotating between syn-and anti-conformers.

X-Ray Crystallography
Data for compounds 3, 4, 5, 10, 11 and 12 were collected as described in the General Experimental section.

Circular Dichroism
Samples were prepared at a concentration of 100 μM in the solvent indicated, and 300 μL of the resulting solution was placed in a 1 mm path length quartz cuvette. Data were acquired between 240 and 400 nm, since below 240 nm CHCl3 absorbs significantly, and preliminary analysis indicated no peaks occurred above 400 nm. A data pitch of 1 nm and continuous scanning speed of 25 nm·min -1 were employed. Each spectrum was acquired twice, and the data averaged. The resulting raw data was smoothed using the Savitsky-Golay method, with a window size of 25 nm. Figure S2. CD spectra of pyrimidine dimer 10 (black) and trimer 13 (blue) and tetramer 16 (red); CHCl3, 100 μM.

Homo-Pyrimidine Series
Dimer 10 and trimer 13 display qualitatively similar spectra, with strong minima for each at ~250 nm and ~300 nm. Tetramer 16 retains the minimum at 250 nm observed for dimer and trimer 10 and 13, but gains a strong new minimum at ~280 nm and maximum at ~300 nm. Considered alongside the computation and ROESY data outlined in the main text (Figure 4), this may suggest that the minimum at ~280 nm and maximum at ~300 nm observed for the tetramer are diagnostic of the formation of a (P)-helix in the pyrimidine-(S)-imidazolidin-2-one oligomers. The intensity of CD observed for both the pyridazine dimer 11 and trimer 14 was significantly less than that of the equivalent pyrimidines, making them challenging to interpret. Trimer 14 exhibits a minimum at ~295 nm and a maximum at ~265 nm, but dimer 11 shows no clear peaks. The CD spectra of both pyrazine dimer 12 and trimer 15 display no strong or diagnostic features common to both. There may be a maximum below 240 nm for trimer 15, but due to the absorption characteristics of the solvent this could not be fully observed. Trimer 21 gave only weak CD peaks, consistent with the homo-oligomers in the pyridazine and pyrazine series. The only major features are a minimum at ~290 nm, which is in agreement with the putative minimum observed for the pyridazine series at ~295 nm, and a potential maximum below 240 nm, consistent with that suggested for pyrazine homo-trimer 15. The CD spectrum of pyrimidine-pyridazine trimer 22 displays a strong minimum at ~300 nm, consistent with those observed for the pyrimidine homo-dimer and trimer. Its overall appearance resembles an overlay of the pyrimidine and pyridazine dimers 10 and 11. A maximum was also observed at ~275 nm. The CD spectrum of pyrimidine-pyridazine pentamer 20 displays a strong minimum at ~310 nm, consistent with those observed in this region for the pyrimidine homo-dimer and trimer, and the pyrimidine-pyridazine trimer 22. A strong maximum was also observed at ~290 nm, such that the spectrum of 20 resembles that of 22, slightly red-shifted. Both helical structures are consistent with the dipole repulsion hypothesis, and the (P)-conformer has H 9' ↔H 41 and H 12 ↔H 40 distances that would be expected to produce nOe correlations ( Figure S7, bottom left). The fact that these nOes are observed (along with H 9' ↔H 40 ) is consistent with 16 adopting an ensemble of conformers heavily biased towards those where the termini are in close contact and the helix adopted is of (P)-stereochemistry. The alternative (M)-helix is expected to give H 9' ↔H 40 and H 9' ↔H 41 distances much greater than the detection limit of the nOe experiment ( Figure S7, middle right).
Atomic Coordinates of (P)-helical energy minimum of 16