A trefoil knot self-templated through imination in water

The preparation of topologically nontrivial molecules is often assisted by covalent, supramolecular or coordinative templates that provide spatial pre-organization for all components. Herein, we report a trefoil knot that can be self-assembled efficiently in water without involving additional templates. The direct condensation of three equivalents of a tetraformyl precursor and six equivalents of a chiral diamine produces successfully a [3 + 6] trefoil knot whose intrinsic handedness is dictated by the stereochemical configuration of the diamine linkers. Contrary to the conventional wisdom that imine condensation is not amenable to use in water, the multivalent cooperativity between all the imine bonds within the framework makes this trefoil knot robust in the aqueous environment. Furthermore, the presence of water is proven to be essential for the trefoil knot formation. A topologically trivial macrocycle composed of two tetraformyl and four diamino building blocks is obtained when a similar reaction is performed in organic media, indicating that hydrophobic effect is a major driving force behind the scene.

All reagents and solvents were purchased from commercial sources and used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded at ambient temperature using Bruker AVANCE III 400/500 or Agilent DD2 600 spectrometers, with working frequencies of 400/500/600 and 100/125/150 for 1 H and 13 C respectively. Chemical shifts are reported in ppm relative to the residual internal non-deuterated solvent signals (for proton NMR, D2O: δ = 4.70 ppm, CD3SOCD3: δ = 2.50 ppm). High-resolution mass spectra (HRMS) were recorded on a Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). CD spectra were recorded on a Circular Dichroism Spectrometer (Chirascan V100, Applied Photophysics Ltd).

Synthetic procedures
The precursor S1 was synthesized according to the procedure from the literature 1 . The procedure is decribed as follows. 5-bromoisophthalaldehyde (3.00 g, 14.08 mmol) and pyridine-4-boronic acid (2.08 g, 16.90 mmol) were dissolved in a mixture of THF (180 mL) and 2 M aqueous potassium carbonate (42.2 mL, 84.5 mmol).The mixture was then degassed (N2 bubbling, 30 min) before the addition of Pd(PPh3)4 (325.2 mg, 0.28 mmol). The reaction was heated at 80 °C for 24 h. After cooling down the reaction solution to room temperature, the solvent was removed in vacuo. Purification via column chromatography (5:1 EtOAc/hexane) afforded the desired product S1 as a white solid (1.51g, 7.12 mmol, 50%).
1 2+ ·2PF6was obtained by performing counterion exchange to 1 2+ ·2Br -: 1 2+ ·2Brwas dissolved in water at 90 °C, followed by adding NH4PF6 (100 mg), after which a white solid was collected by filtration and washed with water for three times. After drying the solid under vacuum, the pure compound 1 2+ ·2PF6was obtained in close to quantitative yield.

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Supplementary Figure 14. ESI-HRMS of S-2 6+ ·6Br -. The signals labeled in the spectrum correspond to molecular cations that contain five, five and four charges, respectively, by either gaining Brcounterions or losing a proton.
Supplementary Figure 15. UV-Vis absorption spectra of trefoil knots including two enantiomers and the racemic mixture. Normalized partial UV-Vis absorption spectra of the product S-2 6+ ·6Br -, R-2 6+ ·6Brand their racemic mixture recorded in water at 298 K. The racemic cage mixture was self-assembled by using racemic CHDA. Their UV-Vis absorption spectra are almost identical.

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Supplementary Figure 16. CD spectra of trefoil knots including the two enantiomers and their racemic mixture. CD spectra of the product S-2 6+ ·6Br -, R-2 6+ ·6Brand their racemic mixture (0.04 mM) recorded in water. The racemic trefoil knot mixture was self-assembled by condensing a racemic mixture of CHDA and 1 2+ ·2Brin water. Once self-assembled, the trefoil knot, including both enantiomers and racemic mixture, was observed remarkably inert within hours, after dilution of the solution to 0.04 mM.
Supplementary Figure 22. ESI-HRMS of S-3 4+ ·4Br -. The signals labeled in the spectrum correspond to molecular cations that contain four and three charges, respectively.

Supplementary
Supplementary Figure 26. The kinetic formation curve of 2 6 ·6CH3COO -. A plot of the yields of S-2 6 ·6CH3COOversus reaction time. The yields of the trefoil knot at each time were measured by using the 1 H NMR spectroscopic results in Supplementary  Figure 25).

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The studies of the self-assembly kinetics were performed by recording the 1  However, we discovered that S-2 6+ ·6CH3COOwould undergo decomposition partially even at room temperature due to the basicity of CH3COO -, which means that the yields would be more than what we calculated.
Another question is that, what is the kinetic byproducts during the self-assembly? We thus performed the self-assembly of the trefoil knot at room temperature, in order to increase the life-time of the byproducts. Here, H2O/CD3SOCD3 (3:1, v/v)   Supplementary Figure 27. ESI-HRMS of a mixture of S-2 6+ ·6Brand S-X 8+ ·8Br -. The signals labeled in the spectrum correspond to molecular cations that contain seven, and six charges, respectively, which belongs to a [4+8] product S-X 8+ ·8Br -.
Supplementary Figure 32. Solvent effect on the formation of S-2 6+ ·6Br -. 1 H NMR spectra (500 MHz, 298 K) of 1:2 mixture of 1 2+ ·2Br -(2 mM) and (SS)-CHDA (4 mM) recorded after heating the mixture at 80 °C for 12 h in the mixtures of CD3SOCD3 and D2O with different ratios. DMF was added as an internal standard to calculate the yields of the knot. By comparing the integrations of the resonances of DMF (blue arrow) and one proton (red arrow) in the trefoil knot, the yields of the latter were determined.
We further investigated the impact of the D2O/CD3SOCD3 ratio on the yields of the trefoil knot S-2 6+ . We used D2O/CD3SOCD3 mixtures to do the measurement, whose ratios were 2:8, 4:6, 6:4, and 8:2 (Supplementary Figure 32). An internal standard namely DMF was added into the samples to help the calculation of the yields of the trefoil knot S-2 6+ ·6Brin different solvent systems. After the self-assembly systems was heated at 80 °C for 12 h and reached the equilibria, 1 H NMR spectra were recorded (Supplementary Figure 32). With the increasing ratio of D2O/CD3SOCD3, the yield of S-2 6+ ·6Brincreased from very low (i.e., which is too low to be precisely determined by integrating the resonances) to 58%. Such experiment indicates the important role of S28 hydrophobic effects in water. The yield of S-2 6+ ·6Brself-assembled in pure water is difficult to calculate accurately due to the relatively poor water solubility of the tetraformyl precursor 1 2+ ·2Brand trefoil knot S-2 6+ ·6Br -.

Theoretical calculations
The four right-handed trefoil knots in the main text were optimized by using the density functional theory (DFT) at the BP86-D3/6-311G(d) level with the Gaussian 16 package 2 . The solvent effect of water was included with the polarizable continuum model (PCM) using solvent accessible surface.
Supplementary Figure 33. Theoretical calculation. Comparison of the experimental (red traces) and theoretical (yellow traces) spectra including (a) UV-Vis and (b) CD spectra. The theoretical spectra were calculated based on the structure of P-Ᾱ-S-2 6+ which was also predicted by using DFT. The experimental spectra were obtained by using sample S-2 6+ ·6Br -. The experimental ones are well-consistent with the calculated ones. The comparison of theoretical (c) UV-Vis and (d) CD spectra based on the predicted structures, namely P-Ᾱ-S-2 6+ , P-A-R-2 6+ , P-Ᾱ-R-2 6+ and P-A-S-2 6+ , respectively.