Living supramolecular polymerization of fluorinated cyclohexanes

The development of powerful methods for living covalent polymerization has been a key driver of progress in organic materials science. While there have been remarkable reports on living supramolecular polymerization recently, the scope of monomers is still narrow and a simple solution to the problem is elusive. Here we report a minimalistic molecular platform for living supramolecular polymerization that is based on the unique structure of all-cis 1,2,3,4,5,6-hexafluorocyclohexane, the most polar aliphatic compound reported to date. We use this large dipole moment (6.2 Debye) not only to thermodynamically drive the self-assembly of supramolecular polymers, but also to generate kinetically trapped monomeric states. Upon addition of well-defined seeds, we observed that the dormant monomers engage in a kinetically controlled supramolecular polymerization. The obtained nanofibers have an unusual double helical structure and their length can be controlled by the ratio between seeds and monomers. The successful preparation of supramolecular block copolymers demonstrates the versatility of the approach.

Procedure for M3 Seed preparation: M3 Seed solution was obtained by applying sonication to a Cyclohexane/CHCl3 (84:16 v/v) solution of M3 and MeM3 (molar ratio: 3:1; total concentration: 1.2 mM) for 20 minutes at 273 K in a roundbottom flask tightly closed with glass stopcock. M3 Seed was used for further experiments immediately.
General procedure for seeded LSP A 1.2 mM solution of M3 in 84:16 v/v cyclohexane/chloroform in was added to a 1 mm quartz cuvette and annealed for 40 sec at 323 -328 K to ensure complete depolymerisation. After this treatment spontaneous polymerization does not occur for >3 h. The monomer solution was placed into a CD spectropolarimeter with the temperature set to 293 K and thermostated for 10 min. A defined volume of an M3 Seed solution was added and the cuvette content was homogenized by turning the cuvette upside down 5 -10 times and placed back into the CD spectropolarimeter at 293 K.
Notes: 1) For every experiment we prepared fresh M3 Seed solutions (old solutions can be depolymerized as described above); 2) M3 Seed solution was always kept at 273 K.

AFM studies on supramolecular polymers
Samples of supramolecular polymers for AFM were prepared by spin-coating (8000 rpm) on the silicon wafer immediately after 100-fold dilution with cyclohexane. Dilution is necessary to prevent severe bundling. AFM images were recorded for several different areas of the substrate. AFM data was processed using WSxM 5.0 software. 1 Images were analyzed using the ImageJ software, developed at the US National Institute of Health. 2 Lengths of all separate unbundled fibers were used for statistics. In every case, we measured approximately 200 fibers (Supplementary Table 1). Weightaveraged length (Lw) and number-averaged length (Ln) for n objects were calculated according to equations S1 and S2.
The length distribution was characterized by the polydispersity index (PDI) according to: General procedure for preparation of supramolecular block copolymer by seeded LSP A 1.2 mM solution of M5 in 93:7 v/v cyclohexane/chloroform was added to a 1 mm quartz cuvette and was annealed for 40 sec at 323 -328 K to ensure complete depolymerisation. The monomer solution was placed in a CD spectropolarimeter with temperature set to 293 K and thermostated for 10 min. A defined volume of M3 Seed solution was added and the cuvette content was homogenized by turning the cuvette upside down 5 -10 times and placed back into CD spectropolarimeter at 293 K.
Notes: 1) For every experiment we prepared fresh M3 Seed solutions (old solutions can be depolymerized as described above); 2) M3 Seed solution was always kept at 273 K.

Living supramolecular polymerization
To provide evidence for the living nature of supramolecular polymerization we performed a multicycle dilution experiment. 20 µL of M3 Seed solution was added to 200 µL of M3 solution at 293 K, polymerization occurred and was completed within 60 seconds. 100 µL of the obtained polymer solution was added to 100 µL of M3 solution. This procedure was repeated for two more cycles ( Supplementary Fig. 36a). Since after each cycle, the initial concentration of  Fig. 40b).

Thermodynamics of polymerization
To obtain thermodynamic data, we studied thermal depolymerisation of polymers, which proceeds under thermodynamic control unlike the kinetically controlled, cooling-induced polymerization. The The standard values of enthalpy (ΔH°), entropy (ΔS°) and Gibbs free energy were obtained using the van`t Hoff plot ( Supplementary Fig. 55).

General procedure compounds 2 -4
In a 10 mL round-bottom flask pentafluorophenyl ester S1 -S3 (7.7 mmol, 1.9 equiv.) and 1 (4 mmol, 1 equiv.) were dissolved in 3 mL of dry DMF and 2 mL of triethylamine. The reaction mixture was stirred at 120 °C until 1 was completely consumed (ca. 2 -3 hours). Solvents were removed under reduced pressure and the crude material was purified by flash column chromatography (gradient elution CH2Cl2 to CH2Cl2/acetone 8:2). equiv.) and 2,6-lutidine (4.38 ml, 4.1 g, 38 mmol, 2 equiv.) were added subsequently and the reaction mixture was stirred at room temperature. When all starting material was consumed, the reaction mixture was extracted with CH2Cl2. The organic phase was dried over magnesium sulfate, passed through a short celite pad and solvents were removed under reduced pressure. The brown oil, obtained after evaporation, was dissolved in 50 mL of CH2Cl2. TBDMSCl (4.3 g, 28 mmol, 1.5 equiv.) and imidazole (1.9 g, 28 mmol, 1.5 equiv.) was added and the reaction mixture was stirred for 12h at room temperature. The reaction mixture was transferred into a separatory funnel and washed with water (100 mL). The organic phase was dried over magnesium sulfate. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9:1) to give S4 (2.6 g, 55% two steps) as a colourless oil.
The reaction mixture was stirred for 10 minutes at 0 °C and for 2 h at room temperature. The reaction mixture was concentrated under reduced pressure and 100 mL of petroleum ether was added. The precipitate was filtered off and the mother liquor was passed through a silica pad to obtain S6 (1.96 g, 63%) as colourless oil.

(S)-butyl(6-iodo-4-methylhexyl)sulfide (S9)
This compound was prepared following the same procedure as for S6 to give S9 (450 mg, 30%) as colourless oil. General procedure compounds S10 -S12 A 250 mL round-bottom flask was charged with 3,4,5-trihydroxymethylbenzoate (10 mmol, 1 equiv.), the corresponding alkyl halide (35 mmol, 3.5 equiv.), a spatula tip of potassium iodide and 100 mL of dry DMF. Anhydrous Cs2CO3 (42 mmol, 4.2 equiv.) was added and the reaction mixture was stirred at 130 °C until all starting material was consumed (NMR control). The solvent was removed under reduced pressure. The residue was suspended in 150 mL of water and extracted with CH2Cl2 (4*75 mL). The combined organic phase was dried over magnesium sulfate and evaporated under reduced pressure. To the residue were added 50 mL of MeOH, 150 mL of water and sodium hydroxide (0.15 mol, 15 equiv.). The reaction mixture was refluxed until all starting material was consumed (TLC control), cooled down to room temperature, carefully acidified with concentrated hydrochloric acid to pH 1 and extracted with CH2Cl2. The organic phase was dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude material was purified by flash column chromatography (petroleum ether/ethyl acetate 9:1).

General procedure compounds M1 -M5
To a suspension of compound 2 -4 (2 mmol, 1 equiv.) in 2 mL of anhydrous CH2Cl2 was added 2 mL of TFA at 0 °C. The reaction mixture was stirred at room temperature until all starting material was consumed. CH2Cl2 and TFA were removed under vacuum. Pentafluorophenyl ester S13 -S15 (4 mmol, 2 equiv.), 5 mL of DMF and triethylamine 3 mL were added subsequently and the reaction mixture was stirred at room temperature overnight. After removal of solvents under reduced pressure the crude material was purified by flash column chromatography (CH2Cl2 to CH2Cl2/acetone 8:2) to yield the title compound.
Comments on reaction conditions, side products and purification: 1. DMF is needed to completely dissolve starting materials containing all-cis C6H6F5 group and to prevent gelation which sometimes can happen if DCM alone is used as a solvent.
2. To completely remove pentafluorophenol from the reaction mixture, sometimes two purifications by flash column chromatography are needed.
3. For the linker based on γ-aminobutyric acid approximately 13% of butyrolactam was formed as a side product.

Methyl 4-((tert-butoxycarbonyl)(methylamino)butanoate (S17)
A 100 mL heat gun-dried Schlenk flask was charged with γ-(Boc-amino)butyric acid (2 g, 9.9 mmol, 1 equiv.), methyl iodide (6 mL, 99 mmol, 10 equiv.) and 40 mL of dry DMF under argon. NaH (2.36 g of 60% (w%) solution in mineral oil, 60 mmol, 6 equiv.) was added at 0 °C, the reaction mixture was stirred at this temperature for 2 h and ice bath was removed and reaction was stirred at room temperature overnight. Excess of NaH was carefully quenched with water, 50 mL of concentrated aqueous solution of ammoniac was added. The reaction mixture was diluted with 150 mL of water and extracted with ethyl acetate (4*50 mL). The combined organic phase was washed with water (3*50 mL) and brine. The organic phase was dried over sodium sulfate and evaporated under reduced pressure to give S17 (2.2 g, 97%) as yellowish oil ,which was used for the next step without purification. Compound S18 S17 (254 mg, 1.1 mmol, 1 equiv.) was dissolved in CH2Cl2 (1 mL) and TFA (1 mL) was added. The reaction mixture was stirred for 30 minutes and the solvent was removed under vacuum. To the obtained solid material S14 (1g, 1.32 mmol, 1.2 equiv), few crystals of DMAP, CH2Cl2 (2 mL) and TEA (1 mL) were added subsequently. The reaction mixture was stirred for 12h at room temperature.
The solvent was removed under reduced pressure and the crude material was purified by flash column chromatography (petroleum ether/ethyl acetate 8:2) to give S18 (515 mg, 67%) as a colourless oil.

Parametrization
The molecular models of the molecules M2, M3, M4 and MeM3 herein studied, along with the solvent composed by cyclohexane and chloroform were parametrized by using the General Amber Force Field (GAFF), 5 and the partial charges were modelled by using the AM1-BCC method. 6 These parameters were extracted by using the software Antechamber. 7

Molecular dynamics setup
All the simulation herein performed, were run on GROMACS-2018.6 8 patched with plumed-2.5. 9 All systems were simulated for 1 μs of MD at the temperature of 293 K and pressure of 1 atm in explicit TIP3P water molecules 10 in periodic boundary NPT conditions (constant N: number of particles, P: pressure and T: temperature), employing the v-rescale thermostat 11 and the Berendsen barostat (semi-isotropic barostat during the study of fiber stability). 12 A timestep of 2 fs was used in the MD simulations. The electrostatic interactions were treated using particle mesh Ewald (PME). 13 The cutoff lengths of the real summation and of the VdW were set to 1.0 nm. The dynamics of the hydrogens was constrained using the LINCS algorithm. 14 All the systems studied were simulated with a solvent composition 84:16 cyclohexane:chloroform.
While for the studies of conformations and self-assembly the systems after being minimized were equilibrated with short runs followed by the 1μs MD simulation, the MD simulations of the preformed fibers were performed by first pre-equilibrating the "tails" of the monomers, by keeping restrained the atoms of the "core" (Supplementary Fig. 25b) for 10ns. After this it followed 1μs of MD as production run.

Metadynamics
In this work, we employed metadynamics (MetaD) simulations 15 to enhance the exploration of the conformational space of the single monomers. We used two collective variables (CV): CV1, monitoring the orientation of the pentafluorocyclohexane moiety (F5-CHX) with respect to the amide moiety, and CV2, controlling the opening of the "core" of the monomer. CV2 is defined as the radius of gyration of the heavy atoms of the "core", while CV1 is defined as the difference between two distances (dFsH and dHsO, in Supplementary Fig. 25c). dFsH is the distance between geometrical center of the fluorides and the amide nitrogen, while dHsO is the distance between the geometrical center of , which allows to obtain a smoother convergence of the system. 16 During the WT-MetaD run, the bias was deposited along CV1 and CV2 in the form of Gaussianshaped kernels of height 0.8 kJ/mol and σ equal to 0.02 and 0.01 nm along CV1 and CV2 respectively while the deposition rate was 1 ps -1 . The bias factor of this WT-MetaD run was set to 15.

Intrinsic structural dynamics of the fiber
In order to deeper investigate on the dynamic of the M3 and M5 monomers within the fibers, we performed an unsupervised machine-learning analysis that allowed us to classify the arrangement,  Supplementary Fig. 52a), and the sulfurs atoms on M5 ( Supplementary Fig. 52b) that occupy the equivalent position of the alkyl moieties in M3. Based on this description, the behavior of each monomer in the system is represented (and simplified) by the movements and behaviors of these five centers, which are monitored and characterized by the SOAP vectors. This approach was previously demonstrated to be capable of providing remarkably rich insights into the internal structure and dynamics of supramolecular polymers. 18 We carried out this SOAP analysis with the Python package DScribe, 19 setting the input parameters as rcut = 60Å, nmax = 5, lmax = 5, and leaving the other parameters as default.
The SOAP centers could then be classified into different states by applying a clustering algorithm to the SOAP dataset, to obtain the most probable states in the multidimensional descriptor space. First, we reduced the high-dimensional SOAP features via principal component analysis (PCA) by retaining only the first three components. This approach reduced the computational cost for the processing of the data, while maintaining a high level of accuracy (up to ~86% of the information preserved). Linear PCA dimensionality reduction was performed using the Python package Scikit-Learn. 20

Discussion on NMR experiments
MeM3 shows broadening of cyclohexane resonances at ca. 5.5 -4.5 ppm similar to M3 ( Supplementary Fig. 19). Therefore, the broadening of cyclohexane resonances (5.5 -4.5 ppm) in M3 can not be due to the formation of aggregates with intermolecular N-H hydrogen bonds (since no N-H hydrogen bonding is possible for MeM3). It indicates that for both compounds, broadened proton resonances for cyclohexane moiety are due to ring flipping of cyclohexane, which is generally slower for all-cis fluorinated cyclohexanes, 26 and slow equilibrium between multiple folded states in solution as evident from corresponding FESs (Supplementary Fig. 26b and d). To further address this point we performed VT-NMR measurements in chloroform ( Supplementary Fig. 20), in which M3 does not polymerize but folds, as evident from HOESY measurements ( Supplementary Fig. 18

Discussion on seed preparation
During our initial attempts to prepare M3 Seed we noticed that sonication of a 0.9 mM solution (cyclohexane/chloroform 84:16 v/v) at 273 K for 20 min did not lead to homogeneous seeds with low polydispersity, and instead long (ca. 1 µm) bundled fibers were obtained as indicated by AFM ( Supplementary Fig. 27). Moreover, the obtained seeds did not provide reliable reproducibility and length control during seeded living supramolecular polymerization. Therefore, we decided to use