Combined reversible switching of ECD and quenching of CPL with chiral fluorescent macrocycles

A series of chiral fluorescent macrocycles display a remarkable combination of both +/– ECD and strong on/off CPL reversible switching upon cation binding and displacement.


Synthesis and characterization
All reactions involving air sensitive compounds were carried out under N 2 or argon by means of an inert gas/vacuum double manifold line and standard Schlenk techniques using dry solvents (CH 2 Cl 2 , tetrahydrofuran, tetrahydropyran, 1,4-dioxane and Et 3 N). Reactions involving oxygen sensitive reagents were performed using degassed solvents. Unless otherwise stated, all reagents were purchased from commercial sources and used without further purification (e.g. 2-amino-fluorene, 1amino-pyrene, 3-nitro-1,8-naphthalic anhydride, perylene, tert-butyl acetyl chloride).

Optical properties
Optical properties were recorded in analytical grade solvents (CH 2 Cl 2 , acetonitrile). UV-Vis absorption spectra were recorded on a JASCO V-650 spectrophotometer at 20 °C. Electronic circular dichroism (ECD) spectra were recorded on a Jasco J-815 spectropolarimeter at 20 °C in a 1 cmcuvette.
Fluorescence spectra were measured using a Varian Cary 50 Eclipse spectrophotometer. All fluorescence spectra were corrected for the wavelength-dependent sensitivity of the detection. Fluorescence quantum yields  were measured in diluted solutions (at least 5 different concentrations for each sample) with an optical density lower than 0.1 using the following equation: where A is the absorbance at the excitation wavelength (λ), n the refractive index and D the integrated intensity. "r" and "x" stand for reference and sample respectively. The fluorescence quantum yields were measured in acetonitrile relative to phenanthrene (φ = 12.5% in EtOH), anthracene (φ = 27% in EtOH) or coumarine 153 (φ = 38% in EtOH). Excitation of reference and sample compounds was performed at the same wavelength.
The circularly polarized luminescence (CPL) spectra were recorded with the home-made spectrofluoropolarimeter previously described, 1 the samples were excited with an UV (365 nm) LED, using a 90° geometry between excitation and detection.
Ba(ClO 4 ) 2 and NaBAr F salts used for titration experiments were purchased from commercial sources and used without purification.
Spectral data match those reported in the literature. 2a
Spectral data match those reported in the literature. 2b S7

Synthesis of monomeric amides
Neopentylcarboxamides, bearing only one fluorophore, were synthesized and used as monomeric references for the analyses and for comparison with the optical properties of macrocyclic compounds.
Pyrene monomer was used as a monomeric reference for pyrene-18C6, pyrene-18C4 and pyrene-16C4 compounds.
NMI monomer was used as a monomeric reference for NMI-18C6 derivative.

2.2.1.
General procedure for the synthesis of monomeric amides (S1) In a 10 mL flask under nitrogen atmosphere, dry CH 2 Cl 2 (0.1 M) and dry NEt 3 (3 equivalents) were added to 1 equivalent of the aromatic amine. The mixture was cooled down to 0 °C (ice bath) and tert-butyl acetyl chloride (2 equivalents) was added in one portion. The cooling bath was removed and the reaction was allowed to reach 25 °C on its own and stirred for an additional 3 hours. The conversion was followed by TLC analysis. After completion, the reaction was quenched by the addition of water and the mixture was extracted three times with CH 2 Cl 2 . The combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated under vacuum. The pure monomeric amide was obtained after purification by column chromatography.

Synthesis of polyether macrocycles 16C4 and 18C4
Polyether macrocycle 18C6 was synthesized according to previously reported literature. 4 This procedure was adapted to the synthesis of macrocycles 16C4 and 18C4 (See Scheme S1 below). Scheme S1. Synthesis of Unsaturated Macrocycles 18C6, 18C4 and 16C4

Synthesis of polyether macrocycle 16C4
In a 5 mg vial, 1.42 mg (0.0007 mmol, 0.0001 equiv) of Rh 2 (TCPTCC) 4 was dissolved in 1.0 mL of dry THF. In a flame dried Schlenk, adapted with a condenser, under nitrogen atmosphere, was introduced the rhodium solution and the vial was washed with 0.7 mL of dry THF. Then, 10.0 mL of THF (for a total of 11.7 mL) were added, followed by α-diazo-β-keto-methyl-ester 1 (1.00 g, 7 mmol, 1 equiv, c = 0.6 M). The solution was heated at 60 °C for 15 hours. The completion of the reaction was followed by TLC analysis and infrared spectroscopy (2146 cm -1 ). The solvent was removed under reduced pressure. The mixture was filtered through a pad of neutral alumina (solid deposit, 5 EtOAc/CH 2 Cl 2 1:1) and the solvent was evaporated. The residual solid was dissolved in 4 mL of CH 2 Cl 2 and 100 mL of pentane were added to precipitate the macrocycle. The macrocycle was filtered on a filtration funnel and washed with 100 mL of Et 2 O/pentane (1:9) to yield 338 mg (0.91 mmol, 372.41 g/mol, 26%) of 16C4 as a white solid.

Synthesis of polyether macrocycle 18C4
In a 3 mL vial, 1.42 mg (0.0007 mmol, 0.0001 equiv) of Rh 2 (TCPTCC) 4 was dissolved in 1.0 mL of dry tetrahydropyran (THP). In a flame dried Schlenk, adapted with a condenser, under nitrogen atmosphere, was introduced the rhodium solution and the vial was washed with 0.7 mL of dry THP. Then, 10.0 mL of THP (for a total of 11.7 mL) were added, followed by α-diazo-β-keto-methyl-ester 1 (1.00 g, 7 mmol, 1 equiv, c = 0.6 M). The solution was heated at 60 °C for 15 hours. The completion of the reaction was followed by TLC analysis and infrared spectroscopy (2146 cm -1 ). The solvent was removed under reduced pressure. The residual solid was dissolved in 3 mL of CH 2 Cl 2 and 100 mL of pentane were added to precipitate the macrocycle. The macrocycle was filtered and washed with 100 mL of Et 2 O/pentane (1:9) to yield 771 mg (1.93 mmol, 400.47 g/mol, 55%) of 18C4 as a white solid.   (18C6, 18C4 or 16C4) and 3 equivalents of aromatic amine. The mixture was cooled down to -100 °C (EtOH/liquid nitrogen bath). Then 4 equivalents of freshly sublimed t-BuOK were added in one portion. After stirring for 1-2 minutes at -100 °C, the cooling bath was removed and the reaction was allowed to reach 25 °C on its own and stirred for an additional 3 hours. The conversion was followed by TLC analysis and LR-ESI-MS. Upon completion, the reaction was quenched by adding a few drops of methanol and directly purified by column chromatography (SiO 2 ) without further treatment. A second column chromatography (Al 2 O 3 , neutral) could be required. Finally, the resulting oil or solid was purified by selective precipitation (dissolution in a minimal amount of CH 2 Cl 2 or ethyl acetate required for solubility, followed by addition of a large excess of pentane) affording the desired chiral polyether macrocycle.

General remarks
Compounds were resolved by chiral stationary phase HPLC using a semi-preparative CHIRALPAK® IG column. It is worth mentioning that it is necessary to remove traces of Et 2 NH.HCl present in the separated compounds. The residue was thus dissolved in CH 2 Cl 2 , the organic phase was washed three times with H 2 O, dried over anhydrous Na 2 SO 4 , filtered and concentrated under vacuum to afford the pure products.

Summary of separation data
The separation data are summarized in the following table: where t r1 /t r2 are the retention time of the first and the second eluted enantiomer and t 0 is the dead time.
The optical rotation of the enantiopure fractions are collected in the following table:

HPLC traces
For each compound, the data are reported as follows: separation conditions, analytical HPLC trace of the racemate, analytical HPLC trace of the first eluted enantiomer, analytical HPLC trace of the second eluted enantiomer and semi-preparative HPLC trace.        S52 Figure S9. Absorption (continuous lines) and fluorescence (dotted lines) spectra of perylene-18C4 in CH 2 Cl 2 without (red) or with (blue) NaBAr F (2 equiv).

General procedure
In a typical experiment, the ECD spectrum of a solution of enantiopure compound (ca. 10 -5 M) in acetonitrile or CH 2 Cl 2 was recorded in a 1 cm cell. For the complexation experiments, 3.0 equivalents (10 -3 M stock solutions) of NaBAr F in dichloromethane or Ba(ClO 4 ) 2 in acetonitrile were added to the solution and the ECD spectrum was recorded again.  In a typical experiment, ECD/fluorescence/CPL spectrum of a solution of enantiopure compound (ca. 10 -5 M) in CH 2 Cl 2 was recorded. For the complexation experiments, 2 equiv of NaBAr F from a 10 -3 M solution in CH 2 Cl 2 were added to the solution and the spectrum was recorded again. To switch back the system, 2 equiv of 18-Crown-6 from a 10 -3 M solution in CH 2 Cl 2 were added to the solution and the spectrum was recorded again. The procedure was repeated over several cycles.

General remarks
All calculations were run with Gaussian'09 and Gaussian'16 suites of programs using defaults grids and convergence criteria. 7 The input geometry of all compounds was built starting from the X-ray structure of pyrene-18C6 with (S,S) configuration bound to one water molecule. The conformational space was explored by varying manually the macrocycle-C(=O) and N-aryl bonds, and optimizing the resulting structures with DFT at M06-2X-D3/def2-SVP level, 8 including the IEF-PCM implicit solvent model for dichloromethane. 9 All converged structures displayed a clear -stacking interaction between the aromatic rings. TDDFT calculations were run with different combinations of functionals (ωB97X-D, M06-2X, B3LYP, CAM-B3LYP), basis sets (def2-SVP, def2-TZVP) and environment description (in vacuo or with IEF-PCM solvent model for dichloromethane). The number of roots (excited states) varied from 50 to 80 depending on the specific compound. The calculated spectra shown in Figures S36 and S37 were obtained with M06-2X and CAM-B3LYP functionals, which yielded the best agreement with the experimental spectra. ECD plots were generated from Gaussian log files using the program SpecDis. 10 The plotting parameters were chosen on a best-fitting basis, and are listed in the caption of Figure S37.

S71
Because of the structural complexity and flexibility of the 18C6-based macrocycles, ECD calculations were mainly intended for investigating the dependence of ECD spectra on the key structural parameters of the macrocycles.
In Figure S36, four structures of (S,S)-fluorene-18C6 are reported which were obtained upon rotation around the N-aryl and macrocycle-C(=O) bonds. All structures feature a pair of stacked fluorene rings with clear chirality defined by their long axes, i.e. the direction along which the main π-π* transition around 280 nm is polarized. Accordingly, the respective calculated ECD spectra display strong ECD couplets, whose sign depends on the chirality defined by the transition moments. It is apparent that the modification the reciprocal arrangement of the fluorene rings (i.e., exchanging the fluorene in the front with that in the back) causes a sign reversal in the ECD couplet. This rearrangement is caused by concerted rotations around the macrocycle-C(=O) bonds. While differing in sign and intensity, the four spectra are similar to each other in overall shape and position of bands. The weighted average of the four spectra (obtained using computed internal energies) is shown in Figure S37 (left). It consists in a negative ECD couplet, which is in agreement with experimental spectrum for the 1 st eluted enantiomer ( Figure 4C in the main text). The vibrational fine structure in the experimental spectrum cannot be predicted at the current level of calculation, which does not include vibronic effects.
For pyrene-and perylene-18C6 we investigated the impact of switching the ring position on the ECD spectra ( Figure S37, two middle columns). For (S,S)-perylene-18C6 the situation is similar to fluorene-18C6: exchanging the ring position from front to back causes a sign reversal in the ECD couplet, which is also accompanied by a strong intensity decrease. The overall ECD profile consists in a negative ECD couplet, as experimentally found for the 2 nd eluted enantiomer. The situation for pyrene-18C6 is more complicated because the two arrangements -obtained after the rings exchange their positions -are associated with very different ECD profiles. The structure attained from re-optimization of the X-ray geometry is associated with a negative ECD couplet; the second structure with exchanged ring positions is associated instead with a red-shifted positive couplet ( Figure S37). If one assumes that the first conformation is dominant in solution, the combination of the two would yield an overall spectrum similar to that observed for the 2 nd eluted enantiomer ( Figure 4A, main text), namely a positive ECD band at 350 nm (mainly due to the major conformer), flanked by a long-wavelength weak negative band (due to the minor conformer).
Finally, for NMI-18C6 the rotamerism around the N-aryl bond has a pronounced impact on the orientation of the transition moments, polarized either along the chromophore long or short axis (see yellow double arrows in Figure S37). Therefore, we focused our attention on this degree of conformational freedom without switching the reciprocal position of the rings. The two different arrangements (obtained upon 180°-rotation around the N-aryl bonds) display ECD spectra which are almost the mirror image of each other long wavelengths, while having consistent shape and sign at shorter wavelengths ( Figure S37, right). Assuming a coexistence of both structures in solution, this would lead to a signal cancelation at long wavelengths, yielding a positive exciton couplet at short wavelengths. The overall result resembles the ECD measured for the 2 nd eluted enantiomer ( Figure  4D, main text).