Functional group introduction and aromatic unit variation in a set of π-conjugated macrocycles: revealing the central role of local and global aromaticity

π-Conjugated macrocycles are molecules with unique properties that are increasingly exploited for applications and the question of whether they can sustain global aromatic or antiaromatic ring currents is particularly intriguing. However, there are only a small number of experimental studies that investigate how the properties of π-conjugated macrocycles evolve with systematic structural changes. Here, we present such a systematic experimental study of a set of [2.2.2.2]cyclophanetetraenes, all with formally Hückel antiaromatic ground states, and combine it with an in-depth computational analysis. The study reveals the central role of local and global aromaticity for rationalizing the observed optoelectronic properties, ranging from extremely large Stokes shifts of up to 1.6 eV to reversible fourfold reduction, a highly useful feature for charge storage/accumulation applications. A recently developed method for the visualization of chemical shielding tensors (VIST) is applied to provide unique insight into local and global ring currents occurring in different planes along the macrocycle. Conformational changes as a result of the structural variations can further explain some of the observations. The study contributes to the development of structure–property relationships and molecular design guidelines and will help to understand, rationalize, and predict the properties of other π-conjugated macrocycles. It will also assist in the design of macrocycle-based supramolecular elements with defined properties.

. Synthetic route to compound 2e (including the halogenated starting material) and unsuccessful conversion into compound 3e.

Instrumentation
Purification by preparative GPC was carried out on a LaboACE LC-5060 (Japan Analytical Industry Co., Tokyo, JAPAN) recycling GPC system equipped with a JAIGEL-2HR column and a TOYDAD800-S detector. CHCl3 was used as the eluent at a flow rate of 10 mL min -1 .
NMR spectra were recorded at 400 MHz for 1 H and 101 MHz for 13 C on a Bruker AV-400 spectrometer.
High-resolution mass spectrometry was carried out on systems from Thermo Scientific for atmospheric pressure chemical ionization (APCI) and Waters for electrospray ionization (ESI). While the Thermo Scientific system gives the actual mass of the ionized compounds, the Waters system is calibrated to give the mass of the neutral compounds. This was considered when calculating the m/z values for comparison with the measurements.

Synthesis of compounds 1a-e
The procedures for synthesis of compounds 1a-e were inspired and adapted from previous reports. [2][3][4] 1.3.1 α 1 ,α 4 -Dioxo-1,4-benzenediacetic acid, 1,4-diethyl ester (1a) 1,4-Diiodobenzene (3.50 g, 10.6 mmol, 1.0 equiv.) was dissolved in 150 mL dry degassed THF under nitrogen and cooled to -78 °C with an acetone/dry ice bath. t-BuLi (1.7 M in pentane, 25 mL, 42.5 mmol, 4.0 equiv.) was then added slowly by cannula transfer under vigorous stirring. The resulting mixture was kept at -78 °C for 2 h and was then cooled to -94 °C with an acetone/N2(liq.) bath for the quick addition of diethyl oxalate (14 mL, 105 mmol, 9.9 equiv.). The reaction was kept at -94 °C for 1 h and was then allowed to slowly warm to r.t. After stirring at r.t. for 1 h, the reaction was poured into 2 M aqueous HCl (200 mL) and extracted three times with CH2Cl2. The combined organic phases were extracted with 2 x 150 mL sat. aqu. Na2SO3 solution and dried over MgSO4. The solvent was evaporated under reduced pressure and the remaining diethyl oxalate was distilled off under high vacuum. The compound was purified by column chromatography on silica gel using CH2Cl2/petroleum ether (1:1) as eluent. For removing trace amounts of diethyl oxalate, the now crystalline product was suspended in hexane and filtered off, yielding compound 1a as pure, slightly yellow crystals (1.30 g, 4.67 mmol, 44%). 1 H NMR (400 MHz, CDCl3): δ = 8.16 (s, 4H), 4.47 (q, J = 7.1 Hz, 4H), 1.44 (t, J = 7.2 Hz, 6H) ppm. 13  mixture was kept at -78 °C for 2 h and was then cooled to -94 °C with an acetone/N2(liq.) bath for the quick addition of diethyl oxalate (14 mL, 105 mmol, 9.9 equiv.). The reaction was kept at -94 °C for 1 h and was then allowed to slowly warm to r.t. After stirring at r.t. for 1 h, the reaction was poured into 2 M aqueous HCl (200 mL) and extracted three times with CH2Cl2. The combined organic phases were dried over MgSO4, the solvent was evaporated under reduced pressure, and the remaining diethyl oxalate was distilled off under high vacuum. The compound was purified by column chromatography on silica gel using CH2Cl2/petroleum ether (1:1) as eluent. For removing trace amounts of diethyl oxalate, the now crystalline product was suspended in hexane and filtered off, yielding compound 1b as pure white powder (2.24 g, 6.82 mmol, 64% 2,6-Dibromoanthracene (2.00 g, 5.95 mmol, 1.0 equiv.) was dissolved in dry Et2O (120 mL) under nitrogen and cooled to -78 °C with an acetone/dry ice bath. n-BuLi (2.5 M in hexanes, 9.5 mL, 23.8 mmol, 4.0 equiv.) was then added slowly under vigorous stirring. The resulting mixture was kept at -78 °C for 1 h and was then allowed to warm to r.t. After 2 h at r.t., the reaction was cooled to -94 °C with an acetone/N2(liq.) bath for the quick addition of diethyl oxalate (8.0 mL, 58.9 mmol, 9.9 equiv.).
The reaction was kept at -94 °C for 1 h and was then allowed to slowly warm to r.t. After stirring at r.t.
for 1 h, the reaction was poured into 2 M aqueous HCl (200 mL) and extracted three times with CH2Cl2.
The combined organic phases were dried over MgSO4 and the solvent was evaporated under reduced pressure. The resulting crude product was filtered over a thick pad of silica using petroleum ether/CH2Cl2 (1:1) to elute diethyl oxalate and other impurities before eluting the pure product with CH2Cl2. After evaporation of the solvent, compound 1c was obtained as pure yellow powder (0.69 g, 1.3.4 α 1 ,α 6 -Dioxo-1,6-pyrenediacetic acid, 1,6-diethyl ester (1d) 1,6-Dibromopyrene (3.00 g, 8.33 mmol, 1.0 equiv.) was dissolved in dry THF (150 mL) under nitrogen and cooled to -78 °C with an acetone/dry ice bath. n-BuLi (1.6 M in hexanes, 20.3 mL, 32.5 mmol, 3.9 equiv.) was then added slowly under vigorous stirring. The resulting mixture was kept at -78 °C for 1 h and was then allowed to warm to r.t. After 2 h at r.t., the reaction was cooled to -94 °C with an acetone/N2(liq.) bath for the quick addition of diethyl oxalate (11 mL, 82.5 mmol, 9.9 equiv.). The reaction was kept at -94 °C for 1 h and was then allowed to slowly warm to r.t. After stirring at r.t. for 1 h, the reaction was poured into 2 M aqueous HCl (100 mL) and extracted three times with CH2Cl2.
The reaction was kept at -94 °C for 1 h and was then allowed to slowly warm to r.t. After stirring at r.t.
for 1 h, the reaction was poured into 2 M aqueous HCl (200 mL) and extracted three times with CH2Cl2.
The combined organic phases were dried over MgSO4 and the solvent was evaporated under reduced pressure. The resulting crude product was recrystallized from EtOH, yielding compound 1e as pure, bright yellow powder (0.93 g, 2.46 mmol, 55%

Synthesis of compounds 2a-e
The synthesis of compounds 2a-e was carried out using standard procedures for the saponification of carboxylic esters. The procedure for the synthesis of 2c-e was further inspired by previous reports.

Synthesis of compounds 3b-d
The synthesis of compounds 3b-d was carried out by adapting previously reported procedures. 6,7 Notes: (i) Compound 3a was obtained from commercial suppliers. (ii) The synthesis of compound 3e was not successful following the procedure used for the synthesis of compounds 3b-d.

Synthesis of the π-conjugated macrocycles
The macrocycles were prepared adapting a previously reported procedure. 7

General procedure
To a mixture of the respective dioxodiacetic acid 2a-d (1.0 equiv.) and diacetic acid 3a-d (1.0 equiv.) in varying amounts of dry degassed THF under nitrogen, triethylamine (9.5 equiv., Et3N) and acetic anhydride (15 equiv., Ac2O) were added. The reaction mixture was heated to reflux under nitrogen for 3 days before the respective alkyl bromide (44 equiv., RBr), the respective alcohol (82 equiv., ROH), and 1,8-diazabicyclo [5.4.0]undec-7-ene (21 equiv., DBU) were added. After refluxing for another 24 h, the reaction was allowed to cool to r.t., poured into 1 M aqueous HCl (350 mL), extracted three times with CHCl3, and dried over MgSO4. Evaporation of the solvent under reduced pressure gave the crude product, which was split into two fractions for purification by gel permeation chromatography (GPC).
Each fraction was dissolved in CHCl3 and filtered through a 0.2 µm syringe filter before loading the GPC column with the resulting solution (CHCl3 was used as eluent for the GPC).

Alternative procedure using a syringe pump for the addition of the dioxodiacetic and diacetic acid:
To a refluxing mixture of Et3N (9.5 equiv.) and Ac2O (15 equiv.) in dry degassed THF (400 mL) under nitrogen, the respective dioxodiacetic acid (1.0 equiv.) and diacetic acid (1.0 equiv.) dissolved in 20 mL dry degassed THF were added over 10 h (2 mL h -1 ) using a syringe pump. The reaction was refluxed for 3 days (starting with the addition of the reactants) before adding the respective alkyl bromide (44 equiv., RBr), the respective alcohol (82 equiv., ROH) and DBU (21 equiv.) and proceeding with next steps in accordance with the general procedure described above.

Synthesis of macrocycle BCyc-Et following the alternative procedure (using a syringe pump):
The same amounts of reagents and reactants as for the synthesis following the general procedure were used. Purification by GPC and evaporation of the solvent afforded macrocycle BCyc-Et (93.6 mg, 0.095 mmol, 17%) as a dark yellow oil, which slowly crystallised over time to give a yellow powder.

Macrocycle NCyc-Et
Synthesis of macrocycle NCyc-Et following the general procedure:

Macrocycle ACyc-Et
Synthesis of macrocycle ACyc-Et following the general procedure: Important note: Although we found the synthesis of all precursors and all other macrocycles to be reproducible, we struggled to re-synthesize ACyc-Et in later attempts. However, as highly interesting effects were observed for this macrocycle in both the computations and experiments, we decided not to omit the available data here.

Voltammetric measurements
Details provided in the methods section of the paper.

Electroreduction and -oxidation of the macrocycles at arbitrary concentration in 1,2-dichloroethane
Electroreduction: For BCyc-Et, PCyc-Et, and PCyc-Hx, the mid-peak potential vs. Fc/Fc + (the midpoints between the anodic and cathodic peak potentials) were determined and are provided in the main part of the paper. For NCyc-Et and ACyc-Et, where the peak potentials could not be determined, the inflection-point potentials were determined by differentiating the cyclic voltammograms ( Figure   S45).

Electrooxidation:
As the electrooxidation was chemically irreversible for all macrocycles, the inflection-point potentials were determined by differentiating the cyclic voltammograms ( Figure S45).
The measurements were carried out at arbitrary concentration as the small amounts available for some of the macrocycles prevented the preparation of solutions with defined concentration. While the concentration has an impact on the current, the redox potential does not depend on the concentration.

Electroreduction of BCyc-Et at defined concentrations
Cyclic voltammetry of BCyc-Et solutions in 1,2-dichloroethane (DCE), N,N-dimethylformamide (DMF), and propylene carbonate (PC), all containing 0.1 M NBu4PF6 as supporting electrolyte, were performed with 2 mm diameter platinum disk electrode. The obtained voltammograms ( Figure S46) suggest two quasi-reversible electroreduction steps. With the intention of getting an indication of the stoichiometry and kinetic parameters of these steps, simulated voltammograms were fitted to the obtained voltammograms using DigiSim 3.03b software, assuming two consecutive one-electron reductions steps: Similarly to non-substituted BCyc (also known as PCT), 9 the most reduced form in the assumed process  Table S1.. Other parameters for the simulations: parameters according to the abovementioned mechanism. The parameters found by fitting are listed in gray cells in Table S1. Hydrodynamic radii (rH) were calculated from fitted diffusion coefficients and solvent viscosities using the Stokes-Einstein equation. Additional peaks of low current magnitude that are visible in the recorded voltammograms can be caused by adsorption of BCyc-Et on the platinum electrode.
Contrary to the previous approach used for the analysis of the voltammograms of BCyc, where diffusion coefficients were obtained from steady-state voltammograms recorded with an ultramicroelectrode (UME), here the diffusion limiting current could not be unambiguously read from voltammograms recorded with a 25 μm diameter UME ( Figure S47). This is due to the slower kinetics of the electron transfer for BCyc-Et than for BCyc. Therefore, diffusion coefficients (D) of BCyc-Et were obtained by fitting to voltammograms recorded with a bigger (2 mm diameter) electrode. To verify the procedure of finding D by fitting cyclic voltammograms recorded at a larger disk electrode, we applied the procedure to the voltammograms of BCyc. Diffusion coefficients of BCyc obtained this way differ less than 10% from the values obtained from the UME measurements.

Discussion of the simulation results
The hydrodynamic radii obtained from fitting voltammograms simulated under the assumption of two one-electron reduction steps (rH in Table S1) roughly correspond to the size of the macrocycle.
However, estimating the hydrodynamic radius from the molecular geometry is difficult and neglects that interactions with solvent molecules can increase the actual hydrodynamic radius. Such interactions can be expected particularly for molecules with polar functional groups, such as ester groups.
Under the assumption of two two-electron reduction steps, larger hydrodynamic radii would be obtained from the simulations, which do not correspond well with the bare macrocycle but may in line with an increased hydrodynamic radius because of interactions with solvent molecules.

Electrochemical measurement set-up
A three-electrode set-up was used for all electrochemical measurements. All electrochemical measurements were carried out in a quartz EPR tube (Wilmad, outer diameter 4 mm), sealed with a suba-seal rubber septum ( Figure S49). The working (WE) and counter electrodes (CE) were two Teflon-coated Pt wires (ADVENT, purity 99.99%, bare wire diameter = 0.125 mm), coiled to enhance their surface area. The working electrode was encased in a glass capillary to avoid short-circuiting and maintain the wire at the bottom of the EPR tube that is inserted into the EPR cavity. An Ag/AgCl reference electrode (RE, calibrated using a master saturated calomel electrode SCE) was used for aqueous electrochemical measurements. Teflon-coated silver wire (ADVENT, purity 99.99%, bare wire diameter = 0.125 mm) was used as a pseudo-reference electrode (pseudo-RE) for the measurements in DCE, with 5 mM ferrocene (Fc) added to calibrate the potential. The EPR tube was purged with nitrogen gas prior to electrochemical measurements performed with a µAutolabIII potentiostat in combination with NOVA software. Figure S49. Spectroelectrochemical cell assembled in an EPR tube. The tube is inserted into the EPR setup, but only the bottom 2 mm of the tube are placed in the actual EPR cavity.

EPR measurement set-up
Continuous wave X-band EPR measurements were performed at room temperature in an X-band Super High Sensitivity Probehead ER 4122SHQE and an EMX-T-DU/L Bruker spectrometer.
The bottom 2 mm of the EPR tube containing the electrochemical cell, i.e. only the working electrode (see Figure S49), was placed inside the EPR cavity (resulting in Q values of approx. 1300-4500 due to the conductivity of the sample). After insertion of the EPR tube into the cavity and optimisation of its position, the electrodes were connected to the potentiostat and the electrochemical and EPR measurements were carried out simultaneously.

Sample preparation
BCyc-Et or 4-amino-TEMPO was dissolved in 0.4 ml DCE to a final concentration of 5 mM and supplemented with 0.1 M NBu4PF6. Aqueous electrochemical measurements with 4-amino-TEMPO (5 mM) were carried out in 0.5 M Na2CO3 in milliQ water, adjusted to pH 8.0.

EPR spectroelectrochemical measurements of 4-amino-TEMPO
To test the measurement set-up, electrochemical EPR measurements were first carried out on the EPR active compound 4-amino-TEMPO under aqueous conditions. As can be seen in Figure S50A, the cyclic voltammogram showed a redox potential Em, pH 8.0 = 0.631 V vs. Ag/AgCl (0.836 V vs. SHE) that is consistent with previously reported values (Em, pH 7.0 = 0.874 V vs. SHE). 15 The in operando EPR measurements show that the nitroxide EPR signal changes upon the redox reaction, but it does not disappear completely ( Figure S50B), which is expected given that the redox reaction occurs just at the electrode. The corresponding experiment under non-aqueous conditions (in DCE) showed more sluggish electron transfer, but redox cycling is nonetheless evident from the in operando electrochemical EPR measurements ( Figure S50C).

EPR spectroelectrochemical measurements of BCyc-Et
In contrast to the measurements of 4-amino-TEMPO, in operando electrochemical EPR measurements with the BCyc-Et only gave EPR spectra with flat lines (Figure S51). Although the CV ( Figure S51A) and the control measurements with 4-amino-TEMPO ( Figure S50) suggest that the set-up needs to be further improved for optimal in-operando measurements, they enable us to conclude that it is highly unlikely that a paramagnetic intermediate is formed during redox cycling of BCyc-Et, corroborating that it does not proceed in single electron transfer steps.
Measurements at various scan rates including 5 mV s -1 (the scan rate used for the measurements shown in Figure S50) were carried out, with no EPR signal appearing in any of these measurements. In situ chronoamperometry coupled to EPR measurements was also carried out. A potential range of -2.5 V to -1.0 V (vs. Fc/Fc + ) was investigated by holding the potential for more than 300 seconds at different values while recording EPR spectra. In agreement with the result obtained from in situ CV-EPR measurements, no EPR signal was observed over the scanned potential range.

Current densities
For BCyc and BCyc-Et, we also computed current densities using the gauge-including magnetically induced current (GIMIC) method 16 based on PBE0/def2-SVP computations carried out in Turbomole 7.4. 17 Currents were integrated along a plane bisecting the C=C double bond in a vinylene unit. These results are presented in Table S5. Table S5 also   BCyc-Et 6-BCyc-Et 6+