Title: Cross-conjugation controls the stabilities and photophysical properties of heteroazoarene photoswitches

Azoarene photoswitches are versatile molecules that interconvert from their E-isomer to their Z-isomer with light. Azobenzene is a prototypical photoswitch but its derivatives can be poorly suited for in vivo applications such as photopharmacology due to undesired photochemical reactions promoted by ultraviolet light and its relatively short half-life (t1/2) of the Z-isomer (2 days). Experimental and computational studies suggest that these properties (λ of the E isomer and t1/2 of the Z-isomer) are inversely related. We identified isomeric azobisthiophenes and azobisfurans from a high-throughput screening study of 1700 azoarenes as photoswitch candidates with improved λ and t1/2 values relative to azobenzene. We used density functional theory to predict the activation free energies, reaction free energies, and vertical excitation energies of the Eand Z-isomers of 2,2and 3,3-substituted azobisthiophenes and azobisfurans. The half-lives depend on whether the heterocycles are π-conjugated or cross-conjugated with the diazo π-bond. The 2,2-substituted azoarenes both have t1/2 values on the scale of 1 hour, while the 3,3-analogues have computed half-lives of 40 (thiophene) and 230 (furan) years. The 2,2-substituted heteroazoarenes have significantly higher λ absorptions than their 3,3-substituted analogues: 76 nm for azofuran and 77 nm for azothiophene.


Introduction
Photoswitches are molecules that are reversibly and chemically interconverted between two states with light. Azobenzene is the most widely studied photoswitch and has a well-documented synthesis via diazonium coupling reactions. 1 The relatively small size of azobenzene, along with the significant chemical and structural changes resulting from isomerization, has enabled its use for the spatiotemporal control of proteins, lipids, neurotransmitters, neurons, and carbohydrates. [2][3][4][5][6][7][8][9][10][11][12][13][14] Phenyl functionalization can improve photophysical properties for applications in chemical biology [15][16] and materials science. [17][18][19][20][21] Scheme 1 shows the photochemical E → Z conversion of azobenzene and the subsequent Z → E thermal reversion. Azobenzene requires ultraviolet light (320 nm) to promote the E → Z isomerization, limiting its utility in vivo due to undesired lightpromoted dimerizations, destruction of living tissue from UV-light, and low light penetrability in living tissue. 11,[22][23] Further, the meta-stable Z-isomer thermally reverts to the E isomer and has a half-life (t1/2) of 2 days at room temperature in acetonitrile. This relatively short half-life results in an unstable photostationary state (PSS), thus limiting photoswitch efficacy where bistability is neded. 8 As such, an ideal photoswitch features a long-lived t1/2 (e.g., weeks to months), an E-isomer λ max in the visible range, and well-separated λ max values for the E and Z isomers to prevent bimodal isomerization. Scheme 1. The azobenzene photoswitch reaction. The E → Z isomerization (top) occurs photochemically under UV light. The Z → E reversion (bottom) occurs thermally or photochemically under visible light.
Woolley and Fuchter reported that λ max absorptions are redshifted into the visible range by replacing phenyl groups with heteroaryl rings (benzodioxanes 24 , diazinines 25 , and imidazoles 26 ) and by functionalizing the aryl groups. 24,[27][28][29][30][31][32][33][34][35][36][37] These heteroazoarenes have relatively high E → Z photochemical reaction yields, ranging between 46-98, and Z-isomer half-lives ranging from 1 second up to 46 years. The range in E-isomer λ max for these heteroazoarenes is 310-720 nm, pushing them deep into the visible light range. There is an inverse relationship between λ max and t1/2 for these heteroazoarenes, those with the highest t1/2 have the smallest λ max . Scheme 2 shows heteroazoarenes with heteroaryl rings, and the heteroazoarenes of each type with the longest t1/2 values are shown in Figure 1. Thermal Z → E reactions can proceed through inversion or rotation mechanistic pathways. [38][39][40][41] Past computational studies have found that the inversion mechanism is preferred for azoarene thermal Z → E isomerization reactions. 33 Thiophene-containing heteroazoarenes have well-separated E and Z absorption bands and a high yield of Z-isomer for the E → Z photoisomerization reaction (94-98%). 2-hemi-azothiophene and its derivatives have been studied by Wegner, Dreuw, and Wachtveitl 31-32 (Scheme 2b), and two (3,3) bis-azothiophenes ( Figure 1) have been studied by Perry. 45 The unsubstituted 2-hemiazothiophene (Scheme 2b, left) has a Z-isomer t1/2 of 7 hours in acetonitrile. Wegner and Heindl 31 showed that electron-donating groups (EDG) at the phenyl para position (Me and OMe groups) of 2-hemi-azothiophenes lower the t1/2 of the Z-isomer to 0.5 and 1.9 hours (methyl and methoxy substituent, respectively). Electron withdrawing groups (EWGs) increase the t1/2 to 14.3 and 17.8 hours (CF3 and CN, respectively). Substitution of the thiophene ring with an electron-donating group (OMe) lowered the t1/2 to 2.8 hours. Installing push-pull substituents (OMe and CN groups) on an azothiophene lowered the t1/2 to 9 minutes. The λ max is 365 nm for the unsubstituted hemiazothiophene and 405 nm for the push-pull hemi-azothiophene. All hemi-thiophene E-isomer λ max values are red-shifted towards the visible range, an improvement over azobenzene. Figure 1 shows the heteroazoarenes photoswitches of each type with the longest measured half-lives. Although recent studies have increased the Z-isomer t1/2 or the E isomer λ max , experimental and theoretical studies on heteroazoarenes generally show an inverse relationship between t1/2 and λ max . 29,[46][47][48] The reason for this relationship has not been well defined; there is no clear connection between how the E-isomer λ max relates to the Z-isomer half-life. We have computed the thermal and photophysical properties of isomeric bis-heteroazoarenes photoswitches (two azobisfurans and two azobisthiophenes) using density functional theory (DFT) and time-dependent density functional theory (TD-DFT). The Z isomers of 1, 2, 3, and 4 are shown in Figure 2. Watchtveitl and co-workers initially reported derivatives of 3-Z. [31][32] The (3,3)-azobisthiophene (4) and one derivative with ester groups substituted at the 2-position were synthesized, and UV/Vis spectra and Z-isomer half-lives were measured by Perry and co-workers. 49 These molecules had t1/2 and λ max values of 38 days and 316 nm (unsubstituted azothiophene) and 14 days and 350 nm (estersubstituted azothiophene).
The four heteroazoarenes presented in this study (1, 2, 3, and 4) were obtained from the results of a high-throughput virtual screening (HVTS) study, which generated 1700 heteroazoarenes molecules for the VERDE materials database 50 . We calculated the λ max and t1/2 values for all molecules, which highlighted the 77 and 76 nm difference in λ max between the (2,2) and (3,3) substituted structures and a 10 6 range in half-lives.

Results and discussion
We performed IRC calculations and geometry optimizations to obtain the reactive conformers for the lowest energy transition states of azofurans 1-TS and 2-TS, and azothiophenes 3-TS and 4-TS. Figure 3 shows the optimized reactants 1-Z and 2-Z, along with their optimized transition structures 1-TS and 2-TS, and Figure 4 shows the energies and geometries of transition structures of (3-4)-Z, (3-4)-TS. 2-Z has an isomerization barrier that is 8.5 kcal mol -1 higher than the corresponding azofuran 1-Z. We sought to understand how the electronic structures of (1-4)-Z affect the 10 6 -fold range in half-lives. The transition states feature coplanar aryl groups; we quantify this coplanarity with an out-of-plane angle (θ). θ is the angle between two normal vectors to the planes of the aryl rings. indicating increased π-character, while the other C-N bond is nearly unchanged from 1-TS to 1-Z (1.38 Å and 1.36 Å, respectively). The relative planarity between the two rings (θ) is significantly different for 1-TS and 2-TS. The two furan rings in 1-TS are fully coplanar (θ=0°), maximizing the π-conjugation between them. 2-TS is a cross-conjugated azofuran, which implies that πconjugation is between part of the aromatic π-system of the blue furan with the diazo bond throughout the reaction. While 2-TS also involves a rehybridization of one of the N-atoms, the furans are not coplanar (θ=34°) because the rings are not electronically communicating through the diazo bond. The difference in the C-O bond lengths in 2-TS is 0.03 Å (1.35 and 1.38 Å), which suggests that the delocalization of oxygen lone pairs substantially decreases. The C-N bonds flanking the N=N bond are 1.42 and 1.32 Å. As such, azoarenes featuring cross-conjugation do not have full π-delocalization across the diazo bond. This phenomenon results in the 8.5 kcal mol -1 ΔΔG ‡ of these isomeric azofurans. The (2,2)-diazofurans are 10 6 -fold more reactive than the (3,3)-diazofurans. We now turn our attention to the azothiophenes 3 and 4. . The shorter C-N bond length in 3-Z is consistent with an aromatic C-N bond, and the longer C-N bond lengths in 4-Z are consistent with single bonds. The shorter C-N bonds and the longer N=N bond indicate that 3-Z has more electron delocalization between the two thiophene rings than 4-Z.
The ΔG ‡ for 3-TS is 22.6 kcal mol -1 , and the ΔG ‡ for 4-TS is 30.1 kcal mol -1 . The difference in activation free energies (ΔΔG ‡ ) is 7.5 kcal mol -1 , corresponding to half-lives of one hour and 40 years, respectively. Transition structures 3-TS and 4-TS involve rehybridization about one of the N atoms and have nearly linear NNC2 and NNC3 angles (179° and 178°, respectively). The N=N bonds are shorter in 3-TS (1.25 Å) and 4-TS (1.24 Å) relative to their corresponding reactants (1.26 Å and 1.25 Å, respectively). These bond lengths shorten as the azothiophenes reach their respective transition structure geometries, resulting from the rehybridization of one of the N atoms.
The C-C bond lengths in the thiophene rings range from 1.36 to 1.45 Å, which is consistent with aromatic C-C bond lengths in thiophene rings. The C-S bonds are 1.71-1.76 Å in 3-Z but diverge in 3-TS (1.70-1.84 Å) because one of the sulfur lone pairs is delocalized through the transition structure. This phenomenon results in the zwitterionic electronic structure shown in Figure 7. The shorter bond length resembles a C-S double bond, 51 while the longer bond corresponds to a broken C-S π-bond (the aromatic C-S bonds in the crystal structure of thiophene are 1.74 Å). 51 The red thiophene ring has two nearly identical C-S bond lengths (with lengths of 1.  1-E and 3-E have fully delocalized π-systems across the diazo bond, while 2-E and 4-E show interrupted π-electron density due to their cross-conjugated nature. Cross-conjugated molecules have incomplete π-delocalization, which has been shown to impact photophysical properties by increasing molecular band gaps and lowering λ max absorption. [52][53][54][55] Cross-conjugation has also been found to affect the reversibility and activation free energy barriers of Diels-Alder and thiol addition reactions. [56][57][58] The delocalization effect in 1-E and 3-E leads to higher-lying HOMOs and lower-lying LUMOs relative to 2-E and 4-E, leading to increased HOMO-LUMO gaps. The HOMO for 1-E is -6.18 eV, 0.41 eV higher than the HOMO for 2-E (-6.59 eV), and the HOMO for 3-E is -6.32 eV, 0.25 eV higher than the HOMO for 4-E (-6.58 eV). The LUMO for 1-E is -2.64 eV, which is 0.47 eV lower than the LUMO for 2-E (-2.17 eV), and the LUMO for 3-E is -2.81 eV, 0.53 eV lower than the LUMO for 4-E (-2.27 eV). The HOMO-LUMO gaps of 1-E and 2-E are 3.54 eV and 4.42 eV (a 0.88 eV difference), and the same gaps are 3.51 eV and 4.31 eV for 3-E and 4-E, a difference of 0.80 eV.

Conclusion
We have used DFT to determine the photophysical properties and thermal stabilities of four bis-diazoarene photoswitches. We predict that the thermal half-lives of the isomeric Z-isomers range from hours to years. The (3,3)-substituted isomers (2 and 4) have cross-conjugated πsystems, significantly affecting the photophysical and thermal properties. This truncated πconjugation leads to large optical gaps, thus requiring UV light for photoswitching (300 nm for 2-E and 314 nm for 4-E). Those with fully π-conjugated systems (1-E and 3-E) contain relatively small optical gaps, which enable visible light photoswitching (376 nm and 391 nm, respectively). The extent of π-conjugation strongly influences the transition structures and thermal half-lives of the Z-isomers. 1-Z and 3-Z have a small ΔG ‡ , which translates to a t1/2 of one hour. 2-TS and 4-TS are higher in energy due to the limited π-delocalization, and the t1/2 is in the range of 40-230 years.

Computational methods
We performed DFT calculations to predict the activation free energies (ΔG ‡ ) of (1-4-Z). We computed the structures and energies of the Z-isomer, E-isomer, and transition structure for each reaction. First, the transition structures were optimized using the EZ-TS code recently reported by our group. 59 After locating the lowest energy transition states, we ran intrinsic reaction coordinate (IRC) calculations and optimized the reactive conformers corresponding to the reactant (Z-isomer) and product (E-isomer) for each thermal Z → E isomerization. All calculations were performed using the Gaussian 16 software. 60 The PBE0 61 /6-31+G(d,p) 62 model chemistry was used for all geometry optimizations and frequency calculations. Vertical excitation energies were calculated using (TD-DFT) 63 with the ωB97X-D 64 /6-31+G(d,p) model chemistry in IEFPCM water . 65