Fluorinated Azobenzenes Switchable with Red Light

Abstract Molecular photoswitches triggered with red or NIR light are optimal for photomodulation of complex biological systems, including efficient penetration of the human body for therapeutic purposes (“therapeutic window”). Yet, they are rarely reported, and even more rarely functional under aqueous conditions. In this work, fluorinated azobenzenes are shown to exhibit efficient E→Z photoisomerization with red light (PSS660nm >75 % Z) upon conjugation with unsaturated substituents. Initially demonstrated for aldehyde groups, this effect was also observed in a more complex structure by incorporating the chromophore into a cyclic dipeptide with propensity for self‐assembly. Under physiological conditions, the latter molecule formed a supramolecular material that reversibly changed its viscosity upon irradiation with red light. Our observation can lead to design of new photopharmacology agents or phototriggered materials for in vivo use.

But the respective aldehyde 4 exhibited unusual behavior. We observed that E-4 undergoes substantial E!Z photoisomerization (PSS 660 nm = 75 % Z-4) upon irradiation with red light (λ max = 660 nm) (Table 1). To assure that the emission shoulder of our light source does not overlap with the green light range, we used a 630 nm-cutoff filter SCHOTT RG-630.
Based on analogous reports for other classes of photoswitches, we hypothesized that the bathochromic shift in the absorption range can be attributed to the extended conjugated π-electron system of the chromophore. To check this hypothesis, we have prepared two further aldehyde derivatives -the bis-aldehyde 5 from the alcohol 3, and the non-conjugated aldehyde 7 from the alcohol 6. The conjugated bis-aldehyde 5 showed even more pronounced photoisomerization (PSS 660 nm = 82 % Z-5) under red light irradiation (λ max = 660 nm, filter cut-off < 630 nm) (Figure 1), while the non-conjugated aldehyde 7, as well the alcohols 2, 3, or 6, did not switch significantly under these conditions, as compared with the photostationary states (PSS) achieved with the reverse isomerization elicit by violet light (PSS 407 nm ) (Table 1).
At higher concentrations (e. g. 1.5 mM in DMSO), the absorption peak tail above 600 nm, responsible for the photoisomerization of E-4 and E-5 with red light, is clearly visible (Figure 2). The S 0 -S 1 absorption bands of both photoisomers, which in azobenzene systems are commonly identified as n-π* transitions, exhibit in our case sufficient separation (from 25 nm for 6 to 51 nm for 5) to selectively address each photoisomer with visible light. This is important, as the distribution of photoisomers in PSS at a given wavelength is governed by the ratio of the products of molar attenuation coefficients and quantum yields for each isomer. For example, at 630 nm the ɛ 630 nm of E-5 is 11.5 M À 1 cm À 1 , while the ɛ 630 nm of E-5 is 1.47 M À 1 cm À 1 . The ratio of both values satisfyingly corroborates with the observed Z/E-photoisomer ratio in PSS (82 % of Z-5) obtained upon irradiation with the red LED, and indicates that the difference in quantum yields for both photoisomers is not critical in the demonstrated case.
Next, we have investigated thermal stability of the aldehydes Z-4 and Z-5 in MeCN at 60°C. The half-life of Z-4 under these conditions -10.8 h -was comparable to other TFAB derivatives, [21b] while the half-life of Z-5 -3.2 h -was similar to the value of the unsubstituted azobenzene ( Figure S5). [21b] To gain more understanding on the observed photophysical properties, we have corroborated our experimental results with theoretical calculations on all E-and Z-isomers, respectively. The results of ground state optimization show that aldehyde substituents in para position lead to an extended π-conjugation, which causes a shift of both the HOMO (n) and LUMO (π*) levels  -isomers). Upon optimization of their structure we discovered that compounds 4 and 5 are capable of photoisomerization with red light (> 630 nm), which expands the scope of their potential in vivo applications; b) UV-Vis spectra of the bis-aldehyde 5 (38 μM in MeCN). The E!Z photoisomerization is more efficient with red (λ max = 660 nm, cut-off filter < 630 nm PSS 660 = 82 % Z-5) than with green light (λ max = 532 nm, PSS 523 = 67 % Z-5). (see also Table 1); inset: optical demonstration of the photochromism of 5; 407 nm-9 mW/cm 2 , 523 nm-7 mW/cm 2 , 660 nm (with filter)-56 mW/cm 2 . Table 1. The percentage of Z photoisomers in photostationary states (PSS) obtained upon irradiation with violet, green, or red light of the compounds 2-7 determined by 1 H NMR spectroscopy. to lower energies. The decrease in the energy level of the LUMO (π*) is more pronounced than for the HOMO (n), which results in a significantly lower HOMO-LUMO gap for the compounds 4 and 5 compared to the non-conjugated aldehyde 7, unsubstituted TFAB, or sp 3 -substituted derivatives 2, 3 or 6 ( Figure 3,  In all cases, the y-axis depicts the molar attenuation coefficient ɛ (M À 1 cm À 1 ). The spectra of pure Z-isomers (red lines) have been calculated from spectra registered for samples irradiated 15 min with 523 nm LED with concomitant determination of the E/Z-ratio by 1 H NMR (procedure described in Supporting information, pages S77-S92, Figures S12-S40).
are in qualitative agreement with the experimentally observed shifts in the measured UV-Vis spectra ( Figure S9). Then we wondered, if our observation can be implemented into a material, which properties are reversibly triggered with red light. Incorporation of aldehydes, like 4 or 5, into smart materials or systems of biological interest would be complicated. Thus we hypothesized that similar bathochromic shift may occur for other conjugated sp 2 -substituents, more suitable as linkers, -in particular C=C bonds. To verify that, we have coupled the bis-aldehyde 5 upon base-catalyzed condensation with two equivalents of a Bocprotected cyclic dipeptide cyclo(Gly-Lys), followed by complete removal of the residual Boc groups.
The resulting symmetric unsaturated TFAB derivative 8 also photoisomerized upon exposure on red light (λ max = 660 nm, > 630 nm) (PSS 660 nm = 61 % (Z)-8) (Table 1), with the Z-8 half-life of 17 min at 60°C (Figure S6, left). This is considerably shorter than the aldehydes 4 or 5. Nonetheless, the half-life of 14.8 h for the same Z-8 measured at 20°C ( Figure S6, right) is still comparable with other red light-switchable azobenzenes. The compound 8 remained stable during 10 cycles of forth-and-back switching with alternate 660 nm and 470 nm irradiation ( Figure S2-S4). In presence of 10 mM reduced glutathione (a standard mimic of intracellular reduction potential) under physiological conditions at 25°C roughly half of the compound 8 was degraded within 10 hours ( Figure S41). Therefore, it has to be kept in mind, that eventual intracellular applications of this chromophore have to be time-limited.
Next, we wanted to investigate self-assembly properties of 8 in aqueous solutions, and potential macroscopic effects which can be triggered upon its exposure on red light. Compound 8 suspended in water, 200 mM aq. NaCl, or Ringer solution under physiological conditions, and shortly boiled, yielded gel-like opaque viscous material at the concentration range between 10 g/L and 20 g/L (1-2 wt%) of E-8 (Table S20-S22). The presence of salts significantly enhanced its melting temperature.
However, its low mechanical stability -quantified with rheological experiments ( Figure S26) -was different from the stability of hydrogels formed by the gelators 1 a-c under analogous conditions. Upon irradiation with red light (660 nm, with filter > 630 nm, 56 mW/cm 2 ), this material rapidly (5 min) dissipates to non-viscous, opaque fluid (Figure 4). This fluid returns to the initial viscosity upon short boiling, which thermally restores the E-isomer.
The structure of this material has been investigated with electron microscopy techniques ( Figure 5, Figure S42-S56). In the non-irradiated material, we observed mostly μm-long thick structures ( Figure 5A), contrary to previously investigated hydrogels formed from 1 a-c, consisted of a dense network of small fibers. Upon irradiation with red light, along with the viscosity change, we observed almost total decay of the thick structures ( Figure 5B). They were, however, restored upon thermally induced back-isomerization ( Figure 5C). The morphology of freeze-dried  Table S3) corroborates with the experimentally observed bathochromic shift of the absorption maximum in the E-5. EWG aldehyde substituents in 5 stabilize all orbitals (π, n, and π*) in comparison to 3. However, the stabilization is most pronounced for the π* (LUMO) orbital, most likely due to the extended conjugation in the π-orbital system depicted in the orbital contour.
SEM and air-dried TEM samples was also characterized for various solvent compositions ( Figure 5E-F).
In addition, 1 H-NMR transverse relaxation (T 2 ) measurements were used to investigate the microstructure of the gel as it is directly linked to the molecular mobility (1-10 nm) of the components. The mobility can be quantitatively assessed by the decay of the transverse magnetization using time-domain NMR techniques. Herein, a magic sandwich echo (MSE) pulse sequence was used to refocus the initial transverse magnetization (100 ms) of rigid components in combination with a CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence to refocus the magnetization of more mobile components up to 1 s. As shown in Figure S58a distinct decay of the FID by 80 % in the first 100 ms is observed, which indicates a highly polycrystalline nature of the microstructure and corroborates with the structures observed with electron microscope (Figure 5a).
Comparison between 8 and the hydrogelators 1 a-c indicates the crucial role of a flexible linker between the azobenzene and the peptide fragment for efficient selfassembly of fibrous structures and the resulting hydrogel formation. A similar issue has been already discussed for hydrogelation of short linear photochromic peptides decorated with azobenzenes. [26] In conclusion, we demonstrated the efficient E!Z photoisomerization of tetra-ortho-fluoroazobenzenes substituted with sp 2 -hybridized conjugated substituents with red light within the "therapeutic window" (> 630 nm). These results were successfully corroborated with calculations. Importantly, TFAB derivatives with saturated substituents, or a fluorinated azobenzene substituted with an sp 2 -hybridized carbon at the meta-position were essentially inert to red light within our experimental setup.
The new chromophore has been coupled with a cyclic dipeptide motif. The resulting compound, structurally analogous to previously described hydrogelators, formed a viscous material in aqueous media under physiological conditions. This material was reversibly dissipated to non-viscous fluid with red light, and recovered its initial form upon thermal equilibration. The microscopic structure indicated its polycrystalline nature, which is reversibly dismantled upon photoisomerization. This example demonstrated that the red light-switchable conjugated TFAB photochrome can be incorporated into larger molecules, using a C=C bond linker, remains stable under physiological conditions, and can elicit macroscopic effects upon irradiation with red light. Thus, this photochrome is suitable for applications in new biocompatible materials, or in photopharmacology agents, comprising these operational inside human organisms, with reservation to its slow decomposition upon exposure to glutathione.  infrastructural support of our research. Open access funding enabled and organized by Projekt DEAL.