General Principles for the Design of Visible‐Light‐Responsive Photoswitches: Tetra‐ortho‐Chloro‐Azobenzenes

Abstract Molecular photoswitches enable reversible external control of biological systems, nanomachines, and smart materials. Their development is driven by the need for low energy (green‐red‐NIR) light switching, to allow non‐invasive operation with deep tissue penetration. The lack of clear design principles for the adaptation and optimization of such systems limits further applications. Here we provide a design rulebook for tetra‐ortho‐chloroazobenzenes, an emerging class of visible‐light‐responsive photochromes, by elucidating the role that substituents play in defining their key characteristics: absorption spectra, band overlap, photoswitching efficiencies, and half‐lives of the unstable cis isomers. This is achieved through joint photochemical and theoretical analyses of a representative library of molecules featuring substituents of varying electronic nature. A set of guidelines is presented that enables tuning of properties to the desired application through informed photochrome engineering.


Introduction
Molecular photoswitches form the basis of light-responsive systems that are designed to enable reversible control of function with high spatiotemporal resolution. [1] They have found application in remotely manipulating biological systems, [2,3] smart materials, [4,5] and molecular machines. [6,7] In particular,t heir potential in biomedical context, along the principles of photopharmacology, [8][9][10][11] evoked considerable interest in recent years.T he available panel of molecular photoswitches features many established architectures that mainly rely on double bond isomerisation (azobenzenes, [12] azoheteroarenes, [13] stilbenes, [14] hemithioindigos [15] ), electrocyclisation (diarylethenes [16] ), or mixed mechanisms (spiropyrans [17] ). Furthermore,v arious novel designs [18] have appeared during the last decade,i ncluding donor-acceptor Stenhouse adducts (DASAs), [19,20] hydrazone [21] -a nd acylhydrazone [22] -based switches,B F 2 -coordinated azo compounds, [23] diazocines, [24] indigos, [15,25] and iminothioindoxyls. [26] Thed evelopment of new molecular photoswitches is largely driven by the challenge of enabling the use of visible, and red or even near-IR (NIR) light for operation in both directions. [27][28][29] This is relevant especially in biological applications,w here red/NIR light enables deep (1 cm) tissue penetration without the toxic effects induced by higher energy light. [30] Thesuccessful application of the new visible-light-responsive photoswitches depends on establishing their design principles,b ased on the understanding of the interplay between the nature of the substituents and the key photochemical properties.T his understanding is enabled through synthesis,s pectroscopic studies,a nd theoretical investigations. [31][32][33] It ultimately allows both the tuning of these properties,and the effective choice of substituents determining the function of the photoresponsive unit in ab iological system, material, or amolecular machine.
Here we present as ystematic spectroscopic and theoretical investigation into the photochemistry of tetra-orthochloro-azobenzenes,with the aim to provide aguide for their design. Te tra-ortho-substituted azobenzenes emerged as privileged light-responsive molecular photoswitches,w ith good absorption band separation and half-lives of the metastable cis isomer in the range that enables multiple applications. [27][28][29][34][35][36] Among them, azobenzenes with all four ortho positions substituted with chlorine atoms ( Figure 1A), have already enabled using green and even red light to control peptide conformation, [27] antibiotic potency, [37] ion channel activity, [38][39][40] and the function of nucleic acids [41] and ion receptors [42,43] for controlling the transport through biological membranes [44] (Figure 1D). However,w hile for normal azobenzenes several relationships between structure and photochemical properties have been defined, only little systematic information is available for the tetra-ortho-substituted systems,making their design largely atrial-and-error endeavor.T he main difference between those switches and classical azobenzenes comes from the fact that, for their operation in both directions,v isible-light absorption bands are used, which correspond to weakly allowed transitions of np*c haracter ( Figure 1B,C). This presents ac hallenge for informed design of photoresponsive units for applications and necessitates the systematic study on parameters that govern the key properties,s uch as band separation, switching efficiency,p hotostationary state distributions and thermal stability of the metastable isomer.

Results and Discussion
Alibrary of ten compounds with different groups in one of the para positions was designed, spanning the range of Hammet s para constants from the most electron-donating (ÀNMe 2 , s para = À0.83) to the most electron-withdrawing (ÀNO 2 , s para =+ +0.78). We focused on the para-substituents, since meta ones show less pronounced resonance effects,and all ortho positions are occupied in the studied molecules. Furthermore,inall the applied molecules ( Figure 1D), parasubstituents are used.
Thes ynthesis of tetra-ortho-substituted azobenzenes is known to be challenging due to the highly sterically congested nature of the central N=Nd ouble bond that is surrounded with four large chlorine substituents.T his limits the use of classical methods for azobenzene synthesis,s uch as the Baeyer-Mills reaction, [45] diazonium coupling [27] or oxidative coupling of anilines, [46] and has inspired the development of methods better suited for these targets:l ate-stage CÀH chlorination [38,47,48] and the reaction of diazonium salts with lithiated aromatic compounds,r eported recently by our group. [49] Here,weuse the latter method to prepare aversatile library of tetra-ortho-chloro-azobenzenes ( Figure 2, Table 1), additionally highlighting the robustness of this method. Furthermore,t his substrate scope was acquired without the use of transition metals,instead using aSmiles rearrangement to synthesize compound 1 (see the Supporting Information).
Forthe photochemical evaluation, we have chosen DMSO as the solvent, because it facilitates the solubility needed throughout the analytical methods used (UV/Vis spectrophotometry,N MR spectroscopy) and, with its intermediate polarity,i ta pproximates well both organic and aqueous systems well. Even more importantly,inphotopharmacology it is often used as as olvent for stock solutions,w hich after irradiation are diluted into aqueous media for biological evaluation. [37,[50][51][52] Hence,i ti so ften the photochemistry in DMSO that determines the properties of molecules in final applications.
Thep hotochemistry of tetra-ortho-substituted azobenzenes in the visible range of the electromagnetic spectrum is related to the presence of S 0 -S 1 absorption bands that are traditionally associated with n-p*transitions. [53] Theinstallation of ortho substituents induces as ignificant distortion of the geometry,w hich in turn, allows for the separation of the S 0 -S 1 absorption bands of the two isomers and thus enables their selective excitation with light of specific wavelengths. [29] This selective addressing is crucial, because the ratio of molecular attenuation coefficients e of the two forms at the irradiation wavelength is one of the two key factors (the other being the ratio of quantum yields f for the photoisomerisation in both directions) determining the photostationary state distribution (PSD) of isomers under irradiation at that wavelength. A) The trans-isomer can be switched to the cis isomer using green or red light. The metastable cis isomer can be switched back using violet or blue light. B) The spectra of both isomers feature the high energy absorption band in the UV region, associated with the symmetry-allowed p-p*t ransition, and alow energy band in the visible region, associated with the weakly allowed n-p*t ransition. C) The operation of tetra-ortho-chloro-azobenzenes with visible light is enabled due to the separation of n-p*b ands. D) Examples of application of tetra-ortho-chloro-azobenzenes for visible-light regulation of processes in biology and supramolecular chemistry.
Thespectra of compounds 1-10 are presented in Figure 2 and their properties are summarized in Table 1. In almost all cases,w eh ave observed n-p*a bsorption bands for the trans isomer in the l = 450-465 nm region. Only for compound 1, which features avery strong electron-donating ÀNMe 2 group, we did not observe aw ell-resolved band in this region, probably due to the overlap with av ery strong p-p*b and.
Thep osition of the bands was well reproduced theoretically (Table 1).
To shed more light onto the experimental results,wehave performed theoretical calculations on all trans isomers (see the Supporting Information for details). It should be noted that all compounds trans-1-10 strongly depart from the planarity of standard azobenzenes,w ith a4 8.88 8 twisting of Figure 2. Visible-lightb and separation in compounds 1-10 for the trans isomers (blue spectra) and cis isomers (orange spectra) in DMSO. The xaxis depicts the wavelength l (nm) and the y-axis depicts the molar attenuation coefficient e (M À1 cm À1 ). On the right side of each spectrum, apanel is provided showing the band separation in more detail in the 550-650 nm range. The spectra of pure cis isomers were calculated by irradiation of the sample in [D 6 ]DMSO with l exc = 526 nm (FWHM = 90 nm) until reaching PSS. 1 HNMR spectra of aliquots (0.6 mL) were then taken to determine the cis/trans ratio, followed by calculationo fthe cis spectra based upon the molar extinction coefficients of the trans-species (see the Supporting Information for full spectra). The pie charts show the content of cis isomer at PSS that can be achieved under irradiation with l exc = 426 nm (blue chart), l exc = 526 nm (green chart) or 625 nm (red chart) LEDs, as determined by NMR spectroscopy in [D 6 ]DMSO (see the SupportingInformation).
the aromatic rings with respect to the diazo bond in 4 ( Figure 3A). In the Supporting Information, Table S1, we provide the transition energies determined with TD-DFT and with additional CC2 corrections for all structures (see the Supporting Information for technical details). Forcompound 4,t he best estimate for the S 0 -S 1 excitation is 466 nm, in obvious agreement with the experimental value 457 nm (see Table 1). As can be seen in Figure 3B,this transition has an np*topology,mainly localized on the diazo bond, though it is slightly dipole-allowed due to the above-mentioned nonplanarity (f = 0.05). According to theory,t his transition is separated by more than 1eVf rom the following excitation (S 0 -S 2 , f = 0.03), and even by 1.5 eV from the intense S 0 -S 4 absorption (f = 0.41). Amongst all studied compounds,t he most red-shifted n-p*t ransition should occur in the NMe 2bearing compound 1 (497 nm, f = 0.11) according to theory, but in that case it is likely buried under the very probable S 0 -S 2 excitation (f = 0.86), that is much closer-lying than in compound 4.T he second most red-shifted n-p*t ransition returned by theory is obtained for the NO 2 -substituted compound 10 (482 nm, f = 0.09), which fits the experimental ordering (see Table 1). As can be seen in Figure 3B,t he addition of strong donating or accepting groups does not fundamentally change the nature of the transition, although one notice small red lobes (accepting character) on the nitro group of compound 10.F or 1,i nc ontrast, it is mostly the planarization on one side of the compound that accounts for the improved delocalization and the observed red-shift, rather than the direct donating nature of the amino moiety.
Upon irradiation with green and red light, we consistently observe the emergence of ah ypsochromically shifted band, mostly in the l = 440-455 nm region, which corresponds to the cis isomer.T he calculations indicate that the unsubstituted cis-4 is 26.3 kJ mol À1 less stable than its trans counterpart, with ag eometry rather typical for these structure ( Figure 3A). Thel owest excited state conserves its n-p* character ( Figure 3C), and the CC2-corrected vertical excitation energy of 434 nm (f = 0.03), is again close to the experimental value (441 nm, see Table 1). Them ost redshifted transition computed in the cis series is obtained for 1 (461 nm, f = 0.15), which is again consistent with experimental data. As can be seen in Figure 3C,t he geometry of cis-1 resembles closely the one of cis-4 but the lone pair of the amino group now recovers its clear donating character (blue lobe) explaining the red-shift. Data for the other compounds can be found in the Supporting Information.
Photostationary state distributions (PSDs) that can be achieved under irradiation with visible light are of crucial importance for applications,e specially in biological context where the two isomers are expected to have different potency in, for example,binding to the cellular target. [11] Only in very rare cases [55,56] it is possible to design molecules in which the stable isomer is almost inactive at agiven concentration, while the irradiation leads to the metastable isomer which is orders  of magnitude more potent, thereby making the application virtually independent from PSD.Inthe majority of cases, [9,57] the difference in potencyismuch less pronounced, requiring high efficiencyfor switching in both directions.
In series 1-10,weobserve (Figure 2, green pie charts) that under irradiation with green light (l = 526 nm) for switching in the forward (trans to cis)direction, aphotostationary state with the distribution containing 46-62 % cis isomer can be attained. Theonly exceptions were compound 1 (likely due to the band overlap) and compound 10,which is possibly due to short half-life of the cis isomer,w hose back-isomerization competes with the photochemical transformation towards this isomer.H owever, as the use of red light is of much more biological relevance,w ea lso evaluated the PSDs under l = 625 nm irradiation (Figure 2, red pie charts). To our delight, we observed distributions mostly exceeding 80 % cis,which is also consistent with negligible absorptivity of this isomer at wavelengths corresponding to red light. Again, lower values observed for compounds 1 and 10 can be explained by the substantial band overlap in this spectral region ( Figure 2). Altogether,the limitation that remains to be solved for tetraortho-chloro-azobenzenes,s imilarly to almost all available molecular photoswitches,i st he overall low red-light absorptivity of the trans form, which is one to two orders of magnitude lower than that of wavelengths corresponding to green light, leading to prolonged irradiation times [37] and sometimes compromising the PSD in cases where fast thermal back-isomerization of the metastable state is ac ompeting process (for example,i nt he case of compound 10).
In this context, the quantum yield of the forward isomerization becomes important, potentially determining the usefulness of red-light operation of aphotoswitch in abiological context. While in the studied series of molecules no general trends can be observed (f t!c = 38 AE 16 %) (Table 1), we note that quantum yields observed for compounds with strong electron-donating substituents (compounds 1 and 2, f t!c = 34-63 %) are somewhat higher than for those with electronwithdrawing groups (9,10, f t!c = 18-20 %), although additional studies are still needed to confirm this trend. In ab roader context of photochemical process efficiency, we also analysed the photoswitching cross section (Table 1) under green light irradiation (that is,t he product of the quantum yield f and molar attenuation coefficient e at l exc = 526 nm, the maximum emission of the green LED used here). In general, values in the useful order of magnitude (10 2 -10 3 ) were found, again with the strong EDG-substituted compounds 1 and 2 showing the highest efficiency.T he same trends are observed for irradiation with red light (l exc = 625 nm, Table 1), albeit with cross sections in the 10 0 -10 1 order of magnitude.
Ther everse (cis to trans)s witching was studied by irradiation with blue light (l exc = 426 nm, Table 1). We were delighted to see that for most of the compounds it was possible to recover > 80 %ofthe trans isomer (Figure 2, blue pie charts). This highlights the good dynamic range that can be achieved with tetra-ortho-chloro-azobenzenes 2-9,w hich can be switched between containing 82-90 % trans isomer under blue light irradiation and 83-90 % cis isomer under red light irradiation. Compound 10 features the best PSD under blue light (95 % trans), but its forward switching is less pronounced (see above). Strikingly,d ue to the overlap with as trong p-p*b and in the blue region of the spectrum, the behavior of compound 1 is essentially inverted, as it can be most efficiently switched in the forward direction with blue light (66 % cis isomer) and in the reverse direction with green light (78 % trans isomer). Thanks to quantum yields exceeding 30 %a nd strong absorptivities of all the studied compounds at l = 426 nm, the reverse switching is an efficient process,w ith cross sections in the 10 3 -10 4 order of magnitude. Figure 3. A) Representation of the DFT optimal geometries for the two stable isomers as well as inversion and rotation transition states, together with relative free energies in parenthesis (in kJ mol À1 )a nd key dihedral angles for 4;B )electron density difference plot for the lowest transitions in three selected trans compounds. The blue and red lobes indicate regions of decrease and increase of density upon excitation, respectively. Representation threshold 15 10 À4 au;C)same for the cis isomers;D)spin density difference for the rotationt ransitions tate of 4 as given by BS-DFT.R epresentation threshold 10 10 À3 au.
Them ain motivation behind the introduction of tetraortho-substituted azobenzenes has been the possibility to achieve visible-light-switching without compromising the half-life or the metastable isomer,w hich was the typical drawback of the more established azobenzene architectures substituted with both an electron-withdrawing and electrondonating substituent in the para positions (push-pull systems). [29] Indeed, our data (Table 1) for the tetra-orthochloro-azobenzenes confirm that for most of the studied para-substituents (compounds 2-9), the half-life of the cis isomer is between 4t o3 8d,w hich for all practical purposes translates to bistable systems in biological applications, meaning that the thermal cis-trans isomerisation can often be neglected for s p between À0.27 and 0.54. However, compounds with strongly electron-donating and -withdrawing groups (such as compounds 1 and 10)f eature much lower stability of the cis isomer,a ne ffect especially pronounced for compound 10,w here ah alf-life of about 1h was measured.
Thet hermal back-isomerization in an azobenzene can typically take place through an inversion or ar otation mechanism, and both have been found here through theoretical investigation ( Figure 3A;S upporting Information, Table S1). Forall investigated compound, the latter mechanism yields am ore stabilized transition state and rotation is therefore the most favored pathway.W en ote that this mechanism comes with ar upture of the p bond, and we therefore used broken-symmetry DFT to investigate it, which lead to the expected spin distribution ( Figure 3D). The theoretical back-isomerization barriers are listed in Table 1 and it can be seen that they are significantly smaller than their experimental counterpart, but that the trends are nicely reproduced. Indeed, excluding the compound substituted with aC F 3 group,w eo btain ad etermination coefficient, R 2 , between experiment [DG exp ]a nd theory [DG calc ]o f0 .82. Compound 9 proved to be most difficult for theoretical assessment. At this stage,i tm ight be interesting to take aspecific look at azobenzenes 1 and 10,asthey are substituted with the prototypical strong donor (NMe 2 )a nd acceptor (NO 2 )g roups.A sm ight be appreciated, both groups experimentally show quite small and similar t 1/2 .H owever,t he underlying reasons are different. Indeed, in compound 1,the cis form is essentially non-stabilized, with ar elative free energy of 31.4 kJ mol À1 as compared to the trans form, which is much higher than in the non-substituted case (26.3 kJ mol À1 ). The cis-to-trans barrier is small owing to this lack of stabilization (Supporting Information, Table S1). In contrast, for compound 10,t he relative free energy of cis as compared to the trans structure,2 6.3 kJ mol À1 ,i se ssentially unchanged from the unsubstituted case,b ut the rotational transition state is itself much more stabilized (Supporting Information, Table S1). Forthis reason, abalance needs to be found between the relative stabilities of the two isomers and the stabilization of the rotational TS itself.
Altogether,t he data presented herein enables the formulation of certain general rules for the design of tetra-orthochloro-azobenzene photoswitches for specific applications (Figure 4). In situations where long half-lives of the cis isomer are required, for example,w hen the effects of both isomers of ap hotopharmacological agent on ac ell line for longer time are studied, the use of substituents from the middle of the Hammet s p scale is recommended (such as those in compounds 4 and 6), as it provides the metastable state that persists for multiple weeks,s imilarly to those observed for hemiindigo photoswitches that also respond to red-light irradiation. [58,59] Conversely,w hen life-times on the scale of hours are desired, as in the case of photoswitchable antibiotics that are activated prior to administration and then should spontaneously lose their activity, [60] the strongly electron-withdrawing groups (for example,c ompound 10) are favored.
In photopharmacology,afew azobenzene-based bioactive molecules have been described in which the cis isomer shows potencyseveral orders of magnitude higher than trans. [55,56] In such cases,the photostationary state distribution that one can achieve is of less importance,aseven low concertation of the metastable state will result in localized activation. Here,t he use of tetra-ortho-chloro-azobenzenes with strong electrondonating groups (for example,compound 1)isrecommended, as it offers the most efficient activation with visible light. However,such systems are so far scarce,and most often, the difference in potency of the photoisomers is limited, which requires that high photostationary states are achieved. Especially in these cases,the intermediate substituents (such as compounds 3-8)s hould be considered.

Conclusion
We present herein as ystematic analysis of the photochromism of tetra-ortho-chloro-azobenzenes,a ne merging class of visible-light operated photoswitches with great potential for use in biological and material sciences.T heir versatility,u nderlined by the possibility to tune their photochemical properties towards the desired application, renders them ah ighly useful tool in as till limited repertoire of molecular photoswitches that respond to low energy green and red light. Detailed understanding of the influence that substituents play on key photochemical properties and thermal isomerization barriers,aspresented here,will enable successful design of functional, photoresponsive systems.I n al ong-term perspective,t hese insights provide am ajor step towards using light for the efficient regulation of biological processes with outstanding spatiotemporal precision.