Orthogonal photoswitching in a multifunctional molecular system

The wavelength-selective, reversible photocontrol over various molecular processes in parallel remains an unsolved challenge. Overlapping ultraviolet-visible spectra of frequently employed photoswitches have prevented the development of orthogonally responsive systems, analogous to those that rely on wavelength-selective cleavage of photo-removable protecting groups. Here we report the orthogonal and reversible control of two distinct types of photoswitches in one solution, that is, a donor–acceptor Stenhouse adduct (DASA) and an azobenzene. The control is achieved by using three different wavelengths of irradiation and a thermal relaxation process. The reported combination tolerates a broad variety of differently substituted photoswitches. The presented system is also extended to an intramolecular combination of photoresponsive units. A model application for an intramolecular combination of switches is presented, in which the DASA component acts as a phase-transfer tag, while the azobenzene moiety independently controls the binding to α-cyclodextrin.

L ight as an external stimulus has been extensively used in chemistry, biology and material sciences to control processes and properties in a non-invasive manner with high spatiotemporal precision [1][2][3][4][5][6][7] . However, as systems grow more complex, this control often needs to be extended to processes that act simultaneously, using multiple sources of light at different wavelenghts 8 in an orthogonal manner. When Bochet and co-workers 9,10 reported the proof-of-principle for the irreversible wavelength-selective removal of photolabile protecting groups (PPGs; Fig. 1a) in 2000, it paved the way for numerous impressive applications 8 . These include functionalized surfaces 11 , hydrogels 12 and lithography 13 , control of nucleic acids 14 , gene expression 15,16 , regulation of enzyme activity 17,18 and interfering with neuronal processes 19 . Despite the successful use of this concept, light-mediated uncaging is an inherently irreversible process 6 , which limits future applications towards responsive systems both in material and life sciences.
On the other hand, the field of molecular photoswitches exploits the reversibility of the photoswitching process 2,3,20-24 . However, the concept of addressing photoactive molecules in parallel with light of different wavelengths has not been translated from wavelength-selective uncaging of PPGs to independent photoswitching (Fig. 1b). Such orthogonal control has great potential for employment in various fields, as many of the abovementioned applications used for wavelength-selective uncaging would benefit from the reversibility of activation. Furthermore, reversible orthogonal control would enable independent modulation of different material properties and investigation of complex networks of signalling pathways in biological systems.
The wavelength selectivity (orthogonality) of the photoswitching is essential and the lack thereof currently represents a major limiting factor for successful applications (N.B.: throughout this article we refer to orthogonality as a characteristic of a system, where each component of the system can be controlled independently and irrespective of the order of addressing) 25 .
Although the idea of combining two different photoswitches in an intermolecular manner and addressing them independently may seem simple, its execution remains a major challenge 26 . Wavelength-selective control was so far confined to irreversible processes likely because of the fact that many of the most abundantly used photoswitches, such as azobenzenes, diarylethenes and spiropyrans, have overlapping absorption bands in the ultraviolet-visible region 2,8 . Thus, it is difficult to find spectral regions with large differences in photoswitching efficiency (defined, at a given wavelength, as a product of extinction coefficient, e and quantum yield, f). Moreover, the possibility of undesirable energy transfer between the chromophores 27 can lead to a major loss of selectivity. Herein, we present an orthogonal, intermolecular combination of two classes of photoswitches and their intramolecular combination as a first-generation orthogonally addressable, dual functional molecular system. The reported intermolecular combination of photoswitches is based on a donor-acceptor Stenhouse adduct (DASA, Fig. 1c) and an azobenzene (Fig. 1d). Both photoswitches can be independently controlled by irradiation with light of three different wavelengths and by taking advantage of a thermal isomerization process. The modular and experimentally robust combination allows for functionalization of each constituent. A model application for an   intramolecular combination of switches is presented, in which the DASA component acts as a phase-transfer tag, while the azobenzene moiety independently controls the binding to acyclodextrin (a-CD).

Results
Selecting a compatible photoswitch pair. Defining a photochromically compatible pair of photoswitches would constitute the first example of wavelength-selective/orthogonal control of multiple photoswitches in one solution. For this reason, we aimed to find compatible classes of photoswitches ( Fig. 1), especially those that would exhibit a high complementarity in the 300-800 nm spectral region where photoswitches normally operate (Fig. 1e). We chose azobenzene photoswitches as one class: they have been successfully applied in molecular photocontrol [20][21][22][23][24][28][29][30][31] , are easily accessible and allow for custom modification, functionalization and spectral fine-tuning. Azobenzenes have their main absorption bands located between 300 and 500 nm (p-p* and n-p* transitions; Fig. 1d,e and Table 1) 20,32 . We envisioned that the T-type 2 DASAs (DASA 1 and DASA 2, Fig. 1c) could be used as the complementary class 33,34 since they show very little absorption between 300 and 500 nm (Fig. 1c,e and Table 1) [35][36][37] . Owing to the fact that the reverse switching of DASAs is thermally driven 33,34,36,37 , three different wavelengths would be sufficient for wavelength-selective control. Importantly, for the design of orthogonal systems, one can exploit the tunability of thermal half-lives of the thermodynamically unstable states of the photoswitches in use. By changing the substitution pattern of azobenzenes, both very long and very short thermal half-lives have been attained. Increased thermal stability could be achieved, for example, through ortho-substitution 32,38-41 .
On the other hand, compounds with shortened thermal half-lives can be obtained by tuning of the electron density, through incorporation of both electron-donating groups (for example, -NMe 2 and -OMe), electron-withdrawing groups and combinations of both (push-pull systems) 42 . For the DASAs a clear relation between half-life and structure has not been determined yet 33,34 . However, it has been shown that changing the acceptor can influence the half-life to some extent 33,34 . Methylbarbituric acid-based cyclized DASAs ( . Finally, tuning half-lives of switches is often relatively independent of structural features required for function. This leaves ample space to tune the thermal relaxation rate of switches to magnitudes needed for different applications [20][21][22][23][24][28][29][30][31]43 . Intermolecular combination of photoswitches. Initially, we studied push-pull azobenzene 3 in combination with DASA 1 ( Fig. 1 and Table 1). The 4 0 -methoxy group in azobenzenes increases photostationary states 44 (PSS, defined as the percentage of the thermodynamically unstable isomer that can be produced upon irradiation at a given wavelength), while the ester moiety represents a handle for convenient functionalization. The absorption spectrum of the mixture of 1 and 3 is, to a large degree, a linear combination of the spectra of the separate photoswitches, indicating a low level of intermolecular energy transfer ( Supplementary Fig. 39) 26 . The combination of photoswitches 1 and 3 showed a remarkably high level of wavelength selectivity, apparent in ultraviolet-visible spectra and reversible photochromism plots (Fig. 2). Irradiation of azobenzene 3 led to a selective, nearly quantitative trans-cis isomerization (l ¼ 370 nm, irradiation 1, Fig. 2), which proceeds with a quantum yield of j ¼ 0.15. The relatively long half-life of the cis-isomer of azobenzene 3 (t 1/2 48 h; Table 2) makes it thermally stable on the timescale of the experiment (o1 h). Irradiation of the cis-3 isomer (l ¼ 430 nm, irradiation 2, Fig. 2) allowed selective cis-trans isomerization without affecting photoswitch 1. DASA 1 could be selectively cyclized by longer wavelength irradiation (l ¼ 590 nm, irradiation 3, Fig. 2), without affecting the azobenzene, after which it showed a short thermal half-life for cycloreversion (37 s at room temperature; Table 2). DASA 1 can also be switched selectively (irradiation 5, Fig. 2) without affecting the azobenzene 3 after it has been switched to the cis-isomer (irradiation 4, Fig. 2). By using a broad-spectrum visible light source (white light, Supplementary Table 1 In summary, in a mixture of photoswitches 1 and 3 ( Fig. 2), azobenzene 3 could be selectively switched from trans to cis (irradiation 1) and back from cis to trans (irradiation 2). The same applies for DASA photoswitch 1, which could be selectively cyclized (irradiation 3) and then relaxed back to its thermally stable state. It is important to note that full chromatic orthogonality was observed because both states of 1, as well as the thermal relaxation process, were not affected by light pulses that address 3 (irradiations 1, 2 and 4). Similarly, both states of 3 were not affected by light pulses addressing 1 (irradiations 3 and 5).
Structural scope of photoswitches. Subsequently, we investigated whether other azobenzenes could be used in a two-switch system with compound 1. Towards that end, azobenzenes 4-6 ( Fig. 3 and Table 2) were selected. These photochromes are not only structurally diverse, but they also exhibit very different functional characteristics: the l max of 4 is bathochromically shifted 20,38-42 , whereas l max of 5 and 6 are hypsochromically shifted ( Fig. 3 and Table 2) as compared with compound 3. Moreover, they differ markedly in the half-lives of their thermally unstable cis-isomers as compared with 3, with a shortened half-life for 4 20 We then investigated possibilities for structural diversity of DASAs that can be incorporated in an orthogonal switching system with azobenzenes. In principle, changing the acceptor part of this photochromic compound affects the absorption maximum and thermal half-life, while the donor moiety has little influence on l max , affecting only its solubility 33,34 . DASA 2 (Fig. 3), with a Meldrum's acid-derived acceptor moiety, shows a hypsochromically shifted absorption maximum ( Fig. 3 and Table 2) and its cyclized form exhibits a longer half-life (B130 s; Supplementary Fig. 8), when compared with that of DASA 1 (refs 33,34). We selected azobenzene 6 ( Fig. 3) to study the orthogonality of photoswitching in solution since it is compatible with DASA 2 because of its hypsochromically shifted p-p* absorption band (Fig. 3, Table 2 and Supplementary Fig. 22). Gratifyingly, both switches 2 and 6 can also be selectively addressed in parallel with light of different wavelengths ( Supplementary Figs 49 and 50). The flexibility with regard to the DASA photoswitch highlights the robustness and modularity of the photoswitch combination and bodes well for versatile use in different applications.
Orthogonal photoswitching in an intramolecular system. Apparently, loss of selectivity through energy transfer did not pose a problem for the intermolecular system described thus far, even at high concentrations up to B80 mM (1) and B400 mM (3; Supplementary Figs 51 -53). Therefore, the reversible photocontrol in the intermolecular case is purely dependent on the spectral properties of the two photoswitches involved.
For an intramolecular combination, on the other hand, linker design, geometry and distance of the photochromes become more important 26 . While intramolecular energy transfer between chromophores can drastically reduce the switching selectivity 8,26,27 , it can be partially controlled by adjusting the lifetime of the photo-excited state and/or by changing the design of the linker 8,26,27 . Furthermore, properties that arise from energy transfer and the interplay of the combination of two photoswitches can sometimes be desired. This is the case, for example, in molecular logics 26,[45][46][47][48] , where such hybrid multiswitches (or multiphotochromes) allow (sequential) access to multiple different states 45,46 , usually in combination with external stimuli other than light 49-56 . To probe whether the level of selectivity of the intermolecular combination could be retained in the intramolecular system, or whether new properties could be observed, we combined an azobenzene and a DASA photoswitch into a single molecule. Towards that end, compounds 7 and 8, which differ only in the length of the linker, were synthesized (Fig. 4) and the selectivity of their photoresponsiveness was evaluated (Fig. 5).
Importantly, the selective switching of the azobenzene moiety is retained. However, irradiation with l ¼ 590 nm not only switched the DASA moiety but also affected, to some extent, the azobenzene part in both 7 and 8 (Fig. 5). Generally, improved selectivities were observed with the elongated linker unit (from -(CH 2 ) 4 -to -(CH 2 ) 12 -). In compound 7 (shorter linker), irradiation at l ¼ 370 nm (Fig. 5a) does partially affect the DASA moiety, which is not observed for compound 8 (longer linker). Interestingly, compound 8 shows a slight decrease in addressability of exclusively the DASA photoswitch with l ¼ 590 nm, but not with white light (Fig. 5b). No such effect is observed for the compound with shorter linker (compound 7). As a control experiment, the intermolecular switching of the corresponding azobenzenes in combination with the DASA photoswitch 1 was studied and orthogonality was observed ( Supplementary Figs 54-58). Modulation of phase transfer and supramolecular interactions. We proceeded to apply this intramolecular system for controlling the location and the function of the molecule. Previously reported extraction experiments of DASA molecules 33,34 established the potential of DASAs as a phase-transfer tag 34,37 . Reversible photoswitching of DASAs is only observed in aromatic solvents. Polar protic solvents (as, for example, methanol or water) stabilize DASA's zwitterionic cyclized state, whereas halogenated solvents favour the elongated triene structure. Photoswitching results in formation of the hydrophilic zwitterionic cyclized form and phase transfer to the aqueous layer 34 . We envisioned exploiting this behaviour to transport photoswitchable cargo that is, an azobenzene. On an independent level of photocontrol, the isomerization of the azobenzene could be used to manipulate a certain function, for example, the well-known host-guest binding to CDs (especially a-CD) [57][58][59][60] . Therefore, we set off to evaluate a model system with dual functionality (Fig. 6) in which the DASA moiety of 8 controls the phase where the molecule resides (location), whereas the azobenzene moiety controls the supramolecular binding to a-CD (function). In toluene, the azobenzene moiety of compound 8 could be switched selectively between trans-and cis-forms (Fig. 5b). Upon cyclization of the  Towards that end, we established the phase-transfer behaviour of DASA in the intramolecular system 8. Inspired by traditional logP I (oct/wat) measurements 61 , a functional phase-transfer system was designed to operate at pH 9 (using a saturated aqueous NaHCO 3 solution to adjust the pH Fig. 7). The extraction experiment described in Fig. 7 establishes the photoswitching and light-controlled phase-transfer behaviour of compound 8. Compound 8 is dissolved in toluene and the organic layer is underlayed with water (pH 9). Upon irradiation of the biphasic mixture (stage I, Fig. 7) with l ¼ 365 nm, the azobenzene part of 8 is selectively switched, as can be observed by ultraviolet-visible spectroscopy (stage II, Fig. 7). Subsequent irradiation with white light results in a successful phase transfer of 8 to the aqueous layer (stage III, Fig. 7). Separation of the biphasic mixture and addition of dichloromethane to the aqueous phase results in back-extraction and recolouration of the organic solution (stage IV, Fig. 7), taking advantage of the short half-life of the DASA 1-type moiety. Importantly, the geometry of the azobenzene moiety can be controlled without affecting the DASA moiety in the organic phase (Fig. 7). Isolation of phase-transferred compound 8 and photoswitching in water (pH Z9, K 2 CO 3 ) established reversibility with negligible fatigue Having established the light-induced phase transfer of compound 8 through DASA cyclization (light-control of location), we studied the possibility of controlling host-guest binding of compound 8 in water (light control of function). CDs are well known as host molecules with a multitude of studied guest molecules 57,58,60 . a-CD binds trans-azobenzenes but not cisazobenzenes 59,62 .
Compound 8 was isolated after phase transfer and was then subjected to binding studies with a-CD in water (pH Z9; ref. 57). A clear change in the absorption spectrum upon titration of the host was observed (Supplementary Fig. 63) for the thermally adapted compound 8, indicating the binding of trans-azobenzene moiety of 8 to a-CD. For irradiated samples, this effect was markedly reduced (Supplementary Figs 64-67), suggesting a much weaker binding of the cis-azobenzene moiety of 8 to a-CD. Photoswitching is retained even in the bound state, where interestingly different PSS values are observed ( Supplementary  Figs 68-70). Thus, by switching the azobenzene moiety, the affinity for the a-CD host can be tuned (Fig. 8).
Overall, the azobenzene moiety of compound 8 can be independently controlled by light in the organic phase with irradiation at l ¼ 365 nm (trans-cis isomerization; Figs 5b and 7) and l ¼ 430 nm (cis-trans isomerization; Fig. 5b). Upon switching of the DASA moiety with white light, compound 8 undergoes phase transfer to the basic aqueous layer. Herein, the transazobenzene moiety binds to a-CD more strongly than the cisazobenzene one. Compound 8 can be recovered from the aqueous layer by back-extraction to dichloromethane. In summary, compound 8 incorporates the two independently photocontrolled, functionally different capabilities for phase transfer, induced by DASA cyclization and host-guest binding, dependent on the geometry of the azobenzene moiety.

Discussion
In conclusion, an intermolecular two-switch system consisting of a DASA and an azobenzene was developed by a rational, spectra-guided design. The robustness of the photoswitch combination is illustrated by the compatibility of differently substituted azobenzenes, as well as DASAs. The flexibility in functional group substitution emphasizes the generality and modularity of this approach and may potentially also allow combination with other classes of photoswitches or PPGs. Furthermore, control of thermal relaxation allows specific tailoring of the switch to the needs of the system. Recent non-orthogonal applications of independent switches in the same solution highlight the great potential of our approach [63][64][65][66][67][68] . The intramolecular photoswitching extends the applicability of this combination, which was exemplified by a dual functional molecule where both the binding to a-CD and phase transfer can be controlled with different wavelengths of light. The intramolecular combination shows interesting switching behaviour dependent on the linker length-an aspect that will be studied in-depth in the future. The presented study show levels of orthogonal selectivity that are unparalleled both for wavelength-selective uncaging 8 and multiphotochromes 26 . It represents a major step towards the 0.7 Thermally adapted 1.2 equiv. α-CD 2.5 equiv. α-CD 8.7 equiv. α-CD 11.2 equiv. α-CD 12.4 equiv. α-CD 18.7 equiv. α-CD 24.9 equiv. α-CD 31.1 equiv. α-CD Thermally adapted 365 nm PSS 0.9 equiv. α-CD 1.8 equiv. α-CD 3.5 equiv. α-CD 6.9 equiv. α-CD 13.5 equiv. α-CD 19.9 equiv. α-CD 26.1 equiv. α-CD 32.0 equiv. α-CD 37.8 equiv. development of future orthogonal and reversible photoswitchable tools that can be used for non-invasively interfering with and the manipulation of multifunctional molecular systems. Future research should focus on overcoming the strong solvent dependence of the photoswitching of the DASAs and orthogonal switching in the near infrared region of the spectrum [38][39][40][41] . Determination of quantum yields. The quantum yield was determined by following initial photoswitching kinetics (percentage of switching B10%) as decrease in absorbance at l max and using the light intensity determined from standard ferrioxalate actinometry 69 . A more detailed description can be found in Supplementary Methods.

Methods
Photoswitching experiments. Each mixture of compounds was analysed by measuring ultraviolet-visible spectra of the different accessible states and by kinetic measurement of the time-dependent reversible photochromism. Henceforth, each photoswitch was followed at its l max . Importantly, samples were irradiated during measurements at an B90°angle to the light path of the ultraviolet-visible spectrometer with distances from light source and their respective intensities at this distance detailed in Supplementary Tables 1 and 2 Data availability. The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information Files.