Switchable protection and exposure of a sensitive squaraine dye within a redox active rotaxane

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Switchable protection and exposure of a sensitive squaraine dye within a redox active rotaxane © The Author(s) 2024 Published version Wasternack, Janos; Schröder, Hendrik V.; Witte, J. Felix; Ilisson, Mihkel; Hupatz, Henrik; Hille, Julian F.; Gaedke, Marius; Valkonen, Arto M.; Sobottka, Sebastian; Krappe, Alexander; Schubert, Mario; Paulus, Beate; Rissanen, Kari; Sarkar, Biprajit; Eigler, Siegfried; Resch-Genger, Ute; Schalley, Christoph A. Wasternack, J., Schröder, H. V., Witte, J. F., Ilisson, M., Hupatz, H., Hille, J. F., Gaedke, M., Valkonen, A. M., Sobottka, S., Krappe, A., Schubert, M., Paulus, B., Rissanen, K., Sarkar, B., Eigler, S., Resch-Genger, U., & Schalley, C. A. (2024). Switchable protection and exposure of a sensitive squaraine dye within a redox active rotaxane. Communications Chemistry, 7, Article 229. https://doi.org/10.1038/s42004-024-01312-1 2024 communications chemistry Article https://doi.org/10.1038/s42004-024-01312-1 Switchable protection and exposure of a sensitive squaraine dye within a redox active rotaxane Check for updates Janos Wasternack 1, Hendrik V. Schröder 1, J. Felix Witte 2,MihkelIlisson 1, Henrik Hupatz 1, Julian F. Hille1, Marius Gaedke1, Arto M. Valkonen 3, Sebastian Sobottka4, Alexander Krappe 5, Mario Schubert 6, Beate Paulus2, Kari Rissanen 3, Biprajit Sarkar4,7, Siegfried Eigler 5, Ute Resch-Genger 8& Christoph A. Schalley 1 In nature, molecular environments in proteins can sterically protect and stabilize reactive species such as organic radicals through non-covalent interactions. Here, we report a near-infrared fluorescent rotaxane in which the stabilization of a chemically labile squaraine fluorophore by the coordination of a tetralactam macrocycle can be controlled chemically and electrochemically. The rotaxane can be switched between two co-conformations in which the wheel either stabilizes or exposes the fluorophore. Coordination by the wheel affects the squaraine’s stability across four redox states and renders the radical anion significantly more stable—by a factor of 6.7—than without protection by a mechanically bonded wheel. Furthermore, the fluorescence properties can be tuned by the redox reactions in a stepwise manner. Mechanically interlocked molecules provide an excellent scaffold to stabilize and selectively expose reactive species in a co-conformational switching process controlled by external stimuli. From RNA enclosed in capsid shells1,2to reactive molecular centers and activesites3burieddeepwithinshieldingproteins, confinementinprotective environments is a fundamental strategy in nature to mitigate undesired side reactions of labile species4,5.Inspecific molecular environments within proteins, for instance, reactive moleculessuchasorganicchromophores, radicals, or ions are not only sterically shielded, but are also stabilized through non-covalent interactions. Prime examples are β-barrels hosting labile chromophores in fluorescent proteins6or organic radical ions stabilized through hydrogen bonding in flavoenzymes7and photosynthetic reaction centers8. It remains a formidable challenge for synthetic chemists to stabilize reactive species (e.g., organic radicals) without interfering with the molecules’functions to enable applications in biomedical imaging9, sensing10,or proximity labeling11. In contrast to classical covalent protecting group strategies, encapsulation by a receptor offers a supramolecular approach for the stabilization of reactive guests by non-covalent interactions (Fig. 1a). A host molecule, such as a macrocycle, confines a reactive guest and acts as a dynamically bound ‘supramolecular protecting group’. The protection strength is determined by energetic stabilization upon binding inside the host and steric shielding. The abundance of free, unprotected guest is significantly decreased upon binding inside a confined space12–16. An alternative approachto increase stability involves the incorporation of reactive sites into mechanically interlocked molecules (MIMs) as seen for synthetic17–21 and natural22–26 examples like the lasso peptide microcin J2527 (Fig. 1b). An advantage compared to a non-interlocked complex is that the mechanical bond prevents dissociation into subcomponents and thus conformationally locks the stabilizing structure. For example, some mechanically interlocked peptides display mechanical bonds with a low degree of conformational freedom, equipping them with an unusually high physicochemical stability compared to their non-interlocked counterparts22,27. A synthetic example was reported by SMITH et al., who appliedprotectionwithinMIMsforsensitivesquaraine dyes. Squaraine (Sq) dyes are important near infrared (NIR) fluorescent dyes with broad application in bioimaging28–31. NIR dyes offer low absorption interference by biological tissues, deeper tissue penetration, reduced scattering, and among them, Sq are of particular interest due to their highly intense NIR absorption 1Institut für Chemie und Biochemie, Freie Universität Berlin, Arnimallee 20, 14195 Berlin, Germany. 2Institut für Chemie und Biochemie, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany. 3University of Jyvaskyla, Department of Chemistry, Survontie 9 B, 40014 Jyväskylä, Finland. 4Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 34-36, 14195 Berlin, Germany. 5Institut für Chemie und Biochemie, Freie Universität Berlin, Altensteinstraße 23A, 14195 Berlin, Germany. 6Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany. 7Institut für Anorganische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany. 8Bundesanstalt für Materialforschung und -prüfung (BAM), Biophotonics, Richard Willstätter Straße 11, 12489 Berlin, Germany. e-mail: [email protected] Communications Chemistry | (2024) 7:229 1 1234567890():,; 1234567890():,; and emission bands31–33. However, Sq suffer from their vulnerability to nucleophilic attack at the dye’s electrophilic C 4 O 2 core, resulting in color fading28. Their enclosure by HUNTER-VÖGTLE type tetralactam macrocycles encircling the Sq core through NH ∙∙∙O hydrogen bonds is a useful means of protection against nucleophilic attack28. A particularly interesting aspect of MIMs is that co-conformational flexibility can be intentionally designed into a molecular structure, enabling controlled large-amplitude motions of their subcomponents. This property has made MIMs preferred scaffolds for the construction of artificial molecular switches and machines34–38 mimicking dynamic conformational changesfound in biological nanomachines. In SMITH’SSqrotaxanes, addition of chloride salts leads to the displacement of the macrocycle from the Sq station, thus switching off the protection28–30. To make such switchable protection multi-stimuli responsive is the aim of this work. Besides switching by chloride addition and removal, we demonstrate that electrochemical switching can also be realized in this Sq rotaxane. We show that the energetic stabilization and steric shielding of the reactive Sq fluorophore within a conformationally dynamic MIM can be altered by electrochemical and chemical stimuli. These external stimuli either increase the stabilization by the wheel or induce a co-conformational change, resulting in the controllableexposureofthisreactivesite(Fig.1c). Furthermore, the rotaxane exists in four oxidation states, which can be utilized for tuning of the optoelectronic properties of those dyes. Results and discussion Preparation and structure of Rot [2]Rotaxane Rot comprises a HUNTER-VÖGTLE type tetralactam macrocycle TLM and a threaded and mechanically interlocked bis(aminothienyl) squaraine axle. This macrocycle was chosen because the pyridyl group facilitates binding and preorganisation during the synthesis of Rot and also improves solubility of the macrocycle39. Wheel TLM and axle Ax form a threaded complex, pseudo[2]rotaxane psRot in dichloromethane with an association constant of K a =520±160M -1 at 293 K as determined by 1H NMR titration (SI section S2Fig. S17). Slow evaporation of dichloromethane solutionsofAx and ofpsRot yieldedsinglecrystalssuitableforXRDanalysis. The 1:1 complex stoichiometry was confirmed by solid-state XRD structures of the psRot and Ax (Figs. 2and 4g). The structure of psRot is similar to reported Sq tetralactam rotaxanes (SI section S4 Fig. S25)28,30,40.TheSq bound by four NH ∙∙∙O hydrogen bonds (2.21 Å) and the bond lengths within the neutral Sq within psRot are very close to the ones in free Ax.Rot was obtained in 32% yield from the solution of psRot in an end-capping approach via copper-catalyzed azide-alkyne HUISGEN cycloaddition of a sterically demanding trityl stopper (Figs. 3band8).Themoststablecoconformation SqRot0involvesthewheelencirclingthesquarainecoreby forming NH ∙∙∙ O hydrogen bonds. Chloride switching of neutral Rot Addition of chloride salts to a dichloromethane solution of Rot results in a significantly increased fluorescence intensity, likely caused by shuttling of the wheel to the vicinityof the triazol group, thereby exposing the squaraine core (Figs. S21–S23). This shuttling behavior has been observed in similar tetralactam rotaxanes28,40.Thefluorescencequantumyieldsaswellasthe maxima of absorption and emission of the fluorescent states of Ax and Rot were measured (SI section S3, Table S1). Ax0displays fluorescence with an emission maximum of λ max (Ax0) = 678 nm and an absolutely measured fluorescence quantum yield of Φ(Ax0) = 6.7% and Rot0displays Fig. 1 | Different protecting strategies. a Encapsulation of a labile guest by a host molecule results in an inclusion complex in which the guest is sterically shielded from unwanted side reactions with a compound X. bLasso peptide microcin J2527 as example of drastically increased physicochemical stability by mechanical bonding. cElectrochemical switching of protection by a macrocycle encircling a labile squaraine dye in a [2]rotaxane. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 2 fluorescence with an emission maximum of λ max (Rot0)=683nm and a quantumyieldofΦ(Rot0) = 4.0%.ThelowerquantumyieldofRot0couldbe attributed to lower absorption of the shielded Sq or partial absorption of the emitted light by the wheel. Addition of 50 equiv. Bu 4 NCl to a solution of Ax0leads to a decreased quantum yield of Φ(Ax0⊃Cl− ) = 5.9%, while under the same conditions the quantum yield of Rot0increases to Φ(Rot0⊃Cl− ) = 4.2%. The increased fluorescence intensity in TriazRot0⊃Cl− thus likely results from increased absorbance and reduced quenching by the wheel of the deshielded squaraine. The observed properties and the chloride induced translocation of the wheel are in good alignment with similar systems28,40. The chloride affinity of Rot0was determined by 1HNMR (K a =84±12M -1)andfluorescence titrations (K a =90±20M -1) using n- Bu 4 NCl (Figs. S19–S22). Figure 3shows the 1H NMR spectra of Rot before and after addition of 59 equiv. n-Bu 4 NCl. Binding of chloride induces the co-conformational change to TriazRot0⊃Cl−in which the wheel TLM binds a chloride ion and the triazole station, as indicated by characteristic shifts of the NH signals ε:Δδ(ε)=0.2ppmandο:Δδ(ο)=−1.7 ppm, and the aromaticsignalsχ:Δδ(χ) = 0.3 ppmand σ:Δδ(σ) = 0.2 ppm.Abindingmodeof chloride in similar tetralactam - triazol rotaxanes through CH ∙∙∙Cl− ∙∙∙HN hydrogen bonds between the triazole’s CH group and the NH groups of the tetralactam has been reported41,42. Quantum chemical investigation of TriazRot0⊃Cl−by MD simulations usingCREST, indicatehydrogenbonding between theamideNH protons of the isophthalamide and chloride, while the pyridyl bis amide participates in hydrogen bonding towards the triazol group. Weak hydrogen bonds between the triazol proton m and chloride are indicated by the comparatively low shift of m: Δδ(m) = 0.1 ppm. This is in line with the calculated binding motif in which the triazol is tilted away from the bound chloride anion (SI section S9 Fig. S42). The presence of only one set of broadened signals suggests that translational exchange of the wheel between the two triazoles is faster than the NMR time scale. Precipitation of the chloride by addition of Na[B(Ph-3,5-(CF 3 ) 2 ) 4 ] leads to back shuttling of the wheel onto the Sq. Reversibility of the shuttling was again confirmed by fluorescence spectroscopy (Fig. S23). Redox switching of Rot We hypothesized that a similar switching between co-conformations might be realized by electrochemistry. LEIGH and co-workers reported an electrochemically switchable hydrogen-bonded molecular shuttle43. We recently developed a series of tetrathiafulvalene rotaxanes, which are electrochemically switchable between different co-conformations44–53.(Quasi) reversible redox reactions have been reported for some Sq derivatives54. Cyclic voltammetry in 1,2-dichloroethane (Fig. 4bandTableS2) reveals that Ax0undergoes an irreversible one-electron reduction to the radical anion Ax●−(E pc -1 =−0.99 V against the decamethylferrocene/ decamethylferrocenium couple) as well as two reversible one-electron oxidations, to the radical cation Ax●+(E 1/2 1=0.45V)anddicationAx2+ (E 1/2 2= 0.95 V). These three redox reactions are most likely localized on the thiophene-squaraine π-system54. Scan rate-dependent plots of the anodic peak potential (SI section S5 Figs. S26 and S29) indicate that the reduction of the axle (Ax0→Ax●− )is followed by an irreversible chemical reaction step in a so-called EC i mechanism. For Sq●− , Quantum chemical calculations at various levels of density functional theory (DFT) (SI section S9) indicate elongated O-C1- bonds and an increase of electron density on the oxygen atoms (Fig. 4aand h, Table S4). In Rot●− , both effects result in stabilization of the NH ∙∙∙O hydrogen bonds between Sq●−and wheel by 42 kJ/mol (Table S7). In addition to the electronic stabilization of the wheel–Sq●−interaction, the steric shielding of Sq●−by TLM contributes to an observed increase in lifetime.Incontrastto the (quasi)irreversible reduction of Ax0,thereduction of Rot0is reversible on the CV timescale (scan rate of 100 mV/s), further supporting the significant stabilization of the radical anion Sq●−by rotaxanation. The lifetime of the radical anion Rot●−was investigated by CV- based digital simulations (SI section S5 Fig. S29). Fitting the experimental voltammograms at different scan rates according to an EC i mechanism yields an average radical lifetime of 1.7 s (Ax●− ) and 10.8 s (Rot●− ). However, the anodic peak (E pa )ofRot was overlayed by incipient solvent/ electrolyte decomposition. Thus, the life-time ratio τ Rot /τ Ax > 6.7 ± 2.8 is considered to be a lower boundary of the stabilization effect caused by rotaxanation. Organic radicals are typically highly reactive species with short lifetimes55. They were categorised by INGOLD according to their lifetimes as transient radicals (lifetimes < 1.44 ms) and persistent radicals (lifetimes > 1.44 ms)56,57. For example the benzyl radical is transient with a lifetime below 1 ms57,58. The drastic impact of thermodynamic and steric factors on radical stability are illustrated by the existence of bench stable radicals like TEMPO. Supramolecular encapsulation is a common means of stabilisation of transient and persistent radicals4,43,55,59–64.Aprominent example being the drastic stabilisation of a persistent tetrazine radical anion with a lifetime of 2 h through encapsulation by two cyanostar macrocycles leading to a complex with a 360-fold lifetime of 30 d64.Ourcasethus represents the stabilisation of a more reactive persistent radical. In contrast tothe strengthening of the wheel–axle interaction in Rot by the reduction, oxidation reduces the electron density of Sq as indicated by calculated electrostatic potential surface maps (ESPs), resulting in a weakened wheel–axle interaction in Rot●+and Rot2+(Fig. 4a and d and S43, Tables S4 and S7). The one-electron oxidation potential of Rot0is indeed anodically shifted by ΔE 1/2 1=+90 mV compared to free Ax0, indicating that the Sq–wheel interaction energetically hampers the first oxidation (Fig. 4e, f). We investigated the co-conformational equilibrium between SqRot and TriazRot in four oxidation states by exploring the co-conformational landscapes using the CREST code65,66 by GRIMME and subsequent evaluation of energetic stabilities employing various density functional approximations (Fig. 5and Table S7). Whereas the neutral Sq station is energetically favored over the triazole station by 29 kJ/mol, the first oxidation renders the triazole station slightly more favorable than the Sq●+station by 3.5 kJ/mol. Hence, one-electron oxidation results in molecular shuttling (SqRot0→TriazRot●+) Fig. 2 | Single Crystal XRD structure of psRot with selection of bond lengths (thermal displacement parameters at 50% probability level). The perspective is indicated by the point of view icon next to the schematic depiction. The macrocycle is positionally disordered in the crystal and the pyridine dicarboxylic amide and isophthalic diamide can replace each other in their positions. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 3 and a co-conformational distribution in which the wheel preferably adopts a distal position to Sq (Fig. 5). Gradual oxidation of Rot0in an electron paramagnetic resonance (EPR) spectroelectrochemical experiment (dichloromethane, 298 K) gave anisotropic signalat g= 2.003 (Fig. 4c) confirmingits transition into a stable radical cation Sq●+. A low temperature spectrum at 223 K reveals the underlying hyperfine structureof the signal partially. The best fit,which is in excellent agreement with the experimental spectrum, was obtained with a simulationusingag-valueof 2.004 and hyperfinecoupling totwoequivalent 14N nuclei and three sets of equivalent 1H nuclei totaling 14 hydrogen nuclei Fig. 3 | Structure of rotaxane Rot and chloride-induced shuttling. a Equilibrium between Ax,TLM and psRot & schematic depictions of these species. bMolecular structure of rotaxane Rot with schematic depiction. cPartial 1H NMR spectrum of SqRot0.dPartial 1H NMR spectrum of TriazRot0⊃Cl−after addition of 59 equiv. n-Bu 4 NCl. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 4 Fig. 4 | Opto-electrochemical investigation of the electronic and co-conformational properties of Ax and Rot. a Electrostatic potential surface maps of Ax in four different oxidation states. bCyclic voltammograms (100 mV/s) of Ax and Rot. The second scan cycle is shown. The additional peaks at approximately −0.1 V in the second scan cycle, the i pa /i pc ratio, and the non-linear behavior of i pa at different scan rates (inset) indicate an EC i mechanism for the one-electron reduction of Ax. cEPR spectra of Ax•+and Rot•+(d) Spin density plots the radical species Ax•+, isovalue: 0.02 a.u. eDifferential pulse voltammograms (10 mV/s scan rate, 25 mV modulation amplitude, 50 ms modulation time, 5 mV step potential, 0.5 s interval time) of Ax, Rot, and Rot after addition of excess n-Bu 4 NCl. The second oxidation reaction of the rotaxane in presence of chloride appears irreversible in both the DPV and CV. fEnergy diagram, illustrating the stabilizing and destabilizing effects of the macrocycle and the presence of chloride on the first oxidation potentials of the Sq in Ax &Rot. Energies were calculated from (a) redox potentials: ΔG=n∙F∙E,( b) binding constants: ΔG=−R∙T∙ln(K a ) and (c) according to HESS’law ΔGc=ΣΔGa&b. gThe SCXRD-structure of Ax (thermal displace parameters at 50% probability level). hAnnotation of discussed bond lengths (from DFT calculations) and rotational barriers within the bis(aminothienyl)squaraine core. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 5 (four 1Hthienyl,two14Namine,eight1HN-C 1H 2 ) (Table S3). Supported by a spin density plot derived from DFT calculations, the experimental data indicate that the radical in Sq●+is delocalized over the thiophene-Sq system (Fig.4dand Fig. S43). The low temperature EPR spectrum of singly oxidized Rot●+shows very similar spectral features indicating virtually no electronic effect of the wheel on the electronic coupling of the axle-centered radical cation. Similar findings have been observed for other hydrogen-bonded radicals4and could result from weakened wheel–Sq interactions in Rot●+ and/or subsequent co-conformational changes as indicated by the electrochemical experiments. The second one-electron oxidation (Rot●+/Rot2+)occursatasimilar potential compared to that of the free Ax●+(ΔE 1/2 2=+10 mV), indicating that hydrogen bonding between Sq and the wheel in Rot●+is significantly weakened compared to the neutral state (Fig. 4a, e, and f and 5). Consequently, the second oxidation of the deshielded Sq energetically resembles thefreeaxlemorethanthefirst oxidation, as no further conformational equilibria contribute to the stabilization or destabilization of the system. Further evidence was derived from differential pulse voltammetry utilizing Rot0,bothintheabsenceandpresenceofoneequivalentn-Bu 4 NCl(Fig. 4e). As the chloride-binding wheel in complex TriazRot0⊃Cl−adopts a distal position to Sq, the first oxidation reaction displays a potential (E 1/2 1= 0.43 V) similar to free Ax0. This illustrates that the increased oxidation potential in Rot0is indicative of the wheels position: The first oxidation potential of Rot0is higher than that of free Ax0, because the stabilization energy in Rot0arising from rotaxanation and the intramolecular wheel–Sq hydrogen bonds, needs to be compensated upon oxidation. Asthe chloride-addition alreadyleadsto a co-conformationTriazRot0⊃Cl−in which the Sq is deshielded, the binding energy of the macrocycle is compensatedfor by the interactionbetweenthe wheel, triazole andchloride. The subsequent oxidation of the deshielded Sq thus requires a similar potential as the free axle. Further, the slightly lower oxidation potential of Rot0in the presence of chloride suggests a stabilization of the oxidized Rot●+⊃Cl−by the strongly coordinating chloride anion67. Further oxidation to Rot2+⊃Cl− is irreversible, likely caused by a nucleophilic attack at the now sterically deprotected Sq core28. Theoretical calculations show doubly oxidized Rot2+to have a pronounced energetic bias towards the triazole station of 18 kJ/mol (Fig. 5and Table S7). The reduced electron density and thus, significantly lower H-bond accepting capability of Sq2+is illustrated by electrostatic potential surface maps of the stoppered axle as found within Rot. The negative charge accumulation at Sq within Rot●−and Rot0and conversely, the lack of negative charge at Sq in Rot●+and Rot2+are depicted in Fig. S43. Thus, the H-bondaccepting ability of Sq decreasesdrastically from Sq●−toSq2+,while more negative charge is retained at the triazole groups in the oxidized states. In accordance with the electrochemical and quantum chemical data the interactionbetween Sq and thewheelarealready weak enoughin Rot●+that the wheel adopts a position close to the triazole motif. Quantum chemical investigation of this binding motif suggests H-bonding between two NH protons of the wheel and the electron rich N atoms of the triazol unit (Fig. S42). To investigate the oxidation induced shuttling (SqRot0→TriazRot2+)by 1D and 2D NMR experiments, we developed a reliable method to obtain chemically stable solutions of Ax2+and Rot2+. Initial attempts using NOPF 6 as oxidant in dichloromethane-d 2 resulted in partial precipitation. In acetonitrile, the resulting dye solutions decompose within minutes. The solubility and lifetime in dichloromethane-d 2 of the Sq2+species could be drastically increased (time scale of days) by addition of the weakly coordinating anion [Al(O-C(CF 3 ) 3 ) 4 ]−([pf]− ) as Li[pf] during the oxidation with NOPF 6 .Rot2+did not display a stabilizing effect compared to the dicationic Ax2+, as expected for the Sq being exposed in this state (see below for stability study). Oxidation reactions for both, Ax and Rot, are reversible, as demonstrated by using tin powder as reductant in an UV-Vis titration (SI section S7 Fig. S34). The 1H NMR spectrum of Ax2+displays multiple sets of signals which can be attributed to rotational isomers of Ax2+(Supporting Information Section S1, S6 and S7). Quantum chemical calculations reveal a four-fold increase of rotational barriers in Ax2+compared to Ax0(SI section S6 Table S4 and section S9 Table S5). At room temperature these rotations are free in Ax0andhinderedinAx2+leading to the observed spectral behavior. The much lower rotational barriers of Ax0and Rot0cause decoalescence of most signals only below −30 °C (SI section S8). Based on the calculated barriers for Ax0, the rotation around the bonds between the Sq and the thiophene units should be frozen first, due to their higher barrier. NOE cross peaks between proton signals of one of triazoles and methyl groups of the macrocycle indicate the wheel to be located at the triazole in Rot2+(Figs. S6–S10). Multiple sets of signals and extensive peak broadening Fig. 5 | Structural investigation of the most stable co-conformational space of Rot. a Calculated most stable co-conformations of Rot in four different oxidation states with schematic depictions of each state. bCo-conformational electrochemical and chemical equilibrium between six states of Rot. Only the most stable coconformations of each state are depicted. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 6 in the 1D and 2D NMR spectra reveal a slow shuttling motion of the wheel between the two triazoles.Additionally, the non-bound side of the axlelikely displays rotational isomerism like Ax2+. The stability of both oxidized states, Rot●+and Rot2+,onalaboratory time scale allowed us to study their optical properties (Fig. 6and S34 and SI section S3). The first oxidation of Rot0is accompanied by a hypsochromic shift of the most intense absorbance band, Rot0(λ max = 665 nm) to Rot●+ (λ max = 601 nm). The strongest band is further shifted by the second oxidation from Rot●+(λ max = 601 nm) to Rot2+(λ max = 509 nm). The emission band of Rot0(λ max = 683 nm, Φ(Rot0) = 4.0%) is close to the bands of similar reported bis-(aminothienyl)squaraine rotaxanes68. Due to the disproportionation equilibrium from Rot•+into Rot0and Rot2+,strong emission bands of the neutral and doubly charged species limit the detection of potential fluorescence of the radical cation. Dicationic Rot2+ (λ max = 570 nm, Φ(Rot2+) = 0.6%) shows weak fluorescence, a photograph of the luminescence is depicted as inset in Fig. 6.InlinewiththeTriazRot2+ co-conformation, the quantum yield is almost equal to that of Ax2+ (λ max = 548 nm, Φ(Ax2+) = 0.5%) (SI section S3 Fig. S24 and Table S1). The change of absorption properties was supported by time-dependent DFT calculations, suggesting that charge-transfer effects upon excitation play a role in Ax0and Ax●+. The difference density of the bright state displays a charge transfer from Sq to the neighboring thiophene moieties (Table S6). To further elucidate the protection and deprotection in the neutral and +2 oxidation states, bleaching of the squaraine core in Ax0,Rot0,Ax2+and Rot2+byattackofnucleophiles wasinvestigated using UV-Vis spectroscopy (SI section S10 Fig. 43). Tetramethylammonium hydroxide in dichloromethane proved effective in bleaching Ax2+and Rot2+,whileRot0was unaffected (Fig. S44b–d). While the half-life of Ax2+(≈10 min) and Rot2+ (≈5 min) under these conditions is comparable, Rot0was stable (Fig. S44a). Because the squaraine core in the neutral rotaxane is shielded by the macrocycle from nucleophilic attack, no significant bleaching occurs. In line with the suggested co conformation TriazRot2+,thedeshieldedSq 2+core bleaches similarly quickly as in the free Ax2+. The shielding effect of the macrocycle has been demonstrated for similar systems28. As demonstrated above, low concentrations of hydroxide are not sufficient to bleach Rot0. At higher concentrations of OH− ,the possibility of shuttling by binding of hydroxide in a similar motive as with chloride,cannotbeexcluded.Thus,forthecomparisonofthe stability ofAx0 and Rot0, sterically demanding potassium tbutoxide was used (Fig. S44f, g). To increase the solubility of the nucleophile, 5% of tBuOH were added to the solvent. In this mixture, free Ax0was bleached (≈12 min), while Rot0was affected only very little, indicating the expected protection of the squaraine core within the bound wheel (Fig. S44e). Summary and conclusion In this proof-of-concept study, we show that a co-conformationally dynamic rotaxane can serve as scaffold to either stabilize or expose highperforming dye molecules used for bioimaging applications, in this case a near‐infrared squaraine dye. [2]Rotaxane Rot can be reversibly switched between four different redox states, resulting in two different co-con- formations, in which the squaraine dye is either shielded (oxidation state 1 and 0) or exposed (oxidation state +1and+2). Shielding results in an increase of the dye’s lifetime, whereas the exposed states display physicochemical and optoelectronic properties similar to the non-complexed dye. At the same time, the squaraine-centered redox reactions allow for a stepwise tuning of the emission between 570 and 683 nm of the fluorescent Rot0 and Rot2+states. A potential application involves the transport of labile fluorophores—stabilizedbymechanicalbonding—intoa location wherethe co-conformational equilibrium of the rotaxane is triggered leading to the exposure of the fluorophore or a different reactive species. Such a system could be useful for applications in bioimaging (e.g. for multiplexing), sensing, and proximity labeling. Methods General NMR experiments were performed on JEOL ECX 400, JEOL ECP 500, Bruker AVANCE 500, JEOL ECZ 600, and Bruker AVANCE 700 instruments.Residual solvent signals were usedastheinternal standards. All shifts are reported in ppm and NMR multiplicities are abbreviated as s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad). High-resolution ESI mass spectra were recorded on an Agilent 6210 ESI-TOF mass spectrometer. HPLC grade solvents were used for sample preparation and the sampleswere introduced into the ion source with a flow rate of 2–4 µL/min. UV/Vis spectra were recorded on a Varian Cary 50 Bio spectrometer with a xenon lamp. Fluorescence spectra were recorded on Infinite®MNano +(Tecan Deutschland GmbH, Crailsheim, Germany) and PerkinElmer Fl 6500 spectrometers. Suprasil glass cuvettes with pathlengths of 1 cm were used. Photoluminescence quantum yields (Φ)were measured with an integrating sphere setup from Hamamatsu (Quantaurus- QY C11347-11). All measurements were performed at room temperature using 10 mm × 10 mm long neck quartz cuvettes. All reagents and solvents were obtained from commercial sources and used without further purification. Lithium tetrakis(perfluoro-tertbutoxy) aluminate was provided by Prof. Dr. Ingo Krossing and Malte Sellin (University of Freiburg, Germany). Dry solvents were purchased from Acros Organics. Deuterated solvents were purchased from Eurisotop and Deutero. Deuterated dichloromethane was dried by vigorous stirring with calcium hydride for 2 weeks at room temperature. N-Ethyl-N-(2-(prop-2-yn-1-yloxy)ethyl)thiophen-2-amine69,4-trityl- phenylazide42 and tetralactam macrocycle TLM70 were synthesized according to literature procedures. Thin-layer chromatography was performed on silica gel coated plates with fluorescent indicator F254 (Macherey-Nagel). For column chromatography, silica gel (0.04-0.063 mm, Macherey-Nagel) was used. Synthesis Bis(aminothienyl)squaraine Axle Ax68 Bis(aminothienyl)squaraine axle Ax was prepared according to a modified literature procedure68 (Fig. 7): A mixture of toluene (750 mL) and n-butanol (250 mL) was azeotropically dried by refluxing on a DEANSTARK trap (oil bath 145 °C) for 2 h. The collected water was drained and the solvent mixture was cooled under N 2 -atmosphere. N-Ethyl-N-(2- (prop-2-yn-1-yloxy)ethyl)thiophen-2-amine (9.7 g, 47 mmol, 2.0 equiv.) and squaric acid (2.7 g, 24 mmol, 1.0 equiv.) were added. A color change from yellow to deep green to deep blue to black was observed. The reaction mixture was heated to reflux under N 2 on the DEAN-STARK trap (oil bath 145 °C) for 8 h. The solvents were removed under reduced pressure, and the metallic green residue was purified by column chromatography using dichloromethane-acetone (0% →30% acetone) as Fig. 6 | UV-Vis (solid) spectra of Rot in three oxidation states and fluorescence (dashed) spectra of Rot0and Rot2+.The inset photographs on each curve depict a vial containing dichloromethane solution of each species, the counterion is [pf]  . https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 7 eluent. Analytically pure Ax was obtained as a metallic green solid (6.50 g, 13 mmol, 56% yield), the analytical data are consistent with the reported values (Figs. S1, S2, and S16)68. 1HNMR(600 MHz, CD 2 Cl 2 ): δ=7.88(d,J= 4.7 Hz, 2 H, d), 6.31 (d, J=4.7Hz,2H,e), 4.17 (d, J=2.4Hz,4H,k), 3.79 (t, J=5.4Hz,4H,j), 3.69 (t, J= 5.4 Hz, 4 H, i), 3.61 (q, J= 7.2 Hz, 4 H, g), 2.50 (t, J= 2.4 Hz, 2H, m), 1.30 (t, J= 7.2 Hz, 6 H, h)ppm. 13C{1H} NMR (151 MHz, CD 2 Cl 2 ): δ= 178.2, 171.5, 170.0, 138.1, 115.6, 109.3, 79.6, 75.1, 67.4, 58.9, 54.2, 50.4, 12.2 ppm. HRMS (ESI): m/z calculated for C 26 H 28 N 2 O 4 S 2 [M]●+496.1501, found 496.1513. Squaraine-TLM Rotaxane Rot Rotaxane Rot was prepared in the following manner (Fig. 8): A 25-ml dried Schlenk flask was charged with tetralactam macrocycle TLM (190 mg, 0.20 mmol, 1.0 equiv.) and Ax (200 mg, 0.40 mmol, 2.0 equiv.) under N 2 counterflow. Dry dichloromethane (8 mL) was added, the flask was sealed, and the mixture was stirred for 1 h at room temperature. After that, 4-trityl- phenylazide (350 mg, 0.96 mmol, 4.8 equiv.), Cu(ACN) 4 BF 4 (25 mg, 0.08 mmol,0.4 equiv.) and tris((1-benzyl-4-triazolyl)methyl)amine (TBTA) (43 mg, 0.08 mmol, 0.4 equiv.) were added under N 2 counterflow. The flask wassealedagainandstirredovernightatroomtemperature.Themixture was purified by silica column chromatography using step gradient elution (0.8%→2.6%methanolindichloromethane).Rot elutedwith1.3% →1.5% methanol content. Analytically pure Rot was obtained as a deep blue crystalline solid (140 mg, 0.06 mmol, 32% based on TLM) (Fig. S11, 12, and 16). 1HNMR(600 MHz,CD 2 Cl 2 ):δ= 10.34(b, NH,2H,ε),9.18(b,1 H,χ), 8.76 (b, NH,2H,ο), 8.42 (d, J=7.8Hz,2H,β), 8.31 (d, J= 1.4 Hz, 2 H, σ), 8.09(t,J= 7.8 Hz,1 H,α),7.98(s,2 H,m), 7.64(d, J= 8.8 Hz,4 H, o),7.51(d, J= 4.7 Hz, 2 H, d), 7.45 (d, J= 8.8 Hz, 4 H, p), 7.25-7.30 (m, 24 H, t&u), 7.20-7.23 (m, 6 H, v), 6.83 & 6.82 (2x s, 8 H, ι&ι’), 6.17 (d, J= 4.7 Hz, 2 H, e), 4.62 (s, 4 H, k), 3.72 (t, J=5.4Hz,4H,j), 3.58 (t, J=5.4Hz,4H,i), 3.49 (q, J=7.2Hz,4H,g), 2.20–2.37 (m, 8 H, μ), 2.07 & 2.06 (2x s, 24 H, θ&θ’), 1.61 (m, 8 H, ν), 1.49 (m, 4 H, ξ), 1.39 (s, 9 H, φ), 1.19 (t, J= 7.2 Hz, 6 H, h) ppm. 13C{1H} NMR (175 MHz, CD 2 Cl 2 ): δ= 179.2, 169.9, 167.7, 164.7, 162.6, 153.7, 149.7, 148.4, 147.9, 147.4, 146.7, 145.2, 139.3, 137.9, 135.4, 135.2, 135.0, 134.5, 132.7, 131.8, 131.6, 131.3, 128.8, 128.2, 126.6, 125.7, 125.4,125.1,123.3,121.4,120.0,114.1, 109.3,67.7,65.3, 64.8,53.8, 50.3,45.2, 35.6, 34.7, 31.5, 30.1, 26.8, 23.4, 19.1, 19.0, 12.1 ppm. HRMS (ESI): m/z calculated for C 139 H 137 N 13 O 8 S 2 [M]●+2180.0154, found 2180.0108. Electrochemical switching Cyclic voltammetry (CV) (Figs. S26 and S27) and differential pulse voltammetry (DPV) (Fig. S27 and Table S2) were performed on an Autolab Fig. 7 | Adapted procedure for the preparation of squaraine Ax.55. Fig. 8 | Capping-synthesis of Rot by CuII catalyzed azide alkyne HUISGEN cycloaddition of trityl phenyl azide with the pseudo[2]rotaxane of Ax and TLM in dichloromethane. TBTA: tris((1-benzyl-4-triazolyl)methyl)amine. https://doi.org/10.1038/s42004-024-01312-1 Article Communications Chemistry | (2024) 7:229 8