Solvent Induced Helix Folding of Defined Indolenine Squaraine Oligomers

Abstract A protecting group strategy was employed to synthesise a series of indolenine squaraine dye oligomers up to the nonamer. The longer oligomers show a distinct solvent dependence of the absorption spectra, that is, either a strong blue shift or a strong red shift of the lowest energy bands in the near infrared spectral region. This behaviour is explained by exciton coupling theory as being due to H‐ or J‐type coupling of transition moments. The H‐type coupling is a consequence of a helix folding in solvents with a small Hansen dispersity index. DOSY NMR, small angle neutron scattering (SANS), quantum chemical and force field calculations agree upon a helix structure with an unusually large pitch and open voids that are filled with solvent molecules, thereby forming a kind of clathrate. The thermodynamic parameters of the folding process were determined by temperature dependent optical absorption spectra.


Introduction
The helix structure is one of the most important supramolecular elements in chemistry. [1] It plays a decisive role in the superstructure of proteins, polysaccharides, DNA, and RNA but was also found in purely artificial supramolecular assemblies of low molecular weight molecules, in coordination polymers and also in oligomers and polymers of covalently linked organic monomers. [2] Among the latter are polyisocyanides, [3] polyisocyanates, [4] meta-phenylene oligomers, [5] oligo(m-phenylene ethynylene)s, [6] oligo-β-aminoacids, [7] heterocyclic [8] and non-heterocyclic [9] aromatic amide oligomers, just to name a few parent structures. All these oligomers/polymers may adopt helix structures by a folding process. [10] In many cases the helix folding depends on specific conditions such as temperature, [11] solvent, [6,12] (metal) ion concentration, [13] pHvalue, [14] hydrogen bonding [15] or chiral templates such as carbohydrates. [15b,16] However, in only very few cases the parent monomer units in the main chain of the folding oligomers/ polymers are dyes with an intrinsic strong absorption in the visible or near infrared (NIR) optical range. [17] The reason for that is the lack of suitable dyes which possess a structure that is intrinsically prone to helix formation. T. Nebeshima et al. described a BODIPY trimer that folds to one helix turn upon titration with cesium ions. [13a] Werz. et al. [18] also suggested a helix structure for oligo-BODIPYs where the relative orientation of the monomers is locked by ethyl substituents. A special case is the linear meso-linked oligo-Zn-porphyrin in which the helicity is induced by stepwise turning the porphyrin plane around the connecting axis induced by interaction of the side chain substituents. [19] Though aggregates [20] and polymers [21] of squaraine dyes are well known, they have not yet been explored in the context of folding processes. Their optical properties [21c,22] -a narrow and intense absorption band in the red to NIR spectral range and a high fluorescence quantum yield -make them an ideal candidate for studying modification of optical properties upon changes of the mutual orientation of chromophores within either a supramolecular aggregate or covalently bound oligomers or polymers. Thus, the control of supramolecular folding structure would enable to tune optical properties for potential applications in e. g. solar cells, sensors, biolabels, and nonlinear optics.
In earlier work [21c] we made the puzzling observation that a polymer based on the cisoid squaraine SQ shows in some solvents such as CHCl 3 a red-shift of the main absorption band compared to the SQ monomer but in other solvents such as acetone a blue-shift (see Figure S4 in the Supporting Information). The SQ monomer was described first by E. Terpetschnig et al. [22d,23] in the late 90ies and, unlike most other squaraine dyes, possesses a bent structure (Figure 1). Following Kasha's exciton theory, [24] for a linear arrangement of monomer units within a polymer strand one expects that the lowest energy exciton state is allowed but all other states within the exciton manifold are forbidden. Thus, we interpreted the observed redshift of the SQ polymer as being due to exciton interactions of the localized transition moments in a J-type (= head-to-tail) manner. However, the blue-shift in other solvents such as DMF or acetone was explained by a solvent dependent helix folding. This sounds reasonable in view of the cisoid structure of SQ which allows connecting two squaraines with an about 120°a ngle via a biaryl bond. The twist around the biaryl axis then controls the helix pitch in return. Calculations showed that approximately three squaraines were needed for a turn. And indeed, cyclic trimers of SQ which we synthesised in earlier works also indicate that for a helix turn about three monomers are necessary. [21c,25] While for the SQ polymer we investigated photoinduced energy transfer processes in great detail, [26] the exact structure and the folding conditions remained unexplored. However, this knowledge is necessary in order to use the folding for tuning optical properties for further applications. Therefore, in this study we will address the question of how many SQ monomers are necessary to form a helix, what is the helix pitch and the helix diameter and we will also give information about the folding thermodynamics. Thus, we will explore the chain length dependence [27] of the optical spectroscopic properties in various solvents. We will learn that the longer SQ oligomers indeed form a quite unique helical structure which is -unlike practically all other known helix structure of π-systems -very loose with a large helix pitch. In order to prove that, we will present a strategy to synthesise monodisperse SQ oligomers up to the nonamer (see Figure 1) which requires a delicate protecting group technique based on Suginome's [28] 1,8-diaminonaphthalene protected boronic acid, which has been applied for the synthesis of small phenylene dendrons before. [29] Results Synthesis of Oligosquaraines SQ n : The syntheses of SQ monomer, dimer and trimer were already reported by us recently. [30] In order to synthesise longer monodisperse SQ n oligomers up to the nonamer via a Suzuki-Miyaura coupling we employed a protecting group for boronic acids developed by Suginome et al. [28a,c] These authors used 1,8-diaminonaphthalene (dan) to protect aryl boronic acids which can easily be deprotected in an acidic environment. Thus, starting from the symmetric squaraine pinacol boronic ester SQ-Bpin 2 iterative cross coupling with SQ-BrBdan at both sides yielded the oddnumbered SQ oligomers, see Figure 2, lower part. Since after deprotection the oligosquaraine boronic acids are difficult to purify and to characterise, intermediates were characterised at the step of the dan protected boronic acids. [29a,31] In the last step, coupling of the oligosquaraine boronic acids with SQ-Br terminated the oligomer chains. Thus, the bifunctional squaraine SQ-BrBdan is the key building block for chain elongation and was synthesised from the appropriate indolenine and squaric acid ester according to Figure 2, upper part. The indolenines were appended by rac-3-7-dimethyloctyl chains to ensure solubility. The even-numbered oligomers were prepared in a similar way as the odd-numbered oligomers starting with a symmetrically Bdan substituted SQ dimer (see Supporting Information). Yields for deprotection and double cross coupling vary between about 20-90 % per step. The details of each synthetic step can be found in the Supporting Information.

Absorption spectroscopy
The SQ oligomers were dissolved in CHCl 3 and their absorption spectra were measured, see Figure 3a. The absorption spectrum of SQ monomer shows an intense peak at 14300 cm À 1 with a Chemistry-A European Journal much smaller shoulder at higher energies caused by vibronic progression. For SQ 2 and SQ 3 the lowest energy absorption increases in intensity and shifts towards lower energy compared to SQ. At the higher energy side the shoulders are also more intense and structured. As recently discussed, [30,32] this is a consequence of exciton coupling between monomer localised states in a J-type manner, that is, the localised transition moments are arranged in a head-to-tail orientation. Here, the in-phase combination of transition moments refers to the most stable eigenstate and possesses the highest intensity. The higher energy exciton eigenstates (S 2 for SQ 2 and S 2 and S 3 for SQ 3 ) also show some intensity which, for the S 2 state, is caused by structural disorder, that is, rotation around the biaryl axis results in conformers where the transition moments are no longer in head-to-tail orientation but adopt a significant angle relative to each other. While the absorption spectra of the smaller SQ n chromophores with n = 1-3 are almost identical irrespective of the solvents (see Figure 2a and b), the absorption spectra of the higher SQ n oligomers (n > 3) show a strong solvent dependence that can be classified into either J-type or H-type aggregate behavior, depending on the shift of the most intense absorption band: in CHCl 3 the J-type behavior leads to an increase of intensity and bathochromic shift of the lowest energy band with increasing oligomer length. More specific, the lowest energy band shifts from 14300 cm À 1 for SQ to 12700 cm À 1 for SQ 9 . However, in acetone a clear H-type behavior is seen (see Figure 3b) where the highest energy band of the exciton manifold shifts towards higher energies (e. g. to 15500 cm À 1 for SQ 9 ) and gains intensity with increasing chain length. Comparison with the spectra of a related polymer with X n = 50 shows that in CHCl 3 the spectra are practically identical but in acetone the absorption maximum of the polymer is still   www.chemeurj.org 19000 cm À 1 ) vs. oligomer length shows for both solvents a linear correlation (see Figure S1d) and S2d) in the Supporting Information) which proves that no other states besides those generated by exciton coupling are involved in this energy range.

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In the broad range of tested solvents ranging from hydrocarbons, aromatic solvents, chlorinated solvents, polar aprotic solvents, alcohols, and ethers the higher SQ oligomers either show a clear J-type (see Figure 3c and e) or H-type behavior (see Figure 3d and f) for SQ 9 . [33] There is only one exception, benzonitrile (PhCN), which displays H-type spectra at rt but Jtype behavior at elevated temperature indicating a temperature dependent reversible folding process. Thus, in the following we concentrate on the optical properties of the SQ oligomers in CHCl 3 , acetone and PhCN. Since concentration dependent measurements in the range of 10 À 6 -10 À 8 M in PhCN and of 10 À 4 -10 À 6 M in acetone did not reveal any intermolecular aggregation effects, the different optical behavior in CHCl 3 and acetone must be due to a different oligomer structure in the respective solvent (see Figure S3 in the Supporting Information). The most extremes conceivable are a linear chain and a helix structure. The former is expected to show J-type behavior, the latter H-type behavior. However, structural disorder such as the formation of rotamers around the biaryl axes might blur the pure J/H-type spectroscopic appearance of the oligomers in the respective solvent. This interpretation is based on our earlier work on SQ polymers. [21c,26] When looking at the evolution of absorption maxima in CHCl 3 it is obvious that all oligomers adopt a chain structure, see inset in Figure 3a. This is different for acetone solutions of the oligomers. The inset in Figure 3b shows a close similarity of the energy of absorption maxima in acetone and in CHCl 3 solution up to the tetramer. However, for the higher oligomers SQ 5 to SQ 9 the absorption maximum in acetone switches to the highest exciton state, indicating partial helix formation for SQ 5 and almost complete helix formation for SQ 9 as the spectral differences between SQ 8 and the latter are minor.

Helix folding
While in CHCl 3 the absorption spectra of the higher oligomers display clear J-type behavior and in acetone distinct H-type behavior, those in PhCN possess H-type features but upon heating, the spectra change gradually and reversibly and display the typical J-type behavior. This allows determining the thermodynamic parameters of the chain-helix interconversion. In Figure 4 the temperature dependent spectra of SQ 9 are given as the most prominent example, those of SQ 4 -SQ 8 can be found in Figure S8 in the Supporting Information. Clearly, at low temperature (268 K) we find H-type spectra and at elevated temperature (413 K) J-type spectra. An almost perfect isosbestic point proves that only two species are involved in the equilibrium. Therefore, we fitted the temperature dependent extinction coefficient at the maximum absorption of the Hband at 15200 cm À 1 by assuming a simple two-state interconversion equilibrium C G H H with Equation 1, see inset in Figure 4a), thus, we assumed a fully cooperative coil-helix transformation. [34] Here, ɛ H is the extinction coefficient of the pure helix (H) and ɛ C that of the pure chain (C) structure.
The thereby evaluated enthalpy ΔH ranges between À 15.7 kJ mol À 1 for the tetramer to À 33.7 kJ mol À 1 for the octamer, the respective entropy between À 33.3 J mol À 1 K À 1 and À 101.6 J mol À 1 K À 1 (see Figure 4b). Thus, helix formation is exothermic but disfavored by entropy, as anticipated for a more ordered structure. A closer inspection of the enthalpy and entropy, however, reveals a distinct step-like behavior, that is, these values are very similar for the pentamer to heptamer but distinctly more negative for the octamer and nonamer. The highest values are obtained for the tetramer. We assume that this is due to the number of helix turns that may form in each oligomer. Assuming about three monomers per helix turn (see above), the pentamer to heptamer may form two turns while the octamer and nonamer may form three turns. In Figure 4b),

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Full Paper doi.org/10.1002/chem.202101063 the corresponding enthalpy and entropy is also normalised by the number of monomers which gives an enthalpy of roughly À 4 kJ mol À 1 and an entropy of À 11 J mol À 1 K À 1 per monomer. The Gibbs free energy is on the order of À 2 to À 6 kJ mol À 1 depending on oligomer length. As we observe either a clear Jor H-type behaviour in all other solvents and no temperature dependence of the spectra, the absolute values of the Gibbs free energy are expected to be much larger there. Indeed, measuring absorption spectra of SQ 9 in CHCl 3 /acetone mixtures ranging from pure CHCl 3 to pure acetone in 5 %vol fraction steps (see Figure S10 in the Supporting Information) yielded equilibrium constants and, thereby, Gibbs free energies referring to each particular solvent mixture. [11,34] Extrapolating the ΔG dependence to pure CHCl 3 and to pure acetone gave ΔG = + 13.4 kJ mol À 1 and À 12.4 kJ mol À 1 , respectively, for the helix formation process, compared to À 3.4 kJ mol À 1 in PhCN, see Figure S11 in the Supporting Information.

Helix structure
While optical spectra and thermodynamic considerations speak for a helix formation of longer SQ oligomers in acetone, the geometric dimensions need to be elucidated. Simple 1D and 2D NMR methods proved to be unhelpful as the oligoquaraines show rather complex spectra in CD 2 Cl 2 or acetone-d 6 and strongly temperature dependent spectra in PhCN-d 5 (see Figure S45-47 in the Supporting Information). For this reason, we performed diffusion ordered DOSY NMR measurements in acetone-d 6 and in CD 2 Cl 2 (see Figure S48-56 in the Supporting Information). The latter was chosen as the squaraine solutions showed somewhat sharper and better resolved signals in CD 2 Cl 2 than in CDCl 3 . Nonetheless, the optical spectroscopic behavior is almost identical in the two solvents. From the DOSY NMR measurements we calculated the hydrodynamic radii R 0 of the oligomers from the corresponding diffusion coefficients D by the Stokes-Einstein equation (Equation S1 in the Supporting Information). While different equations [35] according to different hypothetical geometries can be used to calculate a hydrodynamic radius, here we resort to the simple Stokes-Einstein equation which will at least yield comparable results within the SQ oligomer series within one solvent. The results are given in Figure 5. Here we can see that in CD 2 Cl 2 the hydrodynamic radius increases with the number of monomer units. This is expected for random coils whose size, on average, increases with the chain length. In acetone, a similar trend is followed up to the tetramer. However, beginning with the pentamer, the curve flattens considerably, that is, the structure of the longer oligomers starts to be more compact due to limited one dimensional growth, in agreement with the suggested helix structure.
In order to gain more precise information on the nanoscale structure we performed small angle neutron scattering (SANS) measurements on SQ 8 in acetone-d 6 (see Figures S57 and S58 in the Supporting Information). The best fit of the scattering data was obtained for a flexible cylinder model with a cross-section radius R c = (0.9 � 0.1) nm, a total length of L = (8.5 � 0.5) nm, and a Kuhn segment length of l K = (2.1 � 0.1) nm. A model independent Guinier plot for small q yields a radius of gyration R G = (1.56 � 0.02) nm, which is in accordance with the flexible cylinder dimensions and with the hydrodynamic radius determined by DOSY. The flexible cylinder structure revealed by SANS confirms the above-mentioned hypothesis of structural disorder such as the formation of rotamers around the biaryl axes blurring the pure J/H-type spectroscopic appearance.
Modelling of the helix structure using the semiempirical AM1 Hamiltonian yields indeed a helix with about 2.7 squaraine dyes per turn which gives an approximate radius of 10 Å (in good agreement with the SANS data) and a separation between the centers of the terminal squaraines of the helix of 50.5 Å (see Figure 6, upper part). The optimized equilibrium biaryl twist angle is about 43°. As the squaraine chromophores are practically stiff and flat units, this biaryl twist angle determines the large helix pitch of about 20 Å. This also is consistent with what appears as Kuhn segment length in the SANS model. Consequently, the helix voids must be filled by solvent molecules. A double helix structure -as seen in e. g., oligometa-phenylenes [2d,36] -which would lead to a denser structure and the replacement of the solvent molecules within the helix, though geometrically possible, can safely be excluded as we did not find any concentration dependence of the absorption spectra. [37] Furthermore, the dimethylmethylene groups in the 3-position of the indolenine heterocycles prevent dense πstacking anyway.
We also simulated a solvated helix by propagating the AM1 optimized helical SQ octamer including the dimethyl octyl side chains in a periodic acetone solvent box using the CHARMM36 force field. Along the 100 ps trajectory several snapshots were optimized with the forcefield. Inside the helical pitch, the acetone solvent molecules orient with their partially positive charged methyl groups towards the electron rich groups of the squaraine, that is, the indolenines flanking the squaric acid core. This forms the first solvation layer between the helix sheets as seen in Figure 6, lower part right. The simulations show, that the layers between two neighbouring sheets are bridged by another singular layer of acetone. This orientation can be seen

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Full Paper doi.org/10.1002/chem.202101063 throughout multiple simulations and appears to the preferred orientation induced by the helix's extensive π-system. While obtaining reliable thermodynamic data from QM/MM calculations is unfortunately out of scope due to the size of the system, this recurring structural motif might stabilise the helix and compensate for the entropic contributions at a given temperature. Using that AM1 structural model we also calculated the absorption spectrum of SQ 8 by the ZINDO method, see Figure 6 lower part left, which is in very good agreement with the experimental spectrum, particularly in view that vibronic progressions were disregarded in the calculations. This emphasized the correctness of our structural model. The large pitch of our structural model also indicates that the blue shift of absorption maxima (H-type) is not caused by parallel arrangement of transition dipole moments in π-stacked squaraines but is because of the gauche conformation [38] of the banana-bent squaraines around the connecting biaryl axis. This can easily be seen when comparing the TD-DFT computed absorption spectra of linear SQ 3 vs. the closed form, see Figure S59 in the Supporting Information.

Discussion and Conclusion
An iterative cross coupling/deprotection strategy allowed the synthesis of monodisperse SQ oligomers and established the chain length dependence of optical spectra, the thermodynamics of helix folding and their geometry. The coil-helix transition of the longer oligomers follows the cooperative model, [39] that is, as a nucleation step, about three monomers are necessary to form a loop until propagation of the oligomer leads to helix formation. AM1 computations, DOSY and SANS measurements agree on an oligomer helix with an unprecedented large pitch (ca. 20 Å) and, consequently, voids between the squaraine dyes that are filled by solvent molecules. Calculated absorption spectra based on this structural model also are in very good agreement with experiment. The obvious question arises what causes the helix formation if direct π-π interaction of stacking aromatic systems as found in e. g. oligo(m-phenylene ethynylene)s can be ruled out at this large helix pitch.
In general, in the most favourable case, helix folding requires partial desolvation, that is, removal of solvent molecules which has to be compensated by attractive interactions

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Full Paper doi.org/10.1002/chem.202101063 between helix segments, be it e. g., hydrogen bonding or πstacking. Furthermore, because helix folding is despite desolvation usually entropically unfavourable, the entropy term must be overcompensated by the enthalpy term. In our case, folding a helix with a small pitch as to allow π-stacking interactions would require strong twisting around the biaryl axes between the squaraine dyes beyond the equilibrium angle of ca. 43°. This should afford a significant amount of extra enthalpy which is unfavourable and thus a loose helix is preferred. [40] The solution experiments with diverse solvents shows that the reason for helix folding is a microscopic solute-solvent interaction which must at least overcompensate the entropy loss of helix formation. The most intriguing observation is that SQ 9 displays distinct J-type behavior in nitrobenzene but H-type behavior in benzonitrile, although both solvents are very similar in almost all their physical properties (see Table S2 in the Supporting Information). This reminds of a bistable situation in which tiny changes of the solute-solvent interaction lead to one of the preferred structures and is typical of a cooperative phenomenon. Almost all other aromatic solvents also induce a J-type behavior as do THF, CHCl 3 and CH 2 Cl 2 and thus may be regarded as "good" solvents. DMF, acetone, alcohols, and less polar solvents such as hydrocarbons and dialkyl ethers induce H-type behavior and are "poor" solvents leading to helix folding. However, titration with hydrogen bonding spermine or octylglucoside [16] did not lead to helix formation of SQ 9 in either CHCl 3 , DCM, toluene, or benzonitrile (see Figure S6 and S7 in the Supporting Information). Furthermore, solutions of SQ 9 in enantiomerically pure (S)-1-phenylethylamine or (S)-2-methylbutanol exhibited J-type and H-type spectra, respectively, but no CD signal at all (see Figure S5 in the Supporting Information). Thus, specific hydrogen bonding phenomena appear not to play a decisive role in the helix folding.
None of the usually employed solvent parameters such as dipole moment, refractive index, permittivity, E N T -values, or Onsager factors [41] proved to be helpful for explaining the observed selective J-or H-type behaviour. However, the observed trend correlates with the dispersion index of the Hansen solubility parameter which characterises the polarisability of a solvent. [42] Thus, solvents with δ D index larger than 17.4 favor the random coil, while those below, the helix. [43] Here also nitrobenzene (δ D = 20.0) and benzonitrile (δ D = 17.4) in fact differ markedly. The latter is obviously right at the border concerning the polarizability influence on the coil-helix equilibrium as shown by the above mentioned temperature dependence of absorption spectra. Another aromatic solvent that has a low Hansen dispersion index is hexafluorobenzene (δ D = 16.9) which indeed also induces the H-type behavior while benzene (δ D = 18.4) with the somewhat larger dispersity index favours the random coil. Therefore, because at the estimated distance of ca. 20 Å, the reason for helix formation cannot be any direct interchromophore interaction, we suppose that the interstitial solvent molecules act as a kind of "glue" which is induced by the static dipole moments of SQ monomers (6.9 D). [44] The dipole moment of SQ is sizable unlike all other squaraines which are centrosymmetrc with a vanishing dipole moment. According to our solvation experiments, the solvents that qualify for helix formation must be either polar (hydrogen bonding or non-hydrogen bonding) or nonpolar, but in any case, possess little polarisability, as characterised by small Hansen dispersion indices. Thus, the enthalpy measured above does not represent the helix formation itself but the stabilisation of interstitial solvent by the folded helix. Such an enhanced solvent order was observed e. g., for polymers in hydrocarbon solutions up to a distance of 2 nm, [45] theoretically predicted for several diverse solvents between graphene sheets, [46] on silica surfaces, [47] and for nanoparticles [48] up to a distance of 2 nm. Furthermore, clathrate formation of solvents between alky side chains of aggregated perylene diimide helices was observed to control the helicity of the aggregate. [49] Molecular mechanics simulations of a helix hexamer with freely optimised acetone solvent molecules indeed indicate such a preferred solvent order, see Figure 6. Therefore, it is conceivable that solvent molecules adopt a long-range order between the turns of the SQ n helix with a pitch of 2 nm stabilizing the entire solvated helix structure, thus forming a kind of clathrate. Therefore, we conclude that longer squaraine oligomers form helix-solvent clathrates as an unusual supramolecular entity that has a strong impact on the absorption spectra by exciton coupling interactions. Such a behavior is unprecedented for dye oligomers to the best of our knowledge but should be considered in other supramolecular dye arrangements as well.