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Influence of Hydrophobicity on Excitonic Coupling in DNA-Templated Indolenine Squaraine Dye Aggregates

Control over the strength of excitonic coupling in molecular dye aggregates is a substantial factor for the development of technologies such as light harvesting, optoelectronics, and quantum computing. According to the molecular exciton model, the strength of excitonic coupling is inversely proportional to the distance between dyes. Covalent DNA templating was proved to be a versatile tool to control dye spacing on a subnanometer scale. To further expand our ability to control photophysical properties of excitons, here, we investigated the influence of dye hydrophobicity on the strength of excitonic coupling in squaraine aggregates covalently templated by DNA Holliday Junction (DNA HJ). Indolenine squaraines were chosen for their excellent spectral properties, stability, and diversity of chemical modifications. Six squaraines of varying hydrophobicity from highly hydrophobic to highly hydrophilic were assembled in two dimer configurations and a tetramer. In general, the examined squaraines demonstrated a propensity toward face-to-face aggregation behavior observed via steady-state absorption, fluorescence, and circular dichroism spectroscopies. Modeling based on the Kühn–Renger–May approach quantified the strength of excitonic coupling in the squaraine aggregates. The strength of excitonic coupling strongly correlated with squaraine hydrophobic region. Dimer aggregates of dichloroindolenine squaraine were found to exhibit the strongest coupling strength of 132 meV (1065 cm–1). In addition, we identified the sites for dye attachment in the DNA HJ that promote the closest spacing between the dyes in their dimers. The extracted aggregate geometries, and the role of electrostatic and steric effects in squaraine aggregation are also discussed. Taken together, these findings provide a deeper insight into how dye structures influence excitonic coupling in dye aggregates covalently templated via DNA, and guidance in design rules for exciton-based materials and devices.


Supporting Information 2: Electrophoretic Analysis
Nondenaturing PAGE was carried out to evaluate the formation of DNA HJ templaitng squaraine aggregates. The 15% non-denaturing electrophoresis gel, 1

Supporting Information 3: Thermal Denaturation
Thermal denaturation (melting) profiles of squaraine-DNA constructs were recorded using a Varian Cary5000 spectrophotometer equipped with a thermal probe (Agilent Technologies Cary Temperature Controller G9808). Prior to measurements, samples were degassed for 5 min at room temperature in a vacuum centrifuge. Samples were equilibrated at 25 °C for 2 min before starting a temperature ramp of 1 °C/min over a temperature range from 25 °C to 85 °C. Absorption was monitored at 260 nm. The melting temperatures (Tm) of squaraine-DNA constructs were determined from the first derivatives of sigmoidal melting curves. The visual inspection of the first derivative profile revealed the presence of a minor melting transition at a lower temperature (with the exception of SQ-Sl3 transverse dimer). As such, the main melting temperatures were determined by fitting two Gaussian curves to the first derivative in OriginPro 2019 (Figs. S2 and S3).
The melting temperatures of squaraine-modified DNA HJs were compared with melting temperature of unlabeled HJ as a control. 1 The melting temperatures are summarized in Table S1.    Figure S4. Acquired steady-state absorption spectra converted to extinction of squaraine-DNA constructs in 1× TBE, 15 mM MgCl2 at room temperature. The squaraine-DNA construct concentration was 1.5 M. Figure S5. Squaraine monomer fluorescence normalized to dye peak maximum of squaraine-DNA constructs in 1× TBE, 15 mM MgCl2 at room temperature. The squaraine-DNA construct concentration was 0.5 M.

Supporting Information 6: Fluorescence and Fluorescence Suppression in Squaraine Aggregates
Fluorescence spectra were collected and scaled by the absorptance at the excitation maximum. The absorptance is defined as where A is absorptance at the excitation wavelength (λexc), Ttransmitted light, and absabsorption at λexc. The area under emission curve of each aggregate was integrated in OriginPro2019. Obtained areas of integration were used to calculate fluorescence suppression according to the following formula: Figure S6. Scaled fluorescence emission of squaraine monomer, adjacent dimer, transverse dimer, and a tetramer recorded in 1× TBE, 15 mM MgCl2 containing 0.5 μM HJ DNA construct at room temperature. The aggregates were excited at exc = 630 nm. The fluorescence was normalized by the absorptance at the excitation wavelength.  Figure S7. Acquired full CD spectra of the squaraine-DNA constructs recorded in 1× TBE, 15 mM MgCl2 at room temperature. The squaraine-DNA construct concentration was 1.5 μM.

Supporting Information 8: Fitting Absorption and CD using KRM Model
When intermolecular distances are much greater than dye size, the excitonic hopping parameter Jm,n can be expressed in a point dipole-dipole interaction form [2][3][4] representing the interaction between a pair of molecular transition dipoles μm and μn for the dyes at sites m and n: ,the position vector between the dye centers dyes m and n. (further R1,2 between two dyes) In the extended dipole approximation, 5 the Coulomb energy between a pair of dipoles is given in Standard International units by: where δ is the oscillating point charge in Coulombs, 0 is the permittivity of the vacuum ( 0 = 8.85 × 10 − 12 m − 3 kg − 1 s 4 A 2 ), is the relative dielectric constant of the medium, and are the location of the "+" and "-"charges on molecule 1 and similarly for molecule 2. The distance units are meters and the exchange energy Jm,n unit are Joules.
The transition dipole moment is given by μ = δl where l is the distance in meters between the two point charges on a given molecule, and is given by = 2 where n is the index of refraction of the medium.
Then the equation (S2) can be rewritten as: The quantity J0 has units of J·m 3 .
When the position vectors r and s are rewritten as: And when| − | ≫ , equation (eq S3) reduces to: Thus, 0 is the same constant in both the point dipole-dipole approximation and in the extended dipole approximation.

S17
The theoretical absorbance as a function of energy, for comparison with experimental data, was computed from the line spectra obtained by diagonalizing the system Holstein-like Hamiltonian 6 by convolution with a Gaussian as: Where the is the transition rates between the ground state and the state having the eigenenergy , and is a linewidth.
Similarly, the CD absorbance as a function of energy was computed as: The phenomenological constants used in the theoretical model are shown in Tbl. S4. To fit the monomer and then dimers and a tetramer data, we considered the length of the squaraine transition dipole moment to be 1.3 nm (which is slightly shorter than the length of an indolenine squaraine dye), the linewidth to be 0.028-0.034 eV, and number of vibronic states nv to be 3.
The transition dipole moment was determined using the following expression: 7 01 = 9.58 × 10 −2 ( (2 2 +1)  Table S5, and its values was used as a fitting parameter in the calculations of squaraine dimers and a tetramers.
As a measure the goodness of the fits, we evaluated the overlap integrals of the experimental spectra with the theoretical spectra (Tbl. S6). Letting ∫ , ( ), , ℎ ( ) , , ( ), , ℎ ( ) denote respectively the experimental absorbance spectrum, theoretical absorbance spectrum, experimental CD spectrum, and theoretical CD spectrum where E is energy, the normalized absorbance overlap integral of the spectrum is defined by: As an overall goodness parameter, we introduce: In addition, mean-square deviation of absorbance and CD were utilized: = ∑ [ , ( ) − , ℎ ( )] 2 (eq S12) = ∑ [ , ( ) − , ℎ ( )] 2 (eq S13) where w1, w2, w3, and w4 are user selected weights. For a typical run the weights were chosen as w1 = 1, w2 = w3 = 0, and w4 = w5 = 1. A center-to-center distance R between two dyes was calculated as: A slip angle s was calculated as: Where n1 is a unit orientation vector given by: = sin( 1 )cos( 1 )̂+ sin( 1 )sin( 1 )̂+ cos( 1 ) And where is a unit vector connecting dye centers is: The oblique angle between two vectors was calculated as: = cos −1 [sin( 1 ) sin( 2 ) cos( 1 ) cos( 2 ) + sin( 1 ) sin( 2 ) sin( 1 ) sin( 2 ) + cos( 1 ) cos ( 2 )]. Extracted Jm,n values and calculated geometric parameters for dye aggregates are summarized in the Tbls. S8 -S13.  r -the ratio of theoretical to experimental values of the ratio of the max abs CD peak height to max absorbance peak height OIAB -normalized overlap integral for the experimental and theoretical absorbance curves OICD -normalized overlap integral for the experimental and theoretical CD spectra OItot -mean of OIAB and OICD MSDabs -absorbance spectrum mean-square deviation MSDcd -CD spectrum mean-square deviation wabscdrms -weighted mean-squared deviation between the experimental and theoretical ABS and CD spectra

Supporting Information 9: Influence of Electrostatic Interactions
To evaluate contribution of electrostatic forces on excitonic coupling strength in the squaraine aggregates, we calculated a ground state static dipole moment (a property responsible for permanent dipole-dipole interactions), polarizability (a property responsible for interactions involving induced dipoles), and surface area (a property related to polarizability and hydrophobic effect) in squaraine dyes. These properties were plotted as a function of J1,2 (Fig. S14, Tbl. S14). We did not observe strong relationships between these properties and J1,2 indicating that permanent dipole-dipole and induced-dipole interactions not being the major factors in excitonic coupling. The role of permanent dipole-dipole interactions appeared to be the least pronounced especially for the adjacent dimers because the excitonic coupling strength, in general, decreased as the static dipole increased (Fig. S14a). However, the squaraine SQ-Sl2 with the highest static dipole moment warrants attention. With the strongest static dipole moment among the examined squaraines, SQ-Sl2 exhibits a strong coupling (71.3 meV) in the transverse dimer configuration, and weaker coupling in the adjacent dimer configuration (64.3 meV). This can be explained with a parallel or antiparallel alignment of dyes maintained in each dimer configuration. In the transverse dimer, dyes might be forced (by a steric factor and/or DNA conformation) to adopt an antiparallel alignment which is beneficial for the dyes with a large static dipole in order to avoid repulsion and form attractive Coulomb interactions. And in the adjacent dimer, dyes might be forced to adopt a parallel alignment destabilizing the dimer if dye's static dipole moment is large. On the other hand, dyes with small static dipole like SQ-H2, SQ-Cl2 and SQ-Sl5 do not form strong dipole-dipole interactions and so less sensitive to dimer (anti)parallel alignment.
Polarizability responsible for induced-dipole interactions including dispersion appears to play a more prominent role for hydrophobic squaraines (Fig. S14cd). Comparable polarizabilities of hydrophilic dyes SQ-Sl2, SQ-Sl3 and SQ-Sl5 do not correlate with the difference in the excitonic coupling strength in their dimers. However, higher polarizability of SQ-Cl2 and SQ-Me2 compared to that of SQ-H2 might explain stronger excitonic coupling in the dimers of the former.
Finally, larger surface areas of SQ-Sl3 and SQ-Sl5 might explain stronger coupling between these dyes than expected from their hydrophobicity (Fig. S14ef). Hydrophobic effect may play a strong role in minimizing a large surface area of SQ-Sl3 and especially SQ-Sl5 while polar sulfo groups maintain contact with water.  In addition, we examined electron density distribution via electrostatic potential surfaces (Fig.  S15). The electrostatic potential is the energetic magnitude of interaction between a +1 positive charge and each point of molecular surface of constant electron density. In this regard, the S35 electrostatic potential surface serves as an equivalent to charge distribution. Electrostatic potentials revealed centrosymmetric electron density distribution in accordance with the D-A-D pattern characteristic for indolenine squaraines (where D is an electron-donor moiety and A is an electronacceptor moiety). Squaraine SQ-Cl2 was characterized by the most neutral electrostatic potential surface and a lack of static dipole moment. Figure S15. Electrostatic potential surfaces of custom squaraine dyes scaled to ±0.07 e/a.u.³. Red is the most negative electrostatic potential, blue is the most positive electrostatic potential, and green is zero electrostatic potential. The values of ground state dipole moments in Debye calculated for dyes in water are provided.
All electrostatic potential surfaces and static polarizability data were generated via DFT using Gaussian16 8 and GaussView 5.0.9. Chemical structures of squaraine dyes were created in Avogadro software 9 where dye geometries were initially approximated using the universal force field (UFF). 10 Next, the molecular structures in gas state were optimized using M06-2X exchange correlation functional 11 and the 6-31+** basis set. To generate an electrostatic potential surface, a volume was generated using 80 3 points for the volume for both the electrostatic potential and electron density with a self-consistent field. The isosurface was generated using a molecular orbital isovalue of 0.02 e/a.u.³ with a density isovalue of 0.004 e/a.u.³ The color scale was fixed for all electrostatic potential surfaces with -7e-2 at the red value and 7e-2 at the blue value.