A membrane intercalating metal-free conjugated organic photosensitizer for bacterial photodynamic inactivation

Photodynamic inhibition (PDI) of bacteria represents a powerful strategy for dealing with multidrug-resistant pathogens and infections, as it exhibits minimal development of antibiotic resistance. The PDI action stems from the generation of a triplet state in the photosensitizer (PS), which subsequently transfers energy or electrons to molecular oxygen, resulting in the formation of reactive oxygen species (ROS). These ROS are then able to damage cells, eventually causing bacterial eradication. Enhancing the efficacy of PDI includes the introduction of heavy atoms to augment triplet generation in the PS, as well as membrane intercalation to circumvent the problem of the short lifetime of ROS. However, the former approach can pose safety and environmental concerns, while achieving stable membrane partitioning remains challenging due to the complex outer envelope of bacteria. Here, we introduce a novel PS, consisting of a metal-free donor–acceptor thiophene-based conjugate molecule (BV-1). It presents several advantageous features for achieving effective PDI, namely: (i) it exhibits strong light absorption due to the conjugated donor–acceptor moieties; (ii) it exhibits spontaneous and stable membrane partitioning thanks to its amphiphilicity, accompanied by a strong fluorescence turn-on; (iii) it undergoes metal-free intersystem crossing, which occurs preferentially when the molecule resides in the membrane. All these properties, which we rationalized via optical spectroscopies and calculations, enable the effective eradication of Escherichia coli, with an inhibition concentration that is below that of current state-of-the-art treatments. Our approach holds significant potential for the development of new PS for controlling bacterial infections, particularly those caused by Gram-negative bacteria.


Figure S2
13 C NMR in DMSO-d 6

Computational methods
To ensure the full reproducibility of our computational results, we provide here a complete account of our setup. The optoelectronic properties of BV-1 have been investigated in the framework of (time dependent) density functional theory using the ORCA suite of programs. 1,2 The starting configurations of the molecule have been found using a conformer-rotamer search algorithm based on the GFN2-xTB tight-binding Hamiltonian. [3][4][5] A few lowest-energy conformers have been then fully optimized using a revised PBE0 hybrid functional with 37.5% of exact exchange, 6 suitable for accurately describing the strong charge-transfer excitation which dominates the absorption spectrum of BV-1, and the D3BJ dispersion correction. 7 The Kohn-Sham orbitals have been expanded on a Gaussian-type def2-TZVPP basis set. 8 The corresponding def2/J basis was also used as an auxiliary basis set for Coulomb fitting in a resolution-of-identity/chain-of-spheres (RIJCOSX) framework.
Time-dependent DFT calculations have been performed on lowest-energy conformers using the same functional and the same basis sets; a large basis of 600 vectors connecting occupied and unoccupied Kohn-Sham orbitals has been used for the calculations of the first 30 electronic transitions of the absorption spectrum of the molecule. Solvent effects have been included in the calculations through the linear-response conductor-like polarizable continuum model (LR-CPCM). 9 Absorption and emission spectra in different solvents, including vibrational contributions, have been calculated using the excited-state dynamics (ESD) module implemented in ORCA. 10 Excited-state absorption (ESA) spectra have been calculated using the expectation value formalism, and are shown as Gaussian convolution of electronic transitions (FWHM = 4000 cm -1 ). These transitions have been calculated both in a vertical configuration, i.e. in the ground state optimized geometry, as well as in the adiabatic configuration, i.e. in the S1 (and T1) excited-state optimized geometry. The vertical transitions can be interpreted as the limit for t=0, which correspond to the time-gated spectra taken at short probe delays before the conformational relaxation occurs. The adiabatic transitions, on the other hand, represent the limit for longer probe delays, reproducing the condition of the completely relaxed system. In all the examined solvents, we observe a blue shift of the ESA transitions going from the vertical to the adiabatic configuration that is consistent with the results of ultrafast spectroscopy measurements. Note that the computed theoretical values are shifted at higher energies with respect to the experimental values, due to computational details. The optimized geometries in Cartesian coordinates (in Å) of the BV-1 ground and S1 excited states in water are reported below.

Figure S8
HOMO and LUMO orbitals of BV-1 calculated at the revPBE0@def2-TZVPP level of theory and density difference between the ground and S1 excited states in the geometry of the ground state (vertical). The red arrow indicates the displacement of charge density from blue to red regions upon GS→S1 excitation (charge transfer excited state). 18

Figure S10
Jablonski diagrams of singlet and triplet excited states of BV-1 in vacuum and in solution (dichloromethane, ethanol and water). Vertical (calculated in the ground-state structure) and adiabatic (in the S1 excited-state structure) distributions are shown. Possible intersystem conversion (ISC) pathways are also indicated by dashed lines.

Figure S11
Transient absorption dynamics of BV-1 at different probe wavelengths.

Figure S12
Excited-state absorption (ESA) from the S1 (left panels) and T1 (right panels) excited states in three solvents (dichloromethane, ethanol and water). ESA have been calculated both in the vertical (in the ground-state structure) and in the adiabatic (in the S1 excited-state structure for S1 ESA and in S1 and T1 structures for T1 ESA, labelled as ad1 and ad2, respectively) transition geometries. The