Photophysical Properties and Protein Binding Studies of Piperazine-Substituted Anthracene-BODIPY Dyads for Antimicrobial Photodynamic Therapy

Abstract

This work presents the synthesis, characterisation, photophysical properties, time-resolved spectroscopic behaviour, and biological evaluation of two structurally distinct heavy-atom-free BODIPY-anthracene dyads (BDP-1) and the newly designed 2,6-bis[1-(tert-butyl) 4-(prop-2-yn-1-yl) piperazine-1,4-dicarboxylate] BODIPY-anthracene (BDP-2), incorporating 2,6-alkynyl-piperazine substituents for potential application in antimicrobial photodynamic therapy. BDP-1 exhibits absorption and emission maxima at 507 nm and 516 nm, respectively, with a Stokes shift of 344 cm^-1 in dichloromethane (DCM), characteristic of unsubstituted BODIPYs. In contrast, BDP-2 undergoes a red-shift in the absorption maximum to 552 nm (Stokes shift of 633 cm^-1), which is attributed to the extended conjugation from the introduction of the alkyne groups. Time-resolved infrared spectroscopy confirmed efficient spin-orbit charge transfer intersystem crossing, and nanosecond transient absorption studies confirmed the formation of a long-lived triplet state for BDP-2 (up to 138 µs in MeCN). A binding constant (K_b) of 9.6 × 10⁴ M^-1 was obtained for BDP-2 when titrated with bovine serum albumin (BSA), which is higher than comparable BODIPY derivatives. BDP-2 displayed improved hemocompatibility compared to BDP-1 (<5% haemolysis of human erythrocytes up to 200 μg·mL^-1). Antimicrobial activity of BDP-1 and BDP-2 was most potent when irradiated at 370 nm compared to the other wavelengths employed. However, BDP-2 did not retain the potent (6 log) and rapid (within 15 min) eradication of Staphylococcus aureus achieved by BDP-1 under irradiation at 370 nm. These findings demonstrate the rational design of BDP-2 as a biocompatible, and heavy-atom-free BODIPY offering promise for targeted antimicrobial photodynamic therapeutic applications.

Keywords: BODIPY; anthracene dyad; antimicrobial activity; fluorescence; heavy-atom-free; photodynamic therapy; triplet state.

Figure 1

BODIPY structures in this work.

Scheme 1

Synthetic pathway to BDP-2.

Figure 2

Absorption and emission spectra for BDP-1 and BDP-2 in DCM.

Figure 3

TRIR spectra of BDP-1 in CDCl₃, excited at 510 nm (time range, 0.2–8000 ps).

Figure 4

TRIR of BDP-2 in CDCl₃, excited at 540 nm (time range, 0–250 ns).

Figure 5

TRIR spectra following excitation of BDP-2 in CDCl₃ at 400 nm (0–5 ps): (a) In the fingerprint region, (b) in the alkynyl region, and (c) the fingerprint region from 5 to 9000 ps. (d) In the alkynyl region from 5 to 9000 ps.

Figure 6

Nanosecond transient absorption (ns-TAS) of BDP-2 in DCM, following excitation at 532 nm (0–750 µs).

Figure 7

Fluorescence quenching curves of BSA (50 µM) in PBS solution in the presence of different amounts (0–40 µM) of BDP-2. Insert: Stern–Volmer plot of the fluorescence titrations.

Figure 8

In vitro haemolysis studies of human erythrocytes for (a) BDP-1 and (b) BDP-2 at varying concentrations under dark conditions and when irradiated at 370 nm. Values shown are the mean ± SEM for triplicate assays. Statistically significant differences between the light and dark series are shown: *** p ≤ 0.0001. The red dotted line indicates 5% haemolysis.

Figure 9

Photodynamic bactericidal activity of BDP-1 (a) and BDP-2 (b) against two bacterial species. Assays containing 10% DMSO (controls) or compounds (100 μg·mL⁻¹) and approximately 10⁶ CFU·mL⁻¹ bacteria were incubated in the dark or irradiated for 60 min at 370 nm. Values shown are mean ± SEM for three separate assays performed in triplicate. The Y axis is logarithmic scale. Statistically significant differences between the reduction in CFU/mL for light-treated series (control minus BDP-treated) against E. coli vs. S. aureus are shown: * p ≤ 0.05, *** p ≤ 0.0001. For BDP1 with light treatment, bars representing zero CFU/mL are not visible.

Figure 10

Photodynamic bactericidal activity of (a) BDP-1 and (b) BDP-2 against S. aureus (ATCC 25923). Assays containing 10% DMSO (controls) or compounds (100 μg·mL⁻¹) and approximately 10⁶ CFU·mL⁻¹ bacteria were incubated in the dark or irradiated at 370 nm for 15, 30, and 60 min. Values shown are mean ± SEM for three separate assays performed in triplicate. The Y axis is a logarithmic scale. Statistically significant differences between the reduction in CFU/mL for the light-treated series (control minus BDP-treated) are shown: *** p ≤ 0.001, * p ≤ 0.05. For BDP1 with light treatment, bars representing zero CFU/mL are not visible.

Figure 11

Effect of irradiation wavelength on antimicrobial activity of (a) BDP-1 and (b) BDP-2. Bacterial killing activity at 100 μg/mL is shown against S. aureus (ATCC 25923). Assays were incubated in the dark or irradiated for 60 min at 370, 525, 550, and 570 nm. Values shown are mean ± SEM for three separate assays performed in triplicate. Statistically significant differences between the reduction in CFU/mL for light-treated series (control minus BDP-treated) are shown: *** p ≤ 0.001.