Hydrogen Bond Acceptors and Additional Cationic Charges in Methylene Blue Derivatives: Photophysics and Antimicrobial Efficiency

Photodynamic inactivation of bacteria (PIB) by efficient singlet oxygen photosensitizers might be a beneficial alternative to antibiotics in the struggle against multiresistant bacteria. Phenothiazinium dyes belong to the most prominent classes of such sensitizers due to their intense absorption in the red-light region (λ abs, max ca. 600–680 nm, ε > 50000 L mol−1 cm−1), their low toxicity, and their attachment/penetration abilities. Except simple substituents like alkyl or hydroxyalkyl residues, nearly no modifications of the phenothiaziniums have been pursued at the auxochromic sites. By this, the properties of methylene blue derivatives and their fields of application are limited; it remains unclear if their potential antimicrobial efficacy may be enhanced, also to compete with porphyrins. We prepared a set of six mainly novel methylene blue derivatives with the ability of additional hydrogen bonding and/or additional cationic charges to study the substituents' effect on their activity/toxicity profiles and photophysical properties. Direct detection of singlet oxygen was performed at 1270 nm and the singlet oxygen quantum yields were determined. In suspensions with both, Gram-positive and Gram-negative bacteria, some derivatives were highly active upon illumination to inactivate S. aureus and E. coli up to 7 log10 steps (99.99999%) without inherent toxicities in the nonirradiated state.

over a period of 2h, keeping the temperature between 2-5°C. After stirring over night at room temperature, the solvent and the excess amine were removed at reduced pressure. The crude material was purified by column chromatography with silica gel using chloroform/methanol 10:1  6:1 as the eluent, to give the bromide salt of 11 as colourless solid (1.83 g, 7.26 mmol, 73 %).

General procedure I: Synthesis of boc-protected methyleneblue derivatives
To a solution of 3-Dimethylaminophenothiazinium triiodide (10, 1.24 g, 2 mmol) in dichloromethane (500 mL) was added dropwise a solution of triethylamine (0.3 g, 0.4 mL, 3 mmol) in dichloromethane (50 mL). After stirring for 5 minutes the appropriate amine (6 mmol) in dichloromethane (250 mL) was added over a period of 2 h. The solution was stirred over night at room temperature and was then washed with water (3x 250 mL). The organic layer was dried over MgSO 4 and the solvent was evaporated at reduced pressure not exciding a water bath temperature of 40°C. The crude material was purified by repeated flash chromatography with silica gel using dichloromethane/ethanol 10:1 as the eluent.
The TFA salt from the former step was dissolved in double distilled water (5 mL) and was passed through the column (height 10 cm, diameter 1 cm) of anion exchanger (Amberlite IRA-958) eluting with water (40 mL). The aqueous solution was lyophilized to give the product as dark blue solid.

General procedure III: Synthesis of asymmetric methyleneblue derivatives
To a solution of 3-Dimethylaminophenothiazinium triiodide (10, 1.24 g, 2 mmol) in dichloromethane (360 mL) was added dropwise a solution of the appropriate amine (12 mmol) in dichloromethane (40 mL) over a period of 1 h. The solution was stirred over night at room temperature and was then washed with water (1x 200 mL). The organic layer was dried over MgSO 4 and the solvent was evaporated at reduced pressure not exciding a water bath temperature of 40°C. The crude material was dissolved in dichloromethane (10 mL) and precipitated by addition of diethylether (50 mL). The precipitate was settled by the aid of a centrifuge, the supernatant was decanted off and the dark blue residue was purified by repeated flash chromatography with silica gel.
The methyleneblue derivative (0.5 mmol) was dissolved in hydrochloric acid (1M, 10 mL) and lyophilized. A solution of this mixed salt was dissolved in double distilled water (6 mL) and was slowly passed through a column (height 10 cm, diameter 1 cm; transferred with 4 mL dilute hydrochloric acid 0.1%) of the conditioned anion exchanger (Amberlite IRA-958) eluting with dilute hydrochloric acid (40 mL, 0.1 %). The aqueous solution was lyophilized to give the product as dark blue solid.  (5)