A novel set of symmetric methylene blue derivatives exhibits e ff ective bacteria photokilling – a structure – response study †

This study focuses on the structure–response relationship of symmetrically substituted phenothiazinium dyes. Four hydrophilic derivatives with the ability of additional hydrogen bonding (5, 6) or additional electrostatic interaction (3, 4) were synthesized, photophysically characterized and compared to the parent compound methylene blue (MB, 1) and a lipophilic derivative (2) without additional coordination sites. Derivative 5 was most effective against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli reaching a maximum photodynamic efficacy of >5log10 steps (≥99.999%) of bacteria killing in 10 minutes (5 μM, 30 J cm) without inherent dark toxicity after one single treatment with the incoherent light source PDT1200 (λmax = 660 nm, 50 mW cm ). Interestingly, one derivative with two additional primary positive charges (3) showed selective killing of Escherichia coli (5 μM, 30 J cm, 4log10 steps inactivation (≥99.99%)) and no antimicrobial effect on Staphylococcus aureus. This might allow the development of a new generation of photosensitizers with higher antimicrobial efficacy and selectivity for future applications.

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Accepted Manuscript
Photochemical & Photobiological Sciences 3 INTRODUCTION: Multiple drug resistance is one of the upcoming threats of our century. 1 This means, the disease-causing microorganism is able to resist different antimicrobials, especially antibiotics, but also antifungal or antiviral drugs. 2 Thus many conventional antibacterial strategies fail and there exists an increasing spread of multi-resistant bacteria. 3,4 Besides the development of novel antibiotics, other methods for controlling the spread of pathogenic bacteria have been extensively studied. [5][6][7][8] Especially useful are disinfection methods, which minimize selection-pressure, unlike antibiotics. One of the most promising methods is the photodynamic inactivation of bacteria (PIB). 4 The bacteria are incubated with per se non-toxic dyes (photosensitizers, PS). After a short time of incubation, the PS can be excited by visible light. This leads to the production of highly reactive oxygen species (ROS) directly at the bacteria during illumination, which oxidatively damage cellular structures such as membranes or DNA and as a result the bacteria are killed 9 . Among the ROS generated, it is well acknowledged that singlet oxygen ( 1 O 2 ) plays the major role in these photodynamic reactions. 10 Phenothiazinium dyes belong to the most prominent class of such PS due to their absorption in the red region of the visible spectrum (ε > 5·10 4 L·mol −1 ·cm −1 , λ max ~ 660 nm), their low dark toxicity and their attachment and penetration abilities. 10,11 MB 1 and other phenothiazinium derivatives have shown promising antimicrobial photodynamic efficacy towards bacteria such as Staphylococcus aureus (S. aureus), 12 Escherichia coli (E. coli) 13,14 and methicillin resistant S. aureus. 15 These PS are also effective against funguses such as Candida species, 16,17 tropical pathogens 18 and viruses, 19 and are therefore applied in the antimicrobial field. MB 1 and its known derivatives have shown to achieve a log 10 -reduction of >5 log 10 steps (>99.999 %) of bacteria at light doses up to 30 J cm -2 , using intensities of 125 mW cm -2 in a concentration range of 2 to 10 µM in suspension. 20 Phenothiazinium compounds are frequently used for PIB in oral treatments, especially in endodontics. Incubation times and total illumination times differ, but are usually in the minute 4 time scale. [21][22][23][24][25] In all these cited studies an antimicrobial efficacy of 5 to 6 log 10 reduction was achieved. In nearly all the studies covering phenothiazinium compounds their dark toxicity became apparent. The authors observed at least 1 log step of reduction of the number of bacteria without illumination. For clinical application dark toxicity is a critical factor. It is a key factor of each photosensitizer that the photodynamic process can be controlled by light.
The synthesis of MB 1 and other phenothiazinium derivatives was summarized by Wainwright et al. 26 Mostly simple substituents like alkyl, alkylaryl or hydroxyalkyl residues were introduced at the auxochromic sites. [27][28][29] Examples of phenothiazine dyes with highly polar substituents or additional coordination sites are rare. 30 Only one example carrying multiple positive charges was presented, but no antimicrobial efficacy or selectivity was published. 31 Structure response relationships between these substitution patterns also comparing lipophilic examples are missing.
Gram-negative bacteria like E. coli are more difficult to inactivate by PIB. This is a result of their altered cell wall structure and cellular architecture in comparison to Gram-positive bacteria. 32 As there is no need of the PS penetrating the cell membrane for good PIB efficacy , 33 a hydrophilic character of the dye is not a disadvantage, as long as it is ensured that most of the PS cannot be washed away of the target organism. Additional electrostatic binding sites or the ability to establish additional hydrogen bonds support stronger and faster attachment to the cell wall of bacteria. In addition, these structural features may also lead to desirable selectively and/or increased efficacy against Gram-negative bacteria. In contrast to also effective but dark toxic lipophilic dye amphiphiles, which penetrate the cell wall and localize intracellular, 34 hydrophilic dyes should not penetrate the bacterial wall. Disorders in the membrane or interaction with DNA or RNA are avoided and therefore dark toxicity is on a lower level.
Recently, we reported on phenothiazinium derivatives with one altered substituent on the auxochromic sites (Fig. 2). 35 With the ability of additional hydrogen bonding and/or 5 additional cationic charges the derivatives have shown to be highly effective upon illumination against S. aureus and E. coli with up to 7 log 10 steps without inherent dark toxicities. The additional positive charges in the substituents also proved to be advantageous suppressing aggregation and therefore enabled to expand the therapeutic concentration window. Six membered ring substituents enhanced the photostability of the compounds. In general a singlet oxygen quantum yield (Φ ∆ ) of 30 -40 % was determined for compound 7 -  The scope of published studies only covers lipophilic to moderately hydrophilic phenothiazinium derivatives. Until now, hydrophilic or very hydrophilic derivatives were not covered systematically.

12.
Aim of the present study is to investigate the antibacterial effect of additional positive charges or hydrogen bond acceptors on symmetrically substituted, now hydrophilic phenothiazine derivatives (Fig. 1). More polarity of the molecules might cause the PS to remain outside the cell, suppressing dark toxicity. Positive charges might though lead to better attachment to the exterior of the cell and fast antimicrobial action. 33 In addition, improved and selective killing of Gram-negative bacteria may occur. Two substituents in the lead structure MB 1 were changed in order to examine the influence of these substituents on photophysical properties such as Φ ∆ , stability and aggregation. Furthermore the new set of symmetrically 3,7substituted phenothiazinium derivatives is compared with the asymmetrically one-fold substituted derivatives published earlier by our group to establish a structure-relationship of these novel set of compounds. 35 Few of the compounds are known as an iodide or bromide salt, but not as a chloride. As iodides can react with singlet oxygen, we consequently investigated the chloride salts of all compounds for the first time.

General materials and methods
Commercial reagents and starting materials were purchased from Acros Organics, TCI

Synthesis and Purification of the compounds
MB chloride was purchased from Sigma Aldrich (Germany) and was used without further purification.

Synthesis of boc-protected methyleneblue derivatives
The boc-protected amine (2 mmol) was added dropwise to a well-stirring solution of phenothiazinium tetraiodide monohydrate (730 mg, 1 mmol) in methanol (100 mL) at room temperature under nitrogen. The reaction mixture was stirred for 6 h and evaporated under reduced pressure to leave a dark residue, which was immediately used for the second step without further purification. To a solution of this salt in dichloromethane (200 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 second portion of the boc-protected amine (3 mmol) in dichloromethane (100 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. (17)
The solution was transferred to four blue caps, the product was precipitated by addition of diethylether (13.5 mL per tube) and centrifuged. The solution was decanted off the precipitate, it was resuspended in diethylether (15 mL per tube) and settled by centrifugation again. The solvent was decanted off and the residue was dried at reduced pressure without heating. 13

Ion exchange protocol for methyleneblue derivatives
The column was packed with ion exchanger (Amberlite IRA-958). The resin was rinsed with acidic sodium chloride solution (10 % aqueous NaCl cont. 0.1 % HCl, 100 mL) and conditioned with dilute hydrochloric acid (0.1 %) / acetonitrile / methanol (3:1:1). The MB derivative (0.5 mmol) was dissolved in hydrochloric acid (1M, 10 mL) and lyophilized. A solution of this mixed salt was dissolved in hydrochloric acid (0.1 %) / acetonitrile / methanol (3:1:1) (6 mL) and was slowly passed through a column (height 10 cm, diameter 1 cm; transferred with 4 mL of the solvent mixture to the conditioned anion exchanger (Amberlite IRA-958) eluting with 20 mL of the solvent mixutre. The solvents methanol and acetonitrile were evaporated at reduced pressure not exciding a water bath temperature of 40 °C. The aqueous solution was lyophilized to give the product as dark blue solid. Lithuania) was used, which has a frequency of f = 1 kHz. Upon irradiation, the transmission T was recorded. The absorption was calculated by the following equation:
These settings are comparable with the settings that were used by Felgentraeger et al. to examine asymmetrically substituted phenothiazinium derivatives which are also compared in this study. 35 For the uptake / attachment experiments of (3), the bacteria (OD = 0.6) were incubated with 10 µM of (3) for 60 min (500 µL bacterial suspension + 500 µL of (3) in H 2 O, in Eppendorf tubes 1.5 mL) to get a final concentration of 5 µM. Upon incubation, the bacteria were centrifuged (13000 rpm, 5 min) and the absorption of the supernatant was determined.
Subsequently a washing step was performed, the bacterial pellet was resuspended and again centrifuged and the absorption of the second supernatant was recorded.
The polarity of the PS was estimated by measuring the octanol-water coefficient. Distribution of 1·10 -4 mol of each phenothiazinium salt between both phases was measured by UV/Vis spectroscopy after 10 minutes of stirring at room temperature.

Direct measurement of singlet oxygen
In order to produce singlet oxygen, the respective PS were filled in a quartz cuvette and For the uptake / attachment experiments of (3), the bacteria were centrifuged (13000 rpm, 5 min) after incubation and a washing step was performed afterwards. This procedure was performed twice. Subsequently, the bacteria were irradiated and treated as mentioned above. In this study we used chloride (Cl -) as the respective counterion for all positively charged derivatives, because iodide (I -) as the counterion can react with singlet oxygen to triiodide. 41 Therefore iodide as the counterion may consume singlet oxygen and influences the photodynamic action of the PS. No influence of chloride as a counterion on ROS generation is known.

Synthesis
Compound 2-I 34 and compound 6-I 42 are literature known as iodide salt (see scheme 3). 34,43 We decided to investigate 2-I as the chloride salt (2).
Phenothiazinium tetraiodide (14), 36 freshly prepared from phenothiazine (13), was converted to MB (1) (scheme 1) and a variety of bis-fold substituted derivatives (schemes 2, 3) by reaction with secondary amines in dichloromethane. Here, we investigated the aggregation of the novel derivatives within a concentration range from 10 µM to 200 µM (Table 1, Fig. 3). In Table 1 the absorption maximum of each derivative is specified. The peak of (3) at 617 nm does not match the peak of (7), the corresponding asymmetric derivative. The other molecules have the same absorption maxima as the asymmetric analogues. The peak of (5) and (6) match the absorption peak of (1) within experimental accuracy, as do the corresponding asymmetric derivatives 11 and 12, respectively, which were analyzed by Felgentraeger et al.. 35 Compared to the asymmetric 25 derivative 12, (6) does not show any dimerization within experimental accuracy. Further, (4) does not show any dimerization effect such as the asymmetric analogue (10). However, (3) shows a weak red shift with increasing concentration (Fig. 3

left). Long term experiments
have shown that the aggregation state of (3) is stable, because the absorption maxima did not shift over time (6 months The lipophilic derivative 2 shows the formation of an absorption peak at 627 nm and the main peak, which is assigned to the monomer at 679 nm, is diminished with increasing PS concentration. This effect is known as hypochromicity. Both effects are known for (1) (vide supra). In line with this, (5) shows similar spectroscopic behavior as (2), but has a much weaker aggregation tendency than the latter. The hypochromic effect is observed at higher concentration, than for (2). Consequently, the new symmetric and hydrophilic derivatives 3 -6 show not such a pronounced (3,5) or no aggregation behavior (4, 6) such as MB 1, which might influence the phototoxic efficacy of the respective dyes in a positive manner. The suppressed tendency to aggregate is also beneficial, because a larger concentration span can be used without negative influence on the photophysics of the compounds. impact on the degradation of MB 1. 47 The authors suggest that the decomposition of (1) and its derivatives is, to a large extent, not due to an O 2 oxidation reaction but most likely an excited state reaction, such as the reaction of the long-lived triplet state with another molecule or the solvent. 47 The triplet state of (1) might be able to abstract an electron or H atom to form MB • and OH • radicals (type-I mechanism of action), which in turn, react with bacteria membranes to form lipid hydroperoxides leading to membrane damage. 47   asymmetrically substituted phenothiazinium derivatives. The molecular weight of each molecule is given as g/mol. λ abs, max describes the absorption maxima of the respective dyes, the dimerization was measured in a concentration range between 10 µM and 200 µM, the photostability is described by the ratio of the height of the absorption maxima upon irradiation to the height of the absorption maxima before irradiation with 250 000 laser pulses at 600 nm equals a total energy of 16.25 J (power was 65 mW for 250 s).

27
In our study the phenothiazinium derivatives were diluted to a final concentration of 5 µM and irradiated at 600 nm for 250 s with a power of 65 mW yielding an energy of 16.25 J.
Hereby, the same amount of light energy per time unit is absorbed by each derivative.
Photostability was recorded by absorption spectroscopy. The value to estimate photostability was given with the ratio of the absorption maxima after and before irradiation (Table 1). It was shown that only (3) is photoinstable for energies up to 16.25 J (Fig. 4). occurs of the whole ring system, as commonly known for MB 1. 26 In contrast, simple alkyl chains as in (2) are less in risk to be oxidized and to degrade, resulting in a far higher stability.
Six-membered rings are known to be very stable moieties in organic chemistry. Ring-opening and degradation is even more hampered. Thus, the cyclic substituents in (4) and (6) are more 30 stabilized and less accessible to photodegradation by the common mechanism and show the highest photostabilities of all compounds presented.
Singlet oxygen quantum yield (Φ ∆ ), effective singlet oxygen toxicity and polarity of the derivatives.
The main advantage of determining Φ ∆ of a PS by the direct measurement of singlet oxygen photons at 1270 nm compared to indirect detection of singlet oxygen by spectroscopic probes is that no other radicals are detected with this method. 50 Table 2  The polarity of the PS was estimated by measuring the octanol-water coefficient. Distribution of 1·10 -4 mol of each phenothiazinium salt between both phases was measured by UV/Vis spectroscopy after 10 minutes of stirring at room temperature.   TMPyP (10 µM) as reference PS whereas Φ ∆ of (1) is displayed as literature value. 51 The quantum yields of the PS are shown in the following order (Eq. 1) and are summarized in Table 2: However, Chen et al. proposed that although singlet oxygen is highly important, the rate of bacteria inactivation is determined by the binding of the dye to the bacteria. 47 In our study we evaluated the effective phototoxicity of singlet oxygen also taking the emission of the light source, the absorption of the PS and Φ ∆ into account. 32 In order to estimate the effectiveness of singlet oxygen that is generated by the absorbed light energy of the different derivatives in the phototoxicity experiments, the values of the emission spectrum "Em" of the light source were convolved with the values for the absolute absorption "Abs" of the respective dyes for the spectral region between 500 nm and 800 nm and multiplied with the Φ ∆ of the phenothiazinium derivatives. 35 Hereby, the absorption of the respective dyes at a concentration of 10 µM was measured. According to the following formula (Eq. 2) an effective toxicity, that might be caused by singlet oxygen, "Eff. Tox. of 1 O 2 " was predicted for each PS: This formula accounts for the effective absorbed energy (i.e. the sum of the product of emission and absorption) of every PS that is used to generate singlet oxygen.

Photobiological studies and photodynamic inactivation of bacteria (PIB)
In our study we investigated the phototoxicity of symmetrically substituted phenothiazinium derivatives against S. aureus and E. coli as representatives for Gram-positive and Gramnegative bacteria.

Mode of action of the phenothiazinium derivatives.
The bacteria were incubated either for 10 min or for 60 min with the respective dyes. The log 10 -reduction of the respective novel PS molecules are presented in Table 3 10'  Different concentrations of the respective PS (1) to (6) were applied and the toxic efficacy is described in steps of log 10 -reduction, "-" means a reduction of < 1 log 10 steps (< 90 %), < 3:  concentrations of the respective PS 1 to 6 were applied and the toxic efficacy is described in steps of log 10 -reduction, "-" means a reduction of < 1 log 10 steps (< 90 %),  In general, Gram-positive bacteria are better accessible by PS, due to their cytoplasmic membrane being surrounded by a relatively porous layer of peptidoglycan and lipoteichoic acid. 54 In succession, they are more easily inactivated by antimicrobial photodynamic therapy than Gram-negative bacteria. 32 Figure 5 shows the inactivation of E. coli by derivative 3. In contrast to this general model,  55 Polycyclodextrin structures can saturate with high concentrations of MB 1. 56 The binding strength to (1) is strongly enhanced, when the sugar moieties carry negatively charged substituents like carboxylates. 57 The stability of these dyes in cyclodextrins is also enhanced, by a protective effect of the water-shielding sugar planes. A similar binding event may take place on the surface of E. coli. The phenothiazinium derivatives diffuse to the area of negative charge accumulation on the lower end of the sugar chains and bind to the lipopolysaccharides. 58 This can be explained as follows: (3) is strongly bound by the multiple electrostatic interactions with its positively charged groups and is then "included" by the dense sitting sugar planes protecting the chromophore from destructive influences of the medium. Upon irradiation a sufficient concentration of singlet oxygen is generated close to the thin cell wall of the Gramnegative bacterium causing much more severe damage to the thick layer in S. aureus. Due to the additional stabilization of the dye by the assumed "inclusion", the PS is photodynamically active over a longer period of time, when sitting on the surface of the Gram-negative bacteria.
If the compound is localized on the exterior, a stronger photobleaching can be assumed. The first supernatants show that (3) attaches to both bacterial strains.
It is well acknowledged that positive charges at a PS lead structure allow better attachment to the exterior wall of bacteria. Surprisingly, and as mentioned above, S. aureus was not affected by the PIB treatment when (3) is used. Therefore, further experiments were conducted in order to examine the structure-response relationship of this compound in particular. Uptake / attachment experiments show that the compound attaches to both bacterial strains, Grampositive and Gram-negative bacteria, in a comparable amount (Fig. 6). In the first supernatant it can be seen that slightly more dye is detached from S. aureus, but within experimental 39 accuracy this does not explain the fact that S. aureus is not inactivated at all (Fig. 5). The phototoxicity studies that were performed with additional washing steps show the same effect as the phototoxicity experiments in suspension, E. coli was inactivated whereas S. aureus was not. The above mentioned explanation regarding localization, stabilization and attachment reflects a possible mechanism being responsible for our observations. However, further studies are in progress and shall be presented in a publication in the future, as improving PIB against Gram-negative species is an important issue in current research. 59 Derivative 6, an analogue with cyclic ether substituents, showed better efficacy in comparison to (4), which carries a positively charged nitrogen atom in the six-membered rings located at 3-and 7-position of the phenothiazinium chromophore (Fig. 7).
The compounds 5 and 6 proved to be the most potent examples investigated in this study, with an antimicrobial efficacy of > 7 log 10 steps at only 5 µM PS concentration at the given irradiation conditions (Fig. 7). They are characterized by a good balance between lipophilicity and hydrophilicity, and have the ability to develop hydrogen bonds in addition to the bond by its positively charged center part. This enables good penetration and adhesion of the dyes in the peptidoglycan structure and also in the lipopolysaccharide layer. Thus, the cell wall of Gram-negative, as well as the one of Gram-positive bacteria is saturated with enough PS to cause quick and severe damage to both species upon illumination. 42 Correlation of the phototoxicity tests with the singlet oxygen quantum yield.
In Eq. 3 the order of Φ ∆ of the different derivatives is displayed. The most effective PS in regard to singlet oxygen generation in our study is (4), followed by MB 1 (Table 2). It can clearly be depicted from the toxicity tests that the quantum yields cannot be correlated to the log 10 -reduction. Exemplarily, despite its high Φ ∆ , (4) has a low phototoxic efficacy compared to (1), (5), (6) and (2). Vice versa, (6), exhibiting the lowest Φ ∆ in this study, showed a higher photokilling compared to (4). Using the herein presented calculated values for the effective toxicity for singlet oxygen ("Eff. Tox. of 1 O 2 ", table 2), which additionally takes the absorbed light into account, a better correlation between predicted singlet oxygen toxicity and our toxicity data was possible: Eff. Tox. of 1 O 2 , order of effectivity: (1) ≈ (5) > (6) ≈ (2) > (4) > (3) The toxicity data show the highest effect with (1), (5) and (6), which is reflected by the value for Eff. Tox. of 1 O 2 . Also, (2) shows a high toxicity, corresponding to the predicted effective 1 O 2 toxicity, but since a substantial dark toxicity adds to this killing effect it will not be taken into further consideration for the correlation. The two lowest values for 1 O 2 toxicity, determined for (3) and (4), reflect the low photokilling of these two PS. A value for predicting the overall toxicity, however, clearly must take many more features, like PS binding ability,

CONCLUSIONS:
The four hydrophilic derivatives with the ability of additional hydrogen bonding (5, 6) or additional electrostatic interaction (3,4) showed fast and effective antimicrobial action against bacteria. With Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli the most effective derivative 5 reached a maximum efficacy of > 5 log 10 steps (≥ 99.999 %) of bacteria killing in 10 minutes (5 µM, 30 J cm -2 ) after one single treatment with the incoherent light source PDT1200 (λ max = 660 nm, 50 mW cm -2 ). In contrast to the parent compound 1 and the lipophilic derivative 2 they showed no inherent dark toxicity.
In this study we confirmed, that phenothiazinium derivatives with cyclic substituents at the auxochromic positions are more stable than acyclic analogs. All hydrophilic derivatives showed good photostability and neglectable aggregation behavior. Additional positive charges are advantageous to suppress aggregation of the compounds. In the concentration range up to 200 µM no aggregation can be observed especially for compound (4) and (6). This might improve the PIB application by extension of the therapeutic concentration window.
In addition we identified one derivative with unique antimicrobial selectivity. Compound 3, comprising two additional primary positive charges, was selective and effective against Gram-negative Escherichia coli (5 µM, 4 log 10 steps inactivation) over Gram-positive Staphylococcus aureus (100 µM, < 1 log 10 steps inactivation). After correlation to the photophysical and chemical properties of the PS, a reasonable explanation might be that (3) is strongly bound by the multiple electrostatic interactions with its positively charged groups and is then "included" by the dense sitting sugar planes on the surface of the Gram-negative bacterium. Upon irradiation a sufficient concentration of singlet oxygen might be generated close to the thin cell wall of E.coli causing much more severe damage than to the thick layer in S. aureus.
Ongoing experiments aim at more insight of the proposed mode of action, which will be the focus of a subsequent publication.
This new generation of phenothiazinium derivatives might allow the development of more efficient PS with shorter process times and higher antimicrobial activity in comparison to MB 1 and its well-known derivatives.