Improving photodynamic inactivation of bacteria in dentistry: Highly effective and fast killing of oral key pathogens with novel tooth-colored type-II photosensitizers

Article abstract of DOI:10.1021/jm4019492

Increasing antibiotic resistances in microorganisms create serious problems in public health. This demands alternative approaches for killing pathogens to supplement standard treatment methods. Photodynamic inactivation of bacteria (PIB) uses light activated photosensitizers (PS) to generate reactive oxygen species immediately upon illumination, inducing lethal phototoxicity. Positively charged phenalen-1-one derivatives are a new generation of PS for light-mediated killing of pathogens with outstanding singlet oxygen quantum yield Φ_Δ of >97%. Upon irradiation with a standard photopolymerizer light (bluephase C8, 1260 ± 50 mW/cm²) the PS showed high activity against the oral key pathogens Enterococcus faecalis, Actinomyces naeslundii, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans. At a concentration of 10 μM, a maximum efficacy of more than 6 log₁₀ steps (≥99.9999%) of bacteria killing is reached in less than 1 min (light dose 50 J/cm²) after one single treatment. The pyridinium substituent as positively charged moiety is especially advantageous for antimicrobial action.

Full text of DOI:10.1021/jm4019492

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Article
Improving photodynamic inactivation of bacteria in
dentistry: Highly effective and fast killing of oral key
pathogens with novel tooth-colored type-II photosensitizers
Andreas Spaeth, Christoph Leibl, Fabian Cieplik, Karin Lehner, Johannes
Regensburger, Karl-Anton Hiller, Wolfgang Bäumler, Gottfried Schmalz, and Tim Maisch
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4019492 • Publication Date (Web): 02 Jun 2014
Downloaded from http://pubs.acs.org on June 9, 2014
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Improving photodynamic inactivation of bacteria
in dentistry: Highly effective and fast killing of
oral key pathogens with novel
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toothꢀcolored typeꢀII photosensitizers
Andreas Späth #*, Christoph Leibl ‡, Fabian Cieplik ‡, Karin Lehner $, Johannes
Regensburger †, Karl-Anton Hiller ‡, Wolfgang Bäumler †, Gottfried Schmalz ‡, Tim Maisch
†
# Department of Organic Chemistry, University of Regensburg, Germany
‡ Department of Operative Dentistry and Periodontology, University Medical Center
Regensburg, Germany
† Department of Dermatology, University Medical Center Regensburg, Germany
* Corresponding Author (eꢀmail: andreas.spaeth@chemie.uniꢀregensburg.de; phone: +49ꢀ
941ꢀ943ꢀ4087)
KEYWORDS: singlet oxygen, photodynamic inactivation, PIB, 7ꢀperinaphthenone, phenalenꢀ
1ꢀone, antimicrobial, photosensitizer, typeꢀII, oral key pathogen, dental, toothꢀcolored
ABSTRACT: Increasing antibiotic resistances in microorganisms involve serious problems in
public health. This demands alternative approaches for killing pathogens to supplement
standard treatment methods. Photodynamic inactivation of bacteria (PIB) uses light activated
photosensitizers (PS) to generate reactive oxygen species immediately upon illumination,
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inducing lethal phototoxicity. Positively charged phenalenꢀ1ꢀone derivatives are a new
generation of photosensitizers for lightꢀmediated killing of pathogens with outstanding singlet
oxygen quantum yield Φꢁ of > 97%. Upon irradiation with a standard photopolymerizer light
(bluephase® C8, 1260 ± 50 mW/cm2) the photosensitizers showed high activity against the
oral key pathogens Enterococcus faecalis, Actinomyces naeslundii, Streptococcus mutans and
Aggregatibacter actinomycetemcomitans. At a concentration of 10 µM a maximum efficacy
of more than 6 log10 steps (≥ 99.9999 %) of bacteria killing is reached in less than one minute
(light dose 50 J/cm2) after one single treatment. The pyridinium substituent as positively
charged moiety is especially advantageous for antimicrobial action.
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INTRODUCTION: Increasingly stringent hygiene standards and the worldwide spread of
infectious nosocomial diseases1 have led to a high interest in compounds, methods and
applications, which prevent the growth of multiresistant microbes. The search for alternatives
to antibiotic treatment is of enormous significance for the treatment of pathogen infections,
especially in the wake of the appearance of the first strains of multiresistant bacteria (MRSA)
and arising vancomycin resistant strains (VRSA) in 2002.2
Although the need for novel antimicrobial active agents exists, nearly no new antibiotics are
being developed, due to exploding development costs and the loss of profitability in this
marketing sector.3,4 Even worse, halfꢀlife times until resistances develop have gotten shorter
when compared to the situation 10 years ago.4 Furthermore in March 2013 the Nature News
Blog highlighted the importance of immediate international action to combat drugꢀresistant
bacteria which will otherwise pose a “catastrophic threat”.5
One alternative treatment is photodynamic inactivation of bacteria (PIB),6 which uses
chromophors, called photosensitizers (PS), which can transfer either charge (typeꢀI) or energy
(typeꢀII) to oxygen to generate reactive oxygen species (ROS) after being illuminated with
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visible light. In case of a typeꢀII reaction the highly reactive singlet oxygen is generated
which kills bacteria via oxidative burst.7
6
Most commonly used photosensitizers are phenothiazines like methylene blue8, which
predominantly act via typeꢀI mechanism of action, or porphyrines like TMPyP9 and their
derivatives or analogs.10 In any case, nearly no novel classes of photosensitizers have been
developed in the last years except fullerenes11.
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ROS like superoxide (O2 ), hydrogen peroxide (H2O2) or the free hydroxyl radical (HO•) are
•−
ubiquitous in bacterial life.12 Bacteria express superoxide dismutases (SOD), catalases and
peroxidases as a defense mechanism against this kind of oxidative stress. These enzymes are
able to catalyze the reaction of ROS to H2O and molecular oxygen,13 resulting in a defense
mechanism effective against the active agents generated by typeꢀI mechanism of the
photodynamic processes. Signs of this exist, such as the fact as expression of SOD increased
in Staphylococcus aureus after treatment with oxidative stressꢀgenerating agents14 and SODꢀ
activity of S. aureus was increased after photodynamic treatment with Protoporphyrin IX in
PIBꢀsusceptible strains.15 A strainꢀdependent phenomenon of PIBꢀtreated S. aureus isolates
became apparent: In 40 methicilinꢀresistant S. aureus (MRSA) and 40 methicillinꢀsensitive S.
aureus (MSSA) strains were found four strains being resistant to PIB by protoporphyrine
diarginate.16 In contrast, no defense mechanism against singlet oxygen is known to date. This
fact emphasizes the benefit of new photosensitizer classes acting only via typeꢀII mechanism
(generation of singlet oxygen) to fight the rising numbers of multiresistant bacteria efficiently
and avoid any development of resistance against PIB derived oxidative stress induced by
oxygen radicals (typeꢀI mechanism of action).
In the search for a new leadꢀstructure for active compounds for medical treatments, nature can
serve as a source of inspiration. Plants defend themselves from pathogenic infections by
inducing biosynthesis of antimicrobial secondary metabolites, termed phytoalexins.17 One
class are phenalenꢀ1ꢀones and related compounds biosynthesized by higher plants and also
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fungi. Some rare examples are active by drug action alone, i.e. by inhibition of succinate
dehydrogenase. Some plant extracts were found to be enriched in antifungal phytoalexins, in
which the most potent active substances are phenylꢀphenalenꢀ1ꢀone derivatives. Anigorufone
(3a) and its natural analogue 2ꢀmethoxyꢀ9ꢀphenylꢀphenalenꢀ1ꢀone (3b) show high
leishmanicidal activity (EC50 < 60 ꢂg/mL).18
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But the occurrence of phenalenꢀ1ꢀone chromophors in plants also suggests that they respond
to attacks of pathogens by biosynthesizing photosensitizers, which are able to use solar energy
for defense using singlet oxygen via illumination.19 A wide range of naturally occurring
phenalenꢀ1ꢀones and related compounds can be found.20
OH
O S O
OR
O
O
O
(1)
(2)
(3a) R = H
(3b) R = Me
Figure 1: Phenalenꢀ1ꢀone (1), its derivatives phenalenꢀ1ꢀoneꢀ2ꢀsulfonic acid (2), the
phytoalexin anigorufone (3a) and its analog 2ꢀmethoxyꢀ9ꢀphenylꢀphenalenꢀ1ꢀone (3b)
From the abundance of the phenalenꢀ1ꢀone chromophore in nature, a low dark toxicity of this
structure can be expected. It has already been shown that phenalenꢀ1ꢀone does not contribute
significantly to the mutagenicity or carcinogenicity of combustion emission extracts.21
Phenalenꢀ1ꢀone or 7ꢀperinapthenone (PN, 1), is a sensitizer of singlet oxygen with a quantum
yield close to one in solvents of different polarities. It has established itself as universal
standard for the quantitative determination of singlet oxygen in solution.22 For the first time
Nonell et al. presented a water soluble derivative, 1HꢀPhenalenꢀ1ꢀoneꢀ2ꢀsulfonic acid (2).
Sulfonation of PN strongly stabilized the chromophor against photobleaching and did not
influence its effectiveness as an extremely efficient singlet molecular oxygen sensitizer (phi
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delta Φꢁ = 97 %).23 But its negative charge prevents its use as an antimicrobial photosensitizer
in aqueous surroundings, since positive charges are essential for adhering to or penetration
through the cell walls of pathogens24.
9
Following these findings, we have made it our mission to utilize the excellent properties of
phenalenꢀ1ꢀones for antimicrobial photodynamic therapy by attaching a positive charge to the
system and examining it for various applications. With its almost neutral color, the
photosensitizer is especially suitable for use in dentistry.
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We present a series of phenalenꢀ1ꢀone (7ꢀperinaphthenone, PN) derivatives with excellent
solubility in water, high photostability in the therapeutic time window and a positive charge
for good adherence to the cell walls of pathogens. This new generation of photosensitizers for
photodynamic inactivation of bacteria shows a singlet oxygen quantum yield Φꢁ of > 97%.
PIB is used in dentistry as a supportive tool in periodontology and for endodontic treatments.
However, the most widely used PS in clinical practice at this time are phenothiazine dyes like
methylene blue (MB) and toluidine blue (TBO)25, which are unpopular with the patients´
esthetic feeling due to their strong blue color.26 In periodontal application this may lead to a
temporary staining of the oral soft tissue for at least a few hours.27 However, when PIB with
phenothiazines is done in endodontics, this may lead to persistent blue staining of dental
structure via diffusion of the PS into dentinal tubules with a consequential need for further
treatment of decoloration with appropriate chemical compounds.28 In contrast, our novel PNꢀ
derivatives (5 ꢀ 9, see scheme 1) have a toothꢀlike color with no esthetic limitations for their
use as PS in the oral cavity.
In this study, PIB with the new generation of PNꢀderivatives (5 ꢀ 9) was tested against oral
key pathogens: Gramꢀpositive Enterococcus faecalis (EF), Actinomyces naeslundii (AN),
Streptococcus mutans (SM), and Gramꢀnegative Aggregatibacter actinomycetemcomitans
(AA).
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Enterococcus faecalis is the prevalent species associated with secondary root canal infections
after failed endodontic treatment.29 Moreover, Enterococci are known for their resistances to
diverse antimicrobial agents.30 Actinomyces naeslundii and Streptococcus mutans are referred
to as early colonizers in the formation of dental plaque.31 Streptococcus mutans is also wellꢀ
known as key species in the pathogenesis of dental caries.32 Aggregatibacter
actinomycetemcomitansis is known to be a cause of for severe forms of periodontitis, the soꢀ
called localized aggressive periodontitis (LAP).33
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The aim of this study is (a) to investigate the properties of the novel photosensitizers for PIB
in dentistry, and (b) to find a effectivenessꢀrelationship of the different derivative structures
based on 7ꢀperinaphthenone in order to select a lead candidate for further development of the
novel photosensitizer class.
RESULTS AND DISCUSSION
Preparation of photosensitizers and investigation of their properties
As a simple and reliable approach to install the positive charge in phenalenꢀ1ꢀon (7ꢀ
perinaphthenone, 1), chloromethylation of the structure leading to (4), followed by alkylation
of a tertiary amine or a pyridine with this compound, is very suitable. Substitution in this
manner either with the pyridinium moiety (9, SAPYR) or with trialkylammonium salts (5 to 8)
gives five new photosensitizers:
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N+
Cl
Cl
O
R
O
O
a
c
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(5) R = CH3; 73 %
(1)
(4)
(6) R = C4H9; 94 %
b
83 %
Cl
(7) R = CH2CH2OH; 87 %
(8) R = CH(CH3)Ph; 81 %
N+
O
(9)
Scheme 1: Synthesis of phenalenꢀ1ꢀone derivatives (4 - 9); conditions: a) HCl(aq), HOAc,
H3PO4, HCHO, 120°C, over night, 36 %; b) pyridine, DMF, RT, over night, then 50°C, 5 h,
83 %; c) tertiary amine, DMF, RT, over night, then 50°C, 2 – 10 h, 73 – 94 %
The products are obtained by conversion of (4) in methanol, ethanol, acetone or N,Nꢀ
dimethylformamide (DMF) with excess amine at slightly elevated temperature in good to
excellent yields (73 – 94 %). The purification is only in need of a short column/plug filtration
of (4). Purification of the PN salts was achieved by precipitation with diethylether and reꢀ
precipitation from ethanol with diethylether, until the UV/Vis spectra showed constant
absorption. SAPYR (9) was additionally washed with iceꢀcold dichloromethane. The purity of
all compounds was checked by NMR and HPLCꢀMS and was ≥ 98 %. This represents a
straight forward and easy synthesis and purification protocol for the preparation of a variety
of positive charged 7ꢀperinapthenone derivatives in larger scale.
Analysis of the structures clearly showed the substituent being located at the αꢀposition to the
carbonyl. The proton signal for this position disappeared in comparison to the proton NMR
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spectrum of the starting material phenalenꢀ1ꢀone (1). We also detected a weak long range
coupling of the substituent´s methylene group to the proton in βꢀposition to the carbonyl in
the COSY spectra of compounds 5 and 9 in D2O. In addition, the coupling pattern of the
aromatic naphthalene subsystem is not affected in any of the prepared compounds after
substitution (all spectra can be found in the supporting information).
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Photophysics, Singlet-Oxygen Generation Ability and Aggregation
Compounds 5 to 9 were investigated for their photophysical stability and absorption
characteristics, singletꢀoxygen generation ability and aggregation behavior. All measurments
were conducted in Millipore water (18 Mꢃ). The molar extinction coefficients for compounds
5 to 9 are similar and match the value of 9800 ± 400 L·mol−1·cm−1 within the error margins
(Fig. 2).
The light source for photodynamic treatment is commercially available. A handꢀheld lightꢀ
curing unit for polymerization of dental resins was used (bluephase® C8 IvoclarVivadent).
However, it has to be considered, that there is only a partial overlap of the emission spectrum
of this blueꢀlight emitting lamp and the absorption spectra of the respective PNꢀderivatives
used for the present study (approx. 5 % of applied energy will be absorbed) (Fig. 2). Though,
this light source was selected, as it is widely applied in dental practice. Furthermore, it does
not emit radiation in wavelengths of the UV range that might cause mutagenicity against
bacterial cultures or surrounding tissues. Nevertheless, development of a light source that
obtains a better overlap with PNꢀderivatives has to be considered for further studies, in order
to advance PIBꢀefficacy.
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14000
12000
10000
8000
6000
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0
60
50
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10
0
ε (SAPYR)
BluePase
(1360mW)
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Wavelength [nm]
Figure 2: Emission spectrum of bluephase® C8 IvoclarVivadent in comparison to absorption
of SAPYR (9)
Nevertheless, all compounds investigated in this study are excellent singlet oxygen generators
with quantum yields Φꢁ > 97 %. Singlet oxygen could be clearly identified by its life time at
an emission wavelength of 1270 ± 10 nm and the typical finger print signal recorded at
different wavelengths around 1270 nm (Fig. 3).
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Figure 3: 3DꢀPlot of the timeꢀ and spectrally resolved singlet oxygen luminescence (“finger
1
print” of O2) generated by PNꢀderivative 5 (left) and typical singlet oxygen luminescence
signal upon excitation at 405 nm (right bottom). All measurements were conducted in air
saturated H2O at 25°C. Singlet oxygen is generated after excitation and detected in the range
of 1150 to 1400 nm with a decay time ≈ 3.5 µs for all derivatives and the measured
wavelengths.
(9)
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0
60
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0
300 320 340 360 380 400 420 440 460 480 500
Wavelength [nm]
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(5)
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Wavelength [nm]
(6)
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300 320 340 360 380 400 420 440 460 480 500
Wavelength [nm]
Figure 4: Photostability measurements of (5), (6) and (9) in a quartz cuvette with an
irradiation at 405 nm with 125 ± 3 J laser energy; time values in the graphs legend are given
in minutes
All compounds show photobleaching, which is approx. 10 % for most of the examples upon
irradiation with laser energy at 405 nm for 5 minutes, equals 32 J (Fig. 4). These are quite
stringent conditions in comparison to the irradiation with the BluePhase C8 lamp for 20 s,
40 s and 120 s. In comparison to the laser beam irradiation, the bleaching of the
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photosensitizers can be neglected when using the lamp. Due to the overall short illumination time
4
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of 120 s photobleaching seems not a relevant factor for antimicrobial testing
.
7
8
9
The potential pathway for the photoꢀdegradation of the phenalenꢀ1ꢀone chromophor with
involvement of 1O2 for these chemical structures may be explained as follows: Degradation of
the molecules 5 to 9 is expected to start with an elimination of the positively charged moiety.
We assume, this takes place by an analog mechanism to Hofmann elimination forming a
tertiary amine and an alkene.34 Following this mechanism, the alkylꢀdimethylammonium
group is more easily eliminated (Scheme 2, (a)). The elimination of trimethylammonium
group and the pyridinium moiety is more difficult, because no corresponding alkene can be
formed. This explains the different photostabilities of the novel PS (Fig. 4).
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We hypothesize the process is followed by a degradation of the chromophor starting from the
ketonꢀbearing ring. By comparison of aromatic systems known from the literature concerning
their reactivity with oxygen species, a high stability of the two condensed aromatic rings in
the phenalenꢀ1ꢀone structure can be derived.35 This part of the molecule can be seen as stable
versus reactions like epoxidation or peroxidation by singlet oxygen under the given
conditions. The more reactive (“vulnerable”) part is certainly the α,βꢀunsaturated Michael
system. As long as the PS´s (5 – 9) double bond is blocked by their charged substituent
located at the αꢀposition, attack of the electrophilic singlet oxygen in a 2+2 cycloaddition
reaction36 is hampered by the altered electronics and the higher steric hindrance (scheme 2).
This is in good accordance to the finding, that substitution of (1) stabilizes the system.23
Attack of the Michael systems double bond in the not substituted chromophor phenalenꢀ1ꢀone
(1), or the products mentioned above, can take place more easily. Tertiary amines (for
example 10) are also accessible to oxidation. After the double bond has been substituted by
singlet oxygen (12), the resulting CꢀC single bond is quickly cleaved forming two aldehyde
groups (13).37 This process finally leads to breakdown and bleaching of the chromophor
(Scheme 2, (b)).
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(a)
N+
N
5
6
7
O
O
[O2]
+
8
9
ꢀ H2O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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53
54
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58
59
60
(10)
(11)
(6)
O
O
O
(b)
OHC
CHO
O
O
[1O2]
(1)
(12)
(13)
Scheme 2: Initial steps in the assumed breakdown pathway for the photosensitizer structures;
(a) elimination of an alkene in (6) under oxidative conditions and warming (Hofmann
elimination); (b) substitution of phenalenꢀ1ꢀone (1) by singlet oxygen and oxidative bond
cleavage
Aggregation of the PN photosensitizers was studied over a wide concentration range via
UV/Vis spectroscopy (Fig. 5) and by temperature dependent NMR studies (see Fig. 6). The
beginning of aggregation was observed in the millimolar concentration range.
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5
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7
8
9
12000
11000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
10µM
100µM
200µM
500µM
1000µM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
300 320 340 360 380 400 420 440 460 480 500
Wavelength [nm]
Figure 5: Absorption spectra of (9) within a concentration range of 100 – 1000 µM in H2O;
within the error margins no dimerization can be observed in the concentration range covered.
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5
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7
8
9
Figure 6: Aromatic region of the proton NMR spectra of (5) in concentration of 1 mM (4), 5
mM (3), 10 mM (2) and 20 mM (1) in D2O; xꢀvalues (f1) in ppm relative to external standard
TMS, 1 ppm = 400 Hz. The spectra show dimerisation beginning in the millimolar
concentration range.
10
11
12
13
14
15
16
17
18
19
20
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22
23
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50
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54
55
56
57
58
59
60
Aggregation can be neglected below the millimolar range (< 1 mM) and is thus no critical
factor for the biological studies. Typical therapeutic concentrations in PIB match the
micromolar range (1 to 500 µM).
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Polarity and Stability
4
5
6
7
8
The polarity of the novel photosensitizers was estimated by measuring the octanolꢀwater
coefficient. Distribution of 1*10ꢀ4 mol of each PN salt between both phases was measured by
UV/Vis spectroscopy after 10 minutes of stirring at room temperature. Table 1 summarizes
the results and gives data for the photophysical measurements as described above:
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
Loss in
Photosensitizer
Singlet
oxygen
quantum
octanol /
water
λmax
absorption emission
[nm](c)
[nm]
λmax
absorption
[%] after
5 / 20
R
O
coefficient
log P
(d)
yield Φꢁ
mins(e)
Residue R =
Cl
(4) (a) 356 ꢀ 410 487 ± 5
(5) (b) 360 ꢀ 417 488 ± 5
n.d.
n.d.
+ 1.3
ꢀ 1.4
N+
1.03 ± 0.05
10 / 36
N+
(6) (b) 364 ꢀ 413 489 ± 5
(7) (b) 360 ꢀ 418 491 ± 5
1.03 ± 0.05
1.06 ± 0.05
33 / 89
66 / 74
ꢀ 0.8
ꢀ 1.3
N+
OH
N+
(8) (b) 362 ꢀ 418 492 ± 5
(9) (b) 363 ꢀ 410 489 ± 5
1.05 ± 0.05
1.02 ± 0.05
16 / 53
12 / 38
ꢀ 0.1
ꢀ 1.3
N+
Table 1: Crucial physical parameters of the phenalenꢀ1ꢀone derivatives; conditions: at 25 °C,
(a) in EtOH, (b) in Millipore water, (c) all compounds show a broad absorption maximum in
this region, which cannot be resolved in distinct maxima, (d) 7ꢀperinaphthenoneꢀ2ꢀsulfonic
acid (PNS) with a Φꢁ = 1.03 ± 0.10 served as reference PS, (e) excitation with 125 ± 3 J of
laser light at 405 nm
pH stability was determined by recording UVꢀVis spectra of compounds 5 and 9 in buffer
solutions at different pH values after incubation for 5 and 30 minutes. The novel derivatives
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5
show excellent stability in acidic medium (down to pH = 2), but decompose slowly in alkaline
solutions with pH > 9 (see supporting information for spectra).
6
7
8
9
The temperature stability of compounds 5 and 9 was investigated by temperature dependent
NMR studies in D2O (Fig. 7). No change appeared in the spectrum after heating to 90 °C for
more than 10 minutes. The compounds are perfectly temperature stable in solution in the
therapeutic time window:
10
11
12
13
14
15
16
17
18
19
20
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7: Temperature stability of 5 by NMR; temperature steps: room temperature (1),
50 °C (2), 70 °C (3) and 90 °C (4), then cooling to room temperature (5), c = 70 mM; xꢀvalues
(f1) in ppm relative to external standard TMS, 1 ppm = 400 Hz.
The shift of the spectrum in the aromatic region of more than 0.5 ppm again indicates
aggregation in the millimolar range (c = 70 mM).
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Photodynamic Inactivation of Bacteria (PIB):
4
5
6
7
8
PIB was performed against oral key pathogens in suspension using different concentrations of
each PNꢀderivative (0 µM, 1 µM, 5 µM, 10 µM, 100 µM, 250 µM) and obtaining irradiation
periods of 20 s, 40 s and 120 s. Hereby all PNꢀderivatives were evaluated against Gramꢀ
positive EF, whereas (9) and (7) were also tested against Gramꢀpositive SM and AN and
Gramꢀnegative AA. Results were evaluated by the method of Miles, Misra and Irwin.42
In all cases, illumination alone or treatment with PNꢀderivative only (dark control) resulted in
no decrease of the colony forming units (CFU) compared to untreated control groups. Figure
8 shows the results of the PIB efficacy using derivative 9 against all oral pathogens in
9
10
11
12
13
14
15
16
17
18
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20
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25
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59
60
suspension. Incubation of EF, SM, AA and AN with different concentrations (0 ꢀ 250 µM) of
(9) caused a decrease in viability upon illumination (Fig. 8). (9) at 5 µM exhibited already a
disinfection efficacy of ≥ 99.999 % (corresponding to ≥ 5 log10 reduction of CFU) upon
illumination for 120 s. Higher concentrations of (9) did not increase further the antibacterial
efficacy of PIB. Furthermore PIB efficacy was as well dependent from the applied light of 25
J/cm2, 50 J/cm2 or 150 J/cm2 in samples of SM
, AN and EF. For example, (9) concentration of
5 µM achieved a killing efficacy of between 3 and 5 log10 using a light doses of 50 J/cm2 and
> 6 log10 using a light dose of 150 J/cm2 Light alone does not have any effect on the viability
of SM, AN and EF. In contrast, samples of AA exhibited a strong lightꢀtoxicity without any
(9). There was a reduction up to 1, 2 and 6 log10 steps of CFU of AA after irradiating for 20 s,
40 s and 120 s, respectively (Fig. 8). Killing efficacy against AA was not enhanced by PIB
using (9) concentrations up to 250 µM.
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5
6
7
8
9
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+1
1e+0
(9), S. mutans, Incubation 10 s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
1
5
10
100
250
conc [ꢀM]
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+1
1e+0
(9), A. actinomycetemcomitans, Incubation 10 s
0
1
5
10
100
250
conc [ꢀM]
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5
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7
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9
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+1
1e+0
(9), E. faecalis, Incubation 10 s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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37
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40
41
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46
47
48
49
50
51
52
53
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55
56
57
58
59
60
0
1
5
10
100
250
conc [ꢀM]
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
1e+1
1e+0
(9), A. naeslundii, Incubation 10 s
0
1
5
10
100
250
conc [ꢀM]
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8
9
Figure 8: Results of photodynamic inactivation of four different bacteria by (9) using the
handꢀheld dental lightꢀcuring unit (bluephase® C8, IvoclarVivadent, Schaan, Liechtenstein);
Output-intensity of 1260 ± 50 mW/cm2, irradiation for 20 s, 40 s or 120 s, corresponding to
applied energies of 25 J/cm2, 50 J/cm2 and 150 J/cm2, respectively. Surviving colonies were
counted 24 h later. Grey column: controls without illumination. Coloured columns: (9) +
light activation, yellow 20 s, blue 40 s and green 120 s illumination, respectively. Black solid
line corresponds to a reduction of 3 log10 steps (99.9 % killing efficacy), black dashed line to
a reduction of 5 log10 steps in bacterial numbers (99.999 % killing efficacy) related to
controls without illumination and without (9). (n = 3 independent experiments: bars represent
the median including 25 – 75 % quartiles)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PIBꢀresults with all PNꢀderivatives against EF are summarized in Table 2.
Enterococcus faecalis
0 ꢁM
1 ꢁM
ꢀ
ꢀ
5 ꢁM
ꢀ
<3
>6
10 ꢁM 100 ꢁM
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
<3
>6
>6
>6
>6
>6
(9)
(5)
(6)
(7)
(8)
<3
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
ꢀ
<3
>6
ꢀ
<3
>6
>6
>6
>6
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
ꢀ
<3
≈3
<3
<3
>6
>6
>6
>6
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
<3
<3
>6
>6
>6
>6
20 s
40 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
<3
<3
>6
<3
<3
>6
>6
>6
>6
120 s
Table 2: PIBꢀresults with all PNꢀderivatives against EF. (-) means < 1 log10 step reduction of
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5
the number of bacteria. This was defined as virtually no efficacy. Threshold for the evaluation
of effectiveness was 3 log10 steps (99.9 % inactivation).
6
7
8
9
Overall, derivative 9 showed the best killing efficacy compared to the other PNꢀderivatives,
whereas (7) was the least effective photosensitizer. PIB with (9) or (5) caused a pronounced
decrease of CFU against all tested species, whereby (9) was more effective at lower
concentrations and illumination periods (Fig. 8). (9) as the most potent PS reached more than
6 log10 steps of bacteria reduction up to total eradication in only 40 seconds. At concentrations
of 10 µM sterilization conditions can be realized.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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33
34
35
36
37
38
39
40
41
42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Depending on the tested species, (9) revealed an efficacy of PIBꢀkilling of ≥ 5 log10 steps
CFU (≥ 99.999 % = disinfection) at concentrations effective from 5 µM and illumination time
of either 120 s or even 40 s (AN, AA), whereas higher concentrations of (5) or (8) or longer
irradiation times were necessary to obtain similar results.
An overview of PIBꢀkilling rates against all four species using (9) and (7) can be found in
table 3.
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7
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9
Enterococcus faecalis
0 ꢁM
1 ꢁM
ꢀ
ꢀ
5 ꢁM
ꢀ
<3
>6
10 ꢁM
<3
>6
100 ꢁM
>6
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
(9)
(7)
>6
>6
<3
>6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
<3
<3
>6
>6
>6
>6
Streptococcus mutans
0 ꢁM
1 ꢁM
ꢀ
ꢀ
5 ꢁM
<3
<3
10 ꢁM
<3
>6
100 ꢁM
>6
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
(9)
(7)
>6
>6
<3
>6
>6
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
ꢀ
<3
>6
>6
>6
>6
Actinomyces naeslundii
0 ꢁM
1 ꢁM
ꢀ
ꢀ
5 ꢁM
<3
>5
10 ꢁM
>5
>5
100 ꢁM
>5
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
(9)
(7)
>5
>5
>5
>5
>5
20 s
40 s
120 s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<3
>5
<3
>5
>5
>5
>5
>5
Aggregatibacter actinomycetemcomitans
0 ꢁM
<3
<3
1 ꢁM
<3
5 ꢁM
<3
10 ꢁM
>6
>6
100 ꢁM
>6
20 s
40 s
120 s
(9)
(7)
<3
>6
>6
>6
>6
>6
>6
>6
20 s
40 s
120 s
ꢀ
<3
>3
ꢀ
<3
>6
<3
<3
>6
<3
>3
>6
>3
>6
>6
Table 3: PIBꢀkilling rates against all four species by compound 9 and 7. (-) means < 1 log10
step reduction of the number of bacteria. This was defined as virtually no efficacy. Threshold
for the evaluation of effectiveness was 3 log10 steps (99.9 % inactivation).
The photoꢀstability of (7) was the lowest in all compounds. Due to the short treatment time
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5
6
7
8
employed in this study, we assume that small antimicrobial activity of (7) might be partially
caused by decomposition. However, the polarity caused by the additional polar hydroxylꢀ
group should play the key role.
9
Although light activated reduction of AA by the novel photosensitizers was efficient and the
dominant process for the bacteria killing, AA was the only species, where treatment with light
alone led to a reduction of CFU. This lightꢀtoxicity can be explained due to the presence of
endogenous photosensitizers in AAꢀcells.38
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
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40
41
42
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46
47
48
49
50
51
52
53
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55
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60
CONCLUSION:
Equipping the singlet oxygen standard 7ꢀperinaphthenone with the ability to bind to the
surface of pathogens led to novel antimicrobial active and very effective photosensitizers with
outstanding single oxygen quantum yield, which can be used to improve PIB in dental
practice. This was accompanied with the successful development of a straight forward and
simplified synthesis and purification protocol for the novel compounds. The preparation of a
variety of positive charged 7ꢀperinapthenone derivatives is now possible in a larger scale with
good to excellent yields (73 – 95 %). All derivatives showed high stability over a wide range
of changing pH (2 – 9), good temperatureꢀstability and photostability in the therapeutic time
window for typical PIB applications.
The light activation of the new generation of PNꢀderivatives 5 to 9 achieved antibacterial
efficacy against oral key pathogens of 99.999% (AN) or even 99.9999 % (AA
concentrations of at least 5 µM of the respective PS and an illumination period of 120 s or 40
s (5 µM: AA AN) for the most efficient derivate (9, “SAPYR”).
, EF, SM) at
,
A polar substituent seems to cause a decrease in effectiveness, whereas a pyridinium moiety is
advantageous for antimicrobial action. A suboptimal balance between charge and lipophilicity
can be an explanation for a weaker and slower attachment to the cell wall. In addition,
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9
derivatives with a substituent based on a quaternary dimethylammonium moiety tend to lower
photostability. The trimethylammonium and the pyridinium substituent are more stable, due to
reduced elimination ability of these residues. With compound 9, or „SAPYR“, it was recently
demonstrated, that this typeꢀII photosensitizer exhibits a high killing efficacy (reduction of >
5 log10 CFU) against monospecies and polyspecies biofilms.39 Compound 9 may serve as lead
candidate for further studies.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
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58
59
60
Even though the absorption spectra of the PN derivatives only accounted for a 5% overlap
with the emission spectrum of the light source used, PIB with the novel class of PS was
already able to compete with the technological standard (> 5 log10 pathogen reduction) in a
fast and efficient process, elucidating the enormous capability of this new generation of
photosensitizers. Nevertheless, further studies with more appropriate light sources are
necessary to optimize PIB efficiency using these new photosensitizers.
Ongoing studies in our lab extending our investigations into this interesting molecule class
towards other key pathogens while also investigating a library of substitution patterns, will
show the scope and limitations of a pure singlet oxygen generator for antimicrobial
photodynamic therapy.
EXPERIMENTAL SECTION
General materials and methods
Commercial reagents and starting materials were purchased from Acros Organics, Alphaꢀ
Aesar, TCI Europe, Fluka, Merck, Frontier Scientific or SigmaꢀAldrich and used without
further purification. The dry solvents acetone, dichloromethane, dimethylsulfoxide and
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9
dimethylformamide were purchased from Roth (RotiDry Sept) or SigmaꢀAldrich (puriss.,
absolute), stored over molecular sieves under nitrogen and were used as received.
Thin layer chromatography (TLC) analyses were performed on silica gel 60 Fꢀ254 with 0.2
mm layer thickness and detection via UV light at 254 nm / 366 nm or through staining with
ninhydrin in ethanol. Flash column chromatography was performed on Merck silica gel (Si 60
40ꢀ63 ꢂm) either manually or on a Biotage® solera™ flash purification system. Column
chromatography was performed on silica gel (70–230 mesh) from Merck.
10
11
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Melting points were measured on a SRS melting point apparatus (MPA100 Opti Melt) and are
uncorrected.
NMR spectra were recorded on Bruker Avance 300 (1H 300.13 MHz, 13C 75.47 MHz, T =
300 K), Bruker Avance 400 (1H 400.13 MHz, 13C 100.61 MHz, T = 300 K), Bruker Avance
600 (1H 600.13 MHz, 13C 150.92 MHz, T = 300 K) and Bruker Avance III 600 Kryo (1H
600.25 MHz, 13C 150.95 MHz, T = 300 K) instruments. The chemical shifts are reported in
δ[ppm] relative to external standards (solvent residual peak). The spectra were analyzed by
first order, the coupling constants
J are given in Hertz [Hz]. Characterization of the signals: s
= singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet, psq = pseudo
quintet, dd = double doublet, dt = doublet of triplets, ddd = double double doublet. Integration
is determined as the relative number of atoms. Assignment of signals in 13Cꢀspectra was
determined with 2Dꢀspectroscopy (COSY, HSQC and HMBC) or DEPT technique (pulse
angle: 135 °) and given as (+) for CH3 or CH, (–) for CH2 and (Cq) for quaternary Cq. Error of
reported values: chemical shift 0.01 ppm (1H NMR) and 0.1 ppm (13C NMR), coupling
constant J 0.1 Hz. The solvents used for the measurements are reported for each spectrum.
IR spectra were recorded with a BioꢀRad FTꢀIRꢀFTS 155 spectrometer. Fluorescence spectra
were recorded on a ‘Cary Eclipse’ fluorescence spectrophotometer and absorption spectra on
a “Cary BIO 50” UV/VIS/NIR spectrometer from Varian. All measurements were performed
in 1 cm quartz cuvettes (Hellma) and UVꢀgrade solvents (Baker or Merck) at 25 °C.
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Mass spectra were recorded on Varian CHꢀ5 (EI), Finnigan MAT95 (EIꢀ, CIꢀ and FABꢀMS),
Agilent QꢀTOF 6540 UHD (ESIꢀMS, APCIꢀMS), Finnigan MAT SSQ 710 A (EIꢀMS, CIꢀ
MS) or Thermo Quest Finnigan TSQ 7000 (ESꢀMS, APCIꢀMS) spectrometer. Xenon serves
as the ionization gas for FAB.
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Synthesis and Purification of the compounds
The purity of all synthesized compounds was determined by NMR spectroscopic methods
(Bruker Avance 300, DMSOꢀd6) and HPLCꢀMS confirming a purity of > 98%.
Phenalen-1-one (1)40
βꢀNaphthole (10.4 g, 72 mmol), glycerine (20.0 mL, 270 mmol) and sodium 3ꢀ
nitrobenzenesulphonate (12.4 g, 65 mmol) in 36 mL conc. sulfuric acid and 24 mL water was
heated to 140 °C for 1 h. The resulting black tar was poured onto ice and diluted with water.
After crystallization overnight in the fridge, the supernatant was discarded and the product
was extracted with warm toluene (60°C, 10x 50 mL). The pure product crystallized as yellowꢀ
brown solid while evaporating the solvent under reduced pressure to give 4.12 g (22.8 mmol,
32 %).
1H-NMR (400 MHz, CDCl3): δ[ppm] = 8.62 (dd,
J = 1.0 Hz, J = 7.4 Hz, 1H), 8.19 (dd, J =
1.2 Hz, 8.1 Hz, 1H), 8.02 (dd,
J = 1.1.Hz, J = 8.3 Hz, 1H), 7.75 (m, 3H), 7.58 (m, 1H), 6.73
(d,
J
= 9.8 Hz, 1H). ꢀ 13C-NMR (100 MHz, CDCl3): δ[ppm] = 185.8 (q), 141.9 (+), 135.1 (+),
132.4 (q), 132.1 (+), 131.6 (+), 130.6 (+), 129.7 (q), 129.4 (+), 128.0 (q), 127.7 (q), 127.3 (+),
126.8 (+); ꢀ MS (ESIꢀMS, CH2Cl2/MeOH + 10 mmol NH4OAc): e/z (%) = 181.1 (100, MH+);
ꢀ MF: C13H8O; ꢀ MW: 180.2 g/mol
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2-Chlormethyl-1H-phenalen-1-one (2)
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Phenalenꢀ1ꢀone 1 (5.40 g, 30 mmol) and paraformaldehyde (16.50 g, 72.0 mmol) in 120 mL
acetic acid and 75 mL phosphoric acid (85 %) was heated to 110 °C. Hydrochloric acid (36 %
w/w, 80 mL) was added over a period of 30 min. The brown solution was heated for further 8
h. After cooling to room temperature, 250 mL of ice cold distilled water were added. The
reaction mixture was carefully neutralized with a saturated solution of K2CO3. The product
was extracted with dichloromethane (3x 100 mL). Further purification was carried out by
flash chromatography (silica gel, dichloromethane/petroleum ether 1:1, Rf = 0.3) and gave
2.37 g of bright yellow, fine powder (10.41 mmol, 36 %).
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M.p.: 133 °C; ꢀ1H-NMR (300 MHz, CDCl3): δ[ppm] = 8.58 – 8.53 (dd,
Hz, 1H), 8.14 ꢀ 8.08 (dd, = 1.0 Hz, = 8.1 Hz, 1H), 7.96 – 7.91 (dd, = 0.6 Hz,
1H), 7.81 (s, 1H) 7.74 – 7.65 (m, 2H), 7.56 – 7.47 (dd, = 7.0 Hz; = 1.2 Hz; 1H), 4.62 (s,
J
= 1.2 Hz,
J = 7.3
J
J
J
J
= 8.2 Hz,
,
J
J
2H, ꢀCH2ꢀ); ꢀ 13C-NMR (75 MHz, CDCl3): δ[ppm] = 183.4 (q), 140.3 (+), 135.4 (q), 135.2
(+), 132.2 (+),132.0 (q), 131.9 (+), 130.8 (+), 128.8 (q), 127.2 (+), 127.1 (q), 127.0 (q), 126.8
(+), 41.5 (ꢀ); ꢀ IR (neat): ν[cmꢀ1] = 3040 (w), 2969 (w), 2843 (w), 1635 (s), 1622 (s), 1594 (s),
1568 (s), 1507 (m),1422 (m), 937 (m), 789 (s), 767 (s); ꢀ MS (ESIꢀMS, CH2Cl2/MeOH + 10
mmol NH4OAc): e/z (%) = 229.0 (100, MH+); ꢀ HR-MS (ESI): m/z calcd. for C14H10ClO
(MH+): 229.0415. Found: 229.0414 (MH+); ꢀ MF: C14H9OCl; ꢀ MW: 228.7 g/mol;
General procedure I: Synthesis of positively charged phenalen-1-one derivatives
2ꢀ(Chloromethyl)ꢀ1Hꢀphenalenꢀ1ꢀone (4) (230 mg, 1 mmol) was dissolved in DMF (2 mL).
The appropriate tertiary amine or pyridine (10 mmol) in DMF (2 mL) was added drop wise
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