Enhanced anti-microbial effect through cationization of a mono-triazatricyclodecane substituted asymmetric phthalocyanine

Article abstract of DOI:10.1016/j.jinorgbio.2018.10.001

Antimicrobial photodynamic therapy (aPDT) is an effective way to combat infectious diseases and antibiotic resistance. Photosensitizer is a key factor of aPDT and has triggered extensive research interest. In this study, a new asymmetric Zn(II) phthalocyanine mono-substituted with a triazatricyclodecane moiety (compound 3) and its cationic N-methylated derivative (compound 4) were synthesized. Their photodynamic antimicrobial activities were evaluated using bioluminescent bacterial strains. Compound 3 showed phototoxicity only toward the Gram-positive bacteria, whereas the cationic derivative compound 4 exhibited strong anti-bacterial activity against both Gram-positive and Gram-negative strains. These bacterial species were eradicated (>4.0 logs or 99.99% killing) at appropriate concentrations of compound 4 with 12.7 J/cm² of red light, demonstrating compound 4 as a potent aPDT agent.

Full text of DOI:10.1016/j.jinorgbio.2018.10.001

JournalofInorganicBiochemistry189(2018)192–198
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Enhanced anti-microbial effect through cationization of a
mono-triazatricyclodecane substituted asymmetric phthalocyanine
Huajian Lina, Jincan Chenb, Yaxin Zhangb, Azeem Ullab, Jianyong Liua, Fan Linc,
⁎
Longguang Jianga, Mingdong Huanga,b,
b
c
a
College of Chemistry, Fuzhou University, Fujian 350118, China
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Fuzhou, Fujian 350002, China
Qingdao Sundynamic Technology Co., Ltd, Qingdao, Shandong 266000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antimicrobial photodynamic therapy (aPDT) is an effective way to combat infectious diseases and antibiotic
resistance. Photosensitizer is a key factor of aPDT and has triggered extensive research interest. In this study, a
new asymmetric Zn(II) phthalocyanine mono-substituted with a triazatricyclodecane moiety (compound 3) and
its cationic N-methylated derivative (compound 4) were synthesized. Their photodynamic antimicrobial activ-
ities were evaluated using bioluminescent bacterial strains. Compound 3 showed phototoxicity only toward the
Gram-positive bacteria, whereas the cationic derivative compound 4 exhibited strong anti-bacterial activity
against both Gram-positive and Gram-negative strains. These bacterial species were eradicated (> 4.0 logs or
99.99% killing) at appropriate concentrations of compound 4 with 12.7 J/cm2 of red light, demonstrating
compound 4 as a potent aPDT agent.
Cationic phthalocyanine
Photosensitizer
Antimicrobial photodynamic therapy (aPDT)
Bioluminescent bacteria
Synthesis
1. Introduction
Moreover, the triazatricyclodecane is water soluble, and will be pro-
tonated at aqueous solution, potentially rendering the conjugate aqu-
Phthalocyanines have many important industrial applications, in-
cluding dyes and pigments in fabric, demonstrating that they are safe
and environment-friendly materials. They also possess remarkable sta-
bility and unique photochemical and photophysical properties, broad-
ening their uses in many high technology fields, including semi-
conductor materials [1,2], solar cells [3,4], optical data storage [5],
chemical sensors [6,7], oxidation-reduction catalysts [8–11] and pho-
tocatalysts [12], as well as photodynamic therapy (PDT) for anti-
microbial [13–15] or antitumor applications [16–18]. The emergence
of multi-resistant bacteria due to the over-use of antibiotics has become
a global challenge [19,20]. Phthalocyanines have emerged as a new
class of photosensitizer possessing potent antimicrobial effect, even
toward drug-resistant bacterial strains separated from hospitals
[21–24].
eous solubility and positive charges in a weakly acidic environment.
The positive charge is a common property of antimicrobial agents,
which allows the adsorption and binding to bacterial surface that carry
large amount of negative charges [25,26]. We also wanted to avoid the
positional isomer on ZnPc during conjugation because single compound
is a key for approval by regulatory agent, should the compound pro-
ceeds to clinical trial stage.
It turned out that compound 3 was not water soluble, and did not
show desirable antimicrobial effect toward bacterial strains. We suspect
that the compound
3 was not protonated at aqueous solution.
Quaternization of aliphatic or aromatic nitrogen atom at the end of the
synthetic pathway of phthalocyanine is a common way to prepare ca-
tionic phthalocyanine [27]. Most of the published cationic phthalo-
cyanines with quaternary amine groups exhibit excellent photodynamic
antimicrobial effect [28–32]. Moreover, it was discovered recently by
Hamblin group that aPDT can be greatly enhanced by the addition of
simple inorganic salts especially iodine ion at micromolar concentration
[33]. Thus, we carried out methylation on the tertiary amine group of
compound 3 in a hope to form an cationic phthalocyanine compound 4.
Photophysical and photochemical properties including UV–Vis spectra,
In this study, we designed a new asymmetric Zn(II) phthalocyanine
(ZnPc): triazatricyclo-substituted Zn(II) phthalocyanine (compound 3)
by conjugating with a Nitrogen-rich compound (1,3,5-triazatricyclo
[3.3.1.1(3, 7)] decane-7-amine, Scheme 1). The substituent is quite
bulky in size and can reduce the aggregation of phthalocyanine. Ag-
gregation of Pc typically leads to the quench of photodynamic effect.
⁎
Corresponding author at: College of Chemistry, Fuzhou University, Fujian 350118, China.
E-mail address: HMD_lab@fzu.edu.cn (M. Huang).
https://doi.org/10.1016/j.jinorgbio.2018.10.001
Received 14 June 2018; Received in revised form 29 September 2018; Accepted 1 October 2018
Availableonline06October2018
0162-0134/©2018ElsevierInc.Allrightsreserved.

H. Lin et al.
JournalofInorganicBiochemistry189(2018)192–198
Scheme 1. Synthesis of compound 3 and 4.
photodynamic antibacterial and hemolysis activities of these com-
pounds were characterized and evaluated. Encouragingly, the com-
pound 4 exhibited very potent antimicrobial effect.
quite difficult. The purified carboxy compound 2 was then conjugated
to compound 1,3,5-triazatricyclo [3.3.1.1(3, 7)] decan-7-amine by
amine coupling chemistry. The product was precipitated out with water
and collected by centrifugation, followed by washing with deionized
water, acetonitrile and CH2Cl2, and dried to give blue solid. The com-
pound 3 was further purified by semi-preparative HPLC system. The
structure of compound 3 was fully confirmed by 1H, 13C NMR spectrum
(Figs. S2, S3) and mass spectrometry measurement (Electrospray Ioni-
2. Results and discussion
2.1. Synthesis of compound 3
The target compound (3) was synthesized using the scheme shown
in Scheme 1. The intermediate compound (1) was prepared by statistic
condensation method. Condensation of trimellitic anhydride or phthalic
anhydride in the presence of urea and catalyst at high temperature can
produce tetra-formamido-phthalocyanine or phthalocyanine, respec-
tively. Using a mixture of trimellitic anhydride and phthalic anhydride
at a ratio of 1:7 for condensation, we made a mixture of 2-for-
mamidophthalocyanine zinc (1) and phthalocyanine zinc with only
trace amount presumably polycarboxy substituted phthalocyanine zinc.
The amide group of compound 1 was hydrolyzed to carboxy group,
leading to 2-carboxyphthalocyanine (compound 2), which was sepa-
rated out of the mixture to a high purity (> 90%, Fig. S1). It is im-
portant to carry out the hydrolysis before the purification, because the
separation of the compound 1 out of the condensation mixture was
zation, ESI, Fig. S4, m/z calculated for compound
3
[M + H]+
757.1879, found 757.1846).
2.2. Antimicrobial effects of compound 3
Phthalocyanine and its derivatives are highly potent photo-
sensitizers in photodynamic antimicrobial therapy. They sensitize
oxygen under red light (~670 nm) to generate reactive oxygen species
(ROS) and damage effectively nearby bacteria or cells [34]. In this
study we evaluated the photodynamic antibacterial activity of com-
pound 3 against two luminescent bacterial strains, Gram-positive (S.
aureus) and Gram-negative (E. coli). These are genetically engineered
bacterial strains expressing both luciferase and luciferase substrate, and
are luminescent while alive with bioluminescence intensity (relative
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H. Lin et al.
JournalofInorganicBiochemistry189(2018)192–198
Fig. 1. Photo cytotoxicity of compound 3 against E. coli (a) and S. aureus (b); dark cytotoxicity of compound 3 against E. coli (c) and S. aureus (d); panel (e) shows the
LED array light source used in this assay (λ = ~670 nm, fluency rate = 42.5 mW/cm2, illumination time = 5 min to a light dose of 12.7 J/cm2).
Fig. 2. Photo cytotoxicity of compound 4 against E. coli (a) and S. aureus (b); dark cytotoxicity of compound 4 against E. coli (c) and S. aureus (d) (λ = ~670 nm,
fluency rate = 42.5 mW/cm2, illumination time = 5 min to a light dose of 12.7 J/cm2).
luminescence units, RLU) proportional to the number of live cells, al-
lowing the real time evaluation of photodynamic bacterial inactivation
effect. An array of light-emitting diode (LED, fluency rate = 42.5 mW/
cm2) light source was designed for this assay on 96-well plates (5 min,
to a light dose of 12.7 J/cm2). The advantage of this LED light source is
its capability to illuminate all 96 wells of microplate evenly at the same
time and without generating too much heat, thus reducing the systemic
error of the experiments (Fig. 1). In our assay, the phosphate buffered
saline (PBS) suspensions of bacterial strains S. aureus or E. coli were
added to the 96-well plate at a density of about 106 CFU/ml and in-
cubated at 37 °C in dark for 10 or 15 min (10 min for S. aureus, 15 min
for E. coli) with the compound 3. The compound 3 was first dissolved in
DMSO, and diluted into the assay buffer keeping the final assay con-
centration of DMSO at < 1%. After the illumination, the luminescence
intensity of the wells was monitored for 50 min. As shown in the results
antimicrobial effect against the Gram-negative strains, even at con-
centrations up to 10 μM, but it did have potent antimicrobial effect
against the Gram-positive strains with a IC50 value at nanomolar range
(Fig. 1B). In addition, the dark toxicity measurement of compound 3
was performed under the same conditions. The compound 3 did not
show any noticeable toxic toward these strains in the absence of light at
concentrations up to 10 μM to either for S. aureus or E. coli. We sus-
pected that compound 3 cannot be protonated at neutral aqueous so-
lution presumably due to high rigidity of triazatricyclodecane moiety.
Thus, the methylation of tertiary amine group was carried out in order
to get the cationic phthalocyanine (compound 4) [35].
2.3. Synthesis of compound 4
The cationic compound 4 was synthesized through methylation of
compound 3 in anhydrous DMF using excess amount of iodomethane
of antibacterial assay, the compound
3 did not show potent
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H. Lin et al.
JournalofInorganicBiochemistry189(2018)192–198
(Scheme 1). Interestingly, only one tertiary amino group was methy-
lated despite the used of excess amount of iodomethane during the
reaction, as shown by both proton NMR spectra (Fig. S5) and ESI mass
spectra (Fig. S6). We do not know why only one tertiary amino group
was methylated. It is likely that the bulky nature of the triaza-
tricyclodecane moiety prevents further methylation.
2.4. Antimicrobial efficacy of compound 4
The cationic compound 4 now exhibited high cytotoxicity toward
both types of bacteria with IC50 values at nanomolar range (7.2 nM and
200 nM for S. aureus and E. coli, respectively, Fig. 2). This observation
can be attributed to the difference in morphology of their membranes.
The Gram-negative species contains an inner cytoplasmic membrane
and an out membrane which are separated by the periplasmic region,
covered by a tightly organized outer cell wall that imparts a high per-
meability barrier for the photosensitizer. The Gram-positive strains has
a membrane covered by a relatively porous cell wall composed of
peptidoglycan and lipoteichoic acid that allows photosensitizer to bind
or cross. It was found that, most of the neutral, anionic or cationic
photosensitizers can efficiently kill Gram-positive bacteria, whereas
only cationic photosensitizer are able to kill Gram-negative species
[36,37].
Fig. 4. Photo cytotoxicity and dark cytotoxicity of compound 4 against E. coli
and S. aureus (λ = ~670 nm, fluency rate = 42.5 mW/cm2, illumination
time = 5 min to a light dose of 12.7 J/cm2).
mammalian cells. For future clinical application, additional long term
toxicity and systemic toxicity evaluation need to be carried out.
3. Experimental
3.1. Materials and instruments
Colony counting method was further used to validate the anti-
microbial efficacy (Fig. 3), and we found that compound 4 killed > 4
logs of bacteria (> 99.99%) of S. aureus at a concentration of 1.6 μM,
and induced similar anti-bacterial effect against E. coli at 8.0 μM
(Figs. 4, S8). These results are consistent with the measurements using
luminescent bacteria.
All the reactions were carried out under the atmosphere of nitrogen.
The beta-carboxy Zn(II) phthalocyanine compound 2 was synthesized
in our lab according to our previously published method [42]. 1,3,5-
Triazatricyclo [3. 3. 1. 1 (3, 7)] decan-7-amine, O-benzotriazole-N,N,
N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU), N,N-diiso-
propylethylamine (DIPEA), dimethyl sulfoxide-d6 and dry DMF (dried
with molecular sieves) were purchased from the J & K Scientific LTD.
The common solvents such as acetonitrile, CH2Cl2 and diethyl ether
were purchased from the Sinopharm Chemical Reagent Co., Ltd. and
dehydrated before use. All organic reagents purchased from commercial
source were of analytical purity. The planar light-emitting diode (LED,
In addition, the dark toxicity measurement of compound 4 was
performed under the same conditions in dark. The compound 4 did not
showed noticeable dark toxicity toward these strains at concentrations
up to 10 μM for either S. aureus or E. coli.
2.5. UV–Vis and fluorescence spectra of compounds 3 and 4
λ = 660 nm
25 nm, 42.46 mW/cm2) light source used in the in-
Both compounds 3 and 4 did not have any solubility in water. In
DMSO, they showed UV–Vis spectra similar to each other with a B-band
at 346–347 nm, a vibronic band at 606 nm, and an intense sharp Q-
band at 677 nm (Fig. 5). The absence of absorption at 630 nm demon-
strated that they were non-aggregated in the organic solvent. Both
compound 3 and 4 showed a strong fluorescence emission at 690 nm in
DMSO (exc = 610 nm, Fig. 5). The molar extinction coefficients data of
the two compounds was available in supporting information (Table S1).
activation experiments was custom-made by Uniglory Electronics (HK)
Co., Ltd. The light intensity was measured by Spectra-Physics Model
407A Power Meter.
1H and 13C NMR spectra were recorded on Bruker Avance III 400 and
Bruker Avance III 500 spectrometers (400 and 500 MHz) in deuterated
solvent and the spectras were referenced internally by using the residual
solvent (DMSO-d6) resonances relative to SiMe4. Electrospray ionization
(ESI) mass spectra were recorded on a Thermo Finnigan LCQ Deca XP Max
mass spectrometer. The compounds were dissolved in DMSO and the
methyl alcohol was used as the mobile phase. UV–Vis and fluorescence
spectrum were measured with a BioTek Synergy 4 multi-mode microplate
reader in a 96-well plate under room temperature.
2.6. Safety of compound 4 toward blood red cells
A molecule containing high positive charge density will induce
toxicity to mammalian cells and restrict its clinical applications
[38–40]. To evaluate whether compound 4 will show toxicity toward
mammalian erythrocytes, we carried out hemolysis assay with red
blood cells of healthy mice at compound concentrations between 0.1
and 10 μM which was dissolved in PBS containing 1% DMSO [41]. We
observed negligible lytic activities at these concentrations (Fig. 6).
These results demonstrated the potential safety of compound 4 for the
3.2. Synthesis
3.2.1. Synthesis of compound 3
The compound 2 (80.6 mg), HBTU (100.7 mg) and DIPEA (46.9 mg)
were added to a solution of anhydrous DMF (40 ml), and then stirred at
Fig. 3. Antimicrobial effect of compound 4 against S.
aureus (a) and E. coli (b). S. aureus (B) or E. coli (D)
were incubated with compound 4 (1.6 and 8.0 μM
respectively) for 10 or 15 min, for S. aureus and E.
coli, respectively, followed by illumination with red
light at a dose of 12.7 J/cm2, and counted on agar
plates. A and C were the controls (no compound 4
but with light).
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H. Lin et al.
JournalofInorganicBiochemistry189(2018)192–198
Fig. 5. (a) UV–Vis spectra of compounds 2, 3 and 4 in DMSO. (b) Fluorescence spectrum of compounds 2, 3 and 4 in DMSO (exc = 610 nm).
40 h in the absence of light. After removing the volatiles in vacuo, the
remained solution was dumped into diethyl ether (about 1:5, v/v) to
give the blue precipitate, which was collected by centrifugation at
10000 rpm, washed with diethyl ether, ethyl acetate and acetonitrile,
and then dried in lyophilizer to give blue solid. The structure of com-
pound 4 was fully confirmed by proton NMR spectrum (Fig. S5) and
mass spectrometry measurement (Electrospray Ionization, ESI, Fig. S6),
which are available in supporting information. UV–Vis (DMSO):
λmax = 677 nm, HRMS (ESI): m/z calculated for C41H31N12OZn+
[M − I]+ 771.2035, found 771.2043. 1H NMR (400 MHz, DMSO-d6,
ppm): δ = 9.57 (s, 1H, Pc-Hα), δ = 9.00–9.28 (m, 7H, Pc-Hα), δ = 8.96
(s, 1H, - HN-CO-), δ = 8.59 (d, J = 8.0 Hz, 1H, Pc-Hβ), δ = 8.02–8.27
Fig. 6. Hemolysis assay of cationic compound 4 (0.1–10 μM) using mice ery-
throcytes. Erythrocytes incubated with distilled water were used as the positive
control.
(m, 6H, Pc-Hβ), δ = 4.26–4.54 (m, 4H, N-CH2-N+), δ = 3.99 (d,
J = 8.0 Hz, 2H, N-CH2-N), δ = 3.69 (s, 2H, C-CH2-N+), δ = 3.39 (s, 3H,
CH3-N+), δ = 2.59 (s, 4H, C-CH2-N).
room temperature for about 40 min, followed by the addition of 1, 3, 5-
triazatricyclo [3. 3. 1. 1(3, 7)] decan-7-amine (33.5 mg) and further
reaction at 37 °C overnight (about 17 h). The mixture was dumped into
water (about 1:4, v/v) to give the blue precipitate, which was collected
by centrifugation at 10,000 rpm, washed with deionized water, acet-
onitrile and CH2Cl2, and then dried in a lyophilizer to give blue solid
compound 3 (69.2 mg, 60.6%). The purity of compound 2 and com-
pound 3 were confirmed on HPLC by C18 column. Both compounds
were eluted with H2O/DMF gradient (50–100%, containing 0.1% TFA)
in 30 min. The HPLC chromatograms of compounds 2 and 3 showed
that they have high purities (> 95%). The final product compound 3
was eluted faster (22 min vs 24 min) on C18 column compared to its
precursor compound 2, indicating higher hydrophilicity of compound 3
compared to its precursor. The structure of compound 3 was fully
confirmed by 1H, 13C NMR spectrum (Figs. S2, S3) and mass spectro-
metry measurement (Electrospray Ionization, ESI, Fig. S4), which are
available in supporting information. UV–Vis (DMSO): λmax = 677 nm,
HRMS (ESI): m/z calculated for C40H29N12OZn+ [M + H]+ 757.1879,
found 757.1846. 1H NMR (400 MHz, DMSO-d6, ppm): δ = 9.49 (s, 1H,
Pc-Hα), δ = 8.86–9.17 (m, 7H, Pc-Hα), δ = 8.54 (d, J = 8.0 Hz, 1H, Pc-
Hβ), δ = 8.47 (s, 1H, - HN-CO-), δ = 7.98–8.19 (m, 6H, Pc-Hβ),
δ = 4.00–4.46 (m, 6H, N-CH2-N), δ = 3.87 (s, 6H, N-CH2-C). 13C NMR
(500 MHz, DMSO-d6, ppm): δ = 167.96, 153.15, 153.10, 152.96,
152.87, 152.31, 152.16, 151.53, 151.01, 139.42, 138.00, 137.91,
137.86, 137.31, 135.62, 129.63, 129.56, 129.51, 128.66, 122.57,
122.55, 122.49, 122.35, 122.08, 72.90, 59.60, 43.50 (some of the sig-
nals were overlapped).
3.3. Bacteria strain and culture conditions
The bioluminescent bacterial strains Gram-positive S. aureus, lumi-
nescent Staphylococcus aureus Xen29 were purchased from Shanghai
Biofeng Company (China). The Gram-negative bacteria, Escherichia coli
(E. coli DH5α), were transformed with bioluminescent plasmid
(pAKlux2.1) in our laboratory. They were also grown in Luria-Bertani
medium (LB medium) at 37 °C overnight. Then 100 μl of suspension of
the bioluminescent bacteria was added into fresh medium (10 ml) to
grow again until their optical density at 600 nm (OD600) reached about
0.6 (108 CFU/ml). The luminescent bacteria were further diluted with
PBS to give a relative luminescence intensity of roughly 2.0 × 105 for E.
coli and 1.0 × 105 for S. aureus in opaque white 96-well plate and read
with Synergy 4 multi-mode microplate reader. The luminescence in-
tensity was proportional to the amount of live bacteria within certain
range.
The Gram-positive S. aureus ATCC6538 and Gram-negative E. coli
ATCC25922 were grown in LB medium at 37 °C with agitation of
220 rpm for 7 h firstly. Then 60 μl of the suspension was added into
fresh medium (2 ml) to grow again (about 7 h for S. aureus, and 6 h for
E. coli) to mid log phase. The cells were harvested at 4000 rpm for
5 min, washed twice and re-suspended with PBS. The bacteria suspen-
sion (about 106 CFU/ml) was obtained after the dilution of 100 folds
with PBS.
3.4. Photodynamic inactivation of bacteria
The photosensitizers (PS) were dissolved in DMSO (1 ml) to 2.0 mM
and kept in the absence of light until use. After dilution with water by
10 folds, it was further diluted into concentration gradients with
DMSO:water (1:9). These PS gradients (20 μl each) were transferred
3.2.2. Synthesis of compound 4
Iodomethane (1.5 ml) was added to a mixture of compound 3
(30.0 mg) in anhydrous DMF (10 ml), and then stirred at 40 °C for about
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H. Lin et al.
JournalofInorganicBiochemistry189(2018)192–198
into opaque 96-well microplates containing bacteria suspension (about
106 CFU/ml, 180 μl). Final concentration of DMSO was kept to be low
(1%) and nontoxic to the bacteria. Then the bacteria were incubated at
37 °C in the dark (10 min for S. aureus, and 15 min for E. coli). After that,
Conflicts of interest
There are no conflicts to declare.
the microplate was illuminated with
a
planar LED light source
Acknowledgements
(λ = 660 nm
25 nm, fluency rate = 42.5 mW/cm2) for 5 min to give
a light dose of 12.7 J/cm2. After the illumination, the luminescence
intensity of the microplate was monitored on microplate reader for
50 min with 2 min interval (Fig. S7). The survival fractions Sl were
calculated as following formula:
Our research work is financially supported by grants from National
Key R&D Program of China (2017YFE0103200), and National Natural
Science Foundation of China (31370737, 31400637, 31570745,
31670739).
Sl = (Le/Lc) × 100%
Ethical approval
Here, Le and Lc were luminescence intensity of the experimental
and control group, respectively.
This article does not contain any studies with human participants or
animals performed by any of the authors.
To measure antimicrobial effect on Gram-positive S. aureus
ATCC6538 and Gram-negative E. coli ATCC25922, the PS was added to
bacteria at various concentrations in transparent 96-well microplate
under the same condition, incubated (10 min for S. aureus, and 15 min
for E. coli), and illuminated for 5 min using our LED light source. To
count the number of alive bacteria, 100 μl of the bacteria suspension
from each well was taken out for serial dilution with PBS. Then 100 μl
of the each diluted bacteria suspension was added into agar plate and
incubated at 37 °C overnight for counting of viable colonies (the agar
plates were prepared with LB medium one day in advance). The sur-
vival fractions S were calculated as following formula:
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2018.10.001.
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