A Report of Minor Research Project Triangulenium cation–silica microparticle conjugates as an efficient tool for the photosensitised disinfection of water contaminated by bacterial pathogens Ms. SEENA SEBASTIAN, (1933-MRP/14-15/KLMG034/UGC-SWRO) Assistant Professor, Department of Chemistry Assumption Autonomous College Changanacherry, Kottayam, Kerala, 686101 Submitted to University Grants Commission, New Delhi March 2017
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A Report of Minor Research Project
Triangulenium cation–silica microparticle conjugates as an efficient
tool for the photosensitised disinfection of water contaminated by
bacterial pathogens
Ms. SEENA SEBASTIAN,
(1933-MRP/14-15/KLMG034/UGC-SWRO)
Assistant Professor, Department of Chemistry
Assumption Autonomous College
Changanacherry, Kottayam, Kerala, 686101
Submitted to
University Grants Commission, New Delhi
March 2017
Abstract
Photosensitized processes have been shown to offer favourable perspectives for both the
treatment of several kinds of infectious diseases and addressing some environmental problems of
high scientific and societal impact. The photosensitized production of singlet oxygen can be used
for the disinfection of waste water contaminated with bacterial pathogens. A suitable choice of
the photosensitizer leads to the inactivation of a broad spectrum of pathogens. An extensive drop
in microbial cell survival can be often achieved by irradiation with intrinsically non-damaging
visible light in the presence of photosensitiser doses markedly lower than those found to be
phototoxic for host cells or tissues, as well as for non-target organisms. The trioxatriangulenium
carbocation is a member of the triphenylmethane dye family. Triangulenium cations are
remarkably stable carbenium ion in crystalline form as well as in solution such as in water,
acetonitrile, dichloromethane etc. The aqueous solutions are stable in the pH range of 1-9 so it is
less susceptible to nucleophilic attack. The most interesting property is that its absorption bands
is in the visible spectral region and has very good excited state properties with high fluorescence
and phosphorescence quantum yields. Changing the bridge atom in the trioxatriangulenium
cation from oxygen to nitrogen leads to the formation of triazatriangulenium cation with
significantly increased cation stability. The triazatriangulenium cations have moderate singlet
oxygen quantum yield. In this project triazatriangulenium cation and its derivatives are used as
photosensitizers for water disinfection. The synthesis of heterosensitizers gain attention in recent
times and found application in many fields including photosensitised disinfection of waste water
as they have many advantages over the homogenious photosensitizers. Among the hetero
sensitizers silica gel supported organic compounds received great attention. Immobilization of
organic functional group on silica surfaces produces modified silica.
The main objective of this work is to synthesize a hetrosensitizer in which
triazatriangulenium supported on silica. The silica is functionalised with amino groups using the
aminopropyl trimethoxy silane and methoy trimethyl silanes. The starting material for the
synthesis of triazatriangulenium cation is tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate
.The tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate is cyclised with amino functionalized
silica resulting in the loading of triazatriangulenium on the silica surface. The singlet oxygen
production capacity of the triazatriangulenium-silica microparticle conjugates can be monitored
using the molecular probe disodium 9,10 anthracene dipropionic acid. The disinfection studies
are also conducted inorder to determine the effectiveness of the synthesiszed
triazatriangulenium-silica microparticle conjugates for the waste water treatment. All the four
types of synthesiszed triazatriangulenium-silica microparticle conjugates produced the singlet
oxygen as it is evident from the photobleaching of molecular probe disodium 9, 10 anthracene
dipropionic acid. By the use of four types of synthesiszed triazatriangulenium-silica
microparticle conjugates there is large decrease in the survival of bacterial pathogens as it is
evident from the disinfection studies.
INTRODUCTION
The availability of drinking water is a critical problem for an important part
of the world population, mainly located in third-world countries where over one billion people
suffer diseases related to waterborne microorganisms. Classical water disinfection techniques
such as chlorination, and less-common alternatives based on other oxidizing reagents (ozone,
chlorine dioxide, etc.) or physical treatments (membrane filtration or UV-C illumination), are
typically used in urban or industrial areas. However, they are difficult to apply in isolated
regions, especially those in poorer countries, due to the lack of infrastructures.
Photodynamic microbial inactivation in the presence of cationic photosensitising
agents, aimed at the protection of the environment and improvement of its quality, can find a
particularly useful application for the disinfection of microbiologically polluted waters. The
aqueous milieu can facilitate a real-time electrostatic interaction between the positively charged
groups of the photosensitiser molecule and the array of negatively charged moieties at the
surface of many types of microbial cells, so that the photoinactivation of such cells can be
performed by irradiation after incubation times of the order of minutes, when no appreciable
accumulation of the photosensitiser in other cell types has occurred.
In this project triazatriangulenium cation and its derivatives are used as photosensitizers
for water disinfection. The trioxatriangulenium carbocation is a member of the triphenylmethane
dye family. Its trivial name is trioxatriangulenium and has the acronym TOTA+. Triangulenium
cations are remarkably stable carbenium ion in crystalline form as well as in solution such as in
water, acetonitrile, dichloromethane etc. The aqueous solutions are stable in the pH range of 1-9
so it is less susceptible to nucleophilic attack. The most interesting property is that its absorption
bands is in the visible spectral region (λmax, MeCN = 450 nm).and has very good excited state
properties with high fluorescence and phosphorescence quantum yields. Changing the bridge
atom in the trioxatriangulenium cation from oxygen to nitrogen significantly increases the cation
stability.
Trioxatriangulenium (TOTA) cation exhibits a triplet energy of 228 kJM-1 and a
quantum yield of intersystem crossing of 0.57 in water. The triplet state may therefore
transfer efficiently its energy to molecular oxygen to produce singlet oxygen.
Azatriangulenium cations (TATA) have similar photophysical properties and exhibit a robust
photochemical and thermal stability compared to trioxatriangulenium (TOTA) cation.
Triangulenium cations are efficient sensitizers for singlet oxygen generation.
The photodynamic inactivation of bacterial cell comprises the action of three
components: a photosensitizing agent (PS), a light source of an appropriate wavelength (artificial
light or sunlight) and oxygen. Two oxidative mechanisms of photoinactivation (PI) are
considered to be implicated in the inactivation of the target cells. The type I pathway involves
electron/hydrogen atoms-transfer reactions from the PS triplet state with the participation of a
substrate to produce radical ions while the type II pathway involves energy transfer from that
triplet state to molecular oxygen to produce singlet oxygen (1O2). Both processes lead to highly
toxic reactive oxygen species (ROS) such as 1O2 and free radicals, able to irreversibly alter vital
components of cells resulting in oxidative lethal damage. The main advantages of photodynamic
inactivation are the non-target specificity, the few side effects, the prevention of the regrowth of
the micro organisms after treatment and the lack of development of resistance mechanisms due
to the mode of action and type of biochemical targets.
MATERIALS AND METHODS
Solvents used were of reagent grade and used without further purification. Reagents were
purchased from Sigma-Aldrich and used as received. Absorption spectra were recorded using
Evolution 201 UV-visible spectrophotometer. Emission spectra were recorded using Fluoromax-
3 spectrophotometer. IR spectra were recorded on JASCO 4100 model, FTIR spectrometer. The
1H NMR spectra were recorded on 400 MHz on Bruker FT-NMR spectrometer with
tetramethylsilane(TMS) as internal standard. Chemical shifts were reported in parts per million
(ppm) downfield to tetramethylsilane.Molecular mass was determined by Waters 3100 mass
detector with an Electro-Spray-Ionization unit.
Synthesis of Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate
3.1.1 Preparation of 2, 6, 2’, 6’, 2’’, 6’’-hexamethoxytriphenylcarbinol.
Scheme 1
A solution of phenyllithium was prepared by addition of
bromobenzene (4.56 g, 33.28 mmol) in dry ether (15 mL) to lithium wire (0.52 g, 72.04 mmol).
1,3- dimethoxybenzene (6 g, 35.71 mmol) in dry benzene (25 mL) was added followed by
N,N,N’,N.’tetramethylenediamine (0.0298 g, 0.0256 mmol) and the mixture was stirred at 0oC
for 2hr under nitrogen to give a white suspension of 2,6– dimethoxyphenyllithium. To this
suspension was added diethyl carbonate (1.21 g, 10.95 mmol) dissolved in benzene (35 mL). The
mixture was heated at 80 oC for 12 hr in an inert atmosphere to give a brown solution. The
cooled reaction mixture was poured into 100 mL of water. The phases were separated and the
water phase was extracted with dichloromethane. The organic layer is concentrated and the
compound was precipitated by the addition of diethyl ether (100 mL).
Synthesis of Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate
Scheme 2
Aqueous HBF4 solution(50%,1.25ml,20mmol) was added to a solution of
tris(2,6-dimethoxyphenyl carbinol(2.9g,6.6mmol) in absolute ethanol (50ml).Diethyl ether(50ml)
was then added, followed by petroleum ether. The dark-blue precipitate formed was collected by
filtration and thoroughly washed with diethyl ether, yielding 3.6g of greenish black crystals.
Functionalisation of silica surface
Silica surface was functionalised with (3-aminopropyl)-trimethoxysilane
and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy silane based on
the reported procedure Prior to functionalization the silica material was refluxed in water (25 ml
per g of support) for 1 h. After the water treatment the material was collected by filtration and
washed with toluene (20 ml/g).The wet material was suspended in toluene (100 ml/g) and the
majority of the remaining water was removed during 2 h of azeotropic distillation (2.5 ml/g).
After cooling to ambient temperature pure APTMS (3.6 ml/g),MTMS (2.8 ml/g) or both silanes
were added to the slurry. In the latter case MTMS was added before APTMS. The total amount
of silane was kept constant at 19 mmol of reactive silane per g of support. Mixtures of APTMS
and MTMS are identified by the molar ratio of the silanes in the reaction mixture. For example, 1
g of support with APTMS/MTMS 1:5 was reacted with 0.7 ml (3.2 mmol) APTMS and 2.2 ml
(15.8 mmol) MTMS. The mixture was vigorously stirred for 14 h at room temperature. Then the
solid was filtered off, redispersed in fresh toluene (100 ml/g) and refluxed for 1 h. The solid was
collected by filtration and washed with isopropanol (20 ml/g).In one experiment a Soxhlet
extraction was performed at this stage. The functionalized material was placed in a Soxhlet filter
and treated with a 2:1 diethyl ether: acetonitrile mixture for 24 h. The functionalized material
was dried at 373 K in an evacuated oven.
Synthesis of triazatriangulenium silica microparticle conjugates
The triazatriangulenium silica microparticle conjugates are synthesized by
adding activated amino surface functionalized silica to a solution of tris (2, 6 dimethoxyphenyl)
carbenium tetrafuroborate in NMP under inert condition. The functionalized silicas were
characterized by the elemental analysis of CHN, Infrared spectroscopy was performed using KBr
pellets and a spectral range of 4000 to 400 cm, elemental analysis, SEM, TEM images.
Photophysical studies
The production of singlet oxygen by the triazatriangulenium-silica
particles was determined using the molecular probe disodium 9, 10-anthracene dipropionic acid
(ADPA). Typically, 50 mg of the triazatriangulenium– silica particle conjugate was suspended in
10 ml water. Then, 250 μl of this suspension was diluted to 1 ml with water and ADPA (23 μl,
5.5 mM) was added. The particles were exposed to LED light for 150 min with UV-visible
absorption spectra recorded every 30 min. Control experiments were performed using blank
silica micro particles. The same experiment was performed with sunlight also.
Photosensitization studies of microbial cell cultures with the triazatriangulenium-silica
particles
Irradiated and unirradiated samples of E. coli (10 ml final volume) were
prepared by adding a suitable volume of the triazatriangulenium- silica microparticle conjugates
to the cell suspensions, in order to achieve a final photosensitiser concentration of 10 μM. The
samples thus obtained were incubated at 37 °C in the dark for 5 min and then irradiated for 30
min with sun light. Unirradiated and irradiated bacterial cells were serially 10-fold diluted in
growth medium and the number of colonies found after 18–24 h incubation at 37 °C was
counted.
RESULTS AND DISCUSSION
Functionalisation of silica surface
FT-IR spectroscopy
FT-IR spectra were collected on FT-IR Spectrometer using KBr pellets in
the frequency range of 4000–400 cm-1. It was performed to study the structures and identify the
functional groups of raw silica, silica functionalized with APTMS and with the mixture of
APTMS and MTMS. All the FT-IR spectra present similar features. The intense and broad band
appearing at 1000–1235 cm–1 is corresponding to the asymmetric stretching vibrations of Si–O–
Si and the one at 794 cm–1 is due to the symmetric stretching vibration of Si–O–Si. The
characteristic band at 3300–3600cm–1 is responsible for the broadening Si–OH stretching
vibration and the isolated hydroxyl groups (water or alcohol) stretching by hydrogen bonding.
The characteristic bands located at 3300–3400 cm–1and 1479–1640 cm–1
were assigned to the stretching and bending vibrations of aliphatic amine (N–H) groups,
respectively, for the amino functionalized silica particles, was absent in silica indicates the
successful grafting of aminosilane into the silica surface.
By comparing the FTIR spectra of raw silica and silica functionalized with
APTMS and with the mixture of APTMS and MTMS new bands at 2941cm–1 and 2853 cm–1 are
observed for amine functionalized particles. These are the characteristic peaks of aminosilanes.
The presence of asymmetric and symmetric stretching vibrations of –CH2 on functionalized
silica particles indicates the grafting of aminopropyl groups of APTMS on the surface of raw
silica.
Elemental Analysis
Silica gels were functionalized with amino groups using (3-aminopropyl)-
trimethoxysilane and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy
silane. Results of elemental analysis of the functionalized silica gel samples are summarized in
table 1
Sample C: (mmol/g) N: (mmol/g) H : (mmol/g)
Silica functionalized
with APTMS
5.46
1.26
18.77
Silica functionalized
wit mixture of
APTMS+MTMS
4.07
0.71
18.1
Table 1. Elemental analysis amino functionalized silica gel
Scanning electron microscopy
The SEM images of silica functionalized with APTMS and with the
mixture of APTMS and MTMS are shown in figure3.
Figure1 .SEM images of (a) silica functionalized with APTMS (b) silica functionalized with
APTMS and MTMS.
Synthesis of triazatriangulenium-silica particles.
Four types of triazatriangulenium silica particles were prepared.(1)Silica
surface modified with aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium
tetrafluroborate was cyclised.(2) Silica surface modified with aptms and monoaza derivative was
prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are
cyclised with ethanolamine.(3) silica surface modified with aptms and mtms using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised.(4) Silica surface modified with aptms
and mtms using this monoaza derivative was prepared with Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.
FT-IR spectroscopy
(a) Silica surface modified with aptms alone and using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised.
The characteristic stretching and bending vibrations of aliphatic amine
groups observed in the region 1479cm-1 to 1640cm-1 was absent in the FT-IR spectrum of
synthesized triazatriangulenium silica particle insted the band at 1649cm-1 indicates the presence
of triazatriangulenium on the silica surface.
(b) Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with
ethanolamine.
The characteristic stretching and bending vibrations of aliphatic amine groups observed
in the region 1479cm-1 to 1640cm-1 was absent in the FT-IR spectrum of synthesized
triazatriangulenium silica particle insted the band at 1644cm-1 indicates the presence of
triazatriangulenium on the silica surface
(c) silica surface modified with aptms and mtms using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised.
The characteristic stretching and bending vibrations of aliphatic amine groups observed in the
region 1473cm-1 to 1634cm-1 was absent in the FT-IR spectrum of synthesized
triazatriangulenium silica particle insted the band at 1649cm-1 indicates the presence of
triazatriangulenium on the silica surface.
(d)Silica surface modified with aptms and mtms using this monoaza derivative was
prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are
cyclised with ethanolamine.
The characteristic stretching and bending vibrations of aliphatic amine groups observed in the
region 1473cm-1 to 1634cm-1 was absent in the FT-IR spectrum of synthesized
triazatriangulenium silica particle insted the band at 1648cm-1 indicates the presence of
triazatriangulenium on the silica surface.
Elemental Analysis
Silica gels were functionalized with amino groups using (3-aminopropyl)-
trimethoxysilane and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy
silane.This functionalized silica were used for cyclising Tris(2,6-dimethoxyphenyl)carbenium
tetrafluroborate.Thus triazatriangulenium compounds were loaded on the silica surface.
Sample C: (mmol/g) N: (mmol/g) H : (mmol/g)
S1
7.71
1.95
30.7
S2
5.91
2.32
20.8
S3
6.57
0.96
23.1
S4
7.03
2.14
27.7
Table 2. Elemental analysis of triazatriangulenium loaded amino functionalized silica.
S1- Silica surface modified with aptms alone and using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised.
S2- Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.
S3- silica surface modified with aptms and mtms using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised
S4- Silica surface modified with aptms and mtms using this monoaza derivative was prepared
with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with
ethanolamine
Scanning electron microscopy
Figure2. SEM images of (a) Silica surface modified with aptms (b) Silica surface modified with
aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(c)
Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.
Figure3. SEM images of (a) Silica surface modified with aptms and mtms (b) Silica surface
modified with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium
tetrafluroborate was cyclised(c) Silica surface modified with aptms and mtms monoaza
derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two
sides are cyclised with ethanolamine.
TEM IMAGES
Figure4. TEM images of (a) Silica surface modified with aptms alone and using this Tris(2,6-
dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified with aptms
and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate
further two sides are cyclised with ethanolamine(c) Silica surface modified with aptms and mtms
monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate
further two sides are cyclised with ethanolamine.
Photochemical characterization of the photosensitizing materials
The absorption maximum in the visible region of the of the triazatriangulenium was found to be
525nm in acetonitrile solution but when supported on the silica surface modified with aptms
changed to 485nm. The strong hypsochromic shift is a consequence of the less polar
environment of the silica surface and, probably some contribution of rigidochromism. Its
emission maximum is 557 nm in acetonitrile solution and 562 nm when this sensitizer is
immobilized in the silica surface modified with aptms.
Figure 5.Absorption spectra of (a) Silica surface modified with aptms alone and using this
Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified
with aptms and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium
tetrafluroborate further two sides are cyclised with ethanolamine. (c) Silica surface modified
with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was
cyclised(d) Silica surface modified with aptms and mtms monoaza derivative was prepared with
Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with
ethanolamine.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
300 400 500 600 700
Ab
so
rba
nc
e
Wavelength,nm
a
b
c
d
Figure6.Emission spectra of (a) Silica surface modified with aptms alone and using this
Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified
with aptms and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium
tetrafluroborate further two sides are cyclised with ethanolamine. (c) Silica surface modified
with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was
cyclised(d) Silica surface modified with aptms and mtms monoaza derivative was prepared with
Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with
ethanolamine.
Singlet oxygen production
The singlet oxygen production of the triazatriangulenium-silica particle during exposure
to LED was monitored by the molecular probe ADPA. In the presence of singlet oxygen, the
ADPA is photobleached, through the conversion to the endoperoxide, leading to a reduction in
the absorbance bands of the ADPA probe. Fig. 7a shows the change in absorbance intensity of
the ADPA bands upon irradiation with the LED light. Over a period of 60 min, the bands
0
100
200
300
400
500
600
700
800
900
1000
500 550 600 650 700
Inte
ns
ity
Wavelength,nm
a
b
c
d
decrease, suggesting that singlet oxygen is generated. The results obtained when blank silica
particles were left in the light of the LED in the presence of ADPA are shown in Fig. 7b. In this
instance the absorbance bands of the ADPA do not decrease significantly, indicating that singlet
oxygen is not produced by the blank silica particles. The control experiment shows that the
triazatriangulenium is essential for the generation of singlet oxygen. Experiments using the free
triazatriangulenium (Fig. 7c) confirm that the triazatriangulenium is an effective photosensitiser
as the ADPA is completely converted to the endoperoxide by singlet oxygen in the 60 min
period. A comparison of the data shown in Fig. 7a–b is shown in Fig. 7d. Over the 60 min
irradiation period, singlet oxygen is generated by both the triazatriangulenium silica particle and
the free triazatriangulenium as evidenced by the decrease in the absorption band of the ADPA at
400 nm. While the rate of singlet oxygen production is greater for the, triazatriangulenium
clearly shows that singlet oxygen is produced by the triazatriangulenium silica particles. The
singlet oxygen production of all the triangulenium supported samples were monitored using
ADPA.In all the four cases the the photobleaching of ADPA was observed.
Figure7. Production of singlet oxygen in the presence of LED light by (a) Triazatriangulenium