Axial ligand modified high valent tin(IV) porphyrins: Synthesis, structure, photophysical studies and photodynamic antimicrobial activities on Candida albicans

Article abstract of DOI:10.1039/c5ra09343k

Herein, we report the synthesis, structure, photophysical properties and photodynamic antimicrobial activities on Candida albicans of axial ligand modified high valent tin(IV) porphyrins, namely Sn^IV(OH)₂T(4-CMP)P (1), Sn^IV

Full text of DOI:10.1039/c5ra09343k

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: R. Soman, D.
Raghav, S. Sujatha, R. K and C. Arunkumar, RSC Adv., 2015, DOI: 10.1039/C5RA09343K.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Axial Ligand Modified High Valent Tin(IV) Porphyrins: Synthesis,
Structure, Photophysical Studies and Photodynamic Antimicrobial
Activities on Candida Albicans
Rahul Somana, Darpan Raghavb, Subramaniam Sujathaa, Krishnan Rathinasamyb* and
Chellaiah Arunkumara*
The photophysical studies, fluorescence imaging, single crystal x-ray structure analysis and DFT
calculations revealed that the compounds 2 and 3 have shown enhanced phototoxicity towards
Candida albicans compared to 1.

RSC Advances
Page 2 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Axial Ligand Modified High Valent Tin(IV) Porphyrins: Synthesis,
Structure, Photophysical Studies and Photodynamic Antimicrobial
Activities on Candida Albicans
Rahul Somana, Darpan Raghavb, Subramaniam Sujathaa, Krishnan Rathinasamyb* and
Chellaiah Arunkumara*
Herein, we report the synthesis, structure, photophysical properties and photodynamic
antimicrobial activities on Candida albicans using axial ligand modified high valent tin(IV)
porphyrins, namely SnIV(OH)2T(4ꢀCMP)P (1), SnIV(OH)2T(4ꢀCP)P (2) and SnIV(Oꢀ
NO2Ph)2T(4ꢀCMP)P (3). The newly synthesized porphyrins were characterized by various
spectroscopic methods and single crystal Xꢀray diffraction analysis. The crystal structure
analysis of precursor porphyrin, SnIV(Cl)2T(4ꢀCMP)P and 3 are well stabilized by various
intermolecular interactions and porphyrin 3 shows complementary bonding interactions
between the nitro groups (NꢁꢁꢁO) forming one dimensional array. The fluorescent lifetime of 3
is less compared to other porphyrins which indicates that there is a considerable interaction
between the tin(IV) porphyrin core and nitrophenyl system and this could be the reason for its
significant phototoxicity. This is further evident from Xꢀray crystallography and DFT
calculations. In the presence of light, tin(IV) porphyrins significantly inhibited the growth of
C. albicans in liquid as well as in the solid agar medium and the growth inhibitory effects
were very less under dark conditions. The porphyrin internalisation as well as localization
within the Candida cells was examined by fluorescence microscopic analysis. Our results
suggest that the mechanism behind the photodynamic inactivation of C. albicans could be
through the generation of singlet oxygen species within the cells.
1

Page 3 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Introduction
Photodynamic antimicrobial chemotherapy1 (PACT), a new treatment modality,
follows similar principles as that of photodynamic therapy (PDT) that utilizes nontoxic
components such as light, oxygen and a photosensitizing agent, promoting microbial
eradication by the production of reactive oxygen species such as singlet oxygen, superoxide
and hydroxyl radicals.2 Photosensitizer is generally allowed to get internalized within the
prokaryotic or eukaryotic cells of interest. When the cells containing photosensitizers are
exposed to light of a specific wavelength, it gets excited to singlet excited state and
undergoes intersystem crossing, reaches triplet energy state. While coming back to its ground
state, the photosensitizer produces singlet oxygen from cellular oxygen, which causes cell
damage leading to cell death.2 (Scheme 1a)
Scheme. 1 (a) Mechanism for the photodynamic antimicrobial chemotherapy; (b) Schematic representation of
tin(IV) porphyrins, 1−3 in this study.
Porphyrin based photosensitizers are widely accepted because it shows promising
broad spectrum of activity against microorganisms.3 C. albicans is an opportunistic fungal
pathogen which can affect vital areas of the body and its infections are more frequent in
immunocompromised individuals.4 Such severe infections are difficult to treat and were
usually cured with azoles which target the fungal cell membranes.5 However, there is a
growing interest towards finding better alternatives to treat infections caused by C. albicans
as these are developing resistance towards established azoles.6 The ability of photosensitizers
to cause photodynamic damage depends on their photophysical properties. A good
photosensitizer preferably has high triplet state quantum yields and long triplet state
lifetimes.7 Recently, tin(IV) porphyrins have shown much attention owing to their interesting
photophysical properties.8 They are very stable, ease of synthesis, diamagnetic and usually
sixꢀcoordinated with transꢀdiaxial ligands.9 High valent tin(IV) porphyrins with chlorine as
2

RSC Advances
Page 4 of 30
View Article Online
DOI: 10.1039/C5RA09343K
its axial ligands such as tin(IV) ethyl etiopurpurin and tin(IV) octaethylbenzochlorin were the
two well established secondꢀgeneration photosensitizers.10 In this line, we are inspired to
explore the synthesis, structure and photophysical properties of axial ligand modified tin(IV)
porphyrins (1−3) (Scheme 1b) with hydroxyl and 4ꢀnitrophenolate moieties. To better
understand the electronic transitions that takes place in SnIV(Cl)2T(4ꢀCMP)P, 1 and 3,
computational studies were performed. The photodynamic antimicrobial chemotherapy of C.
albicans using the porphyrins, 1−3 were also studied. The cytotoxic effects of porphyrins
were assessed in C. albicans cells by agar well diffusion and broth assay. Microscopic
analysis and antifungal assays were done in order to confirm the internalization of
compounds as well as for demonstrating the phototoxicity induced by these compounds in C.
albicans.
Experimental
Materials and methods
Chemicals employed for the synthesis were obtained as AR grade and used as received.
Solvents were purified using the available literature methods.11 The reference compound,
mesoꢀtetrakis(4ꢀNꢀmethylpyridyl)porphyrin, R [H2T(4ꢀNMP)P] was prepared according to
the literature procedure.12 Chemicals used for antimicrobial studies were purchased from
HiMedia and Sigma Aldrich. Optical absorption spectra were recorded at room temperature
1
on Shimadzu UVꢀ2450 spectrophotometer and H NMR spectra were taken using a Bruker
Avance III 400 MHz spectrometer. Fluorescence spectra and quantum yield measurements
were performed using a Perkin Elmer LS 55 luminescence spectrophotometer at 25.0 ± 0.5
°C. Fluorescence lifetime measurements were calculated from timeꢀresolved fluorescence
intensity decays using a Horiba Scientific Fluoromaxꢀ4 spectrofluorometer with data station
software in time correlated single photon counting mode. A pulsed lightꢀemitting diode
(Horiba NanoLEDꢀ560) was used as an excitation source. This LED generates optical pulse
at 564 nm of pulse duration 1.5 ns. LED profile (instrument response function) was measured
at the excitation wavelength using a Ludox 530 (colloidal silica) as the scatterer. To optimize
the signalꢀtoꢀnoise ratio, 10000 photon counts were collected in the peak channel. Photoꢀ
irradiations were done using a General electric Quartz line lamp (300 W). Light intensities
were measured with a pyroelectric detector (RJPꢀ735 from Laser Probe) which is connected
3

Page 5 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
to an energy ratio meter (RJꢀ7620 from laser probe). The study was conducted for an
irradiation time of 300 min and the average light fluence rate was 95 ꢀJ/cm2).
Synthesis of tin(IV) porphyrins
Synthesis
of
trans-dihydroxy[5,10,15,20-tetrakis(4'-carboxymethylphenyl)
porphyrinato] tin(IV), SnIV(OH)2T(4-CMP)P, 1
The precursor, 5,10,15,20ꢀtetrakis(4'ꢀcarboxymethylphenyl)porphyrin [H2T(4ꢀ
CMP)P] was synthesized using modified Lindsey method13 and the crude product was
purified by silica gel column chromatography using acetone / chloroform as eluent.
Evaporation of the solvent and recrystallization from chloroform / hexane results the desired
product with 40 % yield. UVꢀVisible data in THF, λmax (log ε / M−1 cm−1): 417 (6.12), 514
1
(4.88), 549 (4.50), 591 (4.33), 646 (4.12). H NMR data in CDCl3 (δ in ppm) (400 MHz):
8.82 (s, 8H, βꢀpyrroleꢀH), 8.44–8.46 (d, 8H, J = 8.00 Hz, oꢀphenylꢀH), 8.28–8.31 (d, 8H, J =
8.00 Hz, mꢀphenylꢀH), 4.11 (s, 12H, pꢀcarboxymethylꢀH), −2.81 (s, 2H, iminoꢀH).
Tin metallation was performed as per the reported method14 using stannous chloride
as metal ion carrier in pyridine. Excess water was added to precipitate the product which was
then filtered. The crude product was reꢀdissolved in chloroform, passed through anhydrous
Na2SO4 and purified using neutral alumina in chloroform / acetone mixture. The unreacted
free base porphyrin was collected initially, after which transꢀdichloro[5,10,15,20ꢀtetrakis(4'ꢀ
carboxymethylphenyl) porphyrinato] tin(IV), [SnIV(Cl)2T(4ꢀCMP)P] was separated out from
the column. The solvent was evaporated to dryness under vacuum and the yield was noted as
80 %. 1H NMR data in CDCl3 (δ in ppm) (400 MHz): 9.19 (s, 8H,
βꢀpyrroleꢀH), 8.51–8.53 (d,
8H, J = 8.00 Hz, oꢀphenylꢀH), 8.40–8.42 (d, 8H, J = 8.00 Hz, mꢀphenylꢀH), 4.13 (s, 12H, pꢀ
carboxymethylꢀH). This product was reꢀdissolved in tetrahydrofuran and reacted with slight
excess of potassium carbonate (1:50) in minimum quantity of water for 6 hrs. After
evaporation of solvent, the residue was reꢀdissolved in acetone / chloroform mixture and then
passed through anhydrous Na2SO4. It was then purified using neutral alumina to give 1 and
the yield was found to be 75 %. UVꢀVisible data: λmax (log ε / M−1 cm−1), (a) in THF: 427
1
(5.97), 561 (4.89), 600 (4.76); (b) in acetone: 423 (5.86), 555 (4.47), 595 (4.20). H NMR
data in CDCl3 (δ in ppm) (400 MHz): 9.10 (s, 8H, βꢀpyrroleꢀH), 8.50–8.52 (d, 8H, J = 8.00
Hz, oꢀphenylꢀH), 8.41–8.43 (d, 8H, J = 8.00 Hz, mꢀphenylꢀH), 4.11 (s, 12H, pꢀ
carboxymethylꢀH).
4

RSC Advances
Page 6 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Synthesis
of
trans-dihydroxy[5,10,15,20-tetrakis(4'-carboxyphenyl)porphyrinato]
tin(IV), SnIV(OH)2T(4-CP)P, 2
To the THF solution of 1, potassium hydroxide (20 equiv.) was added and the
resultant solution was refluxed for 24 hrs. The precipitated product was filtered to get the
purple solid residue and further treated with 0.01 N HCl (Caution: If the acid concentration is
slight excess, demetallation occurs) to obtain the final product. The porphyrin layer was
extracted with ethyl acetate, dried under vacuum and the yield was 60 %. UVꢀVisible data:
λmax (log ε / M−1 cm−1), (a) in THF: 430 (5.84), 563 (4.69), 602 (4.18); (b) in acetone: 425
1
(5.14), 561 (3.97), 601 (3.87). H NMR data for 2 in DMSOꢀd6 (δ in ppm) (400 MHz): 9.32
(s, 8H, βꢀpyrroleꢀH), 8.47–8.49 (d, 8H, J = 8.00 Hz, oꢀphenylꢀH), 8.44–8.46 (d, 8H, J = 8.00
Hz, mꢀphenylꢀH). ESIꢀmass data of 2: 939 [Mꢀ2H]+ (Calcd. 941).
Synthesis of trans-dinitrophenolate [5,10,15,20-tetrakis(4'-carboxymethylphenyl)
porphyrinato] tin(IV), SnIV(O-NO2Ph)2T(4-CMP)P, 3
Porphyrin, 1 and 4ꢀnitrophenol in nitrobenzene was magnetically stirred at 100−120
°C for 4 hrs. The formed porphyrin phenolate complex was crystallized using hexane to
afford dark red single crystals of 3 after a week time. UVꢀVisible data: λmax (log ε / M−1
cm−1), (a) in THF: 425 (5.84), 558 (4.73), 597 (4.29); (b) in acetone: 421 (6.17), 553 (4.84),
1
592 (4.58). H NMR data for 3 in CDCl3 (δ in ppm) (400 MHz): 9.06 (s, 8H,
β
ꢀpyrroleꢀH),
8.45–8.47 (d, 8H, J = 8.00 Hz, oꢀcarboxymethylphenylꢀH), 8.14–8.16 (d, 8H, J = 8.00 Hz, mꢀ
carboxymethylphenylꢀH), 6.54–6.55 (d, 8H, J = 7.20 Hz, mꢀnitrophenylꢀH), 4.07 (s, 12H, pꢀ
carboxymethylꢀH), 1.74–1.76 (d, 8H, J = 7.20 Hz, oꢀnitrophenylꢀH).
Crystal structure determination
Xꢀray quality single crystals of compounds, SnIV(Cl)2T(4ꢀCMP)P and 3 were obtained
by slow vapour diffusion method using chloroform / hexane and nitrobenzene / hexane
respectively. Red crystals were mounted on a glass capillary with suitable size and the crystal
data was collected on a Bruker AXS Kappa Apex II CCD diffractometer with graphite monoꢀ
chromated Mo Kα radiation (λ = 0.71073 Å) at 298 K. The reflections with I > 2σ(I) were
employed for structure solution and refinement. The SIR9215 (WINGX32) program was used
for solving the structure by direct methods. Successive Fourier synthesis was employed to
complete the structures after fullꢀmatrix least squares refinement on |F|2 using the
SHELXL9716 software.
5

Page 7 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Hirshfeld surface analysis
To analyse and quantify the various intermolecular interactions present in the crystal
structure of tin(IV) porphyrins, we have generated the Hirshfeld surfaces and 2D fingerprint
plots using Crystal Explorer 3.1.17
Microorganism and growth conditions
C. albicans strain CAF4ꢀ2 was obtained from Molecular genetics laboratory, School
of Biotechnology, National Institute of Technology Calicut. The organism was grown
aerobically at 37 °C in a standard YPD medium containing 1 % yeast extract, 2 % peptone, 2
% dextrose18. Since the strain is ura–, the growth medium was supplemented with 25 ꢂg/mL
uridine.19
Agar well diffusion assay for determining the photodynamic growth inhibition of C.
albicans
YPD Broth and YPD agar supplemented with uridine were prepared and sterilized by
autoclaving. C. albicans was initially grown overnight in 5 mL YPD Broth in a shaking
incubator. Shaking was maintained at 200 rotations per minute (rpm) while the temperature
o
was kept constant at 37 C. YPD Agar containing petri plates were prepared by pouring the
sterile YPD Agar into sterile petri plates to a uniform depth of 4 mm which is equivalent to
approximately 25 mL in a 90 mm plate. The medium was allowed to solidify in the plates and
the overnight culture of C. albicans was spread evenly on the top of the solidified agar with
the help of a sterile cotton swab. Wells were made in the agar plates for addition of the
porphyrin solutions. Three different concentrations of all the porphyrins were added in equal
volumes. The organism was allowed to grow aerobically at 37 oC in light or dark conditions.
After 12 hrs of incubation, the zone of inhibition was calculated by using the formula given
below:20
I (Diameter of the inhibition zone in mm x 100)
% of inhibition =
90 (Diameter of the Petriꢀplate in mm)
6

RSC Advances
Page 8 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Photodynamic inactivation of C. albicans in broth assay: Determination of half maximal
inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) values of
porphyrins
The C. albicans cells were inoculated in YPD liquid medium containing different
concentrations of porphyrins or 0.2 % DMSO (solvent control). The growth inhibition of C.
albicans was studied by monitoring absorbance at 600 nm (A600), at different time intervals
(0, 15, 30, 60, 90, 120, 150, 180, 240 and 300 min) in the presence of 3 different
concentrations of all the compounds (10, 25 and 50 ꢀM) and 20 ꢀg/mL of fluconazole as a
positive control. A comparative study of the same was done by varying the experimental
conditions in light and dark. The A600 value of control where no compound was added was
subtracted with A600 value obtained after adding the compound. The same was followed in
all the cases where compounds were added. The percentage inhibition of Candida growth
was calculated by using the equation:21
% of inhibition = [1−(XA600 / CA600)] x 100
where, XA600 represents the A600 value of X ꢀM compound treated culture at the different
time points. CA600 represents the A600 of the control culture at the same time. The percentage
of growth inhibition was determined for 5 hrs after the addition of porphyrins to the C.
albicans culture. The IC50 values of compounds were determined by plotting the percentage
inhibition of cell growth against compound concentration.21
C. albicans cells were grown for 4 hrs in the absence and presence of the compounds
in light and dark conditions. The lowest concentration of the compound that inhibited the
visible growth of the C. albicans was considered as the MIC. The concentration of the
compound, in which there was no visible growth was diluted and spread on to the agar plates
and incubated for additional 12 hrs at 37 °C. The number of colonyꢀforming units was
calculated by counting the colonies on each plate.21
Cellular uptake profiling of porphyrins in C. albicans cells
In order to establish a time profile for intracellular accumulation of the compounds, a
spectrofluorimetric analysis was carried out. C. albicans cells treated with different
porphyrins were allowed to grow in 5 mL liquid culture. The cultures were incubated in a
shaking incubator in the dark. After 30, 60, 90, 120, 150, 180, 240 and 300 minutes, aliquots
7

Page 9 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
of cells were harvested by centrifuging at 6000 rpm for 5 minutes. The supernatant liquid was
discarded and the cell pellet was washed thrice with 1 mL of 1X PBS in order to remove the
uninternalized porphyrins. After this, cells were gently resuspended in 1X PBS and then were
transferred into 1 mL fluorescence cuvette. A fluorescence spectral analysis was carried out
by exciting the samples at 550 nm.22
Wide field fluorescence microscopic analysis
For studying the intracellular localization and phototoxicity of the compounds, two
sets of C. albicans cells were grown in liquid medium. One set of cells were cultured and
grown by incubating in dark for 6 hrs. Another set was initially grown in the dark for 6 hrs
and was then exposed to light and grown for 3 more hrs. The cells were then harvested and
washed thrice with 1X PBS in order to remove the excess unꢀinternalised compounds. As per
the spectrofluorimetric analysis, the porphyrins get excited around 550 nm and give emission
around 600 nm. Hence, for this study the compounds were excited using the green filter (490ꢀ
510 nm). C. albicans cells were stained with Hoechst 33342 (10 ꢂg/mL followed by 30
minutes incubation) to visualize the DNA23. The fluorescence imaging was performed using
LEICA DM5000B fluorescent microscope (Germany) at 100x magnification (oil immersion)
and the images were processed by using ImageJ (NIH, USA).
Confocal fluorescence microscopic studies
The C. albicans cells grown in YPD medium as described above were incubated in dark with
the compounds for 6 hrs. The cells were then harvested and washed thrice with 1X PBS in
order to remove the excess unꢀinternalised compounds. Cells were then mounted on clean
glass slides and sealed for microscopic analysis.24 The confocal fluorescence imaging was
performed using Confocal fluorescent microscope (Germany) at 63x magnification. To
confirm the internalization of the compounds, a progressive confocal optical slicing approach
was employed. Images were sliced progressively at every 0.8 ꢂm depth and the
internalization of the compounds was analysed. Images were obtained by merging bright field
and fluorescent images and further processed by using ImageJ (NIH, USA).
8

RSC Advances
Page 10 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Measurement of singlet oxygen
1,3ꢀDiphenylisobenzofuran (DPBF) was used as a selective singlet oxygen (1O2) acceptor,
which was bleached upon reaction with 1O2 generated by porphyrin donors. DPBF (1.7 x 10ꢀ7
M) solutions without or with porphyrin derivatives (6.6 x 10ꢀ7 M) were prepared in DMSO
under dark conditions. These solutions were irradiated at room temperature and under gentle
magnetic stirring. The breakdown of DPBF was monitored by measuring the decrease in
absorbance at 410 nm at preꢀestablished irradiation intervals.25 From this plot, the rates of 1O2
production of porphyrins 1−3 relative to those of H2TPP and R were determined.
Results and Discussion
Synthesis
The precursor, H2T(4ꢀCMP)P was prepared using the modified procedure of Lindsey
et al.13 and their tin(IV) complexes, SnIV(Cl)2T(4ꢀCMP)P, 1 and 2 were prepared by the
variant literature methods.14 Synthesis of 3 was done by condensing 1 with 4ꢀnitrophenol
(Scheme 2). Synthesized porphyrins were isolated, purified by column chromatography and
characterized by UVꢀVisible, 1H NMR spectroscopic methods, mass spectrometry and single
crystal Xꢀray diffraction analysis.
Scheme 2. General synthesis of tin(IV) porphyrins, 1−3.
9

Page 11 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Photophysical properties
The photophysical properties such as optical absorption, steady state emission,
fluorescence quantum yield and fluorescence decay profile were studied for tin(IV)
porphyrins 1–3, SnIV(OH)2TPP and the porphyrin ligand H2T(4ꢀCMP)P in THF medium. The
electronic absorption spectra of tin(IV) porphyrins exhibit an intense Soret (B) band
corresponds to S0 → S2 transition around 425 nm and two visible (Q) bands around 560 and
600 nm which corresponds to Q(1,0) and Q(0,0) peaks of S0 → S1 transition.26 It is observed that
there is a marginal red shifted absorption spectrum for 2 and a marginal blue shifted
absorption spectrum for 3 compared to that of 1 in THF as well as acetone media (Fig. S1).
The steadyꢀstate fluorescence of porphyrins was performed in order to study their electronic
properties in excited state. The normalized absorption and emission spectrum of porphyrin 3
is shown in Fig. 1.
On excitation near the Soret band, all the tin(IV) porphyrins exhibit two wellꢀdefined
emission bands near 605 nm (band 1) and 657 nm (band 2) which corresponds to S1 → S0
transition and the spectral profile is comparable with the reported hexaꢀcoordinated tin(IV)
porphyrin systems.9,26 Also, there is a very weak emission corresponding to S2 → S0 transition
at 434, 462 and 489 nm for porphyrins, SnIV(OH)2TPP, 1 and 2 respectively (Table 1).
Fig. 1 Normalized absorption (solid line) and emission (dashed line) spectra of 3 in THF at 298 K. Emission
spectra was recorded by exciting the Soret region.
10

RSC Advances
Page 12 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Table 1 Photophysical data of porphyrins recorded in tetrahydrofuran at 300 K.
Porphyrin
H2T(4ꢀCMP)P
Absorption
max (nm)
Fluorescencea Quantum Yield
Fluorescence
λ
λ
max (nm)
lifetime, τ (ns)
Φ
f (S1-S0)
0.057
417, 514, 549,
591, 646
425, 560, 600 434, 604, 656
427, 561 600 462, 605, 657
430, 563, 602 489, 605, 658
605, 656
650, 716
10.10b
SnIV(OH)2TPP
0.044
0.017
0.008
0.010
01.48c
01.75d
0.90d
1, SnIV(OH)2T(4ꢀCMP)P
2, SnIV(OH)2T(4ꢀCP)P
3, SnIV(OꢀNO2Ph)2T(4ꢀCMP)P 425, 558, 597
0.66d
aFluorescence spectra of porphyrins were obtained as a function of λex in the Soret band region. bExcited at 460
c
nm and emission monitored at 650 nm in toluene. Excited at 460 nm and emission monitored at 600 nm in
toluene. dExcited at 460 nm and emission monitored at 600 nm in DMF.
The S1 → S0 fluorescence quantum yield (Φf) of the tin(IV) porphyrins were also measured
using H2TPP as the reference27 (Φf = 0.11). As seen from the data given in table 1, there is a
quenching of fluorescence in 2 and 3 compared to 1 which may be due to the excited state
processes such as enhanced internal conversion, intersystem crossing etc.26 As anticipated,
the metalloporphyrins show low quantum yields compared to the free ligand, H2T(4ꢀCMP)P
which indicates that the metal coordination may induce the radiationless decay rate.28 The
fluorescence decay profiles (λexc = 460 nm and λem = 600 nm) of porphyrins measured by
timeꢀcorrelated single photon counting (TCSPC) technique shown that the fluorescent life
time for 3 is shorter than that of 1 which supports that the tin(IV) porphyrin and nitrophenyl
system considerably interact with each other (Fig. 2).29 This is further confirmed by the
crystal structure analysis of 3 (Fig. 4b) which indicates that there is an π−π interaction
between porphyrin plane and 4ꢀnitrophenyl system due to their close proximity. The
fluorescence decay times were summarized in table 1 and the S1 lifetimes (τ) of porphyrins,
1−3 were determined to be 1.75, 0.90 and 0.66 ns respectively. The τ of porphyrin 2 is
comparable with the reported tin(IV) porphyrin, SnIV(OH)2THP and its value is 0.91 ns.30
11

Page 13 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 2 Fluorescence decay curves of 1 and 3 observed at 600 nm along with IRF measured in THF. (λexc = 460
nm)
Crystal structure description of porphyrins, SnIV(Cl)2T(4-CMP)P and 3
Single crystal XRD analysis showed that the porphyrins, SnIV(Cl)2T(4ꢀCMP)P and 3
crystallized in triclinic Pꢀ1 system (Table 2). The asymmetric unit of SnIV(Cl)2T(4ꢀCMP)P
consists half a molecule of porphyrin and an acetonitrile molecule in which the porphyrin
plane is found to be almost planar in nature. The ORTEP and molecular crystal packing
diagrams of SnIV(Cl)2T(4ꢀCMP)P are shown in Fig. 3. The solvent molecule (CH3CN) acts as
a bridge connecting two neighbouring porphyrin molecules through strong hydrogen bonding
between nitrogen of the acetonitrile with the βꢀpyrrole hydrogen [(Solvent)NꢁꢁꢁH(βꢀpyrrole), 2.733
Å] of one molecule and with phenylꢀhydrogen [(Solvent)NꢁꢁꢁH(phenyl), 2.631 Å] of the another
molecule which is viewed along ‘bc’ plane and depicted in Fig. 3b. Other intermolecular
interactions which makes the compound SnIV(Cl)2T(4ꢀCMP)P to form the two dimensional
array of molecules viewed down ‘ab’ plane (Fig. 3c) involving SnꢀClꢁꢁꢁC(βꢀpyrrole) (3.368 Å)
and OꢁꢁꢁH bonding [(carbonyl)OꢁꢁꢁH(βꢀpyrrole), 2.421 Å and (carbonyl)OꢁꢁꢁH(carboxymethyl), 2.634 Å].
Overall, the crystal structure of SnIV(Cl)2T(4ꢀCMP)P is well stabilized by various
intermolecular interactions described above.
Fig. 4a and 4b represents the top and sideꢀon view of ORTEP diagrams of 3
respectively. From the sideꢀon view, it is clear that the porphyrin core is planar and the two
axial ligands (4ꢀnitrophenyl ring) are present in the apex positions which make an angle of
123.18 (14)o between the porphyrin mean plane and 4ꢀnitrophenyl ring through oxygen atom
of the phenolate.
12

RSC Advances
Page 14 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Table 2 Summary of crystal structure data of porphyrins.
SnIVCl2T(4-CMP)P
3
Formula
C56H42Cl2N6O8Sn
C64H44N6O14Sn
Formula weight
CCDC
Crystal System
Space Group
Density, mg/m3
a [Å]
b [Å]
c [Å]
α [deg]
β [deg]
1116.55
948482
Triclinic
Pꢀ1
1239.74
974215
Triclinic
Pꢀ1
1.468
1.276
7.8170(3)
10.9187(4)
15.3971(6)
93.089(2)
99.167(2)
102.119(2)
1263.26(8)
0.71073
1
10.6568(5)
12.5048(5)
13.3413(5)
73.218(3)
76.096(4)
74.284(4)
1612.99(12)
0.71073
1
γ [deg]
V [Å3]
λ, Å
Z
Temperature, K
No. of unique
reflections
GOF on F2
293(2)
4958
293(2)
6337
1.081
0.0288
0.0778
1.099
0.0349
a
R1
wR2
b
0.0865
[a]R1 = Σ||Fo|–|Fc||/Σ|Fo|; Io > 2σ (Io). [b]wR2 = [Σ w(Fo –Fc2)2/Σw(Fo ) ]
.
2
2 2 1/2
Fig. 4c represents the three dimensional network arrangement of 3 (viewed down ‘a’ axis)
depicting various intermolecular interactions, viz., (carbonyl)OꢁꢁꢁH(carboxymethyl), 2.582 Å,
(carbonyl)OꢁꢁꢁH(phenyl), 2.384 Å, (nitro)OꢁꢁꢁH(βꢀpyrrole), 2.587 Å, (βꢀpyrrole)CꢁꢁꢁH(phenyl), 2.759 – 2.884 Å,
(βꢀpyrrole / phenyl)CꢁꢁꢁH(carboxymethyl), 2.885 Å, (nitro)NꢁꢁꢁO(nitro), 3.002 Å and each interactions are
present in the asymmetric unit of 3 in two numbers. Interestingly, the nitro group present in
the apex positions are involving complementary bonding interactions between the nitrogen of
one molecule with the oxygen of another molecule making a one dimensional array of
molecules (Fig. 4d).
13

Page 15 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 3 (a) ORTEP diagram of SnIV(Cl)2T(4ꢀCMP)P with atomic numbering (solvent molecule, acetonitrile is not
shown for clarity; thermal ellipsoids shown at 40 % probability level); Molecular crystal packing diagram of
SnIV(Cl)2T(4ꢀCMP)P (b) depicting NꢁꢁꢁH bonding viewed along ‘bc’ plane; (c) depicting ClꢁꢁꢁC and OꢁꢁꢁH
bonding viewed down ‘ab’ plane.
The SnIV−O bond length in 3 is compared with various tin(IV) porphyrins available in the
literature31ꢀ33 and the data are presented in table 3. It is seen that the observed bond length in
3 (2.066 Å) is comparable with the tin(IV) porphyrins containing phenolic BODIPY as well
as 2ꢀnitrophenol as axial ligands.
14

RSC Advances
Page 16 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 4 ORTEP diagrams of 3 (a) top view and (b) side view (thermal ellipsoids shown at 40 % probability level;
the aryl rings are omitted for clarity in side view of the molecule); (c) Molecular crystal packing diagram of 3
depicting various intermolecular interactions (viewed down ‘a’ axis); (d) One dimensional array of molecules
involving complementary NꢁꢁꢁO bonding (viewed along ‘ab’ plane).
Table 3 The SnIV−O bond length in various tin(IV) porphyrins.
SnIV−O (Å)
2.066
S. No
Axial Ligand
4ꢀnitrophenolate
phenolate
4ꢀnitrophenolate
phenolic BODIPY
2ꢀnitrophenolate
Porphyrin
3
1
2
3
4
5
2.055
2.083
2.062
2.070
TTP
TTP
TPP
TTP
15

Page 17 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Hirshfeld surface analysis
In order to further quantify the various intermolecular interactions in porphyrins
SnIV(Cl)2T(4ꢀCMP)P and 3, Hirshfeld surfaces (HSs) and their associated fingerprint plots
were calculated using Crystal Explorer 3.1.17 The short and long close contacts can be
visualized by colorꢀcoding, where the colour intensity indicates the relative strength of the
interactions: red regions represent closer contacts and negative dnorm value; blue regions
represent longer contacts and positive dnorm value; and white regions represent the distance of
contacts is exactly the van der Waals separation and with a dnorm value of zero. The HSs of
both the porphyrins SnIV(Cl)2T(4ꢀCMP)P and 3 shows intense red spots due to the close OꢁꢁꢁH
contacts whereas faint red spots are observed for NꢁꢁꢁH and CꢁꢁꢁH contacts for SnIV(Cl)2T(4ꢀ
CMP)P and 3 respectively (Fig. 5). The 2Dꢀfingerprint plot provides the decomposition of
Hirshfeld surfaces into contribution of different intermolecular interactions present in crystal
structure which features spike of various length and thickness.34 A closer inspection at the
FPs reveal that the major intermolecular interactions in both the complexes are mainly CꢁꢁꢁH
[21.5 % in SnIV(Cl)2T(4ꢀCMP)P; 15.1 % in 3], HꢁꢁꢁH [33.9 % in SnIV(Cl)2T(4ꢀCMP)P; 30.1
% in 3] and OꢁꢁꢁH [16.5 % in SnIV(Cl)2T(4ꢀCMP)P; 27.6 % in 3] contacts.
Fig. 5 Hirshfeld surfaces of porphyrins, SnIV(Cl)2T(4-CMP)P and 3 (a, d) top view and (b, e) side view with
dnorm mapped ranging from ꢀ0.28 (blue) to 3.49 (red); (c, f) Fingerprint plots of SnIV(Cl)2T(4-CMP)P and 3 with
di and de ranging from 1.0 to 2.8 Å. Close contacts are labeled as follows: CꢁꢁꢁH (1), HꢁꢁꢁH (2), OꢁꢁꢁH (3), NꢁꢁꢁH
(4) and HꢁꢁꢁCl (5).
16

RSC Advances
Page 18 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Photodynamic inactivation of C. albicans using tin(IV) porphyrins
Agar well diffusion assay for determining the photodynamic growth inhibition of C.
albicans
Earlier studies have shown the ability of C. albicans to exhibit multidrug resistance.35 The
photodynamic effect of porphyrins was assessed by performing zone of inhibition assay. It
was found to be dependent on the photosensitization environment as the compounds which
were illuminated with visible light showed maximum zone of inhibition as compared to the
compounds which were left unilluminated. Fig. 6 indicates the drug concentration and its
corresponding zone of inhibition in dark and light conditions. According to our study,
compound 3 gave the maximum percentage inhibition in light in a concentration dependent
manner i.e. around 24.4 % inhibition at 50 ꢂM (30.9 ꢂg/mL) concentration. Also, compounds
1, 2 and the reference compound R showed a high percentage inhibition in a concentration
dependant manner upon illumination. In dark, the zone of inhibition of the compounds is
much lesser as compared to light illuminated. All the compounds showed a higher percentage
of inhibition as compared to the standard antifungal drug fluconazole, which implicates the
possible use of these compounds against multidrug resistant strains of C. albicans.
Fig. 6 Comparison of the percentage of inhibition by different compounds under light and dark conditions.
Percentage of inhibition was found to increase with the increase in the concentrations of the compounds. a, b,
and c refer to the concentrations 10, 25 and 50 µM respectively. All compounds showed higher percentage of
inhibition as compared to the standard antifungal drug fluconazole.
Photodynamic inactivation of C. albicans in broth assay: Determination of IC50 and
MIC values of porphyrins
Inhibition of growth of C. albicans cells in broth was studied under light and dark conditions.
All the compounds show phototoxic effects in a time and concentration dependant manner.
Porphyrins 1, 2, 3 and R inhibited the growth of C. albicans with an IC50 value of 14.7 ꢂM,
17

Page 19 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
9.1 ꢂM, 9.0 ꢂM and 9.4 ꢂM respectively (Fig. 7). Interestingly, all the synthesized
compounds exhibited lower IC50 values as compared to that of fluconazole (17.5 ꢂM). Also,
there was a significant difference between the percentage inhibition of porphyrins under light
and dark conditions. Compounds, 1−3 exhibit a higher percentage of inhibition when exposed
to light, however in dark the percentage of inhibition is significantly lower.
Fig. 7 C. albicans cells were grown in YPD broth medium supplemented with uridine, with varying
concentrations of different porphyrins or DMSO (0.2 %). The study was performed in dark and light conditions.
Growth curves for C. albicans cells in the absence (♦) and presence of 10 ꢀM (❚), 25 ꢀM (▲) and 50 ꢀM (x)
porphyrins are shown in the graph. The figure also shows the percentage inhibition of growth of C. albicans in
the presence of different porphyrins in varying concentrations after 5 hrs of growth under light and dark
conditions. The IC50 mentioned in the figures corresponds to the samples which were illuminated with light.
18

RSC Advances
Page 20 of 30
View Article Online
DOI: 10.1039/C5RA09343K
The concentration of compound at which lesser or no colonies observed was considered as
the MIC.36 Porphyrins at a concentration of 50 ꢂM exhibits nearly complete inhibition of cell
growth when exposed to light. Very few colonies (2ꢀ20) observed in the petri plates in which
C. albicans cells treated with the compounds and exposed to light were plated. Importantly,
compound 3 showed the least number of isolates. Comparatively more number of colonies
(40ꢀ70) was observed when compound treated C. albicans cells, cultured under dark, were
plated on the solid medium (Fig. 8). This shows that growth inhibitory effect of our
compounds was maximum when the cells were exposed to light, explaining the
photodynamic mode of action for our compounds.
Fig. 8 Figure comparing the number of isolates obtained after plating the C. albicans cells, treated with 50 ꢂM
of porphyrins, cultured in light and dark conditions. Treated C. albicans, cultured in light, gave lesser number of
colonies, upon plating. In general, C. albicans cells treated with different porphyrins gave lesser number of
colonies upon plating as compared to the untreated cells.
Cellular uptake profiling of the porphyrins in C. albicans cells
The internalization profile indicates that the rate of internalization of all the compounds
increased with time. It was found that compound 3 showed maximum internalization and
compound 1, the minimum internalization. These results are in compliance with those
obtained from photodynamic inactivation experiments performed in this study in which
compound 1 showed a higher IC50 value as compared to other compounds. In the light of the
above observations, it is reasonable to suggest that the slow and inefficient internalization of
compound 1 could be the reason for its least potency compared to other compounds under
study (Fig. 9).
19

Page 21 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 9 The cellular uptake profiling study results reveals the slow intake of all porphyrins into the Candida cells.
Compound 3 showed a better uptake profile than other compounds. Compound 1 showed relatively less
internalisation as compared to the other compounds.
To study the internalization of porphyrins, a fluorescent microscopic analysis of C. albicans
CAF4ꢀ2 cells was carried out by incubating the cells with tin(IV) porphyrins (Fig. 10). The
cells exposed to all the three porphyrins exhibit fluorescence when excited using the green
filter excitation, which confirms the internalisation of the compounds. Hoechst 33342 was
used to stain the nucleus. The fluorescence of all the compounds was uniformly distributed
throughout the cells, indicating their cytoplasmic localisation. These results also confirm that
the cytotoxic effect on C. albicans was specifically due to the internalisation of the
porphyrins.
20

RSC Advances
Page 22 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 10 Internalization of porphyrins in C. albicans CAF4ꢀ2. Cells were incubated with the solvent control (DMSO) or with
different porphyrins, and were grown for 6 hrs and processed for microscopy as given in the materials and methods. (A)
bright field image (B) Hoechst staining (C) porphyrin fluorescence (D) merged.
Confocal fluorescence microscopic studies
In order to confirm the internalisation of porphyrins in C. albicans cells, a progressive
confocal microscopic analysis was performed. The cells were sliced optically through Zꢀaxis
with 0.8 ꢂm depth. Compound 3 shows the best internalisation profile (Fig. 11). The C.
albicans cells exposed to 3 were optically sliced. Red fluorescence of 3 increased
progressively and reached maximum value at a depth of 2.4−4.8 µm (Fig 11 EꢀG) within the
cells. Red fluorescence is negligible on the upper and lower sections of the cells (Fig 11 A, B
21

Page 23 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
and J), showing that there is no or less binding of the compound with the cell membrane. In
all the cases, red fluorescence increases as the cells were sliced deeper, confirming the
internalization and cytoplasmic localization of the compounds. All the other compounds
showed proper internalisation (data not shown). This data is in support with the results
obtained from fluorescent wide field imaging.
Fig. 11 Internalisation of compound 3 by progressive confocal optical slicing of C. albicans. Images were
obtained by merging bright field and fluorescent images. AꢀJ: As the 0.8 ꢂm slices progress deeper within the
cells, the intensity of red fluorescence increased and reached a maximum at the middle portion, indicating
efficient compound internalization i.e. within the cell's cytoplasm.
It is well documented that photosensitizers like porphyrins were known to interfere with the
mitochondrial membrane potential and release the inner mitochondrial membrane enzymes
like cytochrome c and activate cell death pathways.37 Because of the bright cytoplasmic
staining, we were not able to clearly differentiate the internalization of the synthesized
porphyrins to the other cellular organelles like mitochondria or vacuoles which is also quite
possible.
Photogeneration of singlet oxygen
The photodynamic inactivation of C. albicans could be through the generation of singlet
oxygen species within the cells. This can be visualized using 1,3ꢀdiphenylisobenzofuran
1
(DPBF) which was able to capture the O2 generated by the porphyrins.2c,25 The relationship
1
between the change in absorbance by DPBF and irradiation time reflects the O2 yield of the
compounds. The absorbance of DPBF at 410 nm decreased in the presence of porphyrin with
1
increasing irradiation time and from the slope of the line, the relative rate of O2 generation
1
1
was compared. Higher the line slopes, higher the O2 yield. From the plot, the order of O2
22

RSC Advances
Page 24 of 30
View Article Online
DOI: 10.1039/C5RA09343K
generation rate was found to be 3 > 2 > 1 > R > H TPP (Fig. 12). The results are in good
2
agreement with the antifungal assays and fluorescence microscopic results.
Fig. 12 Plot of change in absorbance of DPBF vs. irradiation time in the presence of porphyrins.
DFT calculations for HOMO-LUMO gap in porphyrins
In order to understand more about the electronic transitions, we have performed DFT
calculations at the BP86/def2ꢀSVP level of theory38 using Gaussian 0939 program package for
compounds using their crystallographic information files (CIF) obtained from the single
crystal Xꢀray diffraction studies. For comparison, we have optimized the structures of H2TPP
and H2T(4ꢀNMP)P. The highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) of free base porphyrins, H2TPP, H2T(4ꢀNMP)P and tin(IV)
porphyrins, SnIV(Cl)2T(4ꢀCMP)P, 1 and 3 are shown in the Fig. 13. For free base porphyrins,
the HOMO and LUMO is a typical πꢀtype orbital on the porphyrin core. Hence, the electronic
transition is a wellꢀknown π → π* type transition upon excitation. In the case of
SnIV(Cl)2T(4ꢀCMP)P, the HOMO is mainly based on the porphyrin core and axial ligand
chlorine has lesser contribution. Here, the HOMOꢀ1 is porphyrin based orbital and the
HOMOꢀ2 and HOMOꢀ3 is concentrated on the axial ligand (Fig. S9). For 1, the HOMO
doesn’t have any contribution from the axial ligand, whereas the HOMOꢀ1, which is slightly
lesser in energy than HOMO (ꢁE = 0.05 eV) have more contribution from the hydroxyl
moiety (Fig. S10). For 3, the HOMO has major contribution from 4ꢀnitrophenolate moiety
and also the underlying orbitals, HOMOꢀ1 and HOMOꢀ2 has contribution from the axial
ligand (Fig. S11). In all the above structures the LUMO is a π based orbital on the porphyrin
core.
23

Page 25 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
Fig. 13 Orbital energies (in eV) at the BP86/def2ꢀSVP level of theory for the compounds. The nature of highest
occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) and the HOMOꢀLUMO
gap of the compounds are shown.
On analyzing the electronic transitions based on the nature of orbitals in tin(IV) porphyrins,
there is a charge transfer (CT) from chlorine atom, hydroxyl and 4ꢀnitrophenolate group to
the porphyrin core in SnIV(Cl)2T(4ꢀCMP)P, 1 and 3 respectively. Since chlorine has less
contribution in the HOMO of SnIV(Cl)2T(4ꢀCMP)P, π → π* is more favorable and the extend
of CT will be less since the orbitals corresponding to CT (HOMOꢀ2 and HOMOꢀ3) (Fig. S9)
is stabilized much on comparing with HOMO. Upon photon excitation, both π → π* and CT
transition are possible in 1, which are energetically more favorable because the HOMO has a
πꢀtype character and the HOMOꢀ1 has contribution from the hydroxyl fragment, ꢁE[HOMO−
= 0.05 eV. However, 3 shows the most prominent charge transfer from the 4ꢀ
(HOMO−1)]
nitrophenolate to porphyrin core which is the cause for the intersystem crossing (ISC), in turn
resulting in the higher population of lower energy triplet state.40 Our experimental results
which also correlates with the theoretical observations that 3 shows more phototoxicity which
can be attributed to more CT which in turn results in the generation of more singlet oxygen.
Hence, the enhanced phototoxicity of 3 is in concordance with the DFT calculations. The
results of all the above studies show that the newly synthesized porphyrins 2 and 3 possibly
the better candidates for treating C. albicans infections.
24

RSC Advances
Page 26 of 30
View Article Online
DOI: 10.1039/C5RA09343K
Conclusions
In summary, high valent tin(IV) porphyrins, 1−3 were synthesized, characterized and their
photophysical properties were studied. Xꢀray crystal structure determination of porphyrins
SnIV(Cl)2T(4ꢀCMP)P and 3 were achieved successfully and the result shows that there is π−π
interaction between the porphyrin plane and 4ꢀnitrophenyl system in 3 due to their close
proximity. We also explored the potential applications of these newly synthesized tin(IV)
porphyrins towards photodynamic antimicrobial chemotherapy (PACT) in C. albicans. The
light and dark experiments carried out in agar plates and broth media supports the
photodynamic inactivation of C. albicans. Porphyrins, 1−3 showed lower IC50 values than the
standard drug fluconazole. Also, 2 and 3 possess high cellular penetration and cytoplasmic
binding compared to 1. Wide field and confocal fluorescence microscopic experiments are
evident for porphyrin internalisation throughout the cytoplasm which is the main reason
behind the induced phototoxicity. The higher activity of 3 is further supported by DFT
calculations which reveal the most prominent charge transfer from 4ꢀnitrophenolate to
porphyrin core. This charge transfer increases the population of lower energy triplet state
leading to the generation of more singlet oxygen. To conclude, porphyrins 1−3 exhibited
significant antimicrobial activities, and especially 2 and 3 could be developed as new
antifungal agents for treating C. albicans infections.
Acknowledgements
The Department of Science and Technology (DST), New Delhi is gratefully acknowledged
for the financial support to SS (SR/WOSꢀA/CSꢀ146/2011) and CA (SB/EMEQꢀ016/2013).
We express our sincere thanks to Prof. T. N. Guru Row and Mr. Vijith Shetty for one of the
single crystal data collections. Authors are thankful to Dr. Babu Varghese, SAIF, IIT Madras
for another single crystal data collection, structure solution and refinement. We acknowledge
Ms. Indu, P, Ms. Rojisha, V. C and Dr. P. Parameswaran, Theoretical and Computational
Chemistry Laboratory, Department of Chemistry, NIT Calicut for the DFT calculations. We
1
would like to thank Prof. I. Ibnusaud for providing H NMR facility at Institute for Intensive
Research in Basic Sciences (IIRBS), MG University, Kottayam. We thank Ms. Agisha and
Dr. R Suseela Bhai for providing wide field microscope facility at Indian Council of
Agricultural Research (ICAR) − Indian Institute of Spices Research (IISR), Calicut. Our
thanks due to Mr. Vimal, G and Dr. Santhosh for providing Fluoromaxꢀ4 fluorescence
25

Page 27 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
spectroscopy facility at School of Pure and Applied Physics, MG University, Kottayam. We
also extend our thanks to Dr. Anilkumar P. R. for providing confocal microscopy facility at
Tissue culture laboratory, Sree Chitra Tirunal Institute for Medical Sciences and Technology
(SCTIMST), Trivandrum.
Note and References
aBioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of
Technology Calicut, Kozhikode, Kerala, India
arunkumarc@nitc.ac.in (C. Arunkumar)
–
673 601; Eꢀmail address:
bSchool of Biotechnology, National Institute of Technology Calicut, Kozhikode, Kerala,
India – 673 601; Eꢀmail address: rathin@nitc.ac.in (K. Rathinasamy)
†Electronic supporting information: Additional figures, 1H NMR and mass spectra.
References
1. M. Wainwright, J. Antimicrob. Chemother., 1998, 42, 13.
2. (a) E. D. Sternberg, D. Dolphin and C. Bruckner, Tetrahedron., 1998, 54, 4151; (b) L. M.
Giroldo, M. P. Felipe, M. A. de Oliveira, E. Munin, L. Procopio Alves and M. S. Costa,
Laser. Med. Sci., 2009, 24, 109; (c) S. C. Karunakaran, P. S. Babu, B. Madhuri, B.
Marydasan, A. K. Paul, A. S. Nair, K. S. Rao, A. Srinivasan, T. K. Chandrashekar, Ch.
M. Rao, R. Pillai and D. Ramaiah, ACS Chem. Biol., 2012, 8, 127.
3. (a) M. M. Gois, C. Kurachi, E. J. B. Santana, E. G. O. Mima, D. M. P. Spolidório, J. E. P.
Pelino and V. S. Bagnato, Laser. Med. Sci., 2010, 25, 391; (b) D. A. Caminos, M. B.
Spesia, P. Pons and E. N. Durantini, Photochem. Photobiol. Sci., 2008, 7, 1071.
4. (a) M. J. McCullough, B. C. Ross and P. C. Reade, Int. J. Oral Maxillofac Surg., 1996,
25, 136; (b) G. Molero, R. D. Orejas, F. N. García, L. Monteoliva, J. Pla, C. Gil, M.S. –
Pérez and C. Nombela, Internatl. Microbiol., 1998, 1, 95.
5. C. Onyewu, J. R. Blankenship, M. D. Poeta and J. Heitman, Antimicrob. Agents
Chemother., 2003, 47, 956.
6. (a) D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod and J. Bille, Antimicrob.
Agents Chemother., 1995, 39, 2378; (b) S. A. Flowers, B. Colon, S. G. Whaley, M. A.
Schuler and P. D. Rogers, Antimicrob. Agents Chemother., 2015, 59, 450
7. E. S. Nyman and P. H. Hynninen, J. Photochem. Photobiol. B., 2004, 73, 1.
26

RSC Advances
Page 28 of 30
View Article Online
DOI: 10.1039/C5RA09343K
8. (a) K. P. Ghiggino, N. K. Giri, J. Hanrieder, J. D. Martell, J. Müller, M. F. Paige, B.
Robotham, J. Szmytkowski and R. P. Steer, J. Phy. Chem. A., 2013, 117, 7833; (b) S.
Dey, P. Mondal and S. P. Rath, New J. Chem., 2015, 39, 4100.
9. (a) D. P. Arnold and J. Blok, Coord. Chem. Rev., 2004, 248, 299; (b) V. S. Shetti, Y.
Pareek and M. Ravikanth, Coord. Chem. Rev., 2012, 256, 2816.
10. (a) B. W. Pogue, R. W. Redmond, N. Trivedi and T. Hasan, Photochem. Photobiol.,
1998, 68, 809; (b) D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer.,
2003, 3, 380; (c) M. R. Detty, S. L. Gibson and S. J. Wagner, J. Med. Chem., 2004, 47,
3897; (d) W. M. Sharman, C. M. Allen and J.E. Van Lier, Drug Discov. Today., 1999, 4,
507.
11. D. D. Perrin and W. L. F. Armarego, Purification of Organic Solvents, Pergamon Press,
Oxford, 1988.
12. J. S. Lindsey, I. C. Schreiman, H. C. Hsu, P. C. Kearney and A. M. Marguerettaz, J. Org.
Chem., 1987, 52, 827.
13. J. S. Lindsey and R. W. Wagner, J. Org. Chem., 1989, 54, 828.
14. (a) D. P. Arnold, J. Chem. Edn., 1988, 65, 1111; (b) M. J Crossley, P. Thordarson and R.
AꢀS. Wu, J. Chem. Soc. Perkin Trans. 1., 2001, 2294; (c) P. Bhyrappa, C. Arunkumar and
J. J. Vittal, J. Chem. Sci., 2005, 117, 139.
15. (a) A. G. Altomare, G. Cascarano, C. Giacovazzo and A. Gualardi, J. Appl. Crystallogr.,
1993, 26, 343; (b) L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837.
16. G. M. Sheldrick, SHELXL97; University of Goettingen: Goettingen, Germany, 1997.
17. (a) S. K. Wolff, D. J. Grimwood, J. J. McKinnon, M. J. Turner, D. Jayatilaka and M. A.
Spackman, Crystal Explorer 3.1 (2013), University of Western Australia, Crawley,
Western Australia, 2005ꢀ2013; http://hirshfeldsurface.net/CrystalExplorer; (b) M. A.
Spackman and D. Jayatilaka, CrystEngComm., 2009, 11, 19.
18. D. M. Hampsey, G. Das and F. Sherman, J. Biol. Chem., 1986, 261, 3259.
19. W. A. Fonzi and M. Y. Irwin, Genetics., 1993, 134, 717.
20. J. V. Bennett, J. L. Brodie, E. J. Benner and W. M. M. Kirby, Appl. Microbiol., 1966, 14,
170. (b) S. S. Magaldi, C. MataꢀEssayag, H. De Capriles, C. Perez, M. T. Colella, C.
Olaizola and Y. Ontiveros. Int. J. Infect. Dis., 2004, 8, 39.
21. D. Rai, J. K. Singh, N. Roy and D. Panda, Biochem. J., 2008, 410, 147.
22. S. Makhseed, M. Machacek, W. Alfadly, A. Tuhl, M. Vinodh, T. Simunek, V. Novakova,
P. Kubat, E. Rudolf and P. Zimcik, Chem. Commun., 2013, 49, 11149.
27

Page 29 of 30
RSC Advances
View Article Online
DOI: 10.1039/C5RA09343K
23. (a) P. Appadurai and K. Rathinasamy, Toxicol. Lett., 2014, 225, 66; (b) D. Suresh, M. S.
Balakrishna, K. Rathinasamy, D. Panda and S. M. Mobin, Dalton Trans., 2008, 2812; (c)
S. A. G. Lambrechts, M. C. G. Aalders and J. Van Marle, Antimicrob. Agents
Chemother., 2005, 49, 2026; (d) J. M. Bliss, C. E. Bigelow, T. H. Foster and C. G.
Haidaris, Antimicrob. Agents Chemother., 2004, 48, 2000; (e) S. Oriel and Y. Nitzan.
Photochem. Photobiol., 2012, 88, 604.
24. K. Rathinasamy, B. Jindal, J. Asthana, P. Singh, P. V. Balaji and D. Panda, BMC
Cancer., 2010, 10, 213.
25. M. O. L. Lourenço, B. A. Iglesias, M. R. P. Pereira, H. Girão, R. Fernandes, M. G. P. M.
S. Neves, J. A. S Cavaleiro, and J. P. C. Tomé. Dalton Trans., 2015, 44, 530.
26. (a) D. R. Reddy and B. G. Maiya, J. Porphyrins Phthalocyanines., 2002, 6, 3; (b) A. A.
Kumar, L. Giribabu, D. R. Reddy and B. G. Maiya. Inorg. Chem., 2001, 40, 6757.
27. (a) P. G. Seybold and M. Gouterman, J. Mol. Spectrosc., 1969, 31, 1; (b) O. Ohno, Y.
Kaizu and H. Kobayashi, J. Chem. Phys., 1985, 82, 1779.
28. T. Zoltan, F. Vargas, V. Lopez, V. Chavez, C. Rivas and A. H. Ramirez, Spectrochim.
Acta Part A Mol. Biomol. Spectrosc., 2015, 135, 747.
29. H. J. Kim, K.ꢀM. Park, T. K. Ahn, S. K. Kim, K. S. Kim, D. Kim and H.ꢀJ. Kim, Chem.
Commun., 2004, 2594.
30. N. Venkatramaiah, B. Ramakrishna, A. R. Kumar, N. Veeraiah and R. Venkatesan, J.
Alloys Compd., 2012, 513, 318.
31. S. J. Langford, A. P. Marcia, K. J. Lee, K. J. Macfarlane and J. A. Weigold, J. Incl.
Phenom. Macrocycl. Chem., 2001, 41, 135.
32. T. Lazarides, S. Kuhri, G. Charalambidis, M. K. Panda, D. M. Guldi and A. G.
Coutsolelos, Inorg. Chem., 2012, 51, 4193.
33. G. D. Fallon, M. A. P. Lee, S. J. Langford and P. J. Nichols, Org. Lett., 2002, 4, 1895.
34. (a) F. R. Kooriyaden, S. Sujatha, B. Varghese and C. Arunkumar, J. Fluor. Chem., 2015,
170, 10; (b) R. Soman, S. Sujatha and C. Arunkumar, J. Fluor. Chem., 2014, 163, 16.
35. (a) L. Millon, A. Manteaux, G. Reboux, C. Drobacheff, M. Monod, T. Barale and Y. M.
Briand, J. Clin. Microbiol., 1994, 32, 1115; (b) R. Franz, S. L. Kelly, D. C. Lamb, D. E.
Kelly, M. Ruhnke and J. Morschhäuser, Antimicrob. Agents Chemother., 1998, 42, 3065.
36. T. Beuria, P. Singh, A. Surolia and D. Panda. Biochem. J., 2009, 423, 61.
28