Synthesis, structure, electrochemical, DNA interaction and antimicrobial studies of fluorinated trans-dicationic pyridinium porphyrins

Article abstract of DOI:10.1039/c6ra09148b

A series of trans-pyridyl porphyrins, 5,15-di(pentafluorophenyl)-10,20-bis(2′/3′/4′-pyridyl)porphyrin (1-3), trans-dicationic pyridinium porphyrins, 5,15-di(pentafluorophenyl)-10,20-bis(2′/3′/4′-N-methylpyridyl)porphyrins (4-6) and their copper(ii) and zi

Full text of DOI:10.1039/c6ra09148b

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Synthesis, Structure, Electrochemical, DNA Interaction and Antimicrobial
Studies of Fluorinated Trans-Dicationic Pyridinium Porphyrins
Jagadeesan Ramesh, Subramaniam Sujatha∗ and Chellaiah Arunkumar∗
Bioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of Technology
Calicut, Kozhikode, Kerala, India - 673 601.
Abstract:
A series of trans-pyridyl porphyrins, 5,15-di(pentafluorophenyl)-10,20-bis(2ꞌ/3ꞌ/4ꞌ-pyridyl)porphyrin
(1−3), trans-dicationic pyridinium porphyrins, 5,15-di(pentafluorophenyl)-10,20-bis(2ꞌ/3ꞌ/4ꞌ-N-
methylpyridyl)porphyrins (4−6) and their copper(II) and zinc(II) derivatives were synthesized. These
compounds were characterized using various spectroscopic methods, electrochemical and single crystal
X-ray crystallographic studies. The trans-dicationic porphyrin derivatives exhibit red shifted absorption
spectra over the simple pyridyl porphyrins. The reduction potentials of trans-pyridyl porphyrins are more
positive than those of MTPPs. Crystal structure of 3c is forming networks of molecules through Zn-N
coordination displaying large number of channels. The intermolecular interactions involving fluorine
contributes considerably to the crystal packing in all the structures which was further confirmed by
computational Hirshfeld surface analysis. The dicationic porphyrins were further explored for its DNA
interaction abilities and antimicrobial activities. The UV-visible and fluorescence spectroscopic titrations
indicate that the porphyrins bind with the calf thymus DNA by outside groove binding mode with or
without self-stacking. The intrinsic binding constants Kb of these dicationic porphyrins to DNA was
found to be in the range of 105−106 M−1. The results reveal that among the three sets of porphyrins (4−6),
the 3-/4-pyridyl derivatives display higher DNA binding activities compared to the 2-pyridyl analogues.
The photocleavage experiments disclose that the porphyrins employ 1O2-mediated mechanism in cleaving
DNA and the freebase and zinc(II) derivatives show better photoinduced cleavage ability compared to its
copper(II) analogues. The dicationic porphyrins also show significant antimicrobial activities than those
of non-fluorinated analogues.
Keywords: Trans-dicationic pyridyl/pyridinium porphyrins, Electrochemical studies, Crystal
structure analysis, DNA interaction studies, Antimicrobial studies.
________________________________________________________________________
*Corresponding author. Fax: 0495-2287250
E-mail addresses: sujatha@nitc.ac.in (Dr. S. Sujatha); arunkumarc@nitc.ac.in (Dr. C. Arunkumar)
1

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Introduction
Synthetic molecules that interact efficiently with nucleic acids are well known as
artificial nucleases. They are highly stable, low cost and site specific alternatives to natural
nucleases which help to improve the essential understanding of mechanistic characteristics of
enzyme action, to develop new biotechnological tools and also in nucleic acid targeting
therapeutics. Porphyrin based artificial nucleases are very important due to their application in
photodynamic therapy (PDT).1,2 They are fascinating molecules because of their unique
spectroscopic, geometric and physicochemical properties and also find use in various fields
including material chemistry and biomimetics.3 In particular, cationic porphyrins enter the cells
readily and mainly interact with DNA through three major binding modes involving
intercalation, outside groove binding and outside binding without self-stacking depending on the
DNA base composition, choice of peripheral substituents and the nature of central metal on the
porphyrin.4 When porphyrins are photo activated, singlet oxygen (1O2) is produced by energy
transfer from the photo excited sensitizer to the ground state oxygen which is known as type II
mechanism and it is more important in PDT. The excited photosensitizer can act as a reducing
agent in the formation of reactive oxygen species such as superoxide radical, hydrogen peroxide
or hydroxyl radical.3
Fiel and coworkers first investigated the interaction of cationic porphyrins with the
negatively charged backbone of DNA either by electrostatic binding or intercalation.5 The
cationic porphyrins act as a bifunctional compounds and due to the electrostatic interaction
between the positively charged porphyrin periphery and anionic phosphate backbone of nucleic
acid, effective interaction with DNA is possible. And, the binding modes can be studied using
UV-Visible titration, fluorescence, viscometry etc. and the DNA cleavage ability is usually
detected by agarose gel electrophoresis.6-9 Porphyrins bearing pyridyl/pentafluorophenyl
substituents at the porphyrin periphery have been widely used as photosensitizer in PDT due to
their high triplet quantum yields and also efficient catalyst for various redox reactions.10-12 The
pyridyl substituted porphyrins can also act as important building blocks for supramolecular self-
assemblies.13,14 Also, fluorinated porphyrins show enhanced pharmacokinetic properties
compared to its non-fluorinated analogues and in fact they mimic the biological functions of
enzyme cytochrome P450.15-17 In order to explore the combined properties of a porphyrin
containing both pentafluorophenyl group as well as pyridyl group at the porphyrin periphery, we
2

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synthesized the porphyrins namely, trans-pyridyl porphyrins, 5,15-di(pentafluorophenyl)-10,20-
bis(2ꞌ/3ꞌ/4ꞌ-pyridyl)porphyrin (1−3), trans-dicationic pyridinium porphyrins, 5,15-
di(pentafluorophenyl)-10,20-bis(2ꞌ/3ꞌ/4ꞌ-N-methylpyridyl)porphyrins (4−6) and their copper(II)
and zinc(II) derivatives using 5-(pentafluorophenyl) dipyrromethane and 2-/3-/4-pyridine
carboxaldehyde as a precursors (Fig. 1). Their photophysical, electrochemical properties, DNA
interaction abilities as well as its antimicrobial activities have also been explored.
F
F
R
N
F
F
N
N
N
F
N
M
N
F
N
F
F
(R1)
(R2)
(R3)
R
F
F
R =
+
R = R1; M = 2H, 1a
R = R1; M = Cu(II), 1b
R = R1; M = Zn(II), 1c
R = R2; M = 2H, 2a
R = R2; M = Cu(II), 2b
R = R2; M = Zn(II), 2c
R = R3; M = 2H, 3a
R = R3; M = Cu(II), 3b
R = R3; M = Zn(II), 3c
+
N
+
N
N
R = R4; M = 2H, 4a
R = R4; M = Cu(II), 4b
R = R4; M = Zn(II), 4c
R = R5; M = 2H, 5a
R = R5; M = Cu(II), 5b
R = R5; M = Zn(II), 5c
R = R6; M = 2H, 6a
R = R6; M = Cu(II), 6b
R = R6; M = Zn(II), 6c
(R4)
(R5)
(R6)
Fig. 1. Chemical structures of trans-pyridyl and trans-dicationic pyridinium porphyrins under study.
Experimental
Materials and methods
nalytical grade reagents / chemicals were purchased and used as such without any
A
further purification. Solvents were purified as per the standard procedures18 before its use for any
chemical reaction. Calf thymus DNA was purchased from Sigma Aldrich. The supercoiled pBR
322 DNA and ethidium bromide was purchased from Merck. UV-Visible spectra were recorded
at room temperature in DMF using a Shimadzu UV-2450 double beam spectrophotometer and
1H NMR spectra were taken in a Bruker Avance III 400 MHz spectrometer. Mass spectra were
recorded under ESI/HR-MS at 61,800 resolution using Thermo Scientific Exactive mass
spectrometer (Thermo Fischer Scientific, Bremen, Germany). Fluorescence spectra were
measured using a Perkin Elmer LS 55 luminescence spectrophotometer. Cyclic voltammetric
measurements of the porphyrin samples were performed on a CH-instruments Inc, USA (Model:
CHI660E) equipped with potentiostat / galvanostat with Fourier Transform AC Voltammeter.
The electrochemical cell consists of a three-electrode cell assembly; a glassy carbon working
electrode, standard Calomel electrode (SCE) as reference electrode and a platinum wire as the
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auxiliary electrode. The concentrations of the porphyrin samples were employed as ~ 0.5 mM.
All the measurements were performed at 25 °C in DMF under N2 atmosphere using 0.1 M
tetrabutylammonium hexafluorophosphate (TBAHFP) as the supporting electrolyte. Single
crystal structure X-ray data collections were performed on a Bruker AXS Kappa Apex II CCD
diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 298 K.
Synthesis of trans-porphyrins
Synthesis of 5,15-di(4ꞌ-pyridyl)-10,20-bis(pentafluorophenyl)porphyrin and their
metal derivatives
The precursor, 5-(pentafluorophenyl)dipyrromethane was prepared using the available
literature method.19 5-(pentafluorophenyl)dipyrromethane (0.1 g, 3.2 x 10-4 mol) and 4-
pyridinecarboxaldehyde (0.034 g, 43.2 x 10-4 mol) were added to a dry double neck round-
bottom flask with 350 mL of CH2Cl2 and N2 gas purged for 10 min. Then, trifluoroacetic acid
(TFA, 1.5 mL) was added and the contents were stirred under N2 at ice cold temperature (~ 5 °C)
for 30 min. To that, about 0.14 g of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was
added and the reaction mixture was stirred at room temperature for an additional 1 h. After that,
the solution was quenched with saturated NaHCO3 and extracted with CHCl3. Compound was
purified by silica column chromatography using chloroform-acetone mixture and was dried
under vacuum. The porphyrin, 3a was yielded as a purple solid (0.046 g; Yield: 36 %). The
copper(II) (3b) and zinc(II) (3c) complexes were synthesized in chloroform/methanol mixture
using the corresponding metal acetate and the yields were found to be quantitative (93-95 %).
UV-Visible data of porphyrins in DMF at 298 K, λmax (log ε / M−1 cm−1): 3a, 412 (5.41), 507
(4.21), 582 (3.71); 3b, 411 (5.40), 537 (4.13); 3c, 421(5.24), 553 (3.85). 1H NMR data of 3a (400
MHz, CDCl3): δ (ppm) 9.08 (d, 4H), 8.91 (d, 4H), 8.87 (d, 4H), 8.17 (d, 4H), -2.92 (s, 2H). ESI
mass data of 3a (C42H18N46F10): 796.62 (Calcd.), 797.15 (Found). Fluorescence spectral data of
porphyrins in DMF at 298 K (λex at 415 / 416 nm), λem for 3a: 640 (s), 706 (s); λem for 3c: 600
(s), 649 (s). ESI-mass data of 3a: 795.17 (calcd: 796.62). Similarly, the 2ꞌ-/3ꞌ-pyridyl and its
metal complexes (1a−1c, 2a−2c) were synthesized.
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Synthesis of 5,15-di(4ꞌ-N-methylpyridyl)-10,20-bis(pentafluorophenyl)porphyrin and
their metal derivatives
5,15-di(pyridyl)-10,20-bis(pentafluorophenyl)porphyrin (0.020 g, 2.5 x 10-5 mol) was
taken in a dry double neck round-bottom flask containing 30 mL of CHCl3. Methyl
trifluoromethanesulfonate (23 µL, 2 x 10-4 M) was added in drops for 5 min and the contents
were stirred vigorously for another 30 min. The precipitate was filtered through sintered crucible
(G4) and thoroughly washed with CHCl3 to remove any other impurities. The desired product, 6a
was obtained in 92 % yield (0.026 g). The copper(II) (6b) and zinc(II) (6c) complexes were
synthesized as discussed earlier and the yields were 93-95 %. UV-Visible data of porphyrins in
DMF at 298 K, λmax (log ε / M−1 cm−1): 6a, 417 (5.36), 509 (4.29), 583 (3.85); 6b, 416 (5.38),
541 (4.26); 6c, 426 (5.21), 557 (4.13). 1H NMR data (400 MHz, Acetone-d6): δ (ppm), 6a, 9.64-
9.09 (m, 16 H), 5.00 (s, 6H), -2.95 (s, 2H); 6c, 9.57 (s, 4H), 9.93 (s, 4H), 9.10 (d, 8H), 4.98 (s,
6H). Fluorescence spectral data of porphyrins in DMF at 298 K (λex at 415 / 416 nm), λem for 6a:
646 (s), 707 (s); λem for 6c: 616 (s), 654 (s). ESI-mass data of 6a: 811.17, loss of two triflate
anions and a methyl group (calcd: 1124.82). Similarly, the porphyrins, 4a−4c and 5a−5c were
synthesized.
Single crystal X-ray structure determination
Upon mounting the appropriate sizes of the single crystals of zinc(II) porphyrins (1a, 2a,
3c, 6a and 6c) on a glass capillary, the crystal structure data collections were achieved on a
Bruker AXS Kappa Apex II CCD diffractometer. The reflections with I > 2σ(I) were employed
for structure solution and refinement. The structure was solved using SIR9220 (WINGX32)
program by direct methods. And, structure refinement was done using the SHELXL9721 software
by successive Fourier synthesis on |F|2 using the full-matrix least squares refinement.
Hirshfeld surface analysis
To analyse the intermolecular interactions present in the crystals, Hirshfeld surface
analysis and generation of 2D fingerprint plots were obtained using Crystal Explorer 3.122 based
on results of single crystal X-ray diffraction analysis.
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DNA binding studies
The concentration of the calf thymus (CT) DNA was determined by using the average
1
1
extinction coefficient value of 6600 M− cm− of a single nucleotide at 260 nm.23 The stock
solution (100 mM) of compound 4−6 were prepared in 6 % DMF, 50 mM Tris Buffer and 50
mM NaCl were diluted in millipore water for further use. Spectroscopic titrations were carried
out to determine the binding affinity between DNA and porphyrins under study (4−6).
Absorption titrations were carried out in the presence of fixed concentration of porphyrins (3
ꢀM) and titrated with varying concentration of DNA (0–3 ꢀM).24 The fluorescence emission
spectra of freebase and copper(II) dicationic porphyrins were recorded in the range of 450-800
nm (λexc = 411−426 nm). The change in fluorescence intensity was observed by titrating the
fixed amount of cationic porphyrins (1 µM) with varying concentration of CT-DNA (0−1 µM).25
The relative binding of porphyrins to DNA (12 ꢀM) was studied by fluorescence spectroscopy
using ethidium bromide (EB) bound to DNA solution in Tris-buffer (pH = 7.0).26 The
fluorescence intensity at 490-800 nm (λex = 480 nm) of EB (7.5 ꢀM) with an increasing amount
of the complex concentration (0-12 ꢀM) was recorded. The final volume of the reaction mixture
was made to 3 mL by adding 10 mM of Tris-HCl buffer (pH 7.2).
DNA cleavage experiments
The cleavage of supercoiled pBR 322 DNA was studied by agarose gel electrophoresis.27
The pBR 322 DNA (0.2 ꢀg) was treated with the porphyrins of two different concentrations (5
and 10 ꢀM) and the reactions were carried out in 50 mM Tris-HCl buffer (pH = 7.2, [NaCl] = 50
mM), and then the solutions were irradiated with a UV illuminator (365 nm, 15 W) at 25 °C for
60 min. The samples were then analyzed by electrophotolysis for 1-2 hours at 75 V on 1 %
1
agarose gel in TBE buffer. The gel was stained with 0.5 ꢀg mL− ethidium bromide (EB) and
photographed under UV light using Gel Doc 100 system, Bio-Rad; Hercules, CA. All
experiments were performed at least triplicate.
Measurement of singlet oxygen
A steady-state method was implemented to find the singlet oxygen quantum yield28,
using 1,3-diphenylisobenzofuran (DPBF) as the oxygen scavenger and meso-
tetrakis(tetraphenyl) porphyrin (H2TPP) as the reference. DPBF (50 ꢀM) solutions without or
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with porphyrin derivatives (0.5 ꢀ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 pre-established
1
irradiation intervals. From this plot, the rates of O2 production of porphyrins under study
relative to those of H2TPP were determined. Light intensities were measured with a pyroelectric
detector (RJP-735 from Laser Probe) which was connected to an energy ratio meter (RJ-7620
from Laser Probe and the average light fluence rate was 95 µJ/cm2. The singlet oxygen quantum
yield29,30 of the porphyrins with respect to the reference was calculated using the equation:
ɸ(1O2)comp = ɸ(1O2)ref (mcomp/mref) (Fref/Fcomp
)
where m is the slope of a plot of difference in change in absorbance of DPBF (at 410 nm) with
the irradiation time, and F is the absorption correction factor (F= 1 – 10-OD).
Antimicrobial and Cytotoxicity assessment
The antimicrobial activity of the 4-pyridyl (3a–3c) and 4-N-methylpyridyl porphyrins
(6a–6c) were performed using the literature method31 against the MTCC cultures which were
obtained from Microbial culture collection, Chandigarh, India are as follows: Two gram-positive
Staphylococcus aureus, Bacillus subtilis and two gram-negative Pseudomonas aeruginosa and
Escherichia coli bacterial strains and a fungal strain Candida albicans were used. The ligand
and their metal complexes were dissolved in dimethylsulphoxide (DMSO) to a stock solution
of 210-560 ꢀM. The sample at three different concentrations (40-160 ꢀM) was loaded onto
the sterile discs on agar plates directly. Plates swabbed with the bacteria culture were
incubated at 35−37 °C for 24 hours. At the end of the incubation period, inhibition zones
formed on the medium were evaluated in mm and studies were performed in duplicate.
Solvent control test was also performed in order to study the effect of dimethylsulphoxide on
the growth of microorganism and it showed no activity against microbial strains. The standard
antimicrobial drug (tetracycline) was also screened under similar condition for comparison.
Cytotoxicity of the tested compounds were performed by means of a colorimetric assay
(MTT assay) using Vero (African green monkey epithelial kidney) cells, obtained from King
Institute, Chennai, India. The assay was carried out after 48 h of cell incubation in the medium,
containing compounds at concentrations ranging from 10 to 100 ꢀg/mL. The cells were cultured
in open vented 25 cm3 culture flasks (Corning, NY) in a standard horizontal laminar flow
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chamber and incubated at 37 °C in an atmosphere of 5% CO2 and 95% air and were provided
with the growth medium of 90% Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal
Bovine Serum (Hi-Media®) and 5% Streptomycin-Penicillin (Sigma-Aldrich, USA). A quantity
of 0.5 − 1 × 104 cells/mL was seeded into a 96-well plate and after 24 or 48 h, the cells were
washed and maintained with different concentrations of samples (10-100 µg/mL) and incubated
for 48 h at 37 °C under 5% CO2 atmosphere. The extent of MTT reduction was measured
spectrophotometrically at 570 nm and a reference at 630 nm with a microplate reader. The
percentage of inhibition of the cells are measured using the formula,
Mean OD of untreated cells (control) − Mean OD of treated cells
x 100
Percentage of inhibition =
Mean OD of untreated cells (control)
The minimal inhibitory concentration (MIC) of compounds under antimicrobial study
were determined using the standard method of Wariso and Ebong.32 The compounds were
dissolved in DMSO, the final concentration of sample after dilution are in the range of 100 - 1.56
mg/ml. The MIC value corresponds to the lowest concentration that inhibited the growth after 24
h at 37 °C.
Results and discussion
Consuming 5-(pentafluorophenyl)dipyrromethane and 2/3/4-pyridinecarboxaldehyde, the
desired trans-pyridylporphyrins, 5,15-di(pentafluorophenyl)-10,20-bis(2′/3′/4′-pyridyl)porphyrin
were synthesized using the modified procedure of Lindsey et al.33 and their copper(II) and
zinc(II) metal complexes were prepared by the variant literature method.34
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F
F
F
F
F
CHO
F
R
F
F
F
F
F
F
N
F
i) TFA, N2 atm
ii) NaOH
F
R
CHO
HN
+
NH
N
F
N
H
F
TFA, DDQ
F
R
HN
NH
F
F
F
1a; 2-NP
2a; 3-NP
3a; 4-NP
i) CF3SO3CH3 / CH3I
ii) MII(OAc)2
CHCl3, MeOH
N
N
N
MII(OAc)2
R =
CHCl3, MeOH
CH3
F
+
R
F
F
F
F
R
F
F
N
F
N
F
M
N
N
F
N
N
F
M
N
F
N
F
F
F
R
F
F
2CF3SO3− / 2I−
F
+
R
H3C
F
F
M = 2H, Cu(II) and Zn(II)
4a-4c; 2-NMP
5a-5c; 3-NMP
M = Cu(II) and Zn(II)
1b-1c; 2-NP
2b-2c; 3-NP
6a-6c; 4-NMP
3b-3c; 4-NP
Scheme 1. Synthesis of trans-pyridyl porphyrins and its dicationic pyridinium derivatives.
N-methylation of trans-pyridyl porphyrins was performed using methyl
trifluoromethanesulfonate in CHCl3 medium to yield the corresponding trans-dicationic
pyridinium porphyrins. All the synthesized porphyrins (Scheme 1) were isolated, purified by
column chromatography and characterized by spectral, electrochemical methods and single
crystal X-ray diffraction analysis.
Optical absorption data of porphyrins, 1−6 were measured in DMF, exhibited an intense
Soret (B) and 2−4 visible (Q) bands and these absorption spectral features are similar to that of
corresponding 5,10,15,20-tetraphenylporphyrins, TPPs.34 It is also observed that there is a
marginal blue shifted absorption spectra for copper(II) complexes and considerable red shift of
8−10 nm for zinc(II) complexes compared to that of the free ligand (Fig. 2a).
Fig. 2. (a) Optical absorption and (b) fluorescence emission spectra of 1a−6a in DMF at 298 K.
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The fluorescence spectra of porphyrins, 1−6 were measured using DMF as solvent and
figure 2b shows the fluorescence emission spectra of various trans-porphyrins under study. On
excitation at the Soret band, the free ligands show two well defined emission peaks around 640-
646 nm and 704-708 nm whereas the zinc(II) derivatives exhibit around 597-616 nm and 649-
654 nm. Interestingly, the zinc(II) derivative of dicationic 4-pyridinium porphyrin, 6c shows a
red shift of 16 nm compared to its free ligand 4-pyridylporphyrin, 3c (Table 1).
Table 1. Photophysical data of trans-pyridyl and trans-dicationic pyridinium porphyrins, 1−6 recorded in DMF.
Absorption, λmax (nm)
Cu(II), b
Fluorescencei, λmax (nm)
Cu(II)ii, b
Zn(II), c
Porphyrin
2H, a
Zn(II), c
2H, a
412, 508, 583, 639
414, 509, 542, 584, 640
412, 507, 582
415, 507, 583
416, 508, 583
411, 537, 568
412, 539, 571
411, 537
414, 542, 574
415, 575
416, 541
421, 552
422, 553
421, 553
425, 555
426, 556
426, 557
642, 706
642, 708
640, 706
640, 704
639, 705
646, 707
649, 710
654, 712
649, 707
650, 710
655, 711
653, 704
597, 651
602, 653
600, 649
600, 651
597, 650
616, 654
1
2
3
4
5
6
417, 509, 583
iFluorescence spectra of the porphyrins were obtained as a function of λex in the Soret band; iiLess intense
fluorescence peaks were observed for the Cu(II) complexes.
Electrochemical studies
The redox potentials of trans-porphyrins, 1−6 were measured by cyclic voltammetry and
compared to those of simple tetraphenylporphyrins, MTPPs [M = 2H, Cu(II) and Zn(II)] (Table
2). A representative cyclic voltammograms of selected porphyrins, 2a, 5a, 3c and 6c are shown
in figure 3. The trans-pyridyl porphyrins, 1−3 undergo two reversible one electron reduction
reaction, forming the anion and dianion similar to that of MTPP counter parts. As predicted, the
reduction potentials of trans-pyridyl porphyrins are more positive when compared to MTPPs.32
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Fig. 3. Overlayed cyclic voltammograms of (a) 2a, 5a and (b) 3c, 6c in DMF containing 0.1 M tetrabutylammonium
hexafluorophosphate (TBAHFP) as the supporting electrolyte with a scan rate of 0.1 V/s.
Among the pyridyl porphyrins, the 4-pyridyl derivatives show more positive value of
250-300 mV than 2-/3-pyridyl analogues (150-250 mV) reflecting the net electron richness of the
porphyrin core. The trans-dicationic pyridinium porphyrins, 4−6 show four electron reduction
which occurs at the conjugated π-ring system (−986 to −1680 mV) as well as at the
methylpyridyl substituents (−577 to −910 mV).32 In the case of 4c and 5a−c, only three electron
reduction process is observed possibly attributed to solvent effect.33 Due to the presence of
electron withdrawing / positively charged substituents at the porphyrin periphery, the possible
oxidation process was not observed in the studied compounds.35,36
Table 2. Comparison of reduction potentials (mV) for 1a−6c in DMF using 0.1 M TBAHFP at 25oC.
Compound Redn (1) Redn (2) Redn (3) Redn (4)
H2TPP
CuTPP
ZnTPP
1a
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-1108
-1190
-1335
-851
-990
-1183
-810
-943
-
-799
-923
-1058
-1093
-986
-987
-1532
-1714
-1726
-1349
-1492
-1594
-1302
-1453
-
-1263
-1486
-1554
-1325
-1579
-1252
1b
1c
2a
2b
2c*
-
-
-
3a
3b
-
3c
4a
-543
-687
-
-872
-832
-834
4b
4c#
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5a#
5b#
5c#
6a
-
-
-
-738
-800
-910
-703
-780
-879
-1240
-1215
-1264
-1284
-1319
-1469
-1426
-1663
-
-1457
-1556
-1680
-577
-676
-722
6b
6c
*Not soluble in DMF; #First reduction was not observed.
X-ray crystallographic studies
Single crystal XRD analysis of porphyrins, 1a, 2a, 3c, 6a and 6c was done using the
suitable size of single crystals which were grown at room temperature using appropriate solvents
and the data are given in table 3. All the compounds were crystallized in monoclinic system with
various space groups as listed in table 3. The asymmetric unit of structures, 1a, 2a, 3c, 6a and 6c
contain half molecule of porphyrin with no solvent molecule, five water molecules, one THF
−
−
molecule, a CF3SO3 anion and a CF3SO3 anion respectively.
(a)
(b)
(c)
(d)
(e)
Fig. 4. ORTEP diagrams of (a) 1a, (b) 2a, (c) 3c, (d) 6a and (e) 6c. (Thermal ellipsoids are shown at 30 %
probability level; Solvent molecules were not shown for clarity). Atom colours: Gray – Carbon; Blue – Nitrogen;
Green – Fluorine; Brown – Zinc; White – Hydrogen.
Interestingly, the zinc(II) centre in 6c is hexa-coordinated with the inner core nitrogens
and two methanolic oxygens in its apex positions. The lattice THF molecule in 3c shows the
well-known envelope conformation as observed earlier.37 The ORTEP and molecular crystal
packing diagrams of porphyrins are shown in figure 4 and 5 respectively. Remarkably, there are
no interactions on pyridyl nitrogen with any other atom on neither of the phenyl ring nor in the
porphyrin periphery of the structures 1a and 2a. Unlike the reported36 crystal structure of 5,15-
di(4ꞌ-pyridyl)-10,20-bis(pentafluorophenyl) porphyrin having weak π−π interactions between the
porphyrin macrocycles leading to form a π–stacking of molecules, 1a (2-pyridyl) and 2a (3-
pyridyl) do not have such interactions, instead the porphyrin molecules in both structures are
arranged nearly in an orthogonal fashion (Fig. 5a and 5b).
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Table 3. X-ray crystal structure data of porphyrins, 1a, 2a, 3c, 6a and 6c.
1a
2a
3c
6a
6c
Empirical formula C42H18F10N6 C42H26F10N6O4 C50H32F10N6O2Zn C46H24F16N6O8S2 C48H30F16N6O8S2Zn
fw
CCDC no.
colour
crystal system
space group
a, Å
b, Å
c, Å
α, (deg)
β, (deg)
γ, (deg)
T (K)
796.62
1434316
Black
Monoclinic Monoclinic
P 21/c
15.113(3)
10.4895(16) 6.7009(3)
11.628(2)
90
112.085(9)
90
293(2)
0.71073
1708.1(5)
4
1.549
2397
868.69
1443805
Brown
1004.18
1021573
Purple
Monoclinic
C 2/c
23.891(4)
13.986(4)
14.697(3)
90
105.084(15)
90
293(2)
0.71073
4742(2)
4
1156.83
1058021
Purple
Monoclinic
P 21/n
13.5879(17)
11.0738(10)
17.130(2)
90
113.223(4)
90
1252.27
1041762
Red
Monoclinic
P 21/n
9.1236(4)
14.0611(7)
19.6345(10)
90
100.7510(10)
90
C 2/c
31.4898(16)
22.5181(12)
90
122.093(2)
90
293(2)
0.71073
4025.4(4)
4
293(2)
293(2)
λ, Å
0.71073
2368.7(5)
2
1.622
4175
0.71073
2474.7(2)
2
volume (Å3)
Z
Dcalcd (mg/m3)
no. of unique
reflections
1.433
3949
1.407
4170
1.681
6110
no. of parameters 263
refined
372
361
495
364
GOF on F2
0.957
0.0595
0.0718
1.051
0.0518
0.1392
1.041
0.0545
0.1472
1.137
0.0779
0.1632
1.028
0.0547
0.1371
b
R1
c
wR2
aR1 = Σ||Fo|–|Fc||/Σ|Fo|; Io > 2σ (Io). bwR2 = [Σ w(Fo –Fc2)2/Σw(Fo ) ] .
2
2 2 1/2
In contrast, the crystal structure of 3c is forming an interesting network of molecules
through Zn-N coordination showing a large number of channels present in only two directions
(Fig. 5d) that are occupied by the solvent (THF) molecules. Notably, the dimension of the cavity
in the existing system is smaller compared to the zinc porphyrin-MOF reported with the
tetratopic ligand which has larger dimensions present in all the three directions.38 The average
Zn-Npyrrole and Zn-Npyridine distances are found to be 2.059 (3) and 2.401 (11) Å. This array of
porphyrin molecules is exhibiting an orthogonal fashion of arrangements between them.
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 5. Two / three dimensional array of molecules, (a) 1a forming through FꢁꢁꢁF, HꢁꢁꢁH, CꢁꢁꢁC and CꢁꢁꢁH interactions
viewed down ‘a’ axis; (b) 2a forming through CꢁꢁꢁF and FꢁꢁꢁH interactions viewed down ‘b’ axis; (c) 3c forming
through NꢁꢁꢁH, CꢁꢁꢁH, CꢁꢁꢁN and NꢁꢁꢁN interactions viewed down ‘a’ axis; (d) 3c forming cavity filled with solvent
THF molecules viewed down ‘bc’ plane; (e) 6a forming through FꢁꢁꢁF, FꢁꢁꢁH and CꢁꢁꢁH interactions viewed down
‘bc’ plane; (f) 6c viewed down ‘b’ axis.
Figure 5c and 5d represents the molecular crystal packing of 3c with cavity in the
absence and presence of solvent THF molecules at the cavities. Interestingly, the solvated THF
molecule present inside the cavities of 3c was not involved in any kind of intermolecular
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interactions with the porphyrin molecule. The packing diagram of 6a and 6c are also shown in
figure 5e and 5f in which the former forms a one dimensional array of molecules through many
intermolecular interactions listed in table 4. Interestingly, the intermolecular interactions in 6c
are mainly through the solvated methanol molecules and trifluoromethanesulfonate anions
present in the crystalline lattice and also partly through FꢁꢁꢁH interactions. Both zinc(II) centre in
3c and 6c is hexa-coordinated and exhibits nearly planar geometry and the mean plane deviation
of core atoms is found to be 0.2389 Å and 0.0497 Å respectively. The pentafluorophenyl ring
and 4′-pyridyl / 4′-N-methylpyridyl ring make dihedral angles of 66.99°, 67.66° for 3c and
87.69°, 57.03° for 6c with the porphyrin mean plane.
Table 4. Distances (in Å) for different types of interactions in porphyrins, 1a, 2a, 3c, 6a and 6c.
Interactionsi
(pyrrole)C−HꢁꢁꢁC(pyrrole)
(ph)C−HꢁꢁꢁO(sol)
(pyrrole)C−HꢁꢁꢁO(sol)
(ph)C−HꢁꢁꢁO(OTf)
(N-methyl)C−HꢁꢁꢁO(OTf)
(pyrrole)C−HꢁꢁꢁF(ph)
(ph)C−HꢁꢁꢁF(ph)
(N-methyl)C−HꢁꢁꢁF(ph)
(sol)C−HꢁꢁꢁF(ph)
(ph)CꢁꢁꢁO(sol)
1aii
2aii
3cii
6aii
6cii
2.841, 2.885 (8)
-
-
-
-
-
-
-
-
-
-
-
-
2.870 (4)
-
-
-
-
-
-
2.451 (2)
2.670 (4)
-
-
-
-
2.370 – 2.641 (6)
-
2.611 (2)
2.448 (2)
2.600 (2)
2.539 (4)
2.519 (4)
2.613, 2.653 (8)
-
-
-
-
2.443 (4)
2.513 (4)
-
-
-
-
-
-
-
-
2.639 (2)
3.064 (2)
(ph)CꢁꢁꢁO(OTf)
-
-
-
-
3.044 – 3.149 (6)
(ph)CꢁꢁꢁF(ph)
-
3.01, 3.047 (8)
-
-
3.005, 3.065 (4)
(ph)CꢁꢁꢁF(OTf
-
-
-
-
3.120 – 3.150 (6)
(sol)OꢁꢁꢁO(OTf)
-
-
-
-
2.799 (2)
FꢁꢁꢁF
2.919 (4)
-
-
2.802 (4)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.008 (2)
2.935 (2)
(sol)O−HꢁꢁꢁO(OTf)
(sol)O−HꢁꢁꢁS(OTf)
(ph)C···C(pyrrole)
(ph)C‒F···C(Pyrrole)
(ph)C‒H···H(ph)
(ph)C···C(ph)
(ph)C‒H···N(pyrrole)
(ph)C···N(pyrrole)
(ph)N···N(pyrrole)
3.332 (4)
3.137 (4)
2.255 (2)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.208 (2)
-
-
-
-
2.682, 2.726 (8)
3.219 (4)
3.054, 3.068 (4)
2.870, 2.873 (8)
(ph)C‒H···C(pyrrole)
i
ii
Different types. Value in parenthesis gives the number of interactions present in the molecule.
It is also noted that 6c is prevented from the ZnꢁꢁꢁN(pyridyl) and N(pyridyl)ꢁꢁꢁC interactions
because of the N-methylation on the nitrogen of the pyridyl ring. As seen earlier, there are no
direct π−π as well as intramolecular interactions in 1a, 2a, 3c, 6a and 6c. And, their crystal
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packing consists of a number of intermolecular interactions involving carbon, fluorine, hydrogen,
oxygen, nitrogen and sulphur, viz., (ph/pyrrole/sol)C−HꢁꢁꢁF, (ph)C−HꢁꢁꢁC(pyrrole) (ph)C−HꢁꢁꢁO(sol/OTf)
(ph)CꢁꢁꢁO(sol/OTf) (ph)CꢁꢁꢁF(sol/OTf) (sol)OꢁꢁꢁO(OTf) (sol)O−HꢁꢁꢁO(OTf) (sol)O−HꢁꢁꢁS(OTf), FꢁꢁꢁF, etc (Table 4).
,
,
,
,
,
,
The weak intermolecular interactions present in porphyrins 1a, 2a, 3c, 6a and 6c were
analyzed and quantified through Hirshfeld surface analysis using Crystal Explorer 3.1 (Fig. 6).
The close contacts can be visualized by colour-coding short or long contacts, the colour and its
intensity indicating the relative strength of the interactions. The molecular HSs generated for the
porphyrins have different shapes reflecting different kind of crystal packing modes and is further
evident from the geometrical analysis (Table 4). The HSs mapped with dnorm highlights intense
red spots for HꢁꢁꢁH, CꢁꢁꢁH, ZnꢁꢁꢁN, OꢁꢁꢁH and OꢁꢁꢁS/H close contacts respectively for 1a, 2a, 3c, 6a
and 6c. The interactions involving fluorine such as FꢁꢁꢁH/C/F/O features medium/less intense red
spots indicate that those interactions are weaker and longer compared to our earlier reports on
tetra substituted fluorinated porphyrins.37,39 The breakdown of 2D fingerprint plots (FPs) of the
HSs for porphyrins under study reveals that the interactions involving fluorine are 49-58 %
which demonstrates the influence of halogen atoms on the intermolecular interactions which in
turn stabilizes the crystal packing.
(i)
(ii)
(iii)
(iv)
(v)
(a)
1a
2a
3c
6a
6c
(b)
(vi)
(vii)
(viii)
(ix)
(x)
Fig. 6. (a) Hirshfeld surfaces mapped with dnorm ranging from -0.15 Å (red) to 1.76 Å (blue) and (b) 2D fingerprint
plots, (i and vi) 1a; (ii and vii) 2a; (iii and viii) 3c; (iv and ix) 6a; (v and x) 6c.
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DNA binding studies
Electronic spectral titrations
To investigate the interaction of cationic porphyrins to DNA, the UV-Vis titrations were
performed by fixing porphyrin concentration at 3 ꢀM while varying the DNA concentration (Fig.
7). All the cationic porphyrins showed different extent of hypochromism (37−64 %) as well as
bathochromic response (1−5 nm) at the Soret band which indicates that these porphyrins bind to
DNA. It is well known that the porphyrins upon binding with DNA through intercalation results
in hypochromism (up to 40 %) with red shift (> 10 nm) whereas a low hypochromism and small
red shift is the signature of outside binding without self-stacking while a large hypochromism
with a small red shift is indicative of outside binding with self-stacking.24a The changes in the
absorption spectra of porphyrins 4−6 in the absence and presence of CT-DNA were presented in
Table 5.
Table 5. UV-Vis spectral titration parameters for porphyrins, 4−6 with CT-DNA.
Kb (M−1)
Porphyrin
Hypochromicity, H (%)
ꢁλ (nm)
DP-2-NMPPa
0.0
0.0
7.0
3.5
4.5
1.0
16.20
39.70
15.70
58.42
60.10
52.95
2.50 x 104
8.39 x 104
4.35 x 105
2.96 x 105
8.47 x 105
7.02 x 105
DP-3-NMPPa
DP-4-NMPPa
4a
4b
4c
5.0
2.0
4.5
4.0
2.5
4.5
61.87
43.85
64.02
43.35
45.30
37.20
1.83 x 106
1.50 x 106
1.24 x 106
1.38 x 106
2.00 x 106
1.75 x 106
5a
5b
5c
6a
6b
6c
aDP-2/3/4-NMPP correspond to 5,15-di(phenyl)-10,20-bis(2
′
-3
porphyrins which are taken from ref: Bioorg. Med. Chem., 2006, 14, 2956-2965
′
-4′-N-methylpyridyl)
In order to further quantify the binding affinities of porphyrins to DNA, the intrinsic
binding constant, Kb has been determined based on the absorbance titration data using the
following equation:40
[DNA] / (εa - εf) = [DNA] / (εb - εf) + 1 / Kb (εb - εf)
where εa, εf, εb correspond to Aobs/[porphyrin], the extinction coefficient for the free porphyrin,
and the extinction coefficient for the porphyrin in the fully bound form, respectively. In the plot
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of [DNA] / (εa - εf) versus [DNA], Kb is given by the ratio of slope to intercept. The binding
1
constants obtained for the porphyrins 4−6 are in the range of 2.96 x 105 − 2.00 x 106 M− .
Interestingly, the Kb values obtained are higher than the reported non-fluorinated cationic
pyridinium porphyrins41 which discloses that the pentafluorophenyl groups play an essential role
to control DNA interaction abilities. Moreover, the binding constant of 6a−6c are almost tenfold
higher than the reported Ru(II) analog of trans-porphyrin (1.1 x 105 ) that contain both 4-pyridyl
as well as fluoro substituents at its periphery.42 The results clearly signal that among the three
sets of porphyrins (4−6), the binding affinity of 3-/4-N-methylpyridyl derivatives are higher
compared to 2-N-methylpyridyl derivatives presumably due to the more effective electrostatic
interaction between the positively charged porphyrin and the phosphate moiety of nucleotide in
the former than the later.43
Fig. 7. Absorption spectra of pH 7.2 buffer solutions of (a) 4a and (b) 5c in the presence of increasing amounts of
CT DNA. [porphyrin] = 3 µM, [DNA] = 0 – 3 µM. Inset: Plot of [DNA] / (ꢁε) vs. [DNA]
Emission studies
The freebase and zinc(II) porphyrins emit luminescence in Tris buffer at pH 7.2. The 2-
N-methylpyridyl and 3-N-methylpyridyl freebase porphyrins exhibit two emission bands around
636 nm and 699 nm respectively whereas the corresponding Zn(II) derivatives show two
emission bands around 600 and 650 nm respectively. The 4-N-methylpyridyl derivatives shows
red shifted emission bands compared to the 2-N-methylpyridyl and 3-N-methylpyridyl
derivatives at 648 and 703 nm respectively for the free ligand and 623 and 652 nm for the Zn(II)
complex. With the incremental addition of DNA, there is a decrease in fluorescence intensity
without much wavelength shift which can be attributed to self-stacking of the porphyrin
molecules along the DNA surface (Fig. 8).25a
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Fig. 8. Fluorescence emission spectra of porphyrins, (a) 4a and (b) 6c with increasing concentration of CT DNA.
[porphyrin] = 3 µM, [DNA] = 0 – 1 µM. λex = 480 nm.
Fluorescence quenching experiments were carried out by adding the porphyrins to CT
DNA pre-treated with Ethidium bromide (EB). EB is an efficient DNA intercalator that emits
strong fluorescence upon binding to DNA.44 When EB is replaced or excluded from the interior
hydrophobic environments of the DNA double helix by the third molecule, the fluorescence of
the EB–DNA system is quenched. Notably, all the porphyrins exhibit remarkable decrease in
intensity of fluorescence which indicates that the porphyrins were able to push the intercalated
EB out of the nucleotide cavities. The quantitative information of the fluorescence quenching by
porphyrins were obtained using the following Stern-Volmer equation:45
F0/F = 1 + KSV[Q] = 1 + Kqτ0[Q]
where, F0 and F represent the steady-state fluorescence intensity in the absence and presence of
the quencher (porphyrin). [Q] is the quencher concentration, Ksv is the Stern-Volmer quenching
rate constant which is obtained by the ratio of slope to the intercept of the diagram F0/F vs.
[porphyrin]. Subsequently, the approximate quenching constant (Kq) can be calculated from the
equation, Ksv = Kq.τ0. The lifetime of the fluorophore (CT-DNA-EB) is τ0 (19.2 ns) in the
absence of the quencher. The value of Kq is in the order of 1012 M-1 S-1 which is much higher
than the maximum diffusion collisional quenching rate of various quenchers with
biomacromolecules46,47(≈ 2.0 × 1010 M-1 S-1) (Table 6), showing the existence of a static
quenching mechanism rather than dynamic collision. Since, Ethidium bromide is a very strong
intercalator, the formation of non-fluorescent (EB–DNA)–porphyrin adduct could be the reason
for the static quenching.
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Fig. 9. Emission spectra of EB bound to DNA in the presence of porphyrins, (a) 4a (λex = 411 nm) and (b) 5a (λex
=
412 nm) with increasing concentration of CT DNA. [DNA] = 12 µM, [EB] = 7.5 µM, [porphyrin] = 0 − 12 µM.
Inset: Fluorescence quenching curve of EB bound DNA by the porphyrins.
The fluorescence quenching efficiency is also calculated from the fluorescence quenching
curves and it is found that the highly efficient 3-pyridyl/4-pyridyl derivatives quench the
fluorescence around 50-56 %.
Table 6. The Stern–Volmer quenching parameters for DNA–Ethidium bromide by porphyrins in Tris-buffer at pH =
7.0.
Porphyrin
KSV (M-1), 104
Kq (M-1 S-1), 1012
4a
4b
4c
5a
5b
5c
6a
6b
6c
5.31
2.76
8.25
4.30
2.18
1.14
5.73
2.98
7.33
3.82
4.58
2.39
6.58
3.43
9.63
5.02
5.87
3.06
DNA photocleavage studies
The porphyrins, 4−6 in DMF-Tris buffer containing NaCl (pH = 7.2) and aqueous
solutions of circular plasmid DNA (pBR 322) were irradiated using a 15 W transilluminator at
365 nm for 60 min. The irradiated samples were subjected to gel electrophoresis in order to
determine the photocleavage ability of the porphyrins.
Fig. 10. Gel electrophoresis of pBR 322 DNA in the absence (lane 1) and presence (lanes 2-7) of porphyrins 4a, 4b
and 4c at the dark (a) and after irradiation (b). [DNA] = 0.2 ꢀg; [porphyrin] = 5 ꢀM for lanes 2, 3 and 4. [porphyrin]
= 10 ꢀM for lanes 5, 6 and 7. Photo irradiations were conducted using a UV illuminator (365 nm, 15 W) for 60 min.
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Fig. 11. Gel electrophoresis of pBR 322 DNA in the absence (lane 1) and presence (lanes 2-7) of porphyrins 5a, 5b
and 5c (a) and 6a, 6b and 6c (b) after irradiation. [DNA] = 0.2 ꢀg; [porphyrin] = 5 ꢀM for lanes 2, 3 and 4.
[porphyrin] = 10 ꢀM for lanes 5, 6 and 7. Photo irradiations were conducted by using a UV illuminator (365 nm, 15
W) for 60 min.
Figure 10 illustrates the concentration dependent gel electrophoresis experiment for
complexes 4a−4c in dark (left) as well as light (right), where lane 1 represents DNA in the
absence of porphyrin. Figure 11 elucidates the photocleavage experiments of porphyrins, 5 and
6. The results indicate that the porphyrins show better activity under light compared to the dark
condition. Unlike the reported48 water soluble porphyrin Zn-TTHPP, our zinc(II) derivatives
show better photo induced cleavage ability. Moreover, the free ligands also show efficient DNA
damage than that of copper(II) counter parts.
Fig. 12. Effect of scavengers on the cleavage of DNA in presence of porphyrins: lane 1: DNA control; lane 2: DNA
+ 4a; lane 3: DNA + 4a + NaN3 (50 mM); lane 4: DNA + 4a + DMSO (50 mM); lane 5: DNA + 4c; lane 6: DNA +
4c + NaN3 (50 mM); lane 7: DNA + 4c + DMSO (50 mM).
In order to recognize the DNA photocleavage mechanism of porphyrins, we examined
the influence of scavengers such as NaN3 and DMSO.49 Photocleavage experiments with NaN3
(singlet oxygen quencher) was carried out and the cleavage of plasmid DNA was inhibited
whereas in presence of DMSO, a hydroxyl radical scavenger, the degree of cleavage is almost
unaltered. This indicates that not the hydroxyl radical but the singlet oxygen is involved in the
DNA photocleavage mechanism (Fig. 12).
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Measurement of singlet oxygen species
The singlet oxygen involved DNA cleavage can be further visualized using 1,3-
diphenylisobenzofuran (DPBF) which is an singlet oxygen quencher (Fig. 13).28-30 The
1
relationship 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
1
porphyrin with increasing irradiation time and from the slope of the line, the relative rate of O2
generation was compared. From the slope values, it is observed that the singlet oxygen yield of
freebase porphyrins and its zinc(II) derivatives are higher and the values are comparable with the
reported tricationic zinc(II) porphyrin29 whereas it is very high compared to the N-confused
porphyrin.30 The zinc(II) porphyrin 5c showed a very high singlet-oxygen quantum yield of 0.93
compared with 0.14 for its copper(II) analogue 5b in DMSO. The measured luminescence
lifetimes are also comparable with the reported cationic porphyrins. Notably, the results are in
good agreement with that of gel electrophoresis experiments.
Fig. 13. Decomposition of DPBF by compounds, (a) 4a−4c; (b) 5a−5c and (c) 6a−6c. Porphyrin (0.5 ꢀM) and
DPBF (50 µM) were irradiated in DMSO.
Antimicrobial studies
The antimicrobial activity of trans-4-pyridyl (3a−3c) and trans-4-N-methylpyridyl
derivatives (6a−6c) along with H2TPP and standard tetracycline were performed using well
diffusion method at three different concentrations (40-160 ꢀM) against the MTCC cultures.
The results reveal that the fluorinated analogues showed better activity compared to the non-
fluorinated analogue, H2TPP. The growth inhibition observed in zone inhibition assay of metal
ions are negligible compared to the porphyrins. It is also observed that all the porphyrins
showed maximum zone of inhibition at maximum concentration with MIC values in the range of
6.7 to 16.6 mM against the investigated strains (Fig.14 & 15). At the concentration of 63 µM,
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porphyrin 6b showed maximum zone of inhibition of 19 mm and 17 mm against B. subtilis and
E. coli respectively, 6c showed inhibition zone of 18 mm against E. coli. The porphyrin 3c did
not show any activity against B. subtilis and P.aeruginosa. In the case of the fungal strain C.
albicans, maximum activity is found for porphyrin 3b followed by 6b and 6c, no activity is
found for H2TPP and very less activity of 4 mm is found for 3c (Fig. 14). Among the samples
used, highest MIC was 16.6 mM for sample 3a against C. albicans followed by 16.1mM for 3a
against B. subtilis. Lowest MIC was recorded for 6b (6.7 mM) against B. subtilis followed by
porphyrin 6a against E. coli (6.8 mM). Interestingly, the trans-porphyrins under study exhibit
better activity towards the fungi C. albicans compared to the reported electron deficient
fluorinated metalloporphyrins containing trifluoromethylphenyl groups.50 The porphyrin
molecules are likely to alter the structure of cell membrane owing to their affinity to biological
organisms which might be the cause for its biological activity.51
Fig. 14. Antimicrobial studies of the trans-4-pyridyl and trans-dicationic 4-pyridinium porphyrins along with
H2TPP and standard tetracycline (% of error: ± 5).
Cytotoxicity of porphyrins under antimicrobial study in normal Vero cell lines was
performed by means of a colorimetric assay (MTT assay). The results showed no variation in the
morphological features of the cells at 100 µg/mL concentration. Thus, the compounds were
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found to be safe while treated against normal cells and showed very less toxicity (6−10 %) on the
normal Vero cells (Fig. S9).
Fig. 15. The minimum inhibition concentration of trans-4-pyridyl and trans-dicationic 4-pyridinium porphyrins.
4. Conclusions
Trans-pyridyl porphyrins, trans-dicationic pyridinium porphyrins and its metal
derivatives [Cu(II) and Zn(II)] were synthesized, characterized using conventional
spectroscopic methods, electrochemical studies as well as by single crystal X-ray
crystallographic analysis. The title compounds exhibited red shifted absorption spectra
than the simple pyridyl porphyrins. The reduction potentials of trans-pyridyl porphyrins
are more positive than those of MTPPs. X-ray crystal structures of 1a, 2a, 3c, 6a and 6c
indicates that the interactions involving fluorine contributes considerably to the crystal
packing which were further confirmed by computational Hirshfeld surface analysis. The
dicationic porphyrins were further explored for its DNA interaction abilities and
antimicrobial activities. Studies reveal that among the studied porphyrins (
4−6), the 3-/4-
pyridinium derivatives were found to be efficient groove binders with high intrinsic
binding constant (Kb) values compared to its corresponding non-fluorinated analogues
reported earlier. Hence, the introduction of fluorine into the phenyl rings as well as the
position of positive charges on the N-methylpyridinium group of porphyrin greatly
influences their interaction with DNA. The photocleavage experiments disclose that the
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1
porphyrins employ O2-mediated mechanism in cleaving DNA and the freebase, zinc(II)
derivatives show better photoinduced cleavage ability compared its copper(II) analogues.
Among the porphyrins studied, the zinc(II) complex, 6c shows the most promise as a
photosensitizer for PDT. The dicationic porphyrins also show significant antimicrobial
activities than those of non-fluorinated analogues. Thus, the results obtained from the
present work would be further explored in medicinal chemistry as new potent therapeutic
agents.
Supplementary Data
Crystallographic data of the porphyrins, 1a, 2a, 3c, 6a and 6c have been deposited with the
Cambridge Crystallographic Data Centre, CCDC reference numbers are 1434316, 1443805,
1021573, 1058021 and 1041762. Copies of this information may be obtained free of charge from
The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: + 44 1233336 033; e-
mail: deposit@ccdc.cam.ac.uk).
Acknowledgements
SS (SR/WOS-A/CS-146/2011) thank DST, New Delhi for the financial support. We
would like to thank Dr. Babu Varghese, SAIF, IIT Madras for the single crystal data collection,
structure solution and refinement. Authors also thank Dr. Leeba Balan, University of Madras,
Chennai, Tamilnadu for helping us to acquire the antimicrobial studies.
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Synthesis, Structure, Electrochemical, DNA Interaction and
Antimicrobial Studies of Fluorinated Trans-Dicationic Pyridinium
Porphyrins
Jagadeesan Ramesh, Subramaniam Sujatha* and Chellaiah Arunkumar*
Trans-dicationic pyridinium porphyrins: Synthesis, structure, electrochemical, DNA
interaction and antimicrobial studies of fluorinated trans-pyridyl and trans-dicationic pyridinium
porphyrins are reported and the studies indicate that they are superior artificial nucleases than its
non-fluorinated analogues; bind with the calf thymus DNA by outside groove binding mode with
or without self-stacking, also show significant antimicrobial activities than those of non-
fluorinated analogues.