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

Abstract

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(OH)₂T(4-CP)P (2) and Sn^IV(O-NO₂Ph)₂T(4-CMP)P (3). The newly synthesized porphyrins were characterized by various spectroscopic methods and single crystal X-ray diffraction analysis. The crystal structures of the precursor porphyrin, Sn^IV(Cl)₂T(4-CMP)P and 3 are well stabilized by various intermolecular interactions and porphyrin 3 shows complementary bonding interactions between the nitro groups (N⋯O) forming a one-dimensional array. The fluorescence lifetime of 3 is lower 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 X-ray 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 much less under dark conditions. The porphyrin internalization as well as localization within the Candida cells were 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.

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Introduction

Photodynamic antimicrobial chemotherapy¹ (PACT), a new treatment modality, follows similar principles to those of photodynamic therapy (PDT) which utilizes nontoxic components such as light, oxygen and a photosensitizing agent, promoting microbial eradication by the production of reactive oxygen species such as a singlet oxygen, superoxide and hydroxyl radicals.² A photosensitizer is generally allowed to get internalized within the prokaryotic or eukaryotic cells of interest. When the cells containing a photosensitizer are exposed to light of a specific wavelength, it gets excited to a singlet excited state and undergoes intersystem crossing, reaching a triplet energy state. While coming back to its ground state, the photosensitizer produces a singlet oxygen from cellular oxygen, which causes cell damage leading to cell death.² (Scheme 1a).

Scheme 1 (a) Mechanism for photodynamic antimicrobial chemotherapy; (b) schematic representation of tin(IV) porphyrins, 1–3 in this study.

Porphyrin based photosensitizers are widely accepted because they show a 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 are 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 been shown much attention owing to their interesting photophysical properties.⁸ They are very stable, easy to synthesize, diamagnetic and usually six-coordinated with trans-diaxial ligands.⁹ Two high valent tin(IV) porphyrins with chlorine as their axial ligands, tin(IV) ethyl etiopurpurin and tin(IV) octaethylbenzochlorin, were the well established second-generation photosensitizers.¹⁰ On 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 4-nitrophenolate moieties. To better understand the electronic transitions that take place in Sn^IV(Cl)₂T(4-CMP)P, 1 and 3, computational studies were performed. The photodynamic antimicrobial chemotherapy of C. albicans using porphyrins 1–3 was also studied. The cytotoxic effects of the porphyrins were assessed in C. albicans cells by agar well diffusion and a broth assay. Microscopic analysis and antifungal assays were done in order to confirm the internalization of compounds as well as to demonstrate the phototoxicity induced by these compounds in C. albicans.

Experimental

Materials and methods

The chemicals employed for the synthesis were obtained as AR grade and used as received. The solvents were purified using the available literature methods.¹¹ The reference compound, meso-tetrakis(4-N-methylpyridyl)porphyrin, R [H₂T(4-NMP)P], was prepared according to the literature procedure.¹² The chemicals used for the antimicrobial studies were purchased from HiMedia and Sigma Aldrich. Optical absorption spectra were recorded at room temperature on a Shimadzu UV-2450 spectrophotometer and ¹H 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 time-resolved fluorescence intensity decays using a Horiba Scientific Fluoromax-4 spectrofluorometer with data station software in time correlated single photon counting mode. A pulsed light-emitting diode (Horiba NanoLED-560) was used as an excitation source. This LED generates an optical pulse at 564 nm with a pulse duration of 1.5 ns. The LED profile (instrument response function) was measured at the excitation wavelength using a Ludox 530 (colloidal silica) as the scatterer. To optimize the signal-to-noise ratio, 10 000 photon counts were collected in the peak channel. Photo-irradiations were done using a general electric quartz line lamp (300 W). 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). The study was conducted for an irradiation time of 300 min and the average light fluence rate was 95 μJ cm⁻².

Synthesis of tin(IV) porphyrins

Synthesis of trans-dihydroxy[5,10,15,20-tetrakis(4′-carboxymethylphenyl) porphyrinato] tin(IV), Sn^IV(OH)₂T(4-CMP)P, 1. The precursor, 5,10,15,20-tetrakis(4′-carboxymethylphenyl)porphyrin [H₂T(4-CMP)P], was synthesized using a modified Lindsey method¹³ and the crude product was purified by silica gel column chromatography using acetone/chloroform as the eluent. Evaporation of the solvent and recrystallization from chloroform/hexane results the desired product with a 40% yield. UV-visible data in THF, λ_max (log ε/M⁻¹ cm⁻¹): 417 (6.12), 514 (4.88), 549 (4.50), 591 (4.33), 646 (4.12). ¹H NMR data in CDCl₃ (δ in ppm) (400 MHz): 8.82 (s, 8H, β-pyrrole-H), 8.44–8.46 (d, 8H, J = 8.00 Hz, o-phenyl-H), 8.28–8.31 (d, 8H, J = 8.00 Hz, m-phenyl-H), 4.11 (s, 12H, p-carboxymethyl-H), −2.81 (s, 2H, imino-H).

Tin metallation was performed as per the reported method¹⁴ using stannous chloride as the metal ion carrier in pyridine. Excess water was added to precipitate the product which was then filtered. The crude product was re-dissolved in chloroform, passed through anhydrous Na₂SO₄ and purified using neutral alumina in a chloroform/acetone mixture. The unreacted free base porphyrin was collected initially, after which trans-dichloro[5,10,15,20-tetrakis(4′-carboxymethylphenyl) porphyrinato] tin(IV), [Sn^IV(Cl)₂T(4-CMP)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, β-pyrrole-H), 8.51–8.53 (d, 8H, J = 8.00 Hz, o-phenyl-H), 8.40–8.42 (d, 8H, J = 8.00 Hz, m-phenyl-H), 4.13 (s, 12H, p-carboxymethyl-H). This product was re-dissolved in tetrahydrofuran and reacted with a slight excess of potassium carbonate (1 : 50) in a minimum quantity of water for 6 h. After evaporation of the solvent, the residue was re-dissolved in an 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%. UV-visible data, λ_max (log ε/M⁻¹ cm⁻¹): (a) in THF: 427 (5.97), 561 (4.89), 600 (4.76); (b) in acetone: 423 (5.86), 555 (4.47), 595 (4.20). ¹H NMR data in CDCl₃ (δ in ppm) (400 MHz): 9.10 (s, 8H, β-pyrrole-H), 8.50–8.52 (d, 8H, J = 8.00 Hz, o-phenyl-H), 8.41–8.43 (d, 8H, J = 8.00 Hz, m-phenyl-H), 4.11 (s, 12H, p-carboxymethyl-H).

Synthesis of trans-dihydroxy[5,10,15,20-tetrakis(4′-carboxyphenyl) porphyrinato] tin(IV), Sn^IV(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 h. 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 in 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%. UV-visible data, λ_max (log ε/M⁻¹ cm⁻¹): (a) in THF: 430 (5.84), 563 (4.69), 602 (4.18); (b) in acetone: 425 (5.14), 561 (3.97), 601 (3.87). ¹H NMR data for 2 in DMSO-d₆ (δ in ppm) (400 MHz): 9.32 (s, 8H, β-pyrrole-H), 8.47–8.49 (d, 8H, J = 8.00 Hz, o-phenyl-H), 8.44–8.46 (d, 8H, J = 8.00 Hz, m-phenyl-H). ESI-mass data of 2: 939 [M − 2H]⁺ (calcd 941).

Synthesis of trans-dinitrophenolate [5,10,15,20-tetrakis(4′-carboxymethylphenyl) porphyrinato] tin(IV), Sn^IV(O-NO₂Ph)₂T(4-CMP)P, 3. Porphyrin, 1 and 4-nitrophenol in nitrobenzene were magnetically stirred at 100–120 °C for 4 h. The formed porphyrin phenolate complex was crystallized using hexane to afford dark red single crystals of 3 after a week. UV-visible data, λ_max (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), 592 (4.58). ¹H NMR data for 3 in CDCl₃ (δ in ppm) (400 MHz): 9.06 (s, 8H, β-pyrrole-H), 8.45–8.47 (d, 8H, J = 8.00 Hz, o-carboxymethylphenyl-H), 8.14–8.16 (d, 8H, J = 8.00 Hz, m-carboxymethylphenyl-H), 6.54–6.55 (d, 8H, J = 7.20 Hz, m-nitrophenyl-H), 4.07 (s, 12H, p-carboxymethyl-H), 1.74–1.76 (d, 8H, J = 7.20 Hz, o-nitrophenyl-H).

Crystal structure determination

X-ray quality single crystals of compounds Sn^IV(Cl)₂T(4-CMP)P and 3 were obtained by a slow vapour diffusion method using chloroform/hexane and nitrobenzene/hexane respectively. The red crystals were mounted on a glass capillary with a suitable size and the crystal data was collected on a Bruker AXS Kappa Apex II CCD diffractometer with graphite monochromated Mo K_α radiation (λ = 0.71073 Å) at 298 K. The reflections with I > 2σ(I) were employed for structure solution and refinement. The SIR92 (ref. 15) (WINGX32) program was used for solving the structure by direct methods. Successive Fourier synthesis was employed to complete the structures after full-matrix least squares refinement on |F|² using the SHELXL97 (ref. 16) software.

Hirshfeld surface analysis

To analyse and quantify the various intermolecular interactions present in the crystal structure of tin(IV) porphyrins, we generated Hirshfeld surfaces and 2D fingerprint plots using Crystal Explorer 3.1.¹⁷

Microorganism and growth conditions

C. albicans strain CAF4-2 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, and 2% dextrose.¹⁸ Since the strain is ura⁻, the growth medium was supplemented with 25 μg mL⁻¹ 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 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 °C in light or dark conditions. After 12 h of incubation, the zone of inhibition was calculated using the formula given below:²⁰

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 μM) and 20 μg mL⁻¹ of fluconazole as a positive control. A comparative study of the same compounds was done by varying the experimental conditions in the light and dark. The A₆₀₀ value of the control where no compound was added was subtracted from the A₆₀₀ value obtained after adding the compound. The same process was followed in all the cases where compounds were added. The percentage inhibition of the Candida growth was calculated using the equation:²¹

% of inhibition = [1 − (X_A600/C_A600)] × 100

where X_A600 represents the A₆₀₀ value of X μM of the compound treated culture at the different time points. C_A600 represents the A₆₀₀ value of the control culture at the same time. The percentage of growth inhibition was determined for 5 h after the addition of porphyrins to the C. albicans culture. The IC₅₀ values of the compounds were determined by plotting the percentage inhibition of cell growth against the compound concentration.²¹

C. albicans cells were grown for 4 h 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 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 an additional 12 h at 37 °C. The number of colony-forming 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 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 1× PBS in order to remove the un-internalized porphyrins. After this, the cells were gently re-suspended in 1× PBS and then were transferred into a 1 mL fluorescence cuvette. A fluorescence spectral analysis was carried out by exciting the samples at 550 nm.²²

Wide field fluorescence microscopy analysis

For studying the intracellular localization and phototoxicity of the compounds C. albicans cells were grown in a liquid medium for 6 h. The cells were then harvested and washed thrice with 1× PBS in order to remove the excess un-internalized compounds. As per the spectrofluorimetric analysis, the porphyrins get excited around 550 nm and undergo emission around 600 nm. Hence, for this study the compounds were excited using a green filter (490–510 nm). C. albicans cells were stained with Hoechst 33342 (10 μg mL⁻¹ followed by 30 minutes incubation) to visualize the DNA.²³ The fluorescence imaging was performed using a LEICA DM5000B fluorescence microscope (Germany) at 100× magnification (oil immersion) and the images were processed using ImageJ (NIH, USA).

Confocal fluorescence microscopy studies

The C. albicans cells grown in YPD medium as described above were incubated in the dark with the compounds for 6 h. The cells were then harvested and washed thrice with 1× PBS in order to remove the excess un-internalized compounds. The cells were then mounted on clean glass slides and sealed for microscopic analysis.²⁴ The confocal fluorescence imaging was performed using a confocal fluorescence microscope (Germany) at 63× magnification. To confirm the internalization of the compounds, a progressive confocal optical slicing approach was employed. Images were sliced progressively at every 0.8 μm of depth and the internalization of the compounds was analysed. The images were obtained by merging the bright field and fluorescence images and were further processed using ImageJ (NIH, USA).

Measurement of singlet oxygen species

1,3-Diphenylisobenzofuran (DPBF) was used as a selective singlet oxygen (¹O₂) acceptor, which was bleached upon reaction with ¹O₂ generated by porphyrin donors. DPBF (1.7 × 10⁻⁷ M) solutions without or with porphyrin derivatives (6.6 × 10⁻⁷ 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 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(4-CMP)P, was prepared using the modified procedure of Lindsey et al.¹³ and their tin(IV) complexes, Sn^IV(Cl)₂T(4-CMP)P, 1 and 2, were prepared by the variant literature methods.¹⁴ The synthesis of 3 was done by condensing 1 with 4-nitrophenol (Scheme 2). The synthesized porphyrins were isolated, purified by column chromatography and characterized by UV-visible and ¹H NMR spectroscopy methods, mass spectrometry and single crystal X-ray diffraction analysis.

Scheme 2 General synthesis of tin(IV) porphyrins, 1–3.

Photophysical properties

The photophysical properties such as optical absorption, steady-state emission, fluorescence quantum yield and the fluorescence decay profile were studied for tin(IV) porphyrins 1–3, Sn^IV(OH)₂TPP and the porphyrin ligand H₂T(4-CMP)P in THF medium. The electronic absorption spectra of tin(IV) porphyrins exhibit an intense Soret (B) band which corresponds to the S₀ → S₂ transition around 425 nm and two visible (Q) bands around 560 and 600 nm which correspond to the Q_(1,0) and Q_(0,0) peaks of the 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 in acetone media (Fig. S1†). Steady-state fluorescence of the porphyrins was performed in order to study their electronic properties in the excited state. The normalized absorption and emission spectra of porphyrin 3 are shown in Fig. 1.

Fig. 1 Normalized absorption (solid line) and emission (dashed line) spectra of 3 in THF at 298 K. Emission spectrum was recorded by exciting the Soret region.

On excitation near the Soret band, all the tin(IV) porphyrins exhibit two well-defined emission bands near 605 nm (band 1) and 657 nm (band 2) which correspond to the S₁ → S₀ transition and the spectral profile is comparable with the reported hexa-coordinated tin(IV) porphyrin systems.^9,26 Also, there is a very weak emission corresponding to the S₂ → S₀ transition at 434, 462 and 489 nm for porphyrins Sn^IV(OH)₂TPP, 1 and 2 respectively (Table 1).

Table 1 Photophysical data of porphyrins recorded in tetrahydrofuran at 300 K

Porphyrin	Absorption λmax (nm)	Fluorescence^a λmax (nm)	Quantum yield Φf (S1–S0)	Fluorescence lifetime, τ (ns)
a Fluorescence spectra of porphyrins were obtained as a function of λex in the Soret band region.b Excited at 460 nm and emission monitored at 650 nm in toluene.c Excited at 460 nm and emission monitored at 600 nm in toluene.d Excited at 460 nm and emission monitored at 600 nm in DMF.
H2T(4-CMP)P	417, 514, 549, 591, 646	650, 716	0.057	10.10^b
Sn^IV(OH)2TPP	425, 560, 600	434, 604, 656	0.044	1.48^c
1, Sn^IV(OH)2T(4-CMP)P	427, 561, 600	462, 605, 657	0.017	1.75^d
2, Sn^IV(OH)2T(4-CP)P	430, 563, 602	489, 605, 658	0.008	0.90^d
3, Sn^IV(O-NO2Ph)2T(4-CMP)P	425, 558, 597	605, 656	0.010	0.66^d

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The S₁ → S₀ fluorescence quantum yields (Φ_f) of the tin(IV) porphyrins were also measured using H₂TPP as the reference²⁷ (Φ_f = 0.11). As seen from the data given in Table 1, there is a quenching of the 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(4-CMP)P which indicates that the metal coordination may induce the radiationless decay rate.²⁸ The fluorescence decay profiles (λ_exc = 460 nm and λ_em = 600 nm) of the porphyrins measured by the time-correlated single photon counting (TCSPC) technique show that the fluorescence lifetime of 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 a π–π interaction between the porphyrin plane and the 4-nitrophenyl system due to their close proximity. The fluorescence decay times are 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 value of porphyrin 2 is comparable to that of the reported tin(IV) porphyrin, Sn^IV(OH)₂THP and its value is 0.91 ns.³⁰

Fig. 2 Fluorescence decay curves of 1 and 3 observed at 600 nm along with the IRF measured in THF (λ_exc = 460 nm).

Crystal structure description of porphyrins, Sn^IV(Cl)₂T(4-CMP)P and 3

Single crystal XRD analysis showed that the porphyrins, Sn^IV(Cl)₂T(4-CMP)P and 3 crystallized in a triclinic P1̄ system (Table 2). The asymmetric unit of Sn^IV(Cl)₂T(4-CMP)P consists of 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 Sn^IV(Cl)₂T(4-CMP)P are shown in Fig. 3. The solvent molecule (CH₃CN) acts as a bridge connecting two neighbouring porphyrin molecules through strong hydrogen bonding between the nitrogen of the acetonitrile and the β-pyrrole hydrogen [_(solvent)N⋯H_(β-pyrrole), 2.733 Å] of one molecule and the phenyl-hydrogen [_(solvent)N⋯H_(phenyl), 2.631 Å] of the other molecule which is viewed along the ‘bc’ plane and depicted in Fig. 3b. Other intermolecular interactions which make the compound Sn^IV(Cl)₂T(4-CMP)P form the two-dimensional array of molecules viewed down the ‘ab’ plane (Fig. 3c) involve Sn–Cl⋯C_(β-pyrrole) (3.368 Å) and O⋯H bonding [_(carbonyl)O⋯H_(β-pyrrole), 2.421 Å and _(carbonyl)O⋯H_{(carboxymethyl)}, 2.634 Å]. Overall, the crystal structure of Sn^IV(Cl)₂T(4-CMP)P is stabilized well by various intermolecular interactions described above.

Table 2 Summary of crystal structure data of porphyrins

	Sn^IVCl2T(4-CMP)P	3
a R1 = ∑||Fo| − |Fc||/∑|Fo|; Io > 2σ(Io).b wR2 = [∑w(Fo^2 − Fc^2)^2/∑w(Fo^2)^2]^1/2.
Formula	C56H42Cl2N6O8Sn	C64H44N6O14Sn
Formula weight	1116.55	1239.74
CCDC	948482	974215
Crystal system	Triclinic	Triclinic
Space group	P1̄	P1̄
Density [mg m^−3]	1.468	1.276
a [Å]	7.8170(3)	10.6568(5)
b [Å]	10.9187(4)	12.5048(5)
c [Å]	15.3971(6)	13.3413(5)
α [deg]	93.089(2)	73.218(3)
β [deg]	99.167(2)	76.096(4)
γ [deg]	102.119(2)	74.284(4)
V [Å^3]	1263.26(8)	1612.99(12)
λ [Å]	0.71073	0.71073
Z	1	1
Temperature [K]	293(2)	293(2)
No. of unique reflections	4958	6337
GOF on F^2	1.081	1.099
R1^a	0.0288	0.0349
wR2^b	0.0778	0.0865

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Fig. 3 (a) ORTEP diagram of Sn^IV(Cl)₂T(4-CMP)P with atomic numbering (solvent molecule, acetonitrile, is not shown for clarity; thermal ellipsoids shown at 40% probability level); molecular crystal packing diagram of Sn^IV(Cl)₂T(4-CMP)P (b) depicting N⋯H bonding viewed along the ‘bc’ plane; (c) depicting Cl⋯C and O⋯H bonding viewed down the ‘ab’ plane.

Fig. 4a and b represent the top and side-on view of the ORTEP diagrams of 3 respectively. From the side-on view, it is clear that the porphyrin core is planar and the two axial ligands (4-nitrophenyl ring) are present in the apex positions which make an angle of 123.18 (14)° between the porphyrin mean plane and the 4-nitrophenyl ring through the oxygen atom of the phenolate.

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 the side view of the molecule). (c) Molecular crystal packing diagram of 3 depicting various intermolecular interactions (viewed down the ‘a’ axis). (d) One-dimensional array of molecules involving complementary N⋯O bonding (viewed along the ‘ab’ plane).

Fig. 4c represents the three-dimensional network arrangement of 3 (viewed down the ‘a’ axis) depicting various intermolecular interactions, viz., _(carbonyl)O⋯H_{(carboxymethyl)}, 2.582 Å, _(carbonyl)O⋯H_(phenyl), 2.384 Å, _(nitro)O⋯H_(β-pyrrole), 2.587 Å, _(β-pyrrole)C⋯H_(phenyl), 2.759–2.884 Å, _{(β-pyrrole/phenyl)}C⋯H_{(carboxymethyl)}, 2.885 Å, and _(nitro)N⋯O_(nitro), 3.002 Å, and each interaction is present twice in the asymmetric unit of 3. Interestingly, the nitro group present in the apex positions involves complementary bonding interactions between the nitrogen of one molecule with the oxygen of another molecule making a one-dimensional array of molecules (Fig. 4d).

The Sn^IV–O bond length in 3 is compared with various tin(IV) porphyrins available in the literature^31–33 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 2-nitrophenol as axial ligands.

Table 3 The Sn^IV–O bond length in various tin(IV) porphyrins

S. no.	Sn^IV–O (Å)	Axial ligand	Porphyrin
1	2.066	4-Nitrophenolate	3
2	2.055	Phenolate	TTP
3	2.083	4-Nitrophenolate	TTP
4	2.062	Phenolic BODIPY	TPP
5	2.070	2-Nitrophenolate	TTP

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Hirshfeld surface analysis

In order to further quantify the various intermolecular interactions in the porphyrins Sn^IV(Cl)₂T(4-CMP)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 color-coding, where the colour intensity indicates the relative strength of the interactions: red regions represent closer contacts and a negative d_norm value; blue regions represent longer contacts and a positive d_norm value; and white regions represent the distance of contacts being exactly the van der Waals separation and with a d_norm value of zero. The HSs of both the porphyrins Sn^IV(Cl)₂T(4-CMP)P and 3 show intense red spots due to the close O⋯H contacts whereas faint red spots are observed for the N⋯H and C⋯H contacts for Sn^IV(Cl)₂T(4-CMP)P and 3 respectively (Fig. 5). The 2D fingerprint plot provides the decomposition of the Hirshfeld surfaces into the contribution of the different intermolecular interactions present in the crystal structure which features spikes of various length and thickness.³⁴ A closer inspection of the FPs reveal that the major intermolecular interactions in both the complexes are mainly the C⋯H [21.5% in Sn^IV(Cl)₂T(4-CMP)P; 15.1% in 3], H⋯H [33.9% in Sn^IV(Cl)₂T(4-CMP)P; 30.1% in 3] and O⋯H [16.5% in Sn^IV(Cl)₂T(4-CMP)P; 27.6% in 3] contacts.

Fig. 5 Hirshfeld surfaces of porphyrins, Sn^IV(Cl)₂T(4-CMP)P and 3, (a and d) top view and (b and e) side view with d_norm mapped ranging from −0.28 (blue) to 3.49 (red); (c and f) fingerprint plots of Sn^IV(Cl)₂T(4-CMP)P and 3 with d_i and d_e ranging from 1.0 to 2.8 Å. Close contacts are labeled as follows: C⋯H (1), H⋯H (2), O⋯H (3), N⋯H (4) and H⋯Cl (5).

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 a zone of inhibition assay. It was found to be dependent on the photosensitization environment as the compounds which were illuminated with visible light showed a maximum zone of inhibition as compared to the compounds which were left un-illuminated. 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 the light in a concentration dependent manner i.e. around 24.4% inhibition at a 50 μM (30.9 μg mL⁻¹) concentration. Also, compounds 1, 2 and the reference compound R showed a high percentage inhibition in a concentration dependant manner upon illumination. In the dark, the zone of inhibition of the compounds is much less as compared to that of the compounds in the light. 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. The 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 a higher percentage of inhibition as compared to the standard antifungal drug fluconazole.

Photodynamic inactivation of C. albicans in a broth assay: determination of the IC₅₀ and MIC values of the porphyrins

Inhibition of the 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 μM, 9.1 μM, 9.0 μM and 9.4 μM respectively (Fig. 7). Interestingly, all the synthesized compounds exhibited lower IC₅₀ values as compared to that of fluconazole (17.5 μM). 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 the 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 μM (▮), 25 μM (▲) and 50 μM (×) porphyrins are shown in the graphs. The figure also shows the percentage inhibition of the growth of C. albicans in the presence of different porphyrins in varying concentrations after 5 h of growth under light and dark conditions. The IC₅₀ values mentioned in the figure correspond to the samples which were illuminated with light.

The concentration of the compound at which few or no colonies were observed was considered as the MIC.³⁶ Porphyrins at a concentration of 50 μM exhibit nearly complete inhibition of cell growth when exposed to light. Very few colonies (2–20) were observed in the Petri plates in which the C. albicans cells treated with the compounds and exposed to light were plated. Importantly, compound 3 showed the least number of isolates. Comparatively a greater number of colonies (40–70) was observed when compound treated C. albicans cells, cultured under dark conditions, were plated on the solid medium (Fig. 8). This shows that the growth inhibitory effect of our compounds was at a 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 μM of porphyrins, cultured in light and dark conditions. Treated C. albicans cells, cultured in the light, gave a lower number of colonies upon plating. In general, C. albicans cells treated with different porphyrins gave a lower 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 the maximum internalization and compound 1 the minimum internalization. These results are in compliance with those obtained from the photodynamic inactivation experiments performed in this study in which compound 1 showed a higher IC₅₀ value as compared to the other compounds. In light of the above observations, it is reasonable to suggest that the slow and inefficient internalization of compound 1 could be the reason for it showing the least potency compared to the other compounds under study (Fig. 9).

Fig. 9 The cellular uptake profiling study results reveal the slow intake of all porphyrins into the Candida cells. Compound 3 shows a better uptake profile than the other compounds. Compound 1 shows relatively less internalization as compared to the other compounds.

To study the internalization of the porphyrins, a fluorescence microscopic analysis of C. albicans CAF4-2 cells was carried out by incubating the cells with tin(IV) porphyrins (Fig. 10). The cells exposed to all three of the porphyrins exhibit fluorescence when excited using the green filter excitation, which confirms the internalization 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 localization. These results also confirm that the cytotoxic effect on C. albicans was specifically due to the internalization of the porphyrins.

Fig. 10 Internalization of the porphyrins in C. albicans CAF4-2. Cells were incubated with the solvent control (DMSO) or with different porphyrins, and were grown for 6 h and processed for microscopy as given in the materials and methods section. (A) Bright field image, (B) Hoechst staining, (C) porphyrin fluorescence, and (D) merged.

Confocal fluorescence microscopy studies

In order to confirm the internalization of the porphyrins in C. albicans cells, a progressive confocal microscopy analysis was performed. The cells were sliced optically through the Z-axis with 0.8 μm depth. Compound 3 shows the best internalization profile (Fig. 11). The C. albicans cells exposed to 3 were optically sliced. Red fluorescence of 3 increased progressively and reached a maximum value at a depth of 2.4–4.8 μm (Fig. 11E–G) within the cells. Red fluorescence is negligible on the upper and lower sections of the cells (Fig. 11A, B and J), showing that there is no or less binding of the compound with the cell membrane. In all cases, red fluorescence increases as the cells are sliced deeper, confirming the internalization and cytoplasmic localization of the compounds. All the other compounds showed proper internalization (data not shown). This data is in support with the results obtained from fluorescence wide field imaging.

Fig. 11 Internalization of compound 3 by progressive confocal optical slicing of C. albicans. Images were obtained by merging the bright field and fluorescence images. A–J: as the 0.8 μm 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 are known to interfere with the mitochondrial membrane potential, 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 from the other cellular organelles like mitochondria or vacuoles, which is also quite possible.

Photogeneration of singlet oxygen species

The photodynamic inactivation of C. albicans could be through the generation of singlet oxygen species within the cells. This can be visualized using 1,3-diphenylisobenzofuran (DPBF) which was able to capture the ¹O₂ generated by the porphyrins.^2c,25 The relationship between the change in absorbance by DPBF and the irradiation time reflects the ¹O₂ yield of the compounds. The absorbance of DPBF at 410 nm decreased in the presence of porphyrin with an increasing irradiation time and from the slope of the line, the relative rate of ¹O₂ generation was compared. The higher the line slopes, the higher the ¹O₂ yield. From the plot, the order of the ¹O₂ generation rate was found to be 3 > 2 > 1 > R > H₂TPP (Fig. 12). The results are in good agreement with the antifungal assays and fluorescence microscopy results.

Fig. 12 Plot of change in absorbance of DPBF vs. irradiation time in the presence of porphyrins.

DFT calculations for the HOMO–LUMO gap in porphyrins

In order to understand more about the electronic transitions, we have performed DFT calculations at the BP86/def2-SVP level of theory³⁸ using the Gaussian 09 (ref. 39) program package for compounds using their crystallographic information files (CIFs) obtained from the single crystal X-ray diffraction studies. For comparison, we have optimized the structures of H₂TPP and H₂T(4-NMP)P. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the free base porphyrins, H₂TPP, H₂T(4-NMP)P and tin(IV) porphyrins, Sn^IV(Cl)₂T(4-CMP)P, 1 and 3 are shown in Fig. 13. For free base porphyrins, the HOMO and LUMO are typical π-type orbitals on the porphyrin core. Hence, the electronic transition is a well-known π → π* type transition upon excitation. In the case of Sn^IV(Cl)₂T(4-CMP)P, the HOMO is mainly based on the porphyrin core and the axial ligand chlorine has a lesser contribution. Here, the HOMO−1 is a porphyrin based orbital and the HOMO−2 and HOMO−3 are concentrated on the axial ligand (Fig. S9†). For 1, the HOMO doesn’t have any contribution from the axial ligand, whereas the HOMO−1, which is slightly lower in energy than the HOMO (ΔE = 0.05 eV) has more contribution from the hydroxyl moiety (Fig. S10†). For 3, the HOMO has a major contribution from the 4-nitrophenolate moiety and also the underlying orbitals, HOMO−1 and HOMO−2 have a contribution from the axial ligand (Fig. S11†). In all the above structures the LUMO is a π based orbital on the porphyrin core.

Fig. 13 Orbital energies (in eV) at the BP86/def2-SVP level of theory for the compounds. The nature of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) and the HOMO–LUMO 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 the chlorine atom, hydroxyl and 4-nitrophenolate group to the porphyrin core in Sn^IV(Cl)₂T(4-CMP)P, 1 and 3 respectively. Since chlorine has less contribution in the HOMO of Sn^IV(Cl)₂T(4-CMP)P, a π → π* transition is more favorable and the extent of the CT will be less since the orbitals corresponding to the CT (HOMO−2 and HOMO−3) (Fig. S9†) are stabilized in comparison with the HOMO. Upon photon excitation, both the π → π* and CT transitions are possible in 1, which are energetically more favorable because the HOMO has a π-type character and the HOMO−1 has contribution from the hydroxyl fragment (ΔE_{[HOMO–(HOMO−1)]} = 0.05 eV). However, 3 shows the most prominent charge transfer from 4-nitrophenolate to the porphyrin core which is the cause for the intersystem crossing (ISC), which in turn results in the higher population of the lower energy triplet state.⁴⁰ Our experimental results correlate 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 species. 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 are possibly the better candidates for treating C. albicans infections.

Conclusions

In summary, high valent tin(IV) porphyrins, 1–3 were synthesized and characterized, and their photophysical properties were studied. X-ray crystal structure determination of the porphyrins Sn^IV(Cl)₂T(4-CMP)P and 3 was achieved successfully and the results show that there is a π–π interaction between the porphyrin plane and the 4-nitrophenyl 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 evidence for porphyrin internalization 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 4-nitrophenolate to the porphyrin core. This charge transfer increases the population of the lower energy triplet state leading to the generation of more singlet oxygen species. To conclude, porphyrins 1–3 exhibited significant antimicrobial activity, 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/WOS-A/CS-146/2011) and CA (SB/EMEQ-016/2013). 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 would like to thank Prof. I. Ibnusaud for providing a ¹H NMR facility at the Institute for Intensive Research in Basic Sciences (IIRBS), MG University, Kottayam. We thank Ms Agisha and Dr R. Suseela Bhai for providing a wide field microscope facility at the 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 a Fluoromax-4 fluorescence spectroscopy facility at the School of Pure and Applied Physics, MG University, Kottayam. We also extend our thanks to Dr Anilkumar P. R. for providing a confocal microscopy facility at Tissue culture laboratory, Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Trivandrum. We would also like to thank Dr Md. Anaul Kabir, Molecular Genetics Laboratory, SBT, NIT Calicut for providing the C. albicans strain CAF4-2 for the study.

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Footnote

† Electronic supplementary information (ESI) available: Additional figures, ¹H NMR and mass spectra. CCDC 948482 and 974215. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra09343k

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