A novel axially palladium(II)-Schiff base complex substituted silicon(IV) phthalocyanine: Synthesis, characterization, photophysicochemical properties and photodynamic antimicrobial chemotherapy activity against Staphylococcus aureus

Polyhedron 173 (2019) 114135

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A novel axially palladium(II)-Schiff base complex substituted silicon(IV) phthalocyanine: Synthesis, characterization, photophysicochemical properties and photodynamic antimicrobial chemotherapy activity against Staphylococcus aureus Pinar Sen ⇑, Tebello Nyokong ⇑ Institute for Nanotechnology Innovation, Department of Chemistry, Rhodes University, PO Box 94, Grahamstown 6140, South Africa

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 11 July 2019 Accepted 21 August 2019 Available online 31 August 2019

In this study, a novel silicon(IV) phthalocyanine is reported for the first time as a phthalocyanine derivative bearing axially a palladium(II)-Schiff base complex. The photophysical and photochemical properties of the new Si(IV)Pc, such as absorption, fluorescence, singlet oxygen quantum yields, triplet state quantum yields and exited state lifetimes were measured in DMSO. The new silicon phthalocyanine displayed very low fluorescence, showing efficient intersystem crossing resulting in high triplet and high singlet oxygen quantum yields in DMSO. When compared with the unsubstituted SiPcCl2, the singlet oxygen quantum yield value (UD = 0.47) in relation to the triplet quantum yield (UT = 0.82), which is an important determinant for PDT applications, increased. The photodynamic antimicrobial chemotherapy activity (PACT) of new Si(IV)Pc towards Staphylococcus aureus was determined in comparison to the unsubstituted SiPcCl2. The results of the photodynamic antimicrobial effect study demonstrated that the Pd(II) complex substituted SiPc (5) possesses excellent photodynamic activity with a reduction percentage value of 99.94% and a log red value of 3.26. Ó 2019 Elsevier Ltd. All rights reserved.

Keywords: Silicon(IV) phthalocyanine Schiff base-Palladium complex Photodynamic antimicrobial therapy S. aureus Photophysicochemical properties

1. Introduction After the World Health Organization reported that bacteria show multi-drug resistance, which is encountered as the most important problem for human health, photodynamic antimicrobial chemotherapy (PACT) has gained increasing interest as an alternative bactericidal technique [1,2]. PACT is a light-controlled therapy and offers more promising strategies to prevent and kill pathogenic bacteria compared with conventional antibiotic approaches [3,4]. The mechanism of the PACT process is based on the photo-inactivation of microorganisms through singlet oxygen produced with the reaction of non-toxic dyes called photosensitizers (PS) and visible light in the presence of oxygen [5]. The singlet oxygen, produced by energy transfer from the triplet state PS to molecular oxygen, reacts with the biomolecules and performs a photodynamic process which is responsible for killing the bacteria [6]. PSs play an important role in this process since they absorb the light and produce toxic species such as singlet oxygen. Some of the ⇑ Corresponding authors. Fax: +27 46 6225109. E-mail addresses: (T. Nyokong).

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(P.

https://doi.org/10.1016/j.poly.2019.114135 0277-5387/Ó 2019 Elsevier Ltd. All rights reserved.

Sen),

[email protected]

features for ideal antimicrobial PS are high singlet oxygen production resulting from high triplet-state quantum yields and a high molar extinction coefficient, specifically in the red and near-infrared UV–vis spectral regions (600–800 nm range), for a maximum light penetration [7]. Phthalocyanines, as heterocyclic macrocyclic aromatic compounds, are known as potential PSs, meeting these mentioned requirements for photodynamic applications [8,9], in addition to the use in various fields such as dye-sensitized solar, non-linear optical devices, sensors, catalysis [10]. Considering the fact that the structure of PSs may be a key factor in determining their antimicrobial activity, several kinds of phthalocyanines with different central metals and as neutral, anionic and cationic structures have been reported [11–14]. Among the studied phthalocyanines, axially substituted silicon (IV) phthalocyanines (SiPcs) show promising antibacterial effects [15–17]. As SiPcs can provide axial substitution, they help to reduce aggregation and increase the solubility in organic solvents, and this structural property provides a significant advantage for PDT applications [18]. Schiff bases came into existence by the condensation reaction of a primary amine and an aldehyde. They are the most studied

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P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

ligands as building blocks in coordination chemistry since they can generate very stable complexes with various heavy metal ions [19]. Metal complexes of Schiff bases, providing metal-nitrogen bonding, are formed by the complexation of Schiff bases and have had importance in medicinal chemistry for many years [20]. They have gained considerable attention because of their biological activities against bacteria, fungi and certain type of tumours [21]. The influences of metal ions on biological activities, such as antibacterial, antifungal, anticancer and antioxidant, have been taken into consideration and well documented [22]. In this context, palladium(II) complexes of Schiff base ligands have been reported as bio-active agents following the success of cis-platin, which is used antitumor drugs worldwide [23–25]. In this study, we have prepared the axially Schiff base palladium(II) complex substituted silicon(IV) phthalocyanine as a combination of two functional molecules, resulting in a trinuclear phthalocyanine derivative. To the best of our knowledge, this kind of molecule has not been reported to date. The photophysicalchemical properties of the obtained new structure were determined and the photodynamic antibacterial activity against Staphylococcus aureus was determined, comparing the results with the unsubstituted SiCl2Pc to observe the effect of the metal complex when incorporated into the silicon(IV) Pc. 2. Experimental 2.1. Chemicals 4-Tert-butyl-phenol, SnCl4, 2,6-lutidine, paraformaldehyde, 1,3diamino-2-propanol, PdCl2, NaH, ethanol, acetonitrile, chloroform (CHCl3), triethylamine (TEA), toluene, cyclohexane, diethylether and 1,3-diphenylisobenzofuran (DPBF) were purchased from Sigma Aldrich. All solvents were dried and purified as reported by Perrin and Armarego [26] before use. Silica gel (Merck grade 60) was used for the column chromatography. Phosphate-buffered saline (PBS) solution pH 7.4 was prepared using the appropriate amounts of Na2HPO4 and KH2PO4 in ultra-pure water from Millipore water, ELGA, Veolia water PURELAB, flex system (Marlow, UK). Nutrient agar and agar bacteriological BBL Muller Hinton broth were purchased from Merck and S. aureus (ATCC 25923) was obtained Davies Diagnostics.

Photo-irradiations for singlet oxygen studies were done using a General Electric Quartz line lamp (300 W). A 600 nm glass cut off filter (Schott) and a water filter were used to filter off ultraviolet and infrared radiation respectively. An interference filter (Intor, 670 nm with a band width of 40 nm) was additionally placed in the light path before the sample. Light intensity was measured with a POWER MAX5100 (Molelectron detector incorporated) power meter. The Modulight ML7710-680-RHO laser system (680 wavelength probe) was employed for the PACT studies. For the PACT studies, various irradiation periods at 0.5 W cm
2 irradiance, resulting in energy doses ranging from 0.9 kJ cm
2 to 3.6 kJ cm
2 corresponding to time periods ranging from 30 to 120 min, were employed. 2.3. Synthesis The syntheses of 5-tert-butyl-2-hydroxybenzaldehyde (1) and silicon phthalocyanine dichloride (SiPcCl2) (4) have been reported elsewhere [27,28]. The obtained spectroscopic results are in agreement with the literature data. 2.3.1. Synthesis of N,N0 -bis(5-tert-butyl-salicylidene)-1,3-diamino-2propanol (H3L) (2) 5-Tert-butyl-2-hydroxybenzaldehyde (1) (0.5 g, 2.8 mmoL) was dissolved in 20 mL ethanol, then 1,3-diamino-2-propanol (0.126 g, 1.4 mmoL) dissolved in 20 mL of ethanol was added dropwise to the solution. The reaction mixture was purged with argon and the reaction mixture stirred under reflux for 8 h. The reaction for the formation of the Schiff base (2) was monitored by thin layer chromatography (TLC) (CHCl3/EtOH 10/0.1). After the reaction completed, the solution was evaporated to 1/3 volume. The formed precipitate was filtered off, washed with ethanol and dried in a vacuum desiccator. Yield: 75% (0.430 g). FT-IR (UATR-TWOTM) m max/cm
1: 3378 (OH), 3304 (OH), 3053 (Ar, CAH), 2954–2867 (Aliph, CAH), 1634 (C@N), 1612–1513 (Ar, CAC), 1491–1392 (CAC), 1266 (tert, CAN), 1241 (Asym., Ar-O-), 1179 (CAN), 1051 (Sym., Ar-O-), 828. 1 H NMR (DMSO) d (ppm): 8.30 (s, 2H, ACH@N), 7.43 (s, 2H, ArH), 7.35 (d, 2H, ArH), 6.83 (d, 2H, ArH), 5.24 (s, 2H, OH), 4.59 (s, 1H, AOH), 3.77 (d, 4H, ACH2), 3.39–3.31 (m, 1H, ACH), 1.27 (s, 18H, ACH3). 13C NMR (CHCl3) d (ppm): 167.86, 155.44, 141.26, 129.85, 126.32, 123.55, 116.53, 69.97, 63.27, 34.15, 31.91. (MALDI-TOF) m/z: 411.527 [M+1]+. Anal. Calc. for C25H34N2O3 (%): C, 73.14; H, 8.35; N, 6.82; O, 11.69; Found (%): C, 73.09; H, 8.38; N, 6.78.

2.2. Instruments Fluorescence emission spectra were recorded on a Varian Eclipse spectrofluorometer with a 1 cm quartz cell at room temperature. Infrared spectra were recorded on a Bruker ALPHA FT-IR spectrometer with a platinum attenuated total reflectance (ATR) sampling accessory. Absorption spectra were recorded on a Shimadzu UV-2550 spectrophotometer. Fluorescence lifetimes were measured using a time correlated single photon counting setup (TCSPC) (FluoTime 300, Picoquant GmbH). The excitation source was a diode laser (LDH-P-670 driven by PDL 800-B, 670 nm, 20 MHz repetition rate, 44 ps pulse width, Picoquant GmbH). Triplet quantum yield values were obtained using a laser flash photolysis system having a LP980 spectrometer with a PMT-LP detector and an ICCD camera (Andor DH320T-25F03). The signal from the PMT detector was recorded on a Tektronix TDS3012C digital storage oscilloscope. The excitation pulses were produced by a tunable laser system consisting of a Nd:YAG laser (355 nm, 135 mJ/4–6 ns), pumping an optical parametric oscillator (OPO, 30 mJ/3–5 ns) with a 420–2300 nm (NT-342B, Ekspla) wavelength range. Acquisition of 1H NMR spectra was carried out on a Bruker AMX600 MHz NMR spectrometer. Mass analysis was performed on a Bruker AutoFLEX III Smartbeam TOF/TOF mass spectrometer.

2.3.2. Synthesis of [N,N0 -bis(5-tert-butyl-salicylidene)-1,3-diamino-2propanol]palladium(II) (3) In a 100 ml round bottom flask, the ligand (2) (0.2 g, 0.48 mmoL) was dissolved in acetonitrile (20 mL). A solution of PdCl2 (0.088 g, 0.50 mmol) in 20 mL acetonitrile was added dropwise to the mixture, followed by stirring for 30 min at room temperature under an argon atmosphere. After this, TEA (0.147 mL, 1.05 mmoL) was added to the resulting solution; the reaction mixture was stirred under reflux for 12 h. The completion of the complexation reaction was monitored by TLC (CHCl3/EtOH 10/0.1) and then it was allowed to cool. The solution was evaporated to 1/3 volume and the precipitate formed by addition of cyclohexane was collected by centrifugation. The collected compound was washed with cyclohexane and diethylether, and dried. Yield: 70% (0.175 g). FT-IR (UATR-TWOTM) m max/cm
1: 3335 (OH), 3062 (Ar, CAH), 2956–2868 (Aliph, CAH), 1614 (C@N), 1542 (Ar, CAC), 1449–1363 (CAC), 1262 (tert, CAN), 1214 (Asym., Ar-O-), 1123 (CAN), 1061 (Sym., Ar-O-), 827. 1H NMR (DMSO) d (ppm): 8.32 (s, 2H, ACH@N), 7.35 (s, 2H, ArH), 7.17 (d, 2H, ArH), 6.98 (d, 2H, ArH), 4.45–4.41 (m, 1H, ACH), 3.61 (t, 4H, ACH2), 3.76 (s, 1H, OH), 1.69 (s, 18H, ACH3). 13C NMR (CHCl3) d (ppm): 164.97, 152.42, 141.67, 126.25, 124.07, 121.81, 114.84, 68.14, 42.48,

P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

33.83, 31.68. (MALDI-TOF) m/z: 533.155 [M+H2O+1]+. Anal. Calc. for C25H32N2O3Pd (%): C, 58.31; H, 6.26; N, 5.44; O, 9.32; Pd, 20.67; Found (%): C, 58.28; H, 6.35; N, 5.39. 2.3.3. Synthesis of the silicon(IV) phthalocyanine (5) A mixture of [N,N0 -bis(5-tert-butyl-salicylidene)-1,3-diamino2-propanol]palladium(II) (3) (0.100 g, 0.194 mmoL) and silicon phthalocyanine dichloride (4) (0.04 g, 0.064 mmol) in toluene (40 ml) was stirred and then sodium hydride (5 mg, 0.194 mmol) was added to this mixture under an inert argon atmosphere. The resulting mixture was stirred under reflux at 110 °C for 2 days. After cooling to room temperature, the mixture was filtered off and the solvent was evaporated in a vacuum. The crude product was further purified by column chromatography (silica gel, CHCl3/ THF//TEA-85/15/1) to give 5 as a green solid. Yield: 18% (0.019 g). FT-IR (UATR-TWOTM) m max/cm
1: 3053 (Ar, CAH), 2952–2865 (Aliph, CAH), 1612 (C@N), 1531 (Ar, CAC), 1439–1363 (CAC), 1260 (tert, CAN), 1208 (Asym., Ar-O-), 1121 (CAN), 1077 (Sym., Ar-O-), 1064 (Si-O), 829, 734. 1H NMR (DMSO) d (ppm): 8.45 (s, 4H), 7.95 (m, 8H), 7.35–7.04 (m, 8H), 6.87 (s, 4H), 6.68 (d, 4H), 6.58 (d, 4H), 3.78 (m, 8H), 3.14 (m, 2H), 1.29 (s, 36H). UV–Vis (DMSO-1  10
5 M) kmax (nm) (log e): 680 (4.5), 355 (4.36). (MALDI-TOF) m/z: 1582.718 [M+H2O]+, 1085.674 [M+H2O-C25H31N2O2Pd]+. Anal. Calc. for C82H78N12O6Pd2Si (%): C, 62.79; H, 5.01; N, 10.72; O, 6.12; Pd, 13.57; Si, 1.79; Found (%): C, 62.44; H, 5.06; N, 10.58. 2.4. Antimicrobial studies The photodynamic antimicrobial studies of the Pcs 4 and 5 against Staphylococcus aureus were performed through the viable cell count method. Firstly, a stock culture of S. aureus was grown aerobically in 6 mL of nutrient broth under incubation at 37 °C on a rotary shaker (200 rpm) to obtain the bacteria culture. The incubation was maintained until the optical density of the culture recorded at 600 nm was determined to be around 0.6. The resulting microbial solution was centrifuged to remove the broth culture and the bacteria colonies were washed three times with PBS. Then, a stock solution of the bacteria cultures was prepared by diluting to 1/1000 (106 colony forming unit (CFU/mL). The antimicrobial photoactivity of the phthalocyanines were determined at 2.5 lM bacteria concentration and each Pc (4/5) was prepared by dissolving in water containing 5% DMSO, corresponding to 0.312–40 lM concentrations in 6 mL of PBS. The prepared suspensions containing the bacteria and Pcs at different concentrations were tested with the plate method over agar to observe the optimum application concentration. The optimum application concentrations of Pcs were determined as 10 lM for 4 and 0.625 lM for 5 from the agar plates as a result of the CFU/mL counting, depending on the degree of reduction in bacteria colonies by exposure to light from 30 to 120 min. After the bacteria/photosensitizer suspensions at the mentioned specific concentrations were determined, each prepared bacteria/photosensitizer suspension was incubated in an oven equipped with a shaker for 30 min in the dark at 37 °C before plating. The photoinactivation with the Si(IV) Pcs (4 and 5) was evaluated with 30 min irradiation time intervals up to 120 min. While a part of the incubated bacterial suspensions was kept in the dark, the other part of the suspension was irradiated at the Q-band maximum of the photosensitizers, using methods previously reported [29]. The results of the performed experiments were determined by plating over agar after irradiation and keeping the experiments in the dark for a certain period of time. Also, control treatments were performed without photosensitizer, both with irradiation and in the dark, in order to determine the effect of light and sol-

3

vents. All experiments were carried out three times. The data for CFU/mL were converted to the logarithmic form.

3. Results and discussion 3.1. Synthesis and characterization The reaction steps carried out for the target compound are summarized in Scheme 1. The synthesis of 5-tert-butyl-2-hydroxybenzaldehyde (1) was achieved by starting with the commercially available 4-tertbutyl-phenol and applying the procedure for obtaining aldehydes in the ortho-position of aromatic phenols [28]. The new ligand (2), containing a bis-imine group and acting as N2O2-chelating, was synthesised by the usual condensation reaction of the aldehyde derivative (1) and 1,3-diamino-2-propanol in ethanol at reflux temperature. The reaction of PdCl2 with the ligand (2) was conducted in acetonitrile in the presence of TEA to give the palladium Schiff base complex (3). Finally, the reaction of SiPcCl2 (4) and [N,N0 -bis(5-tert-butyl-salicylidene)-1,3-diamino-2-propanol]palladium(II) (3) provided the silicon(IV) phthalocyanine (5), using NaH in toluene. Common spectroscopic methods, such as FT-IR, 13C NMR, 1H NMR, UV/Vis and MS, together with elemental analysis, were employed for determining the proposed structures. All the spectroscopic data confirmed the expected compounds. In the FT-IR spectrum of the condensation product 2, the first significant difference is that the carbonyl vibration of the aldehyde functional group at around 1640 cm
1 disappeared and an imine vibration (AC@N) band appeared at 1634 cm
1. In the FT-IR spectrum of 3, the C@N peak originating from compound 2 shifted to a lower frequency, appearing at 1614 cm
1 upon complexation, which is confirmation for the coordination of the ligand through the azomethine nitrogen atom [30]. Also, the vibrational band observed at 3378 cm
1 due to Ar-OH in the free ligand disappeared in the Pd(II) complex due to deprotonation. The aliphatic –OH vibration band of diaminopropanol is seen in the Pd(II) complex (3), indicating that the –OH group of the diaminopropanol does not take part in coordination [30]. In the FT-IR spectrum of 5, the AC@N peaks of the Pd(II) complex appeared at 1612 cm
1 and the –OH vibration belonging to compound 3 at 3335 cm
1 disappeared, providing evidence for the formation of 5. The most significant result was the observation of a Si-O band at 1064 cm
1 [31]. A MALDI-TOF mass spectrometer was utilized for acquisition of the mass spectra of the products. The expected molecular ion peaks were obtained at m/z 411.527 [M+1]+ for 2, m/z 533.155 [M+H2O +1]+ for 3 and 1582.718 [M+H2O]+ for 5. The fragment ion peak belonging to SiPc (5) was also observed at m/z 1085.674 [M +H2O-C25H31N2O2Pd]+, corresponding to the removal of one Pd(II) complex from the axial site, as seen in Fig. 1. UV–Vis spectroscopy is one of the first methods used for the structural characterization of phthalocyanines. Phthalocyanines, with an 18 p-electron conjugated aromatic system, show characteristic bands in their absorption spectra. The strongest of these is the Q band, which results from the chromophoric system and expresses an absorption in the visible region between 600 and 700 nm. The other main absorption band, which is the so-called B band, is in the UV region at about 300–350 nm. The Q band is attributed to the p-p* transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) of the Pc ring. The B-band arises from deeper p levels to the LUMO [32]. The comparative absorption spectra of the Pd(II) complex 3 and Pc 5 were studied in DMSO at 1  10
5 M concentrations (Fig. 2). In

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P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

OH

OH

OH

OH H2N

O

N

NH2 ii

i

N

OH

HO

2 iii

1 OH N

N Pd

O

O

3 iv Cl O

O

N

Pd

N N

N

N Si

N

N N

N N

O

Cl N

4

N

N Si

N

N N

N N

O N

N Pd

O

O

5 Scheme 1. Synthesis route: (i) SnCl4, 2,6-lutidine, toluene, paraformaldehyde, reflux, 12 h, (ii) ethanol, reflux, 2 h, (iii) PdCl2, acetonitrile, TEA, reflux, 12 h, (iv) NaH, toluene, reflux, 2 days.

the UV–vis spectrum of the 3, the most distinctive absorption band appear at around 400 nm, which is ascribed to metal-to ligand charge transfer transitions [33]. The electronic absorption spectrum of the SiPc (5) is typical for non-aggregated phthalocyanines, showing an intense Q band at 680 nm, which is indicative for the metallated phthalocyanines, as expected [32], together with two vibronic bands at 613 and 654 nm, and the B (or Soret) band at 355 nm. Also, the metal-to ligand charge transfer transition resulting from the Pd(II) complex was observed between 400 and 450 nm in the UV–vis spectrum of 5. When comparing to the UV–vis spectrum of the SiPc (5) with the unsubstituted SiPcCl2 (4), the Q band of Pd(II) complex substituted SiPc is 8 nm red shifted in DMSO (Table 1). 1 H NMR and 13C NMR spectra have supplied satisfactory information verifying the proposed structures. In the 1H NMR spectrum of 2, the disappearance of the HC@O proton signal as distinct from 1 and the appearance of an imine proton peak at d 8.30 ppm indi-

cate that the condensation reaction has occurred to give the imine compound. The phenolic OH and aliphatic OH protons, originating from diamino propanol, were observed at d 5.35 and 4.59 ppm, respectively. The methylene protons were observed at d 3.77 and 3.39–3.31 ppm and the methyl protons on the benzene ring were at d 1.27 ppm. In the 1H NMR spectrum of 3, the disappearance of the signal due to the phenolic OH protons originating from the free ligand (2) indicates the coordination of phenolic oxygen atom to the Pd (II) ion. The azomethine ACH@NA proton of the ligand (2) shifted to high field in complex 3 at d 8.32 ppm. In the 1H NMR spectrum of compound 5, the appearance of the new peaks in the aromatic region at d 7.95–6.58 ppm are proof of axial ligation. When the structures obtained were evaluated by 13C NMR spectroscopy, the peak at d 167.86 ppm belonging to the carbon atom of the imine group is characteristic proof for the existence of Schiff base formation for compound 2. The aromatic and

P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

5

Fig. 1. MALDI-TOF MS spectra of 2 (a), 3 (b) and 5 (c).

aliphatic carbon peaks were observed in the ranges d 155.44–116.53 and 69.97–31.91 ppm, respectively. The azomethine carbon atom of complex 3 appeared at d 164.97 ppm upon coordination to the Pd(II) ion. The aromatic and aliphatic carbon peaks were observed in the ranges d 152.42–114.84 and 68.14–31.68 ppm, respectively.

4. Physicochemical properties 4.1. Fluorescence quantum yields (UF), emission and lifetimes The fluorescence emission, absorption and excitation spectra of the newly synthesized SiPc compound are shown in Fig. 3.

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P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

standard and unsubstituted SiPcCl2 (UF = 0.41) in DMSO (Table 1). This indicates the presence of the Schiff base metal complex as an axial ligand, where Pd, as a heavy atom, allows inter-system crossing [38]. The fluorescence lifetime (sF) of the axially-disubstituted Schiff base Pd(II) complex silicon phthalocyanine (5) was determined from the exponential fitting of the fluorescence decay curves by utilizing the TCSPC (time correlated single photon counting) method, which is a counting process in DMSO (Fig. S6 in the supporting information). The determined sF value is 4.46 ns for 5, which is smaller than that for the unsubstituted SiPcCl2 (sF = 5.06 ns). The fluorescence lifetime values are directly proportional to the fluorescence quantum yields, meaning a fluorophore having a high fluorescent quantum yield has a high fluorescence lifetime [39]. When comparing SiPc 5 with SiPcCl2 a good correlation is seen. Fig. 2. Absorption spectra of the palladium complex (3) and the SiPc (5) in DMSO (concentration 1  10
5 mol.L
1).

The fluorescence behaviour of SiPc 5 was studied in DMSO by comparing it with the unsubstituted SiPcCl2 (4) and the related data are listed in Table 1. Upon excitation at 608 nm, the fluorescence emission wavelength of the studied Pc (5) was determined to be 682 nm. The emission profile is a mirror image of the absorption spectrum for 5. The almost symmetric appearance of the emission and absorption spectra indicate that the emission corresponds to similar absorption transitions [34]. The excitation spectrum of compound 5 is slightly blue-shifted compared to its absorption spectrum. This observation could be regarded an inconsiderable shift in comparison with its absorption spectra. The closeness of the wavelengths in the absorption and excitation spectra could be attributed to the fact that there is no change in the molecule upon excitation [35]. In addition, the observed Stokes shift was 7 nm for 5 in DMSO, which is similar to the unsubstituted SiPcCl2 and is typical for MPc complexes (Table 1). The fluorescence quantum yield (UF) and fluorescence lifetime (sF) are also important for photodynamic applications. UF is described as the ratio of the number of photons emitted through fluorescence, to the number of photons absorbed by the fluorophore, which indicate the efficiency of the radiation-induced process. The fluorescence quantum yields of the obtained Pcs were calculated using a method reported in the literature, using Eq. (1) [36]:

UF ¼ UF ðStdÞ

F  AStd  n2 F Std  A  n2Std

ð1Þ

In this equation, F and FStd are the areas under the emission curves of the Pcs and standards, respectively. A and AStd indicate the absorbance values corresponding to the excitation wavelength of the sample and standard, respectively. The refractive indices of the solvents used for the solution of the studied samples and standard are respectively expressed as n2 and n2Std. The unsubstituted ZnPc was utilized as a standard with the value UF = 0.2 [37]. The fluorescence quantum yield (UF) of the SiPc (5) was measured in DMSO. The UF value of 5 was determined as 0.015 (Table 1). The UF value of 5 is much lower than those of the ZnPc

4.1.1. Singlet oxygen quantum yields (UD) The most important factor that determines whether a photosensitizer is applicable to PDT applications is the efficiency of its singlet oxygen generation. Singlet oxygen (1O2) production is basically based on the energy transfer between a triple excited state and the ground state of oxygen, in addition to substituents on the photosensitizer and solvents which could quench singlet oxygen. The singlet oxygen quantum yield (UD) was determined by the comparative chemical method, which is based on the chemical quenching of DPBF upon oxidation by singlet oxygen generated during irradiation of 5 in DMSO (Fig. S7 in the supporting information). The quenching of DPBF was monitored with an UV–Vis spectrophotometer, showing a gradual decrease in absorbance upon light irradiation in the presence of SiPc 5. The same procedure was applied for the unsubstituted ZnPc (UD = 0.67 in DMSO) as a reference [40] for comparison purposes, using the experimental set-up as described in the literature [41]. The novel Si(IV) Pc (5) bearing the Schiff base-Pd(II) complex produced higher singlet oxygen generation in DMSO with a UD value of 0.47 compared to the axially unsubstituted SiPcCl2 (UD = 0.18). This observation could be attributed to the heavy metal effect of the palladium atom which enhances intersystem crossing to the triplet state, resulting in high triplet and singlet oxygen quantum yields [35]. An electronic transition from a singlet to a triplet excited state within a molecule is a spin-forbidden process and as such occurs inefficiently for many compounds. In order for an effective transition between states to occur, spin–orbit perturbation is generally required [42]. Enhanced spin–orbit perturbations can be accomplished by adding a heavy atom directly to the molecule (an internal heavy atom effect), by placing the molecule in an environment containing heavy atoms (an external heavy atom effect) or by linking groups containing heavy atoms to the main structure (an external heavy atom effect) [43,44]. When compared to previously published di-axially substituted Si(IV) phthalocyanine studies, 5 was found to be more effective in terms of production of singlet oxygen [45,46]. At the end of the chemical degradation experiment, while the singlet oxygen quenchers showed degradation, there was no change in the Qband, indicating the stability of 5.

Table 1 Photophysicochemical parameters of the complexes. Compound

Solvent

Abs

Exc.

Em.

UF

Stokes shift DStokes, nm

sF (ns)

UD

UT

sT (ls)

SiPc (5) SiPcCl2 (4)

DMSO DMSO

680 672

675 674

682 680

0.015 0.41

7 6

4.46 5.06

0.47 0.18

0.82 0.38

235 155

P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

4.1.2. Triplet quantum yield (UT) and lifetimes (sT) One of the most common transitions of an excited photosensitizer is intersystem crossing to populate its triplet state. The triplet quantum yield is a centrally important parameter in photodynamic applications since the efficiency of a phthalocyanine as a photosensitizer is dependent on its triplet state quantum yield (UT) and lifetime (sT). PSs performing efficient intersystem crossing and generating a high triplet state population and lifetime cause high singlet oxygen production, which is toxic for cells. The triplet quantum yield (UT) was measured by exciting SiPc 5 and the axially unsubstituted SiPcCl2 (4) at the Q-band wavelength by utilizing the unsubstituted ZnPc (UT = 0.65) [47] as a reference, as described in the literature [44]. The samples and the standard were dissolved in deaerated DMSO. The Si(IV) Pc (5) exhibited a triplet quantum yield with a value of 0.82, which is higher than the axially unsubstituted SiPcCl2 with a value of 0.38, indicating that 5 exhibits a more efficient intersystem crossing as a result of the heavy atom effect. The triplet lifetime decay curve of the SiPc (5) (as an example) is shown in Fig. S8 in the supporting information. An exponential decay curve was used to fit the experimental data. The triplet lifetime (sT), which is much longer than the fluorescence lifetime, was determined as 235 ls for 5 and 155 ls for 4. The triplet quantum yields and triplet state lifetimes are shown in Table 1.

Fig. 3. Normalized absorption, excitation and emission spectra of the SiPc (5) in DMSO (excitation wavelength: 608 nm).

7

4.2. Antimicrobial activity The photodynamic activities of 4 and 5 were studied against Staphylococcus aureus, which is a gram-positive bacterium. The unsubstituted SiPcCl2 (4) was studied for comparative purposes in order to identify the effect of the Pd(II)-Schiff base complex on the photodynamic antimicrobial activity when it introduced as an axial ligand in 5. The PACT experiment performed for 4 was not effective at lower concentrations. This is why compound 4 was prepared at much a higher concentration than 5. The photodynamic inactivation of the Pcs in terms of the decreased cells were quantified as Log reductions (Fig. 4.) and the reduction percentage as opposed to viable cell percentage (Fig. 5) The reduction in bacteria colonies were calculated by using Eq. (2) [14]:

log reduction ¼ log10 ðAÞ
log10 ðBÞ

ð2Þ

where A and B are the number of viable microorganisms before and after treatment with light or in the dark, respectively. While the SiPcCl2 (4) did not exhibit dark toxicity, the SiPc (5) showed dark cytotoxicity against S. aureus after being kept for 120 min (Fig. 5). This difference for compound 5 could be attributed to carrying the Pd(II) complex in the axial position, since

Fig. 5. Phototoxicity studies S. aureus in the presence of the SiPcs (4 and 5) with light and in the dark.

Fig. 4. Survival curves of S. aureus in the presence of the SiPcs (4 and 5) with light and in the dark.

8

P. Sen, T. Nyokong / Polyhedron 173 (2019) 114135

Table 2 Percentage reduction and log reduction of S. aureus after irradiating for 120 min. Compound

Concentration (lM)

Percentage reduction

Log reduction

SiPc (5) SiPcCl2 (4)

0.625 10

99.94 98.86

3.26 1.94

palladium complexes are kinetically labile and naturally qualify as metallodrugs [48]. Control experiments showed that the viability of bacteria was insignificantly affected by illumination (data not shown). The Pd-complex substituted SiPc (5) displayed the higher potency with a log reduction value of 3.26, more than that of the unsubstituted SiPcCl2 (4) with a log reduction value of 1.94, as a result of radiation with light (Table 2). Photodynamic antimicrobial therapy is based on the ability of singlet oxygen production of PS. This is related to why SiPc (5) has a much better efficacy despite being applied in a 16 times smaller concentration. It also relates to the antimicrobial effect of the palladium complex itself [46]. The survival percentage (viable colonies) of the bacteria is stated by the number of colonies remaining alive after treatment of the cells. Fig. 5 represents the bar graph of the survival percentage of S. aureus performed with light and in the dark for compounds 4 and 5. When the results were evaluated in terms of the reduction percentage of the cells, the reduction percentage value of 5 reached 99.94% after exposure to light for 120 min (Table 2). 5. Conclusion A new type of silicon(IV) phthalocyanine carrying a Pd(II)-Schiff base complex at the axial position was successfully synthesized and characterized in this work. The new phthalocyanine was characterized by common spectroscopic methods, such as 1H NMR, 13C NMR, FT-IR, UV–Vis and MS and elemental analysis. After the photophysicochemical properties were determined, the potential of the photodynamic antimicrobial effect of the novel Pc on Staphylococcus aureus was investigated. The results of the antimicrobial activities indicated that the newly synthesized SiPc (5) exhibit higher phototoxicity against bacterial pathogens at much lower concentrations than that of the unsubstituted SiPc (4). Acknowledgements This work was supported by the Department of Science and Technology (DST) Innovation, National Research Foundation (NRF) South Africa and Sasol Inzalo Foundation through DST/NRF South African Research Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology (UID 62620) as well as Rhodes University. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.poly.2019.114135. References [1] Y. Liu, R. Qin, S.A.J. Zaat, E. Breukink, M. Heger, Antibacterial photodynamic therapy: overview of a promising approach to fight antibiotic-resistant bacterial infections, J. Clin. Trans. Res. 1 (2015) 140, https://doi.org/ 10.18053/jctres.201503.002. [2] M. Wainwright, Photodynamic antimicrobial chemotherapy (PACT), J. Antimicrob. Chemother. 42 (1998) 13. [3] R. Bonnett, Chemical Aspects of Photodynamic Therapy, Gordon and Breach Science, Canada, 2000.

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