Synthesis of Conjugated Polymer Based in Zn(II) Porphyrin Bearing Terminal Alkynyl Groups as Photosensitizer ^†

Abstract

5,10,15,20-tetrakis-[4-(ethynyl)phenyl]porphyrin (TEP) was synthesized from the condensation of 4-(ethynyl)benzaldehyde and pyrrole catalyzed by boron trifluoride diethyl etherate in dichloromethane (DCM). After oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and purification, TEP was obtained in 34% yield. This porphyrin was metaled with Zn(II) acetate in DCM/methanol to produce the complex ZnTEP in 98% yield. A homocoupling reaction of terminal alkynes of ZnTEP to diynes was used to synthesize conjugated polymer organogel (ZnTEPP). The reaction was co-catalyzed by PdCl₂(PPh₃)₂ and CuI in tetrahydrofuran. The solvent was evaporated to obtain xerogel and the SEM images showed microporous structure. In addition, spectroscopic and photodynamic studies of ZnTEPP indicated that the porphyrin unit retains its properties as a photosensitizer. Thus, this polymer is an interesting material with potential applications in forming photoactive aseptic surfaces.

1. Introduction

In hospitals, surfaces are one of the main possible reservoirs of bacteria, which can cause a notable incidence of nosocomial infections [1]. For this purpose, photodynamic inactivation (PDI) of microorganisms has been proposed to eliminate bacteria. This therapy uses a photosensitizer, visible light and oxygen to produce highly reactive oxygen species (ROS), which can react with several cell components. These molecular modifications induce a loss of biological functionality that causes cell death [2]. In most PDI studies, photosensitizers are added to a microbial suspension from a homogeneous solution. In this methodology, after PDI treatment the photosensitizer remains in the place of action, producing an undesired photodynamic effect and contaminating the medium. In addition, under these conditions the photodynamic agent is difficult to recover for reuse in subsequent applications. This drawback can be avoided by using photosensitizers chemically bound to a support [3]. Thus, porphyrins immobilized on a surface have been proposed for the inactivation of microorganisms, considering economic and ecological subjects. In this sense, the coating of surfaces with photosensitizers (PSs) that are immobilized in a film is of great interest for maintaining aseptic surfaces in public health [4].

In this study, 5,10,15,20-tetrakis-[4-(ethynyl)phenyl]porphyrin (TEP) was synthesized by acid catalyzed condensation of 4-(ethynyl)benzaldehyde and pyrrole. This porphyrin was treated with Zn(II) to form the complex ZnTEP. A homocoupling reaction of terminal alkynes of ZnTEP to diynes was used to obtain the conjugated polymer organogel (ZnTEPP). After evaporation of the solvent, xerogel was obtained, which is a type of solid-formed gel that has a microporous structure and larger surface area together with very smaller pore sizes. Moreover, spectroscopic and photodynamic studies of ZnTEPP indicated that the porphyrin unit retains its properties as PS. Thus, this polymer is an interesting material with potential applications to form photoactive aseptic surfaces.

2. Materials and Methods

2.1. Materials

Chemicals were obtained from Sigma–Aldrich (Milwaukee, WI, USA). They were used without further purification. Tryptic soy (TS) broth and agar from Britania (Buenos Aires, Argentina) were used in microbial cultures. Microtiter plates (96-well) were acquired to Deltalab (Barcelona, Spain). Organic solvents (GR grade) from Merck (Darmstadt, Germany) were distilled and maintained on molecular sieves. Ultrapure water was obtained from a Labconco (Kansas City, MO, USA) equipment model 90901-01.

2.2. Instrumentation

Proton nuclear magnetic resonance spectra were achieved on a FT-NMR Bruker Avance DPX400 spectrometer (Bruker BioSpin, Rheinstetten, Germany). Mass spectra were attained on a Bruker micrO-TOF-QII (Bruker Daltonics, Billerica, MA, USA) equipped with an ESI source (ESI-MS). Scanning electron microscopy (SEM) images were obtained with a field emission scanning electron microscope FE-SEM (Σigma Zeiss, Oberkochen, Germany) with a thin Cr film on the sample surface and an acceleration voltage of 5 kV. UV-visible absorption spectra were recorded on a Shimadzu UV-2401PC spectrometer (Shimadzu Corporation, Tokyo, Japan), while fluorescence spectra were carried out on a Spex FluoroMax spectrofluorometer (Horiba Jobin Yvon Inc., Edison, NJ, USA). Fluence rates were obtained with a Radiometer Laser Mate-Q (Coherent, Santa Clara, CA, USA). Photooxidation of anthracene derivative was performed with a Cole-Parmer illuminator 41720-series (Cole-Parmer, Vernon Hills, IL, USA) with a 150 W halogen lamp. Optical filters (GG455 cutoff filter) were used to select a wavelength range between 455 and 800 nm (44 mW/cm²).

2.3. Synthesis

5,10,15,20-Tetrakis(p-ethynylphenyl)porphyrin (TEP). To a solution of 4-(ethinyl)benzaldehyde (260 mg, 2.0 mmol) and pyrrole (140 µL, 2.0 mmol) in 350 mL of dichloromethane (DCM) was added 70 µL of BF₃^.OEt₂. The reaction mixture was stirred at room temperature. After 3 h, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 340 mg, 1.5 mmol) was added and the mixture was stirred for 1 h under reflux. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography (silica gel, n-hexane/DCM (7:3)) to yield 121 mg (34%) of TEP. ¹H-NMR (CDCl₃, TMS) δ (ppm) −2.80 (brs, 2H, pyrrole NH), 3.37 (s, 4H, C≡CH,), 7.94 (d, 8H, J = 8.00 Hz, 3,5-ArH,), 8.19 (d, 8H, J = 8.00 Hz, 2,6-ArH), 8.88 (s, brs, pyrrole-H, 8H). ESI-MS (m/z) 711.2556 [M + H]⁺ (711.2549 calculated for C₅₂H₃₀N₄ + H).

Zn(II) 5,10,15,20-tetrakis(p-ethynylphenyl)porphyrin (ZnTEP). TEP (30 mg, 0.042 mmol) was dissolved in 10 mL of DCM. Then, 5 mL of a saturated solution of Zn(II) acetate in methanol was added. The mixture was stirred for 2 h under argon at room temperature. The organic phase was washed three times with 15 mL of water. The organic solvent was evaporated under reduced pressure to obtain 32 mg (98%) of ZnTCP. ¹H-NMR (CDCl₃, TMS) δ (ppm) 3.39 (s, 4H, C≡CH,), 7.92 (d, 8H, J = 8.00 Hz, 3.5-ArH,), 8.18 (d, 8H, J = 8.00 Hz, 2,6-ArH), 8.84 (s, brs, pyrrole-H, 8H). ESI-MS (m/z) 773.1693 [M + H]⁺ (773.1684 calculated for C₅₂H₂₈N₄Zn + H).

Zn(II) 5,10,15,20-tetrakis(p-ethynylphenyl)porphyrin polymer (ZnTEPP). A sample of ZnTEP (20 mg, 0.026 mmol) was suspended in 1.1 mL of anhydrous tetrahydrofuran (THF). Then, 2 mg of PdCl₂(PPh₃)₂ (0.3 µmol) and 1 mg of CuI (5 µmol) were added to this solution. After these, compounds were dissolved by sonication and 21 µL of TEA was added to start the reaction. The solution was kept without stirring under a nitrogen atmosphere in dark at room temperature for 48 h. Then, the polymer obtained was washed in 10 mL of THF to remove the reaction residues and the ZnTEP monomers that did not polymerize. A portion of organogel (10 mg) was dried under vacuum to form xerogel.

2.4. Spectroscopic Studies

UV-visible absorption and fluorescence emission measurements in N,N-dimethylformamide (DMF) were achieved as previously described [4]. Fluorescence emission spectra were recorded by exciting the samples at λ_exc = 550 nm. The fluorescence quantum yield (Φ_F) of each porphyrin was calculated using Zn(II) 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (ZnTMP) as a reference (Φ_F = 0.049) in DMF [5].

2.5. Determination of O₂(¹Δ_g) Production

9,10-Dimethylanthracene (DMA, 35 µM) and the photosensitizer in DMF (2 mL) were irradiated with light at 455–800 nm. The kinetics of DMA photooxidation were analyzed by the decrease in the absorption band at λ_max = 378 nm. The observed rate constants k_obs^DMA of DMA oxidation and quantum yields of O₂(¹Δ_g) production (Φ_Δ) of the porphyrins were obtained as previously reported [4]. ZnTMP was used as a reference (Φ_Δ = 0.73) [5].

3. Results and Discussion

3.1. Synthesis of Porphyrins and Polymer

The synthetic procedure to obtain porphyrins are shown in Scheme 1. TEP was synthesized from the condensation of 4-(ethynyl)benzaldehyde and pyrrole catalyzed by BF₃^.OEt₂ in DCM. After oxidation with DDQ and purification by flash column chromatography (silica gel), TEP was obtained in 38% yield. This is an appropriate yield for the synthesis of symmetrically substituted porphyrin derivatives [6]. Then, TEP was metaled with Zn(II) acetate in DCM/methanol to produce the complex ZnTEP in 98% yield. This porphyrin bears four ethynylphenyl substituents at the meso position. Homocoupling reaction of terminal alkynes of ZnTEP to diynes was used to synthesize conjugated polymer organogel ZnTEPP (Scheme 2). The reaction was co-catalyzed by PdCl₂(PPh₃)₂ and CuI in THF. The polymer was washed with THF to obtain the organogel ZnTEPP. The solvent contained in this polymer was evaporated to obtain xerogel [7].

3.2. SEM Images

Figure 1 shows the SEM images of the ZnTEPP polymer. First, an aliquot of the organogel polymer was deposited on a glass surface as a film and the solvent was evaporated at room temperature. Images of this material are illustrated in Figure 1A. As can be seen, SEM image shows a structure of superimposed scales that cover the surface. On the other hand, a representative SEM image of a portion xerogel polymer is shown in Figure 1B. The porous structures observed in the xerogel are due to the removal of the solvent THF captured in the interstices of the organogel. Therefore, xerogel can retain the original shape but with a very contracted size.

3.3. Photophysical Characterization

The UV-visible absorption spectra of ZnTEPP and its constitutional porphyrin ZnTEPP in DMF are shown in Figure 2A. These spectra were also compared with that for ZnTMP, which was used as a reference. The main optical characteristics of these compounds are summarized in

Table 1. Spectroscopic and photodynamic properties of ZnTMP, ZnTEP and ZnTEPP in DMF.

PS	λSoret (nm)	εSoret a	λem(nm)	ΦF b	kobsDMA (s−1) c	ΦΔ d
ZnTMP	426	4.07 × 105	608	0.049 ± 0.004	(2.02 ± 0.02) × 10−2	0.73 ± 0.03 e
ZnTEP	428	5.56 × 105	612	0.030 ± 0.003	(1.60 ± 0.02) × 10−2	0.57 ± 0.03
ZnTEPP	446	-	620	0.008 ± 0.002	(0.53 ± 0.01) × 10−4	0.019 ± 0.004

^a Molar absorption coefficient (Lmol⁻¹cm⁻¹), ^b fluorescence quantum yield, ^c observed rate constants for the photooxidation reaction of DMA, ^d quantum yield of O₂(¹Δ_g) production, ^e from Ref. [5]. Values represent the mean ± standard deviation of three separate experiments.

. The spectra of ZnTEP and ZnTMP show the typical Soret band around 420 nm and Q-bands in the 500–600 nm range, characteristic of the corresponding Zn(II) substituted porphyrins [8]. The sharp absorption of Soret bands indicated that porphyrins are dissolved as a monomer in this medium. Furthermore, the polymer ZnTEPP essentially retained the spectroscopic properties of the porphyrin-based chromophore, despite being an extensively aggregated system. The UV-visible absorption observations also confirm the polymerization of ZnTEP. The Soret and Q bands of ZnTEPP exhibit a red-shifted maximum of around 18 nm in comparison with those of ZnTEP in DMF, together with a small broadening of both bands. These results indicate only a slight interaction between the porphyrin units in the polymer structure [4].

Fluorescence emission properties of ZnTEP and ZnTMP were compared in DMF as indicated in Figure 2B. The spectra show two bands around 610 and 670 nm, which are typical for similar meso-substituted Zn(II) porphyrin derivatives [4,8]. These emission bands have been assigned to Q_x(0-0) and Q_x(0-1) transitions. This is characteristic of porphyrins with D_2h symmetry, indicating that the vibronic structure of the tetrapyrrolic macrocycle remains practically unchanged upon excitation [9]. The emission spectrum of ZnTEPP is characterized by a broad emission band centered at 620 nm and a second band at 680 nm. Both bands are red-shifted with respect to the monomer in solution, in agreement with the fact that the Q bands in the film are also red-shifted. In addition, ZnTEPP presented good emission proprieties indicating that the spectroscopic characteristics of the porphyrin based-chromophore were retained in the polymeric matrix. These results also indicate that the porphyrin can be embedded in the polymer without substantial aggregation. These minor spectral changes in absorbance spectrum and the fluoresce properties of the polymer suggest that the π-π stacking between the porphyrin cores is impede and only takes place a weak interaction. The fluorescence quantum yields (Φ_F) of the porphyrins were determined using ZnTMP as a reference (Table 1). The values of Φ_F for ZnTEP and ZnTEPP agree with those previously reported for similar structures [4,8].

3.4. Photodynamic Properties

Photooxidation of DMA induced by ZnTEP and ZnTEPP was determined in DMF. The reaction was followed by the decay of the DMA band at 378 nm due to the formation of the 9,10-endoperoxide product [4,5]. Since DMA quenches O₂(¹Δ_g) by chemical reaction, it was used as an approach to determine the ability of these PSs to produce O₂(¹Δ_g) [10]. The values of k_obs^DMA were calculated from first-order kinetic plots of the DMA absorption vs. time. Additionally, the results were compared with those using ZnTMP as a reference. Table 1 summarized the values of k_obs^DMA calculated from the first order kinetic plots. The value of k_obs^DMA for the reaction sensitized by ZnTEP is lower than that obtained for ZnTMP. In addition, the values of Φ_Δ were determined comparing the kinetic data with that of the reference. The Φ_Δ value obtained for ZnTEP was slight lower than the expected for a Zn(II) porphyrin derivative [8]. ZnTEP is a low polar porphyrin and this tetrapyrrolic macrocycle can be not completely dissolved as a monomer in DMF, reducing the production of O₂(¹Δ_g). Photooxidation induced by ZnTEPP can be considered appropriate, since O₂(¹Δ_g) generation occurs in the polymeric structure.

4. Conclusions

The symmetrically meso-substituted porphyrin TEP was synthesized from the condensation of 4-(ethynyl)benzaldehyde and pyrrole catalyzed by acid in good yield. This porphyrin was metaled with Zn(II) acetate to form the complex ZnTEP. A homocoupling reaction of the terminal alkynes of ZnTEP to diynes was used to synthesize the conjugated polymer organogel ZnTEPP. Moreover, the solvent was removed to obtain xerogel. SEM images of ZnTEPP showed microporous structures. The ZnTEPP polymer retains the spectroscopic characteristics of the porphyrin-based chromophore despite being an extensively polymeric system. In addition, photodynamic studies indicated that the porphyrin unit in ZnTEPP retains its properties as PS, which was able to produce O₂(¹Δ_g). Thus, this polymer is an interesting material with potential applications in forming photoactive aseptic surfaces.