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Porphyrin-Coumarin Dyads: Investigation of Photophysical Properties and DNA Interactions Fan Cheng, Hua-Hua Wang, Jaipal Kandhadi, Fang Zhao, Lei Zhang, Atif Ali, Hui Wang, and Hai-Yang Liu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02292 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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The Journal of Physical Chemistry

Porphyrin-Coumarin Dyads: Investigation of Photophysical Properties and DNA Interactions Fan Cheng,[a] Hua-Hua Wang,[a] Jaipal Kandhadi,[b] Fang Zhao, [b] Lei Zhang,[b] Atif Ali,[a] Hui Wang,*[b] Hai-Yang Liu*[a]. a. Department of Chemistry, Key Laboratory of Functional Molecular Engineering of Guangdong Province, South China University of Technology, Guangzhou, Guangdong 510641, China b. State Key Laboratory of Optoelectronics Materials and Technologies, Sun-Yat Sen University, Guangzhou, Guangdong 510275, China

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Abstract: Two new nonconjugated porphyrin-coumarin dyads with different orientations with respect to donor-acceptor entities and their zinc complexes were synthesized. Single-crystal structures of the freebase porphyrin-coumarin dyads were successfully resolved. The absorption spectra of the dyads were linear combinations of the spectra of their corresponding monomers, indicating a negligible electronic communication between the coumarin and porphyrin moieties. However, the fluorescence emission of the coumarin entity in all the dyads was quenched significantly compared to that of pristine coumarin, and this effect was attributed to intramolecular energy transfer from the coumarin to the porphyrin. The energy transfer kinetics from the coumarin to the porphyrin were shown to be fast (kFörster = 1.13×1013 s-1 for the ortho-isomer and 5.13×1011 s-1 for the para-isomer in DMF) and efficient (transfer efficiency ca. 96–97%). Transient absorption studies showed that the excited state decay process (S2→S1*, S1*→S1, S1→S0 and S1→T1) of the para-isomer was faster than that of the ortho-isomer in DMF. All the synthesized dyads were tested for their interactions with ct-DNA and photocleavage activity toward PBR322-DNA. The results revealed that all the dyads interacted with ct-DNA via only an external groove-binding mode; the binding constants were calculated to be 3.24×105 (3a), 3.05×105 (3b), 3.04×105 (4a), and 4.88×105 (4b), and the photocleavage activity was in the order 4b < 3b < 4a < 3a. Furthermore, only the zinc complexes of the porphyrin-coumarin dyads could be absorbed by tumor cells (A549). These complexes were mainly localized in the cytoplasm, exhibited red fluorescence, and showed low cytotoxicity toward all the tumor cell lines tested. The results showed that these zinc complexes of the porphyrin-coumarin dyads have potential applications in fluorescence imaging.

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1. INTRODUCTION

Porphyrins can be considered to be a versatile class of π-conjugated tetrapyrrolic macrocycles with interesting photophysical and biological properties. These macrocycles have specific applications in light harvesting1, 2 and photodynamic therapy.3, 4 In the last several decades, many different porphyrin derivatives have been prepared via the peripheral functionalization of meso-substituted porphyrins aimed to enhance the biological5 and photophysical properties of these molecules.6 Coumarins are an another group of biologically relevant heterocyclic compounds, which also possess interesting biological properties, such as anticancer,7 antiHIV,8 antimicrobial,9 and anti-inflammatory activities.10 Coumarins also exhibit good photophysical properties and are useful for making laser dyes,11 making chemical sensors,12 light harvesting13 and making fluorescent probes for biological imaging.14 In recent years, research on porphyrin-coumarin compounds has focused on organic lightemitting diodes (OLEDs), fluorescence probes and light harvesting applications, which have attracted much interest.15 A series of conjugated16, 17 or nonconjugated porphyrin-coumarin compounds have been prepared,17, 18, 19 and efficient intramolecular energy transfer (ET) from the coumarin moiety to the porphyrin core has been observed for both types of dyads. It was reported that porphyrin-coumarin dendrimers are suitable for OLED applications,16, 20 while some porphyrin-coumarin dyads are suitable for use as fluorescent probes of thiols in living cells.21 To date, several efforts have been devoted to the development of new porphyrincoumarin functional molecules. Previously, we have reported the synthesis of corrolephenothiazine22,

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and porphyrin-phenothiazine dyads22 and found that the relative

orientation or connection mode between corrole/porphyrin and phenothiazine affects the photophysical activity and bioactivity markedly. As an extension of our work on the porphyrin donor-acceptor system, we report here the synthesis and photophysical properties of two new nonconjugated porphyrin-coumarin dyads and their zinc complexes (Scheme 1).


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The relative locations of porphyrin and coumarin were tuned by the hydroxyl group on the phenyl ring. The DNA interactions, photonuclease activity and in vitro antitumor activity of the synthesized porphyrin-coumarin dyads were also investigated. To the best of our knowledge, this is the first report of the bioactivity of porphyrin-coumarin dyads.

HO NH N

N

BrCH2CH2Br

NH

HN

DMF, K2CO3

N

OH

O

O NH

N HN

DMF, K2CO3

O

N

2a: ortho2b: para-

CH2Cl2

O

N

N Zn N

N

3a: ortho3b: para-

O O

O

Br

1a: ortho1b: para-

Zn(OAc)2

N HN

O O

4a: ortho4b: para-

Scheme 1. Synthesis scheme of the porphyrin-coumarin dyads and their zinc complexes.

2. EXPERIMENTAL

2.1 Materials and Methods. All reagents purchased from commercial sources without further purification unless otherwise indicated. UV-visible absorption spectra were measured using a 3900H (Hitachi, Japan) in a 1-cm optical path length quartz cell at room temperature. Fluoresce emission spectra were measured on a F-4500 (Hitachi, Japan) fluorescence spectrophotometer. The femtosecond transient absorption experiment was performed as previously reported.24 A CHI-660E (ChenHua, China) electrochemical analyzer used to obtain cyclic voltammograms (CVs). Gel electrophoresis was performed on a DYCP-31CN (Liuyi, China) electrophoresis cell and then analyzed using a Gel Doc XR system (Bio-Rad, US). Intracellular location was visualized by an Axio Observer Z1 (Zeiss, Germany) fluorescent microscope. HR-MS spectra were recorded on an Esquire HCT PLUS (Bruker, Germany). 1H NMR spectra were recorded on an AVANCE III HD 400M spectrometer (Bruker, Germany) in CDCl3 or DMSO-d6 solution.


4

O O

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All biological experiments used Ultrapure MilliQ water. A red LED lamp was used as a light source, and the distance between the light source and the surface of the sample was kept constant (10 cm) in all the experiments. The LED irradiance was measured by a TES-1330A luxmeter (TES, Taiwan) at the plate surface; the irradiance dose used for the present work was 3 mw·cm-2, and the light dose reached 22 J·cm-2 after irradiation for 2 h. Cell lines of human hepatocellular carcinoma (Bel-7402), human lung cancer (A549), human cervical cancer (SiHa and HeLa), human gastric cancer (Sgc-7901), and Rattus norvegicus pheochromocytoma (Pc-12) were purchased from the American Type Culture Collection. Cancer cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Gibco, US). 2.2 Fluorescence Quantum Yield. Luminescence quantum yield (ΦF) values were measured in dilute N,N-dimethylformamide (DMF) solutions with an absorbance below 0.1 by using Eq. 1, where A(λ) is the absorbance at the excitation wavelength (λ); n is the refractive index; and I is the integrated luminescence intensity. Subscripts “r” and “s” stand for reference and sample, respectively. ΦFs Ar (λ ) Is n2s = × × ΦFs As (λ ) Ir n2r

(Eq. 1)

5,10,15,20-Tetraphenylporphyrin (TPP) in aerated toluene was used as a standard with ΦF = 0.11.25 2.3 Fluorescence Lifetime. Fluorescence lifetimes of the porphyrin and dyads were measured by time-correlated single-photon counting using a FLSP920 combined fluorescence lifetime and steady-state spectrometer (Edinburgh Instruments, UK) with an EPL 405 pulse diode laser (Edinburgh Instruments, UK) with a spectral FWHM of 405 ± 10 nm and pulse width of 58.8 ps at a 10-MHz repetition rate. Fluorescence decay of the porphyrin and dyads was observed at 650 nm. Fluorescence decay curves were fitted by (Eq. 2): I = I ⁄ (Eq. 2)


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where It is the intensity at time t; I0 is a normalization term (the pre-exponential factor); and τ is the lifetime. Highly diluted colloidal silica (Ludox) in water was used as a scattered solution to acquire the instrument response function (IRF). Data acquisition was conducted until the maximum count in a single channel reached at least 104. All experiments were performed by using quartz cuvettes at room temperature for air-equilibrated solutions. The fluorescence lifetime of coumarin was measured with a laser excitation wavelength of 310 nm, and fluorescence decay was observed at 410 nm. 2.4 Singlet Oxygen Quantum Yield. For the singlet oxygen generation experiment, an aerated solution of 1,3-diphenylisobenzofuran (DPBF) (20 µM) and the photosensitizer (0.5 µM) in DMF (3 mL) was irradiated at 650 nm under an LED lamp at 25 °C for 60 s intervals. The reaction of DPBF with 1O2 was monitored by measuring the decreasing intensity of the absorption band at 418 nm over time. The singlet oxygen quantum yield was calculated by using (Eq. 3).26 Φ ( 1O 2) Por = Φ ( 1O 2) TPP ×

m Por F TPP × m TPP F Por

(Eq. 3)

where the superscripts ‘Por’ and ‘TPP’ denote examines complexes and tetraphenylporphyrin (TPP), respectively; Φ (1O2) is singlet oxygen quantum yield; m is the slope of a plot of the difference in the change in absorbance of DPBF (at 418 nm) against the irradiation time; and F is the absorption correction factor, which is given by F = 1 – 10–OD (OD at the irradiation wavelength). 5,10,15,20-Tetraphenyl porphyrin (TPP) in aerated DMF was used as a standard with Φ∆ = 0.6.27 2.5 Femtosecond Transient Absorption Measurements. The femtosecond timeresolved absorbance difference spectrometer employs a regenerative Ti: sapphire amplifier laser with 500-Hz repetition (Legend Elite USP HE+, Coherent, 35 fs, 800 nm) as the


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primary laser source. The output beam was split into two. One beam, with a power of 6 µJ/pulse was focused onto pure water to generate a white light continuum as a probe beam. The other beam was frequency doubled by a 150-µm BBO crystal to generate a 400-nm pump beam and then passed a translation stage. An optical parametric amplifier (OPerA Solo) was used to generate a 405-nm pump beam when the concentration of the sample was lowered to 10 µM. A mechanical chopper was employed to modulate the pump repetition frequency to 1/2 the probe repetition rate. The pump and probe pulses were focused to diameters of 500 and 200 µm, respectively, at the flow cell interface by two planoconcave mirrors, and the probe pulse was recorded by a fiber spectrometer (Avantes, AvaSpec_ULS2048L-USB2) in external trigger mode. The polarization of the 400-nm pump beam was set to the magic angle (54.7°) with respect to the probe beam. The optical path in the samples was 2 mm. The pump energy was 2 µJ/pulse. The UV-visible absorption spectra of the samples before and after the experiments showed almost no change. The reported uncertainties in the fit parameters are estimated at 90% confidence limits based on the reproducibility observed by fitting a number of independently measured transients. 2.6 Theoretical Calculations. DFT geometry optimizations were carried out using the B3LYP method; the 6-31g(d,p) basis set was used for C, H, O, and N atoms, and the Zn atom used the lanl2DZ basis. Frequency analysis confirmed that the obtained geometries were genuine global minimum structures. The Gaussian G09 package used to perform all calculations.28 2.7 Cyclic Voltammetry. All CVs were obtained in DMF solutions containing 0.1 M tetrabutylammonium perchlorate (TBAP) and complexes (1 mM) under a nitrogen atmosphere at the ambient temperature. The scan rate was 100 mV/s. A three-electrode system consisting of a glassy carbon working electrode, a platinum wire counter electrode and a saturated Ag/AgNO3 electrode as the reference electrode was employed. The


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Ag/AgNO3 electrode contained 1 M TBAP in DMF. Half-wave potentials (E1/2) for reversible or quasireversible redox processes were calculated as E1/2 = (Epa + Epc)/2, where Epa and Epc represent the anodic and cathodic peak potentials, respectively. The E1/2 value for the ferrocene couple under these conditions was 0.47 V. 2.8 DNA Binding Experiments. Biological-grade calf thymus DNA (ct-DNA) was obtained from Sigma-Aldrich (US). All DNA binding experiments involving ct-DNA stock solution were performed in 5 mM Tris-HCl buffer solution (5 mM Tris, 50 mM NaCl, pH = 7.2). A solution of ct-DNA in the Tris-HCl buffer gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9, indicating that the DNA was sufficiently free of protein.29 The concentration of ct-DNA was determined based on the concentration of the nucleotide and was calculated using an extinction coefficient of 6600 M-1·cm-1 at 260 nm. The absorbance titration experiments were carried out with a complex concentration of 5 µM in 5% DMF, varying the ct-DNA concentration from 0 to 6.3 µM. All the samples initially pre-equilibrated with ct-DNA for 5 min before recording each spectrum. Fluorescence quenching experiments were carried out with the successive addition of 0–0.5 µM ct-DNA to the complexes (10 µM). These samples were excited at 430 nm, and their emission was observed between 500 to 800 nm. Viscosity. Viscosity measurements were performed using a stopwatch and an Ubbelohde viscometer maintained at (30.0 ± 0.1) °C in a thermostatic bath. The flow times of the ctDNA solutions (100 µM) with varying concentrations of the complexes (0, 2, 4, 6, 8, 10 µM) were measured five times with a stopwatch after the solutions were incubated for 5 min in the thermostatic bath, and the average flow times were obtained. The data were presented as (η/η0)1/3 versus [complex]/[DNA], where η0 and η are the relative viscosities of DNA in 5 mM Tris-HCl buffer and in the presence and absence of the complex, respectively.30,

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Specific viscosity values were calculated from the observed flow time of the DNA solutions


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(t) corrected for the flow time of the buffer alone (t0). Specific viscosity values were calculated from the observed flow times using (Eq. 4).32 η = (t - t0)/t0 (Eq. 4) The control experiment was carried out on ethidium bromide (EB) using the same method. Viscosity measurements that are sensitive to changes in DNA length are regarded as the least ambiguous and most critical test of binding modes in solution. Classical intercalative binding generally results in a significant increase in DNA viscosity owing to elongation of the DNA helix upon insertion of planar aromatic chromophores between the base pairs. In contrast, nonclassic intercalation or external binding does not decrease or lead to any change in the DNA viscosity under the same conditions. Molecular Docking Analysis. The crystal structure 1BNA d(CGCGAATTCGCG)2 was downloaded from the Protein Data Bank (PDB ID: 1BNA) (http://www.rcsb.org./pdb). AutoDock Tools (ADT) v1.5.6 was used for molecular docking studies. The water molecules were removed from 1BNA before performing docking calculations. Atomic charges for DNA were calculated using the Kollman method, and polar hydrogens were added before computing Gasteiger charges. Grid dimensions (60 Å × 60 Å × 60 Å) were defined to include both the minor and major grooves of DNA. A genetic algorithm available in ADT was employed for docking. By default, the ligand was considered to be flexible, and the DNA was considered to be rigid. 2.9 DNA Cleavage Experiments. Tris(hydroxymethyl)aminomethane (Tris), sodium chloride, boric acid (H3BO3), DMSO, L-histidine, agarose gel-loading buffer, and pBR322 DNA were purchased from Shanghai Sangon Company (China) and were of biological grade. All DNA cleavage experiments were carried out in 50 mM Tris-HCl buffer solution (50 mM Tris, 18 mM NaCl, pH = 7.2). TBE buffer (89 mM Tris, 89 mM H3BO3, 20 mM EDTA, pH = 8.3) was used for electrophoresis.


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Cleavage of supercoiled DNA (0.1 µg) was carried out at 37 °C in 50 mM Tris-HCl buffer in a total volume of 10 µL. Varying concentrations of complexes (dissolve in DMF) and pBR322 DNA (Shanghai Sangon Company) were incubated under red light for 2 h, and then, loading buffer was added (2 µL). The superhelical DNA (pBR322 DNA) was loosely bound after being treated with the compound, and the supercoiled DNA and loose DNA ran at different rates during agarose gel electrophoresis. The rates of electrophoresis were in the following order: supercoiled DNA (form I) > linear DNA (form III) > nicked circular DNA (form II). The results of the cleavage reactions were monitored by 1% agarose gel electrophoresis at 70 V for 2 h. Finally, the gel was photographed on a Gel Doc XR system (Bio-Rad) after being stained with a 1 mg·L−1 EB solution. The cleavage mechanism was studied after the addition of different additives, such as NaN3, L-histidine, and 1,4-diazabicyclo(2.2.2)octane (DABCO) as quenchers of singlet oxygen; DMSO, KI and tert-BuOH as scavengers of hydroxyl radicals; benzoquinone (BQ) to eliminate superoxide radicals (O2·-); and ethylenediaminetetraacetic acid (EDTA) as a metal-chelating agent. 2.10 Cytotoxicity Assays. An MTT assay was carried out using 5 × 103 cells in 100 µL of medium seeded on 96-well plates and grown overnight at 37 °C in a 5% CO2 incubator. Serial dilutions of the porphyrin-coumarin dyads ranging from 0.8–100 µM in DMSO were added to the monolayer. Control wells were prepared by addition of culture medium (100 µL, 5‰ DMSO). The plates were incubated under normal conditions for 48 h. The cultures were assayed after the addition of 20 µL of 5 mg·mL−1 MTT and incubation for 4 h at 37 °C. Finally, the MTT-containing medium was aspirated, and a buffer (100 µL) containing DMSO (50%) and sodium dodecyl sulfate (20%) was added to solubilize the MTT formazan. The optical density of each well was measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC50 values determined by plotting the percent cell viability


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versus concentration on a logarithmic graph and determining the concentration at which 50% of the cells remained viable relative to the control. Each experiment was repeated at least three times to obtain the mean values. 2.11 Intracellular Location. A549 cells were placed in 24-well culture plates (4 ×104 cells per well) and grown overnight at 37 °C in a 5% CO2 incubator. Then, the test compounds were added to the wells. The plates were incubated at 37 °C in a 5% CO2 incubator for 24 h. Upon completion of incubation, the wells were washed three times with PBS. After removing the culture medium, the cells were stained with DAPI and imaged by fluorescence microscopy. 2.12 Synthetic Procedures. The starting compound 5-(2',4'-hydroxyphe-nyl)-10,15,20triphenylporphyrin 1a and 1b with a monohydroxyl group were synthesized according to the procedures reported in the literature.33 The 5-[2',4'-(1-bromoethoxyphenyl)]-10,15,20triphenylporphyrin 2a and 2b were synthesized following a previously reported method.34 Synthetic routes for the porphyrin-coumarin dyads and their zinc complexes are shown in Scheme 1. 2.12.1 Synthesis of Dyads 3a and 3b. The porphyrin-coumarin dyad 3a was synthesized by reacting 2a with excess 7-hydroxycoumarin, using K2CO3 to provide an alkaline environment in dry DMF and stirring at room temperature for 48 h. The progress of the reaction was monitored by thin-layer chromatography (TLC), and a new green spot was observed below the porphyrin 2a on a TLC plate after development with 50% dichloromethane and 50% hexane. After the reaction, the product was washed several times with dichloromethane and saturated salt water, and the organic phase was collected. The crude product was purified by silica gel chromatography with dichloromethane/hexane (3:1) as the eluent, and the porphyrin-coumarin dyad 3a was obtained as a purple solid after


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recrystallization from dichloromethane and hexane at an 89% yield. The dyad 3b was synthesized by using the same method and obtained at a yield of 91%. 3a: HR-MS calcd for C55H39N4O4: 819.2966; found: m/z 819.2972 [M+1]+; 1H NMR (400 MHz, CDCl3) δ 8.88 (dd, J = 9.2, 4.5 Hz, 4H, β-H), 8.77 (dd, J = 14.4, 4.7 Hz, 4H, βH), 8.36 – 8.12 (m, 6H, phenyl-H), 8.02 – 7.95 (m, 1H, phenyl-H), 7.88 – 7.70 (m, 10H, phenyl-H), 7.45 – 7.29 (m, 2H, phenyl-H), 5.58 (d, J = 2.1 Hz, 1H, coumarin-H), 5.07 (d, J = 9.4 Hz, 1H, coumarin-H), 4.97 (d, J = 9.4 Hz, 1H, coumarin-H), 4.70 (dd, J = 8.6, 2.2 Hz, 1H, coumarin-H), 4.20 (s, 2H, methylene-H), 3.98 (d, J = 8.6 Hz, 1H, coumarin-H), 3.55 (s, 2H, methylene-H), -2.99 (s, 2H, inner-H). 3b: HR-MS calcd for C55H39N4O4: 819.2966; found: m/z 819.2971 [M+1]+; 1H NMR (400 MHz, CDCl3) δ 8.86 (d, J = 3.5 Hz, 8H, β-H), 8.23 (d, J = 6.5 Hz, 6H, phenyl-H), 8.13 (d, J = 7.8 Hz, 2H, phenyl-H), 7.76 (d, J = 6.7 Hz, 9H, phenyl-H), 7.60 (d, J = 9.4 Hz, 1H, coumarin-H), 7.36 (d, J = 8.1 Hz, 1H, coumarin-H), 7.28 (d, J = 8.0 Hz, 2H, phenyl-H), 6.95 (d, J = 8.4 Hz, 2H, coumarin-H), 6.27 (d, J = 9.5 Hz, 1H, coumarin-H), 4.53 (dd, J = 21.2, 2.9 Hz, 4H, methylene-H), -2.75 (s, 2H, inner-H). 2.12.2 Synthesis of Zinc Dyads 4a and 4b. The porphyrin-coumarin dyad 3a (30.0 mg, 0.037 mmol) and zinc acetate (67 mg, 0.37 mmol) were dissolved in dichloromethane (DCM) (20 mL) and stirred at room temperature for 30 min. After the end of the reaction, the crude product was filtered, dried, evaporated and purified by silica gel chromatography with DCM/MeOH (100:1) used as the eluent. The zinc porphyrin-coumarin dyad 4a was obtained as a purple-pink solid after recrystallization from CH2Cl2 and hexane at a 94% yield. The complex 4b was synthesized using the same method and obtained at a similar yield of 95%. 4a: HR-MS calcd for C55H37N4O4Zn: 881.2101; found: m/z 881.2108 [M+1]+; 1H NMR (400 MHz, DMSO) δ 8.83 – 8.62 (m, 8H, β-H), 8.32 – 8.01 (m, 6H, phenyl-H), 7.90 (d, J = 7.3 Hz, 1H, phenyl-H), 7.79 (s, 10H, phenyl-H), 7.58 (d, J = 8.4 Hz, 1H, phenyl-H), 7.38 (t, J


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= 7.4 Hz, 1H, phenyl-H), 7.01 (d, J = 9.5 Hz, 1H, coumarin-H), 6.33 – 6.19 (m, 2H, coumarin-H), 5.84 (d, J = 9.5 Hz, 1H, coumarin-H), 5.76 (d, J = 8.5 Hz, 1H, coumarin-H), 4.32 (s, 2H, methylene-H), 3.76 (s, 2H, methylene-H). 4b: HR-MS calcd for C55H37N4O4Zn: 881.2101; found: m/z 881.2102 [M+1]+; 1H NMR (400 MHz, DMSO) δ 8.85 – 8.75 (m, 8H, β-H), 8.23 – 8.13 (m, 6H, phenyl-H), 8.05 (dd, J = 20.3, 8.9 Hz, 3H, phenyl-H), 7.80 (s, 9H, phenyl-H), 7.69 (d, J = 8.6 Hz, 1H, phenyl-H), 7.35 (d, J = 8.0 Hz, 2H coumarin-H), 7.12 (dd, J = 18.9, 16.8 Hz, 2H, coumarin-H), 6.33 (d, J = 9.5 Hz, 1H, coumarin-H), 4.58 (s, 4H, methylene-H). 2.13 X-ray Crystallography. Single crystals of C55H38N4O4 (3a and 3b) were grown by slow evaporation of solutions of the complex in DCM and hexane at 4 °C. A suitable crystal was selected, and data collection was carried out on an Xcalibur Sapphire3 Gemini Ultra diffractometer. The crystal was kept at 150 K during data collection. Using Olex2, the structure was solved with the olex2.solve structure solution program using charge flipping and refined with the XL refinement package using least squares minimization. All nonhydrogen atoms of the complex were refined anisotropically. The hydrogen atoms in this structure were located via the difference Fourier map and constrained to ideal positions in the refinement procedure; the CCDC number of 3a is 1553272, and the CCDC number of 3b is 1540739. The crystal structure refinement parameters for 3a and 3b are given in Table 1.


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Table 1. Crystal Data and Structure Refinement of Complexes 3a and 3b Identification code Empirical formula Formula weight Temperature/K Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° Volume/Å3 Z ρcalc g/cm3 µ/mm-1 F(000) Crystal size/mm3 Radiation 2θ range for data collection/° Index ranges Reflections collected Independent reflections Data/restraints/parameters Goodness-of-fit on F2 Final R indexes [I>=2σ (I)] Final R indexes [all data] Largest diff. peak/hole / e Å-3

3a C55H38N4O4 818.89 150 triclinic P-1 10.9637(3) 11.3625(3) 18.6473(6) 100.429(3) 97.103(3) 106.298(3) 2155.05(12) 2 1.262 0.638 856.0 0.4 × 0.2 × 0.1 Cu Kα (λ = 1.54184) 8.32 to 148.8 -13 ≤ h ≤ 9, -12 ≤ k ≤ 14, -23 ≤ l ≤ 20 15466 8537 [Rint = 0.0296, Rsigma = 0.0363] 8537/0/576 1.054 R1 = 0.0784, wR2 = 0.2130 R1 = 0.0939, wR2 = 0.2247 1.12/-0.67

3b C55H38N4O4 818.89 150 triclinic P-1 10.8525(3) 12.7577(5) 16.0539(5) 75.621(3) 74.784(3) 78.133(3) 2054.01(12) 2 1.324 0.670 856.0 0.4 × 0.2 × 0.2 Cu Kα (λ = 1.54178) 5.82 to 122.16 -12 ≤ h ≤ 9, -14 ≤ k ≤ 14, -18 ≤ l ≤ 17 17854 6241 [Rint = 0.0296, Rsigma = 0.0316] 6241/7/712 1.033 R1 = 0.0459, wR2 = 0.1184 R1 = 0.0515, wR2 = 0.1227 0.35/-0.32

3. RESULTS AND DISCUSSION

3.1 Synthesis. The synthetic procedure of porphyrin-coumarin dyads is shown in Scheme 1. All dyads were well characterized by spectroscopic analysis, including

1

H NMR

spectroscopy, HR-MS (Figure S1-S11) and X-ray crystallography (see Experimental Section). The X-ray single-crystal structure of dyads 3a and 3b are shown in Figure 1, and the relevant crystallographic data for these compounds are summarized in Table 1. Two dyads crystallized in the P-1 space group. The porphyrin and coumarin moieties of dyad 3a are folded, and the center-to-center distance between the donor and acceptor is 4.0 Å. However, the structure of dyad 3b is stretched; a much longer center-to-center distance of 15.7 Å was observed. The fixed bond length and bond angle in the dyad lead to dyad 3b being stretched compared to the others. This finding shows that the connection mode strongly affects the relative orientation between the porphyrin and coumarin moieties.


14

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Figure 1. Molecular structures of complex 3a (top) and 3b (bottom) (hydrogen atoms are omitted for clarity; displacement parameters are drawn at 50% probability level). The 1H NMR spectrum of dyad 3a shows that all the coumarin protons are shifted upfield (Figure 2). This effect is caused by the ring current effect of the porphyrin. The methylene proton (Hg, Hf) also exhibits an upfield shift, indicating that the porphyrin and coumarin moieties are in close proximity. In contrast, the coumarin and methylene protons in dyad 3b were affected very little by the shielding effect from the porphyrin. These observations suggest that the solution conformations of these dyads are similar to those in the crystal structures.


15


Page 16 of 42

Figure 2. 1H NMR spectra (400 Hz) of coumarin and dyads at room temperature.

3.2 Photophysical Properties. The steady-state absorption spectra of the metal-free dyads (3a, 3b) and Zn dyads (4a, 4b) were measured in DMF, and the spectral behavior of these dyads was compared with that of their individual monomeric units 1a and 1b (Figure 3). Spectral data, including maximum absorption wavelength and molar extinction coefficients (ε), are summarized in Table 2. All the dyads exhibited intense Soret bands from 418 nm to 426 nm with a shoulder at 400 nm, with the electronic transition assigned to the second excited state (S2). In addition, the dyads exhibited four Q-bands located at 500–650 nm, with a lower intensity than that of the Soret band, and these electronic transitions were attributed to the first excited state (S1). These characteristic bands are similar to those for compounds with porphyrin groups. The absorption band at approximately 320 nm corresponds to the coumarin moiety. Furthermore, coordination with zinc led to a redshift of the Soret band to 426 nm, and only two Q-bands were observed. In addition, unlike the


16

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conjugated bridge dyads, for which the absorption was red shifted by 10-20 nm due to the enhancement of electron delocalization, we only observed slight changes in our noncovalent bridge cases. The absorption spectra of these dyads are consistent with the linear combination of the spectra of their corresponding monomers, suggesting that there is weak or negligible electronic interaction between the porphyrin and coumarin units in their ground states.35

Figure 3. Normalized UV-vis spectra of 1 µM porphyrin, dyads and coumarin in DMF at 298 K. Table 2. Physicochemical Properties Of Dyads Absorption [nm] (ε [103 M-1 cm-1])

Emission

Stokes Shift

[nm]

[nm]

Complex Soret

Q-band

τ [ns]

ΦF

Φ∆

Cou

324

─

─

─

─

─

393

69

0.19

0.08 36

─

1a

─

417 (503)

514 (24)

547 (9)

590 (5)

649 (5)

654

5

11.44

0.19

─

3a

320 (41)

418 (370)

514 (22)

548 (12)

590 (11)

645 (9)

652

7

11.46

0.12

0.42

4a

318 (42)

426 (715)

558 (27)

600 (12)

─

─

610

10

2.21

0.42

0.31

1b

─

418 (484)

515 (23)

551 (8)

592 (3)

649 (3)

654

5

11.55

0.19

─

3b

320 (33)

418 (422)

513 (18)

549 (10)

591 (7)

648 (6)

654

6

10.89

0.18

0.44

4b

324 (37)

426 (547)

559 (21)

600 (12)

─

─

612

12

1.85

0.44

0.31

ε, molar absorption coefficient; ΦF, fluorescence quantum yield; τ [ns], fluorescence lifetime; ΦF, fluorescence quantum yield; Φ∆, singlet oxygen quantum yield.


17


Page 18 of 42

Figure 4. Normalized fluorescence spectra (λ = 420 nm) of 1 µM dyads in DMF at 298 K. The steady-state fluorescence spectral studies of the dyads along with their monomers were carried out in DMF (Figure 4), and the spectral characteristics, including emission maxima and quantum yields, are summarized in Table 2. The fluorescence of the ortho-isomers (3a, 4a) was significantly quenched compared with that of the monomer, but the para-isomers (3b, 4b) exhibited much lower fluorescence quench. This result suggests that the ortho-dyads possess stronger intramolecular interaction between the porphyrin and coumarin moieties than the para-dyads. Fluorescence quantum yield measurements were performed at an excitation wavelength of 420 nm. The fluorescence quantum yield of the zinc dyad was observed to be higher than that of the free-base dyads, while the fluorescence lifetime of the zinc complex 4a (2.21ns) was significantly shorter than that of the metal-free complex 3a (11.46 ns). The fluorescence lifetimes in different solvents are listed in Table S1. It can be seen the fluorescence lifetime increased with increasing solvent viscosity for all the tested compounds. For the singlet oxygen quantum yield, there was no substantial difference between the ortho- and para-dyads (Table 2). However, free-base dyads exhibit much higher singlet oxygen quantum yields than their zinc complexes. This finding is consistent with the much higher fluorescence of zinc complexes, indicating the low intersystem crossing rate constants after the insertion of the zinc atom into the free-base dyad.


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To further check the effect of the relative orientation between the porphyrin and coumarin moieties to the excited state photophysical properties of the dyads, the femtosecond transient absorption (TA) spectra of the porphyrin-coumarin dyads 3a and 3b were recorded. The spectra were monitored from 415 to 750 nm by excitation at 405 nm. Figure 5 shows the chirp-corrected time-resolved differential TA spectra of dyad 3a in DMF. From delay times of -0.14 ps to 0.8 ps, the spectra exhibit a rapid increase in an intense positive excited state absorption (ESA) band centered at ca. 440 nm with a sharp concave peak at 458 nm. The 458-nm concave peak can be assigned as the impulsive stimulated Raman gain signal from the C-H stretching modes of the solvent [34]. Meanwhile, an intense negative band in the 415−430 nm region (B-band) and four weak concave bands in the 500−700 nm region (Qband) were caused by ground-state bleaching (GB), which can be clearly seen by comparing the spectrum with the ground-state absorption spectrum of dyad 3a shown in the upper panel. The change in the TA spectrum is much slower from delay times of 2 to 1000 ps. These observations indicate that there are several excited species in the system after photoexcitation. Dyad 3b has a similar TA spectrum to dyad 3a (Figure S13). The TA spectra exhibit no distinct broadening peaks, suggesting that there is no photoinduced charge separation species in the system.37 Figure 6 shows the time profile at 440 nm, and the profile exhibits a three-term exponential fit, corresponding to species lifetimes of τ1, τ2 and τ3. Since τ3 is within the nanosecond time scale, this term may be assigned to the sum of S1→ S0 (fluorescence) and the S1→T1 (intersystem crossing, ISC) decay process. On the other hand, the shorter lifetimes τ1 and τ2 may be attributed to the S2→S1* internal conversion (IC) decay and the S1*→S1 vibration relaxation (VR) process. Interestingly, the ortho-dyad 3a exhibited significantly long lifetimes for all the excited state species. This effect may be caused by the π-π interaction between the porphyrin and coumarin in this dyad.


19

Intensity / a.u.

0.04

∆OD

(b)

ESA

0.00 GB

-0.06

GB

-0.14ps 0.1ps 0.14ps 0.4ps 0.8ps

GB

SRG GB

(c)

0.06 ∆OD

Page 20 of 42

(a)

0.08

0.00 0.06

-0.14ps 2ps 70ps 1000ps

0.00 -0.06 450

500

550 600 Wavelength / nm

650

700

Figure 5. Femtosecond transient absorbance of 5 µM dyad 3a in DMF: (a) Ground-state absorption (black solid line) and steady fluorescence (red solid line) in DMF; (b) transient spectrum from -0.14 ps to 0.8 ps; (c) Transient spectrum from 2 ps to 1000 ps.

dyad 3a

0.05

τ 1 = 0.46 ps

440nm

0.05

τ 1 = 0.29 ps

∆OD

τ 2 = 35.0 ps τ 3 = 6253 ps

τ 2 = 27.6 ps

τ 3 = 2698 ps

0.00

-200

dyad 3b

440nm

∆OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

absorbance / a.u.


0.00

0

200

400

600

800 1000 1200 1400

-200

0

200

Time / ps

400

600

800 1000 1200 1400

Time / ps

Figure 6. Time profiles at a wavelength of 440 nm of 5 µM dyads in DMF after being subjected to a 405-nm pump beam. The red lines correspond to the three exponential fitting results.

3.3 Energy Transfer from Coumarin to Porphyrin in the Dyads. Figure 7a shows quenched emission spectra of 3a (λex = 320 nm) compared to those of the individual constituent coumarin between 330-550 nm, and the quenching efficiency Q can be estimated using Eq. 6. The results are listed in Table 3, and the quenching efficiency Q ranges from 96% to 97%, indicating that the intramolecular ET or charge transfer occurred from coumarin to porphyrin. On the other hand, Figure 7b and Figure S12 show that the absorption spectra of


20

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the dyads were similar to their excitation spectra (emission monitored at 650 nm), and there is a large area of overlap between the fluorescence spectrum of 7-hydroxycoumarin and the absorption spectrum of the reference porphyrin (gray areas in Figure 7c and Figure S12), which is further evidenced by the distance of 4-17 Å from the coumarin to porphyrin obtained from the crystal structure and by theoretical calculation (Table S5). Furthermore, the fluorescence lifetime of the dyads is obviously shorter than that of the monomer coumarin, as shown in Figure 7d and Table S2. All of these results suggest the occurrence of intramolecular fluorescence resonance energy transfer (FRET) from the singlet state of the coumarin to the porphyrin. All of above evidence showed that intramolecular FRET occurs between singlet states in bichromophoric D−A systems, often via the Förster mechanism (or dipole-dipole interactions).38 The free-energy changes (∆GEN) accompanying ET by the dipole-dipole mechanism from Por-Coumarin*1 to Por*1-Coumarin were calculated by (Eq. 5)39 and are listed in Table 3; the E0-0 (0-0 spectroscopic transition energy) values were estimated from an overlap of their absorption and emission spectra (Cou: 3.60 ev; 3a: 2.02 ev; 3b: 2.02 ev; 4a: 2.13 ev; 4b: 2.13 ev). An energy level diagram depicting the photochemical events is shown in Figure 8.

(

)

(

∆G EN = − E0 − 0 Cou ∗ + E0 − 0 Por ∗

Q=

) (Eq. 5)

Φ ref − Φ dyad

κ obs =

Φ ref

(Eq. 6)

Q (1 − Q )

τ ref

(Eq. 7)


21


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Figure 7. Superposition of the normalized absorption and fluorescence spectra in DMF at 298 K. (a) Normalized fluorescence spectra of 5 µM coumarin and the dyad in DMF (λex = 320 nm). (b) Overlay of the absorption and excitation spectra of dyad 3a in DMF (λem = 650 nm). (c) The absorption and fluorescence spectra (λex = 320 nm) of the porphyrins and coumarin are in red and blue, respectively. The overlaps are shaded in gray. Note that the folded conformation drawn for the dyads is only to make them fit within the graphs. (d) Fluorescence decay of the dyads (λex = 320 nm, λem = 410 nm) in DMF. The driving forces for the photoinduced ET (∆GEN) from Cou* to Por for all the dyads were found to be exothermic, which was more pronounced for the free-base dyad than the zinc dyad (Table 3). The rates of fluorescence quenching (kobs) were calculated by Eq. 7, where τref is the fluorescence lifetime of the donor. The kFörster values were calculated by Eq. 8, where ϕ and τ are the emission quantum yield and lifetime (see Table 2); Rcc is the donoracceptor center-to-center distance (from Table S5); η is the refractive index of the solvent; and JFörster is the overlap integral, which can be evaluated according to Eq. 9. JFörster values for the dyads were calculated using PhotochemCAD software,40 and the obtained values are tabulated in Table 3. κ2 is the orientation factor, and the value of κ2 was determined by Eq.


22

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10,41 where γ1 is the angle between the donor dipole and R (the vector from the donor to the acceptor); γ2 is the angle between the acceptor dipole and R; and α is the angle between the donor and acceptor dipoles (Table S6).42, 43 In the case of free-base dyads, the calculated JFörster values were found to be 2.21×10-13 cm3 mol-1, and for the zinc dyads, the values were a little smaller than those for the free-base dyads and ranged from 1.72×10-13 to 2.09×10-13 cm3 mol-1. The rate of Förster ET (kFörster) was found to be 1.13×1013 s-1for the free-base dyad 3a and 5.13×1011 s-1 for the free-base dyad 3b; it can be observed that the ET of the ortho-PorCou dyad was faster than that of the para-Por-Cou dyad. k Forster =

8.8 ×10 28 κ 2φ D J Forster n 4τ DR 6

∫ F (v )ε (v )ε ∫ F (v )dv

−4

J Forster =

(Eq. 8)

dv

(Eq. 9)

= ( − 3cosγ cosγ ) (Eq. 10) From a careful examination of Figure 7a, it can be observed that upon excitation of dyad 3a at 320 nm in DMF, the emission corresponding to the coumarin moiety was efficiently quenched, and there was an emission band corresponding to porphyrin moiety at approximately 655 nm and a shoulder band at approximately 720 nm. Under these conditions, the equimolar solution of dyad 3b was excited at 320 nm, and a very weak emission for the porphyrin was observed at approximately 655 nm. This finding suggests that in terms of the intramolecular ET from the coumarin to the porphyrin moiety, dyad 3a is more efficient than dyad 3b, which is due dyad 3a having a shorter D-A distance than dyad 3b. This result shows that the distance between the donor and acceptor is an important factor for intramolecular ET. Similar experiments performed on zinc dyads indicate that metal-free dyad is more efficient than the zinc dyad. In conclusion, the ortho-dyad has a shorter D-A distance and exhibits faster and more efficient intramolecular ET than the para-dyad. However, in this study, while


23


Page 24 of 42

dyads coordinated with the metal zinc exhibited decreased ET efficiency, these dyads exhibited increased ET speed.

Figure 8. Energy level diagram showing energy transfer reactions from the singlet excited state of the coumarin moiety to the porphyrin of Por-Cou. Table 3. Energy Transfer Data for the Dyads kobs Dyad %Q J (cm3 mol-1) (109 s-1) 97 170 2.21×10-13 3a 97 170 2.21×10-13 3b 96 126 2.09×10-13 4a 96 126 1.72×10-13 4b

K2

kFörster (s-1)

η (DMF)

0.2 0.8 0.1 1.7

1.13×1013 5.13×1011 6.63×1013 6.55×1011

1.430 1.430 1.430 1.430

∆GEN (ev) -1.58 -1.58 -1.47 -1.47

3.4 Cyclic Voltammetry. The electrochemical properties of the porphyrin-coumarin dyads 3a and 3b and the zinc complexes 4a and 4b as well as the reference porphyrins 1a and 1b were further examined by cyclic voltammetry. CVs are shown in Figure 9, and the redox data vs. the ferrocene/ferrocenium couple (Fc/Fc+) data are compiled in Table S3. For the porphyrin, three reduction peaks (−0.498, −0.988 and −1.591 V) and an oxidation (1.022 V) were observed. The reduction peak at −0.498 V probably occurred due to rearrangement in the adsorbed layer,44 and the reduction peaks at −0.988 and −1.591 V can be assigned to the reduction of the porphyrin ring; the oxidation wave indicates oxidation of the porphyrin ring.45 Compared with the porphyrin, dyad 3a showed two similar reduction peaks (−1.0975 and −1.711 V) and an oxidation peak (1.085 V), which were attributed to the porphyrin-based


24

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reductions and oxidation, respectively. Additional reduction peaks (−1.887 V) and an additional oxidation peak (1.419 V) were attributed to coumarin. While the zinc dyad 4a still exhibited rearrangement in the adsorbed layer, showing a reduction peak (-0.697 V), only one reversible reduction peak (-1.497 V) could be assigned to reduction of the porphyrin ring, the anticipated second porphyrin- and coumarin-based reductions could probably not be observed due to their occurrence beyond the solvent window. Two reversible oxidation peaks (0.809 and 1.110 V) corresponding to complex 4a were observed, and these peaks were attributed to the first and second porphyrin-based oxidations, respectively. There was a slight difference between the voltages of the waves in the case of the ortho- and para-substituted dyads. The electrochemical properties discussed above clearly suggest the existence of negligible π–π interactions between the porphyrin and coumarin. Therefore, the intramolecular interactions of ortho-isomer contribute to the π-π stacking of the coumarin and porphyrin components in dyad 3a.

Figure 9. Cyclic voltammograms of the complexes in DMF (V vs. Ag/AgNO3; Pt electrode as a working electrode; TBAP, 0.1 M; DMF).

3.5 DFT Calculation. DFT was used to optimize the geometry of the dyads (Figure S14), and the calculations matched well with the experimental data; the selected bond lengths and angles are summarized in Table S4. The calculated C-N bond lengths of 3b are consistent with the X-ray data, for example, the calculated bond length for DN1-C19 is 1.37662 Å, and the


25


Page 26 of 42

experimental value is 1.37620 Å. The four C-N-C angles of the crystal were found to be 110.67, 105.49, 110.70, and 105.47°, which are well matched with the calculated data (110.69, 105.45, 110.70, and 105.47°). In general, the optimized geometry of the para-isomer 3b was better matched with the crystal structure than that of 3a. A comparison of the calculated distances and angles with the structural data is presented in Table S5. The calculated Dc-c (15.7 Å) of the para-isomer 3b matched well with the structural data (15.5 Å), but the calculated Dc-c (7.3 Å) of the ortho-isomer 3a was much longer than the value obtained from the X-ray structural data (4.0 Å). Such a difference is due to the conventional DFT-B3LYP calculation did not include dispersion correction,46 which is very important for ortho-isomer 3a. After performing dispersion correction in DFT-B3LYP calculation, the obtained theoretical Dc-c of 3a is 3.6 Å (Figure S15), matching well with the X-Ray structural data (4.0 Å). The center-to-center distance between the porphyrin and coumarin moieties increased in the following order: 3a (4.0 Å) < 4a (4.8 Å) < 3b (15.7 Å) < 4b (16.1 Å). Figure 10 shows the TD-DFT-calculated spectrum of dyad 3a together with the individual transitions. The calculated Q-band is formed by two transitions, with absorption maxima at 608 and 653 nm, which correspond to the HOMO-1→ LUMO+1 (18%), HOMO → LUMO (81%) and HOMO-1 → LUMO (32%), HOMO → LUMO+1 (68%) transfers. The intense Soret band is also a result of two transitions, with absorption maxima at 439 and 443 nm, which represent the HOMO-1 → LUMO+1 (72%), HOMO → LUMO (16%) and HOMO-1 → LUMO (61%), HOMO → LUMO+1 (29%) transfers. These transitions occur on the porphyrin part of the molecule, without the participation of the coumarin part. Another less intense band at 300-400 nm is composed of many transitions, with absorption maxima at 373 nm, which represents the HOMO-5 → LUMO (64%), HOMO-3 → LUMO (21%) transfer. Among these transitions, the absorption at 340 nm corresponds to the HOMO-1 → LUMO+2 (100%) transfer. This finding suggests that while a charge transfer (CT) characteristic from


26

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the porphyrin to the coumarin part of the molecule can be observed, the signal is weak. Furthermore, the same results were obtained for the other dyad in this study.

Figure 10. (a) Computed TD-DFT spectrum of dyad 3a with the inclusion of DMF as a solvent. (b) Frontier molecular orbitals of dyad 3a optimized at the B3LYP/6-31G(d,p) level.

3.6 DNA Binding Experiments. 3.6.1 Absorption Spectroscopic Studies. Electronic absorption spectroscopy is an efficient method to determine the binding between complexes and DNA. As shown in Figure 11, the absorption spectra of complex 3a exhibited 26% hypochromism and a 6-nm redshift from 432 nm to 438 nm. Similar binding behavior was observed for complex 3b (Figure S16), which exhibited 29% hypochromism and a 5-nm redshift. The zinc complexes 4a and 4b were exhibited 24.4% and 19.6% hypochromicity in the absorption spectra, both exhibiting 3nm bathochromic shifts of their maximum absorption peaks (Figure S16). All the complexes exhibited small red shifts and low levels of hypochromism, indicating a likely external binding mode between the porphyrins and ct-DNA.47 To quantitatively determine the binding strength of the DNA, the binding constants between the complexes and ct-DNA were calculated by (Eq. 11), and the values obtained were 3.24×105 (3a), 3.05×105 (3b), 3.04×105 (4a), and 4.88×105 (4b). These observations indicate that the porphyrin-coumarin dyads interacted strongly with the bases of the DNA.48  DNA  DNA 1 =  + εb − εf Kb ( ε b − ε f

εa − εf

)

(Eq. 11)


27


Page 28 of 42

where εa is the apparent extinction coefficient of complex in the presence of DNA; εf and εb are the extinction coefficients of the complex when free and fully bound to DNA, respectively; [DNA] is the concentration of ct-DNA in the base pairs; From the fit plots of [DNA]/(εa - εf) versus [DNA], the binding constant Kb was obtained from the ratio of the slope to the intercept.

Figure 11. Absorption spectral changes of dyad 3a (5 µM) upon the addition of ct-DNA (1: 0; 2: 1×10−7; 3:3.0×10−7; 4: 7.0×10−7; 5: 15.0×10−7; 6: 3.1×10−6.). The inset shows the plot of [DNA]/(ɛa - ɛf) versus [DNA].

3.6.2 Fluorescence Spectroscopic Studies. As reported for other porphyrins, if the compound is inserted into the DNA base pair, the luminescence is enhanced. This phenomenon is ascribed to shielding of the compound by the ct-DNA and quenching of water molecules. In contrast, the collisions between molecules can reduce the emission intensity; the fluorescence intensity decreased significantly, and this effect was caused by the selfaccumulation of the porphyrin molecules on the DNA surface, which may indicate an external binding mode. As shown in Figure 12, the fluorescence intensity of the porphyrin-


28

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coumarin dyads decreased gradually upon addition of ct-DNA in buffer, which is likely due to an external binding mode between the dyads and ct-DNA, which is consistent with the absorption measurements.

Figure 12. Changes in the emission spectra of the complexes upon the addition of ct-DNA. (a) Complex 3a, (b) complex 3b, (c) complex 4a, (d) complex 4b. The arrow shows the change in intensity with increasing complex concentration.

3.6.3 Viscosity Studies. Viscosity experiments are among the most critical methods used to determine the DNA-binding mode in the absence of crystallographic and NMR data.49 An increase in viscosity is ascribed to an increase in the length of the DNA helix due to intercalation. A decrease in viscosity occurs due to a decrease in the effective length of the DNA, perhaps due to external binding. For example, EB is a normal DNA intercalator, and compared with the effect of the addition of EB, the viscosity of the ct-DNA does not change significantly with the addition of increasing amounts of 3a (Figure 13), suggesting that 3a may bind to DNA via an external binding mode, which is consistent with the result of the spectroscopic analysis.


29


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Figure 13. Effect of increasing amounts of EB, 3a, 3b, 4a and 4b on the relative viscosities of the ct-DNA.

3.6.4 Molecular Docking Analysis. To further confirm this result, we also performed a molecular docking analysis. Molecular docking is an attractive tool that predicts the binding modes of small molecules at target active sites. When two DNA strands are coiled together, they form two different grooves in the B-DNA structure, which run parallel to the backbone, namely, the major groove and the minor groove, As shown in Figure 14, the docked complex exhibited preferential binding at the groove site over the intercalation site. In addition, the complex exhibited binding in the major groove of the DNA via π-π interactions. From the results of the crystal structure analysis and theoretical calculations, the high steric hindrance effect of the compound may explain this result.


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Figure 14 Molecular docking of dyads with DNA 3.6.5 DNA Cleavage Experiments. The complex photocleavage activities were examined by using plasmid pBR322 DNA. A mixture of the porphyrin-coumarin dyad and the plasmid DNA (0.1 µg) in 10 µl of buffer (5% DMF) was irradiated under an LED for 2 h. Agarose gel electrophoresis patterns for the photocleavage of the DNA are shown in Figure 15. Lanes 1-2 are the control DNA with or without illumination, and lane 3 is the DNA treated with the porphyrin-coumarin dyads without illumination; almost no DNA cleavage was observed in lanes 1-3. The amount of nicked circular DNA (form II) increased upon addition of the porphyrin-coumarin dyad (lane 4-8); the supercoiled DNA completely degraded to form II upon addition of each of the porphyrin-coumarin dyads at different concentrations (lane 8, 3a:130 µM; 3b:155 µM; 4a:140 µM; 4b:160 µM; additional details in Table S7). All the dyads exhibited efficient DNA cleavage activity under red light; the DNA photocleavage activity of the dyads exhibited the following order: 4b < 3b < 4a < 3a. The photocleavage activity of the ortho-porphyrin-coumarin dyad was better than that of the para-porphyrincoumarin dyad. From the above results, the photocleavage activity was inconsistent with the singlet oxygen quantum yield. To investigate the cleavage mechanism, further experiments were carried out using various additives (Figure 16). The addition of NaN3 or DABCO, a scavenger of singlet oxygen (1O2), has a minor effect on DNA cleavage. The DNA photocleavage restrictions were caused by DMSO (dimethyl sulfoxide), and tert-BuOH, a scavenger of hydroxyl radicals (•OH), had no effect on the DNA cleavage. BQ was added to eliminate the superoxide radical (O2·─), which effectively inhibited the photocleavage activity. These results strongly suggest that the cleavage mechanism of the porphyrin-coumarin dyad may involve superoxide radicals and singlet oxygen. Furthermore, EDTA was added as a chelating agent to shield the effect of zinc, resulting in inhibition of the photocleavage activity. Lane 0


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showed a small amount of DNA cleavage after addition of Zn(OAc)2, which is consistent with the result shown in lane 9, suggesting that the cleavage mechanism of the zinc dyad also involves the metal ion.

Figure 15. Photocleavage of pBR322 DNA by 3a (a), 3b (b), 4a (c) and 4b (d) under irradiation at 650 nm. Lane 1: DNA alone (no hv), lane 2: DNA alone (hv 2 h), lane 3: complex at 80 µM (no hv), lanes 4-8: complexes at different concentrations (hv 2 h).

Figure 16. Mechanisms of photocleavage of pBR322 DNA by porphyrin-coumarin dyads. Lane 0, DNA + Zn(OAc)2; Lane 1, DNA control; Lane 2, DNA + dyads; Lane 3, DNA + dyads + NaN3; Lane 4, DNA + dyads + L-histidine; Lane 5, DNA + dyads + DABCO; Lane 6, DNA + dyads + KI; Lane 7, DNA + dyads + DMSO; Lane 8, DNA + dyads + tert-BuOH; Lane 9, DNA + dyads + BQ; Lane 10, DNA + dyads + EDTA; All lanes were irradiated with LED light for 2 h.

3.7 In vitro Cytotoxicity and Cellular Uptake Studies. The in vitro cytotoxicity was tested using the MTT assay. Six cancer cell lines (Bel-7402, A549, SiHa, Hela, Sgc-7901, and PC-12) were examined in this study. The IC50 values of all the synthesized porphyrincoumarin dyads were much greater than 100 µM for all the tested cancer cell lines, under


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both dark and illuminated conditions. The cell viability data from the treatment of A549 cancer cells with the porphyrin-coumarin dyads are shown in Figure 17a; nearly 80% of the cancer cells survived in the dark or upon exposure to light after photodynamic therapy (PDT). This finding suggests that the dyads possess low cytotoxic activity. To check whether the porphyrin-coumarin dyads could be absorbed by cancer cells, the A549 cell line was selected for an uptake test, and the results are shown in Figure 17b. The blue fluorescence from the DAPI-stained nuclei, and the red luminescence is from the absorbed porphyrin-coumarin dyads. No red fluorescence could be observed when the free-base porphyrin dyads 3a and 3b were incubated with the tumor cells; however, the corresponding zinc complexes exhibited significant red fluorescence. This result suggests that only zinc complexes of the porphyrin dyads 4a and 4b could be absorbed by the tumor cells. The overlay of the fluorescence images shows that these zinc-porphyrin complexes are mainly localized in the cytoplasm. The low photocytotoxicity of the zinc complexes of the porphyrin-coumarin dyads is probably due to the low singlet oxygen quantum yield of these dyads (Table 2).

Figure 17. (a) Cell viability plots as obtained from the MTT assay in A549 cells treated with the dyads and kept in the dark or exposed to light (light dose = 22 J·cm-2). (b) Cellular uptake of A549 cells exposed to 50 µM dyads 3a, 3b, 4a and 4b for 24 h. (blue: nucleus, red: dyad, 200× magnification)


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CONCLUSIONS

We synthesized ether-bond-linked porphyrin-coumarin dyads and their zinc complexes. Spectroscopic analysis and DFT calculations showed that intermolecular ET occurred from the coumarin to porphyrin subunit very efficiently due to the high spectral overlap between the emission spectrum of the coumarin subunit and the absorption spectrum of the porphyrin moiety. Porphyrin analogs are candidates for PDT, so the porphyrin-coumarin dyads were assayed for DNA fragmentation properties. We found that the substitution position on the coumarin in the porphyrin-coumarin dyads influenced DNA cleavage. The ortho-Por-Cou dyad was more efficient than the para-Por-Cou dyad, and the free-base dyads were more efficient than the zinc complexes. Furthermore, all the dyads bound to DNA via an external binding mode. Unfortunately, the hybrid molecules did not exhibit the expected photocytotoxicity. In vitro experiments showed that the porphyrin-coumarin dyads had low PDT activity in all the tumor cells tested, probably because of the low quantum yield of the singlet oxygen and the high molecular weight. The results obtained here are promising for the design of new porphyrin-coumarin dyads for light harvesting and biological imaging applications.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 1

H NMR, HR-MS graph, detailed of spectral analysis, CV graph, DFT calculation and

photocleavage DNA data (PDF) AUTHOR INFORMATION


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Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Fan Cheng: 0000-0003-0794-9211 Jaipal Kandhadi: 0000-0002-6621-8016 Hai-Yang Liu: 0000-0002-1793-952X Notes The authors declare no competing financial interest. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 21671068, 81400023), National Basic Research Program (973 Program) of China under Grant 2013CB922403 and the Open Fund of State Key Laboratory of Optoelectronic Materials and Technologies (Sun Yat-sen University) (No. OEMT-2015-KF-05).

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