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Phenazine Embedded Copper (II) Complex as a Fluorescent Probe for the Detection of NO and HNO with Bioimaging Application Abu Saleh Musha Islam, Mihir Sasmal, Debjani Maiti, Ananya Dutta, Sholanki Ganguly, Atul Katarkar, Sumana Gangopadhyay, and Mahammad Ali ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00010 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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ACS Applied Bio Materials

Phenazine Embedded Copper (II) Complex as a Fluorescent Probe for the Detection of NO and HNO with Bioimaging Application Abu Saleh Musha Islam †, Mihir Sasmal †, Debjani Maiti †, Ananya Dutta †, Sholanki Ganguly†, Atul Katarkar ‡, Sumana Gangopadhyay ⊥, and Mahammad Ali *†,§ † Department

of Chemistry, Jadavpur University, Kolkata 700 032, India; E-mail: [email protected], [email protected] ‡ Department

of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges, Switzerland

⊥ Department

of Chemistry, Gurudas College, Narkeldanga, Kolkata 700 054

§ Vice-Chancellor,

[email protected]

Aliah University, ll-A/27, Action Area II, Newtown, Action Area II, Kolkata, West Bengal 700160;

KEYWORDS • Sensing of NO and HNO • Exogenous and Endogenous intracellular detection of NO and HNO •DFT calculation

ABSTRACT: We report a novel phenazine embedded fluorescent probe (PIP) which on complexation with Cu(II) ion forming [(PIP)CuII(Cl)] becomes non-fluorescent, but regenerates fluorescence on selective reaction with NO and HNO over different biologically reactive oxygen and nitrogen (ROS/RNS) species under physiological conditions. The fluorescence intensity of PIP gets quenched due to the formation of [(PIP)CuII(Cl)] complex, which regenerates the fluorescence by 67 and 84 % on reaction either with NO or HNO respectively in the presence of other biological reducing species. A details photophysical properties of PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] have been studied by density functional theory (DFT) calculations. The recognition efficacy of [(PIP)CuII(Cl)] for exogenous and endogenous NO and HNO in A549 and RAW 264.7 cells with flow cytometry application have also been demonstrated successfully.

INTRODUCTION Reactive oxygen (ROS) and nitrogen (RNS) species have shown many biological significances.1 Nitric oxide (NO) is one of such species which is produced in the biological system by the nitric oxide synthases (NOS)2 through the oxidation of L-arginine to L-citruline. NO behaves as a biological signaling agent and plays crucial roles in vasodilation, neurotransmission and the immune response and also in many physiological and pathological processes.3,4 In addition, NO has also been associated with several diseases like amyotrophic lateral sclerosis, Parkinson, ischemic brain injury (stroke), Alzheimer, and Huntington diseases. 5 On the other hand, Nitroxyl (HNO) is one-electron reduced and protonated form of NO generated by oxidative degradation of L-arginine via NOS 6,7 and also by the reduction of NO in the cytochrome c, xanthine oxidase, mitochondria etc.8 In the physiological conditions, HNO shows different chemical and biological properties. It reacts with thiols in aldehyde dehydrogenase leading to the inhibition of enzyme’s activity.8 Additionally, HNO is used clinically as a deterrent for alcohol use, inhibiting platelet aggregation, increasing heart muscle contractility and conferring vasoprotective effects.9 HNO acts as a both electrophilic and nucleophilic agent. When HNO acts as an

electrophile, it exhibits myocardial antihypertrophic, superoxide-suppressing actions and cGMP-elevating effects.10 However, when it behaves as a nucleophile; it coordinates and reduces metal ions. According to these findings, it is obvious that NO, as well as HNO, play critical roles in biology. Therefore, for the detection of NO and HNO in living biological systems is a challenging work and has attracted greater attention of the chemists. Some reactive nitrogen species like ONOO also attract attention11,12 because it is closely related to bioactive molecules like NO and HNO. There are too many methods to be applied till to date for the recognition of NO and HNO involve EPR (electron paramagnetic resonance), colorimetry, electrochemical and HPLC (high performance liquid chromatography) methods 13-15 etc. However, these analytical technics are time-consuming precluding rapid in situ detection of NO and HNO in living systems. By contrast, fluorescent technics is modern and flourishing technique for small molecule sensors due to its simplicity, low cost, excellent selectivity, high sensitivity, real-time analysis and great potential for bio imaging.16-18 Most of the previously reported fluorescent probes for the detection of NO were

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based on: (i) organic probes and (ii) metal complex probes. There are a number of important organic fluorescent probes for NO sensing that have been developed by the different research groups, which are mainly based on (i) formation of triazole from ortho aromatic diamines 19-23, (ii) oxidative deamination 24,25, (iii) diazo ring formation 2629, (iv) N-nitrosation reaction 30-32 and (v) 1,3,4-oxadiazole formation from thiosemicarbazide.33 However, for the metal complex based probes Lippard group constructed several metal-complexes of the probes for the detection of NO. The metal ions involved in these processes include copper(II),34-36 cobalt(III),37,38 ruthenium(II)39, rhodium(III),40 and iron(III).38 The general strategy for NO sensing by metal complexes involve the coordination of a metal ion to the fluorophore resulting quenching of fluorescence intensity in the initial step, which is further be restored by the reduction of metal centre by NO; sometimes with the release of the fluorophore (Scheme 1).41-47

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fluorescence quenching. However, on further treatment with NO and HNO, [(PIP)CuII(Cl)] is reduced to [(PIP)CuI] leading to a remarkable turn-on response. For NO and HNO sensing we have functionalized a phenazine fluorophore due to its planarity may exhibit potent anti-tumor, antiparasitic and anti-microbial activities. Phenazines exhibits absorption and emission bands in the region of 400-700 nm. Therefore, it gives an added advantage of tissue penetration depth. The present probe seems to be promising because it is highly selective and sensitive towards NO and HNO compared to other copper-based NO/HNO sensors. Scheme 2. Strategies for the HNO detection (a) Cu (II) to Cu (I) reduction reaction of HNO probe, and (b) Staudinger ligation based HNO probes. (FL/R= fluorophore).

Scheme 1. (a) Fluorophore displacement by NO, (b) CuII reduction by NO and (c) Ligand nitrosation via CuII reduction by NO. (FL= fluorophore, M= Metal, L= Ligand)

EXPERIMENTAL SECTION

In addition, the detection of HNO involves two distinct methods: (i) reduction of paramagnetic Cu(II) to a diamagnetic Cu(I) with a simultaneous turn-on response and (ii) Staudinger ligation. The unpaired electron in a d9 orbital of Cu(II) can quench the fluorescence of a fluorophore by photoinduced electron transfer (PET) process in the Cu–fluorophore complex. The further addition of HNO induces the transformation of Cu(II) to Cu(I) thereby blocking the PET process which causes restoration of fluorescence 48-52 (Scheme 2). On the other hand, in the Staudinger ligation process a 2 (diphenylphosphino)benzoate can react with HNO to promote aromatic hydroxylation of the fluorophore leading to turn on fluorescent response 53-61 by intramolecular charge transfer (ICT) mechanism (Scheme 2). Inspired by Lippard, Yoon and James works,50,51 we have developed a fluorophore (PIP) (Scheme 3) which on complexation with Cu(II) forms [(PIP)CuII(Cl)] resulting

Materials. The materials like salicylaldehyde, K2CO3, 2-Picolyl chloride, KI and 2,3-diamino-phenazine are of reagent grade and purchased from Sigma Aldrich. Unless otherwise stated, nitrate/chloride salts of all cations, sodium salts of anions and different reactive species (vide infra) were purchased from Sigma Aldrich. Solvents like ethanol, dimethyl formamide, methanol, acetonitrile etc. (Merck, India) were dried before use by adopting standard methods. Physical Measurements. To record the Fourier transform infrared (FT-IR) spectra in the range 400–4000 cm-1 on KBr pellets an IR 750 series-II FT-IR (Nicolet Magna) spectrophotometer was used. The electronic and fluorescence spectra were recorded in the same way as discussed previously.33 The electrospray ionization mass spectra ESI-MS+ (m/z) of probe (PIP), [(PIP)CuII(Cl)] and [(PIP)CuI] complexes were generated on mass spectrometer (Model: QTOF Micro YA263). To run 1HNMR spectra Bruker 300 and 500 MHz spectrophotometers were used in dimethyl sulfoxide (DMSO)-d6 using tetramethylsilane (δ = 0) as an internal standard. 13C NMR spectra were recorded on a 100 MHz spectrophotometer. The X-band electron paramagnetic resonance (EPR) spectra were generated from an EPR


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Spectrometer (Model: JEOL, JES-FA 200). The reaction of [(PIP)CuII(Cl)] complex towards NO and HNO at different pH were recorded in the pH range 2–11, which was measured on a precalibrated digital pH meter (Model: Systronics 335, India) using buffers of pH 4, 7 and 10. Synthesis: Preparation of 2-(Pyridin-2-ylmethoxy) benzaldehyde (1): The compound 1 was prepared by a slight modification of a literature procedure.62 Salicylaldehyde (10 mmol, 1.23 g) and K2CO3 (18 mmol, 2.52 g) were suspended in dry MeCN (60 mL) and was heated to reflux for 40 min. 2-Picolyl chloride (11.5 mmol, 1.89 g) and a catalytic amount of KI (0.15 g) was added to the above reaction mixture, which was further refluxed for 12 h which was cooled, filtered and volume was reduced to 15 mL. It was then diluted with water (40 mL) and pH was adjusted to 4 by 1 M HCl and extracted with dichloromethane (DCM; 2 × 40 mL). The pH was further adjusted to 8 by 4.0 M Na2CO3 solution and extracted with DCM (3 × 40 mL). The combined organic phase was dried over anhydrous Na2SO4, and then vacuum evaporated to get a pale-yellow solid. The crude solid product was recrystallized in MeOH/DCM (8:2, v/v) to give the desired NMR grade pure product as an off-white crystalline solid (66 % yield). 1H NMR (in CDCl3, 300 MHz) (δ, ppm): 10.63 (s, -CHO, 1H), 8.64 (d, 1H), 7.87 (m, 1H), 7.58 (m, 2H), 7.30 (t, 1H), 7.10 (t, 2H), 5.34 (s, 2H) (Figure S1). Synthesis of 2-[2-(Pyridin-2-ylmethoxy)-phenyl]-1Himidazo[4,5-b]phenazine (PIP): Both 2,3-diaminophenazine (0.21 g, 1 mmol) and 2-(Pyridin-2-ylmethoxy)benzaldehyde (0.213 g, 1 mmol) were dissolved in DMF (8 mL). The resulting solution was stirred under reflux for 16 h which on slow cooling down to room temperature precipitation occurs which was filtered, washed with hot ethanol three times and then recrystallized from DMF–H2O to get a brown powdery product (0.29 g, 78% yield). 1H NMR (in CDCl3, 500 MHz) (δ, ppm): 13.83 (s, -NH, 1 H) 9.14 (s, 1 H), 8.75 (d, 2H), 8.32 (m, 2H), 7.80 (s, 2H), 7.54 (m, 7H), 5.48 (s, 1H) (Figure S2). 13C NMR (in CDCl3,100 MHz) (δ, ppm): 158.29, 157.12, 155.19, 149.56, 148.38, 142.59, 141.08, 140.91, 140.74, 137.49, 133.09, 131.80, 129.72, 129.50, 129.22, 129.11, 123.67, 122.66, 121.16, 118.74, 115.69, 113.52, 105.92, 70.96 (Figure S3). ESIMS+ (m/z): 404.0792 (PIP+H+) (Figure S4). Preparation of Complex [(PIP)CuII(Cl)]: Copper(II) chloride dihydrate, CuCl2·2H2O (65 mg, 0.5 mmol) and PIP (20 mg, 0.5 mmol) were dissolved in 10 ml of freshly distilled MeCN. The probe (PIP) solution was added to the CuCl2 solution and left stirring at room temperature for 2h. A precipitate formed which was filtered under suction and washed with acetonitrile solution to yield pure brown solid. ESI-MS+ (m/z): 507.320 ([(PIP)CuII(Cl)] + Li+) or (PIP—Cu2+) (Figure S5). Preparation of Sample Solutions: For UV-Vis and fluorescence titrations the stock solution of the probe

PIP, and NO were prepared by the reported method.33 As well as the solutions of ONOO–, •OH, HNO and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were prepared by the reported method.33 All the experiments were carried out in (5:5 v/v) CH3CN/HEPES buffer (10 mM) at pH 7.4. For fluorescence titration the probe concentration was taken 10 M and a variable concentration (0-250 µM) of Cu2+ solution was added incrementally in a regular interval of concentration. Then the fluorescence data were recorded for each solution. Similarly, titration of 10 µM [(PIP)CuII(Cl)] complex was carried out by the gradual addition of NO from 0 to 80 µM and HNO from 0 to 70 µM keeping [(PIP)CuII(Cl)] at 10 µM. Density Functional Theory (DFT) Calculations: Geometries of PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] were optimized at B3LYP/6-31G(d) level using a Gaussian 09 W software package.63 The ground state optimized geometries of PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] showed no imaginary frequency. Time dependent density functional theory (TDDFT)64,65 with the B3LYP density functional associated with the conductor-like polarizable continuum model (CPCM)66-68 was applied for the study of the low-lying excited states of the PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] in CH3CN using the optimized geometry of the ground state. The vertical excitation energies of the lowest 40 singlet states are also computed. Cell Cytotoxicity assay: A549 cell and murine macrophages cells Raw 264.7 were grown in Dulbecco's modified Eagle's (DMEM) medium provided with 10% FBS and 1% antibiotic at 37 °C with 5% CO2.69 Cell viability of the probe [(PIP)CuII(Cl)] in A549 and Raw 264.7 cells were analyzed with gradual increase in concentration of PIP—Cu2+ ([(PIP)CuII(Cl)]) complex, in the range 10-100 µM/ml for 24 h by MTT assay.33 Cell incubation, imaging and flow cytometry analysis: Cell incubation and imaging were performed by the previously reported method.33,69 NO monitoring at exogenous and endogenous were performed with A549 and Raw 264.7 cells respectively. Cells were seeded on glass coverslip. A549 cells were incubated with probe PIP (5 µM) and Cu2+ (5 µM) for 30 min, subsequently treated with DEA-NONOate (NO donor, 5 µM and 10 µM) for 30 min. The coverslip washing were performed with 1x PBS, 3 times and mount on slide. Likely, A549 cells were incubated with probe PIP (5 µM) and Cu2+ (5 µM) for 30 min, consequently treated with Angeli’s salt (HNO donor, 5 µM and 10 µM) for 30 min. The coverslip washing were performed with 1x PBS, 3 times and mount on the slide. The Raw 264.7 cells incubated with probe PIP (5 µM) and Cu2+ (5 µM) for 30 min, subsequently co-stimulated with or without LPS (1.0 mg/mL) and IFN-γ (1000 U/mL) for 6 h, also with NO scavenger PTIO (2-Phenyl-4,4,5,5tetramethylimidazoline-1-oxyl 3-oxide) was used to ensure accuracy of the experiment in a similar setting. Live cell images were collected by using fluorescence microscope (Carl Zeiss, Germany). 1×106 Raw 264.7 cells seeded in T25 flask (BD Falcon) at 37 °C. Cells were further incubated with probe PIP (5 µM) and Cu2+ (5 µM)



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for 30 min, subsequently co-stimulated with or without LPS and IFN-γ for 6 h and also with NO scavenger PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). Treated and untreated control cells were washed with ice cold 1X PBS and resuspended in 500 μl of binding buffer and flow cytometric analysis were carried out with FACS LSR (Becton Dickinson). Flow cytromety analysis was done by using Flowing Software version 2.5.1.

RESULTS AND DISCUSSION The fluorescence probe PIP was synthesized according to Scheme 3 and characterized by various spectroscopic techniques like 1H-NMR (Figures S1 and S2), 13C-NMR (Figure S3) and Mass spectra (Figure S4) Scheme 3. Scheme for the Synthesis of PIP.

Figure 1. (a) Fluorescence spectral changes of PIP (10 μM) in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4 by the gradual addition Cu2+ (0-250 μM); (b) Plot of (F-F0)/(F∞-F0) vs [Cu2+].

Photophysical Properties of [(PIP)CuII(Cl)] towards NO and HNO: When PIP was excited at 400 nm, it displayed a strong fluorescence, which might be due to the involvement of lone pair of electrons on NH- nitrogen atom in strong conjugation throughout the 1HImidazo[4,5-b]phenazine moiety. But, when coordination of 1H-Imidazo[4,5-b]phenazine moiety occurs through N— (generated by deprotonation of NH group) to Cu2+ this conjugation 50 is mostly blocked. A process like electron or energy transfer may occur from the phenazine fluorophore to chelated Cu2+ leading to quenching of fluorescence (Scheme 4). The paramagnetic effect of Cu2+ may also be responsible for such fluorescence quenching. Fluorescence Sensing of Cu(II) by PIP: A solution of PIP (10 μM) in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4 exhibited strong fluorescence at 560 nm when excited the probe at 400 nm. Upon incremental addition of CuCl2 (0250 μM) to this solution the integrated fluorescence intensity of PIP was greatly quenched (Figure 1). From these titration data the formation constant50 (Kf) was determined which was found to be (6.83  0.05) × 106 M-1 (Figure 1). The UV-Vis spectrum of the PIP was also recorded in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4, which displayed a well-defined band at 407 nm. Upon gradual addition of Cu2+ up to 250 M, there is a gradual decrease in absorbance at 407 nm with a blue shift to 400 nm (Figure S6) indicating the formation of a complex between PIP and Cu2+. The 1:1 stoichiometry for the complex formation between PIP and Cu2+ is confirmed by the JOB’s plot (Figure S7) and ESI-MS+ (m/z): 507.4146 ([(PIP)CuII(Cl)] + Li+) (Figure S5). The selective fluorescence quenching of PIP by Cu2+ over different transition metal ions (Figure S8) as well as anions (Figure S9) was ascertained by no/negligible perturbation of fluorescence intensity of PIP by these cations and anions.

Now PIP—Cu2+ complex reacts with nitric oxide (NO) under aerobic conditions, the fluorescence intensity of the PIP—Cu2+ complex increases significantly (5-fold) at 560 nm on excitation at 400 nm. To investigate the interaction further, we have carried out a concentration dependent titration for the reaction between PIP—Cu2+ complex (10 µm) and NO (0-80 µm) by adding 1.74 x 10-3 M aqueous NO solution in 5:5(v/v) CH3CN/HEPES buffer at pH 7.4 (Figure. 2a). An excellent linear curve up to 70 μM was observed for a plot of FI vs. [NO], after which a saturation in FI was observed (Figure. 2b). The equilibrium constant (Kf) for the reaction of PIP—Cu2+ complex with NO was determined by following eqn 1 under a special condition where 1>> c*xn, and eqn. 1. turns to y= a+b*c*x with n = 1. 𝑦=

𝑎 + 𝑏 × 𝑐 × 𝑥𝑛

(1) 1 + 𝑐 × 𝑥𝑛 Here a and b are FIs of the PIP—Cu2+ complex in the absence and presence of excess NO (≥ 70 µM), respectively and c = Kf. The slope (b*c) gives the formation constant (c = Kf) as (1.05 ± 0.04) x 104 M-1 (R2=0.997). The PIP—Cu2+ complex is interestingly also sensitive towards HNO where the treatment of the PIP—Cu2+ complex ([(PIP)CuII(Cl)]) with Angeli’s salt induced a dramatic increase in fluorescence intensity (6-fold) (Figure. 3a).


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Figure 2 (a) Fluorescence spectral changes of PIP—Cu2+ complex (10 μM) with NO (0-80 μM) in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4, λex=400 nm, λem=560 nm; (b) plot of F.I vs [NO]; (c) UV expose image (I) PIP—Cu2+ complex, (II) PIP—Cu2+ complex + NO.

Figure 3 (a) Fluorescence titration of PIP—Cu2+ complex (10 μM) with HNO (0-60 μM) in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4, λex=400 nm, λem=560 nm; (b) plot of F. I vs [HNO]; (c) UV exposes image (I) PIP—Cu2+ complex, (II) PIP—Cu2+ complex + HNO.

In the titration of the PIP—Cu2+ complex (10 µm) with HNO (0-60 µm) in 5:5(v/v) CH3CN/HEPES buffer at 25 ˚C and at pH 7.4, fluorescence intensity of PIP—Cu2+ complex increases at 560 nm on excitation at 400 nm. A plot of F.I vs. [HNO] gives an excellent linear curve (Figure. 3b) which was solved by using the eqn. 1 under special conditions, 1>> c*xn, with n = 1 which leads to Kf = (1.34 ± 0.16) x 104 M-1 (R2=0.998). In order to evaluate the real-time sensing performance of PIP—Cu2+ complex toward NO and HNO the time dependent fluorescence responses were investigated (Figure S10a,b). At 560 nm the fluorescence intensity increases with time, attains a maximum at ~700 sec and then remains constant for more than 900 sec for both NO and HNO reactions toward PIP—Cu2+ complex. These observations clearly reveal that the PIP—Cu2+ complex has the potential for real-time monitoring of NO and HNO in biosystems. Reactivity with ROS and RNS: Exclusivity in the detection of analyte is imperative for a real-time sensor system. Therefore, the fluorescence response of PIP—Cu2+ complex was investigated with the treatment of different RNS and ROS (Figure 4). There’s no significant change in fluorescence of PIP—Cu2+ was observed upon addition of the different ROS and RNS including oxidizing agents such as HOCl, H2O2 and ONOO–. Not only that the TEMPO radical, •OH, O2–, AA, DHA, GSH, Cys and NO+ also fail to enhance the fluorescence of PIP—Cu2+ sensor. But the addition of Angeli’s salt (Na2N2O3) and NO on PIP—Cu2+ sensor a 6-fold and 5-fold increase in fluorescence intensity respectively was observed. In aqueous solution Angeli’s salt is decomposed to from HNO and NO2–.52 To eliminate that NO2– was not participating in the process of fluorescence enhancement of PIP—Cu2+ complex NaNO2 was externally added to the PIP—Cu2+ sensor. It was interesting to see that NaNO2 does not influence the increase in fluorescence intensity of the PIP—Cu2+ sensor.

Figure 4 (a) Histogram (b) fluorescence spectral plot for the PIP—Cu2+ complex toward different reactive species in (5:5 v/v) CH3CN/HEPES buffer at pH 7.4, ex= 400 nm, em = 560 nm.



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Scheme 4. Schematic presentation of ON-OFF-ON fluorescence response of PIP. On the basis of these emission studies, we can safely conclude that the sensor PIP—Cu2+ is highly sensitive and selective towards HNO and NO only. We have also investigated the above selectivity study upon PIP—Fe3+ complex with different RNS, ROS, NO and HNO due to the fact that Fe3+ also slightly quenches the emission of the PIP probe. But it was interesting to see that no significant change in fluorescence of PIP—Fe3+ observed (Figure S11). The effect of pH on the Stability of PIP—Cu2+ complex : For biological applications the study of the stability of the fluorophore probe on pH is very important. Thus, the dependence of fluorescence intensities of PIP and PIP—Cu2+ at different pH values was investigated (Figure S12). From pH 2 to 10 no noticeable change in fluorescence of PIP was perceived, though it is highly fluorescent within this range of pH. On the other hand, PIP—Cu2+ is very weakly fluorescent but no dramatic change in fluorescence intensity was also observed within the range pH 2-10. So it could be concluded that both PIP and PIP—Cu2+ complex are stable within the studied pH range, making the probe PIP—Cu2+ suitable for monitoring NO/HNO in the biological system. Mechanism of HNO and NO Sensing: On the addition of NO (8 equivalents) and HNO (6 equivalents) to the PIP—Cu2+ sensor a ~5-fold and ~6-fold increase in fluorescence intensity was observed, respectively. Not only that the quantum yield of PIP—Cu2+ complex (φ= 0.023) also increases in addition of NO (φ= 0.126) and HNO (φ= 0.142), whereas the quantum yield (φ) of free PIP is 0.185. From the HRMS study of the reaction between [(PIP)CuII(Cl)] complex (507.320 ([(PIP)CuII(Cl)] + Li+) and excess NO a new peak is observed at 465.9980 corresponding to ([(PIP)CuI] species (Figure 5), which provide a good confirmation for a turn on the fluorescence response due to reduction of paramagnetic Cu2+ (a quencher) ion to a diamagnetic Cu+ (a nonquencher) ion. This fluorescence response mechanism is in conformity with other Cu2+ complex based NO/HNO sensors like Cu(II)-CP1, [Cu(Ds-en)2] and [Cu(Ds-AMP)2] as reported by Lippard group.34-36 So the sensing mechanism of PIP—Cu2+ complex with NO can be elucidated: (1) nitric oxide reduces Cu2+ ion in [(PIP)CuII(Cl)] to Cu+ ion, itself being converted to NO+; and (2) the oxidized NO+ is released and [(PIP)CuI] (PIP—

Cu+) complex remains in solution. The mechanism is presented in Scheme 1b. The strongest support for the formation of [(PIP)CuI] complex is the nearly perfect matching of emission (Figure S13) and absorbance (Figure S14) spectra of PIP in the presence of CuCl under identical reaction conditions. The most interesting part of these observations is that treatment of NO with PIP did not give any change in fluorescence (Figure S9), which demonstrates the significance of Cu(II) ion as an electron acceptor. The reaction with [(PIP)CuII(Cl)] and NO is as follows eqn. 2 : [(𝑃𝐼𝑃)𝐶𝑢II(𝐶𝑙)] + 𝑁𝑂→[(𝑃𝐼𝑃)𝐶𝑢I] + 𝑁𝑂 + (2) 2+ Therefore, after the reaction of PIP—Cu complex with NO the paramagnetic effect of Cu(II) is turned off and diamagnetic Cu(I) species is formed; as a result an enhancement in fluorescence is observed (Scheme 4). In the case of HNO recognizing process, the interaction between PIP—Cu2+ and HNO generates NO in the first step, which reduces another paramagnetic Cu(II) center in PIP—Cu2+ leading to formation of NO+ and Cu(I) as follows eqn. 3 and 4 : 70 [(𝑃𝐼𝑃)𝐶𝑢II(𝐶𝑙)] + 𝐻𝑁𝑂→[(𝑃𝐼𝑃)𝐶𝑢𝐼] + 𝑁𝑂 II

𝐼

(3)

+

[(𝑃𝐼𝑃)𝐶𝑢 (𝐶𝑙)] + 𝑁𝑂→[(𝑃𝐼𝑃)𝐶𝑢 ] + 𝑁𝑂 (4) The reduction of a paramagnetic Cu(II) center to a diamagnetic Cu(I) restores the quenched fluorescence of [(PIP)CuII(Cl)] complex and PIP remains coordinated to the Cu(I). The formation of [(PIP)CuI] complex is confirmed by ESI-MS+ (m/z) peak at 465.9892 (Figure 5). The reduction of Cu(II) to Cu(I) is confirmed by the EPR spectra where [(PIP)CuII(Cl)] in CH3CN gives the axial signal with g⊥= 2.09, g∥= 2.38 due to S = 1/2 for Cu(II) ion, but upon reaction with HNO the axial signal vanishes almost to the baseline (Figure S15). Lippard et al. designed a Cu-based benzoresorufin50 functionalized fluorophore containing a secondary amine group, for the detection of NO and HNO. The emission response of the sensor to NO and HNO is observed due to the reduction of a paramagnetic Cu(II) to form a diamagnetic Cu(I) for both the NO and HNO reactions. Another N-substituted 1,8-naphthalimide Cu based chromophore51 was reported by James and co-worker for the detection of dual analyte NO and HNO; the fluorescence turn on response mechanism is based not only on the reduction of


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Figure 5. HRMS (ESI-MS+, m/z) spectra for ([(PIP)CuI]) complex. (a) [(PIP)CuII(Cl)] + NO and (b) [(PIP)CuII(Cl)] + HNO.

Figure 6. Optimized molecular structures of PIP, PIP—Cu2+ complex ([(PIP)CuII(Cl)]) and PIP—Cu+ complex ([(PIP)CuI]). Table 1. Some vertical excitation energies and oscillator strengths (fcal) of some lowlying excited singlets from TDDFT// B3LYP/6-31G(d) calculations of PIP, PIP—Cu2+ complex ([(PIP)CuII(Cl)]) and PIP—Cu+ complex ([(PIP)CuI]). PIP and Cu Complexs

Electronic

PIP PIP—Cu2+

PIP—Cu+

Composition

Excitation Energy

Oscillator strength (fcal)

CI

λexp (nm)

Assign

S0 → S3

HOMO–1→ LUMO

3.12 eV (396.25 nm)

0.8144

0.69212

407

ILCT

S0 → S17

HOMO–1→ LUMO+1

3.17 eV (389.92 nm)

0.9680

0.66633

400

MLCT/ILCT

transition

S0 → S7

HOMO-5 → LUMO

0.10632

MLCT

HOMO-4 → LUMO

0.25930

MLCT

HOMO-3 → LUMO

3.07 eV (402.62 nm)

0.3576

HOMO-2 → LUMO


0.54513 0.29849

406

MLCT MLCT


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Cu(II) center, but also the formation of N-nitroxyl product. Till to date only these two Cu complexes are developed for the detection of dual analyte NO and HNO. Here, our phenazine based Cu(II) complex [(PIP)CuII(Cl)] also behave as a dual sensor for NO and HNO following the same mechanism as reported by Lippard and coworkers. Theoretical studies: Ground state geometries and molecular orbital properties: DFT calculations were employed to gain some insights into the coordination mode of the PIP with Cu2+ ion and to know how the metal binding affinity changes their sensing behaviour. The optimized geometries of PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] are depicted in Figure 6. The modeled geometries have a C1 point group and in the case of [(PIP)CuII(Cl)] the central Cu2+ ion assumes a distorted tetrahedral geometry. All the calculated Cu–N distances in [(PIP)CuII(Cl)] complex fall in the range 1.84–1.93 Å and the Cu–O bond distances is 2.08 Å. Due to the formation of [(PIP)CuII(Cl)] and [(PIP)CuI] complexes, some C–C, C–N and C–O bond lengths are slightly changed. Table S1 and S2 describes the change in bond lengths and bond angles respectively in PIP, [(PIP)CuII(Cl)] and [(PIP)CuI] complexes formation. In the case of PIP at ground state the electron density on HOMO-1, HOMO and LUMO reside mainly in 1HImidazo[4,5-b]phenazine moiety. The energy gap between the HOMO and LUMO of PIP is 3.00 eV (Figure 7). In the ground state the electron density of [(PIP)CuII(Cl)] complex is mainly localized on HOMO-1, HOMO, LUMO and LUMO+1 molecular orbitals. Whereas electron density on HOMO-1, HOMO and LUMO arise from metal d orbital together with ligand (PIP) π orbital contributions, while on LUMO+1 comes from ligand (PIP) π and π* orbital contributions. The HOMO, LUMO energy gap for [(PIP)CuII(Cl)] complex becomes 1.15 eV (Figure 7). The energy differences between HOMO and LUMO of PIP and [(PIP)CuII(Cl)] complex clearly indicate that the affinity of PIP towards Cu2+ ion to form a complex. For the reduced [(PIP)CuI] complex electron clouds, mainly come from HOMO-5, HOMO-3, HOMO-2 molecular orbitals of the 1H-Imidazo[4,5-b]phenazine moiety along with metal d orbital. The electron clouds on HOMO, HOMO-4 comes from the metal d orbital only and LUMO comes from ligand (PIP) π and π* orbitals. The HOMO–LUMO energy gap is 2.71 eV (Figure 7) which is nearly close to PIP. The composition of orbitals helps us to understand the nature of electronic transition and thereby absorption spectra of both the PIP and its CuII/I-complexes.

Figure 7. Frontier molecular orbital of PIP, PIP—Cu2+ and PIP—Cu+ complex.

The [(PIP)CuII(Cl)] and [(PIP)CuI] complexes display absorption bands at 400 nm and 406 nm respectively (Figure s14) in (5:5 v/v) CH3CN/HEPES buffer solution at room temperature and the corresponding calculated absorption bands are found at 390 and 403 nm respectively. There is excellent closeness of these calculated bands to our experimental results (Table 1). These absorption bands can be assigned to the S0→S17 and S0→S7 transitions, for [(PIP)CuII(Cl)] (Figure 8) and [(PIP)CuI] (Figure 9) complex respectively. Both the transitions are originated from an admixture of MLCT and ILCT transition.

Figure 8. Frontier molecular orbitals of PIP and PIP—Cu2+ complex for absorption.

The free ligand displayed an absorption band at 407 nm in (5:5 v/v) CH3CN/HEPES buffer at room temperature which is ILCT character. From the TDDFT calculation this assignment was well supported by the absorption band at 396.25 nm (Table 1). This band is assigned to S0→S3 electronic transitions (Figure 8).


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NO and HNO Detection in Living Cells: To investigate the sensing capability of [(PIP)CuII(Cl)] (PIP—Cu2+) sensors for the detection of NO and HNO fluorescence live cell imaging studies were carried out. The cytotoxicity of the [(PIP)CuII(Cl)] complex was assessed in Raw 264.7 and A549 cells (Figure 10). The cell lines showed evidence of well tolerability to [(PIP)CuII(Cl)] complex, indicating the its usefulness as NO and HNO sensor in live cells.

respectively (Figure 11b,c) but no reactivity towards H2S, GSH and Cys was observed. The selectivity of [(PIP)CuII(Cl)] complex towards HNO and NO in response to the detection of different reactive species were further analyzed in A549 cells (Figure s16). Moreover, [(PIP)CuII(Cl)] complex showed only excellent fluorescence response towards NO and HNO. Likely, endogenously induced NO sensing of [(PIP)CuII(Cl)] complex was evaluated in Raw 264.7 cells. The cells were pre-incubated with [(PIP)CuII(Cl)] followed by NO induction with LPS and IFN-γ for 6h. Stimulated cells showed fluorescence compared to non-stimulated cells and NO scavenger PTIO (200 mM) (Figure 12 a,b). Furthermore, the Raw 264.7 cells with the above mention setting, evaluate the detection of NO using flow cytometric analysis. LPS+IFN-γ stimulated cells show shift in fluorescence peak compared to non-stimulated cells and NO scavenger PTIO (200 mM) (Figure 12 c). In conclusion, Probe [(PIP)CuII(Cl)] physiologically displays sensitive and selective fluorescence turn-on responses towards NO and HNO. Thus, [(PIP)CuII(Cl)] should be an excellent nontoxic fluorescent cellular tracker of HNO and NO. CONCLUSION

Figure 9. Frontier molecular orbitals of PIP—Cu+ complex for absorption.

In conclusion a phenazine based Cu-complex, [(PIP)CuII(Cl)], was synthesized for the selective detection of NO and HNO. [(PIP)CuII(Cl)] complex exhibit ~5 and ~6 fold fluorescence enhancement upon treatment with NO and HNO respectively and the emission was observed at ~560 nm, a suitable region for bioimaging applications. The [(PIP)CuII(Cl)] complex was also found to be almost nontoxic and cell permeable, making it useful for imaging exogenous and endogenous NO and HNO in A549 and Raw 264.7 macrophage cells with the possibility of flow cytometric application. DFT calculations were also employed to gain insight into the coordination mode of the PIP with Cu2+ ion and to know how the metal binding affinity changes their sensor property.

Figure 10. Cytotoxicity of the probe PIP—Cu2+ by MTT assay.

Exogenously induced NO and HNO sensing feasibility of [(PIP)CuII(Cl)] complex was assessed in A549 cells. The stability of [(PIP)CuII(Cl)] fluorescence signal in cells were validated upto 60 min of post incubation (Figure 11 a). The live cells were treated with probe [(PIP)CuII(Cl)] followed by NO donor DEA-NONOate and HNO donor Angeli’s salt separately showing intracellular fluorescence contrast to [(PIP)CuII(Cl)] complex (Figure 11 a). Approximately, 3.9-4.6 fold and 5.5-6.1 fold increase in intracellular fluorescence was observed compared to [(PIP)CuII(Cl)] after the treatment with NO and HNO



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Figure 11. Exogenious detection of NO and HNO (a) Control experiment shows excellent stability towards 5 µM [(PIP) CuII (Cl)] (PIP—Cu2+) complex in A549 cells imaged at every 30 min subsequently. 5 µM [(PIP)CuII(Cl)] complex were incubated in A549 cells for 30 min followed by incubated with DEA-NONOate (5 µM and 10 µM) for 30 min and Angeli’s salt (5 µM and 10 µM) for 30 min. Observation of intracellular fluorescence arises due to interaction of NO and HNO with [(PIP)CuII(Cl)] compared to H2S, GSH and Cys which shows very weak/no fluorescence. Images were captured at 40X objective. (b) Representative plots indicating the intracellular fluorescence response of [(PIP)CuII(Cl)] towards NO and HNO on treatment with DEA-NONOate and Angeli’s salt respectively compared to H2S, GSH and Cys (Mean ± SD). (c) Representative plots indicating the fold of fluorescence enhancement for the reaction of [(PIP)CuII(Cl)] with NO and HNO generated from DEANONOate and Angeli’s salt respectively. The data expressed as mean ± SD, n=3, p value ≤ 0.05.


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Figure 12. Endogenous detection of NO and flow cytometry analysis (A) Raw 264.7 cells were incubated with [(PIP)CuII(Cl)] without LPS and stimulated with LPS+IFN-γ for 6h and with NO scavenger PTIO. Fluorescence images were taken at 40X objective. (B) Representative plots of fluorescence intensity of [(PIP)CuII(Cl)] - NO reactions on LPS+IFN-γ stimulation compared to without LPS+IFN-γ and NO scavenger PTIO. (C) Flow cytometry analysis of NO using [(PIP)CuII(Cl)] complex. The dot plot with the forward (FSC-H) versus side (SSC-H) scatter and histogram plot showing gated Raw 264.7 macrophage cells positive for NO. Overlap histogram shows a positive shift of fluorescence intensity peak in LPS+IFN-γ induced NO captured by [(PIP)CuII(Cl)] complex compared to without LPS+IFN-γ and NO scavenger PTIO. Flow cytromety analysis was done by using Flowing Software version 2.5.1. The data expressed as mean ± SD, n=3, p value ≤ 0.05.

ASSOCIATED CONTENT Supporting Information “This material is available free of charge via the Internet at http://pubs.acs.org.” Fluorescence and characterization data of compounds PIP, PIP—Cu2+ and PIP—Cu+ complexes, pH effect, DFT computational data.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Funding Sources This work was funded by the West Bengal DST (Ref. No.809(Sanc)/ST/P/ S&T/4G-9/2104) and CSIR New Delhi (Ref.01(2896)/17/EMR-II), India.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT A S M Islam gratefully acknowledges CSIR, New Delhi for the fellowship (SRF).

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