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Dyes and Pigments 102 (2014) 315e325

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Dyes and Pigments

journal homepage: www.elsevier .com/locate/dyepig

Microwave assisted synthesis, photophysical and redox properties of(phenothiazinyl)vinyl-pyridinium dyes

Luiza G�ain�a a, Ioana Torje a, Emese Gal a, Alexandru Lupan a, Cristina Bischin a,Radu Silaghi-Dumitrescu a, Grigore Damian b, Peter Lönnecke c, Castelia Cristea a,*,Luminita Silaghi-Dumitrescu a

a “Babes-Bolyai” University, Faculty of Chemistry and Chemical Engineering, RO-400028 Cluj-Napoca, Romaniab “Babes-Bolyai” University, Faculty of Physics, M. Kog�alniceanu 1, RO-400082 Cluj-Napoca, Romaniac Leipzig University, Institut für Anorganische Chemie, Johannisallee 29, D-04103 Leipzig, Germany

a r t i c l e i n f o

Article history:Received 27 June 2013Received in revised form19 September 2013Accepted 30 October 2013Available online 7 November 2013

Keywords:PhenothiazineAlkyl-pyridinium cationMicrowaves assisted organic synthesisUVeVis absorptionDFTProoxidant reactivity

* Corresponding author. Tel.: þ40 264 593833; faxE-mail address: castelia@chem.ubbcluj.ro (C. Crist

0143-7208/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.dyepig.2013.10.044

a b s t r a c t

New cationic cyanine dyes, containing electron donor 10-alkyl-phenothiazine and electron acceptorpyridinium units connected through a vinylene bridge were conveniently synthesized by microwavesassisted condensation of phenothiazine carbaldehyde with methylpyridinium salts in dry media. Theirphotophysical properties revealing strong absorption bands in 440e470 nm region ( 3z 104e105) andlarge Stokes shifts of fluorescence emission (610e750 nm) in solid state were supported by computa-tional data using TDDFT level of theory. The redox behavior of PVP was studied by means of electro-chemical and biocatalytical oxidation processes.

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1. Introduction

Experimental and theoretical studies regarding the photo-physical and electrochemical properties of various organic com-pounds containing extended p-conjugated electron systemsenabled the preparation of molecular components suitable fordiverse advanced photonic applications. Derivatives containing thephenothiazine core displayed characteristic UVeVis absorption andemission properties, as well as low and reversible oxidation po-tentials and thus, taking benefit of the summative electronreleasing effects of the nitrogen and sulfur atoms attached to thearomatic rings, phenothiazine appears as a potential heterocycliccandidate in the design of pushepull chromophore systems. Only afairly weak electronic communication between heterocycliccores was observed in alkynyl bridged diphenothiazinyl com-pounds [1], but dumbbell-shaped diphenothiazines bridged byconjugatively linked (hetero)aromatic moieties proved to be redox-active and strongly luminescent [2]. Linear vinylene linked

: þ40 264 590818.ea).

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oligophenothiazines were tested as fluorescent probes for chemo-sensors [3] and phenothiazinyl merocyanines synthesized fromphenothiazinyl aldehydes and N-methylrhodanine or indan-1,3-dione displayed a broad range of fine-tunable redox properties,deep-colored absorption bands and intense emission with largeStokes shifts [4]. Phenothiazinyl rhodanylidene acetic acid mer-ocyanine dyes [5] and phenothiazinyl-vinyl-bipyridine ligandand its Ru(II) complexes [6] were successfully tested as chromo-phores for applications in dye sensitized solar cells. Terpyridinyl-styryl-phenothiazine ligand and its complexes exhibited good oneand two photon excited fluorescence properties [7]. (Pyridinyl)vi-nyl-phenothiazine as pushepull chromophore system providedfluorophore-switching and potential near infrared sensor applica-tion [8] as well as biomarkers in cell biology [9].

Continuing our preoccupation for the identification of newphenothiazine based chromophores with potential applications inmaterials science or molecular biology [10] in this work we bringevidences of new cationic cyanine dyes obtained by the conden-sation of phenothiazine-carbaldehyde with methylpyridiniumsalts. The exploitation of the advantages induced by the dielectricheating allowed us the development of a facile, rapid and moreecofriendly microwave assisted synthetic protocol. Experimental

Table 1Crystal data and structure refinement.

3 7

Formula C24H25IN2S C24H25IN2SFormula weight 500.42 500.42Temperature [K] 130(2) K 130(2) KCrystal system Monoclinic MonoclinicSpace group P21/n C2/cUnit cella [pm] 1735.95(3) 3225.40(9)b [pm] 749.54(1) 747.54(1)c [pm] 1740.71(3) 2087.92(6)a [�] 90� 90�

b [�] 103.144(2)� 117.908(4)�

g [�] 90� 90�

Volume [nm3] 2.20561(6) 4.4487(2)Z 4 8r(calc.) [mg/m3] 1.507 1.494m [mm�1] 1.558 1.545F(000) 1008 2016Crystal size [mm3] 0.2 � 0.1 � 0.03 0.4 � 0.4 � 0.2QMineQMax [�] 2.97e30.51 2.90e30.51Index ranges �24 � h � 24 �46 � h � 46

�10 � k � 10 �10 � k � 10�24 � l � 24 �29 � l � 29

Reflections collected 32,948 38,113Indep. refl. [R(int)] 6735 [0.0366] 6796 [0.0301]Completeness to QMax 99.9% 99.9%TMax � TMin 1 � 0.95929 1 � 0.90006Restraints/parameters 0/353 80/364Goof 1.048 1.041R1I>2sigma(I)/R1(all) 0.0261/0.0361 0.0263/0.0344wR2I>2sigma(I)/wR2(all) 0.0509/0.0544 0.0574/0.0608Residual e-density [e/�A3] 0.411/�0.377 0.732/�0.493

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325316

evidences of photophysical properties of the new (phenothiazinyl)vinyl-pyridinium (PVP) dyes were brought by means of UVeVisabsorption/emission spectroscopy and were completed by theo-retical DFT computational studies. Their redox behavior wasemphasized by electrochemical and biocatalytic oxidationexperiments.

2. Experimental

2.1. Materials and methods

Flash chromatography was performed on silica gel 60 (particlesize 0.032e0.063 mm). Thin layer chromatography was performedon Merck DCAlufolien, silica gel 60 F254 and components werevisualized by UV VL-4LC.

The melting points were determined in capillaries with anElectrothermal 9100 instrument. Elemental analysis was carriedout using Thermo Flash EA 1112 CHN analyzer.

2.1.1. Spectral measurementsNMR spectra (1D, DEPT, 2D-COSY, 2D-HSQC and 2D-HMBC)

were recorded at room temperature on Bruker Avance instruments(1H/13C: 400 MHz/100 MHz or 300 MHz/75 MHz) in solution(deuteriated solvents (CDCl3 or DMSO).

EI-MS spectra were recorded on a GC-MS QP 2010 Shimadzumass spectrometer.

UVeVis absorption spectra were recorded in solvent with aPerkin Elmer Lambda 35 spectrometer; Fluorescence emissionspectra were recorded in solid state with a Perkin Elmer PL 55spectrophotometer.

Stopped-Flow spectrometer SFM-300/S BioLogic Science In-struments with standard configuration for UVeVis absorbanceoptical mode was employed for fast mixing and observation of thekinetics of biocatalytic oxidation reactions.

IR spectra were recorded on a Bruker Vector 22 FT-IR spec-trometer with scanning between 4000 and 600 cm�1 using ATRsampling of the neat substance.

The X ray crystallographic data (Table 1) were collected using aCCD Oxford Xcalibur S diffractometer, MoKa radiation(l ¼ 71.073 pm), u-scan mode. Data reduction was carried out withCrysAlisPro including empirical absorption correction with SCALE3ABSPACK [11]. The structures were elucidated by direct methodsusing SIR 92 [12] and were refined using SHELXL-97 [13]. Aniso-tropic refinement of all non-hydrogen atoms with the exception oftheminor 14% disordered part in 7. The cationic part of compound 7was found to be disordered over two positions with a ratio of0.86(1):0.14(1). For 3 a difference-density Fourier map was used tolocate all Hydrogen atoms at the final stage of the structuredetermination, whereas for 7 all H-atoms were calculated onidealized positions using the riding model. Structure figures weregenerated with ORTEP and DIAMOND [14]. Thermal ellipsoids aredrawn at 50% probability if not otherwise mentioned and in Fig. 1below only the major 86% fraction of the disordered molecule 7 ispresented. CCDC 946801 (3) and 946802 (7) contains the supple-mentary crystallographic data for this paper. The data can be ob-tained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12Union Road, Cambridge CB2 1EZ, UK; fax: (þ44)1223-336-033; ordeposit@ccdc.cam.uk).

EPR spectra were measured at liquid nitrogen temperaturewith a BRUKER-BIOSPIN EMX spectrometer operating at the X-band(9.5 GHz). The EPR parameters were: 100 KHz modulation fre-quency, microwave power 10 mW, modulation amplitude 2 G; timeconstant 10 ms; scan time 60 s; number of scans 5; receiver gain104. Spectral simulations were performed using a rigid limitsimulation POWFIT program with simplex optimization method[15].

2.1.2. Electrochemical measurementsThe oxidation potentials were determined by cyclic voltamme-

try measurements using a Potentiostat/Galvanostat/ZRA Reference600, with a typical three-electrode setup composed of platinum(1 mm) working electrode, platinum and Ag/AgCl as auxiliary andreference electrodes. The electrolyte was 0.1 M tetrabutylammo-nium hexafluorophosphate in dry dichloromethane purged withargon prior to a measurement. Ferrocene/ferrocenium (Fc/Fcþ) wasemployed as internal standard.

2.2. Synthesis

All chemicals used were of reagent grade. Microwaves assistedsyntheses were performed in a CEM Discover microwave reactor(300 W maximum power, mono-mode irradiation, pressurizedreaction vessel, software control) using 20 mL sealed reactionvessels.

10-methyl-10Hphenothiazin-3-carbaldehyde was prepared ac-cording to our previously reported microwaves assisted procedure[16]

N-alkyl-methylpyridinium salts were prepared according toliterature procedures.

N-methyl-2-methylpyridinium iodide m.p. ¼ 223 �C frommethanol (224 �C, ethanol, [17]) EI-MS m/z:107(100%).

N-ethyl-2-methylpyridinium iodide m.p. ¼ 122 �C from dii-sopropyl ether (123 �C, diethyl ether, [17]) EI-MS m/z: 121(43%).

N-butyl-2-methylpyridinium iodide m.p ¼ 101 �C from diiso-propyl ether (98 �C, ethanol, [17]) EI-MS m/z 149(35%).

N-hexyl-2-methylpyridinium iodide light yellow oil, yield 45%from petroleum ether [light-yellow oil [18]] EI-MS m/z 177(58%).

Scheme 1. Condensation of 10-methyl-phenothiazin-3-carbaldehyde with alkyl-pyridinium iodides; reaction conditions: a) convective heating: piperidine, iPr-OH, 82 �C, b) mi-crowave irradiation in dry media: solid support basic Al2O3, 100 �C.

Table 2Experimental conditions applied in the synthesis of PVP 1e9.

Cpd Temperature [�C] Time [h] Yield [%]

D MWa D MWa D MWa

1 82 100 25 1 70 982 82 100 25 1 35 403 82 100 30 1 42 504 82 100 35 1 29 305 82 100 25 1 69 706 82 100 25 1 65 657 82 100 30 1 48 508 82 100 35 1 45 509 82 100 35 1 42 45

a On solid support (basic Al2O3).

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325 317

N-methyl-4-methylpyridinium iodide m.p. ¼ 147 �C frompropan-2-ol [152e153 �C from propan-2-ol [19]]. EI-MS m/z107(100%).

N-ethyl-4-methylpyridinium iodide m.p. ¼ 113 �C from hep-tane. (113 �C, [20]) EI-MS m/z 121(100%).

N-butyl-4-methylpyridinium iodide: brown oil, (brown oil,[21]) EI-MS m/z 149(100%).

N-methyl-2,6-dimethylpyridinium iodide: white crystals,m.p. ¼ 237 �C from methanol. [236e237 �C, [22]]. EI-MS m/z121(42%) [M � HI].

N-ethyl-2,6-dimethylpyridinium iodide: brown crystals,m.p. ¼ 212e214 �C from heptane [23]. EI-MS m/z 135 (26%),[M � HI].

2.2.1. Condensation of 10-methyl-10H-phenothiazine-3-carbaldehyde with N-alkyl-methylpyridinium salts

(Phenothiazinyl)vinyl-pyridinium PVP dyes containing electrondonor 10-alkyl-phenothiazine and electron withdrawing pyr-idinium units connected through a vinylene bridge were synthe-sized in good yields by Knoevenagel condensation of 10-methyl-phenothiazin-3-carbaldehyde with alkyl-pyridinium salts in thepresence of catalytic amounts of bases (Scheme 1). For the opti-mization of the reaction conditions two alternative processingtechniques were considered: the traditional convective heating(reflux in i-propanol) versus the dielectric heating induced by themicrowave irradiation of the reaction mixture in solvent, or insolvent-free conditions (neat reagents and adsorbed on aluminarespectively).

2.2.1.1. General procedure for the preparation of PVP 1e9 underconvective heating. To a solution of 10-methyl-10H-phenothiazine-3 carbaldehyde (1 mmol) in isopropyl alcohol (15 mL) was addedthe N-alkyl-methylpyridinium salt (1.1 mmol) and catalyticamounts of piperidine. The reaction mixture was refluxed understirring during the time indicated in Table 2; the reaction progresswas monitored by TLC. The crude precipitate was filtered, washedwith ethanol and than purified by several recrystallizations fromethanol and acetonitrile (10:1).

2.2.1.2. General procedure for the preparation of PVP 1e9 underdielectric heating.10-Methyl-10H-phenothiazine-3-carbaldehyde (0.8 mmol) and N-alkyl-methylpyridinium salt (0.8mmol for PVP 1e7, or 0.4mmol forPVP 8, 9) were solved in DCM (50 mL), basic aluminum oxide (4 g)was added and then the solvent was further removed by vacuumdistillation using a rotary evaporator. The dry reaction mixture wasintroduced in a quartz reaction vessel which was sealed and sub-jected to microwaves irradiation for 1 h at 100 �C. The crudeproduct was extracted with a mixture of ethanol and acetonitrile(10:1) and further purified by recrystallization from ethanol:ace-tonitrile (10:1). The reaction yields are presented in Table 2.

(E)-N-methyl-2-[(10-methyl-10H-phenothiazine-3-yl)2-vinyl] pyridinium iodide (1)

Orange powder, m.p. ¼ 255 �C (from ethanol and acetonitrile).1H NMR (300 MHz, DMSO-d6, d, ppm): 3.29 (s, 3H, eNeCH3),

4.26 (s, 3H, ]NeCH3), 6.98e6.90 (m, 3H, Ptz-H), 7.16e7.10 (m, 2H,Ptz-H), 7.40 (d, 1H, 3Jtrans ¼ 15.9 Hz, HC]), 7.57 (d, 1H, 3J ¼ 8.4 Hz,Ptz-H), 7.70e7.66 (m, 2H, Py-H), 7.80 (d, 1H, 3Jtrans ¼ 15.9 Hz, HC]),8.38e8.33 (m, 2H, Py-H, Ptz-H), 8.77 (d, 1H, 3J ¼ 6 Hz, Py-H). 13CNMR (75 MHz, DMSO-d6, d, ppm): 35.9, 46.5, 115.2, 115.5, 115.6,121.6, 123.1, 123.6, 124.8, 124.9, 126.3, 127.3, 128.5, 129.8, 130.2,142.3, 144.3, 144.6, 146.3, 147.8, 153.0. IR (nmax, cm�1): 724 (s), 1063

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325318

(s), 1337 (s), 1565 (m), 3475 (s). UVeVis (DMSO): lmax( 3) ¼ 446 nm(1.43 � 104). MS (EI-MS) m/z 330(25%), [M � HI], 316(100%), [Me

Me]. Anal. Calcd. For C21H19IN2S (458.03): C 55.03, H 4.18, I 27.69, N6.11, S 7.00; Found: C 54.03, H 4.24, N 5.85, S 7.55.

(E)-N-ethyl-2-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]pyridinium iodide (2)

Red powder, m.p.¼ 255e257 �C (from ethanol and acetonitrile).1H NMR (400 MHz, DMSO-d6, d, ppm): 14.6 (t, 3H, J ¼ 7.2 Hz, e

CH3), 3.38 (s, 3H,eNeCH3), 4.80 (q, 2H, J¼ 7.2 Hz,]NeCH2), 7.00e7.06 (m, 3H, Ptz-H), 7.20 (d, 1H, 3J ¼ 6.9 Hz, Ptz-H), 7.25 (t, 1H,J ¼ 6.9 Hz, Ptz-H), 7.50 (d, 1H, 3Jtrans ¼ 15.9 Hz, HC]), 7.67 (dd, 1H,3J ¼ 8.4, 4J ¼ 2 Hz, Ptz-H), 7.82 (d, 1H, 4J ¼ 2 Hz, Ptz-H); 7.88 (td, 1H,3J ¼ 6.2 Hz, 4J ¼ 2.4 Hz, Py-H), 7.90 (d, 1H, 3Jtrans ¼ 15.9 Hz, HC]),8.44e8.50 (m, 2H, Py-H), 8.92 (d, 1H, 3J ¼ 6.3 Hz, Py-H). 13C NMR(100 MHz, DMSO-d6, d, ppm): 15.5, 35.4, 53.1, 114.5, 114.6, 115.0,121.1, 122.5, 123.1, 124.9, 125.1, 125.8, 126.8, 127.9, 129.2, 129.8,142.4, 143.9, 144.1, 144.8, 147.2, 151.7. IR (nmax, cm�1): 706 (w), 1026(s), 1337 (m), 1566 (m), 3482 (s). UVeVis (DMSO): lmax( 3) ¼ 444 nm (1.15 � 104). MS (EI-MS): m/z 344(24%), [M � HI], 316(100%) [M-Me]. Anal. Calcd. For C22H21IN2S (472.05): C 55.94, H4.48, I 26.86, N 5.93, S 6.79; Found: C 55.66, H 4.50, N 5.83, S 7.46.

(E)-N-butyl-2-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]pyridinium iodide (3)

Red crystals, m.p.¼ 233e235 �C (from ethanol and acetonitrile).1H NMR (300 MHz, DMSO-d6, d, ppm): 0.90 (t, 3H, J ¼ 7.5 Hz,

CH3), 1.34 (m, 2H, J¼ 7.5 Hz, CH2), 1.78 (m, 2H, J¼ 6.9 Hz, CH2), 3.38(s, 3H, eNeCH3), 4.79 (t, 2H, J ¼ 7.2 Hz), 6.98e7.05 (m, 3H, Ptz-H),7.18 (d, 1H 3J ¼ 7.2 Hz, Ptz-H), 7.24 (t, 1H, 3J ¼ 7.2 Hz, Ptz-H), 7.50 (d,1H, 3Jtrans ¼ 15.6 Hz, HC]), 7.68 (d, 1H, 3J ¼ 8.4 Hz, Ptz-H), 7.77 (s,1H, Ptz-H), 7.86e7.88 (m, 1H, Py-H), 7.9 (d, 1H, 3Jtrans ¼ 15.6 Hz, ]HC), 8.42e8.47 (m, 2H, Py-H), 8.91 (d, 1H, 3J ¼ 6 Hz, Py-H). 13C NMR(75 MHz, DMSO-d6, d, ppm): 14.0, 19.2, 32.2, 36.0, 57.7, 115.1, 115.2,115.5, 121.6, 123.0, 123.6, 125.2, 125.8, 126.4, 127.3, 128.4, 129.8,130.2, 142.8, 144.5, 145.7, 147.7, 152.4, 156.6. IR (nmax, cm�1): 706(w), 1062 (m), 1336 (m), 1566 (m), 3467 (s). UVeVis (DMSO): lmax( 3) ¼ 445 nm (1.08 � 104). MS (EI-MS) m/z 372(5%), [M � H],316(100%), [M � HI]. Anal. Calcd. For C24H25IN2S (500.08): C 57.60,H 5.04, I 25.36, N 5.60, S 6.41; Found: C 57.62, H 5.09, N 5.54, S 6.75.

(E)-1-hexyl-2-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]pyridinium iodide (4)

Red powder, m.p ¼ 223e225 �C (from ethanol and acetonitrile).1HNMR (300MHz, DMSO-d6, d, ppm): 0.81 (t, 3H, J¼ 7 Hz, CH3),

1.24 (m, 6H, CH2), 1.77 (m, 2H), 3.30 (s, 3H, eNeCH3), 4.81 (t, 2H,3J ¼ 7.2 Hz, ]NeCH2), 6.97e7.03 (m, 3H, Ptz-H), 7.21 (d, 1H,3J ¼ 8.6 Hz, Ptz-H), 7.24 (t, 1H, 3J ¼ 8.6 Hz, Ptz-H), 7.69 (d, 1H,3Jtrans ¼ 15.9 Hz, ]CH), 7.69 (d, 1H, 3J ¼ 8.8 Hz, Ptz-H), 7.78 (s, 1H,Ptz-H), 7.84e7.89 (m, 1H, Py-H), 7.92 (d, 1H, 3Jtrans ¼ 15.9 Hz,]CH),8.43e8.52 (m, 2H, Py-H), 8.96 (d, 1H, 3J ¼ 6 Hz, Py-H). 13C NMR(75 MHz, DMSO-d6, d, ppm): 14.2, 22.3, 25.4, 30.0, 31.0, 35.9, 57.8,115.1,115.2,115.5,121.6,123.0,123.6,125.2,125.8,126.4,127.3,128.4,129.8, 130.2, 142.8, 144.5, 145.7, 147.7, 152.4, 156.6. IR (nmax, cm�1):716 (w), 1083 (m), 1331 (w), 1566 (w), 2252 (w), 3426 (s). UVeVis(DMSO): lmax ( 3) ¼ 445 nm (1.50 � 104). MS (EI-MS) m/z: 400(5%),[M � HI], 316(100%) [M�C6H13]. Anal. Calcd. For C26H29BrN2S(480.12): C 64.86, H 6.07, Br 16.60, N 5.82, S 6.66; Found: C 64.33, H6.14, N 5.67, S 7.48.

(E)-1-methyl-4-[2-(10-methyl-10H-phenothiazine-3-yl)vi-nyl] pyridinium iodide (5)

Dark green crystals, m.p. ¼ 280 �C (from ethanol andacetonitrile).

1H NMR (400 MHz, DMSO-d6, d, ppm): 3.37 (s, 3H, eNeCH3),4.24 (s, 3H, ]NeCH3), 6.99e7.06 (m, 3H, Ptz-H), 7.19 (dd, 1H,3J ¼ 9.2 Hz, 4J ¼ 2 Hz, Ptz-H), 7.25 (td, 1H, J ¼ 8 Hz, J ¼ 1.2 Hz, Ptz-H), 7.4 (d, 1H, 3Jtrans ¼ 16.4 Hz, CH]), 7.57e7.59 (m, 2H, Ptz-H),7.91 (d, 1H, 3Jtrans ¼ 16.4 Hz, ]CH), 8.13 (d, 2H, 3J ¼ 6.8 Hz, Py-

H), 8.8 (d, 2H, 3J ¼ 6.8 Hz, Py-H). 13C NMR (75 MHz, DMSO-d6,d, ppm): 35.4, 46.7, 114.8, 115.0, 121.1, 121.1, 122.5, 122.9, 123.0,125.6, 126.8, 127.9, 128.8, 129.5, 139.5, 144.1, 144.8, 146.9, 152.5. IR(nmax, cm�1): 705 (w), 1025 (m), 1336 (m), 1619 (s), 3444 (s). UVeVis (DMSO) : lmax( 3) ¼ 455 nm (1.60 � 104). MS (EI-MS) m/z:330(3%), [M � HI], 316(100%), [M-Me]. Anal. Calcd. ForC21H19IN2S (458.03): C 55.03, H 4.18, I 27.69, N 6.11, S 7.00;Found: C 55.04, H 4.22, N 6.03, S 7.66.

(E)-1-ethyl-4-[2-(10-methyl-10H-phenothiazine-3-yl)2-vinyl] pyridinium iodide (6)

Red powder; m.p ¼ 115e117 �C (from ethanol and acetonitrile).1H NMR (300 MHz, DMSO-d6, d, ppm): 1.04 (t, 3H, J ¼ 7.2 Hz,

CH3), 3.32 (s, 3H, eNeCH3), 4.5 (q, 2H, 3J ¼ 7.2 Hz, CH2), 6.94e7.00 (m, 3H, Ptz-H), 7.13e7.23 (m, 2H, Ptz-H), 7.4 (d, 1H,3Jtrans ¼ 16.5 Hz, CH]), 7.56 (m, 2H, Ptz-H), 7.92 (d, 1H,3Jtrans ¼ 16.5 Hz, ]CH), 8.14 (d, 2H, 3J ¼ 6 Hz, Py-H), 8.9 (d, 2H,3J ¼ 6 Hz, Py-H). 13C NMR (75 MHz, DMSO-d6, d, ppm): 16.6, 35.9,55.5, 115.3, 115.5, 121.5, 121.6, 122.9, 123.5, 123.8, 126.2, 127.3,128.4, 129.3, 130.0, 140.1, 144.1, 144.6, 147.4, 153.3. IR (nmax,cm�1): 967 (w), 1049 (s), 1333 (w), 3650 (m). UVeVis (DMSO):lmax( 3) ¼ 457 nm (1.62 � 104). MS (EI-MS) m/z: 344(3%),[M � HI], 316(100%), [M-Ee]. Anal. Calcd. For C22H21IN2S(472.05): C 55.94, H 4.48, I 26.86, N 5.93, S 6.79; Found: C 52.55,H 4.92, N 5.51, S 7.24.

(E)-1-butyl-4-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]pyridinium iodide (7)

Dark red crystals; m.p ¼ 240e242 �C (from ethanol andacetonitrile).

1H NMR (300 MHz, DMSO-d6, d, ppm): 0.9 (t, 3H, 3J ¼ 7.4 Hz,CH3), 1.24e1.31 (m, 2H, CH2), 1.86 (m, 2H, CH2), 3.34 (s, 3H, NeCH3),4.47, (t, 2H, 3J ¼ 7.2 Hz), 6.96e7.04(m, 3H, Ptz-H), 7.16 (d, 1H,3J ¼ 7.6 Hz, Ptz-H), 7.22 (t, 1H, 3J ¼ 8 Hz, Ptz-H), 7.4 (d, 1H,3Jtrans ¼ 16.2 Hz, CH]), 7.56e7.59 (m, 2H), 7.93 (d, 1H, 3J ¼ 16.2 Hz,]CH), 8.15 (d, 2H, 3J ¼ 6.6 Hz, Py-H), 8.89 (d, 2H, 3J ¼ 6.6 Hz, Py-H).13C NMR (75 MHz, DMSO-d6, d, ppm): 13.8, 19.2, 32.9, 35.9, 59.7,115.3, 115.5, 121.5, 121.6, 123.0, 123.5, 123.8, 126.2, 127.3, 128.4,129.4, 130.0, 140.2, 144.4, 144.6, 147.4, 153.4. IR (nmax, cm�1): 763(w), 1082 (m), 1333 (m), 3359 (m). UVeVis (DMSO):lmax( 3) ¼ 458 nm (1.66 � 104). MS (EI-MS) m/z: 372(2%),[M � HI],316(74%), [M�C4H9]. Anal. Calcd. For C24H25IN2S (500.08): C 57.60,H 5.04, I 25.36, N 5.60, S 6.41; Found: C 57.52, H 5.06, N 5.53, S 7.10.

1-methyl-2,6-bis{(E)-2-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]}pyridinium iodide (8)

Dark red powder; m.p. ¼ 200e202 �C (from ethanol andacetonitrile).

1H NMR (300 MHz, DMSO-d6, d, ppm): 3.36 (s, 3H, eNeCH3),4.24 (s, 3H, ]NeCH3), 6.99e7.03 (m, 2H, Ptz-H), 7.22 (m, 2H, Ptz-H), 7.51 (d, 1H, 3Jtrans ¼ 15.6 Hz, CH]); 7.62e7.69(m, 2H, Ptz-H,and CH]), 7.77 (s, 1H, Ptz-H), 8.15 (d, 2H, 3J ¼ 7.5 Hz, Py-H), 8.31(t, 1H, 3J¼ 7.5 Hz, Py-H). 13C NMR (75MHz, DMSO-d6, d, ppm): 35.9,41.9, 119.8, 120.3, 122.0, 126.4, 127.8, 128.1, 128.3, 131.0, 132.1, 133.2,134.74, 134.76, 146.3, 147.5, 149.4, 152.2, 158.4. IR (nmax, cm�1): 716(w), 1083 (m), 1291 (w), 1563 (w), 2252 (w), 3457 (s). UVeVis(DMSO): lmax( 3) ¼ 468 nm (2.31 � 104). MS (EI-MS) m/z: 567(2%)[M � HI] 553 (100%), [M�Me]. Anal. Calcd. For C36H30IN3S2(695.09): C 62.15, H 4.35, I 18.24, N 6.04, S 9.22; Found: C 58.27, H4.51, N 5.64, S 9.54.

1-ethyl-2,6-bis{(E)-[2-(10-methyl-10H-phenothiazine-3-yl)vinyl]} pyridinium iodide (9)

Dark red powder; m.p. ¼ 216e218 �C (from ethanol andacetonitrile).

1H NMR (300 MHz, DMSO-d6, d, ppm): 1.47 (t, 3H, 3J ¼ 6.9 Hz, eCH3), 3.38 (s, 3H, eNeCH3), 4.85 (q, 2H, 3J ¼ 6.9 Hz, eCH2), 6.99e7.05 (m, 2H, Ptz-H), 7.19e7.72 (m, 2H, Ptz-H), 7.53 (d, 1H,3Jtrans ¼ 15.6 Hz, eCH]), 7.40 (d, 1H, 3J ¼ 8.7 Hz, Ptz-H), 7.71 (d, 1H,

Fig. 1. ORTEP plots of the molecular structures of PVP a) 3 b) 7.

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325 319

3Jtrans ¼ 15.6 Hz, eCH]), 7.80 (s, 1H, Ptz-H); 8.21 (d, 2H, 3J ¼ 8.1 Hz,Py-H), 8.35 (t, 1H, 3J ¼ 8.1 Hz, Py-H). 13C NMR (75 MHz, DMSO-d6, d,ppm): 14.8, 35.9, 42.1, 115.0, 115.5, 116.4, 121.7, 123.0, 123.5, 124.1,126.2, 127.3, 128.5, 129.9, 130.1, 142.3, 143.1, 144.7, 147.5, 152.9. IR(nmax, cm�1): 723 (m), 1082 (w), 1328 (w), 1464 (w), 2253 (w), 3415(s).UVeVis (DMSO): lmax( 3)¼ 470 nm (2.51�104).MS (EI-MS)m/z:581(1%), [M � HI], 553(71%), [M-Et]. Anal. Calcd. For C37H32IN3S2(709.11): C 62.62, H 4.54, I 17.88, N 5.92, S 9.04; Found: C 59.57, H4.73, N 5.65, S 9.64.

2.3. DFT calculations

Geometries constructed with Gaussview [24] have been opti-mized with the hybrid meta GGA M06-2x functional [25] asimplemented in the Gaussian software package [26] using the 6-31G(d,p) basis set. Frequency analysis has been performed in orderto ensure that the optimized geometries are genuine minima. UVeVis spectra have been computed using the time-dependent DFTmethod (100 states) with the same functional and basis set [27,28]

2.4. Biocatalytic oxidation

Hemoglobin was purified as previously described [29]. Proteinswere manipulated in phosphate buffer saline (PBS) unless otherwisestated. Hemoglobin concentrations are given per heme rather thanper tetramer. Myoglobin (from horse heart, Merck, Germany) wasusedwithout further purification. Bloodwas freshly extracted from ahealthy female volunteer in compliance with the requirements ofthe bioethics visa of the Babes-Bolyai University, and diluted withPBS (phosphate buffer saline) as indicated in Figure legends.

Autooxidation measurements were carried out at 37 �C, moni-toring the transformation of the physiologically-useful oxy form of

Fig. 2. Intermolecular p

hemoglobin and myoglobin into the toxic ferric (“met”) forms, at630 nm (for the met form) and 574/581 nm (for oxy hemoglobin/myoglobin, respectively). Experiments were carried out in PBS,with 25 mM globin (per heme) and 50 mM amine derivative. Alter-natively, instead of pure Hb solutions, blood was used, diluted withPBS so as to reach an absorbance level equivalent to 24 mM globin.

Ascorbate peroxidase assays (Table 5) follow the protocol pre-viously described [30], where the enzymatic oxidation of ascorbateby hydrogen peroxide in the presence of Hb and acetate buffer ismonitored by UVeVis spectroscopy at 290 nm, where all absor-bance change was due to the ascorbate.

Dioxygen affinity of Hb was measured at room temperature inPBS buffer. The deoxy form of Hb was obtained by purging argon inan anaerobic cuvette. The solution was titrated with different vol-umes of PBS until full saturation with oxygen of Hb.

3. Results and discussion

3.1. Synthesis of PVP dyes

A comparison between the optimized reaction conditionsapplicable in the synthesis of PVP 1e9 (Table 2), emphasizethe advantage of microwave assisted synthesis in dry mediaover the classical convective heating technique: comparablereaction yields were obtained in tremendously reduced (25:1)reaction time under microwaves irradiation in sealed reactionvessel.

The structures of PVP were assigned according to high resolu-tion NMR spectra. In the 1H NMR spectra of each PVP 1e9 thesignals of the vinyl protons appeared highly deshielded and split indoublet with vicinal coupling constants 3J ¼ 15.6 Hz-16.5 Hz,pointing towards geometrical trans-isomers.

-stacking for PVP 3.

Table 3Experimental, computed photophysical and electrochemical data for PVP 1e9.

labs.[nm] lemb [nm] Xo

c [nm] Egopt [eV] ELUMO [eV] EHOMO [eV] EHOMOeLUMO E1/2

d [mV] E [hartree] Ecation radical [hartree]

Exp ( 3M�1 cm�1) Calcd (f)a

1 446 (1.43 � 104) 515.4 (0.58) 610 532.9 2.32 �4.93 �8.64 3.71 853 �1318.747 �1318.422322 (1.40 � 104) 361.3 (0.44)

351.9 (0.12)309.3 (0.24)

2 444 (1.15 � 104) 508.0 (0.57) 631 531.0 2.33 �4.86 �8.60 3.74 848 �1358.046 �1357.722320 (1.12 � 104) 359.1 (0.21)

352.1 (0.33)307.4 (0.23)

3 445 (1.08 � 104) 505.6 (0.55) 630 532.3 2.32 �4.81 �8.56 3.75 862 �1436.637 �1436.314320 (1.07 � 104) 357.9 (0.18)

350.9 (0.35)306.5 (0.22)

4 445 (1.50 � 104) 501.9 (0.56) 643 535.8 2.31 �4.79 �8.57 3.78 889 �1515.227 �1514.905320 (1.47 � 104) 355.8 (0.28)

348.8 (0.26)306.1 (0.22)

5 455 (1.60 � 104) 538.3 (0.69) 749 548.1 2.26 �5.01 �8.53 3.52 836 �1318.749 �1318.428332 (1.40 � 104) 375.0 (0.63)

319.8 (0.09)318.1 (0.12)

6 457 (1.62 � 104) 533.2 (0.71) 648 546.6 2.27 �4.95 �8.50 3.55 842 �1358.049 �1357.730333 (1.40 � 104) 372.4 (0.64)

318.1 (0.12)316.3 (0.10)

7 458 (1.66 � 104) 529.1 (0.73) 668 542.8 2.28 �4.90 �8.47 3.57 848 �1436.641 �1436.322335 (1.42 � 104) 370.5 (0.66)

316.8 (0.16)8 468 (2.31 � 104) 501.1 (1.32) e 553.3 2.24 �4.50 �8.28 3.78 806 �2349.641 �2349.331

331 (1.64 � 104) 350.5 (0.78)272.2 (0.16)

9 470 (2.51 � 104) 488.2 (1.20) e 552.6 2.24 �4.43 �8.28 3.85 835 �2388.936 �2388.627333 (1.70 � 104) 344.3 (0.73)

276.8 (0.13)269.5 (0.17)

a TDDFT.b In solid state excited at 450 nm.c Assuming u2

32 w (hu � Eg)2 [33].d Cyclic voltammetry.

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325320

Single crystals of regioisomers 3 and 7 grown from ethanol weresubjected to X-ray diffraction analysis (Fig. 1), which confirmed theE configuration of the double bond linking the heterocyclic unitswith a measured bond length of 133.8(2) pm and 134.3(3) pmrespectively.

The phenothiazine units show folding angles of 146.57(5)� for 3and 146.9(2)� for 7 (obtained using atoms C(1)eC(6) and C(7)eC(12) for the calculation of least-square planes) and quasi-equatorial orientations of the N-methyl substituent (typical for10-methyl-phenothiazine derivatives [31]). The substituted ben-zene ring and the vinyl bridge are coplanar, while the pyridiniumunit is quasi-coplanar (torsional angle q ¼ 8.29� in 3 and q 18.56� in7 respectively), thus affording a major orbital overlap of the adja-cent p-systems and consequently the delocalization and trans-mission of electron density between the heterocyclic units(electronic coupling through a bridging ligand may be estimated asa function of the twisting and can be rather accurately approxi-mated by a cos q law [32]).

As expected for aromatic ring systems, the intermolecular con-tact distances are dominated by p-stacking interactions for both 3and 7. Fig. 2 illustrates the detectable almost perpendicular “T-shaped” CH/p bonds (with the shortest C/C distances in therange 361e362 pm) and parallel pep interactions (with theshortest distances in the range 350e366 pm for C/C and 344e353 pm for C/N respectively).

3.2. Photophysical properties

3.2.1. UVeVis absorption spectra of PVPThe electronic absorption spectrum of each PVP 1e9 (Fig. 3a)

contains absorption bands situated in the UV region (200e250,322e335 nm) as well as in the visible region (444e470 nm).Bathochromic shifts of 9e14 nm are observable for the visible ab-sorption bands of PVP 5e7 as compared to PVP 1e4, correlatedwiththe chromophore system of 4-substituted pyridinium salts. Sym-metrical PVP 8 and 9 containing two vinyl-phenothiazine chro-mophore units in the molecular structure exhibited a noticeablehiperchromic effect.

Red shifts of the visible absorption band of PVP were recordedwith decreasing of the solvent polarity (e.g. for 3 lmax 445 nm inDMSO is shifted to 470 nm in DCM as shown in Fig. 3b). This weaksolvatochromism suggests a difference in dipoles between theground and the excited states.

The optical band gap (Egopt) of each PVP 1e9 was estimated byextrapolation at the low energy side of the absorption edge (Xo nm)[33] and it appears reasonably correlated with the theoreticalHOMOeLUMO gap as listed in Table 3.

3.2.2. UVeVis emission spectra of PVPUpon excitation with the longest absorption wavelength,

each PVP in solid state exhibited an emission band situated in the

a)

300 400 500 600 7000.0

0.5

1.0

1.5

2.0

2.5 1

2

3

4

5

6

7

8

9Ab

sorb

ance

Wavelenght [nm]

b)

400 500 600 7000.0

0.2

tolueneDCMacetoneDMSOMeOH

Abso

rban

ce

Wavelenght [nm]

Fig. 3. UVeVis absorption spectra of PVP a) 1e9 10�4 M in DMSO. b) 3 10�5 M in different solvents.

200 300 400 500 600 700 800 9000

100200300400500600700800900

10001100

Inte

nsit

y [a

.u.]

Wavelenght [nm]

1

2

3

4

5

6

7

8

9

Fig. 4. UVeVis excitation and fluorescence emission spectra of PVP 1e9 in solid stateat ambient temperature.

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325 321

visible region (610e750 nm as presented in Table 3) with variableintensities, except for 8 and 9 characterized by negligible emissionproperties. As it may be seen in Fig. 4, PVP 3 and 4 containing longalkyl chains produced the most intense fluorescence emissions,may be favored by the weak intermolecular p-stacking in-teractions (as shown Fig. 2 for PVP 3 in). A tighter package of themolecules in the crystals of PVP 7, or the presence of a smallmethyl group in PVP 1, 5, significantly reduced the fluorescenceemissions. Large Stokes shifts were observable in each case as atypical behavior of phenothiazine containing chromophore; thelargest Stokes shift value (9100 cm�1) was recorded in the case of5 (unfortunately for a low intensity emission band). In solutionall PVP exhibited fluorescence emissions with extremely low

a b

Fig. 5. Cyclic voltammograms (recorded in DCM, 20 �C, v ¼ 100 mV/s, electrolyte: nBu4Nferrocene/ferrocenium (Fc/Fcþ) as internal standard, for PVP: a) 1, b) 7, c) 9.

quantum yields (much below 1%), regardless the polarity of thesolvent employed.

3.3. Redox properties

3.3.1. Electrochemical oxidation of PVPElectrochemical data were collected by means of cyclic vol-

tammetry experiments conducted by scanning in the anodic (upto 1.5 V) and cathodic (up to �0.2 V) region on PVP 1e9 dissolvedin dichloromethane. PVP exhibited up to three oxidation peaks asshown in Fig. 5aec. The quasi-reversible oxidation peaks (EpceEpa ¼ 70e120 mV) situated at E1/2 ¼ 806e889 mV (Table 3) areconsistent with the typical redox processes of the phenothiazinecore and also supported by relevant theoretical predictions suchas the shape and energy of the filled frontier orbitals predomi-nantly located on the phenothiazine unit. The enhanced oxida-tion potential values as compared to 10-methyl-10H-phenothiazine (E0/þ1 ¼ 767 mV) are consistent with an electrondonor effect of the phenothiazine moiety. A satisfactory correla-tion between the energy of HOMO and the oxidation potentialcan be observed in Table 3 (e.g. PVP 8 revealed the highest HOMOenergy value (�8.28 eV) and the lowest oxidation potential(806 mV), while PVP 4 characterized by lower HOMO energyvalue (�8.57) exhibited an enhanced oxidation potential(889 mV)).

3.3.2. Biocatalytic oxidationFig. 6 reveals that PVP 1e9 are rapidly oxidized by H2O2 in

the presence of horseradish peroxidase (HRP). For phenothia-zine derivatives, such oxidations are known to lead to radicalcations and eventually to sulfoxides and sulfones [34]. Inter-estingly, the exceptions are precisely PVP 8 and 9 featuring one

c

þPF6�, Pt working electrode, Pt counter electrode, Ag/AgCl reference electrode) using

Table 4UVeVis absorption maxima for the enzyme-oxidized forms of PVP 1e9 in acetatebuffer, pH 5.

PVP lintermediary [nm] lfinal [nm] 3[M�1 cm�1]

1 324; 505 388 3.9 � 104

2 321; 505 387 4.2 � 104

3 322; 505 390 5.9 � 104

4 322; 505 388 5.3 � 104

5 340; 515 402 3.2 � 104

6 345; 515 404 3.4 � 104

7 342; 515 405 4.9 � 104

8 e 381; 586 e

9 e 416 e

Table 5The effect of 1, 5 and 8 on ascorbate peroxidase activity of Hb (*) and on the oxygenaffinity of Hb (**).

PVP Hb (%)* P50 (%)** n**

1 �9.4 (�4) þ3 (�1) 1.62 (�0.02)5 þ7.5 (�0.7) �8 (�8) 1.66 (�0.16)8 þ32 (�1) þ42 (�4) 1.88 (�0.3)

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325322

phenothiazine unit in excess over the other compounds; here,although UVeVis changes can be noted upon enzymatictreatment, these changes do not appear to lead to well-definedoxidation products via isosbestic points and instead, featureslight increases in absorbance throughout the entire domain,indicative of colloidal aggregation; on the other hand, for

Fig. 6. UVeVis spectra collected upon mixing PVP 1e9 (50 mM) with H2O2 (800 mM) and HRPas catalyst instead of HRP (results not shown).

these two compounds the starting UVeVis spectra arealready distinctly different from the single-phenothiazine series1e7.

Fig. 7 illustrates how the initial transition, from 1 to its firstoxidized species, observed in Fig. 6 immediately upon manualmixing, starts on the millisecond timescale and is characterized bya clear isosbestic point. Hence, there appear to be two processesinduced by hemoprotein-catalyzed oxidation on PVP 1e9: a veryfast one (reaction completed in less than 5 s), and an ensuing muchslower one. Table 4 lists maxima and extinction coefficients for thetwo newly produced species in each case.

Fig. 8 reveals that free radical signals can be detected in samplesfrozen immediately after mixing compounds 1, 5 and 8 withperoxidase and H2O2 (20 s). Intense signals were observed for 1 and5, and much smaller signals were recorded for 8. Samples frozenafter 30 min reaction time reveal almost entire loss of the freeradical. These data suggest that the intermediary product ofoxidation, accumulated inw4 s after mixing, is a cation radical. ForPVP 1, mass spectra of the intermediary product (not shown) werefound to be identical to those of the starting compound, whilespectra collected from a reaction mixture aged 30 min, revealed anadditional peak at 254 m/z (C15H12NOSþ), indicative of a pheno-thiazine sulfoxide.

The shapes of the ERP spectra of free radicals obtained from PVP1, 5 and 8 were very similar.

Good agreement between experimental and simulated EPRspectra was obtained by taking into consideration the values ofanisotropic g and A tensors characteristic for nitrogen or sulfurcentered radicals (e.g. experimental and simulated EPR spectragenerated from PVP 5 as presented in Fig. 8). Best fit magnetic

(30 nM), in acetate buffer (50 mM), pH 5. Similar data were obtained using hemoglobin

Fig. 7. UVeVis spectra of 1 (12.5 mM), collected over 4.7 s after mixing with H2O2

(800 mM) and HRP (30 nM), in acetate buffer (50 mM) pH 5.

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325 323

parameters suggested a local magnetic field characterized by anaxial symmetry (AxzAy). The differences between the spectracollected at different reaction time are related to the prevalence ofone of the radical species. For each case, after 20 s reaction time thepredominant species appears to be the nitrogen centered radical.

The autooxidation rate of Hb (Fig. 9a) was almost unaffected by1e9; the ANOVA test gave p ¼ 0.417. In the case of Mb (Fig. 9b) theeffects were stronger for PVP 1, 5 and 8.

Contrary towhat was seen on pure Hb in Fig. 9a, intact red bloodcells are clearly affected by almost all PVP (Fig. 10), suggesting theexistence of pathways leading to oxidative stress and to hemoglo-bin autooxidation but not initiated by direct exclusive interactionbetween Hb and PVP 1e9.

Table 5 illustrates that the ascorbate peroxidase activity andoxygen affinity of Hb are enhanced by 8, but no significantly effect

a)

N centered radical

Fig. 8. Experimental and simulated EPR spectra of radicals generated by biocatalytic oxid

was obtained for 1 and 5. This difference correlates well with thealready noted tendency of 8 to aggregate in the HRP reaction, andsuggests that binding of 8 on the surface of Hb, as due to anincreased hydrophobicity and/or tendency to self-aggregatecompared to the other compounds, may on one hand acceleratethe production of free radicals and on the other hand decrease theaffinity of Hb for oxygen.

3.4. Computational study

3.4.1. DFT rationalization of UVeVis spectraThe optical absorption spectrum was simulated using the time-

dependent DFT method (GGA M06-2x(6-31G(d,p))) taking intoaccount the lowest spin-allowed singletesinglet transitions for theoptimized molecular geometries. Frequency analysis has beenperformed in order to ensure that the optimized geometries aregenuine minima.

An inspection of the electron distribution in the molecular or-bitals of each PVP indicate that the frontier filled orbitals HOMOand HOMO � 1 appear located predominantly on the phenothia-zine unit, whereas the unoccupied molecular orbitals LUMO andLUMOþ 1 are located predominantly on the pyridine core (in Fig.11plots of the frontier molecular orbitals of 1 are depicted).

Table 3 summarizes selected data on computed singletesingletexcitations of PVP1e9 in the rangedetected in theUVeVis absorptionspectra, together with computed energies of frontier molecular or-bitals. In reasonable agreement with experiment (an error of cca60 nm), allowed electronic transitions are predicted in the 490e515 nm and 300e360 nm regions. The lowest energy transition

b)

S centered radical

ation of PVP 5 with H2O2 a) after 20 s reaction time, b) after 30 min reaction time.

Fig. 9. The autooxidation rate of a) Hemoglobin, b) Mioglobin, measured over 4 h, at 37 �C in PBS, in the presence of 50 mM PVP 1e9.

Fig. 10. The autooxidation rate of blood measured over 18 h, at 37 �C in PBS, in thepresence of 50 mM PVP 1e9.

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325324

originating from HOMO/ LUMO is a charge-transfer (CT) transitione hence the extinction coefficient observed experimentally andconfirmedby the relatively large computed oscillator strength (f). Thehigher energy absorptions relying on excitation processes involvingHOMO� 1/ LUMO, HOMO/ LUMOþ 1 and HOMO� 2/ LUMOtransitions respectively, have thesame typeofCTcharacter. Thebandspredicted to appear below 300 nm show significant contributionsfrom intra-phenothiazine p / p* transitions; such contributionsbeing also present in the lower energy bands but distinctly weaker ascompared to the CTexcitations (7 times less for the500-nmband, and2.5 times less for the 300e360 nm bands).

The experimental solvatochromism was also corroborated bythis theoretical approach. The electron distribution in the LUMOwas tentatively assigned to a less polar excited state (with higherelectron density on the pyridinium core) and thus better stabilized

Fig. 11. Plots of frontier molecular orbitals of 1 and maxima for UVeVis absorption bands orlevel of theory.

by the DCM than the more polar ground state represented byelectrons distribution in HOMO.

3.4.2. DFT rationalization of differences in reactivityTable 3 lists the computed energies resulted by DFT geometry

optimizations on PVP 1e9 and on their corresponding cation rad-icals. The electron affinities of the cation-radicals follow a monot-onous trend, and seem to decrease with the size of the system e asexpected. The ionization potentials of PVP 8 and 9 are distinctlylower than those of 1e7, suggesting an increased reactivity towardsHRP-type oxidants. Yet, 8 and 9 show unremarkable changes inUVevis spectra when exposed to HRP, unlike the well-defined in-termediates formed by 1e7. The general increase in absorbanceupon oxidation of 8 and 9, across the entire spectrum, suggestsfacile aggregation into colloidal precipitate ewhich would hamperaccumulation of free radical species in solution.

3.5. Conclusions

PVP dyes can be advantageously prepared by microwave assis-ted synthesis in dry media, which offers a high reaction rate, lessenergy consumption and a convenient way of avoiding the use ofvolatile organic solvents.

The strong absorptions in the UV and Vis regions, the day lightfluorescence emission in solid state and the oxidability of thedescribed PVP, qualify these dyes as good candidates for furtherstudies in materials science; furthermore, their capacity of bindingto proteins and additional prooxidant reactivity point towards ap-plications involving biological activity.

iginating from the lowest spin-allowed singletesinglet transitions computed at TDDFT

L. G�ain�a et al. / Dyes and Pigments 102 (2014) 315e325 325

Acknowledgments

Financial support from the Romanian Ministry of Education andResearch (Grant ID PCCE 140/2008) is gratefully acknowledged.This work was also possible with the financial support of theSectorial Operational Program for Human Resources Development2007-2013, co-financed by the European Social Fund, under theproject POSDRU/107/1.5/S/76841 entitled “Modern DoctoralStudies: Internationalization and Interdisciplinarity”.

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