Synthesis and characterization of electrochemiluminescent ruthenium(II) complexes containing o-phenanthroline and various α-diimine ligands

Talanta 62 (2004) 595–602

Synthesis and characterization of electrochemiluminescent ruthenium(II) complexes containing o-phenanthroline and various ␣-diimine ligands Byeong Hyo Kim a,∗ , Do Nam Lee a , Hae Jin Park a , Jung Hyo Min a , Young Moo Jun a , Se Jong Park b , Won-Yong Lee b,1 a

Department of Chemistry, Kwangwoon University, Seoul 139-701, South Korea b Department of Chemistry, Yonsei University, Seoul 120-749, South Korea

Received 4 April 2003; received in revised form 11 August 2003; accepted 2 September 2003

Abstract A series of o-phenanthroline-substituted ruthenium(II) complexes containing 2,2 -dipyridyl, 2-(2-pyridyl)benzimidazole, 2-(2-pyridyl)-Nmethylbenzimidazole, 4-carboxymethyl-4 -methyl-2,2 -dipyridyl, and/or 4,4 -dimethyl-2,2 -dipyridyl ligands were synthesized and examined as potent electrochemiluminescent (ECL) materials. The characteristics of these complexes, regarding their electrochemical redox potentials and relative ECL intensities for tripropylamine were studied. As found in a 2,2 -bipyridyl-substituted ruthenium(II) complexes, a good correlation between the observed ECL intensity and the donor ability of ␣-diimine ligands was observed, i.e., the ECL intensity of the Ru(II) complex decreased with an increase in the ligand donor ability. The ECL efficiency increased as the number of substitutions of o-phenanthroline (o-phen) to metal complexes increased. © 2003 Elsevier B.V. All rights reserved. Keywords: Electrochemiluminescence; Ruthenium complexes; ␣-Diimine ligand

1. Introduction Recently, the analytical usefulness of electrochemiluminescence (ECL) has been recognized and exploited [1]. Transition metal complex-based ECL has been used for the analysis of a wide range of compounds [2–6] such as oxalate [6], alkylamines [7], amino acids [8–10], NADH [11,12], and organic acids [13,14]. In particular, the ECL observed with the tris(2,2 -dipyridyl)ruthenium, [Ru(bpy)3 2+ ] system is the most intense and the best characterized [15]. ECL emission of the Ru(bpy)3 2+ /amine systems presumably arises from the energetic electron transfer reaction between the electrogenerated Ru(bpy)3 3+ and a strong reducing intermediate formed by the one-electron oxidation of amines [6,7]: Ru(bpy)3 2+ → Ru(bpy)3 3+ + e− Ru(bpy)3 3+ + reductant → [Ru(bpy)3 2+ ]∗ + product [Ru(bpy)3 2+ ]∗ → Ru(bpy)3 2+ + light (610 nm) ∗

Corresponding author. Tel.: +82-2-940-5247; fax: +82-2-942-4635. E-mail addresses: [email protected] (B.H. Kim), [email protected] (W.-Y. Lee). 1 Co-corresponding author. 0039-9140/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2003.09.001

In general, the emission of luminescent transition metal complexes always arises from the lowest excited state, which is either a metal to ligand charge transfer (MLCT) or localized ␲–␲∗ transition [16,17]. The ECL characteristics of transition metal complexes strongly depend on the metals and ligands of the complex. Previously, we examined the effects of ligands in a series of 2,2 -bipyridyl-substituted ruthenium(II) complexes containing different ␣-diimine ligands on ECL intensity and found an interesting relationship between the donor ability of the ligand and the ECL properties of ruthenium(II) complexes [18,19]. A number of ruthenium(II) complexes containing o-phenanthroline (o-phen) and related ligands have been synthesized [20–29] and utilized as fluorescent pH sensor [27], DNA binding reagent [28], and light switch [29]. However, only a couple of research groups have described the ECL behavior of Ru(o-phen)3 2+ and its derivatives. In those reports, Ru(o-phen)3 2+ produced more intense ECL emission than Ru(bpy)3 2+ [20,21], however, the effects of ligands on ECL efficiency have also not been studied comprehensively. Therefore, to develop more sensitive ECL complexes and to study the relationship between ECL intensity and the

596

B.H. Kim et al. / Talanta 62 (2004) 595–602 CH3 H N

N

N

N

N

o-phen

N

H 3C

N

N

bpy

PBIm-Me C H3

N

N

N

PBIm-H

N

dmbpy

H 3C

(CH2)nCOOH

N

N

mbpy-(CH2)nCOOH

Fig. 1. Chemical structures of ligands used in this study.

ligand effect, o-phenanthroline (o-phen) was introduced to ruthenium(II) complexes containing ␣-diimine ligands such as 2,2 -dipyridyl (bpy), 2-(2-pyridyl)benzimidazole (PBImH), 2-(2-pyridyl)-N-methylbenzimidazole (PBIm-Me), 4, 4 -dimethyl-2,2 -dipyridyl (dmbpy), 4-carboxymethyl-4 methyl-2,2 -dipyridyl (mbpy-CH2 CO2 H), and 4-carboxypropyl-4 -methyl-2,2 -dipyridyl (mbpy-(CH2 )3 CO2 H) as shown in Fig. 1. The results show that it may be possible to tune ECL properties by introducing ligands with different donor abilities to metal complexes and synthesis of new ECL labels for immunoassay.

2. Experimental 2.1. Materials All reactions were carried out under a dry nitrogen atmosphere unless otherwise stated. Solvents were purchased and dried by the standard method. Most of the chemical reagents were purchased from Aldrich Chemical Co. and used as received without further purification in most cases. 4-(Carboxymethyl)-4 -methyl-2,2 -bipyridine [30], 4-(3-carboxypropyl)-4 -methyl-2,2 -bipyridine [30], cis-Ru(bpy)2 Cl2 ·2H2 O [31], and [RuCl2 (p-cymene)]2 [32] were prepared according methods in the literature. 2.2. Instrumentation 1H

NMR spectra were recorded on a 300 MHz Bruker instrument. Chemical shifts are reported in ppm relative to residual solvent as an internal standard. GC/MS was recorded on a HP 6890 mass spectrometer and FAB mass was recorded on a JMS-DX303 (JEOL Co.). Infrared spectra (IR) were recorded on a Nicolet 205 FT-IR and UV spectra were recorded on a Shimadzu UV-240 or a Sinco S-3100. Flow injection analysis (FIA) was performed with the ECL detection system described previously [19]. A Gilson Miniplus 3 peristaltic pump was used to deliver a buffered carrier stream. Injection was performed using a standard low-pressure injection valve equipped with a 100 ␮l injection loop. All connecting tube had an inner diameter of 0.25 cm. The potential of the working electrode was con-

trolled by using an EG&G Princeton Applied Research 273A potentiostat. The flow cell was assembled from a conventional LC–EC dual platinum electrode (Bioanalytical Systems) and placed against a transparent Plexiglas window for the detection of ECL emission. A 1.5 ␮m Teflon spacer was inserted between the electrode and the Plexiglas to create a 100 ␮l flow cell volume. In addition to the dual working electrode, a stainless steel counter electrode at the cell outlet and a silver quasi-reference electrode were employed. The assembled flow cell was placed directly in front of the photon multiplier tube (PMT) window. The entire flow cell was enclosed in a light-tight box. The photon multiplier tube was a Hamamatsu Photonics H5784 optical sensor module in conjunction with a linear chart recorder to record the output. Static mode was examined with the same PMT apparatus. The ECL measurement used a conventional three-electrode configuration. A glassy carbon electrode (Bioanalytical Systems) with a 3 mm diameter was used as the working electrode along with the Pt wire auxiliary electrode and the Ag/AgCl (3 M NaCl) reference electrode. 2.3. Experimental conditions In the FIA experiments, the dual platinum electrode was polished prior to each experiment with 0.05 ␮m alumina, sonicated, and rinsed with methanol followed by water. The flow cell was assembled and placed in the FIA system. The buffered carrier stream flow rate was 2.0 ml/min. The working electrode was held at a potential of +1.3 V (versus the Ag quasi-reference electrode). Ru(II) complex solution and tripropylamine (TPA) solutions were prepared in the same 50 mM pH 7.0 phosphate buffer. TPA solutions (1.0 mM) were mixed with 1.0 mM Ru(II) complex solutions (1:1 v/v). A mixture of Ru(II) complex and TPA was injected and passed through the cell. Blank injections were made in all studies. Blanks were prepared by mixing the given concentration of Ru(II) complex solution and the same buffer (1:1 v/v). Corrected ECL signals were obtained by subtracting the ECL signals for blank solutions from the observed ECL signals for TPA. For all studies, the ECL signal was calculated on the basis of the maximum peak height. In the static mode, the working electrode was cleaned prior to each experiment by repeated potential cycling

B.H. Kim et al. / Talanta 62 (2004) 595–602

(0∼+1.2 V) in 0.2 mM sulfuric acid. During the course of the ECL measurement, the potential of the working electrode was cycled from 0.5 to +1.3 V with a scanning rate of 100 mV/s. 2.4. Syntheses 2.4.1. General procedure for the preparation of [Ru(L)(L )2 ](PF6 )2 (L,L = PBIm-H, PBIm-Me, bpy, dmbpy, o-phen) A solution of [RuCl2 (p-cymene)]2 (153.1 mg, 0.25 mmol) and L (0.5 mmol) in ethanol (5 ml) was stirred for 2 h. Next, 10 ml of distilled water containing L (1 mmol) was added to the solution, which was then refluxed for an additional 14–70 h. The reaction was monitored by TLC. After cooling, ethanol was removed under reduced pressure and the residue was treated with a saturated aqueous solution of NH4 PF6 , which gave a red precipitate. The solid was filtered and recrystallized from acetone/ethyl acetate. Dark red crystals were obtained in 69–96% yield. 2.4.1.1. [Ru(o-phen)2 (PBIm-H)](PF6 )2 (1). 80% yield; TLC (sat. NaCl/MeOH) Rf 0.28; UV (acetone) 470 nm; IR (KBr) νmax 3068 (aromatic CH), 1604, 1425; 1 H NMR (300 MHz; acetone-d6 ) δ 5.41 (d, 1H, J = 8.3 Hz, aromatic), 6.56–6.62 (m, 1H, aromatic), 6.97–7.02 (m, 1H, aromatic), 7.20–7.24 (m, 1H, aromatic), 7.59 (d, 1H, J = 8.1 Hz, aromatic), 7.72–7.79 (m, 3H, aromatic), 7.87–7.92 (m, 2H, aromatic), 7.99–8.04 (m, 1H, aromatic), 8.29–8.41 (m, 7H, aromatic), 8.50 (d, 1H, J = 8.1 Hz, aromatic), 8.60–8.77 (m, 5H, aromatic); FAB MS m/z 800 (M-PF6 − -H+ )+ , 655 (M-2PF6 − -H+ )2+ , 476 (Ru(o-phen)(PBIm-H))+ , 461 (Ru(o-phen)2 )+ , 282 (Ru(o-phen)+H+ )+ . 2.4.1.2. [Ru(o-phen)(PBIm-Me)2 ](PF6 )2 (2). 86% yield; UV (acetone) 465 nm; IR (KBr) νmax 3072 (aromatic CH), 2963 (aliphatic CH), 1604, 1511, 1480; 1 H NMR (300 MHz; acetone-d6 ) δ 4.53 (s, 6H, N-methyl), 5.58–5.60 (m, 2H, aromatic), 6.84 (ddd, 2H, J = 1.1, 7.2, 8.4 Hz, aromatic), 7.34 (ddd, 2H, J = 1.1, 7.2, 8.4 Hz, aromatic), 7.47 (ddd, 2H, J = 1.3, 5.7, 8.1 Hz, aromatic), 7.80 (d, 2H, J = 7.8 Hz, aromatic), 7.86–7.90 (m, 2H, aromatic), 8.19–8.28 (m, 6H, aromatic), 8.56 (dd, 2H, J = 1.3, 5.2 Hz, aromatic), 8.73 (dd, 2H, J = 1.3, 8.1 Hz, aromatic), 8.90–8.92 (m, 2H, aromatic); FAB MS m/z 845 (M-PF6 − )+ , 700 (M-2PF6 − )2+ , 490 (Ru(PBIm-Me)(o-phen))+ . 2.4.1.3. [Ru(o-phen)2 (PBIm-Me)](PF6 )2 (3). 69% yield; UV (acetone) 458 nm; IR (KBr) νmax 3087 (aromatic CH), 2924 (aliphatic CH), 1631, 1596, 1425, 1335; 1 H NMR (300 MHz, acetone-d6 ) δ 4.63 (s, 3H, N-methyl), 5.65 (d, 1H, J = 8.4 Hz, aromatic), 6.91 (ddd, 1H, J = 0.9, 7.3, 8.4 Hz, aromatic), 7.37–7.47 (m, 2H, aromatic), 7.71–7.93 (m, 5H aromatic), 8.09–8.11 (m, 1H, aromatic), 8.19–8.25 (m, 1H, aromatic), 8.29 (dd, 1H, J = 1.3, 5.3 Hz, aro-

597

matic), 8.36–8.43 (m, 5H, aromatic), 8.52–8.56 (m, 2H, aromatic), 8.72–8.76 (m, 2H, aromatic), 8.80 (dd, 2H, J = 1.1, 8.4 Hz, aromatic), 8.93 (d, 1H, J = 8.4 Hz, aromatic); FAB MS m/z 816 (M-PF6 − )+ , 671 (M-2PF6 − )2+ , 460 (Ru(o-phen)2 -H+ )+ , 310 (Ru(PBIm-Me))+ , 282 (Ru(o-phen)+H+ )+ . 2.4.1.4. [Ru(o-phen)(dmbpy)2 ](PF6 )2 (4) [33]. 87% yield; TLC (sat. NaCl/MeOH) Rf 0.2; UV (acetone) 455 nm; IR (KBr) νmax 3084 (aromatic CH), 2920 (aliphatic CH), 1619, 1480, 1421; 1 H NMR (300 MHz, acetone-d6 ) δ 2.50–2.60 (m, 12H, methyl), 7.18–7.19 (m, 2H, aromatic), 7.37–7.38 (m, 1H, aromatic), 7.44–7.46 (m, 2H, aromatic), 7.64–7.66 (m, 2H, aromatic), 7.81–7.83 (m, 1H, aromatic), 7.89–7.97 (m, 3H, aromatic), 8.39–8.43 (m, 3H, aromatic), 8.66–8.80 (m, 6H, aromatic); FAB MS m/z 795 (M-PF6 − )+ , 650 (M-2PF6 − )2+ , 466 (Ru(dmbpy) (o-phen))+ . 2.4.1.5. [Ru(o-phen)(bpy)2 ](PF6 )2 (5) [33,34]. 80% yield; UV (acetone) 453 nm; IR (KBr) νmax 3084 (aromatic CH), 1631, 1608, 1464, 1445, 1425; 1 H NMR (300 MHz, acetone-d6 ) δ 7.31–7.36 (m, 2H, aromatic), 7.55–7.61 (m, 2H, aromatic), 7.83–7.91 (m, 4H, aromatic), 8.06–8.23 (m, 6H, aromatic), 8.36–8.40 (m, 4H, aromatic), 8.76–8.82 (m, 6H, aromatic); FAB MS m/z 739 (M-PF6 − )+ , 594 (M-2PF6 − )2+ , 438 (Ru(bpy)(o-phen))+ . 2.4.1.6. [Ru(o-phen)2 (dmbpy)](PF6 )2 (6). 88% yield; UV (acetone) 451 nm; IR (KBr) νmax 3084 (aromatic CH), 2917 (aliphatic CH), 1616, 1476, 1429, 1243; 1 H NMR (300 MHz, acetone-d6 ) δ 2.55 (s, 6H, methyl), 7.27 (dd, 2H, J = 0.9, 5.7 Hz, aromatic), 7.72 (dd, 2H, J = 5.1, 8.4 Hz, aromatic), 7.81 (d, 2H, J = 5.7 Hz, aromatic), 7.98 (dd, 2H, J = 5.1, 8.4 Hz, aromatic), 8.25 (dd, 2H, J = 1.2, 5.1 Hz, aromatic), 8.38 (d, 2H, J = 9.0 Hz, aromatic), 8.42 (d, 2H, J = 9.0 Hz, aromatic), 8.55 (dd, 2H, J = 1.2, 5.1 Hz, aromatic), 8.71 (d, 2H, J = 0.9 Hz, aromatic), 8.72 (dd, 2H, J = 1.2, 8.4 Hz, aromatic), 8.84 (dd, 2H, J = 1.0, 8.3 Hz, aromatic); FAB MS m/z 937 (M+H+ ), 792 (M-PF6 − +H+ )+ , 646 (M-2PF6 − )2+ , 465 (Ru(o-phen)(dmbpy))+ , 461 (Ru(o-phen)2 )+ , 282 (Ru(o-phen)+H+ )+ . 2.4.1.7. [Ru(o-phen)2 (bpy)](PF6 )2 (7) [34]. 96% yield; UV (acetone) 451 nm; IR (KBr) νmax 3087 (aromatic CH), 1631, 1608, 1472, 1445, 1421; 1 H NMR (300 MHz, acetone-d6 ) δ 7.42–7.47 (m, 2H, aromatic), 7.74 (dd, 2H, J = 5.4, 8.4 Hz, aromatic), 7.96–8.04 (m, 4H, aromatic), 8.18 (ddd, 2H, J = 1.5, 6.6, 8.7 Hz, aromatic), 8.26 (dd, 2H, J = 1.2, 5.4 Hz, aromatic), 8.40–8.41 (m, 4H, aromatic), 8.55 (dd, 2H, J = 1.2, 5.4 Hz, aromatic), 8.74 (dd, 2H, J = 1.2, 8.7 Hz, aromatic), 8.83–8.87 (m, 4H, aromatic); FAB MS m/z 763 (M-PF6 − )+ , 618 (M-2PF6 − )2+ , 460 (Ru(o-phen)2 -H+ )+ , 437 (Ru(o-phen) (bpy))+ .

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2.4.2. Preparation of [Ru(o-phen)3 ](PF6 )2 (8) A solution of o-phen (216.3 mg, 1.2 mmol) and [RuCl2 (p-cymene)]2 (122.5 mg, 0.2 mmol) in 15 ml of ethanol–water (1:2) mixture was refluxed for 14 h. After cooling, the solvent was removed under reduced pressure to give a fine red solid, which was treated with a saturated aqueous NH4 PF6 solution to give a red precipitate. Recrystallization in acetone/ethyl acetate resulted in dark red crystals (91%). 2.4.2.1. [Ru(o-phen)3 ](PF6 )2 (8) [33]. UV (acetone) 448 nm; IR (KBr) νmax 3080 (aromatic CH), 1619, 1588, 1584, 1421; 1 H NMR (300 MHz, acetone-d6 ) δ 7.73–7.79 (m, 6H, aromatic), 8.34–8.39 (m, 12H, aromatic), 8.76 (d, 6H, J = 8.3 Hz, aromatic); FAB MS m/z 787 (M-PF6 − )+ , 642 (M-2PF6 − )2+ , 460 (Ru(o-phen)-H+ )+ . 2.4.3. General procedure for the preparation of [Ru(L)2 (L )](PF6 )2 (L = bpy, o-phen, L = mbpy-CH2 CO2 H, mbpy-(CH2 )3 CO2 H) A suspension of L (0.15 mmol) and cis-RuL2 Cl2 ·2H2 O (0.1 mmol) in 10 ml of ethanol–water (1:1) mixture was degassed for 15 min and then refluxed under nitrogen for 3 h. After cooling, ethanol was removed under reduced pressure, the residue was treated with a saturated aqueous NH4 PF6 solution, and a precipitate quickly formed. After filtering and washing with cold water, the product was recrystallized from acetonitrile/ethanol and red crystals were obtained. 2.4.3.1. [Ru(bpy)2 (mbpy-CH2 CO2 H)](PF6 )2 (9). 50% yield; UV (EtOH) 463 nm; IR (KBr) νmax 3400 (OH), 3050 (aromatic CH), 1710 (CO), 1620 (aromatic C=C), 1600, 1480, 1460, 1440, 1420, 1240; 1 H NMR (300 MHz, acetone-d6 ) δ 2.52 (s, 5H, methyl), 7.34–7.36 (m, 2H, aromatic), 7.48–7.55 (m, 4H, aromatic), 7.78–7.80 (m, 4H, aromatic), 7.98–8.01 (m, 2H, aromatic), 8.11–8.18 (m, 4H, aromatic), 8.63 (bd s, 2H, aromatic), 8.74–8.77 (m, 4H, aromatic); FAB MS m/z 787 (M-PF6 − +H+ )+ , 743 (M-2PF6 − -CO2 +H+ )2+ , 597 (M-2PF6 − -CO2 )+ , 413 (Ru(bpy)2 )+ , 257(Ru(bpy))+ . 2.4.3.2. [Ru(o-phen)2 (mbpy-CH2 CO2 H)](PF6 )2 (10). 48% yield; UV (acetone) 448 nm; IR (KBr) νmax 3421 (OH), 3084 (aromatic CH), 2917 (aliphatic CH), 1724 (CO), 1619, 1433, 1406, 1239; 1 H NMR (300 MHz, acetone-d6 ) δ 2.52 (s, 5H, methyl), 7.22–7.24 (m, 1H, aromatic), 7.66–7.77 (m, 4H, aromatic), 7.91–7.97 (m, 2H, aromatic), 8.19–8.21 (m, 2H, aromatic), 8.31–8.39 (m, 4H, aromatic), 8.50–8.51 (m, 2H, aromatic), 8.66–8.69 (m, 4H, aromatic), 8.78–8.81 (m, 3H, aromatic); FAB MS m/z 835(M-PF6 − )+ , 645 (M-2PF6 − -CO2 )+ , 462 (Ru(o-phen)2 +H+ )+ , 281(Ru(o-phen))+ . (11). 2.4.3.3. [Ru(o-phen)2 (mbpy-(CH2 )3 CO2 H)](PF6 )2 77% yield; UV (acetone) 448 nm; IR (KBr) νmax 3445 (OH), 3087 (aromatic CH), 2924 (aliphatic CH), 1724 (CO), 1616, 1429; 1 H NMR (300 MHz, acetone-d6 ) δ 1.89–1.95 (m,

2H, methyl), 2.32 (t, 2H, J = 7.3 Hz, methyl), 2.50 (s, 3H, methyl), 2.85 (t, 2H, J = 7.7 Hz, methyl), 7.23–7.29 (m, 2H, aromatic), 7.66–7.71 (m, 2H, aromatic), 7.74–7.83 (m, 2H, aromatic), 7.90–7.96 (m, 2H, aromatic), 8.19–8.22 (m, 2H, aromatic), 8.32–8.39 (m, 4H, aromatic), 8.51–8.52 (m, 2H, aromatic), 8.67–8.81 (m, 6H, aromatic); FAB MS m/z 864 (M-PF6 − )+ , 718 (M-2PF6 − )2+ , 463 (Ru(o-phen))2+ , 282 (Ru(o-phen))+ .

3. Results and discussion 3.1. Syntheses Since Ru(o-phen)3 2+ and its derivatives are known to produce more intense emission than non-substituted Ru(bpy)3 2+ , it is worth studying Ru(o-phen)3 2+ -related Ru complexes by differentiating those with one or two o-phen ligand(s) from those with the other ligands shown in Fig. 1 to elucidate the ligand-dependency of ECL efficiency in terms of the electronic effect of the ligands. Thus, various novel ruthenium complexes containing o-phenanthroline and/or the other above-mentioned ligands were prepared by the reaction of cis-Ru(o-phen)2 Cl2 ·2H2 O or [RuCl2 (p-cymene)]2 . In addition, by introducing acid-derived bipyridine to ruthenium complexes, novel ECL label complexes such as Ru(o-phen)2 (mbpy-(CH2 )n CO2 H)2+ were synthesized for the use in immunoassays. All of the prepared complexes were fully identified, and their electrochemical and ECL properties were examined and characterized in comparison to those of the well-known Ru(bpy)3 2+ complex. To optimize the reaction conditions, divergent synthetic strategies were used by modifying the solvent system, reaction time and sequence of ligand addition. In most cases, we adopted [RuCl2 (p-cymene)]2 as the starting complex (Scheme 1A) because of its stability and ready availability, while Ru complexes containing acid-functionalized were prepared starting from Ru(bpy or o-phen)2 Cl2 ·2H2 O (Scheme 1B). Thus, Ru(II) complexes containing α-diimine ligands, [Ru(o-phen)n (L)3−n ]2+ (L = PBIm-R, bpy, dmbpy, R = H, Me, n = 1, 2, 3) were obtained through the sequential reaction of [RuCl2 (p-cymene)]2 with L or o-phen in ethanol, H2 O, or H2 O/EtOH, as shown in Scheme 1A. When 1 eq. of L or o-phen was applied to [RuCl2 (p-cymene)]2 in EtOH solution at room temperature, [RuCl2 (p-cymene)(o-phen)] or [RuCl2 (p-cymene)(L)] was obtained quite selectively and used without further purification to introduce other ligands. By refluxing [RuCl2 (p-cymene)(o-phen)] or [RuCl2 (p-cymene)(L)] with 2 eq. of an alternative ligand in aqueous solution followed by NH4 PF6 treatment, [Ru(o-phen)n (L)3−n ]2+ were obtained in yields of 69–96% (Table 1). A symmetrical complex such as [Ru(o-phen)3 ]2+ was obtained in 91% yield by refluxing [RuCl2 (p-cymene)]2 with 3 eq. of o-phen in aqueous EtOH (v/v = 2:1) followed

B.H. Kim et al. / Talanta 62 (2004) 595–602

599

Scheme 1. Table 1 Synthesis of Ru(II) complexes containing o-phen ligand(s) Entry

[Ru(L)n (L )](PF6 )2

L

L

n

Yield (%)a

1 2 3 4 5 6 7 8

[Ru(o-phen)2 (PBIm-H)](PF6 )2 (1) [Ru(o-phen) (PBIm-Me)2 ](PF6 )2 (2) [Ru(o-phen)2 (PBIm-Me)](PF6 )2 (3) [Ru(o-phen) (dmbpy)2 ](PF6 )2 (4) [Ru(o-phen) (bpy)2 ](PF6 )2 (5) [Ru(o-phen)2 (dmbpy)](PF6 )2 (6) [Ru(o-phen)2 (bpy)](PF6 )2 (7) [Ru(o-phen)3 ](PF6 )2 (8)

o-phen PBIm-Me o-phen dmbpy bpy o-phen o-phen o-phen

PBIm-H o-phen PBIm-Me o-phen o-phen dmbpy bpy –

2 2 2 2 2 2 2 3

80b 86b 69b 87 [33] 80 [33,34] 88b 96 [34] 91 [33]

a b

Isolated yield. New complex.

by NH4 PF6 treatment (Table 1, entry 8). The reaction time was regulated based on the disappearance of ligand on TLC. These new complexes are completely soluble in acetone or acetonitrile, but less soluble in ethanol or water. When introducing ligands containing acid derivatives such as mbpy-(CH2 )n CO2 H (n = 1, 3) to ruthenium, mbpy-(CH2 )n CO2 H (n = 1, 3) were synthesized by known methods [30], and cis-Ru(bpy)2 Cl2 ·2H2 O and cis-Ru(1,10-phen)2 Cl2 ·2H2 O were used as starting complexes (Scheme 1B) as mentioned above. The results are summarized in Table 2. By refluxing cis-Ru(bpy)2 Cl2 ·2H2 O Table 2 Synthesis of new Ru(II) complexes containing mbpy-(CH2 )n CO2 H (n = 1, 3) Entry

[RuL2 (mbpy-(CH2 )n CO2 H)](PF6 )2

Yield(%)a

1 2 3

[Ru(bpy)2 (mbpy-CH2 CO2 H)](PF6 )2 (9) [Ru(o-phen) 2 (mbpy-CH2 CO2 H)](PF6 )2 (10) [Ru(o-phen)2 (mbpy-(CH2 )3 CO2 H)](PF6 )2 (11)

50 48 77

a

Isolated yield.

and cis-Ru(o-phen)2 Cl2 ·2H2 O with acid ligand in aqueous EtOH solution (v/v = 2:1) followed by NH4 PF6 treatment, [RuL2 (mbpy-(CH2 )n CO2 H)] (PF6 )2 was recrystallized from acetone/ethyl acetate in yields of 48–77% (Table 2). These acid complexes were expected to be especially useful as ECL immunoassay labels for detecting proteins and oligonucleotides. 3.2. Electrochemical and ECL characteristics 3.2.1. Electrochemical and ECL properties of Ru(II) compounds containing 2-(2-pyridyl)-N-methylbenzimidazole ligand Cyclic voltammograms of all the ruthenium(II) complexes containing o-phenanthroline and 2-(2-pyridyl)benzimidazole ligand were obtained in 50 mM phosphate buffer at pH 7 with a small amount of acetonitrile (<20% (v/v)) showed a quasi-reversible one electron process for Ru(II)/Ru(III) oxidation–reduction with half-wave potentials in the range of 0.92 < E1/2 < 0.99 V versus Ag/AgCl (3 M NaCl)

600

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Table 3 Electrochemical and ECL properties of Ru(II) complexes containing o-phen ligand(s) Entry

Ru(II) cpd

λ (nm)a

Epa (V)b

Epc (V)b

Ep (mV)b

ECL(%)c,d

1 2 3 4 5 6 7 8

[Ru(o-phen)2 (PBIm-H)](PF6 )2 (1) [Ru(o-phen) (PBIm-Me)2 ](PF6 )2 (2) [Ru(o-phen)2 (PBIm-Me)](PF6 )2 (3) [Ru(o-phen) (dmbpy)2 ](PF6 )2 (4) [Ru(o-phen) (bpy)2 ](PF6 )2 (5) [Ru(o-phen)2 (dmbpy)](PF6 )2 (6) [Ru(o-phen)2 (bpy)](PF6 )2 (7) [Ru(o-phen)3 ](PF6 )2 (8)

470 465 458 455 453 451 451 448

0.61 0.94 1.02 1.01 1.12 1.11 1.15 1.16

0.54 0.88 0.96 0.95 1.06 1.05 1.09 1.09

70 60 60 60 60 60 60 70

0 11 28 46 133 94 162 194

± ± ± ± ± ± ±

0.61 1.4 2.5 6.9 4.9 8. 3 9.9

a

Acetone. All potentials were determined at room temperature in 50 mM phosphate buffer (pH 7.0) with a small amount of acetonitrile (<20%, v/v) at a glassy carbon electrode versus Ag/AgCl (3 M NaCl). c ECL relative to [Ru(bpy) ](PF ) (100%). 3 6 2 d Mean ± S.D., n = 5. b

(E1/2 = (Epa + Epc )/2). This behavior was confirmed by the fact that the separation of the anodic and cathodic peak potentials, Ep , and the ratios of anodic to cathodic currents, Ipa /ipc , were close to 60 mV and 1.0, respectively (Table 3). A methyl group on the 2-(2-pyridyl)benzimidazole ligand (PBIm-Me) made the oxidation potential shift anodically by around 410 mV compared to that obtained with Ru(II) complex containing PBIm-H ligand (e.g., [Ru(o-phen)2 (PBIm-H)](PF6 )2 ). This indicates that the addition of a methyl group to the imidazole ligand makes the donor ability of the PBIm-Me ligand weaker than that of PBIm-H, resulting in an anodic potential shift. ECL experiments were carried out in Ru(II) complex solutions containing tripropylamine as a coreactant in the FIA system. ECL emissions were obtained for each of the complexes upon sweeping the potential sufficiently positive to oxidize both the complex and tripropylamine. ECL intensities of Ru(II) compounds containing PBIm-Me ligand were still quite small (Table 3, entries 2, 3; 11, 28%) compared to that of [Ru(bpy)3 ](PF6 )2 complex. 3.2.2. Electrochemical and ECL properties of Ru(II) compounds containing o-phenanthroline ligand The electrochemical properties of Ru(II) complexes containing o-phenanthroline ligand were quite similar to those of the [Ru(bpy)3 ](PF6 )2 complex. However, the half-wave potential of [Ru(o-phen)3 ](PF6 )2 complex was shifted anodically by 10 mV compared to those observed with [Ru(bpy)3 ](PF6 )2 complex [19]. As shown in Table 1, as the number of the o-phen ligands added to the Ru(II) complex increased, the oxidation potential shifted anodically compared to Ru(bpy)3 (PF6 )2 . This relationship indicates that the donor ability of the o-phen ligand is slightly weaker than that of the bpy ligand, resulting in an anodic potential shift. The ECL intensities of the Ru(II) compounds containing the o-phen ligand were significantly greater than those of the corresponding Ru(II) compounds containing the 2,2 -bipyridyl ligand.

3.2.3. Electrochemical and ECL properties of Ru(II) compounds containing 4,4 -dimethyldipyridyl ligand As shown in Table 3, the methyl group on the bpy ligand (e.g., [Ru(o-phen)2 (dmbpy)](PF6 )2 , entry 6) shifted the oxidation potential cathodically by approximately 40 mV compared to that obtained in Ru(II) complex containing bpy ligand (e.g., [Ru(o-phen)2 (bpy)](PF6 )2 , entry 7). A similar tendency was observed with [Ru(o-phen)(dmbpy)2 ](PF6 )2 and [Ru(o-phen)(bpy)2 ](PF6 )2 (entries 4 and 5). Moreover, the cathodic shift was enhanced (110 mV) as the number of dmbpy ligands increased to two. This indicates that the addition of a methyl group to the dipyridyl ligand increases the electron density of the Ru(II) ion relative to that of dipyridyl alone. Thus, the donor ability of the dmbpy ligand is stronger than that of the bpy ligand, resulting in a cathodic potential shift. However, the addition of a methyl group to the bpy ligand reduces the ECL intensity compared to those observed with Ru(II) complexes containing dipyridyl ligand (e.g., Ru(bpy)3 (PF6 )2 complex). 3.2.4. Electrochemical and ECL properties of Ru(II) compounds containing mbpy-(CH2 )n CO2 H (n = 1, 3) ligand Ru(bpy)3 2+ ECL has become important for the detection of proteins and oligonucleotides in immunoassays and DNA probe assays. Since a preliminary ECL immunoassay was reported by the Bard group using a Ru-chelate with an N-hydroxysuccinimide residue (Ru-chelate), which enables protein or DNA to be labeled [35], further studies on new Ru-chelate labels have been reported by IGEN [36] and Perkin Elmer [37]. To identify new ECL labels and to investigate the effect of the ligand on the ECL intensity of Ru-chelates, ruthenium(II) complexes containing 4-carboxymethyl-4 -methyl-2,2-dipyridyl (mbpy-CH2 CO2 H) ligand have been synthesized, in which the carboxylic acid group of the ligand can be coupled with an amino group of a protein or DNA through a conventional coupling reaction such as the 1,3-dicyclohexylcarbodiimide (DCC) reaction [38].

B.H. Kim et al. / Talanta 62 (2004) 595–602

601

Table 4 Electrochemical and ECL properties of Ru(II) complexes containing mbpy-(CH2 )n CO2 H (n = 1, 3) Entry

Ru(II) cpd

λ (nm)a

Epa (V)b

Epc (V)b

Ep (mV)b

ECL (%)c,d

1 2 3

[Ru(bpy)2 (mbpy-CH2 CO2 H)](PF6 )2 (9) [Ru(o-phen) 2 (mbpy-CH2 CO2 H)](PF6 )2 (10) [Ru(o-phen)2 (mbpy-(CH2 )3 CO2 H)](PF6 )2 (11)

463 448 448

1.07 1.07 1.07

0.99 1.01 1.01

80 60 60

85 ± 4.3 136 ± 6.5 169 ± 8.5

a

Acetone. All potentials were determined at room temperature in 50 mM phosphate buffer at pH 7.0 with less than 20% v/v of acetonitrile at a glassy carbon electrode versus Ag/AgCl (3 M NaCl). c ECL relative to [Ru(bpy) ](PF ) (100%). 3 6 2 d Mean ± S.D., n = 5. b

As shown in Table 4, the half-wave potentials of Ru(II) complexes containing mbpy-(CH2 )n CO2 H (n = 1, 3) ligands were alike. However, the ECL intensities of the Ru(II) complexes containing an o-phen ligand were greater than those of Ru(II) complexes containing a bpy ligand. In addition, the ECL intensities increased as the alkyl chain length of mbpy-(CH2 )n CO2 H ligand increased. 3.3. Relationship between the observed ECL intensities and ligand properties of Ru(II) complexes The energy gap law states that radiationless processes become more efficient as the emitting state approaches the ground state [16,17]. In UV absorbance measurements, strong MLCT bands were observed at 440–460 nm for all of the Ru(II) complexes tested. As expected previously [39], the MLCT band energies of [Ru(o-phen)2 L](PF6 )2 decreased as the donor property of the ligand increased. The donor property of the ligands increases in the order of o-phen < bpy < dmbpy < mbpy-CH2 CO2 H ∼ mbpy-(CH2 )3 CO2 H < PBIm-Me < PBIm-H. In addition, it is evident that the ECL intensities of the Ru(II) complexes increase as weaker electron donating ligands are introduced (ECL intensity order; PBIm-H < PBIm-Me < dmbpy < bpy < o-phen). Based on the above electrochemical properties, UV absorbance, and ECL experiments, it is likely that a stronger donor ligand in a Ru(II) complex causes a decrease in the MLCT band energy of the complex (red-shift in UV absorbance), and thus making radiationless processes more efficient, finally contributing, in part, to the decreased ECL emission. However, the raditionless process alone does not determine the ECL efficiencies of the metal complexes. Luminescence life time and quantum yield are all affecting the ECL efficiencies. Therefore, further studies on photoluminescence are under way in this direction.

4. Conclusions We synthesized a series of ruthenium(II) complexes containing o-phenanthroline ligand in good yields, including seven new complexes (1, 2, 3, 6, 9, 10, and 11), and studied relationship between the observed ECL intensities

and ligand properties of Ru(II) complexes. More intense ECL emissions were observed for ruthenium(II) complexes containing 4,4 -dimethyldipyridyl, 4-carboxymethyl-4 -methyl-2,2 -dipyridyl, and o-phenanthroline ligand. Especially, the ligand o-phenanthroline had a greater effect on ECL than 2,2 -dipyridyl. A good correlation was observed between the ECL intensity and the donor ability of ligands as well as the number of substitutions in the complex. The newly prepared Ru(L)2 (mbpy-(CH2 )n COOH)(PF6 )2 (L = bpy, o-phen, n = 1, 3) can be used as a sensitive label for ECL immunoassay and DNA probe assay.

Acknowledgements Financial supports from the Korea Science and Engineering Foundation (R01-2000-00050) and partly from Kwangwoon University in 2003 are greatly acknowledged.

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