Strongly Circularly Polarized Emission from Water-Soluble Eu(III)- and

Apr 7, 2017 - Determination of Equilibrium Constants with the HYPERQUAD Suite of Programs Talanta 1996, 43, 1739– 1753 DOI: 10.1016/0039-9140(96)019...
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Strongly Circularly Polarized Emission from Water-Soluble Eu(III)and Tb(III)-Based Complexes: A Structural and Spectroscopic Study Marco Leonzio,† Andrea Melchior,*,‡ Georgina Faura,‡ Marilena Tolazzi,‡ Francesco Zinna,§,∥ Lorenzo Di Bari,*,§ and Fabio Piccinelli*,† †

Luminescent Materials Laboratory, DB, Università di Verona, and INSTM, UdR Verona, Strada Le Grazie 15, 37134 Verona, Italy Dipartimento Politecnico di Ingegneria e Architettura, Laboratorio di Tecnologie Chimiche, Università di Udine, via Cotonificio 108, 33100 Udine, Italy § Dipartimento di Chimica e Chimica Industriale, Università di Pisa, via Moruzzi 13, 56124 Pisa, Italy ‡

S Supporting Information *

ABSTRACT: Water-soluble Eu(III) and Tb(III) complexes with N,N′-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexaneN,N′-diacetic acid (H2bpcd) have been synthesized and characterized in their racemic and enantiopure forms. The ligand has been designed to bind Ln(III) ions, providing a dissymmetric environment able to solicit strong chiroptical features while at the same time leaving a few coordination sites available for engaging further ancillary ligands. Potentiometric studies show that Ln(III) complexes have a relatively good stability and that at pH 7 the [Ln(bpcd)]+ species is largely dominant. DFT calculations carried out on the (S,S)[Y(bpcd)(H2O)5]+ complexes (the closed-shell equivalents of [Eu(bpcd)(H2O)5]+ and [Tb(bpcd)(H2O)5]+) indicate that the two trans-O,O and trans-Npy,Npy configurations are equally stable in solution and present two coordinated water molecules. This is in agreement with the hydration number ∼2.6 determined by luminescence lifetime measurements on Tb(III) and Eu(III) complexes. A detailed optical and chiroptical spectroscopic characterization has been carried out and reveals that the complexes display an efficient luminescence in the visible spectral range accompanied by a strong CPL activity. A value for glum (around 0.1 on the top of the 546 nm band) for the Tb-based complex has been found. This is one of the highest glum values measured up to now for chiral Tb complexes. These results suggest that in principle Tb(bpcd)Cl is suitable to be employed as a CPL bioprobe for relevant analytes in aqueous media.

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INTRODUCTION Lanthanide complexes (Ln(III) = Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Yb(III)) play a pivotal role in several fields of biomedical sciences, such as medical diagnostics and optical imaging,1−3 luminescent sensing,4−8 and magnetic resonance imaging,9−11 Thanks to time-gated detection, it is possible to isolate the typical long emission lifetimes of Ln(III) ions (Eu(III) and Tb(III) in the millisecond range; Sm(III) and Dy(III) in the microsecond range) from autofluorescence in complex microenvironments such as cells, tissues, and living animals.12 Usually, when a lanthanide complex is designed for biomedical applications, a strong overall luminosity or brightness (B) is required. As B = εΦ, with ε being the molar absorption coefficient and Φ the luminescence quantum yield, sizable brightness values are expected if the ligand strongly absorbs the exciting light and efficiently transfers the excitation energy to the lanthanide ion (antenna ef fect). A proper ligand choice is also required in order to ensure biocompatibility and high values of the intrinsic quantum yield (ΦLn). Stable Ln(III) complexation under physiological conditions must be provided to avoid both © 2017 American Chemical Society

the release of the toxic free metal ion and, at the same time, the intrusion of solvent molecules on the inner coordination sphere, which would increase the nonradiative quenching of the Ln(III) excited state by the multiphonon relaxation process. Luminescent complexes of Eu(III) and Tb(III) in aqueous solution have been extensively exploited in the biomedical field, as their excited states (in particular those of the Tb(III) ion) are less sensitive to nonradiative vibrational quenching caused by high-energy oscillators (such as OH). When the environment around Ln(III) is chiral, the metal may display circularly polarized luminescence (CPL), a chiroptical phenomenon, which is rapidly gaining interest in the literature, thanks to its biomedical and technological applications.13−16 Lanthanides can display highly polarized emission in terms of dissymmetry factors or glum = 2(IL − IR)/(IL + IR) (IL and IR being left- and right-polarized intensities, respectively). High values of the emission dissymmetry factors17 are essential in order to Received: February 16, 2017 Published: April 7, 2017 4413

DOI: 10.1021/acs.inorgchem.7b00430 Inorg. Chem. 2017, 56, 4413−4421

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Inorganic Chemistry ensure practical applications; these values are commonly reached by lanthanides but are almost precluded by nonaggregated, purely organic molecules.18 A further ligand feature is to modulate the Ln(III) environment, in response to the presence of an analyte, thus triggering a spectroscopic signal (luminescence or CPL on/off or change: i.e., optical or chiroptical switches). Induced CPL upon binding to Ln chiral bioanalytes is of particular use to signal selectively the presence of chiral species in solution, such as certain proteins19 or chiral ions: e.g., lactate and sialic acid.14 Recently, Tircsó et al.20 proposed a novel Gd(III) complex with great potential as a contrast agent, characterized by a strong kinetic inertness (superior by 2−3 orders of magnitude in comparison to nonmacrocyclic MRI contrast agents approved for clinical use). They employed the new ligand H4cddadp (N,N′-bis[(6-carboxy-2-pyridyl)methyl]cyclohexanediamineN,N′-diacetic acid) (Chart 1) that is the chiral version of

Chart 2. Eu(III) and Tb(III) Complexes Discussed in This Worka

a

The ligand has been prepared in S,S, R,R, and racemic forms.

ligand protonation and complex formation constants need to be determined. DFT calculations, which have been previously applied successfully to obtain structural and thermochemical data for f-block metal complexes,11,23,24 are used in order to obtain the most probable structures of the complexes in solution.

Chart 1. Ligands Discussed in the Present Work

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EXPERIMENTAL SECTION

EuCl3·6H2O, TbCl3·6H2O, and YbCl3·6H2O (Aldrich, 98%) were stored under vacuum for several days at 80 °C and then transferred into a glovebox. Eu(bpcd)Cl (or Tb(bpcd)Cl or Yb(bpcd)Cl) has been synthesized by adding to a water solution (10 mL) of the ligand (80 mg, 0.19 mmol, as the R,R, S,S, or rac isomer) an equimolar quantity of EuCl3·6H2O (or TbCl3·6H2O or YbCl3·6H2O) (0.19 mmol; 69 mg for Eu, 71 mg for Tb, and 74 mg for Yb salts, respectively). The pH value was adjusted to 6.5− 7 with KOH (0.2 M in water). The solution was stirred overnight at room temperature, and then the water was completely removed by evaporation and ethanol (10 mL) was added. The reaction mixture was stored overnight at 0 °C, and then a white solid was separated by filtration. Ethanol was removed under reduced pressure, and a white solid (Eu(bpcd)Cl, Tb(bpcd)Cl, or Yb(bpcd)Cl) was obtained in good yield. Eu(bpcd)Cl: yield 50%. ESI-MS (scan ES+; m/z): 563 (100%); 561 (92%) ([Eu(bpcd)]+). Anal. Calcd for C22H26EuN4O4Cl·H2O (MW 615.90): C, 42.90; H, 4.58; N, 9.10; O, 12.99. Found: C, 42.83 ; H, 4.55; N, 9.07; O, 12.88. Tb(bpcd)Cl: yield 52%. ESI-MS (scan ES+; m/z): 569 ([Tb(bpcd)] + ), 601 ([Tb(bpcd) (CH 3 OH)] + ). Anal. Calcd for C22H26TbN4O4Cl·H2O (MW 622.86): C, 42.42; H, 4.53; N, 9.00; O, 12.84. Found: C, 42.40 ; H, 4.47; N, 8.93; O, 12.79. Yb(bpcd)Cl: yield 54%. ESI-MS (scan ES+; m/z): 584 (100%); 585 (74%); 583 (65%) ([Yb(bpcd)]+), 616 (100%); 617 (74%); 615 (65%) ([Yb(bpcd)(CH3OH)]+). Anal. Calcd for C22H26YbN4O4Cl·H2O (MW 636.97): C, 41.48; H, 4.43; N, 8.80; O, 12.56. Found: C, 41.44; H, 4.40; N, 8.75; O, 12.50. Elemental Analysis. Elemental analyses were carried out by using a EACE 1110 CHNOS analyzer. Potentiometric Titrations. The NaOH and HCl stock solutions were prepared with Fixanal (Fluka Analytical) and ultrapure water (>18 MΩ cm) from a Milli-Q system (ELGA Purelab UHQ). Stock solutions of Eu(III) and Tb(III) were prepared by dissolving the chloride hexahydrate salts (Sigma-Aldrich) in Milli-Q water. The lanthanide content in the stock solutions was determined by EDTA titration and using xylenol orange as indicator. The ionic strength of all solutions used in this study was adjusted to 0.1 M by using the appropriate amounts of NaCl. Free acid concentrations in lanthanide solutions were checked by Gran’s method.25 The protonation constants of the ligand and the formation constants of its metal complexes were determined via potentiometric titration. The temperature in the titration cell was maintained at 25.0 ± 0.1 °C by means of a circulatory bath. Electromotive force (emf) values were measured with a combined glass electrode (Metrohm Unitrode 6.0259.100) and collected by a computer-controlled apparatus (Amel

H 4 bp e d a ( N , N ′ - b i s [ ( 6 - c a r b o x y - 2 - p y r i d y l ) m e t h y l ] ethylenediamine-N,N′-diacetic acid) (Chart 1). The ligand H4cddadp binds the metal ion in a 8-fold coordination, and one water molecule completes the inner coordination sphere. The high stability of this complex (that presumably should also be found in the Tb- and Eu-based analogues) and the relatively high values of the glum factor shown by several DACH (1,2cyclohexanediamine)-based Tb(III) and Eu(III) complexes (around 0.2)21 led us to investigate the chiroptical activity of the new Eu(III) and Tb(III) complexes 1 and 2, respectively (shown in Chart 2), in which the ligand H2bpcd (N,N′-bis(2pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N′-diacetic acid) is similar to H4cddadp (Chart 1). With the purpose to employ the complexes 1 and 2 as chiroptical probes, the metal ion in the coordination compound should not be saturated from a coordinative point of view, facilitating the analyte−complex interaction. This is the reason the two carboxylic units bound to the pyridine rings have been removed from the design of the ligand backbone (compare the molecular structures of H4cddadp and H2bpcd, Chart 1). In this contribution, the synthesis and optical and chiroptical spectroscopic characterization of complexes 1 and 2 (Chart 2) are presented. As far as biosensing is concerned, the knowledge of the Ln(III) species present at a given pH is key information to understand the nature of the emissive complex.22 Therefore, the 4414

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Instruments, 338 pH Meter). Before each titration, the electrode was calibrated by acid−base titrations with standard HCl and NaOH solutions. The carbon dioxide content in solution was checked by Gran’s method.25 Protonation studies were performed on solutions containing the ligand (typical concentrations (0.8−1) × 10−3 M). Complexation studies with Ln(III) (Ln = Eu(III), Tb(III)) ions were carried out at 1/1 and 2/1 ligand/metal ratios. Protonation and complex formation constants were obtained by processing the experimental data with the Hyperquad26 program. DFT Calculations. As the paramagnetic Eu(III) and Tb(III) complexes are rather difficult to model computationally, the analogues of the diamagnetic Y(III) ion were studied. It has been shown that Y(III) complexes may serve as suitable models for the Eu(III) analogues,27 consistent with the fact that its ionic radius differs from that of Eu(III) ion by 0.05 Å.28 As the ligand is hexadentate and, in principle, the metal ion can be up to 9-coordinated, 5 water molecules were also included in the starting model to obtain minimum energy geometries. For comparison, similar complexes with the larger La(III) ion were also studied. The La(III) ion is 9-coordinated in water,29 while complexes can be up to 10-coordinated;30 therefore this can provide the upper limit to the number of inner-sphere water molecules allowed by the ligand arrangement around the ion. Geometry optimizations of the [La(bpcd)(H2O)5]+ and [Y(bpcd)(H2O)5]+ isomers were carried out at the DFT level under vacuum using the B3LYP31,32 exchange−correlation functional. The 6-31+G(d) basis set was employed for the ligand atoms, while Y(III) and La(III) ions were described by the quasi-relativistic small core Stuttgart−Dresden pseudopotential33 and the relative basis set. This level of theory was previously demonstrated to provide correct geometries and thermochemical properties, maintaining the calculation feasible also with similar complex systems.11,23,34 All final geometries were checked to be minima by vibrational analysis. Solvent effects were included by means of the PCM model.35 All calculations were carried out with the Gaussian09 program.36 ESI-MS. Electrospray ionization mass spectra (ESI-MS) were recorded with a Finnigan LXQ Linear Ion Trap (Thermo Scientific, San Jose, CA, USA) operating in positive ion mode. The data acquisition was under the control of Xcalibur software (Thermo Scientific). A MeOH solution of sample was properly diluted and infused into the ion source at a flow rate of 10 μL/min with the aid of a syringe pump. The typical source conditions were as follows: transfer line capillary at 275 °C; ion spray voltage at 4.70 kV; sheath, auxiliary, and sweep gas (N2) flow rates at 10, 5, and 0 arbitrary units, respectively. Helium was used as the collision damping gas in the ion trap set at a pressure of 1 mTorr. Luminescence and Decay Kinetics. Room-temperature luminescence was measured with a Fluorolog 3 (Horiba-Jobin Yvon) spectrofluorometer, equipped with a Xe lamp, a double-excitation monochromator, a single-emission monochromator (Model HR320), and a photomultiplier in photon counting mode for the detection of the emitted signal. All spectra were corrected for the spectral distortions of the setup. In decay kinetics measurements, a xenon microsecond flashlamp was used and the signal was recorded by means of a multichannel scaling method. True decay times were obtained using the convolution of the instrumental response function with an exponential function and the least-squares-sum-based fitting program (SpectraSolve software package). Circularly Polarized Luminescence. CPL spectra were recorded with a homemade spectrofluoropolarimeter described previously.37 The spectra were recorded in CD3OD or H2O in 2.0−3.0 mM (for Tb(bpcd)Cl) or 8.0 mM (for Eu(bpcd)Cl), under 254 nm irradiation using a 90° geometry between the excitation and the detection directions. Before carrying out the measurements on the single enantiomers, we verified that the racemates provided 0-signal over each spectral window we investigated. NIR-CD Spectra. NIR-CD spectra were recorded with a Jasco J200D spectropolarimeter on a 0.03 M CD3OD solution in a 1 cm cell. The spectra were corrected by subtracting the solvent baseline. The instrument was calibrated using as a standard Yb(DOTMA) solution in H2O.38

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RESULTS AND DISCUSSION Synthesis and Spectroscopic Characterization. The H2bpcd ligand (Chart 1) was synthesized by following a literature procedure.39 The synthesis of the Eu(III) and Tb(III) complexes 1 and 2 (as both ligand enantiomers and as the racemate) was performed in good yields, as described in the Experimental Section, and the purity of the compounds was checked by ESI-MS and elemental analysis. The UV/vis absorption spectrum of the Eu(III) complex (1) in water is shown in Figure 1, together with that of the ligand

Figure 1. UV absorption spectra of the complex 1 (Eu(bpcd)Cl; red circles) and of H2bpcd (black line) in water. [Eu(bpcd)Cl] = [H2bpcd] = 0.1 mM.

(H2bpcd). The band around 260 nm (typical of the π → π* transition of the pyridine rings) shows a molar absorption coefficient (ε) in the range 4000−5000 M−1 cm−1 for both ligand and complex and, as expected, it is red-shifted upon Eu(III) complexation. Moreover, its vibrational progression becomes more evident. The absorption spectra of the ligand and its sodium salt (obtained by deprotonation of the carboxylic units) are identical (data not shown). As the spectrum of the Tb(III) complex is completely superimposable with that of Eu(III), it is not reported. As can be seen from inspection of the excitation and emission luminescence spectra, upon excitation of the ligand (λexc = 265 nm), the typical luminescence of both Eu(III) and Tb(III) ions, stemming from the f−f transitions, was detected (Figure 2). H2bpcd can be considered a good sensitizer of Eu(III) and Tb(III) luminescence. Eu(III) ion is considered a valuable spectral probe, since from its luminescence emission spectrum it is usually possible to identify the number of species and the point symmetry of its environment. In detail, in the case of the 5D0 → 7F0 (0−0) emission transition of Eu(III), both the ground and excited states are degenerate, which results, in principle, in a 1:1 correspondence between the number of peaks in the emission spectrum and the number of distinct Eu(III) ion environments.40−42 In addition, the ligand crystal field is capable of splitting each emission band of the other terms into several components, whose number is directly connected with the point symmetry of the Eu(III) ion.43 The spectrum of Eu(bpcd)Cl (Figure 2a), in water solution, shows the typical profile of the emission from the 5D0 level of the 4415

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Table 1. Observed Excited State Lifetimes for Eu(III) and Tb(III) Complexes τobs (observed lifetime), ms complex

H2O

D2 O

Eu(bpcd)Cl Tb(bpcd)Cl

0.30(1) 0.94(1)

1.70(1) 2.15(1)

coordination environments are expected for Eu(III) and Tb(III) complexes. Species in Solution and Structures of the Aquo Complexes. The protonation constants of H2bpcd obtained from the best fit of the experimental emf data (Figure 3, as an example) are given in Table 2. In the same table, also the values available in the literature for the ligands in Chart 1 are reported.46

Figure 3. Titration curves for the ligand alone (blue ■, 0.95 mM) and in the presence of lanthanide ions in a 1:1 molar ratio (red ◆, Eu, 0.66 mM; green ▲, Tb, 0.64 mM): a = (mol of free NaOH)/(mol of ligand); m = (mol of free NaOH)/(mol of Ln(III) ion). Only some of the experimental points are reported in the plot. Full lines are calculated with the stability constants in Table 2.

Figure 2. Steady-state luminescence excitation (left) and emission (right) spectra of (a) Eu(III) complex (1 mM Eu(bpcd)Cl in water) and (b) Tb(III) complex (1 mM Tb(bpcd)Cl in water). The same emission spectra are obtained upon direct excitation of the metal ions.

The resulting protonation constants for H2bpcd are indicative of two fairly strongly acidic and two weakly acidic sites. The first protonation constant (Table 2) for H2bpcd is slightly higher than that found for the analogous H2bped ligand. This difference in ligand basicity could be due to the structural rigidity of the cyclohexyl backbone in comparison to the more flexible ethylenic backbone and has already been observed when H4cddadp and H4bpeda protonations are compared (Table 2).20 Variable pH UV−vis titration (Figure S2 in the Supporting Information) suggests that the first protonation (log K1 = 9.72) can be assigned to an aliphatic amine group, while the second protonation (log K2 = 5.87) is related to a pyridyl nitrogen as the absorbance at λ 260 nm increases as this species begins to form at pH ∼7. The other protonations involve an acetate and the remaining pyridyl group, as suggested by the continuous increase in the absorbance also below the pH at which the H2bpcd species reaches its maximum concentration. This protonation sequence is in agreement with what was suggested previously for H2bped on the basis of a UV−vis and NMR spectroscopic study.46 The titration curves reported for 1:1 Ln(III):ligand ratios (Figure 3) present similar profiles and show a buffering effect up to 4 equiv of hydroxide. The best fit of all the experimental data shows that only the species [Ln(bpcd)]+ and [Ln(bpcd)(OH)]

Eu(III) ion in a strongly distorted environment, with a dominant 5 D0 → 7F2 hypersensitive band. In addition, the presence of one and three components in the region of 5D0 → 7F0 and 5D0 → 7F1 bands, respectively, calls for the existence of only one Eu(III) environment with C1, Cs, C2 and C2v possible symmetries. As the ligand around the metal ion is chiral, Cs or C2v can be ruled out and the possible point symmetry for Eu(III) in the complex will be C1 or C2. The discussion on the Eu(III) luminescence spectrum will be reclaimed later in the text. The 5D0 and 5D4 excited state lifetimes for Eu(III) and Tb(III), respectively, have been calculated from the luminescence decay curves (Figure S1 in the Supporting Information) in H2O and D2O (Table 1). All of the curves were well fitted to a single exponential function, which would suggest the presence of only one emitting species. By using Horrock’s equation,44,45 it is possible to calculate the number of water molecules in the close proximity of the metal ion (hydration number). In both cases, a hydration number of 2.6−2.7 is found and, for this reason, similar 4416

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Table 2. Protonation Constants (log Kj) for the Ligand H2bpcd and Formation Constants (log β) for the Complexes with Eu(III) and Tb(III) at 25 °Ca reaction L + H ⇆ HL HL + H ⇆ H2L H2L + H ⇆ H3L H3L + H ⇆ H4L L + Eu ⇆ EuL L + Eu ⇆ EuL(OH) + H L + Tb ⇆ TbL L + Tb ⇆ TbL(OH) + H L + Gd ⇆ GdL L + Gd ⇆ GdL(OH) + H

H2bpcd (μ = 0.1 M (NaCl))

H2bpedb (μ = 0.15 M (NaCl))

H4cddadpc (μ = 0.15 M (KCl))

9.72(1) 5.87(4) 2.94(7) 2.22(7)

log Kj 8.67 (8.84) 5.53 (5.63) 3.11 (3.02)