Chiral lanthanide hexaazamacrocycles for circularly polarized luminescence, high relaxivity and magnetic resonance imaging

Abstract

Multifunctional lanthanide complexes have been extensively studied in recent years owing to their widespread applications in physics, chemistry and biology, including quantum information processing, molecular spintronics and theranostics. The multifunctionality includes chirality, luminescence, magnetism, ferroelectricity, fluorescence/magnetic resonance imaging etc. Although various N- and O-donor ligands have shown the ability to synthesize these complexes, Schiff bases are still the most widely used in constructing air-stable species. Herein, we report the facile gram-level synthesis of three pairs of lanthanide macrocycle enantiomers via an in situ [2 + 2] imine condensation with a trivalent lanthanide ion as a template. Eu(III)-based compounds, R/S-Eu and R/S-Eu-Ph₃PO, both show efficient circularly polarized luminescence (CPL) with maximum dissymmetry factors (g_lum) of 0.098 and 0.110, respectively. The latter exhibited stronger emission intensity and a longer luminescence lifetime than the former due to the lower vibrational coordination structures around the Eu(III) ion. Gd(III)-based species, R/S-Gd, possess a relatively high relaxivity of up to 35.04 and 34.09 mM⁻¹ s⁻¹ for R- and S-enantiomers, benefitting from the presence of one coordinated water molecule and abundant intermolecular H-bonds. In addition, the results of the MTT assay and in vitro experiments demonstrated the low toxicity and efficient MRI of Gd(III)-chelates in A549 cells.

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Introduction

Lanthanide macrocyclic compounds have exhibited versatile applications across various fields, including display devices, information storage, magnetic resonance imaging (MRI) and molecular theranostics by virtue of their excellent luminescence properties, enormous magnetic anisotropy, high thermostability/air-stability and facile modification.^1–5 They can be mainly divided into the following families according to the type of organic ligand (Fig. 1): DOTA derivates,⁶ porphyrin,^7,8 phthalocyanine,⁹ crown ether,¹⁰ Schiff-base,¹¹ metallacrown¹² and heteroatom-assisted macrocycles.¹³ Among them, Schiff-base lanthanide macrocycles are probably the easiest to obtain via a metal-template reaction between a primary amine and a carbonyl. In particular, the commercially available chiral primary amines, e.g. (1R,2R)-1,2-diphenylethylenediamine, (1R,2R)-1,2-diaminocyclohexane and (R)-1-phenylethan-1-amine (Fig. 1), facilitate the construction of multifunctional lanthanide compounds.^14,15 Comparatively, chiral DOTA,¹⁶ porphyrin¹⁷ and phthalocyanine¹⁸ usually involve more complicated synthesis and purification. In addition, Schiff-base macrocycles offer a simpler method to construct multiple-decker lanthanide sandwich complexes for magnetic interaction studies compared to phthalocyanine derivates.^19,20

Fig. 1 Representative macrocyclic ligands for lanthanide ions and commercially available chiral primary amines.

For circularly polarized luminescence (CPL), chiral lanthanide compounds with suitable sensitizing ligands usually exhibit a much higher dissymmetry factor (g_lum) than organic molecules by virtue of most magnetically allowed f–f transitions,²¹ which expands the application of lanthanide probes with CPL detection to biological assays.²² Recently, a more comprehensive evaluating tool, i.e. CPL brightness (B_CPL), involving two additional parameters, absorption extinction coefficient, ε and quantum yield, φ, besides g_lum, was proposed to quantify the performance of CPL emitters.²³ Among all chiral lanthanide compounds, Eu(III)-based compounds are the most studied giving the largest g_lum value to date, up to 1.38.²⁴ One of the most efficient strategies to enhance CPL activity is increasing the rigidity of the complex,²⁵e.g. the recently reported heterobimetallic binolate Er(III) complex²⁶ and spinolate Tb(III) complex.²⁷ In addition, substituents, auxiliary ligands and external stimuli can also influence CPL properties, which has been clearly demonstrated by a series of enantiopure Eu(III) macrocycle complexes.²⁸ Both the chiral substituents on the macrocycle scaffolds and external static magnetic fields enabled an evident increase in g_lum. It is worth noting that apart from improving CPL performance, some macrocyclic ligands, e.g. DOTA derivates, also enable the exceptional water solubility of complexes, which is advantageous for their biological applications.

In terms of the size of the macrocycle cavity, penta- and hexa-azamacrocycles exhibited the ability to encapsulate trivalent lanthanide ions in the equatorial plane, giving rise to complexes with high symmetry.^11,29 Recently, such lanthanide macrocycles have received increasing attention in the field of single-molecule magnets (SMMs), making some impressive achievements,³⁰e.g. the current record-holder of the anisotropy barrier for air-stable SMMs, RRRR/SSSS-Dy-D_6hF₁₂.³¹ In 2022, Shanmugam et al. reported a discrete bifunctional macrocyclic Schiff-base Ce(III) complex that combines ferroelectricity with field-induced SMM behavior.³² It is noteworthy that such encapsulations can also prevent the proximity of high-energy oscillators to lanthanide ions to some extent, therefore enhancing the luminescence properties.³³ In addition, since the Gd-DOTA compound was clinically approved as a contrast agent for human use in 1989, macrocyclic Gd(III) chelates have been greatly studied in the MRI contrast agent field due to their improved safety (more kinetically inert than acyclic species) and satisfactory relaxivity.² From a molecular structural point of view, excellent luminescence properties and high relaxivity seem to be contradictory as the latter usually needs inner-sphere coordinated water (vide infra) while the former does not, with the presence of an O–H oscillator accelerating the non-radiative quenching pathways. Herein, we report the one-pot synthesis of lanthanide hexaazamacrocycle enantiomers, [Ln(L^N6)Cl₂CH₃OH][Ln(L^N6)Cl₂H₂O]Cl₂ (Ln = Eu and Gd, L^N6 is the chiral Schiff-base macrocycle derived from the imine condensation between 2,6-diformylpyridine and (1R,2R)/(1S,2S)-1,2-diphenylethylenediamine; R/S-Eu and R/S-Gd). Eu(III) complexes show strong chiroptical properties (|g_lum| = 0.1, B_CPL = 71.45 M⁻¹ cm⁻¹), while Gd(III) analogues possess relatively high relaxivity up to 35.04 and 34.09 mM⁻¹ s⁻¹ for the R- and S-enantiomer, respectively, and exhibit efficient MRI at low concentrations. In addition, another pair of Eu(III) enantiomers, [Eu(L^N6)(Ph₃PO)₂(ClO₄)][ClO₄]₂ (R/S-Eu-Ph₃PO), with the same equatorial macrocycle as R/S-Eu, was also prepared, showing enhanced luminescence and chiroptical properties by virtue of lower-vibrational coordination structures around Eu(III) ions. To the best of our knowledge, this is the first study of chiral hexaazamacrocycles that support both strong circularly polarized luminescence (CPL) and high relaxivity as well as in vitro MRI.

Experimental

Chemicals and synthesis

EuCl₃·6H₂O, GdCl₃·6H₂O, (1R,2R)-1,2-diphenylethylenediamine and (1S,2S)-1,2-diphenylethylenediamine were purchased from Sigma-Aldrich. 2,6-Diformylpyridine was synthesized as reported previously.³⁴ All solvents were commercially available and used as received without any further purification. All experiments were performed under aerobic conditions. R/S-Eu and R/S-Gd were synthesized by the following procedure: LnCl₃·6H₂O (4 mmol) was dissolved in 80 ml of CH₃OH at room temperature and then homochiral diphenylethylenediamine (8 mmol) and 2,6-diformylpyridine (8 mmol) were added. The mixture was heated under reflux for 8 h. After cooling to room temperature, the solvent was removed on a rotary evaporator, affording the target compound as a white solid in quantitative yield. The single crystals suitable for X-ray measurement can be obtained by the slow diffusion of Et₂O into CH₃OH solution at room temperature for several days. R/S-Eu-Ph₃PO was prepared by two steps: firstly, the precursor was synthesized using a similar method to R/S-Eu except that Eu(ClO₄)₃·6H₂O was used; then, 1 mmol (ca. 1.07 g) precursor and 2 mmol Ph₃PO (0.56 g) were dissolved in 25 ml of CH₃OH and then heated at 65 °C for 10 h. After that, the system was allowed to cool to room temperature within 24 h, giving light yellow crystals at the bottom of the vial (yield: 51% based Eu).

Instruments and measurements

Single-crystal X-ray data for R/S-Ln and R/S-Eu-Ph₃PO were collected at 180 K on a Bruker SMART APEX diffractometer (Bruker AXS, Ettlingen, Germany) equipped with graphite-monochromatized Mo Kα radiation (λ = 0.71073 Å). The structures were solved and refined in Olex2 by intrinsic phasing (SHELXT) and full-matrix least-squares on F² (SHELXL), respectively.^35–37 All non-hydrogen atoms were refined with anisotropic thermal parameters. All hydrogen atom positions were calculated geometrically and refined using the riding model. The crystal data have been deposited at the Cambridge Structural Database (CCDC 2313857, 2313859, 2313860, 2313872, 2326392, 2326394, and 2328816†). UV-vis spectra were recorded in moderate rate mode on a SHIMADZU UV-1750 UV-vis spectrophotometer. The photoluminescence excitation and emission spectra and luminescence decay lifetimes were collected on an Edinburgh FLSP-920 at room temperature. The overall luminescence quantum yields were determined using an integrating sphere on a fluorescence spectrophotometer (C9920-2, Hamamatsu Photonics K. K., Japan). CPL and total luminescence measurements were performed on a JASCO CPL-300 with a bandpass of 10 nm. The ¹H-NMR and ¹³C-NMR spectra were recorded at 500 and 126 MHz, respectively, on a Bruker AV. Chemical shifts are given relative to TMS. High-resolution mass spectra (HRMS) were obtained on an IonSpec Ultima 7.0 T FT-ICR-MS (IonSpec, USA) using ESI and MALDI as an ionization method. The static magnetic properties were explored by variable-temperature direct current (dc) magnetic susceptibility measurement in the temperature range of 1.9–300 K and under a 1000 Oe dc field on a Quantum Design MPMS XL-7 SQUID magnetometer. Thermogravimetric analysis (TGA) spectra were recorded on a NETZSCH STA 449F5 instrument in the range of 30–800 °C at a heating rate of 10 K min⁻¹ under N₂ conditions. The relaxation rates of R/S-Gd aqueous solutions with different concentrations were determined on a 1.2 T MRI scanner (Shanghai, China). The cytotoxicity of gadodiamide hydrate and R/S-Gd was assessed using a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the lung cancer cells (A549 cells) were seeded at 10 000 cells per well in a 96-well plate and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U mL⁻¹ penicillin–streptomycin for 24 h. After washing with PBS three times, the fresh culture medium containing various concentrations of gadodiamide hydrate and R/S-Gd was added to the cells and continually incubated for 24 h. Finally, the cells were washed with PBS three times and cultured in 100 μL of fresh medium containing 10 μL of 0.5 mg mL⁻¹ MTT. After 4 h of incubation, the supernatant was replaced with 100 μL of dimethyl sulfoxide (DMSO) and shaken for 10 min. The optical intensity (OD) at 490 nm was read using a PowerWave XS2 microplate reader. The untreated cells served as the control samples, and the cell viability was calculated by dividing the OD of drug-treated samples by the OD of control samples. For in vitro T₁-weighted MRI studies, A549 cells were seeded in a 6-well plate for 24 h and incubated with various concentrations of gadodiamide hydrate and R/S-Gd for another 24 h. After being washed with PBS three times, the cells were detached using trypsin and concentrated via centrifugation (1000 rpm, 5 min). The cells were immobilized using 1% agarose for MRI testing.

Results and discussion

Syntheses, crystal structures and magnetism

R/S-Ln were in situ synthesized via a [2 + 2] imine condensation reaction between (1R,2R)/(1S,2S)-1,2-diphenylethylenediamine and 2,6-diformylpyridine with LnCl₃·6H₂O (Ln = Eu, Gd) as a template in CH₃OH at 65 °C, while R/S-Eu-Ph₃PO were prepared using two steps: (i) Eu(III)-macrocycle precursors were synthesized using Eu(ClO₄)₃·6H₂O as a template; (ii) the coordinated solvent molecules at the axial positions in the precursors were replaced by Ph₃PO (Scheme 1). The crystalline samples suitable for single-crystal X-ray diffraction (SCXRD) were obtained by slow vapor diffusion and slow cooling approaches for R/S-Ln and R/S-Eu-Ph₃PO, respectively. The SCXRD structural analysis suggested that R/S-Ln and R/S-Eu-Ph₃PO crystallized in the chiral polar space group, P2₁, and the chiral non-polar space group, P2₁2₁2₁, respectively (Tables S1–S3†). The asymmetric unit of R/S-Ln contains two independent cationic lanthanide macrocycles while for R/S-Eu-Ph₃PO, only one cationic Eu(III) macrocycle, [Eu(L^N6)(Ph₃PO)₂(ClO₄)]²⁺, was observed. As shown in Fig. 2, Eu(III) and Gd(III) ions are both nine-coordinate and encapsulated in the Schiff-base macrocycle, giving a hula-hoop local coordination geometry determined by continuous shape measurement (Tables S11–S13†). Therefore, all complexes have the same equatorial ligand, but for axial positions, they are occupied by two chlorides and one H₂O or CH₃OH molecule for R/S-Ln and three oxygen atoms from two Ph₃PO and one ClO₄⁻ for R/S-Eu-Ph₃PO, respectively. Given that enantiomeric lanthanide complexes generally show little difference in the first coordination environment, here, we only described the structural characteristics of S-Ln and S-Eu-Ph₃PO. The important structural parameters of these six compounds can be found in Tables S5–S10.† For S-Eu, the average Eu–N bond length is respectively 2.623 and 2.620 Å for Eu1 and Eu2. The Eu–Cl distances are 2.725(3) and 2.678(3) Å for Eu1 and 2.686(3) and 2.716(3) Å for Eu2, respectively. The axial Cl–Eu–Cl bond angles for Eu1 and Eu2 are similar, with a difference of < 3° (Table S8†). It is evident that the largest difference in the coordination environment between Eu1 and Eu2 is the axially coordinated oxygen atoms which come from H₂O and CH₃OH molecules with Eu–O distances of 2.434(6) and 2.518(7) Å, respectively. The Eu1⋯Eu2 distance is found to be 10.862 Å. We found that S-Eu-Ph₃PO possesses slightly longer Eu–N bond lengths, averaged at 2.630 Å, compared to S-Eu, suggesting that the replacement of axial ligands can also influence the equatorial crystal field. The average Dy–O distance from Ph₃PO, 2.309 Å, is shorter than the length of Dy–O_ClO4⁻ of 2.469 Å, due to the strong affinity of Ph₃PO to lanthanide ions, which has also been demonstrated by other luminescent lanthanide compounds with Ph₃PO as the antenna.^33,38 The axial O_Ph3PO–Dy–O_Ph3PO angle is 145.27°, which is comparable to Cl–Eu–Cl angles in S-Eu. For S-Gd, axial Gd–Cl distances are very similar for Gd1 and Gd2, averaging 2.690 and 2.691 Å, respectively. The equatorial Dy–N bond lengths range from 2.578(6) to 2.679(6) Å for Gd1 and 2.571(7) to 2.688(6) Å for Gd2. The axial Gd–O distances, 2.416(5) Å for Gd1–O_H2O and 2.501(6) Å for Gd2–O_CH3OH, are little shorter than those for S-Eu. The Gd1⋯Gd2 distance is 9.669 Å, which is obviously shorter than that of Eu1⋯Eu2. In addition, the molecular packing of the complexes revealed the presence of abundant hydrogen bonds in the lattice, which is beneficial for relaxivity and luminescence (vide infra, Fig. S1†). The static magnetic properties of R/S-Gd were determined (Fig. S2 and S3†), giving the respective χ_MT values at 300 K of 15.41 and 15.20 cm³ K mol⁻¹, which are close to the theoretical value of 15.76 cm³ K mol⁻¹ for two isolated spin-only Gd(III) ions. Fig. 3 and S4, S5† display the perfect mirror-image CD spectra of these three enantiomeric pairs of R/S-Eu, R/S-Gd and R/S-Eu-Ph₃PO in CH₃OH with intense Cotton effects at 255 and 305 nm deriving from the equatorial macrocyclic Schiff-base, demonstrating their chiral nature. It should be noted that these macrocyclic compounds are stable up to 240 °C, as revealed by TGA analysis (Fig. S6 and S7†).

Scheme 1 Synthetic route of R/S-Ln (Ln = Eu and Gd) and R/S-Eu-Ph₃PO.

Fig. 2 The ball-and-stick representation of the crystal structures of S-Eu (a), S-Gd (b) and R/S-Eu-Ph₃PO (c). Color code: orange, Dy; red, O; blue, N; green, Cl; purple, P; white, H.

Fig. 3 CD spectra of R/S-Eu in CH₃OH (c = 1.0 × 10⁻⁵ mol L⁻¹).

Photophysical properties

The excitation and luminescence spectra of S-Eu were collected in CH₃OH solution at 1.0 × 10⁻⁵ mol L⁻¹ at room temperature. Fig. 4 shows the excitation spectrum of S-Eu with a ligand-centered band between 275 and 350 nm when monitoring the transition ⁵D₀ → ⁷F₂ of the Eu(III) ion at 617 nm. It can be clearly seen that two strong bands in the 275–350 nm range are located at the same wavelength in the absorption spectrum (Fig. 4 and S8†). The energy of the triplet state (T₁) located on the ligands was determined to be ca. 20 367 cm⁻¹ by the zero-phonon component in the phosphorescence spectrum of the Gd(III) species (Fig. S8†). This value is 3117 cm⁻¹ higher than the acceptance level of the Eu(III) ion. All these indicated efficient energy transfer from the ligand to the Eu(III) ion and suppressed back energy transfer from the Eu(III) ion to the ligand as the above energy gap is in the optimum range of 2000–5000 cm⁻¹.^8,39–41 Upon exciting the solution at 306 nm, all characteristic emission bands of the Eu(III) ion associated with ⁵D₀ → ⁷F_J (J = 0–5) transitions were clearly observed (Fig. 4). The spectrum was dominated by three bands which correspond to the transitions from the excited state ⁵D₀ to ⁷F₁ (591 nm), ⁷F₂ (612 nm), and ⁷F₄ (702 nm). In addition, it is evident that the emission intensities in H₂O are strongly weaker than those in CH₃OH, which can be expected because more O–H oscillators exist in the former that induce more serious nonradiative quenching processes. Quantum yields and luminescence lifetimes in both solutions were determined, up to 18.5% and 0.65 ms in CH₃OH and 7.2% and 0.33 ms in H₂O (Fig. S9 and S10†). As shown in Fig. 4, S-Eu-Ph₃PO showed similar optical properties and comparable quantum yields (Fig. S11 and S12†) to S-Eu but stronger emission intensity and longer luminescence lifetimes, i.e. 1.37 ms in CH₃OH and 0.38 ms in H₂O, under the same measurement conditions, as non-radiative quenching by O–H vibrations from coordinated water molecules in S-Eu was eradicated via replacing H₂O with Ph₃PO.

Fig. 4 Absorption and excitation spectra (left), emission spectra (middle) and decay curves (right) of S-Eu (top) and S-Eu-Ph₃PO (right) in CH₃OH and H₂O at room temperature, c = 1.0 × 10⁻⁵ mol L⁻¹.

Circularly polarized luminescence spectra of two Eu(III) macrocycle enantiomers were also recorded in CH₃OH solution, which showed near-perfect mirror image signals (Fig. 5). It can be clearly seen that a strong CPL signal was observed at 597 nm, which is common for most chiral Eu(III) compounds and due to the special electronic and magnetic ⁵D₀ → ⁷F₁ transitions.²³ The luminescence dissymmetry factor, g_lum, defined by 2(I_L − I_R)/(I_L + I_R) (where I_L and I_R are the emission intensities of left- and right-circularly polarized light, respectively), and B_CPL, were used to evaluate the CPL properties. As shown in Fig. S13 and S14† for the g_lum plots, S-Eu-Ph₃PO exhibited larger |g_lum| values of the ⁵D₀ → ⁷F₁ and ⁵D₀ → ⁷F₃ transitions than S-Eu. The |g_lum| values of the above two transitions of S-Eu-Ph₃PO increased by 10% compared to those of S-Eu, indicating that (i) the homochiral equatorial macrocycle is mainly responsible for the CPL activity; (ii) the replacement of solvent molecules enhances the molecular rigidity to some extent, therefore improving the CPL properties. The |g_lum| values for the transition of ⁵D₀ → ⁷F₁ are respectively 0.098 and 0.110 for S-Eu and S-Eu-Ph₃PO. The CPL brightness for these two compounds was also calculated and the median B_CPL values, 71.45 and 30.89 M⁻¹ cm⁻¹, for the ⁵D₀ → ⁷F₁ transition (λ_exc 329 nm, λ_em 597 nm) were obtained (Table S15†), which are comparable to Eu(PyBox)TTA₃,⁴² CsEu(hfbc)₄ ⁴³ and Eu(BnMeH22IAM).⁴⁴

Fig. 5 (Left) CPL spectra and total luminescence traced in the background of R/S-Eu (top) and R/S-Eu-Ph₃PO (bottom) at room temperature in CH₃OH solution (c = 1.0 × 10⁻⁵ mol L⁻¹). Excitation at 329 nm. Bandpass: 10 nm; (middle) HR-ESI-MS of R/S-Gd in CH₃OH/H₂O solution, confirming the presence of the Gd(III) macrocycle; (right) ¹H NMR (top) and HMQC (bottom) of the diamagnetic Y(III) macrocycle, R-Y, in CD₃OD.

Relaxivity and in vitro MRI of Gd(III) chelates

Hydrophilic gadolinium(III) contrast agents, often affectionately called “gado” by medical workers,⁴⁵ have greatly expanded the utility of MRI which is a noninvasive clinical tool without ionizing radiation.^46,47 They can shorten the longitudinal relaxation time (T₁) of water protons in their vicinity by virtue of the slow electron relaxations and fast water exchange kinetics of gadolinium(III) ions in the S = 7/2 ground state, therefore affording brighter MR signals.⁴⁸ In the design of novel Gd(III)-based MR contrast agents (GBCAs), there have been several long recognized design principles (vide infra) at the molecular scale for high relaxivity (r₁), mainly involving three intrinsic physical parameters: the hydration number (q), the rotational correlation time (τ_R) and the water exchange rate (1/τ_m).⁴⁹ Other molecular factors, e.g. electron relaxation and the Gd–H(water) distance (r_Gd–H), are usually not considered, as the former contributes little to r₁ in high magnetic fields while the latter is relatively difficult to control via chemical synthesis although it exhibits the inverse six power to T_1m (T₁ of the coordinated water molecule).² According to the following eqn (1)–(4), if a GBCA has q = 0, its relaxivity mainly comes from outer-sphere water molecules that are hydrogen bonded to the GBCA. Under this circumstance, polar groups will play a vital role in transmitting the relaxation.⁵⁰ For GBCAs with q > 0, the contribution of inner-sphere relaxation to r₁ should be considered. Theoretically, the greater the q value, the higher the inner-sphere relaxivity; however, more coordinated water molecules will greatly destroy the stability of chelates, increasing the risk of releasing toxic Gd(III) ions.^2,51 At present, almost all commercially approved GBCAs are macrocyclic chelates with a nine-coordinate Gd(III) ion and q = 1. For τ_R and τ_m, it should be noted that (i) τ_R dominates τ_c and further determines T₁; (ii) for small GBCAs, τ_m ≪ T_1m. Therefore, increasing r₁ can be achieved by slowing down the rotation of the compound. A straightforward and effective way is to increase the molecular weight of the compound by constructing a polynuclear structure or binding to macromolecules, e.g. serum albumin.^3,52,53

As mentioned above, the Gd–O_H2O distances are respectively 2.416(5) Å and 2.424(5) for S-Gd and R-Gd (Table S3†), which are shorter than the values in Gd-DOTA (2.463 Å) and Gd-DPTA (2.448 Å).^6,54,55 In addition, from the packing modes of solid structures shown in Fig. S4,† it was clearly observed that the Gd1-macrocycle interacts with the Gd2-macrocycle via an O–H⋯O–H⋯O–H⋯Cl hydrogen-bond bridge, forming a dimeric structure, and the coordinated water molecule also produces O–H⋯O–H hydrogen bonding interactions with two free CH₃OH molecules. The presence of abundant hydrogen bonds not only promotes second-sphere relaxation but also reduces the rate of molecular rotation.²

r₁ = r^IS₁ + r^OS₁ (1)

Where r^IS₁ and r^OS₁ are inner-sphere and outer-sphere relaxivity, respectively; C is a physical constant; τ_C is a correlation time for magnetic fluctuation and is composed of three parts: τ_R, the water residency time, τ_m and the electron relaxation time, T_1e.

One should be cautioned that although single-crystal X-ray diffraction confirmed the presence of a single aqua ligand that directly bonds to the Gd(III) ion (Fig. 3), the q value should be determined in solution by other physical methods, e.g. electron-nuclear double resonance spectroscopy and luminescence lifetime measurements of isostructural Eu(III) or Tb(III) complexes.^56,57 The luminescence lifetimes of the ⁵D₀ → ⁷F₂ transition for the S-Eu complex in H₂O and D₂O are 0.33 ms and 1.7340 ms, respectively (Fig. 4 and S15†). Therefore, the hydration state for the Gd(III) complex was estimated from the equation proposed by Horrocks et al.⁵⁸ The obtained q value was 2.22, suggesting that the CH₃OH molecule coordinated to Gd2 was replaced by the water molecule.

The above structural characteristics imply a high r₁ for Gd(III) compounds. Before attempting the relaxivity rate characterization, the stability of R/S-Ln in solution was examined by NMR measurements. Given that the chemical shifts of paramagnetic lanthanide compounds span a wide range and are not even observed,⁵⁹ we prepared the analogous Yb(III) complex, R-Y (Tables S4 and S14†). As shown in Fig. 5, the ¹H and ¹³C NMR spectra of R-Y in CD₃OD clearly showed 7 proton resonances and 9 carbon signals, indicating a single species with high symmetry in solution. Integration of the ¹H spectrum gives the formula of the species, [Y(L^N6)Cl₂]⁺. In addition, all ¹H and ¹³C signals were accurately assigned combined with the HMQC spectrum. The HR-ESI-MS of S-Gd showed sharp peaks with +1 charge states, corresponding to Gd(III) macrocycles with different axial ligands (Fig. 5, middle). The MALDI-TOF-MS of the same compound exhibited one set of isotope peaks, indicating the species [Gd(L^N6)Cl₂]⁺ (Fig. S16†). The HR-ESI-MS spectrum of S-Eu-Ph₃PO and the ¹H spectrum of its isostructural Y(III) compound confirmed the presence of only one species of [Eu(L^N6)(Ph₃PO)₂(ClO₄)]²⁺ in solution (Fig. S17†), which is consistent with the solid structure determined by SCXRD. All results and analysis revealed that such macrocyclic compounds possess good solution stability. Fig. 6 and S18† illustrate the concentration-dependent behavior of the longitudinal relaxation rate (1/T₁) of Gd(III) compounds at 25 °C and 1.2 T. A linear fit of the data gives an r₁ value of 35.04 mM⁻¹ s⁻¹ for R-Gd and 34.09 mM⁻¹ s⁻¹ for S-Gd, respectively, i.e. each Gd(III) ion possesses a relatively high r₁, ca. 17 mM⁻¹ s⁻¹. This value is much larger than that of commercially approved Gd(III)-based small molecules and comparable to that of some macromolecular compounds. In order to explore the potential of Gd(III)-based species in T₁-weighted MRI, we selected the commercially available Gd(III) MRI contrast agent, gadodiaminde hydrate, for comparison and first evaluated their cytotoxicities by the MTT assay. The results presented in Fig. S19† clearly show that the viabilities of A549 cells exceed 90% after incubation with various concentrations of R/S-Gd and gadodiaminde hydrate for 24 h, demonstrating that R/S-Gd do not exhibit obvious cell toxicity. Then, in vitro T₁-weighted MRI at 1.2 tesla was performed with A549 cells after incubation with the above three Gd(III) compounds for another 24 h and the results suggest that R/S-Gd possess comparable MR signal values to gadodiaminde hydrate (Fig. 6).

Fig. 6 (a) Relaxivity determined from concentration-dependent T₁-weighted MR measurements at 1.2 T (bottom) of S-Gd at room temperature; (b) T₁-weighted MRI of gadodiaminde hydrate (grey), S-Gd (soft red) and R-Gd (light blue) after incubation with A549 cells at various concentrations of Gd(III). The error bars mean the standard derivation (n = 3).

Conclusions

In conclusion, three pairs of mononuclear lanthanide macrocyclic enantiomers, R/S-Eu, R/S-Gd and R/S-Eu-Ph₃PO, were prepared via a facile [2 + 2] imine condensation with lanthanide ions as the template. Their enantiomeric relationship was verified by the mirror-symmetric solid structure determined from SCXRD and CD spectra. S-Eu exhibited brilliant photoluminescence and efficient CPL with an emission quantum yield, luminescence lifetime and luminescence dissymmetry factor of 18.5%, 0.65 ms and 0.098, respectively, in CH₃OH solution at room temperature, while isostructural Gd(III) chelates displayed one of the highest relaxivities to date, ca. 17 mM⁻¹ s⁻¹ per Gd(III) ion, low cell toxicity and efficient MRI at low concentrations. By employing Ph₃PO to replace H₂O and CH₃OH molecules located at the axial positions of S-Eu, a longer luminescence lifetime was achieved in S-Eu-Ph₃PO, i.e. 1.37 ms in CH₃OH solution, benefitting from the eradication of non-radiative quenching by high energy O–H vibrations in the above solvent molecules. Biodistribution studies and in vivo MRI of Gd(III) species are underway.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

We thank the National Natural Science Foundation of China (22201279 and 22171100) for financial support.

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Footnote

† Electronic supplementary information (ESI) available: Crystallographic data, quantum yield, magnetic characterization etc. CCDC 2313857 (S-Eu), 2313859 (R-Eu), 2313860 (R-Gd), 2313872 (S-Gd) 2326392 (R-Eu-Ph₃PO), 2326394 (S-Eu-Ph₃PO) and 2328816 (R-Y). For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4qi00275j

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