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The macrocyclic ligand 3,6,9-tris(methylenebutyl phosphonic acid)-3,6,9-15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene (PCTMB) was synthesized and complexes of Eu3+, Tb3+, and Gd3+ studied by X-ray crystallography, luminescence, and relaxometry. In the crystal these complexes are dimeric and possess 8-coordinate Ln3+ centers that are linked by bridging phosphonates. The rigidity introduced by the pyridyl nucleus forces the EuPCTMB and TbPCTMB to adopt a twisted snub disphenoid (TSD) coordination geometry. Examination of the 5D0 → 7F0 luminescent transition of EuPCTMB in the solid state confirmed the existence of a single distinct Eu3+ coordination environment, whereas two Eu3+ coordination environments were observed in aqueous solution. Lifetime analysis of aqueous TbPCTMB solutions determined that q = 0.1 and q = 1.0 for the two coordination environments and Stern-Volmer quenching constants (KSVτ = 1101 M−1, KSVΦ = 40780 M−1) support the presence of a monomer/dimer equilibrium. Relaxivity studies of GdPCTMB in H2O/CH3OH exhibited a concentration dependency (0.02 mM –10.00 mM) ranging from r1 = 7.0 mM−1s−1 to 4.0 mM−1s−1 consistent with the trend observed by luminescence.
The lanthanide series of metal ions possess a diverse array of physical properties that have been the source of many technological innovations over the years.1 In particular, lanthanides have become invaluable components for contrast enhancement media used in nuclear medicine, magnetic resonance imaging and as optical probes. Equally important for advancing their use in medicine has been the molecular architecture of tailored ligand systems that can provide an optimal coordination environment for maximum stability; a requirement for rendering the metal ion inert and non-toxic for in vivo applications. Furthermore, the organic ligand framework can function to enhance photo-physical properties2–5 and modulation of water exchange kinetics in the chelate structure.6–8 For biological applications, the cumulative body of knowledge has clearly demonstrated that the high thermodynamic and kinetic inertness of chelates formed between Ln3+ ions and polyaza-macrocyclic ligand systems is a paramount feature for safeguarding long term chelate integrity.9 Of considerable interest in this regard is the family of chelates derived from 1,4,7,10-tetraazacyclododecane (cyclen) which is a familiar signature of many MRI and radiopharmaceutical contrast agents in clinical use and under development.
The versatility of cyclen-based ligands for lanthanide coordination is evidenced by the many related anologs present in the literature that are designed for enhancing various aspects of performance through structural “fine tuning”. A noteworthy example reported over 25 years ago is the 12-membered tetraaza-macrocyclic ligand which incorporates a pyridine nucleus within the macrocyclic ring, pyclen.10 The aminocarboxylic acid derivative of pyclen, PCTA, is known to form stable lanthanide chelates (log KLnL = 20.39),11 its Gd3+ chelate was also considered as a neutrally charged, general perfusion MRI contrast agent.12 Surprisingly, however, these fascinating derivatives have received far less attention than their cyclen-based counterparts. Recently pyclen-based ligands have been rediscovered through several reports that survey the effect of structural morphology upon water exchange kinetics in lanthanide chelates; a potentially useful tool for optimizing the performance of MR contrast media.13–15 In addition, these same ligands have been found to possess rapid chelation kinetics under very mild conditions thus stimulating renewed interest for nuclear medicine applications where the time required for complexation to occur is an important concern.11
Our interest in the pyclen family of chelates stems from their unique multi-modal imaging potential and the ability to control in vivo tissue targeting through alterations of ligating functionality. In particular it has been demonstrated that incorporation of a phosphonate ester ligating functionality provides an efficient means of altering biodistribution properties and for selective targeting of cancer.16 In the case of highly lipophilic phosphonate chelates the formation of non-covalent dimers has been inferred to be an important aspect of the cellular targeting process. To gain a better understanding of the chemistry responsible these observations we undertook a study of the solid state and solution behaviour of a pyclen-phosphonate chelate which has been suggested to dimerize in aqueous media. Herein is presented a detailed analysis of the solid state crystal data and solution dynamics that are relevant to the design of targeted diagnostic and therapeutic agents structured around the lanthanide chelate.
All solvents and reagents were purchased from commercial sources and used as received unless otherwise stated. 1H, 13C and 31P NMR spectra were recorded on a Varian Mercury or Bruker Avance spectrometer operating at 299.99, 75.43 and 121.44 MHz, respectively. Infrared spectra were recorded on a Perkin Elmer 1600 Series FTIR. 3, 6, 9, 15-tetraazabicyclo[9.3.1] pentadeca-1(15), 11, 13-triene (pyclen) was prepared using previously published methods.10
Paraformaldehyde (1.2 g, 38.2 mmol) was added to a solution of 3, 6, 9, 15-tetraazabicyclo[9.3.1]pentadeca-1(15), 11, 13-triene (2.5 g, 12.1 mmol) in THF (30 mL) and the resulting slurry stirred at ambient temperature for 30 minutes. Tri-n-butyl phosphite (9.6 g, 38.2 mmol) was then added and the turbid reaction mixture stirred for an additional 72 hours at ambient temperature. The resulting homogeneous reaction mixture was concentrated in vacuo to give a pale yellow, viscous oil (10.0 g). The crude product was purified by column chromatography on basic alumina (5 × 26 cm) eluting with chloroform. Two fractions were collected (2 column volumes) beginning with the second column volume of eluent. After concentration in vacuo the product was isolated as colorless oil (9.06 g, 92%). 1H NMR (300 MHz, CDCl3) δ = 0.87 (m, CH3, 18H), 1.35 (m, CH2, 12H), 1.59 (m, CH2, 12H), 2.62 (m, CH2, 4H), 2.74 (m, CH2, 10H), 3.05 (d, CH2, 2JPH 10 Hz, 4H), 4.01 (m, OCH2, 12H), 7.17 (d, 3-Ar, 3JH-H 8 Hz, 2H), 7.58 (t, 4-Ar, 3JH-H 8 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ = 13.5 (CH3), 13.52 (CH3), 18.6 (CH2), 18.7 (CH2), 32.2 (d, CH2, 3JPC 6 Hz), 32.5 (d, CH2, 3JPC 6 Hz), 50.1 (d, CH2, 2JPC 8 Hz), 51.2 (d, CH2, 1JPC 157 Hz), 51.2 (d, CH2, 2JPC 10 Hz), 51.9 (d, CH2, 1JPC 157 Hz), 60.8 (CH2), 65.6 (d, CH2, 3JPC 6 Hz), 67.2 (d, 2JPC 6 Hz), 123.0 (3-Ar), 137.0 (2-Ar), 157.2 (4-Ar); 31P[1H] NMR (121.44 MHz, CDCl3) δ = 24.94 (2P), 24.96 (1P).
m/z: (ESI+); 826 (100% [M + H]+.
A solution of the hexa-ester 1 (9.06 g, 11.36 mmol) and KOH (7.65 g, 136.3 mmol) in water (100 mL) and 1,4-dioxane (30 mL) was heated to reflux for 18 hours. The reaction mixture was then cooled and filtered while warm. The filtrate was concentrated to give a solid which was suspended in 5:1 v/v CHCl3/MeOH (200 mL) and heated to reflux. The hot solution was filtered and the solvent removed from the filtrate in vacuo to afford an off-white solid. The residue was taken up into CHCl3 (60 mL) with stirring followed by the addition of CH3CN in small portions until the solution became slightly turbid. Upon cooling with continued stirring in an ice bath a white precipitate was observed. The precipitate was isolated by filtration and washed with CH3CN. After drying in vacuo the title compound was isolated as a colorless solid (4.14 grams, 49%). mp: 150–160 °C, dec.; 1H NMR (300 MHz, D2O) δ = 0.78 (m, CH3, 9H), 1.22 (m, CH2, 6H), 1.43 (m, CH2, 6H), 2.47 (m, CH2, 6H), 2.81 (m, CH2, 10H), 3.65 (d, CH2, 2JPH 10 Hz, 4H), 4.84 (m, OCH2, 6H), 7.22 (d, 3-Ar, 3JH-H 8 Hz, 2H), 7.63 (t, 4-Ar, 3JH-H 8 Hz, 1H); 13C NMR (75 MHz, D2O) δ = 15.9 (CH3), 21.3 (CH2), 35.2 (CH2), 35.2 (CH2), 52.0 (CH2), 52.9 (CH2), 55.9 (d, 1JP-C 144 Hz), 62.5 (CH2), 67.2 (d, -OCH2, 2JP-C 6 Hz), 126.3 (3-Ar), 141.7 (2-Ar), 159.7 (4-Ar); 31P[1H] (121.44 MHz, D2O) δ = 20.59; IR υmax/cm−1: 1211 (P=O), 1068 (P-O-C); m/z: (ESI+); 695 (100% [M + H3 + K]+; Anal. Found C 35.4 H 5.9 N 5.7, C26H48K3N4O9P3•2.5CH3OH•2KOH requires C. 35.5 H 6.3 N. 5.8.
K3PCTMB (352 mg, 0.4 mmol) was dissolved in water (5 mL) and the strongly basic solution adjusted to pH 5 via drop-wise addition of 6N HCl. An aqueous LnCl3 solution (0.4 mmol in 1.5 mL) was then added to the ligand in 200 μL aliquots. The reaction was stirred throughout and the pH maintained close to 5 by addition of a 1N KOH solution. Following the addition of each LnCl3 aliquot, chelation progress was monitored by RP- HPLC (Phenomenex PRP-1 C18 column (4.6 × 250 mm), 1 mL/min, 80/20 v/v methanol:water, λ = 266 nm; ligand tR = 6 min., chelate tR = 10 min.). When all the free ligand was found to be consumed the aqueous solution was filtered through a 0.2 μm syringe filter and lyophilized to give the chelate as a flocculent white solid. Each chelate was then dissolved in a minimum of boiling water and, upon cooling was found to crystallize as a colorless solid.
m/z: (ESI+); 807 (100%, [EuLH]+, an appropriate isotope pattern was observed); IR υmax/cm−1: 1234 (P=O), 1054 (P-O-C); Anal. Found: C 34.9 H 6.1 N 6.2 C26H48N4O9 KP3Eu•3H2O requires C 34.8 H 6.1 N 6.2.
m/z: (ESI+); 812 (100% [GdLH]+, an appropriate isotope pattern was observed); IR υmax/cm−1: 1235 (P=O), 1054 (P-O-C); Anal. Found: C 35.2 H 6.2 N 6.3 C26H48N4O9 KP3Gd•2H2O requires C 35.3 H 6.0 N 6.3.
m/z: (ESI+); 813 (100% [TbLH]+, an appropriate isotope pattern was observed); IR υmax/cm−1: 1234 (P=O), 1054 (P-O-C); Anal. Found: C 34.5 H 6.0 N 6.1 C26H48N4O9 KP3Tb•3H2O requires C 34.5 H 6.0 N 6.2.
A Leica Z microscope was used to identify a suitable colorless needle 0.3mm × 0.08mm × 0.03mm from a representative sample of crystals of the same habit. The crystal was coated in a cryogenic protectant (paratpne), and was then fixed to a loop which in turn was fashioned to a copper mounting pin. The mounted crystal was then placed in a cold nitrogen stream (Oxford) maintained at 110K.
A BRUKER SMART 1000 X-ray three-circle diffractometer was employed for crystal screening, unit cell determination and data collection. The goniometer was controlled using the Smart software suite (Microsoft operating system). The sample was optically centered with the aid of a video camera such that no translations were observed as the crystal was rotated through all positions. The detector was set at 5.0 cm from the crystal sample (CCD-, 512×512 pixel). The X-ray radiation employed was generated from a Mo sealed X-ray tube (Kα = 0.70173Å with a potential of 50 kV and a current of 40 mA) and filtered with a graphite monochromator in the parallel mode (175 mm collimator with 0.8 mm pinholes).
Dark currents were obtained for the appropriate exposure time of 10 sec and a rotation exposure was taken to determine crystal quality and the X-ray beam intersection with the detector. The beam intersection coordinates were compared to the configured coordinates and changes were made accordingly. The rotation exposure indicated acceptable crystal quality and the unit cell determination was undertaken. Forty data frames were taken at widths of 0.3° with an exposure time of 10 seconds. Over 200 reflections were centered and their positions were determined. These reflections were used in the auto-indexing procedure to determine the unit cell. A suitable cell was found and refined by nonlinear least squares and Bravais lattice procedures and reported. The unit cell was verified by examination of the hkl overlays on several frames of data including zone photographs. No super-cell or erroneous reflections were observed.
After careful examination of the unit cell a standard data collection procedure was initiated. This procedure consists of collection of one hemisphere of data collected using omega scans, involving the collection over 2400 0.3° frames at fixed angles for , 2θ, and χ (2θ = −28°, χ = 54.73°), while varying omega. Each frame was exposed for 20 sec and contrasted against a 20 sec. dark current exposure. The total data collection was performed for duration of approximately 15 hours at 110 K. No significant intensity fluctuations of equivalent reflections were observed. After data collection the crystal was measured carefully for size, morphology and color.
Steady state and lifetime measurements were performed on an Edinburgh Instruments FL/FS900CDT Fluorometer equipped with a 450W xenon arc lamp and a 100W μF 920H flash lamp. High resolution emission spectra (ΔJ = 0) were recorded from 578.5–582.0 nm using a 0.05 nm emission monochromator step size (λex= 270 nm). Full emission spectra were recorded from 525–725 nm using a 0.25 nm step size. For Stern-Volmer static and dynamic quenching constant measurements λex= 270 nm, λem= 616 nm. Solutions of Eu-PCTMB were prepared at 0.01 mM with varying quencher concentration (Nd-PCTMB) ranging from 0.0 mM – 0.04 mM. All solutions were stirred for one hour at 60 °C then equilibrated for 4 hours at room temperature prior to measurements.
Pyclen was synthesized according to procedure described by Stetter et al.10 Subsequently, pyclen was reacted with paraformaldehyde and tributyl phosphite in THF to afford the hexa-butyl phosphonate 1. Selective hydrolysis of the ester intermediate to the mono ester was achieved under basic hydrolysis conditions using 1 equivalent of KOH per phosphonate in dioxane and water. The hydrolysis reaction was followed using 31P NMR by monitoring the disappearance di-alkyl phosphonate ester resonances at 25 ppm and the appearance of the mono-ester resonances at approximately 20 ppm. Upon completion the reaction mixture was concentrated and the crude product crystallized as the potassium salt from MeOH/CH3CN. Lanthanide chelates were then prepared by acidifying an aqueous solution of K3PCTMB (pH 5) followed by aqueous LnCl3 in small aliquots. The pH was maintained between 5 and 6 by addition of small amounts of a KOH solution and the chelation reaction was monitored by RP-HPLC. When all the free ligand was consumed the aqueous solution was freeze dried and crystallized from hot water to afford X-ray quality crystals.
X-ray diffraction of single crystals of Eu1 and Tb1 revealed that both chelates crystallized in the P space group (Table 1). The two chelates are isostructural and have 2 chelate molecules and 9 waters of crystallization in the unit cell. The PCTMB ligand occupies 7 of the 8 coordination sites of each metal ion with water excluded from the inner coordination sphere by a fourth coordinating phosphonate monoester. The source of this eighth ligand is the neighbouring chelate molecule of the unit cell in much the same way as is observed for the analogous cyclen-based systems 2 and 317, 18 wherein the metal ions of Eu2 and Tb3 are sandwiched between 4 nitrogen and 4 oxygen donor atoms (Figure 1). Although superficially this appears similar to many macrocyclic octa-coordinate Ln3+ chelates of cyclen-based phosphonate (DOTP)19 and phosphinate ligands, Ln2-6,20–22 there are significant differences between the coordination environment in those chelates and the chelates of PCTMB. These differences stem primarily from the nature of the macrocyclic ring. Cyclen adopts a square conformation, defined by Dale’s nomenclature23 as [3,3,3,3], in which the nitrogen atoms are located on the sides of the ring and each ethylene bridge adopts a gauche conformation.24–26 It has been observed that if the conformation of cyclen is distorted into a another conformation significant loss of chelate stability can sometimes result.27 In the case of pyclen, however, the rigidity of the pyridine group dictates that one side of the macrocycle must incorporate 4 bonds and so a [3,3,3,3] conformation is impossible. Instead the macrocyclic ring adopts a [4,2,4,2] ring conformation with the nitrogen atoms located in the centre of each side (Figure 2). The structure of the pyclen ring in Eu1 (Figure 1) closely resembles that reported previously for the macrocycle alone.28
The incorporation of the pyridyl group into the macrocycle also has one further consequence for the chirality of the macrocyclic ring. Cyclen may adopt one of two conformations, each of which is chiral; either (δδδδ) or (λλλλ) conformation may be adopted according to the helicity of its ethylene bridges. Each ethylene bridge in cyclen adopts the same helicity as the others within the cyclen ring. These two conformations of cyclen may interconvert through a ring flipping motion. In contrast, the conformation of each N-C-C-N bridge in pyclen alternates around the ring, such that pyclen adopts a (δλδλ) conformation. Because (δλδλ) pyclen is the mirror image of (λδλδ) pylcen (Figure 2), pyclen is achiral. This means that the chelates of pyclen have one fewer elements of chirality than their related cyclen-based chelates. This is significant because pyclen, once it has become part of a chelate, is unable to flip its conformation, as cyclen can, owing to the presence of the fused pyridyl ring. This means that despite being structurally rigid in a chelate, pyclen, unlike rigid cyclen derivatives,29–31 adopts one major low energy conformation.
It is informative to compare the structures of EuPCTMB and TbPCTMB with those of the cyclen-based chelates structures Eu2 and Tb3; some selected geometrical parameters of these chelates are presented in Table 2. Comparing these structures it can be seen that the two ethylene bridges of pyclen adopt gauche conformations, comparable with the cyclen derivatives. On the opposite side of the ring however, pyclen is much more strained, a consequence of including pyridine in the system. Because of the planar arrangement of the pyridine ring and its substituents in the 2- and 6- positions, the N-C-C-N torsion angles on this side of the macrocycle are approximately half that of a gauche conformation. The result is that the nitrogen atoms in the 4- and 10- positions are oriented below the level of those in the 1- and 7- positions. In contrast to cyclen, the nitrogen atoms of pyclen are not co-planar, lying about 0.4 Å above or below the mean plane (Table 2). Not surprisingly the nitrogen of the pyridine ring (N-1) lies furthest from this plane. This in turn distorts the donor oxygen atom plane of EuPCTMB and TbPCTMB by a similar amount. As a consequence, the chelates of PCTMB are unable to assume the twisted square antiprismatic (TSAP) coordination geometry adopted by the chelates of Eu2 and Tb3. Instead the EuPCTMB and TbPCTMB are found to adopt a twisted snub dispheniod (TSD) coordination geometry (Figure 3). In so doing, the chelates of PCTMB are able to maintain metal-donor atom bond distances comparable to those observed in the chelates Eu2 and Tb3.
Although the coordination geometry of EuPCTMB and TbPCTMB are different from those of the cyclen based phosphinate and phosphonate derivatives 2-6 the phosphonate groups remain successful at excluding water from the inner coordination sphere. Lukeš and co-workers32 have suggested that a critical parameter in achieving this goal is the O-M-O bond angle, β (Table 3). If this angle becomes tighter than 136° then the vacant coordination site on the metal ion becomes too sterically encumbered to accommodate a water molecule. The parameters collected in Table 3 seem to confirm this observation with angles (β) significantly smaller than 136° observed for all q = 0 chelates; a category that includes all the phosphonate and phosphinate derivatives. Indeed the narrowest O-M-O angles (β) of all are observed in the cases of EuPCTMB and TbPCTMB and arise from the different coordination geometry observed in these chelates. Although the LnPCTMB chelates have the smallest β angles, of all the chelates collected in Table 3 they also have the largest β′ angles, notably the O-M-O angles are much larger than the critical 136° required by Lukes et al. for water coordination, and yet these chelate remain q = 0. These large differences in O-M-O angles are partly the result of the [4,2,4,2] ring conformation observed in these chelates that also results in a very narrow N-M-N bite angle, which, it should be noted, is not entirely symmetrical. The presence of the pyridine ring results in one side of the macrocycle being brought closer to the metal ion, with the other, more flexible, side bowed out slightly. This is sharp contrast to the near perfect symmetry observed in the structure of GdDOTP.19
In chelates of DOTA and DOTAM, both of which possess one bound water molecule (q = 1), the Ln3+ ion lies much closer to the 4 oxygen donor atoms than it does to the 4 nitrogen donors. For these chelates the metal ion is typically found to lie approximately 7/10 of the distance (d/c, Table 3) to the mean oxygen atom plane, irrespective of which coordination geometry (SAP or TSAP) is observed.33 In contrast the d/c ratios for the q = 0 cyclen-based phosphonate and phosphinate chelates (Table 3) are much smaller (typically ≈ 0.62), indicating that the Ln3+ ion lies significantly closer to the mean nitrogen plane. Significantly, the d/c ratios of EuPCTMB and TbPCTMB are in line with those observed for these cyclen-based phosphonate and phosphinate systems. Although the distance between the mean nitrogen and oxygen atom planes is about 0.15 Å greater in the phosphonate and phosphinate systems compared with the acetate and amide systems, the metal also lies closer to the nitrogen plane in absolute terms, by about 0.1 Å. The crystal structures of the Ln3+ chelates of ligands 3 and 4 and their d/c ratios provides some insight into the relationship between the position of the Ln3+ ion and the hydration state (q value) of the chelate. The crystal structures of Ln3 chelates across the lanthanide series reveal that the d/c ratio falls across the series, reaching a minimum of about 0.61 at europium and remaining more or less constant thereafter. Thus, the chelates of the smaller heavy lanthanides Eu3, Tb3, Er3 Yb3, Eu4 and Y4, all of which are q = 0, have small d/c ratios, between 0.61 and 0.63.18 Nd3 has slightly larger d/c ratio, 0.65, but a small O-M-O bond angle, 129°, and is also q = 0.18 However, chelates of larger Ln3+ ions La4, La3 and Ce3 are q = 1 and have d/c ratios comparable to those of DOTA and DOTAM chelates, d/c = 0.68 – 0.70.18, 21 The smaller of the two O-M-O bond angles (β) in the chelates of La3 and Ce3 are 135°,18 a shade narrower than the cut-off proposed by Lukeš and co-workers,32 and yet a water molecule is still able to coordinate with the metal center. The crystal structure of Pr3, presented subsequently,34 provides reason for pause at this point; it is presented as a q = 1 chelate and yet has β = 129.7° and β′ = 136.4° and a relatively small d/c ratio of 0.65. It is not until one considers that the reported water bond distance, 2.820 Å, is longer than is normally considered a bonding interaction that we can really understand this chelate. Pr3 is, in reality, probably q = 0 but is able to maintain an interaction with a water molecule as a result of its d/c ratio. This evidence suggests that when the Ln3+ ion in aza-crown based chelates such as these is octa-coordinate and a water molecule is absent from its coordination sphere, the Ln3+ ion moves towards the amines of the macrocycle in search of increased electron density. The question as to which comes first, movement of the Ln3+ ion or departure of the water molecule, is something of a ‘chicken and the egg problem’. However it seems clear that this has implications for understanding dissociative water exchange processes during which it now seems likely that the position of the Ln3+ ion fluctuates according to the hydration state of the chelate. It also implies that the d/c ratio may provide a better delineation between chelates that can be hydrated (q = 1) and those that cannot (q = 0) given that the O-M-O angle which may vary significantly, even within the same chelate. It seems that one may conclude from this that a d/c ratio much smaller than 0.68 will result in water being excluded from the inner coordination sphere, but that until this ratio is smaller than 0.65 non-bonding interactions between metal and water are still possible.
Owing to the absence of chirality in the pyclen ring, the chirality of an LnPCTMB chelate is determined by the orientation of the pendant arms (Δ or Λ) and the configuration at phosphorus (R- or S-). The prochiral phosphorus atom of the ligand becomes chiral upon coordination with the metal ion, so either configuration may be result from the synthesis of the chelate. Studies into the solution and solid state structures of Ln4 and Ln5 chelates have shown an interdependence of the orientation of the pendant arms and the configuration at phosphorous.20, 37 A single C4-symmetric coordination isomer predominates in solution for Ln4 and Ln5 chelates, while crystal structure data reveals that a Λ orientation of the pendant arms is associated with an R- configuration at phosphorous and a Δ orientation with an S- configuration at phosphorus.20, 21 This observation is reminiscent of that observed in α-substituted acetate derivatives of cyclen.29, 38–40 In sharp contrast, these two elements of chirality do not exhibit any such interdependence in the Ln6 chelates, despite the apparent structural similarity of the ligand systems.22 Multiple isomers of Ln6 chelates are observed in solution and, at least in the crystal, the pendant arms bind cooperatively (i.e. with the same helicity) even though the configuration at phosphorous alternates RSRS- around the ring.
The structures of LnPCTMB, Ln2 and Ln3 chelates in the crystal share some common features (Figure 4 and supplementary figures S1 and S2). Each chelate is present in a dimer. The dimer is associated by two bridges in which one phosphonate of each chelate coordinates to both Ln3+ ions of the dimer. For each of these chelates the dimer is made up of two chelate molecules that are enantiomers of one another. The pendant arms bind cooperatively, with one molecule of the dimer exhibiting a Δ orientation and its partner a Λ orientation. However, when it comes to the configuration at phosphorous these chelates resemble Ln6 chelates more than either the Ln4 or Ln5 chelates. Clearly, the configuration at phosphorous is strongly influenced by the nature of the phosphorous R-substituent. In the structures of LnPCTMB, Ln2, and Ln4 the preferred conformation of the pendant arm is with the R-substituent of phosphorous in a gauche position with respect to the nitrogen of the macrocycle (Figure 5). However, in the case of Ln3 chelates the R-substituent exhibits a preference for a position anti- to the nitrogen of the macrocycle, with only the middle arm adopting the gauche conformation preferred in the chelates of PCTMB, 2 and 4.
In both LnPCTMB and Ln3 chelates phosphorus adopts RSR-/SRS- configurations going around the macrocyclic ring. In contrast, the configuration at phosphorous in Ln2 chelates is RRS-/SSR-, this despite the interdependence with pendant arm orientation observed in the analogous tetra-phosphinate chelates Ln4 and Ln5. Notably, it is the pendant arm that bridges the two chelate molecules that has the inverted configuration at phosphorous and it is only through this inversion of configuration that the pendant arm is able to make a second oxygen donor atom available for coordination in the dimeric structure (Supplementary Figure S1). It appears that in the case of Ln2, the energy penalty incurred by placing the phosphorous R-substituent into an anti- position is more than compensated for by the drop in energy, and increase in enthalpy, associated with dimerization of the chelate.
It is less clear why the central pendant arm of LnPCTMB and Ln3 chelates should adopt an anti- conformation. One explanation may lie in a phenomenon exhibited by both chelates and clearly visible by inspection of Figure 2; by inverting the configuration of the middle pendant arm these chelates are able to bind a second sphere water molecule in a pincer action. Hydrogen bonded by both non-bridging phosphonates this water molecule lies closest to the metal ion, 5.574 Å from the Eu3+ ion and 5.476 Å from Tb3+ and may have a residence lifetime on the chelate long enough to have significance in relaxometric studies of the Gd3+ chelate. Notably this pincer binding action used to hold water molecule is absent from the structures of Ln2 chelates. As a result the 4 second sphere water molecules of Ln2 chelates are hydrogen bonded to just one phosphinate each and lie further from the metal ion, over 6.5 Å away.
The dimeric forms of Ln2 and Ln3 chelates that are observed in the crystal structure have also been shown to persist in the solution state.17, 18 The presence of dimeric structures in solution may be beneficial, as in the case of luminescent probes, or detrimental, as in the case of an MRI contrast agent. For these reasons it is important to understand the behaviour of the dimeric structure of PCTMB chelates in solution. LnPCTMB chelates are minimally soluble in water at room temperature. However, upon heating to reflux it is possible to obtain a chelate solution that is 1 – 2 mM, which persists after cooling. Solutions of EuPCTMB and TbPCTMB are brilliantly luminescent when irradiated with common UV sources which provide a convenient tool for probing their sensitized photo-physical properties.
Rapidly exchanging water molecules in the inner-coordination sphere of a gadolinium ion contribute significantly to its overall relaxivity. Clearly then a chelate that excludes all water molecules from the inner-coordination sphere is likely to be ineffective as an MRI contrast agent. Relaxivity (r1) is the measure of how effective a contrast agent and is defined as the increase in longitudinal relaxation rate per unit concentration of contrast agent. Relaxivity is usually determined by measuring the longitudinal relaxation rate (R1 which = 1/T1) of solutions of the contrast agent at different concentrations. A linear regression analysis then affords the relaxivity as the slope of the line. Accordingly, the longitudinal relaxation rates of solutions of GdPCTMB were measured over the concentration range 0.06 – 6.0 mM. Measurements were performed in 2:1 v/v water/methanol solution, owing to the poor solubility of the chelate in water at concentrations as high as 6.0 mM. The results, shown in Figure 6 on a logarithmic axis for greater clarity, reveal that R1 is not linearly dependent upon the concentration. This non-linearity is most usually observed if a structural change, that alters relaxivity, occurs as a result of a change in concentration. Fitting data in the concentration ranges 0.06 – 0.2 mM and 2.0 – 6.0 mM confirms this observation. The data in each of these two concentration ranges fits well to a straight line (Figure 6). Data fitting in the high concentration range affords a value of r1 = 4.1 mM−1s−1. In contrast, the low concentration range affords a value of r1 = 7.0 mM−1s−1.
These observations are consistent with dissociation of the dimeric structure of GdPCTMB, observed in the crystal structures of EuPCTMB and TbPCTMB, as the concentration of the chelate is decreased. The relaxivity of GdPCTMB obtained at higher concentrations (r1 = 4.1 mM−1s−1 at >2.0 mM) is significantly higher than that observed for Gd2 (r1 = 1.9 mM−1s−1 at 0.1 mM) which was shown to exist solely as a dimer across the entire concentration range studied.17 Parker and co-workers described Gd2 as an entirely outer-sphere chelate owing to this dimerization phenomenon in solution.17 It seems reasonable to believe that at higher concentration, above 2.0 mM, GdPCTMB exists solely in a dimeric form in solution; the enhanced relaxivity of this chelate can then be ascribed to the effect of water molecules in the second hydration sphere. As described earlier LnPCTMB chelates appear to bind water molecules in the second-hydration sphere using two phopshonate mono-esters in a pincer action; this not only decreases the distance of closest approach of molecules in the second hydration sphere but may reasonably be expected to increase their residence lifetime on the chelate. It is now well established that a long lived second-hydration sphere in Gd3+ complexes can lead to substantial relaxivities,41 even in the absence of an inner-hydration sphere; GdDOTP is just one example of this.42 It then appears that as the concentration of GdPCTMB decreases the chelate begins to dissociate, permitting water into the inner coordination sphere. By allowing water into the inner coordination sphere relaxivity the relaxivity of the chelate is increased to 7.0 mM−1s−1. It seems apparent that LnPCTMB chelates, unlike the analogous Ln2 and Ln3 chelates, do not persist as doubly bridged dimers in solution over a wide concentration range. Rather, the extent of dimerization in LnPCTMB chelates is highly concentration dependent, a conclusion supported by the results of luminescent studies on EuPCTMB.
The luminescent properties of Eu3+ can provide valuable insight into the speciation of its chelates in solution. In addition to allowing the hydration state (q value) to be determined using Horrocks’ method,43, 44 later modified by Parker and co-workers,45 the 5D0 → 7F0 transition of the Eu3+ emission spectrum provides a single line for each Eu3+ species present in solution, the result of the non-degeneracy of both the 5D0 and 7F0 states. Providing that the energy of this transition is different for each species in solution it is possible to examine each species individually by following this transition. Examining this transition has been used with considerable success in examining hydration equilibria in Eu3+ chelates.46 However, in order to examine the 5D0 → 7F0 transition it is necessary to acquire the emission spectrum with high spectral resolution, 0.1 nm or better. The high resolution emission spectrum of EuPCTMB in the crystalline phase (Figure 7, bottom) is characteristic of a single Eu3+ coordination environment. A single 5D0 → 7F0 transition is observed and the 3 transitions expected for the 5D0 → 7F1 transition of a chelate of this type39 are visible and clearly spaced. Dissolution in water at 0.4 mM significantly changes the nature of the emission spectrum (Figure 7 top); most clearly the two lines observed for the 5D0 → 7F0 transition indicate that two Eu3+ coordination environments are now present. The line at 580.4 nm corresponds closely to the single line observed for the dimer in the crystalline phase at 580.2 nm. The new line, observed at 579.8 nm, may be ascribed to the presence of a hydrated form of the Eu3+ chelate in solution.
Hydration of a Ln3+ center in this new species was confirmed by determining the q values using an adaptation of Horrocks’ method.45 Owing to the poor solubility of the chelates in aqueous solution the hydration state determination was performed on the more emissive Tb3+ chelate. Following excitation at 280 nm and monitoring emission at 545 nm the decay of Tb3+-based luminescence from a 0.15 mM solution of TbPCTMB was monitored. Double exponential decay of the Tb3+ excited state was observed for TbPCTMB. For chelates with a single hydration state a single exponential decay is expected; a double exponential decay is indicative of the presence of two different Tb3+ hydration states. Fitting the luminescent decay curve to a double exponential model (Table 4 and Supplementary Figure S3) revealed that two species were present, with the more prevalent species a q = 0 and the less prevalent q = 1.
The simplest explanation for these results is that dissolution in water leads to dissociation of the dimeric structure observed in the crystal and that in solution a mixture of discrete monomer and dimer are present. However, it is important to keep in mind that the emission spectrum of Eu3+ affords information limited to the immediate coordination environment of the Eu3+ ion. In the event that a bridging phosphonate were replaced by a water molecule this change would most likely be reflected by a change in the emission spectrum of Eu3+ to a change in the spectrum and in particular the ΔJ = 0 transition. A simple monomer/dimer equilibrium would be expected to be characterized by a change in the relative intensities of the two ΔJ = 0 transitions that was directly dependent upon the chelate concentration.
The Eu3+ ΔJ = 0 transitions in the emission spectra of EuPCTMB were examined as a function of the concentration of the chelate in solution (Figure 8). Over the concentration range 4.3 × 10−1 M to 4.3 × 10−4 M two lines were observed for this transition. Of these, the line at 580.2 nm (ascribed to the dimeric structure, vide infra) is more intense at higher concentrations and, as the solution becomes more dilute, this line becomes relatively less intense. The relative intensity of the line at 579.8 nm (ascribed to a hydrated Eu3+ ion, vide infra) is found to increase as the solution becomes more dilute. However, the trend of these changes is non-linear with respect to chelate concentration which would seem to indicate that a complicated set of equilibria exist in solutions of LnPCTMB chelates. It is also worth noting that given the millisecond timescale of the 5D0-7F0 measurements, all observable species must be exchanging at rate slower than 100Hz.
Examination of the 5D0 → 7F0 transition in Eu3+ chelates is unlikely to be able to distinguish between the presence of a simple monomer/doubly bridged dimer equilibrium and a more complex equilibrium that also involves a singly bridged dimer (Chart 2). This latter scenario would involve a species that had two Eu3+ coordination environments neither of which could be readily distinguished from a monomer/doubly bridged dimer equilibrium. To probe the extent of dimerization in aqueous solution the Stern-Volmer quenching of EuPCTMB by NdPCTMB was assessed using a similar procedure to that reported by Morrow et. al.47 Stern-Volmer quenching constants of KSVτ = 1101 ± 61 M−1 and KSVΦ = 40780 ± 2531 M−1 were thus obtained. Given that the KSVτ is predominantly determined by dynamic, or collisional, quenching it is to be expected that this value would remain largely constant regardless of the extent of dimerization. KSVΦ is a reflection both static and dynamic quenching, the static component of which will only be present when dimerization causes an increase in the rate of excited state deactivation. Thus, in a purely monomeric system KSVΦ should have the same value as KSVτ; however, in this case the large disparity between the two values is a clear indicator of dimerization. From the extremely high KSVΦ value we can conclude that the extent of dimerization far exceeds the levels of doubly bridged dimer that one may expect from Figure 8 and that substantial quantities of singly bridged dimer are present in aqueous solution.
This conclusion is consistent with the relaxometric results. Care should be taken not to draw parallels that are too close between these two sets of experiments because different solvent systems were employed in each case; Senanayake et al. observed substantially different speciation of Ln2 upon addition of methanol to an aqueous solution of the chelate.17 Nonetheless, if a decrease in chelate concentration leads to progressively more singly bridged dimer, in which one Gd3+ ion is hydrated, then this would reasonably be expected to increase relaxivity. The relatively large hydrodynamic volume of this species would enhance τR, while allowing water access to the inner hydration sphere of one Gd3+ ion of the dimer would introduce an effectively large inner-sphere contribution to relaxivity. We may postulate that this is the origin of the concentration dependent relaxivity of GdPCTMB.
The Ln3+ chelates of PCTMB provide a fascinating insight into the behaviour of macrocyclic Ln3+ chelates and in particular those of triphosphonate-based ligands. In the crystal the chelates exist as doubly bridged dimers in which all water is excluded from the inner coordination sphere of the Ln3+ ion. This behaviour closely parallels that of related cyclen-based phosphinate systems, Ln2 and Ln3. However, in solution the behaviour is substantially different from the cyclen-based systems. Although Ln2 and Ln3 chelates are reported to exist exclusively as dimers in aqueous solution the speciation of solutions of LnPCTMB chelates is not only complex but highly concentration dependent. There can be little doubt that a certain amount of dimer dissociation occurs in solution, this gives rise to observation of a q = 1 species in the Tb3+ chelate, an additional line in the 5D0 → 7F0 transition of the Eu3+ emission spectrum and enhanced relaxivity of the Gd3+ chelate at low concentrations. The results of Stern-Volmer experiments show that in addition to mixture of monomer and doubly-bridged dimer, a substantial proportion of singly-bridged dimer (Chart 2) must also be present. Ultimately the considerable relaxivity afforded by the Gd3+ chelate and strong emissive properties of the Eu3+ and Tb3+ chelates of PCTMB, along with the known tumor targeting properties of these chelates,16 affords a system of considerable interest for further study in vivo imaging and therapy applications.
The authors thank the National Institutes of Health (EB-04285), Portland State University and Oregon Health and Science University for financial assistance. We also thank Joseph Reibenspies of Texas A&M University for acquiring x-ray structural data.
Supporting Information Available: Images of the crystal structure of Ln2 and Ln3; fitting of the luminescent decay and steady state spectra of TbPCTMB; Stern-Volmer plots; crystallographic data (.cif files) of the two crystal structures. This material is available free of charge via the Internet at http://pubs.acs.org. CCDC 752333 & 752334 contains the supplementary crystallographic data for this paper. These data can be obtained, free of charge, from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.