DOI: 10.1002/chem.201402103
Full Paper
& Actinides
Highly Luminescent and Stable Hydroxypyridinonate Complexes: A Step Towards New Curium Decontamination Strategies Manuel Sturzbecher-Hoehne, Birgitta Kullgren, Erin E. Jarvis, Dahlia D. An, and Rebecca J. Abergel*[a]
Abstract: The photophysical properties, solution thermodynamics, and in vivo complex stabilities of CmIII complexes formed with multidentate hydroxypyridinonate ligands, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), are reported. Both chelators were investigated for their ability to act as antenna chromophores for CmIII, leading to highly sensitized luminescence emission of the metal upon complexation, with long lifetimes (383 and 196 ms for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), respectively) and remarkable quantum yields (45 % and 16 %, respectively) in aqueous solution. The bright emission peaks were used to probe the electronic
Introduction Over the past decade, increasing efforts have been applied to the development of medical countermeasures against nuclear and radiological threats.[1, 2] One consequence of such threats is the potential for internal contamination of humans with radioactive material, posing significant health risks.[3] Curium is one of five actinide (An) elements with medical significance among the radioactive contaminants listed by the U.S. Food and Drug Administration (FDA).[4] However, the scarcity and high specific activity of Cm isotopes[5] have limited the study of their chemical, structural, and biological properties. Hydroxypyridinonate ligands have long been investigated for their propensity to increase the in vivo elimination rates of An metal ions, such as PuIV and AmIII.[6–9] Two ligands are undergoing preclinical development as potential therapeutics for An decorporation: the octadentate 3,4,3-LI(1,2-HOPO), which links four 1-hydroxy-pyridin-2-one (1,2-HOPO) units through a spermine scaffold, and the tetradentate 5-LIO(Me-3,2-HOPO), which attaches two Nmethyl-3-hydroxy-pyridin-2-one (Me-3,2-HOPO) subunits onto a linear ether backbone by amide linkages (Figure 1).[9] Both compounds display very different molecular structures, acidities, and denticities, resulting in distinguishable safety and [a] Dr. M. Sturzbecher-Hoehne, B. Kullgren, E. E. Jarvis, D. D. An, Dr. R. J. Abergel Chemical Sciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720 (USA) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402103. Chem. Eur. J. 2014, 20, 9962 – 9968
structure of the 5f complexes and gain insight into ligand field effects; they were also exploited to determine the high (and proton-independent) stabilities of the corresponding CmIII complexes (log b110 = 21.8(4) for 3,4,3-LI(1,2-HOPO) and log b120 = 24.5(5) for 5-LIO(Me-3,2-HOPO)). The in vivo complex stability for both ligands was assessed by using 248Cm as a tracer in a rodent model, which provided a direct comparison with the in vitro thermodynamic results and demonstrated the great potential of 3,4,3-LI(1,2-HOPO) as a therapeutic CmIII decontamination agent.
pharmacology profiles as well as different coordination chemistry properties. Nonetheless, they are both expected to act as very efficacious Cm decorporation agents, based on their reported affinities for trivalent lanthanide (Ln) ions and on their in vivo AmIII decorporation properties.[9–12] Another essential property of these ligands is their capacity to sensitize the luminescence emission of selected LnIII cations through the formation of stable complexes and the so-called “antenna” effect.[13–19] In this sensitization process, luminescence of the Ln ion proceeds through the excitation of the ligand and subsequent intramolecular energy transfer from a triplet excited state or a singlet intra-ligand charge-transfer excited state of the ligand to the metal ion (Figure 1).[20] With its lowest triplet excited state determined at 24,390 cm1, 3,4,3-LI(1,2-HOPO) is known to sensitize a large number of LnIII ions, including Eu, Tb, Sm, Yb, Pr, and Nd, with quantum yields up to 7 %.[13, 14] In contrast, the triplet excited energies of Me3,2-HOPO derivatives are too low to sensitize cations such as EuIII ; however, 5-LIO(Me-3,2-HOPO) has been used to sensitize Yb, Pr, Nd, and Ho, giving rise to near infrared emission.[15, 19] Based on these results and on the relative electronic energy levels of metals from the 4f and 5f series, the 1,2-HOPO and Me-3,2-HOPO ligands have the potential to act as antenna chromophores for CmIII.[21, 22] In such case, spectroscopic characterization of the corresponding complexes could not only provide information on the localization of the best accepting states for the CmIII ion and on the effects of the larger spinorbit coupling and smaller electrostatic interactions of the actinides in comparison to the lanthanides, it could also be utilized to characterize solution thermodynamics parameters for CmIII complexes with both experimental chelating agents.
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Full Paper the analogue reactions with YbIII and NdIII.[19] Based on its known relatively efficient EuIII sensitization and on the similar energetics of EuIII and CmIII,[23] the octadentate 1,2-HOPO ligand was expected to sensitize CmIII luminescence to a similar extent. However, the energy transfer between 3,4,3-LI(1,2-HOPO) and CmIII is remarkable, resulting in a much brighter complex than its EuIII equivalent, with a luminescence quantum yield of 45 % (see the Supporting Information, Figure S1), which is more than six times higher than that for the EuIII complex.[14] The characteristic CmIII emission corresponds to the intense 6D7/2 !8S7/2 hypersensitive transition and results in bright orange luminescence, with peaks at lem = 610, 589, and 579 nm (nem = 16 383, 16 957, and 17 284 cm1, respectively; Figure 2).[21] This structured three-peak emission is due to ligand field splitting of the emitting state, J = 7/2, as the spherical symmetry of the CmIII half-filled 5f7 configuration should only result in a small splitting.[21, 24] The most intense emission band at 610 nm (95.8 %) is assigned as originating from the lowest Stark level of the J = 7/2 excited state, and the higher energy splittings at 590 nm (3.9 %) and 579 nm (0.3 %) are the remaining Stark components.
Figure 1. Top: Structures of 3,4,3-LI(1,2-HOPO) (left) and 5-LIO(Me-3,2-HOPO) (right). Bottom: Simplified Jablonski diagram depicting the sensitization of EuIII, CmIII, and YbIII by 3,4,3-LI(1,2-HOPO) or 5-LIO(Me-3,2-HOPO).
Therefore, taking advantage of this energy-transfer process that is very well described in LnIII complexes but still barely explored for AnIII complexes, the coordination properties of the CmIII complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were investigated. The in vivo behavior of these outstandingly stable complexes was then probed by using an established rodent model to confirm the potential of these ligands as future Cm decorporation agents. Finally, this study also suggests that the precision design of ligands for actinide complexation with targeted photophysical and luminescence sensitization properties may provide a better understanding of felement coordination systems.
Table 1. Summary of photophysical parameters for CmIII complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO).[a]
1
1
lmax [nm], emax [M cm ] lexc [nm] lem [nm] tobs {H2O} [ms] tobs {D2O} [ms] Ftot (H2O) q
[CmIII(3,4,3-LI(1,2-HOPO))]
[CmIII(5-LIO(Me-3,2-HOPO))2]
316, 14 900 320 610, 589, 579 383 38 463 46 0.45 0.05 0.8 0.1
345, 27 900 345 608, 591, 577 196 20 222 22 0.16 0.02 2.5 0.2
[a] The uncertainties were determined from the standard deviation between at least two independent experiments performed in aqueous buffered solutions (0.1 m HEPES, pH 7.4).
Results and Discussion Photophysical characterization The photophysical properties of the CmIII complexes formed in situ with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were first investigated in buffered aqueous solutions at pH 7.4; the relevant parameters are summarized in Table 1. The electronic absorption spectrum of [CmIII(3,4,3-LI(1,2-HOPO))] displays an absorption maximum due to p!p* transitions at lmax = 316 nm (emax = 14 900 m1 cm1). This is the same energy as that observed for the corresponding complexes of the central elements in the Ln series (PrIII to YbIII).[13] Similarly, complexation of 5-LIO(Me-3,2-HOPO) with CmIII results in a broad electronic envelope in the UV region, centered at lmax = 345 nm (emax = 27 900 m1 cm1), the exact same energy as that reported for Chem. Eur. J. 2014, 20, 9962 – 9968
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Figure 2. Luminescence spectra of 1.27 mm [CmIII(3,4,3-LI(1,2-HOPO))] (dashed line) and 1.53 mm [CmIII(5-LIO(Me-3,2-HOPO))2] (solid line) upon excitation at 320 nm and 345 nm, respectively, in 0.1 m HEPES, pH 7.4, 25 8C. The inset shows time-resolved luminescence decay measurements for both complexes at their maximum emission.
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Full Paper More surprising was the effective energy transfer between 5LIO(Me-3,2-HOPO) and the CmIII ion, with a 16 % quantum yield (see the Supporting Information, Figure S1), in contrast to the corresponding EuIII complex, which does not show any emission. Bright orange luminescent peaks were observed at lem = 608, 591, and 577 nm (nem = 16 445, 16 929, and 17 319 cm1, respectively; Figure 2) for the CmIII complex formed with the tetradentate Me-3,2-HOPO ligand at pH 7.4. However, for this sample, the main peak at 608 nm is broadened by a shoulder at 604 nm (16 559 cm1), indicative of an underlying second species (the presence of this second species is discussed further with the measured lifetimes and stability constants). Time-resolved analysis of the emitting complexes at pH 7.4 revealed monoexponential decays with respective lifetimes of 383 and 196 ms, for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2HOPO). Applying a method derived by Kimura et al.,[25] the number of inner sphere water molecules was determined as q = 0.8 for 3,4,3-LI(1,2-HOPO), suggesting a nine-coordinate CmIII ion. Although the octadentate ligand coordinates through eight oxygen donor atoms (as previously demonstrated by Xray diffraction characterization of 1,2-HOPO and Me-3,2-HOPO metal complexes[18, 19]), one additional water molecule is interacting in the primary coordination sphere, which may lead to either a monocapped square antiprismatic or a trigonal prismatic structure, as known for LnIII and AnIII.[26, 27] For the 5LIO(Me-3,2-HOPO) complex, the parameter q was calculated as 2.5 at pH 7.4 and 2.3 at pH 8.9 (tobs = 206 ms; see the Supporting Information, Figure S2), leading to a ten-coordinate complex. The coordination number is higher for the complex formed with 5-LIO(Me-3,2-HOPO), in comparison with 3,4,3LI(1,2-HOPO), as the 2:1 ligand/metal ratio required to fully coordinate the metal with this tetradentate ligand results in higher flexibility of the coordination sphere and less steric hindrance around the metal core, facilitating access of two water molecules. This coordination number of ten most likely corresponds to a bicapped square antiprismatic structure.[28, 29] Timeresolved luminescence of the CmIII complexes was also measured in H2O/D2O mixtures, with increasing content of D2O, for both ligands at pH 7.4, resulting in a linear increase of the lifetimes up to 463 and 222 ms for 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO), respectively, in 100 % D2O (see the Supporting Information, Figure S2). Based on the results of a large number of previous Ln luminescence studies with ligands containing 1,2-HOPO and Me3,2-HOPO units, the sensitization process for the CmIII complexes investigated here is attributed to the antennae effect.[16, 20] The lowest T1 excited states of 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were previously reported at 24 390 cm1 and 18 657 cm1, respectively.[14, 19] Although an intramolecular energy transfer from the triplet state of the chromophore could only occur with the lowest-lying excited state of CmIII (6D7/2 at 17 095 cm1) for 5-LIO(Me-3,2-HOPO), several high-energy CmIII excited states could be populated in the case of 3,4,3-LI(1,2-HOPO) (up to 6I9/2 at 23 120 cm1). The energy gap between T1 and the closest accepting state of CmIII would therefore be 1270 or 1562 cm1 for 3,4,3-LI(1,2-HOPO) or 5-LIO(Me-3,2-HOPO), respectively, implying that the energy Chem. Eur. J. 2014, 20, 9962 – 9968
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transfer should be more efficient in the case of the octadentate ligand, as observed experimentally. Although measured in methanol, the highest quantum yield previously reported for a CmIII complex (50 %) is that of a complex formed with a chiral octadentate cage incorporating four 2-hydroxy-isophthalamide (IAM) metal-binding groups.[30] The lowest triplet state of the bidentate IAM chromophore being known as 23 300 cm1, this ligand is assumed to sensitize Cm through the 6I9/2 accepting excited state, similarly to 3,4,3-LI(1,2-HOPO), a process that would indeed result in an energy transfer of similar efficiency. The bathochromatic shift of the emission maxima observed during complexation of LnIII cations is typically in the 0–3 nm range. However, this redshift seems to be much larger for the strong emitting transition of CmIII, 6D7/2 !8S7/2. Similarly to reports for IAM ligands in methanol, shifts of about 14 and 16 nm are observed in the complexation of CmIII by 5-LIO(Me3,2-HOPO) and 3,4,3-LI(1,2-HOPO), respectively, with 593.8 nm as the reference for aqueous CmIII.[31] More importantly, this shift is characteristic of the nephelauxetic effect, and its magnitude may therefore be correlated with the diminution of electrostatic repulsion effects around the metal center and the extent of covalent bonding owing to ligand interactions in the first coordination sphere.[32] The difference in emission maxima for the two investigated ligands would then indicate a stronger covalent character in the 3,4,3-LI(1,2-HOPO) complex. However, this effect may be due to a combination of factors, including the different coordination numbers (9 for 3,4,3-LI(1,2-HOPO) and 10 in the case of 5-LIO(Me-3,2-HOPO)), the chelate effect (octadentate vs. tetradentate ligand), or the difference in aromaticity and acidity of the chromophores (1,2-HOPO vs. Me3,2-HOPO). Solution thermodynamics The sensitized emission of the CmIII ion in the complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) was used to determine their respective stability constants through direct spectrofluorimetric titrations at low micromolar concentrations. For these titrations, the ligands were added to CmIII under acidic conditions (pH 1.1 for 3,4,3-LI(1,2-HOPO) and pH 1.5 for 5-LIO(Me-3,2-HOPO)) and the resulting solutions were forward-titrated with KOH (to pH 4.0 and 11.3, respectively) and backward titrated with HCl. After import of the data sets of emission spectra (lem = 570–630 nm) at varying pH into the refinement program, HypSpec,[33] analysis was performed by nonlinear least-squares refinements, and the resulting thermodynamic parameters are summarized in Table 2. In the 3,4,3-LI(1,2-HOPO) titrations, an immediate rise in emission intensity at 610 nm, corresponding to the formation of the [CmIII(3,4,3-LI(1,2-HOPO))] complex, was observed from pH 1.1 to pH 3, after which no spectral change occurred (Figure 3). Analysis of these spectral changes afforded the determination of an exceptionally high proton-independent stability constant: log b110 = 21.8. For the evaluation of the 5-LIO(Me-3,2-HOPO) CmIII-complex formation constants, UV/Visible absorption and emission spec-
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Full Paper Table 2. Protonation and CmIII complex formation constants for 3,4,3LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO).[a] Species
m, L, h
log bmlh
References
3,4,3-LI(1,2-HOPO) LH3 LH22 LH3 LH4 [CmIIIL] pCmIII7.4
0, 0, 0, 0, 1,
1, 1 1, 2 1, 3 1, 4 1, 0
6.6 12.3 17.3 21.2 21.8 0.4 22.7 0.4
[14] [14] [14] [14] This work This work
5-LIO(Me-3,2-HOPO) LH LH2 [CmIIIL] + [CmIIIL2H] [CmIIIL2] [CmIIIL2(OH)2]3 pCmIII7.4
0, 0, 1, 1, 1, 1,
1, 1 1, 2 1, 0 2, 1 2, 0 2, 2
7.1 13.1 16.1 0.1 31.5 0.5 24.5 0.5 5.0 0.1 20.1 0.5
[34] [34] This This This This This
work work work work work
Figure 4. Fluorimetric titration of [CmIII(5-LIO(Me-3,2-HOPO))2] (3 mm) by KOH in water. I = 0.1 (KCl), T = 25 8C. During the course of the titration, the emission maximum shifts from 604 nm to 608 nm, indicating the formation of two emitting species. The inset shows the corresponding UV/Vis spectral data.
[a] The uncertainties were determined from the standard deviation between three independent titrations.
Figure 3. Fluorimetric titration of [CmIII(3,4,3-LI(1,2-HOPO))] (2.7 mm) by KOH in water. I = 0.1 (KCl), T = 25 8C. Inset: Increase in emission intensity at 610 nm upon raising the pH (data points), which reflects complex formation, with the corresponding HypSpec fit (solid line).
tra were monitored simultaneously and correlated with the pH. A typical redshift of the strong p!p* transition was observed in the UV/Vis spectrum, characteristic of the binding of the metal ion to the ligand, accompanied by an increase in intensity upon pH increase (Figure 4).[11] The emission spectra showed the formation of two species over the course of the titration, with a maximum peak for the first species at 604 nm and a consecutive redshift to 608 nm for the second species, which is in excellent agreement with the recorded broad emission spectra at pH 7.4 (Figure 2). Four stability constants were established, with [CmIII(5-LIO(Me-3,2-HOPO))2H] (log b121 = 31.5) and [CmIII(5LIO(Me-3,2-HOPO))2] (log b120 = 24.5) being the predominant species at physiologically relevant pH (Table 2). The intensity of the peak at 608 nm increased further in basic conditions (pH > 9), indicative of the coordination of hydroxide ions in an emissive complex with an even higher quantum yield. This increase Chem. Eur. J. 2014, 20, 9962 – 9968
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in luminescence sensitization is most likely caused by the hydrolysis of the diaquo-complex, reducing two high-energy O H vibrational overtones that normally quench the efficiency of the energy transfer from the antenna ligand to the metal.[35] Two different lifetimes were recorded between pH 5.5 and 7.0, confirming the sequential formation of two different CmIII complexes of 5-LIO(Me-3,2-HOPO) that display 1:1 and 1:2 metal/ ligand ratios, respectively (see the Supporting Information, Figure S3). Additionally, the lifetime measurements indicated that the 1:1 complex has a significantly lower quantum yield than the 1:2 species. At pH 7.4, the monoexponential decay of the 1:2 metal/ligand complex indicates that this species is predominant, with 33 % of the 1:1 metal/ligand complex present in solution. In addition to benefiting from a higher denticity that facilitates CmIII complexation, 3,4,3-LI(1,2-HOPO) also displays a lower basicity (quantified from the sum of the log Ka values associated to only those protonation steps that result in the neutral ligand species, Slog Ka = 21.2) than 5-LIO(Me-3,2-HOPO) (Slog Ka = 26.2 for two coordinating molecules).[14, 34] To allow comparisons between those ligands as well as with others, despite their varying denticity and/or acidity, the conditional stability constant pCmIII was calculated as a function of pH for a standard set of conditions (see the Supporting Information, Figure S4; initial concentrations: [Cm] = 1 mm, [L] = 10 mm). As expected, the octadentate ligand (pCmIII7.4 = 22.7(1)) exhibits a much higher affinity for CmIII than 5-LIO(Me-3,2-HOPO) (pCmIII7.4 = 20.1(1)), not only at pH 7.4, but over the pH range 2.0–10. In addition, it is consistently more efficient than the current standard ligand for Cm sequestration, the octadentate pentaacetic acid derivative, diethylene triamine pentaacetic acid (DTPA), in binding CmIII (pCmIII7.4 = 21.1) over the pH range 2.0–8.0. Finally, the similar affinities of 5-LIO(Me-3,2-HOPO) and DTPA for CmIII over a wide pH range (2.0–7.0), despite their different denticities, demonstrate the instrumental role played by high-affinity metal-binding groups in the chelation process. It is also important to note that the stability of the CmIII complex
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Full Paper of 3,4,3-LI(1,2-HOPO) falls well within the range observed in corresponding lanthanide complexes (pM7.4 from 17.2 for LaIII to 23.1 for YbIII), and is equivalent to that of the ErIII complex.[13] In vivo CmIII complex stability To evaluate the ability of the ligands, 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO) as well as DTPA, to form Cm complexes stable enough to withstand competition and metal exchange with biological ligands, an in vivo stability experiment was conducted. Cm–ligand complexes were formed in situ (Ligand/Cm ratio > 106) to mimic the complexes involved in a potential decorporation process, and administered parenterally to groups of five mice. The mice were euthanized 24 h after the metal injection, and blood, tissues, and excreta were radioanalyzed for Cm content. The Cm excretion profiles at 24 h for the complexes are shown in Figure 5, and the corresponding Cm body distribution results are provided in the Supporting Information, Table S1. The complexes formed with the octadentate 3,4,3-LI(1,2HOPO) and DTPA were quantitatively excreted (approximately 98 % of the injected doses were recovered after 24 h), largely contrasting with the high retentions of noncomplexed Cm,[36] and indicating high in vivo complex stabilities. A major difference was observed in the excretion pathways: whereas 3,4,3LI(1,2-HOPO) promoted excretion predominantly through the fecal pathway (87 % of the total excretion), the DTPA complex was found mostly in urine (80 % of the total excretion), suggesting that these complexes remain stable through excretion. On the other hand, the complex formed with the tetradentate 5-LIO(Me-3,2-HOPO) was not stable enough to be fully excreted (with 58 % of total excretion through the fecal pathway), as 14 % of the injected Cm was retained at 24 h. Such discrepancy
in excretion patterns between acetate and hydroxypyridinonate ligands has been reported previously for other metal complexes, including EuIII, AmIII, and PuIV.[9, 13, 37, 38] This result suggests ligand-dependent biochemical pathways in the metabolism of these complexes and ultimately in the mechanism of decorporation, probably owing to the respective ionization potential and membrane permeability properties of the ligands. Overall, these results corroborate the thermodynamic stability of the CmIII complexes of DTPA, 3,4,3-LI(1,2-HOPO), and 5-LIO(Me-3,2-HOPO): although all three ligands form highly stable complexes in vitro and in vivo, the octadentate hydroxypyridinonate ligand, 3,4,3-LI(1,2-HOPO), has a higher affinity for CmIII, which enhances its capability to compete with biological ligands such as proteins and bone matrices, as compared with DTPA and the tetradentate 5-LIO(Me-3,2-HOPO). It is important to evaluate whether the decorporation properties of the ligands follow similar trends. To that purpose, the respective decorporation efficacy of the ligands will be assessed by using established animal models and protocols.
Conclusion We report the sensitization of CmIII luminescence by two antenna ligands, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO). Those ligands were chosen for the described spectroscopic and thermodynamic studies because of their high affinity for actinide ions and their potential therapeutic applications as decorporation agents. The in vitro and in vivo stability results obtained here are the first direct confirmation that hydroxypyridinonate chelating agents may target CmIII for sequestration and decontamination purposes. However, the peculiar photophysical properties of the formed CmIII complexes have also revealed very efficient intramolecular energy-transfer processes owing to the unique electronic structure of the CmIII ion. While further studies will necessitate the systematic evaluation of different scaffolds and metal-binding units, it is now evident that tuning the molecular structure of such ligands to explore the coordination chemistry of CmIII could be very powerful in targeting different excited states of the actinide ion, regulating the luminescence sensitization process, and assessing the covalent character of these species.
Experimental Section General
Figure 5. Retention and excretion of 248Cm after injection of 248Cm–ligand complexes into mice. Groups of five mice were injected intraperitoneally with a single dose (0.2 mL injection volume) of 248Cm–ligand complexes (0.23 kBq, 1 mmol of ligand) and were euthanized after 24 h. Data, expressed as percent of injected 248Cm dose (% ID, mean SD), were normalized to 100 % material recovery for each five-mouse group. Excreta of each fivemouse group were pooled; therefore, standard deviations are not available for excretion data. Chem. Eur. J. 2014, 20, 9962 – 9968
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All chemicals were obtained from commercial suppliers and were used as received. The ligands 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2HOPO) were synthesized by Synthetech, Inc. (Albany, OR, USA) and AMRI, Inc. (Albany, NY, USA), respectively, following previously reported procedures, and used as received.[39, 40] Diethylene triamine pentaacetic acid (DTPA) was obtained from Sigma–Aldrich (StLouis, MO, USA). All aqueous solutions were prepared by using deionized water purified through a Millipore Milli-Q reverse osmosis cartridge system, and the pH was adjusted as needed with concentrated HCl or KOH. Stock solutions of ligands were prepared by direct dissolution of a weighed portion of ligand in water prior to each set of experiments. The CmCl3 solution was prepared in
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Full Paper standardized 1 m HCl and had a 95.78 % 248Cm, 4.12 % 246Cm, 0.06 % 245 Cm, 0.02 % 244Cm/247Cm isotopic distribution by atom percentage.
Photophysics UV/Visible spectroscopy: UV/Vis absorption spectra for titrations were recorded on an Ocean Optics USB 2000 spectrophotometer (slit 5 nm, grating 1200 grooves/mm, blaze holographic UV) equipped with a PX-2 pulsed xenon. The spectra were measured with a peak sleeved 1 cm path dip probe (Ocean Optics, Inc.) to maintain low volume measurements and dilution factors. For quantum yield measurements, spectra were recorded on a Varian Cary 6000i double beam absorption spectrophotometer, using quartz cells of 1.00 cm path length. Luminescence spectroscopy: Luminescence spectra for titrations were acquired on an Ocean Optics USB 4000 spectrophotometer (slit 50 mm, grating 600 grooves/mm, blaze 400 nm) equipped with a HPX-2 high-powered xenon light source with a fiber optic scanning monochromator. Samples were pumped through a loop in an asymmetric fluorescence cell (2 10 mm, 40 mL) with a peristaltic pump after each reagent incremental addition. All other emission spectra were recorded on a HORIBA Jobin Yvon IBH FluoroLog-3 spectrofluorimeter, used in steady state mode. A continuous xenon lamp (450 W) was used as the light source. Luminescence lifetimes were determined on a HORIBA Jobin Yvon IBH FluoroLog-3 spectrofluorimeter, adapted for time-correlated single photon counting (TCSPC) and multichannel scaling (MCS) measurements. A sub-microsecond Xenon flashlamp (Jobin Yvon, 5000XeF) was used as the light source, with an input pulse energy (100 nF discharge capacitance) of approximately 50 mJ, yielding an optical pulse duration of less than 300 ns at full width at half maximum (FWHM). Spectral selection was achieved by passage through a double grating excitation monochromator (2.1 nm mm1 dispersion, 1200 grooves/mm). Emission was monitored perpendicular to the excitation pulse, again with spectral selection achieved by passage through a double grating excitation monochromator (2.1 nm mm1 dispersion, 1200 grooves/mm). A thermoelectrically cooled single photon detection module (HORIBA Jobin Yvon IBH, TBX-04-D), incorporating fast rise time photo-multiplier tubes (PMT), wide bandwidth preamplifier, and picosecond constant fraction discriminator, was used as the detector. Signals were acquired by using an IBH DataStation Hub photon counting module and data analysis was performed by using the commercially available DAS 6 decay analysis software package from HORIBA Jobin Yvon IBH. Goodness of fit was assessed by minimizing the reduced chi squared function, and visual inspection of the weighted residuals. Each trace contained at least 5 000 points, and the estimated error on the reported lifetime values is 10 %. Quantum yields were determined as previously described.[14] The q values were calculated by using Equation (1):[25]
q ¼ 0:65 kobs 0:88
ð1Þ
each titration)[41] was used with a Metrohm Titrando 907 (Metrohm) to measure the pH of the experimental solutions. In the incremental titration setup, the Metrohm autoburet was used to add incremental volumes of acid or base standard solutions to the titration cell, and the instruments were fully automated and controlled by using the Tiamo software from Metrohm. All thermodynamic measurements were conducted at 25 8C. Incremental spectrophotometric and spectrofluorimetric titrations: The experimental titration setup was similar to previously described systems.[13] Solutions were assembled from a stock solution of ligand, CmCl3, and the supporting electrolyte solution (0.1 m KCl), with resulting metal concentrations of up to 3 mm. Solutions were forward and backward titrated by the incremental addition of either carbonate-free 0.1 m KOH or 0.1 m HCl titrant (up to 3 cycles) followed by a time delay for equilibration of 100 s. Integration times for the UV/Vis spectra were set to 50 0.1 s, and emission spectra were acquired over 5 20 s for 3,4,3-LI(1,2-HOPO) and 2 50 s for 5-LIO(Me-3,2-HOPO) titrations. Both ligands were excited near their respective maximum absorption wavelength. Buffering of the solution was ensured by the addition of acetic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mm). An average of 60–120 data points were collected in each direct complex titration, each data point consisting of a pH measurement and luminescence and/or UV/Vis spectra over the pH ranges 1.3 to 4.0 and 1.5 to 11.3 for 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO), respectively. All spectra were corrected for dilution. Each titration was performed at least three times, in less than 3 h and under positive Ar gas pressure. Data treatment: All thermodynamic data sets were imported into the refinement program HypSpec[33, 42] and analyzed by nonlinear least-squares refinement. All equilibrium constants were defined as cumulative formation constants, bmlh according to Equation (2), where the metal and ligand are designated as M and L, respectively.
mMþlLþhH Ð ½Mm Ll Hh ; bmlh ¼
½Mm Ll Hh ½Mm ½Ll ½Hh
ð2Þ
All metal and ligand concentrations were held at estimated values determined from the volume of standardized stock solutions. All species formed with the ligands L were considered to have significant absorbance to be observed in the UV/Vis spectra and were therefore included in the refinement process. For the spectrofluorimetric titrations, only the metal–ligand complexes were considered to have significant emission to be observed in the emission spectra. The refinements of the overall formation constants included the previously determined ligand protonation constants[14, 34] in each case and the metal hydrolysis products, the equilibrium constants of which were fixed to the literature values.[43] The pM values, defined as the negative logarithm of the free metal concentration in equilibrium with complexed and free ligand, at a fixed pH (7.4 for physiological conditions) and for fixed ligand and metal concentrations of 10 mm and 1 mm, respectively, were calculated by using the program Hyss.[44, 45]
Solution thermodynamics General considerations: All titrant solutions were degassed by boiling for 1 h while being purged under Ar. Carbonate-free 0.1 m KOH was prepared from Baker Dilut-It concentrate and was standardized by titrating against 0.1 m HCl. Solutions of 0.1 m HCl were similarly prepared and were standardized by titrating against tris(hydroxymethyl) aminomethane (TRIS). A Micro Combi Electrode (Metrohm) glass electrode (response to [H + ] was calibrated before Chem. Eur. J. 2014, 20, 9962 – 9968
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In vivo complex stability evaluation The procedures and protocols used in the presented in vivo study were reviewed and approved by the Institutional Animal Care and Use Committee of the Lawrence Berkeley National Laboratory and were performed in AAALAC accredited facilities. The animals used were young adult female Swiss-Webster mice (78 1 days old, 32 2 g). Three groups of five mice were injected intraperitoneally
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Full Paper (ip) with a single dose of Cm–ligand complex (0.23 kBq, 1 mmol of ligand). The Cm–ligand complex solutions formed with the ligands DTPA, 3,4,3-LI(1,2-HOPO), and 5-LIO(Me-3,2-HOPO) were prepared in situ by mixing and incubating the appropriate quantities of Cm and ligand in 0.14 m NaCl, pH 6, to reach a radioactivity of 0.23 kBq and total ligand concentration of 5.0 m (1 mmol) per mouse dose (0.2 mL). All solutions were filter-sterilized (0.22 mm) prior to administration. After injection of the Cm tracer solution, mice were weighed, identified, and housed in groups of five in plastic stock cages lined with a 0.5 cm layer of highly absorbent low-ash pelleted cellulose bedding (Alpha-dry) for separation of urine and feces. All mice were given water and food ab libitum and were euthanized 24 h after the tracer injection. Experiments were managed as metabolic balance studies, in which blood, all tissues, and excreta were analyzed for Cm by liquid scintillation counting on a Packard Tri-Carb model B4430 from PerkinElmer Corporation (Shelton, CT, USA). The methods of sample collection, preparation, radioactivity measurements, and data reduction have been published previously.[7, 46–48] Material recoveries were higher than 96 %.
Acknowledgements We thank Prof. Kenneth N. Raymond, Dr. Norman M. Edelstein, and Dr. David K. Shuh for helpful discussions. Spectroscopic characterization of Cm complexes and solution thermodynamic studies were supported by the Director, Office of Science, Office of Basic Energy Sciences, and the Division of Chemical Sciences, Geosciences, and Biosciences of the U.S. Department of Energy at LBNL under Contract No. DE-AC02–05CH11231. The in vivo experiments were supported by the National Institutes of Health (RAI087604Z) through the U.S. Department of Energy under Contract No. DE-AC02–05CH11231. Keywords: actinides · curium luminescence · thermodynamics
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energy
transfer
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[1] D. R. Cassatt, J. M. Kaminski, R. J. Hatchett, A. L. DiCarlo, J. M. Benjamin, B. W. Maidment, Radiat. Res. 2008, 170, 540 – 548. [2] W. H. Moos, G. N. Shankar, Drug Dev. Res. 2012, 73, 229 – 231. [3] National Council on Radiation Protection and Measurements, Management of Persons Contaminated with Radionuclides Handbook: Recommendations of the National Council on Radiation Protection and Measurements, National Council on Radiation Protection and Measurements, Bethesda, Md., 2009. [4] US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, 2006. [5] G. J. Lumetta, M. C. Thompson, R. A. Penneman, P. G. Eller, in Chem. Actin. Trans. Elem. (Eds.: L. R. Morss, N. M. Edelstein, J. Fuger), Springer Netherlands, Dordrecht, 2011, pp. 1397 – 1443. [6] R. J. Abergel, P. W. Durbin, B. Kullgren, S. N. Ebbe, J. Xu, P. Y. Chang, D. I. Bunin, E. A. Blakely, K. A. Bjornstad, C. J. Rosen, Health Phys. 2010, 99, 401 – 407. [7] P. W. Durbin, B. Kullgren, S. N. Ebbe, J. Xu, K. N. Raymond, Health Phys. 2000, 78, 511. [8] R. J. Abergel, K. N. Raymond, Hemoglobin 2011, 35, 276 – 290. [9] D. I. Bunin, P. Y. Chang, R. S. Doppalapudi, E. S. Riccio, D. An, E. E. Jarvis, B. Kullgren, R. J. Abergel, Radiat. Res. 2013, 179, 171 – 182. [10] P. Y. Chang, D. I. Bunin, J. Gow, R. Swezey, W. Shinn, D. K. Shuh, R. J. Abergel, J. Chromatogr. Sep. Tech. 2011, 04, DOI: 10.4172/21577064.1000196. [11] M. Sturzbecher-Hoehne, G. J.-P. Deblonde, R. J. Abergel, Radiochim. Acta 2013, 101, 359 – 366.
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[12] G. J.-P. Deblonde, M. Sturzbecher-Hoehne, R. J. Abergel, Inorg. Chem. 2013, 52, 8805 – 8811. [13] M. Sturzbecher-Hoehne, C. Ng Pak Leung, A. D’Alo, B. Kullgren, A.-L. Prigent, D. K. Shuh, K. N. Raymond, R. J. Abergel, Dalton Trans. 2011, 40, 8340 – 8346. [14] R. J. Abergel, A. D’Aleo, C. Ng Pak Leung, D. K. Shuh, K. N. Raymond, Inorg. Chem. 2009, 48, 10868 – 10870. [15] E. G. Moore, G. Szigethy, J. Xu, L.-O. Plsson, A. Beeby, K. N. Raymond, Angew. Chem. 2008, 120, 9642 – 9645; Angew. Chem. Int. Ed. 2008, 47, 9500 – 9503. [16] E. G. Moore, A. P. S. Samuel, K. N. Raymond, Acc. Chem. Res. 2009, 42, 542 – 552. [17] E. G. Moore, J. Xu, C. J. Jocher, I. Castro-Rodriguez, K. N. Raymond, Inorg. Chem. 2008, 47, 3105 – 3118. [18] E. G. Moore, M. Seitz, K. N. Raymond, Inorg. Chem. 2008, 47, 8571 – 8573. [19] E. G. Moore, J. Xu, S. C. Dodani, C. J. Jocher, A. D’Alo, M. Seitz, K. N. Raymond, Inorg. Chem. 2010, 49, 4156 – 4166. [20] A. D’Alo, F. Pointillart, L. Ouahab, C. Andraud, O. Maury, Coord. Chem. Rev. 2012, 256, 1604 – 1620. [21] W. T. Carnall, J. Chem. Phys. 1992, 96, 8713. [22] W. T. Carnall, K. Rajnak, J. Chem. Phys. 1975, 63, 3510 – 3514. [23] T. Kimura, R. Nagaishi, Y. Kato, Z. Yoshida, Radiochim. Acta 2001, 89, 125. [24] R. E. Sykora, Z. Assefa, R. G. Haire, T. E. Albrecht-Schmitt, Inorg. Chem. 2005, 44, 5667 – 5676. [25] T. Kimura, G. R. Choppin, J. Alloys Compd. 1994, 213 – 214, 313 – 317. [26] D. E. Bugaris, R. Copping, T. Tyliszczak, D. K. Shuh, J. A. Ibers, Inorg. Chem. 2010, 49, 2568 – 2575. [27] R. Liminga, S. C. Abrahams, J. L. Bernstein, J. Chem. Phys. 1977, 67, 1015 – 1023. [28] M. N. Akhtar, A. J. Smith, J. Chem. Soc. Chem. Commun. 1969, 705b – 706. [29] P. Thury, Inorg. Chem. 2011, 50, 1898 – 1904. [30] G.-L. Law, C. M. Andolina, J. Xu, V. Luu, P. X. Rutkowski, G. Muller, D. K. Shuh, J. K. Gibson, K. N. Raymond, J. Am. Chem. Soc. 2012, 134, 15545 – 15549. [31] G. Tian, N. M. Edelstein, L. Rao, J Phys Chem. A 2011, 115, 1933 – 1938. [32] N. M. Edelstein, R. Klenze, T. Fanghanel, S. Hubert, Coord. Chem. Rev. 2006, 250, 948 – 973. [33] P. Gans, A. Sabatini, A. Vacca, HypSpec, Leeds, U.K., Florence, Italy, 2008. [34] J. Xu, E. Radkov, M. Ziegler, K. N. Raymond, Inorg. Chem. 2000, 39, 4156 – 4164. [35] J.-C. G. Bnzli, Chem. Rev. 2010, 110, 2729 – 2755. [36] F. Mntrier, D. M. Taylor, A. Comte, Appl. Radiat. Isot. 2008, 66, 632 – 647. [37] B. Kullgren, E. E. Jarvis, D. D. An, R. J. Abergel, Toxicol. Mech. Methods 2013, 23, 18 – 26. [38] E. E. Jarvis, D. D. An, B. Kullgren, R. J. Abergel, Drug Dev. Res. 2012, 73, 281 – 289. [39] D. L. White, P. W. Durbin, N. Jeung, K. N. Raymond, J. Med. Chem. 1988, 31, 11 – 18. [40] J. Xu, B. Kullgren, P. W. Durbin, K. N. Raymond, J. Med. Chem. 1995, 38, 2606 – 2614. [41] P. Gans, B. O’Sullivan, Talanta 2000, 51, 33 – 37. [42] P. Gans, A. Sabatini, A. Vacca, Talanta 1996, 43, 1739 – 1753. [43] A. E. Martell, R. M. Smith, R. J. Motekaitis, NIST Standard Reference Database 46, n.d.. [44] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, HYSS, Leeds, U.K., Florence, Italy, 2009. [45] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev. 1999, 184, 311 – 318. [46] P. W. Durbin, N. Jeung, B. Kullgren, G. K. Clemons, Health Phys. 1992, 63, 427 – 442. [47] P. W. Durbin, B. Kullgren, J. Xu, K. N. Raymond, Radiat. Prot. Dosim. 1994, 53, 305 – 309. [48] P. W. Durbin, B. Kullgren, J. Xu, K. N. Raymond, P. G. Allen, J. J. Bucher, N. M. Edelstein, D. K. Shuh, Health Phys. 1998, 75, 34 – 50. Received: February 10, 2014 Published online on July 7, 2014
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& Actinides
Highly Luminescent and Stable Hydroxypyridinonate Complexes: A Step Towards New Curium Decontamination Strategies Manuel Sturzbecher-Hoehne, Birgitta Kullgren, Erin E. Jarvis, Dahlia D. An, and Rebecca J. Abergel*[a]
Abstract: The photophysical properties, solution thermodynamics, and in vivo complex stabilities of CmIII complexes formed with multidentate hydroxypyridinonate ligands, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), are reported. Both chelators were investigated for their ability to act as antenna chromophores for CmIII, leading to highly sensitized luminescence emission of the metal upon complexation, with long lifetimes (383 and 196 ms for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), respectively) and remarkable quantum yields (45 % and 16 %, respectively) in aqueous solution. The bright emission peaks were used to probe the electronic
Introduction Over the past decade, increasing efforts have been applied to the development of medical countermeasures against nuclear and radiological threats.[1, 2] One consequence of such threats is the potential for internal contamination of humans with radioactive material, posing significant health risks.[3] Curium is one of five actinide (An) elements with medical significance among the radioactive contaminants listed by the U.S. Food and Drug Administration (FDA).[4] However, the scarcity and high specific activity of Cm isotopes[5] have limited the study of their chemical, structural, and biological properties. Hydroxypyridinonate ligands have long been investigated for their propensity to increase the in vivo elimination rates of An metal ions, such as PuIV and AmIII.[6–9] Two ligands are undergoing preclinical development as potential therapeutics for An decorporation: the octadentate 3,4,3-LI(1,2-HOPO), which links four 1-hydroxy-pyridin-2-one (1,2-HOPO) units through a spermine scaffold, and the tetradentate 5-LIO(Me-3,2-HOPO), which attaches two Nmethyl-3-hydroxy-pyridin-2-one (Me-3,2-HOPO) subunits onto a linear ether backbone by amide linkages (Figure 1).[9] Both compounds display very different molecular structures, acidities, and denticities, resulting in distinguishable safety and [a] Dr. M. Sturzbecher-Hoehne, B. Kullgren, E. E. Jarvis, D. D. An, Dr. R. J. Abergel Chemical Sciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720 (USA) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402103. Chem. Eur. J. 2014, 20, 9962 – 9968
structure of the 5f complexes and gain insight into ligand field effects; they were also exploited to determine the high (and proton-independent) stabilities of the corresponding CmIII complexes (log b110 = 21.8(4) for 3,4,3-LI(1,2-HOPO) and log b120 = 24.5(5) for 5-LIO(Me-3,2-HOPO)). The in vivo complex stability for both ligands was assessed by using 248Cm as a tracer in a rodent model, which provided a direct comparison with the in vitro thermodynamic results and demonstrated the great potential of 3,4,3-LI(1,2-HOPO) as a therapeutic CmIII decontamination agent.
pharmacology profiles as well as different coordination chemistry properties. Nonetheless, they are both expected to act as very efficacious Cm decorporation agents, based on their reported affinities for trivalent lanthanide (Ln) ions and on their in vivo AmIII decorporation properties.[9–12] Another essential property of these ligands is their capacity to sensitize the luminescence emission of selected LnIII cations through the formation of stable complexes and the so-called “antenna” effect.[13–19] In this sensitization process, luminescence of the Ln ion proceeds through the excitation of the ligand and subsequent intramolecular energy transfer from a triplet excited state or a singlet intra-ligand charge-transfer excited state of the ligand to the metal ion (Figure 1).[20] With its lowest triplet excited state determined at 24,390 cm1, 3,4,3-LI(1,2-HOPO) is known to sensitize a large number of LnIII ions, including Eu, Tb, Sm, Yb, Pr, and Nd, with quantum yields up to 7 %.[13, 14] In contrast, the triplet excited energies of Me3,2-HOPO derivatives are too low to sensitize cations such as EuIII ; however, 5-LIO(Me-3,2-HOPO) has been used to sensitize Yb, Pr, Nd, and Ho, giving rise to near infrared emission.[15, 19] Based on these results and on the relative electronic energy levels of metals from the 4f and 5f series, the 1,2-HOPO and Me-3,2-HOPO ligands have the potential to act as antenna chromophores for CmIII.[21, 22] In such case, spectroscopic characterization of the corresponding complexes could not only provide information on the localization of the best accepting states for the CmIII ion and on the effects of the larger spinorbit coupling and smaller electrostatic interactions of the actinides in comparison to the lanthanides, it could also be utilized to characterize solution thermodynamics parameters for CmIII complexes with both experimental chelating agents.
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Full Paper the analogue reactions with YbIII and NdIII.[19] Based on its known relatively efficient EuIII sensitization and on the similar energetics of EuIII and CmIII,[23] the octadentate 1,2-HOPO ligand was expected to sensitize CmIII luminescence to a similar extent. However, the energy transfer between 3,4,3-LI(1,2-HOPO) and CmIII is remarkable, resulting in a much brighter complex than its EuIII equivalent, with a luminescence quantum yield of 45 % (see the Supporting Information, Figure S1), which is more than six times higher than that for the EuIII complex.[14] The characteristic CmIII emission corresponds to the intense 6D7/2 !8S7/2 hypersensitive transition and results in bright orange luminescence, with peaks at lem = 610, 589, and 579 nm (nem = 16 383, 16 957, and 17 284 cm1, respectively; Figure 2).[21] This structured three-peak emission is due to ligand field splitting of the emitting state, J = 7/2, as the spherical symmetry of the CmIII half-filled 5f7 configuration should only result in a small splitting.[21, 24] The most intense emission band at 610 nm (95.8 %) is assigned as originating from the lowest Stark level of the J = 7/2 excited state, and the higher energy splittings at 590 nm (3.9 %) and 579 nm (0.3 %) are the remaining Stark components.
Figure 1. Top: Structures of 3,4,3-LI(1,2-HOPO) (left) and 5-LIO(Me-3,2-HOPO) (right). Bottom: Simplified Jablonski diagram depicting the sensitization of EuIII, CmIII, and YbIII by 3,4,3-LI(1,2-HOPO) or 5-LIO(Me-3,2-HOPO).
Therefore, taking advantage of this energy-transfer process that is very well described in LnIII complexes but still barely explored for AnIII complexes, the coordination properties of the CmIII complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were investigated. The in vivo behavior of these outstandingly stable complexes was then probed by using an established rodent model to confirm the potential of these ligands as future Cm decorporation agents. Finally, this study also suggests that the precision design of ligands for actinide complexation with targeted photophysical and luminescence sensitization properties may provide a better understanding of felement coordination systems.
Table 1. Summary of photophysical parameters for CmIII complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO).[a]
1
1
lmax [nm], emax [M cm ] lexc [nm] lem [nm] tobs {H2O} [ms] tobs {D2O} [ms] Ftot (H2O) q
[CmIII(3,4,3-LI(1,2-HOPO))]
[CmIII(5-LIO(Me-3,2-HOPO))2]
316, 14 900 320 610, 589, 579 383 38 463 46 0.45 0.05 0.8 0.1
345, 27 900 345 608, 591, 577 196 20 222 22 0.16 0.02 2.5 0.2
[a] The uncertainties were determined from the standard deviation between at least two independent experiments performed in aqueous buffered solutions (0.1 m HEPES, pH 7.4).
Results and Discussion Photophysical characterization The photophysical properties of the CmIII complexes formed in situ with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were first investigated in buffered aqueous solutions at pH 7.4; the relevant parameters are summarized in Table 1. The electronic absorption spectrum of [CmIII(3,4,3-LI(1,2-HOPO))] displays an absorption maximum due to p!p* transitions at lmax = 316 nm (emax = 14 900 m1 cm1). This is the same energy as that observed for the corresponding complexes of the central elements in the Ln series (PrIII to YbIII).[13] Similarly, complexation of 5-LIO(Me-3,2-HOPO) with CmIII results in a broad electronic envelope in the UV region, centered at lmax = 345 nm (emax = 27 900 m1 cm1), the exact same energy as that reported for Chem. Eur. J. 2014, 20, 9962 – 9968
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Figure 2. Luminescence spectra of 1.27 mm [CmIII(3,4,3-LI(1,2-HOPO))] (dashed line) and 1.53 mm [CmIII(5-LIO(Me-3,2-HOPO))2] (solid line) upon excitation at 320 nm and 345 nm, respectively, in 0.1 m HEPES, pH 7.4, 25 8C. The inset shows time-resolved luminescence decay measurements for both complexes at their maximum emission.
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Full Paper More surprising was the effective energy transfer between 5LIO(Me-3,2-HOPO) and the CmIII ion, with a 16 % quantum yield (see the Supporting Information, Figure S1), in contrast to the corresponding EuIII complex, which does not show any emission. Bright orange luminescent peaks were observed at lem = 608, 591, and 577 nm (nem = 16 445, 16 929, and 17 319 cm1, respectively; Figure 2) for the CmIII complex formed with the tetradentate Me-3,2-HOPO ligand at pH 7.4. However, for this sample, the main peak at 608 nm is broadened by a shoulder at 604 nm (16 559 cm1), indicative of an underlying second species (the presence of this second species is discussed further with the measured lifetimes and stability constants). Time-resolved analysis of the emitting complexes at pH 7.4 revealed monoexponential decays with respective lifetimes of 383 and 196 ms, for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2HOPO). Applying a method derived by Kimura et al.,[25] the number of inner sphere water molecules was determined as q = 0.8 for 3,4,3-LI(1,2-HOPO), suggesting a nine-coordinate CmIII ion. Although the octadentate ligand coordinates through eight oxygen donor atoms (as previously demonstrated by Xray diffraction characterization of 1,2-HOPO and Me-3,2-HOPO metal complexes[18, 19]), one additional water molecule is interacting in the primary coordination sphere, which may lead to either a monocapped square antiprismatic or a trigonal prismatic structure, as known for LnIII and AnIII.[26, 27] For the 5LIO(Me-3,2-HOPO) complex, the parameter q was calculated as 2.5 at pH 7.4 and 2.3 at pH 8.9 (tobs = 206 ms; see the Supporting Information, Figure S2), leading to a ten-coordinate complex. The coordination number is higher for the complex formed with 5-LIO(Me-3,2-HOPO), in comparison with 3,4,3LI(1,2-HOPO), as the 2:1 ligand/metal ratio required to fully coordinate the metal with this tetradentate ligand results in higher flexibility of the coordination sphere and less steric hindrance around the metal core, facilitating access of two water molecules. This coordination number of ten most likely corresponds to a bicapped square antiprismatic structure.[28, 29] Timeresolved luminescence of the CmIII complexes was also measured in H2O/D2O mixtures, with increasing content of D2O, for both ligands at pH 7.4, resulting in a linear increase of the lifetimes up to 463 and 222 ms for 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO), respectively, in 100 % D2O (see the Supporting Information, Figure S2). Based on the results of a large number of previous Ln luminescence studies with ligands containing 1,2-HOPO and Me3,2-HOPO units, the sensitization process for the CmIII complexes investigated here is attributed to the antennae effect.[16, 20] The lowest T1 excited states of 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) were previously reported at 24 390 cm1 and 18 657 cm1, respectively.[14, 19] Although an intramolecular energy transfer from the triplet state of the chromophore could only occur with the lowest-lying excited state of CmIII (6D7/2 at 17 095 cm1) for 5-LIO(Me-3,2-HOPO), several high-energy CmIII excited states could be populated in the case of 3,4,3-LI(1,2-HOPO) (up to 6I9/2 at 23 120 cm1). The energy gap between T1 and the closest accepting state of CmIII would therefore be 1270 or 1562 cm1 for 3,4,3-LI(1,2-HOPO) or 5-LIO(Me-3,2-HOPO), respectively, implying that the energy Chem. Eur. J. 2014, 20, 9962 – 9968
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transfer should be more efficient in the case of the octadentate ligand, as observed experimentally. Although measured in methanol, the highest quantum yield previously reported for a CmIII complex (50 %) is that of a complex formed with a chiral octadentate cage incorporating four 2-hydroxy-isophthalamide (IAM) metal-binding groups.[30] The lowest triplet state of the bidentate IAM chromophore being known as 23 300 cm1, this ligand is assumed to sensitize Cm through the 6I9/2 accepting excited state, similarly to 3,4,3-LI(1,2-HOPO), a process that would indeed result in an energy transfer of similar efficiency. The bathochromatic shift of the emission maxima observed during complexation of LnIII cations is typically in the 0–3 nm range. However, this redshift seems to be much larger for the strong emitting transition of CmIII, 6D7/2 !8S7/2. Similarly to reports for IAM ligands in methanol, shifts of about 14 and 16 nm are observed in the complexation of CmIII by 5-LIO(Me3,2-HOPO) and 3,4,3-LI(1,2-HOPO), respectively, with 593.8 nm as the reference for aqueous CmIII.[31] More importantly, this shift is characteristic of the nephelauxetic effect, and its magnitude may therefore be correlated with the diminution of electrostatic repulsion effects around the metal center and the extent of covalent bonding owing to ligand interactions in the first coordination sphere.[32] The difference in emission maxima for the two investigated ligands would then indicate a stronger covalent character in the 3,4,3-LI(1,2-HOPO) complex. However, this effect may be due to a combination of factors, including the different coordination numbers (9 for 3,4,3-LI(1,2-HOPO) and 10 in the case of 5-LIO(Me-3,2-HOPO)), the chelate effect (octadentate vs. tetradentate ligand), or the difference in aromaticity and acidity of the chromophores (1,2-HOPO vs. Me3,2-HOPO). Solution thermodynamics The sensitized emission of the CmIII ion in the complexes formed with 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO) was used to determine their respective stability constants through direct spectrofluorimetric titrations at low micromolar concentrations. For these titrations, the ligands were added to CmIII under acidic conditions (pH 1.1 for 3,4,3-LI(1,2-HOPO) and pH 1.5 for 5-LIO(Me-3,2-HOPO)) and the resulting solutions were forward-titrated with KOH (to pH 4.0 and 11.3, respectively) and backward titrated with HCl. After import of the data sets of emission spectra (lem = 570–630 nm) at varying pH into the refinement program, HypSpec,[33] analysis was performed by nonlinear least-squares refinements, and the resulting thermodynamic parameters are summarized in Table 2. In the 3,4,3-LI(1,2-HOPO) titrations, an immediate rise in emission intensity at 610 nm, corresponding to the formation of the [CmIII(3,4,3-LI(1,2-HOPO))] complex, was observed from pH 1.1 to pH 3, after which no spectral change occurred (Figure 3). Analysis of these spectral changes afforded the determination of an exceptionally high proton-independent stability constant: log b110 = 21.8. For the evaluation of the 5-LIO(Me-3,2-HOPO) CmIII-complex formation constants, UV/Visible absorption and emission spec-
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Full Paper Table 2. Protonation and CmIII complex formation constants for 3,4,3LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO).[a] Species
m, L, h
log bmlh
References
3,4,3-LI(1,2-HOPO) LH3 LH22 LH3 LH4 [CmIIIL] pCmIII7.4
0, 0, 0, 0, 1,
1, 1 1, 2 1, 3 1, 4 1, 0
6.6 12.3 17.3 21.2 21.8 0.4 22.7 0.4
[14] [14] [14] [14] This work This work
5-LIO(Me-3,2-HOPO) LH LH2 [CmIIIL] + [CmIIIL2H] [CmIIIL2] [CmIIIL2(OH)2]3 pCmIII7.4
0, 0, 1, 1, 1, 1,
1, 1 1, 2 1, 0 2, 1 2, 0 2, 2
7.1 13.1 16.1 0.1 31.5 0.5 24.5 0.5 5.0 0.1 20.1 0.5
[34] [34] This This This This This
work work work work work
Figure 4. Fluorimetric titration of [CmIII(5-LIO(Me-3,2-HOPO))2] (3 mm) by KOH in water. I = 0.1 (KCl), T = 25 8C. During the course of the titration, the emission maximum shifts from 604 nm to 608 nm, indicating the formation of two emitting species. The inset shows the corresponding UV/Vis spectral data.
[a] The uncertainties were determined from the standard deviation between three independent titrations.
Figure 3. Fluorimetric titration of [CmIII(3,4,3-LI(1,2-HOPO))] (2.7 mm) by KOH in water. I = 0.1 (KCl), T = 25 8C. Inset: Increase in emission intensity at 610 nm upon raising the pH (data points), which reflects complex formation, with the corresponding HypSpec fit (solid line).
tra were monitored simultaneously and correlated with the pH. A typical redshift of the strong p!p* transition was observed in the UV/Vis spectrum, characteristic of the binding of the metal ion to the ligand, accompanied by an increase in intensity upon pH increase (Figure 4).[11] The emission spectra showed the formation of two species over the course of the titration, with a maximum peak for the first species at 604 nm and a consecutive redshift to 608 nm for the second species, which is in excellent agreement with the recorded broad emission spectra at pH 7.4 (Figure 2). Four stability constants were established, with [CmIII(5-LIO(Me-3,2-HOPO))2H] (log b121 = 31.5) and [CmIII(5LIO(Me-3,2-HOPO))2] (log b120 = 24.5) being the predominant species at physiologically relevant pH (Table 2). The intensity of the peak at 608 nm increased further in basic conditions (pH > 9), indicative of the coordination of hydroxide ions in an emissive complex with an even higher quantum yield. This increase Chem. Eur. J. 2014, 20, 9962 – 9968
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in luminescence sensitization is most likely caused by the hydrolysis of the diaquo-complex, reducing two high-energy O H vibrational overtones that normally quench the efficiency of the energy transfer from the antenna ligand to the metal.[35] Two different lifetimes were recorded between pH 5.5 and 7.0, confirming the sequential formation of two different CmIII complexes of 5-LIO(Me-3,2-HOPO) that display 1:1 and 1:2 metal/ ligand ratios, respectively (see the Supporting Information, Figure S3). Additionally, the lifetime measurements indicated that the 1:1 complex has a significantly lower quantum yield than the 1:2 species. At pH 7.4, the monoexponential decay of the 1:2 metal/ligand complex indicates that this species is predominant, with 33 % of the 1:1 metal/ligand complex present in solution. In addition to benefiting from a higher denticity that facilitates CmIII complexation, 3,4,3-LI(1,2-HOPO) also displays a lower basicity (quantified from the sum of the log Ka values associated to only those protonation steps that result in the neutral ligand species, Slog Ka = 21.2) than 5-LIO(Me-3,2-HOPO) (Slog Ka = 26.2 for two coordinating molecules).[14, 34] To allow comparisons between those ligands as well as with others, despite their varying denticity and/or acidity, the conditional stability constant pCmIII was calculated as a function of pH for a standard set of conditions (see the Supporting Information, Figure S4; initial concentrations: [Cm] = 1 mm, [L] = 10 mm). As expected, the octadentate ligand (pCmIII7.4 = 22.7(1)) exhibits a much higher affinity for CmIII than 5-LIO(Me-3,2-HOPO) (pCmIII7.4 = 20.1(1)), not only at pH 7.4, but over the pH range 2.0–10. In addition, it is consistently more efficient than the current standard ligand for Cm sequestration, the octadentate pentaacetic acid derivative, diethylene triamine pentaacetic acid (DTPA), in binding CmIII (pCmIII7.4 = 21.1) over the pH range 2.0–8.0. Finally, the similar affinities of 5-LIO(Me-3,2-HOPO) and DTPA for CmIII over a wide pH range (2.0–7.0), despite their different denticities, demonstrate the instrumental role played by high-affinity metal-binding groups in the chelation process. It is also important to note that the stability of the CmIII complex
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Full Paper of 3,4,3-LI(1,2-HOPO) falls well within the range observed in corresponding lanthanide complexes (pM7.4 from 17.2 for LaIII to 23.1 for YbIII), and is equivalent to that of the ErIII complex.[13] In vivo CmIII complex stability To evaluate the ability of the ligands, 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO) as well as DTPA, to form Cm complexes stable enough to withstand competition and metal exchange with biological ligands, an in vivo stability experiment was conducted. Cm–ligand complexes were formed in situ (Ligand/Cm ratio > 106) to mimic the complexes involved in a potential decorporation process, and administered parenterally to groups of five mice. The mice were euthanized 24 h after the metal injection, and blood, tissues, and excreta were radioanalyzed for Cm content. The Cm excretion profiles at 24 h for the complexes are shown in Figure 5, and the corresponding Cm body distribution results are provided in the Supporting Information, Table S1. The complexes formed with the octadentate 3,4,3-LI(1,2HOPO) and DTPA were quantitatively excreted (approximately 98 % of the injected doses were recovered after 24 h), largely contrasting with the high retentions of noncomplexed Cm,[36] and indicating high in vivo complex stabilities. A major difference was observed in the excretion pathways: whereas 3,4,3LI(1,2-HOPO) promoted excretion predominantly through the fecal pathway (87 % of the total excretion), the DTPA complex was found mostly in urine (80 % of the total excretion), suggesting that these complexes remain stable through excretion. On the other hand, the complex formed with the tetradentate 5-LIO(Me-3,2-HOPO) was not stable enough to be fully excreted (with 58 % of total excretion through the fecal pathway), as 14 % of the injected Cm was retained at 24 h. Such discrepancy
in excretion patterns between acetate and hydroxypyridinonate ligands has been reported previously for other metal complexes, including EuIII, AmIII, and PuIV.[9, 13, 37, 38] This result suggests ligand-dependent biochemical pathways in the metabolism of these complexes and ultimately in the mechanism of decorporation, probably owing to the respective ionization potential and membrane permeability properties of the ligands. Overall, these results corroborate the thermodynamic stability of the CmIII complexes of DTPA, 3,4,3-LI(1,2-HOPO), and 5-LIO(Me-3,2-HOPO): although all three ligands form highly stable complexes in vitro and in vivo, the octadentate hydroxypyridinonate ligand, 3,4,3-LI(1,2-HOPO), has a higher affinity for CmIII, which enhances its capability to compete with biological ligands such as proteins and bone matrices, as compared with DTPA and the tetradentate 5-LIO(Me-3,2-HOPO). It is important to evaluate whether the decorporation properties of the ligands follow similar trends. To that purpose, the respective decorporation efficacy of the ligands will be assessed by using established animal models and protocols.
Conclusion We report the sensitization of CmIII luminescence by two antenna ligands, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO). Those ligands were chosen for the described spectroscopic and thermodynamic studies because of their high affinity for actinide ions and their potential therapeutic applications as decorporation agents. The in vitro and in vivo stability results obtained here are the first direct confirmation that hydroxypyridinonate chelating agents may target CmIII for sequestration and decontamination purposes. However, the peculiar photophysical properties of the formed CmIII complexes have also revealed very efficient intramolecular energy-transfer processes owing to the unique electronic structure of the CmIII ion. While further studies will necessitate the systematic evaluation of different scaffolds and metal-binding units, it is now evident that tuning the molecular structure of such ligands to explore the coordination chemistry of CmIII could be very powerful in targeting different excited states of the actinide ion, regulating the luminescence sensitization process, and assessing the covalent character of these species.
Experimental Section General
Figure 5. Retention and excretion of 248Cm after injection of 248Cm–ligand complexes into mice. Groups of five mice were injected intraperitoneally with a single dose (0.2 mL injection volume) of 248Cm–ligand complexes (0.23 kBq, 1 mmol of ligand) and were euthanized after 24 h. Data, expressed as percent of injected 248Cm dose (% ID, mean SD), were normalized to 100 % material recovery for each five-mouse group. Excreta of each fivemouse group were pooled; therefore, standard deviations are not available for excretion data. Chem. Eur. J. 2014, 20, 9962 – 9968
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All chemicals were obtained from commercial suppliers and were used as received. The ligands 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2HOPO) were synthesized by Synthetech, Inc. (Albany, OR, USA) and AMRI, Inc. (Albany, NY, USA), respectively, following previously reported procedures, and used as received.[39, 40] Diethylene triamine pentaacetic acid (DTPA) was obtained from Sigma–Aldrich (StLouis, MO, USA). All aqueous solutions were prepared by using deionized water purified through a Millipore Milli-Q reverse osmosis cartridge system, and the pH was adjusted as needed with concentrated HCl or KOH. Stock solutions of ligands were prepared by direct dissolution of a weighed portion of ligand in water prior to each set of experiments. The CmCl3 solution was prepared in
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Full Paper standardized 1 m HCl and had a 95.78 % 248Cm, 4.12 % 246Cm, 0.06 % 245 Cm, 0.02 % 244Cm/247Cm isotopic distribution by atom percentage.
Photophysics UV/Visible spectroscopy: UV/Vis absorption spectra for titrations were recorded on an Ocean Optics USB 2000 spectrophotometer (slit 5 nm, grating 1200 grooves/mm, blaze holographic UV) equipped with a PX-2 pulsed xenon. The spectra were measured with a peak sleeved 1 cm path dip probe (Ocean Optics, Inc.) to maintain low volume measurements and dilution factors. For quantum yield measurements, spectra were recorded on a Varian Cary 6000i double beam absorption spectrophotometer, using quartz cells of 1.00 cm path length. Luminescence spectroscopy: Luminescence spectra for titrations were acquired on an Ocean Optics USB 4000 spectrophotometer (slit 50 mm, grating 600 grooves/mm, blaze 400 nm) equipped with a HPX-2 high-powered xenon light source with a fiber optic scanning monochromator. Samples were pumped through a loop in an asymmetric fluorescence cell (2 10 mm, 40 mL) with a peristaltic pump after each reagent incremental addition. All other emission spectra were recorded on a HORIBA Jobin Yvon IBH FluoroLog-3 spectrofluorimeter, used in steady state mode. A continuous xenon lamp (450 W) was used as the light source. Luminescence lifetimes were determined on a HORIBA Jobin Yvon IBH FluoroLog-3 spectrofluorimeter, adapted for time-correlated single photon counting (TCSPC) and multichannel scaling (MCS) measurements. A sub-microsecond Xenon flashlamp (Jobin Yvon, 5000XeF) was used as the light source, with an input pulse energy (100 nF discharge capacitance) of approximately 50 mJ, yielding an optical pulse duration of less than 300 ns at full width at half maximum (FWHM). Spectral selection was achieved by passage through a double grating excitation monochromator (2.1 nm mm1 dispersion, 1200 grooves/mm). Emission was monitored perpendicular to the excitation pulse, again with spectral selection achieved by passage through a double grating excitation monochromator (2.1 nm mm1 dispersion, 1200 grooves/mm). A thermoelectrically cooled single photon detection module (HORIBA Jobin Yvon IBH, TBX-04-D), incorporating fast rise time photo-multiplier tubes (PMT), wide bandwidth preamplifier, and picosecond constant fraction discriminator, was used as the detector. Signals were acquired by using an IBH DataStation Hub photon counting module and data analysis was performed by using the commercially available DAS 6 decay analysis software package from HORIBA Jobin Yvon IBH. Goodness of fit was assessed by minimizing the reduced chi squared function, and visual inspection of the weighted residuals. Each trace contained at least 5 000 points, and the estimated error on the reported lifetime values is 10 %. Quantum yields were determined as previously described.[14] The q values were calculated by using Equation (1):[25]
q ¼ 0:65 kobs 0:88
ð1Þ
each titration)[41] was used with a Metrohm Titrando 907 (Metrohm) to measure the pH of the experimental solutions. In the incremental titration setup, the Metrohm autoburet was used to add incremental volumes of acid or base standard solutions to the titration cell, and the instruments were fully automated and controlled by using the Tiamo software from Metrohm. All thermodynamic measurements were conducted at 25 8C. Incremental spectrophotometric and spectrofluorimetric titrations: The experimental titration setup was similar to previously described systems.[13] Solutions were assembled from a stock solution of ligand, CmCl3, and the supporting electrolyte solution (0.1 m KCl), with resulting metal concentrations of up to 3 mm. Solutions were forward and backward titrated by the incremental addition of either carbonate-free 0.1 m KOH or 0.1 m HCl titrant (up to 3 cycles) followed by a time delay for equilibration of 100 s. Integration times for the UV/Vis spectra were set to 50 0.1 s, and emission spectra were acquired over 5 20 s for 3,4,3-LI(1,2-HOPO) and 2 50 s for 5-LIO(Me-3,2-HOPO) titrations. Both ligands were excited near their respective maximum absorption wavelength. Buffering of the solution was ensured by the addition of acetic acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mm). An average of 60–120 data points were collected in each direct complex titration, each data point consisting of a pH measurement and luminescence and/or UV/Vis spectra over the pH ranges 1.3 to 4.0 and 1.5 to 11.3 for 3,4,3-LI(1,2-HOPO) and 5LIO(Me-3,2-HOPO), respectively. All spectra were corrected for dilution. Each titration was performed at least three times, in less than 3 h and under positive Ar gas pressure. Data treatment: All thermodynamic data sets were imported into the refinement program HypSpec[33, 42] and analyzed by nonlinear least-squares refinement. All equilibrium constants were defined as cumulative formation constants, bmlh according to Equation (2), where the metal and ligand are designated as M and L, respectively.
mMþlLþhH Ð ½Mm Ll Hh ; bmlh ¼
½Mm Ll Hh ½Mm ½Ll ½Hh
ð2Þ
All metal and ligand concentrations were held at estimated values determined from the volume of standardized stock solutions. All species formed with the ligands L were considered to have significant absorbance to be observed in the UV/Vis spectra and were therefore included in the refinement process. For the spectrofluorimetric titrations, only the metal–ligand complexes were considered to have significant emission to be observed in the emission spectra. The refinements of the overall formation constants included the previously determined ligand protonation constants[14, 34] in each case and the metal hydrolysis products, the equilibrium constants of which were fixed to the literature values.[43] The pM values, defined as the negative logarithm of the free metal concentration in equilibrium with complexed and free ligand, at a fixed pH (7.4 for physiological conditions) and for fixed ligand and metal concentrations of 10 mm and 1 mm, respectively, were calculated by using the program Hyss.[44, 45]
Solution thermodynamics General considerations: All titrant solutions were degassed by boiling for 1 h while being purged under Ar. Carbonate-free 0.1 m KOH was prepared from Baker Dilut-It concentrate and was standardized by titrating against 0.1 m HCl. Solutions of 0.1 m HCl were similarly prepared and were standardized by titrating against tris(hydroxymethyl) aminomethane (TRIS). A Micro Combi Electrode (Metrohm) glass electrode (response to [H + ] was calibrated before Chem. Eur. J. 2014, 20, 9962 – 9968
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In vivo complex stability evaluation The procedures and protocols used in the presented in vivo study were reviewed and approved by the Institutional Animal Care and Use Committee of the Lawrence Berkeley National Laboratory and were performed in AAALAC accredited facilities. The animals used were young adult female Swiss-Webster mice (78 1 days old, 32 2 g). Three groups of five mice were injected intraperitoneally
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Full Paper (ip) with a single dose of Cm–ligand complex (0.23 kBq, 1 mmol of ligand). The Cm–ligand complex solutions formed with the ligands DTPA, 3,4,3-LI(1,2-HOPO), and 5-LIO(Me-3,2-HOPO) were prepared in situ by mixing and incubating the appropriate quantities of Cm and ligand in 0.14 m NaCl, pH 6, to reach a radioactivity of 0.23 kBq and total ligand concentration of 5.0 m (1 mmol) per mouse dose (0.2 mL). All solutions were filter-sterilized (0.22 mm) prior to administration. After injection of the Cm tracer solution, mice were weighed, identified, and housed in groups of five in plastic stock cages lined with a 0.5 cm layer of highly absorbent low-ash pelleted cellulose bedding (Alpha-dry) for separation of urine and feces. All mice were given water and food ab libitum and were euthanized 24 h after the tracer injection. Experiments were managed as metabolic balance studies, in which blood, all tissues, and excreta were analyzed for Cm by liquid scintillation counting on a Packard Tri-Carb model B4430 from PerkinElmer Corporation (Shelton, CT, USA). The methods of sample collection, preparation, radioactivity measurements, and data reduction have been published previously.[7, 46–48] Material recoveries were higher than 96 %.
Acknowledgements We thank Prof. Kenneth N. Raymond, Dr. Norman M. Edelstein, and Dr. David K. Shuh for helpful discussions. Spectroscopic characterization of Cm complexes and solution thermodynamic studies were supported by the Director, Office of Science, Office of Basic Energy Sciences, and the Division of Chemical Sciences, Geosciences, and Biosciences of the U.S. Department of Energy at LBNL under Contract No. DE-AC02–05CH11231. The in vivo experiments were supported by the National Institutes of Health (RAI087604Z) through the U.S. Department of Energy under Contract No. DE-AC02–05CH11231. Keywords: actinides · curium luminescence · thermodynamics
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