Biochimie (1992) 74, 777-783 © Soci6t6 fran~:aise de biochimie et biologie mol6culaire / Elsevier, Pads

777

Lanthanide complexes of aminophosphonates as shift reagents for 7Li and 23Na NMR studies in biological systems R R a m a s a m y l, M M C A

C a s t r o 2, D M d e F r e i t a s 3, C F G C G e r a l d e s 2 .

1Department of Chemistry, University of Texas at Dallas, Richardson, TX 75083, USA, 2Department of Biochemistry and Center of Neurosciences, University of Coimbra, 3000 Coimbra, Portugal; 3Department of Chemistry, Loyola University Chicago, 6525 N Sheridan Road, Chicago, IL 60626 USA

(Received 27 May 1992; accepted 15 July 1992)

Summary - - A systematic NMR characterization of various Dy(lll) complexes of linear and macrocyclic aminophosphonates as 7Li

and 23Na NMR shift reagents for biological systems was undertaken. Their efficacy as shift reagents (SR) was tested under constant aqueous solution ionic strength conditions at pH 7.5 as a function of p = [SR] / [M+]. Further characterization of the two best SRs, Dy(PcPcP)27- and Dy(DOTp)5-, led to the conclusion that, although quite sensitive to solution pH and the presence of alkali metal ions and Mg2+ and Ca2+, these complexes were stable towards hydrolysis by phosphatases. The lack of precipitation of its solutions in the presence of Ca2+, allowed the choice of Dy(DOTp)5- as the best overall SR for biological studies. Other SRs, like Dy(TTHA)3-, although less sensitive to pH and to divalent ions, require significantly higher concentrations to yield the same shifts, leading to large bulk susceptibility artifacts in perfused tissues and organs. lanthanide shift reagents / metal NMR / ion concentration / NMR of cells

Introduction

The highly diagnostic and non-destructive nature of nuclear magnetic resonance (NMR) spectroscopy provides a powerful tool to study the role of metal ions in cells and tissues. With the advent of aqueous lanthanide shift reagents (SR) it has been possible to resolve the intra- and extracellular cation NMR signals in cells and tissues [1-6]. Several paramagnetic lanthanide complexes have been characterized and employed as shift reagents for NMR active physiological alkali metal ions [1-6]. In brief, these shift reagents are thermodynamically stable, chemically inert, possess a net negative charge at physiological pH, are membrane impermeable, and induce chemical shifts in the extracellular cation resonances sufficient to clearly resolve them from the intracellular cation resonances. Until recently bistripolyphosphatedysprosium(III) (Dy(PPPh 7-) and triethylenetetraaminehexaacetate" dysprosium(Ill) (Dy(TTHA)3-) have been the most commonly used SR for cells and tissues [7-9]. Dy(PPP)27- produces large 23Na and 7Li shifts in systems free of calcium, but in the presence of the *Correspondence and reprints

levels of calcium required for normal cell/tissue function, the induced shifts are too small [7]. Studies have shown that at physiological concentrations, Ca2+ competes with Dy3+ for tripolyphosphates (ppps-) and forms Ca(PPP) 3- complexes [3, 10]. Moreover, Dy(PPP)27- is quickly hydrolysed in systems containing phosphatases [11], leading to the release of Dy3+ which is toxic to cells and tissues. By contrast, Dy(TTHA)3- is relatively non-toxic but the shifts induced are much smaller. For this reason relatively higher concentrations of i3y(TTHA)3- are required to resolve cation NMR resonances in perfused tissues [12, 13] and in live animals [14, 15] leading to problems caused by large bulk susceptibility shifts [16]. Recently, Tm(DOTP) 5- and Dy(bPPPpob)5- have been reported as 23Na shift reagents for perfused heart studies [17-19]. The paramagnetic shifts induced by these complexes on 23Na+ resonances are larger than Dy(TTHA)3-, but less than Dy(PPP)27-. However, in the presence of these reagents (3-5 mM range) the intra- and extracellular pools of Na ÷ in perfused hearts [12, 19] can be distinguished clearly. In particular, Tm(DOTP) 5- and Dy(DOTP) 5- are resistant to hydrolysis by phosphatases in biological systems [20]. The new 23Na+ shift reagent, 1, 4, 7, 10-tetraazacyclododecane-N,N',N",N'"-tetramethylenephosphonate-

778 thulim(Ill) (Tm(DOTP) 5-) is the only one to provide well resolved spectra, from perfused hearts while maintaining normal heart function [10]. For this reason is has recently been used for 23Na+ spectroscopy and imaging studies of live animals [21-23]. In this paper, we report the N M R characterization in aqueous solution of Dy(III) complexes of a variety of phosphonate ligands shown i , figure 1 including Dy(DOTP)S- as shift reagents for 7Li and 23Na N M R studies in biological systems. This shift characterization is only qualitative, as opposed to the much more quantitative characterization published for Dy(TTHA) 3-) [24].

Materials and methods Lithium chloride (LiCI), dysprosium chloride (DyCl3), triethylenetetraaminehexaacetic acid (H6TTHA), nitrilotris(methylene)triphosphonate (NTP), sodium triphosphate (NasPPP), glucose, tetramethylammonium chloride, tetramethylammonium hydroxide,

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deuterium oxide (D20), sodium chloride (NaCI), potassium chloride (KCI), magnesium chloride (MgCl2), calcium chloride (CaCl,), and thulium chloride (TmCl3) were supplied by Aldrich Chemical Company (Milwaukee, Wl). Hepes [4-(2, hydroxyethyl)-l-piperazineethanesulfonic acid] was obtained from Sigma Chemical Company (St Louis, MO). Alkaline phosphatase was supplied by Boehringer Mannheim. Bis(dihydroxyphosphinylmethyl)phosphinic acid (HsPcPcP) was synthesized by Medichem Research Inc (Chicago, IL). l, 4, 7triazacyclononane-N,N',N"-tris(methylene)-phosphonic acid (H6NOTP) and 1, 4, 7, 10-tetraazacyclododecane-N,N', N",N'"tetra(methylenephosphonic) acid (H6DOTP) were synthesised as reported earlier [20, 25-28l. Diethylenetriaminepentamethylenephosphonic acid (HIoDTPP) was obtained from Monsanto Chemical Company (St Louis, MO). All chemicals were used as received except sodium triphosphate which was recrystaflized three times from 40% ethanol. Lanthanide complexes of NOTP and DOTP were prepared by in situ methods as previously described [20, 25, 26], while those of NTP and DTPP were prepared by similar procedures described in literature [29]. Lanthanide complexes of PcPcP were prepared in a similar manner as those used for triphosphate complexes. Hepes solutions adjusted to pH 7.5 with tetramethylammonium hydroxide were used to buffer the solutions containing the shift reagents. Ionic strength adjustments were made using tetramethylammonium chloride as previously described [ 10]. 7Li, 23Na, and 3~p measurements were made at 116.5, 79.4, and 121.4 MHz respectively on a Varian VXR-300 NMR spectrometer. Some 23Na measurements were also made at 52.9 MHz on a Varian XL-200 NMR spectrometer. Both instruments were equipped with a 10-mm multinuclear probe and a variable temperature unit. 60 ° pulses were used for metal NMR studies. The 23Na and 7Li NMR shifts were measured in the absence and presence of shift reagents. As usual, upfield paramagnetic shifts are reported as positive, which is the opposite of the convention for the NMR chemical shift scale. The spectrometer was always field-frequency locked on the 2H resonance of D20 in each sample, thus enabling to negate bulk magnetic susceptibility shifts accompanying the addition of Dy(Ill) or Tin(Ill) since D20 suffers no hyperfine shift [16]. For 31p NMR studies, 45 ° pulses were used with 0.72-s pulse delay at a probe temperature of 37°C.

DOTI=

Results and discussion 7Li÷ and 23Na+paramagnetic shifts induced by phosphonate complexes of dysprosium

NTP

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:poa zN~IGHz)z~N~(CH z)z~N

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Fig 1. Structure of the iigands used in this study.

The efficacy of the phosphonate complexes of dysprosium as shift reagents for 7Li + and 23Na+ N M R studies was tested under constant ionic strength conditions, at pH 7.5. The concentration of LiCl or NaCl was held constant at 5 m M and the ionic strength at 0.36. This concentration for Na + is much lower than in plasma, whereas for Li + it is on the upper limit o f the levels in tissues of manic-depressive patients receiving lithium carbonate. This choice was based on the need to compare the in vitro efficacy of the different SRs for Li + and Na +. The application of the Na + results to physiological conditions, as far as competition with the other

779 ions is concerned, is not hindered, as a much higher Na ÷ concentration would be favorable to this ion in such a competition. The shift reagent concentrations varied from 3 to 30 mM. The paramagnetic shifts induced by the phosphonate complexes are plotted against the stoichiometric mole ratio of shift reagent to Li ÷ or Na ÷, p, in figure 2. The shifts induced in the 7Li÷ and 23Na÷ NMR resonances by Dy(PcPcP)27- and Dy(DOTP)S- are significantly larger than those induced by Dy(NTP)3-, Dy(NOTp)3-, and Dy(DTPP) 6--, under similar conditions. The observed induced shifts might have been slightly larger if the solution were free of added salt. However, the observed shifts are close to their maximum value since a bulky, noncompetitive organic counter cation, tetramethylammonium, was used. Dy(NTP)3- and Dy(DTPP) 6-- do produce large shifts at higher pH (data not shown), but at physiological pH the shifts are small. Based on these studies Dy(PcPcP)27- and Dy(DOTP)S- were chosen for further characterization for NMR studies under physiological conditions.

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pH Dependence of ZLi÷NMR shifts induced by phosphonate shift reagents

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The pH dependence of 7Li÷ shifts induced by Dy(PcPcP)27- and Dy(DOTP)S- is shown in figure 3, where these reagents are compared with Dy(PPP)27-. The shifts exhibited by Dy(PcPcP)27- and Dy(DOTP) 5are dependent on pH between 6 and 8.5. The Dy(PcPcP)27- induced 7Li÷ shifts decrease for pH < 7.5 in a similar manner to those induced by Dy(PPP)27- [10]. Comparing ppps- and PcPcP 5-, the two ligands have values for the two highest pKa's of 8.9, 6.26, and 8.8, 5.2 [30], respectively. Similarly, one would expect that the highest pKa's for the Dy 3÷ complexes of those two ligands would be in the same range. Hence the decreased 7Li ÷ N M R shifts observed below pH 7.5 for Dy(PcPcP)27- resemble the behaviour observed for Dy(PPP)27- [10]. In the case of Dy(DOTP)S-, the decrease of the 7Li ÷ induced shift at pH < 8 is due to stepwise protonation of the complex in this pH range [20]. This protonation process is, however, more complex than previously described as corresponding to the formation of DyH(DOTP) 4- and DyH2(DOTP)3- [20], and will be described in a future publication. In fact, evidence exists that these complexes can aggregate into dimers and tetramers upon changes in p H [ 17, 31 ]. The pH dependence of the X3Na+ shifts induced by 5 mM Dy(DOTP)5- in a sample containing 5 mM Na ÷ was also studied. The observed shifts are maximum above pH 9.5 and drop sharply below pH 8.5 to zero at pH 5.5 (data not shown). This titration curve reflects the protonation of the bound phosphonates in the complex as the pH decreases. At pH 2 the proto-

-301

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Fig 2. Dependence of paramagnetic 7Li÷ and 23Na÷ NMR shifts induced by several shift reagents on the ratio p = [SR]/[M ÷] (M ÷ = Li ÷ or Na÷). Full and broken lines indicate data for Na÷ and Li ÷, respectively. The symbols for the ligands Dy(PcPcP)27- (~), Dy(DTPP) 6- (X), Dy(DOTP) 5(o), Dy(NTP)3- (/3, and Dy(NOTP) 3- (V) are indicated in parentheses. Each point represents the average of 23Na÷ or 7Li÷ NMR measurements on three separate samples. In all cases, the agreement was within 0.02 ppm. Li÷ concentration was held constant at 5 mM while the shift reagent concentrations varied. All solutions were buffered with 50 mM Hepes and also contained 10 mM glucose and 17% D20. The pH was adjusted to 7.5 with tetramethylammonium hydroxide. Ionic strength was adjusted to 0.36 with tetramethylammonium chloride so that all samples had the same ionic strength as that of 30 mM Dy(PcPcP)27-. NMR conditions are described in Materials and methods. nated species present remain intact, not releasing the Dy 3÷ ion, but the Na ÷ affinity for this species is considerably reduced, as observed for Tm(DOTP) 5- [17].

Competition between Li +and other cations for phosphonate shift reagents In physiological solutions, the divalent cations Ca 2+ and Mg 2+ can effectively compete with Li + for binding

780 to the shift reagent and drastically decrease the 7Li + NMR isotropic shifts. Figure 4 shows the effects of Ca :+ and Mg 2+ on the absolute magnitudes of the 7Li + shifts induced by Dy(DOTp)5- and Dy(PcPcP)27-. The induced paramagnetic shifts of the above shift reagents are quite sensitive to the presence of Ca 2+ and Mg2+. A 5 mM Dy(PcPcP)27- solution turns opalescent upon addition of mmol amounts of Ca2+. In fact, precipitation occurs when the Ca 2+ concentration is higher than 3 mM, suggesting that Ca2+ is competing for the ligand in Dy(PcPcP)27-, possibly via a scrambling mechanism. No such observation was made for DOTP complexes of Tm(lll) or Dy(Ill) [ 17, 20]. Monovalent cations present in physiological solutions can effectively compete with Li + for the shift reagent. Moreover, during lithium carbonate psychia-

,61 13 12

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18

16

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[.2+]lmM

Fig 4. Competition between Ca :'+ (1:1) or Mg2+ (A) and Li+ for D y ( P c P c P ) 2 7- (open symbols) and Dy(DOTP)5- (closed symbols). The shift reagents' counter cation was tetramethylammonium ion. LiCI concentration was held constant at 5 hiM. The concentrations of Dy(DOTP)5- and Dy(PcPcP),7were maintained at 5 mM.

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Fig 3. pH Dependence of paramagnetic shifts afforded by Dy(DOTP)-S- (a), Dy(Ppp):7- (~l), and Dy(PcPcp)27- ("0. Li+ concentration was held constant at 5 mM in the three curves. The concentrations of Dy(DOTP)5-, Dy(PPP)J- and Dy(PcPcP)27- were kept constant at 5 mM. The reported pH values are not corrected for the deuterium isotope effect, The pH dependence of 7Li+ shifts was found to be reversible by adding tetremethylammonium hydroxide and HC! above and below pH 7.5, respectively.

tric treatment, Na + and K + are present at much higher concentrations compared to Li +. Since the shift reagents are known to bind monovalent cations with a fairly weak labile interaction, it was therefore necessary to investigate the effects of Na + and K + on the absolute magnitudes of the shifts induced in the ~Li+ NMR resonances. Figure 5 compares the effects of these ions on the shifts induced in the 7Li + resonance by Dy(DOTP)5- and Dy(PcPcP).,7-. The shifts caused by Dy(PcPcP):7- are sensitive to the presence of monovalent cations, but the observed shift is less pronounced than in the presence of divalent cations. In the presence of competing monovalent cations, Li + ions may be forced to exchange between various sites in the second coordination sphere of the shift reagent. Because each site has a specific 0 value, the geometric term in the pseudocontact shift, G = (3 cos20--1)/r3, will be partially averaged because of the rapid exchange of Li + between different sites. Hence, the induced paramagnetic shifts, which are dominated by

781 the pseudo-contact contribution, will decrease [ 10]. In any case, the 7Li+ shifts induced by shift reagents are still quite substantial despite the presence of competing cations like Na +, K +, Ca2*, and Mg 2+. Based on figures 4 and 5, the relative order of cation competition ability for Dy(PcPcP)27- and Dy(DOTP)S- at pH 7.5 can be predicted to be Ca2+ > Mg2+ >> Li + > Na + > K +. As expected, the divalent cations have a much higher affinity than monovalent cations for the shift reagents. The decreasing order of association constants for monovalent cations follows the increasing order of ionic radii, supporting the electrostatic model for shift reagent-cation interaction in solution.

_L t = 90min

t = 30min

Hydrolytic stability of phosphonate shift reagents in biological systems containing phosphatases Figure 6 depicts the time dependencies of the 3]p NMR spectra of solutions of Dy(PcPcP)27- and Dy(PPP)27- in the presence of rat liver homogenates containing alkaline phosphatase. The alp NMR

t = 0min I

300

I

I

200 100

I

0

A I

ppm

300

I 200

I 100

I 0

pprn

Fig 6. Time dependence of 31p NMR spectra of (A) 10 mM Dy(PPP)a7- and (B) l0 mM Dy(PcPcP)27- in the presence of rat liver homogenates containing alkaline phosphatases.

16

ItL , 14 13 12

11 §obsl pprn

10 9 8

? 6

5 4

0

20

40

60

80

100

120

140

[M*3 Ir~M Fig 5, Competition between Na* (~) or K* (A) and Li* for

Dy(PcPcP27- (open symbols) and Dy(DOTP)S- (closed symbols). Same experimental conditions as for figure 4.

spectra of Dy(PcPcP)27- clearly indicate that the shift reagent is not hydrolysed when incorporated in the suspension containing rat liver homogenates. The hydrolytic stability is presumably due to the lack of P-O-P linkages in Dy(PcPcP)J- However, we do observe a decrease in intensities of the chelate ~esonances and an increase in the intensities of the free ligand resonances, suggesting that the complex is dissociating in solution to give Dy3+ and free ligand. The chemical inertness of the complex towards hydrolysis in the presence of rat liver homogenates is confirmed by the absence of 3]p NMR resonances typical of phosphonate degradation products. The inorganic phosphate resonance observed in the case of the Dy(PcPcP)27- sample is an impurity present from the start of the experiment, whose area did not change during the experiment. An inset of the spectra of free ligand PcPcP 5- (fig 6B) also indicated the presence of inorganic phosphate, thus confirming that inorganic phosphate was an impurity and not a degradation product. The chelate Dy(PPP)27- is hydrolysed very rapidly when placed in the suspension containing rat liver homogenates. The P-O-P bonds in Dy(PPP)gare very susceptible to attack by alkaline phosphatase and pyrophosphatases [11 ]. Compared to Dy(PPP)JDy(PcPcP)27- is chemically inert towards hydrolysis by alkaline phosphatase as seen in this study.

782

Conclusions Recently, we d~:monstrated in red blood cells [32, 33], as did Seo et al i34] in salivary glands, that a modified inversion recovery (MIR) pulse sequence can be used to discriminate intracellular from extracellular cations without the use of shift reagents. This method requires T~ values of intracellular cations to be quite different from those in the extracellular pool. Because some cations in several biological systems (eg 23Na+) do not meet the above criteria, the shift reagent NMR method continues to enjoy the status as the method of choice in several types of studies of biological systems. Among the shift reagents commonly used, those based on aminophosphonate ligands show some important advantages relative to those based on oligophosphate ligands, eg Dy(Ppp)_,7-, namely hy-lrolytic stability of the C-P bonds towards phosphatase activity, or relative to those based on aminocarboxylate ligands, eg Dy(TTHA)3-, namely higher affinity towards the alkali metal cations as a consequence of their higher negative charge. However, this later property also causes some disadvantages, such as perturbations of cell membrane potentials and cation distributions in the intra- and extracellular pools [36]. The shift reagents based on the tetraazamacrocyclic tetraphosphonate ligand, Ln(DOTP)5-, combine the above advantages with high stability in solution and no precipitation in the presence of Ca2÷, which are important disadvantages displayed by the Dy(PPP)27and Dy(PcPcP),.7- complexes [101. Therefore, despite their high negative charge and shift sensitivity to pH and competing cations, the Ln(DOTP).~- chelates show the best combined properties as shift reagents for 7Li÷ and 2.aNa÷ studies of biological tissues and live animals 117, 18, 20-231.

4 5 6 7 8 9

l0 I1

12

13

14

Acknowledgments 15

RR acknowledges Loyola University of Chicago, graduate school for a dissertation fellowship. The authors thank Dr Ramanujam of the Medical College, Wisconsin, for providing fresh liver homogenates. MMCAC and CFGCG thank INIC (Portugal) for financial support.

16

References

17

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18 19

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