[Yb(AAZTA)(H2O)]−: an unconventional ParaCEST MRI probe.

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[Yb(AAZTA)(H

O)]

: an unconventional ParaCEST MRI probe

Daniela Delli Castelli,

_{Lorenzo Tei}

_{, Fabio Carniato}

_{, Silvio Aime}

_{* and Mauro Botta}

_*

coordinated water molecule of [YbAAZTA(H2O)]- by 1H-NMR and

CEST-MRI studies. This made it possible for the first time to exploit the bound water molecule of a LnIII _{complex with carboxyilic donor}

groups for CEST imaging. This result envisages the devolopment of a new family of thermodynamically stable ParaCEST probes based on anionic chelates.

Gadolinium(III) complexes represent the most widely used class of contrast agents (CAs) for Magnetic Resonance Imaging applications (MRI), thanks to several favourable properties, including their distinctive ability to effectively reduce the longitudinal relaxation time T1 of bulk water protons.1 An

alternative modality to generate contrast that is attracting increasing attention relies on the Chemical Exchange Saturation Transfer (CEST) effect.2 _{CEST is a relatively new MRI contrast}

approach in which exogenous or endogenous compounds containing exchangeable protons are selectively irradiated and indirectly detected with enhanced sensitivity by measuring (imaging) the decrease of the bulk water signal.3

A subclass of CEST agents, dubbed ParaCEST, is represented by complexes of paramagnetic metal ions such as FeII_{, Co}II_{, Nd}III_,

PrIII_{, Eu}III_{, Tb}III_{, Dy}III_{, Tm}III _{and Yb}III_.4,5 _{From a coordination}

chemistry point of view, Ln-based ParaCEST agents (LnPC) are entirely analogous to the Gd-based CAs (GdCA). As for the GdIII

complexes, LnIII _{ions in LnPC generally form eight- or}

nine-coordinated chelates with ligands containing oxygen and amine nitrogen donor atoms.6 _{What differentiates them is the marked}

preference of GdIII _{for negatively charged oxygen atoms of}

carboxylate/phosphonate groups instead of the presence of neutral oxygen atoms (carboxoamide/hydroxyl groups) that characterize LnPC. However, we should point out that some Ln-based CEST agents exist with negatively charged groups but their CEST properties are not associated with the coordinated water. The fine-tuning of the rate of exchange (kex = 1/M) of

the coordinated water molecule(s) represents one of the key

factors for the optimization of the performance of both GdCA T1 probes and LnPC contrast agents.7 Typically, T1 agents require

bound water molecules in the fast exchange regime (kex  108 s -1_{) to achieve improved efficacy (high relaxivity),}8 _{whereas for}

ParaCEST agents, which exploit the coordinated water as source of mobile protons, optimal kex values of the bound

water molecule(s) lie in the range 102_-103 _s-1_.9

The chemical nature of the donor groups of the chelator, hence the charge density at the Ln(III) centre, greatly affects the exchange rate of the bound water molecule.10 _{As negatively}

charged donor atoms usually accelerate the exchange rate of the water molecules in the inner coordination sphere of the Ln(III) ion, complexes containing only carboxylic donor groups have never been used for CEST applications (as far as the bound water molecule is concerned).11 _{However, the charge}

density at the metal centre also significantly affects the stability of the complexes. In particular, more basic ligands (e.g. with negatively charged carboxylate donors) tend to form complexes with higher stabilities.12 _{High thermodynamic}

stability (and possibly kinetic inertness) is a mandatory requirement for the in vivo use of GdCA or LnPC,13 _{as the}

release and accumulation of the metal ion is highly harmful for the organism. It follows that the clinical translation of LnPC systems entails as a prerequisite the development of ligands containing negatively charged oxygen atoms and the occurrence of the slow-exchange condition for the bound water molecule.14

The bound water molecule as a source of mobile protons might be preferred over protons belonging to the ligand as its

1_{H NMR resonance typically shows the largest chemical shif}

and hence a greater CEST potential. To the best of our knowledge, no lanthanide(III) complex with negatively charged donor oxygen atoms has been reported to be characterized by the presence of a coordinated water molecule under slow exchange conditions suitable for ParaCEST applications. In a very recent study, Helm and co-workers found that the TmIII

This journal is © The Royal Society of Chemistry 20xx Chem. Commun., 2016, 00, 1-3 | 1

a._{Department of Molecular Biotechnology and Health Sciences, Molecular Imaging}

Centre, University of Torino, Via Nizza 52, 10126 Torino, Italy. E-mail: silvio.aime@unito.it

b._{Dipartimento di Scienze e Innovazione Tecnologica (DiSIT), Università degli Studi del}

Piemonte Orientale “Amedeo Avogadro”, Viale T. Michel 11, I-15121 Alessandria, Italy. E-mail: mauro.botta@uniupo.it

†Electronic Supplementary Information (ESI) available: [experimental details]. See DOI: 10.1039/x0xx00000x

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6-amino-6-methylperhydro-1,4-diazepine tetraacetic acid) bearing a methyl 4-nitrophenylcarbamate moiety in the 6-position ([Tm(AAZTA-Ph-NO2)]-, Figure 1) displays a kex value of 1.4 × 104

s-1 _{for the inner sphere water molecule, nearly four order of}

magnitude lower than that measured for the corresponding GdIII _complex.15 _{Interestingly, a change from two to one in the}

number q of inner sphere water molecules was observed in the [Ln(AAZTA-Ph-NO2)]- complexes moving across the series from

Gd3+ _{to Tm}3+_.

Figure 1. Chemical structures of [(AAZTA)]- _{and [ (AAZTA-Ph-NO2)]}-_.

This result is surprising, not only for the magnitude of the effect but because contrary to previous findings where kex was

found to increase with decreasing cation size.16 _{Such an}

unexpected behaviour has stimulated us to explore the possibility of using the [Ln(AAZTA)]- _{family as ParaCEST agents.}

In the present work, a detailed study on [Yb(AAZTA)]- _was

carried out by performing variable temperature high resolution

1_{H NMR spectra at 14 T, proton relaxometric measurements at}

0.94 T and CEST measurements as a function of concentration at 7 T. The choice of the YbIII _{is motivated both by the fact that}

it represents one of the most used Ln3+ _{cations for CEST}

applications and for the expected lower kex value for the

heavier lanthanides, as reported for the AAZTA-Ph-NO2

chelates.17

AAZTA is a heptadentate polyaminocarboxylate pseudo macrocyclic chelator and its GdIII _{complex possesses a relaxivity}

higher than that of the clinically approved GdCAs, thanks to the presence of two coordinated water molecules characterized by a fast exchange rate (298

M = 90 ns). Unlike most other

heptadentate ligands, the thermodynamic stability of the LnIII

complexes is quite comparable to that of octadentate chelates.18 _{These favourable characteristics have promoted the}

use of [Gd(AAZTA)]- _{in preclinical MRI studies in vivo.}19

The high resolution NMR spectrum of an aqueous solution of [Yb(AAZTA)]-_{, reported in Figure 2, features a broad peak at}

83 ppm that disappears in a D2O solution. This signal has a

relative area equivalent to that of the other peaks of the spectrum, corresponding to the -CH2 groups of the ligand. The

large paramagnetic shif, the peak area and the H/D exchange suggest to assign the resonance to a bound water molecule in slow exchange with the bulk, on the NMR timescale. As further confirmation, the Z-spectrum of a solution of [Yb(AAZTA)]- _has

been recorded at 293 K and 7 T (Figure 3). The curve clearly shows a saturation transfer at 83 ppm that highlights the presence of a pool of slowly exchanging protons. Given the importance of this result, which implies a change of the state of hydration of the Ln-AAZTA complexes across the series, we

have sought an additional and independent experimental support.

Figure 2. High field portion of the 1_{H NMR spectrum of a 20 mM aqueous}

solution of [Yb(AAZTA)]- _{recorded at 14 T and 278 K.}

Figure 3. Z-spectrum of a 13.6 mM aqueous solution of [Yb(AAZTA)]

at neutral pH acquired at 7 T and 293 K. Irradiation time 1 s, irradiation power 48 T.

At low magnetic field strengths, the relaxation enhancement of inner-sphere water molecules induced by the paramagnetic complexes of LnIII _{cations other than Gd}III _is

dominated by the dipolar interaction that is modulated only by the electronic relaxation time T1e.1 This parameter assumes

very short values (ca. 0.1 ps) and shows a very little dependence on the chemical structures of the chelates. Being the outer sphere contribution very similar for different complexes of comparable size, it turns out that relaxivity has a negligible frequency dependence up to about 60 MHz.1

Moreover, for the complexes of a given metal ion with different ligands it roughly scales with the value of q. We have measured the longitudinal relaxation rate R1 at 30 MHz and 298 K for 52.5

mM aqueous solutions of the complexes of Dy3+ _{e Yb}3+ _with

DTPA and AAZTA. It is known that the [Ln(DTPA)]2- _complexes

are isostructural along the series and exhibit one inner sphere water molecule (q = 1).20 _{In the case of AAZTA, it has been}

reported that the GdIII _{and Dy}III _{complexes have a hydration}

number of two.18 _{The data, plotted in Figure 4, clearly indicate}

a change of the hydration state in the AAZTA chelates from DyIII

(q = 2) to YbIII _{(q = 1). This result implies that the [Ln(AAZTA)]}

-2 | Chem. Commun., -2016, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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[Ln(AAZTA-Ph-NO2)]- chelates.15

Figure 4. Plot of the longitudinal relaxation rates of 52.5 mM aqueous

solutions of [Dy(AAZTA)]-_{, [Yb(AAZTA)]}-_{, [Dy(DTPA)]}2- _{and [Yb(DTPA)]}2- _{at 30}

MHz and 298 K

A recently published paper by Baranay et al. also supports

this conclusion. The authors refer to a crystallographic structure of [Lu(AAZTA)]- _{featuring just one bound water}

molecule.21

To assess the CEST sensitivity of [Yb(AAZTA)]-_,

measurements of the percentage of saturation transfer (ST %) on solutions at different concentration have been carried out at pH 7 and 293 K, along with the corresponding ST images at 3 T (Figure 5). From these experiments, we can estimate a sensitivity threshold of ca. 1 mM for this probe.

The rate of water exchange at 293 K has been assessed either by line shape analysis of the 1_{H NMR spectrum (k}

ex =

6.33×103 _{± 6.7×10}2 _s-1_{) or by fitting the Z-spectrum to the Bloch}

equations (kex = 6.67×103 ± 5.2×102 s-1). The kex values

estimated through the two different procedures are virtually identical and differ only by a factor of two from that calculated from relaxometric data for [Tm(AAZTA-Ph-NO2)]- at 298 K. The

exchange rate is remarkably slow, especially in consideration of the fact that the corresponding GdIII _{complex has a k}

ex value

four orders of magnitude larger. For Ln(III) ions comparable values are usually found in the case of neutral or cationic complexes.7 _{To find an in-depth explanation of the reasons for}

this behaviour, structural data for the complexes between Dy(III) and Lu(III) would also be very useful as well as variable pressure relaxation data. However, it is very likely that the change in the hydration state across the series is accompanied by a change in the mechanism and hence in the rate of exchange.

In conclusion, the data shown here demonstrate that it is possible to measure coordinated water exchange rates in the useful range for CEST applications even in the case of anionic Ln(III) complexes with ligands possessing negatively charged oxygen atoms. In fact, the eight-coordinated Yb(III) complex with the heptadentate AAZTA ligand represents the Yb complex with the slowest coordinated water exchange rate ever reported. This unexpected result is of considerable importance as it paves the way to the possible development of Ln-based MRI probes that exploit the CEST effect and consist of thermodynamically stable complexes and therefore more easily usable for in vivo applications.

Figure 5. Top: Saturation Transfer (ST %) dependence on the concentration of

[Yb(AAZTA)]-_{. Bottom: MR-CEST images of a phantom containing five}

solutions of [Yb(AAZTA)]- _{of decreasing concentration at pH = 7: A = 13.6}

mM; B = 6.8 mM; C = 3.4 mM; D = 1.7 mM; E = 0.85 mM. The power of the irradiation pulse is 48 T.

Acknowledgements

This work was carried out under the EU COST Action CA15209 “European Network on NMR Relaxometry”. M.B., L.T. and F.C. acknowledge the support of the Università del Piemonte Orientale (Research Grant 2016).

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