This is the accepted version of the journal article: Porcar Tost, Oriol; Pallier, Agnès; Esteban-Gómez, David; [et al.]. «Stability, relaxometric and computational studies on Mn2+ complexes with ligands con- taining a cyclobutane scaffold». Dalton transactions, Vol. 50, Issue 3 (January 2021), p. 1076-1085. DOI 10.1039/D0DT03402A This version is available at https://ddd.uab.cat/record/273922 under the terms of the license Please do not adjust margins Dalton Transactions FULL PAPER Stability, relaxometric and computational studies on Mn2+ complexes with ligands containing a cyclobutane scaffold Received 00th January 20xx, Accepted 00th January 20xx Oriol Porcar-Tost,a Agnès Pallier,b David Esteban-Gómez,c Ona Illa,a Carlos Platas-Iglesias,*c Éva Tóth,*,b and Rosa M. Ortuño*,a DOI: 10.1039/x0xx00000x The stability constants of Mn2+ complexes with ligands containing a trans-1,2-cyclobutanediamine spacer www.rsc.org/ functionalized with picolinate and/or carboxylate functions were determined using potentiometric titrations (25 ºC, 0.1 M KCl). The stability constant of the complex wih the hexadentate ligand containing four acetate groups (L14-, log KMnL = 10.26) is improved upon replacing one (L24-, log KMnL = 14.71) or two (L34-, log KMnL = 15.81,) carboxylate groups by picolinates. The [Mn(L1)]2- complex contains a water molecule coordinated to the metal ion in aqueous solutions, as evidenced by 1H NMRD studies and 17O chemical shifts and transverse relaxation rates. The 1H relaxivities determined at 60 MHz (3.3 and 2.4 mM -1s-1 at 25 and 37 °C, respectively) are comparable to those of monohydrated complexes such as [Mn(edta)] 2-. The exchange rate of the inner-sphere water molecule (kex298 = 248 ×106 s-1) is slightly lower than that of the edta4- analogue. DFT calculations (M11/def2-TZVP) suggest that the water exchange reaction follows a dissociatively activated mechanism, providing activation parameters in reasonably good agreement with the experimental data. DFT calculations also show that the 17O hyperfine coupling constant A/ is affected slightly by changes in the Mn-Owater distance and the orientation of the water molecule with respect to the Mn-O vector. These concerns reinforced the exploration of other, more Introduction biocompatible paramagnetic metal ions which are able to form stable complexes with high relaxivity. Mn2+ is an obvious The search for efficient and nontoxic contrast agents for candidate to replace Gd3+. It is a good relaxation agent due to magnetic resonance imaging (MRI) is a highly active field. Their its d5 electronic configuration that results in slow relaxation value in diagnostics in general and, specifically, in theranostics times of the electron spin,3,4 while being an essential element, confers these compounds with a great relevance in medicine its toxicity profile is better than that of gadolinium.5 In recent and biomedical research.1 years, many comprehensive studies have been conducted on All clinical agents are complexes of Gd3+, which is explained Mn2+ complexes to relate the stability, kinetic and relaxometric by the great relaxation efficiency of this paramagnetic metal properties to structural elements and denticity of the chelating ion allowing for high image contrast. Their stability and kinetic ligands, and some highly effective Mn2+-based compounds inertness, however, might be limiting factors for clinical have been lately reported as promising potential contrast applications, because of the elevated toxicity of gadolinium agents.3,6,7 potentially released from the complexes in vivo. Nephrogenic It is an experimental fact that Mn2+ complexes of typical systemic fibrosis, a pathology directly linked to Gd injections, poly(amino carboxylate) ligands investigated in the context of and Gd accumulation in the brain and bones of patients with MRI have considerably lower kinetic inertness than typical frequent exposure to contrast-enhanced MRI, have recently Gd3+ chelates. This is likely related to the reduced charge-to- led to serious restrictions in their clinical use.2 radius ratio of Mn2+ compared to Gd3+.8 The rigidity of the chelators has been recognized to improve resistance to dissociation, as it was evidenced some years ago for the a. Departament de Química, Universitat Autònoma de Barcelona, 08193 cyclohexyl derivative [Mn(cdta)]2¯ (Scheme 1). 9 , 10 Likewise, Cerdanyola del Vallès, Barcelona, Spain. E-mail: rosa.ortuno@uab.es both the trans-1,2-cyclohexylene backbone and the pyridine b. Centre de Biophysique Moléculaire, UPR 4301, CNRS, Universitéd’Orléans, rue Charles Sadron, 45071 Orléans Cedex 2, France. E-mail: eva.jakabtoth@cnrs.fr function are crucial and rigidifying structural features to c. Universidade da Coruña, Centro de Investigacións Científicas Avanzadas (CICA) provide slow dissociation of the [Mn(pyc3a)]¯ complex3 and Departamento de Química, Facultade de Ciencias, 15071, A Coruña, Galicia, Spain. E-mail: carlos.platas.iglesias@udc.es (Scheme 1), which has successfully passed to in vivo † Electronic supplementary information (ESI) available: Potentiometric experiments. 11 Very recently, a 2,4-pyridyl-disubstituted experiments and structural details of the structures obtained with DFT. See bispidol ligand and its Mn2+ chelate have been reported. The DOI: 10.1039/x0xx00000x Mn2+-bispidine complex has shown an unprecedented kinetic This journal is © The Royal Society of Chemistry 2016 Dalton Trans., 2016, 00, 1-14 | 1 Please do not adjust margins Please do not adjust margins FULL PAPER Dalton Transactions inertness provided by its highly rigid structure. It is Results and discussion monohydrated, has good relaxation efficiency, and preliminary Thermodynamic stability. The protonation constants of the ligands biodistribution studies showed rapid renal clearance when determined at 25 °C in 0.1 M KCl were reported previously, and injected into healthy mice.12 they are subsequently 9.66, 5.84, 3.06, 2.08 and 1.71 for L14-, 9.58, In a previous work, we have investigated three highly rigid 6.00, 3.78, 2.32 and 2.07 for L14- and 8.89, 6.61, 4.26, 2.97 and 2.79 ligands containing a cyclobutane ring in the backbone, namely for L34-.13 Herein we report the stability constants of the H4cbdta (H4L1), H4cbddapa (H4L2) and H4cbddapa (H4L3) corresponding Mn2+ complexes determined under the same (Scheme 1), 13 which are hexa-, hepta-, and octadentate, conditions using potentiometric titrations (Fig. S1, ESI†). The respectively. With Gd3+, they form hydrated complexes that stability and protonation constants of the complexes are compared show relaxivity values and kinetic inertness comparable with with data reported for related systems in Table 1. 16-18 The stability constant Table 1. Stability and protonation constants of [Mn(L1)]2-, [Mn(L2)]2-, [Mn(L3)]2- and related Mn2+ complexes (25 °C, 0.1 M KCl). log KMnL log KMnHL pMnf [Mn(L1)]2- 10.26 + 0.01a 4.07 + 0.02 a 6.50 [Mn(L2)]2- 14.71 + 0.01a 3.25 + 0.01a 8.76 [Mn(L3)]2- 15.81 + 0.07a 3.60 + 0.05a,b 9.62 [Mn(edta)]2- 12.46;c 12.61d 2.95;c 2.90d 7.83 [Mn(cdta)]2- 14.32;c 14.69d 2.90;c 2.42d 9.90 [Mn(pyc3a)]- 14.14e 2.43e 8.58 a This work. b A second protonation constant with value logK MnH2L = 3.35+0.05 was also calculated. c Data from Ref 16. d Data from Ref 17. e Data from Ref 18. f Defined as –log[Mn2+]free at pH 7.4 for [Mn2+]tot = [L]tot = 10 -5 Scheme 1. Chemical structure of the ligands discussed in this work. M. some relevant Gd3+ complexes such as chelates with octapa4¯, of [Mn(L1)]2- is two logK units lower than that of the edta4- complex, cddadpa4¯, or dtpa5¯ (octapa4- = 6,6′-[(ethane-1,2-diyl- and four logK units lower than those of the cyclohexyl derivatives bis((carboxymethyl)azanediyl))bis(methylene)]-dipicolinic acid; dtpa5- = diethylenetriamine-pentaacetate). 14 Although the [Mn(cdta)]2- and [Mn(pyc3a)]-. Replacing one of the carboxylate thermodynamic stability of the picolinate containing groups of L14- by a picolinate unit promotes a dramatic increase of complexes [Gd(L2)]¯ and [Gd(L3)]¯ was similar, their the stability constant, an effect that is related, at least in part, with dissociation kinetics was very different and the monohydrated the increased ligand denticity. Increasing further ligand denticity by [Gd(L3)]¯ was considerably more labile than the bishydrated incorporating a second picolinate unit in [Mn(L3)]2- results in an [Gd(L2)]¯, as a result of the significant kinetic activity of the additional increase of the stability constant by 1 logK unit. The protonated picolinate function to promote dissociation of the stability constant of the [Mn(L3)]2- complex is rather high for a Mn2+ complex. This was an uncommon example of kinetic-inertness complex, comparable to those of macrocyclic complexes such as enhancement by decreasing ligand denticity. Furthermore, a [Mn(do2a)].19 structural analysis by means of density functional theory (DFT) The stabilities of complexes formed with ligands of different calculations suggested that the large bite angle imposed by the basicities are generally compared by using pM values (pMn rigid trans-1,2-cyclobutanediamine spacer could allow the coordination of larger metal ions providing chelates potentially = -log[Mn2+]free), which for Mn2+ complexes are typically calculated apt for biomedical applications. at pH 7.4 for 10-5 M total concentrations of both the ligand and the In this work, we present stability, relaxometric and metal ion. 20 The results confirm that the [Mn(L1)]2- complex computational studies on [Mn(L1)]2¯, [Mn(L2)]2¯ and presents a rather low stability compared with [Mn(edta)]2-, [Mn(L3)]2¯ complexes. Several picolinate-derivatives have [Mn(cdta)]2- and [Mn(pyc3a)]-. The pMn value of the complex with been previously investigated for Mn2+ complexation;6,15 the heptadentate L24- is however comparable or higher than those of present work ascertained again the role of the picolinate [Mn(edta)]2- and [Mn(pyc3a)]-. The complex with octadentate L34- is functions in L2 and L3 to increase complex stability by even more stable, with a pMn value comparable to that of increasing the ligand denticity. [Mn(L1)]2¯ has been studied in [Mn(cdta)]2-. These results demonstrate that the incorporation of more depth regarding the water exchange rate and compared picolinate groups into the ligand scaffold is beneficial in terms of with related Mn2+ complexes described in the literature. The increasing complex stability. The speciation diagrams calculated experimental results have been complemented with DFT with the thermodynamic stability constants highlight this effect. calculations to understand the influence of the rigidity of the Indeed, the [Mn(L1)]2- complex is stable only above pH  6, while ligand spacer on the water exchange process and to provide below this pH dissociation occurs, together with the formation of a helpful information for the rational design of Mn2+ chelates as protonated complex species (Fig. 1). [Mn(L2)]2- is stable in a broader potential MRI contrast agents with improved characteristics. pH range, forming a protonated form below pH  6, and dissociating significantly only below pH 4. The [Mn(L3)]2- complex 2 | Dalton Trans., 2016, 00, 1-14 This journal is © The Royal Society of Chemistry 2016 Please do not adjust margins Please do not adjust margins Dalton Transactions FULL PAPER dissociates even at lower pH (below pH 3.5), while forming two Relaxivity and 17O NMR measurements. The efficacy of a protonated species. The considerably higher stability of potential contrast agent in vitro is determined by its proton [Mn(L2)]2- compared to [Mn(L1)]2- is likely related to the relaxivity (r1), which denotes the enhancement of the coordination of all seven donor atoms of L24- to the metal ion, as longitudinal relaxation rate of water proton nuclei promoted supported by DFT calculations (Fig. S2, ESI†). In [Mn(L3)]2-, DFT by the paramagnetic ion at 1 mM concentration. Among the calculations suggest that the metal ion is seven-coordinated, with three Mn2+ complexes, only [Mn(L1)]2- is expected to contain the metal coordination environment being fulfilled by the two inner sphere water, therefore relaxivities were measured for amine N atoms, the donor atoms of the picolinate groups and an this chelate. The relaxivity determined for [Mn(L1)]2- at pH 7.4 oxygen atom of one of the carboxylate groups. The second (37 ⁰C, 60 MHz) is 2.4 mM-1 s-1, a value that compares well carboxylate group likely remains uncoordinated (Fig. S3, ESI†). with those reported for monohydrated complexes of similar size, such as [Mn(edta)]2-, [Mn(cdta)]2- and [Mn(pyc3a)]- (2.1 – 2.2 mM-1 s-1 at pH 7.4, 37 ⁰C and 60 MHz, Table 2).3,9 r1 decreases with increasing temperature, as usually observed for small Mn2+ complexes, which implies that relaxivity is limited by fast rotation. Table 2. Relaxivity values of [Mn(L1)]2- and related complexes taken from the literature.a r1, 25 °C r1, 37 °C q [Mn(L1)]2- 3.3 2.4 1 [Mn(edta)]2- 2.8b 2.2c 1 [Mn(cdta)]2- - 2.1c 1 [Mn(pyc3a)]- - 2.1c 1 a Data recorded at 60 MHz, 1.4 T. b Data from Ref 9. c Data from Ref. 3 8 6 relaxivity / mM-1s-1 4 2 0 0.04 0.4 4 40 proton Larmor frequency / MHz Figure 2. 1H NMRD profiles recorded from a 3.29 mM [Mn(L1)]2- solution at 25 °C () and 37 °C (▲) at pH = 7.4. A 15% ligand excess was used to ensure full complexation of the metal ion. The lines represent a fit as explained in the text. The 1H NMRD profiles recorded from an aqueous solution of [Mn(L1)]2- at 25 and 37 °C present a dispersion in the range 2- 40 MHz, which is typical of small Mn2+ complexes (Figure 2).10 A second faint dispersion at 0.1 MHz that could be related to the presence of a small amount of free Mn2+, as the [Mn(H2O)6]2+ complex presents a second dispersion in this region with a sharp increase of relaxivity (r1  21 and 64 mM-1 s-1 at 1 and 0.01 MHz, respectively, 25 °C).21,22 However, the amount of free Mn2+ present in solution under the conditions used to record NMRD profiles is below 0.1 M, as calculated on the basis of the thermodynamic stability constant. Thus, this slight relaxivity increase at low field could rather originate from a small scalar contribution, as observed for a few Mn2+ complexes other than the aqua ion.23 The relaxivity measured Figure 1. Species distribution curves of the Mn2+:L4- systems (L = L1, L2 or at 1 MHz and 25 °C was used to estimate the number of L3). [Mn2+]tot = [L]tot = 10-3 M. coordinated water molecules q using the empirical equation This journal is © The Royal Society of Chemistry 2016 Dalton Trans., 2016, 00, 1-14 | 3 Please do not adjust margins Please do not adjust margins FULL PAPER Dalton Transactions Eq (1), which relates the relaxivity to q and the molecular The kex298 value determined for [Mn(L1)]2- (248±12106 s-1) lies weight of the complex:24 in between those reported for [Mn(edta)]2- (kex298 = 471106 s-1)9 and [Mn(cdta)]2- (kex298 = 220106 s-1). 29 The 𝑟1 (1 MHz) 𝑞= −3) (1) [Mn(pyc3a)]- complex presents a somewhat lower water 9.16{1−𝑒 (−2.97×𝐹𝑊×10 } exchange, presumably due to its reduced negative charge. The water exchange process for most of these complexes is This estimation gives a q value of 1.0, which confirms the characterized by positive values of the activation entropy (S‡), monohydrated state of Mn2+ in [Mn(L1)]2-. This suggests the which suggests a dissociatively activated mechanism. For coordination of the ligand in a hexadentate manner in addition [Mn(L1)]2-, the activation entropy has a small negative value to the coordinated water molecule, as observed in the X-ray which points to an interchange mechanism; however, we structures of the [Mn(edta)]2- and [Mn(cdta)]2- complexes.25 should note that S‡ can be obtained with a large error. Therefore, if available, the activation volumes should be rather used to conclude on the mechanism. The positive activation volume reported in the literature for [Mn(cdta)]2- (V‡ =+9.4 cm3 mol-1) 30 confirmed the dissociative character of the water exchange reaction. The V‡ value reported for [Mn(edta)]2- is however considerably lower (+3.4 cm3 mol-1), suggesting that the rigidity of the ligand spacer influences the mechanism of water exchange. The value of the hyperfine coupling constant A/ obtained from the fit (-37.1±0.6 106 rad s-1) is very similar to those reported for related systems,9,29 which confirms that the number of coordinated water molecules assumed in the analysis is correct (q = 1). In order to obtain information on the rotational dynamics of the complex, the 1H NMRD data have been analysed by using the Solomon-Bloembergen-Morgan (SBM) theory and fixing the rate and the activation enthalpy of the water exchange to the values determined from the 17O NMR measurements. Relaxivities only above 3.5 MHz have been included in this fit, within the validity of the SBM approach to describe dynamic processes like water exchange and rotational motion. In addition, some other parameters have been also fixed in the fit: the distances between Mn2+ and the water protons in the inner and outer coordination Figure 3. Reduced transverse 17O NMR relaxation rates (top) and 17O NMR chemical shifts (bottom) of a 4.9 mM solution of [Mn(L1)]2- at pH = 7.0. The sphere, rMnH and aMnH to 2.83 and 3.6 Å, respectively, and the lines represent the simultaneous fit. diffusion coefficient and its activation energy to DMnH298 = 26×1010 m2 s-1 and EDMnH = 18 kJmol-1. For the parameters describing One of the most important parameters that affect the relaxivity of electron spin relaxation we calculated v298 = 37±2 ps and 2 = paramagnetic metal complexes is the exchange rate of the (0.7±0.1)×1020 s-2). The most important information obtained from coordinated water molecule(s) with bulk water. Water exchange is this fit is the rotational correlation time, R298 = 74±2 ps (ER = 25 relatively slow in the [Mn(H2O)6]2+ complex (kex298 = 28  106 kJmol-1), which is reasonable for a small molecular weight chelate. s-1),21, 26 and it is generally accelerated by coordination of The fitted curves are presented in Figure 2. polyaminopolycarboxylate ligands. We thus measured 17O Factors affecting complex stabilities and the hyperfine coupling NMR transverse relaxation rates and chemical shifts on an constant A/. In a recent paper, Gale et al. reported a method to aqueous solution of [Mn(L1)]2- at pH 7.0. The transverse determine the hydration number of Mn2+ complexes using relaxation rates are dominated by the scalar mechanism, transverse 17O NMR measurements.29 This method relies on the which depends on the hyperfine coupling constant, A/, the assumption that the hyperfine coupling constant does not change water exchange rate, kex298, and the longitudinal relaxation significantly depending on the co-ligands coordinated to the metal time of the electron spin, T1e. 27 The reduced transverse ion. (We should note that this method is not applicable to relaxation rates (T2r) increase with decreasing temperature [Mn(L1)]2- since no clear maximum is observable for the (Figure 3), a situation that is characteristic of systems in the temperature-dependent 17O transverse relaxation rates). The fast exchange regime. 28 Under these conditions, T2r is data reported in Table 3 show that the A/ values determined by determined by all of these three parameters. On the other this series of related complexes are relatively similar, with hand, the reduced 17O NMR shifts r provide direct [Mn(cdta)]2- showing the lowest value (-26.4106 rad s-1)28 and information on A/. The simultaneous fit of the chemical shift [Mn(L1)]2- the highest (-37.1106 rad s-1). and relaxation data provided the parameters listed in Table 3. 4 | Dalton Trans., 2016, 00, 1-14 This journal is © The Royal Society of Chemistry 2016 Please do not adjust margins Please do not adjust margins Dalton Transactions FULL PAPER Table 3. Parameters obtained in the fitting of 17O NMR data of [Mn(L1)]2- compared to literature data for [Mn(edta)]2-, [Mn(cdta)]2- and [Mn(pyc3a)]-. [Mn(L1)]2- [Mn(edta)]2- [Mn(cdta)]2- [Mn(pyc3a)]- 298 𝑘𝑒𝑥 / 106 s-1 248±12 410a / 471b 140c / 220d 89.6e H / kJ mol ‡ -1 21.5±1.8 36.6a / 33.5b 42.5c / 33.2d 37.2e S‡ / J K-1 mol-1 -12±6 +43a +54c +32 V‡ / cm3 mol-1 - +3.4a +9.4c - AO/ħ/ 106 rad s-1 e -37.1±0.6 -36.6a / -33.5b -26.4c / -31.4d -28.7e 1/T1e298 (106 s-1) 30±3 - - - a Data from Ref 28. b Data from Ref 9. c Data from Ref 30. d Data from Ref 29. e Data from Ref 3. Table 4. 17O hyperfine coupling constants and water exchange parameters of the coordinated water molecules obtained with DFT calculations for the [Mn(L1)]2-, [Mn(edta)]2-, [Mn(cdta)]2- and [Mn(pyc3a)]- complexes.a [Mn(L1)]2- [Mn(edta)]2- [Mn(cdta)]2- [Mn(pyc3a)]- rMnO / Å 2.253 2.260 2.271 2.253 rMnO (TS) / Å 3.382 3.339 / 2.760 b 3.316 3.320 H‡ / kJ mol-1 28.0 27.95 / 15.6 25.3 28.6 S‡ / J K-1 mol-1 +6.1 +7.4 / -31.9b +0.2 +2.95 G‡298 / kJ mol-1 26.2 25.75 / 25.1b 25.2 27.7 AO/ħ/ 106 rad s-1 e -37.3 -35.8 -36.0 -39.8 298 𝑘𝑒𝑥 / 106 s-1a 163 198 / 255b 241 90 a Data obtained from calculations on the [Mn(L1)(H O)] 2-·5H O, [Mn(edta)(H O)] 2-·5H O, [Mn(cdta)(H O)] 2-·5H O and [Mn(pyc3a)(H2O)]-·5H2O 2 2 2 2 2 2 systems. b Activation parameters obtained for the associative pathway (see text). explicit water molecules and incorporating a continuum solvation model.31 The inclusion of an explicit second-sphere solvation shell is necessary to provide a better description of the bond distances and 17O A/ values of the coordinated water molecule.32 Furthermore, this second solvation sphere is also required to study the water exchange of the coordinated water molecule (see below). The optimized structure of the [Mn(L1)(H2O)]2-·5H2O system is shown in Figure 4. The cyclobutane unit adopts a puckered conformation to alleviate steric strain, with C-C-C angles in the range 86.4 to 87.8° (a planar structure is characterised by angles of 90°). 33 The conformation adopted by the ligand is typical of edta type ligands, providing a capped trigonal prismatic coordination, in which the coordinated water molecule occupies the capping position.30 The same computational approach was used to obtain the Figure 4. Structure of the [Mn(L1)(H2O)]2-·5H2O system optimized at the optimized structures of the complexes with edta 4-, cdta4- and M11/def2-TZVP level. Hydrogen atoms bonded to carbon atoms have been pyc3a3- (Figures S4-S6, ESI†). The calculated distances of the omitted for clarity. Bond distances are provided in Table S1 (ESI†). metal coordination environments are presented in Table S1 (ESI†). The comparison of the distances obtained with DFT and To further analyse the factors that affect A/ values in these those observed in the X-ray structures of the [Mn(cdta)]2- and complexes, we performed DFT calculations at the [Mn(edta)]2- complexes evidences a good agreement between M11/def2-TZVP level (see computational details below). In the experimental and calculated data.25 In this series, a these calculations we used the cluster-continuum approach, in remarkable difference is observed in the Mn-N bond distances which solvent effects are considered by including a number of which are 0.08 Å longer for [Mn(L1)]2- complex than for the other This journal is © The Royal Society of Chemistry 2016 Dalton Trans., 2016, 00, 1-14 | 5 Please do not adjust margins Please do not adjust margins FULL PAPER Dalton Transactions complexes. These relatively long distances are likely responsible for energy minimum in the potential energy surface that lacks any the lower stability of [Mn(L1)]2- and are associated to the more inner-sphere water molecule. The transition state that related open bite angle of the diaminocyclobutane spacer. This effect the two energy minima was subsequently optimized. Similar becomes evident upon comparing the N-Mn-N angles involving the calculations provided the transition states characterizing the amine N atoms, which amount to 78.6, 74.2, 73.7 and 72.9° for the dissociative mechanism for the [Mn(edta)]2-, [Mn(cdta)]2- and complexes of L1, edta4-, cdta4- and pyc3a3-, respectively. [Mn(pyc3a)]- complexes. The frequency analysis confirmed the The optimized structures of the [Mn(L1)]2-, [Mn(edta)]2-, and nature of the optimized transition states as saddle points and [Mn(pyc3a)]- complexes present very similar distances between provided the zero point energies and thermal terms required to the oxygen atom of the coordinated water molecule and the calculate the enthalpy and entropy of the systems, giving access to metal ion (rMnO), while this distance is slightly longer for activation parameters. The results are shown in Table 4. The [Mn(cdta)]2-. The corresponding A/ values obtained with DFT activation parameters can be subsequently used to estimate the are also very similar, ranging from -35.8 106 to -39.8 106 rad 298 values, which are also listed in Table 4. We want to stress that 𝑘𝑒𝑥 s-1 (Table 4). The Mn-Owater distances calculated for the calculation of exchange rates is a difficult problem, in particular [Mn(edta)]2- and [Mn(cdta)]2- are in good agreement with due to the approximations used in the thermochemistry analysis those observed in the solid state for the ammonium salts and the limited number of explicit water molecules introduced in (2.241 and 2.272 Å, respectively), which also evidence a longer our models. Nevertheless, it is gratifying to see that the calculated bond in the latter.25 298 values are in rather good agreement with the experimental 𝑘𝑒𝑥 To evaluate the impact of rMnO variation on the values of A/, values. we performed additional calculations on the [Mn(L1)(H2O)]2-·5H2O system. The potential energy of this system was investigated by varying the rMnO distance around the equilibrium value from 2.20 to 2.31 Å, using steps of 0.1 Å. Furthermore, we also explored the effect of the orientation of the coordinated water molecule with respect to the Mn-O axis, which was found to affect the A/ values of Gd3+ complexes.34 The orientation of the water molecule can be defined by the angle  that form the Mn-Owater and Owater-CH vectors, where CH represents the centroid of the line connecting the two H atoms of the coordinated water molecule. A  angle of 90° therefore indicates that the bisector of the coordinated water molecule is perpendicular to the Mn-Owater vector, while for  angle of 180° the water molecule and the metal ion are on the same plane. The equilibrium structure of [Mn(L1)(H2O)]2-·5H2O shows a  angle of 129.0°. This angle was varied in steps of approximately 0.5° from 124 to 131°. Thus, the variation of rMnO and  using relaxed potential energy surface scans generated 144 geometries for which the 17O A/ values were calculated. The results of our DFT calculations show that the absolute value of A/ increases as the Mn-Owater distance becomes shorter, as would be expected (Figure 5). A shorter distance favours delocalization of the spin density of the unpaired electrons of the metal ion to the observed nucleus, generating excess of  spin density at the latter. The calculations also show that A/ is affected by the orientation of the water molecule, its absolute value increasing as the  angle increases. The analysis of the natural bond orbitals (NBOs) shows that the lone pair of the coordinated water molecule directed to the metal ion possesses an increasing s character Figure 5. Top: Hyperfine coupling constant A/ calculated for the [Mn(L1)(H2O)]2-·5H2O system at the M11/def2-TZVP level. Bottom: as  increases. This allows a more efficient pathway for the Variation of the %s contribution of the water lone pair directed to the metal spin density to sit on the 17O nucleus through spin polarization. ion with the tilt angle, calculated for three different Mn-O distances: 2.31 Å Factors affecting water exchange. The water exchange process (squares); 2.25 Å (circles); 2.20 Å (triangles). in the family of complexes investigated here was further analysed by using DFT calculations. For this purpose, we first explored the potential energy surface of the [Mn(L1)(H2O)]2-·5H2O system by enlarging the Mn-Owater distance in steps of 0.05 Å. This eventually led to a second 6 | Dalton Trans., 2016, 00, 1-14 This journal is © The Royal Society of Chemistry 2016 Please do not adjust margins Please do not adjust margins Dalton Transactions FULL PAPER Conclusions We have reported a combined experimental (potentiometric, 17O NMR and 1H relaxometric) and theoretical (DFT) study to characterize Mn2+ complexes formed with highly rigid, open- chain chelators containing carboxylate or picolinate coordinating functions and a cyclobutane ring in the ligand skeleton. The ligands L24- and L34-, bearing one or two picolinate functions, respectively, form Mn2+ complexes of a considerably higher thermodynamic stability than the tetracarboxylate analogue L14-, as expected on the basis of the increased number of ligand donor atoms of the picolinates. The cyclobutane ring is responsible for the lower stability constant of [Mn(L1)]2- as compared to [Mn(edta)]2- or [Mn(cdta)]2-, and this was also corroborated with DFT calculations which indicate longer Mn-N distances and more open N-Mn-N angles for the cyclobutane-derivative. Figure 6. Structure of the transition state characterizing the associative Monohydration of [Mn(L1)]2- has been confirmed by NMRD mechanism in [Mn(edta)(H2O)]2-·5H2O system (M11/def2-TZVP level). Hydrogen atoms bonded to carbon atoms have been omitted for clarity. The and 17O chemical shift data. The water exchange on Mn(1)-O(5) and Mn(1)-O(6) distances are 2.760 and 2.324 Å, respectively). [Mn(L1)]2- is faster than that on [Mn(edta)]2- but slower than on [Mn(cdta)]2-. The DFT study presented here provides insight into different Our calculations predict positive values for S‡, as expected for parameters that govern the relaxation properties of dissociative mechanisms,35 though smaller than the experimental [Mn(L1)]2- and closely related derivatives. The calculated 17O ones. Furthermore, the Mn-Owater distances calculated for the A/ values present a good agreement with the experimental transition states (> 3.3 Å) are also indicative of a dissociative data and fall in a rather narrow range. The spin density at the mechanism, in which the coordinated water molecule leaves 17O nucleus is affected by both the Mn-O water distance and the the metal coordination environment before the incoming orientation of the water molecule with respect to the water molecule approaches the metal ion. Attempts to model Mn-Owater vector. Nevertheless, our calculations indicate that the associative mechanism failed in all cases, except for the A/ values are little affected by these structural changes, [Mn(edta)(H2O)]2-·5H2O system. In this latter case, we could supporting their use to determine the hydration state of Mn2+ characterize the transition state responsible for the associative complexes.29 DFT was also used to estimate the activation water exchange pathway (Fig 6), in which the entering water parameters for the dissociative pathway of the water molecule approaches the metal ion (2.760 Å) while the leaving exchange reaction in [Mn(L1)]2-, [Mn(edta)]2-, [Mn(edta)]2- and water molecule is still coordinated (2.324 Å). This transition [Mn(pyc3a)]-. The lower water exchange of [Mn(pyc3a)]- appears to state leads to a minimum energy structure in which the metal be related to a higher H‡, associated to a strong interaction ion is eight-coordinated, with Mn-Owater distances of 2.429 and between the metal ion and the coordinated water molecule. Our 2.393 Å. We note that while not very common, well calculations also suggest that the associative pathway plays a role in characterized eight-coordinated Mn2+ complexes have been the water exchange reaction in [Mn(edta)]2-. reported.36 The corresponding activation parameters (Table 4) are characterized by a negative value of S‡ and a lower value of H‡, as would be expected. 37 Surprisingly, the dissociative and Experimental Section associative pathways are characterized by similar free energies of Sample preparation and potentiometric titrations. Mn2+ activation at 298 K, and thus it is likely that the two of them play a concentration was determined by titrating the metal solutions with role in water exchange. This would be in agreement with the small standardized Na2H2EDTA in urotropine buffer (pH 5.6 – 5.8) in the positive value of S‡ determined for [Mn(edta)(H2O)]2- compared presence of xylenol orange as an indicator. The [MnL]2¯ complexes to [Mn(cdta)(H2O)]2- (Table 3). Thus, the faster water exchange were prepared by mixing the ligand and the metal and adjusting the of the [Mn(edta)(H2O)]2- complex is likely related to the pH to 7. presence of both the associative and dissociative water Stability constants of complexes and their protonation exchange mechanisms. The presence of a rigidifying spacer constants are described and defined in Equations (2) and (3). (cyclobutane or cyclohexane) in the ligand appears to inhibit the associative pathway. The DFT results shown in Table 4 also (2) suggest that the lower exchange rate of [Mn(pyc3a)(H2O)]¯ is due to a higher H‡, presumably related to a stronger electrostatic interaction between the coordinated water molecule and the metal (3) ion, due to the reduced overall negative complex charge. Where [M], [L], and [ML] are the equilibrium concentrations of free metal ion, deprotonated ligand, and deprotonated complex, This journal is © The Royal Society of Chemistry 2016 Dalton Trans., 2016, 00, 1-14 | 7 Please do not adjust margins Please do not adjust margins FULL PAPER Dalton Transactions 2 ℎ𝜐‡ respectively. Experimental data were refined using the computer Γ𝑛 = 1 + 24 ( ) (5) 𝑘𝐵 𝑇 software Hyperquad 2008. 38 Species distribution plots were calculated taking the experimental constants using the computer The nature of the energy minima and transition states was software HySS.39 The ionic product of water used at 25 ºC was pKw = confirmed with frequency analysis. Bulk solvent effects (water) 13.77, while the ionic strength was kept at 0.1 M. Fixed values were were considered with the integral equation formalism variant of the used for pKw and total concentrations of metal, ligand, and acid. See polarizable continuum model (IEFPCM). 48 Natural bond orbital ESI† for more details. (NBO) analysis was carried out with the NBO program (version 3.1),49,50 as included in Gaussian. 1H NMRD and 17O NMR measurements. 1H NMRD profiles of an aqueous 3.29 mM [Mn(L1)]2¯ solution (pH = 7.4) were measured at 25 and 37 °C on a Stelar SMARTracer Fast Field Cycling NMR Conflicts of interest relaxometer (0.00024-0.24 T, 0.01-10 MHz, 1H Larmor frequency) and a Bruker WP80 NMR electromagnet adapted to variable field There are no conflicts to declare. measurements (0.47-1.88 T, 20-80 MHz), controlled by the SMARTracer PC-NMR console. The temperature was controlled by a VTC91 temperature control unit and maintained by a gas flow. The Acknowledgements temperature was determined by previous calibration with a Pt resistance temperature probe. To avoid any free Mn2+, 15% L14- O.P.-T. thanks Universitat Autònoma de Barcelona for a excess was used. predoctoral fellowship and for a travel grant. Financial support from Variable-temperature 17O NMR measurements of a 4.9 mM Spanish Ministerio de Economía y Competitividad (grant CTQ2016- aqueous solution of [Mn(L1)]2¯ at pH = 7.0 were performed on a 77978-R, AEI/FEDER, UE) is gratefully acknowledged. C. P.-I. and D. Bruker Advanced 400 MHz spectrometer using a 10 mm BBFO E.-G. thank Centro de Supercomputación de Galicia (CESGA) for probe (9.4 T, 54.2 MHz) in the temperature range 1–75 °C. The providing the supercomputing facilities. temperature was calculated according to published calibration routines with ethylene glycol and MeOH. Acidified water (HClO4, pH 3.3) was used as diamagnetic reference. Transverse 17O relaxation References times were obtained by the Carl-Purcell-Meiboom-Gill spin-echo technique. To eliminate susceptibility corrections to the chemical 1 (a) The Chemistry of Contrast Agents in Medical Magnetic shifts, the sample was placed in a glass sphere fixed in a 10 mm Resonance Imaging, second Ed.; A. Merbach, L. Helm and E. NMR tube. To improve sensitivity, H217O (10 % H217O, CortecNet) Tóth, Eds.; Wiley, Weinheim, 2013; (b) Theranostics and Image Guided Drug Delivery; M. Thanou, Ed; RSC, Cambridge, 2018. was added to achieve ~1% 17O content in the sample. See, for instance (a) T. Grobner, Nephrol. Dial. Transplat., 2006, Computational details. All calculations described in this work were 21, 1104; (b) T. Kanda, T. Fukusato, M. Matsuda, K. Toyoda, H. performed with the Gaussian 16 program package (version B.01).40 Oba, J. Kotoku, T. Haruyama, K. Kitajima and S. Furui, Full geometry optimisations of the [Mn(L1)(H2O)]2-·5H2O, [Mn(L2)]2-, Radiology, 2015, 276, 228; (c) R. J. McDonald, J. S. McDonald, D. [Mn(L3)]2-, [Mn(edta)(H2O)]2-·5H2O, [Mn(cdta)(H2O)]2-·5H2O and F. Kallmes, M. E. Jentoft, D. L. Murray, K. R. Thielen, E. E. [Mn(pyc3a)(H2O)]-·5H2O systems were performed using DFT Williamson and L. J. Eckel, Radiology, 2015, 275, 772. calculations with the M11 functional, which pertains to the family 3 E. M. Gale, I. P. Atanasova, F. Blasi, I. Ay and P. Caravan, J. Am. of hybrid-meta GGA functionals. M11 is a range-separated Chem. Soc., 2015, 137, 15548. functional of the Minnesota family that uses 100% Hartree-Fock 4. I. Bertini, F. Briganti, Z. Xia and C. Luchinat, J. Magn. Reson., exchange for large interelectronic distances and 42.8% at short 1993, 101, 198. ranges, and was found to be particularly well suited to compute 5 (a) D. Pan, A. H. Schmiedr, S. A. Wickline and G. M. 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