Differences in the Relaxometric Properties of Regioisomeric Benzyl

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Differences in the Relaxometric Properties of Regioisomeric BenzylDOTA Bifunctional Chelators: Implications for Molecular Imaging Lauren Rust,†,# Katherine M. Payne,†,# Fabio Carniato,‡ Mauro Botta,‡ and Mark Woods*,†,§ †

Department of Chemistry, Portland State University, 1719 SW 10th Avenue, Portland, Oregon 97201, United States Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale “Amedeo Avogadro”, Viale T. Michel 11, I-15121 Alessandria, Italy § Advanced Imaging Research Center, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, United States Downloaded via UNIV AUTONOMA DE COAHUILA on May 4, 2019 at 18:50:36 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: The bifunctional chelator S-2-(4-isothiocyanatobenzyl)1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-1,4,7,10-tetraacetate (IBDOTA) is on paper the most attractive of the commercially available bifunctional chelators for magnetic resonance imaging (MRI) applications. The preserved DOTA scaffold is known to produce extremely kinetically and thermodynamically robust chelates with the Gd3+ ion. Also, ligation through four acetate pendant arms should ensure that the rapid water exchange kinetics so, crucial to the function of an MRI contrast agent are retained. However, upon ligation of the Gd3+ ion, IB-DOTA differentiates into two distinct isomers defined by the positions of the benzylic substituent (corner or side). A relaxometric analysis of these two isomers revealed marked differences in the property and behavior of the two chelates. Most notably the side isomer is found to be substantially more likely to aggregate in aqueous solution than its corner counterpart. This aggregation results in higher relaxivity for the side isomer versus the corner isomer, an observation that potentially obscures the impact of differences in water exchange kinetics between the two isomers. The side isomer is composed of a significant fraction of a twisted square antiprismatic coordination geometry that exchanges water more rapidly than optimal (τM = 7 ns) for maximizing relaxivity. The impact of this excessively fast exchange is not observed in the relaxivity of the side isomer only because in isolation this chelate tumbles much more slowly than the corner isomer. However, this situation is not expected to persist when the chelate is employed in a typical bioconjugate. These results imply that the corner isomer of IBDOTA may represent a better choice of bifunctional chelator for bioconjugation applications in which a large macromolecule is to be tagged for MRI applications.

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INTRODUCTION

The presence of the isothiocyanate group is a disadvantage when studying the physicochemical properties of the chelates of IB-DOTA, owing to its propensity to slowly hydrolyze in aqueous solution. For this reason studying the more stable precursor chelate, S-2-(4-nitrobenzyl)-DOTA (NB-DOTA), is preferred; the identity of the para- aromatic substituent will have minimal effects on the properties of the chelate. Although NB-DOTA was first reported in 19926,7 the first report of its solution state properties, behavior, and stability did not appear until 2004.5 That study overlooked a key aspect of the structural chemistry of LnNB-DOTA chelates. It has since been unambiguously demonstrated that NB-DOTA produces, upon introduction of the metal ion, two isomeric chelates differentiated by the regiochemistry of the nitrobenzyl substituent: either corner or side.8,9 Given the way in which bifunctional chelators are most commonly deployed, the

Bifunctional chelators and in particular the commercial availability thereof have revolutionized the fields of molecular imaging and bioconjugation.1−4 A wide selection of bifunctional chelators is now available allowing various functional groups within biomolecules to be tagged with a range of metal ions. S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-1,4,7,10-tetraacetate (IB-DOTA) (Figure 1) remains one of the more important bifunctional chelators. In part this is because it reacts quickly, cleanly, and selectively with primary amines, such as that of a lysine side chain, under mild conditions. But also because of its capacity to form thermodynamically stable and kinetically robust chelates with a large range of metal ions, including larger ions such as the lanthanides.5 The favorable chelation properties of IB-DOTA with Gd3+ make it the bifunctional chelator of choice (certainly among the commercially available options) for applications involving tagging biomolecules for use in magnetic resonance imaging (MRI) research. © XXXX American Chemical Society

Received: March 26, 2019 Revised: April 19, 2019

A

DOI: 10.1021/acs.bioconjchem.9b00223 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

are reported. The potential for regioisomerism to influence the effectiveness of the chelate as a contrast agent in bioconjugates produced from IB-DOTA is examined with a view to determining whether regioisomerism is a consideration in the development of bioconjugates for MRI applications.

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RESULTS AND DISCUSSION It is now well established that the TSAP coordination isomer of GdDOTA-type chelates exhibit water exchange kinetics that are 50−100 times faster than those of the SAP isomer.10−12 This water exchange process is central to the function of the chelate as a contrast agent for MRI. Contrast agents operate by shortening the relaxation time constants of the solvent water in the tissues to which they are distributed. By T1-weighting, Gd3+ chelates are able to make the regions to which they are distributed appear brighter in MR images. This T1-shortening effect arises from modulation of the dipole−dipole interactions between the unpaired electrons of Gd3+ and proximate water protons. Since dipole−dipole interactions are strongly distance dependent, water molecules that are bound directly to the metal ion will be more efficiently relaxed. Exchange of water between the inner coordination sphere of Gd3+ and the bulk solvent will cause an overall shortening of the solvent water T1. Clearly for this effect to be efficient, water exchange should be rapid. This suggests that the TSAP coordination isomer would afford a more effective contrast agent, and given the SAP/ TSAP ratios given in Figure 2, that the side isomer would be superior as a component of an MRI contrast agent.13 However, very fast water exchange has unanticipated, and detrimental, consequences for chelate hydration which can reduce the effectiveness of the contrast agent.17,18 Relaxometic Investigations into the Discrete Regioisomeric Chelates. Samples of the corner and side isomers of GdNB-DOTA were prepared by the reaction of H4NB-DOTA with GdCl3 (pH 5.5) followed by separation by reversed-phase HPLC purification, as described previously.5,8 Whole water exchange can be determined by measuring the transverse 17O relaxation rate constant (R2p) of a solution of the Gd3+ as a function of temperature. This method requires Gd 3+ concentrations of several 10s of mM and so is only practicable on freely soluble chelates: in general the low molecular weight chelate rather than a bioconjugate. The 17O R2p profile of the corner isomer exhibits the usual bell-shaped curve that maximizes at about 305 K (Figure 3A), consistent with

Figure 1. Connectivity structure of the bifunctional chelators H4IBDOTA and H4IBDOTMA, their more stable analogues H4NBDOTA and H4NB-DOTMA, and their conjugate H4BP-DOTA and H4BP-DOTMA.

production of two chemically distinct regioisomers during the chelation reaction has implications for the behavior of bioconjugates derived from IB-DOTA. In general bifunctional chelators are conjugated to the biomolecule prior to the introduction of the metal ion. This produces a bioconjugate incorporating a single DOTA ligand structure. However, when the metal ion is introduced, this single ligand structure will differentiate into two structurally distinct regioisomeric chelates and therefore two distinct bioconjugates. It is likely to be very difficult, and in many cases simply impossible, to separate or even discriminate between the corner and side isomers of these bioconjugates. Our most recent study of LnNB-DOTA chelates demonstrated that the two regioisomers of LnNB-DOTA possess chemically distinct structures that are not in exchange.8 Furthermore, the distribution of SAP and TSAP coordination isomers was found to differ between the two regioisomersthe four possible solution state structures of LnNB-DOTA chelates are shown in Figure 2. The SAP/ TSAP ratio in Gd3+ DOTA-type chelates is known to play an important role in governing the water exchange kinetics of the chelate.10−12 This is in turn expected to strongly influence the relaxometric properties (effectiveness as a contrast agent) of any bioconjugate derived from GdIB-DOTA.13 The physicochemical properties of each regioisomer of GdNB-DOTA

Figure 2. Wedge representations following Dale’s conventions14−16 of the corner and side isomers of LnNB-DOTA (X = NO2) chelates and the coordination isomers adopted by each. The mole fraction of each coordination isomer observed for the Eu3+ chelate (xEu) is also given. B


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Table 1. Fitting Parameters for the 17O R2p NMRD Profiles of the Aqueous Solutions of Corner and Side Isomers of GdNB-DOTAa side [Gd]/mM A/ℏ/106 rad s−1 τM298/ns ΔHM/kJ mol−1 a

corner

SAP

TSAP

8 −3.4 188 45.7

5.4 −3.5 250 48.3

6.6 −3.5 7 28.1

[Gd] = 8 mM and 12 mM, respectively.

case exchange is actually so fast that this isomer may suffer from the negative effects that very rapid water dissociative exchange kinetics has on chelate hydration.17 For these reasons it is unclear whether the side isomer of GdNB-DOTA will benefit from its more rapid water exchange kinetics or suffer from a loss of hydration. To probe this question the nuclear magnetic relaxation dispersion (NMRD) profiles of the corner and side isomers of GdNB-DOTA were recorded at three temperatures (Figure 4). NMRD profiles examine the effectiveness of a contrast agent (expressed in terms of its relaxivity, r1) as a function of the Larmor frequency. The NMRD profiles of both regioisomers of GdNB-DOTA are typical of those of low molecular weight chelates (Figure 4A,C). Relaxivity is higher and flat in the low field region, it disperses just above 1 MHz, and thereafter relaxivity falls gently with increasing B0. Significantly the relaxivity of the side isomer is higher than that of the corner isomer at all fields and temperatures. This difference is more pronounced at lower temperatures and fields; as the temperature increases the difference becomes smaller until at 310 K the relaxivity of the two isomers is very similar. Measuring relaxivity (by linear regression over a concentration range 2.0−0.2 mM, Supporting Information, Figure S1) as a function of temperature demonstrates unequivocally that both chelates are in the fast exchange regime (Figure 4B,D).18 This means that the difference in relaxivity can arise from only differences in the electron spin relaxation parameters, Δ2 and τV (at low fields); the rotational correlation time, τR; and/or the chelate’s hydration state, q/r6 (defined as the number and position of water molecules exchanging with the chelate). The difference in water exchange kinetics between the two chelates will not affect relaxivity (with the exception of its influence on q/r6).24 To analyze these NMRD data we used the same approach as in previous systems in which an extremely rapidly exchanging chelate was present:18 the value of q/r6 was decreased (by increasing the value of rGdH) for chelates that exhibit extremely rapid exchangethe TSAP isomers. The value of rGdH (the Gd−H distance) is unknown in solution, and the choice of value is somewhat arbitrary. Consequently, all fits of NMRD data possess a qualitative component; rGdH = 3.0 Å is widely regarded as a reasonable value for chelates, such as the SAP isomers, that are in rapid exchange. To account for the effect of extremely rapid water exchange in the TSAP isomer, the Gd− H distance is extended to rGdH = 3.1 Å.17,18 This value is intended to qualitatively represent the effect that increasing water exchange could have on the hydration state,17 rather than reflecting a quantitative assessment of that effect. Since the extremely rapidly exchanging TSAP isomer is found to make up 55% and 5% of the side and corner isomer populations, respectively, a weighted average of the two rGd−H values was used in each fitting. Because relaxivity scales

Figure 3. Temperature dependence of 17O transverse relaxation rate constants of the corner (A) and side (B) regioisomers of GdNBDOTA.

moderately fast water exchange kinetics. Fitting this curve to theory19 allows the water residence lifetime to be determined as a weighted average of the two coordination isomers present in solution, affording a value of τM = 188 ns. Although the corner isomer predominates as the more slowly exchanging SAP (∼95%), it exhibits more rapid water exchange than the parent chelate GdDOTA (τM = 244 ns)20 which adopts the SAP isomer to a lesser extent (∼80%).21,22 The water exchange kinetics of chelates from nitrobenzyl-cyclen are commonly found to be faster than those of analogues derived from the unsubstituted parent cyclen.5,10,11 In contrast to the corner isomer, the 17O R2p profile of the side isomer (Figure 3B) is not a simple bell curve. As temperature decreases R2p increases, maximizing about 305 K before decreasing and then increasing again at temperatures below 280 K. This type of profile is characteristic of systems comprised of roughly comparable proportions of two isomeric forms that have very different water exchange kinetics.23 Because the data exhibit clear trends arising from each coordination isomer, it is possible to fit these data to a two component model extracting τM values for both the SAP and TSAP isomer (Table 1). As expected the TSAP isomer is found to have more rapid water exchange than the SAP isomer (τM = 7 ns versus 250 ns). However, neither SAP nor TSAP isomer was found to have water exchange kinetics considered optimal for developing a high relaxivity contrast agent.24 The exchange kinetics of the side isomer (τM = 12.4 ns, weighted average) are substantially more rapid, and closer to optimal, than those of the corner isomer (τM = 188 ns). Significantly, the more rapidly exchanging TSAP-side isomer has water exchange kinetics that are slightly faster than is generally considered optimal by many interpretations of Solomon− Bloembergen−Morgan (SBM) theory.25−29 However, in this C


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Figure 4. Water proton relaxometry of the corner (filled circles) and side (open diamonds) isomers of GdNB-DOTA. A and C: Nuclear magnetic relaxation dispersion (NMRD) profiles recorded at 283 (blue), 298 (black), and 310 K (red). B and D: The temperature dependence of the longitudinal relaxivity at 20 MHz.

proportionally with q/r6, the decrease in hydration state associated with the side isomer will have the effect of lowering the relaxivity relative to the corner isomerthe opposite of what is observed experimentally. A small but real difference in the electron spin relaxation characteristics of the two chelates is apparent from examination of the low field region of the NMRD profiles. The difference in relaxivity between the two isomers is substantially larger at low fields than at high fieldspointing to a difference in electron spin relaxation characteristics between the corner and side isomers. The side isomer appears to exhibit slightly longer electron spin relaxation time constants. However, a difference in electron spin relaxation characteristics cannot explain the difference in high field relaxivity between the two isomers evident in both the NMRD and temperature profiles. This leads to the unexpected conclusion that these two isomeric chelates must tumble at different rates in solution. These conclusions are borne out by the results of fitting the NMRD profiles to SBM theory. The SAP and TSAP isomers of DOTA-type chelates have been shown to possess similar electron spin relaxation characteristics. So it is surprising to find that the trace of the zero-field splitting (ZFS) tensor (Δ2) is noticeably larger for the side isomer than for the corner isomer (Table 2). This difference cannot be attributed to the difference in SAP/TSAP ratio between the two isomers.30 Since the factors that govern electron spin relaxation remain poorly understood, it must be presumed that the disparity in Δ2 arises from differences in way that the nitrobenzyl substituent distorts the ligand structure. This may give rise to differences in the ligand field that alter the ZFS. Fitting also confirms perhaps the most surprising conclusion about the two chelates: the apparent difference in τR. As discrete low molecular weight chelates, the corner and side isomers presumably have comparable hydrodynamic volumeswhich would be expected to yield similar rates of tumbling in solution. It is known that the value of rGdH chosen during the fitting of an NMRD profile can affect the

Table 2. Fitting Parameters for the NMRD Profiles of the Corner and Side Isomers of GdNB-DOTA in Aqueous Solutiona rGdH/Å τM298/ns τR298/ps ΔHR/kJ mol−1 Δ2/1019 s−2 τv298/ps ΔHV/kJ mol−1

corner

side

3.005b 188 90 17 0.86 19.7 1.0

3.055b 12.4c 120 16 1.2 18 1.0

Parameters fixed in this analysis were a = 4.0 Å and D298 = 2.24 × 10−5 cm2 s−1. The hydration state (q/r6) was adjusted to account for the presence of very rapidly dissociatively exchanging TSAP isomers by fixing q = 1 and varying the value of rGdH as given in the table. b Weighted average value of rGdHSAP = 3.0 Å and rGdHTSAP = 3.1 Å previously used when fitting systems with very rapidly exchanging TSAP isomers. The SAP/TSAP ratio is assumed to be the same as previously reported for the Eu3+ chelates. cWeighted average value derived from the 1/τM values of the SAP and TSAP isomers determined from the 17O R2p profiles. a

value of τR that is obtained31the two parameters are mutually compensatory. However, the choice of rGdH value was not found to qualitatively affect the result that different τR values are obtained for each isomerfitting both profiles with rGdH = 3.0 Å also yields different tumbling rates for each isomer (τR = 85 and 101 ps for the corner and side isomer, respectively); it is only the magnitude of the effect that is affected by the choice of rGdH. The different relaxivities observed for the corner and side isomers of GdNB-DOTA can only reasonably be explained by a difference in τR. This also accounts for the comparable relaxivities observed at 310 K. As the temperature increases, the rate of water exchange also increases. A decrease in τM alone will leave relaxivity unaffected unless exchange is already extremely rapid. However, exchange in the TSAP-side (55%) is extremely fast, and reducing τM will D


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corner and TSAP-corner isomers of TbNB-DOTMA tumble in solution at comparable rates.30 However, a comparison of corner and side isomers has not previously been conducted by Curie relaxation studies. Comparison was based on a T2 analysis of each of the most shifted axial ring protons (axS) of the SAP-side, TSAP-side, and SAP-corner isomers of TbNBDOTA. The T2 value for this proton was measured by line width analysis at three field strengths: 9.4, 11.5, and 14.1 T. The TSAP-corner isomer was not measured owing to its low equilibrium abundance in solution.8 The field dependence of the transverse relaxation rate constants was analyzed according the method described by Aime et al.: the values of rTbH were taken from the crystal structures of TbTCE-DOTA and TbDOTMA, respectively,12 and a value of 0.43 ps was used for T1e in each case.34 Fits of the data are shown in Figure 5, and the values of τR obtained are shown in Table 3.

increase the negative impact of exchange on the hydration state (q/r6) of the side isomer. Because the corner isomer (95% SAP) exchanges much more slowly, there is no impact on hydration (q/r6). Thus, the relaxivity of the side isomer falls more quickly with rising temperature than does that of the corner isomer (Figure 4B,D). By 310 K the effects of decreased q/r6 and longer τR in the side isomer are almost exactly offset by one another, and the relaxivities of the two isomers are essentially the same. Nonetheless, this finding that isomeric chelates should tumble at different rates is so surprising that it merits further independent study. Comparison of Rotational Correlation Time Constants (τR). The most likely reason that the corner and side isomers of GdNB-DOTA tumble at different rates in solution is that self-association of the chelate is occurring in aqueous solution. If the extent of self-association is different between the two isomers, then one will be found to tumble more slowly in solution than the other. A Stern−Volmer approach is commonly used to probe self-association in Ln3+ chelates.32,33 In these experiments the isostructural Nd3+ chelate is typically used as a quencher of emission from the corresponding Eu3+ chelate. By necessity Stern−Volmer experiments must be carried out at low concentrations (∼10−5 M), whereas relaxometric measurements are carried out at significantly higher concentrations, ∼10−3 M: thus the situation that exists for relaxometric measurements may not persist in the Stern− Volmer experiment. Adding 0−4 equiv of the NdNB-DOTA to a 10 μM solution of the Eu3+ chelate afforded almost identical static and dynamic quenching constants (KSVΦ and KSVτ, respectively) for the two regioisomers (Supporting Information, Figure S3). This would indicate the there is no appreciable difference in self-association between the two isomers at concentrations ∼10−5 M. However, any intermolecular interaction is likely to be a weak pi-stacking or hydrophobic interaction, and there is a substantial difference in concentration between the two experiments. These results cannot therefore be used to eliminate the possibility of selfassociation in the relaxometric experiments. Efforts to obtain direct evidence of self-association through absorbance or fluorescence measurements focused on the nitrobenzyl group at higher concentrations failed to provide convincing evidence either for or against self-association. A more direct method of assessing τR is through the field dependence of the Curie relaxation contribution.30,34 This method has been employed previously to determine values of τR for chelates of Tb3+. Comparing chelates in which the Ln3+ ion identity or ligand structure are different may not always be justified since Ln−H distance and magnetic anisotropy are likely to vary from chelate to chelate.35 In this study these considerations are less of a concern owing to the common ion (Tb3+) chelates and isomeric ligands involvedthe Tb−H distances are expected to be comparable (Table 3). This approach has been used previously to establish that the SAP-

Figure 5. Dependence of the transverse relation rate constant of the most shift axial (axS) proton on the square of the B0 field for three isomers of TbNB-DOTA: SAP-corner (closed red circles), TSAP-side (open blue diamonds), and SAP-side (open blue circles). G = 10−4 T. Data were measured at 9.4, 11.5, and 14.1 T at 298 K.

The values of τR determined by Curie relaxation are in almost precise agreement with those determined by NMRD fitting (Table 2 vs Table 3). Given that the value of τR obtained by fitting NMRD data depends strongly upon the chosen value of rGdH, the good agreement between these two measures strongly supports the choice of rGdH value used in fitting the NMRD profile. The chosen values of rGdH (3.0 and 3.1 Å for the SAP and TSAP isomers, respectively) were used to represent the effect that very rapid dissociative water exchange has upon the chelate hydration;18 they were not used because physical evidence pointed to these exact values. However, the close correlation of tumbling rate constants afforded by these two independent techniques strongly suggests that these values are good representations of hydration in these chelates. The results of both NMRD and Curie relaxation experiments show an unambiguous difference in the molecular tumbling between the corner and side isomers. Assuming that the difference in the molecular tumbling between the corner and side isomers arises from the degree to which each chelate self-associates, this difference could be exaggerated by

Table 3. Rotational Correlation Time Constants (298 K) for the Corner and Side Isomers of Tb3+ Chelates Determined by Curie Relaxation Are Markedly Different isomer

rTbH (Å)

τR (ps)

SAP − corner TSAP − side SAP − side

3.73 3.69 3.73

90 ± 2 116 ± 1 125 ± 3 E


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Further evidence for greater self-association of the side isomer is apparent from the relaxivities of the GdBP-DOTA isomers (Table 4). Above the CMC the relaxivity of the side isomer is 78% greater than that of the corner isomer, a finding that is supported by inspection of the NMRD profile (Figure 7). This substantial difference in relaxivity is achieved in spite

increasing the hydrophobicity of the substituent. The biphenyl derivatives of other related chelates are known to aggregate strongly, forming micelles above certain concentrations.18,36 Introducing a biphenyl into the substituent should accentuate the differences in the behavior of the two regioisomers. Coupling of the biphenyl moiety to the chelate was achieved by reaction of the commercially available bifunctional chelator IB-DOTA with 4-phenylbenzyl amine in 1:1 (v/v) H2O (pH 9)/dioxane (24 h).18 In the same pot an excess of GdCl3 hydrate was added, and the pH dropped to 5.5 by addition of 1 M hydrochloric acid. The two regioisomers of the GdBPDOTA chelate afforded by this reaction were separated and purified by preparative RP-HPLC. In common with previously reported biphenyl conjugates,18,36 the water proton longitudinal relaxation rate constant (R1) is not linearly dependent upon the concentration of either isomer of GdBP-DOTA (Figure S2, Supporting Information). Two regimes are evident for both isomers: a high relaxivity regime above the critical micelle concentration (CMC), and a lower relaxivity regime below the CMC. Fitting these data as previously described18 affords the relaxivity in each regime as well as the CMC (Table 4). It is notable that

Figure 7. NMRD profiles of the corner (blue) and side (red) isomers of GdBP-DOTA at pH 6.6, 298 K recorded at concentration above the CMC (closed triangles: [GdL] = 1.51 mM (corner) and 1.48 mM (side)) and below the CMC (open circles: [GdL] = 0.15 mM (corner) and 0.20 mM (side).

Table 4. Critical Micelle Concentrations and Relaxivity Values Determined above and below the CMC from the Data in Figure S2 for the Corner and Side Isomers of GdBPDOTA r1 mM−1 s−1 (20 MHz, 298 K) isomer

CMC (mM)

micelle diameter (nm)a

below CMC

above CMC

corner side

0.90 1.10

10 18

8.3 11.6

16.4 29.2

of the fact that the side isomer of GdNB-DOTA is found to have a reduced hydration state (vide infra). The higher relaxivity of the side isomer can be attributed almost entirely to a longer τR value that arises from its greater propensity toward self-association, which in turn affords larger micelles. The NMRD profiles of both isomers at concentrations below the CMC exhibit subtle but highly instructive differences (Figure 7). Below the CMC the relaxivity of the side isomer is again higher than that of the corner isomer (Table 4). However, the attribution of this difference in relaxivity to a difference in τR is evident from the NMRD profiles: slowly tumbling Gd3+ chelates exhibit a characteristic “hump” at high fields that is strongly indicative of a long τR value. Although not pronounced (like those of the profiles recorded above the CMC), there is a clear hump in the NMRD profile of the side isomer below the CMC that is not present in the profile of the corner isomer. A quantitative analysis of these NMRD data buttresses this conclusion with the value of τR obtained for the side isomers 38% longer than that obtained for the corner isomer (Table 5).

a

Hydrodynamic dynamic diameter determined by DLS.

the CMC of the side isomer is 22% larger than that of the corner isomer. This difference is not paralleled in the data obtained for other closely related chelates.18,36 The difference in CMC may reflect a difference in the size of the micelles produced by each isomer. Dynamic light scattering (DLS) was used to confirm that the side isomer produces not just larger micelles but micelles that are almost twice as big as those produced by the corner isomer (Figure 6). These observations are consistent with a greater propensity of the side isomer toward self-association.

Table 5. Fitting Parameters for the NMRD Profiles of the Corner and Side Isomers of GdBP-DOTAa rGdH/Å τM298/ns Δ2/1019 s−2 τv298/ps τR298/ns

corner

side

3.005 188 1.10 19 0.13

3.055 12.4 1.08 17 0.19

Recorded below the CMC. Parameters fixed in this analysis were q = 1, rGdH and τM298; and the outer sphere parameters a = 4.0 Å (the distance of closest approach of an outer sphere water) and DGdH298 = 2.24 × 10−5 cm2 s−1. a

Figure 6. Dynamic light scattering shows that the micelles produced by the side isomers (blue) of GdBP-DOTA are much larger than those produced by the corner isomer (red). F


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CONCLUSIONS The differentiation of macrocyclic substituted bifunctional chelators into corner and side isomers during chelation is more than a mere technical detail. There are real practical implications for the production of two discrete and different chelates from a single bifunctional chelator. There are significant physicochemical differences between the corner and side isomers that have the potential to significantly impact the performance of the resulting bioconjugate in MRI applications. Throughout this work the side isomer is observed to afford higher relaxivity, but this situation need not necessarily persist in all applications. The higher relaxivity of the side isomer is due to the greater degree of self-association of this isomer when a hydrophobic substituent is present. Although this effect could result in higher binding constants for bioconjugates when the side isomer is coupled to small targeting ligands, if GdIB-DOTA is used to label a truly massive macromolecule (e.g., a protein) chelate tumbling could be governed primarily by that of the macromolecule. In these circumstances it is reasonable to presume that the side isomer is likely to offer a significantly less effective contrast agent than the corner isomer, even though water exchange appears closer to optimal. The 55% of the side isomer that adopts a very rapidly exchanging TSAP isomer (τM = 7 ns, much shorter than optimal) will suffer from the reduced hydration state (q/r6) associated with systems in very rapid dissociative exchange.17 This will directly reduce the relaxivity that can be achieved from bioconjugates of GdIB-DOTA derived from the side isomer. It is important to appreciate that the effect of exchange on q/r6 is limiting reducing relaxivity relative to what could be achieved for a chelate tumbling at a given rate. In the studies presented herein, the side isomer tumbles more slowly that the corner isomer; this is a function of the extent of self-association and not an intrinsic feature that will carry over to bioconjugates. Upon incorporation into a bioconjugate, a chelate will experience the effect of both global (that of the conjugate has a whole) and local rotation (rotation around the linker between chelate and biomolecule). The extent to which each of these processes govern relaxivity is also expected to depend upon the molecular structures involved, including the regioisomerism of the chelate.36 However, on the basis of the water exchange characteristics determined in this work the most effective bioconjugates for MRI applications are likely to be obtained if only the corner isomer is isolated and employed in the bioconjugation step.

with a calibrated copper-constantan thermocouple (uncertainty of ±0.1 K). Additional data points in the range 20−70 MHz were obtained on a Stelar relaxometer equipped with a Bruker WP80 NMR electromagnet adapted to variable field measurements (15−80 MHz proton Larmor frequency). Sixteen experiments of two scans were used for the T1 determination for each field. The exact complex concentration was determined by the BMS shift method at 11.7 T.37 17O NMR measurements were recorded on a Bruker Avance III spectrometer (11.7 T) equipped with a 5 mm probe and a standard temperature control unit. Aqueous solutions of the complexes at neutral pH and containing 2.0% of the 17O isotope (Cambridge Isotope) were used. The observed transverse relaxation rates were calculated from the signal width at a half-height. Low field relaxation measurements were made on a 0.47 T Bruker MiniSpec MQ20 using an inversion recovery pulse sequence with full temperature control. Critical micelle concentrations were determined by fitting data to eq 1 where R1obs = the measured longitudinal relaxation rate constant in the presence of the Gd3+ chelate; R1d = the measured longitudinal relaxation rate constant in the absence of the Gd3+ chelate; r1n.a. = the relaxivity of the nonaggregated chelate; r1a. = the relaxivity of the aggregated chelate; CMC = critical micelle concentration; C = the concentration of the Gd3+ chelate. R1obs − R1d = R1p = (r1n.a. − r1a) × CMC + r1a × C

(1)

1

H NMR spectra were recorded on a Bruker Avance IIa spectrometer operating at 400.13 MHz using a 5 mm broadband probe, and Bruker Avance III spectrometers operating at 500.13 and 600.13 MHz. The temperature was controlled using the installed variable temperature controller model 2416 with BCU-05 chiller. For Curie relaxation experiments samples were prepared in D2O at 5 mM concentrations and flamed sealed into the tube. Line-widths were determined by a line fitting analysis using the NUTS software program from ACORN NMR. All lines were fitted a Lorentzian/Gaussian function. Photophysical Measurements. Emission and excitation spectra were collected on a PTI Quantamax 300 phosphorimeter utilizing a Xe-flash lamp and PMT detector. Absorbance spectra were obtained on a UV-3600 Shimadzu UV−vis NIR spectrophotometer. Stern−Volmer experiments were conducted on 10−5 M solutions of EuNB-DOTA using NdNBDOTA as a quencher and direct excitation of Eu3+ at 397 nm, monitoring at 597 nm. GdNB-DOTA chelates were examined over the concentration range 0.2−2 mM. Emission spectra were acquired using excitation at λex = 270, 297, and 340 nm, and excitation spectra were acquired using λem = 400, 535, and 590 nm. Dynamic light scattering experiments were carried out on a Malvern Zetasizer NanoZS equipped with a He−Ne laser operating at λ = 633 nm. 2.5 mM aqueous solutions of chelates were run at 298 K using the internally calibrated 0.6−6 mm particle size range.

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EXPERIMENTAL PROCEDURES General Remarks. All solvents and reagents were purchased from commercial sources and used as received. H4NB-DOTA and H4IB-DOTA were purchased from Macrocyclics. The Nd3+, Eu3+, Gd3+, and Tb3+ chelates were prepared, and the regioisomers were separated and purified using previously published methods.8 Relaxometric and NMR Studies. The 1/T1 nuclear magnetic relaxation dispersion (NMRD) profiles of water protons were measured on a fast field-cycling Stelar SmarTracer relaxometer (Mede, Pv, Italy) over a continuum of magnetic field strengths from 0.00024 to 0.25 T (corresponding to 0.01−10 MHz proton Larmor frequencies). The relaxometer operates under computer control with an absolute uncertainty in 1/T1 of ±1%. The temperature control was carried out using a Stelar VTC-91 airflow heater equipped

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00223. G


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(8) Payne, K. M., and Woods, M. (2015) Isomerism in BenzylDOTA Derived Bifunctional Chelators: Implications for Molecular Imaging. Bioconjugate Chem. 26, 338−344. (9) Webber, B. C., and Woods, M. (2012) Structural Analysis of Isomeric Europium(III) Chelates of NB-DOTMA. Inorg. Chem. 51, 8576−8582. (10) Woods, M., Botta, M., Avedano, S., Wang, J., and Sherry, A. D. (2005) Towards the rational design of MRI contrast agents: a practical approach to the synthesis of gadolinium complexes that exhibit optimal water exchange. Dalton Trans., 3829−3837. (11) Woods, M., Kovacs, Z., Zhang, S., and Sherry, A. D. (2003) Towards the rational design of magnetic resonance imaging contrast agents: isolation of the two coordination isomers of lanthanide DOTA-type complexes. Angew. Chem., Int. Ed. 42, 5889−5892. (12) Woods, M., Aime, S., Botta, M., Howard, J. A. K., Moloney, J. M., Navet, M., Parker, D., Port, M., and Rousseaux, O. (2000) Correlation of Water Exchange Rate with Isomeric Composition in Diastereoisomeric Gadolinium Complexes of Tetra(carboxyethyl)dota and Related Macrocyclic Ligands. J. Am. Chem. Soc. 122, 9781− 9792. (13) Wiener, E. C., Abadjian, M.-C., Sengar, R., Vander Elst, L., Van Niekerk, C., Grotjahn, D. B., Leung, P. Y., Schulte, C., Moore, C. E., and Rheingold, A. L. (2014) Bifunctional Chelates Optimized for Molecular MRI. Inorg. Chem. 53, 6554−6568. (14) Dale, J. (1973) Exploratory calculations of medium and large rings. 1. Conformational minima of cycloalkanes. Acta Chem. Scand. 27, 1115−1129. (15) Dale, J. (2007) Multistep conformational interconversion mechanisms. Top. Stereochem. 9, 199−270. (16) Dale, J. (1980) The conformation of free and complexed oligoethers. Isr. J. Chem. 20, 3−11. (17) Webber, B. C., and Woods, M. (2014) The confluence of structure and dynamics in lanthanide(III) chelates: how dynamics help define structure in solution. Dalton Trans. 43, 251−258. (18) Avedano, S., Botta, M., Haigh, J. S., Longo, D. L., and Woods, M. (2013) Coupling Fast Water Exchange to Slow Molecular Tumbling in Gd3+ Chelates: Why Faster Is Not Always Better. Inorg. Chem. 52, 8436−8450. (19) Helm, L., Morrow, J. R., Bond, C. J., Carniato, F., Botta, M., Braun, M., Baranyai, Z., Pujales-Paradela, R., Regueiro-Figueroa, M., Esteban-Gómez, D., Platas-Iglesias, C., and Scholl, T. J. (2018) In Contrast Agents For MRI: Experimental Methods (Pierre, V. C., and Allen, M. J., Eds.) Royal Society of Chemistry, Cambridge, Chapter 2. (20) Powell, D. H., Ni Dhubhghaill, O. M., Pubanz, D., Helm, L., Lebedev, Y. S., Schlaepfer, W., and Merbach, A. E. (1996) Highpressure NMR kinetics. Part 74. Structural and Dynamic Parameters Obtained from 17O NMR, EPR, and NMRD Studies of Monomeric and Dimeric Gd3+ Complexes of Interest in Magnetic Resonance Imaging: An Integrated and Theoretically Self-Consistent Approach. J. Am. Chem. Soc. 118, 9333−9346. (21) Aime, S., Botta, M., Fasano, M., Marques, M. P. M., Geraldes, C. F. G. C., Pubanz, D., and Merbach, A. E. (1997) Conformational and Coordination Equilibria on DOTA Complexes of Lanthanide Metal Ions in Aqueous Solution Studied by 1H-NMR Spectroscopy. Inorg. Chem. 36, 2059−2068. (22) Aime, S., Botta, M., and Ermondi, G. (1992) NMR study of solution structures and dynamics of lanthanide(III) complexes of DOTA. Inorg. Chem. 31, 4291−4299. (23) Aime, S., Botta, M., Garda, Z., Kucera, B. E., Tircso, G., Young, V. G., and Woods, M. (2011) Properties, Solution State Behavior, and Crystal Structures of Chelates of DOTMA. Inorg. Chem. 50, 7955− 7965. (24) Wahsner, J., Gale, E. M., Rodriguez-Rodriguez, A., and Caravan, P. (2019) Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem. Rev. 119, 957−1057. (25) Bloembergen, N. (1957) Proton relaxation times in paramagnetic solutions. J. Chem. Phys. 27, 572−3.

Relaxivity determinations: plots of R1 vs [Gd3+], for both NB and BP substituted chelates. Additional information about Stern−Volmer experiments (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected]. Phone: +1 503 725 8238 or +1 503 418 5530. ORCID

Mark Woods: 0000-0003-4890-7544 Author Contributions #

L.R. and K.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Portland State University and the National Institutes of Health (DK119945) (M.W.) and Università del Piemonte Orientale (Ricerca Locale 2016) (M.B. and F.C.) for financial support of this work.

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ABBREVIATIONS DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-1,4,7,10tetraacetate; NB-DOTA, S-2-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-1,4,7,10-tetraacetate; IB-DOTA, S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecaneN,N′,N″,N‴-1,4,7,10-tetraacetate; BP-DOTA, S-2,2′,2″,2‴-(2(4-(3-([1,1′-biphenyl]-4-ylmethyl)thioureido)benzyl)1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-1,4,7,10-tetraacetate

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REFERENCES

(1) Gai, Y., Sun, L., Lan, X., Zeng, D., Xiang, G., and Ma, X. (2018) Synthesis and Evaluation of New Bifunctional Chelators with Phosphonic Acid Arms for Gallium-68 Based PET Imaging in Melanoma. Bioconjugate Chem. 29, 3483−3494. (2) Makris, G., Radford, L. L., Kuchuk, M., Gallazzi, F., Jurisson, S. S., Smith, C. J., and Hennkens, H. M. (2018) NOTA and NODAGA [99mTc]Tc- and [186Re]Re-Tricarbonyl Complexes: Radiochemistry and First Example of a [99mTc]Tc-NODAGA Somatostatin ReceptorTargeting Bioconjugate. Bioconjugate Chem. 29, 4040−4049. (3) Spreckelmeyer, S., Ramogida, C. F., Rousseau, J., Arane, K., Bratanovic, I., Colpo, N., Jermilova, U., Dias, G. M., Dude, I., Jaraquemada-Pelaez, M. d. G., et al. (2017) p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-Single Photon Emission Computed Tomography Imaging. Bioconjugate Chem. 28, 2145−2159. (4) Tripepi, M., Capuana, F., Gianolio, E., Kock, F. V. C., Pagoto, A., Stefania, R., Digilio, G., and Aime, S. (2018) Synthesis of High Relaxivity Gadolinium AAZTA Tetramers as Building Blocks for Bioconjugation. Bioconjugate Chem. 29, 1428−1437. (5) Woods, M., Kovacs, Z., Kiraly, R., Brücher, E., Zhang, S., and Sherry, A. D. (2004) Solution Dynamics and Stability of Lanthanide(III) (S)-2-(p-Nitrobenzyl)DOTA Complexes. Inorg. Chem. 43, 2845−2851. (6) McMurry, T. J., Brechbiel, M., Kumar, K., and Gansow, O. A. (1992) Convenient Synthesis of Bifunctional Tetraaza Macrocycles. Bioconjugate Chem. 3, 108−117. (7) Renn, O., and Meares, C. F. (1992) Large-Scale Synthesis of the Bifunctional Chelating Agent 2-(p-Nitrobenzyl)-1,4,7,10-Tetraazacyclododecane-N,N′,N″,N‴- Tetraacetic Acid, and the Determination of Its Enantiomeric Purity by Chiral Chromatography. Bioconjugate Chem. 3, 563−569. H


Article

Bioconjugate Chemistry (26) Bloembergen, N., and Morgan, L. O. (1961) Proton relaxation times in paramagnetic solutions. Effects of electron spin relaxation. J. Chem. Phys. 34, 842−850. (27) Bloembergen, N., Purcell, E. M., and Pound, R. V. (1948) Relaxation effects in nuclear magnetic resonance absorption. Phys. Rev. 73, 679−712. (28) Solomon, I. (1955) Relaxation processes in a system of two spins. Phys. Rev. 99, 559−565. (29) Solomon, I., and Bloembergen, N. (1956) Nuclear magnetic interactions in the HF molecule. J. Chem. Phys. 25, 261−266. (30) Borel, A., Bean, J. F., Clarkson, R. B., Helm, L., Moriggi, L., Sherry, A. D., and Woods, M. (2008) Towards the rational design of MRI contrast agents: electron spin relaxation is largely unaffected by the coordination geometry of gadolinium(III)-DOTA-type complexes. Chem. - Eur. J. 14, 2658−2667. (31) Muller, R. N., Raduchel, B., Laurent, S., Platzek, J., Pierart, C., Mareski, P., and Vander Elst, L. (1999) Physicochemical characterization of MS-325, a new gadolinium complex, by multinuclear relaxometry. Eur. J. Inorg. Chem. 1999, 1949−1955. (32) Amin, S., Voss, D. A., Jr., Horrocks, W. D., and Morrow, J. R. (1996) Restoration of catalytic activity by replacement of a coordinated amide group: synthesis and laser-induced luminescence studies of the phosphate diester transesterification catalyst [Eu(NBAC)]3+. Inorg. Chem. 35, 7466−7467. (33) Kiefer, G. E., and Woods, M. (2009) Solid State and Solution Dynamics of Pyridine Based Tetraaza-Macrocyclic Lanthanide Chelates Possessing Phosphonate Ligating Functionality (LnPCTMB): Effect on Relaxometry and Optical Properties. Inorg. Chem. 48, 11767−11778. (34) Aime, S., Barbero, L., Botta, M., and Ermondi, G. (1992) Determination of metal-proton distances and electronic relaxation times in lanthanide complexes by nuclear magnetic resonance spectroscopy. J. Chem. Soc., Dalton Trans., 225−228. (35) Suturina, E. A., Mason, K., Geraldes, C. F. G. C., Chilton, N. F., Parker, D., and Kuprov, I. (2018) Lanthanide-induced relaxation anisotropy. Phys. Chem. Chem. Phys. 20, 17676−17686. (36) Webber, B. C., Cassino, C., Botta, M., and Woods, M. (2015) Aggregation in Amphiphilic Macrocycle-Substituted Gd3+ DOTAType Chelates Is Affected by the Regiochemistry of Substitution. Inorg. Chem. 54, 2085−2087. (37) Corsi, D. M., Platas-Iglesias, C., van Bekkum, H., and Peters, J. A. (2001) Determination of paramagnetic lanthanide(III) concentrations from bulk magnetic susceptibility shifts in NMR spectra. Magn. Reson. Chem. 39, 723−726.

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Differences in the Relaxometric Properties of Regioisomeric Benzyl

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