Investigation of Two Zr-p-NO₂Bn-DOTA Isomers via NMR and Quantum Chemical Studies

Abstract

A combination of NMR studies and quantum chemical calculations were employed to investigate the structure and energetics of Zr⁴⁺ chelates of pNO₂Bn-DOTA. We have demonstrated that two discrete regioisomeric chelates are generated during the complex formation. The nitrobenzyl substituent can adopt either an equatorial corner or side position on the macrocyclic ring. These regioisomers are incapable of interconversion and were isolated by HPLC. The corner isomer is more stable than the side, and the SAP conformer of both regioisomers is energetically more favorable than the corresponding TSAP conformer.

Graphical Abstract

Two regioisomers of Zr-p-NO₂Bn-DOTA were isolated and characterized by NMR studies and quantum chemical calculations. As opposed to the side regioisomer, the corner regioisomer exists exclusively as the SAP isomer.

INTRODUCTION

In recent years, ⁸⁹Zr emerged as a promising radiotracer for immuno-positron emission tomography (immuno-PET). Immuno-PET utilizes IgG antibodies as targeting vectors, which require long periods to fully accumulate at the target site in vivo. There is a good match between ⁸⁹Zr 78.4 h half-life and the biodistribution of IgG antibodies for immuno-PET. A bifunctional chelator must be used to sequester ⁸⁹Zr to antibodies. The hydroxamate-based iron chelator Desferrioxamine B and its derivatives are widely used for ⁸⁹Zr radiolabeling of antibodies due to its relative simplicity in bioconjugation and radiolabeling steps. Desferrioxamine (DFO) has three hydroxamate groups involved in the coordination of the zirconium ion. While use of DFO analogs has allowed the radiolabeling of antibodies with Zr in proof-of-concept studies, concerns remain regarding the in vivo stability of those complexes ^[1]. This instability has been observed in several animal studies with uptake of ⁸⁹Zr in the bone, indicating complex dissociation, likely due to unsaturation of Zr⁴⁺ coordination sphere.

In recent years, a number of chelators with a higher number of coordination sites for ⁸⁹Zr radiolabeling has been published ^[2] and among them are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ^[3], DFO analogues with an additional hydroxamic acid entity (DFO*) ^[4] and DFO square ^[5].

A suitable bifunctional chelator for Zr⁴⁺ ions is the macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, which has the reactive functionality to link the antibody. For example, the commercially available primary amine reactive S-2-(4-isothiocyanatobenzyl)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-NCSBn-DOTA) forms a thiourea linkage with antibodies in such a way that the ligand structure remains unaltered. Isomerism of the macrocyclic complexes plays a significant role for designing a radiometal-based agent by its ability to complicate the analysis of the radiolabeling step. However, due to its unstable nature, it is not practical to study the isomer distribution of p-NCSBn-DOTA. Thus, here we characterize the isomerization of para-nitrobenzyl DOTA (p-NO₂Bn-DOTA), a more stable analogue, (Scheme 1).

Scheme 1.

Structures of p-NO₂Bn-DOTA and p-NCSBn-DOTA ligands.

In this study we report the isomeric composition for zirconium complexes. Two different isomers were formed during complex formation. Those two complexes were then isolated and analyzed to determine the feasibility of their interconversion at room temperature. To our knowledge this is the first report investigating the isomer composition of the complex of para-nitrobenzyl DOTA with Zr⁴⁺ ion.

RESULTS and DISCUSSION

Recently, Wadas et al. described the DOTA complexation with zirconium-91 and zirconium-89, demonstrating superior in-vitro stability of the Zr-89 DOTA against DFO ^{[3, 6]}. Although a p-NH₂Bn-DOTA zirconium complex was described in the patent ^[7], no details of the isomeric composition were reported. It prompted us to explore the feasibility of the preparation of a cold zirconium bifunctional chelator based on the commercially available p-NCSBn-DOTA and its potential application for Zr-89 radiolabeling. p-NCSBn-DOTA offers eight coordination sites for zirconium as well as isothiocyanate functionality for the linking of the antibodies or proteins of interest. The non-radioactive zirconium complex of p-NCSBn-DOTA was prepared by reacting ZrCl₄ with p-NCSBn-DOTA in anhydrous dimethyl sulfoxide in presence of Et₃N at 80 °C. The LC MS analysis showed formation of two major species with the same mass, however they partially hydrolyzed over the course of 24 hours following dissolution after HPLC purification.

To further investigate these species, the zirconium complex of the more stable analog, p-NO₂Bn-DOTA, was formed. It was obtained under refluxing conditions in ethanol using Et₃N as a base. As in the case of the isothiocyanate derivative, the LC MS also showed formation of two major species with the same mass of the zirconium complex (the ratio 65:35). While the species could not be separated by prep-HPLC, semi-prep HPLC purification allowed separation of the peaks. The zirconium complex crystals were obtained from the prep-HPLC fraction (H₂O/MeCN containing 0.05% TFA) stored overnight at room temperature. The molecular representation is shown in Figure 1. Crystallographic data and structure refinement parameters are presented in Table S1. The X-ray structure shows that Zr-p-NO₂Bn-DOTA crystallized as a hydrate with nine water molecules. The crystal packing unit consists of two zirconium complexes.

Figure 1.

Crystal structure of [C₂₃H₂₉N₅O₁₀Zr]₂ × 9 H₂O unit cell

The Zr⁴⁺ ion is coordinated with four nitrogen and four carboxyl oxygen atoms of the ligand. In the Zr unit, the four nitrogen and the four oxygen atoms form square planes that are almost parallel to each other. The Zr (IV) ion is 0.977 Å out of the oxygen plane and 1.340 Å from the nitrogen plane. The Zr-N_cyclen and Zr-O_pendant mean bond lengths are 2.437 Å and 2.118 Å, respectively, and they follow the trend observed for ZrDOTA ^[3]. Upon metal ion coordination, the ethylene groups of the macrocycle can adopt two different gauche orientations (λ and δ), while the acetate arms are twisted around the Zr⁴⁺ in either Δ or Λ orientation. Two diastereomeric forms, Λ(δδδδ)/Δ(λλλλ) and Δ(δδδδ)/Λ(λλλλ), are present in solution, differing in the orientation of the parallel planes formed by the Zr⁴⁺-bound nitrogen and oxygen atoms. The Λ(δδδδ)/Δ(λλλλ) leads to the square antiprism (SAP) isomer, while the Δ(δδδδ)/Λ(λλλλ) corresponds to the twisted square antiprism (TSAP) isomer. The TSAP and SAP isomers can undergo an exchange process either by ring inversion (δδδδ) ↔ (λλλλ) or arm rotation Δ ↔ Λ ^{[8], [9]}. The geometry around the coordination cage of Zr⁴⁺ ion is of the SAP type, where the mean cyclen N-C-C-N and pendant arm N-C-C-O torsion angles are 57.1° and −16.0°, respectively. The S-configuration of the chiral carbon and the δδδδ ring conformation places the nitrobenzyl group into the equatorial position to minimize steric repulsion or torsional strain. The nitrobenzyl can have two possible positions relative to the macrocycle, the “equatorial corner” and the “equatorial side” ^[10]. Figure 2 (top) shows the orientation of the protons in the macrocycle without any substituents. The nitrobenzyl group can replace the eq₂ hydrogen in an unsubstituted macrocycle and be on the same carbon as ax₂ proton to obtain the equatorial corner position, yielding the corner isomer. The side isomer is produced when the nitrobenzyl group replaces the eq₁ proton and is on the same carbon as the ax₁ proton. In the case of Zr-p-NO₂Bn-DOTA, the complex crystallized as the SAP corner regioisomer with the nitrobenzyl group in the equatorial position (eq₂).

Figure 2.

Possible orientations of the equatorial nitrobenzyl group relative to the macrocycle ring (adapted from ^{[10a, 10b, 11]}). The top portion shows the distinction of the macrocyclic protons with no substituents. The bottom portion shows that the “Bn eq corner” position replaces the eq₂ proton with a nitrobenzyl group, while in the “Bn eq side”, the nitrobenzyl group replaces the eq₁ proton.

To determine whether two major species represent the SAP and TSAP isomers of the zirconium complex, the fractions were purified by semi-prep HPLC. Each peak was then isolated and allowed to equilibrate for 48h at RT before being reanalyzed by analytical HPLC. Interestingly, the two peaks did not interconvert, suggesting that the isolated species are regioisomers (corner or side, Figure 2) of the zirconium complex and not the SAP/TSAP. Peak one is the major regioisomer and is more water soluble. Long-term storage (up to 4 months) of the aqueous solutions (pH 6) of the regioisomer revealed no interconversion of either isomer to the other as observed by LCMS and ¹H NMR.

NMR studies and quantum chemical calculations were conducted to assign the two-solution state HPLC peaks to the SAP corner (peak one) and the SAP side isomers (peak two) of Zr-p-NO₂Bn-DOTA. The ¹H spectra of Zr-p-NO₂Bn-DOTA (Figure S3) show substantial differences in the couplings of each isomer. It is worth noting that peak one contains only one set of resonances, while peak two contains two sets with a 95:5 ratio. Due to the lack of symmetry, the proton spectra of the complexes are crowded, and the full assignments were achieved with the aid of HSQC, COSY, HMBC, ROESY, and quantum chemical calculations. The process of the peak assignment was started by acquiring heteronuclear HSQC spectra for each isomer. The easily identifiable axial proton (ax₂) of the corner isomer was found at 3.41 ppm in HSQC spectrum (Figure S4). The results of the HSQC assignments and the COSY data were used to identify the couplings for the substituted and unsubstituted ethylene bridges of the macrocycle. The COSY spectrum of the corner isomer (Figure 3) showed four vicinal couplings for the axial proton (ax₂) ax₂ ↔ eq₁, ax₂ ↔ Bn, ax₂ ↔ Bn’, and ax₂ ↔ ax₁ in the ethylene bridge with the substituent. Additionally, three geminal ax₂ ↔ eq₂ couplings, three vicinal ax₂ ↔ eq₁ and four ax₁ ↔ eq₁ were identified (Figure 3) for those ethylene bridges without any substitutions leading to the nitrobenzyl group occupying the fourth eq₂ position. Further support that the nitrobenzyl group is in the equatorial eq₂ corner position can be obtained from the ROESY spectrum (Figure 4), which has correlations between ax₂ ↔ ac and eq₁ ↔ ac’ protons. Analysis of HSQC and the geminal cross couplings of the acetate protons indicated that those two ac and ac’ protons belong to the same carbon of the acetate arm, while eq₁ belongs to the next ethylene bridge without any substitutions. In addition, correlations of the benzylic proton to both acetate protons of the pendant arm were observed in the 2D-ROESY spectrum, indicating that this is likely a SAP isomer. These observations suggest that in solution, the corner isomer also exists as SAP, as was observed for the solid state. The experimental assignments of the ¹H and ¹³C NMR chemical shifts (δ) for the SAP isomers of the corner and the side regioisomers are summarized in Table S1 along with the theoretically calculated values.

Figure 3.

The ¹H-¹H correlation spectrum (COSY) of the corner isomer of Zr-p-NO₂Bn-DOTA recorded in D₂O (contains 5 μL of CD₃CN) at 600 MHz and 298 K. p-Nitrobenzyl group occupies the eq₂ position and couplings are indicated as ax₁ ↔ eq₁, ax₁ ↔︎ eq_2, Bn ↔︎Bn’, ax₂ ↔︎ eq₂, ax₂ ↔︎ eq_1, ax₂ ↔︎Bn, ax₂ ↔︎ Bn’, ax₂ ↔︎ ax₁.

Figure 4.

ROESY spectrum of the corner isomer of Zr-p-NO₂Bn-DOTA recorded in D₂O (contains 5 μL of CD₃CN), at 600 MHz and 298 K. Correlations are indicated as ax₂ ↔︎ ac, eq₁ ↔ ac’, and Bn ↔︎ac/ac’.

The theoretical calculations were employed to further support that peak one is the SAP corner isomer. Our calculations showed that the values obtained for the calculated average distances of Zr-N_cyclen (2.499 Å) and Zr-O_pendant (2.145 Å) are comparable to the Zr-N_cyclen (2.437 Å) and Zr-O_pendant (2.118 Å) obtained from the X-ray structure. Quantum chemical calculations for Zr-pNO₂Bn-DOTA (Table 1) indicate that the SAP isomer of the corner regioisomer is more stable than the TSAP in both relative Gibbs free energy (ΔG) by 4.9 kcal mol⁻¹ at 298.15 K and relative electronic energy (ΔE) by 3.6 kcal mol⁻¹. These substantial thermochemical stability differences between the two regioisomers likely explain the absence of the TSAP isomer of the corner regioisomer in the NMR spectrum at room temperature (Figure S3 top).

Table 1.

Relative stability of SAP and TSAP of Zr⁴⁺ complexed with p-NO₂Bn-DOTA. The SAP of the corner isomer is calculated to be more stable than the SAP of the side isomer by ΔE = −1.7 kcal/mol and ΔG = −2.5 kcal/mol.

Regioisomer	ΔE (SAP − TSAP) (kcal/mol)	ΔG (SAP − TSAP) (kcal/mol)
Corner	−3.6	−4.9
Side	−1.9	−2.4

In the geometry optimized structure of the SAP corner isomer (Figure 5), the average distances between one proton of the acetate arm close to the adjacent ethylene bridge and ax₂ protons (ax₂ ↔ ac) are 2.023 Å (-NCH contact) and the other acetate proton close to the eq₁ proton of its section with the average distance of 2.207 Å, also -NCH contact. In the geometry optimized structure of the TSAP corner isomer, the average distances between ax₂ protons and one of the acetate protons of its section (a nitrogen and two carbon away, -NCCH contact) are 2.07 Å. ROESY spectrum showed 3.41–4.20 (ax₂ ↔ ac) and 3.03–3.89 (eq₁ ↔ ac’) -NCH contacts along with three additional (3.02–3.98, 3.34–2.64), (3.09–4.08, 2.94–3.76), (3.09–4.13, 2.97–3.80) contacts, and no ac-NCCH contacts. The ROESY contacts are remarkably consistent with the SAP corner isomer assignment. Also, the distances between the benzylic proton and both acetate protons are 2.362 Å and 2.707 Å, supporting the observed difference in the cross peaks’ intensities (Figure 6 (a), Table S2). Together the NMR studies and quantum chemical calculations strongly support peak one being the SAP corner isomer.

Figure 5.

Geometry optimized corner and side isomers of Zr- pNO₂Bn-DOTA at the level of M06-L/6–31+G(d)-SDD in the water reaction field. The value in parenthesis represents the electronic energy (ΔE) relative to that of the corner SAP isomer. The arm rotation of SAP leads to TSAP and vice versa. Hydrogens are not shown. Atoms are colored as follows: green, carbon; blue, nitrogen; red, oxygen; cyan, zirconium.

Figure 6.

Geometry optimized structures of (a) SAP corner, (b) TSAP side and (c) SAP side zirconium complex showing a representative distance between benzylic and acetate protons.

The ¹H proton spectrum of the side isomer is presented in Figure S3 (bottom). Overlap and the minor species (~5%) made the side isomer assignments more challenging compared to the corner isomer. Nevertheless, peak assignment was also started by acquiring the HSQC spectrum. Absence of the interconversion between isolated peaks and peak one’s assignment to the corner regioisomer indicate that peak two is the side isomer with the nitrobenzyl group occupying the eq₁ position (Figure 2). The easily identifiable axial proton (ax₁) of the side isomer was found at 3.38 ppm in HSQC spectrum (Figure S5). In a similar way, the HSQC, COSY, and HMBC data were used to identify the couplings for the substituted and unsubstituted ethylene bridges of macrocycle. As in the case of the corner regioisomer, the COSY spectrum of the side isomer (Figure 7) showed a presence of four vicinal couplings for the axial proton (ax₁) ax₁ ↔ Bn, ax₁ ↔ ax₂, ax₁ ↔ eq₂ and ax₁ ↔ Bn’ for the substituted ethylene bridge. Additionally, three geminal ax₂ ↔ eq₂, three geminal ax₁ ↔ eq₁ couplings and two vicinal ax₁ ↔ eq₂ were identified (Figure 7) for those ethylene bridges without any substitutions. Indirect evidence that the nitrobenzyl group is in the equatorial eq₁ side position can be obtained from the ROESY spectrum (Figure 8). Figure 8 reveals four correlations of ax₂ ↔ ac/ac’ and three of eq₁ ↔ ac’/ac protons. In addition, the correlations of both benzylic protons to the same acetate proton of the pendant arm were detected. This differs from the couplings observed for the corner isomer, where the benzylic proton was coupled to two acetate protons of the pendant arm and ax₂ protons were coupled ac, while eq₁ protons were coupled to ac’ protons.

Figure 7.

The ¹H-¹H correlation spectrum (COSY) of the side isomer (peak 2) of Zr-p-NO₂Bn-DOTA recorded in D₂O (contains 5 μL of CD₃CN) at 600 MHz and 298 K. J-couplings are ax₁ ↔ eq₁, ax₁ ↔︎ eq_2, Bn ↔︎Bn’, ax₂ ↔︎ eq₂, ax₁ ↔︎ Bn_, ax₁ ↔︎ax₂, ax₁ ↔︎ eq₂, ax₁ ↔︎ Bn’.

Figure 8.

ROESY spectrum of the side isomer of Zr-p-NO₂Bn-DOTA recorded in D₂O (contains 5 μL of CD₃CN), at 600 MHz and 298 K. Correlations are indicated are indicated as ax₂ ↔︎ ac/ac’, eq₁ ↔ ac/ac’, and Bn/Bn’ ↔︎ac.

In geometry optimized structures of the SAP side isomers each acetate arm has one hydrogen close to the adjacent ethylene bridge ax₂ (-NCH contact 2.021 Å) and the other acetate proton close 2.205 Å to the eq₁ proton of its section, also -NCH contact. The SAP-side isomer is missing one of the 2.2 Å proximities due to the nitrobenzyl group occupying the eq₁ position. The average distances between the ax₂ protons and one of the acetate protons of its section (a nitrogen and two carbon away, -NCCH contact) are 2.07 Å for TSAP corner and 2.047 Å for TSAP side conformers (Table S2). Peak two has all -NCH proximities and is consistent with the SAP isomer, with the lack of ac-NCCH contacts excluding the TSAP isomers. However, peak two has only one ROESY contact of the most shifted acetate proton of the arm to the axial proton (2.98–3.96, ax₂ ↔ ac) and the less shifted proton of the same arm coupled to the equatorial proton (2.55–3.51, eq₁ ↔ ac’). Three other ROESY contacts are in reversed order where more shifted are coupled to eq₁ protons (2.87–3.69, 2.97–3.96), (2.77–3.93, 2.77–3.61), (2.91–3.71).

The orientation of the nitrobenzyl group relative to the pendant arm is indicative of the presence of either the TSAP or the SAP isomer. For the geometry optimized Zr-p-NO₂Bn-DOTA TSAP structure, the closest distances between the acetate and the benzylic protons are ac-Bn = 2.454 Å and ac’-Bn’ = 2.555 Å (Figure 6 (b), Table S3), while for the SAP structure, the closest are ac-Bn = 2.130 Å and ac-Bn’= 2.403 Å (Figure 6 (c), Table S3). The ROESY spectrum is consistent with the latter, SAP structure, showing contacts between an acetate proton and both benzylic protons (Bn and Bn’). Quantum chemical calculations for Zr-p-NO₂Bn-DOTA (Fig. 5 bottom) also indicate that the SAP side isomer is more stable than TSAP by ΔG of 2.4 kcal mol⁻¹ at 298.15 K and by ΔE of 1.9 kcal mol⁻¹. Taken together, the NMR studies and quantum chemical calculations are consistent with peak two being the SAP isomer with the nitrobenzyl group occupying the side position. Additionally, the minor species present in the spectra of peak two may be from the TSAP isomer, considering the smaller ΔG (2.4 kcal/mol) between the SAP and the TSAP of the side isomer compared to that of the corner isomer (4.9 kcal/mol). However, no analysis or further purification was attempted on the minor species.

The difference in the orientation of the nitrobenzyl group relative to the macrocycle ring for the corner and the side regioisomers is also detected in 1D ROESY spectra of the nuclei in close proximity to the ortho aromatic protons (Figure S6). Both spectra show the proximity of the benzylic (Bn and Bn’) protons as well as the protons in the substituted ethylene bridge: ax₂ (3.41 ppm), ax₁ (3.62 ppm) and eq₁ (2.64 ppm) protons for the corner isomer and ax₁ (3.38 ppm), ax₂/eq₂ (2.98 ppm) protons for the side isomer. However, the corner isomer spectrum also shows the acetate protons near the nitrobenzyl group corresponding to 2.996 Å and 4.015 Å distances obtained from the geometry optimized SAP corner isomer (Table S2).

The QM calculations are consistent with the assignment of the corner and the side regioisomers to the SAP structure. Correlation plots of the calculated (SAP and TSAP) versus experimental ¹³C NMR chemical shifts (CS) of the macrocyclic carbons for the regioisomers of the corner and side also support the structural assignment to the SAP isomers (Figure S7). The calculations correctly predict the carbon of the acetate arm nearest the substitution and the NC adjacent carbon to the substitution to have the lowest chemical shifts of similar carbons for both regioisomers. The notable chemical shift changes between the corner and side isomers are also observed. The shifts CH₂ of the acetate arm nearest the benzyl substitution were predicted at 70.9 ppm for the corner and at 63.7 ppm for the side isomers. The change of the 7.2 ppm between the structures is identical to the experimentally found change.

The formation of the corner and side isomers has also been observed for the lanthanide complexes of p-NO₂BnDOTA ^[12], p-NO₂Bn-DOTMA ^[13] and the copper (Cu-64) complex of p-NO₂BnNOTA ^[10c]. These reports and our findings indicate that introduction of the nitrobenzyl group into the polydentate ligand system generates regioisomers during the complexation step with transition and rare earth metal ions and need to be taken into consideration if those ligands are used to obtain contrast agents.

CONCLUSION

p-NO₂Bn-DOTA ligand produced two regioisomeric chelates during the complexation step with the Zr⁴⁺ ions. NMR studies were consistent with the two regioisomers differing in the corner or side positioning of the nitrobenzyl group, which was supported by quantum chemical calculations. These calculations also indicate that the SAP isomer of the corner and the side regioisomer is more stable than the TSAP in both relative Gibbs free energy and relative electronic energy. While substantial thermochemical stability differences between the TSAP and the SAP of the corner regioisomer explain the absence of the TSAP isomer of the corner regioisomer in the NMR spectrum at room temperature, that was not the case for the side regioisomer. The implication for p-NCSBn-DOTA, used as a bifunctional chelator for Zr-89 radiolabeling, is that the complexation step will generate a distribution of the corner and the side isomers.

Experimental Section

Reagents and methods

p-NO₂Bn-DOTA and p-NCSBn-DOTA were purchased from Macrocyclics (TX, USA) and all other reagents were obtained from Sigma Aldrich. The deuterated solvents were purchased from Cambridge Isotope Laboratories (MA, USA). Liquid chromatography-mass spectrometry (LC-MS) was performed on an Agilent 1200 Series instrument equipped with LC/MSD XT Agilent Technologies system. An Eclipse Plus C18 column (4.6 × 50 mm; 5 μm) was used. Solvent A was 0.05% TFA in water, Solvent B was 0.05% TFA in acetonitrile, and a linear gradient of 5% B to 95% B over 8 min and further maintained for 1 min at a flow rate of 0.5 mL min⁻¹ was used. Preparative HPLC was performed on an Agilent 1200 Series instrument. An Agilent 5 Prep – C18 column was used. Solvent A was 0.05% TFA in water, Solvent B was 0.05% TFA in acetonitrile, and a linear gradient of 0% B to 50% B over 16 min, followed by increase to 95% B and further maintained for 4 min at a flow rate of 20 mL min⁻¹ was used. The ¹H, ¹³C, COSY, HSQC, HMBC, and ROESY spectra were measured at 25 °C on a Bruker NEO 600 MHz NMR with an 5mm ¹H,¹³C,¹⁵N,²H cryoprobe. Chemical shifts are reported in parts per million (δ) and are referenced to ¹³CD₃CN=1.47 ppm for ¹³C and CHD₂CN=2.06 ppm for ¹H.

Preparation of Zr-pNO₂Bn-DOTA.

pNO₂Bn-DOTA (10mg, 14.6 μmol) was dissolved in ethanol (400 μL) and triethylamine (20 μL) was added. Zirconium chloride (3.4 mg) was added to the reaction mixture. The reaction was refluxed for 2 hours. The reaction was purified by preparative HPLC by the method described in the methods section. m/z (ESI-MS+): [M+H⁺] 626.0.

Quantum Chemistry

All quantum chemical calculations were done with Gaussian 16 ^[14] on the neutral Zr-complexes. We employed M06-L ^[15] for geometry optimization and energy calculations. B3LYP was utilized for calculating the proton shielding tensors with the gauge-independent atomic orbital (GIAO) method. ^[16] For the Zr atom, the pseudopotential SDD ^[17] for 28 core electrons and the SDD basis set were used for the valence electrons; for all other atoms, 6–31+G(d) basis set was utilized for geometry minimization, and 6–311+G(2d,p) was used for NMR calculations on the geometry optimized at the level of M06-L/6–31+G(d). It is noted that the present chemical shift calculations with the pseudo potential neither are gauge invariant ^[18] nor incorporate the relativistic effect of the heavy atom Zr on the shielding tensors of neighboring light atoms ^[19]. The reference proton and ¹³C chemical shifts for TMS were 31.88 ppm and 182.47 ppm, respectively. Water solvent effect was adopted for all calculations using the Polarizable Continuum Model (PCM) as implemented in Gaussian 16 software. Frequency calculations were performed to verify the minima and Gibbs free energy was calculated at 298.15 K.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this study. Deposition Number <url href=“https://www.ccdc.cam.ac.uk/services/structures?id=doi:10.1002/ejic.202300439“> 2255436 (for Zr-p-NO₂Bn-DOTA)</url> contains the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe <url href=“http://www.ccdc.cam.ac.uk/structures“>Access Structures service</url>.