Zwitterionic MRI contrast agents with enhanced relaxivity, stability and reduced renal retention

Abstract

Concerns over in vivo Gd(III) retention have recently prompted the development of Gd(III)-based contrast agents (GBCAs) with high-relaxivity and stability. This work describes the synthesis of a novel modular and enantiomerically pure chelator, (R,R,R)-Bn₃-PCTAZA 8 allowing modification via Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC). Two zwitterionic GBCAs, Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a, were prepared by CuAAC and analysed for important physicochemical properties and complex stability. Both zwitterionic complexes have improved kinetic inertness compared to the clinically relevant GBCA gadopiclenol 3. In addition, Gd-SB₃-PCTA 11a revealed an 18% increase in relaxivity (r₁) compared to gadopiclenol 3. Gd-NOx₃-PCTA 12a, has a slightly lower relaxivity but the highest kinetic stability among all complexes tested. Both agents maintain high relaxivity at clinically relevant magnetic field strengths (1.4 T and 7 T) and have high inertness under transmetallation conditions, acidic conditions and in human serum. These favourable characteristics are due to enhanced second-sphere hydration of the kosmotropic periphery of the complexes. Gd-SB₃-PCTA 11a revealed a significantly reduced retention in mice kidneys compared to gadopiclenol 3. The zwitterionic GBCAs Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a are thus promising candidates for the development of next-generation contrast agents for magnetic resonance imaging (MRI).

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Introduction

Gd(III)-based contrast agents (GBCAs) are widely used for magnetic resonance imaging (MRI). However, concerns about their long-term safety have led to the withdrawal of some derivatives and are a motivation for the development of safer contrast agents.¹ GBCA administration has been linked to nephrogenic systemic fibrosis (NSF) in patients with impaired renal function.^1–3 In addition, recent studies have demonstrated that repeated administration of certain GBCAs results in the accumulation of Gd(III) even in patients with normal renal function and may result in unwanted physiological effects.^4–8 The accumulation of free Gd(III) is a consequence of Gd(III)-release from GBCAs and regulatory authorities have thus restricted the use of GBCAs containing acyclic Gd(III) chelators, which form Gd(III)-complexes with limited in vivo stability.^9,10 However, even GBCAs containing macrocyclic chelators lead to accumulation of Gd(III) in vivo. This Gd(III)-retention is not due to a release of free Gd(III) from the complexes but rather a consequence of an unwanted accumulation of the complete GBCAs in certain tissues (particularly the kidney).^11,12 The accumulation of GBCAs is notable considering the efficient clearance of these compounds from the body. To reduce these risks there is an ongoing search for new GBCAs with higher complex stability, improved clearance, and higher relaxivity.^13,14 High T₁-relaxivity GBCAs at equal concentrations induce greater relaxation rate changes, enhancing T₁-weighted image contrast more effectively and allowing for lower dosing than agents with lower relaxivity.¹⁵

A promising strategy to increase relaxivity is to increase the inner-sphere hydration number q of the Gd(III) complex. However, the increase of inner sphere water molecules is typically associated with a loss of kinetic and thermodynamic stability of the complex. Most stable Gd(III)-complexes of macrocyclic chelators have therefore a hydration number of q ∼ 1.

A notable exception is gadopiclenol 3 (Fig. 1), a highly stable Gd-complex with a hydration number q = 2.^16–18 Gadopiclenol is a chiral Gd(III) complex based on the macrocyclic chelator PCTA and has a relaxivity more than twice as high as the relaxivity of other GBCAs in clinical use.¹⁹ PCTA derivatives are thus a promising platform for the development of next-generation GBCAs combining high relaxivity with stability.¹⁹

Fig. 1 Structures of three clinically used GBCAs: Gd-DTPA 1 (Magnevist®), Gd-DOTA 2 (Dotarem®) and gadopiclenol 3 (Vueway®, Elucirem®).

The introduction of zwitterionic groups (“zwitterionization”), such as sulfobetaines or amine N-oxides is an additional strategy to enhance the longitudinal relaxivity of GBCAs. The decoration of Gd-DOTA with zwitterions increased its longitudinal relaxivity significantly.^20,21 This enhancement was attributed to two main factors: first, the presence of zwitterions accelerated the exchange rate of inner-sphere water molecules by reducing their mean residence time. Second, the kosmotropic properties of these groups increased the hydrodynamic diameter of the complex and promoted the formation of an extended hydration layer.²² Together, these effects caused a 2- to 2.5-fold increase in relaxivity compared to non-zwitterionic analogues.^20,21 In addition, zwitterionization of drugs can lead to favourable pharmacokinetic properties because it leads to efficient renal clearance and decreases unwanted tissue retention.^23,24

This study describes the synthesis and physicochemical evaluation of two zwitterionic GBCAs (Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a). Both have been synthesized from the chiral azide-functionalized PCTA derivative (R,R,R)-Bn₃-PCTAZA 8. The approach addresses the following research questions: (1) Can zwitterionic groups enhance the relaxivity of Gd-PCTA complexes? (2) Are zwitterionic Gd-PCTA complexes thermodynamically and kinetically stable? (3) What is the hydration number of the zwitterionic complexes? (4) What is the influence of zwitterionic groups on the in vivo Gd-retention after repeated dosage?

Results and discussion

Synthesis of zwitterionic chelators and corresponding complexes

The new clickable chelator (R,R,R)-Bn₃-PCTAZA 8 (Scheme 1) was prepared via alkylation of 3,6,9-triaza-1(2,6)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (7, pyclen) with triflate (S)-6 using a modified procedure for the alkylation of cyclen.²⁵ Triflate (S)-6 was available in enantiomerically pure form from alcohol (S)-5, which was prepared from L-malic acid in five steps.^25,26 Alkylation of pyclen 7 with triflate (S)-6 gave the azide-functionalized chelator (R,R,R)-Bn₃-PCTAZA 8 in high yield. Notably, the yield of the alkylation step increased substantially when the triflate was added at 0 °C instead of room temperature. (R,R,R)-Bn₃-PCTAZA 8 was prepared in gram quantities with this procedure. The alkylation of pyclen with triflate 8 gives stereoisomerically pure (R,R,R)-Bn₃-PCTAZA 8 according to NMR and HPLC. This indicates a pure S_N2-mechanism and parallels our observations with the alkylation of cyclen via the same reaction.²⁷ The preparation of (R,R,R)-Bn₃-PCTAZA 8 via alkylation with (S)-triflate 6 is thus advantageous compared to other approaches, which lead to mixtures of stereoisomers and thus cumbersome separations and low overall yields of the final pure stereoisomers.¹⁹ The modification of (R,R,R)-Bn₃-PCTAZA 8 via CuAAC (click-chemistry) was performed according to an established protocol for the preparation of zwitterionic DOTA-derivatives with the alkynes 14 and 15.^20,21 Particular attention should be paid to the amount of alkyne used in the CuAAC reactions. In the case of the sulfobetaine alkyne 14, 3.30 equiv. were sufficient to achieve complete conversion to Bn₃-SB₃-PCTA 9. The moderate isolated yield of 53% is a consequence of the high polarity of the compound, which led to problems in the chromatographic separation of Bn₃-SB₃-PCTA 9 from salts. HPLC-MS analysis of the crude product indicated quantitative formation of 9 with no significant formation of side products. In contrast, a large excess (18.0 eq.) of alkyne N-oxide 15, was required to achieve complete and clean conversion to Bn₃-NOx₃-PCTA 10. The reason is a competing copper-catalysed N-O-deoxygenation of alkyne 15 or the click-product 10.²⁸ Using lower amounts of alkyne N-oxide 15 or extending the reaction time for the CuAAC beyond 2 h, consistently led to complex mixtures containing deoxygenated by-products together with the desired product Bn₃-NOx₃-PCTA 10. A reaction time of approximately 1 h was optimal and gave reproducible and clean conversions to 10.

Scheme 1 Synthesis of the azide-functionalised chelator (R,R,R)-Bn₃-PCTAZA 8 and its subsequent conversion into the zwitterionic chelators 9 and 10, followed by complexation with Gd(III) and Eu(III).

The benzyl esters of Bn₃-SB₃-PCTA 9 and Bn₃-NOx₃-PCTA 10 were hydrolysed under alkaline conditions using NaOH in a THF/H₂O mixture. The reaction mixture was neutralised by treatment with an acidic ion-exchange resin, which was subsequently removed via filtration, affording the zwitterionic chelators SB₃-PCTA and NOx₃-PCTA in almost quantitative yields. Complexation was performed with GdCl₃ or EuCl₃ at room temperature in aqueous 1 M NH₄OAc buffer (pH 5.5). The resulting complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a were obtained in good yields over two steps. The complexation was monitored by HPLC-MS, and notably, no free ligand was detected after 10 minutes, indicating a rapid and efficient complexation of both cations.

Eu(III) complexes allow the determination of the inner-sphere hydration number q via luminescence lifetime measurements.²⁹ Complexes Eu-SB₃-PCTA 11b and Eu-NOx₃-PCTA 12b are thus important model compounds for physicochemical characterisation.

It is notable that all zwitterionic complexes had a water solubility greater than 0.5 M. The log D_7.4 values of the zwitterionic GBCAs were assessed using a standard shake flask-method (n-octanol/PBS). However, quantification was not feasible, as extraction into the n-octanol phase was below the HPLC-DAD detection limit. A log D of −4.2 has been reported for gadopiclenol 3, but the authors noted that the method used reaches its practical limit for compounds of such high hydrophilicity.¹⁶ Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 11b are even more polar than gadopiclenol 3. We measured the RP-HPLC retention times with identical separation parameters: gadopiclenol 3 eluted at 9.8 min, whereas Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 11b eluted at 9.3 min and 2.3 min respectively. This trend indicates that both zwitterionic derivatives are significantly more hydrophilic than gadopiclenol.

Kinetic and thermodynamic stability of zwitterionic Gd(III) complexes

Kinetic inertness is a key determinant of the in vivo stability of GBCAs, as it governs the resistance of the complex to decomplexation processes under physiological conditions. Dissociation of macrocyclic lanthanide complexes occurs via protonation of the complex, leading to the formation of a diprotonated intermediate, which then undergoes loss of Gd(III). This process of decomplexation can be monitored by spectrophotometric quantification of free Gd(III) using complexometric titration with Arsenazo III.^16,30,31 The stability of Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a was assessed in aqueous solution at pH 1.2 and compared to that of gadopiclenol 3 as well as Gd-DOTA 2 (gadoteric acid) under identical conditions.

As previously reported by Robic et al., gadopiclenol 3 is significantly more stable than Gd-DOTA 2 and far more stable than the more labile GBCAs gadodiamide and gadobutrol.¹⁶ This stability trend was confirmed for gadopiclenol 3 and Gd-DOTA 2 in our experiments as depicted in Fig. 2. Notably, the zwitterionic complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a are more stable under acidic conditions than gadopiclenol 3. After 21 days, gadopiclenol 3 was completely dissociated, while Gd-SB₃-PCTA 11a released only 62% and Gd-NOx₃-PCTA 12a just 16% of its Gd(III) content (Fig. 2).

Fig. 2 (A) Released free gadolinium over time for Gd-SB₃-PCTA 11a, Gd-NOx₃-PCTA 12a, gadopiclenol 3 and Gd-DOTA 2 under acidic conditions (pH 1.2) at 37 °C, as determined by complexometric titration with arsenazo III. (B) Time-dependent relative longitudinal relaxation time T₁(t)/T₁(t₀) of gadolinium complexes (1.25 mM) in presence of ZnCl₂ (1.25 mM) in phosphate-buffered saline (PBS, pH 7.4, 37 °C), measured at 1.4 T. Measurements were performed at 0 h, 3 h, 1 day, 3 days, 7 days and 15 days. (C) Stability of Gd(III) complexes in human serum at 37 °C. Measurements were performed at 0 days, 1 day, 3 days, 7 days and 14 days.

The dissociation data of the Gd(III) complexes were evaluated under the assumption that both zwitterionic and non-zwitterionic complexes follow pseudo-first order dissociation kinetics. Linearisation according to the equation ln(A_t/A₀) = −k_obs·t followed by linear regression, gave the dissociation rate constants k_obs. The parameter A_t denotes the concentration of dissociated complex at time t and A₀ the initial concentration of the complex.

Comparison of the observed dissociation rate constants and the total amount of Gd(III) released after 21 days revealed significant differences in the kinetic stability of the complexes. The most stable compound, Gd-NOx₃-PCTA 12a had a dissociation rate that was more than 36-fold lower than that of Gd-DOTA 2 and 15-fold lower than that of gadopiclenol 3. Notably, Gd-NOx₃-PCTA 12a also dissociated 5.5 times more slowly than the sulfobetaine-analogue Gd-SB₃-PCTA 11a, highlighting a substantial difference in complex stability among zwitterionic complexes. However, both new complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a are exceptionally stable under acidic conditions with dissociation rates 3 or 15 times lower respectively than that of gadopiclenol 3.

In summary, these findings reveal a stabilising effect of zwitterionic modifications on the kinetic inertness of the complexes. They suggest that the zwitterionic hydration shell may contribute to an intramolecular pH buffering, thereby stabilising the complex under strongly acidic conditions by protecting the macrocyclic coordination center from protonation and subsequent Gd(III) release.

In contrast to the in vitro evaluation of kinetic stability, the assessment of thermodynamic stability of GBCAs is more challenging, as it cannot be easily extrapolated to in vivo conditions due to the complex composition of biological media, containing numerous competing endogenous complex ligands and metal cations (e.g. Fe(III), Ca(II), Zn(II), Cu(II)).^32,33 Upon decomplexation, free Gd(III) may bind to proteins or precipitate with endogenous anions.³⁴ A robust and widely accepted method for evaluating GBCA stability under physiologically relevant conditions involves transmetalation with Zn(II) in phosphate buffer.^16,33,35 The zwitterionic complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a as well as gadopiclenol as non-zwitterionic reference were challenged with excess Zn(II) in phosphate-buffered saline (PBS, pH 7.4) at 37 °C. Longitudinal relaxation rates were monitored at the following time points: 0 h, 3 h, 1 day, 3 days, 7 days and 15 days. It is important to note that the Zn(II) solution must be freshly prepared with ZnCl₂ and added immediately to the chelator solutions to avoid the formation of zinc phosphate precipitates.³² The zwitterionic complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a, as well as gadopiclenol 3, showed no signs of instability as the measured longitudinal relaxation rates remained constant throughout the observation period of 15 days. These results are consistent with previous studies of gadopiclenol¹⁶ and suggest that Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a are at least as stable as gadopiclenol 3 against transchelation.

These observations were further confirmed by a stability assay in human serum at 37 °C. All Gd(III) complexes were incubated for 15 days and gadolinium release was quantified at 0 h, 1 day, 3 days, 7 days and 15 days by complexometric titration with Arsenazo III. No loss of Gd(III) was observed for any of the zwitterionic complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a or for gadopiclenol 3. The molecular integrity of Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a was verified after 15 days of incubation via LC/MS. At least in serum, we have thus no evidence for reductive biotransformation of the N-oxide 12a. This fact is notable because certain amineoxides have been reported to be reduced in biological media.³⁶

Relaxivity at 1.4 T and 7 T

The relaxivity of GBCAs depends on several factors, including hydration number q, the mean residence time of inner-sphere water molecules, the electronic relaxation time and the rotational correlation time of the complex.^37,38 Relaxivity arises from the combined contributions of inner-sphere (IS) and outer-sphere (OS) interactions, as described by the following equation: R₁ = R_{1,inner-sphere} + R_{1,outer-sphere}. Here R_{1,inner-sphere} refers to the longitudinal relaxation rate originating from inner-sphere interactions and R_{1,outer-sphere} accounts for the contribution from outer-sphere interactions. The inner-sphere component refers to the relaxation of water molecules directly bound to the Gd(III) ion, whereas the outer-sphere contribution results from transient interactions between the complex and bulk water molecules not directly bound to the metal centre. An effective strategy to enhance the longitudinal relaxation rate R₁ is to increase the inner-sphere contribution by raising the hydration number q. According to the Solomon–Bloembergen–Morgan (SBM) theory, R₁ is directly proportional to the hydration number q: where q_Gd refers to the number of inner-sphere water molecules coordinated per Gd(III), [GBCA] is the concentration of the contrast agent, [H₂O] is the bulk water concentration, T_1m is the longitudinal relaxation time of the inner-sphere water protons and τ_m is the mean residence time of these coordinated water molecules.³⁹

Another key factor contributing to the overall relaxivity of a GBCA is the outer-sphere component, which can be modulated by parameters such as molecular size, rotational correlation time and electronic relaxation time.^38,39 As small molecules, GBCAs typically exhibit rapid rotational diffusion, which limits their relaxivity. The outer-sphere contribution can be enhanced by increasing the hydrodynamic size of the complex, thereby reducing its rotational correlation time and resulting in higher relaxivity. For gadopiclenol 3, Robic et al. proposed that the isoserinol arms contribute to an increased hydrodynamic size.¹⁶ In parallel, based on the observations by Botta et al. a second coordination sphere contribution has been suggested, as the highly polar polyhydroxy groups may enhance the number of water molecules associated to the complex.²² These observations are further supported by the findings of Holzapfel et al. on chiral DOTA-based complexes with q = 1.²⁰ It was demonstrated that not only the spatial expansion of the complex through the introduction of side arms significantly influences relaxivity, but also the incorporation of zwitterionic and highly hydrophilic moieties has a substantial impact on the observed high relaxivity.²⁰

Relaxometric studies of the two novel zwitterionic GBCAs Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a were performed at two different and clinically relevant field strengths (1.4 T and 7 T). The longitudinal relaxivity r₁ of both zwitterionic complexes, in comparison with gadopiclenol as a reference, was investigated at a field strength of 1.4 T (60 MHz, 37 °C, water) using a standard inversion-recovery pulse sequence. The spin lattice relaxation time (T₁) was determined and plotted as the reciprocal (1/T₁) against the concentration of the respective complexes. The concentrations of the complexes were precisely determined by inductively coupled plasma mass spectrometry (ICP-MS).

For Gd-SB₃-PCTA 11a a longitudinal relaxivity of r_1,1.4T = 16.29 ± 0.22 mM⁻¹ s⁻¹ was observed, representing an 18% increase compared to gadopiclenol (r_1,1.4T = 13.82 ± 0.05 mM⁻¹ s⁻¹). Gd-NOx₃-PCTA 12a had a slightly lower relaxivity of r_1,1.4T = 12.69 ± 0.07 mM⁻¹ s⁻¹, representing an 8% decrease compared to gadopiclenol (Fig. 3).

Fig. 3 (A) Longitudinal relaxivity (r₁) determined from plotting the reciprocal relaxation time 1/T₁ versus the concentration of Gd-SB₃-PCTA 11a, Gd-NOx₃-PCTA 12a and gadopiclenol 3 in H₂O at 37 °C and 1.4 T (60 MHz) (B) longitudinal relaxivity (r₁) determined from plotting the reciprocal relaxation time 1/T₁ versus the concentration of Gd-SB₃-PCTA 11a, Gd-NOx₃-PCTA 12a and gadopiclenol 3 in H₂O at room temperature and 7 T (300 MHz) (C) representative MR images acquired at 7 T at multiple inversion times (TI) ranging from 50 ms to 3200 ms of Gd-SB₃-PCTA 11a, Gd-NOx₃-PCTA 12a and gadopiclenol 3 in a phantom holder and surrounded by water. Yellow circles represent the regions of interest for five different concentrations of each compound used for subsequent quantitative analysis. (D) Color-coded R₁ relaxation rate map acquired at 7 T with an inversion-recovery spin-echo sequence with multiple inversion time delays for five different concentrations of each compound.

To evaluate the field dependence of relaxivity, additional measurements were conducted at 7 T (300 MHz, room temperature, water), a magnetic field strength increasingly relevant in preclinical and high-resolution clinical imaging. These measurements were performed using a preclinical MR imager by measuring a slice-selective 2D inversion recovery spin echo sequence with inversion times ranging from 50 ms to 3200 ms (Fig. 3).

Both zwitterionic complexes showed a magnetic field dependence comparable to gadopiclenol, maintaining high relaxivity values even at 7 T. For Gd-SB₃-PCTA 11a a longitudinal relaxivity of r_1,7T = 14.28 ± 0.22 mM⁻¹ s⁻¹ was measured, while Gd-NOx₃-PCTA 12a exhibited a relaxivity of r_1,7T = 9.98 ± 0.17 mM⁻¹ s⁻¹. Compared to the values at 1.4 T this corresponds to a field dependent relaxivity reduction of 12% for Gd-SB₃-PCTA 11a, 23% for gadopiclenol 3 and 21% for Gd-NOx₃-PCTA 12a (Table 1).

Table 1 Molecular weights, longitudinal relaxivity (r₁) values at 1.4 T and 7 T and q values for Gd-SB₃-PCTA 11a, Gd-NOx₃-PCTA 12a, gadopiclenol 3, Gd-PCTA and Gd-DOTA 2

Parameter	Gd-SB3-PCTA	Gd-NOx3-PCTA	Gadopiclenol	Gd-PCTA	Gd-DOTA
a H2O, 37 °C.b H2O, room temperature.c Determined via luminescence lifetime measurements of the Eu(III) complexes.d 1.5 T, 37 °C.e Napolitano et al.^19f Robic et al.^16
M [g mol^−1]	1357.39	1039.23	970.10	534.63	558.64
r1,1.4T [mM^−1 s^−1]	16.29 ± 0.22^a	12.69 ± 0.07^a	13.82 ± 0.05^a	4.9^d^e	3.3^d^e
r1,7T [mM^−1 s^−1]	14.28 ± 0.13^b	9.98 ± 0.17^b	10.61 ± 0.18^b (10.68^f)	—	—
q	2.08^c	2.23^c	2^e	2^e	1^e

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In summary, Gd-SB₃-PCTA 11a has a consistently higher relaxivity than the benchmark GBCA gadopiclenol 3 at a magnetic field strength of 1.4 and 7 T. Its superior performance is attributed to enhanced hydration caused by the sulfobetaine side chains, which appear to provide more effective kosmotropicity than the polar isoserinol sidechains present in gadopiclenol 3. This gain in hydration likely contributes not only to a larger hydrodynamic radius, thereby slowing rotational diffusion, but may also promote dynamic equilibria involved in inner-sphere und outer-sphere processes.

Structure-stability relationships of zwitterionic Gd-PCTA complexes

As mentioned above, Gd-NOx₃-PCTA 12a has a slightly lower relaxivity compared to Gd-SB₃-PCTA 11a and gadopiclenol 3. An influence of the stereochemistry as proposed by Napolitano et al.¹⁹ for stereoisomers of gadopiclenol can be excluded here, because both derivatives Gd-NOx₃-PCTA 12a and Gd-SB₃-PCTA 11a are based on the same (enantiomerically pure) scaffold. Instead, two other possible explanations come to mind: (1) the N-oxide sidechain functionalities might contribute to Gd-coordination, which could reduce the number of inner-sphere water molecules to q < 2 (Scheme 2). This effect would reduce the relaxivity of Gd-NOx₃-PCTA 12a and could also explain the high stability of this complex under acidic conditions. The process is not unlikely because N-oxides are good complex ligands for oxophilic metals.⁴⁰ Sulfobetaines in turn are only weak ligands, which would explain the difference to Gd-SB₃-PCTA 11a. (2) The difference in relaxivity might be due to the more compact molecular structure of Gd-NOx₃-PCTA 12a as a consequence of the small N-oxide groups. Molecular size and shape have an impact on the relaxation properties of GBCAs explaining the reduced relaxivity of Gd-NOx₃-PCTA 12a.

Scheme 2 Proposed equilibrium between the q = 2 and q = 1 hydration states of Eu-NOx₃-PCTA 12b.

To check the first hypothesis mentioned above, the hydration number q was determined according to the method described by Beeby et al. and Supkowski et al.^29,41 For this purpose, the luminescence lifetimes τ of the corresponding Eu(III) complexes, Eu-SB₃-PCTA 11b and Eu-NOx₃-PCTA 12b, were measured in both H₂O and D₂O.

The hydration number q was then calculated using the following equation where τ(H₂O) represents the excited-state lifetimes in H₂O and τ(D₂O) in D₂O.

The values obtained are given in Table 2. They reveal that both Eu(III) complexes have almost the same hydration number q ∼ 2. Displacement of an inner-sphere water molecule by one of the N-oxide side chain groups is therefore not responsible for the lower relaxivity of Gd-NOx₃-PCTA 12a. Instead, the more compact molecular structure of the N-oxide 12a compared to sulfobetaine 11a explains the slightly lower relaxivity.

Table 2 Luminescence lifetimes τ and hydration numbers q of Eu-SB₃-PCTA 11b and Eu-NOx₃-PCTA 12b, determined by time-resolved emission spectroscopy in H₂O and D₂O

Complex	τ(H2O) [ms]	τ(D2O) [ms]	q
Eu-SB3-PCTA 11b	0.348 ± 0.002	1.445 ± 0.012	2.08
Eu-NOx3-PCTA 12b	0.357 ± 0.0006	2.061 ± 0.021	2.23

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In vivo gadolinium kidney retention

In a first attempt to assess the impact of zwitterionic modifications on in vivo Gd(III) retention, mice were treated with the most promising zwitterionic GBCA Gd-SB₃-PCTA 11a and gadopiclenol 3 as a current clinical reference, in analogy to an in vivo study design reported by Di Gregorio et al. (Fig. 4).⁴² Animals received ten consecutive doses of 0.1 mmol kg⁻¹, corresponding to a cumulative dose of 1 mmol kg⁻¹. After a 17-day clearance phase following the last administration organs were harvested and analysed. Previous studies revealed the highest retentions of Gd(III) after GBCA administration in the kidney cortex of both animals and humans.^11,43 The impact of a long-term Gd(III) retention in the kidney on patient health is unclear, but certainly an undesirable side effect of clinical GBCA use. The residual renal Gd(III) content was therefore quantified by ICP-MS, revealing amounts of 5.89 ± 0.26 nmol g⁻¹ Gd(III) for the reference compound gadopiclenol 3 and 2.97 ± 0.62 nmol g⁻¹ for the zwitterionic derivative Gd-SB₃-PCTA 11a. The introduction of zwitterionic groups led thus to a reduction of kidney gadolinium retention of 50%.

Fig. 4 Study design of the in vivo kidney retention of Gd-SB₃-PCTA 11a and gadopiclenol 3 with a total dose of 1 mmol kg⁻¹ and a clearance phase of 17 days after last administration and Gd(III) retention in the kidney of animals treated with 3 and 11a as measured by ICP-MS.

Conclusion

GBCAs are an important component of clinical diagnostics using MRI. However, the long-term Gd(III) retention in patients treated repeatedly with GBCAs has led to safety concerns. The next generation of GBCAs should therefore have the highest possible relaxivity (enabling low dosage), high complex stability (no release of Gd(III) in vivo), and the lowest possible gadolinium retention in patients.

This study describes the synthesis of novel zwitterionic GBCAs based on the chelator PCTA. Bn₃-PCTAZA 8 is a central intermediate bearing three azide groups for CuAAC. The alkylation of pyclen gave the stereoisomerically pure (R,R,R)-configured product. Compared to other known protocols for the preparation of chiral PCTA derivatives, this eliminates the laborious separation of stereoisomers after synthesis. Sulfobetaines and N-oxides were introduced as zwitterionic groups via a CuAAC reaction. The resulting complexes Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a have high thermodynamic and kinetic stability, superior to that of gadopiclenol 3 (the most stable clinically used GBCA). The complexes 11a and 12a are exceptionally stable in acidic media (pH 1.2 for days), in human serum, and towards transmetalation with Zn(II).

The hydration number was determined to be q ∼ 2 for both zwitterionic compounds based on the luminescence lifetime of corresponding Eu(III) complexes 11b and 12b. The resulting relaxivities of the zwitterionic GBCAs Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a are therefore much higher than those of the clinically frequently used gadoteric acid. With a relaxivity of r_1,1.4T = 16.29 ± 0.22 mM⁻¹ s⁻¹, the sulfobetaine 11a has a 18% higher relaxivity than gadopiclenol 3 (currently the clinically used GBCA with the highest relaxivity). The increased relaxivity compared to gadopiclenol is likely due to the strong hydration of the zwitterions, which increases the hydrodynamic diameter of the compounds. The decoration of the GBCAs with zwitterions also led to a 50% reduction in gadolinium deposition in the kidneys of mice treated repeatedly with 11a compared to gadopiclenol 3.

Our study has several limitations, which need to be addressed in future work. NMRD measurements could shed more light on the contribution of zwitterionic groups on inner- and outer-sphere effects influencing relaxivity. In vivo experiments have been restricted to a small cohort of animals and sex differences have not been addressed so far. A more comprehensive analysis of the biodistribution is therefore needed.

However, the results obtained suggest that zwitterionic GBCAs such as Gd-SB₃-PCTA 11a and Gd-NOx₃-PCTA 12a have the potential to serve as safer contrast agents for MRI with high stability, high relaxivity, and low renal Gd(III) retention.

Experimental section

General

The sulfobetaine alkyne 14 was synthesised according to the procedure reported by Niu et al., while the synthesis of the alkyne N-oxide 15 has also been previously described in the literature.⁴⁴ In our hands, the most reliable method for obtaining a stable and analytically pure form of the alkyne N-oxide 15 involved the oxidation of N,N-dimethylpropargylamine (13) with meta-chloroperoxybenzoic acid (mCPBA) following a modified version of the procedure reported by Galán et al.⁴⁵ As the alkyne N-oxide 15 has been reported to be unstable even at low temperatures but was found to be stable in aqueous solution, we recommend immediate dissolution in water upon isolation, yielding a 3 M aqueous stock solution. This solution proved to be stable at 4 °C over at least two months and provided a convenient concentration for CuAAC reactions in mixed DMF/H₂O systems, in which all reactants were sufficiently soluble. All commercially available reagents and starting materials were purchased from Sigma Aldrich, TCI, abcr or BLDpharm and were used without further purification. Gadopiclenol 3 was purchased as Vueway® from Bracco Imaging SpA as a 0.5 mmol mL⁻¹ aqueous solution. Non-deuterated solvents in HPLC grade were purchased from VWR chemicals and deuterated solvents were purchased from Deutero GmbH. Water was purified using an ELGA PURELAB Classic UV water system. Reactions were monitored via HPLC-MS or TLC (Macherey Nagel TLC aluminum sheets, ALUGRAMSIL G UV254, 2.5 cm × 7.5 cm). Spots were visualized under UV light and/or by staining with a basic aqueous KMnO₄ solution.

Medium pressure liquid chromatography was performed on automated systems using prepacked cartridges (Interchim). For the use of alkaline alumina an empty cartridge was filled with alkaline alumina for chromatography. Normal phase chromatography was performed using a Biotage Isolera Prime system and for reversed flash chromatography an Interchim PuriFlash 430 system with MeCN/H₂O containing 0.1% formic acid as the mobile phase.

NMR analyses were performed on Bruker Avance III HD 600 MHz and Bruker Avance I 400 MHz spectrometers. Chemical shifts (δ) are expressed in parts per millions (ppm). High-resolution mass spectrometry (HRMS) was performed on an Agilent 6230 ESI-TOF coupled with an Agilent HPLC 1200 series HPLC system.

Analytical HPLC-MS was performed on an Agilent HPLC system 1260 Infinity II with a Macherey Nagel NUCLEODUR C18 Gravity-SB, 3 µm, 100 × 2 mm column linked to a Bruker Ion Trap mass spectrometer with an ESI ionization source.

Relaxivity measurements at 1.4 T

The determination of the longitudinal relaxivity at 60 MHz (1.4 T) was conducted based on the spin lattice relaxation time (T₁) using a Bruker Minispec mq60 analyzer at 37 °C. T₁ measurements were performed using the standard inversion recovery pulse sequence (180°–τ–90°) at a temperature of 37 °C ± 0.1 °C. For each measurement 20 unevenly spaced data points were acquired after inversion times between 5 ms and 400 ms. r₁ was obtained by plotting the inverse relaxation time 1/T₁ versus the concentration of the complexes and slope determination via the linear regression function in OriginLab 2022. The concentration of the complexes was determined via ICP-MS of the highest sample concentration.

Relaxivity measurements at 7 T

Relaxation time measurements at 7 T were conducted using a preclinical MRI scanner (Bruker Biospec 70/30, Ettlingen) at room temperature with a volume receive coil of an inner diameter of 40 mm. Five PCR tubes containing each 320 µL volume of different aqueous sample solutions (0.05–2 mM) were positioned in a holder within a 50 mL Falcon tube filled with water, aligned parallel to the magnet bore. All imaging sequences utilized transversal orientation.

Following an initial survey scan, two distinct T₁-measurement protocols were implemented. A 2D Look Locker EPI-based sequence provided quick qualitative visual T₁ inspection within 4 minutes. This protocol employed inversion times (TI) ranging from 20 to 770 ms in 50 ms increments, with TR 5000 ms and TE 9.9 ms. K-Space acquisition utilized six segments with 8 averages, 10° RF pulses, and a 200 kHz readout bandwidth. Imaging parameters included a 32 mm field of view, 128 × 128 matrix, and 1.2 mm slice thickness.

Subsequently, a slice-selective 2D inversion recovery spin echo sequence was acquired over approximately 2 hours. This protocol incorporated eleven inversion times (50, 80, 120, 160, 200, 400, 600, 800, 1200, 2000, and 3200 ms) with TE 7.0 ms, TR 5000 ms, and an 80 kHz readout bandwidth. Three slices of 1 mm thickness were obtained with 0.5 mm inter-slice gaps and 30 mm field of view with a 128 × 128 matrix.

Relaxation time analysis was conducted using qMapIt, an in-house quantification software that extends ImageJ functionality. Inversion recovery datasets underwent model function fitting: (eqn (4)) with T₁ = R₁⁻¹ via a nonlinear least-squares Dog–Leg algorithm, generating amplitude A and relaxation time T₁ maps. The inversion recovery data was smoothed before fitting with a Median filter of 0.5 pixel to reduce Gibbs-ringing.

Automated contour detection identified individual tube boundaries, with segmented regions stored as regions of interest (ROIs) for subsequent quantitative analysis. The complete analytical workflow was orchestrated through a Python script executed within a Jupyter notebook environment, which seamlessly integrated Fiji and qMapIt operations via PyImageJ.

Inductively coupled plasma mass spectrometry (ICP-MS)

Gadolinium concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS). Prior to ICP-MS measurements, 10 µL of the samples were digested in 1 mL of nitric acid (ROTIPURAN® Supra, 69%) for 24 h at room temperature in pre-cleaned tubes. Afterwards, all samples were filled up to 12 mL with ultrapure water. The digested samples were then diluted 1 : 100 prior to analysis. The measurements were performed on an Agilent Technologies 7800x ICP-MS (Agilent Technologies Inc., Santa Clara, USA) equipped with a quadrupole mass analyzer. Prior to measurement, the ICP-MS setup was tuned with a mixture of Ce, Co, Li, Tl and Y at a concentration of 1 ppb (Agilent Technologies Inc., Santa Clara, USA). External calibration was performed using mixed element standards purchased from Merck KGaAA and PerkinElmer® Inc. Calibration solutions containing Gd at concentrations of 0 to 1000 ppb were prepared freshly. Quantitation was performed by external calibration, corrected by internal standard (115 In for Gd). To ensure stability during the measurement a quality control sample containing 1 ppb Gd was measured.

Kinetic stability in acidic medium

The kinetic stability of the complexes was evaluated by incubating aqueous solutions (8 µmol L⁻¹) of the Gd(III) complexes in hydrochloric acid (pH 1.2) at 37 °C. The Gd(III) concentration of the solutions was confirmed by ICP-MS. The extent of decomplexation was quantified via UV-vis spectroscopy following complexometric titration with Arsenazo III. For each measurement a 1 mL aliquot of the acidic solution was mixed with 39 µL of an aqueous Arsenazo III solution (0.53 mmol L⁻¹) and 100 µL of an aqueous sodium acetate solution (1 mol L⁻¹). Following the absorption at 654 nm was measured using a 1 cm quartz cuvette at time points 1 day, 2 days, 5 days, 10 days, 15 days and 21 days.

Transmetallation/dechelation stability assay in the presence of phosphate and Zn²⁺

Pre-warmed to 37 °C solutions of the Gd(III) complexes (2.5 mM) and ZnCl₂ (2.5 mM) in aqueous 1 × PBS buffer (pH 7.4) were mixed in a 1 : 1 ratio and the spin lattice relaxation time T₁ was measured immediately to define the time point t = 0 min. The mixtures were then incubated at 37 °C and T₁ was recorded at 3 h, 1 day, 2 days, 3 days, 7 days and 15 days. All T₁ measurements were performed at 37 °C ± 0.1 °C using a Bruker Minispec mq60 analyser employing the standard inversion recovery pulse sequence. The T₁ values were subsequently plotted as a ratio relative to the initial value measured at t = 0 min.

Stability in human serum at 37 °C

The stability of the Gd(III) complexes was evaluated by incubating a solution of the complexes in human serum at 37 °C using a thermocycler followed by komplexometic determination with Arsenazo III. Human serum (Capricorn Scientific, male, type AB) was supplemented with sodium azide (1.7 mmol L⁻¹) as a microbial inhibitor prior to use. For each measurement, a 50 µL aliquot of the incubation mixture was diluted with 950 µL H₂O to obtain a final concentration of 8 µmol L⁻¹ of total gadolinium. Subsequently, 39 µL of an aqueous Arsenazo III solution (0.53 mmol L⁻¹) were added and the amount of free Gd(III) was quantified as described above. Measurements were performed at defined time points (0 h, 1 day, 3 days, 7 days and 14 days).

Luminescence lifetime measurements and q value determination

Excitation, emission and lifetime measurements were performed on an Agilent Cary Eclipse fluorescence spectrometer using a 1 cm quartz cell. Luminescence decay was recorded with an excitation wavelength of λ_exc = 271 nm and an emission wavelength of λ_em = 615 nm. The luminescence lifetime was determined by monoexponential fitting of the data in OriginLab 2022. Hydration numbers q were calculated following the protocol by Beeby et al. and Supkowski et al.^29,41

In vivo kidney retention study

All animal experiments described in this manuscript were conducted in accordance with the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines) and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Washington University in St. Louis. The laboratory conducting the experiments is accredited under protocol number 24-0832-02.

For in vivo evaluation of renal gadolinium retention male C57BL/6J mice (n = 2, 10–12 weeks, 25–30 g) received 10 consecutive intravenous injections of gadopiclenol 3 and Gd-SB₃-PCTA 11a via the tail vein at 0.1 mmol kg⁻¹ each (100 µL, saline, pH 7.4) over 10 days. Seventeen days after the final administration, animals were euthanised, organs were harvested and kept frozen until organ digestion and ICP-MS analysis was performed as described in the following. All animals showed no changes in body weight or behaviour following compound administration.

Kidney digestion and ICP-MS analysis

Kidneys (n = 2) were transferred into pre-cleaned tubes, weighed and spiked with dysprosium as internal standard (Thermo Fisher Specpure™, Dy plasma standard, 1008 ± 8 µg mL⁻¹) to reach 100 µg L⁻¹ in the final solution used for ICP-MS. For digestion, 2 mL of nitric acid (ROTIPURAN® Supra, 69%) were added and samples were incubated at 37 °C for 18 h. The digests were diluted to 10 mL with MilliQ H₂O, followed by a 1 : 2 dilution. To remove residual particulates, the solutions were centrifuged at 3500 × g for 15 min, decanted and subsequently analysed by ICP-MS in triplicates under the same conditions as described before.

Synthesis

Triflate 6. To a solution of (S)-benzyl-4-azido-2-hydroxybutanoat (5) (1.54 g, 6.57 mmol, 1.00 eq.) in CH₂Cl₂ (65 mL) at −20 °C was added 2,6-lutidine (1.14 mL, 9.85 mmol, 1.50 eq.) followed by the dropwise addition of trifluoromethanesulfonic anhydride (1.55 mL, 9.20 mmol, 1.40 eq.). During the addition the solution became orange, then redish. The solution was stirred at −20 °C for 1 h, the cooling bath was switched to an ice bath and the solution was stirred at 0 °C for 1 h. All volatiles were removed in vacuo (water bath: room temperature) and the crude product was obtained as a yellowish oil. The crude product was purified via flash chromatography (loaded as solution in CH₂Cl₂ (6 mL), 30 µm spherical silica gel, Interchim cartridge F0040, 100% CH₂Cl₂ isocratic). (S)-Benzyl-4-azido-2-(triflyloxy)-butanoate (2) (2.32 g, 96%) was obtained as an orangeish oil. ¹H-NMR (CDCl₃, 600 MHz) δ = 7.41–7.36 (5H, m, Ph-H̲), 5.30–5.26 (3H, m, Ph-CH̲₂, BnO₂C-CH̲), 3.54–3.42 (2H, m, CH₂-CH̲₂-N₃), 2.28–2.19 (2H, m, CH̲₂-CH₂-N₃).¹⁹F-NMR (CDCl₃, 564 MHz) δ = −74.7 (OTf).

(R,R,R)-Bn₃-PCTAZA 8. To a solution of pyclen 7 (273 mg, 1.32 mmol, 1.00 eq.) in anhydrous MeCN (10 mL) was added Cs₂CO₃ (1.51 g, 4.63 mmol, 3.50 eq.) and the suspension was cooled via an ice bath. Following a solution of (S)-benzyl-4-azido-2-(triflyloxy)-butanoate (6) (1.60 g, 4.37 mmol, 3.30 eq.) in anhydrous MeCN (25 mL) was added dropwise over 30 min. The reaction mixture was warmed to room temperature and stirred at this temperature for 2 h. Afterwards the solvent was removed in vacuo and the residue was dissolved in sat. aq. NaHCO₃ solution (30 mL) and CH₂Cl₂ (40 mL). The phases were separated and the aq. phase was extracted with CH₂Cl₂ (3 × 30 mL). The combined org. phases were dried over Na₂SO₄, the solvent was removed in vacuo and the crude product was obtained as an orange oil. The crude product was purified via flash chromatography (loaded as solution in CH₂Cl₂ (4 mL), 15 µm spherical silica gel, Interchim cartridge F0040, 100% CH₂Cl₂ for 1 CV then gradient to 100% MeCN in 10 CV, 100% MeCN for 3 CV). (R,R,R)-Bn₃-PCTAZA 8 (1.11 g, 98%) was obtained as an orangeish viscous oil. Rf (CH₂Cl₂/MeOH/Et₃N 95 : 4.5 : 0.5) = 0.60. HRMS (ESI) m/z [M+H]⁺ calcd for C₄₄H₅₂N₁₃O₆⁺: 858.4158, found: 858.4117. ¹H-NMR (CDCl₃, 600 MHz) δ = 7.60 (1H, t, ³J_H,H = 7.7 Hz, C_pyridine-H_para to N_pyridine), 7.41–7.27 (15H, m, CH₂-Ph-H̲),7.10 (2H, d, ³J_H,H = 7.6 Hz, C_pyridine-H̲ meta to N_pyridine), 5.22 (2H, d, ²J_H,H = 12.5 Hz, Ph-CH̲₂-), 5.18 (2H, d, ²J_H,H = 12.3 Hz, Ph-CH̲₂-), 5.03 (2H, s, Ph-CH̲₂-), 3.94 (2H, d, ²J_H,H = 11.9 Hz, Py-CH̲₂-N), 3.75 (2H, d, ²J_H,H = 11.9 Hz, Py-CH̲₂-N), 3.52 (2H, m, BnO₂C-CH̲- next to pyridine), 3.46–3.35 (4H, m, CH₂-CH̲₂-N₃ sidearms next to pyridine), 3.21–3.14 (3H, m, BnO₂C-CH̲ opposite pyridine, CH₂-CH̲₂-N₃ sidearm opposite pyridine), 2.90–2.81 (3H, m, CH̲₂-CH₂-N₃), 2.20–2.13 (3H, m, CH̲₂-CH₂-N₃), 2.12–1.98 (3H, m, N-CH̲₂-CH₂-N, N-CH₂-C(H̲)H-N), 1.93–1.85 (2H, m, N-CH̲₂-CH₂-N), 1.84–1.78 (1H, m, N-CH₂-C(H̲)H-N), 1.57–1.52 (1H, m, N-CH₂-C(H̲)H-N), 1.32–1.26 (1H, m, N-CH₂-C(H̲)H-N). ¹³C-NMR (CDCl₃, 150 MHz) δ = 172.9 (C_carbonyl sidearm opposite pyridine), 172.4 (C_carbonyl sidearms next to pyridine), 158.3 (quart. C_pyridine), 137.6 (quart. C_phenyl), 135.9 (C_para to N_pyridine), 128.9 (C_phenyl), 128.8 (C_phenyl), 128.7 (C_phenyl),128.6 (C_phenyl), 128.5 (C_phenyl), 128.4 (C_phenyl), 122.9 (C_meta to N_pyridine), 66.6 (Ph-C̲H₂ sidearms next to pyridine), 66.3 (Ph-C̲H₂ sidearm opposite pyridine), 63.8 (BnO₂C-C̲H-, sidearms next to pyridine), 63.0 (BnO₂C-C̲H-, sidearm opposite pyridine), 57.9 (pyridine-C̲H₂-), 52.9 (C̲H₂-CH₂-N₃), 50.5 (C̲H₂-CH₂-N₃), 48.4 (CH₂-C̲H₂-N₃ sidearms next to pyridine), 48.3 (CH₂-C̲H₂-N₃ sidearm opposite pyridine), 30.0 (N-C̲H₂-C̲H₂-N), 29.8 (N-C̲H₂-C̲H₂-N). t_R (C18 Gravity-SB, method 1): 18.2 min.

Alkyne N-oxide 15. Under nitrogen atmosphere a solution of 3-dimethylamino-1-propyne (13) (2.00 g, 24.0 mmol, 1.00 eq.) in anhydrous CH₂Cl₂ (24 mL) was cooled to 0 °C via an ice bath and subsequently a solution of mCPBA (75% with H₂O, 5.54 g, 24.0 mmol, 1.00 eq.) in anhydrous CH₂Cl₂ (24 mL) was added in a single portion. The reaction mixture was warmed to room temperature and stirred at this temperature for 3 h. Afterwards the reaction mixture was passed through a plug of alkaline alumina. The plug was first eluted with CH₂Cl₂ followed by CH₂Cl₂/MeOH 95 : 5. All volatiles were removed in vacuo (bath temperature at room temperature), the product was dried under high vacuum and 3-dimethylamino-1-propyne N-oxide 15 (1.98 g, 83%) was obtained as a colourless solid. As the product was temperature-sensitive and unstable in its solid form, it was immediately dissolved in MilliQ H₂O to yield a stable 3 M aq. stock solution which was stored at 4 °C. HRMS (ESI) m/z [M+H]⁺ calcd for C₅H₁₀NO⁺: 100.0757, found: 100.0761. ¹H-NMR (D₂O, 300 MHz) δ = 4.20 (s, 2H, CH̲₂), 3.35 (s, 1H, C≡CH̲), 3.30 (s, 6H, CH̲₃).

Bn₃-NOx₃-PCTA 10. Under nitrogen atmosphere a mixture of DMF/H₂O (7 : 1, 1.80 mL) was degassed by purging the solution with nitrogen gas for 15 min under vigorous stirring. Afterwards CuI (8.90 mg, 47.0 µmol, 0.10 eq.) and sodium ascorbate (18.5 mg, 93.0 µmol, 0.20 eq.) were added and the degassing process was allowed to proceed for further 5 min. Following (R,R,R)-Bn₃-PCTAZA 8 (0.40 g, 0.47 mmol, 1.00 eq.) as a solution in DMF (0.25 mL) and an aq. solution of 3-dimethylamino-1-propyne N-oxide (15) (3 M in H₂O, 2.8 mL, 8.39 mmol, 18.0 eq.) were both added in one portion. The reaction mixture was warmed to 55 °C and stirred at this temperature for 65 min. All volatiles were removed in vacuo and the crude product was obtained as a yellowish wax. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (2 mL), 15 µm C18AQ silica, Interchim cartridge F0012, 100% H₂O 5 CV, then gradient to 100% MeCN in 10 CV, 100% MeCN 5 CV). After lyophilization Bn₃-NOx₃-PCTA 10 (392 mg, 73%) was obtained as a beige lyophilisate. HRMS (ESI) m/z [M+H]⁺ calcd for C₅₉H₇₈N₁₆O₉⁺: 1154.6133, found: 1154.6100. ¹H-NMR (D₂O, 600 MHz) δ = 8.31 (1H, s, H̲_triazole sidearm opposite pyridine), 8.11 (2H, s, H̲_triazole sidearms next to pyridine), 7.95 (1H, t, ³J_H,H = 7.81 Hz, C_pyridine-H̲ para to N_pyridine), 7.43–7.27 (13H, m, CH₂-Ph-H̲ sidearms next to pyridine, C_pyridine-H̲ meta to N_pyridine, CH₂-Ph-H sidearm opposite pyridine), 7.20 (2H, dd,³J_H,H = 7.71 Hz, CH₂-Ph-H̲), 7.02 (2H, dd, ³J_H,H = 7.51 Hz, CH₂-Ph-H̲), 5.12 (2H, m, Ph-CH̲₂-, sidearm opposite pyridine), 5.05–4.87 (4H, m, Ph-CH̲₂-, sidearms next to pyridine), 4.75–4.70 (2H, m, CH₂-CH̲₂-triazole sidearm opposite pyridine), 4.65–4.60 (4H, m, CH̲₂-NOx, sidearms next to pyridine), 4.56 (2H, s, CH̲₂-NOx sidearm opposite pyridine), 4.45–4.22 (m, 4H, CH₂-CH̲₂-triazole sidearms next to pyridine), 4.00–3.97 (5H, m, BnO₂C-CH̲-), 3.84 (2 H, m, Pyridine-CH̲₂), 3.45 (2H, m, Pyridine-CH̲₂), 3.25 (12H, d, ⁴J_H,H = 7.35 Hz, N(CH̲₃)₂-O sidearms next to pyridine), 3.22 (6H, d, ⁴J_H,H = 3.86 Hz, N(CH̲₃)₂-O sidearm opposite pyridine), 2.94–2.59 (8H, m, N-C₂H̲₄-N), 2.42–2.36 (4H, m, CH-CH̲₂), 2.26–2.14 (2H, m, CH-CH̲₂). ¹³C-NMR (D₂O, 150 MHz) δ = 173.0 (C_carbonyl), 155.4 (quart. C_pyridine), 143.3 (C_pyridine-H para to N_pyridine), 136.8 (quart. C_triazole sidearms opposite pyridine), 136.5 (quart. C̲_triazole sidearms next to pyridine), 135.1 (quart. C_phenyl sidearms next to pyridine), 133.7 (quart. C̲_phenyl sidearm opposite pyridine), 129.2, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 127.8, 127.5 (C_phenyl, C_triazole-H), 123.03 (C̲_pyridine meta to N_pyridine), 68.6 (Ph-C̲H₂ next to pyridine), 67.5 (BnO₂C-C̲H-), 63.8 (C̲H₂-NOx next to pyridine), 63.7 (C̲H₂-NOx opposite pyridine), 58.2 (Ph-C̲H₂ opposite pyridine), 56.5 (N⁺-(C̲H₃)₂-O sidearms next to pyridine), 56.4 (N⁺-(C̲H₃)₂-O sidearm opposite pyridine), 52.8 (pyridine-C̲H₂), 52.0 (pyridine-C̲H₂), 47.8 (CH₂-C̲H₂-triazole sidearms next to pyridine), 47.5 (CH₂-C̲H₂-triazole sidearms opposite pyridine), 42.28 (C̲H₂-CH₂-triazole), 29.4 (N-C̲₂H₄-N), 24.1 (N-C̲₂H₄-N). t_R (C18 Gravity-SB, method 1): 11.7 min.

Bn₃-SB₃-PCTA 9. Under nitrogen atmosphere DMF/H₂O 7 : 1 (8 mL) was degassed by purging the solution with nitrogen gas for 15 min under vigorous stirring. Afterwards CuI (8.90 mg, 47.0 µmol, 0.10 eq.) and sodium ascorbate (18.5 mg, 93.0 µmol, 0.20 eq.) were added and the degassing process was allowed to proceed for further 5 min. Following (R,R,R)-Bn₃-PCTAZA 8 (400 mg, 466 µmol, 1.00 eq.) as a solution in DMF (1.5 mL) and sulfobetaine alkyne 14 (316 mg, 1.64 mmol, 3.30 eq.) were both added in one portion. The reaction mixture was warmed to 50 °C and stirred at this temperature for 41 h during which the reaction solution became dark brown. All volatiles were removed in vacuo and the crude product was obtained as a brownish waxy solid. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (2 mL), 15 µm C18AQ silica, Interchim cartridge F0012, 100% H₂O 5 CV, gradient to 100% MeCN in 10 CV, 100% MeCN 5 CV). After lyophilization Bn₃-SB₃-PCTA 9 (365 mg, 53%) was obtained as colourless lyophilisate. HRMS (ESI) m/z [M+2H]²⁺ calcd for C₆₈H₉₈N₁₆O₁₅S₃²⁺: 737.3275, found: 737.3293. ¹H-NMR (D₂O, 600 MHz) δ = 8.45, 8.43 (s, 2H, H̲_triazole sidearms next to pyridine), 8.26 (s, 1H, H̲_triazole sidearm opposite pyridine), 7.97–7.94 (m, 1H, C_pyridine-H̲ para to N_pyridine), 7.42–7.02 (m, 17H, CH₂-Ph-H̲, C_pyridine-H̲ meta to N_pyridine), 5.11–4.86 (m, 6H, Ph-CH̲₂), 4.70–4.31 (m, 13H, N⁺(CH₃)₂-C̲H₂-triazole, Pyridine-CH̲₂, BnO₂C-C̲H), 4.00–3.40 (m, 8H, N-CH̲₂-CH̲₂-N), 3.37–3.27 (m, 6H, N⁺(CH₃)₂-C̲H₂-CH₂), 3.09–3.02 (m, 18H, N-(C̲H₃)₂-O), 2.98–2.60 (m, 12H, CH₂-CH̲₂-SO₃, CH-CH̲₂), 2.49–2.20 (m, 12H, CH̲₂-CH₂-SO₃, CH-CH̲₂). ¹³C-NMR (CDCl₃, 150 MHz) δ = 172.8 (C_carbonyl),170.9 (C_triazole-H), 155.6 (HMBC, quart. C_pyridine), 143.1 (HSQC, C_pyridine-H_para to N_pyridine), 135.4, 135.2, 135.1 (quart. C̲_triazole and quart. C_phenyl), 129.3, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 127.5 (C_phenyl-H and C_pyridine-H), 123.1 (C_phenyl-H), 120.8 (HSQC, C_phenyl-H), 68.4 (Ph-C̲H₂), 67.5 (BnO₂C-C̲H-),67.3 (triazole-C̲H₂-N⁺(CH₃)₂), 61.8 (pyridine-C̲H₂), 57.7, 57.6 (N⁺(CH₃)₂-C̲H₂-CH₂), 50.4, 50.3, 50.2 (N-(C̲H₃)₂-O), 47.8, 47.7, 47.2 (CH₂-C̲H₂-SO₃, N-CH₂-C̲H₂-triazole), 32.7, 29.7, 29.4 (HSQC, N-C̲₂H₄-N, N-C̲H₂-CH₂-triazole), 18.3, 18.2 (C̲H₂-CH₂-SO₃). t_R (C18 Gravity-SB, method 1): 13.2 min.

Gd-NOx₃-PCTA 12a. At room temperature Bn₃-NOx₃-PCTA 10 (250 mg, 216 µmol, 1.00 eq.) was dissolved in H₂O (5 mL) and following a solution of NaOH (104 mg, 2.60 mmol, 12.0 eq.) in H₂O (1 mL) was added dropwise. The yellowish solution was stirred at room temperature for 23 h before Dowex® 50 W X8 hydrogen form, 100–200 mesh (500 mg) was added and the suspension was stirred until the pH was found to be neutral. The resin was removed via filtration, washed with H₂O (5 mL) and all volatiles were removed in vacuo. The residue was taken up in a solution of GdCl₃ hexahydrate (84.4 mg, 227 µmol, 1.05 eq.) in 1 M aq. NH₄OAc buffer (11 mL, pH 5.5) and the reaction mixture was stirred at room temperature for 3 h. Afterwards all volatiles were removed in vacuo (bath temperature: 55 °C) and the crude product was obtained as a yellowish oily residue. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (1.5 mL), 15 µm C18AQ silica, Interchim cartridge F0012, 3 CV 100% H₂O, then gradient to 100% MeCN in 10 CV, then 100% MeCN holding for 3 CV). After lyophilization Gd-NOx₃-PCTA 12a (172 mg, 77%) was obtained as a colourless lyophilisate. HRMS (ESI) m/z [M+2H]²⁺ calcd for C₃₈H₅₈GdN₁₆O₉²⁺: 520.1902, found: 520.1951. t_R (C18 Gravity-SB, method 1): 2.3 min.

Gd-SB₃-PCTA 11a. Bn₃-SB₃-PCTA 9 (250 mg, 170 µmol, 1.00 eq.) was dissolved in THF (1 mL) and a solution of NaOH (81.4 mg, 2.04 mmol, 12.0 eq.) in H₂O (5 mL) was added dropwise. The yellowish solution was stirred at room temperature for 23 h before Dowex 50 W X8 hydrogen form, mesh 100–200 (350 mg) was added and the suspension was stirred until the pH was found to be neutral. The resin was removed via filtration, washed with THF/H₂O 1 : 5 (5 mL) and all volatiles were removed in vacuo. The residue was taken up in a solution of GdCl₃ hexahydrate (66.2 mg, 178 µmol, 1.05 eq.) in 1 M aq. NH₄OAc buffer (11 mL, pH 5.5) and the reaction mixture was stirred at room temperature for 1.5 h. All volatiles were removed in vacuo (bath temperature: 55 °C) and the crude product was obtained as a yellowish solid. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (1 mL), 15 µm C18AQ silica, Interchim cartridge F0012, 3 CV 100% H₂O, then gradient to 100% MeCN in 10 CV, then 100% MeCN holding for 3 CV). After lyophilization Gd-SB₃-PCTA 11a (195 mg, 85%) was obtained as a colourless lyophilisate. HRMS (ESI) m/z [M+H+Na]²⁺ calcd for C₄₇H₇₆GdN₁₆NaO₁₅S₃²⁺: 690.6983, found: 690.6971. t_R (C18 Gravity-SB, method 1): 9.2 min.

Eu-NOx₃-PCTA 12b. Bn₃-SB₃-PCTA 9 (14 mg, 12.0 µmol, 1.00 eq.) was dissolved in H₂O (1 mL) and a solution of NaOH (5.8 mg, 145 µmol, 12.0 eq.) in H₂O (0.5 mL) was added dropwise. The yellowish solution was stirred at room temperature for 17 h before Dowex 50W X8 hydrogen form, mesh 100–200 (80 mg) was added and the suspension was stirred until the pH was found to be neutral. The resin was removed via filtration, washed with H₂O (3 mL) and all volatiles were removed in vacuo. The residue was taken up in a solution of EuCl₃ (3.3 mg, 13.0 µmol, 1.05 eq.) in 1 M aq. NH₄OAc buffer (1 mL, pH 5.5) and the reaction mixture was stirred at room temperature for 3 h. All volatiles were removed in vacuo (bath temperature: 55 °C) and the crude product was obtained as a yellowish solid. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (0.2 mL), 15 µm C18AQ silica, Interchim cartridge F0004, 4 CV 100% H₂O, then gradient to 100% MeCN in 10 CV, then 100% MeCN holding for 3 CV). After lyophilization Eu-NOx₃-PCTA 12b (6 mg, 48%) was obtained as a colourless lyophilisate. HRMS (ESI) m/z [M+2H]²⁺ calcd for C₃₈H₅₉EuN₁₆O₉²⁺: 518.1926, found: 518.1952. t_R (C18 Gravity-SB, method 1): 2.0 min.

Eu-SB₃-PCTA 11b. Bn₃-SB₃-PCTA 9 (19.5 mg, 13.0 µmol, 1.00 eq.) was dissolved in H₂O (1 mL) and a solution of NaOH (6.4 mg, 159 µmol, 12.0 eq.) in H₂O (0.5 mL) was added dropwise. The yellowish solution was stirred at room temperature for 18 h before Dowex® 50W X8 hydrogen form, mesh 100–200 (80 mg) was added and the suspension was stirred until the pH was found to be neutral. The resin was removed via filtration, washed with H₂O (3 mL) and all volatiles were removed in vacuo. The residue was taken up in a solution of EuCl₃ (3.6 mg, 14.0 µmol, 1.05 eq.) in 1 M aq. NH₄OAc buffer (1 mL, pH 5.5) and the reaction mixture was stirred at room temperature for 3.5 h. All volatiles were removed in vacuo (bath temperature: 55 °C) and the crude product was obtained as a yellowish solid. The crude product was purified via reversed phase flash chromatography (loaded as solution in H₂O (0.2 mL), 15 µm C18AQ silica, Interchim cartridge F0004, 4 CV 100% H₂O, then gradient to 100% MeCN in 10 CV, then 100% MeCN holding for 3 CV). After lyophilization Eu-SB₃-PCTA 11b (10 mg, 56%) was obtained as a colourless lyophilisate. HRMS (ESI) m/z [M+2H]²⁺ calcd for C₄₇H₇₇EuN₁₆O₁₅S₃²⁺: 677.2059, found: 677.2052. t_R (C18 Gravity-SB, method 1): 9.3 min.

Author contributions

Conceptualization: JVF, WM; methodology, investigation, data curation: LFVS, VRS, MGK, DL, MO, DLJT; formal analysis: LFVS, MGK, MO; project administration, resources: JVF, WM; writing – original draft: LFVS, WM; writing – review & editing: all authors; project administration: JVF, WM; supervision: NFT, MF, WM.

Conflicts of interest

JVF is a cofounder and CEO of Curadel Pharma. JVF and WM have filed a patent application on zwitterionic chelators (WO 2025059025 (A1) 2025-03-20).

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5tb02336j. This material contains NMR spectra and LC/MS data for new compounds and luminescence measurements of Eu-complexes.

Acknowledgements

Proof reading of the manuscript by Antje Wagner is acknowledged. We thank the NMR-facility at the department of chemistry for support with NMR-analysis. This work was supported by the Fraunhofer Internal Programs under Grant No. Attract 178-600040 to NF and VRS. We acknowledge financial support from the Open Access Publication Fund of Universität Hamburg.

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