Improved Efficacy of Synthesizing *M^III-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio-and Physicochemical Study

Abstract

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [⁶⁸Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H₂O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)]⁻ (M^III= Ga^III, Ce^III, Eu^III, Y^III, and Lu^III) complexes were investigated in H₂O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H₂O/EtOH mixtures (0–70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K₁^H and log K₂^H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)]⁻ complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H₂DOTA)]⁺ intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The ratecontrolling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through ${\text{H}}_2\text{O}({*}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}})$ and ${\text{OH}}^{-}({*}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}})$ assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the ^M(HL)k_H2O values increase from Ce^III to Lu^III. However, the logK_M(HL) ^H protonation constants, analogous to the log K^H₂ value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes.

INTRODUCTION

Radiometal/Chelator Complexes.

Complexation of the radioactive metal ion *M by an adequate polydentate chelator L (also referred to as the chelator C) or its bifunctional version (BFC) covalently coupled to a biological targeting vector (TV) is an extremely important consideration in the design and construction of metal-based radiopharmaceuticals. For diagnostic and therapeutic applications in nuclear medicine, trivalent radiometals *M^III are typically chelated by macrocyclic polyamino polycarboxylic chelators, especially DOTA (H₄DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its derivatives, to form high-stability complexes. However, DOTA and its derivatives generally form complexes with M³⁺ ions exceedingly slowly at room temperature, limiting their applications for isotopes with short half-lives. On the other hand, once formed, the *M^III-L complexes, in particular the *M^III-DOTA complexes, guarantee high in vivo thermodynamic stability and kinetic inertness; these properties have been systematically investigated for nonradioactive trivalent metal ions such as lanthanide(III) cations used in magnetic resonance or optical imaging.¹

Slow-formation (i.e., radiolabeling) kinetics may become a critical issue for short-lived radionuclides like gallium-68 (t_1/2 = 67.71 min). For this reason, detailed evaluation and optimization of experimental conditions were conducted to increase the radiolabeling efficiency. Gallium-68 complex formation can be accelerated by increasing the temperature.^2,3 Elevated temperatures (e.g., 90–95 °C) are commonly used to label DOTA-conjugated somatostatin analogues or DOTA affibodies/antibodies with gallium-68, yttrium-90, indium-111, or lutetium-177, providing nearly 100% radiolabeling efficiency. With gallium-68, nearly quantitative radiolabeling (ca. 90–95%) occurs within 10 min under these conditions. Depending on the biological TV, microwave-assisted synthesis can alternatively be used to facilitate *M^III-DOTA-TV complex formation.⁴

Considering slow complex formation, high yields require sufficient excess of the chelating agent. In contrast, high apparent molar radioactivity of the radiopharmaceutical is preferred depending on the type of diagnostic or therapeutic application.⁵ For example, imaging and therapy of tumor cells overexpressing G-protein-coupled transmembrane receptors require high apparent molar activities because of the limited number and affinity of receptors on the target site. The apparent molar radioactivity is the ratio of the product radioactivity per mole chelator by taking into account the amounts of labeled and nonradiolabeled compounds. The molar amount of the radiometal cations is generally ultralow, representing, for example, 5.860 × 10¹² atoms (9.731 × 10⁻¹² mol) for 1 GBq of gallium-68 and 8.285 × 10¹⁴ atoms (1.376 × 10⁻⁹ mol) for 1 GBq of lutetium-177. In contrast, nonradioactive metal impurities (such as Fe^II/Fe^III ions present in the radiometal stock solution and/or the chemicals and glassware used), although low in concentration in the sense of “conventional” chemistry, can efficiently compete with the radiometal for the chelators. Because those impurities are usually not avoidable or reducible, larger amounts of the “chelator” component are required, which, in turn, affects the apparent molar radioactivity of the final radiopharmaceutical.⁵

Formation of Metal Complexes in Water (H₂O) and Binary H₂O/Organic Solvent Mixtures.

The synthesis of metal-based radiopharmaceuticals is generally performed in an aqueous medium, where the M^III ions exist as aqua complexes. Because of its molecular properties (small size, inherently polar, and polarizable), H₂O is an excellent, versatile solvent. It can act as a hydrogen-bond acceptor as well as a donor, allowing formation of a variety of structures that can easily adjust to changes in conditions.⁶ In aqueous media, a well-defined number of H₂O molecules directly surround the metal ion and are bound in the inner (or first) coordination sphere of the M^III ion to form the hydrated [M(OH₂)_n]³⁺ aqua complex, where n is the hydration (or coordination) number. A second shell of less ordered H₂O molecules surrounds the first hydration sphere, forming a second (or outer) hydration sphere. The ordering of H₂O molecules continuously decreases from the first hydration sphere to the bulk solvent.^7,8

The complex formation reaction of M^III ions is generally rapid. The mechanisms of complex formation reactions are usually different for smaller, mono- or bidentate and larger multidenate chelators. The formation rate with smaller chelators is controlled by the H₂O loss from the outer-sphere complex formed between the hydrated metal ion and the chelator. The formation rate of complexes with multidentate chelators is often determined by formation of the first chelate ring. The formation of macrocyclic M^III complexes is generally rather slow because of the highly preorganized nature of these chelators as well as the slow reorganization of the metal−donor bonds in the intermediates, which may play a significant role in formation of the fully encapsulated complex.⁹ The complexes of M^III ions with DOTA and its derivatives are known to form very slowly in the range of pH 4–6, which is unfavorable for the complexation of short-lived radioisotopes. As was recently reported, the reaction of gallium-68 with DOTA-TOC [edotreotide; DOTA(0)-Phe(1)-Tyr(3))octreotide] in an ethanol (EtOH)/H₂O solvent mixture occurs more rapidly than that in pure H₂O, making radiolabeling possible at lower temperatures.¹⁰

In an early publication, a mechanism for the formation of DOTA complexes with lanthanide(III) (Ln^III) ions through a long-lived intermediate [LnH₂(DOTA)]⁺ in which the metal ion interacts only with the deprotonated carboxylate arms of the macrocyclic chelator was suggested.¹¹ A similar diprotonated [Gd(H₂HP-DO3A)]⁺ intermediate has been evidenced in the Gd³⁺-HP-DO3A reaction system.¹² The formation of protonated intermediates may take place in the reaction of several divalent metal ions with the DOTA ligand.¹³ The diprotonated [LnH₂(DOTA)]⁺ intermediate is formed in a fast preequilibrium, and the rate-determining step of complex formation is concerted deprotonation of the N-donor atoms and penetration of the Ln^III ion into the cavity.^14,15 It is worth noting that the Ln^III ions involved in this slow formation reaction are extremely labile; i.e., the rate constants characterizing the H₂O exchange rates of aqueous Ln^III ions (10⁹ s⁻¹) are among the fastest known.^16,17

The solvation of metal ions and chelators in mixed H₂O/organic media is more complicated than the hydration in aqueous media because the ratio of H₂O/organic molecules in the inner-sphere [M(H₂O)_xS_y] depends on both the H₂O/organic solvent ratio and the binding energy difference between the M^III–H₂O and M^III–organic solvent interactions. In addition, steric factors might also be important.^7,8 The H₂O molecules are strongly bound to the metal ions and may remain in the inner sphere even at high organic solvent/H₂O ratios; therefore, interpretation of the kinetic effect of mixed solvent systems is by no means straightforward.¹⁸

The presence of large amounts of organic solvent will also affect intra- and intermolecular interactions. It has been demonstrated that organic solvents such as isopropyl alcohol (iPrOH) can disrupt the hydrogen bonds between H₂O molecules, thereby acting as a structure breaker.^19,20 Furthermore, the conformation of peptide chains in DOTA–peptide or protein conjugates may also be influenced by solute–H₂O interactions, in particular where −NH₂ or −COOH functionalities are concerned.²¹

We recently observed that the presence of EtOH in mixtures utilized to purify ⁶⁸Ge/⁶⁸Ga-generator eluates substantially increased the radiolabeling efficacies for [⁶⁸Ga]Ga-DOTA-TOC derivatives compared to pure aqueous solutions.¹⁰ Considering the favorable effect of EtOH on the formation reaction, the aim of this work was to systematically investigate the effect of nonaqueous solvents on radiometal–chelator complex formations. We included the trivalent radiometals gallium-68, scandium-44, and lutetium-177 and screened mixtures of H₂O and organic solvents such as EtOH, iPrOH, and acetonitrile (MeCN). We studied the rate of complex formation with DOTA by varying the nonaqueous solvent/H₂O ratio, amount of the chelator, temperature, and reaction time.

In order to understand the physicochemical background behind the rate-increasing effect of the organic solvents, we carried out systematic kinetic studies on the formation of [M(DOTA)]⁻ (M = Ga^III, Ce^III, Eu^III, Y^III, and Lu^III) complexes in H₂O/EtOH mixtures (up to 70 vol % EtOH). We could rationalize the effect of EtOH on the reaction rates by determining the protonation constants of the DOTA chelator and [M(H₂DOTA)]⁺ intermediates in H₂O/EtOH mixtures. The solvation of Ga^III, Sc^III, and Y^III in mixed H₂O/EtOH solutions was also tested by gallium-71, scandium-45, and yttrium-89 NMR via measurement of the T₁ relaxation times of the nuclei.

RESULTS AND DISCUSSION

Radiolabeling of DOTA and DOTA Conjugates with the Trivalent Metallic Radionuclides Gallium-68, Scandium-44, and Lutetium-177.

DOTA as a single chelator was used as the model compound because of its prevalent clinical role as a chelator in therapeutic (e.g., [⁹⁰Y]Y/[¹⁷⁷Lu]Lu-DOTA-TOC/DOTA-TATE) or diagnostic (e.g., Gd-DOTA; [⁶⁸Ga]Ga-DOTA-TOC) agents. To determine the effect of additional organic solvents in the reaction mixture, the radiolabeling conditions were selected so that the radiochemical yield for gallium-68 labeling would be low (~50%) at relatively low temperature (70 °C) in a pure aqueous solution. These conditions were also adopted for scandium-44 and lutetium-177. This radiolabeling profile was taken as a reference for pure aqueous systems at 10 nmol of DOTA, and any influence of organic solvents on the radiolabeling yields was directly determined by a comparison to the yield obtained under these conditions.

[⁶⁸Ga]Ga-DOTA.

Table 1 shows the radiochemical yields of radiolabeling DOTA with gallium-68 in H₂O or H₂O/organic solvent mixtures (30 vol % EtOH, MeCN, or iPrOH) in percentage as well as normalized values relative to the radiochemical yield of radiolabeling in a pure aqueous solution. For all investigated nonorganic solvents, a small but highly reproducible increase of the radiolabeling yields by a factor of 1.3 was observable after 15 min of reaction time. This relative increase (nonaqueous system compared to a pure aqueous system) is even more distinctive at shorter reaction times. For example, after 5 min of reaction time, the relative increase values are 3.3 (EtOH), 3.4 (MeCN), and 3.6 (iPrOH). The most obvious enhancement is observed within 3 min of reaction time using MeCN [3.7] and iPrOH [4.5] and at 5 min for EtOH [3.3]. After 10 min, a plateau is achieved, and no significant further increase in radiolabeling can be observed for the nonaqueous systems, while complex formation continues to progress in the aqueous system. Figure 1 illustrates the impact of different amounts of EtOH (0–40 vol %) on the [⁶⁸Ga]Ga-DOTA complex formation at 70 °C. A 2.1-fold increase in the complex formation yields is observed in the presence of as low as 10 vol % EtOH within 5 min of reaction time. A further increase in the EtOH content up to 40 vol % resulted in a 3.5-fold increase. After a 15 min reaction time, the relative increase observed for different amounts of EtOH approximates the same limit, around 80%. The highest impact of the nonaqueous solvent can be observed at 40 vol % solvent content and short reaction times (<5 min).

Table 1.

[⁶⁸Ga]Ga-DOTA Radiolabeling Yields in Percentage (%) and Normalized (in Square Brackets) Values Relative to the Radiochemical Yields Obtained at 70 °C in a Pure Aqueous Solution and in Mixtures with Various Organic Solvents (30 vol%) with 10 nmol of DOTA Depending on the Reaction Time^a

time (min)	radiolabeling yields (%) and factors of increase (in square brackets)
	H2O	EtOH	MeCN	iPrOH
1	5.7 ± 1.1 [1]	9.0 ± 0.7 [1.6]	6.7 ± 0.4 [1.2]	12.0 ± 1.3 [2.1]
3	13.7 ± 2.2 [1]	42.7 ± 3.8 [3.1]	51.3 ± 2.2 [3.7]	63.3 ± 2.4 [4.6]
5	21.0 ± 2.0 [1]	68.3 ± 2.2 [3.3]	71.0 ± 1.3 [3.4]	74.7 ± 0.9 [3.6]
10	51.0 ± 3.3 [1]	79.7 ± 0.4 [1.6]	81.3 ± 0.4 [1.6]	82.3 ± 0.4 [1.6]
15	66.3 ± 2.9 [1]	84.0 ± 0.7 [1.3]	83.7 ± 0.9 [1.3]	86.7 ± 0.4 [1.3]

^aThe dramatic increase observed for a short reaction time, such as 3 min, is indicated in boldface.

Figure 1.

Complex formation kinetics of [⁶⁸Ga]Ga-DOTA in the presence of 0−40 vol % EtOH (10 nmol DOTA, 70 °C, n = 3).

The dramatic increase in the reaction yields in EtOH-containing solvent mixtures suggests that gallium-68 labeling of DOTA is more effective in H₂O/EtOH mixtures compared to the currently used standard aqueous system (“standard” here refers to pure aqueous solutions and reaction temperatures of 95 °C). Because the standard synthesis of 68Ga-DOTA-conjugated radiopharmaceuticals is typically performed at 95 °C, we decided to investigate whether the presence of an organic solvent would improve the rate of complex formation at 95 °C compared to reactions run at 70 °C. Because DOTA conjugated to small TVs can usually be labeled with gallium-68 under those temperatures (95 °C) at sufficiently high concentrations of the chelator conjugate to afford over 90% yields, our assumption was that it might be possible to achieve high radiolabeling yields at much lower concentrations of the chelator if mixed solvent systems were used. The dependence of the radiochemical yield of [⁶⁸Ga]Ga-DOTA on the concentration of DOTA in the presence of 40 vol % EtOH after 5 min of radiolabeling is shown in Figure 2.

Figure 2.

Complex formation yields of [⁶⁸Ga]Ga-DOTA depending on the DOTA concentration for the pure aqueous system and in a solution containing 40 vol % EtOH ([DOTA] = 0.1−20 μM, 95 °C, 5 min, n = 3).

When larger amounts such as 63.3 nmol (i.e., 20 μM concentration) of DOTA were used, the radiochemical yields were similar for the pure aqueous H₂O (92.2%) and the H₂O/EtOH (95.2%) system, and in this case, no significant gain in the radiochemical yield was found in the presence of EtOH. However, the radiochemical yields drop faster in the pure aqueous system than in the H₂O/EtOH mixture with decreasing DOTA concentrations. For example, while labeling yields in pure aqueous systems drop from 92.2% to 84.8% to 63.7% when the DOTA concentration decreases from 20 to 10 to 2 μm, the corresponding yields in 40 vol % EtOH systems are 95.2%, 93.7°%, and 85.2%, respectively. Figure 2 clearly demonstrates that, in the presence of 40 vol % EtOH, the complex formation yields of [⁶⁸Ga]Ga-DOTA are higher at 10 μM DOTA than that obtained in a pure aqueous solution containing twice as much DOTA (20 μM).

[⁴⁴Sc]Sc-DOTA.

DOTA was labeled with scandium-44 as described for [⁶⁸Ga]Ga-DOTA in solvent systems containing different amounts (0–40 vol %) of EtOH, iPrOH, or MeCN. Figure 3 shows the results obtained in the presence of 30 vol % of these solvents in comparison to the conventional radio-labeling procedure performed in pure ammonium acetate (amac) buffer (0.25 M, pH 4.0). Analogous to the radiolabeling experiments with gallium-68, significantly improved scandium-44 radiolabeling yields were observed in the presence of an organic solvent. Under these conditions, yields of up to 91.0% (EtOH; 20 min) were achieved, which represents a 2-fold increase compared to the yields obtained in the pure aqueous buffer system (43.9%, 20 min).

Figure 3.

Complex formation kinetics of [⁴⁴Sc]Sc-DOTA in the presence of 30 vol % nonaqueous solvents (10 nmol DOTA, 70 °C, n = 3, 0.25 M amac buffer, pH 4).

Table 2 shows the radiolabeling yields of [⁴⁴Sc]Sc-DOTA in solutions containing 10–40 vol % EtOH and iPrOH relative to the yield obtained in a pure aqueous buffer solution.

Table 2.

Increase of ⁴⁴Sc-Radiolabeling Yields Depending on the Amount of Organic Solvent (0−40 vol% EtOH and iPrOH) and the Reaction Time Given as Normalized Values Relative to the Yield Obtained in a Pure Aqueous Buffer Solution

time (min)	EtOH (vol %)				iPrOH (vol %)
	10	20	30	40	10	20	30	40
1	1.1	2.1	5.6	5.4	1.4	2.6	4.1	5.2
3	1.4	2.4	4.4	4.1	1.5	2.5	3.6	4.0
5	1.4	2.2	3.3	3.2	1.4	2.3	3.0	3.2
10	1.2	2.0	2.6	2.6	1.3	2.0	2.4	2.6
20	1.2	1.7	2.1	2.1	1.1	1.7	2.0	2.1

At short reaction times (<5 min) and in mixtures containing 30% or more organic solvent, the effect of EtOH slightly exceeds that of iPrOH. For example, at 3 min reaction time, the use of 30 and 40 vol % EtOH afforded 4.4- and 4.1-fold increases, respectively, while the corresponding increase was 3.6- and 4.0-fold with 30 and 40 vol % iPrOH, respectively. For lower solvent concentrations (<30 vol %), the order was reversed. For longer reaction times (>5 min), the impact of both solvents on radiolabeling was more or less the same for each concentration and showed only a modest improvement over the shorter reaction times.

[¹⁷⁷Lu]Lu-DOTA.

The β-emitting radionuclide lutetium-177 (t_1/2 = 6.73 days) is frequently used in peptide receptor radionuclide therapy. Being a lanthanide, Lu³⁺ forms stable complexes with DOTA and its derivatives, and these chelators are frequently used in the construction of lutetium-177 radiopharmaceuticals. In the present work, DOTA was radiolabeled with lutetium-177 in H₂O/EtOH mixtures (0–40 vol %) of EtOH after determination of the baseline conditions (70 °C, chelator–metal ratio 10:1, 0.1 M sodium acetate buffer, pH 8, 30 min).

Figure 4 shows the relative increase of the radiolabeling yields of [¹⁷⁷Lu]Lu-DOTA with increasing percentage of EtOH (10−30 vol %) present in the solvent mixture. At short reaction times of less than or equal to 5 min, the effect of EtOH on the yields somewhat exceeds those observed at longer reaction times above 5 min. For example, at 2 min the relative increase achieved by using 30 vol % EtOH is 2.1, while at 10 min, it is 1.8.

Figure 4.

Complex formation kinetics of [¹⁷⁷Lu]Lu-DOTA in the presence of 0−30 vol % EtOH (10 nmol DOTA, 70 °C, n = 3, 0.1 M sodium acetate buffer, pH 8).

Formation of M^III Complexes with DOTA Chelator in H₂O/EtOH Mixtures.

To understand and explore the impact of organic solvents on the thermodynamic and kinetic properties of the M^III-DOTA systems, systematic studies on the protonation equilibria of DOTA and the formation rates of [M(DOTA)]⁻ (M^III = Ga^III, Ce^III, Eu^III, Y^III, and Lu^III) complexes in H₂O/EtOH mixtures (10, 40, and 70 vol % EtOH) were carried out. We propose a reaction mechanism for the formation of [M(DOTA)]⁻ complexes in the H₂O/EtOH solvent system, which is also supported by multinuclear NMR studies. The solvation of Ga^III, Sc^III, and Y^III ions in mixed H₂O/EtOH solutions was also tested by ⁷¹Ga, ⁴⁵Sc, and ⁸⁹Y NMR by measuring the T₁ relaxation time of the nuclei.

Protonation Equilibria of DOTA in H₂O/EtOH Mixtures.

The protonation scheme of DOTA in pure aqueous solutions is well-known.²² In the present work, the protonation constants of DOTA, defined by eq 1, have been determined by pH potentiometry in H₂O/EtOH mixtures. The log K_i ^H values are shown in Figure 5 (standard deviations are shown with error bars) and Table S2.

$$K_i^{\text{H}}=\frac{[{\text{H}}_i\text{L}]}{[{\text{H}}_{i-1}\text{L}][{\text{H}}^{+}]}$$ (1)

where i = 1, 2,…, 6. The data presented in Figure 5 and Table S2 indicate that the log K₁ ^H and log K₂ ^H values associated with the protonation of two opposite macrocyclic ring N atoms²² decrease with the increase of the EtOH content. This observation is in agreement with literature data reporting that the protonation constants of N atoms in ethylenediaminetetraacetic acid (EDTA) chelator decrease with an increase of the MeOH content of up to around 80 m/m % MeOH. Interestingly, above 80–90 m/m % MeOH, the protonation constants of the N atoms start to rise.²³

Figure 5.

Dependence of the protonation constants (log K_i^H) of DOTA on the EtOH content in H₂O/EtOH mixtures at 25 °C in 0.15 M NaCl ([DOTA] = 2.00 mM; log K_l^H, black ◆; log K₂^H, blue ■; log K₃^H, red ▲; log K₄^H, green ●; log K₅^H, pink ◀; log K₆^H, brown ▼)

The basicity of the ring N atoms of DOTA might be influenced by four effects: (i) the electrostatic repulsion between the protonated N atoms, which reduces the basicity of the remaining macrocylic N atoms; (ii) hydrogen-bonding interaction between the protonated N atom and the negatively charged carboxylate group, which increases the basicity of the N atom, and any potential barrier for this hydrogen-bond formation would decrease the basicity; (iii) the formation of a relatively stable [Na(DOTA)]³⁻ complex (log K_NaL = 4.38),²⁴ which results in a drop in the basicity; (iv) EtOH has a significantly lower proton dissociation constant than H₂O, which also decreases the basicity of the macrocyclic N atoms. The sum of all of these effects will then lead to the gradual decrease of the log K₁ and log K₂ values of DOTA with increasing concentration of EtOH. The log K₃ ^H, log K₄ ^H, logK₅ ^H, and log K₆ ^H values are related to protonation of the carboxylate groups. These values are essentially constant (Figure 5), with the exception of log K₄ ^H, which slightly increases with increasing EtOH concentration. A similar behavior was reported for the EDTA carboxylates, whose protonation constants monotonously increase with increasing concentration of methanol (MeOH).²³

Formation of [M(DOTA)] Complexes in H₂O/EtOH Mixtures (M^III = Ga^III, Ce^III, Eu^III, Y^III, and Lu^III).

It is well-known that the complexes of the macrocyclic, rigid DOTA are formed slowly with trivalent metal ions.^14,15 The slow formation of complexes may cause difficulties when the complex is used as a radiopharmaceutical. The formation mechanism of DOTA complexes is fairly well-known. The slow complexation rate of DOTA is largely due to its rigid structure and can be described by the following mechanism. The metal ion has to enter the coordination cage formed by the four ring N atoms and the four O atoms of the acetates attached to the N atoms. The formation of the fully formed, in-cage complex is hindered by the protonation of two macrocyclic ring N atoms below pH 7, but the four acetates will coordinate to the metal ion to form a diprotonated intermediate, [Ln(H₂DOTA)]⁺, in which the Ln^III ion is situated outside the coordination cage. In addition to the four acetates, four or five H₂O molecules also coordinate to the metal ion.^14,15,25,26 The complex formation is completed by removal of the two protons from the coordination cage, which is followed by rearrangement of the intermediate to the final [Ln(DOTA)]⁻ complex. The rate-determining step is probably the loss of the last proton from the [Ln(HDOTA)] intermediate.¹⁵

In the present work, we studied the formation kinetics of [M(DOTA)]⁻ complexes (M^III = Ga^III, Ce^III, Eu^III, Y^III, and Lu^III) in H₂O/EtOH mixtures containing 10, 40, and 70 vol % EtOH. The formation of [Ce(DOTA)]⁻ and [Eu(DOTA)]⁻ was followed spectrophotometrically by observing the absorption bands at 320 and 250 nm, respectively. The formation of [Ga(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu-(DOTA)]⁻ was followed by monitoring the release of H⁺ from DOTA (indicator method).²⁷ The composition of the diprotonated [Ce(H₂DOTA)]⁺ intermediate in aqueous solution was proven previously by pH-potentiometric titration¹⁴ and also by other methods.^15,25,26 The detection of this intermediate as well as the monitoring of the complex formation in H₂O/EtOH mixtures was performed by spectrophotometry. CeCl₃ and H₄DOTA were reacted in equimolar quantities in 10 and 70 vol % EtOH, and the UV spectra were recorded as a function of time (over 120 and 50 min), as shown in Figures 6 and 7.

Figure 6.

Absorption spectra of the Ce³⁺-DOTA system ([Ce³⁺] = [DOTA] = 1.0 mM, [N-methylpiperazine] = 0.01 M, 10 vol % EtOH, pH 4.46, l = 0.874 cm, 0.15 M NaCl, 25 °C).

Figure 7.

Absorption spectra of the Ce³⁺-DOTA system ([Ce³⁺] = [DOTA] = 0.1 mM, 70 vol % EtOH, [N-methylpiperazine] = 0.01 M pH 4.51, l = 10 cm, 0.15 M NaCl, 25 °C).

The intensity of the band at 296 nm decreases, while that at 320 nm increases over time. These spectra are essentially identical with those obtained in aqueous solutions. These experiences clearly demonstrate that the structure of the diprotonated intermediate [Ce(H₂DOTA)]⁺ is the same in H₂O and 10 and 70 vol % EtOH solutions. Similar phenomena were observed during the spectroscopic studies of the formation of [Eu(DOTA)]⁻. The formation of [Y(DOTA)]⁻, [Lu(DOTA)]⁻, and [Ga(DOTA)]⁻ was also followed by ¹H NMR spectroscopy (Figures S3–S5). The appearance of isosbestic points in the ¹H NMR spectra of these systems clearly indicates the formation of a [M(H₂DOTA)]⁺ intermediate, which is slowly transformed to the final [M(DOTA)]⁻. It should be noted that in the pH range of these studies (pH 2.0) diprotonated [Ga(H₂DOTA)]⁺ complexes are present in equilibrium (two carboxylate groups are protonated in the [Ga(H₂DOTA)]⁺ complexes).²⁸

The kinetic studies on the formation of [Ce(DOTA)]⁻ and [Eu(DOTA)]⁻ were also performed in the presence of excess Ce^III and Eu^III under pseudo-first-order conditions. The concentrations of Ce^III and Eu^III were 5−40 times higher than those of DOTA ([DOTA] = 2.0 × 10⁻⁴ M), and under these conditions, the rate of complex formation can be expressed by eq 2.

$$\frac{\text{d}{[\text{ML}]}_{\text{t}}}{\text{d}t}=k_{\text{obs}}{[\text{L}]}_{\text{t}}$$ (2)

where [ML]_t is the concentration of the [M(DOTA)]⁻ complex formed, [L]_t is the total concentration of the DOTA chelator at a given time point, and k_obs is a pseudo-first-order rate constant. The formation reactions were studied at different pH values by varying the metal ion concentrations. The k_obs versus [M^III] curves (Figures S6–S11) are saturation curves indicating the formation of [M(H₂DOTA)]⁺ intermediates. The thermodynamic stability of these intermediates is characterized by a stability constant defined by eq 3.

$$K_{\text{M}({\text{H}}_2\text{L})}=\frac{[\text{M}({\text{H}}_2\text{L})]}{[{\text{M}}^{\text{III}}][{\text{H}}_2\text{L}]}$$ (3)

where [M(H₂L)] is the concentration of the [M(H₂DOTA)]⁺ intermediate and [H₂L] is the concentration of the H₂DOTA²⁻ chelator. The $K_{M{\text{H}}_2\text{L}}$ stability constants of these intermediates are relatively high, so the k_obs values obtained even at lower metal-ion excess are close to the saturation value. The rate-determining step of the reaction is the deprotonation and rearrangement of the intermediate, followed by the entrance of the M^III ion into the macrocyclic cage:

$$\frac{\text{d}{[\text{ML}]}_{\text{t}}}{\text{d}t}=k_{\text{obs}}{[\text{L}]}_{\text{t}}=k_{\text{f}}[\text{M}({\text{H}}_2\text{L})]$$ (4)

where [M(H₂L)] is the concentration of the [M(H₂DOTA)]⁺ intermediate and k_f is the rate constant characterizing the deprotonation and rearrangement of the intermediate to the [M(DOTA)]⁻ complex. Taking into account the protonation constants of DOTA (Table S2), the stability constant of [M(H₂DOTA)]⁺ intermediate (eq 3), and eq 4, the pseudo-first-order rate constant can be expressed by eq 5.

$$k_{\text{obs}}=\frac{k_{\text{f}}{K}_{\text{M}({\text{H}}_2\text{L})}{K}_1^{\text{H}}{K}_2^{\text{H}}[{\text{M}}^{\text{III}}]{[{\text{H}}^{+}]}^2}{{\alpha }_{\text{H}}+K_{\text{M}({\text{H}}_2\text{L})}{K}_1^{\text{H}}{K}_2^{\text{H}}[{\text{M}}^{\text{III}}]{[{\text{H}}^{+}]}^2}$$ (5)

where ${\alpha }_{\text{H}}=1+K_1^{\text{H}}[{\text{H}}^{+}]+K_1^{\text{H}}{K}_2^{\text{H}}{[{\text{H}}^{+}]}^2+\dots +K_1^{\text{H}}{K}_2^{\text{H}}{K}_3^{\text{H}}{K}_4^{\text{H}}{K}_5^{\text{H}}{K}_6^{\text{H}}{[{\text{H}}^{+}]}^6$ The stability constant of the [M(H₂DOTA)]⁺ intermediates and the k_f rate constants have been calculated from the fitting of the pseudo-first-order rate constants obtained at various pH and [M^III] values to eq 5. The obtained stability constants are shown in Table S3. The k_f rate constants for the formation of [Ce(DOTA)]⁻ and [Eu-(DOTA)]⁻ complexes are presented in Figures S12 and S13 as a function of [OH⁻].

The formation rates of [Ga(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu(DOTA)]⁻ have also been studied under pseudo-first-order conditions that were ensured by the presence of a large excess of DOTA ([Ga^III] = [Y^III] = [Lu^III] = 2.0 × 10⁻⁴ M; [DOTA]_t = (1.0−6.0) × 10⁻³ M). In these cases, the rate of formation reactions can be expressed by eq 6.

$$\frac{\text{d}[\text{ML}]}{\text{d}t}=k_{\text{obs}}{[{\text{M}}^{\text{III}}]}_{\text{t}}$$ (6)

where [ML] is the concentration of the [Ga(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu(DOTA)]⁻ complexes formed, [M^III]_t is the total concentration of species containing Ga^III, Y^III, and Lu^III ions, and k_obs is a pseudo-first-order rate constant. The formation reaction of [Ga(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu(DOTA)]⁻ was investigated by varying the concentrations of DOTA at different pH values. As expected, the k_obs versus [DOTA]_t curves (Figures S14–S22) are saturation curves indicating the formation of the [M(H₂DOTA)]⁺ intermediates.¹⁵ The rate-determining step of the reactions is the deprotonation and rearrangement of the [M(H₂DOTA)]⁺ intermediates followed by the entrance of the metal ion into the cavity of the DOTA chelator:

$$\frac{\text{d}[\text{ML}]}{\text{d}t}=k_{\text{obs}}{[{\text{M}}^{\text{III}}]}_{\text{t}}=k_{\text{f}}{[*\text{M}({\text{H}}_2\text{L})]}_{\text{t}}$$ (7)

where [M(H₂L)]_t is the concentration of the [M(H2DOTA)]⁺ intermediate and k_f is the rate constant characterizing the deprotonation and rearrangement of the intermediate to the final [M(DOTA)]⁻ complex. The concentration of the noncomplexed chelator can be expressed by eq 8 using the protonation constants of the DOTA chelator (Table S2).

$${[\text{DOTA]}}_{\text{free}}=[{\text{H}}_2\text{DOTA}](1+K_3^{\text{H}}[{\text{H}}^{+}]+K_3^{\text{H}}{K}_4^{\text{H}}{[{\text{H}}^{+}]}^2+\dots +K_3^{\text{H}}{K}_4^{\text{H}}{K}_5^{\text{H}}{[{\text{H}}^{+}]}^3)=(1+a_{2\text{H}})[{\text{H}}_2\text{DOTA}]$$ (8)

where ${\alpha }_{\text{2H}}=K_3[{\text{H}}^{+}]+K_3{K}_4{[{\text{H}}^{+}]}^2+K_3^{\text{H}}{K}_4^{\text{H}}{K}_5^{\text{H}}{[{\text{H}}^{+}]}^3+K_3^{\text{H}}{K}_4^{\text{H}}{K}_5^{\text{H}}{K}_6^{\text{H}}{[{\text{H}}^{+}]}^4$. Taking into account the hydrolysis of the M^III ion, the total metal-ion concentration can be expressed by eq 9:

$${[{\text{M}}^{\text{III}}]}_{\text{t}}=[\text{M}({\text{H}}_2\text{L})]+[\text{M}(\text{OH})]+[\text{M}{(\text{OH})}_2]+[\text{M}{(\text{OH})}_3]+[{\text{M}}^{\text{III}}]$$ (9)

Under the experimental conditions (pH 2.5−7.0), hydrolysis of the Ga³⁺ ion may occur, resulting in the formation of [M(OH)]²⁺, [M(OH)₂]⁺, and M(OH)₃ species; i.e., OH⁻ ions may compete with the DOTA for the Ga^III ions. However, in the cases of Y^III and Lu^III, hydrolysis can be neglected at pH < 7.²⁹ Taking into account the protonation constants of DOTA (Table S2 and eq 8), the stability constant of the [M-(H₂DOTA)]⁺ intermediate (eq 3), the total concentration of the M^III ion (eq 9), and eq 7, the pseudo-first-order rate constant can be expressed by eq 10.

$$k_{\text{obs}}=\frac{\frac{k_{\text{f}}{K}_{\text{M}({\text{H}}_2\text{L})}{[\text{L}]}_{\text{t}}}{1+{\alpha }_{2\text{H}}}}{1+\frac{K_{\text{M}({\text{H}}_2\text{L})}{[\text{L}]}_{\text{t}}}{1+{\alpha }_{2\text{H}}}+{\alpha }_{\text{OH}}}$$ (10)

where [L]_t is is the total concentration of the DOTA chelator and ${\alpha }_{\text{OH}}={\beta }_1^{\text{OH}}/[{\text{H}}^{+}]+{\beta }_1^{\text{OH}}/{[{\text{H}}^{+}]}^2+{\beta }_3^{\text{OH}}/{[{\text{H}}^{+}]}^3(\text{log}{\beta }_1^{\text{OH}}=-2.97,\text{log}{\beta }_2^{\text{OH}}=-5.92\text{\hspace{0.17em}and\hspace{0.17em}log}{\beta }_3^{\text{OH}}=-8.2{\text{for the Ga}}^{\text{III}}\text{ion)}$.²⁹ The pseudo-first-order rate constants determined at various pH and [DOTA] values (Figures S14–S22) were fitted to eq 10, and the stability constant of the [M(H₂DOTA)]⁺ intermediates ($K_{M{\text{H}}_2\text{L}}$) and the k_f rate constants were calculated (for the Y^III and Lu^III complexes, α_OH = 0). The stability constants of the [Ga(H₂DOTA)]⁺, [Y(H2DOTA)]⁺, and [Lu(H₂DOTA)]⁺ intermediates ($K_{M{\text{H}}_2\text{L}}$) are presented in Table S3. The calculated k_f rate constants obtained for formation of the [Ga(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu-(DOTA)]⁻ complexes are shown in Figures S23–S25 as a function of [OH⁻]. The kinetic data that we obtained indicate that the k_f values increase with an increase of [OH⁻] and [EtOH]. According to the reaction mechanism proposed for the formation of [M(DOTA)]⁻ complexes, the di- and monoprotonated intermediates exist in equilibrium. The dependence of the k_f values on [OH⁻] can be interpreted by formation of the kinetically active monoprotonated [M-(HDOTA)] intermediates through dissociation of the diprotonated [M(H₂DOTA)]⁺ intermediates in an equilibrium characterized with the K_M(HL)H protonation constant (eq 11). The rate-controlling step of complex formation involves the H₂O- or OH⁻ -assisted deprotonation and rearrangement of the monoprotonated [M(HDOTA)] intermediates to the final [M(DOTA)] complex (Scheme 1).¹⁵

$$K_{\text{M}(\text{HL})}{}^{\text{H}}=\frac{[\text{M}({\text{H}}_2\text{L})]}{\text{[M}(\text{HL})][{\text{H}}^{+}]}$$ (11)

Scheme 1.

Formation Mechanism of M(DOTA) Complexes

Considering the significantly lower polarity (lower relative permittivity) and higher pK_a of the EtOH molecule relative to H₂O (the concentration of the EtO⁻ anion is extremely low in the range pH 2.5−7.0), it can be assumed that deprotonation of the monoprotonated [M(HDOTA)] intermediates takes place via H₂O (as a weak Bronsted base) and OH⁻-assisted pathways even in the presence of large amounts of EtOH. According to the proposed reaction mechanism, the formation rate of the [M(DOTA)]⁻ complexes can be given by eq 12.

$$\frac{\text{d}[\text{ML}]}{\text{dt}}=k_{\text{f}}{[\text{M}({\text{H}}_2\text{L})]}_{\text{t}}{=}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}\text{[M}(\text{HL})][{\text{H}}_2\text{O}]{+}^{\text{M}(\text{HL})}{k}_{\text{OH}}\text{[M}(\text{HL})][{\text{OH}}^{-}]$$ (12)

By considering the total concentration of the intermediates ([M(H₂L)]_t = [M(HL)] + [M(H₂L)]), the definition of the K_M(HL) protonation constant (Scheme 1), the concentration of H₂O molecules, and the ionic product of H₂O (K_w) in EtOH solutions (Table S1), the k_f rate constant can be expressed by eq 13.

$$k_{\text{f}}=\frac{{}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}[{\text{H}}_2\text{O}]{+}^{\text{M}(\text{HL})}{k}_{\text{OH}}{K}_{\text{w}}/[{\text{H}}^{+}]}{1+K_{\text{M}(\text{HL})}{}^{\text{H}}[{\text{H}}^{+}]}$$ (13)

This equation was used for fitting of the k_f values to determine the ${}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}$ and ${}^{\text{M}(\text{HL})}{k}_{\text{OH}}$ rate constants and the $K_{\text{M}(\text{HL})}{}^{\text{H}}$ protonation constants that characterize the formation of [M(DOTA)]⁻ complexes in H₂O and 10, 40, and 70 vol % EtOH solutions. The ${}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}$ and ${}^{\text{M}(\text{HL})}{k}_{\text{OH}}$ rate constants and the $K_{\text{M}(\text{HL})}{}^{\text{H}}$ protonation constants obtained from the fitting are shown in Table 3.

Table 3.

Rate (k) and Equilibrium Constants (K) Characterize the Formation of [Ga(DOTA)]⁻, [Ce(DOTA)]⁻, [Eu(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu(DOTA)]⁻ Complexes in H₂O and 10, 40, and 70 vol % EtOH Solutions (0.15 M NaCl, 25°C)

		H2O	10 vol % EtOH	40 vol % EtOH	70 vol % EtOH
	[H2O] (mol L−1)	55.6	50.1	33.9	16.9
	Kw	1.4 × 10−14	1.1 × 10−14	5.4 × 10−15	2.3 × 10−15
[Ce(DOTA)]	M(HL)kH2O (M−1 s−1)	0.34a	0.42 ± 0.03	0.43 ± 0.04	0.4 ± 0.1
	M(HL)kOH (M−1 s−1)	(1.9 × 107)15	(1.1 ± 0.5) × 107	(1.5 ± 0.3) × 107	(9 ± 3) × 106
	log KM(HL)H	8.64a	8.9(1)	8.5(2)	7.9(3)
[Eu(DOTA)]	M(HL)kH2O (M−1 s−1)		1.46 ± 0.09	1.49 ± 0.05	1.4 ± 0.1
	M(HL)kOH (M−1 s−1)
	log KM(HL)H		8.7(1)	8.4(2)	7.8(2)
[Y(DOTA)]	M(HL)kH2O (M−1 s−1)	1.49 ± 0.08	1.47 ± 0.07	1.37 ± 0.09
	M(HL)kOH (M−1 s−1)	(4.8 ± 0.8) × 109	(4.2 ± 0.7) × 109	(4.4 ± 0.09) × 109
	log KM(HL)H	8.60(3)	8.47(2)	8.13(5)
[Lu(DOTA)]	M(HL)kH2O (M−1 s−1)	4.70 ± 0.08 (Yb3+: 4.4)a	4.64 ± 0.09	4.68 ± 0.05
	M(HL)kOH (M−1 s−1)	(1.1 ± 0.3) × 109	(1.0 ± 0.4) × 109	(1.5 ± 0.6) × 109
	log KM(HL)H	8.40(2) (Yb3+: 8.4)a	8.36 (2)	8.15(1)
[Ga(DOTA)]	M(HL)kH2O (M−1 s−1)	0.82 ± 0.07	1.00 ± 0.09	0.90 ± 0.05
	M(HL)kOH (M−1 s−1)	(1.3 ± 0.1) × 1011	(9 ± 1)×1010	(1.0 ± 0.4) × 1011
	log KM(HL)H	7.16(4)	7.09(3)	6.75(3)

^aReference 15.

The ${}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}$ rate constants that characterize the H₂O- assisted deprotonation and rearrangement of the [M-(HDOTA)] intermediates to the final [M(DOTA)]⁻ complexes increase from Ce^III to Lu^III, whereas the ${}^{\text{M}(\text{HL})}{k}_{\text{OH}}$ values are independent of the size of the Ln^III ions. The ${}^{\text{M}(\text{HL})}{k}_{{\text{H}}_2\text{O}}$ value obtained for [Ga(DOTA)]⁻ is smaller than those acquired for [Eu(DOTA)]⁻, [Y(DOTA)]⁻, and [Lu-(DOTA)]⁻, which can be explained by the different size and coordination number of Ga^III and Ln^III ions (Ga^III, 0.62 Å; Ln^III, 0.97−1.16 Å). The protonation constants $(K_{\text{M}(\text{HL})}{}^{\text{H}})$ of the monoprotonated intermediates decrease with a decrease of the size of the M^III ions and with an increase of [EtOH]. Reduction of the $K_{\text{M}(\text{HL})}{}^{\text{H}}$ values with decreasing M^III size is due to the larger electrostatic repulsion between the protons on the ring N-donor atoms and the smaller M^III ions in the [M-(H₂DOTA)]⁺ intermediates. The decrease of the $K_{\text{M}(\text{HL})}{}^{\text{H}}$ values with increasing [EtOH] can be interpreted by a decrease of the basicity of the ring N-donor atoms in the [M-(H₂DOTA)]⁺ intermediates. The basicity of the macrocyclic N-donor atoms of the free DOTA chelator analogously decreases in EtOH solutions (Table S2). On the basis of the kinetic data, the faster formation of [M(DOTA)]⁻ complexes in EtOH solutions can be rationalized by a decrease of the log $K_{\text{M}(\text{HL})}{}^{\text{H}}$ values. Deprotonation of the diprotonated [M-(H₂DOTA)]⁺ intermediates seems to play a crucial role in the complex formation because the lower log $K_{\text{M}(\text{HL})}{}^{\text{H}}$ values lead to higher concentrations of the kinetically active monoprotonated [M(HDOTA)] intermediates. To highlight the effect of EtOH on the formation rate of [M(DOTA)]⁻ complexes, the k_obs rate constants characterizing the formation of [Ga(DOTA)]⁻ and [Lu(DOTA)]⁻ at pH 4.0 and 25 °C in the presence of 3.4 μM DOTA chelator in H₂O and in 10 and 40 vol % EtOH solutions were calculated using eqs 10 and 13. The k_obs rate constants that characterize the formation of [Ga(DOTA)]⁻ and [Lu(DOTA)]⁻ at pH 4.0 and 25 °C in the presence of 3.4 μM DOTA chelator were found to be 4.83 × 10⁻⁴, 8.00 × 10⁻⁴, and 1.49 × 10⁻³ s⁻¹ for [Ga(DOTA)]⁻ and 3.32 × 10⁻⁵, 3.86 × 10⁻⁵, and 5.00 × 10⁻⁵ s⁻¹ for [Lu(DOTA)]⁻ in H₂O and in 10 and 40 vol % EtOH solutions, respectively. The extent of complex formation is shown in Figure 8. The calculated k_obs rate constants and the data in Figure 8 show that, in the presence of 3.4 μM DOTA at pH 4.0, the increase of [EtOH] from 0 to 40 vol % results in approximately 3 and 1.5 times higher formation rate for [Ga(DOTA)]⁻ and [Lu(DOTA)]⁻, respectively.

Figure 8.

Formation of [Ga(DOTA)]⁻ (A) and [Lu(DOTA)]⁻ (B) complexes in H₂O and in 10 and 40 vol % EtOH solutions ([DOTA] = 3.4 μM, pH 4.0, 25 °C, 0.15 M NaCl).

Hydration/Solvation of Ga^III, Sc^III, and Y^III Ions in H₂O/EtOH Mixtures.

Because the complex formation occurs by the interaction of hydrated/solvated metal ions with the chelator, some knowledge of the hydration/solvation of M^III ions in H₂O and in mixed solutions would be very helpful to better understand the mechanism of complex formation. Because the number and chemical nature of donor atoms coordinating to the M^III ion may influence the NMR chemical shift and relaxation time of the M^III nuclei, we studied the hydration/solvation of Ga^III, Sc^III, and Y^III ions by multinuclear NMR spectroscopy in H₂O/EtOH mixtures. The NMR studies were focused on the measurement of the longitudinal relaxation times (T₁) of Ga^III, Sc^III, and Y^III ions in H₂O/EtOH mixtures because we anticipated that the coordination symmetry as well as the nature of the solvating species would significantly affect the T₁ relaxation times. ⁷¹Ga, ⁴⁵Sc, and ⁸⁹Y NMR spectra of Ga(NO₃)₃, ScCl₃, and YCl₃ solutions obtained in H₂O and in 10, 40, and 60 vol % EtOH solutions are shown in Figures S26–S28. The T₁ values measured in H₂O and in 10, 40, and 60 vol % EtOH solutions are summarized in Table 4.

Table 4.

T₁ Values (ms) of Ga^III, Sc^III, and Y^III Ions in H₂O and in 10, 40, and 60 vol % EtOH Solutions

	H2O	10 vol % EtOH	40 vol % EtOH	60 vol % EtOH
GaIII (1.4 M HNO3)	5.5	4.5	2.2	1.5
ScIII (1.0 M HNO3)	2.1	1.4	0.6	0.5
YIII (0.5 M HNO3)	2.2 × 106	7.6 × 105	5.4 × 105	3.7 × 105

Generally, the exchange between the [M(H O)_x]³⁺ and [M(H₂O)_x-1(X)]³⁺ species formed by solvation of the aqua M^III complexes is fast on the NMR time scale, so the chemical shifts of the observed signals represent a weighted average of the shifts of the different species involved in the specific solvation. Taking into account the lower affinity of the EtOH molecule to M^III ions, we assumed that some of the inner-sphere H₂O molecules were replaced by EtOH ([M-(H₂O)_x-n(EtOH)_n]³⁺). The presence of EtOH slightly influences the chemical shifts of ⁷¹Ga, ⁴⁵Sc, and ⁸⁹Y NMR signals of the solvated ions, which can be interpreted by the formation of [M(H₂O)_x-n(EtOH)_n]³⁺ species (Ga^III, x = 6; Sc^III, x = 8; Y^III, x = 8), which are in fast exchange with the [M(H₂O)_x]³⁺ aqua complex. The obtained NMR data are also consistent with the assumption that the presence of EtOH molecules in the inner sphere can influence the T₁ values of Ga^III, Sc^III, and Y^III ions. Because the relaxation of ⁷¹Ga and ⁴⁵Sc nuclei takes place by a quadrupolar mechanism, the decrease of T₁ values can be explained by the lower symmetry of [M(H₂O)_x−n(EtOH)_n]³⁺ (Ga^III, x = 6; Sc^III, x = 8) species compared to the corresponding aqua ions. The dominant T₁ (spin−lattice) relaxation mechanism of a ⁸⁹Y^III aqua ion is a combination of spin rotation and the more efficient dipolar interaction with the coordinated H₂O and solvent protons. The observed marked decrease of T₁ in the presence of EtOH is likely due to the replacement of some of the inner-sphere H₂O molecules by EtOH, which decreases the contribution of spin rotation relaxation but speeds up dipolar relaxation. It is also likely that breaking of the symmetrical coordination environment of the Y^III ion amplifies the contribution of other relaxation mechanisms, in particular, chemical shift anisotropy.³⁰

CONCLUSIONS

It has been previously reported that nonaqueous solvents can influence the solvation and chelation of metal ions.¹⁰ In the present study, we investigated this phenomenon in the context of radiopharmaceutical chemistry. Here we provide compelling experimental evidence that performing the chelation in a mixture of H₂O and a polar organic solvent (EtOH, iPrOH, and MeCN) can significantly speed up the complex formation and improve the radiolabeling yield. This is an important consideration, especially when the chelation is performed with an extremely low concentration of the radiometal, as is commonly the case in radiopharmaceutical synthesis. The following factors were found to have a significant influence on the chelation.

Impact of the Organic Solvent.

For a given reaction system and temperature, the addition of 40, 30, 20, or even 10 vol % of a nonaqueous solvent like EtOH, MeCN, or iPrOH has a direct and reproducible influence on the complex formation and radiolabeling yields. Depending on the solvent and reaction time, the addition of a nonaqueous solvent facilitates the formation reaction. The effect can be quite significant, in particular for short reaction times (≤10 min). The effect of the organic solvent depends on both the chelator and radiometal. For example, we observed the order EtOH < MeCN < iPrOH (t < 10 min) for ⁶⁸Ga-DOTA complex formation, while the order was reversed (EtOH > MeCN > iPrOH; t < 10 min) for the formation of [⁴⁴Sc]Sc-DOTA. For a given reaction system and temperature, the impact of different nonaqueous solvents grows with its increasing concentration. The greatest impact can be observed for reaction times of <10 min, which are especially of interest for radiolabeling with short-lived radiometals such as gallium-68, in which case the shorter reaction times can afford significant extra radioactivity in the final radiopharmaceutical preparation.

Impact on the Chelator Amount.

For a given reaction system, temperature, and reaction time, the addition of organic solvents increases the radiolabeling yields (Figure 2). Consequently, it is possible to achieve high radiochemical yields (>95%) even when the amount of chelator is reduced by a factor of 10. This translates into a 10-fold increase in the apparent molar radioactivity of the product, which is especially relevant in the context of receptor targeting.

Impact on the Duration of Synthesis.

Applying optimum reaction conditions, one might guarantee constant labeling yields of >98% in routine synthesis. This is well above the current recommendation published by the European Pharmacopoeia.³¹ Such high labeling yields may eliminate the need for radiochemical purification procedures. In practice, the duration of the synthesis from elution of the generator until formulation of the final product will be reduced because of the omission of the purification step. Effectively, this also contributes to an improvement in the final product radioactivity. This is particularly relevant for short-living radio-nuclides such as gallium-68: reducing the reaction time by 10 min results in nearly a 11% gain of radioactivity, i.e., increases the final product radioactivity by 11%.

Impact on the Formation Mechanism of [M(DOTA)]⁻ Complexes.

According to the detailed physicochemical studies presented here, the solvation of the aqua [M(H₂O)_x]³⁺ complexes in the presence of EtOH may result in the formation of [M(H₂O)_x−n(EtOH)_n]³⁺ species (Ga^III, x = 6; Sc^III, x = 8; Y^III, x = 8). However, formation of the [M(DOTA)]⁻ complex occurs through the formation of diprotonated [M(H₂DOTA)]⁺ intermediates in both H₂O and H₂O/EtOH mixtures. The rate-controlling step is the H₂O- and OH⁻ -assisted deprotonation and rearrangement of the monoprotonated [M(HDOTA)] intermediates formed in fast equilibrium with the diprotonated [M(H₂DOTA)] intermediates. Thus, hydration/solvation of the M^III ion does not play an important role in the formation rate of [M(DOTA)]⁻ complexes. EtOH or any other organic solvent indirectly accelerates the formation rate of [M-(DOTA)]⁻ complexes by decreasing the protonation constant $(K_{\text{M}(\text{HL})}{}^{\text{H}})$ of the monoprotonated [M(HDOTA)] intermediates. This, in turn, results in the formation of the kinetically active monoprotonated [M(HDOTA)] intermediate in higher concentration, which speeds up formation of the final complex.