Synthesis of a Bifunctional Cross‐Bridged Chelating Agent, Peptide Conjugation, and Comparison of ⁶⁸Ga Labeling and Complex Stability Characteristics with Established Chelators

Abstract

[⁶⁸Ga]Ga³⁺ can be introduced into receptor‐specific peptidic carriers via different chelators to obtain radiotracers for Positron Emission Tomography imaging and the chosen chelating agent considerably influences the in vivo pharmacokinetics of the corresponding radiopeptides. A chelator that should be a valuable alternative to established chelating agents for ⁶⁸Ga‐radiolabeling of peptides would be a backbone‐functionalized variant of the chelator CB‐DO2A. Here, the bifunctional cross‐bridged chelating agent CB‐DO2A‐GA was developed and compared to the established chelators DOTA, NODA‐GA and DOTA‐GA. For this purpose, CB‐DO2A‐GA(tBu)₂ was introduced into the peptide Tyr³‐octreotate (TATE) and in direct comparison to the corresponding DOTA‐, NODA‐GA‐, and DOTA‐GA‐modified TATE analogs, CB‐DO2A‐GA‐TATE required harsher reaction conditions for ⁶⁸Ga‐incorporation. Regarding the hydrophilicity profile of the resulting radiopeptides, a decrease in hydrophilicity from [⁶⁸Ga]Ga‐DOTA‐GA‐TATE (log _D(7.4) of −4.11±0.11) to [⁶⁸Ga]Ga‐CB‐DO2A‐GA‐TATE (−3.02±0.08) was observed. Assessing the stability against metabolic degradation and complex challenge, [⁶⁸Ga]Ga‐CB‐DO2A‐GA demonstrated a very high kinetic inertness, exceeding that of [⁶⁸Ga]Ga‐DOTA‐GA. Therefore, CB‐DO2A‐GA is a valuable alternative to established chelating agents for ⁶⁸Ga‐radiolabeling of peptides, especially when the formation of a very stable, positively charged ⁶⁸Ga‐complex is pursued.

The new backbone‐modified bifunctional cross‐bridged chelating agent CB‐DO2A‐GA was developed, introduced into the peptide Tyr³‐octreotate and compared to the established chelators DOTA, NODA‐GA and DOTA‐GA in terms of ⁶⁸Ga‐radiolabeling efficiency and kinetic inertness of the formed complexes.

Introduction

⁶⁸Ga‐radiolabeled peptides have emerged as important and powerful tools for the specific and sensitive visualization of malignancies of various origin by Positron Emission Tomography (PET) in routine clinical practice.[ ¹ , ² , ³ ]

For labeling purposes, the [⁶⁸Ga]Ga³⁺ ion is introduced into the peptide carriers by means of complexation by a peptide‐conjugated chelating agent. Here, several different chelators can be employed, but the most commonly used chelating agent for ⁶⁸Ga‐introduction into peptides is DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). NODA‐GA ((1,4,7‐triazacyclononane‐1‐glutaric acid‐4,7‐diacetic acid) is also often applied and both DOTA and NODA‐GA are used in human radiotracer applications, forming very stable complexes with [⁶⁸Ga]Ga³⁺.[ ¹ , ⁴ ] Other options are DOTA‐GA (1,4,7,10‐tetraazacyclododececane‐1‐glutaric acid‐4,7,10‐triacetic acid) and NOTA (1,4,7‐triazacyclononane‐1,4,7‐triacetic acid), which have also shown their suitability for stable ⁶⁸Ga‐introduction and tumor PET imaging applications.[ ² , ⁵ , ⁶ ] All complexes formed by the different chelators with [⁶⁸Ga]Ga³⁺ differ in their charge and the number of carboxylates being not involved in complex formation (Figure 1). These two parameters can considerably influence the in vivo pharmacokinetics of the corresponding radiopeptides, as has been shown recently in several very interesting comparative studies on different chelators for ⁶⁸Ga‐peptide‐labeling, where the influence of the specific chelating agent used for [⁶⁸Ga]Ga³⁺ introduction on the pharmacokinetic profile of the respective carrier peptide was determined.[ ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² ]

Figure 1.

Structures of different ⁶⁸Ga‐complexes of high relevance for preclinical and clinical application: [⁶⁸Ga]Ga‐DOTA, [⁶⁸Ga]Ga‐DOTA‐GA, [⁶⁸Ga]Ga‐NOTA, and [⁶⁸Ga]Ga‐NODA‐GA. The complexes differ in overall charge as well as in the number of carboxylic acids being not involved in complex formation (depicted in green).

From these studies it is evident that no prediction is possible as to which chelating agent has the most favorable influence on the pharmacokinetic profile of a specific carrier peptide. On the contrary, it was shown that it depends on the individual peptide used for tumor targeting which chelating agent or corresponding ⁶⁸Ga‐complex results in the most favorable in vivo imaging properties of the radioligand derived from it.

However, some few general conclusions can be drawn when directly comparing different chelators for peptide modification and ⁶⁸Ga‐labeling: i) In most cases, the choice of the chelating agent does not or only negligibly influence the in vitro binding parameters of the respectively modified peptides,[ ⁶ , ⁷ , ⁸ , ¹² ] ii) The negatively charged [⁶⁸Ga]Ga‐DOTA‐GA complex usually decreases non‐specific accumulation in liver and spleen compared to neutral [⁶⁸Ga]Ga‐DOTA and [⁶⁸Ga]Ga‐NODA‐GA complexes,[ ⁵ , ¹⁰ ] whereas its effect on background clearance of the radiopeptide is not consistent, iii) In general, neutral [⁶⁸Ga]Ga‐DOTA[ ⁷ , ⁸ ] and [⁶⁸Ga]Ga‐NODA‐GA[ ⁵ , ⁹ ] complexes show the most favorable influence on in vivo pharmacokinetics with regard to tumor uptake and tumor‐to‐background‐ratios compared to the respective negatively charged [⁶⁸Ga]Ga‐DOTA‐GA analog although it cannot be predicted whether [⁶⁸Ga]Ga‐DOTA or [⁶⁸Ga]Ga‐NODA‐GA is best suited as the mentioned biodistribution properties are dependent on the specific carrier peptide. But although neutral ⁶⁸Ga‐complexes often gave the best results in terms of the in vivo biodistribution profile of the respectively modified peptides, positively charged [⁶⁸Ga]Ga‐NOTA peptides demonstrated highest tumor‐to‐background ratios of all tested agents in some other cases.[ ⁶ , ¹² ]

Thus, it can be assumed that for each peptide‐based radiotracer to be developed, the most suitable chelating agent, resulting in the highest tumor uptake and tumor‐to‐background ratios has to be identified anew. For this purpose, it is a particularly promising attempt to directly compare chelating agents for ⁶⁸Ga‐introduction which form neutral or positively charged complexes with [⁶⁸Ga]Ga³⁺.

A chelating agent that could be a valuable complement to the already mentioned chelators in these evaluations would be a backbone‐functionalized variant of the chelator CB‐DO2A (1,4,7,10‐tetraazabicyclo[5.5.2]tetradecane‐4,10‐diacetic acid) (Figure 2A).

Figure 2.

Depiction of the structures of the chelating agent CB‐DO2A (A), its Ga(III) complex (B), ^[13] the backbone‐modified derivative CB‐DO2A‐GA being suitable for bioconjugation developed in this work (C) and a corresponding [⁶⁸Ga]Ga‐CB‐DO2A‐GA‐peptide conjugate (D).

CB‐DO2A forms positively charged complexes with [⁶⁸Ga]Ga³⁺ which can have a favorable influence on in vivo pharmacokinetics of respectively modified peptides as outlined above. Further, CB‐DO2A should form ⁶⁸Ga‐complexes of high kinetic inertness as was already shown for the CB‐DO2A complex with [⁶⁴Cu]Cu²⁺. ^[13] Although [⁶⁴Cu]Cu²⁺ of course differs considerably in physical properties (such as ion radius and hardness) and as a result in the type of preferred coordinating atoms, it also requires a complete envelopment of the central metal ion to form stable complexes being inert against oxidative processes under in vivo conditions. In this regard, the rigid geometry of the CB‐DO2A chelator, the pre‐formed structural arrangement of the coordinating atoms and the demonstrated complete envelopment of the central ion were shown to be highly favorable ^[14] and the same factors imply that the corresponding Ga(III) complexes (Figure 2B) should also exhibit a high degree of kinetic inertness.

However, no bifunctional derivative of CB‐DO2A has been described so far which would enable its conjugation to a peptidic carrier molecule without compromising the coordination sphere of the ligand upon biomolecule conjugation.[ ¹⁵ , ¹⁶ ]

Therefore, the aim of the present study was to develop a backbone‐modified variant of CB‐DO2A, namely CB‐DO2A‐GA (1,4,7,10‐tetraazabicyclo[5.5.2]tetradecane‐4‐glutaric acid‐10‐acetic acid) (Figure 2C), which allows the CB‐DO2A backbone to be conjugated to a biomolecule carrier without altering its coordination sphere being necessary for stable complexation of Ga(III) (vide supra). Furthermore, the new chelator was introduced into a peptidic ligand and subsequently radiolabeled with [⁶⁸Ga]Ga³⁺. In addition, the in vitro stability of the resulting ⁶⁸Ga‐CB‐DO2A‐GA peptide (Figure 2D) in human serum as well as the inertness of the complex against challenge were investigated. The established chelating agents DOTA, NODA‐GA and DOTA‐GA were evaluated under identical conditions in order to identify potential differences between the chelators in terms of labeling characteristics and kinetic inertness.

Results and Discussion

Synthesis of the new bifunctional, backbone‐modified CB‐DO2A derivative CB‐DO2A‐GA(tBu)₂ (6)

Commonly, chelators are introduced into peptides during solid‐phase peptide synthesis on solid support. For this purpose, tBu‐protected chelator derivatives are usually applied. Thus, a bis‐tBu‐protected CB‐DO2A‐GA analog, CB‐DO2A‐GA(tBu)₂ (6), was synthesized (Scheme 1).

Scheme 1.

Schematic depiction of the reaction pathway towards the bifunctional, backbone‐modified chelator CB‐DO2A‐GA(tBu)₂ (6), applicable for peptide modification during solid‐phase peptide synthesis (SPPS). Conditions: (a) Sodium bromide (3.50 eq.), sodium nitrite (1.8 eq.), hydrobromic acid (1 M, 30.3 mL), 0 °C, 2 h, H₂SO₄ (1.70 eq.), 45 % yield; (b) TBTA (2.20 eq.), dimethylacetamide (2.7 mL), boron trifluoride etherate (0.16 eq.), chloroform/cyclohexane (1/1, v/v), RT, 3d, 81 % yield; (c) CB‐cyclen (1 eq.), K₂CO₃ (1 eq.), MeCN, 0 °C for 5d, RT for 3d, 40 % yield; (d) K₂CO₃ (1.25 eq.), tert‐butyl‐bromoacetate (1.37 eq.), MeCN, RT, 48 h, 59 % yield; (e) H₂, 10 % Pd/C, THF/H₂O (1 : 1, v/v), RT, 18 h, 75 % yield.

First, 5‐benzyl‐1‐tBu‐2‐bromopentanedioate (3) was prepared from L‐glutamic acid‐5‐benzylester (1) via 5‐(benzyloxy)‐2‐bromo‐5‐oxopentanoic acid (2) according to a published protocol ^[17] with only minor modifications. In the first step, the amino functionality of 1 was substituted by bromine, followed by tBu‐protection of the carboxylic acid of 2 using tert‐butyl‐2,2,2‐trichloroacetimidate (TBTA), giving 3 in a good isolated yield of 37 % over both steps.

In the following, CB‐cyclen was first reacted with 3 and afterwards with tert‐butyl‐bromoacetate to give the fully protected intermediate 5 which was de‐benzylated using hydrogen under palladium catalysis to obtain the bis‐tBu‐protected bifunctional chelating agent CB‐DO2A‐GA(tBu)₂ (6). During the synthesis of 4, the main side product was the formation of the bis‐reacted CB‐cyclen adduct although only 1 equivalent of 3 was used. This side reaction limited the isolated yield of 4 to a moderate 40 %. The following reaction of 4 with tBu‐bromoacetate gave – besides the intended product – two further products of unknown identity which could only be partly separated by gravimetric chromatography and necessitated the use of semipreparative HPLC (high performance liquid chromatography) for purification of the product. Nevertheless, 5 could be obtained in a good isolated yield of 59 %. The reductive deprotection of the benzyl‐protected carboxylic acid of 5 to 6 was finally accomplished using standard conditions (H₂/Pd/C), giving 6 in 75 % yield after HPLC purification.

Synthesis of chelator‐TATE‐conjugates 8–11

Tyr³‐octreotate (TATE), a truncated and stabilized analog of the endogenous peptide hormone somatostatin, which binds to somatostatin receptors and is the basis for different important radiotracers for diagnosis and therapy of neuroendocrine tumors of different origin, ^[1] served as carrier peptide for the chelating agents to be studied in direct comparison. The chelator‐TATE‐conjugates were synthesized by first assembling the TATE sequence on solid support using standard solid‐phase peptide synthesis (SPPS) protocols (Scheme 2A) before the conjugation of the protected chelator derivatives DOTA(tBu)₃, DOTA‐GA(tBu)₄, R‐NODA‐GA(tBu)₃, and CB‐DO2A‐GA(tBu)₂ (6) was carried out to the N‐terminally deprotected but otherwise fully protected peptide intermediate 7 still being attached to the solid phase (Scheme 2B). After cleavage of the chelator‐peptide‐conjugates from the resin and simultaneous deprotection under strongly acidic conditions and purification, the chelator‐TATE‐conjugates 8–11 were obtained in yields of 18 %–63 %. The isolated yields varied in terms of coupling efficiency of the respective protected chelator derivative, the amount of side products formed during cleavage/deprotection and how efficiently these could be separated from the products by HPLC purification.

Scheme 2.

Schematic depiction of the synthesis pathway towards the chelator‐TATE‐conjugates 8–11. Reaction conditions: (a) Fmoc‐deprotection using piperidine/DMF (1/1, v/v), 2+5 min; (b) activation of amino acid: 4.0 eq. amino acid, 3.9 eq. HBTU, 4.0 eq. DIPEA, DMF, 2 min, conjugation of activated amino acid: 10 min, ultrasound‐assisted; (c) cyclization: 4 eq. thallium(III) trifluoroacetate, DMF, 45 min; (d) activation of chelator acids DOTA(tBu)₃, DOTA‐GA(tBu)₄), and R‐NODA‐GA(tBu)₃: 2.5 eq. chelator acid, 2.45 eq. HBTU, 5.0 eq. DIPEA, DMF, 2 min, conjugation of activated chelator acid: 60 min, ultrasound‐assisted; activation of the chelator acid CB‐DO2A‐GA(tBu)₂ (6): 1.6 eq. chelator acid, 2.45 eq. PyBOP, 5.0 eq. DIPEA, DMF, 2 min, conjugation of activated chelator acid: 60 min, ultrasound‐assisted; (e) cleavage from resin and simultaneous deprotection: TFA/TIS/H₂O (95/2.5/2.5, v/v/v), 3 h. Isolated yields over all steps: 63 % for 8, 46 % for 9, 36 % for 10 and 18 % for 11. DMF: N,N‐dimethylformamide, HBTU: N,N,N′,N′‐tetramethyl‐O‐(1H‐benzotriazole‐1‐yl)‐uronium‐hexafluorophosphate, DIPEA: N,N‐diisopropylethylamine, PyBOP: benzotriazole‐1‐yl‐oxy‐tris‐pyrrolidinophosphonium hexafluorophosphate, TIS: triisopropylsilane.

⁶⁸Ga‐Radiolabeling of 8–11 to [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11

The radiolabeling of the chelator‐TATE‐conjugates 8–11 was performed using [⁶⁸Ga]Ga³⁺ obtained in form of ⁶⁸GaCl₃ by fractioned elution of a commercial ⁶⁸Ge/⁶⁸Ga‐generator system using HCl (0.1 M, 1.2 mL). After elution of the ⁶⁸GaCl₃, the pH of the solution was adjusted to pH 3.8–4.0 using sodium acetate solution (1.25 M, pH 4.4) before the respective labeling precursor was added. To determine the maximal molar activity achievable for the respective radiolabeled peptide, the amount of activity per labeling experiment was kept constant at 40 MBq while the amount of added precursor was successively decreased from 40 nmol to 0.4 nmol. The reactions were carried out at 95 °C for the cyclen‐macrocycle‐comprising peptides 8, 9 and 11 while the NODA‐GA‐TATE derivative 10 was reacted at 45 °C for 10 minutes.

Maximally achievable molar activities under these conditions were determined to be 30–35 GBq/μmol for 8, 40–45 GBq/μmol for 9, 50–55 GBq/μmol for 10, and 1 GBq/μmol for 11. Of course, these values can be further improved by using higher starting activities, increasing the volume concentration of reactants or by performing the syntheses under microwave assistance. However, for comparative purposes within the experimental setup the values are valid and show considerable differences between the chelating agents.

Whereas the DOTA‐, DOTA‐GA‐, and NODA‐GA‐TATE derivatives 8–10 showed relatively similar maximal molar activities and an almost quantitative incorporation of the [⁶⁸Ga]Ga³⁺ within 10 minutes reaction time (>98 % in all cases), the CB‐DO2A‐GA‐chelator of 11 showed a considerably lower ability to incorporate the [⁶⁸Ga]Ga³⁺. This is on the one hand reflected in the significantly lower maximally achievable molar activity of only 1 GBq/μmol and on the other hand in the slower complexation kinetics observed during the labeling reaction. While in case of 8–10 the reaction was complete within 10 minutes, a ⁶⁸Ga‐incorporation of >95 % could be observed for [⁶⁸Ga]Ga‐11 only after 20 minutes reaction time.

This less efficient ⁶⁸Ga‐incorporation of CB‐DO2A‐GA, which also resulted in the lower achievable molar activity, can be attributed to the highly rigid, cross‐bridged structure of the chelator. This has also been shown before during the ⁶⁴Cu‐radiolabeling of CB‐DO2A and CB‐TE2A in direct comparison to their non‐cross‐bridged counterparts, where lower ⁶⁴Cu‐radiolabeling efficiencies were found for CB‐DO2A and CB‐TE2A (1,4,8,11‐tetraazabicyclo[6.6.2]hexadecane‐4,11‐diacetic acid) compared to DOTA and TETA (1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐tetraacetic acid).[ ¹⁴ , ¹⁸ , ¹⁹ , ²⁰ ] Although [⁶⁸Ga]Ga³⁺ of course differs from [⁶⁴Cu]Cu²⁺ regarding its chemical and physical properties, it is reasonable to assume that the same constraints on efficient complex formation, being induced by the spatial arrangement of the atoms of the chelators, apply to both radiometals. However, this problem can easily be overcome by applying harsher reaction conditions, resulting in comparable radiochemical yields and molar activities for both classes of chelating agents. ^[20]

Furthermore, the mentioned difficulty in terms of radiometal incorporation turns into an advantage when looking at complex stabilities: A highly rigid, cross‐bridged, pre‐organized ligand sphere decelerates radiometal incorporation but results in a complete and very stable envelopment of the radiometal ion, resulting in a high kinetic inertness of the complex against degradation and challenge under in vitro as well as in vivo conditions.[ ¹⁴ , ¹⁸ , ¹⁹ , ²¹ ]

In contrast to the other agents [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐10, [⁶⁸Ga]Ga‐11 was not homogenous but showed multiple peaks during radio‐HPLC analysis (Figure 3). This effect can be explained by the strongly restricted enantiomerization of the [⁶⁸Ga]Ga‐CB‐DO2A‐complex, having been described before for ^natGa‐CB‐DO2A. ^[13] For this complex, a high enantiomerization barrier was determined and explained by the strong metal‐ligand‐bonds, hindering non‐dissociative pseudorotation processes and resulting in inhibited dissociative processes for enantiomerization. Again, this initially disadvantageous effect is of advantage in the context of complex stabilities. Moreover, the observed different enantiomers can be assumed to not influence the pharmacokinetic profile of respectively modified peptides ^[22] and therefore do not have to be considered problematic.

Figure 3.

Analytical quality control radio‐HPLC chromatograms of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11, obtained after radiolabeling without further purification.

Evaluation of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 with regard to log_D(7.4), stability towards serum peptidase degradation and kinetic inertness of the complexes

[⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 were evaluated in terms of their hydrophilicity/lipophilicity profile as reflected in the corresponding log _D(7.4) values. This value is a good indicator for the main metabolization pathway and non‐specific organ uptake during an in vivo application of the radiotracer.[ ²³ , ²⁴ ]

For the determination of the log _D(7.4) values (as well as for the evaluations of the serum stability and complex inertness), the molar activities of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 were adjusted to 1 GBq/μmol for direct comparability of the results before partition experiments were performed between an aqueous phosphate buffer phase at pH 7.4 and 1‐octanol. For the different chelator‐TATE‐derivatives, relatively high differences between the corresponding log _D(7.4) values were found, with the hydrophilicity decreasing from −4.11±0.11 ([⁶⁸Ga]Ga‐9), over −4.09±0.07 ([⁶⁸Ga]Ga‐8) and −3.72±0.02 ([⁶⁸Ga]Ga‐10) to −3.02±0.08 ([⁶⁸Ga]Ga‐11). [⁶⁸Ga]Ga‐9 showed the highest hydrophilicity, as was to be expected due to two carboxylates of the DOTA‐GA ligand being not involved in the complexation of the [⁶⁸Ga]Ga³⁺. The two neutral complexes [⁶⁸Ga]Ga‐8 and [⁶⁸Ga]Ga‐10 showed a somewhat lower hydrophilicity, with the log _D(7.4) value for the DOTA derivative being slightly lower due to one of the carboxyl groups being not involved in radiometal ion complexation. In contrast, the CB‐DO2A‐GA derivative [⁶⁸Ga]Ga‐11 exhibited a significantly lower hydrophilicity, which can be attributed to the overall lower number of charged groups within the chelator structure. This may considerably influence the pharmacokinetic profile of a respectively modified peptide carrier and might result in a decreased kidney uptake combined with a more pronounced biliary clearance. Whether this is an advantage or disadvantage depends on the particular peptide carrier, but has to be considered in the design of the radiotracer.

For the comparative evaluation of the metabolic stability of the ⁶⁸Ga‐labeled chelator‐peptide‐conjugates against complex challenge by serum components, [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 were incubated with commercially available pooled human serum at 37 °C and aliquots of the mixtures were taken after 0, 15, 30, 45, 60, 75, 90, 105 and 120 minutes. After precipitation of the serum proteins and their removal by centrifugation, protein pellets and remaining solutions were measured for radioactivity and the solutions were analyzed by analytical radio‐HPLC. None of the agents showed a considerable liberation of [⁶⁸Ga]Ga³⁺ within the studied time of 2 hours (Figure 4). Of course, liberated [⁶⁸Ga]Ga³⁺ does not necessarily present as unbound radioactivity in the radioanalytical HPLC runs, but is more likely to be found as an increased radioactivity level in the serum protein pellet as [⁶⁸Ga]Ga³⁺ is chemically similar to Fe³⁺ and thus a mimetic of the latter. Due to this effect, unbound [⁶⁸Ga]Ga³⁺ can in part be incorporated into transferrin[ ²⁵ , ²⁶ ] and in this form would get precipitated together with the other serum proteins. Regarding this precipitation of protein‐associated ⁶⁸Ga, we found slight differences between the agents. For the DOTA‐, NODA‐GA‐ and CB‐DO2A‐GA‐TATE derivatives [⁶⁸Ga]Ga‐8, [⁶⁸Ga]Ga‐10 and [⁶⁸Ga]Ga‐11, comparable values for protein‐associated activity of 19.7±2.2 %, 16.9±3.5 % and 15.7±3.6 % were found, respectively. In contrast, the DOTA‐GA‐TATE derivative [⁶⁸Ga]Ga‐9 showed a slightly increased protein‐bound activity fraction of 29.0±3.5 %, which might indicate a slightly higher transchelation of [⁶⁸Ga]Ga³⁺ from DOTA‐GA to serum transferrin compared to the other chelating agents. In this context, the literature data of in vivo studies on [⁶⁸Ga]Ga‐DOTA‐GA‐modified radiopeptides show that the blood‐associated ⁶⁸Ga‐activity fraction was sometimes described to be higher compared to other chelators used[ ⁵ , ⁶ , ⁹ ] but in some cases also to be lower[ ⁷ , ¹¹ ] than for the corresponding [⁶⁸Ga]Ga‐DOTA‐, [⁶⁸Ga]Ga‐NOTA‐, or [⁶⁸Ga]Ga‐NODA‐GA‐comprising analogues. Thus, there is no clear indication of an increased tendency of ⁶⁸Ga‐transchelation to transferrin in case of [⁶⁸Ga]Ga‐DOTA‐GA compared to the other chelating agents.

Figure 4.

Analytical radio‐HPLC chromatograms of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11, obtained at different time‐points of incubation with human serum (A); and graphic depiction of the results of these evaluations, showing the decrease of peptide‐associated [⁶⁸Ga]Ga³⁺ over time (B). For the quantitative assessment of degradation, the radiochemical purity of all agents was normalized to 100 % at the start of the experiments, having been performed thrice for each substance.

In conclusion, a high inertness of the newly developed [⁶⁸Ga]Ga‐CB‐DO2A‐GA‐complex against complex challenge by serum components and transchelation by transferrin could be demonstrated.

Finally, the kinetic inertness of the ⁶⁸Ga‐complexes of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 was determined in direct comparison by DTPA (diethylenetriaminepentaacetic acid) complex challenge experiments, using a 1,000‐fold excess of DTPA as challenging agent. This assay allows to assess the relative inertness of different complexes and is therefore the standard method for the determination of the relative inertness of different complexes, mimicking the challenge of a radiometal complex by endogenous substances being present in high excess during an in vivo application. For this purpose, product solutions of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 were incubated over 120 minutes with a 1,000‐fold excess of DTPA and the extent of transchelation was determined by analytical radio‐HPLC (Figure 5). The results of these evaluations demonstrated high stabilities of all agents against DTPA challenge. However, one complex, namely [⁶⁸Ga]Ga‐DOTA‐GA showed a slightly lower inertness against challenge with a remaining DOTA‐GA‐associated ⁶⁸Ga‐activity of <94 % after 2 h, whereas the other complexes [⁶⁸Ga]Ga‐DOTA, [⁶⁸Ga]Ga‐NODA‐GA and [⁶⁸Ga]Ga‐CB‐DO2A‐GA demonstrated a peptide‐associated ⁶⁸Ga‐percentage of >98 % after the same time. This observation is explainable by the higher number of carboxylates being not involved in [⁶⁸Ga]Ga‐DOTA‐GA complex formation as it has been shown before that additional carboxylates within a chelator being not necessary for radiometal interaction can decrease the inertness of the formed complex. ^[27]

Figure 5.

Analytical radio‐HPLC chromatograms of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 obtained at different time‐points of incubation of the agents with a 1,000‐fold excess of DTPA (A); and graphic depiction of the results of these complex challenge experiments, showing the decrease of peptide‐associated [⁶⁸Ga]Ga³⁺ over time for the different complexes (B). For the quantitative assessment of the inertness of the ⁶⁸Ga‐complexes against challenge, the radiochemical purity of all agents was normalized to 100 % at the start of the experiments, having been performed thrice for each substance.

Thus, the kinetic inertness of [⁶⁸Ga]Ga‐CB‐DO2A‐GA against challenge proved to be comparable to the established complexes studied under identical conditions and is thus sufficiently high for in vivo applications.

Thus, the newly developed bifunctional cross‐bridged chelating agent CB‐DO2A‐GA forms ⁶⁸Ga‐complexes of high stability against serum peptidase degradation and high kinetic inertness against complex challenge, being mandatory for stable ⁶⁸Ga‐incorporation during in vivo imaging applications.

Conclusion

A new bifunctional cross‐bridged chelating agent – CB‐DO2A‐GA – was developed, enabling the conjugation to biomolecules without alteration of the CB‐DO2A coordination sphere of the resulting complex. Its chemical and radiochemical properties were compared to those of the established chelators for ⁶⁸Ga‐radiolabeling, DOTA, NODA‐GA and DOTA‐GA. CB‐DO2A‐GA could be introduced into the chosen TATE peptide carrier without difficulties and the bioconjugates were radiolabeled with [⁶⁸Ga]Ga³⁺. [⁶⁸Ga]Ga‐CB‐DO2A‐GA demonstrated a very high kinetic inertness against degradation and complex challenge under different conditions, being attributable to the rigid cross‐bridged and pre‐organized ligand sphere.

Therefore, CB‐DO2A‐GA can be a valuable alternative to established chelating agents for ⁶⁸Ga‐radiolabeling of peptides, especially when the formation of a positively charged ⁶⁸Ga‐complex is pursued to achieve a tailored pharmacokinetic profile of the resulting peptidic radiotracer.

Experimental Section

General

Chemicals: All chemicals were used without further purification unless otherwise indicated. H₂O for HPLC was produced by the ultrapure water system Aquinity² from membraPure (Hennigsdorf, Germany). Acetonitrile (HPLC grade), trifluoroacetic acid (TFA) (for peptide synthesis) and 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU) were purchased from Carl Roth (Karlsruhe, Germany). TFA (uvasol quality) for HPLC, H₂O (Tracepur quality), hydrochloric acid (30 %, Suprapur quality) and Fmoc‐Thr(tBu)‐Wang resin were purchased from Merck (Darmstadt, Germany). l‐Glutamic acid‐5‐benzylester, sodium nitrite, acetonitrile (anhydrous), dimethylacetamide, boron trifluoride etherate, Palladium on activated charcoal (10 % Pd/C), tert‐butylbromoacetate, thallium(III) trifluoroacetate, N,N‐diisopropylethylamine (DIPEA) and pooled human serum were purchased from SigmaAldrich (Taufkirchen, Germany). Sodium bromide and tert‐butyl‐2,2,2‐trichloroacetimidate were obtained from Thermo Fisher GmbH (Kandel, Germany). Hydrobromic acid (48 % in water) was purchased from VWR (Bruchsal, Germany). The chelators R‐NODA‐GA(tBu)₃, DOTA‐GA(tBu)₄ and DOTA(tBu)₃ as well as CB‐cyclen and DTPA were obtained from CheMatech (Dijon, France). The N _α‐Fmoc‐ and side chain‐protected amino acids for peptide synthesis were obtained from Merck (Darmstadt, Germany) or Carbolution (St. Ingberg, Germany). All other standard chemicals and solvents were obtained from Carl Roth (Karlsruhe, Germany), Sigma‐Aldrich (Taufkirchen, Germany), TCI Deutschland GmbH (Eschborn, Germany, Thermo Fisher (Kandel, Germany) and VWR (Bruchsal, Germany). Deuterated CDCl₃ was purchased from Deutero GmbH (Kastellaun, Germany). [⁶⁸Ga]Ga³⁺ was obtained by fractioned elution of a ⁶⁸Ge/⁶⁸Ga generator (IGG100, Eckert & Ziegler, Berlin, Germany) using 0.1 M HCl.

Instrumentation: Analytical and semipreparative HPLC analyses were performed on a Dionex UltiMate 3000 System (Thermo Fisher, Dreieich, Germany) equipped with either a Chromolith Performance (RP‐18e, 100–4.6 mm, Merck, Darmstadt, Germany) or a Chromolith SemiPrep column (RP‐18e, 100–10 mm, Merck, Darmstadt, Germany). Radio‐HPLC analyses were performed on the same system, being additionally equipped with a radio detector (GabiStar, ElysiaRaytest, Straubenhardt, Germany). All operations were performed with a flow rate of 4 mL/min using H₂O (supplemented with 0.1 % TFA) and MeCN (also supplemented with 0.1 % TFA) as the eluents. The gradient used for analytical and radioanalytical chromatography was typically 0–100 % MeCN within 5 min, whereas gradients for semipreparative chromatography were chosen individually and are reported in the respective preparative section. Nuclear magnetic resonance (NMR) spectroscopy was performed on a 500 MHz‐Varian NMR System spectrometer and the signals of the deuterated solvents were used as references. Matrix‐assisted laser desorption/ionization spectroscopy (MALDI‐MS) was carried out on a Bruker Daltonics Microflex spectrometer and high‐resolution electrospray ionization mass spectroscopy (HR‐ESI‐MS) was performed on a Thermo Finnigan LTQ FT Ultra Fourier Transform Ion Cyclotron Resonance mass spectrometer was used. Radioactivity was measured using an ISOMED 2010 activimeter and gamma counting was performed on a 2480 Wizard system (Perkin Elmer).

Synthesis of CB‐DO2A‐GA(tBu)₂

5‐(Benzyloxy)‐2‐bromo‐5‐oxopentanoic acid 2

2 was synthesized according to literature methods. ^[17] Briefly, L‐glutamic acid‐5‐benzylester (4.0 g, 16.86 mmol) and sodium bromide (6.07 g, 59.01 mmol, 3.5 eq.) were diluted in an aqueous solution of hydrobromic acid (1 M, 30.3 mL). The solution was cooled to 0 °C and sodium nitrite (2.09 g, 30.35 mmol, 1.8 eq.) was added portion wise. The reaction was stirred for 2 h at 0 °C. Afterwards, sulfuric acid (1.53 mL, 28.66 mmol, 1.7 eq.), followed by diethyl ether (30 ml) was added. The aqueous phase was extracted thrice with diethyl ether (10 mL). Then, the combined organic phases were washed with brine and dried with Na₂SO₄. After evaporation of the solvent, the crude product was purified by column chromatography using cyclohexane/ethyl acetate 3 : 1 (v/v) as the eluent. 5 was obtained in a yield of 45.2 % (2.30 g, 7.62 mmol, purity >92 %). ¹H‐NMR (500 MHz, CDCl₃) δ 7.41–7.32 (m, 5H, H _Ar), 5.12 (s, 2H, H‐6), 4.41 (dd, J=8.5, 5.8 Hz, 1H, H‐2), 2.77–2.53 (m, 2H, H‐4), 2.48–2.27 (m, 2H, H‐3). ¹³C‐NMR (126 MHz, CDCl₃) δ 174.22 (C‐1), 172.04 (C‐5), 135.69 (C‐7), 128.77 (C‐9, C‐11), 128.55 (C‐10), 128.43 (C‐8, C‐12), 66.86 (C‐6), 44.19 (C‐2), 31.58 (C‐4), 29.62 (C‐3). HR‐ESI‐MS (m/z) for [M−H]⁻ (calculated): 298.9925 (298.9924).

5‐Benzyl‐1‐tert‐butyl‐2‐bromopentanedioate 3

The synthesis of 3 was carried out according to the literature with minor changes. ^[17] 2 (2.0 g, 6.64 mmol) was dissolved in chloroform (8 mL) under argon atmosphere. A solution of tert‐butyl‐2,2,2‐trichloroacetimidate (2.6 mL, 14.61 mmol, 2.2 eq.) in cyclohexane (8 mL) was added dropwise over 15 min. The formed precipitate was dissolved by the addition of dimethylacetamide (2.7 mL) and boron trifluoride etherate (0.135 mL, 1.06 mmol, 0.16 eq). The reaction mixture was stirred at ambient temperature for three days. After evaporation of the solvents, the organic phase was extracted three times with hexane. Next, the volatile materials were removed under reduced pressure and the crude product was purified by column chromatography using a gradient of cyclohexane/ethyl acetate 20 : 1 to 9 : 1 (v/v) as the eluent. 3 was obtained in a yield of 81.4 % (1.93 g, 5.41 mmol, purity >98 %). ¹H‐NMR (500 MHz, CDCl₃) δ [ppm]=7.40–7.30 (m, 5H, H _Ar), 5.13 (s, 2H, H‐6), 4.24 (dd, J=8.5, 5.9 Hz, 1H, H‐2), 2.62–2.50 (m, 2H, H‐4), 2.41–2.21 (m, 2H, H‐3), 1.47 (s, 9H, H‐14).¹³C‐NMR (126 MHz, CDCl₃) δ [ppm]=172.13 (C‐5), 168.44 (C‐1), 135.85 (C‐7), 128.75 (C‐9, C‐11), 128.48(C‐10), 128.38 (C‐8, C‐12), 82.79 (C‐13), 66.69 (C‐6), 46.79 (C‐2), 31.78 (C‐4), 29.86 (C‐3), 27.88 (C‐14). MALDI‐MS (m/z) for [M+H]⁺ (calculated):474.90 (475.33). HR‐ESI‐MS (m/z) for [M−H]⁻ (calculated): 355.0375 (355.0550).

5‐Benzyl‐1‐tert‐butyl‐2‐(1,4,7,10‐tetraazabicyclo[5.5.2]tetradecan‐4‐yl)pentanedioate 4

CB‐cyclen (100.0 mg, 0.50 mmol) was dissolved in dry acetonitrile (2 mL) under argon atmosphere and potassium carbonate (69.7 mg, 0.50 mmol, 1.0 eq.) was added. A solution of 6 (180.1 mg, 0.50 mmol, 1.0 eq.) in acetonitrile (1 mL) was added dropwise over 5 h while cooling to 0 °C. The reaction mixture was stirred for 5 h at 0 °C and for another 3 days at ambient temperature. Afterwards, the formed salt was removed by filtration. The solution was concentrated by evaporation and the crude product was purified by semipreparative HPLC using a gradient of 0–80 % MeCN+0.1 % TFA in 8 min (R _t=4.43 min). 4 was isolated as yellowish oil in a yield of 39.5 % (117.2 mg, 0.20 mmol, purity >98 %). ¹H‐NMR (500 MHz, CDCl₃) δ 8.73 (s, 3H, NH), 7.39–7.29 (m, 5H, H _Ar), 5.15–5.09 (m, 2H, H‐6), 4.24–4.00 (m, 1H, H‐2), 3.86–2.63 (m, 21H, H‐15–H‐24), 2.61–2.32 (m, 2H, H‐4), 2.31–1.77 (m, 2H, H‐3), 1.49–1.42 (m, 9H, H‐14). ¹³C‐NMR (126 MHz, CDCl₃) δ 173.15 (C‐1), 172.92 (C‐5), 160.99 (q, J=37.8, CO_(TFA)), 135.96 (C‐7), 128.78 (C _ArH), 128.74 (C _ArH), 128.69 (C _ArH), 128.48 (C _ArH), 128.33 (C _ArH), 115.97 (q, J=289.8, CF_3(TFA)), 83.28 (C‐13), 66.64 (C‐6), 65.16 (C‐2), 64.15+63.42+59.09+58.71+58.51+58.04+57.23+56.16+56.13+55.27+55.09+54.55+53.58+52.79+47.00+46.89+44.93+44.81+44.53+44.21+43.24+42.88 (C‐15−C‐24), 31.33 (C‐4), 31.13, 30.88, 28.20 (C‐14), 28.15, 28.13, 25.67 (C‐3). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 474.80 (475.33). HR‐ESI‐MS (m/z) for [M+H]⁺(calculated): 475.3279 (475.3279).

Bn‐CB‐DO2A‐GA(tBu)₂ 5

To a solution of 4 (95 mg, 20.1 mmol) in dry acetonitrile (1.4 mL) were added potassium carbonate (28 mg, 0.20 mmol, 1.25 eq.) and tert‐butylbromoacetate (33 μL, 0.22 mmol, 1.37 eq.). The reaction was stirred for 48 h at ambient temperature under argon atmosphere. The formed salt was removed by filtration. After removal of volatile materials under reduced pressure, the crude product was purified by semipreparative HPLC using a gradient of 25–65 % MeCN+0.1 % TFA in 8 min (R _t=4.57 min). 5 was isolated as yellowish oil in a yield of 58.8 % (67 mg, 0.10 mmol, purity≥91 %).¹H‐NMR (500 MHz, CDCl₃) δ 7.42–7.30 (m, 5H, H _Ar), 5.17–5.07 (m, 2H, H‐6), 4.21–3.41 (m, 2H, H‐25), 3.39–2.81 (m, 21H, H‐2, H‐15–H‐24), 2.63–2.43 (m, 2H, H‐4), 2.21–1.78 (m, 2H, H‐3), 1.47 (s, 9H, CH ₃), 1.45 (s, 9H, CH ₃). ¹³C‐NMR (126 MHz, CDCl₃) δ 172.77 (C‐5), 171.35 (C‐1), 170.56 (C‐26), 159.78 (q, J=39.2, CO_(TFA)) 135.65 (C‐7), 128.89 (C _ArH), 128.86 (C _ArH), 128.85 (C _ArH), 115.53 (q, J=288.0, CF_3(TFA)), 82.68 (C‐27), 82.07(C‐13), 66.92 (C‐6), 65.24 (C‐2), 57.70+57.47+57.28+56.59+56.45+53.78+52.15+50.13+46.70+44.77 (C‐15−C‐24), 30.87 (C‐4), 28.2 (CH₃), 28.27 (CH₃), 26.06 (C‐3). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 589.02 (589.40). HR‐ESI‐MS (m/z) for [M+H]⁺ (calculated): 589.3962 (589.3960).

CB‐DO2A‐GA(tBu)₂ 6

To a solution of 5 (40 mg, 57 μmol) in THF/water 1 : 1 (v/v, 3 mL) was added 10 % Pd/C (10 mg) before the atmosphere was exchanged to hydrogen and stirred for 18 h at ambient temperature. Pd/C was filtered off and THF was removed by evaporation. After purification, 6 was obtained by semipreparative HPLC using a gradient of 0–80 % MeCN+0.1 % TFA in 8 min (R _t=4.57 min) as colorless solid in a yield of 74.6 % (26 mg, 42 μmol, purity >93 %). ¹H‐NMR (500 MHz, CDCl₃) δ 9.27 (s, 1H, OH), 4.22–3.68 (m, 1H, H‐2), 3.54 (dd, J=53.7, 17.9 Hz, 2H, H‐18), 3.45–2.63 (m, 21H, H‐2, H‐8−H‐17), 2.61–2.46 (m, 2H, H‐4), 2.24–1.76 (m, 2H, H‐3), 1.47 (s, 9H, H‐21), 1.45 (s, 9H, H‐7). ¹³C‐NMR (126 MHz, CDCl₃) δ 175.59 (C‐5), 171.57 (C‐1), 170.56 (C‐19), 160.16 (q, J=38.8, CO_(TFA)), 115.67 (q, J=288.2, CF_3(TFA)), 82.68 (C‐20), 82.44 (C‐6), 65.89 (C‐2), 62.77+57.92+57.45+57.27+56.88+56.71+54.38+53.10+46.16+44.85 (C‐8−C‐17), 31.34 (C‐4), 28.25 (CH₃), 28.21 (CH₃), 25.83 (C‐3). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 498.94 (499.35). HR‐ESI‐MS (m/z) for [M+H]⁺ (calculated): 499.6725 (499.3491).

Synthesis of TATE‐chelator‐derivatives 8–11

The synthesis of the TATE‐chelator‐derivatives (chelator‐D‐Phe‐cyclo[Cys‐Phe‐D‐Trp‐Lys‐Thr‐Cys]Thr‐OH) 8–11 was performed according to standard Fmoc‐based solid‐phase peptide synthesis (SPPS) methods. ^[28] Briefly, Fmoc‐Thr(tBu)‐Wang resin was swollen in dichloromethane for 60 min and then rinsed with DMF. The Fmoc‐protecting groups were cleaved with piperidine in DMF (1 : 1, v/v) for a total of 7 min (2+5 min). For peptide coupling, the respective amino acid (4.0 eq.) was first activated by a two‐minute incubation with HBTU (3.9 eq.) and DIPEA (4.0 eq.) in DMF before the coupling reaction was performed in an ultrasonic bath for 10 min at ambient temperature. After conjugation of the last amino acid Fmoc‐D‐Phe‐NH₂, the cyclization of the peptide was performed using thallium(III)trifluoroacetate (4 eq.) in DMF for 45 min prior to final Fmoc‐protecting group removal. After cleavage of the N‐terminal Fmoc‐protecting group, the coupling of the respective chelator (2.5 eq.) was performed using HBTU (2.45 eq.) and DIPEA (5.0 eq.) as described before. Afterwards, the peptide derivatives were cleaved from the resin and simultaneously deprotected by incubating the resin with a solution of TFA/TIS/H₂O 95/2.5/2.5 (v/v/v) for 3 h at ambient temperature. The products were purified by semipreparative HPLC and isolated as white solids after lyophilization. Gradients used for HPLC purification and synthesis yields for each compound are given below.

DOTA‐TATE 8. Gradient for purification: 0–70 % MeCN+0.1 % TFA in 10 min (R _t=5.04 min), yield: 62.6 % (40.4 mg, 28.2 μmol, purity≥99 %). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 1434.92 (1435.60). HR‐ESI‐MS (m/z) for [M+2H]²⁺ (calculated): 718.3053 (718.3047), [M+H]⁺ (calculated): 1435.6060 (1435.6021), [M−H]⁻(calculated): 1433.5824 (1433.5875).

DOTA‐GA‐TATE 9. Gradient for purification: 10–20 % MeCN+0.1 % TFA in 8 min (R _t=6.62 min), yield: 36.1 % (20.4 mg, 13.5 μmol, purity≥99 %). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 1507.16 (1507.62). HR‐ESI‐MS (m/z) for [M+2H]²⁺ (calculated): 754.3160 (754.3153), [M+H]⁺ (calculated): 1507.6283 (1507.6233), [M−H]⁻ (calculated): 1505.6040 (1505.6087).

NODA‐GA‐TATE 10. Gradient for purification: 12–20 % MeCN+0.1 % TFA in 8 min (R _t=5.04 min), yield: 46.1 % (24.4 mg, 17.4 μmol, purity≥99 %). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 1406.46 (1406.57). HR‐ESI‐MS (m/z) for [M+2H]²⁺ (calculated): 703.7914 (703.7919), [M+H]⁺ (calculated): 1406.5796 (1406.5747), [M−H]⁻ (calculated): 1404.5601 (1404.5609).

CB‐DO2A‐GA‐TATE 11. The conjugation of CB‐DO2A‐GA( ^t Bu)₂ 6 to the TATE peptide on solid support was performed using slightly modified conditions compared to the other chelating agents. For this purpose, 6 (1.6 eq.) was pre‐activated using PyBOP (2.45 eq.) and DIPEA (5 eq.) before conjugation. Gradient for purification: 0–50 % MeCN+0.1 % TFA in 10 min (R _t=6.24 min), yield: 17.6 % (3.7 mg, 2.6 μmol, purity≥99 %). MALDI‐MS (m/z) for [M+H]⁺ (calculated): 1417.47 (1417.62). HR‐ESI‐MS (m/z) for [M+2H]²⁺ (calculated): 709.3183 (709.3176), [M+H]⁺ (calculated): 1417.6327 (1417.6280).

⁶⁸Ga‐Radiolabeling of 8–11 to [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11

[⁶⁸Ga]GaCl₃ (450–500 MBq) was obtained by fractioned elution of a ⁶⁸Ge/⁶⁸Ga generator using HCl (0.1 M, 1.2 mL). The pH of the eluate (30–40 MBq, 100–150 μL) was adjusted to pH 3.8–4.0 using sodium acetate buffer (1.25 M, pH=4.4, 50–60 μL). Next, the respective chelator‐TATE derivative 8–11 (0.4–40 nmol) was added and the reaction was incubated at 95 °C (8, 10 and 11) or 45 °C (9) for 10 min. The optimized molar activity was determined by decreasing the amount of used labeling precursor while leaving the amount of [⁶⁸Ga]Ga³⁺ constant until reaction control by radioanalytical‐HPLC revealed incomplete incorporation of the radiometal. Maximum molar activities were determined to be 30–35 GBq/μmol for 8, 50–55 GBq/μmol for 9, 40–45 GBq/μmol for 10, and 1 GBq/μmol for 11.

For the determination of serum stabilities and log _D7.4 values, the molar activities of all tracers [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 were adjusted to 1 GBq/μmol for comparable conditions.

Determination of log _D(7.4) values for [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11

For the determination of the log _D(7.4) values, a solution of the respective tracer [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 (5 μL, 0.7–1 MBq, obtained as described before) was added to a mixture of 1‐octanol (800 μL) and phosphate buffer (795 μL, pH 7.4, 0.05 M). The mixture was vigorously shaken for two minutes. Then, the phases were separated by centrifugation. An aliquot of 200 μL of the organic and the water phase were transferred to gamma counter tubes and measured for activity by γ‐counting. Each experiment was performed thrice, each in triplicate.

Serum stability of [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11

A solution of the respective tracer [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 (125 μl, 20–25 MBq, obtained as described before) was added to commercially available pooled human serum (500 μL) and incubated at 37 °C. At defined time points (t=0, 15, 30, 45, 60, 75 and 90 min), samples of 75 μL were added to ice‐cold ethanol (75 μL). Precipitation of serum proteins was further supported by incubating the samples for 1 min on ice and the resulting suspension was centrifuged for 90 s. The supernatant was collected, the activity of the supernatant and the precipitate measured, and hereafter, the supernatant was analyzed by analytical radio‐HPLC. Experiments were performed thrice for each radioligand.

DTPA challenge experiments with [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11

A solution of the respective tracer [⁶⁸Ga]Ga‐8–[⁶⁸Ga]Ga‐11 (125–150 μL, 18.75 nmol) was added to a solution of DTPA (7.4 mg, 18,750 nmol, 1,000 eq.) in sodium acetate buffer (500 μL, 0.5 M, pH 4.2). The final volume was adjusted to 650 μL by addition of sodium acetate buffer (1.25 M, pH=3.9, 0–25 μL) and the pH of the resulting solution was 4.0. The solutions were incubated at 37 °C and at pre‐defined time points (t=0, 15, 30, 45, 60, 75, 90, 105 and 120 min), the amount of ⁶⁸Ga‐transchelation was determined by analytical radio‐HPLC. Each experiment was performed in triplicate.

Conflict of interest

The authors declare no conflict of interest.

Damerow H., Wängler B., Schirrmacher R., Fricker G., Wängler C., ChemMedChem 2023, 18, e202200495.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.