Special Issue Article Received 4 June 2013,
Accepted 29 October 2013
Published online 5 December 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3153
Bioorthogonal chemistry for 68Ga radiolabelling of DOTA-containing compounds† Helen L. Evans,a Laurence Carroll,a* Eric O. Aboagye,a and Alan C. Spiveyb Copper-catalysed ‘click’ chemistry is a highly utilised technique for radiolabelling small molecules and peptides for imaging applications. The usefulness of these reactions falls short, however, when metal catalysis is not a practically viable route; such as when using metal chelates as radioligands. Here, we describe a method for carrying out ‘click-type’ radiochemistry in the presence of DOTA chelates, by combining 68Ga radiolabelling techniques with well-established bioorthogonal reactions, which do not rely upon metal catalysis. Keywords: Bioorthogonal chemistry; Gallium-68; DOTA; click chemistry; cyclooctyne; azide; tetrazine; norbornene; strained alkene
Introduction
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a Comprehensive Cancer Imaging Centre, Department of Surgery & Cancer, Hammersmith Campus, Imperial College, London, UK b Department of Chemistry, Imperial College London, South Kensington Campus, London, UK
*Correspondence to: Laurence Carroll, Comprehensive Cancer Imaging Centre, Department of Surgery & Cancer, Hammersmith Campus, Imperial College, London, UK. E-mail: [email protected] †
This article is published in the Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on ‘Current Developments in PET and SPECT Imaging’, edited by Jonathan R. Dilworth, University of Oxford and Sofia I. Pascu, University of Bath.
Copyright © 2013 John Wiley & Sons, Ltd.
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Bioorthogonal reactions have become increasingly investigated for the development of new biological and medicinal chemistry technologies. Amongst these, their employment for pre-targeted imaging has received significant attention, with particular focus on the strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, developed by Bertozzi, and the inverse electrondemand Diels-Alder (IDDA) reactions between tetrazines and strained alkenes.1–9 Here, we describe the development of two DOTA-based prosthetic groups for 68Ga-labelling one of which is reactive in SPAAC ligations (i.e. an azide) and the other towards IDDA ligations (i.e. a tetrazine). The utility of these prosthetic groups is illustrated by reactions with nine small molecule partners, under varying mild conditions of solvent, temperature and time. It can be envisioned that these prosthetic groups will be useful for the radiolabelling of biologically-active molecules, with the aim to use this strategy for pre-targeted positron emission tomography (PET) imaging. ‘Click’ chemistry refers to a set of reactions that are able to proceed under mild conditions and can be applied to the efficient synthesis of a broad variety of compounds, as exemplified in the work of Sharpless et al. and Meldal et al.10,11 Click reactions are modular and wide in scope, give consistently high yields, and form products that are stable under physiological conditions. The term is now more specifically applied to the highly regioselective copper (I) catalysed reaction between azides and terminal acetylenes to form 1,4disubstituted 1,2,3-triazoles, also known as the copper-catalysed azide-alkyne cycloaddition (CuAAC).12–14 This type of reaction usually involves the formation of copper(I) in situ from copper (II) salts with sodium ascorbate, and allows the preparation of a wide spectrum of 1,4-triazole products in high yield.15 The reaction has been utilised for numerous radiopharmaceutical applications, including the preparation of RGD,16–18 fluciclatide (and analogues),19,20 octreotide (and analogues)18,21,22 and other radiolabelled peptides for imaging applications. However, the use of the CuAAC reaction for the radiolabelling of peptides with metal isotopes is not feasible as copper(II) has a high affinity for
amines such as lysine, as well as cysteine and carboxylic acid residues, and has been demonstrated to be a useful cation for chelation to macrocycles such as cyclams.10,23–25 Any copper present forms a stable chelate with any unbound macrocycle, resulting in reduced concentration of the copper, and therefore preventing conversion to the triazole products; the use of excess of copper to circumvent this would prevent subsequent radiolabelling of the cyclam. Biocompatibility and fast reaction kinetics have, however, also been demonstrated by SPAAC reactions, which do not rely on copper catalysis, allowing formation of triazoles under mild conditions.6,8,26 The reduced activation energy for SPAAC reactions, relative to analogous non-strained examples, is a consequence of the strain imposed on the sp-hybridised carbons by the ring structure leading to bond angle distortions of about 17° from the idealised 180° angles of a C-C triple bond; these distortions result in comparable angles to those involved in the transition state of the cycloaddition (~160°).27–29 Cyclooctynes are the smallest ring size that can accommodate the alkyne functional group and still be isolated as a stable molecule, and were found to react in a highly selective manner with azides, demonstrating comparable reaction kinetics to the metalcatalysed reaction, but with no apparent toxicity.6 Superior reaction kinetics have now been demonstrated for IDDA
H. L. Evans et al. reactions between 1,2,4,5-tetrazines and strained alkenes, forming a mixture of dihydropyridazine isomers via a Diels-Alder/retroDiels-Alder reaction at rates that are reported to be 200 000 times faster than the SPAAC reaction in aqueous solvents and at ambient temperatures.30–33 The SPAAC and IDDA reactions have therefore been demonstrated to be useful methods for radiolabelling biomolecules, and do not rely on metal catalysis.6,34–36 18 F remains one of the most important isotopes for PET radiolabelling strategies because of its favourable half-life (109.7 min) and low energy, making it optimum in terms of PET image quality.37 However, the chemistry involved with the introduction of the 18F label into large biomolecules remains limited largely to the synthesis of prosthetic groups, whereby 18 F is introduced by coupling through use of the CuAAC reaction.38 The radiosynthesis of 18F labelled azides, such as 2-[18F]fluoroethyl azide may be achieved, and the SPAAC reaction with a series of cyclooctynes has been reported.39 However, the application of this chemistry to in vivo pre-targeted PET imaging has yet to be successfully demonstrated.4 The high volatility of 2-[18F]fluoroethylazide, coupled with its moderate, but not optimal radiochemical yields [~30 % nondecay-corrected (n.d.c)], encouraged us to find a more robust prosthetic group for the radiolabelling of small molecules and peptides using bioorthogonal chemistry.37,39 68 Ga, although having a shorter half-life (67.7 min) than 18F, and emitting a higher energy positron, has been utilised for the synthesis of radiolabelled probes for PET imaging.23 This is largely due to the relatively low cost of 68Ga, using a generator containing the parent radionuclide 68Ge (1/2 life = 271.0 days), which does not rely on the use of an on-site cyclotron facility, and these generators typically allow for the continuous production of high-quality 68Ga for approximately 1 year. The relatively simple and short radiolabelling methods that are used for metal isotopes, relying on chelating ligands attached to a targeting molecule, are more efficient than those for non-metal isotopes, and solid-phase purification methods are often used, minimising the problems associated with isotope decay during synthesis,.40 Here, we describe the design of two molecules based on the DOTA chelate (Figure 1), which have been functionalised with an azide 1 and a tetrazine 2, to allow two comparative bioorthogonal 68Ga labelled reactions to be examined under a variety of different conditions, with a number of different reactive partners. These studies constitute important proof-
of-concept for eventual use of these reactions as tools for pretargeted PET imaging.3,4,41
Experimental Chemistry ‘Cold’, unradiolabelled reference compounds were synthesised, and were used to confirm the products of the radiolabelled reactions (See supporting information for full experimental procedure and characterisation of ‘cold’ reference compounds).
Radiochemistry High-performance liquid chromatography (HPLC) traces for the radiolabelled products are reported in the supporting information. 68 68 GaCl3 was produced by an 18-month old Ge containing generator and was eluted in 0.1 M HCl (2 mL).
Synthesis of [68Ga]DOTA-azide 1 -1
A solution of DOTA-azide 1 (1 mg mL in DMSO) was diluted with a 68 NaOAc buffer solution (pH 4), and the GaCl3 was eluted directly into 68 the reaction vial (~1.5 mCi). The radiosynthesis of [ Ga]DOTA-azide was achieved in 10 min at 90 °C, in >95% radiochemical yield, and the product was obtained in >95% radiochemical purity by passing through a Sep-Pak light C18 cartridge. The mixture was then diluted with water or 68 acetonitrile for application to the Ga-labelled click reactions.
General procedure for
68
Ga-labelled SPAAC reactions
-1
To a 10 mg mL solution of cyclooctyne (3/4) (50 μL) in either H2O 68 (10% DMSO) or MeCN was added [ Ga]DOTA-azide 1 (100 μL), and the mixture was heated to either 37 °C or 90 °C for the given timepoint, without stirring. The progress of the reaction was monitored by radio-HPLC.
Synthesis of [68Ga]DOTA-tetrazine 2 -1
A solution of DOTA-tetrazine 2 (1 mg mL in DMSO) was diluted with a 68 NaOAc buffer solution (pH 6), and the GaCl3 was eluted directly into 68 the reaction vial (~1.5 mCi). The radiosynthesis of Ga DOTA-tetrazine was achieved in 15 min at 90 °C, in >95% radiochemical yield, and the product was obtained in >95% radiochemical purity by passing through a Sep-Pak light C18 cartridge. The mixture was then diluted with water or 68 acetonitrile for application to the Ga-labelled IDDA reactions.
General procedure for
68
Ga-labelled IDDA reactions
-1
To a 10 mg mL solution of alkene (8–13) (50 μL) in either H2O or MeCN 68 was added [ Ga]DOTA-tetrazine 2 (100 μL), and the mixture was heated to 37 °C for the given time-point, without stirring. The progress of the reaction was monitored by radio-HPLC.
Results and discussion Bioorthogonal chemistry with DOTA-azide 1
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Figure 1. Our DOTA-containing prosthetic groups containing an azide (1) and 68 tetrazine (2) functional group, for bioorthogonal radiolabelling using Ga.
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Azides are a useful class of bioorthogonal compound, which can be readily incorporated into a wide range of biological targets and because of their small size impart minimal structural perturbation.42 This functional group has many applications, with the most important of these being for use in click chemistry. As previously discussed, CuAAC reactions are difficult to perform efficiently in the presence of metal chelators, as these species can remove active copper from the reaction mixture. Instead, we designed [68Ga]DOTA-azide 1, with which we carried out a series of bioorthogonal SPAAC reactions, demonstrating their high yields and reproducibility. The radiosynthesis of 1 may be achieved in radiochemical conversions of greater than 95% n.d.c within 10 min, significantly
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 291–297
H. L. Evans et al. higher yielding than the previously synthesised 2-[18F]fluoroethyl azide.37,39 Azide 1 was used for a series of SPAAC reactions with two analogues of aza-dibenzocyclooctyne (Figure 2).27,43,44 These cyclooctynes have been demonstrated as having superior reaction kinetics to the first generation cyclooctynes, owing to the increased strain accruing from the introduction of two aryl rings adjacent to the alkyne, and increased water solubility as the result of inclusion of a nitrogen atom into the cyclooctyne ring. The two cyclooctynes used in this study contained either a carboxylic acid (3) or maleimide functional group (4) to allow conjugation to the target biomolecules. (Figure 3 and 4) The carboxylic acid analogue 3 formed the triazole isomers in radiochemical conversions from the azide 1 of ~95% n.d.c at 37 °C in acetonitrile within 15 min, and higher temperatures (90 °C) allowed for complete conversion to the click products within just 10 min (Table 1). Cyclooctyne 3 was also able to perform fairly well in the SPAAC reaction with the azide in water, but the addition of 10% DMSO was required in order for the compound to dissolve fully. Low conversions to the triazoles were demonstrated in water at 37 °C (15% n.d.c) at short time-points, but heating to 90 °C enabled the triazoles to be formed in near to quantitative yields, possibly attributable to the increased solubility of the cyclooctyne at higher temperatures. The maleimide analogue 4 similarly demonstrated reasonable reactivity with azide 1 in acetonitrile, and gave near to quantitative conversions to the triazoles when heated to 90 °C. This compound demonstrated poor water solubility, even with the addition of 10% DMSO, and so the reactivity of this compound in aqueous media was not successfully demonstrated. In order to demonstrate the usefulness of the SPAAC reaction when radiolabelling with metal chelators, we also decided to compare these results to those obtained using the traditional CuAAC radiolabelling method, by reaction of our azide with an alkyne under standard CuAAC reaction conditions. An octreotate analogue was chosen for this purpose as this peptide is readily
radiolabelled using CuAAC chemistry by reacting with 2-[18F] fluoroethyl azide.21 E-TOCA (5) was therefore reacted with 68Ga azide 1 under the same reaction conditions to those reported for the 18F-labelling procedure,21 and showed no conversion to the triazole product. This lack of reactivity is attributed to the deactivation of the copper catalyst in the presence of excess DOTA chelate from solid-phase purification, a problem that is not observed in the copper-free version of the reaction. Overall, therefore, azide 1 has been demonstrated as a useful prosthetic group for the radiolabelling of cyclooctyne-containing compounds with 68Ga. The SPAAC reaction may be considered as an alternative method for labelling biomolecules with 68Ga, due to the demonstrated difficulties involved in using the CuAAC in this context. These reactions may be particularly useful when organic solvents can be tolerated (e.g. acetonitrile), and pre-targeted labelling may be achievable if the cyclooctyne is attached to a water-soluble biomolecule. Bioorthogonal chemistry with DOTA-tetrazine 2 Recently, the IDDA reaction between tetrazines and strained alkenes has been demonstrated as a potentially useful bioorthogonal ligation method.1,32,34,35 With this in mind, we designed a DOTA with an appended tetrazine motif 2, which could be used to make a comparison with the 68Ga-labelled SPAAC reaction described earlier. The norbornene core has been shown to react rapidly with tetrazines, because of the large amount of strain contained in the alkene functional group, forming the dihydropyradazine products rapidly in aqueous media.1,2 We initially demonstrated this with DOTA-tetrazine 2, by observing almost 80% radiochemical conversion to the dihydropyradazine isomers (Figure 5) within just 10 min at 37 °C in aqueous media, and complete conversion to the four product isomers; within 20 min under the same conditions, using commercially available norbornene 8 as the strained alkene (Table 2).
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68
Figure 2. Radiosynthesis of [ Ga]DOTA-azide 1 and subsequent SPAAC reaction with cyclooctynes to form triazole products.
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H. L. Evans et al.
68
Figure 3. Attempted CuAAC reaction with [ Ga]DOTA-azide 1.
68
Figure 4. Radiosynthesis of [ Ga]DOTA-tetrazine 2 and subsequent reaction with alkenes via the IDDA reaction to form dihydropyridazine products.
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In order to make a direct comparison with the SPAAC reaction, the reaction was also carried out in acetonitrile as solvent. However, rather than observing the expected dihydropyridazine products, a different product was formed, which had a similar retention time to tetrazine 2. This product is a result of a different cycloaddition reaction between the tetrazine and the nitrile moiety of acetonitrile, presumably by a similar mechanism to that proposed for the reaction of tetrazines with isonitriles.45 The effect of altering the side chain on the norbornene was also demonstrated, by reacting tetrazine 2 with a number of fluoroaniline derivatives (Table 2). These were isolated as separate endo and exo regioisomers before treating with the tetrazine. Commercially available norbornene 8 consists primarily of the endo isomer, showing the most impressive
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radiochemical conversions, possibly attributable to its relatively small side chain. Introducing the fluoroaniline side chain appeared to have a pronounced effect on the radiochemical conversion to the dihydropyridazines within 10 min, with the most significant difference observed with the exo isomers 9/11 (~20-25%). The endo 4-fluoroaniline analogue 10 showed a more comparable reaction rate to norbornene 8 (~50% vs. 80% within 10 min at 37 °C), which may be attributed to the reduced steric hindrance, when compared to the exo isomer, between the tetrazine and the double bond in the transition state for the cycloaddition (Figure 6). These compounds also demonstrated poor water solubility compared with compound 8, which may have additionally contributed to their reduced apparent reactivity.
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J. Label Compd. Radiopharm 2014, 57 291–297
H. L. Evans et al. Table 1. Results for the strain-promoted azide-alkyne cycloaddition reaction between [68Ga] 1 and cyclooctynes 3 and 4 R’
Solvent MeCN (1 % DMSO) H2O (10 % DMSO)
MeCN
Temperature (°C)
Time (minutes)
RCY (%)
37 90 37 90
15 10 10 10
94.2 100.0 15.2 97.3
37 50 90
15 15 10
51.4 79.2 99.4
RCY, radiochemical yields.
Figure 5. Four possible dihydropyridazine products formed from the IDDA reaction between tetrazine 2 and norbornene 8.
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In order to demonstrate the biocompatibility of the IDDA reaction, we also tested the reactivity of the tetrazine with L-tryptophan 13, an alkene-containing a-amino acid, which is found in high abundance in the human body. Reaction of tetrazine 2 with this compound under physiological conditions would hinder the use of this strategy in biological applications, such as pre-targeted imaging. However, despite containing a strained alkene, compound 13 did not show any reactivity towards out tetrazine under these conditions, eliminating this as a potential problem, and demonstrating further the reaction’s utility in biological applications. As with azide 1, tetrazine 2 has therefore been demonstrated as a useful prosthetic group for radiolabelling compounds using 68 Ga. The main advantages of the IDDA over the SPAAC reaction
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Tetrazine 2 was also reacted with an alkene, which does not contain any ring strain. Ethyl vinyl ether 12 showed approximately 10% conversion to the dihydropyridazine products from the tetrazine at 37 °C in 10 min, and increasing the temperature to 90 °C increased this conversion to nearly 30%. Although these results are not very impressive when compared with the strain-promoted reaction, and the reaction lacks the same levels of bioorthogonality, this demonstrates the usefulness of the 68Ga-labelled tetrazine for a wider range of labelling applications. It could be envisaged that these conversions could be greatly enhanced by optimisation of the reaction conditions; for example, through use of different co-solvents and by increasing the reaction temperatures and time.
H. L. Evans et al. Table 2. Results for the inverse electron-demand Diels-Alder reaction between tetrazine 2 and a series of alkenes Alkene
Solvent
Temperature (°C)
Time (minutes)
RCY (%)
37 37 37
10 20 10
79.9 93.6 0 (decomposition of tetrazine)
H 2O
37 37
10 20
25.5 39.0
H 2O
37
10
49.2
H 2O
37
10
20.3
H 2O
37 90
10 10
10.5 27.2
H 2O
37 37
10 60
0 0
H 2O MeCN
RCY, radiochemical yields.
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Figure 6. Proposed transition states for the favoured (exo) orientation between the tetrazine and norbornene, demonstrating a possible reason for the lower reactivity of the exo-substituted isomer because of increased steric hindrance.
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J. Label Compd. Radiopharm 2014, 57 291–297
H. L. Evans et al. include the improved compatibility with aqueous media, and the ability to achieve reasonable to high-radiochemical conversions at low temperatures (37 °C). Tetrazine 2 could be used for a number of applications, including labelling biomolecules containing strained alkenes such as norbornenes or trans-cyclooctenes.46
Conclusions Azide 1 and tetrazine 2 have both been demonstrated as useful prosthetic groups for radiolabelling of an array of small molecules via bioorthogonal chemistry. These could be applied to the labelling of suitably functionalised biomolecules, and it may be anticipated that this could be a useful method for pre-targeted imaging. The SPAAC reaction, although suffering from a lower tolerance towards aqueous media, gave promisingly high-radiochemical conversions to the triazoles in acetonitrile. The IDDA reaction is more compatible with physiological conditions, and for this reason may be considered advantageous in a wider range of applications.
Acknowledgements This work was supported by a studentship from the Imperial Cancer Research UK Centre grant (C37990/A12196), and by CR-UK&EPSRC Cancer Imaging Centre at Imperial College London, in association with the MRC and Department of Health (England) grant C2536/A10337 and UK Medical Research Council core funding grant U.1200.02.005.00001.01.
Conflict of Interest The authors did not report any conflict of interest.
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
297
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Copyright © 2013 John Wiley & Sons, Ltd.
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Accepted 29 October 2013
Published online 5 December 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3153
Bioorthogonal chemistry for 68Ga radiolabelling of DOTA-containing compounds† Helen L. Evans,a Laurence Carroll,a* Eric O. Aboagye,a and Alan C. Spiveyb Copper-catalysed ‘click’ chemistry is a highly utilised technique for radiolabelling small molecules and peptides for imaging applications. The usefulness of these reactions falls short, however, when metal catalysis is not a practically viable route; such as when using metal chelates as radioligands. Here, we describe a method for carrying out ‘click-type’ radiochemistry in the presence of DOTA chelates, by combining 68Ga radiolabelling techniques with well-established bioorthogonal reactions, which do not rely upon metal catalysis. Keywords: Bioorthogonal chemistry; Gallium-68; DOTA; click chemistry; cyclooctyne; azide; tetrazine; norbornene; strained alkene
Introduction
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a Comprehensive Cancer Imaging Centre, Department of Surgery & Cancer, Hammersmith Campus, Imperial College, London, UK b Department of Chemistry, Imperial College London, South Kensington Campus, London, UK
*Correspondence to: Laurence Carroll, Comprehensive Cancer Imaging Centre, Department of Surgery & Cancer, Hammersmith Campus, Imperial College, London, UK. E-mail: [email protected] †
This article is published in the Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on ‘Current Developments in PET and SPECT Imaging’, edited by Jonathan R. Dilworth, University of Oxford and Sofia I. Pascu, University of Bath.
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Bioorthogonal reactions have become increasingly investigated for the development of new biological and medicinal chemistry technologies. Amongst these, their employment for pre-targeted imaging has received significant attention, with particular focus on the strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, developed by Bertozzi, and the inverse electrondemand Diels-Alder (IDDA) reactions between tetrazines and strained alkenes.1–9 Here, we describe the development of two DOTA-based prosthetic groups for 68Ga-labelling one of which is reactive in SPAAC ligations (i.e. an azide) and the other towards IDDA ligations (i.e. a tetrazine). The utility of these prosthetic groups is illustrated by reactions with nine small molecule partners, under varying mild conditions of solvent, temperature and time. It can be envisioned that these prosthetic groups will be useful for the radiolabelling of biologically-active molecules, with the aim to use this strategy for pre-targeted positron emission tomography (PET) imaging. ‘Click’ chemistry refers to a set of reactions that are able to proceed under mild conditions and can be applied to the efficient synthesis of a broad variety of compounds, as exemplified in the work of Sharpless et al. and Meldal et al.10,11 Click reactions are modular and wide in scope, give consistently high yields, and form products that are stable under physiological conditions. The term is now more specifically applied to the highly regioselective copper (I) catalysed reaction between azides and terminal acetylenes to form 1,4disubstituted 1,2,3-triazoles, also known as the copper-catalysed azide-alkyne cycloaddition (CuAAC).12–14 This type of reaction usually involves the formation of copper(I) in situ from copper (II) salts with sodium ascorbate, and allows the preparation of a wide spectrum of 1,4-triazole products in high yield.15 The reaction has been utilised for numerous radiopharmaceutical applications, including the preparation of RGD,16–18 fluciclatide (and analogues),19,20 octreotide (and analogues)18,21,22 and other radiolabelled peptides for imaging applications. However, the use of the CuAAC reaction for the radiolabelling of peptides with metal isotopes is not feasible as copper(II) has a high affinity for
amines such as lysine, as well as cysteine and carboxylic acid residues, and has been demonstrated to be a useful cation for chelation to macrocycles such as cyclams.10,23–25 Any copper present forms a stable chelate with any unbound macrocycle, resulting in reduced concentration of the copper, and therefore preventing conversion to the triazole products; the use of excess of copper to circumvent this would prevent subsequent radiolabelling of the cyclam. Biocompatibility and fast reaction kinetics have, however, also been demonstrated by SPAAC reactions, which do not rely on copper catalysis, allowing formation of triazoles under mild conditions.6,8,26 The reduced activation energy for SPAAC reactions, relative to analogous non-strained examples, is a consequence of the strain imposed on the sp-hybridised carbons by the ring structure leading to bond angle distortions of about 17° from the idealised 180° angles of a C-C triple bond; these distortions result in comparable angles to those involved in the transition state of the cycloaddition (~160°).27–29 Cyclooctynes are the smallest ring size that can accommodate the alkyne functional group and still be isolated as a stable molecule, and were found to react in a highly selective manner with azides, demonstrating comparable reaction kinetics to the metalcatalysed reaction, but with no apparent toxicity.6 Superior reaction kinetics have now been demonstrated for IDDA
H. L. Evans et al. reactions between 1,2,4,5-tetrazines and strained alkenes, forming a mixture of dihydropyridazine isomers via a Diels-Alder/retroDiels-Alder reaction at rates that are reported to be 200 000 times faster than the SPAAC reaction in aqueous solvents and at ambient temperatures.30–33 The SPAAC and IDDA reactions have therefore been demonstrated to be useful methods for radiolabelling biomolecules, and do not rely on metal catalysis.6,34–36 18 F remains one of the most important isotopes for PET radiolabelling strategies because of its favourable half-life (109.7 min) and low energy, making it optimum in terms of PET image quality.37 However, the chemistry involved with the introduction of the 18F label into large biomolecules remains limited largely to the synthesis of prosthetic groups, whereby 18 F is introduced by coupling through use of the CuAAC reaction.38 The radiosynthesis of 18F labelled azides, such as 2-[18F]fluoroethyl azide may be achieved, and the SPAAC reaction with a series of cyclooctynes has been reported.39 However, the application of this chemistry to in vivo pre-targeted PET imaging has yet to be successfully demonstrated.4 The high volatility of 2-[18F]fluoroethylazide, coupled with its moderate, but not optimal radiochemical yields [~30 % nondecay-corrected (n.d.c)], encouraged us to find a more robust prosthetic group for the radiolabelling of small molecules and peptides using bioorthogonal chemistry.37,39 68 Ga, although having a shorter half-life (67.7 min) than 18F, and emitting a higher energy positron, has been utilised for the synthesis of radiolabelled probes for PET imaging.23 This is largely due to the relatively low cost of 68Ga, using a generator containing the parent radionuclide 68Ge (1/2 life = 271.0 days), which does not rely on the use of an on-site cyclotron facility, and these generators typically allow for the continuous production of high-quality 68Ga for approximately 1 year. The relatively simple and short radiolabelling methods that are used for metal isotopes, relying on chelating ligands attached to a targeting molecule, are more efficient than those for non-metal isotopes, and solid-phase purification methods are often used, minimising the problems associated with isotope decay during synthesis,.40 Here, we describe the design of two molecules based on the DOTA chelate (Figure 1), which have been functionalised with an azide 1 and a tetrazine 2, to allow two comparative bioorthogonal 68Ga labelled reactions to be examined under a variety of different conditions, with a number of different reactive partners. These studies constitute important proof-
of-concept for eventual use of these reactions as tools for pretargeted PET imaging.3,4,41
Experimental Chemistry ‘Cold’, unradiolabelled reference compounds were synthesised, and were used to confirm the products of the radiolabelled reactions (See supporting information for full experimental procedure and characterisation of ‘cold’ reference compounds).
Radiochemistry High-performance liquid chromatography (HPLC) traces for the radiolabelled products are reported in the supporting information. 68 68 GaCl3 was produced by an 18-month old Ge containing generator and was eluted in 0.1 M HCl (2 mL).
Synthesis of [68Ga]DOTA-azide 1 -1
A solution of DOTA-azide 1 (1 mg mL in DMSO) was diluted with a 68 NaOAc buffer solution (pH 4), and the GaCl3 was eluted directly into 68 the reaction vial (~1.5 mCi). The radiosynthesis of [ Ga]DOTA-azide was achieved in 10 min at 90 °C, in >95% radiochemical yield, and the product was obtained in >95% radiochemical purity by passing through a Sep-Pak light C18 cartridge. The mixture was then diluted with water or 68 acetonitrile for application to the Ga-labelled click reactions.
General procedure for
68
Ga-labelled SPAAC reactions
-1
To a 10 mg mL solution of cyclooctyne (3/4) (50 μL) in either H2O 68 (10% DMSO) or MeCN was added [ Ga]DOTA-azide 1 (100 μL), and the mixture was heated to either 37 °C or 90 °C for the given timepoint, without stirring. The progress of the reaction was monitored by radio-HPLC.
Synthesis of [68Ga]DOTA-tetrazine 2 -1
A solution of DOTA-tetrazine 2 (1 mg mL in DMSO) was diluted with a 68 NaOAc buffer solution (pH 6), and the GaCl3 was eluted directly into 68 the reaction vial (~1.5 mCi). The radiosynthesis of Ga DOTA-tetrazine was achieved in 15 min at 90 °C, in >95% radiochemical yield, and the product was obtained in >95% radiochemical purity by passing through a Sep-Pak light C18 cartridge. The mixture was then diluted with water or 68 acetonitrile for application to the Ga-labelled IDDA reactions.
General procedure for
68
Ga-labelled IDDA reactions
-1
To a 10 mg mL solution of alkene (8–13) (50 μL) in either H2O or MeCN 68 was added [ Ga]DOTA-tetrazine 2 (100 μL), and the mixture was heated to 37 °C for the given time-point, without stirring. The progress of the reaction was monitored by radio-HPLC.
Results and discussion Bioorthogonal chemistry with DOTA-azide 1
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Figure 1. Our DOTA-containing prosthetic groups containing an azide (1) and 68 tetrazine (2) functional group, for bioorthogonal radiolabelling using Ga.
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Azides are a useful class of bioorthogonal compound, which can be readily incorporated into a wide range of biological targets and because of their small size impart minimal structural perturbation.42 This functional group has many applications, with the most important of these being for use in click chemistry. As previously discussed, CuAAC reactions are difficult to perform efficiently in the presence of metal chelators, as these species can remove active copper from the reaction mixture. Instead, we designed [68Ga]DOTA-azide 1, with which we carried out a series of bioorthogonal SPAAC reactions, demonstrating their high yields and reproducibility. The radiosynthesis of 1 may be achieved in radiochemical conversions of greater than 95% n.d.c within 10 min, significantly
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H. L. Evans et al. higher yielding than the previously synthesised 2-[18F]fluoroethyl azide.37,39 Azide 1 was used for a series of SPAAC reactions with two analogues of aza-dibenzocyclooctyne (Figure 2).27,43,44 These cyclooctynes have been demonstrated as having superior reaction kinetics to the first generation cyclooctynes, owing to the increased strain accruing from the introduction of two aryl rings adjacent to the alkyne, and increased water solubility as the result of inclusion of a nitrogen atom into the cyclooctyne ring. The two cyclooctynes used in this study contained either a carboxylic acid (3) or maleimide functional group (4) to allow conjugation to the target biomolecules. (Figure 3 and 4) The carboxylic acid analogue 3 formed the triazole isomers in radiochemical conversions from the azide 1 of ~95% n.d.c at 37 °C in acetonitrile within 15 min, and higher temperatures (90 °C) allowed for complete conversion to the click products within just 10 min (Table 1). Cyclooctyne 3 was also able to perform fairly well in the SPAAC reaction with the azide in water, but the addition of 10% DMSO was required in order for the compound to dissolve fully. Low conversions to the triazoles were demonstrated in water at 37 °C (15% n.d.c) at short time-points, but heating to 90 °C enabled the triazoles to be formed in near to quantitative yields, possibly attributable to the increased solubility of the cyclooctyne at higher temperatures. The maleimide analogue 4 similarly demonstrated reasonable reactivity with azide 1 in acetonitrile, and gave near to quantitative conversions to the triazoles when heated to 90 °C. This compound demonstrated poor water solubility, even with the addition of 10% DMSO, and so the reactivity of this compound in aqueous media was not successfully demonstrated. In order to demonstrate the usefulness of the SPAAC reaction when radiolabelling with metal chelators, we also decided to compare these results to those obtained using the traditional CuAAC radiolabelling method, by reaction of our azide with an alkyne under standard CuAAC reaction conditions. An octreotate analogue was chosen for this purpose as this peptide is readily
radiolabelled using CuAAC chemistry by reacting with 2-[18F] fluoroethyl azide.21 E-TOCA (5) was therefore reacted with 68Ga azide 1 under the same reaction conditions to those reported for the 18F-labelling procedure,21 and showed no conversion to the triazole product. This lack of reactivity is attributed to the deactivation of the copper catalyst in the presence of excess DOTA chelate from solid-phase purification, a problem that is not observed in the copper-free version of the reaction. Overall, therefore, azide 1 has been demonstrated as a useful prosthetic group for the radiolabelling of cyclooctyne-containing compounds with 68Ga. The SPAAC reaction may be considered as an alternative method for labelling biomolecules with 68Ga, due to the demonstrated difficulties involved in using the CuAAC in this context. These reactions may be particularly useful when organic solvents can be tolerated (e.g. acetonitrile), and pre-targeted labelling may be achievable if the cyclooctyne is attached to a water-soluble biomolecule. Bioorthogonal chemistry with DOTA-tetrazine 2 Recently, the IDDA reaction between tetrazines and strained alkenes has been demonstrated as a potentially useful bioorthogonal ligation method.1,32,34,35 With this in mind, we designed a DOTA with an appended tetrazine motif 2, which could be used to make a comparison with the 68Ga-labelled SPAAC reaction described earlier. The norbornene core has been shown to react rapidly with tetrazines, because of the large amount of strain contained in the alkene functional group, forming the dihydropyradazine products rapidly in aqueous media.1,2 We initially demonstrated this with DOTA-tetrazine 2, by observing almost 80% radiochemical conversion to the dihydropyradazine isomers (Figure 5) within just 10 min at 37 °C in aqueous media, and complete conversion to the four product isomers; within 20 min under the same conditions, using commercially available norbornene 8 as the strained alkene (Table 2).
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68
Figure 2. Radiosynthesis of [ Ga]DOTA-azide 1 and subsequent SPAAC reaction with cyclooctynes to form triazole products.
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Figure 3. Attempted CuAAC reaction with [ Ga]DOTA-azide 1.
68
Figure 4. Radiosynthesis of [ Ga]DOTA-tetrazine 2 and subsequent reaction with alkenes via the IDDA reaction to form dihydropyridazine products.
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In order to make a direct comparison with the SPAAC reaction, the reaction was also carried out in acetonitrile as solvent. However, rather than observing the expected dihydropyridazine products, a different product was formed, which had a similar retention time to tetrazine 2. This product is a result of a different cycloaddition reaction between the tetrazine and the nitrile moiety of acetonitrile, presumably by a similar mechanism to that proposed for the reaction of tetrazines with isonitriles.45 The effect of altering the side chain on the norbornene was also demonstrated, by reacting tetrazine 2 with a number of fluoroaniline derivatives (Table 2). These were isolated as separate endo and exo regioisomers before treating with the tetrazine. Commercially available norbornene 8 consists primarily of the endo isomer, showing the most impressive
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radiochemical conversions, possibly attributable to its relatively small side chain. Introducing the fluoroaniline side chain appeared to have a pronounced effect on the radiochemical conversion to the dihydropyridazines within 10 min, with the most significant difference observed with the exo isomers 9/11 (~20-25%). The endo 4-fluoroaniline analogue 10 showed a more comparable reaction rate to norbornene 8 (~50% vs. 80% within 10 min at 37 °C), which may be attributed to the reduced steric hindrance, when compared to the exo isomer, between the tetrazine and the double bond in the transition state for the cycloaddition (Figure 6). These compounds also demonstrated poor water solubility compared with compound 8, which may have additionally contributed to their reduced apparent reactivity.
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H. L. Evans et al. Table 1. Results for the strain-promoted azide-alkyne cycloaddition reaction between [68Ga] 1 and cyclooctynes 3 and 4 R’
Solvent MeCN (1 % DMSO) H2O (10 % DMSO)
MeCN
Temperature (°C)
Time (minutes)
RCY (%)
37 90 37 90
15 10 10 10
94.2 100.0 15.2 97.3
37 50 90
15 15 10
51.4 79.2 99.4
RCY, radiochemical yields.
Figure 5. Four possible dihydropyridazine products formed from the IDDA reaction between tetrazine 2 and norbornene 8.
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In order to demonstrate the biocompatibility of the IDDA reaction, we also tested the reactivity of the tetrazine with L-tryptophan 13, an alkene-containing a-amino acid, which is found in high abundance in the human body. Reaction of tetrazine 2 with this compound under physiological conditions would hinder the use of this strategy in biological applications, such as pre-targeted imaging. However, despite containing a strained alkene, compound 13 did not show any reactivity towards out tetrazine under these conditions, eliminating this as a potential problem, and demonstrating further the reaction’s utility in biological applications. As with azide 1, tetrazine 2 has therefore been demonstrated as a useful prosthetic group for radiolabelling compounds using 68 Ga. The main advantages of the IDDA over the SPAAC reaction
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Tetrazine 2 was also reacted with an alkene, which does not contain any ring strain. Ethyl vinyl ether 12 showed approximately 10% conversion to the dihydropyridazine products from the tetrazine at 37 °C in 10 min, and increasing the temperature to 90 °C increased this conversion to nearly 30%. Although these results are not very impressive when compared with the strain-promoted reaction, and the reaction lacks the same levels of bioorthogonality, this demonstrates the usefulness of the 68Ga-labelled tetrazine for a wider range of labelling applications. It could be envisaged that these conversions could be greatly enhanced by optimisation of the reaction conditions; for example, through use of different co-solvents and by increasing the reaction temperatures and time.
H. L. Evans et al. Table 2. Results for the inverse electron-demand Diels-Alder reaction between tetrazine 2 and a series of alkenes Alkene
Solvent
Temperature (°C)
Time (minutes)
RCY (%)
37 37 37
10 20 10
79.9 93.6 0 (decomposition of tetrazine)
H 2O
37 37
10 20
25.5 39.0
H 2O
37
10
49.2
H 2O
37
10
20.3
H 2O
37 90
10 10
10.5 27.2
H 2O
37 37
10 60
0 0
H 2O MeCN
RCY, radiochemical yields.
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Figure 6. Proposed transition states for the favoured (exo) orientation between the tetrazine and norbornene, demonstrating a possible reason for the lower reactivity of the exo-substituted isomer because of increased steric hindrance.
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H. L. Evans et al. include the improved compatibility with aqueous media, and the ability to achieve reasonable to high-radiochemical conversions at low temperatures (37 °C). Tetrazine 2 could be used for a number of applications, including labelling biomolecules containing strained alkenes such as norbornenes or trans-cyclooctenes.46
Conclusions Azide 1 and tetrazine 2 have both been demonstrated as useful prosthetic groups for radiolabelling of an array of small molecules via bioorthogonal chemistry. These could be applied to the labelling of suitably functionalised biomolecules, and it may be anticipated that this could be a useful method for pre-targeted imaging. The SPAAC reaction, although suffering from a lower tolerance towards aqueous media, gave promisingly high-radiochemical conversions to the triazoles in acetonitrile. The IDDA reaction is more compatible with physiological conditions, and for this reason may be considered advantageous in a wider range of applications.
Acknowledgements This work was supported by a studentship from the Imperial Cancer Research UK Centre grant (C37990/A12196), and by CR-UK&EPSRC Cancer Imaging Centre at Imperial College London, in association with the MRC and Department of Health (England) grant C2536/A10337 and UK Medical Research Council core funding grant U.1200.02.005.00001.01.
Conflict of Interest The authors did not report any conflict of interest.
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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