18F-Labeling Using Click Cycloadditions

Due to expanding applications of positron emission tomography (PET) there is a demand for developing new techniques to introduce fluorine-18 (t 1/2 = 109.8 min). Considering that most novel PET tracers are sensitive biomolecules and that direct introduction of fluorine-18 often needs harsh conditions, the insertion of 18F in those molecules poses an exceeding challenge. Two major challenges during 18F-labeling are a regioselective introduction and a fast and high yielding way under mild conditions. Furthermore, attention has to be paid to functionalities, which are usually present in complex structures of the target molecule. The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and several copper-free click reactions represent such methods for radiolabeling of sensitive molecules under the above-mentioned criteria. This minireview will provide a quick overview about the development of novel 18F-labeled prosthetic groups for click cycloadditions and will summarize recent trends in copper-catalyzed and copper-free click 18F-cycloadditions.


Introduction
For the application in positron emission tomography (PET) [1], fluorine-18 provides ideal nuclear physical characteristics for in vivo imaging. Fluorine-18 offers a half-life of 110 min, a + -branch of 97%, and especially a low + -energy of 635 keV, which is responsible for a very high spatial resolution [2]. The challenges for researchers are to develop convenient 18 F-labeling strategies, which include short reaction times and applicability for sensitive biomolecules. Especially the harsh conditions during direct 18 F-labeling pose an exceeding challenge [3,4]. Therefore, most of the radiolabeling strategies focus on 18 F-containing prosthetic groups, which allow a sensitive and bioorthogonal 18 F-labeling to treat the multitude of functional groups in those bioactive compounds with respect.
The most established method, which fulfills all mentioned criteria, is given by click reactions. Especially the Cu(I)catalyzed variant of the Huisgen 1,3-dipolar cycloaddition of terminal alkynes and azides offers a very powerful reaction with high specificity and excellent yields under mild conditions [5]. As a result, numerous PET tracers have been synthesized using CuAAC in a widespread spectrum of structural varieties of the prosthetic group within the last decade. One of the latest investigations deals with a polar clickable amino acid-based prosthetic group to further improve the pharmacokinetic properties of radiotracers, particularly suitable for peptides and proteins [6].
However, the need of cytotoxic copper during CuAAC has led to the necessity of alternative fast and copper-free click reaction strategies for radiofluorination and additionally enabling pretargeting approaches in living systems. Those so-called strain-promoted click reactions can be carried out between cyclooctyne derivatives and azides (strain-promoted azide-alkyne cycloaddition, SPAAC) [7][8][9][10][11][12][13] or tetrazines (tetrazine-trans-cyclooctyne (TTCO) ligation) [14][15][16][17] as well as between norbornene derivatives and tetrazines [18]. Especially, the TTCO ligation showed promising reaction rates, which makes this click reaction concept very suitable for 18 Flabeling and also for in vivo application in living systems. Very recently, new versions of 18 F-click cycloadditions are added to the range of reactions [19][20][21][22][23][24][25]. In this line, the first 18 Flabeled -lactame became available via a new radio-Kinugasa reaction [21].   As a consequence, click cycloaddition is one of the most frequently applied methods for 18 F-labeling of new bioactive compounds, with or without a catalytic system. This can be impressively illustrated by the fact that over 50 original papers have been published in this research area within the last eight years. Tables 1-3 give an overview of the 18 F-prosthetic groups, the reaction conditions and reaction partners applied for copper-catalyzed, copper-free and other kinds of 18 F-click cycloadditions, respectively. The most important structures of those prosthetic groups are shown in Figures 1, 3, and 5.
In 2006, Marik and Sutcliffe published the application of the CuAAC as an 18 F-labeling strategy for the first time [26]. They radiolabeled three different alkyne precursors in radiochemical yields (RCY) of 36-81%. Afterwards they were  Calculated as sum from all steps, for the 18 F-prosthetic group, respectively, for the overall reaction leading to the click product, starting from fluorine-18.  With respect to the catalytic system copper sulfate in combination with ascorbic acid or sodium ascorbate has mainly been used, whereas only in a few approaches copper(I) iodide was used [37,42]. It has been shown that addition of bathophenanthroline disulfonate (Cu I stabilizing agent) accelerates the 1,3-dipolar cycloaddition [36,38,45]. The very good access to [ 18 F]FEA led to the development of a variety of radiotracers labeled with this prosthetic group, like 18 Fdeoxyuridine [37], 18 F-fluoro-oxothymidine ( 18 F-FOT), or 18 F-fluoro-thiothymidine ( 18 F-FTT) [40] as well as apoptosis markers [36] and several peptide systems [34,44,49]. In 2012, Smith et al. [40] described the reduction of [ 18 F]FEA using copper wire under acidic conditions, which is a possible explanation of the poor yields during some click reactions.
In 2007, Sirion et al. [50] reported for the first time [ 18 F]fluoro-PEG x -derivatives ( = various polyethylene glycol (PEG) ratios) as new 18 F-labeled prosthetic click groups. These compounds showed a reduced volatility and increased polarity compared with other 18 F-labeled prosthetic groups like [ 18 F]FEA or [ 18 F]fluoroalkynes. These properties ease their handling as well as improving the in vivo behavior of the labeled compounds. The compounds showed a longer circulation time and a reduced renal clearance making them very suitable for in vivo application. Sirion et al. described the preparation of different aliphatic and aromatic 18 F-PEGazides and 18 F-labeled alkynes in RCY of 85-94%. As a proof of concept, they carried out cycloadditions with the 18 Flabeled prosthetic groups and the corresponding alkynes, respectively, azides in high RCY of 71-99%. Several other groups continued this work by using the 18 F-labeled PEGylated prosthetic groups for labeling cRGD derivatives [51] and other peptides [53], nanoparticles [52,54], or folates [55].

To increase the lipophilicity and metabolic stability of radiotracers, [ 18 F]fluoro-aryl-based prosthetic groups have been developed and investigated. In 2007, Ramenda et al. [56] published for the first time a 4-[ 18 F]fluoro-N-methyl-N-(prop-2-ynyl)-benzenesulfonamide (p-[ 18 F]F-SA)
, which was obtained in RCY of 32 ± 5%. Subsequently, this prosthetic group was used for radiolabeling an azido-functionalized neurotensin giving a RCY of 66%. Furthermore, the same group used the 18 F-aryl prosthetic group for the labeling of human serum albumin (HSA) [57] and other proteins, phosphopeptides, and L-RNA [58] in good RCY. A pyridine-based 18 F-prosthetic group was first introduced by Inkster et al. [59] [63], who synthesized 4-[ 18 F]fluoro-3-nitro-N-2-propyn-1yl-benzamide ([ 18 F]FNPB) for 18 F-labeling of cRGDfK and a D4 peptide, which was identified as an EGFR targeting ligand. This approach was followed by the synthesis of 1-(azidomethyl)-4-[ 18 F]fluorobenzene by Thonon et al. [64]. They did a multistep radiosynthesis (4 steps), where the fluorine-18 was introduced in the first step. The desired radiolabeled product could be obtained in a RCY of 34% within 75 min and was used itself to label a 4-ethynyl-L-phenylalanine-containing peptide. The same prosthetic group was also employed by Mercier et al. [65] and Flagothier et al. [66] for 18 F-labeling of siRNA. Other structural analog prosthetic groups have also been developed by Mercier et al. [65] and Chun and Pike [67].
To improve the in vivo behavior of peptides with respect to blood clearance and stability, Maschauer and Prante developed 18 F-gluco-derivatives for CuAAC-radiolabeling of Fmoc-L-propargylglycine with a RCY of 60% [68]. They showed that the 18 F-click labeling reaction was more convenient by using the -anomeric derivative of the azides, respectively, alkynes, giving very high RCY of 71 ± 10%.  [78]. Spiro salts were used as precursors, facilitating purification by using solid phase extractions (RP-18 or SiO 2 -cartridges). Both prosthetic groups could be obtained in RCY of about 30% using an automated synthesis module. To avoid Glaser coupling, which has been observed by using [ 18 F]BFP for radiolabeling of peptides, [ 18 F]AFP was used instead. An important observation was the fact that the applied peptide formed very strong complexes with the copper catalyst, which required the use of bispidine as a strong chelating agent to remove cytotoxic copper species.
One of the latest developments describes the synthesis of an 18 F-labeled alanine derivative as a new prosthetic click group, reported by Schieferstein and Ross [6]. In this case, an amino acid-based prosthetic group has been developed to improve the pharmacokinetic profile of 18 F-click-labeled biomolecules. The prosthetic group was obtained in good RCY of 28 ± 5% from a two-step reaction as described in Figure 2. The final 18 F-labeled prosthetic group was subsequently reacted with an azido-RGD as model system in RCY of 75% within 20 min.
Considering the above-mentioned prosthetic groups for radiolabeling with fluorine-18, Table 1 summarizes important properties of those components. It has been shown that the integration of an 18 F-propyl, 18 F-ethyl, or 18 F-aryl moiety can provide an improved metabolic profile and that the glycosylation or PEGylation can further improve the in vivo behavior. Furthermore, for in vivo application a total removal of the copper catalyst is essential. This could be very challenging in the case where peptides or proteins are able to complex copper species from the catalytic system.

Copper-Free 18 F-Click Cycloadditions
Even though a large number of novel radiotracers using click chemistry have been developed, none of them has entered clinical routine to date, apart from 18 F-RGD-K5, which is already used in clinical trials in US. This can be explained by the need of cytotoxic copper during radiotracer syntheses by using copper-catalyzed 1,3-dipolar Huisgen cycloadditions [96]. Thus, there is still a demand for facile (metal-free) and robust 18 F-labeling reactions for the syntheses of radiotracers for imaging of malignancies in vivo. This leads to the development of catalyst-free click-labeling approaches, which spare copper species during labeling steps and even enable in vivo pretargeting concept. Recent developments deal with biocompatible strain-promoted copper-free versions of the alkyne-azide cycloaddition (SPAAC), where the focus has been set on derivatives of cyclooctynes and dibenzocyclooctynes. First approaches focus on the reaction of 18 F-labeled cyclooctynes with azide-bearing biomolecules. On the other hand, in further approaches cyclooctyne-carrying bioactive compounds are used, which can be labeled with different 18 Flabeled azides. In the beginning, only a few studies have been reported due to the complex and low yielding syntheses of strained cyclooctynes [10,12,14]. However, nowadays lots of cyclooctyne derivatives are commercially available, which facilitates the precursor syntheses and opens a wide range of applications.
In 2011 Bouvet et al. [7] published the first example of a SPAAC with 18 F-labeled aza-dibenzocyclooctyne,  [97]. The 18 F-labeled cyclooctyne could be obtained in a RCY of 85% and a purity >95% within 60 min. The evaluation of this building block in healthy Balb/C mice showed 60% of intact compound at 60 min p.i. and had a blood clearance half-life of 53   A cyclooctyne derivative has been conjugated to bombesin (aza-DBCO-BN, 9 steps) with an overall yield of 17% by Campbell-Verduyn et al. [8]. The aza-DBCO-BN was reacted with various 18 F-azides giving RCY of 19-37% within 30 min. In 2011, Arumugam et al. [9] investigated the direct 18 F-labeling of azadibenzocyclooctyne (DBCO) yielding the 18 F-labeled prosthetic group (RCY = 36%). The radiolabeling was followed by a click reaction with an azido-octreotide leading to the 18 F-labeled octreotide in a RCY of 95% within a total reaction time of 1.5 h. In contrast, other working groups used 18 F-cyclooctynes for labeling RDG-derivatives [11] as well as further integrin-specific peptides [10,13].
Another possibility to perform copper-free click reactions is given by the inverse electron demand of the Diels Alder cycloaddition between a cyclooctene and a tetrazine under the release of nitrogen. The so-called tetrazine-transcyclooctene ligation (TTCO ligation) was first published by Li et al. in 2010 [14]. Concerning the instability of the tetrazines, it is more practical to functionalize the biomolecule with a tetrazine followed by the reaction with an 18 F-labeled cyclooctene. The latter are much more suitable for direct 18 Flabeling than tetrazines. For this purpose a nosylate precursor was used for 18 F-labeling of the cyclooctene providing RCY of 71% within 15 min. To investigate the suitability of the 18 Fprosthetic group in click reactions, the 18 F-cyclooctene was reacted with a 3,6-di(2-pyridyl)-S-tetrazine in an excellent RCY of 98% within 10 s, showing its outstanding feasibility for in vivo pretargeting approaches. These fast reaction rates made this approach very attractive that even 11 Clabeling reaction was explored using the inverse electron demand Diels Alder cycloaddition between a cyclooctene and a tetrazine [98]. In 2011, 18 F-labeled cyclooctene was linked to a tetrazine-RGD derivative by Selvaraj et al. [15] with a RCY of 90% within 5 min at room temperature. The resulting 18 F-labeled tracer was tested in in vivo experiments showing a high tumor accumulation, which could selectively be blocked. In 2012, the group of Devaraj et al. [80] published for the first time the in vivo click reaction of [ 18 F]transcyclooctene and a polymer-modified tetrazine (PMT). The radiolabeled peptide 18 F-PMT10 could be obtained in a RCY of 89.2%. Whole body animal PET scans were carried out 3 h p.i., showing renal clearance and a widespread tissue distribution as can be seen in Figure 4. Previously, the same group described the synthesis of an 18 F-labeled cyclooctene with a RCY of 46.1 ± 12.2%. Subsequently, this prosthetic group was clicked with a tetrazine-modified exendin-4 in RCY of 46.7 ± 17.3% [16].

New Developments in 18 F-Click Cycloadditions
The latest developments in metal-free 18 F-click cycloadditions have been reported by Zlatopolskiy et al. [19][20][21] ( After the click [3+2]cycloaddition to various 18 F-labeled model 2-isoxazolines and isoxazoles was successfully tested, the novel method was applied to three different COX-2 inhibitors (indomethacin conjugates) carrying dipolarophilic moieties of cyclononyne, maleimide, and propyne. The resulting products were obtained in moderate to excellent RCY of 81%, 55%, and 35%, respectively. It is noteworthy that, for the propyne derivative, the milder oxidant [bis(acetoxy)iodo]benzene was used to avoid decomposition. Finally, the method was successfully adapted for 18 Flabeling of two model dipeptide conjugates, cyclononyneand norbornene--Ala-Phe-OMe. However, the original cycloaddition using 4-[ 18 F]fluorobenzonitrile oxide did only provide traces of the desired products. Consequently, 4-[ 18 F]fluorobenzonitrile oxide was further treated with chloramine T (CAT) in situ forming the more stable building block N-hydroxy-4-[ 18 F]fluorobenzimidoyl chloride. With the use of high precursor (peptides) amounts, the latter enabled excellent RCY of the 18 F-labeled dipeptides of up to 88% within 10 min at room temperature [20]. Under optimized conditions low precursor amounts of 5 nmol (cyclononyne) and 50 nmol (norbornene--Ala-Phe-OMe) still allowed RCY of 56% and 47%, respectively.
In a very recent report, Zlatopolskiy and coworkers applied their 18 F-labeled nitrone, C-(4-[ 18 F]fluorophenyl)-N-phenyl nitrone, for the first formation of 18 F-labeled -lactames via the CuI-catalyzed Kinugasa reaction [21] ( Table 3). The optimized reactions went smooth under very mild conditions to give the 18 F-labeled model -lactames in high RCY and various isomeric mixtures of the trans-and cis-product. In dependency on the reactivity of the terminal alkynes, the reaction parameters needed (individual) optimization regarding catalyst system, solvent, temperature, and CuI-stabilizing ligands. As a biologically relevant molecule the 18 Flabeled nucleobase chimera was synthesized as potential PET-imaging agent for bacterial infections.
Moreover, the dipeptide -Ala-Phe-OMe was propiolated and used in this radio-Kinugasa reaction to give excellent RCY of 85% of the 18 F-labeled dipeptide under very mild conditions (aqueous solution, room temperature) [21]. Similarly, this new method was successfully transferred to the 18 F-labeling of proteins. Bovine serum albumin (BSA) was conjugated with 3-propiolamidopropyl chloroformate. This propiolated BSA was successfully radiolabeled with fluorine-18 in the radio-Kinugasa reaction.

Conclusions
The field of click cycloadditions had and still has a major impact in 18 F-labeling chemistry. The very mild reaction conditions mostly applicable and the excellent efficiency of all types of these reactions are particularly suitable for 18 Flabeling. Especially, complex and sensitive biomolecules benefit from this methodology. No protection group chemistry is needed and the 18 F-click cycloaddition step provides the final radiotracer.
Besides several new 18 F-labeled radiotracers are available via click cycloadditions, and the metal-free versions even enabled pretargeting concepts by in vivo click. The latest development of a radio-Kinugasa reaction towards the first 18 F--lactames demonstrates the highly active field and the broad applicability of 18 F-click cycloadditions.