Recent Trends in Bioorthogonal Click-Radiolabeling Reactions Using Fluorine-18

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile bioorthogonal conjugation techniques especially for the radiolabeling of biologically active high molecular weight compounds like peptides, proteins or antibodies. Taking into consideration that the introduction of fluorine-18 (t1/2 = 109.8 min) proceeds under harsh conditions, radiolabeling of these biologically active molecules represents an outstanding challenge and is of enormous interest. Special attention has to be paid to the method of 18F-introduction. It should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. For these reasons and due to the high number of functional groups found in these compounds, a specific labeling procedure has to be developed for every bioactive macromolecule. Bioorthogonal strategies including the Cu-assisted Huisgen cycloaddition and its copper-free click variant, both Staudinger Ligations or the tetrazine-click reaction have been successfully applied and represent valuable alternatives for the selective introduction of fluorine-18 to overcome the afore mentioned obstacles. This comprehensive review deals with the progress and illustrates the latest developments in the field of bioorthogonal labeling with the focus on the preparation of radiofluorinated building blocks and tracers for molecular imaging.


Introduction
Positron emission tomography (PET) is an outstanding instrument for the quantification and localization of physiological as well as pathophysiological activities and processes in vivo which were analyzed by tracing the appropriate biochemical fundamentals [1]. The basics of PET rely on the coincidental detection of annihilation photons in nearly 180° originating from positrons of the parent radionuclide inside the tracer [2]. Measurements and quantifications of the tracer distribution were accelerated noninvasively in living organisms. For that purpose, fluorine-18 produced by a cyclotron was chosen as the ideal radionuclide due to its favorable half-life. Furthermore, the low positron energy and short ranges in tissues lead to high image resolution [3]. However, tracers for PET imaging purposes are restricted by the kind of fluorinated molecules that researchers can prepare.
In principle, radiolabeling of biologically active compounds and especially high molecular weight compounds like peptides, proteins and antibodies with fluorine-18 still represents a considerable challenge and has therefore generated immense interest. As a result of harsh reaction conditions for the direct introduction of fluorine-18 at high specific activity levels, novel radiolabeling strategies comprising fluorine-18-containing prosthetic groups, also referred as 18 F-labeling building blocks, are necessary. Due to this fact and due to the multitude of functional groups found in bioactive macromolecules, a labeling strategy has to be developed for almost every one of these compounds. To overcome the above mentioned obstacles, bioorthogonal click-labeling reactions were developed and successfully applied to allow the selective introduction of fluorine-18 under mild conditions, with high radiochemical yields (RCY) and high specific activity (A S ) [4][5][6][7][8].
The concept of bioorthogonal syntheses refers to all reactions which can proceed inside of living systems without any interference with native biochemical processes. Several requirements have to be fulfilled for a reaction to be considered as bioorthogonal [9]. First and most prominently, selective reactions should be applied to avoid side-reactions with other functional groups found in the biological starting compounds. Both functional groups of the participating reaction partners must be inert to the residual biological moieties and should only react with each other. Secondly, the resulting covalent bond should be strong and chemically inert to biological reactions, and, in addition, it should not affect the (native) biological behavior of the desired molecule. Thirdly, the reaction should benefit from fast reaction kinetics in the case that covalent ligations are achieved prior to probe metabolism and clearance and the reaction must be run on the time scale of cellular processes to prevent biological side reactions. This point is especially important both for the work with short-living positron emitters and further for an application of in vivo pre-targeting strategies. At least, the non-toxicity of the reaction partner as well as catalysts are of high importance for in vivo applications, and the reaction should proceed under biological conditions taking into account pH, aqueous environments, and temperature.
Giving consideration to the criteria of bioorthogonal reactions, Kolb, Finn and Sharpless [10,11] defined the click chemistry as a "set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compounds and combinatorial libraries" and pointed out the following requirements for click reactions: modularity, wideness in scope, very high yielding, stereospecificity (but not necessarily enantioselectivity) and simple product isolation (separation from harmless by-products by non-chromatographic methods). In addition to these criteria, these reactions should proceed using simple reaction conditions (solvent free or solvents like water) including simply accessible starting materials, and the final product has to be stable under physiological conditions [12]. Selected examples of click chemistry reactions are pointed out in Scheme 1.

Scheme 1.
Selected examples of chemical conjugations associated with click chemistry. All reactions highlighted in grey fulfill the bioorthogonality criteria. In recent years, the terms "bioorthogonal reactions" and "click chemistry" have entered into the field of radiochemistry and radiopharmacy. Bioorthogonal reactions in general, and click chemistry in particular, are generic terms for a set of labeling reactions, which make use of several selective and modular building blocks and enable chemoselective ligations to radiolabel biologically relevant compounds. In this context, the Cu(I)-mediated triazole formation from azides and terminal alkynes according to the 1,3-dipolar Huisgen cycloaddition is a particularly powerful ligation reaction, due to its high degree of specificity and the biocompatibility of both starting materials. In the same way, the strain promoted Huisgen click-reaction as well as both variants of the Staudinger Ligation were applied. The absence of catalysts makes these reactions together with the inverse Diels-Alder tetrazine-click reaction tremendously attractive for radiolabeling purposes. Neither azides nor phosphanes, alkenes or tetrazines react with other functional groups commonly present in biopolymers like peptides or proteins. Hence, there is no need for protective group chemistry. Moreover, the desired linking moieties (triazoles, amides or cyclooctapyridazines) are metabolically stable under physiological conditions. As a consequence, click chemistry is a very attractive approach for the design and synthesis of novel potent radiotracers for molecular imaging purposes.

Fluorine-18 in General
Fluorine-18, with a medium half-life of 109.8 min, is one of 17 known radioisotopes [13] and it was introduced as the most prominent radionuclide for PET imaging [14][15][16][17][18][19][20][21]. The low positron energy (E β+ = 0.64 MeV) combined with a short range of the positron in tissue (max. 2.4 mm) provides high resolution images. It can be prepared via cyclotron using the 18 O(p,n) 18 F nuclear reaction in a maximum specific activity (A S ) of 63.3 TBq/µmol no carrier added (n.c.a.) as [ 18 F]fluoride or by the basis for nucleophilic ([ 18 F]fluoride) as well as electrophilic ([ 18/19 F]F 2 ) labeling reactions. In general, the respective radiotracers were achieved with a lower A S using electrophilic labeling reactions due to the addition of F 2 -gas into the Ne-target [23]. For a successful application of [ 18 F]fluoride for radiolabeling purposes, its solubility, nucleophilicity and lipophilicity has to be increased. Standard conditions for the nucleophilic introduction of fluorine-18 generally require: (i) a base such as carbonate, bicarbonate, hydroxide or oxalate, (ii) a phase transfer catalyst such as Kryptofix ® K 2.2.2. or tetraalkylammonium salts and finally (iii) a dipolar aprotic solvent such as acetonitrile, DMSO, DMF [24] or alternatively, tert-butanol [25].
In most of the cases, radiolabeling with fluorine-18 is achieved by the bioisosterical exchange of a single hydrogen or OH groups. This is due to the fact that fluorine is absent in most biologically active compounds. Only weak steric disturbances occur because fluorine is only slightly larger (1.47 Å) than protons (1.20 Å) and somewhat smaller than oxygen (1.52 Å). Covalently bound fluorine occupies a smaller volume than methyl, amino, or hydroxyl groups, making it favorable for incorporation [26,27]. Next, the resulting C-F bond (453 kJ/mol) is stronger in contrast to the corresponding C-H bond (432 kJ/mol) or C-O bond (378 kJ/mol) [28]. Further, fluorine can also be involved to form hydrogen bonds, but with the difference that fluorine is only a hydrogen bond acceptor compared to a OH group. Notably, a change of the physiological and biological behavior of the fluorine-containing molecule to the desired biological target is often observed in combination of its metabolic stability and lipophilicity.

Cu-Catalyzed 1,3-Dipolar Huisgen Cycloaddition
1893 was the year of birth of the 1,3-dipolar Huisgen cycloaddition as Arthur Michael described for the first time the preparation of 1,2,3-triazoles from phenylazide and dimethyl but-2-ynedioate [29]. The reaction of hydrogen azide with acetylene to give the corresponding 1,2,3-triazole was first described by Dimroth and Fester 17 years later [30]. Based on this work, Huisgen investigated and specified the character of this reaction as a 1,3-dipolar addition in 1963 [31]. Arbitrary alkynes are used as first reaction partners and serve as dipolarophiles in this reaction. Azides as second reaction partners are ambivalent compounds only describable as zwitterions in an all-octet formula. The driving force for the formation of triazoles in particular is the disappearance of the charges in the azide [32].
Normally, organic azides react with alkynes at high temperatures to give 1,2,3-triazoles in the Huisgen cycloaddition with formation of both 1,4-and 1,5-disubstituted regioisomers. In 2002, the groups of Sharpless et al. [33] and Meldal et al. [34] independently introduced a revolutionary innovation into the field of 1,3-dipolar Huisgen cycloaddition due to the application of Cu(I)-species as catalyst. The so called copper-catalyzed azide alkyne cycloaddition (CuAAC) proceeds faster, delivers the respective 1,4-isomer exclusively and can be performed at ambient temperatures. Both groups assumed a mechanism in which Cu(I) coordinates first to the terminal alkyne forming a copper(I)acetylide. Computer calculations confirmed these presumptions and predicted a nonconcerted mechanism through a six-membered copper(III) metallacycle [35]. Newer findings using continuous-flow analysis cannot exclude a primary addition of the Cu(I) to the azide moiety [36]. Nowadays, Worrel et al. have suggested a new mechanism discovered by utilizing 63 Cu-enriched catalysts. Thus, two copper atoms are incorporated in the catalytic cycle [37] as seen in Scheme 2.

Scheme 2.
Proposed mechanism of the copper-catalyzed 1,3-dipolar Huisgen cycloaddition (CuAAC) catalyzed by two copper atoms. As a result of its favorable properties and reaction conditions, the CuAAC has gained enormous popularity and has been applied in diverse fields of chemistry [38]. Since 2006, the CuAAC is introduced for labeling purposes of biologically and pharmacologically relevant molecules with fluorine-18 (Scheme 3). Up until now, over 60 works have been published using this 1,3-dipolar Huisgen cycloaddition for 18 F-labeling of small organic compounds, peptides, oligonucleotides, proteins and nanoparticles which were partially used for PET studies [5,6,39,40]. The first application of click chemistry for the design and synthesis of radiometal-based radiotracers to form novel multidentate triazole ligand scaffolds for an efficient chelation of the 99m Tc(CO) 3 core was reported by the group of Schibli in 2006 [41]. In 2008, the first application of the CuAAC for the introduction of carbon-11 using [ 11 C]methyl azide was published by Schirrmacher and co-workers [42].
The bioorthogonality of both functional groups used in the Huisgen cycloaddition makes this reaction to connect 18 F-containing building blocks to sensitive bioactive molecules like peptides or proteins highly attractive. Problematically, copper is known to be cytotoxic towards prokaryotic and eukaryotic cells. It is reported that viruses and oligonucleotides are damaged or destroyed in its presence [43]. Other side effects that it is associated with include hepatitis, Alzheimer's disease or neurological disorders. Therefore, it is of enormous importance for in vivo applications to remove the copper completely from the desired radiotracer or to develop copper-free, bioorthogonal alternatives [44].
The first application of the CuAAC in the field of fluorine-18-radiochemistry and -radiopharmacy was published by Marik and Sutcliffe in 2006. They described the synthesis of three 18  In addition to that case, effects of these labeling moieties on PET imaging and pharmacokinetics were evaluated. In conclusion, the [ 18 F]FBA-labeled peptide was obtained in 22 ± 4% RCY and the [ 18 F]FPA-incorporated peptide was prepared in 13 ± 3% RCY. In contrast, labeling using [ 18 F]2b resulted in only 10% RCY for the respective labeled peptide. Therefore, conventional labeling on solid support should be preferred for longer peptides. Additionally, male athymic nude mice bearing α v β 6 -positive and α v β 6 -negative (control) cell xenografts were employed for biodistribution studies. The click-labeled peptide shows similar tumor uptake but a different metabolism than ulterior 18 F-labeled peptides.
In the same year, Kim et al. compared a two-step click labeling approach (method 1) with a direct radiofluorination (method 2) for the labeling of β-azido-D-glucose ( [49]. They pointed out, that the conditions of the nucleophilic substitution of butyne precursor 1a with K[ 18 F]F delivered a by-product (Scheme 7) in a remarkable percentage which was evidenced as vinyl acetylene (9) among the desired 18 [51]. In general, the high affinity of fluorine for silicon allows a facile introduction of fluorine-18 under mild conditions as well. For this purpose, precursor 12 was prepared and labeled with n-dodecyltrimethylammonium [ 18 F] Problematic, the thermodynamically stable F-Si bond is known to be hydrolyzed in water under physiological conditions unless there are no bulky alkyl groups attached to the silicon. Unfortunately, 18

18 Fluoro-PEG X -Derivatives
This part of the review deals with PEGylated 18 F-labeled building blocks. In general, PEGylation describes the process of covalent connection of polyethylene glycol (PEG) moieties to drugs or therapeutic compounds. These compounds are "masked" from the host's immune system and the water solubility of hydrophobic derivatives is enhanced. Due to the increase of the size its circulatory time is often prolonged by reducing renal clearance.
The main advantage of PEGylated 18 F-labeled building blocks is their reduced volatility compared to the smaller counterparts. Hence, these compounds are easier to handle and the corresponding bioconjugates show a higher blood clearance which is important for in vivo studies. In the next step, different functionalized biomolecule-like compounds were successfully labeled within 10 min with an excellent 18 F-conversion of 97-100%. Small non-polar organic compounds needed longer reaction times (30 min) and resulted in limited 18 F-conversion up to 71%. In most of the cases, the two-step reaction was completed within 40 min from the end of bombardment. The sample labeling reaction of azide functionalized deoxythymidine 20 utilizing alkyne [ 18 F]19 is pointed out in Scheme 11. Scheme 11. 18 23 for the preparation of 18 F-CLIO -a fluorine-18-containing trimodal nanoparticle [54]. The basis of this particle consists of an aminated cross-linked dextran iron oxide (CLIO) decorated with diethylene triamine pentaacetic acid. The advantage of 18 F-CLIO was the ability of a triple detection by PET, fluorescence molecular tomography as well as magnetic resonance imaging. The 18 F-nanoparticle was prepared as briefly follows: 18 F-PEG 3 [ 18 F]23 was added to the azido-CLIO in a PBS buffer. This mixture was then treated with CuSO 4 /bathophenanthrolinedisulfonic acid (BPDS, as disodium salt) and sodium ascorbate in distilled water. The reaction mixture was incubated for 40 min at 40 °C and filtrated to remove the resulting 18 F-CLIO from unreacted 18 F-PEG 3 with a RCY of 58% (d.c.).
First evaluations of 18 F-CLIO suspended in the agar phantom pointed out a detection threshold of 0.025 µg Fe/mL for PET-CT imaging. This is approximately 200 times lower than by T2*-weighted MR imaging. The lowest observed concentration of the nanoparticles by MRI was 5 µg Fe/mL (15), demonstrating a PET detection threshold 200 times more sensitive than MRI at current specific activities. A high in vivo stability for 18 F-CLIO was demonstrated due to the low renal and bladder uptake, no excretion via the kidneys was observed.
In 2011, Gill and Marik used optimized conditions and switched the bioorthogonal functionalities for radiolabeling of N-alkynylated peptide 28 (4-pentynoyl-Val-Asp-Asn-Lys-Phe-Asn-Lys-Glu-Nle-Gln-Asn-Ala-Tyr-Ala-Ile-Glu-Ile-Ala-Leu-Leu-Pro-Asn-NH 2 ) using 18 F-PEG 3 -azide [ 18 F]27 (Scheme 13) [55]. After purification by HPLC, 18 F-labeled peptide [ 18 F]29 could be obtained with RCY of 31% and a A S = 1.44 GBq/µmol. The removal of BPDS was difficult using solid phase extraction (SPE) which lowered the chemical purity. Therefore, optimized reaction conditions without BPDS provided 96% conversion after 60 °C for 10 min when using 1.6 µmol Cu(CH 3 CN) 4 PF 6 and 10 nmol of 28 at high concentrations (125 µL). Noteworthy, a maximum A S and RCP were only obtainable when 28 was quantitatively converted to 18 F-labeled peptide [ 18 F]29 due to their similar molar mass and retention times in HPLC and SPE. Furthermore, the copper source should be considered very crucial, since PF 6 can lower the A S due to an isotopic exchange.
In 2012, Lee et al. used the same 18 F-PEG 3 -azide [ 18 F]27 for the radiolabeling of ZnO nanoparticles (20 nm) in order to observe their behavior and accumulation in organic tissues after oral administration using PET [56]. 18 F-labeling of ZnO particles resulted in A S = 0.73 MBq/mg and RCP > 95%. PET images indicated that 18 F and 18 F-ethoxyazide showed radioactivity in the bone and bladder 3 h after oral administration, whereas radioactivity for 18 F-labeled ZnO nanoparticles was seen only in the gastrointestinal (GI) tract. At 5 h post-administration, biodistribution studies demonstrate that 18 F-labeled ZnO nanoparticles showed radioactivity in the lung, liver and kidney including the GI tract. In most of the cases, the isolation of [ 18 F]33 was accomplished by co-distillation with acetonitrile. Notably, tosylate 32 is also co-distilled when using higher temperatures (>90 °C), and 32 reacts with the alkyne precursor as well in the subsequent labeling step. At temperatures below 90 °C the transfer of tosylate 32 to the cooled receiver vial was negligible, but the distillation time had to be extended [58]. Next, the somewhat difficult handling due to the volatility of the building block during the radiosynthesis brought different reaction set-ups in order to make the handling of 18 F-FEA [ 18 F]33 easier. Up to now, the [ 18 F]FEA-synthesis and the following labeling step with the respective alkyne were transferred into a remotely controlled module using a cooling trap [58].
The convenient and fast access of 18 33 for the radiofluorination of a series of 6-halopurines in a one-pot automated synthesis approach [74]. They used the copper chelator bathophenanthrolinedisulfonate to accelerate the cycloaddition reaction. An overview of all these tracers is given in Scheme 15.

18 Fluoro-Aryl Building Blocks
The 18 F-labeling of aryl precursors is a common task for radiochemists. In general, fluoroaryl compounds possess a higher metabolic stability in vivo than fluoroalkyl moieties due to the fluorine bound to the sp 2 -carbon atom. In most cases, the radiolabeled products show a higher lipophilicity due to the aryl moiety and therefore play a key role in the design of lipophilic radiotracers. A multitude of different aryl click building blocks were evaluated in the past. An overview is shown in Scheme 20.  51 was applied for the radiolabeling of an azide functionalized neurotensin (8)(9)(10)(11)(12)(13); the labeled peptide was obtained with a RCY = 66% as determined by radio-HPLC. The in vitro binding affinity (IC 50 ) of 18 F-neurotensin (8)(9)(10)(11)(12)(13) derivative was determined to be 66 nM.
The first approach for radiolabeling with pyridine-based 18 55. An automated synthesis procedure was evaluated for [ 18 F]FPyKYNE and the subsequent radiolabeling of an RGD peptide. The labeled peptide was obtained in a total RCY of 12-18% with a RCP > 99% after 125 min in a two-step, two-reactor process [86].
Finally Next, two approaches for the preparation of (azidomethyl)-[ 18  The group of Thonon et al. [88] used [ 18 F]67 for subsequent click labeling of a 4-ethynyl-L-phenylalanine-containing peptide. For this purpose, the click-conditions were optimized and the ligation reaction was finished in less than 15 min. Later in 2011, the same group presented a general method for the labeling of siRNA [91]. Later, the specific activity for the building block as well as for the labeled peptides was improved. This was possible due to the stoichiometry of the introduction of n.c.a. [ 18 F]fluoride into the precursor. Investigations to determine the A S were done using a rhodamine-N 3 conjugate. After this improvement, it was possible to prepare alkyne-[ 18

18 Fluoro-Gluco-Derivatives
The glycosylation of peptides represents an important tool for the enhancement of their in vivo behavior like blood clearance and stability. For this reason, labeling building blocks based on carbohydrates for the glycosylation of potential radiotracers using the CuAAC were evaluated by Maschauer and Prante in 2009 [98]. In this context, different α-and β-anomeric azides and alkynes were designed for radiolabeling purposes. They demonstrated that the radiolabeling of triflated precursors with fluorine-18 proceed better when using β-anomeric derivatives like 77 (RCY of [ 18 F]78 was 71 ± 10%). Afterwards, a proof-of-principle reaction of the 18 F-building block [ 18 F]78 was accomplished by labeling Fmoc-L-propargylglycine with a RCY of 60% for unprotected building block and a RCY of 76% for the peracetylated building block.
Finally in 2010, they applied [ 18 F]78 as the building block with the highest RCY to label different peptide moieties like RGD or neurotensin derivatized with L-propargylglycine (Scheme 27). RCYs ranged from 17 to 20% and A S = 55-210 GBq/µmol were obtained for the labeled conjugates in a total synthesis time of 75 min [99]. Furthermore, in vivo investigations with the [ 18 F]FGlc-RGD peptide were carried out using U87MG-bearing mice. Because of the glycosylation they had a better blood clearance and stability in α v β 3 -integrin expressing tumors. The specific tumor uptake of [ 18 F]FGlc-RGD was 0.49% ID/g (60 min p.i.).

Strain-Promoted Huisgen Cycloaddition
Strain-promoted and copper-free variants of the Huisgen cycloaddition of cyclic alkynes with azides are of great interest as powerful tools in reagent/catalyst-free bioconjugations. This type of reaction was applied especially for labeling purposes on living cells or in vivo studies due to the mild reaction conditions and the bioorthogonal character expressed by a high specificity and selectivity. Advantageously, the use of cytotoxic Cu(I) was not mandatory in contrast to the CuAAC. Cyclooctyne is the smallest stable cyclic alkyne and has a high reaction potential to react with azides as demonstrated by Blomquist and Liu in 1953 [101] and by Wittig and Krebs in 1961 [102]. The first connection of this type of click-reaction with cyclooctyne connected with a biological application was recognized by Agard et al. in 2004 [103].
With the entry of copper-free 1,3-dipolar cycloaddition reactions in radiopharmacy, several strainpromoted systems, such as cyclooctynes and dibenzocyclooctynes, have been developed for the incorporation of fluorine-18. Additionally, a few examples of radiometal labeling were reported [104].
In general, two strategies were pointed out. The first comprises the preparation of fluorine-18-containing cyclooctyne derivatives which were applied for radiolabeling of azide-functionalized molecules of interest. Alongside, 18 F-azide building blocks were synthesized and used for the labeling of cyclooctyne-bearing bioactive compounds. In general, two regioisomers are obtained in both strategies of this reaction which could indicate a different biological behavior in certain circumstances depending on the remaining molecule residue.
The first example of a biological application in combination with 18  Subsequently, various azide-functionalized sample molecules and bioactive compounds of interest like carbohydrates and geldanamycine were conjugated and radiolabeled with [ 18 F]82 (for an overview see Scheme 30). For the preparation of the non-radioactive references, 1.5 to 2.0 equiv. of respective azides were treated with 1 equiv. of FB-DBCO. All reactions resulted in the formation of two distinct regioisomers (1,4-and 1,5-triazole regioisomer). Products were isolated using HPLC-purification; but in some cases both regioisomers were not separable. Five different reaction conditions were tested for the radiolabeling procedure with [ 18 90 showed no decomposition or radiolysis over 6 h. Moreover, the stability of these products in PBS/saline buffer as well as in rat serum was tested. After 1 h, over 98% of intact radiotracer was found for both in rat serum. Additionally, formulations of both radiotracers were stable over 4 h in buffer.
In 2013, the same group evaluated an integrin α v β 6 -specific peptide and labeled it with fluorine-18 using this click-chemistry approach on the basis of the previously shown results. For that purpose, peptide N 3 -PEG 7 -A20FMDV2 (91) was yielded after azide-modification and preparation via solid-phase peptide synthesis [108]. Peptide 91 selectively targets the integrin α v β 6 -receptor which is located at the cell surface and has been identified as a prognostic indicator for several cancer entities [109,110].  95 and labeling of Tyr 3 -octreotate. 5 5 5 All following works used a change of functionalities to 18 F-labeled azides which underwent a click-reaction with different cyclooctynes connected to the bioactive molecule of interest. The advantage of labeled azides is their versatile applicability. They can be used for Cu-mediated click-reaction, the variants of the Staudinger Ligation and for copper-free click reaction with cyclooctyne. To demonstrate the versatility, the group of Campbell-Verduyn et al. [112]   In 2012, Evans and co-workers demonstrated that CFC reactions can be carried out between [ 18 F]fluoroethylazide ([ 18 F]33) and five derivatives with different cyclooctyne scaffolds as pointed out in Scheme 35 [113]. The process of radiolabeling was optimized. Highest RCYs were obtained when using acetonitrile as solvent, a reaction time of 15 min, and a temperature of 90 °C. In addition, the biological behavior of [ 18 F]33 was analyzed using BALB/c nude mice.  [114]. Four fluorine-18-labeled biologically active peptides for tumor targeting such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP) and apoptosis targeting peptide (ApoPep) were employed for this purpose. Advantageously, the resulting 18 F-peptides were provided as direct injectable solutions without any HPLC purification and/or formulation processes due to the application of a novel azide-functionalized scavanger resin after the labeling procedure. The remaining cyclooctyne precursor was cached with the azidefunctionalized resin for purification purposes; the overview is given in Scheme 36.

Scheme 36.
Overview of the general labeling and purification procedure.
In the first step, the desired peptides (cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP) and apoptosis targeting peptide (ApoPep)) were modified with the cyclooctyne moiety. The next step involved the radiolabeling with [ 18 F]103 in an ethanol/water mixture (v/v = 1/1) at ambient temperature for 15 min. Afterwards, the azide-functionalized scavenger resin was added and the mixture maintained for 20 min to allow the remaining ADIBO-peptide to conjugate with the resin. After filtration and washing of the resin with PBS solution, all peptides were produced within approx. 35

Staudinger Ligation
In 1919, Hermann Staudinger and Jules Meyer investigated the reaction of organic azides with phosphane derivatives [115]. Phosphane imines and elemental nitrogen were obtained from that reaction under absence of water. When water was added, the respective primary amines were yielded. This reaction is known as Staudinger reduction [116]. Conjugations with the respective phosphane imines are possible to execute using other electrophiles like carbonyl compounds instead of water. Based on these results, ligation reactions were developed which allow a bioorthogonal conjugation between azide functionalized molecules and phosphane species [117][118][119][120][121][122][123][124]. Two versions of this ligation type are known [125]. The non-traceless variant was introduced by Bertozzi et al. in the year 2000 [126]. Modified therephtalate compounds with a diphenylphosphanyl residue serve as fundament of this reaction. These modified phosphanes were reacted with organic azides and led to the desired ligation products after hydrolysis. Notably, the formed ligation product still contains the phosphane species which is oxidized during the reaction (Scheme 38: path A).

Scheme 38. Variants of the Staudinger Ligation.
In the same year, the traceless variant of the Staudinger Ligation was independently and simultaneously developed by Raines and co-workers [127] and the group of Bertozzi [128]. In this case, organic azides were used as starting material as well. In contrast to the non-traceless ligation, phosphanes were introduced which are functionalized in the ortho-position relative to the phosphorus on one of the aromatic rings. The phosphane oxide residue which is formed during the reaction is excluded and both reaction partners are connected exclusively via a native amide (peptide) bond (Scheme 38: path B). Therefore, this reaction type is often adapted in peptide syntheses [129,130]. Both ligations proceed in a wide pH range using organic or aqueous solvents.
The non-traceless Staudinger Ligation is seldom used for radiolabeling purposes with fluorine-18. To date, only one application is known. In 2006, the preparation of a fluorine-18 containing phosphane and the ligation to an azide containing carbohydrate was described [131] as shown in Scheme 39. In this case, the labeled glucose derivative served as an alternative for [ 18  In contrast to the non-traceless variant, several examples for the traceless Staudinger Ligation were published in the past. In general, two strategies were pointed out depending on the incorporation of fluorine-18 into the starting material. In the first case, phosphanes were evaluated containing the fluorine-18. For this purpose, biologically active molecules were modified with azide (direct approach). In the second case, the phosphane residue was introduced into the biologically active molecule and the fluorine-18 was connected to an azide functionalized molecule (indirect approach). Both ways are introduced in Scheme 40.

Scheme 40. Direct and indirect approach of the traceless Staudinger Ligation.
The direct approach was intensively investigated by Mamat and co-workers starting in 2009 [132]. For that purpose, phosphanes with various benzoate residues functionalized in the para-position were prepared [133]. Hence, two reaction paths were developed. First, the conventional esterification of phosphanol 107 with benzoyl chlorides 108a-c in the presence of a base or via Steglich esterification (for 111d) delivered the substituted phosphanes 109a-d (yield: 38−92%). However, application of these reaction conditions sometimes led to low yields of the desired phosphanes or the formation of by-products. Due to this fact, functionalized 4-iodophenyl derivatives 110a-h were applied as an alternative. Phosphanes 109a-h were obtained in 58−89% yield from the Pd-catalyzed cross coupling of 110a-h with diphenylphosphane (HPPh 2 ); the findings are summarized in Scheme 41. This approach should serve as a possibility for the introduction of the 4-[ 18 114. In the second step, the respective azide was added to yield the desired radiotracer. This reaction was carried out as a three-step/one-pot procedure starting from ethylene ditosylate as seen in Scheme 42. The phosphane precursor was added in a one-pot two-step procedure Notably, a two-step sequence was required for the preparation of the 18 F-containing phosphane which generally lowers the RCY as well as the A S of the final tracers. For this purpose, a one-step approach for the preparation of fluorine-18 containing phosphane building blocks like [ 18 F]120 (RCY = 65%, d.c.) was developed based on aliphatic phosphanyl esters [135,136]. A chain length of five carbon atoms was chosen for precursor 119. Different bases as well as fluorination agents and solvents were evaluated for optimization. The best results were obtained using [ 18  ) and a A S = 0.9 GBq/μmol (radiochemical purity > 95%); see Scheme 44. Biological data showed that only a modest fraction of the compound crossed the blood-brain barrier, with peak uptake no greater than 0.12% of the injected dose (0.11% ID/g). Ex vivo autoradiographic analysis of prefrontal cortex, striatum and thalamus slices indicated significant differential binding of  The purification step of this labeling reaction was improved using fluorous solid phase extraction (FSPE). For this purpose, derivative 132 was prepared with a fluorous tag [139] (C 4 F 9 chain on the aromatic ring of the phosphane) attached onto the phosphino fragment. Adversely, this compound was faster oxidized to the corresponding phosphine than the non fluorous counterpart. Thus, the purification step of 132 was done under absence of air. The following Staudinger Ligation with 2-fluoroethyl azide using standard conditions led to the formation of 136 in 27% yield after purification of the reaction mixture using FSPE [140]. The decrease in isolated yield could be attributed to the propensity of the phosphane precursor 132 towards oxidation. The radiofluorination was performed in acetonitrile using  Table 2). The purification using FSPE for the labeling of 132 led to an improvement, but HPLC was still necessary to obtain an analytically pure sample due to the breakthrough of fluorous material into the fluorophobic eluted fraction.

Tetrazine-Click
Investigated first by the group of Fox and co-workers, the tetrazine ligation is a fast bioconjugation method based on inverse-electron-demand Diels−Alder reactions [141], a proposed mechanism is pointed out in Scheme 45. The main advantage of this kind of reaction involves the unusually fast reaction rates (k 2 = 2,000 M −1 ·s −1 , solvent MeOH/water: 9/1) without need for catalysis. A further advantage is the non-reversibility due to the loss of elemental nitrogen, in contrast to most other Diels-Alder reactions. These reactions tolerate a broad range of functionality and proceed with high yield in organic solvents, water, buffer, cell media, or lysate. This fast reactivity enables protein modification at low concentration. The resulting cycloocta[d]pyridazines were obtained as conjugation products from the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. Mechanistically, the tetrazine ligation proceeds in two steps. An inverse-demand Diels-Alder reaction occurs followed by a retro-Diels-Alder reaction to eliminate nitrogen gas.

Scheme 45.
Proposed reaction mechanism of the tetrazine-click reaction. Based on computational work by Bach, E-cyclooctene has a highly twisted double bond resulting in a strain energy of 17.9 kcal/mol compared to Z-cyclooctenes or cyclooctane [142]. Based on these results, highly strained trans-derivatives were used as reactive dienophiles. Further, 3,6-diaryl-s-tetrazines serve as dienes which have been substituted in order to resist immediate reaction with water. The fact that the reaction is not only tolerant of water is also important especially for fast radiolabeling reactions. It has also been found that the rate increases in aqueous media. In addition, norbornenes were introduced as dienophiles at second order rates on the order of 1 M −1 ·s −1 in aqueous media. The labeling of live cells [143][144][145] or polymer coupling [146,147] were prominent examples of an application of this kind of conjugation reaction. Alongside, an elegant work concerning the radiolabeling of peptides using 111 In-DOTA were published by Robillard [148] and a modular strategy for the preparation of 64 Cu and 89 Zr radiometalated antibodies was previously published [149].
One of the first studies to apply the tetrazine-click reaction for radiofluorinations was achieved in the year 2010 by the group of Fox and co-workers (Scheme 46) [150]. First, they tried to label tetrazine derivatives with fluorine-18 for a conjugation with the trans-cyclooctene containing bioactive molecule. The instability of the tetrazines (RCY~1%) prompted the group to change the functionalities. Thus, [ 18  Next, the tetrazine residue was functionalized with a maleimide moiety for the radiolabeling of other cyclic RGD peptides without lysine residue [152]. Two strategies were evaluated for that purpose. In the first, the tetrazine-maleimide linker was conjugated with a cysteine-modified RGD peptide and finally labeled with [ 139 was directly reacted with the maleimide functionalized tetrazine. The original RGD peptide was then treated with this conjugate; however, this reaction proceeded within 20 min whereas the first labeling way took only 5 min. In addition, a higher peptide concentration was mandatory for the second way. MicroPET studies of U87MG tumor-bearing mice showed an uptake of 18 F-RGD [ 18 F]144 of approx. 2% ID/g in the tumor which is comparable with the previous findings of this group.
Next, based on previous work [153], the syntheses and in vivo imaging of two 18 F-labeled PARP1 inhibitors were presented by Reiner et al. in 2011 [154,155]. In the first publication, they compared the conventional direct nucleophilic introduction of [ 18 145 An improvement was done regarding the purification of the resulting radiotracer [ 18 F]146 from the precursor 145 [156]. A magnetic TCO resin was developed and used for a subsequent incubation after the radiolabeling step. The following separation was done using a magnet. An overview was pointed out in Scheme 49. This method was applied to successfully remove the excess of precursor 145 and to avoid lengthy HPLC purifications. After separation of the magnetic resin, the radiotracer [ 18  In 2012, an efficient radiofluorination strategy regarding exendin-4 analogues of imaging of beta cells was described (Scheme 50) [157]. For this purpose, (E)-5-(2-[ 18 F]fluoroethoxy)cyclooct-1-ene ([ 18 F]139) was used on the one hand and aminobenzyl tetrazine 149 was functionalized with maleimide 150 for a conjugation with SH-groups of peptides like cysteine. Therefore, a natural occurring lysine at position C12 was exchanged for a cysteine in an exendin-4-affine peptide (E4 C12 ). Next, E4 C12 peptide was modified with the tetrazine containing cross linker 151 to give E4 C12Tz 153 was obtained with > 94% RCP after separation using the aforepublished magnetic scavenger resin.

Miscellaneous
In addition to the classical CuAAC, the 1,3-dipolar cycloaddition of nitrones with alkenes which led to isoxazolidines or further to isoxazoles belongs to the [3+2]-dipolar cycloadditions and was evaluated for radiofluorination purposes by the group of Zlatopolsky et al. in 2012 [158]. Mild reaction conditions, regioselectivity, absence of protection groups, acceptable yields and metabolic stability of the formed isoxazoles are the main advantages. Both starting materials are stable under a wide range of conditions (pH, temperature, microwave irradiation, reagents) [159] and this reaction can be carried out in different solvents, including aqueous media [160,161]. Of high importance is the absence of metal catalysts like Cu(I) in contrast to the classical CuAAC. In the first sample reaction, the fluorine-18 containing nitrone was simply prepared from the appropriate aldehyde [ 18  The next step includes the radiolabeling of three sample molecule. For this purpose, these molecules were modified with a maleimide residue. The subsequent labeling step was carried out in a one-pot synthesis starting from 4-[ 18 F]benzaldehyde ([ 18 F]154), phenyl hydroxylamine (155) and the respective malinide species in RCYs > 80% (Scheme 52). The respective 18 F-labeled diastereomers were formed as endo-and exo-isomers (ratio approx. 2/1 = endo/exo) in all cases and were easy separable.  157 A further improvement of the labeling reaction was done in the same year [162]. In contrast to the first approach, hydroxylamine (as its HCl salt) was used instead of N-functionalized derivatives for the preparation of 4-[ 18  The last experiment contained the labeling of a dipeptide (β-Ala-Phe-OMe) either bound to the cyclooctyne residue or bound to the norbornene moiety. Both labeled peptides [ 18 F]165 and [ 18 F]166 are pointed out in Scheme 54.

Conclusions
This review has summarized the applications and the scope of bioorthogonal click reactions for the introduction of fluorine-18 in the field of radiopharmacy. This kind of reaction has become a very important tool, especially for the radiofluorination of high molecular weight pharmacologically relevant molecules like peptides or proteins but also for the radiofluorination of small compounds of interest. For labeling purposes, fast and high yielding reactions are required and strategies for a convenient purification in an acceptable time span.  139 46 >94 102 n.d. automatically [157] After considering all results which were obtained in the last years a guideline for the choice of building blocks could be generated: first, a building block should be azide-functionalized for universal applicability and to prevent side-reactions like the Glaser coupling. This is important especially for radiolabeling purposes with alkyne functionalized building blocks using the CuAAC. In addition, when utilizing the CuAAC the use of Cu(I) stabilizing agents like TBTA, BTTAA and bathophenanthroline is crucial and should be at least 1.1 eq to the Cu-source. Second, the building blocks should contain 18 F-propyl, 18 F-ethyl or 18 F-aryl groups for a higher metabolic stability. Third, these blocks should be directly radiolabeled by fluorine-18 for shorter preparation times and higher RCY and A S . Further, glycosylations or PEGylations of these building blocks enhance the in vivo availability of the corresponding radiolabeled bioactive molecule. An overview of building blocks is given in Table 5.
Moreover, it is necessary to separate and deplete catalysts like the copper species completely when using the Huisgen-click. This is important especially for in vivo applications due to the formation of complexes of Cu with the respective high molecular weight bioactive molecule during the labeling procedure. In this case and due to the toxic properties of copper species in vivo, copper-free variants should be applied. Thus, the CuAAC should be more preferred rather for small compounds. Last but not least, the non-radioactive reference building blocks as well as the precursors should be conveniently prepared from inexpensive chemicals.
Tetrazine-click as well as the Cu-free variant of the Huisgen cycloaddition show the best results in terms of the labeling time. This is important, especially for the labeling of high molecular weight compounds. Moreover, the removal of Cu or other catalysts is not needed. The size of the building block and the formation of regioisomers is a drawback, but the higher the molar mass of the molecule to be labeled the smaller is the influence of the building block in most of the cases.
The use of cartridges or scavenger resins for a fast separation and purification is a fundamental topic. This allows labeling reaction with high yields, high radiochemical purity and high specific activity in a very short time. A high hydrophilicity of the bioorthogonal building blocks should be considered for the radiolabeling of proteins and antibodies.