Hindawi Publishing Corporation
`BioMed Research International
`Volume 2014, Article ID 361329, 16 pages
`http://dx.doi.org/10.1155/2014/361329
`
`Review Article
`18F-Labeling Using Click Cycloadditions
`
`Kathrin Kettenbach,1 Hanno Schieferstein,1 and Tobias L. Ross1,2
`1 Institute of Nuclear Chemistry, Johannes Gutenberg University Mainz, 55128 Mainz, Germany
`2 Radiopharmaceutical Chemistry, Department of Nuclear Medicine, Hannover Medical School, 30625 Hannover, Germany
`
`Correspondence should be addressed to Tobias L. Ross; ross.tobias@mh-hannover.de
`
`Received 15 March 2014; Revised 29 April 2014; Accepted 1 May 2014; Published 27 May 2014
`
`Academic Editor: Olaf Prante
`
`Copyright © 2014 Kathrin Kettenbach et al. This is an open access article distributed under the Creative Commons Attribution
`License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
`cited.
`
`1/2
`
`Due to expanding applications of positron emission tomography (PET) there is a demand for developing new techniques to
`introduce fluorine-18 (𝑡
`= 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.
`
`1. Introduction
`
`𝛽
`
`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 𝛽+-energy of 635 keV,
`which is responsible for a very high spatial resolution [2].
`The challenges for researchers are to develop convenient 18F-
`labeling strategies, which include short reaction times and
`applicability for sensitive biomolecules. Especially the harsh
`conditions during direct 18F-labeling pose an exceeding chal-
`lenge [3, 4]. Therefore, most of the radiolabeling strategies
`focus on 18F-containing prosthetic groups, which allow a sen-
`sitive and bioorthogonal 18F-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 reac-
`tion 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) [7–13] or tetrazines
`(tetrazine-trans-cyclooctyne (TTCO) ligation) [14–17] as well
`as between norbornene derivatives and tetrazines [18]. Espe-
`cially, the TTCO ligation showed promising reaction rates,
`which makes this click reaction concept very suitable for 18F-
`labeling and also for in vivo application in living systems. Very
`recently, new versions of 18F-click cycloadditions are added
`to the range of reactions [19–25]. In this line, the first 18F-
`labeled 𝛽-lactame became available via a new radio-Kinugasa
`reaction [21].
`As a consequence, click cycloaddition is one of the most
`frequently applied methods for 18F-labeling of new bioactive
`compounds, with or without a catalytic system. This can be
`
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`BioMed Research International
`
`[18F]fluoroethylazide ([18F]FEA)
`
`[18F]fluoroalkyne(s)
`
`[18F]fluoro-PEG x-alkyne(s)
`
`18F
`
`O
`
`x
`
`[18F]ArBF 3
`
`−
`
`HN
`
`F
`
`O
`
`F
`
`F
`
`B
`F
`
`F
`18F
`
`O-propargyl-4-[18F]fluorobenzoate
`([18F]PFB)
`
`O
`
`O
`
`18F
`
`N-propargyl-2-amino-3-[18F]fluoro-
`propionic acid ([18F]serine)
`
`COOH
`18F
`
`NH
`
`18F
`
`N3
`
`18F
`
`n
`
`1-(azidomethyl)-4-[18F]fluorobenzene
`
`N3
`
`18F
`
`1-(3-azidopropyl)-4-(3-
`[18F]fluoropropyl)piperazine
`[18F]AFP)
`
`N3
`
`N
`
`N
`
`18F
`
`4-[18F]fluoro-N-methyl-N-(prop-2-ynyl)-
`benzenesulfonamide ([18F]F-SA)
`
`18F
`
`3,4,5-tri-O-acetyl-2-deoxy-
`2-[18F]fluoroglucopyranosyl azide
`OAc
`
`AcO
`AcO
`
`N3
`
`O
`
`18F
`
`1-(but-3-ynyl)-4-(3-[18F]fluoro-
`propyl)piperazine
`([18F]BFP)
`
`18F
`
`N
`
`N
`
`[18F]FPy 5yne
`
`O
`
`N
`S
`O O
`Figure 1: Lead structures of the most important 18F-prosthetic groups applied for copper-catalyzed click 18F-fluorination.
`
`N
`
`18F
`
`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 18F-prosthetic groups,
`the reaction conditions and reaction partners applied for
`copper-catalyzed, copper-free and other kinds of 18F-click
`cycloadditions, respectively. The most important structures
`of those prosthetic groups are shown in Figures 1, 3, and5.
`
`2. Copper-Catalyzed 18F-Click Cycloadditions
`In the last decade, the copper-catalyzed azide alkyne cycload-
`dition (CuAAC), which has first been reported independently
`by Rostovtsev et al. [81] and Tornøe et al. [82] in 2002, has
`spread over almost all fields of chemistry [83–87], biology
`[88–90], and material science [91, 92]. The great advan-
`tage of this method is given by its outstanding efficiency,
`its regiospecificity, and fast formation of 1,4-disubstituted
`1,2,3-triazoles at ambient temperatures, which is particularly
`
`suitable for 18F-labeling of sensitive biomolecules. In partic-
`ular, the CuAAC enables incorporation of fluorine-18 via a
`prosthetic group under mild and bioorthogonal conditions
`[22–25]. 1,2,3-triazoles were first introduced by Michael, who
`described the formation of a 1,2,3-triazole from a phenylazide
`in 1893 [93]. Following this pioneering work, Dimroth, Fester,
`and Huisgen described this type of reaction as a 1,3-dipolar
`cycloaddition for the first time in 1963 [5].
`In 2006, Marik and Sutcliffe published the application
`of the CuAAC as an 18F-labeling strategy for the first time
`[26]. They radiolabeled three different alkyne precursors in
`radiochemical yields (RCY) of 36–81%. Afterwards they were
`reacted them with azido-functionalized peptides in RCY of
`54–99% and an overall reaction time of 30 min. Thus, they
`could show a new, very fast, efficient, and mild 18F-labeling
`strategy for complex compounds, especially appropriate for
`sensitive biomolecules. Only two years later, the suitability
`of this approach was demonstrated for the 18F-labeling of a
`folate derivative for in vivo tumor imaging with the same
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1021, p. 2
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`
`10–30%n.d.c.[49]
`[48]
`25–87%
`
`70–75min
`30min
`
`BioMed Research International
`
`[47]
`
`[46]
`
`[45]
`
`n.d.
`
`58±4%
`
`55–75%
`
`[44]
`[43]
`28.5±2.5%[42]
`[41]
`90%
`[40]
`8–12%n.d.c.
`n.d.c.
`[39]
`3±2.6%
`40–64%
`
`60±2%
`
`[38]
`
`[37]
`[36]
`[35]
`[34]
`[33]
`
`[32]
`[31]
`
`[30]
`[29]
`[28]
`
`[27]
`[26]
`
`Literature
`
`75±10%
`1–3.4%n.d.c.
`37±3.6%
`47±8%
`65±6%
`powder
`copper
`15–98%with
`respectively
`61–98%
`25–35%
`8.7±2.3%
`52±5%
`27±6%
`30%
`54–99%
`
`CCA
`RCY2
`
`1h
`
`2.5h
`
`1h
`
`60min
`1h
`n.d.
`35min
`n.d.
`90min
`(estimated)
`30min
`
`n.d.
`3h
`n.d.
`
`1h
`
`1.5h
`66min
`30min
`
`75–80min
`30min
`(CCA)
`time1
`reaction
`Overall
`
`FtRGD
`Alkynesofbenzenerings
`
`CuSO4,NaAsc
`
`3-Butynyltriphenylphosphoniumbromide
`
`n.d.
`1step,5–10min68–75%
`15min.
`[18F]FEA:
`steps
`Precursor:2
`
`n.d.
`
`n.d.
`
`sulfonateprecursor
`[18F]FEAfromapolyflourinated
`
`(CuSO4/NaAsc.)
`BPDS-copper(I)
`One-pot
`CuSO4/Asc
`CuI/ascorbate/DIPEA
`CuSO4/Asc
`
`CuSO4/Asc/BPDS
`
`acid/DIPEA
`CuI/ascorbic
`CuSO4/Asc/BPDS
`Cu2+/Asc
`CuSO4/Asc
`
`copperpowder
`ExcessofCu2+/Ascor
`
`CuI
`CuI/Asc
`
`Cu(I)/Asc/2,6-lutidine
`
`CuI/NaAsc/DIPEA
`
`Catalyticsystem
`
`glycine
`tert-butylesterofN-Boc-(S)-propargyl
`6-halopurines
`Alkyne-func.
`RGDfK
`Nitroaromaticsubstrates
`Haloethylsulfoxides
`4-(prop-2-ynyloxy)Benzaldehyde
`Nucleosides
`ICMT-11(automatedsynthesis)
`
`[Tyr3]octreotateanalogues
`
`5-Ethynyl-2󸀠-deoxyuridine
`ApoptosismarkerICMT11
`3-Cyanoquinolinecore
`RGDpeptides
`Caspase3/7SelectiveIsatin
`
`n.d.
`
`55%
`
`71±4%
`50%n.d.c.
`
`18F]FEA)
`
`18F]fluoroethylazide([
`
`[
`
`n.d.
`
`1step,15min
`
`Terminalalkynes
`
`55%
`
`𝛾-(4-azido-butyl)-folicacidamide
`𝛼V𝛽
`azide
`2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl
`Glucopyranosylazide
`N-(3-azidopropionyl)peptides
`
`6specificpeptideA20FMDV2azide
`
`Reactingagent
`
`70–85%
`86±2%
`59±6%
`45±3%
`n.d.
`36–81%
`
`1step,12min
`1step,22min
`1step,15min
`1step,15min
`(estimated)
`1step,15min
`1step,10min
`
`RCY2
`
`time1
`Steps/reaction
`
`6-[18F]fluoro-1-hexyne
`5-[18F]fluoro-1-pentyne
`4-[18F]Fluoro-1-butyne
`4-[18F]fluoro-1-butyne
`18F]fluoroalkynes
`
`[
`
`18F-prostheticgroup
`
`Table1:Summaryoftheprostheticgroups,reactionconditions,andreactionpartnersappliedforcopper-catalyzedclick18F-fluorination.
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1021, p. 3
`
`

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`4
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`BioMed Research International
`
`[62]
`
`[61]
`
`[60]
`
`[59]
`
`[58]
`
`[57]
`[56]
`
`[55]
`
`[54]
`[53]
`
`[52]
`
`[51]
`[50]
`
`Literature
`
`60%/25%
`77%/55–
`55–60%
`66%
`
`8.5%
`
`>95%
`31±6%
`
`58%
`
`52±8.3%
`71–99%
`
`CCA
`RCY2
`
`37±31%
`75±5%
`79±33%and
`88±4%,
`5–20%
`
`12–18%
`
`(estimated)
`1h
`
`85min
`
`125min
`
`24.6±0.5%
`
`276min
`
`18.7%
`
`160min
`
`[63]
`
`87–93%
`
`1h
`
`Azido-peptidescRGDfKandD4peptide
`
`58%
`
`1step,40min
`
`2h
`
`100min
`n.d.
`
`2,6-lutidine
`CuBr/TBTAand
`fluorophosphates/TBTA
`copper(I)hexa
`Tetrakis(acetonitrilo)
`CuSO4/Asc
`
`Cu(I)-TBTA
`
`2.5h
`
`CuAcetate,NaAsc
`
`n.d.
`(estimated)
`2h
`(estimated)
`1h
`(estimated)
`110min
`10–30min
`(CCA)
`time1
`reaction
`Overall
`
`CuSO4/Asc/BPDS
`
`CuSO4/Asc
`
`Catalyticsystem
`
`Azide-functionalizedDNA
`
`N3–(CH2)4–CO–YKRI–OH(BG142)
`
`protein(HAS),oligonucleotide(L-RNA)
`Azide-functionalizedphosphopeptide,
`albumin(HSA)
`Azide-functionalizedhumanserum
`Azide-functionalizedneurotensin
`amide
`𝛾-(11-azido-3,6,9-trioxaundecanyl)folicacid
`ZnOnanoparticlealkynes
`N-alkynylatedpeptide
`
`Nanoparticleazide
`
`E(RGDyK)2azide
`Variousazides
`
`Reactingagent
`
`Table1:Continued.
`
`42%
`
`1step,15min
`
`18F]FPy5yne
`
`[
`
`n.d.
`
`32±5%
`
`labeling:58%
`
`n.d.
`62±4%
`
`57%
`
`65±1.9%
`85–94%
`
`80min
`labeling:1step,
`steps,
`Precursor:3
`labeling:1step
`steps
`Precursor:2
`
`1step,40min
`
`1step,15min
`
`1step,20min
`
`RCY2
`
`time1
`Steps/reaction
`
`18F]F-SA)
`18F]fluoro-N-methyl-N-(prop-
`
`(p[
`2-ynyl)-benzenesulfonamide
`4-[
`
`[18F]PEG-azide
`
`[18F]PEG3-azide
`
`18F-Fluoro-PEG-Alkyne
`
`18F-prostheticgroup
`
`CuSO4/Asc
`
`transglutaminase-reactivepeptide
`Benzylazide,twolysinederivatives,
`
`58±31%
`
`D-aminoacidanalogueofWT-pHLIPazideCu-Acetate/NaAsc
`
`27.5±6.6%
`
`15min
`labeling:1steps,
`steps,
`Precursor:2
`1step,10min
`
`CuSO4/Asc
`
`Azide-functionalizedRGDpeptide
`
`20–35%
`
`20–25min
`
`([18F]FNPB)
`yl-benzamide
`4-[18F]fluoro-3-nitro-N-2-propyn-1-
`
`18F]fluorobenzoate
`
`18F]PFB)
`([
`propargyl4-[
`
`6-[18F]fluoro-2-etynylpyridine
`([18F]FPyKYNE)
`yloxypyridine
`2-[18F]fluoro-3-pent-4-yn-1-
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`

`BioMed Research International
`
`5
`
`[6]
`
`75%
`
`[78]
`
`17–25%
`peptide:
`SNEW
`59–79%
`Aminoacid:
`
`15–30%
`[77]
`n.d.c.
`[76]
`20±10%
`n.d.
`[75]
`n.d.c.
`[74]
`8.6±2.3%
`20–25%n.d.c.[73]
`[72]
`4.1%
`[71]
`16–24%
`[70]
`5–25%
`[69]
`17–20%n.d.c.
`
`[68]
`
`[67]
`
`[65]
`
`[66]
`[65]
`[64]
`
`Literature
`
`60%
`
`n.d.
`
`15±5%
`
`12%
`15±5%
`90%
`
`CCA
`RCY2
`
`145min
`
`CuSO4,Asc
`
`cRDG-azide
`
`2h
`
`30min
`
`1h
`
`90min
`70min
`80–100min
`70–75min
`3h
`75min
`(estimated)
`1.5h
`n.d.
`
`120min
`
`120min
`120min
`90min
`(CCA)
`time1
`reaction
`Overall
`
`CuSO4,Asc
`
`SNEWpeptide
`N-Fmoc-e-azido-Lnorleucine(aminoacid),
`
`CuI/Asc
`
`CuSO4/Asc
`CuBr/TTMA
`CuSO4/Asc
`Cu-Acetate/NaAsc
`
`CuSO4/Asc
`
`CuSO4/Asc
`
`CuSO4/Asc/TBTA
`CuI/NaAsc/DIEA
`
`Catalyticsystem
`
`Alkyne-functionalizedRGD-boronate
`Alkyne-functionalizedbombesin(BBN)
`Alkyne-functionalizedRGD
`cyanoquinoline(EGFR)alkyne
`ETARligandalkyne
`Alkyne-bearingprotein
`RGD-peptidealkyne
`folatealkyne
`neurotensinpeptoid)
`Alkyne-functionalizedpeptides(RDG,
`Fmoc-L-propargylglycine
`
`siRNAalkyne
`
`linkers)
`siRNA-linker(twonewalkyne-bearing
`siRNAalkyne
`4-Ethynyl-𝐿-phenylalanine-peptide
`
`Reactingagent
`
`Table1:Continued.
`
`CCA:clickcycloaddition;(n.)d.c.:(not)decaycorrected;Asc:ascorbate;DIPEA:diisopropylethylamin;TBTA:tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine;n.d.:nodata.
`2Radiochemicalyieldsforthe18F-prostheticgroupstartingfromfluorine-18fortheclickreaction,respectively;decaycorrected,aslongasnotnotedelsewise.
`1Calculatedassumfromallsteps,forthe18F-prostheticgroup,respectively,fortheoverallreactionyieldingtheclickproduct,startingfromfluorine-18.
`18F]serine
`
`31±9%
`[18F]BFP:
`29±5%
`[18F]AFP:
`
`2steps,125min28±5%
`40min
`[18F]BFP:1step,
`40min
`[18F]AFP:1step,
`72h
`BFP:4steps,
`54h
`AFP:4steps,
`2steps,
`
`[
`
`18F]AFP
`
`18F]BFP
`piperazine-based[
`
`[
`
`n.d.
`
`1step,20min
`
`18F]ArBF3−
`
`[
`
`n.d.
`1.3–4.7%
`84%
`52%
`n.d.
`
`71±10%
`around15%
`around40%
`35%
`
`84%
`41%
`34%
`
`RCY2
`
`1step
`
`1step,10min
`
`2step,7.5min
`
`1step,30min
`
`1step,94–188s
`
`4steps,75min
`
`1step,45min
`4steps,75min
`4steps,75min
`
`time1
`Steps/reaction
`
`azide
`18F]fluorogluco-pyranosyl
`3,4,6-tri-O-acetyl-2-deoxy-2-
`
`[
`
`4-[18F]Fluorophenylazide
`[18F](azidomethyl)fluorobenzene
`[18F]fluoropropoxy)benzene
`1-Azido-4-(3-
`
`18F]-
`
`fluorobenzene
`1-(azidomethyl)-4-[
`
`18F-prostheticgroup
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`Petitioner GE Healthcare – Ex. 1021, p. 5
`
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`

`6
`
`BioMed Research International
`
`prosthetic group, 6-[18F]fluoro-1-hexyne [30]. The radiofolate
`was obtained in RCY of 25–35% and was applied to KB-
`tumor bearing mice. A specific tumor accumulation could be
`observed by using the folate receptor (FR) targeting concept.
`Furthermore, Kim et al. used 18F-labeled alkynes as prosthetic
`groups for the 18F-labeling of 2,3,4,6-tetra-O-acetyl-𝛽-D-
`glucopyranosyl azide [27], which in turn was employed to
`6 specific peptide A20FMDV2 [28].
`label the 𝛼V𝛽
`Considering all known clickable prosthetic groups for
`18F-labeling, [18F]fluoroethyl azide ([18F]FEA) is certainly
`one of the most investigated clickable 18F-prosthetic groups.
`Until today, about twenty different manuscripts deal with
`[18F]FEA to radiolabel a broad variety of biomolecules and
`compounds. In 2007, Glaser and ˚Arstad [31] mentioned for
`the first time the preparation of [18F]FEA with a RCY of
`55% using 2-azidoethyl-4-toluenesulfonate as precursor. As
`a proof of concept, they reacted [18F]FEA with different
`terminal alkynes in very good to excellent RCY of 61–
`98%. 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 (CuI stabilizing agent)
`accelerates the 1,3-dipolar cycloaddition [36, 38, 45]. The very
`good access to [18F]FEA led to the development of a variety
`of radiotracers labeled with this prosthetic group, like 18F-
`deoxyuridine [37], 18F-fluoro-oxothymidine (18F-FOT), or
`18F-fluoro-thiothymidine (18F-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 [18F]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
`[18F]fluoro-PEGx-derivatives (𝑥 = various polyethylene gly-
`col (PEG) ratios) as new 18F-labeled prosthetic click groups.
`These compounds showed a reduced volatility and increased
`polarity compared with other 18F-labeled prosthetic groups
`like [18F]FEA or [18F]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 18F-PEG-
`azides and 18F-labeled alkynes in RCY of 85–94%. As a proof
`of concept, they carried out cycloadditions with the 18F-
`labeled prosthetic groups and the corresponding alkynes,
`respectively, azides in high RCY of 71–99%. Several other
`groups continued this work by using the 18F-labeled PEGy-
`lated 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, [18F]fluoro-aryl-based prosthetic groups have
`been developed and investigated. In 2007, Ramenda et al.
`[56] published for the first time a 4-[18F]fluoro-N-methyl-
`N-(prop-2-ynyl)-benzenesulfonamide (p-[18F]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 18F-aryl prosthetic group for the labeling of
`human serum albumin (HSA) [57] and other proteins, phos-
`phopeptides, and L-RNA [58] in good RCY. A pyridine-based
`18F-prosthetic group was first introduced by Inkster et al.
`[59] in 2008 by reacting [18F]FPy5yne with a model peptide
`in RCY of 18.7% and an overall reaction time of 160 min.
`They started from either 2-nitro- or 2-trimethylammonium
`pyridine to synthesize [18F]FPy5yne with a RCY of 42%.
`Furthermore, [18F]pyridine derivatives have been used to
`radiolabel cRGDs [60] and the D-amino acid analog of WT-
`pHLIP [61].
`In 2009, Vaidyanathan et al. [62] presented a pros-
`thetic group based on a 4-[18F]fluorobenzoate. Propargyl-
`4-[18F]fluorobenzoate ([18F]PFB), which could be obtained
`in RCY of 58 ± 31% within 15 min. To investigate the
`labeling properties of this new prosthetic group, numerous
`compounds have been 18F-labeled using [18F]PFB with RCY
`from 37% to 88% and overall reaction times of about
`1 h. Another approach was published by Li et al. in 2012
`[63], who synthesized 4-[18F]fluoro-3-nitro-N-2-propyn-1-
`yl-benzamide ([18F]FNPB) for 18F-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-[18F]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 18F-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 18F-gluco-derivatives for CuAAC-radiolabeling of
`Fmoc-L-propargylglycine with a RCY of 60% [68]. They
`showed that the 18F-click labeling reaction was more con-
`venient by using the 𝛽-anomeric derivative of the azides,
`respectively, alkynes, giving very high RCY of 71 ± 10%.
`One year later, they published the first in vivo evaluation
`of an 18F-labeled RGD peptide labeled with [18F]FDG-𝛽-
`Az in U87MG-tumor bearing mice showing an improved
`blood clearance and stability [65, 66]. Likewise, Fischer et
`al. demonstrated in 2012 that a [18F]fluorodeoxyglycosyl
`folate could be obtained in RCY of 5–25% and subse-
`quent biodistribution and PET-imaging studies showed a
`high and specific uptake of the radiotracer in FR-positive
`tumors [70]. The variety of new 18F-labeling strategies using
`18F-Fluoroglycosylation is the focus of a review article as a
`part of this special issue provided by Maschauer and Prante
`[94].
`As another promising approach, Li et al. presented in
`2013 an alkyne-functionalized aryltri-[18F]fluoroborate for
`radiolabeling azido-bombesin and azido-RGD. The major
`advantage of this method is the two-step, one-pot procedure
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1021, p. 6
`
`

`

`BioMed Research International
`
`7
`
`O
`
`COOMe
`Cl
`
`N
`
`(i)
`
`O
`
`O
`
`O
`
`COOMe
`18F
`
`N
`
`(ii)
`
`(iii)
`
`(iv)
`
`O
`
`O
`
`COOH
`18F
`
`N
`
`COOH
`18F
`
`NH
`
`Figure 2: Radiosynthesis of a new amino-acid based 18F-prosthetic group (N-propargyl-2-amino-3-[18F]fluoro-propionic acid, “[18F]serine”)
`for 18F-CuAAC-labeling of complex biomolecules. (i) [K ⊂ 2.2.2]+/ 18F−, DMSO, 140∘C, 10 min; (ii) hydrochloric acid (3.3 M), 100∘C, 15 min;
`for analytical purposes (sequential deprotection): (iii) sodium hydroxide (3.3 M), 60∘C, 5 min; (iv) hydrochloric acid (3.3 M), 100∘C, 15 min.
`
`providing a water-soluble and noncoordinating aryltri-
`[18F]fluoroborate anion, which provided specific activities up
`to 555 GBq/𝜇mol [75, 76, 95].
`Two new piperazine-based prosthetic groups, 1-(but-3-
`ynyl)-4-(3-[18F]fluoropropyl)piperazine ([18F]BFP) and 1-(3-
`azidopropyl)-4-(3-[18F]fluoropropyl)piperazine ([18F]AFP),
`have recently been developed by Pretze and Mamat [78].
`Spiro salts were used as precursors, facilitating purification
`by using solid phase extractions (RP-18 or SiO2-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 [18F]BFP for
`radiolabeling of peptides, [18F]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 18F-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 18F-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 18F-labeled prosthetic group was subse-
`quently 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 18F-propyl, 18F-ethyl, or 18F-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.
`
`3. Copper-Free 18F-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 18F-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 18F-labeling reactions for the syntheses of radiotracers
`for imaging of malignancies in vivo. This leads to the develop-
`ment 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 dibenzocyclooc-
`tynes. First approaches focus on the reaction of 18F-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 18F-
`labeled 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 18F-labeled aza-dibenzocyclooctyne,
`[18F]FB-DBCO, and a plethora of azides. The 18F-
`labeled building block was synthesized via acylation of
`commercially available N-(3-aminopropionyl)-5,6-dihydro-
`11,12-didehydrodibenzo[b,f ]azocine with N-succinimidyl-4-
`[18F]fluorobenzoate ([18F]SFB), which can be easily prepared
`in an automated synthesis module [97]. The 18F-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 s. Besides,
`the compound was stable in methanol and phosphate buffer
`over 60 min. Subsequently, [18F]FB-DBCO was reacted
`with various azides as proof of principle showing different
`structural complexities. In all reactions, the formation of
`two regioisomers (1,4- and 1,5-triazole) has been observed
`and in some cases a separation of the regioisomers by HPLC
`was impossible. All 18F-labeled radiotracers were obtained
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1021, p. 7
`
`

`

`8
`
`BioMed Research International
`
`[13]
`
`[18]
`
`[80]
`
`[16]
`
`[79]
`[12]
`
`[11]
`[10]
`
`[15]
`
`[9]
`
`[8]
`
`[7]
`
`[14]
`
`Literature
`
`11.9±3.2%
`tetrazine)
`onthe
`(depending
`46–97%
`89.2%invivo
`
`46.7±17.3%
`solvent)
`onCOTand
`(depending
`9.6–97%
`92%
`74±4.8%
`
`90%
`
`95%
`onazide)
`(depending
`19–37%
`69–98%
`
`>98%
`
`CCA
`RCY2
`
`40±4min
`click:
`of[18F]SFB)
`preparation
`(without
`82min
`
`3h
`
`30min.
`
`80min
`202±34min
`
`30min
`
`1.5h
`HPLC)
`(without
`30min
`2h
`HPLC)
`(without
`30min
`(CCA)
`time1
`reaction
`Overall
`
`CCA:clickcycloaddition;DA:DielsAlder;DBCO:aza-dibenzocyclooctyne;TCO:trans-cyclooctyne.
`2Radiochemicalyieldsforthe18F-prostheticgroupstartingfromfluorine-18fortheclickreaction,respectively;decaycorrected,aslongasnotnotedelsewise.
`1Calculatedassumfromallsteps,forthe18F-prostheticgroup,respectively,fortheoverallreactionleadingtotheclickproduct,startingfromfluorine-18.
`[18F]FBA-C6-DBCO
`
`cycloaddition
`Strain-promotedclick1,3-dipolar
`
`6-specificpeptide
`
`𝛼V𝛽
`
`[10]
`
`[10]
`
`cycloaddition
`Inverseelectron-demandDA
`
`cycloaddition
`Strain-promotedclick1,3-dipolar
`
`cyclo-addition
`Inverseelectron-demandDA
`
`cycloaddition
`Strain-promotedclick1,3-dipolar
`
`derivatives)
`(peptide-/bombesin-
`Tetrazine
`tetrazine
`Polymermodified
`exendin-4
`Tetrazinemodified
`
`ethylazide
`[18F]2-fluoro-
`
`cRGD-DBCO
`Alkylazide
`
`Tetrazine-RGD
`N3(TATE)
`Tyr3-octreotide-
`[18F]azides
`Threedifferent
`Variousazides
`
`cyclo-addition
`inverseelectron-demandDA
`
`3,6-diaryl-s-tetrazine
`
`60±17%
`
`1step,52min
`
`norbornene
`[18F]amine-functionalised
`
`46.1±12.2%
`
`1step,102min
`
`[18F]trans-cyclooctene([18F]TCO)
`
`thederivative)
`(dependingon
`20–57%
`63%
`24.5%
`
`[14]
`
`21%
`
`17%
`
`85%
`
`71%
`
`thederivative)
`(dependingon
`30–80h
`6–11steps,
`1step,45min
`1step,30min
`
`[14]
`
`1step,1h
`
`9steps,—
`
`1step,60min
`
`1step,15min
`
`[18F]cyclooctyne
`
`[18F]PEG4azide
`azadibenzocyclo-octyne
`[18F]bifunctional
`
`[18F]TCO
`
`[18F]DBCO
`(bombesin)
`Aza-DBCO-BN
`TCO-derivative:
`[18F]FB-DBCO
`
`[18F]COT
`
`Reactiontype/catalyticsystem
`
`Reactingagent
`
`RCY2
`
`time1
`Steps/reaction
`
`18F-prostheticgroup
`
`Table2:Summaryoftheprostheticgroups,reactionconditions,andreactionpartnersappliedforcopper-freeclickfluorination.
`
` 2738, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1155/2014/361329, Wiley Online Library on [04/02/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`Petitioner GE Healthcare – Ex. 1021, p. 8
`
`

`

`BioMed Research International
`
`9
`
`[18F]
`dibenzocyclooctyne(s)
`
`([18F]DBCO)
`
`N
`
`O
`
`O
`
`HN
`
`N
`
`O
`
`x
`
`18F
`
`O
`
`R`
`
`O
`
`HN
`
`x
`
`N
`
`O
`
`18F
`
`[18F]amine functionalized norbornene ([18F]NFB)
`
`O
`[18F]trans-cyclooctene ([ 18F]TCO)
`
`R
`
`HN
`
`NH
`
`O
`
`NH
`
`18F
`
`O
`
`H
`
`H
`
`18F
`
`O
`
`HN
`
`Figure 3: Lead structures of the most important 18F-prosthetic groups applied for copper-free click 18F-fluorination.
`
`in good to excellent RCY of 69–98% within an overall
`reaction time of about 2 h. However, the reaction rates in
`these cases were much slower compared to other examples
`of bioorthogonal reactions, limiting this new approach for in
`vivo pretargeting applications.
`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 18F-azides giving RCY of 19–37% within
`30 min. In 2011, Arumugam et al. [9] investigated the direct
`18F-labeling of azadibenzocyclooctyne (DBCO) yielding the
`18F-labeled prosthetic group (RCY = 36%). The radiolabeling
`was followed by a click reaction with an azido-octreotide
`leading to the 18F-labeled octreotide in a RCY of 95% within
`a total reaction time of 1.5 h. In contrast, other working
`groups used 18F-cyclooctynes for labeling RDG-derivatives
`[11] as well as further integrin-specific peptides [10, 13].
`Another possibility to perform copper-free click reac-
`tions 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-trans-
`cyclooctene 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 18F-labeled
`cyclooctene. The latter are much more suitable for direct
`18F-labeling than tetrazines. For this purpose a nosylate pre-
`cursor was used for 18F-labeling of the cyclooctene providing
`RCY of 71% within 15 min. To investigate the suitability
`of the 18F-prosthetic group in click reactions, the 18F-
`cyclooctene was reacted with a 3,6-di(2-pyridyl)-S-tetrazine
`in an excellent RCY of 98% within 10 s, showing its outstand-
`ing feasibility for in vivo pretargeting approaches. These fast
`
`reaction rates made this approach very attractive that even
`11C-labeling reaction was explored using the inverse electron
`demand Diels Alder cycloaddition between a cyclooctene
`and a tetrazine [98]. In 2011, 18F-labeled cyclooctene was
`linked to a tetrazine-RGD derivative by Selvaraj