202
`
`Current Radiopharmaceuticals, 2010, 3, 202-223
`
`
`
`The Click Chemistry Approach Applied to Fluorine-18
`
`T. L. Ross*
`
`Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz,
`Germany
`
`Abstract: New methods to introduce fluorine-18 into biomolecules are constantly of great interest. Particularly, the in-
`creasing number of complex structures poses a challenge to 18F-labelling chemistry. Indirect 18F-labelling procedures us-
`ing prosthetic groups are commonly used for multifunctional biologically active compounds; however there is continuous
`demand for new and improved radiofluorination methods. The Cu(I)-catalysed variant of the Huisgen 1,3-dipolar cy-
`cloaddition of terminal alkynes and azides represents a most efficient and powerful reaction referred to as click chemistry.
`This reaction is highly specific and provides excellent yields under very mild conditions. This reaction ideally complies
`with the requirements of fluorine-18 labelling chemistry. Hence, it offers a convenient and efficient new 18F-fluorination
`method which is particularly suitable for prosthetic group labelling. The first few reports using the click approach in fluo-
`rine-18 chemistry already demonstrated the particular feasibility of this approach. This review gives an overview of the
`Cu(I) 1,3-dipolar cycloaddition of terminal alkynes and azides and further describes the first applications of this click re-
`action in fluorine-18 labelling.
`
`Keywords: Click chemistry, 18F-labelling, prosthetic group labeling, fluorine-18, Huisgen 1,3-dipolar cycloaddition.
`
`1. INTRODUCTION
`
`Since H.C. Kolb, M.G. Finn and K.B. Sharpless imple-
`mented the term “click chemistry” in 2001 [1], there has
`been a huge interest in this technology, this is clearly indi-
`cated in the number of click papers published every month.
`This click chemistry has been used in a broad range of
`chemical applications [2-5]; including drug discovery and
`development [6, 7], bioconjugate chemistry [8], peptide
`chemistry [9] and polymer and surface science [10-12].
`
`The term click chemistry covers a certain set of reactions,
`particularly the Cu(I)-catalysed cycloaddition between al-
`kynes and azides, forming 1,4-disubstituted 1,2,3-triazoles is
`the most efficient one [1]. Very mild reaction conditions
`accompanied by high efficiency, high selectivity and excel-
`lent yields make click chemistry particularly suitable for
`biological applications as well as for radiopharmaceutical
`sciences. In addition, the 1,2,3-triazole products are biologi-
`cally stable and remarkably biocompatible. Consequently,
`numerous applications of this click reaction can be found in
`drug development and research towards biologically active
`molecules or materials [6-9].
`
`Since its introduction, the use of click chemistry has had
`a huge impact on many chemical domains, thus it is not sur-
`prising that this approach has recently reached the field ra-
`diopharmaceutical chemistry [13]. Its rising significance in
`this field was highlighted at the “17th International Sympo-
`sium on Radiopharmaceutical Sciences” in Aachen, Ger-
`many, April 2007, where an entire session was devoted to
`
`
`
`*Address correspondence to this author at the Institute of Nuclear Chemistry,
`Johannes Gutenberg-University Mainz, Fritz-Strassmann-Weg 2, 55128
`Mainz, Germany; Tel: 0049/6131/39-25316; Fax: 0049/6131/39-25253;
`E-mail: ross@uni-mainz.de
`
`“click labelling methods” in fluorine-18 chemistry [14]. In
`addition, the first application of click chemistry in carbon-11
`labelling has been recently reported [15]. Aside from reports
`in fluorine-18 and carbon-11 chemistry, it has been also suc-
`cessfully applied for technetium and rhenium labelling pro-
`cedures. Metallic nuclides such as technetium and rhenium
`are usually introduced into biomolecules by chelating sys-
`tems which are linked to the target structure. Interestingly,
`here the 1,2,3-triazole moiety was not only functionalised as
`a linker to couple the chelating system to the biomolecule,
`but also as an active ligand which contributes one nitrogen
`atom to the corresponding chelating system [16]. The appli-
`cation of click chemistry in radiotracer development and
`synthesis has recently been reviewed in a mini-review by
`Mamat et al. [17].
`
`Complex and multi-functionalised biomolecules and
`pharmaceuticals rely on suitable and efficient 18F-labelling
`chemistry. In particular, mild labelling methods are essential
`for sensitive structures, such as peptides and proteins. The
`Cu(I)-catalysed cycloaddition of alkynes and azides succeeds
`under very mild reaction conditions and has already been
`proven an implementable method for 18F-labelling chemistry.
`
`This review will give an overview of the Cu(I)-catalysed
`cycloaddition chemistry of alkynes and azides and how it has
`been employed in fluorine-18 labelling.
`
`2. THE CU(I)-CATALYSED “CLICK” CYCLOADDI-
`TION
`
`Originally, click chemistry referred to a range of highly
`efficient reactions which fulfil a set of stringent criteria, with
`the most important ones being very high yields, wideness in
`scope, stereospecificity, simple purification and work-up
`procedures. Moreover, a high thermodynamic driving force,
`usually greater than 20 kcal/mol, is one of the most essential
`
`
`
`1874-4710/10 $55.00+.00
`
`© 2010 Bentham Science Publishers Ltd.
`
`Petitioner GE Healthcare – Ex. 1015, p. 202
`
`

`

`The Click Chemistry Approach Applied to Fluorine-18
`
`Current Radiopharmaceutical, 2010, Vol. 3, No. 3 203
`
`2
`
`NR
`
`N
`
`N
`
`1
`R
`
`+
`
`2
`
`NR
`
`N
`
`N
`
`R1
`
`R1
`
`+
`
`N
`
`N
`
`R2
`
`N
`
`e
`
`(cid:1)
`
`
`Scheme 1. Thermal 1,3-dipolar cycloaddition of azides and terminal alkynes to form 1,4-isomers (1) and 1,5-isomers (2) of disubstituted
`1,2,3-triazoles without distinct regioselectivity.
`
`1
`
`isomeric ratio ~ 1:1
`
`2
`
`characteristics [1]. Such “spring-loaded” reactions include
`ring-opening reactions of epoxides [18, 19], aziridines [18,
`20, 21], aziridinium ions [22] and episulfonium ions [22];
`formation of ureas, thioureas, aromatic heterocycles, oxime
`ethers, hydrazones and amides; oxidative additions to car-
`bon-carbon multiple bonds such as epoxidation [23], dihy-
`droxylation [24], aziridination [25], sulfenyl halide addition
`and certain Michael additions. Beside this respectable set of
`reactions, the most attractive representatives of click chemis-
`try are the cycloaddition reactions involving heteroatoms,
`such as Hetero-Diels-Alder [26, 27] and 1,3-dipolar cycload-
`ditions [28, 29]. Among the latter, the Cu(I)-catalysed Huis-
`gen 1,3-dipolar cycloaddition [30, 31] of azides and terminal
`alkynes forming 1,2,3-triazoles is outstanding and meets
`ideally the requirements of click chemistry. On this account,
`the Cu(I)-catalysed alkyne-azide cycloaddition became the
`most popular reaction referred to as click chemistry.
`
`Although the starting materials of the Huisgen 1,3-
`dipolar cycloadditions, azides and terminal alkynes, are
`highly energetic species, they show relative inertness to a
`wide variety of conditions, thus they tolerate most biological
`and organic conditions [30, 32]. Azides and alkynes are or-
`thogonal to most other organic functionalities and remain
`unaffected through a number of subsequent molecule trans-
`formations until their unification. Their general inertness
`results from a high kinetic stability: this is demonstrated with
`the uncatalysed cycloaddition which were long reaction
`times and elevated temperatures are required [33, 34]. CAU-
`TION: attention should be paid to some kinds of azides
`which show the tendency to explosive decomposition by the
`loss of N2. Exothermic decomposition is favoured especially
`by metallic azides, small organic azides and organic azides
`with a high density of energetic functionalities (In case of
`organic azides, the rule is such that the number of nitrogen
`atoms must not exceed the ones of carbon and that
`(NC + NO)/NN (cid:4) 3, whereby N is the number of atoms of the
`corresponding indices [35]). Furthermore, certain transition
`metal species such as Fe(III) or Co(III) and strong acids can
`catalyse such azide decomposition. On the other hand, ali-
`phatic azides show a particularly high kinetic stability and
`can be prepared, stored and handled without major safety
`issues [35, 36].
`
`Thermal formations of 1,2,3-triazoles using azides and
`alkynes usually show no distinct regioselectivity and give a
`1:1 mixtures of both the 1,4- and 1,5-disubstituted 1,2,3-
`triazoles regioisomers (Scheme 1) [33, 34]. Regioselectivity
`can be introduced by the use of highly electron-deficient
`terminal alkynes which favour the 1,4-isomers (1), but the
`1,5-regioisomer (2) is still observed [37, 38].
`
`A major breakthrough the Cu(I)-catalysed variant of this
`cycloaddition was discovered independently by the Sharpless
`
`[30] and Meldal [31] groups in 2002. In the presence of a
`Cu(I) source, the reaction of azides and terminal alkynes lead
`solely to the 1,4-regioisomers. The Cu(I)-catalysed reaction
`shows a much higher reaction rate up to 107 times higher
`than the non-catalysed version and provides high yields
`without the need of elevated temperatures [2, 39]. On the
`other hand, ruthenium catalysts such as Cp*RuCl(PPh3)2
`were found recently to lead to the 1,5-disubstituted 1,2,3-
`triazoles instead of the 1,4-regioisomers [40, 41]. The Cu(I)-
`catalysed 1,3-dipolar cycloadditions can be performed under
`very mild reaction conditions. Typically aqueous mixtures
`are ideal solvents and give high yields even at room tempera-
`ture. Additionally, most of the functional groups are toler-
`ated by this type of reaction and thus protection group chem-
`istry can be circumvented. The 1,4-substituted 1,2,3,-
`triazoles are obtained in near-quantitative yields and very
`high purities and therefore requiring only minimal work-up
`or purification procedures [1, 6, 30, 31]. In particular cases,
`Cu(I)-catalysed triazole coupling can be employed under
`physiological conditions on and even in living cells [42-46].
`
`In addition to the mild reaction conditions and the
`physiological compatibility of this reaction the resulting
`1,2,3-triazole moieties show excellent biological properties.
`As a result of certain atom positions and electronic proper-
`ties, the triazole unit can mimic a peptide bond when it is
`used as a linker in biomolecules and the heterocyclic struc-
`ture also shows a much higher hydrolytic and redox stability
`[2, 6, 47]. Beside the rigid ring structure, the triazole back-
`bone leads to the calculated R1-R2 distance of 5.0 (cid:6) (Scheme
`1) which is close to the substituents distance of a classic
`dipeptide of 3.9 (cid:6) [48]. Furthermore, the N(2) and N(3) at-
`oms of 1,2,3-triazoles provide two weak hydrogen-bond ac-
`ceptors and due to the very strong dipole moment the C(5)
`atom is highly polarised and can function as a hydrogen-
`bond donor, thus mimicking the amide proton [49, 50]. The
`similarity of 1,2,3-triazole linkers and peptide bonds have
`been and continue to be investigated for a broad range of
`interesting biological applications, including anti-histamine
`activity [51], anti-bacterial activity [52], anti-HIV activity
`[53] and selective (cid:1)3 adrenergic receptor inhibition [54].
`
`2.1. Mechanism of the Cu(I)-Catalysed 1,2,3-Triazole
`Formation
`
`A suitable mechanism for the Cu(I) catalysed cycloaddi-
`tion of alkynes and azides should be able to explain the im-
`pressive rate enhancement by the catalyst, the wide spectrum
`of different azide and alkyne reactants as well as the broad
`variety of tolerated reaction conditions. In contrast to the
`concerted mechanism of uncatalysed 1,3-dipolar cyclo-
`additions, a stepwise mechanism is proposed for the Cu(I)-
`catalysed variant of this reaction (Scheme 2) [2, 30, 55, 56].
`
`Petitioner GE Healthcare – Ex. 1015, p. 203
`
`

`

`204 Current Radiopharmaceutical, 2010, Vol. 3, No. 3
`
`T. L. Ross
`
`,,_.__
`(- I
`Cu
`'
`L
`L
`2 _e:;
`\
`I
`Cu +
`
`-
`
`-
`R1
`
`N
`
`N
`(±)
`
`N
`\
`
`2
`R
`
`5
`
`C
`
`R1
`
`( ~
`II
`( ~
`
`Cu 2L n
`
`2
`
`Cu 2L n
`
`1
`
`R
`
`R1
`
`8
`-
`-
`N N N
`(±)
`'
`R2
`
`B
`
`4
`
`R1
`
`R1
`
`D
`
`N
`'',~
`N
`Cu2Ln
`
`2
`
`N R
`
`'
`
`-
`
`......---
`
`N
`'',~
`N
`Cu 2Ln
`
`N R
`
`2
`
`8
`
`7
`
`111
`
`1
`R
`
`R1
`
`(
`
`A
`
`CuLn
`
`~
`[CuLn]2
`
`E
`
`R1
`~ -
`
`,:½
`
`N
`
`N N
`
`2
`
`R
`
`1
`
`j
`fr ,,-,
`\./\
`
`N
`
`\\
`----
`
`+
`
`Cu
`
`LL
`
`\I
`
`N
`
`Cu
`
`_;,--
`
`N
`
`R2
`
`6
`
`
`
`Scheme 2. Proposed stepwise mechanism of Cu(I)-catalysed alkyne-azide couplings [2, 56].
`
`When internal alkynes were found to show no reactivity
`under catalytic conditions, Cu(I) acetylides were already
`suggested as a reactive intermediate in the catalytic process
`[30, 31]. Density functional theory (DFT) calculations on
`monomeric Cu(I) acetylides complexes indicate for a step-
`wise mechanism whereas a concerted cycloaddition of the
`Cu(I) acetylide and the azide is strongly disfavoured by a
`activation barrier of 23.7 kcal/mol [55].
`
`Initially, the Cu(I) acetylide complex (4) (step A) is
`formed, thereby the ligands of the Cu(I) species are dis-
`placed. In case of acetonitrile ligands, step A is calculated to
`be slightly endothermic (approx. -0.6 kcal/mol), which be-
`comes exothermic (11.7 kcal/mol) for water ligands [55].
`This is consistent with the experimental results that the rate
`is much higher in aqueous systems where no additional base
`is needed.
`
`Moreover, Cu(I) acetylides are well-known as utilities for
`C-C bond formations [57] and they are most probably result-
`ing from an initial (cid:1)-complexation of the acetylene with the
`Cu(I) species. For such a (cid:1)-coordination, calculations show a
`dramatic drop in the pKa of the terminal alkyne proton by up
`to 9.8 pH units which makes a subsequent deprotonation and
`thus step A accessible in aqueous solutions [55].
`
`In the next step (B), the azide is coordinated by Cu(I) and
`activated for a electrophilic attack of N(3) at C(4) ((cid:2) no-
`menclature according to triazole rings) [2]. While this elec-
`trophilic attack takes place, the Cu-C(5) double bond formed
`with electron donation from a full d-orbital of the copper.
`
`The depicted Cu(I) dimer (5) in is just one of several pos-
`sible structures which are consistent with DFT calculations
`[55]. Kinetic studies found the catalytic process to be second
`order in copper [56, 58]. However, results also indicate that a
`dynamically exchanging family of Cu(I) acetylide complexes
`and even (cid:1)-complexes of alkynes may also be involved [56,
`59, 60]. Multinuclear copper acetylide complexes are com-
`mon [59], but so far only one known example [61] has been
`
`shown to catalyse the 1,2,3-triazole formation [56]. A huge
`excess of acetylene is proposed to lead to saturated coordina-
`tion of the Cu(I) species and the catalytic effect is inhibited.
`Saturated Cu(I) species are unable to coordinate additionally
`to the azide, as a weaker ligand, and the Cu(I) species remain
`catalytically ineffective [56]. In the same way, commercially
`available copper acetylides, which are already saturated by
`acetylenes, show no catalytic effect. Hence, labile ligand
`dissociation seems to be necessary for catalytic activity [2].
`
`As a result of the abovementioned electrophilic attack,
`the metallocycle (6) is generated in step C [2]. Such eight-
`membered metallocycles containing two copper centres are
`already known and have been characterised [62]. In case of
`monomeric copper complexes which lead to six-membered
`metallocycles, calculations indicate a very low energy barrier
`of only 3.2 kcal/mol for the final ring contraction of the met-
`allocycle to the triazolyl-copper (8) (step D) [55]. Although
`the different ring sizes in both mechanisms may possess little
`variation in kinetics, the conversion to the triazolyl-copper
`(8) should be similarly fast. Experimental findings of elec-
`tron-withdrawing substituents on the alkyne which accelerate
`the catalysis are consistent with both proposed mechanisms
`for step C and D, respectively [31, 60].
`
`In the final step (E), the triazolyl-copper (8) is protonated
`and the catalyst is regenerated while the catalytic cycle is
`completed. Studies using deuterated alkynes or deuterated
`solvents, observed both complete loss of deuterium and high
`incorporation of deuterium on the C(5) position of the tria-
`zole, respectively [55, 56]. Accordingly, solvent molecules
`or a protonated additional base can be assumed as proton
`source.
`
`2.2. Generation of the Cu(I) Catalyst
`
`The Cu(I) catalysed 1,3-dipolar cycloaddition shows high
`consistency and efficiency over a very broad range of condi-
`tions, therefore the catalyst should also be stable and effec-
`tive under these conditions. Typically, there are two princi-
`
`Petitioner GE Healthcare – Ex. 1015, p. 204
`
`

`

`The Click Chemistry Approach Applied to Fluorine-18
`
`Current Radiopharmaceutical, 2010, Vol. 3, No. 3 205
`
`OH
`
`CuSO 4
`Na ascorbate
`
`HO
`NN
`OH
`N
`
`S
`
`OH
`
`H
`
`H
`
`H
`
`HO
`
`J-
`
`S
`
`HO
`
`N3
`
`9
`
`10
`
`OH
`
`HO
`
`H
`
`H
`
`H
`
`11
`
`
`
`Scheme 3. Cu(I)-catalysed cycloaddition of 17-ethynylestradiol (9) and (S)-3-azidopropane-1,2-diol (10). The reaction was accomplished in
`water/tert.-butanol (1:1) at room temperature for 12 – 24h using the CuSO4/sodium ascorbate system (1:10 mol%). The pure product (12)
`was achieved by filtration in a yield of 94% [30].
`
`R1
`
`+
`
`N
`
`N
`
`R2
`
`N
`
`e
`
`12
`
`13
`
`Cu nanoclusters
`(0.1 mol-%)
`
`H2O/tert .-BuOH
`25 °C, 18h
`
`R1
`
`2
`
`R
`
`N
`
`N N
`
`14
`
`R1 = C6H5; C6H5(OH)CH; CH2(OH)
`
`= C6H5; C6H5CH2
`R
`Scheme 4. Cycloadditions of various alkynes of type 12 and azides of type 13 catalysed by Cu(0) nanoclusters. The reactions were carried
`out in water/tert.-butanol (2:1) at 25 °C for 18h. The pure 1,4-disubstituted 1,2,3-triazole products (14) were isolated by filtration in yields of
`80 - 99% [74].
`
`2
`
`ples to provide catalytically active Cu(I) species in the reac-
`tion mixture: One is a direct adding a Cu(I) salt or complex
`directly, while the second technique is based on in-situ gen-
`erations of the Cu(I) species. However, the direct addition of
`Cu(I) species requires generally oxygen-free conditions due
`to
`the
`relatively
`instable Cu(I)
`oxidation
`state
`(E ° (Cu2+ (cid:2) Cu+) = + 0.16 V in water). The in-situ ap-
`proach can be further subdivided into the reduction of a
`Cu(II) salt and the oxidation of metallic Cu(0) forms. As a
`major advantage, the in-situ methods do not require an inert
`atmosphere and can be performed in aqueous mixtures under
`“normal” atmosphere.
`
`The in-situ formation of the Cu(I) catalyst by reduction
`of Cu(II) salts can be done either by a reducing agent or by
`comproportionation with copper metal. As mentioned before,
`these systems tolerate aqueous mixtures and “normal” at-
`mosphere. Generally, the systems are very forgiving of
`common reaction conditions and further tolerate most func-
`tional groups. The most commonly used reducing agent is
`sodium ascorbate (3-10 mol-%) in combination with readily
`available and stable Cu(II) salts (1-5 mol-%), such as Cu(II)
`sulphate pentahydrate or Cu(II) acetate. Another reducing
`agent, tris(2-carboxyethyl)phosphane hydrochloride (TCEP)
`has also been used particularly in biological systems [42, 46,
`63]. In similar applications, where the substrate or biological
`moiety is sensitive to commonly used reducing agents, com-
`proportionation with Cu(0) can be easily accomplished by
`the addition of small pieces (i.e. wire or turnings) of copper
`metal [39, 43, 64]. Typically, the in-situ reactions are carried
`out in water-alcohol mixtures (frequently tert.-butanol) at
`room temperature or with a gentle heating. One example of
`the Cu(I)-cataysed coupling of 17-ethynylestradiol (9) and
`(S)-3-azidopropane-1,2-diol (10) is outlined in Scheme 3.
`The reaction was carried out in a 1:1 mixture of water/tert.-
`
`the
`temperature for 12 - 24h using
`butanol at room
`CuSO4/sodium ascorbate system (1:10 mol-%). The isolated
`pure product (12) was obtained by filtration in a yield of
`94% [30]. Generally, the resulting 1,2,3-triazoles can often
`be isolated after simple work-up procedures such as filtration
`or phase extraction in nearly quantitative yields and high
`purities (> 90%) [30, 56, 58, 65-72].
`
`An additional preparation method of catalytic amounts of
`Cu(I) available for the 1,3-dipolar cycloaddition is oxidation
`of copper metal. In a simple experimental procedure, only a
`small piece of copper metal is added to the reaction mixture.
`This oxidative generation effectively offers Cu(I) without the
`need for an additional oxidation agent [30, 55]. The normal
`surface of copper metal contains copper oxides and carbon-
`ates in the patina in adequate amounts to initialise the in-situ
`generation of the catalyst. Usually, these protocols require
`long reaction times of about 12-48 hours and excess of cop-
`per, but the triazole products can be obtained in good yields
`and high purity without copper contaminations [30, 55].
`
`In alternative methods, the use of nanosize copper, cop-
`per nanoclusters and copper nanoparticles save reaction
`times and show similarly high efficiency as other catalyst
`generating methods [73-75]. The dissolution of Cu(0)
`nanosize activated powder into Cu(I) is mediated by amine
`hydrochloride salts and requires slightly acidic conditions of
`about pH 5. Alkynes or azides bearing an amine hydrochlo-
`ride salt in a functional group, can sufficiently provide this
`oxidative dissolution of Cu(0) into Cu(I) without the addition
`of an amine hydrochloride salt [73]. In contrast, when using
`copper nanoclusters, the alkyne-azide coupling is efficiently
`catalysed without amine hydrochloride salt support (Scheme
`4) [74]. Even the formation of active copper acetylides is
`supposed to take place on the surface of the nanoclusters
`rather than in solution, Cu(I) is still most likely the active
`
`Petitioner GE Healthcare – Ex. 1015, p. 205
`
`

`

`206 Current Radiopharmaceutical, 2010, Vol. 3, No. 3
`
`T. L. Ross
`
`FmocHN
`
`+
`
`O
`
`N3
`
`OH
`
`CuI, DIPEA,
`2,6-lutidine
`
`CH3CN, RT, 3h
`
`FmocHN
`
`N
`
`N
`
`N
`
`O
`
`OH
`
`15
`
`16
`
`17
`
`
`
`Scheme 5. Fmoc-N-propargylamine (15) is coupled to (cid:1)-azido D-leucine (16) using CuI as a direct source of Cu(I) in acetonitrile. The bases
`DIPEA and 2,6-lutidine (2 eq. each) were employed. The 1,2,3-triazole product 17 was obtained in a yield of 97% within 3 h at room tem-
`perature [47].
`
`N
`
`N
`
`N
`
`20
`
`NN
`
`Bn
`
`N
`
`N
`
`N
`
`N
`
`Bn
`
`N
`
`TBT A
`
`NN
`N
`
`Bn
`
`Cu(CH3CN)4PF6
`H2O/t er t.-BuOH
`RT , 24h
`
`~
`
`+
`
`N3
`
`18
`
`19
`
`
`tris-
`(19) using
`(18) and benzyl azide
`Scheme 6. Ligand-assisted Cu(I)-catalysed cycloaddition of phenylacetylene
`(benzyltriazolylmethyl)amine (20) (TBTA) as ligand. Only 0.01 equivalents of Cu(CH3CN)4PF6 as catalyst effectively gave 1-benzyl-4-
`phenyl-1H-1,2,3-triazole (21) in a yield of 84% within 24 h at room temperature [83].
`
`21
`
`oxidation state in the reaction mixture [76]. In case of the
`oxidative generation of Cu(I) catalyst, the nanosize materials
`of copper circumvent long reaction times and offer a compa-
`rable broad range in application and similar high yields as
`alternative protocols.
`
`Direct Cu(I) catalyst sources are provided by either Cu(I)
`salts, for instance CuI and CuBr, or coordinated Cu(I) com-
`plexes such as CuOTf · C6H6 [30], [Cu(CH3CN)4]PF6 [30],
`[CuP(EtO)3]I [77, 78] and [Cu(PPh3)3]Br [68, 77, 78]. Direct
`Cu(I) catalysts are usually employed in organic solvent sys-
`tems as aqueous systems cause solubility problems [78].
`Organic protocols are frequently used in polymer applica-
`tions [10, 68, 78, 79].
`
`All these organic-based systems require a nitrogen base
`like triethylamine, diisopropylethylamine (DIPEA), pyridine,
`bipyridine or 2,6-lutidine. In particular, the excess of base
`leads to higher yields within shorter reaction times (Scheme
`5) [30, 31, 47, 80]. While in aqueous systems the deprotona-
`tion is provided by water and no additional base is required,
`in organic systems the base is essential in the catalytic cycle
`for the deprotonation of the initially formed (cid:2)-complex
`which makes the active Cu(I) acetylide complex (4) (step A
`in Scheme 2) available [55, 56]. Regarding the catalytic spe-
`cies, the Cu(I) complexes show particularly high solubility
`and efficiency in the organic systems while Cu(I) salts have
`only limited solubility. However, neither a complete dissolu-
`tion of the catalyst nor the coupling reactants seems to be
`entirely essential, since there are examples where excellent
`yields and purities are obtained with very limited solubilities
`and in heterogeneous reaction mixtures [30]. Major draw-
`backs of direct protocols are: sensitivity of the Cu(I) oxida-
`tion state, sensitivity to reaction conditions and complicating
`side reactions which can be circumvented usually with the
`
`in-situ methods [30, 31, 47]. The use of steric demanding
`bases such as DIPEA and 2,6-lutidine can minimise side-
`product formations, which are assumed to result mainly from
`alkyne homocoupling mediated by unhindered nitrogen
`bases [2, 30, 47, 48, 57, 81]. Overall, the direct Cu(I) cata-
`lysts were successfully employed in a broad range of organic
`systems and the corresponding 1,2,3-triazoles were obtained
`in good yields [30, 31, 47, 48, 68, 77-80, 82].
`
`On the whole, there are several sufficient methods for
`providing catalytically active Cu(I) species in alkyne-azide
`cycloaddition. The more robust systems provide simple pro-
`cedures and high yields. For special cases, alternative proto-
`cols with similar efficiency are available. The optimal sys-
`tems and conditions often depend on the substrates and the
`final application. Generally, the work-up procedures for most
`reactions require only simplest work-up methods and purifi-
`cation techniques.
`
`2.3. Ligand-supported Cu(I)-Catalysed Alkyne-Azide
`Cycloaddition
`
`Although the general protocols of the Cu(I)-catalysed al-
`kyne-azide coupling show high efficiency, in some cases
`faster kinetics are helpful and preferable. Predominantly in
`bioconjugate applications very low concentrations of the
`reactants and the catalyst are required to prevent degradation
`reactions of the biological scaffolds. Some particular hetero-
`cyclic chelators have been found to accelerate the 1,3-dipolar
`cycloaddition, most likely by chelating and stabilising the
`Cu(I) species. Most effective ligands are oligo- or polytria-
`zoles [83] as well as electron-rich bipyridines [58] and
`bis(oxazolinyl)pyridines (pybox) [84]. The commonly used
`ligand tris-(benzyltriazolylmethyl)amine (20) (TBTA), pro-
`vides very high efficiency and thereby reduces the minimum
`catalyst loading as much as tenfold (Scheme 6) [45, 83].
`
`Petitioner GE Healthcare – Ex. 1015, p. 206
`
`

`

`The Click Chemistry Approach Applied to Fluorine-18
`
`Current Radiopharmaceutical, 2010, Vol. 3, No. 3 207
`
`b
`,f~
`,, 0
`
`N
`
`N
`
`N
`
`21
`
`
`
`NaN3
`Cu(0), CuSO 4
`
`H2O/t er t.-BuOH
`125 °C (MW), 10 m in
`
`0
`
`Br
`
`+
`
`111
`
`0
`
`18
`
`22
`
`Scheme 7. Microwave-supported one-pot Cu(I)-catalysed 1,2,3-triazole formation using phenylacetylene (18), benzyl bromide (22) and so-
`dium azide. Cu(0) turnings and CuSO4 in 1:1 H2O/tert.-BuOH gave 93% of 1-benzyl-4-phenyl-1H-1,2,3-triazole (21) within 10 min at
`125 °C (100 W) [39].
`
`the traditional protocols. This approach furthermore enables
`the one-pot creation of interesting polyvalent molecules such
`as dendrimers. Accordingly, starting from in-situ prepared
`small multi-azides and subsequent (multi)-cycloadditions
`with in-situ available alkynes lead to molecules containing
`several 1,4-substituted 1,2,3-triazole moieties. In Addition,
`microwave-assisted one-pot procedures make the triazole
`products obtainable within short reaction times of 10-30 min
`(Scheme 7) [39]. The ability of the alkyne-azide cycloaddi-
`tion to be combined in a one-pot strategy with other reac-
`tions was shown in a one-pot synthesis of various spiro-
`trione-1,2,3-triazoles. The one-pot synthesis successfully
`accomplished four subsequent transformations; a Wittig re-
`action, a Knoevenagel condensation, a Diels-Alder cycload-
`dition and finally a Cu(I)-catalysed 1,2,3-triazole formation
`[90].
`
`2.5. Microwave-assisted Cu(I)-Catalysed 1,2,3-Triazole
`Formation
`
`Although the Cu(I)-catalysed alkyne-azide cycloaddition
`is generally very efficient at room temperature, completion
`often requires reaction times of 12 – 24 hours [30, 31]. The
`reaction can be dramatically accelerated by microwave heat-
`ing and thereby the reaction times are reduced to minutes
`[39, 68, 77, 78, 91-93]. Similar to the traditional methods,
`microwave-assisted alkyne-azide coupling reactions can be
`performed in organic solvents with directly supported Cu(I)
`species [77, 78] as well as in aqueous systems with in-situ
`generated Cu(I) catalysts [39, 68, 91-93]. In case of solubil-
`ity problems in aqueous systems, microwave chemistry can
`provide fast access to the desired 1,2,3-triazole products in
`anhydrous organic solvents with extremely low catalyst load-
`ings [91]. Furthermore, microwave-assistance was also ap-
`plied in one-pot Cu(I)-catalysed alkyne-azide coupling reac-
`tions and gave the desired 1,2,3-triazoles in high yields after
`very short reaction times of 10 – 15 min (Scheme 7) [39].
`Undoubtedly, the microwave approach benefits from very
`short reaction times, whereas the triazole products show no
`significant enhancement in yield or purity. Hence, micro-
`wave heating considerably accelerate the 1,2,3-triazole for-
`mation. However, there is also evidence which suggests that
`the rate of undesired side reactions is increased [2, 77]. In
`case of 18F-labelling applications, time-saving and fast meth-
`ods are highly required. The dramatic rate acceleration of the
`microwave chemistry makes Cu(I)-catalysed alkyne-azide
`coupling very attractive for 18F-labelling.
`
`2.6. Further Protocols of Cu(I)-Catalysed Alkyne-Azide
`Coupling
`
`In a recent report, a new solvent system using dichloro-
`methane as a co-solvent with water in Cu(I)-catalysed
`
`The tertiary amine centre and the triazole moieties in
`TBTA concertedly function as a tetradentate chelating sys-
`tem of the Cu(I) ion and protect Cu(I) from oxidative degra-
`dation. Moreover, the tertiary amine in TBTA provides a
`proton acceptor, thus making additional base in organic sys-
`tems unnecessary [44, 83]. As a result, TBTA has been suc-
`cessfully used in various bioconjugation studies [42-46, 58,
`63, 80].
`
`Other ligands of the bipyridine and the pybox type effec-
`tively accelerate the Cu(I) catalysed alkyne-azide cycloaddi-
`tion. The water-soluble and commercially available bipyri-
`dine bathophenanthrolinedisulfonic acid [1] has been found
`as a suitable alternative to TBTA. In particular, this ligand
`has been shown to exceed the efficiency of TBTA. However,
`this ligand increases the oxygen sensitivity of the Cu(I) cata-
`lysts and therefore oxygen-free conditions are essential [58,
`85]. Further ligands of the chiral pybox family have been
`used as asymmetric auxiliaries [84]. These chiral molecules
`enable enantioselective alkyne-azide cycloadditions, al-
`though moderate selectivity and kinetic resolution are re-
`ported.
`
`In general, certain auxiliary ligands enlarge the adaptabil-
`ity of the 1,3-dipolar cycloaddition of alkynes and azides,
`especially for bioconjugate applications and experiments at
`low reagent concentrations. In addition, chiral pybox ligands
`offer an enantioselective variant of this catalysis, even
`though further investigations towards higher selectivity in
`the kinetic resolution are necessary. Major advantages of the
`ligand-assisted process are faster kinetics and higher reactiv-
`ity at low concentrations of reactants.
`
`2.4. One-pot Protocols of Cu(I)-Catalysed Alkyne-Azide-
`Coupling
`
`The usual safety inconvenience of handling small organic
`azides which tend to rapid decomposition could be circum-
`vented by an in-situ generation of these compounds without
`the need of their isolation. Such one-pot procedures gener-
`ally require specific selectivity in each reaction step with
`minimal side-product formations. Alkyne and azide func-
`tionalities show broad orthogonality to other functional
`groups, thus their cycloaddition is particularly suitable for
`this approach. Organic azides could be generated success-
`fully from sodium azide and the corresponding halides or
`arylsulfonates in-situ; followed by high-yielding cycloaddi-
`tions with the appropriate alkynes [39, 86, 87]. In addition,
`organic azides can also be obtained in-situ from halides via a
`copper-catalysed reaction with sodium azide and catalytic
`amounts of L-proline [88, 89]. Remarkably, the one-pot click
`chemistry protocols show the same excellent efficiency as
`
` 1
`
` CAS. No. 53744-42-6
`
`Petitione