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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nucl Med Biol. Author manuscript; available in PMC 2013 November 1.
Published in final edited form as:
PMCID: PMC3468714

Highly efficient click labeling using 2-[18F]fluoroethyl azide and synthesis of an 18F N-hydroxysuccinimide ester as conjugation agent



Click labeling using 2-[18F]fluoroethyl azide has been proven to be promising methods of radiolabeling small molecules and peptides, some of which are undergoing clinical evaluations. However, the previously reported method afforded low yield, poor purities and under desirable reproducibility.


A vacuum distillation method was used to isolate 2-[18F]fluoroethyl azide, and the solvent effect of acetonitrile (ACN) and dimethylformamide (DMF) on the click labeling using Cu(I) from copper sulfate/sodium ascorbate was studied. The labeling conditions were optimized to radiosynthesize a hydroxysuccinimide ester (NHS).


2-[18F]fluoroethyl azide was isolated by the vacuum distillation method with > 80% yield within 10 min in a “pure” and click-ready form. It was found that the amount of DMF was critical for maintaining high levels of Cu(I) from copper sulfate/sodium ascorbate in order to rapidly complete the click labeling reaction. The addition of bathophenanthrolinedisulfonic acid disodium salt (BPDS) to the mixture of copper sulfate/sodium ascorbate also greatly improved the click labeling efficiency. Through exploiting these optimizations, a base-labile N-hydroxysuccinimide (NHS) ester was rapidly radiosynthesized in 90% isolated yield with good chemical and radiochemical purities.


We have developed a general method to click-label small molecules efficiently using [18F]2 for research and clinical use. This NHS ester can be used for conjugation chemistry to label antibodies, peptides and small molecules as PET tracers.

Keywords: PET, Fluorine-18, fluoroethyl azide, click chemistry, hydroxysuccinimide ester

1. Introduction

The identification of new small-molecule ligands for numerous biological targets has led to the growth of the non-invasive imaging technique positron emission tomography (PET) in both clinical and research fields [1, 2]. PET imaging studies require the radiolabeling of a compound with ideal biological properties in high yield with good chemical and radiochemical purity. Fluorine-18, which is readily available in high specific activity from most medical cyclotrons, is widely used for PET radiolabeling of small molecules because of its small size and favorable decay characteristics (100% positron, t1/2 = 109.7 min). Typical fluorine-18 radiolabeling employs nucleophilic substitution of a good leaving group with [18F]fluoride, which is produced in [18O]water and azeotropically dried in the presence of Kryptofix 222 and potassium carbonate or another base at elevated temperatures [3, 4]. Because this reaction proceeds under harsh and basic conditions, attempts to label temperature- or base-sensitive products may result in a lot of radiolabeled and non-radioactive by-products, poor yields, and high variability. Furthermore, optimization of labeling conditions and the necessary HPLC purification for removal of by-products is costly, time-consuming and requires highly-trained personnel. The widespread dissemination of PET radiopharmaceuticals demands a facile method to label compounds with fluorine-18.

Since the term “click chemistry” was introduced in 2001[5], Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) [6, 7] has been extensively utilized in many fields of chemistry [8], including PET radiolabeling [9]. This Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of terminal alkynes and organic azides affords 1,4-disubstituted 1,2,3-triazoles exclusively, in high yield and under mild conditions. The 1,2,3-triazole group is stable to acid and basic hydrolysis, as well as reductive and oxidative conditions due to its aromaticity. Furthermore, this triazole group is biologically stable [10] and possesses a polarity and size similar to that of an amide bond [11], thereby improving water solubility [12] and allowing the synthesis of a wide-range of compounds with biological potential [13]. Because of these features of the click reaction and the readily accessible building blocks with azide and alkyne, click labeling has become a valuable and powerful tool in PET chemistry [9].

Many fluorine-18 labeled alkynes and azides have been synthesized for labeling of small molecules and peptides as PET tracers using click chemistry [9]. Among the reported compounds used for click labeling of small molecules and conjugation with peptides, 2-[18F]fluoroethyl azide ([18F]2) appeared to be the most promising fluorine-18 labeled azide [14]. Its advantages include:1) it can be used with a wide variety of commercial available or easily synthesized small molecules and pharmaceuticals that contain an alkyne as a building block; 2) 2 is the lowest molecular weight azide that is suitable for labeling with F-18, and facilitates the introduction of a triazole group with the least steric effect to a labeled molecule; 3) the incorporation yield of [18F]2 has been reported to be >95% and it can be distilled/purified from the reaction mixture [14]. [18F]2 has been previously used to successfully label small molecules and peptides [1420]. However, [18F]2 was isolated only in ~50% yield with impurities that are difficult to separate, resulting in low specific activity of final products [14, 16, 17], and the click labeling conditions in the literature sometimes required elevated temperatures or long reaction times [14].

During our labeling of a variety of small molecule PET tracers with [18F]2, a highly efficient method for the isolation by vacuum distillation of [18F]2 was developed, and the subsequent click labeling conditions were optimized in order to afford final products at room temperature within 10 minutes, and with high purity and specific activity after facile HPLC purification. Here we describe this distillation procedure and our exploration of the solvent effects on the click chemistry which facilitated optimization of the reaction. This improved method was demonstrated by the radiosynthesis of a base-labile NHS ester in high yield using the click labeling with [18F]2. Our method will provide an efficient and general procedure for the click labeling of small molecules using [18F]2 as a synthon for PET labeling.

2. Materials and methods

2.1. General

All chemicals were obtained from standard commercial sources and used without further purification. All reactions were carried out by standard air-free and moisture-free techniques under an inert nitrogen atmosphere with dry solvents unless otherwise stated. Flash column chromatography was conducted using silica gel. Melting points were uncorrected. Routine 1H and 13C NMR spectra were recorded at 300 or 400 MHz. All chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS). All coupling constants (J) are given in Hertz (Hz). Splitting patterns are typically described as follows: s, singlet; d, doublet; t, triplet; m, multiplet. ESI/MS was performed on a Waters ZQ 4000 single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) LC-MS interface. High performance liquid chromatography (HPLC) was performed with an ultraviolet detector and a well-scintillation NaI (Tl) detector and associated electronics for radioactivity detection. An Agilent SB-C18 250 × 9.4 mm 5 μ semi-preparative column (A) and an Agilent SB-C18 250 × 4.6 mm 5 μ analytical column (B) were used for preparation and analysis respectively. [18F]Fluoride was produced at Washington University by the 18O(p,n)18F reaction through proton irradiation of enriched (95%) [18O] water in the RDS111 cyclotron. Radio-TLC was accomplished using a Bioscan AR-2000 imaging scanner (Bioscan, Inc., Washington, DC). Published methods were used for the synthesis of compound 1 [21], 2 [14], 3 [22], and 5 [23] according to literature.

2.2. 1. Synthesis of 4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (4)

To a solution of 3 (0.2 g, 1.25 mmol) in DMF (5 mL) was added a solution of 2 (0.2 g, 2.25 mmol) in DMF (5 mL), followed by addition of a mixture of CuSO4·5H2O (70 mg in 0.9 mL water) and sodium ascorbate (500 mg in 1.8 mL water). The reaction mixture was stirred at room temperature for 1 h, and then diluted with water (100 mL). Ethyl acetate (3 × 50 mL) was used to extract the product from the aqueous phase. The combined organic phase was washed by saturated NaHCO3 solution (3 × 20 mL), dried over Na2SO4. After the solvent was removed under reduced pressure, the crude product was purified by silica gel flash chromatography using ethyl acetate (EtOAc) to afford 4 as a white solid (247 mg, 79 %). mp, 76–77 °C; 1H NMR (400 MHz, DMSO-D6) δ 9.84 (s, 1H), 8.28 (s, 1H), 7.84 (d, J = 8.8, 2H), 7.21 (d, J = 8.8, 2H), 5.26 (s, 2H), 4.65–4.88 (m, 4H); 13C NMR (100 MHz, DMSO-D6) δ 191.77, 163.38, 142.65, 132.22, 130.31, 125.72, 115.62, 83.16, 81.49, 61.83, 50.61, 50.42.

2.2.2. Synthesis of 2,5-dioxopyrrolidin-1-yl 4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)benzoate (7)

To a solution of 6 (50 mg, 0.18 mmol) in ACN (0.8 mL) and EtOAc (1 mL) was added a solution of 2 (40 mg, 0.45 mmol) in DMF (1 mL), followed by addition of a mixture of CuSO4·5H2O (2.5 mg in 25 μL water), sodium ascorbate (7.5 mg in 25 μL water) and BPDS (6 mg in 100 μL 4:1 water/DMF). The mixture was stirred at room temperature and the progress monitored by TLC. When silca TLC indicated that the starting material 6 was consumed, the reaction was quenched with water (30 mL) and TFA (100 μL). Ethyl acetate (3 × 20 mL) was used to extract the product, and the organic phase was dried over Na2SO4. After ethyl acetate was removed under reduced pressure, the crude product was purified by silica gel chromatography using ethyl acetate to afford 7 as a white solid (56.4 mg, 85 %). mp, 156.0–157.5 °C; 1H NMR (400 MHz, DMSO-D6) δ 8.28 (s, 1H), 8.01 (d, J = 9.2, 2H), 7.25 (d, J = 9.2, 2H), 5.29 (s, 2H), 4.65–4.88 (m, 4H), 2.84 (s, 4H); 13C NMR (100 MHz, DMSO-D6) δ 170.88, 163.96, 161.68, 142.48, 132.80, 125.79, 117.04, 116.03, 83.15, 81.48, 61.96, 50.62, 50.43, 25.95; ESI/MS: 363.5 (M + H+).

2.3.1. Radiosynthesis of 2-[18F]fluoroethyl azide (2) and vacuum distillation

[18F]fluoride (up to 50 mCi in 100–500 μL [18O]water) was transferred to a BD vacutainer (13 × 75 mm, 5 mL, glass, no additives) containing K222 (5.6 mg, 14.9 μmol) and K2CO3 (1 mg, 7.2 μmol), then the mixture was dried by azeotropic distillation at 105 °C using ACN (3 × 1 mL) under a gentle flow of N2 gas. When the drying was close to finish, the vacutainer was removed from the oil bath and the solvent residue (< 100 μL) was removed by a flow of N2 at room temperature. The vacutainer was capped and connected to a dry ice trap (10 mL Pyrex tube with screw-cap) via Teflon tubing (I.D. 1.0 mm) (see Figure 1). After a solution of 1 (2 mg, 8.3 μmol) in ACN (200 μL) was added to the vacutainer, it was shaken and heated at 88 °C for 5 min, directly followed by vacuum distillation which was achieved by a 50 mL syringe. The distillation lasted for 1 min, after which N2 gas (10 mL) was released to the vacutainer. After this distillation procedure was repeated, the Pyrex tube was removed from the dry ice bath and warmed to room temperature in a water bath for the subsequent click labeling. The processing time is about 10 min from the beginning of the labeling reaction, and the isolated yield is >80 % (n > 30), determined by measuring the distilled activity (decay corrected).

Figure 1
Vacuum distillation of [18F]2

Warning: [18F]2 is extremely volatile and should be handled only in a fume hood. Low boiling point solvents (such as dichloromethane and diethyl ether) are not recommended as TLC solvents. A charcoal trap is sufficient to trap [18F]2 and should be attached to any vents of the system.

2.3.2. General method of click labeling using [18F]2 and CuSO4 and sodium ascorbate as Cu(I) source: radiosynthesis of [18F]4

A solution of CuSO4·5H2O (5 mg, 20.0 μmol) in water (50 μL) and a solution of sodium ascorbate (15 mg, 75.7 μmol) in water (50 μL) were mixed, and when the color of the mixture changed from black to yellow in minutes, the above mixture was added, along with a solution of 3 (1.0 mg, 6.2 μmol) in DMF (200 μL), to the distilled [18F]2 in ACN (200 μL) at room temperature. The reaction mixture was shaken occasionally during the reaction. For analysis, an aliquot of the reaction mixture was diluted in ACN or HPLC mobile phase for analysis by silica gel TLC (developed in ethyl acetate; Rf: 0.0 (unknown), 0.48 ([18F]4), 0.88([18F]2)) or by reversed phase HPLC (column B, 40 % ACN, 60 % water, 0.1 % TFA, 1 mL/min, 272 nm; Retention time (min): 2.2 (unknown), 5.6 ([18F]4), 6.2([18F]2).

2.3.3. Radiosynthesis of 7 using CuSO4, sodium ascorbate and BPDS as Cu(I) source

A solution of CuSO4·5H2O (1 mg, 4.0 μmol) in water (10 μL) and a solution of sodium ascorbate (3 mg, 15.1 μmol) in water (10 μL) were mixed; when the color of the mixture changed from black to yellow, the copper salt solution was mixed with a solution of BPDS (1.2 mg, 2.0 μmol) in 1 : 4 DMF/H2O (20 μL). A portion of above mixture (25 μL), along with 6 (1 mg, 3.66 μmol) in DMF (200 μL), was added to the distilled [18F]2 in ACN (200 μL). After 5 min at room temperature, the reaction mixture was diluted with a solution of 10% ACN, 90% water, 0.1% TFA(3 mL) for reversed-phase HPLC purification using column A and mobile phase (32% ACN, 68% water, 0.1% TFA) at 4 mL/min, and UV at 240 nm. [18F]7 was collected at 15min in 90% yield (decay corrected). The HPLC fraction containing [18F]7 was diluted in water (50 mL), then [18F]7 was isolated by solid-phase extraction using a C18 SepPak by passing the diluted sample through the SepPak. The SepPak was rinsed with water (10 mL), and dried under a stream of N2 gas. [18F]7 can be eluted from the SepPak in DMF for immediate conjugation, or in either dichloromethane or diethyl ether, to afford a dried form of [18F]7 after drying over Na2SO4 and removal of solvents.

3. Results and Discussion

3.1. Radiosynthesis of [18F]2 and vacuum distillation

The radiosynthesis of [18F]2 was carried out as described in the literature under typical labeling conditions (Scheme 1). Up to 95 % analytical yield is expected when carried out at 80 °C for 15 min, and published literature indicated that a flow trap method could be used to isolate [18F]2 from the reaction mixture at 130 °C in 63% distillation efficiency or 54% decay corrected radiochemical yield [14]. Following this protocol for our synthesis of [18F]2, the volatile [18F]2 was unexpectedly difficult to trap, even when employing a liquid nitrogen trap with the recommended flow distillation method. This difficulty led us to seek a more efficient technique. Vacuum distillation is routinely used in organic synthesis for isolation, such as `bulb to bulb' distillation or Kugelrohr distillation. Vacuum can also be employed in chemistry and radiochemistry for transferring liquid materials between containers. Thus, a vacuum “bulb to bulb” distillation method (see Figure 1) was developed for the efficient isolation of [18F]2 from the reaction mixture. At the end of the incorporation reaction of [18F]2, [18F]2 was transferred under vacuum almost instantly, along with the near- or above-boiling point ACN as the carrier, from the reaction vessel via a Teflon tubing to a dry ice-cooled trap. The vacuum (up to 20 torr) was achieved with a 50 mL syringe. Subsequently, nitrogen was released to the reaction vessel to ensure the transfer of any residual [18F]2 to the trap under vacuum. Finally, the trap was warmed in a water bath to room temperature for click labeling. The entire process (incorporation, distillation, and warm-up) required < 10 min to afford [18F]2 in a click-ready form in a minimal volume of ACN (200 μL), which has shown to have great solvent effect on the click labeling (see the discussion below). Because the distillation was carried out in a closed system, almost no [18F]2 escaped from the trap; this method avoids releasing [18F]2 to the environment. To our knowledge, such an application of vacuum in radiochemistry has not been previously reported, and this technique should be useful for the isolation of other volatile radioactive materials from reaction mixtures.

Scheme 1
a. [18F]fluoride, K222 K2CO3, ACN, 80–90 °C

The nucleophilic substitution reaction of 1 with [18F]fluoride (Scheme 1) appears to be very fast and robust. Under the labeling conditions similar to those reported [14], within 2.5 min of heating at 88 °C, the labeling reaction was completed, reaching a plateau in isolated yield (Table 1). Longer reaction time did not increase the yields. In addition, labeling reactions carried out from 80 °C to 90 °C or using 0.5 mg to 2 mg of 1 afforded similar isolated yields (data not shown). The high efficiency of the nucleophilic substitution reaction of 1 clearly benefits from the activation of the adjacent azide group in 1. On average, we were able to isolate [18F]2 in > 80% yield (decay corrected, n > 30) within 10 min in the click-ready form. The distillation efficiency was not determined; however, the high isolation yields suggest that the efficiency is close to quantitative.

Table 1
Isolated yields of [18F]2 vs reaction time

The isolated [18F]2 was analyzed by a reversed phase HPLC with the UV at 220 nm. As shown in Figure 2, [18F]2 (retention time: 7.7 min) was isolated as the only radioactive peak in a very chemically pure form with no major UV peaks around it. This is ideal for the HPLC purification of the click labeling mixture in order to achieve a chemically and radiochemically pure final product with high specific activity. From the standpoint of purity, the vacuum distillation method should be superior to the reported flow distillation in that the flow-trap distillation at 130 °C could, in principal, generate significantly more side-products, including the observed elimination by-product vinyl azide from 1. It should be noted that, low specific activity due to impurities has been reported when the flow-trap distillation is used [14, 16, 17].

Figure 2
HPLC chromatograph of distilled [18F]2

The major UV peak in the distilled reaction mixture of [18F]2 was observed at 12 min (Figure 2). NMR analysis of the reaction mixture in CD3CN suggests that this peak is vinyl azide, an elimination by-product of the labeling reaction. Elimination reaction is a common side-reaction in 18F labeling using nucleophilic substitution of ethylene sulfonate precursors under basic conditions, especially at above 100 °C. The facile formation of vinyl azide during the radiolabeling of 2 may also be due to the activation of azide in 1 as that in the nucleophilic substitution. The low-boiling point vinyl azide was isolated efficiently by the vacuum distillation method. It was estimated by HPLC that 25–40 % starting material 1 was distilled as vinyl azide. Therefore, the large amount of vinyl azide became the limiting reagent in the click labeling, in that the stoichiometry requires more than one equivalent of alkyne substrate in order to complete the click-labeling reaction. However, because of the large difference in retention time between [18F]2 and vinyl azide in HPLC, if the alkyne substrates are small molecules, separation of the click-labeled product of [18F]2 from that of vinyl azide should be easy (e.g. Figure 3). Vacuum-distilled [18F]2 is not suitable to label macromolecules, such as proteins and nanoparticles, with high specific activity, due to stoichiometry limitations.

Figure 3
HPLC chromatograph of [18F]7 purification.

3.2. Click labeling-solvent effect

During the development of click labeling of small molecule PET tracers, we found that small changes in the volume of solvent used (e.g. 200 vs. 300 μL ACN and 200 vs. 250 μL DMF) afforded different results in the completion of the click labeling. This observation led us to explore the solvent effect on click labeling. One advantage of click chemistry is that a variety of solvents can be used. However, we only studied the solvent effect of organic solvents, ACN and DMF, which are commonly used in labeling reactions and are well suited to reversed phase HPLC purification. Copper sulfate and sodium ascorbate, which are the most used source of Cu(I) catalyst for click labeling [9], were used here because they afforded good yield [14] and are easy to work up. The click reaction of [18F]2 and 3 (Scheme 2) was used as the model reaction to investigate the solvent effect, because not only is the propargyl group a good building block for click labeling, but also because benzaldehyde derivatives are common conjugation agents in conjugation chemistry.

The results of solvent effects are shown in Table 2 and and3.3. The reaction mixture was analyzed by silica gel radio-TLC and confirmed by reversed-phase HPLC. Three radioactive peaks were observed as [18F]2, [18F]4, and a small amount of unknown by-product in radio-TLC and HPLC. The nature of the by-product was not identified and it is not known whether it is specific to 3, but it was determined that this peak was not from the decomposition of [18F]2, which was stable under the reaction condition (data not shown). When 1 mg of 3 was used in the model reactions (date not shown), all the click labeling reactions were very fast, except for the reaction using the reported condition [14]; as a result, the effect of solvent on the rate of conversion of [18F]2 was not evident. When the amount of 3 was reduced to 0.1 mg, which in principle will reduce the reaction rate by 100-fold due to the second order kinetics with respect to an alkyne substrate 3 [24], the solvent effect could be observed. As shown in Table 2, increases in the amount of DMF will greatly increase the rate of conversion of [18F]2, while increases in the amount of ACN will decrease the rate. The most important factor in click chemistry appears to be the need to maintain Cu(I) at a high level at all times during reaction [8]. In the model reactions where more DMF was used, the Cu(I) mixture remained in the form of a yellowish suspension of CuSO4·5H2O and sodium ascorbate in the click labeling mixture, and the labeling reaction was always very fast and retaining. When more ACN was added, white solids precipitated out very quickly, and the labeling reaction slowed or even stopped completely. The effect of water on the reaction rate was also studied (Table 3). When more water was used (e.g. 200 μL instead of 100 μL), the reaction mixture became homogenous and colorless, but the reaction slowed down greatly and not retaining. Only when a large amount of DMF was used, the reactions with more water were retaining. All the above observations are apparently related to the form of Cu(I), the mixture of copper sulfate and sodium ascorbate, in the reaction mixture, however, the influence of solvents on the form of Cu(I) is beyond the scope of this paper. Kinetic effects on the rate of click labeling reactions were also observed when the reaction volume varied. This was because of the second order kinetics with respect to alkyne substrate 3 and Cu(I) catalyst [24]. A change in the volume of the reaction or in the concentrations of reactants will result in a tremendous change in the rate of click labeling. Therefore, a minimal amount of solvent is required to complete the click labeling within a short time at room temperature. Obviously, solvents have great effects on the rate of click labeling reactions using [18F]2. It appears that a sufficient amount of DMF is critical for maintaining the level of Cu(I) in the solution, affording fast and retaining click labeling.

Table 2
Solvent effect on the click reaction of [18F]2 and 3 using Cu(I) from copper sulfate and sodium ascorbate
Table 3
Solvent effect (water) on the click reaction of 2 and 3 using Cu(I) from copper sulfate and sodium ascorbate

Bathophenanthrolinedisulfonic acid disodium salt (BPDS) has been used to promote the Cu(I) catalyzed click reaction by stabilizing the Cu(I) catalyst in organic synthesis and bioconjugations. As shown in Table 4, the addition of BPDS to the mixture of copper sulfate and sodium ascorbate greatly increases the rate of click labeling, resulting in complete conversion of [18F]2 within 5 min. However, one potential disadvantage in using BPDS is that it may complicate the HPLC purification of very polar compounds.

Table 4
Click reaction of [18F]2 and 3 using Cu(I)/DPBS as catalyst

3.3. Radiosynthesis of an NHS ester using click chemistry

An N-hydroxysuccinimide (NHS) ester is perhaps the most common acylating agent in conjugation chemistry. N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), as an 18F labeled NHS ester, is the most used prosthetic group for 18F-labeling of peptides and proteins [25, 26]. The synthesis of [18F]SFB requires several steps and affords only 30–50 % yields [24]. Recently, a PEGylated NHS ester was synthesized using Cu(I) catalyzed click labeling, but only in 34 % radiochemical yield from the 18F-PEG-alkyne [27]. To demonstrate the ease of click labeling of base-labile compounds using [18F]2, we synthesized an 18F-labeled NHS ester ([18F]7) using Cu(I) catalyzed click chemistry. Non-radioactive 7 was synthesized as shown in Scheme 3. [18F]7 was synthesized by a similar procedure. When a standard CuSO4·5H2O and sodium ascorbate mixture was used as the Cu(I) catalyst source, the basic reaction mixture resulted in some decomposition of [18F]7. When the more efficient CuSO4·5H2O, sodium ascorbate, and DBPS combination was explored as the Cu(I) catalyst source, click labeling of 6 with [18F]2 in 5 min afforded [18F]7 in 90 % HPLC isolated yield with good chemical and radiochemical purity(see Figure 3 for HPLCs). The total synthesis time of [18F]7 is short: < 10 min for the synthesis of click-ready [18F]2, 5 min for click labeling, and < 20 min for HPLC purification of [18F]7. [18F]7 is stable in the HPLC mobile phase, and can be isolated using standard solid phase extraction techniques either in a dry form (eluted with ether or dichloromethane and followed by evaporation of solvents) or in DMF (< 300 μL). [18F]7 has been used successfully to label small molecules and dendrimers (results will be published elsewhere), and may be superior to [18F]SFB in several aspects, such as facile synthesis, labeling yields, in vivo stability, and the introduction of a triazole group.

Scheme 3
a, N-hydroxysuccinimide, DCC, DCM/Dioxane;

3.4. General considerations of click labeling using [18F]2

In the development of PET radiotracers, we must consider the reaction time, the yield, the easy workup, and facile purification of a labeled compound; a radiolabeled compound with good chemical and radiochemical purities is required to serve as a true tracer for PET. The current method was developed based on these considerations. One-pot reactions have been used to synthesize 18F labeled amino acids with very good results [28], but this method requires much more of the alkyne substrate because of the stoichiometry, and may result in low specific activity and has an increased potential for side reactions, due to basic reaction conditions. The vacuum distillation method described here afforded [18F]2 in only 200 μL ACN; this is critical, because in order to take advantage of the second-order kinetics with respect to alkyne substrates and Cu(I) catalyst, the total reaction volume must be minimized. A sufficient amount of DMF is required to maintain a high level of Cu(I) during the reaction and more than one equivalent of the alkyne substrate is needed to complete the reaction because vinyl azide is the limiting reagent. The addition of water to the reaction mixture may slow down the reaction, and may result in low solubility of lipophilic compounds; a mixture of copper sulfate and sodium ascorbate is the choice of Cu(I) catalyst because of the easy workup and facile HPLC purification. We have employed these procedures with great success when using [18F]2 to label a variety of compounds, including chemically-labile ones. Next to direct nucleophilic substitution using [18F]fluoride, [18F]2 appears to be the most useful 18F labeled synthon to label small molecules both in terms of labeling yields and due to the rapid and efficient synthesis and purification using the procedure that we have developed.

4. Conclusions

A vacuum distillation method was developed to isolate [18F]2 in more than 80% yield within 10 min in a “pure” and click-ready form. It was found that sufficient amount of DMF is critical to maintain the high level of Cu(I) in the reaction mixture to ensure the click labeling is rapidly completed. A base-labile NHS ester (7) was synthesized as an example of this new method in short time in up to 90% isolated yield using [18F]2 and copper sulfate, sodium ascorbate and DPBS as the Cu(I) source. Not only can this method be used to label small molecules in high yield and with easy purification, but also the [18F] labeled click-NHS ester can be used for conjugation chemistry with primary amines. We have developed a general method to label small molecules with fluorine-18 with good radiochemical yield, easy purification and high chemical and radiochemical purities. We believe that this novel method could also be of significance for the automated production of [18F]2.


This study was sponsored by HL13851, AG036045, CA121952, and ER64671. We thank Robert Dennett and Brian Wingbermuehle for the production of [18F]fluoride.


bathophenanthrolinedisulfonic acid disodium salt
ethyl acetate
Kryptofix 222
positron emission tomography
trifluoroacetic acid


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[1] Welch MJ, Redvanly CS, editors. Handbook of Radiopharmaceuticals. John Wiley & Sons, Ltd; New York: 2005.
[2] Stahl A, Wieder H, Piert M, Wester H, Senekowitsch-Schmidtke R, Schwaiger M. Positron emission tomography as a tool for translational research in oncology. Molecular Imaging and Biology. 2004;6:214–24. [PubMed]
[3] Bolton R. Radiohalogen incorporation into organic systems. Journal of Labelled Compounds & Radiopharmaceuticals. 2002;45:485–528.
[4] Lasne MC, Perrio C, Rouden J, Barre L, Roeda D, Dolle F, et al. Chemistry of beta(+)-emitting compounds based on fluorine-18. Contrast Agents Ii. 2002;222:201–58.
[5] Kolb HC, Finn MG, Sharpless KB. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed Engl. 2001;40:2004–21. [PubMed]
[6] Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl. 2002;41:2596–9. [PubMed]
[7] Tornoe CW, Christensen C, Meldal M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem. 2002;67:3057–64. [PubMed]
[8] Meldal M, Tornoe CW. Cu-catalyzed azide-alkyne cycloaddition. Chem Rev. 2008;108:2952–3015. [PubMed]
[9] Glaser M, Robins EG. `Click labelling' in PET radiochemistry. Journal of Labelled Compounds & Radiopharmaceuticals. 2009;52:407–14.
[10] Kuijpers BH, Groothuys S, Soede AC, Laverman P, Boerman OC, van Delft FL, et al. Preparation and evaluation of glycosylated arginine-glycine-aspartate (RGD) derivatives for integrin targeting. Bioconjug Chem. 2007;18:1847–54. [PubMed]
[11] Bock VD, Speijer D, Hiemstra H, van Maarseveen JH. 1,2,3-Triazoles as peptide bond isosteres: synthesis and biological evaluation of cyclotetrapeptide mimics. Org Biomol Chem. 2007;5:971–5. [PubMed]
[12] Kolb HC, Sharpless KB. The growing impact of click chemistry on drug discovery. Drug Discov Today. 2003;8:1128–37. [PubMed]
[13] Tron GC, Pirali T, Billington RA, Canonico PL, Sorba G, Genazzani AA. Click chemistry reactions in medicinal chemistry: applications of the 1,3-dipolar cycloaddition between azides and alkynes. Med Res Rev. 2008;28:278–308. [PubMed]
[14] Glaser M, Arstad E. “Click labeling” with 2-[18F]fluoroethylazide for positron emission tomography. Bioconjug Chem. 2007;18:989–93. [PubMed]
[15] Gaeta A, Woodcraft J, Plant S, Goggi J, Jones P, Battle M, et al. Use of 2-[18F]fluoroethylazide for the Staudinger ligation - Preparation and characterisation of GABA(A) receptor binding 4-quinolones. Bioorg Med Chem Lett. 2010;20:4649–52. [PubMed]
[16] Glaser M, Goggi J, Smith G, Morrison M, Luthra SK, Robins E, et al. Improved radiosynthesis of the apoptosis marker 18F-ICMT11 including biological evaluation. Bioorg Med Chem Lett. 2011;21:6945–9. [PubMed]
[17] Iddon L, Leyton J, Indrevoll B, Glaser M, Robins EG, George AJ, et al. Synthesis and in vitro evaluation of [18F]fluoroethyl triazole labelled [Tyr3]octreotate analogues using click chemistry. Bioorg Med Chem Lett. 2011;21:3122–7. [PubMed]
[18] Leyton J, Iddon L, Perumal M, Indrevoll B, Glaser M, Robins E, et al. Targeting somatostatin receptors: preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET. J Nucl Med. 2011;52:1441–8. [PubMed]
[19] Nguyen QD, Smith G, Glaser M, Perumal M, Arstad E, Aboagye EO. Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-3/7 specific [18F]-labeled isatin sulfonamide. Proc Natl Acad Sci U S A. 2009;106:16375–80. [PubMed]
[20] Smith G, Glaser M, Perumal M, Nguyen QD, Shan B, Arstad E, et al. Design, synthesis, and biological characterization of a caspase 3/7 selective isatin labeled with 2-[18F]fluoroethylazide. J Med Chem. 2008;51:8057–67. [PubMed]
[21] Demko ZP, Sharpless KB. An intramolecular [2 + 3] cycloaddition route to fused 5-heterosubstituted tetrazoles. Org Lett. 2001;3:4091–4. [PubMed]
[22] Hans RH, Guantai EM, Lategan C, Smith PJ, Wan B, Franzblau SG, et al. Synthesis, antimalarial and antitubercular activity of acetylenic chalcones. Bioorg Med Chem Lett. 2010;20:942–4. [PubMed]
[23] Gavrilyuk JI, Wuellner U, Salahuddin S, Goswami RK, Sinha SC, Barbas CF., 3rd An efficient chemical approach to bispecific antibodies and antibodies of high valency. Bioorg Med Chem Lett. 2009;19:3716–20. [PMC free article] [PubMed]
[24] Rodionov VO, Fokin VV, Finn MG. Mechanism of the ligand-free CuI-catalyzed azide-alkyne cycloaddition reaction. Angew Chem Int Ed Engl. 2005;44:2210–5. [PubMed]
[25] Hou S, Phung DL, Lin WY, Wang MW, Liu K, Shen CK. Microwave-assisted one-pot synthesis of N-succinimidyl-4[18F]fluorobenzoate ([18F]SFB) J Vis Exp. 2011 [PubMed]
[26] Vaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F. Nat Protoc. 2006;1:1655–61. [PubMed]
[27] Gill HS, Tinianow JN, Ogasawara A, Flores JE, Vanderbilt AN, Raab H, et al. A modular platform for the rapid site-specific radiolabeling of proteins with 18F exemplified by quantitative positron emission tomography of human epidermal growth factor receptor 2. J Med Chem. 2009;52:5816–25. [PubMed]
[28] McConathy J, Zhou D, Shockley SE, Jones LA, Griffin EA, Lee H, et al. Click synthesis and biologic evaluation of (R)- and (S)-2-amino-3-[1-(2-[18F]fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid for brain tumor imaging with positron emission tomography. Mol Imaging. 2010;9:329–42. [PubMed]