Synthesis and application of 4-[18F]fluorobenzylamine: A versatile building block for the preparation of PET radiotracers

Article abstract of DOI:10.1039/c0ob00255k

A novel synthesis of 4-[¹⁸F]fluorobenzylamine ([ ¹⁸F]FBA) by means of transition metal-assisted sodium borohydride reduction of 4-[¹⁸F]fluorobenzonitrile ([¹⁸F]FBN) is described. This approach could successfully

Full text of DOI:10.1039/c0ob00255k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and application of 4-[18F]fluorobenzylamine: A versatile building
block for the preparation of PET radiotracers†
Ingrid Koslowsky, John Mercer and Frank Wuest*
Received 14th June 2010, Accepted 16th July 2010
DOI: 10.1039/c0ob00255k
A novel synthesis of 4-[18F]fluorobenzylamine ([18F]FBA) by means of transition metal-assisted sodium
borohydride reduction of 4-[18F]fluorobenzonitrile ([18F]FBN) is described. This approach could
successfully be extended to borohydride exchange resin (BER) enabling a viable option for use in
automated syntheses. [18F]FBA was used for the synthesis of 4-[18F]fluorobenzylamine-based thiol
group-reactive prosthetic groups 4-[18F]fluorobenzyl-2-bromoacetamide ([18F]FBBA) and
4-[18F]fluorobenzylamidopropionyl maleimide ([18F]FBAPM). [18F]FBBA and [18F]FBAPM were
obtained in radiochemical yields of 75% and 55%, respectively. Feasibility of using [18F]FBAPM as
novel prosthetic group for peptide and protein labelling was demonstrated with cysteine-containing
tripeptide glutathione (GSH). [18F]FBBA was used for labelling of a fully phosphorothioated 20mer
oligodesoxynucleotide (ODN).
reactive prosthetic group 4-[18F]fluorobenzyl-2-bromo-acetamide
([18F]FBBA).
Introduction
[18F]FBBA has been shown to successfully radiolabel DNA-
type probes such as modified oligonucleotides and peptide nucleic
acids.5 An automated synthesis of this compound employing
a Zymate robot system has also been reported, however with
relatively low radiochemical yields of 3 to 18%.6
4-[18F]Fluorobenzylamine ([18F]FBA) is the key intermediate
in the synthesis of prominent oligonucleotide labelling prosthetic
group [18F]FBBA. However, compared to the dominant role of
other 18F-labelled aryl fluorides, such as [18F]fluorobenz-aldehydes,
[18F]fluorobenzyl halides, and [18F]fluorohalo-benzenes for the
synthesis of 18F-labelled radiotracers, only very few examples are
known where [18F]FBA was utilized. Besides the frequent use of
[18F]FBA for the preparation of [18F]FBBA, [18F]FBA was also
used for the synthesis of 18F-labelled prostaglandins, folic acid and
epidermal growth factor receptor (EGFR) ligand.7
Various approaches for the synthesis of [18F]FBA have been
reported. All syntheses rely on the formation of 4-[18F]fluoro-
benzonitrile ([18F]FBN) and subsequent conversion into [18F]FBA
by means of various reducing agents. Haradahira et al. syn-
thesized [18F]FBA by the reduction of [18F]FBN with borane-
dimethylsulfide as the reducing agent.7b The reported radio-
chemical yield was 39 to 49%. Borane-mediated reduction
of a [18F]FBN derivative was also reported by Vaidyanathan
et al. employing NaBH4/I2 as the BH3 source for reducing 3-
iodo-4-[18F]fluorobenzonitrile in the synthesis of 4-[18F]fluoro-3-
iodobenzyl)-guanidine.8 However, most reports describe synthesis
of [18F]FBA via reduction of [18F]FBN with LiAlH4 under
anhydrous conditions.
Problematically, all these reaction conditions are particularly
difficult to maintain in automated synthesis units where the
transfer of reagents through common lines compromises the
anhydrous conditions required to ensure reduction of the nitrile
group. The use of molecular sieves and sodium sulfate drying
cartridges, and flushing the lines with drying solvents, have been
attempted to improve the radiochemical yield.6,9 Moreover, the
formation of aluminium salts during the reduction can also create
Positron emission tomography (PET) has become a powerful non-
invasive molecular imaging technique which provides functional
information on physiological, biochemical and pharmacological
processes in living subjects.1 Recent advances in probe devel-
opment and imaging technology have placed PET in a unique
position among other molecular imaging methodologies as a truly
translational research approach. In combination with suitable
radiolabeled probes, also referred to as radiotracers, PET offers
exceptional possibilities to study pharmacokinetics, including
metabolism, and modes of action of novel and established drugs
in vivo.2
Among the available PET radionuclides, the short-lived positron
emitter fluorine-18 (18F, t1/2 = 109.8 min) is particularly useful
due to its favorable nuclear and chemical properties.3 Organic
radiochemistry with 18F differs significantly from conventional
chemistry. Radiosyntheses involving the short-lived positron emit-
ter 18F require fast and efficient reactions while considering the
extraordinary stoichiometry resulting from the typically produced
submicromolar amounts of radiolabelled compounds.
18F has found numerous applications for the labelling of both,
small molecules and compounds of high molecular weight like
peptides, proteins, and oligonucleotides. However, incorporation
of 18F into high molecular weight compounds represents a special
challenge. Peptides, proteins and oligonucleotides can usually
not be labelled with 18F at high specific activity directly due
to the required strongly basic reaction conditions at elevated
temperatures. Over the last decades, various bifunctional labelling
precursors, also referred to as prosthetic groups, have been de-
veloped allowing 18F labelling under mild conditions.4 Prominent
examples include the amine group reactive acylating agent N-
succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and thiol group-
Department of Oncology, University of Alberta, 11560 University Ave,
Edmonton, AB T6G 1Z2, Canada. E-mail: wuest@ualberta.ca; Fax: +1
780 432 8483; Tel: +1 780 989 8150
† Electronic supplementary information (ESI) available: HPLC and HR-
MS Spectra. See DOI: 10.1039/c0ob00255k
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problems in a synthesis unit as transfer of the [18F]FBA reaction
mixture can block transfer lines in the synthesis unit. Therefore,
an alternative method for the reduction step which tolerates
the presence of water would be a welcome improvement in the
synthesis of [18F]FBA.
In this work we report on the improved synthesis of [18F]FBA
utilizing the borohydride/transition metal catalyst method for ni-
trile reduction. We also report on a solid-phase approach for nitrile
group reduction using a NaBH4 impregnated resin which, upon
activation with a metal catalyst, rapidly and quantitatively reduces
[18F]FBN to [18F]FBA. Elution from an activated borohydride
exchange cartridge (BER) eliminates precipitates and provides a
more viable option for use in automated synthesis units.
The feasibility of the proposed approach was demonstrated by
the efficient synthesis of [18F]FBA-based thiol group-reactive pros-
thetic groups [18F]FBBA and 4-[18F]fluorobenzylamido-propionyl
maleimide ([18F]FBAPM), respectively (Scheme 1).
Scheme 2 Reaction conditions: (i) [18F]KF, K222, K2CO3, DMSO, 95 ◦C,
15 min; (ii) NaBH4/Co(OAc)2, THF–H2O, room temperature.
a
Table 1 Reduction of [18F]FBN with NaBH4/Co(OAc)2
Entry
NaBH4/mg
Co(OAc)2/mg
Radiochemical yield [%]b
1
2
3
4
5
6c
10
15
25
30
40
10
5
5
10
10
20
5
75
49
61
50
58
>95
a All reactions (except for entry 6) were carried out in THF at room
temperature for 15 min. b Radiochemical yield determined by radio-TLC
representing the percentage of [18F]FBA present in the reaction mixture.
c Reaction was performed in THF–H2O (2 : 1) at room temperature for
5 min.
sealed reaction vial. After purification via solid-phase extraction
(SPE), [18F]FBN was obtained in high radiochemical yields
of >80% and high radiochemical purity greater 95%. Sodium
borohydride in combination with Co(OAc)2 as transition metal
salt was used to reduce the nitrile group of [18F]FBN. For this
purpose, [18F]FBN was eluted with 1 mL of THF from the SPE-
cartridge into a reaction vial containing different amounts of
NaBH4 and Co(OAc)2. The reaction was performed at room
temperature for 5–15 min.
Scheme
1
Thiol group-reactive prosthetic groups [18F]FBBA and
[18F]FBAPM.
[18F]FBBA and [18F]FBAPM were used for the radiolabelling of
a fully phosphorothioated 20mer ODN and cysteine-containing
tripeptide glutathione (GSH), respectively.
The results of the reduction of [18F]FBN with NaBH4/Co(OAc)2
are summarized in Table 1.
Results and discussion
The results in Table 1 clearly show that the reduction is not
quantitative (in the case of entries 1–5), and that the reaction
depends on the NaBH4/Co(OAc)2 ratio and the solvent. Best
results for the reduction in pure THF as the solvent were obtained
with 10 mg of NaBH4 and 5 mg of Co(OAc)2 (entry 1). Extension
of the reaction time to 30 min did not improve the radiochemical
yield. Reduction of the amount of Co(OAc)2 (less than 5 mg)
resulted in a significant drop in radiochemical yield (<25%).
A plausible explanation for the incomplete reaction at larger
amounts of NaBH4 and Co(OAc)2 (entries 2–5) is the fixation of
large amounts of formed Co2B at the glass walls which is then not
available for the reduction. Moreover, larger amounts of Co2B also
favor absorption of substantial amounts of [18F]FBA. However,
significant higher radiochemical yields of >95% were achieved
when pure THF as the solvent was replaced with 2 : 1 mixture of
THF and water (entry 6). Using THF–H2O (2 : 1) as the solvent
accelerates the reaction, and complete conversion of [18F]FBN is
accomplished at room temperature after 5 min. The formed Co2B
appears as a fine crystalline precipitate in THF–H2O without the
tendency to stick on the glass walls. Comparable results were
achieved with CoCl2 as transition metal salt instead of using
Co(OAc)2.
Synthesis of 4-[18F]fluorobenzylamine ([18F]FBA) via transition
metal-assisted NaBH4 reduction
Unlike LiAlH4, NaBH4 is a rather mild reducing agent. NaBH4
readily reduces aldehydes, ketones, and acyl halides, but it is
virtually inert to many other functional groups, including nitrile
groups. However, the reducing potential of NaBH4 is significantly
enhanced in the presence of various transition metal salts like
NiCl2, Co(OAc)2 and CuCl2. In the presence of aqueous solutions
of transition metal salts NaBH4 readily reduces nitrile groups
through the formation of metal borides in situ followed by the
release of hydrogen. Using CoCl2, and NaBH4, Osby et al.
reported that benzylamine could be produced in high yield from
benzonitrile.10 More recently, Khurana et al. demonstrated that
high yields of benzylamine could be obtained in 5 min when
incubated with NiCl2 and NaBH4 in anhydrous ethanol at ambient
temperature.11
The favorable reducing characteristics of NaBH4 in the presence
of transition metal salts for nitrile groups prompted us to set
up the radiosynthesis of [18F]FBA exploiting transition metal-
assisted NaBH4 reduction of [18F]FBN. The synthesis of [18F]FBA
is depicted in Scheme 2.
[18F]FBN was easily prepared by the reaction of 4-cyano-
N,N,N-trimethylanilinium trifluoromethansulfonate 1 with the
powerful nucleophilic radiofluorination agent [18F]KF (generated
by treatment of cyclotron-produced [18F]fluoride with Kryptofix
(K222)/K2CO3) in dry DMSO at elevated temperature (95 ◦C) in a
Synthesis of 4-[18F]fluorobenzylamine ([18F]FBA) using borohydride
exchange resin (BER)-transition metals
All metal catalysts used in the BER study (NiCl2, Co(OAc)2 and
to a lesser extent CuSO4) were able to activate the BER as seen by
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Table 2 Reduction of [18F]FBN with BERa
Radiochemical yield [%]b
Table 3 Effect of N-methyl-morpholine on radiochemical yield for
coupling of [18F]FBA with N-succinimidyl-3-maleimide-propionatea
Entry
N-methyl-morpholine/mL
Radiochemical yield [%]b
Time after addition of [18F]FBN to BER 1 min
3 min
5 min
1
2
3
4
5
0
0
NiCl2
Co(OAc)2
CuSO4
94
74
<1
95
93
4
93
95
10
25
50
100
200
30
83
77
71
a All reactions were carried out in EtOH–H2O at room temperature.
b Radiochemical yield determined by radio-TLC representing the percent-
age of [18F]FBA present in the reaction mixture.
a All reactions were carried out in THF (200 mL) at room temperature for
10 min. b Radiochemical yield determined by radio-TLC representing the
percentage of [18F]FBAPM present in the reaction mixture.
the change in colour of the beads from tan to black. Activation of
BER was only observed with NaBH4 embedded resin prepared in-
house. Commercially available borohydride-containing resin was
not reactive regardless of the metal catalyst or solvents used
(data not shown). The addition of NiCl2 to the BER showed
the most rapid activation (within 10 s), followed by Co(OAc)2
(1 min). Activation of the resin with CuSO4 was observed to occur
only after a prolonged incubation time of 3–5 min. The rate of
conversion of [18F]FBN to [18F]FBA also varied with the catalyst
used. CuSO4 demonstrated the slowest rate of nitrile reduction
with 10% conversion at 5 min. In contrast, 74% of [18F]FBN
had been converted into [18F]FBA with Co(OAc)2 by 1 min,
with 93% and 95% reduction by 3 min and 5 min, respectively.
NiCl2 demonstrated the most rapid rate of reduction with 94%
of the nitrile converted to [18F]FBA after 1 min of incubation
with the BER-transition metal catalyst mixture. The results are
summarized in Table 2.
Once the beads were activated with NiCl2, the activity of
the transition metal/borohydride as reducing agent was rapidly
expended. Conversion was quantitative if the [18F]FBN/ethanol
mixture was added within 1 min after the activation of the BER
resin. Longer wait times prior to the addition of the nitrile
decreased the extent of reduction. Thus, 78% conversion to
[18F]FBA was observed if [18F]FBN was added 3 min after bead
activation. This effect was even more pronounced at later time
points reaching only 16% conversion from [18F]FBN to [18F]FBA
after a wait time of 5 min and less than 1% after a wait time of
15 min.
Scheme 3 Reaction conditions: (i) N-Methylmorpholine, THF, 10 min,
60 ◦C.
morpholine to 100 mL and 200 mL did not improve the radio-
chemical yield (entries 4 and 5). Lower amounts of N-methyl-
morpholine (25 mL) gave only moderate radiochemical yields of
30% (entry 2).
Thus, optimized reaction conditions for the synthesis of
[18F]FBAPM included the use of 2–3 mg of N-succinimidyl-3-
maleimide-propionate and 50 mL of N-methyl-morpholine in THF
(200 mL) at room temperature. The reaction mixture was purified
by means of semi-preparative HPLC to afford 18F]FBAPM
in a radiochemical yield of 55% (decay-corrected based upon
[18F]fluoride) within a total synthesis time of 60–70 min. The
specific activity was determined to be 50–70 GBq/mmol, and the
radiochemical purity exceeded 95%.
[18F]FBA was further used for the synthesis of the prominent
oligonucleotide labelling agent [18F]FBBA (Scheme 4).
Synthesis of prosthetic groups 4-[18F]fluorobenzyl-2-
bromoacetamide ([18F]FBBA) and 4-[18F]fluorobenzyl-
amidopropionyl maleimide ([18F]FBAPM)
Synthesis of novel thiol-reactive prosthetic group [18F]FBAPM
commenced with the synthesis of [18F]FBA according to the
described Co(OAc)2-assisted NaBH4 reduction of [18F]FBN. The
synthesis of [18F]FBAPM is given in Scheme 3.
After Sep-Pak purification, [18F]FBA was treated with N-
succinimidyl-3-maleimide-propionate in the presence of N-
methyl-morpholine. Without the use of N-methyl-morpholine as
auxiliary base, no product formation was observed. The effect
of N-methyl-morpholine upon the acylation reaction between
[18F]FBA and N-succinimidyl-3-maleimide-propionate is summa-
rized in Table 3.
Scheme 4 Reaction conditions: (i) 2-bromoacetyl bromide, CH2Cl2, room
temperature.
[18F]FBBA was rapidly formed upon addition of 2-bromoacetyl
bromide/CH2Cl2, 1/10 (v/v) to [18F]FBA, with near quantitative
conversion (97%) of [18F]FBA to [18F]FBBA within 5 min. A decay-
corrected radiochemical yield of 75% before HPLC purification
was achieved within 60 min which represents a considerable
improvement to the previously reported 18% yield using LiAlH4
as the reducing agent.6
The use of 50 mL of N-methyl-morpholine gave the best
results, and a radiochemical yield of 83% based upon [18F]FBA
was achieved (entry 3). Increase of the amount of N-methyl-
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18F-labelling of glutathione (GSH) with [18F]FBAPM and fully
phosphorothioated 20mer oligodeoxynucleotide (ODN) with
[18F]FBBA
Treatment of ODN with [18F]FBBA at 120 ◦C in phosphate
buffered saline/methanol resulted in the formation of 18F-labelled
ODN. The isolated radiochemical yield after purification with
NAPTM-10 columns was about 30% based upon [18F]FBBA. The
radiochemical purity was greater than 95%, and the specific
activity was determined to be 3.2 GBq/mmol (n = 14).
The labelling properties of [18F]FBAPM as a novel thiol group-
reactive prosthetic group for peptide and protein labelling was
assessed by the reaction with cysteine-containing tripeptide glu-
tathione (GSH) (Scheme 5).
Conclusions
We have developed a convenient and facile method for the
conversion of readily available [18F]FBN into [18F]FBA. The
application of Co(II)/NaBH4 as reducing agent can be extended
to the use of borohydride-exchange resins (BERs) as the hydride
source, which will further facilitate automation of [18F]FBA and
its broader use as a versatile 18F-labelling building block.
Based on the novel synthesis of [18F]FBA we have prepared
[18F]FBAPM as a novel prosthetic group for labelling of cysteine-
containing peptides as exemplified by the reaction with GSH.
[18F]FBA was also used for the preparation of established oligonu-
cleotide labelling agent, [18F]FBBA, for subsequent conjugation
with 5¢-thiol-functionalized oligonucleotides.
Scheme 5 Reaction conditions: (i) phosphate buffer, pH 7.2, room
temperature.
A solution of [18F]FBAPM in EtOH was added to GSH
solutions at various concentrations (1.0 mg mL-1, 100 mg mL-1,
10 mg mL-1, 1 mg mL-1) in phosphate buffer (pH 7.2) at room
temperature. The conversion of maleimide [18F]FBAPM into
[18F]FBAPM-GSH was monitored by radio-TLC reflecting the
percentage of radioactivity area of conjugate [18F]FBAPM-GSH
relative to the total radioactivity area. The results are summarized
in Table 4.
Experimental
General
1H NMR and 13C NMR spectra were recorded on a Varian Inova-
400 at 400 MHz and 100 MHz, respectively. Chemical shifts (d)
were determined relative to the solvent and converted to the TMS
scale. Mass spectra were obtained on a Agilent Technologies 6220
oaTOF by electrospray ionisation. Flash chromatography was
conducted using MERCK silica gel (mesh size 230–400 ASTM).
Thin-layer chromatography (TLC) was performed on Merck silica
gel F-254 aluminium plates, with visualization under UV light
(254 nm). All chemicals were obtained from commercial suppliers
(reagent grade) and used without further purification.
Table 4 Radiolabelling of GSH with [18F]FBAPMa
GSH concentration
1 mg mL-1 100 mg mL-1 10 mg mL-1 1 mg mL-1
% [18F]FBAPM-GSH >95
>95
>95
65
a All reactions were carried out at room temperature for 20 min.
For GSH concentrations >10 mg mL-1, conversion of
[18F]FBAPM was greater 95%. At a lower GSH concentration
of 1 mg mL-1, conversion was incomplete reaching 65%. These
findings are consistent with our previous results dealing with
the labelling of cysteine-containing peptides by means of other
maleimide-containing 18F-labelled prosthetic groups ([18F]FBAM,
[18F]FBOM and [18F]FDG-MHO). This makes [18F]FBAPM an
attractive prosthetic group for the mild and efficient labelling of
cysteine-containing peptides.
Chemical syntheses
4-Fluorobenzylamidopropionyl maleimide (FBAPM). Fluo-
robenzylamine (85 mL, 750 mmol) in DMF (1 mL) was added
dropwise over 2 h to a solution of N-succinimidyl-3-maleimide-
propionate (200 mg, 750 mmol) and N-methyl-morpholine (50
mL) in DMF (500 mL). The reaction mixture was stirred at room
temperature for an additional hour. The solvent was evaporated
under reduced pressure and the residue was washed with 1 N HCl
(2 ¥ 5 mL) followed by water (2 ¥ 5 ml). The residue was dissolved
in dichloromethane and purified by column chromatography
(dichloromethane–MeOH 40/1) to afford 116.5 mg (56%) of the
desired product. 1H NMR (CDCl3, 400 MHz): d 2.57 (t, J = 7.1 Hz,
2 H), 3.86 (t, J = 7.1 Hz, 2 H), 4.38 (d, J = 5.8 Hz, 2 H), 5.86 (s,
1H), 6.71 (s, 2H), 7.00 (m, 2H) 7.24 (m, 2H). 13C NMR (CDCl3,
100 MHz): d 34.3, 34.7, 43.0, 115.6 (d, J = 21.6 Hz), 129.7 (d, J =
7.6 Hz), 133.9 (d, J = 33.0 Hz), 134.2, 169.4, 170.5. HRMS (ESI,
positive): calcd. for C14H13O3N2FNa [M+Na]+ 299.0802, found
299.0802.
Alkylation of a fully phosphorothioated 20mer ODN was
performed with purified [18F]FBBA as shown in Scheme 6.
Coupling of FBAPM with glutathione (GSH). Glutathione
(GSH) (22.3 mg, 72.3 mmol) dissolved in phosphate buffer (300 mL,
pH 7.1) was added to [19F]FBAPM (20 mg, 72.3 mmol) in ethanol
Scheme 6 Reaction conditions: (i) phosphate buffered saline (0.1 M, pH 8,
methanol, 1/1 (v/v), 120 ◦C, 30 min.
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(200mL). The mixture was stirred at room temperature for 60 min.
The product FBAPM-GSH was isolated with preparative TCL
(10% NH4OAc (w/w) in MeOH–CH2Cl2 (3 : 1)) to give 24.0 mg
(57%) of the desired compound. HRMS (ESI, negative): calcd. for
C24H29O9N5FS [M - H]- 582.1675, found 582.1678.
after the addition of [18F]FBN. Alternately, [18F]FBN was added 1,
3, 5, or 15 min after the addition of the transition metal salt to the
resin followed by radio-TLC analysis (SiO2, n-BuOH/HOAc–H2O
(4 : 1 : 1), Rf ([18F]FBA) = 0.4).
Radiosynthesis of [18F]FBAPM. 4-[18F]fluorobenzylamine
([18F]FBA) was prepared via reduction of [18F]FBN with
NaBH4/Co(OAc)2 as described (vide supra). After reduction of
[18F]FBN, the reaction mixture was filtered through a Millipore
filter and diluted with 0.01 N NaOH (15 mL). The solution
was passed through a SepPak cartridge (SepPak Plus tC18
(Waters) to retard approximately 60 to 70% of [18F]FBA at
the cartridge. Other SepPak cartridges (e.g. Oasis (Waters),
Chromafix HR-P (Macherey-Nagel) and Chromafix C18 ec
(Macherey-Nagel) were less effective. The cartridge was dried in
a stream of nitrogen and eluted with THF (2 mL). The volume
of the solvent was reduced to 0.5 mL through gentle heating
and a stream of nitrogen. N-Methylmorpholine (50 mL) and
N-succinimidyl-3-maleimide-propionate (2–3 mg) were added,
and the reaction mixture was heated at 60 ◦C for 10 min. The
reaction mixture was purified by means of semi-preparative
HPLC using gradient elution as indicated in the general section
for radiosynthesis. Product fraction (13–15 min) was collected,
diluted with water (15 mL) and passed through a LiChrolut
RP18 cartridge (500 mg). The cartridge was eluted with ethanol
(1.5 mL) to afford [18F]FBAPM. In a typical experiment, starting
from 750 MBq of [18F]fluoride, 275 MBq of [18F]FBAPM (55%,
decay-corrected) could be prepared within a total synthesis
time of 60–70 min. The specific activity was determined to
be 50–70 GBq/mmol, and the radiochemical purity exceeded
95%. Radio-HPLC analysis: CH3CN/0.1 M triethylammonium
chloride (40/60), tR = 4.4 min.
Radiosyntheses
No-carrier added aqueous [18F]fluoride ion was produced on a TR-
19/9 cyclotron (Advanced Cyclotron Systems, Burnaby, Canada)
by irradiation of [18O]H2O via the 18O(p,n)18F nuclear reaction.
Resolubilization of the aqueous [18F]fluoride was accomplished
R
with Kryptofixꢀ 2.2.2 (K222) and K2CO3. High performance
liquid chromatography (HPLC) analyses were carried out with
a SUPELCOSIL LC-18S column (4.6 ¥ 250 mm, 5 mm) using an
indicated isocratic eluent from a gradient pump L2500 (Merck
Hitachi) with a flow rate of 1 mL min-1. The products were
monitored by a UV detector L4500 (Merck Hitachi) at 254 nm
and by g-ray detection with a scintillation detector GABI (X-
RAYTEST). Semi-preparative HPLC was carried out on a Knaur
chromatography system (Berlin, Germany) equipped with a Phe-
nomenex Nucleosil C18 column (10 ¥ 250 mm, 10 mm), integrated
radioactivity detection and UV detection at 254 nm, using gradient
elution with 0.2% TFA in water (solvent A) and methanol (solvent
B): 0 min 70% A, 30% B; 10–22 min 20% A, 80% B. The flow rate
was 2 mL min-1. Radio-TLC was performed on Merck silica gel
F-254 aluminium plates.
Optimization of 4-[18F]fluorobenzylamine ([18F]FBA) synthesis
via Co(OAc)2-assisted NaBH4 reduction of 4-[18F]fluorobenzo-
nitrile ([18F]FBN). 4-Cyano-N,N,N-trimethyl-anilinium trifluo-
romethansulfonate 1 (1 mg) was dissolved in dry DMSO (300
mL) and added to a vial containing dried [18F]fluoride (50–
150 MBq). The vial was sealed and the mixture was heated at
95 ◦C for 15 min. Water (10 mL) was added, and the mixture
was passed through a Sep-Pak cartridge (Sep-Pak Plus tC18,
Waters). The cartridge was washed with water (5 mL) and
[18F]FBN was eluted with THF (1 mL). Radio-TLC analysis (SiO2,
petroleum ether/ethyl acetate (1 : 1), Rf 0.7) of the eluent indicated
high radiochemical purity (>95%) of [18F]FBN. [18F]FBN was
eluted into a second reaction vial containing various amounts
of Co(OAc)2 and NaBH4. Aliquots were taken and the progress
of the reduction reaction was monitored by radio-TLC (SiO2, n-
BuOH/HOAc–H2O (4 : 1 : 1)). The Rf value of [18F]FBA was 0.4
under these conditions.
Radiolabelling with GSH. [18F]FBAPM in ethanol (25 mL, 5–7
MBq) was added to 500 mL of GSH solution (1.0 mg mL-1 to
1.0 mg mL-1) in phosphate buffer (pH 7.2) and was incubated
at ambient temperature. Conversion was monitored by radio-
HPLC after 20 min. Radio-HPLC analysis: CH3CN/0.1 M
triethylammonium chloride (40/60), tR = 2.3 min for [18F]FBAPM-
GSH.
Synthesis of [18F]FBBA and radiolabelling of ODN. Synthesis
of [18F]FBBA was performed in a GE TRACERlab FXFDG
automated synthesis unit as previously described6 starting from
[18F]FBA prepared via Co(OAc)2-assisted NaBH4 reduction of 4-
[18F]fluorobenzonitrile ([18F]FBN).
[18F]FBBA was purified by normal phase HPLC using a Knaur
chromatography system (Berlin, Germany), equipped with a
Synthesis of 4-[18F]fluorobenzylamine ([18F]FBA) using borohy-
dride exchange resin (BER)-transition metal reduction. BER was
prepared as reported by Sim et al.12 Briefly, to 20 g of Amberlite
IRA-400 anion exchange resin was added 50 mL of 1M NaBH4 in
THF. The mixture was stirred for 15 min at ambient temperature.
The BER was washed thoroughly with distilled water and dried
under vacuum at 60 ◦C for 5 h. The dried resin was stored under
nitrogen atmosphere at 4 ◦C.
R
Waters Prep Nova-Pakꢀ HR Silica column (300 ¥ 7.8 mm;
porosity 6 mm), integrated radioactivity detection and UV detec-
tion at 254 nm (fixed wavelength detector K-200, Knaur, Berlin,
Germany). The radioactive peak corresponding to [18F]FBBA (tR =
8 min) was collected by isocratic elution with dichloromethane–
ethyl acetate (95/5, v/v) at a flow rate of 3 mL min-1. The collected
fraction was concentrated to dryness under a gentle stream of
nitrogen. Radio-TLC demonstrated that semi-preparative HPLC
produced pure 18F]FBBA (determined by co-spotting 18F]FBBA
with the non-radioactive N-(4-fluorobenzyl)-2-bromoacetamide
standard (50% ethyl acetate/heptane; [18F]FBBA Rf 0.4).
The C18 Sep-pak cartridge containing [18F]FBN was eluted with
-
1 mL of anhydrous ethanol. 200 mg of BER (ca. 3 mmol BH4 /g
resin) was combined with 0.06 mmols of Co(OAc)2, NiCl2, or
CuSO4 in 0.5 mL distilled, deionized water. Upon activation of
the bead (evident by black discolouration of the resin), 0.5 mL of
[18F]FBN was added. Radio-TLC was performed at 1, 3, and 5 min
4734 | Org. Biomol. Chem., 2010, 8, 4730–4735
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Typically, 150 to 460 MBq of purified 18F]FBBA was collected
for radiolabeling ODNs.
2 (a) W. C. Eckelman, Nucl. Med. Biol., 2002, 29, 777; (b) H. D. Burns,
T. G. Hamil, Wai-si Eng, B. Francis, C. Fioravanti and R. E. Gibson,
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Lyophilized ODN, 0.25 mg, was reconstituted with 0.5 mL of
purified [18F]FBBA (150 to 460 MBq) in phosphate buffered saline
(PBS) 0.1 M, pH 8:methanol, 1 : 1 (v/v). The mixture was heated
in a vented vial at 120 ◦C for 30 min. Methanol 50%, 0.5 mL,
was added midway through the reaction to maintain volume.
Unreacted [18F]FBBA was separated from 18F-labelled ODN using
a NAPTM-10 column (GE Healthcare, UK) by eluting [18F]ODN
with 1.5 mL PBS 0.01 M, pH 7.2. No further purification was
performed.
Analysis of the radiolabelled ODN was performed with reverse
phase HPLC using a Beckman Coulter Inc system consisting of a
Model 168 Diode Array u.v. module, 260 nm; a Model 126 analyt-
ical dual pump; a radioactivity detector (Ortec, TN): ACE MateTM
Single Channel Analyzer; and a Phenomenex Luna, 10 mm,
10 ¥ 250 mm, C18 column (Torrance, CA, USA) with guard col-
umn. A gradient mobile phase was employed: triethylammonium
acetate 100 mM, pH 7.0 (aq)/acetonitrile 90/10 to 60/40 over
30 min, followed by a washout phase of 30/70 for 5 min at a flow
rate of 3 mL min-1. The concentration of 18F-labelled ODNs was
determined by UV spectroscopy at 260 nm using a Beckman DU
7400 spectrophotometer.
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Acknowledgements
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The authors are grateful to Michaela Grote and Jo¨rn Schlesinger
for their excellent support and contributions to this work. F.W.
thanks the Dianne and Irving Kipnes Foundation for supporting
this work.
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