A simplified protocol for the automated production of succinimidyl 4-[ 18F]fluorobenzoate on an IBA Synthera module

Article abstract of DOI:10.1002/jlcr.1892

The important peptide labelling reagent succinimidyl 4-[ ¹⁸F]fluorobenzoate ([¹⁸F]SFB) has been synthesised in 75-85% decay corrected radiochemical yield using the IBA Synthera platform (IBA Cyclotron Solutions, Louvain-la-neuve, Bel

Full text of DOI:10.1002/jlcr.1892

Note
Received 24 January 2011,
Revised 25 March 2011,
Accepted 29 March 2011
Published online 13 July 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1892
A simplified protocol for the automated
production of succinimidyl 4‐[18F]
fluorobenzoate on an IBA Synthera module
a
a
a
Uwe Ackermann,a,b Shinn Dee Yeoh, John I. Sachinidis, Stan S. Poniger,
*
Andrew M. Scott,a,b,c and Henri J. Tochon‐Danguya,b
The important peptide labelling reagent succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) has been synthesised in 75–85%
decay corrected radiochemical yield using the IBA Synthera platform (IBA Cyclotron Solutions, Louvain‐la‐neuve, Belgium)
with the fluorodeoxyglucose‐integrated fluidic processor nucleophilic and only four reagent vials in a single reactor.
(4‐ethoxycarbonylphenyl) trimethylammonium triflate was used as the labelling precursor and 1 M aqueous tetra-
methylammonium hydroxide for the hydrolysis of the intermediate ethyl 4‐[18F]fluorobenzoate. N,N,N′,N′‐tetramethyl‐O‐
(N‐succinimidyl)uronium tetrafluoroborate (TSTU) was then used to form [18F]SFB from 4‐[18F]fluorobenzoate. By omitting
the addition of acetic acid and introducing a combined hydrolysis/water removal step, the synthesis time was shortened to
58 minutes. After SepPak purification, the radiochemical purity of [18F]SFB was 95.8–98.2%. These simplifications might be
of significance to users of other automated synthesis modules.
Keywords: SFB; automated synthesis; protein labelling; nucleophilic aromatic substitution
further purification. (4‐Ethoxycarbonylphenyl)trimethylammonium
Introduction
triflate and [19F]SFB were synthesised according to literature
Succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) (1) is an important
labelling agent for biomolecules such as proteins, peptides and
antibodies.1–3 [18F]SFB does not require HPLC purification but is
mostly used crude after removal of unreacted [18F]KF kryptofix
2.2.2 complex by C‐18 SepPak purification.4,5
procedures.7,5
For quality control, a Shimadzu HPLC system equipped with a
5 μL injection loop, a SPD‐20A UV‐Vis detector (Shimadzu Scientific
Instruments, Columbia, MD, USA) and two LC‐20 AD solvent pumps
(Shimadzu Scientific Instruments, Kyoto, Japan) for high pressure
mixing of mobile phase was used. The stationary phase was a
Phenomenex Gemini C‐18 (Phenomenex, Torrance, CA, USA), 10 µ
RP column, 150 × 2.3mm. Acetonitrile (A) with 0.1% formic acid and
water (B) with 0.1% formic acid were used as the mobile phase and
a gradient elution technique was used for analysis: 0–
18 minutes: 5–90% A, 18–30 minutes: isocratic 90% A. For
the detection of radioactive compounds, an in‐house built flow‐
through detector with a GM tube was used. Specific radioactivity
was measured by HPLC using a mass standard curve of known
concentrations of [19F]1. FDG‐IFP Nucleophilics were purchased
from ABX advanced biochemical compounds, Germany and were
used without any modifications.
In our laboratory, [18F]SFB has been produced from
(4‐ethoxycarbonylphenyl)trimethylammonium triflate (2) using
an old modified fluorodeoxyglucose (FDG) synthesiser with two
reactor vials and nine reagent vials.6
However, low synthesis yields of 25 15%, a long synthesis time
of 90 minutes and an unreliable synthesis have prompted us to
improve the [18F]SFB synthesis.
Here, we report the synthesis of [18F]SFB from the ester 2 using
a single IBA Synthera module without any modifications with the
standard, unmodified [18F]fluorodeoxyglucose‐integrated fluidic
processor (FDG‐IFP Nucleophilic) and only four reagent vials.
Experimental
General
No‐carrier‐added [18F]fluoride was produced by the 18O(p, n)18F
nuclear reaction with a 10 MeV proton beam generated by the IBA
Cyclone 10/5 cyclotron (IBA Cyclotron Solutions, Louvain‐la‐
neuve, Belgium) in a titanium target using recycled [18O]H2O at
Austin Health, Centre for PET. Typical irradiation parameters were
20 μA for 30 minutes, which produced 5.4–8.1GBq of [18F]fluoride.
Solvents were purchased from MERCK and used as received.
aCentre for Nuclear Medicine and PET, Austin Health, Vic., Australia
bSchool of Medicine, Dentistry and Health Sciences, The University of Melbourne,
Vic., Australia
cLudwig Institute for Cancer Research, Melbourne‐Austin Branch, Vic., Australia
*Correspondence to: Uwe Ackermann, Centre for PET, Austin Health.
Reagents were purchased from Sigma–Aldrich and used without E‐mail: ackerman@petnm.unimelb.edu.au
J. Label Compd. Radiopharm 2011, 54 671–673
Copyright © 2011 John Wiley & Sons, Ltd.

U. Ackermann et al.
Synthesis of [18F]SFB
[18F]SFB solution was then transferred to an in‐house built
reformulation unit where it was diluted with 60 mL of water,
trapped on a C‐18 SepPak and eluted with 1 mL of acetonitrile.
[18F]SFB was synthesised in 58 minutes with decay corrected
radiochemical yields of 80 5% (n = 22) and a specific activity of
80.1–149.1 GBq/µmol at the end of synthesis. After simple
SepPak purification, [18F]SFB had a radiochemical purity of
97 1.2% and the product is obtained in 1 mL of acetonitrile.
Figure 2 shows a typical HPLC trace where the retention time of
[18F]SFB is 18.5 minutes. The retention times for compounds 3
and 4 are 21.6 and 16.8 minutes, respectively, thus showing that
the conversions in each step are almost quantitative.
The shorter synthesis time was achieved because our method
does not require acidification of the crude reaction mixture before
trapping on a C‐18 SepPak and it also combines the hydrolysis of
4‐[18F]fluorobenzoate (3) and the removal of water, which was
added as part of the 1 M aqueous tetramethylammonium
hydroxide solution, in a single step. From our experience with
the previously used, modified FDG synthesiser, we believe that
hydrolysis takes place at the end of the acetonitrile evaporation. It
is also important to note that with the exception of the [18F]KF
kryptofix 2.2.2 complex, evaporation to dryness is not achieved at
any point of this synthesis protocol because of the presence of high
boiling DMSO. Because of the protocol modifications described, it
has been possible to perform the [18F]SFB (1) synthesis using an
unmodified FDG‐IFP Nucleophilic with only four reagent vials. The
FDG‐IFP Nucleophilic is a disposable kit that clips onto the Synthera
module and can be used to perform nucleophilic substitution
reactions followed by base or acid hydrolysis plus cartridge
purification. This kit is commonly used for the synthesis of [18F]FDG,
[18F]FLT, [18F]FAZA or [18F]FMISO. The IFP can be ejected at the end
of synthesis and because all radioactivity is contained within the
IFP, the Synthera module itself can be used for subsequent
radiosyntheses on the same day with a new IFP. The use of IFPs
avoids cross contamination and minimises the radiation exposure
of the operator.
Preparation of the Synthera module:
Vial 1: Eluent (20 mg kryptofix 2.2.2 (53 µmol) and 3.5 mg K2CO3
(25 µmol)) in 0.4 mL of acetonitrile plus 0.2 mL of water
Vial 2: 5mg (4‐ethoxycarbonylphenyl)trimethylammonium triflate
(20 µmol) in 1 mL dimethyl sulfoxide (DMSO)
Vial 3: 20 mg TSTU (66 µmol) in 1 mL acetonitrile
Vial 4: 20 μL of a 1 M aqueous tetramethylammonium hydroxide
(20 µmol) in 4 mL acetonitrile
After loading the reagents, [18F]fluoride from the target was
trapped on a QMA ion exchange cartridge (Waters Corporation,
Milford, MA, USA) and eluted using eluent from vial 1. The kryptofix
complex was dried at 110 °C for 5 minutes and 95 °C for 3 minutes
under vacuum and argon flow. After drying, the precursor was
added from vial 2 and labelling was achieved by heating to 110 °C
for 15minutes. Base solution was then added from vial 4 and the
solution was heated to 90 °C for 15 minutes under vacuum and
argon flow. This achieved hydrolysis of ethyl 4‐[18F]fluorobenzoate
to 4‐[18F]fluorobenzoate during the evaporation step as well as
removal of the water that was added as part of the 1 M aqueous
tetramethylammonium hydroxide solution. N,N,N′,N′‐tetramethyl‐
O‐(N‐succinimidyl)uronium tetrafluoroborate (TSTU) in 1 mL of
acetonitrile was then added from vial 3 and 4‐[18F]fluorobenzoate
converted to [18F]SFB at 110 °C for 5 minutes. The [18F]SFB solution
was then transferred into an in‐house built reformulation module
where it was diluted in 60 mL of water and subsequently trapped
on a Waters C‐18 Plus SepPak (Waters Corporation, Milford, MA,
USA). After washing of the SepPak with 5 mL of water and drying
with nitrogen, [18F]SFB was eluted with 1 mL of acetonitrile.
[18F]SFB was obtained in decay corrected radiochemical yields
of 80 5% (n = 22) and the radiochemical purity was 97 1.2%.
Specific radioactivity was 80.1–149.1 GBq/µmol at the end of
synthesis and the total synthesis time was 58 minutes.
In summary, using the IBA Synthera module, we were able to
Results and discussion
increase the yields of [18F]SFB (1) from 25 15% to 80 5%
The radiolabelling of ethyl 4‐[18F]fluorobenzoate (3) was achieved (n = 22), shorten the synthesis time and significantly improve
by reacting the (4‐ethoxycarbonylphenyl)trimethylammonium the reliability of the synthesis compared with the old modified
triflate (2) precursor with dried [18F]KF kryptofix 2.2.2 complex FDG synthesiser method we have used previously. Compared
in DMSO at 110 °C for 15 minutes. A 1 M aqueous solution of with the recently published [18F]SFB synthesis methods, our
tetramethylammonium hydroxide in 4 mL of dry acetonitrile was synthesis does not require modifications to commercially
then added and the mixture heated to 90°C for 15 minutes under available modules and has the highest yields of all procedures
vacuum and argon flow. This achieved hydrolysis of ethyl 4‐[18F] published.4,8,9 Furthermore, our method delivers [18F]SFB in a final
fluorobenzoate (3) to 4‐[18F]fluorobenzoic acid (4) during the volume of 1 mL of acetonitrile, which will make evaporation to
evaporation process as well as removalofwater throughazeotropic dryness quicker, if dry [18F]SFB is required for the subsequent
distillation with acetonitrile. The resulting 4‐[18F]fluorobenzoate (4) peptide labelling step.
solution in the remaining 1 mL of DMSO was reacted with TSTU
The method of Tang et al. is a manual synthesis that requires C‐18,
at 110 °C for 5 minutes to form [18F]SFB (1) (Figure 1). The crude alumina and SCX cartridges for [18F]SFB purification and produces
O
O
OH
O
O
O
OEt
O
OEt
N
TSTU
NMe4OH
[
18F]KF/Kryptofix
O
DMSO/CH3CN1/1
110°C, 5 min
CH3CN
90°C, 15 min
DMSO
110°C, 15 min
18F
18F
18F
+ NMe3
-OTf
18F]3
[
18F]1
[
18F]4
[
2
Figure 1. Synthesis of succinimidyl 4‐[18F]fluorobenzoate (1).
www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 671–673

U. Ackermann et al.
mV(x100)
AD2
2.5
2.0
1.5
1.0
0.5
0.0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
min
Figure 2. Radio‐HPLC trace of succinimidyl 4‐[18F]fluorobenzoate after C‐18 SepPak purification.
[18F]SFB in 43.8 4.6% yields.4 The synthesis time is given as less radiation exposure. This may be important for busy PET centres
than 60 minutes, which is comparable with our synthesis time of with limited radiochemistry resources.
58 minutes, and [18F]SFB is obtained in 2 mL of acetonitrile. Glaser
et al. have published a manual [18F]SFB synthesis using microwave
Conclusion
assisted labelling of a 4‐N,N,N‐trimethylaniliniumphenylmethanone
trifluoromethanesulphonate precursor.8 This method requires
Using the IBA Synthera module as well as a modified synthesis
SepPak purification of the intermediate p‐[18F]fluorobenzaldehyde,
which is then oxidised with (diacetoxyiodo)benzene in the presence
of N‐hydroxysuccinimide to form [18F]SFB. The synthesis times
were 2.75hours for HPLC purification and 1.67 hours for a SepPak
method that gave [18F]SFB in only 89% radiochemical purity.
Although this synthesis protocol seems less attractive than our
method, we believe that the chemistry approach described by
Glaser et al. is extremely innovative and has significance beyond the
[18F]SFB production. The method published by Scott et al. uses a
Tracerlab FXFN module with very minor modifications and the
synthesis as well as the SepPak purification is fully automated.9
Overall, this method produces [18F]SFB in a shorter time frame
(~45 minutes) but with lower radiochemical yields (38% nondecay
corrected) and in a larger volume of acetonitrile (2 mL) than our
method. The Tracerlab FXFN method uses only 10 mg of TSTU, an
amount we found was too low to achieve complete conversion
of 4‐[18F]fluorobenzoate into [18F]SFB with the Synthera module.
The high radiochemical yields and excellent radiochemical purity
of our synthesis may be due to a longer, 15minutes fluorination
reaction and the subsequent 15 minutes combined hydrolysis/
water removal step. If implemented on the Tracerlab FXFN module,
our method may further improve the [18F]SFB production on this
system. A key advantage of using the Synthera module over the
Tracerlab FXFN module is the use of disposable IFPs. The IFPs can be
ejected at the end of synthesis, thus allowing the operator to use
the Synthera module for subsequent radiotracer productions on
the same day without any cross contamination and minimal
procedure, the decay corrected radiochemical yields of [18F]SFB
have been increased from 25 15% to 80 5% and the synthesis
time has been shortened by 32 minutes. This setup is now routinely
used in our laboratory for the [18F]SFB production. This simplified
protocol can potentially be implemented in other automated
synthesis modules.
Acknowledgements
This work was supported by a grant from the National Health and
Medical Research Council, NHMRC project grant number 469002.
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J. Label Compd. Radiopharm 2011, 54 671–673
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