Facile synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([ 18F]SFB) for protein labeling

Article abstract of DOI:10.1002/jlcr.1481

An efficient preparation of N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) based on a convenient three-step, one-pot procedure is described. [¹⁸F]Fluorination of the precursor ethyl 4-(trimethylammonium triflate)benzoate

Full text of DOI:10.1002/jlcr.1481

Research Article
Received 17 August 2007,
Revised 30 October 2007,
Accepted 31 October 2007
Published online 2 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1481
Facile synthesis of N-succinimidyl
4-[18F]fluorobenzoate ([18F]SFB) for
protein labeling
Ã
G. Tang,a Wenbin Zeng,b Meixiang Yu,b and G. Kabalkaa
An efficient preparation of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) based on a convenient three-step, one-pot
procedure is described. [18F]Fluorination of the precursor ethyl 4-(trimethylammonium triflate)benzoate gave ethyl
4-[18F]fluorobenzoate. Saponification of the ethyl 4-[18F]fluorobenzoate with aqueous tetrapropylammonium hydroxide
yielded the corresponding 4-[18F]fluorobenzoate salt ([18F]FBA), which was then treated with N,N,N,N0-tetramethyl-O-(N-
succinimidyl)uronium hexafluorophosphate. The purified [18F]SFB was used for the labeling of AvastinTM (Bevacizumab)
through [18F]fluorobenzoylation of the Avastin’s a-amino groups. The decay-corrected radiochemical yields of [18F]SFB were
as high as 44% (based on [18F]fluoride (n = 10) with a synthesis time of less than 60 min. [18F]Avastin was produced in decay-
corrected radiochemical yields of up to 42% (n = 5) within 30 min (based on [18F]SFB). The radiochemical purities of [18F]SFB
and [18F]Avastin were greater than 95%.
Keywords: protein labeling; 18F; VEGF; SFB; Avastin; one-pot; radiosynthesis
receptor tyrosine kinases: VEGF receptor VEGFR-1 (Flt-1) and
VEGFR-2 (Flk-1/KDR). VEGFR-1 and VEGFR-2 are found predomi-
Introduction
The application of biomolecules such as proteins, peptides, and
antibodies labeled with positron-emitting nuclides has emerged
as an important field in targeted molecular imaging for in vivo
studies of many physiological and pathological processes.1,2
The incorporation of fluorine-18 into proteins, peptides, and
antibodies usually requires the use of prosthetic groups, also
referred to as bifunctional labeling agents. A large number of
18F-labeled prosthetic groups have been developed that can be
attached to biomolecules via acylation, amidation, imidation,
alkylation reactions, photochemical conjugation, and solid-
phase syntheses.1,3 The acylation approach using N-succinimi-
dyl-4-[18F]fluorobenzoate ([18F]SFB) is one of the most versatile
18F-labeling methods with respect to the in vivo stability and
radiochemical yield.2,3
The published radiosyntheses of [18F]SFB involve a three-step
multi-vessel procedure: (1) [18F]fluorination of an appropriate
aromatic precursor, (2) formation of the 4-[18F]fluorobenzoate
salt ([18F]FBA), and (3) conversion of the salt to [18F]SFB.1 In the
past few years, a number of modifications have been made in an
effort to improve the synthesis of [18F]SFB for routine applica-
tion.1 Recently, two different fully automated preparations of
[18F]SFB on modified commercial modules were described,2,4
but further improvements in the [18F]SFB synthesis would
be beneficial.
nantly on the surface of vascular endothelial cells and bind to
VEGF with high affinity.5,6 There is very low or undetectable
expression of VEGFRs in most normal tissues (with the exception
of renal glomeruli), whereas VEGF is upregulated in most human
tumor types. VEGF expression is associated with tumor
progression and patient survival in a variety of human cancers.
Therefore, VEGF is considered an attractive target for anticancer
diagnosis and therapy. AvastinTM (Bevacizumab) is a recombinant
humanized monoclonal IgG1 antibody (93% human, 7% murine
sequences—molecular weight 149 kDa), which selectively binds
with high affinity to all isoforms of human VEGF and inhibits
the VEGF’s biologic activity through a steric blocking of the
binding of VEGF to its receptors VEGFR-1 and VEGFR-2 on
the surface of endothelial cells.6 Bevacizumab and its analogues
are potentially valuable for therapeutic agents due to their
specific inhibition of tumor angiogenesis (microvascular growth)
and, thereby, inhibition of tumor growth and metastatic disease
progression.5 [18F] Avastin ([18F] Bevacizumab) holds promise
as a Nuclear Medicine diagnostic agent using positron emission
tomography.
aDepartments of Chemistry and Radiology, University of Tennessee Graduate
School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37920, USA
bDepartment of Medicine, University of Tennessee Graduate School of Medicine,
1924 Alcoa Highway, Knoxville, TN 37920, USA
Angiogenesis, the process of new blood vessel growth, plays
a critical role in several physiological and pathological states,
particularly in tumor growth, invasion, and metastasis. Several
growth factors have been implicated in tumor angiogenesis and
one of the most potent positive regulators of angiogenesis is
vascular endothelial growth factor (VEGF). VEGF binds to two
*Correspondence to: G. Kabalka, Departments of Chemistry and Radiology,
University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway,
Knoxville, TN 37920, USA.
E-mail: kabalka@utk.edu
J. Label Compd. Radiopharm 2008, 51 68–71
Copyright r 2008 John Wiley & Sons, Ltd.

G. Tang et al.
In this report we describe an efficient preparation of [18F]SFB C18 Sep-Pak cartridge, a Sep-Pak alumina cartridge and a
based on a convenient three-step, one-pot procedure, consisting Lichrolut SCX cartridge (200 mg, Merck) in series. The Sep-Pak
of [18F]fluorination of the precursor ethyl 4-(trimethylammonium C18 cartridge trapped the [18F]SFB, the Sep-Pak alumina
triflate)benzoate, saponification to generate the [18F]FBA salt cartridge removed the free 18Fꢁ, and the SCX cartridge removed
with tetrapropylammonium hydroxide, and conversion of the the impurities. The cartridges were washed with 10% aqueous
[18F]FBA salt to [18F]SFB. The resultant [18F]SFB was used for MeCN (15 mL) and then the product [18F]SFB eluted with MeCN
labeling the Avastin through [18F]fluorobenzoylation of the (2 mL). The solvent was then removed under a stream of
Avastin’s a-amino groups.
nitrogen at 601C to provide the dry [18F]SFB.
Experimental
Labeling Avastin with [18F]SFB
Avastin (0.20–0.50 mg, 3.3 ꢀ 10ꢁ6 mmol in 20–50 mL of phos-
phate-buffered saline (0.05 M, pH 7.4)) and aqueous 0.1 M
Na2HPO4 (0.2 mL) were added to the dried [18F]SFB residue.
[The [18F]SFB can also be dissolved in a minimal quantity of
acetonitrile or dimethyl sulfoxide (DMSO) prior to the addition of
the protein solution]. The mixture was allowed to react at room
temperature for 15 min and then purified by passing it through
two self-made Sep-Pak Gel Filtration cartridges [prepared by
opening Sep Pak silica cartridges from Waters and filling them
with the Sephadex G-50. The cartridges were washed with 10 mL
of sterile water before use].
General
Ethyl 4-(trimethylammonium triflate)benzoate was synthesized in
our laboratory according to published procedures.7 Sephadex
G-50 was purchased from Sigma-Aldrich. All other reagents used
in the synthesis were commercial products and were used without
further purification unless otherwise indicated. Sep-Pak QMA, Sep-
Pak C18, and Sep-Pak alumina cartridges were purchased from
Waters (Milford, MA) and Lichrolut SCX cartridge was obtained
from Waters (Eschborn, Germany). Thin-layer chromatography
(TLC) was carried out using pre-coated aluminum-backed silica gel
60 F254 TLC plates (E. Merck Company, Darmstadt, Germany) to
verify product purities. All analytical high-performance liquid
Purity Determination of [18F]SFB and [18F]Avastin
chromatography (HPLC) were performed using a Perkin-Elmer Analytical HPLC analysis on
a
Nova-Paks reversed-phase
system and a Nova-Paks reversed-phase Symmetry analytical C18 analytical C18 column (Waters) was used to determine radio-
column (3.9 mm ꢀ 300 mm, 5 mm, Waters) or a PRP-3 reversed- chemical purity of the [18F]SFB at a flow rate of 1 mL/min using
phase column (4.1 mm ꢀ 150 mm, Hamilton Company), consisting the following gradient (the eluent components were A: 0.01 M
of a pump, a variable wavelength UV detector, and a radioflow aqueous H3PO4; B: MeCN): 0.5 min—95%A/5%B; 8 min—90%A/
detector.
10%B; 16 min—10%A/90%B; 1 min—95%A/5%B. The analytical
HPLC on PRP-3 reversed-phase column was used for
a
222 /K[18F]F complex
determining radiochemical purity of [18F]Avastin at a flow rate
of 2 mL/min eluted with the following gradient (A: 0.01 M
aqueous H3PO4; B: MeCN): 0.5 min—95%A/5%B; 28 min—10%A/
90%B; 1 min—95%A/5%B. Radio TLC analysis on a silica gel 60
plate (MeCN/H2O: 95/5, v/v) was also used to confirm radio-
chemical purity. Trichloroacetic acid (TCA) precipitation8 was
used for determination of [18F]Avastin radiochemical purity. A
K222 detection test was performed on the silica gel 60 coated
plate developed with methanol/15% aqueous ammonium
hydroxide (9/1, v/v) using iodine vapor for staining.9 Radio-
chemical stability was monitored using radio TLC and an
analytical HPLC for periods up to 6 h.
K
No-carrier-added [18F]Fꢁ was obtained through the nuclear
reaction 18O(p, n)18F by irradiation of 495% 18O-enriched water
with an 11MeV proton beam using a Siemens RDS-112 negative
ion cyclotron. After the delivery of [18F]Fꢁ from the cyclotron, the
radioactivity was passed through a Sep-Pak light QMA cartridge
to trap [18F]Fꢁ ([18O]water was collected for recycling). [The
Sep-Pak light QMA cartridge was pre-conditioned sequentially
with 5 mL of 0.5 M potassium bicarbonate, 10mL of deionized
water, and 10mL of acetonitrile before use.] The [18F]Fꢁ was
eluted with 1.5 mL of a solution prepared by mixing aqueous
K2CO3 (0.11 g, 0.8 mmol in 1.0 mL of water) with Kryptofix 222
(K222) (0.60 g, 1.6 mmol in 19mL of acetonitrile). The solvent was
evaporated under a stream of nitrogen at 1201C. Azeotropic
drying was repeated twice with 1 mL portions of MeCN to
generate the anhydrous K222/K[18F]F complex.
Results And discussion
Several procedures have been reported for the synthesis of
[18F]SFB. All utilize a three-step, two-pot procedure involving
aromatic nucleophilic substitution of various ammonium triflate
moieties (including ethyl, tert-butyl, and pentamethylbenzyl
4-(trimethylammonium triflate)), benzoates, and no-carrier-
added [18F]fluoride. The substitution reaction is followed by
hydrolysis of the ester protecting group using aqueous
NaOH,10,11 aqueous HCl,4 or trifluoroacetic acid.3,12,13 The
final synthetic step employs activation by N,N,N0,N0-tetra-
methyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU).
When ethyl and tert-butyl esters were used as precursors, two
purification steps were necessary: purification of the intermedi-
ate [18F]FBA and purification of the final product using solid-
phase extraction (cartridges). When the pentamethylbenzyl
ester was used as a precursor, three purification steps were
required.12,13
Radiosynthesis of [18F]SFB
Ethyl 4-(trimethylammonium triflate)benzoate (1) (5.0 mg,
20 mmol) in anhydrous MeCN (1 mL) was added to the dried
K222/K[18F]F and the mixture heated at 901C for 10 min to
produce ethyl 4-[18F]fluorobenzoate (2). The ethyl ester was
subsequently hydrolyzed to form 3 using 20 mL of tetrapropy-
lammonium hydroxide (1.0 M in water) at 1201C for 3 min, and
then the mixture azeotropically dried using MeCN (1 mL).
Subsequently, a solution of N,N,N0,N0-tetramethyl-O-(N-succini-
midyl)uronium hexafluorophosphate (HSTU) (12 mg, 33 mmol) in
MeCN (1 mL) was added and the solution heated at 901C for
5 min. After cooling, 5% aqueous acetic acid (9 mL) and water
(15 mL) were added. The reaction mixture was passed through a
J. Label Compd. Radiopharm 2008, 51 68–71
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

G. Tang et al.
Using [18F]SFB, we labeled Avastin in radiochemical yields up
to 42.3% (decay corrected, n = 5). Adding a small amount of
acetonitrile or DMSO to dissolve the [18F]SFB prior to the
addition of protein did not improve the protein labeling yield
but was somewhat more convenient. Purification of [18F]Avastin
using Sep-Pak Gel Filtration cartridges (two Sephadex G-50
cartridges) greatly reduced the total synthesis time (ꢂ30 min).
The identities of [18F]SFB and [18F]Avastin were confirmed by
comparison with non-radioactive reference compounds. The
radiochemical purity of [18F]SFB was greater than 95%,
confirmed by TLC and HPLC. The retention times of [18F]SFB
and [18F]FBA were ꢂ17 min and ꢂ10 min, respectively. A color
spot test for K222 by TLC did not detect K222 in the final [18F]SFB
solution also. (The detection limit for Kryptofix using the color
spot test is o50 mg/mL.) Solutions of [18F]SFB in MeCN or
CH3OH were stable over a period of hours. The radiochemical
purity of the [18F]Avastin was greater than 95% for all
preparations, confirmed by TLC, TCA, and HPLC. The purification
Figure 1. HPLC chromatogram of the purified [18F]Avastin.
We manually synthesized [18F]SFB (Scheme 1) using ethyl of [18F]Avastin using the Sephadex G-50 column and self-made
4-(trimethylammonium triflate)benzoate (1) as a precursor via a Sep-Pak Sephadex G-50 cartridges produced excellent radio-
three-step, one-pot procedure. [18F]Fluorination of the ammo- chemical purity (495%), as shown in Figure 1. The retention
nium triflate precursor 1 in the solvent MeCN gave 2, which was times of [18F]SFB and [18F]Avastin were ꢂ6 min and ꢂ12 min,
directly used for the hydrolysis reaction. Hydrolysis of 2 with respectively, under the conditions utilized.
aqueous tetrapropylammonium hydroxide yielded the [18F]FBA
salt 3, which was azeotropically dried using MeCN. Reaction of 3
with HSTU, followed by purification using a C18 cartridge, a Sep-
Conclusion
An efficient method for preparing [18F]SFB, one of the most
Pak alumina cartridge, and a cation exchange cartridge in series
yielded the purified [18F]SFB. [18F]SFB was produced in a decay-
corrected radiochemical yield of up to 43.874.6% (based on
versatile 18F-labeled agents used for labeling biomarkers, has
been developed. [18F]Avastin was successfully produced from
Scheme 1
[18F]fluoride; n = 10) in less than 60 min (n = 10). This simplified [18F]SFB. Purification of [18F]Avastin was conducted using
one-pot procedure should be readily adaptable to automated Sep-Pak Gel Filtration cartridges in place of a Gel Filtration
radiosynthesis techniques.
column that further reduced the synthesis time. TLC provided a
In our study, MeCN was used in place of DMSO as solvent for simple and practical method to routinely monitor the progress
the [18F]fluorination reaction and aqueous tetrapropylammo- of both the [18F]SFB radiosynthesis and the protein labeling
nium hydroxide replaced aqueous NaOH in the saponification. reaction. The new one-pot procedure should be readily
These changes allowed us to carry out the three-step radio- adaptable to the automated production of [18F]SFB and
synthesis in one pot because no purification of the [18F]FBA was [18F]Avastin using currently available commercial synthesis
required. The [18F]fluorination reaction had been carried out in modules.
MeCN in earlier studies using the tert-butyl benzoate precursor4
and the pentamethylbenzyl benzoate precursor12 HSTU can also
be used in place of TSTU to achieve similar radiochemical yields.
Acknowledgements
In our hands, our one-pot procedure produced the same yield as This research was supported in part by the Robert H. Cole
those reported earlier1 but in a shorter time.
Foundation.
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J. Label Compd. Radiopharm 2008, 51 68–71

G. Tang et al.
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