Inhibition of radical reactions for an improved potassium tert-butoxide-promoted 11C-methylation strategy for the synthesis of α-11C-methyl amino acids

Article abstract of DOI:10.1002/jlcr.3259

α-¹¹C-Methyl amino acids are useful tools for biological imaging studies. However, a robust procedure for the labeling of amino acids has not yet been established. In this study, the ¹¹C-methylation of Schiff-base-activated α-amino a

Full text of DOI:10.1002/jlcr.3259

Special Issue Article
Received 1 October 2014,
Revised 13 November 2014,
Accepted 7 December 2014
Published online 18 February 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3259
Inhibition of radical reactions for an
improved potassium tert-butoxide-promoted
11
C-methylation strategy for the synthesis
†,‡
11
of α- C-methyl amino acids
Chie Suzuki,a,b Koichi Kato,c,d Atsushi B. Tsuji, Ming-Rong Zhang,
Yasushi Arano,b and Tsuneo Sagaa
a
d
*
α-11C-Methyl amino acids are useful tools for biological imaging studies. However, a robust procedure for the labeling of amino
acids has not yet been established. In this study, the 11C-methylation of Schiff-base-activated α-amino acid derivatives has been
optimized for the radiosynthesis of various α-11C-methyl amino acids. The benzophenone imine analog of methyl 2-amino
butyrate was 11C-methylated with [11C]methyl iodide following its initial deprotonation with potassium tert-butoxide (KOtBu).
The use of an alternative base such as tetrabutylammonium fluoride, triethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene did
not result in the 11C-methylated product. Furthermore, the KOtBu-promoted 11C-methylation of the Schiff-base-activated amino
acid analog was enhanced by the addition of 1,2,4,5-tetramethoxybenzene or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and
inhibited by the addition of 1,10-phenanthroline. These results suggest that inhibition of radical generation induced by KOtBu
improves the α-11C-methylation of the Schiff-base-activated amino acids. The addition of a mixture of KOtBu and TEMPO to a
solution of Schiff-base-activated amino acid ester and [11C]methyl iodide provided optimal results, and the tert-butyl ester
and benzophenone imine groups could be readily hydrolyzed to give the desired α-11C-methyl amino acids with a high
radiochemical conversion. This strategy could be readily applied to the synthesis of other α-11C-methyl amino acids.
Keywords: α-methylation; amino acid; Schiff base; potassium tert-butoxide; PET; electron transfer; radical
of the corresponding Schiff-base-activated amino acid derivatives.
Introduction
The radiolabeling of the core structure of α-methyl amino acids
with 11C could potentially be used as an effective strategy to
Amino acids have been labeled with various isotopes, including
positron emitting radionuclides.1,2 Because amino acids are the
building blocks of proteins and hormones and intermediates
aDiagnostic Imaging Program, Molecular Imaging Center, National Institute of
Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
for neurotransmitters and energy metabolism,1,2 amino acid
analogs labeled with 11C, 18F, and 13N have been explored
bDepartment of Molecular Imaging and Radiotherapy, Graduate School of
Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba
260-8675, Japan
extensively as tool compounds for the imaging of biological
phenomenon by positron emission tomography (PET).
The incorporation of a methyl group at the α-position of
amino acids is one of the strategies used for the design of amino
acid-based PET tracers. Radiolabeled α-methyl amino acids are
generally more stable toward metabolism than their desmethyl
counterparts because their α,α-dialkylated structure makes them
more resistant to transamination, the first step of amino acid
catabolism.3 The metabolic characteristics of radiolabeled
α-methyl amino acids allow them to be used as noninvasive
imaging probes, which can be used to simplify the kinetic
analysis of amino acid transport activity4–9 or investigate the
metabolism of amino acid side chains during the synthesis of
neurotransmitters.10 However, the number of radiolabeled
α-methyl amino acid tracer compounds available for imaging
studies is limited, because the vast majority of these compounds
are synthesized by the incorporation of a radiohalogen into their
side chain.4,5,11 In contrast, L-[11C]-α-methyl tryptophan ([11C]
AMT)12 and 2-amino-[3-11C]isobutyric acid ([3-11C]AIB)13 were
labeled with 11C at their α-methyl group by the 11C-methylation
cDepartment of Integrative Brain Imaging, National Center of Neurology and
Psychiatry, 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-5551, Japan
dMolecular Probe Group, Molecular Imaging Center, National Institute of
Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
*Correspondence to: Koichi Kato, Department of Integrative Brain Imaging,
National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-cho, Kodaira,
Tokyo 187-5551, Japan.
E-mail: katok@ncnp.go.jp
†This article is published in Journal of Labelled Compounds and Radiopharmaceuticals
as a special issue on ‘Bengt Långström’, edited by Antony Gee, Department of
Chemistry and Biology, Division of Imaging Sciences and Bioengineering, Kings College
London, UK and Albert Windhorst, Department of Radiology and Nuclear Medicine,
VU University Medical Center, Amsterdam, The Netherlands.
‡Dedicated to Prof. Dr. Bengt Långström, with deepest appreciation, to celebrate
his outstanding life-long contribution to the field of radiochemistry and on the
occasion of his retirement as editor of the Journal of Labelled Compounds and
Radiopharmaceuticals.
J. Label Compd. Radiopharm 2015, 58 127–132
Copyright © 2015 John Wiley & Sons, Ltd.

C. Suzuki et al.
synthesize a variety of 11C-labeled α-amino acids and evaluate
their biological properties by PET. In this study, we have developed
an optimized procedure for the 11C-methylation of Schiff-base-
activated α-amino acid derivatives that can be used for the
radiosynthesis of a wide variety of α-11C-methyl amino acids.
mixture was stirred at room temperature for 24 h. The reaction mixture
was then filtered, and the filtrate was collected and concentrated in
vacuo to give a residue, which was dissolved in diethyl ether (20 mL).
The resulting solution was washed with brine (3 × 20 mL) and dried over
anhydrous magnesium sulfate. The solvent was removed in vacuo to give
a residue, which was purified by column chromatography over silica gel
eluting with a mixture of ethyl acetate/hexane (1:50 – v/v) to provide 3a
as a colorless oil (765.8 mg, 83.6%). 1H NMR (CDCl3): δ 7.15–7.66 (10H, m,
aromatic), 3.99–4.03 (1H, dd, J = 7.7, 5.5 Hz, CH), 3.72 (3H, s, COOCH3),
1.89–1.97 (2H, m, CH2CH3), 0.83–0.88 (3H, t, J = 7.3 Hz, CH2CH3); 13C
NMR (CDCl3): δ 173.1, 170.6, 139.7, 136.6, 130.2, 129.0, 128.7, 128.4,
128.2, 128.0, 66.9, 52.2, 27.2, 10.7; FAB-MS [M + H]+: m/z 282, found: 282.
Experimental
General
All of the chemicals used in the current study were purchased as the
analytical grade and used without further purification. H-Abu(2)-OtBu-
HCl (2b), H-Ala-OMe (2c), H-Nva-OtBu-HCl (2d), and H-Phe-OtBu-HCl
(2e) were purchased from Watanabe Chemical Industries, Ltd.
(Hiroshima, Japan). (R)-α-Ethylalanine monohydrate5 and (R)-α-
methylphenylalanine monohydrate7 were purchased from Nagase &
Co., Ltd. (Kobe, Japan). Compounds 3c and 4c were prepared as
described previously.13 Solutions of potassium tert-butoxide (KOtBu)
(1 M solution in THF) and tetrabutylammonium fluoride (TBAF) (1 M
solution in THF) were purchased from Sigma Aldrich (Milwaukee, WI,
USA) and Tokyo Chemical Industry Co., Ltd (Tokyo, Japan), respectively.
1,2,4,5-Tetramethoxy benzene (TMB) was synthesized according to a
previously published procedure.14 2,2,6,6-Tetramethylpiperidine-1-oxyl
(TEMPO) and 1,10-phenanthroline (o-phen) were purchased from Sigma
Aldrich and Tokyo Chemical Industry Co., Ltd, respectively. 1H and 13C
NMR spectra were obtained on a JEOL-AL-300 spectrometer (JEOL,
Tokyo, Japan). Chemical shifts (δ) have been reported in units of parts
per million (ppm) relative to tetramethylsilane (0.00 ppm for 1H and 13C),
which was used as an internal reference. Fast atom bombardment-mass
tert-Butyl 2-((diphenylmethylene)amino)butyrate (3b)
Compound 3b was prepared from benzophenone imine and H-Abu(2)-
OtBu-HCl (2b) according to the procedure described earlier for 3a.
Compound 3b was isolated as a colorless oil (824.2 mg, 99.7%). 1H
NMR (CDCl3): δ 7.16–7.83 (10H, m, aromatic), 3.83–3.87 (1H, dd, J = 7.3,
5.1 Hz, CH), 1.83–1.98 (2H, m, CH2CH3), 1.44 (9H, s, C(CH3)3), 0.85–0.89
(3H, t, J = 7.3 Hz, CH2CH3); 13C NMR (CDCl3): δ 171.7, 170.1, 139.9,
136.9, 130.2, 128.9, 128.6, 128.5, 128.1, 128.0, 80.9, 67.5, 28.2, 27.0,
10.7; FAB-MS [M + H]+: m/z 324, found: 324.
tert-Butyl 2-((diphenylmethylene)amino)pentanoate (3d)
Compound 3d was prepared from benzophenone imine and H-Nva-O-
tBu-HCl (2d) according to the procedure described earlier for 3a.
Compound 3d was isolated as a colorless oil (774.6 mg, 96.4%). 1H NMR
(CDCl3): δ 7.16–7.82 (10H, m, aromatic), 3.89–3.93 (1H, t, J = 6.2 Hz, CH),
1.83–1.90 (2H, q, J = 6.6 Hz, CH2CH2CH3), 1.44 (9H, s, C(CH3)3), 1.21–1.37
(2H, m, CH2CH2CH3), 0.82–0.86 (3H, t, J = 7.3 Hz, CH2CH2CH3); 13C NMR
(CDCl3): δ 171.8, 170.0, 139.9, 136.9, 130.2, 128.9, 128.6, 128.5, 128.1,
128.0; FAB-MS [M + H]+: m/z 338, found: 338.
spectrometry (FAB-MS) was conducted on
a JEOL JMS-SX 102A
spectrometer (JEOL). Carbon-11 was produced by an 14N (p, α) 11C nuclear
reaction with a CYPRIS HM18 cyclotron (Sumitomo Heavy Industry, Tokyo,
Japan). HPLC was conducted on a JASCO HPLC system (JASCO, Tokyo,
Japan). Effluent radioactivity was determined with a NaI (Tl) scintillation
detector system (ORTEC, Oak Ridge, TN, USA). Analytical reversed-phase
tert-Butyl 2-((diphenylmethylene)amino)-3-phenylpropanoate (3e)
(RP)-HPLC was performed over
a
COSMOSIL 5C18-AR-II column
Compound 3e was prepared from benzophenone imine and H-Phe-OtBu-
HCl (2e) according to the procedure described earlier for 3a. Compound
3e was isolated as a colorless oil (824.2 mg, 99.7%). 1H NMR (CDCl3): δ
7.60–7.80 (15H, m, aromatic), 4.08–4.12 (1H, dd, J = 8.8, 4.4 Hz, CH),
(150 × 4.6 mm, i. d.; Nacalai Tesque, Kyoto, Japan) at a flow rate of
1 mL/min with a mobile phase consisting of acetonitrile/H2O (70/30 – v/v)
for the methyl ester (system 1) or acetonitrile/H2O (85/15 – v/v) for the
tert-butyl ester (system 2). Analytical hydrophilic interaction 3.12–3.30 (2H, m, CH2Ph), 1.44 (9H, s, C(CH3)3); 13C NMR (CDCl3): δ 171.0,
chromatography (HILIC)-HPLC was performed over a Cosmosil HILIC
column (150 × 4.6 mm, i. d.; Nacalai Tesque) at a flow rate of 1 mL/min
170.5, 139.7, 138.5, 136.5, 130.2, 130.0, 128.9, 128.3, 128.2, 128.1, 128.0,
127.8, 126.3, 81.3, 68.1, 39.7, 28.2; FAB-MS [M + H]+: m/z 386, found: 386.
with
a mobile phase consisting of acetonitrile/30 mM aqueous
Methyl 2-((diphenylmethylene)amino)-2-methylbutanoate (4a)
ammonium acetate solution (80/20 – v/v). Semipreparative HPLC was
performed over a Cosmosil HILIC column (250 × 10 mm, i. d.; Nacalai
Tesque) at a flow rate of 5 mL/min with a mobile phase consisting of
acetonitrile/30 mM aqueous ammonium acetate (80/20 – v/v).
A solution of KOtBu in THF (1.0 M, 1.42 mL, 1.42 mmol) was added to a
solution of 3a (80 mg, 0.28 mmol) in THF (3 mL) in a dropwise manner at
0 °C. Methyl iodide (89.0 μL, 1.42 mmol) was then added to the mixture
in a dropwise manner at 0 °C, and the resulting mixture was stirred for
16 h at room temperature. The solvent was removed in vacuo to give a
residue, which was purified by column chromatography over silica gel
eluting with a mixture of ethyl acetate/hexane (1/50 – v/v) to give
unlabeled 4a as a yellow oil (55.4 mg, 66.0%). 1H NMR (CDCl3): δ
7.15–7.82 (10H, m, aromatic), 3.29 (3H, s, COOCH3), 1.97–2.02 (2H, m,
CH2CH3), 1.39 (3H, s, CCH3), 0.86–1.00 (3H, t, J = 7.7 Hz, CH2CH3); 13C NMR
(CDCl3): δ 175.4, 166.7, 141.3, 137.7, 130.2, 128.7, 128.6, 128.4, 128.1,
127.8, 66.8, 51.4, 33.2, 23.7, 8.6; FAB-MS [M + H]+: m/z 296, found: 296.
Chemistry
Methyl 2-aminobutanoate (2a)
Thionyl chloride (21.1 mL, 291 mmol) was added in a dropwise manner to
a suspension of DL-2-aminobutyric acid (1a, 3.0 g, 29.1 mmol) in methanol
(30 mL) at À30 °C under an atmosphere of N2, and the resulting mixture
was stirred for 3 h at À30 °C. The mixture was then warmed to room
temperature and stirred for 18 h. The solvent was removed in vacuo
to give a residue, which was codistilled from toluene to remove any
residual thionyl chloride. The resulting residue was then crystallized
from a mixture of methanol/diethyl ether to provide 2a as a white solid
(4.01 g, 89.7%). 1H NMR (CDCl3): δ 8.82 (2H, s, NH2), 4.09–4.14 (1H, t,
J = 5.7 Hz, CH), 3.83 (3H, s, COOCH3), 2.08–2.16 (2H, quin, J = 7.7 Hz,
CH2CH3), 1.10–1.15 (3H, t, J = 7.3 Hz, CH2CH3); FAB-MS [M + H]+: m/z 118,
found: 118.
tert-Butyl 2-((diphenylmethylene)amino)-2-methylbutanoate (4b)
Compound 4b was prepared from 3b (100 mg, 0.31 mmol) using methyl
iodide (96.7 μL, 1.55 mmol) and a solution of KOtBu in THF (1.0 M,
1.55 mL, 1.55 mmol) according to the procedure described earlier for
4a. Compound 4b was isolated as a yellow oil (58.6 mg, 56.1%). 1H
NMR (CDCl3): δ 7.20–7.83 (10H, m, aromatic), 1.86–1.94 (2H, m, CH2CH3),
1.31 (9H, s, C(CH3)3), 1.21 (3H, s, CCH3), 1.00–1.05 (3H, t, J = 7.3 Hz,
CH2CH3); 13C NMR (CDCl3): δ 174.3, 166.9, 141.9, 138.9, 129.8, 128.9,
128.6, 128.3, 128.0, 127.9, 80.6, 67.1, 36.6, 28.1, 24.0, 8.8; FAB-MS [M
Methyl 2-((diphenylmethylene)amino)butyrate (3a)
Benzophenone imine (819 μL, 4.88 mmol) and compound 2a (500 mg,
3.25 mmol) were added to dichloromethane (10 mL), and the resulting + H]+: m/z 338, found: 338.
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J. Label Compd. Radiopharm 2015, 58 127–132

C. Suzuki et al.
then treated with a 2.0 M solution of aqueous HCl (150 μL) and held at
room temperature for 90 s. A portion of each mixture was then collected
and analyzed by HILIC-HPLC.
tert-Butyl 2-((diphenylmethylene)amino)-2-methylpentanoate (4d)
Compound 4d was prepared from 3d (100 mg, 0.30 mmol) using
methyl iodide (92.6 μL, 1.48 mmol) and a solution of KOtBu in THF
(1.0 M, 1.48 mL, 1.48 mmol) according to the procedure described
earlier for 4a. Compound 4d was isolated as a yellow oil (26.5 mg,
25.4%). 1H NMR (CDCl3): δ 7.19–7.82 (10H, m, aromatic), 1.75–1.88
(2H, m, CH2CH2CH3), 1.38–1.56 (2H, m, CH2CH2CH3C), 1.32 (9H, s, C
(CH3)3), 1.23 (3H, s, CH3), 0.91–0.96 (3H, t, J = 7.3 Hz, CH2CH2CH3); 13C
NMR (CDCl3): δ 174.2, 166.6, 141.7, 138.7, 129.6, 128.7, 128.5, 128.2,
127.8, 127.7, 80.4, 66.7, 45.6, 27.9, 24.4, 17.4, 14.6; FAB-MS [M + H]+:
m/z 352, found: 352.
Deprotection of compound 4a in DMSO
Following the 11C-methylation process, the solvent was evaporated
under a stream of N2 gas to give a residue, which was dissolved in DMSO
(150 μL) and treated with a 1.0 M solution of NaOH (150 μL). The resulting
mixture was heated at 100 °C for 90 s and then treated with a 2.0 M
aqueous solution of HCl (150 μL). The resulting mixture was held at room
temperature for 90 s before being sampled for analysis by HILIC-HPLC.
Deprotection of compounds 4b, 4d, and 4e
tert-Butyl 2-((diphenylmethylene)amino)-2-methylbutanoate (4e)
The product of the 11C-methylation process was treated with a 2.0 M
aqueous solution of HCl (150 μL), and the resulting mixture was heated
at 100 °C for 5 min. The mixture was then cooled to room temperature
before being sampled for analysis by HILIC-HPLC.
Compound 4e was prepared from 3e (100 mg, 0.26 mmol) using methyl
iodide (81.1 μL, 1.30 mmol) and a solution of KOtBu in THF (1.0 M,
1.30 mL, 1.30 mmol) according to the procedure described earlier for
4a. Compound 4e was isolated as a yellow oil (31.7 mg, 30.6%). 1H
NMR (CDCl3): δ 6.95–7.82 (15H, m, aromatic), 3.08–3.37 (2H, m, CH2CH3),
1.56 (3H, s, CCH3), 1.28 (9H, s, C(CH3)3); 13C NMR (CDCl3): δ 173.8, 166.7,
141.8, 138.8, 137.7, 131.4, 130.2, 128.8, 128.7, 128.2, 128.1, 127.9, 127.8,
126.5, 81.0, 68.0, 49.1, 28.1, 24.0; FAB-MS [M + H]+: m/z 400, found: 400.
Procedure for the remote-controlled synthesis of compound 5
After EOB, the [11C]carbon dioxide was transferred to a reaction vessel
containing a 0.05 M solution of lithium aluminum hydride in THF
(300 μL) and trapped until the solution reached a radioactivity plateau
at 0 °C. The radioactive solution was then heated at 150 °C to remove
THF through evaporation. The resulting residue was then cooled to 0 °C
before being treated with an aqueous solution of HI (57%, 400 μL). The
mixture of radioactive compounds and HI was then heated at 150 °C to
generate [11C]H3I. Gaseous [11C]H3I was distilled from the mixture using
a stream of N2 gas at a flow rate of 30 mL/min and transferred through
ascarite and phosphorus pentoxide columns into the reaction vessel
containing 10 μmol of 3b in THF (150 μL) at À10 °C. After the solution
reached its radioactivity plateau, it was treated with a solution of 10 μmol
KOtBu and 10 μmol TEMPO in THF, and the resulting solution was heated
for 3 min at 20 °C. The reaction mixture was then treated with a 2.0 M
aqueous solution of HCl (150 μL), and the resulting mixture was heated
for 5 min at 100 °C. The mixture was then cooled to room temperature
and purified by semipreparative HILIC-HPLC. The fraction containing 5
(retention time: 10–11 min) was evaporated to dryness to give a radioactive
residue, which was dissolved in water (2 mL). The RCP of the resulting
solution of 5 was determined to be 99.6 0.4% by analytical HILIC-HPLC.
2-Amino-2-methylpentanoic acid (6)
A 4 M solution of HCl in EtOAc (2 mL) was added in a dropwise manner to
a solution of 4d (47.0 mg, 0.13 mmol) in EtOAc (2 mL) at 0 °C, and the
resulting mixture was stirred for 4 h at room temperature. The organic
phase was extracted with water (3 × 5 mL), and the combined aqueous
extracts were distilled to dryness under vacuum to give a residue. The
residue codistilled from acetonitrile under reduced pressure to provide
1
6 as a white solid (9.7 mg, 41.2%). H NMR (CD3OD): δ 1.71–1.96 (2H, m,
CH2CH2CH3), 1.54 (3H, s, CCH3), 1.50–1.26 (2H, m, CH2CH2CH3C),
1.01–0.96 (3H, t, J = 7.3 Hz, CH2CH2CH3); 13C NMR (CD3OD): δ 173.8, 61.0,
40.6, 22.9, 18.0, 14.3; FAB-MS [M + H]+: m/z 132, found: 132.
Radiochemistry
[11C]methyl iodide
[
11C]Methyl iodide ([11C]H3I) was prepared by wet method15 as
described later. After the end of bombardment (EOB), [11C]carbon
dioxide was bubbled into a solution of lithium aluminum hydride in
THF (0.05 M, 500 μL) at 0 °C for ~3 min. After the solvent was removed
at 150 °C, an aqueous solution of hydriodic acid (HI, 57%, 400 μL) was
added to the residue at 0 °C. The resulting [11C]H3I was transferred
through ascarite and phosphorus pentoxide columns under a stream
of N2 gas into THF or dimethyl sulfoxide (DMSO) (1 mL). The
radiochemical purity (RCP) was determined to be >98% by analytical
RP-HPLC using system 1.
Statistical analysis
All of the experiments described in this study were performed in
triplicate, and the resulting data have been presented as the mean
standard deviation. The effects of the additives were evaluated using
ANOVA with Dunnett’s multiple comparison test (Microsoft Excel
software; Microsoft, Seattle, WA, USA).
Results and discussion
General conditions for the 11C-methylation process
The 11C-methylation of Schiff-base-activated α-amino acids
represents a desirable strategy for the generation of radiolabeled
amino acids because the enhanced acidity13,16 of the α-protons
of these compounds facilitates their alkylation. Aldimine analogs
are usually employed as precursors for the synthesis of α,α-
dialkylated amino acids under nonlabeling conditions.
Unfortunately, however, most aldimine derivatives show a low
level of chemical stability, and this represents a significant issue
for PET chemistry, which requires compounds to be stable to the
routine handling conditions required of conventional and
remote-controlled syntheses. Benzophenone imine analogs are
more stable than benzaldimine analogs,17 and benzophenone
imine-type labeling precursors are therefore more suitable for
the radiosynthesis of α-11C-methyl amino acids.
A portion of the [11C]H3I solution (100 μL) containing [11C]H3I (50 MBq)
was added to 50 μL solutions of compounds 3a–e (10 μmol). The
individual mixtures were then treated with a solution of base (i.e., TBAF,
triethylamine (TEA), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or KOtBu;
5, 10, or 20 μmol) in THF (150 μL) both in the presence and absence of
an additive (i.e., TMB, TEMPO, or o-phen; 10 μmol). The resulting mixtures
were held at room temperature for 90 s, and a portion of each mixture
was collected for analysis by RP-HPLC. The radiochemical identity of
the 11C-labeled compounds was verified by comparing their HPLC
profiles with those of the corresponding unlabeled compounds, and
the efficiency of each reaction was evaluated according to the
radiochemical conversion (RCC), which was calculated from the
radiochromatogram after a decay correction.
Deprotection of compound 4a in THF
The products of the 11C-methylation process were treated with a 1.0 M
We initially explored the 11C-methylation of the benzophenone
solution of NaOH (150 μL) at 100 °C for 90 s. The reaction mixtures were imine analog of methyl 2-amino butyrate 3a under various
J. Label Compd. Radiopharm 2015, 58 127–132
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

C. Suzuki et al.
Table 1. Radiochemical conversions for the α-11C-
methylation of 3a
Entry
Precursor
Base (μmol)
Solvent
RCCa (%)
1
2
3
4
5
6
7
8
9
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
TBAF (20)
TBAF (20)
TEA (10)
DMSO
THF
nd
nd
nd
nd
DMSO
DMSO
DMSO
DMSO
DMSO
THF
DBU (10)
KOtBu (5)
KOtBu (10)
KOtBu (20)
KOtBu (5)
KOtBu (10)
KOtBu (20)
21.2 3.0
15.3 4.9
0.9 0.2
14.1 0.6
54.3 6.9
58.8 2.7
THF
THF
10
RCC, radiochemical conversion; TBAF, tetrabutylammonium
fluoride; TEA, triethylamine; DBU, 1,8-diazabicyclo[5.4.0]
undec-7-ene; KOtBu, potassium tert-butoxide; DMSO,
dimethyl sulfoxide; nd, not detected.
aThe RCC data were calculated using the
radiochromatograms obtained by analytical reversed-phase
HPLC following decay correction.
conditions. Several bases were screened against the reaction,
including TBAF, TEA, and DBU, but all of these bases failed to provide
the desired 11C-methylated product (Table 1, entries 1–4). In our
previous report concerning the synthesis of [3-11C]AIB, treatment
of a solution of Schiff-base-activated alanine analogs 3c with TBAF
resulted in the immediate formation of a yellow solution, which
indicated that the substrate was rapidly deprotonated to give the
corresponding anion.13 In the current case, treatment of a solution
of 3a with TBAF, TEA, or DBU did not result in a color change after
90 s, which suggested that 3a was not being deprotonated by these
\bases. These results therefore demonstrated that increasing the
length of the side-chain structure from methyl to ethyl had led to
an increase in the pKa of the α-protons in 3a, making them
resistant to deprotonation by TBAF. Based on this result, we
proceeded to investigate the use of a stronger base for the
deprotonation and subsequent 11C-methylation of 3a. When a
solution of KOtBu was added to a solution of 3a, the reaction
mixture immediately turned red, and the 11C-methylated
compound was obtained following the addition of [11C]H3I
(Figure 1A and C; Table 1, entries 5–10). Having identified a suitable
base for the reaction, we proceeded to screen different solvents
and found that 4a was obtained with a higher RCC when the
reaction was conducted in THF compared with DMSO.
During the course of this work toward the 11C-methylation of 3a,
we observed an unknown by-product peak that was eluted from
the RP-HPLC column just before [11C]H3I (Figure 1C). This peak
was also observed by RP-HPLC analysis when a mixture of [11C]
H3I and KOtBu was held at room temperature for 90 s (data not
Figure 1. RP-HPLC profiles of (A) the ultraviolet absorbance of unlabeled 4a at
254 nm, (B) radioactivity of a solution of [11C]H3I in THF, (C) radioactivity of the
reaction mixture treated with entry 9 in Table 1 (entry 1 in Table 2), and (D)
radioactivity of the reaction mixture treated with entry 3 in Table 2.
shown). KOtBu has been reported to act not only as strong base which is an electron transfer inhibitor,20 or TEMPO, which is a
but also as single-electron donor,18,19 which could result in the radical scavenger, prevented the formation of the by-product
formation of radicals and provide some explanation for (Figure 1D), resulting in an improvement in the RCC of the
the formation of the by-product observed by RP-HPLC. To evaluate KOtBu-promoted 11C-methylation of 3a (Table 2, entries 2 and 3).
the influence of the electron transfer reaction on the α-11C- In contrast, the addition of o-phen, which is an organocatalyst for
methylation of Schiff-base-activated α-amino acid and the electron transfer-related reactions,19 had an adverse impact on
formation of the observed by-products, we investigated the the 11C-methylation reaction of 3a and gave a much lower yield
addition of three different additives to the reaction and compared of the desired product (Table 2, entries 4). Similar tendencies were
the RCCs of the resulting products (Table 2). The addition of TMB, also observed in the KOtBu-promoted 11C-methylation of 3c
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 127–132

C. Suzuki et al.
Table 2. The effect of additives on the radiochemical conversion of the α-11C-methylation reaction
Entry
Precursor
Base (μmol)
Solvent
Additive
RCCa (%)
1
2
3
4
5
6
7
8
3a
3a
3a
3a
3c
3c
3c
3c
3c
3c
3c
3c
KOtBu (10)
KOtBu (10)
KOtBu (10)
KOtBu (10)
KOtBu (10)
KOtBu (10)
KOtBu (10)
KOtBu (10)
TBAF (20)
TBAF (20)
TBAF (20)
TBAF (20)
THF
THF
THF
THF
THF
THF
THF
THF
DMSO
DMSO
DMSO
DMSO
None
TMB
TEMPO
o-phen
None
54.3 6.9
80.6 7.6*
79.4 5.0*
1.2 1.0*
38.9 7.8
45.9 8.5
62.5 3.9*
8.3 3.3*
78.0 9.7
77.6 8.4
76.7 12.7
84.1 4.8
TMB
TEMPO
o-phen
None
TMB
TEMPO
o-phen
9
10
11
12
RCC, radiochemical conversion; TBAF, tetrabutylammonium fluoride; KOtBu, potassium tert-butoxide; DMSO, dimethyl sulfoxide;
TMB, 1,2,4,5-tetramethoxy benzene; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; o-phen, 1,10-phenanthroline.
aThe RCC data were calculated using the radiochromatograms obtained by analytical reversed-phase HPLC following decay correction.
*P < 0.01 compared with the RCC without additive analyzed by ANOVA with Dunnett’s multiple comparison test.
Scheme 1. Synthesis of the α-11C-methyl amino acids. (a) Methanol, thionyl chloride; (b) benzophenone imine, dichloromethane; (c) [11C]H3I, KOtBu, DMSO, or THF; (d) (i)
NaOH, H2O; (ii) HCl, H2O; or (i) HCl, H2O.
(Table 2, entries 5–8). It is noteworthy, however, that the RCC of the
TBAF-promoted 11C-methylation of 3c was not changed by the
addition of TMB, TEMPO, or o-phen (Table 2, entries 9–12). These
results suggest that KOtBu, but not TBAF, would promote the
generation of radicals by a single-electron transfer, which
hampered the 11C-methylation of Schiff-base-activated α-amino
acids. The addition of 10 μmol TEMPO led to the highest RCC of
all of the KOtBu-promoted 11C-methylation reactions.
Taken together, the results of these experiments indicated that
the optimal reaction conditions involved the addition of a mixture
of 10 μmol KOtBu and 10 μmol TEMPO to the solution containing
the Schiff-base-activated precursor and [11C]H3I. With the
optimized conditions in hand, we proceeded to investigate the
11C-methylation of several other benzophenone imine-amino acid
analogs (Scheme 1). The benzophenone imine analog of tert-butyl
2-amino butyrate 3b yielded the desired 11C-methylated product
Table 3. Radiochemical conversions of KOtBu-promoted
α-11C-methylation reactions in the presence of 10 μmol
2,2,6,6-tetramethylpiperidine-1-oxyl
Entry
Precursor
RCCa (%)
1
2
3
4
5
3a
3b
3c
3d
3e
79.4 5.0
91.2 5.1
62.5 3.9
84.7 2.4
53.2 0.7
aThe radiochemical conversion (RCC) data were calculated
using the radiochromatograms obtained by analytical
reversed-phase HPLC following decay correction.
4b with high RCC. The benzophenone imine analogs of amino 4a in THF with a 1.0 M aqueous solution of NaOH at 100 °C for
acids bearing a longer alkyl side chain 3d or aromatic ring 3e also 90 s did not give any of the desired hydrolysis product. We
reacted smoothly under the optimized conditions to give the previously reported the hydrolysis of 4c under the same
desired 11C-methylated products with high levels of reproducibility conditions except that DMSO was used as the solvent instead
and high RCCs (Table 3). These results therefore demonstrated that of THF. DMSO has been reported to increase the rate of ester
this new method involving the treatment of a mixture of Schiff- hydrolysis,21 and we therefore proceeded to investigate the
base-activated precursor and [11C]H3I with KOtBu in the presence hydrolysis of 4a in DMSO by removing the THF in vacuo
of TEMPO could be applied to the α-11C-methylation of various following the 11C-methylation reaction and replacing it with
α-amino acids.
DMSO. The resulting DMSO solution of 4a was then treated with
The hydrolysis of the ester moiety in the 11C-methylated a 1.0 M aqueous solution of NaOH at 100 °C for 90 s, which
products was explored. Surprisingly, treatment of a solution of resulted in the complete hydrolysis of the methyl ester of 4a.
J. Label Compd. Radiopharm 2015, 58 127–132
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

C. Suzuki et al.
Scheme 2. Remote-controlled synthesis of compound 5.
Subsequent removal of the benzophenone imine group by the synthesis of the desired α-11C-methyl amino acids in high RCCs.
treatment of the acid with a 2.0 M aqueous solution of HCl gave This new method for the synthesis of radiolabeled α-11C-methyl
5 with a conversion of 63.8 12.2%. In contrast to the conditions amino acids could be extended to the synthesis of many other
described earlier for the methyl ester, the tert-butyl ester and α-11C-methyl amino acids.
benzophenone imine groups of compounds 4b, 4d, and 4e were
hydrolyzed simultaneously with high RCCs when they were
heated at 100 °C for 5 min following the addition of a 2.0 M
Acknowledgements
solution of aqueous HCl to the reaction mixture of the
11C-methylation reaction. This strategy provided the desired
We would like to thank the staff of the Cyclotron Operation and
Radiochemistry sections for the production of the radioisotope,
technical support for the remote-controlled synthesis, and the
identification of compounds by mass spectral analysis. This work
was supported in part by KAKENHI 23510289 and 24591803 from
MEXT and JSPS.
hydrolyzed products 5, 6, and 7 with RCCs of 85.4 3.9, 90.7
1.7, and 63.5 2.3%, respectively, following the two steps.
Short reaction times and operationally simple procedures are
preferred for remote-controlled 11C-labeling syntheses, and the
simultaneous hydrolyses of the ester and imine groups of the
α-11C-methylated amino acid analogs under acidic conditions
therefore represent a better strategy for this approach. With this
in mind, the use of amino acid analogs protected as the
corresponding tert-butyl esters would be more appropriate for
the remote-controlled radiosynthesis of α-11C-methyl amino
acids than the methyl ester.
Conflict of Interest
The authors did not report any conflict of interest.
Having optimized the 11C-methylation and hydrolysis reactions,
the synthesis of compound 5 was selected as a suitable example
to evaluate the application of this strategy to the remote-
controlled synthesis of radiolabeled α-11C-methylated amino acids
(Scheme 2). Each step in the synthesis of compound 5 was
conducted under the conditions described earlier with only minor
modifications. Thus, the reaction time for the 11C-methylation
reaction was extended from 90 s to 3 min to allow for an increase
in the temperature of reaction mixture to 20 °C, because the
[11C]H3I was dissolved at À10 °C. Upon completion of the reaction,
the desired product 5 was purified by semipreparative HPLC. The
total time required for the synthesis 5 from EOB to formulation,
including the production of [11C]H3I and the purification of 5,
was approximately 40 min. The radioactivity of the final product
was determined to be in the range of 1.88–2.55 GBq, and
radiochemical yield was 10.3 1.2% (decay uncorrected, relative
to the calculated [11C]carbon dioxide) when the synthesis was
started from 18.5 to 22.2 GBq of [11C]carbon dioxide at EOB. After
purification, the RCP was determined to be 99.6 0.4%. The
ultraviolet absorption peak for 1a at 210 nm was not observed
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J. Label Compd. Radiopharm 2015, 58 127–132