Synthesis of L-[5-11C]Leucine and L-α-[5-11C]Methylleucine via Pd0-mediated 11C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging

Article abstract of DOI:10.1002/cmdc.202100255

The efficient synthesis of L-[5-¹¹C]leucine and L-α-[5-¹¹C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd⁰-mediated ¹¹C-methylation and microfluidic hydrogenation. The synthesis of L-[5-¹¹C]leucine and L-α-[5-¹¹C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15–38 % based on [¹¹C]CH₃I, radiochemical purity of 95–99 %, and chemical purity of 95–99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5-¹¹C]leucine or L-α-[5-¹¹C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5-¹¹C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5-¹¹C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].

Full text of DOI:10.1002/cmdc.202100255

Accepted Article
Title: Synthesis of L-[5-11C]Leucine and L-alfa-[5-11C]Methylleucine
via Pd0-mediated 11C-Methylation and Microfluidic
Hydrogenation: Potentiality of Leucine PET Probes for Tumor
Imaging
Authors: Shuhei Takataki, Tsuyoshi Tahara, Mieko Tsuji, Daiki Ozaki,
Nina Shibata, Yoshinobu Hashizume, Masaaki Suzuki,
Hirotaka Onoe, Yasuyoshi Watanabe, and Hisashi Doi
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202100255
Link to VoR: https://doi.org/10.1002/cmdc.202100255
01/2020

10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
11
11
a
Synthesis of L-[5- C]Leucine and L- -[5- C]Methylleucine via
Pd0-mediated 11C-Methylation and Microfluidic Hydrogenation:
Potentiality of Leucine PET Probes for Tumor Imaging
Shuhei Takatani,§[a,d] Tsuyoshi Tahara,§[b,d] Mieko Tsuji,§[d] Daiki Ozaki,§[d] Nina Shibata,[d] Yoshinobu
Hashizume,[e] Masaaki Suzuki,[d,f] Hirotaka Onoe,*[g] Yasuyoshi Watanabe,*[c,d] Hisashi Doi*[a,d]
[a]
Dr. S. Takatani,§ Dr. H. Doi*
Laboratory for Labeling Chemistry
RIKEN Center for Biosystems Dynamics Research
6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Email: hisashi.doi@riken.jp
[b]
[c]
Dr. T. Tahara§
Laboratory for Biofunction Dynamics Imaging
RIKEN Center for Biosystems Dynamics Research
6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Prof. Dr. Y. Watanabe*
Laboratory for Pathophysiological and Health Science
RIKEN Center for Biosystems Dynamics Research
6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Email: yywata@riken.jp
[d]
Dr. M. Tsuji,§ D. Ozaki,§ N, Shibata
Division of Bio-Function Dynamics Imaging
RIKEN Center for Life Science Technologies
6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
Dr. Y. Hashizume
RIKEN Program for Drug Discovery and Medical Technology Platforms
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Prof. Dr. M. Suzuki
[e]
[f]
National Center for Geriatrics and Gerontology
35 Gengo, Morioka, Obu, Aichi 474-8511, Japan
Prof. Dr. H. Onoe*
[g]
Human Brain Research Center
Graduate School of Medicine
Kyoto University
54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
Email: hirotaka.8n@kyoto-u.ac.jp
[§]
These authors contributed equally.
Supporting information for this article is given via a link at the end of the document.
Abstract: The efficient synthesis of L-[5-11C]leucine and L-a-[5-
11C]methylleucine has been investigated using a continuous two-
step sequence of rapid reactions consisting of Pd0-mediated 11C-
methylation and microfluidic hydrogenation. The synthesis of L-[5-
11C]leucine and L-a-[5-11C]methylleucine was accomplished within
40 min with a decay-corrected radiochemical yield of 15–38% based
on [11C]CH3I, radiochemical purity of 95–99%, and chemical purity of
95–99%. The Pd impurities in the injectable solution measured using
inductively coupled plasma mass spectrometry met the international
criteria for human use. Positron emission tomography scanning after
an intravenous injection of L-[5-11C]leucine or L-a-[5-11C]methyl
leucine in A431 tumor-bearing mice was performed. As a result, L-a-
[5-11C]methylleucine was found to be a potentially useful probe for
visualizing the tumor. Tissue distribution analysis showed that the
accumulation value of L-a-[5-11C]methylleucine in tumor tissue was
high [12 ± 3% injected dose/g tissue (%ID/g)].
of the mammalian target of rapamycin (mTOR)[2] and possibly
involved in reducing central fatigue.[3] In the field of positron
emission tomography (PET), several research groups have
reported the synthesis of 11C-labeled leucine from [11C]HCN.[4] In
particular, the method reported by Barrio et al. in 1983 has been
recognized as an early success of the routine synthesis of L-
leucine labeled with 11C at the 1-position of the carboxy carbon
([1-11C]Leu), through a continuous process utilizing a modified
Bucherer–Strecker reaction with [11C]HCN and separation of the
DL-stereoisomers by enzymatic reaction with D-amino acid
oxidase.[4b] These sophisticated synthetic methods used for the
preparation of [1-11C]Leu[4] have been adopted for animal and
clinical PET studies.[5,6] Currently, [1-11C]Leu has attracted
attention as a valuable probe for measuring the regional rate of
cerebral protein synthesis[6] and especially for visualizing
leptomeningeal angioma tumors in Sturge–Weber syndrome.[6k]
In 1987, Uppsala group reported a fundamental synthetic
method for several types of 11C-labeled amino acids by 11C-
alkylation with Schiff base, which was also applicable to labeling
11C at the 3-position of leucine.[7]
Introduction
In light of the increasing recognition of the potential value of 11C-
labeled leucine,[8] we have investigated the efficient synthesis of
leucine labeled with 11C at the 5-position, which involves
terminal 11C-labeling of the leucine carbon framework, because
the precursive [1-11C]Leu undergoes decarboxylation by in vivo
L-Leucine is one of the three branched-chain amino acids
among the nine essential amino acids. L-Leucine plays a vital
role in regulating biological functions and maintaining
homeostasis in the human body, as exemplified by it being a
major component for the biosynthesis of proteins,[1] an activator
1
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10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
metabolism to release [11C]CO2, resulting in a complicated PET
image attributed to the detection of [1-11C]Leu and [11C]CO2.[9] In
addition, our planned strategy included 11C-labeling of a-
methylleucine, an artificial amino acid, from the perspective of
the in vivo metabolic stability of a-alkylated amino acids.[10] In
this study, the synthesis of L-[5-11C]leucine ([5-11C]Leu), and L-a-
[5-11C]methylleucine ([5-11C]MeLeu) was carried out using a
continuous two-step reaction sequence consisting of a rapid
Pd0-mediated 11C-methylation using [11C]CH3I, followed by rapid
reduction utilizing H-Cube®[11], a microfluidic reactor capable of
performing relatively safe catalytic hydrogenation reactions
using hydrogen generated by electrolysis of water. Herein, we
report a practical synthetic procedure for [5-11C]Leu and [5-
11C]MeLeu, and discuss the potential of these probes for tumor
PET imaging through the results of PET studies on A431 tumor-
bearing mice.
obtained (S)-1 with 60–99% enantiomeric excess (ee). The use
of chiral HPLC separation afforded the enantiomerically pure
borane precursor, (S)-1.
O
B
O
O
Pd0-mediated
11C-methylation
11CH3
O
OBn
OBn
R
N
R
N
Cbz
Cbz
Bn
Bn
(S)-1: R = H
(S)-2: R = CH3
[
[
11C]3: R = H
11C]4: R = CH3
Cbz: carbobenzoxy, Bn: benzyl
O
5
3
11CH3
4
2
Reduction and
deprotection by
hydrogenation
1
OH
R NH2
[5-11C]Leu: R = H
[5-11C]MeLeu: R = CH3
Results and Discussion
Scheme
1 outlines our planned synthetic strategy using
Scheme 1. Synthetic strategy for L-[5-11C]leucine ([5-11C]Leu) and L-a-[5-
[11C]CH3I, a widely used 11C-labeling reagent. With the aim of
efficiently synthesizing [5-11C]Leu and [5-11C]MeLeu in
consideration of the short half-life of 11C (20.4 min), we focused
on two-step continuous rapid reactions as follows: (1) Pd0-
mediated 11C-methylation onto an alkene framework using
11C]methylleucine ([5-11C]MeLeu).
[11C]CH3I
and
alkenylborane,[12]
and
(2)
microfluidic
O
O
(a)
hydrogenation. During the hydrogenation step, we accomplished
two reactions at once, namely, the reduction of the alkene and
deprotection of the carbobenzoxy (Cbz) and benzyl (Bn) groups.
Pd0-mediated 11C-methylation[13] and microfluidic hydrogenation
are generally recognized as fast reactions with high reaction
efficiencies, and each reaction can therefore be expected to be
completed within a short reaction time (5 min). From a practical
viewpoint, one-step synthesis by cross-coupling between
[11C]CH3I and an alkylborane substrate (5-demethyl-5-
pinacolborylleucine) would be ideal for the introduction of the
[11C]CH3 group into the terminal alkyl chain of leucine. Even
though some sophisticated C(sp3)–C(sp3) couplings using
[11C]CH3I have been reported,[14] it remains difficult to
substantially overcome the problem of β-hydrogen elimination in
Pd chemistry, which generates unwanted alkenyl by-products.
Therefore, we considered that the two-step continuous reaction
of 11C-methylation onto an alkene, followed by reduction of the
alkene (Scheme 1) would be better and more practical for 11C-
radiolabeling leucines.
OH
OH
R NH
R NH2
Cbz
5: R = H
6: R = CH3
(S)-α-propargylgycine: R = H
(S)-α-propargylalanine: R = CH3
O
(b)
(c)
(S)-1 or (S)-2
OBn
R N
Regarding the benzylation
of 5, epimerization was
occurred partly.
(S)-1 was purified
by chiral HPLC.
Cbz Bn
7: R = H
8: R = CH3
Scheme 2. Preparation of organoborane precursors (S)-1 and (S)-2. Reaction
conditions: (a) benzyl chloroformate, NaHCO3, Na2CO3, H2O, acetone, 17 h,
0 °C to rt [(5) 86%, (6) 65%]; (b) NaH, benzyl bromide, tetrabutylammonium
iodide, DMF, 1 h, 0 °C then overnight, rt, under Ar [(7) 46%, (8) 41%]; (c)
bis(pinacolato)diborane, CuCl, KOAc, LiCl, DMF, overnight, rt, under Ar, [(S)-1
58%, (S)-2, 53%].
The organoborane precursors [(S)-1 and (S)-2] for Pd0-mediated
rapid 11C-methylation were prepared starting from (S)-a-
propargylglycine and (S)-a-propargylalanine, respectively
As shown in Figure 1, we adopted H-Cube® as a commercially
available microfluidic hydrogenation system and built it into our
existing 11C-radiolabeling system. Pd0-mediated 11C-methylation
(Scheme 2). Initially, Cbz was introduced as
a first-line
protecting group to protect the reactive primary amine to yield 5
and 6, respectively, and then, both the Cbz-NH and the
carboxylic acid were protected by the Bn group. Protection of the
primary amine and carboxylic acid is necessary to ensure a
smooth key reaction of Pd0-mediated rapid 11C-methylation. The
copper(I)-catalyzed borylation of terminal alkynes 7 or 8 with
bis(pinacolato)diborane[16] afforded the desired internal
pinacolborane-addition products (S)-1 and (S)-2 in moderate
yields (58% and 53%, respectively).Unfortunately, epimerization
often occurred during the synthesis of (S)-1, which has a-
hydrogen atom. For example, during the preparation of (S)-1, we
using (S)-1 was carried out at 75 °C for 5 min in the absence of
[17]
K2CO3
to avoid epimerization at the a-carbon of the amino
acid structure. Subsequently, the reaction mixture was injected
into the H-Cube® system under the conditions of 10% Pd/C
cartridge (30 mm), ethanol flow of 1.0 mL/min, and H2 gas flow
of 25 mL/min, giving the desired 11C-labeled L-leucine ([5-
11C]Leu) with radioactivity of 2.2–2.7 GBq and decay-corrected
radiochemical
radioactivity of 25–30 GBq (Figure 2).
yield of 23–38% based on [11C]CH3I with
2
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10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
®
(3) H-Cube
RI detector
RI/UV detector
waste
Bubble detector
10% Pd/C
Ar, He
30 mm CatCart®
(1)
AscariteꢀP4O10
Ar, He
waste
filter
11CO2
Mixer of
substrate and H2
H2O
or Saline
Heater unit
HPLC
column
N2
H2 gas
generator
Ar, He
(2)
Vacuum
MSC
(4)
Sterile filter
HI
Ethanol
(5)
H-Cube pump
Pd/C : palladium on activated carbon
CatCart®: catalyst cartridge
(6) Product
HPLC pump
MSC: metal scavenger cartridge
Figure 1. Flowchart of the synthesis of [5-11C]Leu and [5-11C]MeLeu. (1) Preparation of [11C]CH3I, (2) Pd0-mediated rapid 11C-methylation using (S)-1 or (S)-2, (3)
catalytic reduction of [11C]3 or [11C]4 using the H-Cube® system, (4) HPLC purification, (5) concentration under reduced pressure, and (6) radiopharmaceutical
formulation.
11C]Leu and [5-11C]MeLeu were confirmed by co-injection with
their corresponding reference compounds, L-leucine or L-
Radiation
Intensity
methylleucine, using analytical HPLC equipped with a Corona
charged aerosol detector (CAD).
[5-11C]Leu
Radiation intensity
Current intensity
(a)
(b)
[5-11C]Leu
L-leucine
D-leucine
Figure 2. Semi-preparative HPLC chromatogram of the synthesized [5-
11C]Leu.
Figure 3. (a) Chiral HPLC analysis of synthesized [5-11C]Leu using
enantiomerically pure alkenylborane substrate (S)-1 with 99% ee. The
production of its isomer, D-[5-11C]leucine, was not observed. (b) Chiral HPLC
analysis of a mixture of unradiolabeled L-leucine and D-leucine using a Corona
charged aerosol detector. HPLC conditions for (a) and (b): Guard column:
Astec CHIROBIOTIC T, 4.6 mm × 20 mm, 5 µm; column: Astec CHIROBIOTIC,
4.6 mm × 250 mm, 5 µm; mobile phase, acetonitrile/H2O = 50:50; flow rate, 1
mL/min; column temperature, 25 ºC; detectors: Corona charged aerosol
detector and radio-detector; retention time of [5-11C]Leu = 5.0 min.
After the radiopharmaceutical formulation of [5-11C]Leu, the
molar activity was calculated to be 150–200 GBq/µmol (LC-MS
was used for this measurement because the small amount of [5-
11C]Leu cannot be detected using conventional UV-HPLC
methods owing to its weak UV absorption). The radiochemical
and chemical purities were both >95%. The total synthesis time,
including HPLC purification and radiopharmaceutical formulation,
was 38–40 min. When we used (S)-1 with 99%ee as the borane
precursor, we obtained [5-11C]Leu with 99%ee after Pd0-
mediated rapid 11C-methylation and microfluidic hydrogenation
(Figure 3). Thus, we confirmed that Pd0-mediated rapid 11C-
methylation did not cause epimerization during the synthesis.
Similarly, [5-11C]MeLeu was synthesized by the above
mentioned two-step continuous rapid reaction (Figure 4). In this
case, the Pd0-mediated 11C-methylation of (S)-2 was carried out
under standard conditions in the presence of K2CO3[17] because,
in principle, epimerization does not occur at the a-carbon as the
quaternary carbon center. After radiopharmaceutical formulation,
the radioactivity of [5-11C]MeLeu was 0.6–1.2 GBq and the
[11C]CH3I-based decay-corrected radiochemical yield was
calculated to be 6–15%. The molar activity was approximately
60–180 GBq/µmol (LC-MS measurement). The total synthesis
was completed within 40 min and the radiochemical and
chemical purities were both >99%. For product identification, [5-
Radiation
Intensity
[5-11C]MeLeu
Figure 4. Semi-preparative HPLC chromatogram of the synthesized [5-
11C]MeLe.
3
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[11C]Met
a-1
[5-11C]Leu
b-1
[5-11C]MeLeu
c-1
SUV
4.0
a-2
b-2
c-2
0.2
Figure 5. PET summation images of A431 tumor-bearing mice between 30 and 60 min using [11C]Met (a-1: coronal, a-2: sagittal), [5-11C]Leu (b-1: coronal, b-2:
sagittal), and [5-11C]MeLeu (c-1: coronal, c-2: sagittal). The white arrows indicate the A431 tumor tissue; SUV: standardized uptake value (an index of the
accumulation level of radioactivity).[†]
†
The SUV was calculated using the following equation: SUV = r/(a/w) [g/mL], where r is the radioactivity concentration (Bq/mL) measured by the PET scanner
within the region of interest, a is the radioactivity of the injected PET probe [Bq], and w is the weight of the animal [g]. Most of the body has a density close to 1.0
and the SUV is normally regarded as unitless.
[5-11C]Leu and [5-11C]MeLeu were dissolved in saline and then
intravenously injected into the tail vein of A431 tumor-bearing
mice. Figure 5 shows the summation PET images of the whole
body 30–60 min after injection with [5-11C]Leu (injected
radioactivity, 15 MBq; molar activity, 37 GBq/µmol; volume 0.1
mL, at the time of injection) and [5-11C]MeLeu (injected
radioactivity, 15 MBq; molar activity, 76 GBq/µmol; volume, 0.1
mL, at the time of injection). In this study, L-[methyl-
11C]methionine ([11C]Met),[18] a representative amino acid PET
probe for tumor imaging, was used for comparison with [5-
11C]Leu and [5-11C]MeLeu.
The tissue biodistribution analyses of A431 tumor-bearing mice
using PET probes are shown in Figure 7. At 60 min after the
administration of each PET probe, the mice were sacrificed and
collected the blood, brain, heart, liver, kidney, pancreas, tumor,
and other major tissues, followed by measurement of the
remaining radioactivity in each of these tissues. The graphs in
Figure 7 show the radioactivity uptake rate of [11C]Met, [5-
11C]Leu, and [5-11C]MeLeu in the major tissues with the data
expressed as the percentage of injected radioactivity per gram
of tissue (%ID/g).
%ID/g
From the PET images, it was notable that the A431 tumor tissue
was more clearly visible using [5-11C]MeLeu when compared to
[11C]Met or [5-11C]Leu (Figure 5). The time–activity curves of
these three PET probes over 60 min are shown in Figure 6. The
tumor uptake of [11C]Met and [5-11C]Leu reached high
accumulation 6 and 8 min after intravenous administration, and
then gradually decreased to a plateau between 8 and 60 min,
respectively. In contrast, the tumor accumulation of [5-
11C]MeLeu continuously increased over 40 min.
40
35
30
25
20
15
10
5
[5-11C]Leu
[5-11C]MeLeu
[
11C]Met
0
r
y
e
l
s
rt
in
Bra He
e
a
c
ood
ent
num
Liv
Lung
bl
Ovary
Spleen
Kidne
Mus
Pancreas
Plasma
e
Duode
Whol
inal cont
est
Int
%ID/g
400
%ID/g
20
ꢀ
ꢀꢀ
15
10
5
300
200
100
0
0
Urine
Tumor
(A431)
0
10
20
30
40
50
60
Time (min)
Figure 7. Biodistribution analysis of [11C]Met (light blue), [5-11C]Leu (green),
and [5-11C]MeLeu (red) in A431 tumor-bearing mice at 60 min after the
injection (n = 5–6). The radioactivity in each tissue was measured using a g-
well counter. The asterisks represent the values that are statistically different
from the corresponding group (*p <0.05, **p <0.01).
Figure 6. Time–activity curves of [11C]Met (light blue), [5-11C]Leu (green), and
[5-11C]MeLeu (red) accumulation in the tumor region of A431 tumor-bearing
mice during the PET scan.
4
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These three PET probes were mainly accumulated in the
pancreas and A431 tumor tissue, where L-type amino acid
transporter 1 is considered to be highly expressed.[19] According
to a previous report, L-a-methylleucine has a low affinity for L-
type amino acid transporter 1.[20] Regarding this issue of probe
accumulation via amino acid transporters, further biological
study is needed. Interestingly, the accumulation of [5-11C]MeLeu
(12 ± 3%ID/g) in the tumor tissue was approximately two-fold
higher than that of [11C]Met (6 ± 2%ID/g). However, the
accumulation of [5-11C]Leu was less than that of [11C]Met in the
tumor tissue. In particular, the [5-11C]MeLeu values observed in
the liver, spleen, duodenum, and intestinal contents were lower
than those of [11C]Met and the accumulation in the liver was the
lowest. On the other hand, [5-11C]MeLeu was more abundant in
urine than [11C]Met and [5-11C]Leu. These results indicate that
biliary excretion is one of the metabolic pathways for [11C]Met
and [5-11C]Leu, but [5-11C]MeLeu is hard to undergo biliary
excretion and resulted in renal excretion as another different
metabolic pathway. These results could be associated with that
as an artificial amino acid, a-methylleucine has a tendency to be
hardly metabolized in the liver.[10] In addition, the rapid excretion
of [5-11C]MeLeu in urine lowers the amount of [5-11C]MeLeu in
the blood, which may result in clearer PET imaging with a high
S/N ratio not including [5-11C]MeLeu in blood.
imaging of the pancreas. In regarding PET probes of leucine
derivatives, it is of interest to point out that several types of 18F-
labeled leucine derivatives as artificial amino acids have been
recently developed and the possibilities of tumor PET imaging
investigated.[21]
In addition, the Pd impurities in the injection solution of [5-
11C]Leu or [5-11C]MeLeu were confirmed to be ~12 ppb (12 µg/L)
via inductively coupled plasma mass spectrometry (ICP-MS). In
particular, the radioactivity necessary for clinical PET studies
using [5-11C]Leu or [5-11C]MeLeu is estimated to be 150–250
MBq per person. Therefore, the amount of Pd contamination in
the formulated injectable solution corresponds to 10–20 ng,
which safely meets the international criteria for Pd impurities for
human use (permitted daily exposure of Pd in the case of
parenteral injection is 10 µg/d).[22,23] For the purpose of
developing PET radiopharmaceuticals, our synthetic protocols
for 11C-radiolabeling of leucine and its derivatives are essentially
applicable to human PET studies.
Conclusion
We have developed an efficient procedure to synthesize [5-
11C]Leu and [5-11C]MeLeu by Pd0-mediated rapid 11C-
methylation and rapid hydrogenation utilizing a H-Cube® system.
This method not only provides the chemical synthesis of
[11C]leucine but also new 11C-radiolabeling chemistry for
branched alkyl chains, such as isopropyl and sec-butyl groups.
From our PET imaging and biodistribution studies using A431
tumor-bearing mice, [5-11C]MeLeu, as an a-methyl-introduced
artificial amino acid, was confirmed to be a superior potential
probe for tumor imaging compared with [5-11C]Leu and [11C]Met.
Since the Pd contamination value in the injectable solutions of
[11C]leucine met the international criteria for human use, we plan
to perform clinical PET studies in the near future. In addition, we
will investigate other possibilities for D-form probes of 11C-
labeled leucines[24] because D-leucine currently draws clinical
attention from the viewpoint of epilepsy treatment.[25] Further
biological and medicinal studies will be reported in due course.
Figure 8A shows the uptake ratios of the tumor tissue to muscle,
blood, and liver. The ratios of tumor tissue to muscle and blood
of [5-11C]MeLeu were slightly lower than those of [11C]Met. It was
notable that there was a significant difference in the ratio of
tumor tissue to liver between [5-11C]MeLeu and the other probes.
(A)
%ID/g
%ID/g
%ID/g
10
8
10
8
10
8
ꢀ
ꢀꢀ
ꢀꢀ
6
6
6
4
4
4
2
2
2
0
0
0
Tumor/Muscle ratio
Tumor/Blood ratio
Tumor/Liver ratio
[
11C]Met
[5-11C]Leu
[5-11C]MeLeu
(B)
%ID/g
%ID/g
%ID/g
25
20
15
10
5
40
30
20
10
0
40
30
20
10
0
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
Experimental Section
ꢀ
General methods for organic chemistry and PET radiochemistry.
0
1H and 13C NMR spectra were recorded on
a JNM-ECS 400SS
Pancreas/Muscle
ratio
Pancreas/Blood
ratio
Pancreas/Liver
ratio
instrument (JEOL). The chemical shifts in deuterated chloroform and
dimethyl sulfoxide (CDCl3 and DMSO-d6) are given as δ values using
tetramethyl silane as an internal standard. Data were reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad), integration, and coupling constants. High-resolution
mass spectra (HRMS) were measured on an ExactiveTM Plus Orbitrap
mass spectrometer (Thermo Fisher Scientific). Optical rotations were
measured with a JASCO P-2100 polarimeter. All chemicals and solvents
were used as received from commercial sources without further
purification. The room temperature described in this text was
approximately 25 °C. The synthetic procedure and 1H NMR spectra for
compound 5 have been reported in the Ph.D. thesis of B. H. M.
Kuijpers.[26] In the present paper, the full instrumental analysis data of
compound 5 (1H NMR, 13C{1H} NMR, [1H,1H] 2D NMR, and high-
resolution mass spectrum) are summarized in the Supporting Information.
Medium-pressure liquid chromatography (MPLC) was performed on a Hi-
Flash column (YAMAZEN) with EPCLC-AI-580S (YAMAZEN). Semi-
[
11C]Met
[5-11C]Leu
[5-11C]MeLeu
Figure 8. Uptake ratio of
11C]MeLeu (red) in A431 tumor-bearing mice: (A) tumor/muscle, tumor/blood,
and tumor/liver; (B) pancreas/muscle, pancreas/blood, and pancreas/liver. The
asterisks represent the values that are statistically different from the
corresponding group (*p <0.05, **p <0.01).
[
11C]Met (light blue), [5-11C]Leu (green), and
[
Figure 8B shows the uptake ratios of the pancreas to the
muscle, blood, and liver. It is of great interest that the uptake
ratio of pancreas to liver of [5-11C]MeLeu was several-fold higher
than those of the other probes. Therefore, the PET images and
biodistribution analyses confirm that [5-11C]MeLeu was not only
a potential probe for tumor PET imaging, but also useful for
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
preparative purification and purity analysis by HPLC was performed
using a CBM-20A communication bus module (Shimadzu), LC-20AB
liquid chromatograph (Shimadzu), CTO-20AC column oven (Shimadzu),
SPD-M20A UV/VIS detector (Shimadzu), Corona CAD (ESA
Biosciences), and RLC-700 radio analyzer (Aloka). The molar amount of
the 11C-radiolabeled probe was determined from the mass intensity
calibration curve on a QTRAP4500 (AB SCIEX). [11C]Carbon dioxide was
produced by a 14N(p,a)11C nuclear reaction using a Sumitomo CYPRIS
HM-12S cyclotron (Sumitomo Heavy Industries) and then converted into
(4.5H, m), 7.09–7.32 (15H, m); 13C NMR (100 MHz, CDCl3; compound
exists as a 1:1 mixture of rotamers) δ 19.6, 20.4, 52.2, 52.6, 58.2, 59.2,
67.1, 67.3, 67.6, 67.7, 70.6, 70.8, 80.4, 80.8, 127.4, 127.6, 127.8, 128.0,
128.2, 128.3, 128.4, 128.5, 128.8, 135.0, 135.3, 135.9, 136.2, 136.7,
137.0, 155.8, 169.3; HRMS-ESI (m/z): [M
C27H25NNaO4, 450.1676; found, 450.1676.
+
Na]+ calculated for
Benzyl
(S)-2-{benzyl[(benzyloxy)carbonyl]amino}-2-methyl-4-
pentynoate (8).
[
11C]methyl iodide by reduction of 11C]carbon dioxide with lithium
[
aluminum hydride and iodination with hydroiodic acid using a RIKEN
original 11C-radiolabeling system. The microfluidic hydrogenation reaction
was performed on an H-Cube® HC 2-ST 1 (ThalesNano) with a 10%
Pd/C 30 mm CatCart® (ThalesNano) as the catalyst cartridge. A metal-
scavenger material (SiliaMetS® Thiol) was used to eliminate the Pd. The
amount of Pd impurities contained in the injectable solution was
measured by ICP-MS (Agilent 7700x, Agilent Technologies).
The synthetic procedure for compound 8 was similar to that described for
compound 7. Compound 8 was a light-yellow oil (512 mg, 41%).
Characterization data for compound 8: Rf = 0.32 (hexane/ethyl acetate
5/1); 1H NMR (400 MHz, DMSO-d6) δ 1.42 (3H, s), 2.66 (1H, dd, J = 16.4
Hz, 2.7 Hz), 2.98 (1H, t, J = 2.7 Hz), 3.11 (1H, d, J = 16.4 Hz), 4.55 (1H,
d, J = 17.8 Hz), 4.78 (1H, d, J = 17.8 Hz), 5.00–5.12 (2H, m), 5.01 (1H, d,
J = 12.8 Hz), 5.10 (1H, d, J = 12.8 Hz), 7.12–7.36 (15H, m); 13C NMR
(100 MHz, DMSO-d6) δ 21.3, 25.7, 30.7, 47.6, 62.8, 66.7, 74.1, 79.7,
126.1, 126.4, 126.5, 126.9, 127.2, 127.7, 128.0, 128.1, 128.1, 128.2,
128.3, 128.4, 135.5, 136.2, 139.4, 155.5, 172.2; HRMS-ESI (m/z): [M +
Na]+ calculated for C28H28NNaO4, 464.1832; found, 464.1835.
Please see the supporting information for characterization data for
compounds (S)-1, (S)-2, 5, 6, 7, and 8 (1H NMR, 13C{1H} NMR, [1H,1H]
2D NMR, HRMS, and enantiomeric excess analysis), analysis of the
chemical and radiochemical purity, molar activity measurement of L-[5-
11C]leucine and L-a-[5-11C]methylleucine, and measurement of the Pd
impurities in the injection solution.
Benzyl
(S)-2-{benzyl[(benzyloxy)carbonyl]amino}-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-4-pentynoate ((S)-1).
Preparation of organoborane precursors (S)-1 and (S)-2.
A mixture of CuCl (255 mg, 2.57 mmol) and LiCl (109 mg, 2.57 mmol) in
DMF (8 mL) was stirred at room temperature for 1 h under Ar. To this
mixture, a mixture of 7 (1.0 g, 2.34 mmol), bis(pinacolato)diborane (653
mg, 2.57 mmol), and KOAc (253 mg, 2.57 mmol) in DMF (2 mL) was
added. The reaction mixture was stirred at room temperature overnight
under Ar and H2O (20 mL) was added. The resulting solution was
extracted with ethyl acetate (100 mL) and washed with brine (3 × 20 mL).
The organic layer was dried over Na2SO4, the solvent removed in vacuo,
and the crude product purified by MPLC (hexane/ethyl acetate, 100/0 to
80/20) to afford compound (S)-1 as a light yellow oil (758 mg, 58%). The
optical resolution of compound (S)-1 was determined using preparative
HPLC on a chiral stationary-phase column (Column: Daicel Chiralpak IC,
50 × 250 mm; mobile phase, hexane/2-propanol = 90/10; flow rate, 35.4
mL/min; UV detection at 254 nm; column temperature, 40 °C, retention
times, (S)-1 = 24.3 min and (R)-1 = 29.3 min.).
(S)-2-{[(benzyloxy)carbonyl]amino}-2-methyl-4-pentynoic acid (6).
To a solution of (S)-α-propargylalanine (1.0 g, 7.87 mmol, >98.0% ee),
an aqueous solution (30 mL) of NaHCO3 (655.7 mg, 7.87 mmol) and
Na2CO3 (1.67 g, 15.73 mmol), acetone (4.0 mL), and benzyl
chloroformate (1.6 mL, 11.1 mmol) were added slowly at 0 ºC. The
reaction mixture was stirred at room temperature for 17 h. The mixture
was washed with diethyl ether (3 × 10 mL) and the aqueous layer was
adjusted to pH ~2 using 6 N HCl. The resulting solution was extracted
with ethyl acetate (40 mL) and washed with water (3 × 10 mL). The
organic layer was dried over Na2SO and the solvent was removed in
vacuo to afford compound 6 as a colorless oil (752 mg, 65%).
Characterization data for compound 6: 1H NMR (400 MHz, DMSO-d6): δ
1.37 (3H, s), 2.57 (1H, dd, J = 17.3 Hz, 2.5 Hz), 2.86 (1H, t, J = 2.5 Hz),
2.96 (1H, d, J = 17.3 Hz), 3.34 (1H, s), 5.02 (2H, dd, J = 13.6 Hz, 13.6
Hz), 7.27–7.37 (5H, m), 7.72 (1H, s); 13C NMR (100 MHz, DMSO-d6): δ
22.7, 25.9, 57.1, 65.1, 73.4, 80.4, 127.6, 127.8, 128.3, 137.0, 154.9,
Characterization data for compound (S)-1: Rf = 0.30 (hexane/ethyl
acetate 5/1); 1H NMR (400 MHz, CDCl3; compound exists as a 1:1
mixture of rotamers) δ 1.22 (12H, s), 2.72‒2.92 (2H, m), 4.08‒4.20 (1H,
m), 4.37 (0.5H, dd, J = 9.2, 5.8 Hz), 4.61 (0.5H, dd, J = 9.2, 5.6 Hz), 4.70
(1H, d, J = 13.2 Hz), 4.81 (1H, d, J = 12.4 Hz), 4.89–5.23 (3H, m), 5.53
(0.5H, br s), 5.61 (0.5H, br s), 5.84 (0.5H, s), 5.85 (0.5H, s), 7.08-7.30
(15H, m); 13C NMR (100 MHz, CDCl3; compound exists as a 1:1 mixture
of rotamers) δ 24.8, 24.9, 35.6, 36.3, 51.3, 52.2, 59.6, 60.0, 66.7, 66.9,
67.4, 67.6, 83.7, 127.1, 127.3, 127.8, 127.88, 127.94, 128.1, 128.2,
128.3, 128.47, 128.52, 128.6, 133.2, 133.5, 135.5, 135.8, 136.4, 136.6,
137.5, 137.9, 156.1, 156.4, 170.9, 171.1 (the signal for the carbon atom
directly attached to the boron atom was not observed); HRMS-ESI (m/z):
[M + Na]+ calculated for C33H38BNNaO6, 578.2684; found, 578.2684. The
enantiomeric excess was determined by HPLC analysis using a chiral
stationary phase column (Column: Daicel Chiralpak IC, 4.6 × 250 mm;
mobile phase, hexane/2-propanol = 90/10; flow rate, 1.0 mL/min; UV
detection at 254 nm; retention times, (S)-1 7.8 min and (R)-1 9.4 min) to
be 99%ee. [a]2D4 −58.8 (c 0.52, CHCl3).
174.4; HRMS-ESI (m/z): [M
+
Na]+ calculated for C14H16NNaO4,
284.0893; found, 284.0892.
Benzyl (S)-2-{benzyl[(benzyloxy)carbonyl]amino}-4-pentynoate (7).
To a solution of NaH (60%, dispersion in paraffin liquid; 640 mg, 16
mmol) was added a solution of 5 (2.0 g, 8.1 mmol) in DMF (20 mL) and
the resulting mixture stirred at 0 °C for 1 h under Ar. To the mixture,
benzyl bromide (10 mL, 81 mmol) and a solution of tetrabutylammonium
iodide (299 mg, 0.81 mmol) in DMF (3 mL) were added at 0 ºC. The
reaction mixture was stirred at room temperature overnight under Ar and
quenched with H2O (30 mL) at 0 °C. The resulting solution was extracted
with ethyl acetate (150 mL) and washed with brine (3 × 30 mL). The
organic layer was dried over Na2SO4 and the solvent was removed in
vacuo and the crude product purified by MPLC (hexane/ethyl acetate,
100/0 to 80/20) to afford compound 7 as a colorless oil (1.6 g, 46%).
Benzyl
(S)-2-{benzyl[(benzyloxy)carbonyl]amino}-2-methyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4-pentynoate ((S)-2).
Characterization data for compound 7: Rf = 0.30 (hexane/ethyl acetate
5/1); 1H NMR (400 MHz, CDCl3; compound exists as a 1:1 mixture of
rotamers) δ 1.97 (1H, t, J = 2.6 Hz), 2.80–2.97 (2H, m), 4.18 (0.5H, dd, J
= 9.2, 5.2 Hz), 4.29 (0.5H, dd, J = 9.2, 6.0 Hz), 4.45 (0.5H, d, J = 15.2
Hz), 4.46 (0.5H, d, J = 16.4 Hz), 4.70 (0.5H, d, J = 12.4 Hz), 4.94–5.23
The synthetic procedure for compound (S)-2 was similar to that
described for compound (S)-1. Purification was performed using MPLC
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
(hexane/ethyl acetate, 100/0 to 50/50). Compound (S)-2 was a colorless
oil (337 mg, 53%).
impurities and sterilization, [5-11C]MeLeu was dissolved in distilled water
(2.0 mL) or saline (2.0 mL) and then collected in a sterile vial. The pH of
the solution was 7 and the final sample volume was 2.5 mL. The total
synthesis time, starting from the trapping of [11C]CO2 for preparation of
Characterization data for compound (S)-2: Rf = 0.22 (hexane/ethyl
acetate 5/1); 1H NMR (400 MHz, DMSO-d6) δ 1.18 (12H, s), 1.22 (3H, s),
2.43 (1H, br s), 3.03 (1H, br s), 4.12 (1H, d, J = 14.4 Hz), 4.62–4.99 (3H,
m), 4.98 (1H, d, J = 12.6 Hz), 5.11 (1H, d, J = 12.6 Hz), 5.55 (1H, br s),
5.90 (1H, d, J = 2.4 Hz), 7.07–7.35 (15H, m); 13C NMR (100 MHz,
DMSO-d6) δ 21.4, 24.9, 25.3, 48.1, 64.8, 67.0, 84.0, 126.6, 126.8, 127.8,
128,2, 128.3, 128.5, 128.6, 128.8, 128.9, 136.3, 136.7, 136.9, 140.1,
155.9, 173.6 (the signal for the carbon atom directly attached to the
boron atom was not observed); HRMS-ESI (m/z): [M + Na]+ calculated for
C34H41BNNaO6, 592.2841; found, 592.2841. The enantiomeric excess
was determined by HPLC analysis using a chiral stationary phase
column (Column: Daicel Chiralpak IC, 4.6 × 250 mm; mobile phase,
hexane/CH2Cl2 = 25/75; flow rate, 1.0 mL/min; UV detection at 254 nm;
retention times (S)-2 7.3 min and (R)-2 8.9 min) to be 99% ee. [a]2D4 −62.6
(c 0.50, CHCl3).
[
[
11C]CH3I, was within 40 min. The radioactivity, molar activity, and
11C]CH3I-based decay-corrected conversion yield of [5-11C]MeLeu was
0.6–1.2 GBq, 60–180 GBq/µmol, and 6–15%, respectively. The
radiochemical and chemical purities were both >99%. The chemical
identity of 5-[11C]MeLeu was confirmed by co-injection with reference in
analytical HPLC (Guard column: Astec CHIROBIOTIC T, 4.0 mm × 20
mm, 5 µm; column: Astec CHIROBIOTIC T, 4.6 mm × 250 mm, 5 µm;
mobile phase, methanol only; flow rate, 1.0 mL/min; column temperature,
25 °C; detectors Corona CAD and RI detector; retention time, 6.8 min).
General biological procedures.
All experimental protocols were approved by the Animal Care and Use
Committee of RIKEN Kobe Institute (MAH24-03) and were performed
according to the guidelines for animal experiments published by the
National Institutes of Health.[27] Human epidermoid carcinoma A431 cells
were grown in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum and 100 μg/mL penicillin-streptomycin. Five-
week-old female BALB/c-nu/nu mice were obtained from CLEA Japan
(Tokyo, Japan) and maintained for 1 week prior to the experiment. To
establish tumor-bearing mice, A431 cells (5 × 106/100 μL) were injected
subcutaneously into the right shoulder of mice 2 weeks before the PET
scans. Mice were used for PET experiments when the tumor had grown
to ~5–10 mm in diameter.
11C-Radiolabeling.
Synthesis of [5-11C]Leu.
[
11C]CH3I was trapped in a mixed solution of (S)-1 (6.0 mg, 10.8 µmol),
tris(dibenzylideneacetone)dipalladium(0) (3.0 mg, 3.3 µmol), and tri(o-
tolyl)phosphine (4 mg, 13.1 µmol) in DMF (360 µL) and H2O (40 µL) at
room temperature. The mixture was heated to 75 °C for 5 min and then
diluted with ethanol (1.0 mL). The resulting mixture was injected into the
H-Cube® system (catalyst cartridge, 10% Pd/C 30 mm CatCart®; mobile
phase, ethanol; flow rate, 1 mL/min; reaction temperature 75 °C; H2
generation operation in Full H2 mode). After passing through the flow
reactor, the reaction mixture was collected and purified by semi-
preparative HPLC (column: Asahipak NH2P-90 20F, 20 mm × 300 mm, 9
µm; mobile phase, acetonitrile/10 mM CH3COONH4 = 60/40 to 50/50 for
20 min; flow rate, 8 mL/min; UV detection at 254 nm; retention time, 11–
15 min). The fraction of interest was collected in a flask and concentrated
in vacuo. In accordance with the radiopharmaceutical formulation,
including filtration to scavenge the Pd impurities and sterilization, [5-
11C]Leu was dissolved in distilled H2O (2.0 mL) or saline (2.0 mL) and
then collected in a sterile vial. The pH of the solution was 7 and the final
sample volume was 2.5 mL. The total synthesis time, starting from the
trapping of [11C]CO2 for preparation of [11C]CH3I, was approximately 38
MicroPET imaging.
A small-animal PET scanner (microPET Focus220, Siemens Medical
Systems, Erlangen, Germany) was used for imaging studies of the mice.
Before the PET scanning, a catheter was inserted into the tail vein for
probe injection and the animals were kept on a heating pad to maintain a
body temperature of 37 °C. The probes ([5-11C]Leu, [5-11C]MeLeu, and
[
11C]Met) at a concentration of 15 MBq/0.1 mL were injected under 1.5%
isoflurane anesthesia and dynamic PET scans (6 × 10 s, 6 × 30 s, 11 ×
60 s, and 15 × 180 s) were performed during a 60 min period
immediately after the probe injection. Emission data were acquired using
a 3D list-mode method and PET images were reconstructed using the
filtered back-projection algorithm. Maximum intensity projection images
were displayed using the ASIPro software (Concorde Microsystems,
Knoxville, USA). The uptake of the probes in tissues was displayed as
the standardized uptake value (SUV).
min. The radioactivity, molar activity, and
[
11C]CH3I-based decay-
corrected radiochemical yield of [5-11C]Lue was 2.2–2.7 GBq, 150–200
GBq/µmol, and 23–38%, respectively and the radiochemical and
chemical purities were both >95%. The chemical identity of [5-11C]Leu
was identified by HPLC analysis using the co-injection of the unlabeled L-
leucine as a reference (Guard column: Astec CHIROBIOTIC T, 4.0 mm ×
20 mm, 5 µm; column: Astec CHIROBIOTIC T, 4.6 mm × 250 mm, 5 µm;
mobile phase acetonitrile/H2O = 50/50; flow rate, 1.0 mL/min; column
temperature, 25 °C; detectors: Corona CAD and refractive index (RI)
detector; retention time, 5.0 min).
Biodistribution study.
A concentration of 15 MBq of probes in 100 μL of saline was injected into
tumor-bearing mice via the tail vein. The mice were sacrificed under deep
anesthesia with 3.0% isoflurane at 60 min after the injection, and the
blood, urine, brain, heart, lung, liver, kidney, spleen, pancreas,
quadriceps muscle, ovary, duodenum, intestinal contents, and tumors
were then collected, and their volumes and weights were promptly
measured. The radioactivity in the various tissues, whole blood, plasma,
and urine samples were measured using a g-well counter (Wizard 1480;
PerkinElmer, Waltham, USA). The results were expressed as the %ID/g.
Synthesis of [5-11C]MeLeu.
[
11C]CH3I was trapped in a solution of (S)-2 (6.0 mg, 10.8 µmol), K2CO3
(4.0 mg, 28.9 µmol), tris(dibenzylideneacetone)dipalladium(0) (3.0 mg,
3.3 µmol), and tri(o-tolyl)phosphine (4 mg, 13.1 µmol) in DMF/H2O
(360/40 µL) at room temperature. The mixture was heated to 110 °C for 5
min and then diluted with ethanol (1.0 mL). The resulting mixture was
injected and passed through an H-Cube® system (catalyst cartridge: 10%
Pd/C 30 mm CatCart®; mobile phase ethanol, flow rate 1 mL/min,
reaction temperature 75 °C, H2 generation operation in Full H2 mode).
After completion, the reaction mixture was purified by HPLC (Asahipak
NH2P-90 20F, 20 mm × 300 mm, 9 µm; mobile phase, acetonitrile/10
mM CH3COONH4 = 70/30 to 60/40 for 20 min; flow rate, 8 mL/min; UV
detection at 254 nm; retention time, 15–19 min). In accordance with the
radiopharmaceutical formulation, including filtration to scavenge the Pd
Statistical analysis.
Statistical analysis was performed using SPSS software (Windows
software, version 21, SPSS Inc.). All data are presented as the mean
±SD. The differences between each group of [11C]Met, [5-11C]Leu, and
[5-11C]MeLeu with respect to the radiotracer uptake in A431 tumor tissue
(Figure 7 and 8) were analyzed using an unpaired Student’s t-test with
adjustments by the Bonferroni method for multiple comparisons. The
statistical significance was set at p <0.05.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100255
ChemMedChem
FULL PAPER
Tomasi, M. Veronese, A. Bertoldo, C. B. Smith, K. C. Schmidt, J. Cereb.
Blood Flow Metab. 2019, 39, 1849–1863; p) K. C. Schmidt, I. Loutaev,
Z. Quezado, C. Sheeler, C. B. Smith, Neurobiol. Dis. 2020, 143,
104978.
Acknowledgements
This work was supported in part by a consignment expense for
the Molecular Imaging Program on “Research Base for
Exploring New Drugs” from the Ministry of Education, Culture,
Sport, Science, and Technology (MEXT), Japan, Individual
Knowledge Research in FY 2012 Strategic Programs for R&D
(President’s Fund) by RIKEN, and JSPS KAKENHI Grant
Number JP19K17184 (Grant-in-Aid for Young Scientist). We
thank Mr. M. Kurahashi (Sumitomo Heavy Industry Accelerator
Service Ltd.) for operating the cyclotron, Dr. Y. Wada for
technical support in reconstitution of the PET images, and Dr. K.
Takahashi (Nuclear Chemistry Research Team, RIKEN) for ICP-
MS analysis of Pd impurities.
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Conflicts of Interests
There is no conflict of interests to declare.
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Keywords: amino acids • 11C-methylation • microfluidic
hydrogenation • isotopic labeling • tumor imaging
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and (R)-α-propargylalanine by the same process shown in Scheme 1.
O
B
O
O
OBn
N
R
Cbz
Bn
(R)-1: R = H
(R)-2: R = CH3
8
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9
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Entry for the Table of Contents
Efficient synthesis of L-[5-11C]leucine and L-a-[5-11C]methylleucine was accomplished by Pd0-mediated 11C-methylation and
microfluidic hydrogenation. PET imaging showed that L-a-[5-11C]methylleucine is a potentially useful probe for visualizing the tumor.
The synthetic protocols for 11C-labeled leucine and its derivatives are applicable to human PET studies.
10
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