A convenient and reproducible method for the synthesis of astatinated 4-[211At]astato-l-phenylalanine: Via electrophilic desilylation

Article abstract of DOI:10.1039/c8ob02394h

The ²¹¹At-labeled compound, 4-[²¹¹At]astato-l-phenylalanine, is one of the most promising amino acid derivatives for use in targeted alpha therapy (TAT) for various cancers. Electrophilic demetallation of a stannyl precursor is the most widely used approach for labeling biomolecules with ²¹¹At. However, the low acid-resistance of the stannyl precursor necessitates the use of an N- and C-terminus-protected precursor, which results in a low overall radiochemical yield (RCY) due to the multiple synthetic steps involved. In this study, a deprotected organosilyl compound, 4-triethylsilyl-l-phenylalanine, was employed for the direct synthesis of astatinated phenylalanines. ²¹¹At was separately recovered from the irradiated ²⁰⁹Bi target using chloroform (CHCl₃) and N-chlorosuccinimide-methanol (NCS-MeOH) solution. The RCYs of 4-[²¹¹At]astato-l-phenylalanine obtained from the triethylsilyl precursor with the use of ²¹¹At, isolated in CHCl₃ and NCS-MeOH solution, were 75% and 64% respectively. In both cases, the retention time of the 4-[²¹¹At]astato-l-phenylalanine was found to be about 20 min, which showed reasonable correlation with the retention time of non-radioactive 4-halo-l-phenylalanines (4-chloro-, 4-bromo-, and 4-iodo-l-phenylalanine). The one-step reaction examined in this study involved mild reaction conditions (70 °C) and a short time (10 min) compared to the other currently reported procedures for astatination. Electrophilic desilylation was found to be very effective for the labeling of aromatic amino acids with ²¹¹At.

Full text of DOI:10.1039/c8ob02394h

Organic &
Biomolecular Chemistry
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A convenient and reproducible method for the
synthesis of astatinated 4-[211At]astato-L-phenyl-
alanine via electrophilic desilylation
Cite this: Org. Biomol. Chem., 2019,
17, 165
c
Shigeki Watanabe, *†a Mohammad Anwar-Ul Azim,†a,b Ichiro Nishinaka,
Ichiro Sasaki,a,d Yasuhiro Ohshima,a Keiichi Yamada d and Noriko S. Ishiokaa
The 211At-labeled compound, 4-[211At]astato-L-phenylalanine, is one of the most promising amino acid
derivatives for use in targeted alpha therapy (TAT) for various cancers. Electrophilic demetallation of a
stannyl precursor is the most widely used approach for labeling biomolecules with 211At. However, the
low acid-resistance of the stannyl precursor necessitates the use of an N- and C-terminus-protected pre-
cursor, which results in a low overall radiochemical yield (RCY) due to the multiple synthetic steps
involved. In this study,
a deprotected organosilyl compound, 4-triethylsilyl-L-phenylalanine, was
employed for the direct synthesis of astatinated phenylalanines. 211At was separately recovered from the
irradiated 209Bi target using chloroform (CHCl3) and N-chlorosuccinimide-methanol (NCS-MeOH) solu-
tion. The RCYs of 4-[211At]astato-L-phenylalanine obtained from the triethylsilyl precursor with the use of
211At, isolated in CHCl3 and NCS-MeOH solution, were 75% and 64% respectively. In both cases, the
retention time of the 4-[211At]astato-L-phenylalanine was found to be about 20 min, which showed
reasonable correlation with the retention time of non-radioactive 4-halo-L-phenylalanines (4-chloro-,
4-bromo-, and 4-iodo-L-phenylalanine). The one-step reaction examined in this study involved mild
reaction conditions (70 °C) and a short time (10 min) compared to the other currently reported pro-
cedures for astatination. Electrophilic desilylation was found to be very effective for the labeling of aro-
matic amino acids with 211At.
Received 26th September 2018,
Accepted 4th December 2018
DOI: 10.1039/c8ob02394h
rsc.li/obc
amino acid transporters (LATs) are key players in the uptake of
neutral amino acids into cells. Among the four known LAT
Introduction
Amino acids play central roles both as building blocks of pro- families (LAT1, LAT2, LAT3 and LAT4), LAT1 has been receiving
teins and as intermediates in metabolism. The movement of the most attention because of its higher expression in human
amino acids into cells mainly occurs through carrier-mediated cancer tissues,2 including cholangiocarcinoma, multiple
processes.1 Amino acid transport is generally increased in myeloma, and malignant glioma, as well as lung, uterine cervi-
malignant cancer cells compared to normal cells, due to their cal, oral, prostate, and breast cancer.3–5 For this reason, amino
rapid growth and continuous proliferation. This increased acid transporters are attractive targets for diagnostic radio
transport of amino acids across the plasma membrane is facili- imaging and targeted radiotherapy of various cancers, and
tated by the upregulation of amino acid transporters. L-type many radiolabeled amino acid derivatives have been
developed.6–13 Targeted alpha therapy (TAT) is a trustworthy
approach for oncology treatment. Because of the short tissue
range of α particles, they deliver an extremely cytotoxic radi-
ation dose to cancer cells, while surrounding healthy tissue is
spared. This feature of cell-specific irradiation facilitates the
aDepartment of Radiation-Applied Biology, Takasaki Advanced Radiation Research
Institute, National Institutes of Quantum and Radiological Science and Technology
(QST), 1233 Watanuki, Takasaki, Gunma 370-1292, Japan.
E-mail: watanabe.shigeki@qst.go.jp; Fax: +81-27-346-9688; Tel: +81-27-346-9460
treatment of disseminated cancers.14–16 Several potent
α-particle emitters have been attempted for TAT. Among them,
211At has been considered as potentially suitable because of
the following reasons; 211At emits high-energy α-particles with
100% of its decays, has no long-lived α-emitting daughters as
shown in Scheme 1, and has a half-life (t1/2 = 7.21 h) compati-
ble with a variety of molecule carriers.14,17–20 High-energy
bIn vitro Division, National Institute of Nuclear Medicine and Allied Sciences
(NINMAS), Bangladesh Atomic Energy Commission (BAEC), BSM Medical University,
Shahbag, Dhaka-1000, Bangladesh
cTokai Quantum Beam Science Center, Takasaki Advanced Radiation Research
Institute, QST, 2-4 Shirakata, Tokai, Ibaraki 319-1106, Japan
dDivision of Molecular Science, Graduate School of Science and Technology, Gunma
University, 4-2 Aramaki, Maebashi, Gunma 371-8510, Japan
†These authors equally contributed to this work.
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was to demonstrate electrophilic desilylation as a facile and
reproducible method for the no-carrier-added radiohalogena-
tion of amino acid derivatives with 211At at reasonable radio-
chemical yields. In addition, we performed in vitro studies
using a cancer cell line not only to evaluate its possible appli-
cation to TAT, but also to characterize the 211At-labeled
compounds.
Results and discussion
Synthesis of 4-[131I]iodo-L-phenylalanine 2 via electrophilic
desilylation
Since astatine has chemical properties similar to its closest
halogen, iodine, the labeling chemistry investigated for astati-
nation has been largely based on radioiodination. This back-
ground led us to synthesize 4-[131I]iodo-L-phenylalanine 2 from
4-triethylsilyl-L-phenylalanine 5 prior to the synthesis of 4-
Scheme 1 Decay scheme of 211At.
[
211At]astato-L-phenylalanine 1 as shown in Scheme 2.
α-particles with an energy of 5.9 MeV and 7.5 MeV are emitted
in the decay, corresponding to a range of 60–90 µm in human
tissue. In addition, 211At is mainly produced via the 209Bi
(α,2n)211At reaction. Furthermore, 211At is regarded as a
unique α-emitting radionuclide in that it can be produced
using a cyclotron, whereas most α-emitting radionuclides are
produced using a nuclear reactor. Hence, astatinated amino
acid derivatives have great potential as an agent for TAT.
Although classical halogenation methods are available for
the synthesis of radiohalogenated compounds, electrophilic
reactions via a positive form of the halogen or a nucleophilic
substitution via the halide anion have been generally used for
radiohalogenation.21,22 Among various approaches, electrophi-
lic destannylation is one of the most common.21–23 However,
the low acid-resistance of stannyl compounds necessitates the
use of an N- and/or C-terminus-protected precursor, which
results in a low overall radiochemical yield (RCY) due to the
multiple synthetic steps required for synthesis. Although asta-
tinated phenylalanine was synthesized with considerable
radiochemical yields by Cu+-assisted nucleophilic halogen
exchange reactions, harsh conditions were required to com-
plete the reaction, and the separation of the radiohalogenated
product from a chemically similar substrate has been often
challenging.24 It is known that electrophilic desilylation reac-
tions are also available for the preparation of radiohalogenated
compounds.25–27 Previous studies including ours reported that
m-[211At]astatobenzylguanidine (211At-MABG) was successfully
HPLC analysis of the reactant showed a strong radioactive
peak at 19.8 min (data not shown). On the other hand, the
retention time of the corresponding non-radioactive 4-iodo-L-
phenylalanine 9 was observed to be 18.9 min. Because the
dead volume between both detectors were about 0.6 mL (about
700 mm length × 10 mm internal diameter) and the flow rate
in this study was 1 mL min−1, the time lag of both peaks was
estimated to be 0.6 min. The results showed that a strong
radioactive peak observed was compound [131I]2.
Synthesis of 4-[211At]astato-L-phenylalanine 1 via electrophilic
desilylation
Before the synthesis of the astatinated compound, 211At was
isolated with a dry distillation method from an irradiated Bi
target and then eluted with CHCl3 or N-chlorosuccinimide-
methanol (NCS-MeOH) solution. It is known that the chemical
synthesized
from
a
silyl
precursor,
m-trimethyl-
silylbenzylguanidine in a good RCY.27,28 The use of electrophi-
lic desilylation is therefore an alternative for labeling phenyl-
alanine with 211At. Additionally, compared to stannylated
phenylalanine derivatives, silylated phenylalanine with depro-
tected N- and C-termini is much more readily synthesized
because of its higher resistance to acid. This paper describes
the synthesis of 4-[211At]astato-L-phenylalanine, from the
corresponding triethylsilyl derivative of phenylalanine, and
from the tin precursor, N-tert-butoxycarbonyl-4-(tri-n-butylstan-
Scheme 2 Synthesis of 4-[211At]astato- (1) or 4-[131I]iodo-L-phenyl-
nyl)-L-phenylalanine methyl ester. The purpose of this study alanine (2) via electrophilic desilylation or electrophilic destannylation.
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Fig. 1 (a) HPLC profile of and (b) TLC analysis of [211At]1 synthesized from a silylated precursor 5 with 211At in CHCl3 and 4-halo-L-phenylalanine
derivatives.
species of astatine is readily changed by various functions and found a reasonable correlation between the retention time
such as pH, solvent, radiolysis and so on.29,30 Such sensi- and hydrophobicity of the phenylalanine derivatives (astatine,
tivities often cause low efficiencies for isolation or RCYs for iodine, bromine, and chlorine). Also, a single radioactive spot
the synthesis of astatinated compounds. However, CHCl3 and was observed at the Rf value of 0.82 in TLC analysis (Fig. 1B),
NCS-MeOH solution stabilize its chemical species, resulting in which was identical to those of 9 (Rf = 0.75). These results
efficient and reproducible recovery of 211At and synthesis of its strongly suggested that the single radioactive peak was [211At]1.
labeled compounds.29,31 This background encouraged us to After preparative HPLC, RCY was 75% and the radiochemical
synthesize [211At]1 from silyl precursor 5 using 211At in the purity (RCP) was 99%.
abovementioned solutions. In the case of CHCl3 solution in
On the other hand, two peaks were observed at retention
the presence of NCS, HPLC analysis showed a single radio- times of 19.5 and 20.1 min in the case of 211At in NCS-MeOH
active peak at retention time of 20.6 min shown in Fig. 1A. The solution (Fig. 2A). Interestingly, the two peaks merged at a
fact that astatine has no stable isotopes makes it difficult to retention time of 20.5 min after the fractions were kept for
characterize astatinated compounds by conventional analytical 24 h after preparative HPLC (Fig. 2B). Since the peak at the
techniques, because the total amounts of the astatinated com- retention time of 20.1 min was almost the same as that in case
pound is on the order of 10−12 mol (pmol). Therefore, HPLC of 211At–CHCl3 solution, it was probably the desired com-
analyses of the corresponding non-radioactive 4-halogenated pound. On the other hand, the peak at 19.5 min was unknown
phenylalanine derivatives were performed for the characteriz- due to the serious analytical limitations for the characteriz-
ation of the desired compounds. Retention times of other ation of astatinated compound with no stable isotopes.
halo-L-phenylalanines—namely, 4-iodo- (9), 4-bromo- (10), and However, we can assume the following scenario: At+ cations
4-chloro-L-phenylalanine (11)—were 18.9 min, 17.5 min, and probably existed in the NCS-MeOH solution,29 and some of At+
16.8 min, respectively. Our study demonstrated that [211At]1 might be changed to different positive species for some
has almost the same retention time as that of compound 9 reason. Consequently, impurities were generated by reaction
Fig. 2 (a) HPLC profile of [211At]1 synthesized with 211At in NCS-MeOH and (b) purified [211At]1 at 24 hours after reaction.
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Fig. 3 (a) Cellular uptake of [211At]1 into LS180 cells at various time points (n = 4) (b) inhibition of cellular uptake of [211At]1 by L-amino acids or their
analogs (n = 4).
with the positive species, and then the impurities might have as α-methyl phenylalanine and astatinated peptides containing
been converted to the desired compound within 24 hours, aromatic residue via silylated precursors.
probably due to radiolysis. RCY and RCP after preparative
Uptake and inhibition studies
HPLC were 64% and 99%, respectively.
The results of our in vitro studies are shown in Fig. 3. Uptake
of [211At]1 by LS180 colon adenocarcinoma cell was time-
dependently increased and then plateaued at about 20 min
after incubation (Fig. 3A). Inhibition assays using several
amino acids and their derivatives demonstrated that uptakes
Synthesis of 4-[211At]astato-L-phenylalanine 1 via electrophilic
destannlylation
Astatinated [211At]1 was also synthesized from the stannylated
precursor 6 as shown in Scheme 2. Since a tributylstannyl
in the presence of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic
group is easily removed by acid and heat, it is difficult to syn-
acid (BCH), α-methyl-tyrosine (AMT), Leu, Phe, and Tyr were
thesize the deprotected stannyl precursor. Hence, three steps
significantly reduced compared to uptake of the control. On
were needed to obtain the desired compound as shown in
the other hand, the uptake of the astatinated phenylalanine
Scheme 2. Consequently, the overall processing time was
was not inhibited when Ser, Asn, Arg, or α-methyl-aminoisobu-
about 3 hours including reaction and work up, which was
tyric acid (MeAIB) was added to the solution (Fig. 3B). While a
longer than that of the electrophilic desilylation (1 hour). The
previous study reported that uptake of amino acids via LAT-2
was inhibited in the presence of Ser and Asn, uptake of the
astatinated compound was not decreased in this study. These
RCY with the use of 211At isolated in CHCl3 was found to be
10–17%. Meyer et al. also reported the successful synthesis of
astatinated phenylalanine from 6 using 211At, although three
steps were required to synthesize the desired compound in
35–50% overall RCY.24 Development of quick and efficient
labeling approaches is one of the important issues for the syn-
thesis of 211At-labeled compounds for TAT because its half-life
is relatively short and the produced radioactivity is limited and
has to be used without waste as much as possible. In the
results clearly indicate that [211At]1 was incorporated into
cancer cells via LAT-1;31,32 thus these in vitro studies gave us
valuable information for the characterization of astatinated
compounds with no stable isotopes.
Previous studies have reported that survival time of rats
treated with the astatinated phenylalanine was significantly
prolonged,24,33
although
4-[211At]astato-L-phenylalanine
present study, precursor
5 was successfully synthesized
showed a saturable uptake in glioma cells (<1%). This study
demonstrated higher uptake of [211At]1 (13% of applied dose)
after incubating for 30 min, which suggests that [211At]1 has
also potential as a TAT reagent with good therapeutic effect.
because of its high acid-resistance; thereby, electrophilic desi-
lylation successfully afforded [211At]1 in one step with higher
RCYs (64–75%) than those from the corresponding stannylated
precursor. Meyer et al. also reported that a Cu+-catalyzed
nucleophilic halogen exchange reaction could be used for the
synthesis of astatinated phenylalanine.24 Although good RCYs
(65–85%) were obtained with a one-step reaction, the reaction
Conclusions
has some potential drawbacks such as the relatively high temp- Based on the findings, it can be concluded that electrophilic
erature required for the reaction and the possible dilution by desilylation is a more convenient method than the currently
halogen homologue-carriers. These results strongly indicated reported Cu+-assisted nucleophilic reaction for the synthesis of
that electrophilic desilylation is a more promising and repro- 4-[211At]astato-L-phenylalanine, showing competitive RCY
ducible method for the synthesis of astatinated aromatic (65–85%) and RCP (>99%). Electrophilic desilylation with NCS
amino acids. This approach can certainly be applied to the is also very effective for the labeling of aromatic amino acids.
syntheses of other promising phenylalanine derivatives such This method is also applicable for the synthesis of 211At-
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labeled peptides containing aromatic amino acid residues, The solvent was removed under reduced pressure to give the
other types of promising reagent for cancer therapy.
crude
product.
Chromatographic
purification
(SiO2,
hexane : EtOAc = 4 : 1(v/v) as an eluent) gave 4 as a colorless oil
(408 mg, 1.03 mmol, 71% yield). Rf 0.61 (hexane : AcOEt = 3 : 1
(v/v)). 1H NMR (300 MHz, CDCl3) δ 0.76 (q, 6H, J = 7.6 Hz),
0.94 (t, 9H, J = 7.6 Hz), 1.39 (s, 9H), 3.00 (dd, 1H, J = 13.6 Hz,
6.5 Hz), 3.09 (dd, 1H, J = 13.7 Hz, 5.7 Hz), 3.69 (s, 3H), 4.59 (q,
1H, J = 7.6 Hz), 4.96 (d, 1H, J = 8.5 Hz), 7.10 (d, 2H, J = 7.6 Hz),
7.40 (d, 2H, J = 7.8 Hz).
Experimental
All chemicals were of reagent grade with highest purity and
used as purchased. Na[131I]I solution was purchased from
PerkinElmer. NCS was purchased from Tokyo Chemical
Industry Co Ltd. 4-Iodo- (9), 4-bromo- (10), and 4-chloro-L-
phenylalanine (11) were obtained from Watanabe Chemical
Industries. Aluminum-backed sheets (Silica gel 60) were used
for analytical TLC. McolorpHast™ pH-indicator strips (non-
bleeding) were used to check the pH of the reaction mixture. A
centrifugal C-18 solid-phase extraction column, Monospin
C-18, was purchased from GL Science Co. Inc. Reversed-phase
high-performance liquid chromatography (RP-HPLC) analysis
was performed with a system that contained two pumps,
LC-20AD (Shimadzu), a variable-wavelength UV detector fol-
lowed by SPD-20A (Shimadzu), and a γ-ray detector, GABI
PET-Star (Elysia-raytest). Chromatography was performed on a
TOSOH TGKgel ODS-100 V 5 µm column (Size: 250 mm ×
4.6 mm i.d.) at a flow rate of 1 mL min−1, eluted for 40 min
with a linear gradient of water containing 0.1% TFA and aceto-
nitrile containing 0.1% TFA at ratios from 90 : 10 to 10 : 90.
Non-radioactive compounds were detected by the UV-Vis detec-
tor at 254 nm, and radioactivity was measured by the γ-ray
detector with 50–800 keV. TLC analysis of the reaction mixture
was performed using a solvent system of n-BuOH : AcOH : H2O
= 2 : 2 : 1. TLC plates were exposed overnight to imaging plates
(Fujifilm BAS-MP 2040s), and the radioactivity distribution on
the TLC plate was visualized and quantified by a bioimaging
4-Triethylsilyl-L-phenylalanine, 5. Silyl phenylalanine
4
(788 mg, 2.00 mmol) was saponified by treatment with 1 M
NaOH aq. (2.00 mL) in MeOH (10 mL) for 6 h at 0 °C to give
N-Boc-4-triethylsilyl-L-phenylalanine as a colorless oil (747 mg,
98% yield). Rf: 0.05 (hexane : AcOEt = 3 : 1 (v/v)), Rf 0.57
1
(CH2Cl2 : MeOH : AcOH = 92 : 5 : 3 (v/v/v)). H NMR (300 MHz,
CDCl3) δ 0.73–0.82 (9H, m, –Si(CH2C
̲H3̲ )3), 0.93–0.99 (6H, m,
–Si(C
̲H̲
2CH3)3), 1.41 (9H, s, Boc), 3.02–3.08 (1H, m, βCH2),
3.16–3.21 (1H, m, βCH2), 4.60 (1H, m, αCH), 4.92 (1H, bs,
αNH), 7.17 (2H, d, J = 7.7 Hz, aromatic), 7.43 (2H, d, J = 8.0 Hz,
aromatic). APCI-MS, found m/z 378.1, calcd for C20H32NO4Si
378.2 [M − H]−.
N-Boc-4-triethylsilyl-L-phenylalanine (747 mg) was then dis-
solved in 4 M HCl in 1,4-dioxane (3.0 mL) and the solution
was kept for 2 h at room temperature. The mixture was concen-
trated in vacuo and the residue was solidified by cold diethyl
ether. The resulting solid was collected by centrifuge and
dried in vacuo. 4-Triethylsilyl-L-phenylalanine 5 was obtained
as a white solid (295 mg, 54% yield). RP-HPLC tR = 10.17 min
(column: YMC-pack Pro C18 (YMC, 4.6 × 150 mm); eluent,
30–80% acetonitrile (containing 0.1% TFA); flow rate, 1 mL
min−1; detection, UV 254 nm).
N-Boc-4-tributylstannyl-L-phenylalanine methyl ester, 6. The
stannylated precursor 6, was synthesized from 3 (230 mg,
560 µmol) by modified procedures as reported previously.21
Compound 6 was obtained as a colorless oil (186 mg,
326 µmol, 57% yield). Rf 0.46 (hexane : AcOEt = 4 : 1 (v/v)), Rf
0.84 (CH2Cl2 : MeOH = 95 : 5 (v/v)). APCI-MS, found m/z 569.6,
calcd for C27H48NO4Sn 569.4 [M + H]+. 1H NMR (300 MHz,
CDCl3) δ 0.85–0.96 (9H, m, SnBu3), 1.00–1.07 (6H, m, SnBu3),
1.24–1.38 (6H, m, SnBu3), 1.41 (9H, s, Boc), 1.46–1.65 (6H, m,
1
analyzer system, Typhoon FLA 7000 (GE Healthcare). H NMR
Spectra were recorded on an Avance II 300 MHz spectrometer
(Bruker). Chemical shifts are given in ppm relative to respect-
ive solvent peak. Atmospheric pressure chemical ionization
mass spectra (APCI-MS) were recorded on an API-2000 mass
spectrometer (AB SCIEX). The radioactivities of 211At and 131I
used in each reaction and of the HPLC fractions of purified
[
211At]1 and [131I]2 were measured with a curiemeter IGC-7
β
(Hitachi Aloka Medical) or with
(ARC-7001; Hitachi Aloka Medical).
a well-type γ counter
SnBu3), 3.02–3.07 (2H, m, CH2), 3.71 (3H, s, OCH3), 4.58 (1H,
α
α
m, CH), 4.96 (1H, d, J = 7.8 Hz, NH), 7.08 (2H, d, J = 7.5 Hz,
aromatic), 7.38 (2H, d, J = 7.8 Hz, aromatic).
Synthesis of 4-triethylsilyl-L-phenylalanines
N-Boc-4-iodo-L-phenylalanine methyl ester, 3. N-Boc-4-iodo-
Production of 211At
L-phenylalanine methyl ester 3 was synthesized by the same Irradiation was performed using an AVF-930 cyclotron
procedures as reported previously.23
(Sumitomo Heavy Industries, Ltd) installed at the Takasaki Ion
N-Boc-4-triethylsilyl-L-phenylalanine methyl ester, 4. To a Accelerators for Advanced Radiation Application (TIARA) of the
stirred solution of 3 (588 mg, 1.45 mmol) and [Rh(cod)Cl]2 National Institutes for Quantum and Radiological Science and
(2.71 mg, 0.38 mol%) in DMF (10 mL) at room temperature Technology (QST). 211At was produced by bombarding a
under N2 atmosphere, Et3N (0.60 mL, 4.35 mmol, 3.0 eq.) and natural bismuth plate target (245 mg, size, 10 mm × 10 mm ×
triethylsilane (0.46 mL, 2.89 mmol, 2.0 eq.) were added. The 0.25 mm thickness) with 28 MeV α-beams at 3 µA of beam
reaction mixture was stirred for 22 h at 80 °C. The mixture was current for 30 min. 211At was isolated from the irradiated
cooled and then poured into distilled water (50 mL). The target using the previously described dry distillation method.34
product was extracted by EtOAc (50 mL, twice). The organic 211At was eluted with 500–1500 µL CHCl3 or NCS-MeOH solu-
layer was washed with brine and dried over anhydrous Na2SO4. tion (concentration, 1.5 µg µL−1). The radioactivity produced at
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the end of irradiation of 211At was 24 MBq. The radioactivities
recovered at the end of isolation were 13.9 MBq (CHCl3) and
19.4 MBq (NCS-MeOH).
Conflicts of interest
There are no conflicts of interest.
Synthesis of [211At]astato-L-phenylalanine
Acknowledgements
General procedures for the synthesis of 1 from 4-triethyl-
silyl-L-phenylalanine 5. Two solvent systems, 211At eluted in
CHCl3 or in NCS-MeOH, were examined for the synthesis of 1
via the silyl precursor 5. In case of 211At in CHCl3, 200 µL of
the 211At solution (2.4 MBq) was poured into a 1.5 mL-reaction
vial, and then solvent was evaporated with the gentle flow of
N2 gas. After adding precursor 5 (200 µg, 0.72 µmol) in 5 µL
MeOH, NCS (400 µg, 3.0 µmol) in 20 µL MeOH, and 0.3 M of
MeOH–acetic acid solution (20 µL), the mixture was evaporated
to dryness with a gentle N2 flow. For the synthesis of 1 using
211At in NCS solution, 10 MBq of 211At was used without extra
NCS addition to the residue. TFA (20 µL) was then added to
the mixture and heated at 70 °C for 10 min. For the synthesis
of [131I]2, an aliquot of Na[131I]I solution (10 kBq) was used.
Procedures after cooling to room temperature were basically
identical to those for the synthesis of [211At]1.
We are thankful to the staff of the Medical Radioisotope
Application Group at Takasaki Advanced Radiation Research
Institute of National Institutes for Quantum and Radiological
Science and Technology (QST). We are also grateful to Mr
Masashi Koka, Mr Koji Imai, and the staff of the Takasaki Ion
Accelerators for Advanced Radiation Application for operating
the AVF cyclotron. This work is supported in part by JSPS
KAKENHI (17K10459, 18K07625).
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General procedures for the synthesis of 1 from N-Boc-4-(tri-
n-tributylstannyl)-L-phenylalanine methyl ester 6. CHCl3 solu-
tion (200 µL) containing 211At (2.4 MBq) was evaporated by the
gentle flow of N2 gas. The stannyl precursor 6 (200 µg,
0.35 µmol) in 5 µL MeOH, together with 20 µL of EtOH–AcOH
and NCS (400 µg, 3.0 µmol) in 20 µL MeOH, was added to the
residue and then reacted for 15 min at room temperature. The
reactant was diluted with 260 µL of H2O; then, the resultant
solution was passed through a centrifugal C-18 column,
Monospin C-18 (6000 rpm for 3 min). The centrifugal column
was washed with 500 µL of H2O (6000 rpm for 3 min × 2
times). The 211At-labeled compound 7 trapped inside the
column was eluted with 100 µL of acetonitrile. After the eluent
was treated with N2 gas flow for evaporation, TFA (20 µL) was
added and then reacted at r.t. for 20 min. After cooling at
room temperature and evaporation of TFA, 2 M NaOH (50 µL)
was added to the residue, and then the mixture was heated at
100 °C for an additional 45 min. After the mixture was neutral-
ized with 2 M HCl, an aliquot of the solution was used for TLC
analysis, and the remaining solution was injected into
RP-HPLC. The same procedures were used for the synthesis of
[
131I]2 from the stannylated precursor 6.
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Uptake and inhibition studies
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times, and then dissolved in 0.1 M NaOH solution (200 µL).
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