K. Cao et al.
(6 mL, 6 mmol) was added to the residue, and the mixture was extracted
with ethyl acetate (10 mL × 3). Pooled ethyl acetate extracts were dried
over Na2SO4 and filtered, and solvent evaporated under reduced
pressure to give [1-14C]N-benzyloxycarbonyl-β-alanine (355 mg,
83.7 mCi, radiochemical yield 90%) as a white solid. 1H NMR (300 MHz,
[2H6]DMSO) δ 7.20–7.45(m, 5H), 5.01(s, 2H), 3.20(q, J = 6.8 Hz, 2H), 2.38
(t, J = 7.1 Hz, 2H). MS ESI+ [M + H]+ = 224, 226.
[1-14C]β-Alanine, 5
To a 25-mL flask was weighed [1-14C]N-benzyloxycarbonyl-β-alanine
(355 mg, 1.58 mmol, 83.7 mCi) and 10% Pd/C (17 mg, 0.16 mmol). The
flask was equipped with a balloon, degassed, and flushed with nitrogen
three times prior to the addition of water and ethanol (5 mL each),
followed by the introduction of hydrogen gas. The mixture was stirred
at room temperature for 3 h. The reaction mixture was filtered through
Celite, and the bed was washed with water (5 mL × 2). The combined
filtrates were evaporated under a gentle stream of nitrogen to give
138 mg of [1-14C]β-alanine (82.1 mCi, radiochemical yield 98%) as a pale
yellow solid. 1H NMR (300 MHz, [2H6]DMSO) δ 2.81(t, J = 6.3 Hz, 2H), 2.08
(t, J = 6.5 Hz, 2H).
Scheme 1. Synthesis of carbon-14 labeled-N-succinimidyl-3-maleimidopropionate
from K14CN.
overall conversion, inconsistency, and difficulty in purifying
the desired label product.
[1-14C]3-Maleimidopropionic acid, 6
To [1-14C]β-alanine (138 mg, 1.52 mmol, 82.1 mCi) was added maleic
anhydride (162 mg, 1.65 mmol) and acetic acid (8 mL). The reaction
mixture turned cloudy after the first hour. The mixture was stirred at
room temperature for 18 h, and then the cloudy suspension was heated
to 115 °C for 8 h and became a clear solution. The solvent was
evaporated under a gentle stream of nitrogen. The crude product was
dissolved in 50% acetonitrile and purified by preparative HPLC. UV:
305 nm, flow rate: 7 mL/min. Gradient: 0 min, 10% B; 15 min, 50% B;
20 min, 95% B; and 25 min, 10% B. The product retention time was
5.9 min. Pooled fractions containing the desired product were
concentrated under reduced pressure to give [1-14C]3-maleimidopropionic
Because of the difficulties with the initial approach, an alternative
labeling approach was developed. C. A. Townsend and A. Basak
reported the synthesis of carbon-13 labeled β-alanine and
derivatives.6 Following similar chemistry, we successfully prepared
carbon-14 labeled β-alanine from readily available [14C]KCN, see
Scheme 1. For the cyanation reaction of benzyloxycarbonylami-
noethyl-1-methanesulfonate, 3,6 we discovered that the reaction
was completed in 18 h with the addition of 1 eq. of KI, which also
gave better yields. Thus 2-benzyloxycarbonylaminoethyl-1-
methanesulfonate reacted with [14C]KCN in the presence of 1 eq.
of KI that gave a 93% conversion to [1-14C]3-benzyloxycarbonyl-
aminopropionitrile, 3, after purification. Subsequent hydrolysis of
nitrile 3 with NaOH and H2O2 yielded [1-14C]N-benzyloxycarbonyl-
β-alanine, 4, in 90% radiochemical yield. Deprotection of 4 with
hydrogen gas and 10% Pd/C gave [1-14C]β-alanine, 5, in near
quantitative yield. Following the previously reported procedures,
[1-14C]3-maleimidopropionic acid 6 was prepared in 70% yield,
and [1-14C]N-succinimidyl 3-maleimidopropionate, 7, was prepared
in 94% radiochemical yield.1,7,8
1
acid (170 mg, 58 mCi, radiochemical yield 70%) as a white solid. H NMR
(300MHz, [2H6]DMSO) δ 7.01(s, 2H), 3.62(t, J = 7.4Hz, 2H), 2.45(t, 2H, partially
buried under DMSO peak).
[1-14C]N-Succinimidyl 3-maleimidopropionate, 7
To [1-14C]3-maleimidopropionic acid (170 mg, 1.00 mmol) and
N-hydroxysuccinimide (114 mg, 1.00 mmol) was added DMF (5 mL).
The solution was cooled in an ice water bath under nitrogen. N,
N′-diisopropylcarbodiimide (0.31 mL, 2.00 mmol) was added slowly.
The ice water bath was removed 5–10 min after the completion of
the addition. The solution turned pink in color, and a white precipitate
was formed. After stirring at room temperature for 5 h, the reaction
flask was placed in an ice bath for 30 min, and the urea was removed
via filtration. The solid was washed with DMF (3 mL × 3). Combined
filtrates were evaporated under a stream of nitrogen to give the crude
product as an off-white solid. The solid was dissolved in acetonitrile
(15 mL) and the acetonitrile solution was cooled in an ice water bath
for 10 min and filtered. The yellow orange filtrate was evaporated
under a stream of nitrogen to give 324 mg of a gray powder as a
mixture of 27% diisopropyl urea and 73% desired product (54.5 mCi,
radiochemical purity 98%, radiochemical yield: 88%). The product
mixture was used as is for subsequent PEGylation and coupling with
adnectins without detrimental interference from the diisopropyl urea.
1H NMR (300 MHz, [2H6]DMSO) δ 7.04(s, 2H), 3.74(t, J = 6.9 Hz, 2H), 3.04
(t, J = 6.9 Hz, 2H), 2.79(s, 4H).
The final product is a mixture of 27% diisopropyl urea and 73%
of [1-14C]BMPS (determined by 1H NMR). Our earlier attempts of
purification using flash chromatography resulted in very low
recoveries (<20%). The product mixture was used as is for the
coupling with a 40-Kda PEG-NH2 in CH2Cl2 to form PEG-[14C]
Maleimide. The excess [1-14C]N-succinimidyl-3-maleimido-
propionate as well as diisopropyl urea were removed by
ultrafiltration with
a 30,000 molecular weight cutoff. No
interference from diisopropyl urea was observed.
Results and discussion
Conclusion
Our initial synthesis of a carbon-14 labeled linker molecule
started by heating [1,4-14C]maleic acid with β-alanine. The Our previous synthesis of [14C]BMPS suffered from low yields,
product isolated was then reacted with N-hydroxysuccinimide inconsistencies, and a difficult purification. Our new improved
to produce [14C]BMPS, 1.1 This synthesis suffered from low route started with 100 mCi of [14C]KCN to produce 55 mCi of
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 667–669
Cao, Kai
