Preparation and Characterization of [5-13C]-(2S,4R)-Leucine and [4-13C]-(2S,3S)-Valine
1-Isopropyl [4-13C]-(2S,3S)-3-Methyl-N-(trifluoroacetamido)aspart- 1 H, CH), 6.95 (d, JH,H ϭ 8.6 Hz, 1 H, NH) ppm. 13C NMR
FULL PAPER
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ate: H NMR (300 MHz, CDCl3): δ ϭ 1.27 (d, JH,H ϭ 5.9 Hz, 3 (75.5 MHz, CDCl3): δ ϭ 8.2 (C-4), 15.32 (C-5), 21.65 (2 ϫ CH3),
3
1
H, CH3), 1.29 (d, JH,H ϭ 5.9 Hz, 3 H, CH3), 1.36 (dd, JC,H
129.41, JH,H ϭ 7.3 Hz, 3 H, 5-H), 3.08 (m, 1 H, 3-H), 4.80 (dd,
ϭ
39.47 (C-3), 55.78 (C-2), 70.83 (OCH), 115.51 (q, 1JC,F ϭ 287.6 Hz,
3
2
CF3), 157.3 (q, JC,F ϭ 37.7 Hz, CO), 169.08 (C-1) ppm.
3JH,H ϭ 4.0, JH,H ϭ 8.0 Hz, 1 H, 2-H), 5.11 (dq, JH,H ϭ 5.9,
3JH,H ϭ 5.9 Hz, 1 H, OCH) ppm. 13C NMR (75.5 MHz, CDCl3):
δ ϭ 13.17 (C-5), 21.49 (CH3), 21.56 (CH3), 41.68 (d, C-3), 54.23
(C-2), 71.0 (OCH), 118.75 (CF3), 159.10 (CO), 177.68 (CO) ppm.
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Isopropyl [13C]-4-Iodo-3-methyl-2-(trifluoroacetylamino)butanoate
(12a): The same procedure was applied as for 11. The yield was 92
1
%. 1H NMR (300 MHz, CDCl3): δ ϭ 1.05 (dd, JC,H ϭ 127.35,
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3JH,H ϭ 6.9 Hz, 3 H, 5-H), 1.30 (d, JH,H ϭ 6.3 Hz, 3 H, CH3),
1.31 (d, 3JH,H ϭ 6.3 Hz, 3 H, CH3), 2.43 (m, 1 H, 3-H), 2.96 (ddd,
1-Isopropyl (2S,3S)-3-Methyl-N-(trifluoroacetamido)homoserinate:
A solution of N-methylmorpholine (0.31 mL, 2.81 mmol) was ad-
ded dropwise to a three-necked round-bottomed flask charged with
a solution of the protected aspartate ester (0.73 g, 2.56 mmol) in
dry THF (50 mL), equipped with a pressure-equalizing dropping
funnel, under dry argon and cooled to Ϫ50 °C. Subsequently, iso-
butyl chloroformate (0.35 mL, 2.7 mmol) was added slowly using
a syringe and the mixture was stirred for 5 min. The resulting sus-
pension was quickly filtered into a solution of sodium borohydride
(77 mg, 2.1 mmol) in dry THF at Ϫ20 °C and was stirred for 3 h.
The reaction was quenched by addition of a 1:1 acetic acid/water
mixture (1 mL). The solvents were evaporated and the resulting oil
was taken up in ether (50 mL), extracted with water and brine and
dried with MgSO4. The product was purified using column chro-
matography (PE/E, 70:30) with acetic acid (5 drops) added to the
eluent. Yield 400 mg (58 %). 1H NMR (300 MHz, CDCl3): δ ϭ
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3JH,H ϭ 8.3, JH,H ϭ 10.2, JC,H ϭ 3.7 Hz, 1 H, 4-H), 3.20 (ddd,
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3JH,H ϭ 5.5, JH,H ϭ 10.2, JC,H ϭ 5.3 Hz, 1 H, 4-H), 4.83 (ddd,
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3JH,H ϭ 3.8, JH,H ϭ 8.6, JC,H ϭ 4.7 Hz, 1 H, 2-H), 5.11 (sept,
3JH,H ϭ 6.3 Hz, 1 H, CH), 6.95 (d, 3JH,H ϭ 8.6 Hz, 1 H, NH) ppm.
13C NMR (75.5 MHz, CDCl3): δϭ 8.2 (C-4), 15.32 (C-5), 21.65 (2
ϫ CH3), 39.47 (d, 1JC,C ϭ 35.3 Hz, C-3), 55.78 (C-2), 70.83 (OCH),
115.51 (q, 1JC,F ϭ 287.6 Hz, CF3), 157.3 (q, JC,F ϭ 37.7 Hz, CO),
2
169.08 (C-1) ppm.
(2S)-Valine·HCl (1): 11 (1.5 g, 3.9 mmol) was dissolved in meth-
anol, 5 % Pd/C (1.5 g) was added and hydrogen gas was bubbled
through. After TLC indicated complete disappearance of the start-
ing material, the reaction mixture was flushed with argon and fil-
tered through Celite under argon. Subsequently, the solution was
concentrated to dryness and the protected valine was redissolved
in 2-propanol (20 mL). 2 KOH solution (10 mL) was added and
the mixture was stirred for 4 h. The solvent was evaporated and
the residue was redissolved in water. Dowex Hϩ was added and the
mixture was stirred gently for 1 h. The resin was rinsed with water
(100 mL) and eluted with 0.2 NH3. The fractions giving a ninhy-
drin stain were collected and the solvents evaporated. 1 HCl
(10 mL ) was added and the solution lyophilized giving a yield of
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0.77 (d, JH,H ϭ 7.1 Hz, 3 H, 5-H), 1.29 (d, JH,H ϭ 6.2 Hz, 3 H,
CH3), 1.31 (d, 3JH,H ϭ 6.2 Hz, 3 H, CH3), 2.44 (m, 1 H, 3-H), 3.24
3
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(dd, JH,H ϭ 12.1, JH,H ϭ 10.6 Hz, 1 H, 4-H), 3.57 (dd, JH,H
ϭ
ϭ
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12.1, JH,H ϭ 4.9 Hz, 1 H, 4-H), 4.84 (dd, JH,H ϭ 7.8, JH,H
3
2.8 Hz 1 H, 1-H), 5.13 (h, JH,H ϭ 6.2 Hz, 1 H, CH), 7.28 (d,
3JH,H ϭ 7.8 Hz, 1 H, NH) ppm. 13C NMR (75.5 MHz, CDCl3):
δϭ 10.44 (C-5), 21.69 (C-7/C-8), 38.74 (C-3), 53.50 (C-2), 63.91 (C-
1
0.46 g of valine·HCl (77 %). H NMR (300 MHz, D2O): δ ϭ 1.02
1
4), 70.57 (C-6), 115.61 (d, JC,F ϭ 287.3 Hz, C-9), 158.10 (d,
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(d, JH,H ϭ 7.2 Hz, 4-H), 1.04 (d, JH,H ϭ 7.1 Hz, 5-H), 2.35 (dqq,
2JC,F ϭ 38.0 Hz, C-10), 170.06 (C-1) ppm.
3JH,H ϭ 7.2, JH,H ϭ 7.1, JH,H ϭ 4.4 Hz, 1 H, 3-H), 3.94 (d,
3JH,H ϭ 4.4 Hz, 1 H, 2-H) ppm. 13C NMR (75.5 MHz, D2O): δϭ
17.47 (C-4), 17.96 (C-5), 32.58 (C-3), 62.61 (C-2), 183.83 (C-1)
ppm.
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1-Isopropyl [3-13C]-(2S,3S)-3-Methyl-N-(trifluoroacetamido)homo-
serinate: The reaction was performed as for unlabelled homoserine.
1
The yield was 65 %. H NMR (300 MHz, CDCl3): δ ϭ 0.77 (dd,
3JH,H ϭ 7.1, 1JC,H ϭ 126.7 Hz, 3 H, 5-H), 1.29 (d, JH,H ϭ 6.2 Hz,
3
(2S,3S)-Valine·HCl (1a): The procedure as described for the prep-
aration of 1 was repeated with 11a (0.8 g, 2.1 mmol), giving 1a in
a yield of 81 % (0.26 g, 1.7 mmol). 1H NMR (300 MHz, D2O): δ ϭ
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3 H, CH3), 1.31 (d, JH,H ϭ 6.2 Hz, 3 H, CH3), 2.44 (m, 1 H, 3-
H), 3.24 (ddd, 3JH,H ϭ 12.1, 3JH,H ϭ 10.6, 3JC,H ϭ 1.7 Hz, 1 H, 4-
3
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H), 3.57 (ddd, JH,H ϭ 12.1, JH,H ϭ 4.9, JC,H ϭ 1.6 Hz, 1 H, 4-
1
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1.02 (dd, JC,H ϭ 125.1, JH,H ϭ 7.2 Hz, 3 H, 4-H), 1.04 (dd,
H), 4.84 (ddd, 3JH,H ϭ 7.8, 3JH,H ϭ 2.8, 3JC,H ϭ 2.9 Hz 1 H, 1-H),
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3JH,H ϭ 7.1, JC,H ϭ 5.4 Hz, 3 H, 5-H), 2.35 (ddqq, JH,H ϭ 7.2,
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5.13 (H, JH,H ϭ 6.2 Hz, 1 H, CH), 7.28 (d, JH,H ϭ 7.8 Hz, 1 H,
NH) ppm. 13C NMR (75.5 MHz, CDCl3): δ ϭ 10.68 (C-5), 21.51
(C-7/C-8), 38.36 (d, JC,C 35.15 Hz, C-3), 53.84 (C-2), 63.83 (C-4),
70.43 (C-6), 115.61 (d, JC,F ϭ 287.3 Hz, C-9), 158.10 (d, JC,F
38.0 Hz, C-10), 170.08 (C-1) ppm.
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3JH,H ϭ 7.1, JH,H ϭ 4.4, JC,H ϭ 4.4 Hz, 1 H, 3-H), 3.94 (dd,
3JC,H ϭ 4.9, 3JH,H ϭ 4.4 Hz, 1 H, 2-H) ppm. 13C NMR (75.5 MHz,
1
1
D2O): δϭ 17.47 (C-4), 17.96 (C-5), 32.58 (d, JC,C ϭ 35.5 Hz, C-
1
2
ϭ
3), 62.61 (C-2), 183.83 (C-1) ppm.
Diethyl Ethylidenesuccinate (8): Acetaldehyde (0.73 mL, 13.0
mmol) in DCM (5 mL) was slowly added to a stirred solution of 5
(5 g, 11.7 mmol) in DCM (50 mL). The mixture was stirred at room
temp. for 4 h and subsequently extracted with brine. The organic
layer was dried with MgSO4 and the solvents were evaporated. The
resulting raw product was purified by column chromatography (PE/
Isopropyl 4-Iodo-3-methyl-2-(trifluoroacetamido)butanoate (12): A
solution of iodine (0.99 g, 1.99 equiv.) in dry DCM (10 mL ) was
added dropwise to a solution of triphenylphosphane (1.02 g, 2
equiv.) in dry DCM. A mixture of imidazole (0.3 g, 2.2 equiv.) and
protected homoserine (0.5 g, 1.9 mmol), dissolved in dry DCM
(10 mL), was added dropwise to the resulting pale-coloured liquid.
After 2 h, TLC indicated complete conversion. After filtering off
the solids, the product was extracted with 1 HCl solution, water
and brine and dried with MgSO4. The resulting oil was purified
using column chromatography (PE/E, 85:15) with glacial acetic
1
E). Yield: 90 % (2.1 g). H NMR (200 MHz, CDCl3): δ ϭ 1.25 (t,
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3JH,H ϭ 7 Hz, 3 H, CH3), 1.28 (t, JH,H ϭ 7 Hz, 3 H, CH3), 1.84
(d, 3 H, 6-H), 3.35 (s, 2 H, 3-H) 4.15 (q, 3JH,H ϭ 7 Hz, 2 H, OCH2),
3
4.20 (q, JH,H ϭ 7 Hz, 2 H, OCH2), 6.13 (q, 1 H, 5-H) ppm. 13C
NMR (75.5 MHz, CDCl3): δ ϭ 14.1 (C-6), 14.5 (2 ϫ CH3), 32.1
(C-3), 60.7 (2 ϫ CH2), 126.9 (C-5), 140.1 (C-2), 166.8 (CO), 170.7
(CO) ppm.
1
acid (5 drops) added to the eluent. Yield 0.70 g (95 %). H NMR
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(300 MHz, CDCl3): δ ϭ 1.05 (d, JH,H ϭ 6.9 Hz, 3 H, 5-H), 1.30
(d, 3JH,H ϭ 6.3 Hz, 3 H, CH3), 1.31 (d, 3JH,H ϭ 6.3 Hz, 3 H, CH3),
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2.43 (m, 1 H, 3-H), 2.96 (dd, JH,H ϭ 8.3, JH,H ϭ 10.2 Hz, 1 H,
2-Ethylfumaric Acid (9): 8 (1.5 g, 7.5 mmol) was refluxed in toluene
4-H), 3.20 (dd, JH,H ϭ 5.5, JH,H ϭ 10.2 Hz, 1 H, 4-H), 4.83 (dd, (20 mL) in the presence of DBU (1 equiv.) and the rearrangement
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3JH,H ϭ 3.8, 3JH,H ϭ 8.6 Hz, 1 H, 2-H), 5.11 (sept, 3JH,H ϭ 6.3 Hz,
of the double bond was monitored by TLC (PE/E, 90:10). When
Eur. J. Org. Chem. 2003, 4664Ϫ4678
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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