L-aspartate aminotransferase (40 units/ml) and (2R,3R)-[3-
2H1]-b-chloroalanine 17a16 (10 mg, 0.072 mmol); dH (300 MHz,
(2R)-b-Hydroxyethylcysteine (18). Methyl (2R)-N-carbo-
benzyloxy-b-hydroxyethylcysteineate (100 mg, 0.32 mmol) was
heated to reflux in 4 M aqueous sulfuric acid (3 ml) for 4 h.
The solution was allowed to cool, washed with dichloromethane
(2 × 5 ml), and neutralised with aqueous ammonium hydroxide.
This solution was concentrated in vacuo until solid began to
precipitate. Water was added until the residue redissolved and
the pH of the resultant solution was adjusted to 7. The solution
was chromatographed on an anion exchange column (Dowex
1 × 8–200, 2.5 × 15 cm, OH− form) by passing water until
the pH of the eluant became neutral, and eluting the amino
acid with 5% aqueous acetic acid (50 ml). The solvent was
removed in vacuo to yield (2R)-b-hydroxyethylcysteine 18 as a
10% 2HCl in 2H2O, Fig. 3c) 2.74 (2H, br t, J2 ,1 6.1, CH2S), 3.09
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(1H, d, J3S,2 7.4, H-3S), 3.71 (2H, dt, J1 ,2 6.1, J1 ,OH 2.2, CH2OH)
and 4.33 (1H, d, J2,3S 7.4, H-2).
Incubation of (2R,3S)-[3-2H1]-b-chloroalanine (17b) prepared
by total synthesis with L-aspartate aminotransferase in the
presence of b-mercaptoethanol. This was carried out as above
using L-aspartate aminotransferase (40 unit/ml) and (2R,3S)-[3-
2H1]-b-chloroalanine 17b16 (10 mg, 0.072 mmol); dH (300 MHz,
2
2
10% HCl in H2O, Fig. 3b) 2.74 (2H, unresolved dt, CH2S),
3.21 (1H, br s, H-3R), 3.73 (2H, unresolved dt, CH2OH) and
4.34 (1H, d, J2,3R 4.3, H-2).
white solid (26 mg, 45%); mp 188–189 ◦C; [a]D −51.2 (c 2,
32
H2O) [lit31 [a]D −53.3 (c 2, H2O)]; m/z [+ve FAB (glycerol)] 166
([M + H]+); mmax (film)/cm−1 3412 (br, OH, NH) and 1732 (acid);
Independent synthesis of stereospecifically labelled samples of
(2R)-b-hydroxyethylcysteineate
dH (300 MHz,2H2O, Fig. 2a) 2.74 (2H, dt, J2 ,1 5.8, J2 ,OH 2.0,
CH2S), 3.10 (1H, dd, J3S,3R 15, J3S,2 7.3, H-3S), 3.23 (1H, dd,
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Methyl (2R)-N-carbobenzyloxy-b-hydroxyethylcysteineate.
Boron trifluoride diethyletherate (7 drops) was added dropwise
to a suspension of methyl (2S)-N-carbobenzyloxyaziridine-2-
carboxylate 14 (2.11 g, 8.99 mmol) in b-mercaptoethanol (5.8 ml)
with vigorous stirring under nitrogen at room temperature, and
stirring was continued for 46 h. Chloroform (50 ml) was added
to the solution which was washed with 10% aqueous sodium
bicarbonate (2 × 50 ml), water (6 × 50 ml) and brine (50 ml)
and dried (Na2SO4). The solvent was removed in vacuo to yield a
colourless gum which was chromatographed on silica gel, eluting
with petroleum ether–ethyl acetate (11 : 9). Chromatography was
repeated twice to remove a close-running impurity. This yielded
methyl (2S)-N-carbobenzyloxy-b-hydroxyethylcysteineate as a
J3R,3S 15, J3R,2 4.5, H-3R), 3.71 (2H, dt, J1 ,2 5.8, J 2.3, CH2OH)
and 4.33 (1H, ABX, J2,3R 4.5, J2,3S 7.3, H-2); dC (75.5 MHz,
2H2O) 33.1 (C-3), 35.8 (SCH2), 54.1 (C-2), 62.0 (CH2OH) and
170.5 (acid).
(2R,3S)-[3-2H1]-b-Hydroxyethylcysteine (18b). This was
prepared as described above using methyl (2S,3S)-[3-2H1]-N-
carbobenzyloxy-b-hydroxyethylcysteineate (6 mg, 0.022 mmol)
to yield the product 18b (2 mg, 50%); mp 188–189 ◦C; [a]D
30
−50.9 (c 0.05, H2O); m/z [+ve FAB (glycerol + H2O)] 167
([M + H]+); mmax (film)/cm−1 3412 (br, OH, NH,) and 1732
2
2
(acid); dH (250 MHz, 10% HCl in H2O, Fig. 2c) 2.74 (2H,
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t, J2 ,1 5.7, CH2S), 3.21 (1H, d, J3R,2 4.3, H-3R), 3.75 (2H, t, J1 ,2
25
colourless oil, (1.71 g, 61%); [a]D +9.05 (c 1, CHCl3); m/z
5.7, CH2OH) and 4.31 (1H, d, J2,3R 4.3, H-2); dD (38.4 MHz,
(CI) Found 314.1065. C14H19NO5S + H+ requires 314.1062; m/z
[+ve FAB (3-NBA)] 314 ([M + H]+ and 336 ([M + Na]+); mmax
(film)/cm−1 3358–3063 (OH, NH), 1735 (ester), 1719 (urethane)
and 1697 (amide); dH (250 MHz, C2HCl3) 2.71 (2H, m, CH2S),
2.98 (2H, ABX, J3R,3S 14.0, J3R,2 5.0, J3S,2 5.7, H-3), 3.69 (2H,
m, CH2OH), 3.76 (3H, s, OCH3), 4.61 (1H, m, H-2), 5.11 (2H,
OCH2Ar), 5.70 (1H, d, J 5.13, NH) and 7.32–7.35 (5H, s, ArH);
dC (75.5 MHz, C2HCl3) 34.7 (C-3), 36.1 (SCH2), 52.7 (OCH3),
54.0 (C-2), 60.8 (CH2OH), 67.1 (OCH2Ar), 128.1–128.5 (Ar),
136.1 (ipso Ar), 155.9 (urethane) and 171.1 (ester).
10% HCl in H2O) 3.18 (12H, s, 2H-3S); dC (75.5 MHz, 10% 2HCl
2
in H2O) 31.4 (t, C-3), 34.0 (SCH2), 52.3 (C-2), 60.2 (CH2OH)
and 170.4 (acid).
(2R,3R)-[2,3-2H2]-b-Hydroxyethylcysteine (18a). This was
prepared as described above using methyl (2S,3R)-[2,3-2H2]-N-
carbobenzyloxy-b-hydroxyethylcysteineate (8 mg, 0.029 mmol)
◦
30
to yield the product 18a (2.2 mg, 42%); mp 185–188 C; [a]D
−51.4 (c 0.05, H2O); m/z [+ve FAB (glycerol + H2O)] 168 ([M +
H]+); mmax (film)/cm−1 3410 (br, OH, NH) and 1730 (acid); dH
2
2
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(250 MHz, 10% HCl in H2O, Fig. 2b) 2.74 (2H, t, J2 ,1 5.9,
Methyl (2R,3S)-[3-2H1 ]-N-carbobenzyloxy-b-hydroxyethyl-
cysteineate. This was prepared as described above using methyl
(2S,3R)-[3-2H1]-N-carbobenzyloxyaziridine-2-carboxylate 14b
CH2S), 3.08 (2H, s, H-3S) and 3.71 (2H, t, J1 ,2 5.9, CH2OH);
dD (38.4 MHz, 10% HCl in H2O) 3.18 (12H, s, 2H-3R) and 4.12
(12H, s, 2H-2).
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25
(85 mg, 0.36 mmol) to yield the product (45 mg, 40%); [a]D
+9.05 (c 1, CHCl3); m/z [+ve FAB (3-NBA)] 315 ([M + H]+); mmax
(film)/cm−1 3354 (br, OH) and 1718 (urethane); dH (300 MHz,
C2HCl3) 2.73 (2H, m, CH2S), 3.02 (1H, d, J3R,2 4.3, H-3R), 3.71
(2H, m, CH2OH), 3.78 (3H, s, OCH3), 4.63 (1H, m, H-2), 5.14
(2H, OCH2Ar), 5.73 (1H, d, J 6.5, NH) and 7.36–7.38 (5H, s,
ArH); dD (38.4 MHz, CHCl3) 3.00 (12H, s, 2H-3S); dC (75.5 MHz,
C2HCl3) 36.9 (t, C-3) 38.6 (SCH2), 55.2 (CH3O), 56.4 (C-2), 63.2
(CH2OH), 69.7 (OCH2Ar), 131.1–130.6 (Ar), 138.5 (ipso Ar),
158.3 (urethane) and 173.5 (ester).
Incubation of stereospecifically labelled samples of
D-b-chloroalanine with D-amino acid aminotransferase and
b-mercaptoethanol
Incubation of (2S)-b-chloroalanine (19) with D-amino acid
aminotransferase in the presence of b-mercaptoethanol. This
was carried out following the method for the L-aspartate
aminotransferase incubations but using D-amino acid amino-
transferase (40 units/ml) and (2S)-b-chloroalanine 1932 (10 mg,
0.072 mmol). The optical rotation of the white solid was
measured to give the value aD28.5 +0.021; dH (300 MHz, 10%
Methyl (2R,3R)-[2,3-2H2]-N-carbobenzyloxy-b-hydroxyethyl-
cysteineate. This was prepared as described above using methyl
(2S,3S)-[2,3-2H2]-N -carbobenzyloxyaziridine-2-carboxylate
14a (50 mg, 0.21 mmol) to yield the product (16.2 mg, 20%);
2HCl in 2H2O, Fig. 4a) 2.74 (2H, dt, J2 ,1 6.1, J2 ,OH 1.5, CH2S),
2.90 (1H, dd, J3R,2 7.4, J3R,3S 15.2, H-3R), 3.11 (1H, dd, J3S,2 4.5,
J
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3S,3R 15.0, H-3S), 3.61 (2H, dt, J1 ,2 6.1, J1 ,OH 2.2, CH2OH) and
30
[a]D +9.01 (c 1, CHCl3); m/z [+ve FAB (3-NBA)] 316 ([M +
4.23 (1H, dd, J2,3R 7.4, J2,3S 4.5, H-2).
H]+); mmax (film)/cm−1 3354 (OH, br) and 1718 (urethane,
amide); dH (300 MHz, C2HCl3) 2.69 (2H, m, CH2S), 3.01 (1H, s,
H-3S), 3.69 (2H, m, CH2OH), 3.76 (3H, s, OCH3), 5.11 (2H,
OCH2Ar), 5.70 (1H, s, NH) and 7.33–7.34 (5H, s, ArH); dD
(38.4 MHz, CHCl3) 2.92 (12H, s, 2H-3R) and 4.60 (12H, s, 2H-2);
dC (75.5 MHz, C2HCl3) 34.9 (t, C-3) 36.8 (SCH2), 53.4 (OCH3),
54.6 (t, C-2), 61.3 (CH2OH), 67.8 (OCH2Ar), 128.8–129.2 (Ar),
136.6 (ipso Ar), 156.5 (urethane) and 171.7 (ester).
Incubation of (2S,3S)-[3-2H1]-b-chloroalanine (19b) with D-
amino acid aminotransferase in the presence of b-mercapto-
ethanol. This was carried out as above using (2S,3S)-[3-2H1]-
b-chloroalanine 19b16 (10 mg, 0.072 mmol) prepared by total
synthesis; dH (300 MHz, 10% 2HCl in 2H2O, Fig. 4b) 2.74 (2H,
ꢀ
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unresolved dt, CH2S), 3.07 (1H, m, H-3R), 3.82 (2H, dt, J1 ,2
ꢀ
6.0, J1 ,OH 2.1, CH2OH) and 4.23 (1H, d, J2,3R 7.0, H-2).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 3 5 7 – 3 3 6 4
3 3 6 3