2
064
A. Liljeblad et al. / Tetrahedron: Asymmetry 12 (2001) 2059–2066
1
1
(
3
±)-6: H NMR: l (ppm) 0.93 (t, 6H, 2×CH
6
3), 2.05 (s,
Trisodium 2-[(carboxymethyl)oxy]succinate: H NMR:
l (ppm) 2.38 (2×q, 2H, CHCH CO ), 3.75 (q, 2H,
H, CH CONH), 2.15 (m, 1H, CH(CH ) ), 3.75 (s, 3H,
6
6
6
3
3 2
2
2
13
CO CH ), 4.57 (dd, 1H, CH
6
CO Me), 6.15 (d, 1H, NH
6
),
CH O), 3.92 (dd, 1H, CH
6
6
), C NMR: l (ppm) 42.6
H ), 79.9 (CH), 178.7 (CO2),
2
3
2
2
1
3
C NMR: l (ppm) 17.8 (CH(C
6
H ) ), 18.8 (CH(C
6
H ) ),
3 2
(CHC
6
H CO), 69.9 (OC
6
6
6
3
2
2
2
+
2
(
3.1 (C
6
H CONH), 31.2 (C
6
H(CH ) ), 52.0 (C
6
H ), 57.0
180.4 (C6 O ), 180.6 (C6 O ). M =258.
2 2
3
3 2
3
C6
HCO Me), 170.0 (C
6
O Me), 172.7 (C
6
O Me). Elemen-
2
2
2
tal analysis: obs. C, 55.52%; H, 8.88%; N, 8.05%, calcd
C, 55.47%; H, 8.73%; N, 8.09% for C H NO . M =
4.5. Determination of enzymatic regioselectivity
+
8
15
3
1
73.
The mixtures of the trimethyl ester and three dimethyl
monobutyl regioisomers in addition to three dibutyl
monomethyl regioisomers and tributyl ester were
obtained when 3 was dissolved in butanol in the pres-
ence of sulfuric acid as a catalyst and the solution was
stirred at room temperature for 1 h. Analysis by GLC
showed the retention times for the components in the
reaction mixture. Regioselectivity of the enzymatic
reaction was then confirmed by preparing chemically all
three monobutyl regioisomers (8, 24 and 25, Scheme 4)
and comparing the place of the peaks in the chro-
matogram with those of the enzymatic reaction.
4
.3.5. Synthesis of 7. Compound 7 (4.557 g, 22.2 mmol)
was prepared by esterifying (±)-N-acetylmethionine
(
5.402 g, 28.2 mmol) with thionyl chloride (2.46 mL,
3.7 mmol) in methanol (75 mL) and purifying in the
3
1
same manner as compound 6. (±)-7: H NMR: l (ppm)
.96 (s, 3H, CH3CONH), 2.00 (m, 2H, CH CH S) 2.04
s, 3H, CH S), 2.46 (t, 2H, CH CH
1
6
6
2
2
(
6
6
S), 3.71 (s, 3H,
2
3
2
CO CH ), 4.67 (m, 1H, CH
6
CO Me), 6.40 (s, 1H, NH
6
),
2
3
2
1
3
C NMR: l (ppm) 15.4 (C
6
H S), 23.0 (C
H CH S) 51.4 (C
6
H CONH),
3
3
2
9.9 (C
6
H S), 31.6 (C
6
6
H3), 52.4
(C6 HCO Me), 169.9 ( C6 O Me), 172.5 (C6 O Me). Elemen-
2 2 2
2
2
2
tal analysis: obs. C, 46.31%; H, 7.42%; N, 6.91%,
calculated C, 46.81%; H, 7.37%; N, 6.82% for
Synthetic routes to compounds 8, 24 and 25 are
depicted in Scheme 4. a-Methyl malic acid 18 was
synthesized from 16 and the structure confirmed by
preparing intramolecular lactone 20 by known meth-
+
C H NO S. M =205.
8
15
3
22
4.4. Enzymatic reactions
ods. a-Butyl malic acid 19 was synthesized in a similar
manner. Esterifications to methyl butyl esters 22 and 23
were performed in neat alcohol with dicyclohexylcar-
bodiimide (DCC, 1 equiv.) and DMAP (0.05 equiv.).
Compounds were purified by column chromatography
as described in Section 4.3.
The reactions were typically performed as small-scale
experiments where one of the substrates 3–7 (3–5: 0.1
M, 6 and 7: 0.05 M) was dissolved in butanol or water
(
2 mL). The enzyme preparation (as a best compromise
7
5 mg/mL for 3–5 and 40 mg/mL for 6 and 7) was
1
added in order to start the reaction and the mixture was
shaken at room temperature (25°C).
H NMR 22: l (ppm) 0.88 (t, 3H, CH3
6 CH ), 1.33 (m,
2
2H, CH CH
2
CO CH
13.6 (C
6
), 1.56 (m, 2H, CH CH CH
6
2), 2.77 (2×q,
H, CHCH6 CO Me), 3.76 (s, 3H, CH6 ), 4.06 (t, 2H,
2 2 3
13
3
2
3
2
For gram-scale resolution, acylase I on Eupergit C (4.5
g) was added on 3 (1.375 g, 5.9 mmol) in butanol (129
mL) and dihexyl ether (1.379 mL, 5.9 mmol). After 53
h the enzyme was filtered off at 55% conversion. Purifi-
cation by column chromatography using ace-
tone:petroleum ether (2:8) yielded the unreacted
6
CH ), 4.46 (dd, 1H, CH
6
), C NMR: l (ppm)
2
2
2
6
H CH ), 19.0 (CH C
6
H ), 30.4 (CH CH CH2),
2
6
3
2
3
3
2
38.6 (CH6 CO Me), 52.7 (C6 H ), 64.8 (CO C6 H ), 67.2
2 2 3 2 2
+
(C6 H), 170.6 (C6 O Me), 173.7 (C6 O Me). [M+H] =205.
2 2
1
H NMR 23: l (ppm) 0.94 (t, 3H, CH3
6 CH ), 1.36 (m,
2
20
trimethyl ester (S)-3 (0.479 g, 2.1 mmol, e.e. 96%, [h]D
2H, CH CH ), 1.66 (m, 2H, CH CH CH
6
6
2), 2.83 (2×q,
3
2
3
2
−
63.0 (c=1, MeOH)) and a-butyl ester (R)-8 (0.575 g,
2H, CHCH
6
CO Me), 3.25 (d, 1H, CH), 3.72 (s, 3H,
2
2
2
0
2
.1 mmol, e.e. 78%, [h] +49.0 (c=1, MeOH)). Spec-
CH
6
), 4.25 (t, 2H, CO CH
C NMR: l (ppm) 13.5 (C
6
CH ), 4.51 (dd, 1H, CH
H CH ), 18.9 (CH C
6
),
H ),
2
D
3
2
2
2
13
troscopic data for (S)-3 are in accordance with those
for (±)-3. The hydrolysis of (S)-3 (243 mg, 1.04 mmol)
with sodium hydroxide (125 mg, 3.13 mmol) yielded
trisodium (S)-2-[(carboxymethyl)oxy]succinate (258 mg,
6
6
3
2
3
30.4 (CH CH C
65.8 (CO C
6
H ), 38.5 (CH
6
CO Me), 51.9 (C
6
H3),
O Me), 173.7
3
2
2
2
2
6
H ), 67.2 (C
6
H), 170.6 (C
6
2
2
2
(C6 O Me). Elemental analysis: obs. C, 53.17%; H,
2
2
0
1
.00 mmol, e.e. 96%, [h] −20.6 (c=0.2, H O)). Enan-
7.92%, calcd C, 52.93%; H, 7.90% for C H O .
9 16 5
D
2
tiopurity was determined by esterifying the product
back to (S)-3.
Monobutyl esters 24, 25 and 8 were synthesized from
1 (13.3 mL, 0.10 mmol), 22 (20 mg, 0.10 mmol) and 23
2
1
(
(
(
R)-8: H NMR: l (ppm) 0.94 (t, 3H, CH
6
CH ), 1.38
(20 mg, 0.10 mmol), respectively, with the aid of sil-
ver(I) oxide (70 mg, 0.30 mmol) in given organic sol-
vents. The amounts of butyl and methyl 2-iodoacetates
were 97 mg (0.40 mmol) and 60 mg (0.30 mmol),
respectively. Reactions took place for 4 days and the
products were detected by the GLC method and con-
firmed by GLC–MS. The products were not isolated.
3
2
m, 2H, CH CH
6
), 1.64 (m, 2H, CH CH CH
6
2), 2.89
2×q, 2H, CH6 CO Me), 3.72 (s, 3H, CH6 ), 3.75 (s, 3H,
2 2 3
3
2
3
2
CH
6
), 4.18 (m, 2H, CO CH ), 4.31 (q, 2H, CH
6
6
2O), 4.41
2
3
2
2
1
3
(
(
(
(
m, 1H, CH
6
), C NMR: l (ppm) 13.7 (C
6
H CH ), 19.1
H ), 37.6 (CHC
3
CH C
C
6
H ), 30.5 (CH CH C
6
6
H CO), 51.9
3
2
3
(C
2
2
2
6
H ),
52.0
6
H3),
65.4
(CO C
6
H2),
68.1
O2),
3
2
OC6 H CO Me), 75.5 ( C6 H), 170.1 (C6 O ), 170.4 (C6
2 2 2
1
7
70.6 (C
6
O ). Elemental analysis: obs. C, 52.37%; H,
2
Methyl 2-iodoacetate was synthesized from methyl 2-
bromoacetate (6.4 mL, 68 mmol) by dissolving it into
acetone (75 mL) and adding sodium iodide (10.17 g, 68
.24%, C, calcd 52.17%; H, 7.30% for C H O . [M+
1
2
20
7
+
H] =277.