Monatin and Its Stereoisomers: Chemoenzymatic Synthesis and Taste Properties
FULL PAPER
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Phenylethyl isocyanate (102 mg, 0.69 mmol) was added to a solu-
tion of 5 (200 mg, 0.58 mmol) and 60% NaH (24 mg, 0.58 mmol)
in dry THF cooled to 0 °C. The reaction was warmed up to room
temperature and, after 2 h, heated at reflux for 4 h. A solution of
brine was added, the THF evaporated in vacuo and the aqueous
phase extracted with AcOEt, dried with Na2SO4, and the solvent
evaporated. The crude product was purified by flash chromatog-
raphy (hexane/AcOEt, 7:3) and crystallised from cyclohexane/
CH2Cl2 to give 240 mg of pure diester 14 (84%). M.p. 78–80 °C.
Synthesis of Monatin Stereoisomers: The synthesis of compounds
2,[2] 3[14] and 4[2] (Scheme 1) has been described in the literature;
the hydrogenolysis reactions of diacid isoxazolines 9 and 10 have
been reported[2] on the racemic mixture.
Enzymatic Hydrolysis of Diethyl 5-(3-Indolylmethyl)-4,5-dihydrois-
oxazole-3,5-dicarboxylate (4): Protease (250 mg) from Aspergillus
oryzae (Sigma type XXII, 3.6 U/mg) was suspended in a two-phase
system prepared from phosphate buffer (25 mL, 0.1 m, pH 7.0) and
toluene (19 mL). Substrate 4 (1.09 g, 3.2 mmol) in toluene (6 mL)
was added and the pH was kept constant by addition of 0.1 n
NaOH. The reaction was monitored by chiral HPLC analysis of
the organic phase. After 80 h the phases were separated, the organic
phase was dried with Na2SO4 and the solvent removed in vacuo to
obtain 453 mg of 5 (41%). The aqueous phase was acidified to pH
3 with 5% HCl and extracted with AcOEt. The organic phase was
washed with brine, dried with Na2SO4, filtered and the solvent re-
moved under reduced pressure. The crude product was purified by
flash chromatography (eluent: CH2Cl2/MeOH, 7:3) to obtain
242 mg of 6 (24%).
1
[α]2D5 = –7.5 (c = 0.64, MeOH). H NMR (CDCl3): δ = 1.21 (t, J =
7.17 Hz, 3 H, OCH2CH3), 1.35 (t, J = 7.17 Hz, 3 H, OCH2CH3),
1.62 (d, J = 6.80 Hz, 3 H, CH3), 3.21 (d, J = 18.2 Hz, 1 H), 3.42
(2d, J = 15.26 Hz, 2 H), 3.61 (d, J = 18.2 Hz, 1 H), 4.18 (q, J =
7.17 Hz, 2 H, OCH2CH3), 4.28 (q, J = 7.17 Hz, 3 H, OCH2CH3),
5.22 (m, 1 H, CH), 5.82 (d, J = 6.99 Hz, 1 H, NH), 7.10–7.50 (m,
8 H, indole and Ar), 7.52 (d, J = 7.54 Hz, 1 H, Ar), 8.05 (d, J =
8.27 Hz, 1 H, Ar) ppm. MS: m/z (%) = 491 (30) [M – 2], 473 (5),
403 (5), 388 (30), 345 (10), 146 (5), 145 (40), 105 (40), 91 (100), 77
(60).
Diethyl 5-[1–4-Iodobenzoyl)-2,3-dihydro-1H-indol-3-ylmethyl]-4,5-
dihydroisoxazole-3,5-dicarboxylate (15): DCC (400 mg, 1.94 mmol)
and a catalytic amount of DMAP were added to a solution of 4-
iodobenzoic acid (471 mg, 1.9 mmol) in dry CH2Cl2 (5 mL) under
nitrogen. After 1 h at reflux a solution of 5 (98 mg, 0.286 mmol) in
dry CH2Cl2 (2.3 mL) was added, the reaction mixture heated at
reflux for three days. It was then filtered and the solvent evaporated
in vacuo. The crude product was purified by flash chromatography
(hexane/AcOEt, 8:2) to give 98.5 mg of 15 as a white solid (60%).
M.p. (EtOH) 111–114 °C. [α]2D5 = –11.0 (c = 0.79, CH2Cl2). 1H
NMR (CDCl3): δ = 1.25 (t, J = 7.2 Hz, 3 H, CH3), 1.35 (t, J =
7.2 Hz, 3 H, CH3), 3.18 (d, J = 18.5 Hz, 1 H), 3.48 (2d, J = 15.6 Hz,
2 H, indole-CH2), 3.58 (d, J = 18.5 Hz, 1 H), 4.18 (q, J = 6.7 Hz,
2 H, CH2) 4.28 (q, J = 6.7 Hz, 2 H, CH2), 7.10–8.50 (m, 9 H,
indole and ar) ppm. MS: m/z (%) = 574 (5) [M+], 360 (40), 231
(100), 203 (20), 104 (15), 76 (18).
Diethyl (3R)-5-(3-indolylmethyl)-4,5-dihydroisoxazole-3,5-dicarbox-
ylate (5): M.p. 104–105 °C. [α]2D5 = –55.1 (c = 0.59, CHCl3); ee Ͼ
97% (HPLC, Chiralcel OD). 1H NMR (CDCl3): δ = 1.3 (2t, J =
7.2 Hz, 6 H, OCH2CH3), 3.38 (dd, J = 18.7 Hz, 2 H, CH2), 3.47
(dd, J = 15, J = 21.4 Hz, 2 H, indole-CH2), 4.25 (2q, J = 7.2 Hz,
4 H, OCH2CH3), 7.1–7.7 (m, 5 H, indole), 8.1 (br. s, 1 H, NH)
ppm. MS: m/z (%) = 344 (20) [M+], 299 (10), 271 (15), 243 (40),
130 (100).
Ethyl
(3S)-5-(3-Indolylmethyl)-5-carboxy-4,5-dihydroisoxazole-3-
carboxylate (6): M.p. 91–92 °C. [α]2D5 = +3.75 (c = 0.24, MeOH);
1
ee Ͼ 87%. H NMR ([D6]acetone); δ = 1.25 (t, J = 7.3 Hz, 3 H,
OCH2CH3), 2.85 (m, 2 H, indole-CH2), 3.3 (m, 2 H, CH2), 4.25
(q, J = 7.3 Hz, 2 H, OCH2CH3), 6.9–7.7 (m, 5 H, indole) ppm.
MS: m/z (%) = 316 (M+, 20), 272 (40), 255 (18), 203 (15), 181 (15),
167 (15), 130 (100), 103 (5), 77 (5).
3-Ethyl 5-(2-Isopropyl-5-methylcyclohexyl)-5-(2,3-dihydro-1H-indol-
(3R)-5-(3-Indolylmethyl)-4,5-dihydroisoxazole-3,5-dicarboxylic Acid
(7): A solution of KOH 10% (3.54 mmol) was added to a solution
of 5 (405 mg, 1.56 mmol) in 9 mL of aqueous 80% EtOH. After
2.5 h at room temperature and in the dark the mixture was acidified
to pH 3 by addition of 10% HCl, treated with brine and extracted
with AcOEt. The organic phase was dried with Na2SO4, filtered
and the solvent evaporated in vacuo to give 208 mg of diacid 7
3-ylmethyl)-4,5-dihydroisoxazole-3,5-dicarboxylate
(16):
DCC
(55 mg, 0.27 mmol), a catalytic amount of DMAP and (–)-menthol
(46 mg, 0.29 mmol) were added to a stirred solution of 6 (85 mg,
0.27 mmol) in 5.7 mL of CH2Cl2. The reaction mixture was stirred
at room temperature for 3 d. The organic phase was washed with
water and dried with Na2SO4, filtered and the solvents evaporated.
The crude product was purified by flash chromatography (hexane/
AcOEt, 7:3) to give 63 mg of 16 (52%). M.p. (hexane/CH2Cl2) 118–
119 °C. [α]2D5 = –2.4 (c = 1.45, CH2Cl2). 1H NMR (CDCl3): δ =
0.67 (d, J = 6.75 Hz, 3 H, CH3), (d, J = 6.75 Hz, 3 H, CH3), 0.90
(d, J = 6 Hz, 3 H, CH3), 1.28 (t, J = 6.7 Hz, 3 H), 1.20–1.90 (m, 9
H, menthyl), 3.18 (dd, J = 18.7 Hz, 1 H), 3.48 (2d, J = 15 Hz, 2
H, CH2-indole), 3.58 (d, J = 18.7 Hz, 1 H), 4.28 (q, J = 6.7 Hz, 2
H), 4.73 (m, 1 H), 7.10–7.65 (m, 5 H, indole), 8.1 (br. s, 1 H, NH)
ppm. MS: m/z (%) = 454 (40) [M+], 317 (5), 271 (5), 243 (20), 197
(5), 130 (100), 83 (5).
(62%), m.p. 166–168 °C. [α]2D5 = –6.7 (c = 0.18, MeOH). H NMR
1
(CDCl3, [D6]acetone): δ = 3.4 (dd, J = 18.7 Hz, 2 H, CH2), 3.45 (s,
2 H, indole-CH2), 6.8–7.5 (m, 5 H, indole), 10.15 (br. s, 1 H, NH)
ppm. MS: m/z (%) = 244 (18), 227 (20), 199 (10), 154 (37), 131
(20), 130 (100), 103 (10), 89 (10), 77 (18), 63 (10), 51 (10).
(3S)-5-(3-Indolylmethyl)-4,5-dihydroisoxazole-3,5-dicarboxylic Acid
(8): A solution of 10% KOH (1.77 mmol) was added to a solution
of 6 (270 mg, 0.78 mmol) in 6 mL of aqueous 80% EtOH. After
2.5 h at room temperature in the dark the mixture was acidified to
pH 3 by addition of 10% HCl, treated with brine and extracted
with AcOEt. The organic phase was dried with Na2SO4, filtered
and the solvent evaporated in vacuo to give 139 mg of diacid 8
The hydrogenolysis of isoxazoline diacids 7 and 8 gave a mixture
of diastereoisomers 9 (71%) and 10 (60%) respectively. The mix-
tures were resolved by semi-preparative HPLC.
(62%), m.p. 166–168 °C. [α]2D5 = +4.3 (c = 0.11, MeOH). H NMR
1
1
(2R,4R)-Monatin (12): [α]2D5 = –3.85 (c = 0.78, MeOH). H NMR
(CDCl3 with [D6]acetone): δ = 3.4 (dd, J = 18.7 Hz, 2 H, CH2),
3.45 (s, 2 H, indol-CH2), 6.8–7.5 (m, 5 H, indole), 10.15 (br. s, 1
H, NH) ppm. MS: m/z (%) = 244 (18), 227 (20), 199 (10), 154 (37),
131 (20), 130 (100), 103 (10), 89 (10), 77 (18), 63 (10), 51 (10).
(D2O): δ = 2.05 (dd, J = 15.2, J = 11.7 Hz, 1 H, H-3a), 2.70 (dd,
J = 15.2, J = 2.0 Hz, 1 H, H-3b), 3.08 (d, J = 14.7 Hz, 1 H, H-5a),
3.28 (d, J = 14.7 Hz, 1 H, H-5b), 3.63 (dd, J = 11.7, J = 2.0 Hz, 1
Diethyl 5-[1-(1-Phenylethylcarbamoyl)-2,3-dihydro-1H-indol-3-yl- H, H-2,), 7.0–7.8 (m, 5 H, indole) ppm. LCMS (ESI): m/z (%) =
methyl]-4,5-dihydroisoxazole-3,5-dicarboxylate (14): (R)-(+)-1- 293 (100) [M + 1], 274 (5), 257 (10), 121 (40). HRMS (ESI, pos):
Eur. J. Org. Chem. 2005, 1652–1658
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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