1500
A. Arcelli et al. / Tetrahedron: Asymmetry 16 (2005) 1495–1501
5.3.3. (3S,6R,20R)-3-(2,3-Dihydroxy-propyl)-6-methyl-4-
[(S)-1-phenethyl]-morpholine-2,5-dione, 11. The prod-
uct, obtained starting from 7, was isolated as a pure so-
5.4.3. (3S,5R)-5-Hydroxymethyl-3-[(S)-1-phenethyl-
amino]-dihydrofuran-2-one, 15. The product was ob-
tained as a pure wax in 50% overall yield. H NMR: d
1
1
lid (mp 147–148 °C). H NMR: d 1.42 (d, 3H, J = 6.6),
1.42 (d, 3H, J = 6.6), 1.85–2.05 (m, 2H), 3.56 (dd, 1H,
J = 4.5, 12), 3.62 (t, 1H, J = 8.7), 3.81 (dd, 1H, J = 3,
12), 4.13 (q, 1H, J = 6.6), 4.58 (m, 1H), 7.35 (5ArH).
13C NMR: d 24.4, 32.7, 55.1, 57.7, 64.1, 78.4, 127,
127.3, 128.4, 144.7, 178.2. IR (CHCl3) m (cmÀ1) = 3614
(OH), 1772 (C@O). [a]D = À94.4 (c 0.4, CHCl3). Anal.
Calcd for C13H17NO3: C, 66.36; H, 7.28; N, 5.95.
Found: C, 66.65; H, 7.26; N, 5.98.
1.71 (d, 3H, J = 7.2), 2.42 (m, 1H), 2.55 (m, 1H), 3.58
(dd, 1H, J = 3.3, 12.3), 3.9 (dd, 1H, J = 8.1, 10.8), 3.93
(dd, 1H, J = 2.4, 12.3), 4.61 (q, 1H, J = 6.6), 4.84 (m,
1H), 5.14 (q, 1H, J = 7.2), 7.3 (m, 5ArH). 13C NMR
(CD3OD): d 20.1, 20.8, 31, 53.8, 56.1, 64.7, 66.4, 79.9,
128, 128.1, 129.5, 140.9, 175.1, 176.2. [a]D = À39 (c 1,
CH3OH). Anal. Calcd for C16H21NO5: C, 62.53; H,
6.89; N, 4.56. Found: C, 62.72; H, 6.86; N, 4.57.
5.4.4. (3S,5S)-5-Hydroxymethyl-3-[(S)-1-phenethyl-
amino]-dihydrofuran-2-one, 16. The product was iso-
lated as an oil in 48% overall yield. H NMR: d 1.42
5.3.4. (3S,6R,20S)-3-(2,3-Dihydroxy-propyl)-6-methyl-4-
[(S)-1-phenethyl]-morpholine-2,5-dione, 12. The prod-
uct, obtained starting from 8, was isolated as a pure
solid (mp 116–117 °C). 1H NMR: d 1.46 (d, 3H,
J = 6.6), 1.7 (d, 2H, J = 7), 2.5(m, 2H), 3.5(m, 4H),
4.5(m, 1H), 4.6 (q, 1H, J = 6.6), 5.19 (q, 1H, J = 7),
7.4 (m, 5ArH). 13C NMR: d 20, 21.6, 28.5, 53.3, 54.7,
63.8, 64.9, 76.4, 126.7, 128.1, 129, 138.1, 172.2, 174.7.
[a]D = À5 3 c( 1, CHCl3). Anal. Calcd for C16H21NO5:
C, 62.53; H, 6.89; N, 4.56. Found: C, 62.45; H, 6.87;
N, 4.54.
1
(d, 3H, J = 6.6), 1.76 (m, 1H), 2 (m, 1H), 3.49 (dd,
1H, J = 8.4, 10.2), 3.55 (dd, 1H, J = 4.2, 12.6), 3.83
(dd, 1H, J = 3, 12.6), 4.17 (q, 1H, J = 6.6), 4.35(m,
1H), 7.35(A5 rH).
13C NMR: d 24.3, 32.4, 56.2, 58,
63.4, 78, 127.1, 127.5, 128.6, 144.1, 176.9. IR (CHCl3)
m (cmÀ1) = 3620 (OH), 1775(C @O). HPLC–MS: 236.1
[M+1]+, 258.1 [M+Na]+. The product was not isolated
in sufficiently pure form to measure the specific rotation.
5.5. (2R,S)(6S)-4N-[(S)-1-Phenethyl]-2-hydroxy-6-
methyl-1,4-morpholin-5-one, 17
5.4. Conversion of diols 9–12 into c-lactones 13–16
A solution of diol 9–12 (1.5g, 5mmol) in 100 mL of
isopropanol was cooled at 0 °C and then saturated with
NH3 by bubbling for about 30 min. The reaction flask
was stopped and kept for 3 days at rt. After testing by
TLC, the ammonia and the organic solvent were evapo-
rated in vacuo. The residue was submitted to cyclization
by refluxing in 50 mL of toluene/ethanol = 9:1 for 24 h
and the reaction monitored by TLC. The organic sol-
vents were evaporated to dryness under vacuum and
the residue submitted to silica gel chromatography elut-
ing with ethyl acetate.
Compound 17 was obtained in about 80% yield by the
reduction of 1 with 2 M LiBH4 in THF at rt overnight.8
The oily product was isolated as a diastereomeric mixture
1
(ꢀ1:1) nonseparable by silica gel chromatography. H
NMR: d 1.53 (d, 3H, J = 6.9), 1.55 (d, 3H, J = 6.9), 1.56
(d, 3H, J = 6.9), 1.59 (d, 3H, J = 6.9), 2.88 (m, OH),
2.92 (dd, 1H, J = 2.4, 12), 3.03 (dd, 1H, J = 2.7, 12),
3.22 (dd, 1H, J = 7.5, 12), 3.37 (m, OH), 3.46 (dd, 1H, J =
3.9, 12), 4.37 (q, 1H, J = 6.9), 4.58 (q, 1H, J = 6.9), 5.04
(m, 1H), 5.34 (m, 1H), 7.35 (m, 5ArH). 13C NMR: d 15.1,
15.7, 17.8, 18.3, 44.8, 45.6, 49.7, 50, 67.3, 71.8, 80.2, 90.5,
127.1, 127.5, 127.7, 128.5, 128.6, 139.3, 139.4, 168.9,
169.6. HPLC–MS: 236.3 [M+1]+, 258.3 [M+Na]+.
5.4.1.
(3R,5S)-5-Hydroxymethyl-3-[(S)-1-phenethyl-
amino]-dihydrofuran-2-one, 13. The pure product was
isolated as an oil in 45% overall yield. 1H NMR: d
1.43 (d, 3H, J = 6.6), 2.18–2.4 (m, 2H), 3.56 (t, 1H,
J = 8.7), 3.6 (dd, 1H, J = 4.8, 12), 3.82 (dd, 1H, J = 3,
12), 3.84 (q, 1H, J = 6.6), 4.68 (m, 1H), 7.35(m,
5ArH). 13C NMR: d 24, 31.6, 53.7, 56.3, 64.1, 78.4,
5.6. Enzyme kinetics
5.6.1. Materials. a-Glucosidase (EC 3.2.1.20) from
bakerÕs yeast, b-glucosidase (EC 3.2.1.2) from almonds,
a-mannosidase (EC 3.2.1.24) from jack bean, a-galacto-
sidase (EC 3.2.1.22) from green coffee beans, p-nitro-
phenyl glucosides, and 4-(2-hydroxyethyl)-1-piper-
azinethanesulfonic acid and potassium salt (HEPES)
were purchased from Sigma.
126.4, 127.4, 128.7, 143.8, 178. IR (CHCl3)
m
(cmÀ1) = 3622 (OH), 1773 (C@O). [a]D = À4.8 (c 0.6,
CHCl3). Anal. Calcd for C13H17NO3: C, 66.36; H,
7.28; N, 5.95. Found: C, 66.45; H, 7.3; N, 5.95.
5.4.2. (3R,5R)-5-Hydroxymethyl-3-[(S)-1-phenethyl-
amino]-dihydrofuran-2-one, 14. The product was
recovered as an oil in 47% overall yield. H NMR: d
5.6.2. Kinetics. The kinetic hydrolyses of glucosides
were carried out at pH = 6.85in the presence of 0.1 M
of HEPES buffer solution and 0.05–0.2 units of enzyme.
Stock solutions of various inhibitors were prepared by
dissolving the substrates at 0.01–0.04 M concentration
in EtOH, H2O or EtOH/H2O, and EtOH/DMSO mix-
tures. Stock solution (10–100 lL) were added to enzyme
buffered solutions contained in 10–12 cells placed in the
multicell holder accessory of a Cary100 UV spectropho-
tometer and thermostated at 37 0.01 °C for 15min.
Then, appropriate aliquots of a glucoside solution
contained in 10–12 Hamilton syringes, thermostated at
1
1.41 (d, 3H, J = 6.6), 2 (m, 1H), 2.38 (m, 1H), 3.4 (dd,
1H, J = 9, 10.2), 3.62 (dd, 1H, J = 4.8, 12.6), 3.81 (q,
1H, J = 6.6), 3.88 (dd, 1H, J = 3, 12.6), 4.33 (m, 1H),
7.35(5ArH). 13C NMR: d 24.1, 31.4, 54.7, 56.3, 63.5,
78.4, 126.4, 127.6, 128.9, 143.2, 176.6. IR (CHCl3)
m
(cmÀ1) = 3610 (OH), 1770 (C@O). HPLC–MS:
236.1 [M+1]+, 258.1 [M+Na]+. The product was not
isolated in sufficiently pure form to measure the specific
rotation.