20
Compound H was amorphous, [α]
−30.0 3° (c 1.16, CH OH).
3
D
20
Compound 1 (secologanin) was crystallized from isopropanol:hexane, [α]
−84.8 3° (c 0.5, CH OH).
3
D
+
Mass spectrum (m/z, I , %): 389.4 (100) [M] , 227 (17.4), 177.4 (43.5), 149.5 (56.5).
Compound 2 (loganin) was crystallized from hot isopropanol, mp 213-218°C, [α]
rel
20
−109 2° (c 1.0, CH OH).
D
3
Mass spectrum (m/z, I , %): 408.5 (80), 391.7 (22.5), 229.6 (100), 197.3 (10), 179.4 (65).
rel
20
Compound 3 (loganigenin) was amorphous, [α]
−27.0 3° (c 1.12, CHCl ), R 0.23 (CHCl :CH OH 97:3).
f
3 3 3
D
PMR spectrum of loganin aglycon (400 MHz, DMSO-d , δ, ppm, J/Hz): 1.05 (3H, d, J = 8.0, CH ), 1.35 (1H, m, H-9),
6
3
1.65 (2H, m, H-6), 2.05 (1H, dd, J = 6.0, 6.0, H-8), 2.95 (1H, m, H-5), 3.65 (3H, m, OCH ), 3.90 (1H, d, J = 2.0, H-7), 4.45 (1H,
3
d, J = 3.0, OH-7), 4.75 (1H, t, H-1), 7.2 (1H, d, J = 7.0, OH-1), 7.4 (1H, s, H-3).
20
Compound 4 (glucologanin) was amorphous, [α]
−80.0 3° (c 0.2, CH OH).
D
3
Mass spectrum (m/z, I , %): 591.6 (10), 575.3 (4), 391.5 (20), 229 (33), 212 (30), 193 (20), 179 (10), 149.3 (100).
rel
PMRspectrum ofglucologanin (400 MHz, DMSO-d , δ, ppm, J/Hz): 1.2 (3H, s, CH -10), 1.75 (2H, m, H-6), 2.20 (1H,
6
3
m, H-9), 2.65 (1H, m, H-5), 4.0 (2H, s, CH-1′, CH-1″), 4.6 (4H, dm, J = 7.8, CH -6′, CH -6″), 7.1 (1H, s, H-3).
2
2
+
Compound 5. Mass spectrum ofloganin dimer (5) (m/z, I , %): 798.3 (18), 781.4 (6) [M] , 766.2 (6), 601.4 (2), 408.3
rel
(43), 391.1 (10), 229.2 (100), 211.1 (9), 197.2 (7), 179.1 (16), 149.1 (5).
+
Compound 8. Mass spectrum of 8 (m/z, I , %): 422.2 (45), 405.4 (35) [M] , 389.2 (90), 243.2 (100), 225.2 (86), 211.0
rel
(29), 165.3 (49), 151.0 (40).
PMR spectrum (400 MHz, DMSO-d , δ, ppm): 1.2 (3H, s, CH -10), 2.7 (2H, m, H-5, H-11), 3.7 (3H, m, OCH ), 7.3
6
3
3
(1H, s, H-3).
IR spectrum (KBr, ν, cm−1): 1696 (C=O), 1655 (C=O).
Partial Acid Hydrolysis of 4. Glycoside (15 mg) was dissolved in HCl solution (0.2 mL, 9%), and stored for 3 d at
room temperature. Then the hydrolysate was analyzed using TLC and CHCl :CH OH (60:40) with detection by anilinium
3
3
phthalate [11]. Reference samples were D-glucose, cellobiose, and gentiobiose. Sugars in the hydrolysate were D-glucose and
gentiobiose (5:1 ratio). This was a convenient method for analyzing sugars using TLC because it did not require preliminary
removal of acid.
Partial Enzymatic Hydrolysis of Glucologanin (4). Glycoside (50 mg) was dissolved in water (0.5 mL), treated with
enzyme preparation (30 mg) obtained from grapevine snail [12], and stored at room temperature (25°C) for 2 h. The enzyme
was precipitated with ethanol (see above). The filtrate was evaporated to dryness in vacuo. The solid was analyzed by TLC
using CHCl :CH OH (97:3) (detection of loganin aglycon) and CHCl :CH OH (84:16) (detection of loganin). Thus, 2 and 3
3
3
3
3
were identified.
Hydrolysis of Loganin (2). Loganin (2, 0.2 g) was dissolved in water (1.5 mL), treated with enzyme preparation
(0.15 g) obtained from grapevine snail [12], stored in a thermostat for 22 h at 38-40°C, treated with ethanol (9 mL), heated until
clear precipitation of the enzyme, and filtered. The filtrate was evaporated in vacuo. The solid was extracted with hot EtOAc
(2 × 5 mL). The EtOAc solution was cooled to room temperature (+20°C), filtered, concentraed to a volume of about 0.1 mL,
and left overnight for crystallization. The resulting crystals of the aglycon were separated, washed with EtOAc, and dried at
room temperature (+20°C) to afford loganin aglycon (0.1 g).
The solid that was insoluble in EtOAc was analyzed using PC and n-BuOH:HOAc:H O (4:1:2). The chromatographic
2
reference was an authentic sample of D-glucose. Detection used anilinium phthalate and heating [11]. Sugars appeared as
colored spots. It was found that this part of the hydrolysate contained D-glucose.
Synthesis of 6 and 7. Loganin (0.2 g) was dissolved in anhydrous pyridine (1.5 mL), treated with acetic anhydride
(1 mL), stored at room temperature (22-24°C) for 23 h, treated with icewater (10-fold amount relative to the reaction volume),
and stirred with cooling for 2 h. The resulting thick mass was ground with a glass rod until it converted to a crystalline mass
that was stored in the cold (+5°C) for 20 h. The precipitate was separated, thoroughly washed with cold water, and dried. The
resulting mixture of 6 and 7 (0.19 g) was chromatographed over a column (L = 47 cm, D = 1.2 cm) packed with silica gel in
a 1:80 ratio (mixture:silica gel). The eluent was a CHCl :CCl mixture (1:1, 200 mL), then CHCl :CCl + CH OH (1%).
3
4
3
4
3
Fractions containing pure compounds were combined and evaporated to dryness to afford 6 (0.097 g) and 7 (0.035 g).
20
Compound 6, mp 124-125°C (CCl ), [α]
−60.0 4° (c 0.4, CHCl :CH OH 83:17). PMR spectrum (400 MHz,
4
D
3 3
DMSO-d , δ, ppm): 1.15 (1H, m, H-9), 1.9 (15H, m, OCH -2′,3′,4′,6′,7), 3.65 (3H, m, OCH -12), 7.3 (1H, s, H-3). IR spectrum
6
3
3
(KBr, ν, cm−1): 1715 (C=O), 1758 (C=O), 1703 (C=O).
20
Compound 7 was amorphous, [α]
−59.9 4° (c 0.5, CHCl :CH OH 83:17).
D
3 3
43