Glycosylation of vanillin by amyloglucosidase in organic media

Article abstract of DOI:10.1016/j.tetlet.2005.11.114

Glycosylation of vanillin using amyloglucosidase from a Rhizopus mold with D-glucose, D-galactose, D-mannose, maltose, sucrose and D-sorbitol in di-isopropyl ether yielded glycosides in the range 13-53%. NMR spectral data confirmed linking between the phenolic OH of vanillin and C1 and/or C6 of the carbohydrate moieties.

Full text of DOI:10.1016/j.tetlet.2005.11.114

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 695–699
Glycosylation of vanillin by amyloglucosidase in organic media
Ramaiah Sivakumar and Soundar Divakar*
Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore 570 020, India
Received 25 August 2005; revised 17 November 2005; accepted 23 November 2005
Available online 7 December 2005
Abstract—Glycosylation of vanillin using amyloglucosidase from a Rhizopus mold with D-glucose, D-galactose, D-mannose, malt-
ose, sucrose and D-sorbitol in di-isopropyl ether yielded glycosides in the range 13–53%. NMR spectral data confirmed linking
between the phenolic OH of vanillin and C1 and/or C6 of the carbohydrate moieties.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Vanillin 1 (4-hydroxy-3-methoxybenzaldehyde)1 is used
as an additive in food and beverages (60%), considerable
amounts as flavour and fragrances (20–25%) and 5–10%
as an intermediate for pharmaceuticals. It possesses a
wide range of pharmacological activities such as anti-
microbial,2 anticarcinogenic,3 antioxidant,4 antifungal5
and antimutagenic.6 The solubility of vanillin in water
varies from 3 g/l at 4.4 ꢁC to 62.5 g/l at 80 ꢁC.7 Thus
the solubility and bioavailability of 1 limits its pharma-
cological applications. Glycosylation is a useful tool to
improve the water solubility and bioavailability8,9 of
vanillin.
up involved distilling off the solvent and maintaining
the reaction mixture at boiling water temperature for
5–10 min to denature the enzyme. The residue was
repeatedly extracted with chloroform to remove unre-
acted 1 and the dried residue, consisting of vanillin gly-
coside and unreacted mono- or disaccharide, was
subjected to HPLC analysis on an amino-propyl column
(3.9 · 300 mm length), eluted with 80:20 (v/v) acetoni-
trile/water with a flow rate of 1 ml/min and monitoring
with a RI detector. Conversion yields were determined
from HPLC peak areas of the glycoside and free mono-
or disaccharides and expressed with respect to mono- or
disaccharide concentrations. Errors based on the HPLC
measurements were of the order 10%. Glycosides were
isolated using a Sephadex G25 (100 cm · 1 cm) column,
eluting with water and subjected to spectroscopic
characterization.
The preparation of vanillin glycosides has been reported
by cell suspension culture,9 chemical10 and plant cell tis-
sue and organ culture methods.11 However, prepara-
tions by enzymatic methods have not been previously
reported. The present work describes an enzymatic
method using amyloglucosidase from a Rhizopus mold
for the preparation of glycosides with mono- and disac-
charides in a non-polar solvent.
Glycosylation of 1 was carried out with the following
mono- and disaccharides: pentoses—D-ribose and
D-arabinose; hexoses—D-glucose 2, D-galactose 3 and
D-mannose 4; ketoses—D-fructose; and disaccharides—
maltose 5, lactose and sucrose 6; carbohydrate alco-
hols—D-mannitol and D-sorbitol 7. Amyloglucosidase
exhibited maximum activity in non-polar solvents con-
taining a certain minimum amount of water to stabilize
its active conformation in a non-polar solvent compared
to other enzymes.12,13 Hence, glycosylation of 1 was
always carried out in the presence of buffers of certain
pH and concentration, worked out from a series of
reactions conducted with different pH, buffer concentra-
tions, substrate concentration, amyloglucosidase con-
centration and incubation period. Optimized reaction
conditions for the preparation of vanillin glucoside at
equimolar concentrations of 1 and 2 were found to be
A
typical synthesis involved reacting 1 (0.0005–
0.0025 mol) and a mono- or disaccharide (2–7, 0.0005–
0.0025 mol) at reflux with stirring in 100 ml of di-isoprop-
yl ether in the presence of 10–80% (by weight of 2–7)
amyloglucosidase and 0.1–1.0 ml of 10 mM of pH 4.0–
8.0 buffer for a period of 72 h. Refluxing di-isopropyl
ether for 72 h did not produce any peroxides. Work-
Keywords: Amyloglucosidase; Disaccharides; Glycosylation; Mono-
saccharides; Organic media; Vanillin glycosides.
*
Corresponding author. Tel.: +91 821 2515792; fax: +91 821
2517233; e-mail: divakar643@gmail.com
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.11.114

696
R. Sivakumar, S. Divakar / Tetrahedron Letters 47 (2006) 695–699
Table 1. Synthesis of vanillin glycosides with mono- and disaccharides (2–7)a
Glycosides
Glycosylation
yieldb (%)
Product (%
proportionc)
O
O
O
H3C
CH3
O
O
CH3
O
H
O
CH2OH
H
O
H
53
C1a (52), C1b (17) and
C6a (31) mono-glucosides
OH
H
O
HO
O
HO
H
H
H
HO
H
O
H
H
HO
H
H
HO
HO
O
OH
H
OH
H
H
OH
OH
8a vanillin-O-α-D-glucopyranoside
8b vanillin-O-β-D-glucopyranoside
8c vanillin-O-6-α-D-glucopyranoside
O
CH3
H
O
18
C1a galactoside
O
OH
H
OH
H
H
H
O
HOH2C
HO
9 vanillin-O-α-D-galactopyranoside
O
CH
3
O
13
C1a mannoside
H
O
H
H
OH
H
H
OH
OH
O
HOH C
2
10 vanillin-O-α-D-mannopyranoside
O
O
O
CH3
CH3
CH3
H
CH2OH
O
O
O
O
HO
H
H
O
H
H
O
H
O
H
HO
H
H
OH
O
O
OH
HO
29
C1 (42), C6 (30) and C600
(28) mono-maltosides
H
O
OH
H
H
H
H
H
OH
H
H
H
HO
H
H
HO
O
H
OH
O
O
OH
OH
OH
HOH2C
H
O
H
H
OH
H
H
H
H
HO
H
OH
O
OH
OH
H
HOH2C
11a vanillin-O-α-maltopyranoside
11b vanillin-O-6-α-maltopyranoside
11c vanillin-O-6′′-α-maltopyranoside

R. Sivakumar, S. Divakar / Tetrahedron Letters 47 (2006) 695–699
697
Table 1 (continued)
Glycosides
Glycosylation
yieldb (%)
Product (%
proportionc)
O
O
CH3
O
CH3
O
OH
O
H
O
O
HO
HO
H
23
C1 (39) and C600 (61)
mono-sucrosides
H
HO
O
O
HO
H
OH
H
OH
H
HO
O
O
HO
H
HO
HO
O
OH
H
OH
OH
12a vanillin-O-1-sucroside
′′
-
12b vanillin-O-6 sucroside
O
O
O
CH3
CH3
O
CH3
O
O
H
O
H
O
H
O
OH
H
OH
H
OH
OH
H
H
HO
H
HO
H
H
H
HO
H
H
OH
OH
OH
OH
13
C1 (13), C6 (25) mono- and
C1–C6 (62) di-sorbitolides
H
OH
H
H
O
H
OH
H
OH
O
CH3
13a vanillin-O-1-D-sorbitolide
13b vanillin-O-6-D-sorbitolide
O
13c vanillin-O-1,6-di-D-sorbitolide
a 1 and 2–7—1 mmol each; enzyme concentration 40% w/w of 2–7; incubation period—72 h; solvent—di-isopropyl ether; temperature—68 ꢁC.
b Conversion yields were from HPLC with respect to free 2–7. Error in yield measurements is 10%.
c The product proportions were calculated from the area of respective carbon signals.
40% (w/w D-glucose 2) of amyloglucosidase, 1.0 ml of
0.01 M pH 4.0 acetate buffer (corresponding to a con-
centration of 0.01 mM buffer in 100 ml solvent), and
72 h incubation period in di-isopropyl ether at 68 ꢁC.
A maximum yield of 53% was obtained for a mixture
of three mono-glucosides (Table 1): vanillin-O-a-D-
glucopyranoside 8a, vanillin-O-b-D-glucopyranoside 8b
and vanillin-6-O-a-D-glucopyranoside 8c (Scheme 1
and Table 1).
respective CMCs, the proton NMR signals were unusu-
ally broad such that, in spite of recording the spectra at
35 ꢁC, the individual coupling constant values could not
be determined precisely. Also, the carbon signals from
tertiary carbon atoms could not be unambiguously as-
signed. In the case of vanillin-glucosides 8, the downfield
chemical shift value for C1a at 99.2 ppm, C1b at
101.5 ppm and C6 at 68.0 ppm and the corresponding
protons at 4.65, 4.94 and 3.55 ppm indicated that vanil-
lin formed three mono-glucosides with D-glucose at
C1a, C1b and C6.15 With D-galactose, only the a ano-
mer reacted with vanillin, the 13C chemical shift value
for C1a in 9 being 95.8 ppm (1H at 4.22 ppm). D-Man-
nose was also glycosylated (10) at C1a (100.8 ppm).
Maltose, a disaccharide, was converted into three
mono-glycosides 11 with signals at C1a (98.2 and
4.68 ppm), C6a (reducing end—67.2 and 3.54 ppm)
and C600a (non-reducing end—66.1 and 3.69 ppm). In
the product from sucrose, the signals for the C1 (66.0
The vanillin glycosides (Table 1) were subjected to two-
dimensional HSQCT NMR spectroscopy on a Bruker
500 MHz instrument. The glycosides were also tested
for angiotensin converting enzyme inhibitory activities14
and the IC50 values are also shown along with the spec-
tral data.15 Vanillin glycosides are surfactant molecules,
which form micelles above certain critical micellar con-
centrations (CMC). Since the concentrations employed
for NMR measurements are very much higher than their

698
R. Sivakumar, S. Divakar / Tetrahedron Letters 47 (2006) 695–699
O
O
amyloglucosidase
(40% w/w of carbohydrate)
+
CH3
+
CH3
H2O
R - OH
1 ml, pH 4.0 acetate
buffer (0.01 M),
iso
O
O
di- -propyl ether
carbohydrate
OR
OH
2-7
8-13
1
C1 /β, C6 and C1 - C6
α
vanillin glycosides
Scheme 1. Synthesis of vanillin glycosides.
8. Kometani, T.; Tanimoto, H.; Nishimura, T.; Kanbara, I.;
Okada, S. Biosci. Biotechnol. Biochem. 1993, 57, 2192–
2193.
9. Tietze, L. F.; Griesbach, U.; Schuberth, I.; Bothe, U.;
Marra, A.; Dondoni, A. Chem. Eur. J. 2003, 9, 1296–
1302.
10. Reichel, L.; Sckickle, R. Plant Chem. 1943, 76B, 1134–
1137.
11. Sommer, J.; Schroeder, C.; Stockigt, J. Plant Cell Tiss.
Org. Cult. 1997, 50, 119–123.
and 3.49 ppm) position of the fructose moiety, and for
the C600 (66.1 and 3.72 ppm) position of the glucose
moiety showed these to have been glycosylated in the
two products 12. NMR data also clearly showed that
hydrolysis of sucrose to glucose and fructose had
occurred, and the resulting glucose had glycosylated
vanillin at C1b (101.5 and 4.94 ppm) and C6 (68.0
and 3.55 ppm). Maltose was not hydrolyzed. D-Sorbitol,
the open chain sugar alcohol, gave 13, comprising two
mono-glycosides at C1 (67.2 and 3.65 ppm), C6 (66.2
and 3.58 ppm) and one di-glycoside at the C1 and C6
positions (66.5, 3.46–65.5, 3.65 ppm, respectively).
12. Ljunger, G.; Adlercreutz, P.; Mattiasson, B. Enzyme
Microb. Technol. 1994, 16, 751–755.
13. Vic, G.; Thomas, D.; Crout, H. G. D. Enzyme Microb.
Technol. 1997, 20, 597–603.
14. Cushman, D. W.; Cheung, H. S. Biochem. Pharmacol.
1971, 20, 1637–1648.
15. The synthesized glycosides were characterized spectro-
scopically. Only resolvable signals are shown. Some
assignments are interchangeable. Vanillin signals are
primed; non-reducing end sugar units are double primed.
D-Ribose, D-arabinose, D-fructose, D-mannitol and
lactose did not form glycosides perhaps because the
required oxo-carbenium ion intermediate16 was not
formed during the catalytic conversion by amyloglucosi-
dase. Although the D-glucose we employed was an a,b-
anomeric mixture (40:60), the glycosides formed showed
predominant proportions of the a anomer (>80%), indi-
cating the potential for ÔinvertingÕ amyloglucosidase16
(from Rhizopus mold) to convert the majority of b-D-
glucose into its respective a-glycoside.
8a–c: UV (H2O,
k
max): 195.5 nm (r!r*, e195.5
—
2241 Mꢀ1), 279.5 nm (p!p*,
e
279.5—291 Mꢀ1), IR
(stretching frequency): 3358 cmꢀ1 (OH), 1260 cmꢀ1 (gly-
cosidic aryl alkyl C–O–C asymmetric), 1030 cmꢀ1 (glyco-
sidic aryl alkyl C–O–C symmetric), 1408 cmꢀ1 (C@C),
1636 cmꢀ1 (CO), 2933 cmꢀ1 (CH), optical rotation (c 1,
H2O): [a]D at 25 ꢁC = +62.8, ACE activity: IC50
value = 2.46 lmol, 2D-HSQCT (DMSO-d6) C1a-gluco-
side (8a): 1H NMR dppm: (500.13 MHz): 4.65 (H-1a,
5.5 Hz), 3.23 (H-2a), 3.42 (H-3a), 3.78 (H-4a), 3.15
(H-5a), 3.60 (H-6a), 13C NMR dppm (125 MHz): 99.2
(C1a), 72.3 (C2a), 73.5 (C3a), 70.2 (C4a), 72.5 (C5a),
Acknowledgements
The Department of Science and Technology, India, is
gratefully acknowledged for the financial support. R.S.
thanks the Council of Scientific and Industrial Research
(CSIR), New Delhi, India, for providing a Senior
Research Fellowship.
1
60.5 (C6a); vanillin (mono-glucoside 8a): H NMR dppm
6.59 (H-20), 6.20 (H-50), 3.73 (OCH3), 13C NMR dppm
:
111.4 (C20), 114.5 (C50). C1b-glucoside (8b): 1H NMR:
4.94 (H-1b, 5.3 Hz), 2.98 (H-2b), 3.22 (H-3b), 3.68 (H-6b).
13C NMR: 101.5 (C1b), 74.6 (C2b), 76.1 (C3b), 60.8 (C6b),
C6-glucoside (8c): 1H NMR: 4.91 (H-1a), 3.23 (H-2a),
3.20 (H-3a), 3.62 (H-4a), 3.23 (H-5a), 3.55 (H-6a), 13C
NMR: 92.7 (C1a), 72.3 (C2a), 72.6 (C3a), 70.2 (C4a), 75.2
(C5a), 68.0 (C6a); 9: UV (H2O, kmax): 198.0 nm (r!r*,
References and notes
1. Walton, N. J.; Mayer, M. J.; Narbad, A. Phytochemistry
2003, 63, 505–515.
2. Lopez-Malo, A.; Alzamora, S. M.; Argaiz, A. J. Food Sci.
1998, 63, 143–146.
3. Stephan, D.; Peter, K. Nucleic Acid Res. 2003, 31, 5501–
5512.
4. Burri, J.; Graf, M.; Lambelet, P.; Loliger, J. J. Sci. Food
Agric. 1989, 48, 49–56.
5. Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Narbad,
A. J. Agric. Food Chem. 2005, 53, 1769–1775.
6. Kometani, T.; Tanimoto, H.; Nishimura, T.; Okada, S.
Biosci. Biotechnol. Biochem. 1993, 57, 1290–1293.
7. The Merck Index; Merck: USA, 1989.
e
198.0—2909.2 Mꢀ1), 281.0 nm (p!p*, e281.0—517.9 Mꢀ1),
IR (stretching frequency): 3271 cmꢀ1 (OH), 1261 cmꢀ1
(glycosidic aryl alkyl C–O–C asymmetric), 1031 cmꢀ1
(glycosidic aryl alkyl C–O–C symmetric), 1406 cmꢀ1
(C@C), 1664 cmꢀ1(CO), optical rotation (c 1, H2O), [a]D
at 25 ꢁC = +8.82, ACE activity:IC50 value = 2.36 lmol,
2D-HSQCT (DMSO-d6):C1a-glycoside:1H NMR: 4.22
(H-1a), 3.69 (H-2a), 3.52 (H-3a), 3.48 (H-4a), 3.43 (H-
5a), 3.35 (H-6a); 13C NMR: 95.8 (C1a), 69.4 (C2a), 69.9
(C3a), 70.8 (C4a), 71.1 (C5a), 62.0 (C6a); vanillin (mono-
galactoside):1H NMR: 7.38 (H-20), 6.90 (H-50), 7.33 (H-
13
60), 3.86 (OCH3), 9.74 (CHO), C NMR: 129.2 (C10),

R. Sivakumar, S. Divakar / Tetrahedron Letters 47 (2006) 695–699
699
111.3 (C20), 148.6 (C30), 153.5 (C40), 115.9 (C50), 126.4
(12a): 1H NMR: 4.72 (H-100a), 3.68 (H-200a), 3.46 (H-300a),
3.62 (H-400a), 3.65 (H-500a), 3.59 (H-600a), 3.49 (H-1), 3.88
(H-3), 3.89 (H-4), 3.86 (H-5), 3.4 (H-6); 13C NMR: 98.5
(C100a), 71.0 (C200a), 72.2 (C300a), 69.8 (C400a), 72.1 (C500a),
60.5 (C600a), 66.0 (C1), 76.8 (C3), 80.9 (C4), 81.5 (C5), 62.2
(C6); vanillin (mono-sucroside 12a): 1H NMR: 7.22 (H-
(C60), 56.1 (OCH3), 191.4 (CHO); 10:UV (H2O,
k
max):198.5 nm (r!r*,
e
198.5—3401.6 Mꢀ1), 278.0 nm
(p!p*, e278.0—284.4 Mꢀ1), IR (stretching frequency):
3365 cmꢀ1 (OH), 1249 cmꢀ1 (glycosidic aryl alkyl C–O–
C asymmetric), 1030 cmꢀ1 (glycosidic aryl alkyl C–O–C
symmetric), 1406 cmꢀ1 (C@C), 1651 cmꢀ1 (CO),
2940 cmꢀ1 (CH), optical rotation (c 1, H2O): [a]D at
25 ꢁC = ꢀ3.6, ACE activity:IC50 value = 2.31 lmol, C1a-
glycoside:13C NMR (DMSO-d6):100.8 (C1a), 70.5 (C2a),
71.3 (C3a), 67.1 (C4a), 73.8 (C5a), 61.3 (C6a); vanillin
(mono-mannoside):109.4 (C20), 114.72 (C50), 121.9 (C60);
13
20), 6.60 (H-50), 8.35 (H-60), C NMR: 112.8 (C50), 126.3
1
(C60). C600-Glycoside (12b): H NMR: 4.63 (H-100a),
3.08(H-200a), 3.42 (H-300a), 3.15 (H-400a), 3.19 (H-500a),
3.72 (H-600a), 3.48 (H-1), 3.67 (H-3), 3.57 (H-5), 3.46 (H-
13
6), C NMR: 98.6 (C100a), 69.9 (C200a), 72.3 (C300a), 69.8
(C400a), 71.5 (C500a), 66.1 (C600a), 62.3 (C1), 76.5 (C3), 82.2
(C5), 60.5 (C6); 13a–c: UV (H2O, kmax): 193.5 nm (r!r*,
11a–c:UV (H2O,
k
max):194.5 nm (r!r*, e194.5
—
4782.4 Mꢀ1), 278.5 nm (p!p*, e278.5—327.8 Mꢀ1), IR
(stretching frequency):3361 cmꢀ1 (OH), 1265 cmꢀ1 (glyco-
sidic aryl alkyl C–O–C asymmetric), 1024 cmꢀ1 (glycosidic
aryl alkyl C–O–C symmetric), 1412 cmꢀ1 (C@C),
1651 cmꢀ1 (CO), 2930 cmꢀ1 (CH), 1205 cmꢀ1 (OCH3),
optical rotation (c 1, H2O): [a]D at 25 ꢁC = +92.0, ACE
activity:IC50value = 3.65 lmol, 2D-HSQCT (DMSO-d6):
e
193.5—2939.9 Mꢀ1), 273.0 nm (p!p*, e273.0—289.8 Mꢀ1),
IR (stretching frequency): 3386 cmꢀ1 (OH), 1260 cmꢀ1
(glycosidic aryl alkyl C–O–C asymmetric), 1038 cmꢀ1
(glycosidic aryl alkyl C–O–C symmetric), 1409 cmꢀ1
(C@C), 2943 cmꢀ1 (CH), optical rotation (c 1, H2O):
[a]D at 25 ꢁC = +13.9, ACE activity: IC50 value = 1.79
lmol, 2D-HSQCT (DMSO-d6): C1-glycoside (13a): 1H
NMR: 3.65 (H-1), 3.37 (H-2), 3.48 (H-3), 3.57 (H-4), 3.54
(H-5), 3.58 (H-6); 13C NMR: 67.2 (C1), 70.5 (C2), 74.1
(C3), 71.2 (C4), 69.0 (C5), 62.9 (C6); vanillin (mono-
1
C1a-glycoside (11a): H NMR: 4.94 (H-100a, 5.7 Hz), 4.68
(H-1a), 3.25 (H-200a), 3.10 (H-2a), 2.88 (H-300a), 3.20 (H-
3a), 3.65 (H-400a), 3.30 (H-4a), 3.72 (H-5a), 3.60 (H-600a),
13
3.48 (H-6a); C NMR: 100.3 (C100a), 98.2 (C1a), 73.8
1
sorbitolide 13a): H NMR: 7.40 (H-20), 7.20 (H-50), 7.58
13
(C200a), 70.1 (C2a), 74.5 (C300a), 75.1 (C3a), 70.0 (C400a),
79.1 (C4a), 69.8 (C5a), 60.8 (C600a), 60.8 (C6a); vanillin
(H-60), 3.81 (OCH3), 9.75 (CHO), C NMR: 130.5 (C10),
111.2 (C20), 153.8 (C40), 111.1 (C50), 124.5 (C60), 55.9
(OCH3), 191.5 (CHO). C6-Glycoside (13b): 1H NMR: 3.55
(H-1), 3.54 (H-2), 3.44 (H-3), 3.68 (H-4), 3.46 (H-5), 3.58
(H-6); 13C NMR: 63.2 (C1), 70.8 (C2), 72.5 (C3), 73.1
(C4), 68.2 (C5), 66.2 (C6); vanillin (mono-sorbitolide 13b):
1H NMR: 6.88 (H-20), 6.85 (H-50), 7.39 (H-60), 13C NMR:
129.1 (C10), 110.8 (C20), 153.8 (C40), 111.4 (C50), 126.1
(C60). C1–C6 Di-glycoside (13c): 1H NMR: 3.46 (H-1),
3.35 (H-2), 3.36 (H-3), 3.54 (H-4), 3.65 (H-6); 13C NMR:
66.5 (C1), 67.0 (C2), 73.5 (C3), 76.1 (C4), 65.5 (C6);
1
(mono-maltoside,11a): H NMR: 6.26 (H-20), 6.62 (H-50),
13
7.18 (H-60), 3.73 (OCH3), C NMR: 130.0 (C10), 109.5
(C20), 114.2 (C50), 126.8 (C60); C6-glycoside (11b): 1H
NMR: 4.94 (H-100a), 4.88 (H-1a), 3.54 (H-6a), 13C NMR:
100.3 (C100a), 92.4 (C1a), 67.2 (C6a); C600-glycoside (11c):
1H NMR: 4.94 (H-100a), 4.88 (H-1a), 3.69 (H-600a),
13C NMR: 100.3 (C100a), 92.4 (C1a), 66.1 (C600a); 12a,b:
UV (H2O, kmax): 194.0 nm (r!r*, e194.0—6820.8 Mꢀ1),
278.5 nm (p!p*,
e
278.5—423.4 Mꢀ1), IR (stretching
frequency): 3374 cmꢀ1 (OH), 1254 cmꢀ1 (glycosidic aryl
alkyl C–O–C asymmetric), 1026 cmꢀ1 (glycosidic aryl
alkyl C–O–C symmetric), 1412 cmꢀ1 (C@C), 1650 cmꢀ1
(CO), 2936 cmꢀ1 (CH), 1211 cmꢀ1 (OCH3), optical rota-
tion (c 1, H2O): [a]D at 25 ꢁC = +48.6, ACE activity: IC50
value = 35.3 lmol, 2D-HSQCT (DMSO-d6): C1-glycoside
vanillin (di-sorbitolide 13c): H NMR: 6.65, 6.68 (H-50),
1
13
6.69, 6.84 (H-60), C NMR: 129.1, 130.5 (C10), 111.3,
111.7 (C20), 153.5, 153.5 (C40), 115.3, 115.9 (C50), 119.8,
120.3 (C60).
16. Chiba, S. Biosci., Biotechnol., Biochem. 1997, 61, 1233–
1239.