Enzymatic synthesis of selected phenolic and vitamin glycosides

In the present work amyloglucosidase from Rhizopus mold and b-glucosidase
isolated from sweet almond were employed to synthesize few selected phenolic and
vitamin glycosides. The phenols employed possess a hydroxyl group at the 4th position
of phenyl ring along with another hydroxyl or -OCH3 group at the 3rd position besides
possessing a -CH=CH-, -CH2 or -CHO group para to the 4th -OH like vanillin 1, Nvanillyl-
nonanamide 2, curcumin 3, DL-dopa 4 and dopamine 5. The vitamins employed
are riboflavin 43 (vitamin B2), ergocalciferol 44 (vitamin D2) and a-tocopherol 45
(vitamin E). All these vitamins possess OH groups in their structure in the form of ribitol
OH in riboflavin 43, acyclic OH in ergocalciferol 44 and phenolic OH in a-tocopherol
Chapter ONE deals with literature reports on biotransformations, glycosidases,
their sources, structural features of glucoamylase and b-glucosidase, glycosylation
methods, glycosylation mechanism and information on related enzymatically synthesized
glycosides. A detailed report on investigations of some important factors that influence
the glycosidase catalyzed reactions in organic solvents like nature of substrate, nature of
solvent, thermal stability, role of water, kinetic studies of glycosidase catalyzed reactions
and immobilization are presented. Besides, advantages of carrying out glycosylation
using reverse micelles, super critical carbon di-oxide, microwave and response surface
methodology (RSM) have been discussed. This chapter ends with a brief scope of the
present investigation.
Chapter TWO describes materials and methods employed in the present work.
Enzyme and chemicals employed and their sources are mentioned. Glycosylation
procedure encompassing the related analytical and assay procedures are described and
the newer methods wherever necessary have been described in detail.
Chapter THREE describes amyloglucosidase from Rhizopus mold and b-
glucosidase from sweet almond (native/immobilized) catalysed syntheses of selected
phenolic glycosides of vanillin 1, N-vanillyl-nonanamide 2, curcumin 3, DL-dopa 4 and
dopamine 5 with D-glucose 6, D-galactose 7, D-mannose 8, D-fructose 9, D-arabinose 10,
D-ribose 11, maltose 12, sucrose 13, lactose 14, D-sorbitol 15 and D-mannitol 16 by
reflux method in di-isopropyl ether solvent at 68 °C. Reaction parameters were
optimized in terms of incubation period, pH, buffer, enzyme and substrate concentrations
for the synthesis of respective glucosides. Maximum conversion yields obtained for
amyloglucosidase catalyses were: 4-O-(D-glucopyranosyl)vanillin 17a-c - 53%, 4-O-(Dglucopyranosyl)
N-vanillyl-nonanamide 24a-c - 56%, DL-dopa-D-glucoside 34a-d - 62%
and dopamine-D-glucoside 40a-c - 58%. Similarly, maximum glucosides obtained for b-
glucosidase catalyses were: 4-O-(b-D-glucopyranosyl)vanillin 17b - 10%, 4-O-(Dglucopyranosyl)
N-vanillyl-nonanamide 24c - 35%, 1,7-O-(bis-b-D-glucopyranosyl)
curcumin 30 - 44%, DL-dopa-D-glucoside 34b,c - 33% and dopamine-D-glucoside 40b -
65%. Solubility in water of 4-O-(D-glucopyranosyl)vanillin, 4-O-(D-glucopyranosyl)Nvanillyl-
nonanamide and 1,7-O-(bis-b-D-glucopyranosyl)curcumin were found to be 35.2
g/L, 7.7 g/L and 14 g/L respectively.
Under the optimized conditions determined, glycosides of vanillin 1, N-vanillylnonanamide
2, curcumin 3, DL-dopa 4 and dopamine 5 were synthesized with various
carbohydrates molecules. Product glycosides were isolated through column
chromatography and characterized by measuring melting point and optical rotation
besides subjecting them to a detailed spectroscopic investigation by UV, IR, Mass and
2D HSQCT NMR. Phenols underwent glycosylation mostly and in few cases arylation
also with the respective carbohydrates indicated: vanillin 1 - D-glucose 6, D-galactose 7,
D-mannose 8, maltose 12, sucrose 13, lactose 14 and D-sorbitol 15 with conversion
yields for amyloglucosidase catalyses in the 13-53% range and for b-glucosidase in the
6-25% range; N-vanillyl-nonanamide 2 - D-glucose 6, D-galactose 7, D-mannose 8, Dribose
11, maltose 12 and lactose 14 with conversion yields for amyloglucosidase
catalyses in the 9-56% range and for b-glucosidase in the 9-35% range; curcumin 3 - Dglucose
6, D-galactose 7, D-mannose 8 and lactose 14 with conversion yields for b-
glucosidase in the 12-44% range; DL-dopa 4 - D-glucose 6, D-galactose 7, D-mannose 8,
D-sorbitol 15 and D-mannitol 16 with conversion yields for amyloglucosidase catalyses
in the 12-62% range and for b-glucosidase in the 17-33% range and dopamine 5 - Dglucose
6, D-galactose 7 and D-mannose 8 with conversion yields for amyloglucosidase
catalyses in the 32-58% range and for b-glucosidase in the 28-65% range. About 61
individual glycosides were synthesized enzymatically using both the glucosidases, of
which 45 are being reported for the first time. Two-Dimentional NMR studies confirmed
the linking between phenolic OH of aglycon and C1 and/or C1-O-/C6-O- position of the
carbohydrate molecules.
b-Glucosidase exclusively yielded b-glycosides and in very few cases C6-Oarylated
products. However, amyloglucosidase on the other hand showed both C1a and
C1b-glycosylated and/or C1-O-/C6-O-arylated products. In most cases C1 glycosylated
products were detected. Only few carbohydrate molecules showed C1-O-/C6-Oarylation.
D-Sorbitol 15 and D-mannitol 16 gave arylated products by reacting only to the
primary OH groups. No reaction occurred at the secondary hydroxyl groups of the
carbohydrate molecules. Also, only mono glycosylated or mono arylated products were
detected. No carbohydrate molecule gave bis products. Both amyloglucosidase and b-
glucosidase did not catalyze the reaction with D-fructose 9 and D-arabinose 10. Among
the phenols employed only curcumin 3 showed bis glycosylated products. Phenolic OH
at the 4th position readily reacted with the carbohydrate molecules employed and
wherever possible DL-dopa 4 and dopamine 5 underwent reaction at the 3rd phenolic OH
also. Thus water insoluble N-vanillyl-nonanamide 2, curcumin 3 and less water soluble
vanillin 1 were converted to more water soluble glycosides.
Amyloglucosidase from Rhizopus mold and b-glucosidase from sweet almond,
catalysed synthesis of 4-O-(a-D-glucopyranosyl-(1¢®4)D-glucopyranosyl)vanillin was
optimized using response surface methodology. A Central Composite Rotatable Design
involving 32 experiments of five variables: glucosidases 10-50% w/w of maltose,
vanillin 0.5-2.5 mmol, incubation period 24-120 h, buffer concentration 0.04 mM-0.2
mM (0.4-2 mL) and pH 4-8 were employed. Saddle shaped surface plots for both the
enzymes exhibited total reversal of the maltosylation behaviour at a critical cross-over
point corresponding to 30% (w/w maltose) enzyme concentration, pH 6 and a buffer
concentration of 0.125 mM (1.25 mL) implying that a critical enzyme to buffer
concentration and pH dictate the extent of vanillin maltosylation.
Chapter FOUR describes amyloglucosidase and b-glucosidase catalysed
syntheses of glycosides of riboflavin 43 (vitamin B2), ergocalciferol 44 (vitamin D2) and
a-tocopherol 45 (vitamin E). Since ergocalciferol 44 and a-tocopherol 45 are light and
air sensitive, the reaction was carried out in an amber coloured 150 mL round bottomed
flask under nitrogen atmosphere. Work-up and isolation of the compound was also
carried out in dark. Reaction parameters were optimized in terms of incubation period,
pH, buffer, enzyme and substrate concentrations for the syntheses of glucosides of
riboflavin 43, ergocalciferol 44 and a-tocopherol 45. Maximum conversion yields for the
glucosides obtained for amyloglucosidase catalyses were: 5-O-(D-glucopyranosyl)
riboflavin 46a-c - 25% and 20-O-(D-glucopyranosyl)ergocalciferol 53a-c - 42%. With b-
glucosidase the maximum glucoside yields were: 5-O-(b-D-glucopyranosyl)riboflavin
46b - 24% and 6-O-(b-D-glucopyranosyl)a-tocopherol 54 - 23%. Water solubility of 5-
O-(D-glucopyranosyl) riboflavin, 20-O-(D-glucopyranosyl)ergocalciferol and 6-O-(b-Dglucopyranosyl)
a-tocopherol were determined to be 8.2 g/L, 6.4 g/L and 25.9 g/L
respectively.
Under the optimized conditions, glycosides of riboflavin 43, ergocalciferol 44
and a-tocopherol 45 with various carbohydrates like D-glucose 6, D-galactose 7, Dmannose
8, D-ribose 11, maltose 12, sucrose 13 and lactose 14 were synthesised.
Vitamins underwent glycosylation/arylation with the respective carbohydrates indicated:
riboflavin 43 - D-glucose 6, D-galactose 7, D-mannose 8, D-ribose 11, maltose 12,
sucrose 13 and lactose 14 with conversion yields for amyloglucosidase catalyses in the 5-
40% range and for b-glucosidase in the 7-24% range; ergocalciferol 44 reacted only with
D-glucose 6 to give a conversion yield of 42% for amyloglucosidase catalyses and a-
tocopherol 45 - D-glucose 6, D-galactose 7 and D-mannose 8 with conversion yields for
b-glucosidase catalyses in the 11-23% range. Out of 21 individual glycosides prepared,
15 glycosides are reported for the first time. Here also the glycosides were isolated by
column chromatography and characterized by measuring melting point and optical
rotation and by recording UV, IR, Mass and 2D HSQCT spectra. Two-Dimentional
NMR studies confirmed the linking between primary/acyclic/phenolic OH of the aglycon
and the C1 and/or C1-O-/C6-O- position of the carbohydrate molecules.
b-Glucosidase exclusively yielded b-glycosides only and no C6-O-arylated
products were detected. However, amyloglucosidase on the other hand showed both C1a
and C1b-glycosylated and/or C1-O-/C6-O-arylated products. Here also, no reaction
occurred at the secondary hydroxyl groups of the carbohydrate molecules. Also, only
mono glycosylated or mono arylated products were detected. Both amyloglucosidase and
b-glucosidase did not catalyze the reaction with D-fructose 9, D-arabinose 10, D-Sorbitol
15 and D-mannitol 16. Among the vitamins employed ergocalciferol 44 showed
glycosylation/arylation only with D-glucose 6. Thus the water insoluble ergocalciferol 44
and a-tocopherol 45 and less water soluble riboflavin 43 were converted to more water
soluble glycosides thereby improving their potential bioavailability and pharmacological
properties.
Chapter FIVE describes kinetic study of the glucosylation reaction between
vanillin 1 and D-glucose 6 catalyzed by amyloglucosidase from Rhizopus mold leading
to the synthesis of 4-O-(D-glucopyranosyl)vanillin 17a-c in detail. Initial reaction rates
were determined from kinetic runs involving different concentrations of vanillin 1 5 mM
to 0.1 M and D-glucose 6 5 mM to 0.1 M. Graphical double reciprocal plots showed that
kinetics of the amyloglucosidase catalyzed reaction followed Ping-Pong Bi-Bi
mechanism where competitive substrate inhibition by vanillin 1 led to dead-end
amyloglucosidase-vanillin complexes at higher concentrations of vanillin 1. An attempt
to obtain best fit of this kinetic model through computer simulation yielded in good
approximation, the values of four important kinetic parameters: kcat = 35.0 ± 3.2
10-5M/h.mg, Ki = 10.5 ± 1.1 mM, Km D-glucose = 60.0 ± 6.2 mM, Km vanillin = 50.0 ± 4.8
mM.
Chapter SIX describes evaluation of antioxidant and angiotensin converting
enzyme inhibition activity of the enzymatically synthesized phenolic and vitamin
glycosides. About 39 enzymatically prepared phenolic and vitamin glycosides were
subjected to antioxidant activities and 48 glycosides were tested for angiotensin
converting enzyme (ACE) inhibition activity. Both phenolic and vitamin glycosides
exhibited IC50 values for antioxidant activities in the 0.5 ± 0.03 mM to 2.66 ± 0.13 mM
range and ACE inhibition in the 0.56 ± 0.03 mM to 3.33 ± 0.17 mM range. Introduction
of a carbohydrate molecule to the phenolic OH decreased the antioxidant activity.
However, some of the glycosides still possessed substantial amount of antioxidant
activities. Also, comparable ACE inhibition values only were observed between free
phenol/vitamin and the respective glycosides. Best IC50 values (£ 0.75 mM) observed for
antioxidant activity are for: 4-O-(a-D-glucopyranosyl-(1¢®4)b-D-glucopyranosyl)Nvanillyl-
nonanamide 28d - 0.75 ± 0.04 mM, 1,7-O-(bis-D-mannopyranosyl)curcumin
32a,b - 0.75 ± 0.04 mM, 6-O-(D-galactopyranosyl)a-tocopherol 55a,b - 0.72 ± 0.04 mM
and 6-O-(D-mannopyranosyl)a-tocopherol 56a,b - 0.5 ± 0.03 mM. Similarly, best ACE
inhibitory activities for the glycosides (< 0.75 mM) detected were: 4-O-(b-Dglucopyranosyl)
vanillin 17b - 0.61 ± 0.03 mM, 4-O-(D-galactopyranosyl)vanillin 18a,b -
0.61 ± 0.03 mM, 1,7-O-(bis-b-D-galactopyranosyl-(1¢®4)D-glucopyranosyl) curcumin
33a,b - 0.67 ± 0.03 mM and DL-3-hydroxy-4-O-(6-D-sorbitol)phenylalanine 38 - 0.56 ±
0.03 mM. Among the glycosides tested, phenolic glycosides showed better antioxidant
and ACE activities than the vitamin glycosides.
Thus the present investigation has brought out clearly the
glycosylation potentialities of amyloglucosidase from Rhizopus mold and b-
glucosidase from sweet almond in the reaction between selected phenols/vitamins
with structurally diverse carbohydrate molecules employed.