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Enzymatic synthesis of selected phenolic and vitamin glycosides

Sivakumar, R. (2008) Enzymatic synthesis of selected phenolic and vitamin glycosides. PhD thesis, University of Mysore.

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Abstract

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.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Rhizopus; Amino glucosidase; Vanillin; Agiotensin converting enzyme; Antioxidants; Enzymatic synthesis
Subjects: 600 Technology > 08 Food technology > 16 Nutritive value > 04 Vitamins
500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 04 Biosynthesis
Divisions: Fermentation Technology and Bioengineering
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 15 Mar 2010 05:26
Last Modified: 15 Mar 2010 05:26
URI: http://ir.cftri.com/id/eprint/9393

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