Enzymatic transglucosidation14

In case of ^-glucosidation using a large amount of alcohol, the ratio of alcohol, H2O and D-glucose was studied for improvement of conversion yield by Vic and Crout.12 By applying the reported procedure,12 a mixture of D-glucose

(1.1 g), native P-glucosidase (ca. 370 units) or the above-mentioned immobilized P-glucosidase (ca. 1.1 g, corresponding to ca. 370 units), alcohol (18 ml or 18 g) and H2O (2 ml) was incubated for 4 days at 50°C (method I). In case of P-glucosidation using the controlled amount of alcohol, a mixture of D-glucose (1.1 g), the above-mentioned immobilized P-glucosidase (ca. 1.1 g, corresponding to ca. 370 units), alcohol (4 equivalents) in 90% (V/V) ieri-butanol (27 ml) and H2O (3 ml) was incubated for 7 days at 50°C (method II). The results are shown in Table 4. The direct P-glucosidation of 3-methyl-2-buten-1-ol or 2-methyl-2-propen-1-ol using p-nitrophenyl-P-D-glucopyranoside as glycosyl donor under kinetic condition was reported to give the corresponding P-D-glucopyranosides (30) or (31) in 25 or 14% yield, respectively.10 In the present procedure (method I) using high concentration of the alcohol acceptor under equilibrium condition, chemical yield of 30 or 31 was fairly improved to 65 or 51%, respectively (Table 4, entries 2 and

Table 4

Synthesis of P-D-glucopyranosides under equilibrium condition (1)

Immobilized p-glucosidase (EC 3.2.1.21;

alcohol

glu-OR

Entry ROH (eq)

2 3b

6b 7

9 10

Me2C=CHCH2OH (29) Me2C=CHCH2OH (29) Me2C=CHCH2OH (29) CH2=C(Me)CH2OH (35) HO-^^-CH2CH2OH (4)

HO MeO

CHCH2OH (4)

4 glu-OCH2CH=CMe2

4 glu-OCH2CH=CMe2

4 glu-OCH2CH=CMe2

4 glu-OCH2C(Me)=CH2 7

OH OMe

a Native P-glucosidase was used. b The recovered immobilized P-glucosidase was used.

4). On the other hand, in case of the direct (-glucosidation using 4-equivalents of 4-hydroxyphenylethyl alcohol and cinnamyl alcohol congeners in 90% tert-butanol/H2O solution (method II), chemical yields of (-D-glucopyranosides were not always satisfactory and should be improved (Table 4, entries 5-11).

Then, for the purpose of the practical synthesis of naturally occurring (-d-glucopyranosides, direct (-glucosidation of the functionalized alcohol was carried out and the results are shown in Table 5.

When a large amount of benzyl alcohol (29 equivalents) or phenethyl alcohol (24 equivalents) was used as an acceptor for D-glucose in the presence of the immobilized (-glucosidase, benzyl (-D-glucopyranoside (1) or phenethyl (-d-glucopyranoside (14) was obtained in 53 or 34% yield, respectively. Moreover, the same (-glucosidation using the recovered immobilized enzyme afforded 1 or 14 in 52 or 22% yield, respectively. Direct (-glucosidation of allyl alcohol was reported to give 49.15 When this reaction was carried out by the present method I, the yield of 49 was found to be 68% (Table 5, entry 5). When a large amount of aliphatic alcohol including terpene alcohols such as geraniol, nerol and (-)-myrtenol was used as an acceptor for D-glucose in the presence of the immobilized (-glucosidase, a moderate yield of the corresponding (-D-glucopyranosides (7, 9, 50, 51, 52, 53) was given. As the length of the alkyl or alkenyl chain of alcohol becomes long, the yield of (-D-glucopyranoside decreases gradually in spite of an increase in the number of enzyme units. The yield of (3Z)-hexenyl (-D-glucopyranoside (50) was found to be higher than that of n-hexyl (-D-glucopyranoside (7) in spite of the same length of alkyl chain C(6)-alcohol (Table 5, entries 7-10). The yield of the (-D-glucopyranosides was found to be subtly affected by the geometry of the alkenyl chain of alcohol (Table 5, entries 14 and 15). Direct (-glycosidation between 1,6-hexanediol and D-glucose using (-glucosidase (EC 3.2.1.21) from almonds by Vic and Crout12 was reported to be an excellent procedure. On the other hand, enzymatic synthesis of w-hydroxyalkyl and n-alkyl (-galactopyranosides by the transglycosylation reaction using (-galactosidase is also reported.16 When 4 equivalents of 1,8-octanediol was subjected to (-glucosidation using the immobilized (-glucosidase and the recovered immobilized enzyme in a co-solvent system (tBuOH:H2O = 9:1(V/V)),12 moderate yields (entry 20, 19% yield; entry 21, 16% yield) of 8-hydroxyoctyl (-D-glucopyranoside (27) were obtained. When a large amount of 1,6-hexanediol (25 equivalents) or 1,8-octanediol (20 equivalents) was employed for (-glucosidation using the immobilized enzyme, the yield of 25 or 27 was found to be 61 or 58%, respectively (Table 5, entries 18 and 23). When the immobilized enzyme was used in the preparation of 27, the yield of 27 was considerably improved (entry 23, 58% yield) in comparison to that (entry 22, 31% yield) obtained by using native enzyme. The recovered enzyme was also found to be effective (entry 19, 48% yield; entry 24, 43% yield).

The yield of this (-glucosidation would be controlled by the equilibrium of the coordination form of the enzyme. As illustrated in Fig. 3, the reaction site of the enzyme is proposed to be highly hydrophilic. To confirm the relationship

Table 5

Synthesis of P-D-glucopyranosides under equilibrium condition (2)

^OH Immobilized i-glucosidase (EC 3.2.1.21)

alcohol

glu-OR

Entry

ROH (eq)

Method Time (I or II) (days)

glu-OR (yield; %)

1

PhCH2OH (29)

I 4

glu-OCH2Ph

1 (53)

2a

PhCH2OH (29)

I 4

glu-OCH2Ph

1 (52)

3

PhCH2CH2OH (24)

I 4

glu-OCH2CH2Ph

14 (34)

4a

PhCH2CH2OH (24)

I 4

glu-OCH2CH2Ph

14 (22)

5

CH2=CHCH2OH (43)

I 3

glu-OCH2CH=CH2

49 (68)

6b

Me(CH2)5OH (23)

I 4

glu-O(CH2 )5Me

7 (14)

7

Me(CH2)5OH (23)

I 4

glu-O(CH2 )5Me

7 (9)

8a

Me(CH2)5OH (23)

I 4

glu-O(CH2 )5Me

7 (9)

9

(3Z)-MeCH2CH=CH(CH2 )2OH (25)

I 7

glu-O(CH2 )2 CH=CHCH2Me

50 (17)

10a

(3Z)-MeCH2CH=CH(CH2 )2OH (25)

I 7

glu-O(CH2 )2 CH=CHCH2Me

50 (17)

11b

Me(CH2)7OH (18)

I 4

glu-O(CH2 )7Me

9 (5)

12

Me(CH2)7OH (18)

I 4

glu-O(CH2 )7Me

9 (5)

13a

Me(CH2)7OH (18)

I 4

glu-O(CH2 )7Me

9 (8)

14

Geraniol (19)

I 4

glu-OC^H^

51 (11)

15

Nerol (19)

I 4

glu-OC^H^

52 (7)

16

Myrtenol (19)

I 4

glu-OC!oH15

53 (4)

17b

HO(CH2)6OH (25)

I 6

glu-O(CH2 )6OH

25 (68)

18

HO(CH2)6OH (25)

I 6

glu-O(CH2 )6OH

25 (61)

19a

HO(CH2)6OH (25)

I 6

glu-O(CH2 )6OH

25 (48)

20

HO(CH2)8OH (4)

II 7

glu-O(CH2 )8OH

27 (19)

21a

HO(CH2)8OH (4)

II 7

glu-O(CH2 )8OH

27 (16)

22b

HO(CH2)8OH (20)

I 7

glu-O(CH2 )8OH

27 (31)

23

HO(CH2)8OH (20)

I 7

glu-O(CH2 )8OH

27 (58)

24a

HO(CH2)8OH (20)

I 7

glu-O(CH2 )8OH

27 (43)

clogP

-0.106

0.01

0.409

0.938

0.952

1.104

1.333

1.397

1.881

2.915

2.939

2.969

2.969

Yield

61

68

51

65

58

53

34

17

9

4

5

11

7

glu-OR

25

49

31

30

27

1

14

50

7

53

9

51

52

Figure 8: Relationship between hydrophilicity of alcohol and yield of glu-OR.

between the yield of this reaction and the hydrophilicity of the alcohol, the clog P (coefficient log P) value of each alcohol as the indicator of hydrophobicity was calculated. The clogP value was calculated by the CLOGP program (v. 4.71) from BioByte. As shown in Fig. 8, a good relationship between yields of this P-glucosidation and the clog P value of each alcohol is observed. This result suggests that the hydrophilicity of the alcohol influences the coordination to the active site of P-glucosidase and the yield of this P-glucosidation.

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