Synthesis Of Pdglucopyranoside Under Kinetically Controlled Condition78

The success of the glycosidic bond formation depends on the reactive intermediate (enzyme-bound glycosyl cation) being trapped faster by the glycosyl acceptor than by water. We are attracted to this transglycosylation reaction since alcohols as the glucosyl acceptor are better bound at the active site than water. This can be achieved by using predominantly organic reaction media. Research concerning enzymatic reactions in organic media has been extremely active during the last few years. There are two approaches to optimizing the product yield from a given glycosidase in enzymatic glycoside synthesis, i.e., the use of either a high donor or high acceptor concentration.9 High concentration of both is usually impractical due to the solubility limitation. High donor concentration is only practical if the donor is cheap such as glucose. High acceptor concentration is practical if the acceptor is

Figure 3: Plausible mechanism of hydrolysis or ^-D-glycosidation via glycosyl cation.

Figure 3: Plausible mechanism of hydrolysis or ^-D-glycosidation via glycosyl cation.

cheap or can be recovered from the reaction mixture. For the purpose of the synthesis of naturally occurring ^-D-glucopyranosides, the use of equal portions of both glycosyl donor and acceptor alcohols from the synthetic viewpoint is desirable. Our current work has been directed toward establishing which reaction conditions in respect of the glycosyl donor and the enzyme including an immobilized form in aqueous or organic media are better in the synthesis of ^-D-glucopyranosides. In order to determine the effective reaction conditions, the synthesis of benzyl ^-d-glucopyranoside (1) was selected as a model transglycosylation reaction. In order to investigate the best reaction conditions of ^-glucosidation of primary alcohols, screening experiments in respect of the enzymes, glycosyl donors and solvents were carried out. From a screening experiment including the use of immobilized

P-glucosidase, the effective enzyme and glycosyl donor for the synthesis of benzyl P-D-glucopyranoside (1) appeared to be P-glucosidase (EC 3.2.1.21) from almonds and 4-nitrophenyl P-D-glucopyranoside, respectively. Enzymatic glycosi-dation of 20 kinds of primary alcohols and 4-nitrophenyl P-D-glucopyranoside using P-glucosidase from almonds gave stereoselectively the corresponding P-D-glucopyranosides (1-20) in moderate yield, respectively, as shown in Table 1.

Table 1

Preparative scale synthesis of P-D-glucopyranoside using primary alcohols (under kinetic condition)

Entry ROH (eq)

P-Glucosidase/buffer (U ml)-1

1

CH3OH (200)

4.9

2

CH3CH2OH (150)

4.2

3

CH3(CH2 )2OH (10)

4.2

4

CH3(CH2 )3OH (1)

7.0

5

CH3(CH2 )4OH (1.6)

12.0

6

CH3(CH2 )5OH (1.7)

10.0

7

CH3(CH2 )6OH (1)

1.0

8

CH3(CH2 )7OH (1)

0.5

9

(CH3)2CHCH2OH (10)

4.1

10

(CH3)2CH(CH2)2OH (1)

10

11

CH3OCH2CH2OH (10)

4.4

12

CH3CH2OCH2CH2OH (10)

4.3

13

PhCH2OH (1)

12.0

14

Ph(CH2)2OH (1)

15

15

Ph(CH2)3OH (1)

8.0

16

Ph(CH2)4OH (1)

0.5

17

Ph(CH2)5OH (0.5)

0.4

18

Ph(CH2)6OH (0.5)

0.2

19

10

20

Ç^CH2OH

glu-OCH2CH3 3 (64)

glu-OCH2CH(CH3)2 10 (34)

glu-O(CH2)2OCH2CH3 13 (24)

glu-OCH2Ph 1 (21)

Addition of 4-nitrophenyl (-D-glucopyranoside to a solution of the 20 kinds of primary alcohols dissolved in phosphate buffer (pH 5) containing (-glucosidase was carried out over a period of 16-32 h. The reaction can be easily monitored by reverse-phase HPLC and terminated when the formation of the desired product is at a maximum. The results are summarized in Table 1.

The structures of all products were determined by either direct comparison with the corresponding (-glucopyranosides or analysis of 1H- and 13C-NMR data. Identification of the (-configuration of the anomeric center was easily achieved via analysis of the C—H/C-H coupling constant (d, J = 7.8 Hz). The synthetic (-d-glucosides (8, 9, 11 and 14) were identical with those of the reported (-D-glucosides (n-heptyl-(-D-glucopyranoside 8, n-octyl-(-D-glucopyranoside 9, isopentyl-(-D-glucopyranoside 11 and 2-phenylethyl-(-D-glucopyranoside 14), respectively. Chemical yield of (-D-glucopyranosides varied from 2 to 81% depending on the alcohols used. In spite of the moderate chemical yield, (-D-glucopyranoside was the only product in all cases. Prolonged reaction times (>24 h) generally resulted in decreased yields of the (-D-glucopyranoside, presumably due to competing hydrolysis of the product by (-glucosidase.

Then enzymatic glycosidation of three kinds of secondary alcohols, six diols including 1,w-diol and 4-nitrophenyl (-D-glucopyranoside using (-glucosidase from almonds gave stereoselectively the corresponding (-D-glucopyranosides (21-29) in moderate yield, respectively, as shown in Table 2.

The synthetic (-D-glucosides (21 and 29) were identical with those of the reported (-D-glucosides (isopropyl-(-D-glucopyranoside 21 and 2-hydroxybenzyl-(-D-glucopyranoside 29), respectively. In the case of using 1-phenylethanol as a sugar acceptor, a diastereomeric mixture of (-D-glucopyranoside (23) possessing a 42% diastereomeric excess (d.e.) was obtained in 12% yield (entry 3). When five kinds of 1,w-alkanediols were applied in the present enzymatic glycosylation, monoglycosylation products (24-28) were obtained in moderate yield in spite of possessing long methylene side chains (entries 4-8). When salicyl alcohol was applied in the enzymatic glycosylation reaction, only an aliphatic hydroxyl group was active for glycosylation and a phenolic hydroxyl group was unchanged and intact. The presence of an ortho-hydroxyl group seems to have a positive effect on the enzyme-catalyzed glycosylation by the (-glucosidase.

For the purpose of the synthesis of naturally occurring (-D-glucopyranosides, enzymatic glycosidation of nine kinds of functionalized alcohols and 4-nitrophenyl (-D-glucopyranoside using (-glucosidase from almonds was carried out.10 The reaction gave stereoselectively the corresponding (-D-glucopyranosides (30-37) in moderate yield, respectively, as shown in Table 3.

The structures of all products were determined by either conversion to the corresponding acetates or direct comparison with the corresponding natural (-glucosides. Identification of the (-configuration of the anomeric center was easily achieved via the analysis of the C-H/C-H coupling constant. The synthetic (-D-glucosides (30, 33, 34, 35 and 36) were identical with

Table 2

Preparative scale synthesis of P-D-glucopyranoside using secondary alcohols and 1,w-diols (under kinetic condtion)

Table 2

Preparative scale synthesis of P-D-glucopyranoside using secondary alcohols and 1,w-diols (under kinetic condtion)

Entry ROH (eq)

P-Glucosidase/buffer (U ml)-1

Entry ROH (eq)

P-Glucosidase/buffer (U ml)-1

HO(CH2)5OH (1 HO(CH2)6OH (1 HO(CH2)7OH (1 HO(CH2)8OH (1 HO(CH2)9OH (1

Qch2oh

16 2

glu-O(CH2)5OH glu-O(CH2)6OH glu-O(CH2)7OH glu-O(CH2)8OH glu-O(CH2)9OH gul-OCH2-^)

those of the reported naturally occurring P-D-glucosides (3-methyl-2-buten O-P-D-glucopyranoside 30, 4-methoxybenzyl O-P-D-glucopyranoside 33, salidroside (rhodioside) 34, cinnamyl O-P-D-glucopyranoside 35 and 4-methoxycinnamyl-P-D-glucopyranoside 36), respectively.

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