A

Thermoanaerobactor ethanolicus

Figure 6: Enantioselective oxidation of secondary alcohols with secondary alcohol dehydrogenase (SADH), from a thermophilic bacterium (log E is used in the original paper).

Figure 6: Enantioselective oxidation of secondary alcohols with secondary alcohol dehydrogenase (SADH), from a thermophilic bacterium (log E is used in the original paper).

a similar reaction with 2-pentanol raised the Tr to 70 °C, and that with 2-hexanol further raised the Tr to 260 0C. These results suggest that the chain length changes the structure of the rate-determining step.

4. GENERAL APPLICABILITY OF THE "LOW-TEMPERATURE METHOD" EXAMINED

4.1. Application to solketal and other primary and secondary alcohols

The "low-temperature method" has been applied to some primary and secondary alcohols (Fig. 7).2b For example, solketal, 2,2-dimethyl-1,3-dioxolane-4-methanol21 (3) had been known to show low enantioselectivity in the lipase-catalyzed resolution (lipase AK, Pseudomonas fluorescens; E = 16 at 23°C, 27 at 0°C);21a however, the E value was successfully raised up to 55 by lowering the temperature to —40°C (Table 1). Further lowering the temperature rather decreased the E value and the rate was markedly retarded. Interestingly, the loss of the enantioselectivity below —40° C is not caused by the irreversible structural damage of lipase because the lipase once cooled below —40° C could be reused by allowing it to warm higher than —40°C, showing that the lipase does not lose conformational flexibility at such low temperatures.

Similar temperature effect using other racemic alcohols such as 2-hydroxy-methyl-1,4-benzodioxane (4), 2-phenylpropanol (5), and 1-cyclohexylethanol (6) was also observed as shown in Fig. 8, obeying Equation 7. These results suggest that the temperature effect is widely applicable regardless of primary or secondary alcohols and an origin of lipase.

The thermodynamic parameters (AAH and AAS*) and the racemic temperatures Tr (AAHVAAS*)5'6 estimated for these alcohols are shown in Table 2. The results indicate that the enantioselectivity in all of these reactions is governed by the activation-enthalpy differences (AAH*), which originates from the difference in the steric and electronic interactions operating between the fasterand slower-reacting enantiomers in the transition state. The higher enantioselectivity for secondary alcohol 6 comes from the larger negative AAH*, and the low selectivity for 5 depends on the smaller ones. The negative values of AAH are compensated by the negative values of AAS* in all cases.

OH O

Figure 7: Substrates for the lipase-catalyzed resolutions at low temperatures.

Table 1

Temperature effect in the lipase-catalyzed kinetic resolution of solketal

Entry

Temp. (°C)

Lipase (mg)

Time (h)

Ester (% ee)

Alcohol (% ee)

Conv.b

E

1

30

20

3

63

69

0.52

9

2

0

20

6

88

25

0.22

20

3

-10

60

8

84

77

0.48

26

4

-20

60

11

92

32

0.26

31

5

-30

100

16

93

39

0.30

39

6

-40

200c

24

93

63

0.41

55

7

-50

200c

48

74

97

0.57

27

8

-60

200c

48

93

51

0.35

44

a MW =ca. 33 000. Lipase (ca. 1% w/w) is absorbed on Celite. b Calculated from ee(s). c TTN is ca. 3000 at 50% conversion.

a MW =ca. 33 000. Lipase (ca. 1% w/w) is absorbed on Celite. b Calculated from ee(s). c TTN is ca. 3000 at 50% conversion.

403 55 7.4

Figure 8: Correlation between ln E and 1/T for the lipase-catalyzed resolution of 1, 3-6.

403 55 7.4

Figure 8: Correlation between ln E and 1/T for the lipase-catalyzed resolution of 1, 3-6.

Table 2

Thermodynamic parameters for the lipase-catalyzed resolutions (i-Pr2O, vinyl acetate)

Table 2

Thermodynamic parameters for the lipase-catalyzed resolutions (i-Pr2O, vinyl acetate)

Compound

Lipase

AAH* (kcal mol-1)

AAS* (cal deg-1 mol-1)

Tr (°C)

1

PSa

-3.01 ± 0.13

-4.31 ±0.50

425

3

AKb

-3.53 ± 0.20

-7.18 ± 0.79

219

4

AK

-2. 24 ± 0.12

-1.35 ± 0.40

1386

5

PS

-1.13 ± 0.05

-1.77 ± 0.96

365

5

AK

-0. 74 ± 0.05

-1.51 ± 0.19

217

6

PS

-3.73 ± 0.25

-3.02 ±0.92

962

a With vinyl butyrate. b In Et2O.

a With vinyl butyrate. b In Et2O.

4.2. Resolution of (±)-2-hydroxy-2-(pentafluorophenyl)acetonitrile

The "low-temperature method" was then applied to the resolution of (±)-2-hydroxy-2-(pentafluorophenyl)acetonitrile (7) (Fig. 9),2c which is usable for the syntheses of a variety of ethane diols, amino alcohols containing C6F5 groups as novel chiral ligands.22 After screening lipases such as Amano PS and AK, lipase LIP (Pseudomonas aeruginosa lipase immobilized on Hyflo Super-Cel, Toyobo, f OH F 9Ac

FyV^CN L|Pase , fYV"CN

f OH F 9Ac

FyV^CN L|Pase , fYV"CN

Figure 9: Correlation between ln E and 1/T in the lipase LIP-catalyzed transesterification of (±)-7.

Figure 9: Correlation between ln E and 1/T in the lipase LIP-catalyzed transesterification of (±)-7.

Co., Ltd, Japan) was found to be the best choice (E = 113, 1.5 h, 44% conv. at 30°C). The E value was increased regularly by lowering the temperature to 0°C to reach > 427 (4.0 h, 44% conv.), which is a satisfactory and practical level.

4.3. Immobilization of lipase on porous ceramic support (Toyonite) for acceleration2d

As described above, the temperature effect is useful for enhancing the enantioselectivity; however, one problem is the decrease in the reaction rate. For example, although in a lipase AK-catalyzed resolution of solketal, the E value (9 at 30°C, Table 1, entry 1) is increased up to 55 by lowering the temperature to —40°C, 10 times the amount of lipase and 8-fold the reaction time are required as compared with those at 30°C.2b Thus, the rate of acceleration is an important subject especially to make the low-temperature reaction practical.

Acceleration of the reaction rate was attained by using an immobilized lipase on the porous ceramic support (Toyonite).23 The immobilized lipase PS is commercially available as lipase PS-C "Amano" II, which has (methacryloy-loxy)propylsilanetrioxyl bridges on the ceramic surface, and lipases are immobilized on the bridges.23 Immobilization of an enzyme is known to affect the enzyme conformation, rigidity, and aggregation state to alter the enantioselectivity and reactivity.24 As shown in Table 3, the use of the Toyonite-immobilized lipase AK accelerates the reaction from 11 000 (TTN/h, total turnover number per hour) for Celite-immobilized one to 53 000 at 30°C. Observed maximal acceleration is 80 times at —40°C in the case of entry 4 compared with that of entry 3. The great acceleration ability is significant for practical use,25-27 especially at low temperatures. In an organic solvent, lipase molecules usually form aggregation structures, even on Celite, which reduce the activity, while on Toyonite lipases may be highly dispersed by immobilization with organic bridges on the porous ceramic to exert the inherent high activity.

Table 3

Toyonite-immobilized lipase-catalyzed resolution of solketal (±)-3 (vinyl butylate)

Table 3

Toyonite-immobilized lipase-catalyzed resolution of solketal (±)-3 (vinyl butylate)

Entry

Lipase

Organic bridge

E

TTN/h

30°C

—40°C

30° C

40°C

1

AK

Celite

9.0

55

11 000

110

2

AK

Toyonite

3.2

21

53 000

1400

3

PS

Celite

6.8

15

6400

22

4

PS

Toyonite

6.8

15

110 000

1700

4.4. Structural optimization of organic bridges on Toyonite2d

Structure of organic bridges attached on the support is also crucial for the conformational engineering of enzymes. The ability of organic bridges in the low-temperature reaction was then examined by using the substrate of 2-hydroxymethyl-1,4-benzodioxane 4 as shown in Table 4 and Fig. 10.2d Figure 10 shows that choice of organic bridges is crucially important both to accelerate the reaction and to enhance the enantioselectivity. As far as examined for 9a-i, terminal olefin is not necessary, and carbonyl function is requisite. A small structural change of the bridges remarkably affects the temperature effect probably by the difference in a manner of hydrogen bonding between the bridge and functions of

Table 4

Effect of organic bridges on the Toyonite-immobilized lipase-catalyzed resolution of (±)-4

Table 4

Effect of organic bridges on the Toyonite-immobilized lipase-catalyzed resolution of (±)-4

Entry

Organic bridge

E

TTN/h

30°C

—30°C

30°C

—30°C

1

Nonea

18

33

3100

26

2

Noneb

19

31

5300

52

3

9a

8.6

18

7300

130

4

9b

10

17

14 000

280

5

9c

6.6

9.3

14 000

230

6

9d

7.9

.28

8700

280

7

9e

4.8

21

27 000

840

8

9f

11

28

12 000

100

9

9g

6.8

12

57 000

940

10

9h

5.9

6.8

7200

61

11

9ic

-

-

-

-

a Commercially available lipase PS immobilized on Celite. b Toyonite without organic bridges. c Lipase could not be immobilized.

a Commercially available lipase PS immobilized on Celite. b Toyonite without organic bridges. c Lipase could not be immobilized.

Figure 10: Temperature effect on the Toyonite-immobilized lipase-catalyzed resolution of (±)-4 by varying the organic bridges.

Figure 10: Temperature effect on the Toyonite-immobilized lipase-catalyzed resolution of (±)-4 by varying the organic bridges.

lipase molecules. The bridge 9d was the best choice from the points of reaction efficiency and availability.

4.5. Practical resolution of azirine 1 by the "low-temperature method" combined with Toyonite-immobilized lipase and optimized acylating agent2e

In the first attempt of the low-temperature method for azirine-2-methanol 1 using the Celite-immobilized lipase (Scheme 1), the E value was increased from 17 (30°C) up to 99 (—40°C); however, the TTN/h was decreased from 4700 to 210. On the other hand, use of Toyonite D-M-immobilized lipase28 in the reaction remarkably raised the TTN/h to 4200 at —40°C. In contrast, it lowered the E value to 33. Therefore, adjustment of the two conflicting features, the reaction rate and the enantioselectivity, is essential for the practical use of the low-temperature method. For this purpose, the acylating agents29 were screened and found to be highly effective on the E values. The results of the reactions carried out at —40°C are shown in Table 5.2e Elongation of the acyl chain to vinyl butanoate dramatically increased the E value up to 96, a comparable value in the Celite-immobilized lipase, together with a sufficient TTN/h of 3200. Further improvement was attained by using Toyonite 200M-immobilized lipase, which gave the highest E value (130) and TTN/h (7800) with vinyl butanoate at —40°C.

These results indicate that the low-temperature method increases the enantio-selectivity, at least above inversion temperature, and the enantioselectivity and reaction rate can be optimized by the use of Toyonite-immobilized lipase and a suitable acylating agent.

Table 5

Toyonite-immoblized lipase-catalyzed resolution of (±)-1 at -40°C

(S)-1

Entry

Acylating agents

R

Lipasea

E

TTN/h

1

ch3

3a (1)b

A

99

210

2

ch3

3a (1)

B

34

4200

3

CH3(CH2 )2

3b (1)

B

96

3200

4

CH3(CH2 )4

3c (1)

B

54

1600

5

CH3(CH2 )4

3c (2)

B

102

2000

6

CH3(CH2 )6

3d (2)

B

35

1500

7

CH3 (CH2)8

3e (2)

B

40

1500

8

CH3 (CH2 )10

3f (2)

B

44

1800

9

PhCH2CH2

3g (2)

B

71

3600

10

CH2Cl

3h (1)

B

7

5300

11

CH3(CH2 )2

3b (1)

C

130

7800

12

CH3 (CH2 )4

3c (2)

C

77

8600

13

CH3 (CH2)2

3b (2)

D

68

1500

a A: Lipase PS on Celite; B: lipase PS immobilized on Toyonite D-M with 3-(methacryloyloxy)propylsilanetrioxy bridges; C: lipase PS immobilized on Toyonite 200M with 3-(methacryloyloxy)propylsilanetrioxy bridges; D: lipase PS immobilized on Toyonite 200 with 3-(propanoyloxy)hexylsilanetrioxy bridges. b Equivalent of acylation agent.

a A: Lipase PS on Celite; B: lipase PS immobilized on Toyonite D-M with 3-(methacryloyloxy)propylsilanetrioxy bridges; C: lipase PS immobilized on Toyonite 200M with 3-(methacryloyloxy)propylsilanetrioxy bridges; D: lipase PS immobilized on Toyonite 200 with 3-(propanoyloxy)hexylsilanetrioxy bridges. b Equivalent of acylation agent.

Table 5

Toyonite-immoblized lipase-catalyzed resolution of (±)-1 at -40°C

(2R*, 3R*)-3-methyl-3-phenyl-2-aziridinemethanols2h

The lipase-catalyzed resolution of (2R* ,3S*)-3-methyl-3-phenyl-2-aziridine-methanol (±)-11 by using the "low-temperature method" gave synthetically useful (2R,3S)-11 and its acetate (2S,3R)-11a with (2S)-selectivity (E = 55 at -40°C), while a similar reaction of (2R*,3R*)-3-methyl-3-phenyl-2-aziridinemethanol (±)-12 gave (2S,3S)-12 and its acetate (2R,3R)-12a with (2R)-selectivity (E = 73 at —20°C) (Scheme 2). Compound (±)-11 was prepared conveniently via diastereos-elective addition of MeMgBr to t-butyl 3-phenyl-2H-azirine-2-carboxylate, which was successfully prepared by the Neber reaction of oxime tosylate of t-butyl

1) CH3MgBr

Ph C02f-Bu

Lipase

PS-CII, CH: OH (2 Si-selective

Lipase PS-C II

(2S)-selective

Lipase PS-C II

(2S)-selective

Scheme 2: Resolution of (2R*,3S*)- and (2R*,3R*)-3-methyl-3-phenyl-2-aziridinemethanols.

benzoylacetate. As far as we know, a few examples of the lipase-catalyzed reaction for such 2-aziridinemethanols having two stereogenic centers at and y-carbons are known,30 and none of the reaction with aziridine derivatives without N-protection have been reported. As shown in Fig. 11, the reaction of 11 in acetone gave higher enantioselectivity, and the best result (E = 55) was obtained at —40°C. However, further lowering the temperature to —50°C did not increase the E value. In contrast, the temperature modulation in THF increased the E value continuously to —60°C. The temperature effect for compound 12 in acetone is shown in Fig. 12. The inversion temperatures (Tinv) for 11 and 12 are different in acetone.

E 50

30 20

Figure 11: Temperature effect in the lipase-catalyzed resolution of (±)-11 (♦: in acetone; •: in THF; ▲: in Et2O).

E 50

30 20

Figure 11: Temperature effect in the lipase-catalyzed resolution of (±)-11 (♦: in acetone; •: in THF; ▲: in Et2O).

Figure 12: Temperature effect in the lipase-catalyzed resolution of (±)-12 in acetone.

Figure 12: Temperature effect in the lipase-catalyzed resolution of (±)-12 in acetone.

4.7. Resolution of 5-(hydroxymethyl)-3-phenyl-2-isoxazoline2g

Optically active 5-(hydroxymethyl)-3-phenyl-2-isoxazoline 13 is a versatile key intermediate for the syntheses of ^-hydroxy ketones,31 y-amino alcohols,32 and y-amino acids.33 However, the lipase-catalyzed kinetic resolution of isoxazoline (±)-13 has not been reported so far probably because of the low enantioselectivity expected for primary alcohols (Scheme 3). The enantioselectivity was found to be very low (E value = 4-5 in i-Pr2O) at room temperature; however, it could be markedly improved up to an E value of 249 at — 60° C by using lipase PS-C II in acetone, which was the best solvent among those tested (THF, i-Pr2O) (Fig. 13). The temperature effect involving Tinv was markedly influenced by the solvent, acylating agent, and support. Tinv was not observed in the case with vinyl acetate below to —60°C.

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