Crown ethers as regulators of enzymatic reactions: Enhanced reaction rate and enantioselectivity in lipase-catalyzed hydrolysis of 2-cyano-1-methylethyl acetate

Article abstract of DOI:10.1021/jo951598+

Both reaction rate and enantioselectivity in lipase-catalyzed hydrolysis of 2-cyano-1-methylethyl acetate were significantly changed by addition of some crown ethers. Although various crown ethers accelerated the reaction rate, the enantioselectivity greatly depended on the nature of each one. Among 27 crown ethers and 5 polyethers tested, benzo-crown, armed azacrown, and thiacrown ethers offered high enantioselectivity of the lipase-catalyzed hydrolysis. Hydrophobicity and chirality of the crown ether did not affect the hydrolysis behavior, but its concentration greatly influenced enantioselectivity. Two hundred fifty or more times the crown ether, molarity based on the enzyme, was required to attain satisfactorily high reaction rate and enantioselectivity.

Full text of DOI:10.1021/jo951598+

2158
J . Org. Chem. 1996, 61, 2158-2163
Cr ow n Eth er s a s Regu la tor s of En zym a tic Rea ction s: En h a n ced
Rea ction Ra te a n d En a n tioselectivity in Lip a se-Ca ta lyzed
Hyd r olysis of 2-Cya n o-1-m eth yleth yl Aceta te
Toshiyuki Itoh,*,† Yumiko Takagi,‡ Takako Murakami,† Yuji Hiyama,† and Hiroshi Tsukube*,§
Department of Chemistry, Faculty of Education, and Department of Chemistry, Faculty of Science,
Okayama University, Okayama 700, J apan
Received September 1, 1995 (Revised Manuscript Received December 15, 1995X)
Both reaction rate and enantioselectivity in lipase-catalyzed hydrolysis of 2-cyano-1-methylethyl
acetate were significantly changed by addition of some crown ethers. Although various crown ethers
accelerated the reaction rate, the enantioselectivity greatly depended on the nature of each one.
Among 27 crown ethers and 5 polyethers tested, benzo-crown, armed azacrown, and thiacrown
ethers offered high enantioselectivity of the lipase-catalyzed hydrolysis. Hydrophobicity and
chirality of the crown ether did not affect the hydrolysis behavior, but its concentration greatly
influenced enantioselectivity. Two hundred fifty or more times the crown ether, molarity based
on the enzyme, was required to attain satisfactorily high reaction rate and enantioselectivity.
In tr od u ction
oselectivity of the lipase reaction.5 Sih et al. first reported
that dextromethorphan and levomethorphan worked as
enantioselective inhibitors in Candida cylindracea lipase-
catalyzed hydrolysis of (()-arylpropionic acids and (()-
(aryloxy)propionic esters.5a We found that (S)-2-amino-
4-(methylthio)-1-butanol similarly enhanced enantio-
selectivity in Pseudomonas cepacia lipase-catalyzed hy-
drolysis of 2-cyano-1-methylethyl acetate (1).5b Each of
these remarkably improved enantioselectivity but rarely
increased reaction rate. We recently demonstrated that
some crown ethers had potential to enhance not only
enantioselectivity but also reaction rate in the lipase-
catalyzed hydrolysis of acetate 1.1
The synthetic value of lipases has been well recognized
because their reactions proceed efficiently and selectively
under mild conditions.2 Since only a limited number of
lipase-catalyzed reactions are applicable for practical
optical resolution, several methods were recommended
to improve their reaction performance: optimization of
reaction conditions, modification of substrate,3 selection
of nonaqueous media,4 and use of an additive that
regulates lipase reactivity.5 Of these, the additive method
is the most advantageous. It is simple to use, but only a
few compounds have been reported to enhance enanti-
† Department of Chemistry, Faculty of Education, Okayama Uni-
versity.
We examine here a total of 32 crown ethers and their
derivatives as additives in this lipase reaction and
explain our search to optimize the utility of the crown
ethers. Although nitrile 1 was not an appropriate
substrate for the lipase PS employed, addition of some
crown ethers surprisingly enhanced both enantioselec-
tivity and reaction efficiency. The reaction profiles are
investigated in detail to discuss the mechanism behind
the crown ether modification of the lipase performance.
This is the first systematic study of regulation of an
enzymatic reaction by crown ether-type additives, though
they are known as complexing agents for several pro-
teins.6
‡ Department of Chemistry, Faculty of Education, Okayama Uni-
versity, Research Fellow of the J apan Society for the Promotion of
Science. Present address: Department of Chemistry, Faculty of
Teacher Education, Hyogo University of Education, Yashiro-cho, Hyogo
673-14, J apan.
§ Department of Chemistry, Faculty of Science, Okayama Univer-
sity. Present address: Department of Chemistry, Faculty of Science,
Osaka City University, Osaka 558, J apan.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
(1) A preliminary account has appeared: Itoh, T.; Hiyama, Y.;
Betchaku, A.; Tsukube, H. Tetrahedron Lett. 1993, 34, 2617.
(2) (a) Whiteside, G. M.; Wong, C.-H. Angew. Chem., Int. Ed. Engl.
1985, 24, 617. (b) J ones, J . B. Tetrahedron 1986, 42, 3351. (c) Chen,
C.-S.; Sin, C. J . Angew. Chem., Int. Ed. Engl. 1989, 28, 695. (d)
Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114. (e) Santaniello, E.;
Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem. Rev. 1992, 92, 1071
and references cited therein.
Resu lts a n d Discu ssion
(3) For example, see: Itoh, T.; Takagi, Y.; Nishiyama, S. J . Org.
Chem. 1991, 56, 1521 and references cited therein.
Action s of a Cr ow n Eth er Ad d itive. We chose
2-cyano-1-methylethyl acetate (1) and lipase PS as
substrate and enzyme in this study, because nitrile 1 is
a useful chiral building block and lipase PS is applicable
to various substrates.2c,d This purified lipase showed a
single band in a SDS disk gel electrophoresis experiment
and has a molecular weight of 32000.1 It effectively
catalyzed hydrolysis of many unnatural ester substrates
but often exhibited modest enantioselectivity.5b Although
nitrile 1 was not a favorable substrate for lipase PS, some
crown ethers were found to significantly enhance both
enantioselectivity and reaction efficiency in the hydroly-
sis.
(4) (a) Sakurai, T.; Margolin, A. L.; Russell, A. J .; Klivanov, A. M.
J . Am. Chem. Soc. 1988, 110, 7236. (b) Parida, S.; Dordick, J . S. J .
Am. Chem. Soc. 1991, 113, 2253. (c) Parida, S.; Dordick, J . S. J . Org.
Chem. 1993, 58, 3238. (d) Kamat, S. Biotechnol. Bioeng. 1992, 40, 158.
(e) Arroyo, M.; Sinisterra, J . V. J . Org. Chem. 1994, 59, 4410. Control
of enzyme enantioselectivity by pressure change has recently been
reported. (f) Kamat, S. V.; Beckman, E. J .; Russell, A. J . J . Am. Chem.
Soc. 1993, 115, 8845 and references cited therein.
(5) Dextrometrophan,5a (S)-2-amino-4-(methylthio)-1-butanol,5b so-
dium chloride,5c,f calcium chloride,5d potassium chloride,5e and Triton
X-1005g were reported as effective additives. (a) Guo, Z.-W.; Sih, C. J .
J . Am. Chem. Soc. 1989, 111, 6839. (b) Itoh, T.; Ohira, E.; Takagi, Y.;
Nishiyama, S.; Nakamura, K. Bull. Chem. Soc. J pn. 1991, 64, 624. (c)
Tsukube, H.; Betchaku, A.; Hiyama, Y.; Itoh, T. J . Chem. Soc., Chem.
Commun. 1992, 1751. (d) Holmberg, E.; Holmquist, M.; Hedenstrom,
E.; Berglund, P.; Norin, T.; Hogberg, H.-E.; Hult, K. Appl. Microbiol.
Biotechnol. 1992, 35, 572. (e) Khmelnitsky, Y. L.; Welch, S. H.; Clark,
D. S.; Dordick, J . S. J . Am. Chem. Soc. 1994, 116, 2647. (f) Tsukube,
H.; Hiyama, Y.; Betchaku, A.; Itoh, T. J . Org. Chem. 1994, 59, 7014.
(g) Bhaskar, Rao A.; Rehman, H.; Krishnakumari, B.; Yadav, J . S.
Tetrahedron Lett. 1994, 35, 2611.
(6) (a) Odell, B.; Earlam, G. J . Chem. Soc., Chem. Commun. 1985,
359. (b) Reinhoudt, D. N.; Eondebak, A. M.; Nijenhuis, W. F.; Verboom,
W.; Kloosterman, M.; Schoemaker, H. E. Ibid. 1989, 399.
0022-3263/96/1961-2158$12.00/0 © 1996 American Chemical Society

Crown Ethers as Regulators of Enzymatic Reactions
J . Org. Chem., Vol. 61, No. 6, 1996 2159
Ta ble 1. Effect of Cr ow n Eth er Ad d itives on
Lip a se-Ca ta lyzed Hyd r olysis
relative
rate
% ee
of (R)-2
% ee
of S-1
time
c
entry additive (h) (% conv) (c/h) (% yield) (% yield)
E
1
2
3
4
5
6
7
8
9
none
none
none
nonea
nonea
nonea
3
4
5
6
7
8
9
10
11
12
13
14
15
14
60
96
3
18
38
41
8
1.3
0.6
0.4
2.7
1.7
1.4
1.3
1.8
0.8
3.7
1.1
3.5
2.4
2.1
3.3
3.7
4.4
3.6
2.2
4.8
3.9
3.1
2.4
3.8
5.4
3.3
3.6
3.2
4.3
4.2
4.1
3.1
2.8
3.2
2.1
1.9
1.5
3.3
3.7
1.6
1.3
3.7
77 (18)
80 (3.5) 16 (40) 16
17 (48)
9
68 (41)
75 (18)
81 (20)
79 (32)
80 (15)
87 (13)
72 (11)
80 (48)
84 (16)
79 (38)
75 (30)
72 (23)
76 (39)
70 (42)
56 (52)
73 (43)
69 (37)
47 (53)
77 (54)
87 (40)
81 (40)
83 (42)
83 (32)
87 (34)
74 (40)
84 (24)
65 (39)
71 (39)
74 (40)
79 (26)
84 (24)
84 (7)
8 (51)
6 (62)
48 (43) 17
68 (51) 17
18 (61) 11
29 (56) 19
8
8
22
33
14
14
14
13
14
14
14
14
14
14
14
14
14
14
37
46
18
25
11
52
16
45
34
30
46
52
62
50
41
67
54
44
46
42
35
46
50
41
60
59
57
57
37
42
32
46
23
46
52
22
18
52
F igu r e 1.
9 (60)
7
The hydrolysis of 1 was generally carried out in a
nonbuffered aqueous solution to exclude an effect of
complexation between crown ether and metal cation (eq
1). Typically, 5.0 mL of an aqueous lipase solution7 was
added to a solution of (()-1 (65.0 mg, 0.511 mmol),
together with a crown ether additive (33 mol % toward
the substrate) in 0.5 mL of acetone. The resulting
mixture was stirred at 35 °C, and the reaction was
stopped when spots of ester 1 and alcohol 2 became the
same size on a silica-gel TLC plate. The alcohol (R)-2
produced and remaining ester (S)-1 were extracted with
ethyl acetate and separated by silica-gel TLC (hexane/
ethyl acetate ) 2:1). The enantiomeric excess of the
remaining acetate 3 was determined by capillary GPC
analysis using chiral stationary phase (Chiraldex G-Ta).
The enantiomeric excess of alcohol (R)-2 was also deter-
mined by GPC analysis of the corresponding acetate
(Figure 1). Typical results obtained for reaction at 35°C
are summarized in Table 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
89 (32) 28
16 (58) 13
65 (37) 17
40 (38) 11
30 (41)
8
64 (34) 14
77 (36) 13
91 (28) 10
72 (32) 14
48 (29)
95 (29)
9
9
16
17(RS) 14
18(RS) 14
91 (44) 24
68 (39) 28
68 (26) 20
60 (30) 20
46 (43) 18
72 (42) 30
75 (32) 15
59 (32) 22
99 (33) 27
99 (40) 37
99 (21) 34
99 (21) 34
50 (38) 20
61 (55) 21
41 (47) 18
67 (44) 18
25 (57) 15
18(R)
18(S)
19
11
18(RS)a 6.5
19(RS) 14
20
21
22
23
24
25
26
27
14
13
14
14
14
14
13
13
We examined ten crown ethers 3-12, one cryptand 13,
eight heteromacrocycles 15, 16, 20-24, 26, eight armed
macrocycles 14, 17-19, 25, 27-29, and five acyclic
analogs 30-34 as additives (Figure 2). They vary widely
in structures and guest binding properties.
28(RS) 15
28(S)
29
30
31
32
33
34
85 (18)
80 (45)
84 (23)
56 (38)
74 (34)
85 (19)
70 (18)
79 (30)
24
15
14
14
14
14
14
The effect of these crown ethers on the enantioselec-
tivity of the lipase-catalyzed hydrolysis was evaluated
by E value,8 while the relative rate was calculated from
percentage conversion per reaction time compared with
that in the absence of additive.9 It is noteworthy that
the reaction rate was enhanced in the presence of most
additives employed, but the enantioselectivity depended
strongly on the nature of the additive (see Table 1).
Among 32 additives, benzocrown ether (entry 10), armed
azacrown ethers (entries 21-26), and thiacrown ethers
(entries 28-33) apparently enhanced both enantioselec-
tivity and reaction rate. The highest E value recorded
was 37 when the hydrolysis was carried out in the
presence of 1,4,8,11-tetrathiacyclotetradecane (23) (entry
30). Other thiacrown ethers also offered satisfactorily
large E values: 34 for 24; 34 for 25; and 27 for 22.
48 (42)
80 (43) 15
24 (57) 15
18 (55)
84 (33) 22
7
9
a
Reaction was carried out at pH 7.2 in 0.1 M phosphate buffer.
Rea ction P r ofile of Lip a se-Ca ta lyzed Hyd r olysis
in th e P r esen ce of Cr ow n Eth er . The E value is the
most convenient factor by which to evaluate the enanti-
oselectivity in the hydrolysis of racemic substrates. This
is known to be constant when the reaction possesses a
small Km,8 but the E value varied greatly in the reaction
conversion in this hydrolysis (see entries 1-6 in Table
1). This seems due to large Km of the enzymatic reaction
between the lipase and substrate 1.8 We determined E
values in three different reaction media: in pure water,
in a buffer solution, and in an aqueous solution of crown
ether additive (Figure 3). E values obtained were
extremely reduced at over 40% conversion when the
reaction was carried out in the absence of buffer or
additive. This must be a result of reverse reaction.
The reaction in the buffer solution (pH 7.2) also offered
a constantly high E value at 30-50%, while a lower E
value was observed at lower conversion.9 It was note-
worthy that thiacrown 23 maintained satisfactorily high
enantioselectivity even at 60% conversion. Since the
hydrolysis in the presence of thiacrown 23 provided a
much higher E value than those in the buffer solution,
this was confirmed as a useful additive that improves
this enzymatic reaction.
(7) The enzyme content in lipase PS employed here was less than
10% by weight, and the remainder was mostly amorphous inorganic
compounds (Celite). The content of the enzyme molecule is thus
estimated as less than 3.1 × 10-3 mmol/g of lipase PS.
(8) Chen, C. -S.; Fujimoto, Y.; Girdauskas, G.; Sih, C. J . J . Am.
Chem. Soc. 1982, 102, 7294. Chen, C-S.; Wu, S-H.; Girdauskas, G.;
Sih, C. J . Ibid. 1987, 109, 2812. The enantiomeric ratio was calculated
from E ) ln[(1 - c)(1 - ee(S)-1)]/ln[(1 - c)(1 + ee(S)-1)], where c means
conversion ratio and c ) ee(S)-1/(ee(S)-1 + ee(R)-2).
(9) We have reported the E value of this control experiment as 7-8
based on results of 19F NMR analysis of (+)-R-methoxy-R-phenyl-R-
(trifluoromethlyl)acetate (MTPA ester method).1 GPC analysis of all
experiments gave slightly higher E values than those obtained using
MTPA ester method.10 In the MTPA ester method, the remaining
acetate was first converted to alcohol by LAH reduction and then to
the corresponding MTPA ester.5b Since kinetic resolution may occur
during the MTPA formation process, capillary GPC analysis provided
us highly reliable results.
(10) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1972, 95, 512.

2160 J . Org. Chem., Vol. 61, No. 6, 1996
Itoh et al.
F igu r e 3. E value dependence of the lipase-catalyzed hy-
drolysis of 2-cyano-1-methylethyl acetate (1) upon reaction
conversion.
tration for the three cases but changes in the enantiose-
lectivity differed. Addition of 5-33 mol % of thiacrown
23 increased the E value from 16 to 29. Because the E
value showed a decrease in the presence of larger
amounts of 23, 250 or 1650 equiv of thiacrown ether 23,
based on the lipase, were optimum.7 Crown ether 28 and
polyamine 15 acted as regulators in different fashions:
the former changed the E value only slightly, and the
latter decreased it more noticeably. The nature of crown
ether additive clearly had an impact on the reaction
behavior.
Wh y Cr ow n Eth er s Mod ify Lip a se P er for m a n ce.
Crown ethers bind metal cations with varying strength
depending on structural variations. The lipase solution
employed was confirmed to contain the following alkali
and alkaline earth metal cations: Na+, 6.5 × 10-4 mol/
L; K+, 4.6 × 10-4 mol/L; Mg2+, 6 × 10-4 mol/L; Ca2+, 6.3
× 10-4 mol/L. These metal cations are good guests for
crown ethers 17-19 but not for thiacrown ethers 21-
27, though both types of additive enhanced the E value
and reaction rate of the hydrolysis. Some metal salts
were recently reported to influence the catalytic activities
of enzymes,5c-f but crown ether-metal cation complex-
ation may not be involved in this.
The reactivity of an enzyme is known to be significantly
influenced by the nature of a solvent.4 Since polyethylene
glycols and related polyethers provided unique microen-
vironments for proteins, we compared the additive activ-
ity of the crown ether with its hydrophobicity. The
hydrophobicity is a good indicator by which to evaluate
solvent character and is generally characterized by log
P.12 Since the log P values of the employed crown ethers
have not been reported, we estimated “relative hydro-
phobicity” from Rf value of the additive recorded on
reversed phase thin layer chromatography (see Experi-
mental Section). Figure 5 shows plots of E value and
relative hydrophobicity, 1/Rf, for various additives. Al-
though the additives 12, 15, and 25 having polar amino,
amide, and hydroxyl groups exhibited similarly higher
hydrophobicity values, thiacrown ether 25 offered a much
F igu r e 2. List of tested compounds.
Chirality of crown ether did not affect either enanti-
oselectivity or reaction efficiency of the hydrolysis (entries
22-25 and 35-36 in Table 1). We examined two kinds
of chiral crown ethers: crowns 17-19 have asymmetric
carbons on their sidearms; crown 28 has an asymmetric
carbon in the parent crown ring. The similar E values
and reaction rates were typically recorded when (R)-28
and (S)-28 were employed as additives (entries 35 and
36 in Table 1).11 The chirality of the sidearm of crown
18 also slightly affected the reactivity (entries 22-25 in
Table 1). Since no significant difference was observed
between enantiomers of chiral crown ethers, the stere-
ochemistry of the crown ether additives had no effect on
hydrolysis rate or enantioselectivity.
Figure 4, parts a and b, illustrate the effect of additive
concentration on the E value and reaction rate. There
are very interesting contrasts between the additive
activities of crown ethers 23, 28, and 15. The relative
rate increased similarly with increased additive concen-
(11) (S)-28 was derived from (S)-isopropylideneglycerol by a modi-
fication of the published method: Miyazaki, T.; Yanagita, S.; Itoh, A.;
Okahara, M. Bull. Chem. Soc. J pn. 1982, 55, 2005. For the method of
preparation, see reference 5f.
(12) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng.
1987, 30, 81.

Crown Ethers as Regulators of Enzymatic Reactions
J . Org. Chem., Vol. 61, No. 6, 1996 2161
a
F igu r e 6. Induced chemical shift values of 13C NMR of
3-hydroxybutyronitrile 2 by thiacrown 20, 23, and azacrown
15.
b
Crown ethers bind neutral organic molecules in the
crystal states and nonpolar media.13 Since our substrate
1 has a nitrile group, and product 2 has a further
hydroxyl moiety, crown ether may trap such neutral
molecules having acidic moieties in the course of the
reaction. We conducted 13C NMR binding experiments
in CDCl3 solution using polyamine 15 and thiacrowns 20
and 23. These three additives accelerated the hydrolysis,
but only thiacrown 23 enhanced its enantioselectivity.
Figure 6 illustrates typical spectral changes of product
2 in the presence of crown ether additive. Large induced
13C NMR spectral changes were observed at C-3 position
by addition of these crown ethers, while no significant
change was found in the case of substrate 1. It is
suspected that they may bind the product and depress
the reverse reaction. Thiacrown 23 was suggested to
bind product 2 particularly strongly; this may explain
the high additive activity of thiacrown 23. Since the
lipase itself preferentially hydrolyzes (R)-enantiomer of
1,5b (R)-alcohol 2 should be preferentially acylated by the
lipase. However, it is impossible to run the reverse
reaction with 0.1 M acetate as the acetylating agent
because the concentration of water in a 10% aqueous
acetone solution is in excess of 45 M under the experi-
mental conditions. Both reaction rate and enantioselec-
tivity can be enhanced if the reverse reaction occurs
inside the enzyme near or at the entrance of the active
site and is inhibited by trapping of the product with the
crown ether. Large induced shifts of carbon signals for
2 were similarly observed with other additives, 6, 20, 23,
24, and 26. Since these additives mediated highly
enantioselective hydrolysis (see Table 1), the induced
chemical shift provides a good guideline for selection of
a potential additive.
F igu r e 4. (a) Effect of concentration of additive on the E value
of lipase-catalyzed hydrolysis. (b) Effect of concentration of
additive on rate of lipase-catalyzed hydrolysis.
F igu r e 5. Relationship of relative hydrophobicity of additive
(1/Rf) with the E value in hydrolysis.
Molecu la r Ba sis for En h a n ced En a n tioselectiv-
ity. There are three possibilities in additive-enhanced
enantioselectivity of the lipase hydrolysis. (1) The first
is the enantioselective inhibition,5a in which the crown
ether additive may be specifically located near the active
site of the lipase and modify its reactivity. Since the
lipase should provide a chiral cavity, chirality and
hydrophobicity of the crown ether additive should reflect
higher E value than additives 12 and 15. All other
additives showed lower “relative hydrophobicity” values
but their E values varied broadly. Thus, there is no
significant correlation between hydrophobicity and the
E value. Since the reaction rate was also independent
of the relative hydrophobicity, the lipase employed was
believed not to accommodate these additives site-specif-
ically near the active site or allosteric site.
(13) Goldberg, I. In Crown Ethers and Analogs; Patai, S., Rappoport,
Z., Eds.; J ohn Wiley & Sons: Chichester, 1989; p 399.

2162 J . Org. Chem., Vol. 61, No. 6, 1996
Itoh et al.
on the reactivity. Because the lipase hydrolysis did not
depend on the chirality and hydrophobicity of the addi-
tive, this possibility can be ruled out. (2) The second is
inhibition of the reverse reaction, which may occur inside
in the enzyme close to or at the entrance to the active
site, by the presence of crown ethers. The interaction
between the crown ether and product alcohol (R)-2 seems
adequate to inhibit the reverse reaction, and it explains
both accelerations of the hydrolysis rate and enhance-
ment of the enantioselectivity. However, it should be
noted that large amounts of crown ether decreased the
enantioselectivity, although it increased the rate; thus,
another mechanism must be cooperatively involved. (3)
The last possibility is modification of the lipase local
conformation. Enzyme reactivity was sometimes modi-
fied by addition of lipid14 or salt.5d The employed crown
ethers may interact with certain sites of the lipase as
proposed in some proteins, thereby activating the lipase
and changing its enantioselectivity. Previously it was
reported that covalent modification of the arginine side
chain in the lid of lipases from Humicola lanuginosa and
from Rhizomucor miehei decreased their enantioselec-
tivity toward esters of 2-methyldecanoic acid.15 The
crystal structure of Candida rugosa lipase has shown two
different conformations.16 In one, called “closed”, the
helical surface loop partially covers the hydrophobic
crevice containing the active site, while in the other
conformation, called “open”, the surface loop moves and
the crevice is uncovered.17 It was recently reported by
Kazlauskas and his colleagues18 that 2-propanol treat-
ment of Candida rugosa lipase caused modification of
enantioselectivity. 2-Propanol treatment was proposed
to convert the closed form of this lipase to the open form,
thereby enhancing the enantioselectivity.18 Although
Pseudomonas cepacia lipase has not been characterized
in detail, such conformational changes may be induced
by crown ether additive.
While we have not ruled out other possibilities, both
the second and third points mentioned explain our
observations in Figure 4, parts a and b. When crown
ethers remain at the entrance of the lipase, they may
modify local conformation of the lipase, trap the product
alcohol, and accelerate diffusion of the alcohol into the
bulk water phase (Figure 7).
The present study demonstrated that the crown ether
derivatives have the potential to enhance both reaction
rate and enantioselectivity of the lipase-catalyzed hy-
drolysis of 2-cyano-1-methylethyl acetate. These crown
compounds cannot change the original enantioselectivity
of enzyme, but enhance its potential ability to a level at
which the reaction can be used practically.19 This work
represents not only a significant advance in improvement
of lipase-catalyzed organic synthesis but also provides an
interesting combined use of crown ether with enzyme.
The approach is therefore recommended as a new tech-
F igu r e 7. Plausible mechanism of acceleration of the lipase-
catalyzed reaction by thiacrown 23.
nique by which to regulate enzymatic reactions by
chemical reagents.
Exp er im en ta l Section
Ma ter ia ls. Crown ethers were purchased from Aldrich or
J ensen and used without further purification. Azacrown
ethers 17-19 were prepared by reaction of corresponding
unsubstituted azacrown ethers (Aldrich) and (1,2-epoxyethyl)-
benzene. Pseudomonas cepacia lipase was provided by Amano
Pharmaceutical Co., Ltd. (J apan).
En zym e-Ca ta lyzed h yd r olysis. A typical example is
described below: To an acetone solution (0.5 mL) of ester 1
(65.0 mg, 0.511 mmol) and 1,4,8,11-tetrathiacyclotetradecane
(38.4 mg, 0.168 mmol) was added the enzyme solution (5.0
mL), and the resulting mixture was incubated at 35 °C. The
reaction was stopped by addition of small pieces of ice and then
extracted with ethyl acetate, dried over MgSO4, and evapo-
rated to dryness. The product (R)-2 and remaining substrate
(S)-1 were separated by silica-gel TLC (hexane:ethyl acetate
) 2:1). The enzyme solution was prepared in the following
way: A boltex shaking suspension of lipase PS (50 mg) in
deionized water (7.7 mL) was centrifuged at 3000 rpm for 5
min at room temperature, and then 5.0 mL of the supernatant
was immediately used as the enzyme solution. GPC analysis
for determination of % ee of 2-cyano-1-methylethyl acetate was
carried out using a capillary column on chiral phase; Chiraldex
G-Ta, L 0.25 mm × 20 m; carrier gas: He 40 mL/min; temp
(°C): 100; inlet pressure: 1.35 kg/cm2; amount 400 ng;
1
detection, FID; (R)-2-cyano-1-methylethyl acetate: tR 5.2; K′1
2
1.1, (S)-2-cyano-1-methylethyl acetate: tR 9.4; K′2 2.8, K2/K1
) 2.5.
Rela tive Hyd r op h obicity of Cr ow n Com p ou n d s. Rela-
tive hydrophobicity is defined by 1/Rf value recorded on
reversed phase TLC of Merck-LKC18 using a mixed solvent
of acetone and water (4:1). Triplicate TLC experiments were
conducted, and Rf values shown are the average. Crowns 3:
Rf 0.65, 5: Rf 0.38, 7: Rf 0.28. Azacrowns 9: Rf 0.69, 10: Rf
0.79, 12: Rf 0.36. Armed macrocycles 14: Rf 0.63, 17: Rf 0.24,
18: Rf 0.67, 28: Rf 0.18, 29: Rf 0.67. Heteromacrocycle 15:
(14) Okahata, Y.; Ijiro, K. Bull. Chem. Soc. J pn. 1992, 65, 2411;
Hatano, A.; Ijiro, K.; Okahata, Y. The 6th Conference of Synthetic
Organic Chemistry Using Biocatalysts, Seto J apan, J anuary 1994, Abst
No. 2017
(15) Holmquist, M.; Martinelle, M.; Berglund, P.; Clausen, I. G.;
Patkar, S.; Svendsen, A.; Hult, K. J . J . Protein Chem. 1993, 12, 749.
(16) (a) Rubin, B.; J amison, P.; Harrison, D. Lapases: Structure,
Mechanism and Genetic Engineering; Alberghina, L.; Schmid, R. D.;
Verger, R., Eds.; VCH: New York, 1991; pp 63-66. (b) Grochulski, P.;
Li, Y.; Schrag, J . D.; Bouthillier, F.; Smith, P.; Harrison, D.; Rubin,
B.; Cygler, M. J . Biol. Chem. 1993, 268, 12843.
(19) We employed thiacrown 23 as an additive in hydrolysis of
2-substituted-2-propenyl acetates or 1-alkyl-3-cyanopropyl acetates.
Hydrolysis rate of all substrates was accelerated by addition of 5 mol
% of thiacrown 23, but enantioselectivity strongly depended on the
nature of the substrate. Significant enhancement of enantioselectivity
was observed when the original E values were from 7 to 15.
(17) Grochulski, P.; Li, Y.; Schrag, J . D.; Cygler, M. Protein Sci.
1994, 3, 82.
(18) Colton, I. J .; Sharmin, N. A.; Kazlauskas, R. J . J . Org. Chem.
1995, 60, 212.

Crown Ethers as Regulators of Enzymatic Reactions
J . Org. Chem., Vol. 61, No. 6, 1996 2163
Ta ble 2. 13C NMR Sp ectr a of Aceta te 1 in th e P r esen ce of Cr ow n Eth er s (in CDCl3)
additive
(equiv)
C-6(Ac),
ppm
C-4,
ppm
C-2,
ppm
C-3,
ppm
C-1(CN),
ppm
C-5(CO),
ppm
shift at C-3,
ppm
shift at C-1,
ppm
shift at C-5,
ppm
none
5 (2.0)
6 (2.0)
7 (2.0)
19.08
18.90
19.17
19.05
19.03
19.21
19.21
19.08
19.10
19.16
19.01
19.09
19.06
20.83
20.65
20.90
20.78
20.76
20.99
20.98
20.82
20.88
20.91
20.78
20.84
20.78
24.27
24.07
24.36
24.23
24.19
24.41
24.39
24.27
24.29
24.34
24.19
24.25
24.20
65.43
65.27
65.51
65.40
65.37
65.51
65.50
65.38
65.38
65.45
65.34
65.47
65.43
116.17
116.03
116.13
116.09
116.07
116.07
116.23
116.07
116.14
116.16
116.08
116.21
116.16
169.92
169.69
170.04
169.87
169.70
170.10
170.09
169.86
169.92
169.91
169.88
169.96
169.84
reference
-0.16
0.08
-0.03
-0.06
0.08
reference
-0.15
-0.05
-0.08
-0.10
-0.10
0.06
-0.10
-0.04
-0.01
-0.10
0.04
reference
-0.24
0.12
-0.06
-0.22
0.18
0.17
-0.07
0.00
9 (2.0)
15 (2.0)
18 (2.0)
20 (2.0)
23 (2.0)
24 (2.0)
25 (2.0)
32 (2.0)
33 (2.0)
0.06
-0.05
-0.05
0.02
-0.09
0.04
-0.01
-0.05
0.04
0.00
-0.01
-0.09
Ta ble 3. 13C NMR sp ectr a of a lcoh ol 2 in th e p r esen ce of
cr ow n eth er s (in CDCl3)
Rf 0.19. Thiacrowns 20: Rf 0.41, 21: Rf 0.58, 22: Rf 0.82, 23:
Rf 0.41, 24: Rf 0.49, 25: Rf 0.18, 26: Rf 0.37. Acyclic
polyethers 30: Rf 0.83, 31: Rf 0.73, 32: Rf 0.67, 33: Rf 0.83.
Syn th esis of (R)-4,7,10,13-Tetr a oxa -N-(2-p h en yl-2-h y-
d r oxyeth yl)a za cyclop en ta d eca n e (18). To a solution of
1-aza-15-crown-5 (9) (97%, 468 mg, 2.07 mmol) and (R)-(1,2-
epoxyethyl)benzene (249 mg, 2.07 mmol) in dry ether (10 mL)
was added of activated alumina powder20 (1.0 g, activity 1).
The resulting mixture was stirred for 23 days at room
temperature and then filtered through a glass sintered filter
and evaporated to dryness. Silica-gel flash column chroma-
tography (hexane:ethyl acetate:methanol, 4:4:1) gave (R)-18
(530 mg, 1.56 mmol) in 75% yield as colorless oil: [R]23D -34.5°
additive C-4, C-2, C-3, C-1(CN), shift at C-3, shift at C-1,
(equiv) ppm ppm ppm
ppm
ppm
ppm
none 22.41 27.24 63.65 117.80
reference
-0.27
0.36
0.05
-0.58
0.08
0.10
0.08
0.06
0.37
0.16
0.14
0.24
0.33
0.34
0.25
0.44
0.48
0.50
0.50
0.21
0.08
0.12
0.02
-0.12
-0.18
reference
-0.02
-0.26
0.24
0.08
0.09
0.09
0.11
0.12
-0.27
-0.21
-0.26
-0.31
-0.39
-0.23
-0.33
-0.31
-0.38
-0.37
-0.40
-0.36
-0.25
-0.24
-0.20
-0.15
-0.23
5 (2.0) 22.38 27.19 63.38 117.78
6 (2.0) 22.63 27.39 64.00 117.54
7 (2.0) 22.48 27.23 63.70 118.04
9 (2.0) 22.63 27.36 63.07 117.88
15 (0.5) 22.83 27.59 63.73 117.89
15 (1.0) 22.87 27.62 63.75 117.89
15 (1.5) 22.88 27.64 63.73 117.91
15 (2.0) 22.88 27.63 63.71 117.92
18 (2.0) 22.74 27.47 64.02 117.53
20 (0.5) 22.54 27.33 63.81 117.59
20 (1.0) 22.50 27.33 63.79 117.54
20 (1.5) 22.49 27.27 63.88 117.49
20 (2.0) 22.67 27.42 63.98 117.41
21 (2.0) 22.66 27.44 63.99 117.57
22 (2.0) 22.63 27.38 63.90 117.47
23 (0.5) 22.73 27.48 64.08 117.49
23 (1.0) 22.76 27.49 64.13 117.42
23 (1.5) 22.77 27.49 64.14 117.43
23 (2.0) 22.78 27.50 64.15 117.40
24 (2.0) 22.61 27.37 63.86 117.44
25 (2.0) 22.56 27.33 63.73 117.55
26 (2.0) 22.53 27.32 63.77 117.56
27 (2.0) 22.53 27.30 63.67 117.60
32 (2.0) 22.45 27.21 63.53 117.65
33 (2.0) 22.42 27.16 63.46 117.57
1
(c 0.96, CHCl3), H NMR (200 MHz, δ, CDCl3, J ) Hz) 2.47 (1
H, dd, J ) 13.1, 12.9), 2.66 (1 H, dd, J ) 6.1, 5.4), 2.73 (1 H,
OH, s), 2.73 (1 H, t, J ) 4.8), 2.75-2.85 (2 H, m), 3.55-3.73
(16 H, m), 4.61 (1 H, dd, J ) 10.4, 3.1), 7.1-7.4 (5 H, m);
13C NMR (50 MHz, CDCl3, ppm) 55.75, 65.26, 69.91, 70.11,
70.41, 70.54, 70.78, 125.92, 127.13, 128.12, 142.33; IR (neat,
cm-1) 3420, 2900, 1460, 1360, 1120, 940, 710. Anal. Calcd
for C18H29NO5: C, 63.69; H, 8.61; N, 4.13. Found: C, 63.66;
H, 8.60; N, 4.11.
(S)-18 was prepared from (S)-(1,2-epoxyethyl)benzene by
the procedure described above. (S)-18 ; [R]23 +35.4° (c
D
1.01, CHCl3). The spectroscopic data were exactly the same
as (R)-18.
4,7,10,13-Tr ioxa -N-(2-p h en yl-2-h yd r oxyet h yl)a za cy-
clod od eca n e (17) was prepared from crown 8 and (1,2-
epoxyethyl)benzene (oil, 77%): 1H NMR (200 MHz, δ, CDCl3,
J ) Hz) 2.44 (1 H, dd, J ) 12.9, 10.7), 2.58 (1 H, dd, J ) 4.3,
4.0), 2.66 (1 H, dd, J ) 7.1, 4.1), 2.69 (1 H, dd, J ) 13.8, 3.4),
2.83 (1 H, dd, J ) 14.1, 3.4), 2.87 (1 H, dd, J ) 14.2, 3.4), 3.54-
3.72 (12 H+OH, m), 4.63 (1 H, dd, J ) 10.6, 3.3), 7.14-7.3 (2
H, m), 7.3-7.4 (3 H, m); 13C NMR (50 MHz, CDCl3, ppm) 55.68,
64.50, 69.44, 70.44, 70.63, 70.83, 125.86, 127.04, 128.07,
142.32; IR (neat, cm-1) 3430, 3030, 2850, 1450, 1360, 1090,
1030, 910, 700. Anal. Calcd for C16H25NO4: C, 65.06; H, 8.53;
N, 4.74. Found: C, 65.09; H, 8.60; N, 4.70.
dissolved in CDCl3 at a concentration of 0.05-0.06 mol/L.
Results are summarized in Tables 2 and 3.
Ack n ow led gm en t. This research was supported in
part by a Grant-in-Aid to Y. T. as a J SPS Research
Fellow from the Ministry of Education, Science and
Culture of J apan. It was also supported in part by the
Ryobi-Teien Fund for Bioscience and the Nishida Re-
search Fund for Fundamental Organic Chemistry. The
authors are grateful to Dr. Yoshihiko Hirose of Amano
Pharmaceutical Co. for providing information on lipase
PS. They also thank the SC-NMR Laboratory of Okaya-
ma University for the 13C NMR experiment. The
provision of lipases by Amano Pharmaceutical Co. is also
appreciated.
4,7,10,13,16-P en ta oxa -N-(2-p h en yl-2-h yd r oxyeth yl)a za -
cycloocta d eca n e (19) was prepared from crown 10 and (1,2-
epoxyethyl)benzene (oil, 56%): 1H NMR (200 MHz, δ, CDCl3,
J ) Hz) 2.50 (1 H, dd, J ) 12.5, 10.5) 2.63 (1 H, t, J ) 3.0)
2.70 (1 H, t, J ) 4.8) 2.86 (1 H, dd, J ) 11.9, 4.9) 2.90 (1 H,
dd, J ) 13.6, 4.9) 3.5-3.7 (20 H+OH, m) 4.61 (1 H, dd, J )
10.1, 3.0) 7.1-7.4 (5 H, m); 13C NMR (50 MHz, CDCl3, ppm)
53.40, 55.04, 64.52, 69.78, 69.91, 70.29, 70.50, 70.56, 70.64,
70.78, 125.91, 127.16, 128.16, 142.46; IR (neat, cm-1) 3400,
2850, 1440, 1340, 1240, 1100, 930, and 750. Anal. Calcd for
C20H33NO6: C, 62.64; H, 8.67; N, 3.65. Found: C, 62.33;
H,8.72; N,3.68.
Su p p or tin g In for m a tion Ava ila ble: Tables of experi-
mental results detailing the data of Figures 3 and 4, parts a
1
and b, H and 13C NMR and IR spectra for compounds 17-19
NMR Bin d in g Exp er im en ts. 13C NMR studies were
carried out with a Varian VXR-200 spectrometer (SC-NMR
Laboratory of Okayama University). Crown ethers were
(12 pages). This material is contained in libraries on micro-
fiche, immediately follows this article in the microfilm version
of the journal, and can be ordered from the ACS; see any
current masthead page for ordering information.
(20) Yang, L.; Weber, A. E.; Greenlee, W. J .; Patchett, A. A.
Tetrahedron Lett. 1993, 34, 7035.
J O951598+