Preparation of gemini-type amphiphiles bearing cyclitol head groups and their application as high-performance modifiers for lipases

Article abstract of DOI:10.1016/j.carres.2003.12.006

Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N′- [iminobis(trimethylene)]bisquinamide, prepared from

Full text of DOI:10.1016/j.carres.2003.12.006

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 493–501
Preparation of gemini-type amphiphiles bearing cyclitol head groups
and their application as high-performance modifiers for lipases
Yurie Mine,a Kimitoshi Fukunaga,a,* Ken-ichi Samejima,a Makoto Yoshimoto,a
Katsumi Nakaoa and Yoshiaki Sugimurab
aDepartment of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai,
Ube 755-8611, Japan
bGeneral Education Division, Oshima National College of Maritime Technology, Komatsu, Oshima-cho, Yamaguchi 742-2193, Japan
Received 14 June 2003; accepted 8 December 2003
Abstract—Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly
synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N0-[iminobis(trimethylene)]bisquinamide,
prepared from iminobispropylamine and quino-1,5-lactone, and dialkyl N-(3-carboxypropanoyl)-L-glutamates as polar and
hydrophobic components, respectively. Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL) are co-lyophilized with
these synthesized gemini-type amphiphiles, and their transesterification activities in organic solvents are evaluated. The modified
PCL and CRL prepared by using each amphiphile showed highly enhanced and moderately enhanced enzyme activity, respectively.
These results are discussed in terms of the increased preferential exclusion of the hydrophilic heads of the amphiphile and of the
topological view of the amphiphile.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Amphiphile; Axial hydroxy group; Modified lipase; Quinic acid
1. Introduction
For lipase activation, however, the co-lyophilizates4 or
co-precipitates5;6 with amphiphiles that have sugar
alcohol as a hydrophilic moiety were more active than
In recent years, enzymatic catalysis in non-aqueous
media has gained considerable interest as an efficient
preparative approach to biosynthesis, because the
development of enzymes in non-aqueous media has
numerous advantages compared to their use in con-
ventional aqueous media. Although numerous enzymes
are catalytically active in organic media, the activity of
native enzymes in anhydrous organic media is much
lower than that in water. Hence, many methods have
been introduced leading to improved enzyme activity
in organic media.1 One of the most practical and
simple methods of enzyme modification among them is
co-lyophilization with lyoprotectants such as carbohydrates.2;3
those with lactose, sucrose, or
D
-glucitol (sorbitol).4 It
might be suggested that the poorer activating abilities of
carbohydrates seem consistent with the difficulty to open
the lid of the lipase and to crystallize it in the activated
form, because the amphiphiles, which contain a hydro-
phobic tail and a hydrophilic head group, have a high
efficiency in making a lipid–water interface, and in turn
in crystallizing the enzyme in an open lid form upon
co-lyophilization.4 More recently, we have prepared the
gemini or dimeric amphiphiles that have two alkyl
chains and two
D-arabinitol groups connected with a
spacer.7;8 These amphiphiles were about 11-fold more
efficient for activating lipase than conventional single-
head and single-tail amphiphiles. This high enzyme
activity could be explained in terms of steric exclusion of
polyolic heads of the amphiphiles from the non-polar
* Corresponding author. Tel.: +88-836-85-9272; fax: +88-836-85-9201;
e-mail: fukun@yamaguchi-u.ac.jp
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.12.006

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Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
region of protein surface in the aqueous system prior
to freeze-drying.9 The steric exclusion of the sugar is
enhanced as the number of equatorial OH (eq OH)
groups per sugar molecule decreases.10 In fact, it is
known that myo-inositol, one of a typical cyclic polyol,
is strongly repelled from the non-polar region of the
protein surface and displays a very extensive preferential
hydration of the protein different from other linear
polyols.11;12 Since quinic acid is the most readily avail-
able cyclic polyol at present, a chemical synthesis of the
gemini amphiphile containing quinic acid as the heads
was carried out. The preparative details of the synthesis
and the activation behavior of the resulted gemini
amphiphiles toward lipases in non-aqueous media are
reported herein.
the reaction of racemic 6-methyl-5-hepten-2-ol (racemic
sulcatol) with isopropenyl acetate catalyzed by PCL
co-lyophilized with each amphiphile, 9–13, or native pow-
der (control) PCL in diisopropyl ether at 30 ꢁC. From
most of the data points in Figure 1, the yield of (R)-())-
acetyloxy sulcatol in the reaction catalyzed by the PCL-
co-lyophilizate with the amphiphile was approximately
100% after only about 150 min, whereas the native
powder PCL showed little catalytic activity. Initial re-
action rates up to ca. 20% conversion were used to
evaluate the specific activity: mmol hÀ1 (g of lipase)À1
,
and the enantioselectivity was expressed as the
enantiomeric excess (ee) of the enantiopreferred product at
ca. 30% conversion. Table 1 summarizes enzyme activ-
ities and enantioselectivities for the transacetylations by
using the PCL co-lyophilized with each amphiphile at
various amphiphile concentrations. The primary differ-
ence of the molecular structure among the amphiphiles
used in this investigation is in the hydrophile–lipophile
balance (HLB). In Table 2, we list the calculated HLB
values of the amphiphiles, 9–14, from so-called group
2. Results and discussion
2.1. Synthesis of the gemini amphiphiles
numbers assigned to each group on the basis of the
P
The gemini-type amphiphiles, 2-[3-(bis{3-[(1,3,4,5-tetra-
hydroxycyclohexanecarbonyl)amino]propyl}carbam-oyl)-
propionylamino]pentanedioic acid dialkyl esters (9:
didecyl ester; 10: didodecyl ester; 11: ditetradecyl ester;
12: dihexadecyl ester; 13: dioctadecyl ester), were pre-
pared using the previously reported mixed anhydride
method,7;8 substituting N,N0-[iminobis(trimethylene)]-
equation:16 HLB ¼ ðhydrophilic group numbersÞ þ
P
ðlipophilic group numbersÞ þ 7, and the solubilities
of the amphiphiles in water. Tables 1 and 2 illustrate
that the HLB value correlated with the effectiveness of
the amphiphiles as well as the results in our previous
paper.8 The PCL co-lyophilized with 9 (HLB 10.7) from
the aqueous amphiphile solution of 32–65 mM (the
molar ratio of amphiphile/PCL is 100–200, assuming
PCL is 100% pure) showed the largest enzyme activity
for the transacetylation between racemic sulcatol and
isopropenyl acetate in diisopropyl ether, and the enzyme
activity of the PCL-10 (HLB 8.8) or 11-co-lyophilizate
at the aqueous amphiphile solution of 48–65 mM (the
molar ratio is 150–200) was comparable to those of the
gemini amphiphiles in our previous works.7;8 Use of
the amphiphile of HLB below 5 was less effective. It has
been clearly established that lid displacement, which
uncovers the catalytic site, is involved in the mechanism
of interfacial activation of lipases,4;17 and it might be
suggested that the extent of the activation of lipases in
non-aqueous media is directly proportional to the per-
formance of an amphiphile to open its lid in an aqueous
solution prior to co-lyophilization. Thus, this great
enhancement of the PCL activity induced by the co-
lyophilization with amphiphiles of HLB 7–11, can be
accounted for by its HLB value close to 10, at which the
hydrophilic–lipolytic property at the interface just bal-
ances. In turn the performance as an oil–water interface
is maximized. However, the enzyme activity of the PCL-
co-lyophilizate with 9 (HLB 10.7) was 2-fold over that
of its counterpart amphiphile, 14 (HLB 11.3),8 even
though their HLB values are very close [107 vs
53 mmol hÀ1 (g PCL)À1 at a 1:100 molar ratio as shown
in Table 1]. The critical micelle concentration (cmc) of
bis-
bisquiamide (3) as a polar component, in moderate
yields. Lactonization of -())-quinic acid (1) was
D
-gluconamide for N,N0-[iminobis(trimethylene)]-
D
carried out by azeotropic dehydration,6 since the
reported reaction is known to proceed under severe
conditions in low yield.13 The gemini-type amphiphile
bearing linear polyols as heads, 2-(3-{bis[3-(2,3,4,5,6-
pentahydroxyhexanoylamino)propyl]carbamoyl}propionyl-
amino]pentanedioic acid didecyl ester (14)14 was also
prepared for comparison. Compounds 9–13 and com-
pound 14 were purified by a combination of column and
gel-filtration chromatography and by repeated re-
crystallization from 2-propanol, respectively, and their
structures were confirmed by 13C NMR spectroscopy.
Scheme 1 shows the molecular structures of the com-
pounds 1–14.
2.2. Enzyme activity of the gemini amphiphile–lipase
complexes in organic solvent
For this study Pseudomonas cepacia lipase (PCL) and
Candida rugosa lipase (CRL) were selected as lipases,
because serious differences in the enzyme activity
between the PCL- and CRL-co-lyophilizate with poly-
(oxyethylene) detergent, crown ethers, or cyclodextrins
were previously observed.14;15 Figure 1 shows re-
presentatively the time courses of the transacetylation by

Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
495
Scheme 1. The structure of compounds 1–14.
the novel non-ionic gemini amphiphiles has not been
measured yet. However, it has been known that ionic
gemini amphiphiles are more likely than conventional
surfactants to adsorb at the aqueous solution surface
rather than to form micelles.18 Thus, it could be pre-
sumed that when the gemini amphiphile was mixed with
lipase in aqueous buffer solution before co-lyophiliza-
tion, it interacts monomerically with enzyme protein.
Furthermore, this estimate was borne out by the
dependence of the amphiphile-derived activation of lipase
in non-aqueous medium on the aqueous concentration
of the amphiphile as shown in Table 1: if the amphi-
philes are organized as micelles, a sudden increase in the
activation should be expected at a certain con-
centrations.4 Therefore, the factor governing the enzyme
activity is attributable to the abundance of axial
hydroxy groups, which enhance the natural-like char-
acteristics of PCL due to the preferential hydration of
the enzyme protein, by the hydrophilic heads of 14
80
60
40
20
0
(9)
(10)
(11)
(12)
(13)
Control
0
100 200 300 400 500
Reaction time ( min )
Figure 1. Comparison of the time course of the PCL-co-lyophilizate
and non-co-lyophilizate with an amphiphile-mediated transesterifica-
tion between racemic sulcatol (2 mmol) and isopropenyl acetate
(2 mmol) in anhyd diisopropyl ether (4 mL) at 30 ꢁC. Enzymes were
obtained by lyophilization of 10 mg PCL from 10 mL of aq phosphate
buffer (10 mM, pH 7) containing 32 lmol of the corresponding
amphiphile (ꢀ: 9; :10; M: 11; N: 12; ꢀ: 13) or no additive (i.e.,
ꢁ
control PCL powder: ), and enzyme concentration as native PCL in
j
diisopropyl ether was 2.5 mg mLÀ1
.

496
Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
Table 1. Enzyme activities and enantioselectivities of the PCL co-lyophilized with an amphiphile and of the native PCL
Molar quantitiesa (lmol) Molar ratio of amphiphile/PCL Enzyme activity (mmol hÀ1 (g-PCL)À1
)
eeR (%)
b
Co-lyophilized amphiphile
9
16
32
48
65
16
32
48
65
16
32
48
65
16
32
48
65
16
32
48
65
32
––
50
100
150
200
50
61
107
118
124
47
59
75
77
42
52
55
60
29
48
48
47
26
20
17
20
53
1
92
88
88
90
85
85
84
83
88
84
83
82
88
88
88
85
90
87
85
86
87
86
9
9
9
10
10
10
10
11
11
11
11
12
12
12
12
13
13
13
13
14
Nonec
100
150
200
50
100
150
200
50
100
150
200
50
100
150
200
100
––
aPCL (10 mg) was co-lyophilized with the amphiphile of different molar quantities from 10 mL of aq phosphate buffer.
bEnantiomeric excess (ee) of the (R)-())-acetyloxy sulcatol determined by the enantiomeric purity of the product at ca. 30% conversion.
cControl PCL powder.
Table 2. The calculated HLB values and solubilities in water of the
amphiphiles
enhanced as high as 78-fold, whereas, there was only a
5-fold increase for the co-lyophilized CRL with its
diastereomeric amphiphile, D-BIG2C12CA (HLB 9.4)
Amphiphile
HLB value
Solubilities in watera
from the same aqueous concentration.19 The lack of
enhancement for the CRL co-lyophilizate with
9
10.7
8.8
++
+
10
11
12
13
14
6.9
5.0
+
D
-BIG2C12CA was correlated to the inhibited contact of
)
)
++
carbonyl groups of the amphiphile with Tyr69 in the lid
of the CRL by the steric hindrance between potential
oligosaccharide chains attached at Asn351 of the CRL
3.1
11.3
aThe extent of solubility of 30 mg of amphiphile in 10 mL of water at
25 ꢁC was graded from + (fairly soluble) to ++ (easily soluble); );
insoluble.
and polyol chains of the
D-amphiphile, when two
hydrophobic tails of the amphiphile bind to the strands
of the small and large b-sheet situated in N-terminal end
side of the CRL,19;20 as are illustrated in Figure 2. Thus,
it was suggested that by comparing the enhanced
enzyme activity of the CRL co-lyophilizate with 9 (HLB
10.7) or 10 (HLB 8.8) from the aqueous amphiphile
solution of 62 mM (21- or 29-fold, respectively) to that
of the co-lyophilizate with 14 (HLB 11.3) from the same
aqueous concentration (84-fold) (Table 3), the steric
hindrance between potential oligosaccharide chains,
attached at Asn351, of the CRL and cyclic polyol chains
of the present amphiphiles are intermediate between
compared to that of 9.11;12 Regarding enantioselectivity,
enantiomeric specificity of every activated PCL was
unaltered (Table 1). Next, we investigated the depen-
dence of the enzyme activity of the CRL co-lyophilized
with each amphiphile, 9, 10, 12–14, or native powder
CRL. The reaction used for CRL was transbutyrylation
between racemic solketal and vinyl butyrate in anhy-
drous cyclohexane at 30 ꢁC. As shown in Table 3,
moderately enhanced enzyme activities and slightly en-
hanced enantioselectivities were observed in this case. In
our previous paper, it was found that the enzyme activity
of the co-lyophilized CRL with
pentahydroxyhexanoylamino)propyl]carbamoyl}propio-
nylamino)pentanedioic acid didodecyl ester (L-
BIG2C12CA: HLB 9.4) from the aqueous amphiphile
solution of 62 mM (the molar ratio is 200) was greatly
those of
respective low-energy conformer of three amphiphiles
-BIG2C12CA, -BIG2C12CA, and 10, shown in Figure 3,
D
-BIG2C12CA and
L-BIG2C12CA. The
L
-2-(3-{bis[3-(2,3,4,5,6-
L
D
supports this reasoning. The geometry of these mole-
cules was optimized by using MM2 in Chem. 3D of
the Chemical Structure Markup Language package
(Cambridge Software Co.).

Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
497
Table 3. Enzyme activities and enantioselectivities of the CRL co-lyophilized with an amphiphile and of the native CRL
Molar quantitiesa (lmol) Molar ratio of amphiphile/CRL Enzyme activity (mmol hÀ1 (g-CRL)À1
)
eeS (%)
b
Co-lyophilized amphiphile
9
7.7
15
50
100
200
400
50
8
11
21
16
10
12
29
15
9
34
28
39
31
34
29
36
35
34
32
34
36
36
35
37
33
34
32
31
34
38
29
9
9
31
62
9
10
10
10
10
11
11
11
11
12
12
12
12
13
13
13
13
14
Nonec
7.7
15
31
62
100
200
400
50
7.7
15
31
62
100
200
400
50
29
26
34
9
7.7
15
31
62
100
200
400
50
3
23
21
3
7.7
15
31
62
31
––
100
200
400
200
––
5
6
12
84
3
aCRL (10 mg) was co-lyophilized with the amphiphile of different molar quantities from 10 mL of aq phosphate buffer.
bEnantiomeric excess (ee) of the (S)-(+)-butyryloxy solketal determined by the enantiomeric purity of the product at ca. 30% conversion.
cControl CRL powder.
vious paper.14 Racemic sulcatol (6-methyl-5-heptene-
2-ol), racemic solketal (2,2-dimethyl-1,3-dioxolone-4-
Oligosaccharide chain
at Asn 351
methanol) and isopropenyl acetate and vinyl butyrate
were purchased from Aldrich Chemical Co. (Milw, WI,
USA), Tokyo Kasei Kogyo Co. (Tokyo), and Japan
VAM & POVAL (Osaka), respectively. Diisopropyl
Tyr 69 in the lid
Lid
Heads
Amphiphile carbonyl
C
O
HO
Active center
(Ser 209)
ether was purchased from Wako Pure Chemical Co.
(Osaka). All other organic and inorganic reagents were
of the purest grade available from commercial sources.
Tails
β -sheet
3.2. Preparation of ())-quinide (quino-1,5-lactone) (2)
CRL
42
Quinic acid (1) (25 g, 130 mmol) and 100 mL of 2-
methoxyethanol were added to a 300-mL round flask.
The reaction mixture was heated under stirring at 100–
115 ꢁC. Then the solution was diluted with 50-mL of abs
toluene, and again heated under reflux for 3 h, followed
by concentration by azeotropic distillation until the
boiling point of the distillate reached 122 ꢁC. Addition
of 2-methoxyethanol and toluene, followed by azeo-
tropic distillation, was repeated two more times. The
residue was dissolved in 150 mL of boiling EtOH, and
after cooling to room temperature, the solution was left
to stand overnight in a refrigerator. After removing
unreacted needles of 1, the filtrate was concentrated in
vacuo to give a colorless crystalline product, 2, which
was collected by filtration and air dried. Yield: 7.6 g
(34%); mp 185–187 ꢁC, lit. 175–195 ꢁC13; IR (KBr): 3300
(OH), 2900 (CH), and 1780 cmÀ1 (lactone).
(50
33
)
Figure 2. Schematic representation of the interfacial activation of
CRL with an amphiphile.
3. Experimental
3.1. Materials
Two lipases [EC3.1.1.3] from C. rugosa (CRL: 64.6 kD;
Meito Sangyo Co., Nagoya) and P. cepacia (PCL:
31 kD; Amano Enzyme Co., Nagoya) were used. CRL
was employed without further purification. PCL was
employed after the removal of the diatomaceous earth
stabilizer according to procedures described in a pre-

498
Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
Figure 3. Stereoview of three amphiphiles. The geometry of these molecules was optimized by using MM2 in Chem. 3D of Chemical Structure
Markup Language package (Cambridge Software Co.).
3.3. Preparation of N,N0-[iminobis(trimethylene)]-
bis[quinamide] (3)
3.4. Preparation of the gemini amphiphile (9)
To a solution of 4.3 mmol (251 mg) of didodecyl N-(3-
carboxypropanoyl)-L
-glutamate (4)21 dissolved in 50 mL
To a solution of 113 mg (8.6 mmol) of iminobispropyl-
amine in 100 mL of refluxing abs MeOH was added 3.0 g
(17.2 mmol) of 2. After refluxing for 6 h, the mixture was
allowed to warm up to room temperature, evaporated
by a rotary evaporator under vacuum, and the viscous
residue was triturated with 100 mL of CHCl3. The crude
product 3 was collected by vacuum filtration, washed
with cold abs THF, and used without further purifica-
tion.
of dry DMF, was added 4.3 mmol (0.6 mL) of Et3N. The
resulting solution was cooled to 0 ꢁC in an ice–salt bath,
after which 4.3 mmol (0.59 g) of isobutyl chloroformate
was added all at once. After mixing for 30 min, the
mixture was allowed to stand in a refrigerator for 2 h.
The mixed anhydride thus formed was then filtered into
the solution of 4.3 mmol (206 mg) of 3 in 30 mL of dry
DMF, which was warmed to 50 ꢁC (leaving the

Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
499
Et3NÆHCl on the funnel). The mixture was further stir-
red at 50 ꢁC for 2 days, and then the solvent was eva-
porated under high vacuum, by maintaining the
temperature of the bath at 50 ꢁC. Approximately 80 mL
of MeOH were added to the residue. The mixture was
heated until all the material had dissolved. The solution
was allowed to cool slowly to room temperature. The
precipitated unreacted materials were filtered off, and
concentration and cooling of the mother liquor yielded
the crystalline, crude compound. The crude product was
chromatographed on a column of silica gel (Wako Gel
C-300) with MeOH as the eluent. Gel-filtration chro-
matography of the product on a column of Sephadex
LH-20 with MeOH methanol as eluent gave further
purified 9 (234 mg, 55%) as pale-yellow needles: mp 90–
92 ꢁC; IR (KBr): m 3220 (–OH), 1720 (–C@O) and 1620
(–CONH–); 13C NMR data (CDCl3): d 14.0 (CH3– · 2),
23.1 (CH3CH2– · 2), 26.5 (–CH2CH2CH2–O–CO– · 2),
26.6 (–O–CO–CH2CH2CH—), 27.8 (˜CH–CONH–
CH2CH2–CO–N—), 27.9 (–O–CO–CH2CH2CH—), 29.9
(–CH2CH2–O–CO– · 2), 30.0 (CH3(CH2)2CH2(CH2)2
CH2– · 2), 30.3 (CH3(CH2)3CH2CH2– · 2, ˜CH–CO
CH(OH)CH(OH)-(quinic) · 2), 172.0 (–CH2–O–CO–
· 2), 173.6 (˜CH–CONH–CH2CH2–CO–N—), 175.4
(˜CH–CONH–CH2CH2–CO–N—), 178.5 (˜N–CH2CH2
CH2–CONH– · 2). Anal. Calcd for C53H96N4O16: C,
60.90; H, 9.26; N, 5.36. Found: C, 60.72; H, 9.34; N,
5.40.
3.6. Preparation of the gemini amphiphile (11)
Ditetradecanoyl N-(3-carboxypropanoyl)-L-glutamate
(6) (4.3 mmol)21 was treated in an analogous method to
that described above for 9. A combination of column
and gel-filtration chromatography gave 11 as pale-yel-
low needles (303 mg, 64%): mp 122–125 ꢁC; 13C N MR
data (CDCl3): d 14.0 (CH3-2), 23.1 (CH3CH2– · 2), 26.5
(–CH2CH2CH2–O–CO– · 2),
26.6
(–O–CO–
CH2CH2CH—), 27.8 (˜CH–CONH–CH2CH2–CO–N—),
27.9 (–O–CO–CH2CH2CH—), 29.9 (–CH2CH2–O–CO–
· 2), 30.0 (CH3(CH2)2CH2(CH2)6CH2– · 2), 30.3
(–CH2– · 12, ˜CH–CONH–CH2CH2–CO–N—), 30.9
(˜N–CH2CH2CH2–CONH– · 2), 32.5 (CH3CH2CH2–
· 2), 37.8 (˜C(OH)CH2CH(OH)-(quinic) · 2), 41.1 (˜N–
CH2CH2CH2–CONH– · 2), 44.9 (˜N–CH2CH2CH2–
CONH– · 2), 54.7 (–O–CO–CH2CH2CH—), 58.4
(˜C(OH)CH2CH(OH)-(quinic) · 2), 61.4 (˜C(OH)CH2
CH(OH)-(quinic) · 2), 66.7 (–CH2–O–CO– · 2), 67.8
(˜C(OH)CH2CH(OH)-(quinic) · 2), 79.2 (–CH(OH)-
CH(OH)CH(OH)-(quinic) · 2), 172.0 (–CH2–O–CO–
· 2), 173.6 (˜CH–CONH–CH2CH2–CO–N—), 175.4
(˜CH–CONH–CH2CH2–CO–N—), 178.5 (˜N–CH2CH2
CH2–CONH– · 2). Anal. Calcd for C57H104 N4O16: C,
62.16; H, 9.52; N, 5.09. Found: C, 62.10; H, 9.57; N,
5.12.
NH–CH2CH2–CO–N—),
30.9
(˜N–CH2CH2CH2–
CONH– · 2), 32.5 (CH3CH2CH2– · 2), 37.8 (˜C(OH)
CH2CH(OH)-(quinic) · 2), 41.1 (˜N–CH2CH2CH2–
CONH– · 2), 44.9 (˜N–CH2CH2CH2–CONH– · 2),
54.7 (–O–CO–CH2CH2CH—), 58.4 (˜C(OH)CH2
CH(OH)-(quinic) · 2), 61.4 (˜C(OH)CH2CH(OH)-
(quinic) · 2), 66.7 (–CH2–O–CO– · 2), 67.8 (˜C(OH)
CH2CH(OH)-(quinic) · 2), 79.2 (–CH(OH)CH(OH)-
CH(OH)-(quinic) · 2), 172.0 (–CH2–O–CO– · 2), 173.6
(˜CH–CONH–CH2CH2–CO–N), 175.4 (˜CH–CONH–
CH2CH2–CO–N—), 178.5 (˜N–CH2CH2CH2–CONH–
· 2). Anal. Calcd for C49H88N4O16: C, 59.49; H, 8.97; N,
5.66. Found: C, 59.31; H, 9.05; N, 5.70.
3.7. Preparation of the gemini amphiphile (12)
3.5. Preparation of the gemini amphiphile (10)
Dihexadecanoyl N-(3-carboxypropanoyl)-L-glutamate
(7) (4.3 mmol)21 was treated in an analogous method to
that described above for 9. A combination of column
and gel-filtration chromatography gave 12 as pale-yel-
low needles (344 mg, 69%): mp 135–140 ꢁC; 13C N MR
data (CDCl3): d 14.0 (CH3– · 2), 23.1 (CH3CH2– · 2),
26.5 (–CH2CH2CH2–O–CO– · 2), 26.6 (–O–CO–
CH2CH2CH—), 27.8 (˜CH–CONH–CH2CH2–CO–N—),
27.9 (–O–CO–CH2CH2CH—), 29.9 (–CH2CH2–O–CO–
· 2), 30.0 (CH3(CH2)2CH2(CH2)8CH2– · 2), 30.3
(–CH2– · 16, ˜CH–CONH–CH2CH2–CO–N—), 30.9
(˜N–CH2CH2CH2–CONH– · 2), 32.5 (CH3CH2CH2–
· 2), 37.8 (˜C(OH)CH2CH(OH)-(quinic) · 2), 41.1 (˜N–
CH2CH2CH2–CONH– · 2), 44.9 (˜N–CH2CH2CH2–
CONH– · 2), 54.7 (–O–CO–CH2CH2CH—), 58.4
(˜C(OH)CH2CH(OH)-(quinic) · 2), 61.4 (˜C(OH)CH2
CH(OH)-(quinic) · 2), 66.7 (–CH2–O–CO– · 2), 67.8
(˜C(OH)CH2CH(OH)-(quinic) · 2), 79.2 (–CH(OH)-
CH(OH)CH(OH)-(quinic) · 2), 172.0 (–CH2–O–CO–
· 2), 173.6 (˜CH–CONH–CH2CH2–CO–N—), 175.4
Didodecyl N-(3-carboxypropanoyl)-L-glutamate (5)
(4.3 mmol)21 was treated in an analogous method to that
described above for 9. A combination of column and
gel-filtration chromatography gave 10 as pale-yellow
needles (243 mg, 54%): mp 119–123 ꢁC; 13C NMR data
(CDCl3): d 14.0 (CH3– · 2), 23.1 (CH3CH2– · 2), 26.5
(–CH2CH2CH2–O–CO– · 2), 26.6 (–O–CO–CH2CH2
CH—), 27.8 (˜CH–CONH–CH2CH2–CO–N—), 27.9
(–O–CO–CH2CH2CH—), 29.9 (–CH2CH2–O–CO– · 2),
30.0 (CH3(CH2)2CH2(CH2)4CH2– · 2), 30.3 (–CH2– · 8,
˜CH–CONH–CH2CH2–CO–N—), 30.9 (˜N–CH2CH2
CH2–CONH– · 2), 32.5 (CH3CH2CH2– · 2), 37.8
(˜C(OH)CH2CH(OH)-(quinic) · 2), 41.1 (˜N–CH2CH2
CH2–CONH– · 2), 44.9 (˜N–CH2CH2CH2–CONH–
· 2), 54.7 (–O–CO–CH2CH2CH—), 58.4 (˜C(OH)
CH2CH(OH)-(quinic) · 2), 61.4 (˜C(OH)CH2CH(OH)-
(quinic) · 2),
66.7
(–CH2–O–CO– · 2),
67.8
(˜C(OH)CH2 CH(OH)-(quinic) · 2), 79.2 (–CH(OH)-

500
Y. Mine et al. / Carbohydrate Research 339 (2004) 493–501
(˜CH–CONH–CH2CH2–CO–N—), 178.5 (˜N–CH2CH2
CH2–CONH– · 2). Anal. Calcd for C61H112N4O16: C,
63.29; H, 9.75; N, 4.84. Found: C, 63.26; H, 9.78; N,
4.86.
racemic solketal and vinyl butyrate catalyzed by CRL in
cyclohexane, respectively. A general run was as follows:
after organic solvent (4 mL) had been added to the
co-lyophilized enzyme powder, racemic alcohol sub-
strate (2 mmol) was added to the resulting suspension
with magnetic stirring at 30 ꢁC, and then acyl source
substrate (2 mmol) was added to start the reaction. At
30-min intervals, aliquots were withdrawn from the
reaction mixture, and the extent of conversion, and the
enantiomeric purity of the product were determined by
capillary gas chromatography using an FID detector
and a chiral capillary column (Supelco b-DEX 120,
60 m) under the analytical conditions described pre-
viously.7;14 From the GC data, initial reaction rates up
to ca. 20% conversion were used to evaluate the specific
activity: mmol hÀ1 (g of lipase)À1. Conversion was
defined as the percentage of the alcohol substrate which
had reacted to form (R)- and (S)-acyloxy products. The
enantioselectivity was expressed as the enantiomeric
excess (ee) of the enantio-preferred product at ca. 30%
conversion.
3.8. Preparation of the gemini amphiphile (13)
Dioctadecanoyl N-(3-carboxypropanoyl)-L-glutamate
(8) (4.3 mmol)21 was treated in an analogous method to
that described above for 9. A combination of column
and gel-filtration chromatography gave 13 as pale-yel-
low needles (402 mg, 77%); mp 142–145 ꢁC; 13C N MR
data (CDCl3): d 14.0 (CH3– · 2), 23.1 (CH3CH2– · 2),
26.5 (–CH2CH2CH2–O–CO– · 2), 26.6 (–O–CO–
CH2CH2CH—), 27.8 (˜CH–CONH–CH2CH2–CO–N—),
27.9 (–O–CO–CH2CH2CH—), 29.9 (–CH2CH2–O–CO–
· 2), 30.0 (CH3(CH2)2CH2(CH2)10CH2– · 2), 30.3
(–CH2– · 20, ˜CH–CONH–CH2CH2–CO–N—), 30.9
(˜N–CH2CH2CH2–CONH– · 2), 32.5 (CH3CH2CH2–
· 2), 37.8 (˜C(OH)CH2CH(OH)-(quinic) · 2), 41.1 (˜N–
CH2CH2CH2–CONH– · 2), 44.9 (˜N–CH2CH2CH2–
CONH– · 2), 54.7 (–O–CO–CH2CH2CH—), 58.4
(˜C(OH)CH2CH(OH)-(quinic) · 2),
61.4
(˜C(OH)
CH2CH(OH)-(quinic) · 2), 66.7 (–CH2–O–CO– · 2),
67.8 (˜C(OH)CH2CH(OH)-(quinic) · 2), 79.2 (–CH
(OH)CH(OH)CH(OH)-(quinic) · 2), 172.0 (–CH2–O–
CO– · 2), 173.6 (˜CH–CONH–CH2CH2–CO–N—),
175.4 (˜CH–CONH–CH2CH2–CO–N—), 178.5 (˜N–
Acknowledgements
The authors are grateful to Amano Enzyme Co., Ltd.
(Nagoya) and Meito Sangyo Co., Ltd. (Nagoya) for the
provision of PCL and CRL, respectively.
CH2CH2CH2–CONH– · 2).
Anal.
Calcd
for
C65H120N4O16: C, 64.33; H, 9.97; N, 4.62. Found: C,
64.30; H, 10.0; N, 4.65.
References
3.9. Modified enzyme preparation
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Modification by the co-lyophilization of the lipase, CRL
or PCL, was performed as follows: Lipase was dissolved
(1 g LÀ1) in 10 mL of an aqueous phosphate buffer
solution (10 mM, pH 7.0) in a round-bottom flask
(25 mL). After the desired amount of the gemini
amphiphile was added to the lipase solution, mixing was
carried out with a homogenizer (Ultra-Turrax T8.01;
IKA, Staufen) at 20,000 rpm for 2 min in an ice bath,
followed by rapid freezing in liquid nitrogen, and lyo-
philization in a freeze dryer (Model VD-16; TAITEC
Co., Saitama). The light-brown or white solid thus
obtained was employed as the modified lipase. As a
control enzyme powder, lyophilized native lipase was
obtained by lyophilization from the same aqueous buffer
solution in the absence of modifier.
€
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