Enhancement of enantioselectivity in the optical resolution of primary alcohols with modified cyclodextrin colyophilized lipase

Article abstract of DOI:10.1080/00397910701386154

Colyophilization of lipase was carried out with immobilized β-cyclodextrins (β-CyD) bearing methyl, acetyl, benzoyl, and nicotinoyl substituents. The colyophilizates enhanced stereoselectivity in the acylation of several alcohols. The enantioselectivity in the acylation of ethyl-1-hydroxymethyl-phenylphosphine oxide using colyophilized lipase with nicotinoyl-β-CyD increased approximately threefold (from E=34 to E=113). The amphiphilic character of modified CyDs has been found to influence the enhancement of enantioselectivity. Copyright Taylor & Francis Group, LLC.

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Enhancement of Enantioselectivity in the
Optical Resolution of Primary Alcohols with
Modified Cyclodextrin Colyophilized Lipase
Kosei Shioji a , Yoshimitsu Kurauchi a , Keiko Terada a , Yasushi Kodera a
Kentaro Okuma a
a Faculty of Science, Department of Chemistry , Fukuoka University ,
Jonan‐ku, Fukuoka, Japan
&
Published online: 03 Jul 2007.
To cite this article: Kosei Shioji , Yoshimitsu Kurauchi , Keiko Terada , Yasushi Kodera & Kentaro Okuma
(2007) Enhancement of Enantioselectivity in the Optical Resolution of Primary Alcohols with Modified
Cyclodextrin Colyophilized Lipase, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 37:13, 2163-2170, DOI: 10.1080/00397910701386154
To link to this article: http://dx.doi.org/10.1080/00397910701386154
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Synthetic Communicationsw, 37: 2163–2170, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701386154
Enhancement of Enantioselectivity in the
Optical Resolution of Primary Alcohols with
Modified Cyclodextrin Colyophilized Lipase
Kosei Shioji, Yoshimitsu Kurauchi, Keiko Terada,
Yasushi Kodera, and Kentaro Okuma
Faculty of Science, Department of Chemistry, Fukuoka University,
Jonan-ku, Fukuoka, Japan
Abstract: Colyophilization of lipase was carried out with immobilized b-cyclodextrins
(b-CyD) bearing methyl, acetyl, benzoyl, and nicotinoyl substituents. The colyophili-
zates enhanced stereoselectivity in the acylation of several alcohols. The enantioselec-
tivity in the acylation of ethyl-1-hydroxymethyl-phenylphosphine oxide using
colyophilized lipase with nicotinoyl-b-CyD increased approximately threefold (from
E ¼ 34 to E ¼ 113). The amphiphilic character of modified CyDs has been found to
influence the enhancement of enantioselectivity.
Keywords: lipase, modified cyclodextrin, optical resolution, primary alcohols
Hydrolases, especially lipase, have been established in organic synthesis as
catalysts developed from organisms. These catalysts aid the development of
optically active compounds.[1,2] However, the lipase activity in organic
solvents is frequently lower than that in aqueous media.[3,4] It is well docu-
mented that the lyophilization of enzymes causes a loss of enzyme
activity.[5–8] It has been reported that an improvement in the reactivity and
selectivity of the enzyme reaction in organic reaction media can be
achieved by colyophilization with macrocyclic organic additives, such as
cyclodextrins (CyDs). These natural molecular receptors are the subject of
Received September 6, 2006
Address correspondence to Kosei Shioji, Faculty of Science, Department of
Chemistry, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan. E-mail:
shioji@fukuoka-u.ac.jp
2163

2164
K. Shioji et al.
considerable interest in many areas of molecular chemistry.[9] Griebenow
et al. reported that colyophilization of permethylated b-cyclodextrin
increased the activity and enantioselectivity of subtilisin Carlsberg when
compared to the lyophilized powder prepared from buffer alone.[10] Ghanem
and Schurig reported that peracetylated b-cyclodextrin employed as a macro-
cyclic additive enhanced the enantiomeric ratio E and reaction rate in the
Pseudomonas cepacia lipase (PCL)–catalyzed transesterification of 1-(2-
furyl)ethanol in organic solvent.[11] The action of cyclodextrins used as
lipase regulators was interpreted as changing the enzyme conformation, pre-
venting substrate and product inhibition, and/or increasing the lipase solubil-
ity in organic solvent. Although it is important to investigate the correlation
between hydrophobic cavity size and hydrophilic rim, no modified CyDs
were used for the enzyme-catalyzed reaction. Previously, we reported that
lipase AK(pseudomonas fluorescence lipase)–catalyzed optical resolution
afforded optically active alkyl(1-hydroxymethyl)phenylphosphine oxides
and the corresponding phosphine boranes.[12,13] However, the optical resol-
ution was not effective for all of the phosphorus compounds investigated.
Because enantioselectivity in the enzyme-catalyzed reaction was dominated
by the binding between the substrate and the enzyme catalytic site, the
optical resolutions of the phosphorus compounds are influenced by the intro-
duction of substituents at the phosphorus atom. The treatment of lipase in the
presence of modified CyDs is expected to improve the resolution of those
compounds. Herein we report the utility of perbenzoylated and pernicotinoy-
lated b-CyDs as additives to enhance selectivity in the lipase AK–catalyzed
enantioselective transesterification of phosphine oxides (Figure 1).
RESULTS AND DISCUSSION
Perbenzoylated b-CyD (1b) was prepared according to literature methods,[14]
whereas pernicotinoylated b-CyD (1c) was prepared by a modified method.
The introduction of benzoyl groups to b-CyD is expected to increase the
overall hydrophobicity of the molecule. It also appears that the hydrogen
Figure 1. Schematic representation of modified CyD 1.

Optical Resolution with Colyophylized Lipase
2165
Scheme 1.
bonding ability of nicotinoylated CyD 1c is higher than that of 1b. Colyophi-
lizations of Pseudomonas fluorescence (Amano AKG) lipase with modified
b-CyDs were carried out by literature methods.[11] Modified CyDs were
prepared with a 1:6 weight ratio of enzyme to CyD. The enhancement
ability was measured by esterification of the secondary alcohol, 1-(2-furyl)
ethanol, with vinyl acetate in cyclohexane using the colyophilized lipase
AKG as a catalyst, affording (R)-acetate 3 and recovered (S)-alcohol 1
(Scheme 1). The enantiopreference of colyophilized lipase AKG was deter-
mined by comparison of the retention times in the corresponding gas chromato-
graphy (GC) studies. These results are summarized in Table 1. The reaction of
2 with a colyophilizate of 1 was faster than using lyophilized lipase AKG alone.
We applied this method to the transesterification of racemic 1-hydroxy-
alkylphosphine oxides 4. The E-values using tetrahydrofuran (THF) were
higher than those of cyclohexane (1.4–1.6). The esterifications of 4 in THF
at 368C afforded enantiomerically enriched (R)-5 and (S)-4. These results
are summarized in Table 2. The reaction of 4a using colyophilized lipase
with acetyl CyD 1a resulted in an enhancement of the E-values. Moreover,
by using colyophilized lipase with nicotinoylated CyD 1c, the enantioselectiv-
ity in the reaction of the ethyl derivative 4a was fourfold larger than that
obtained using lyophilized lipase AKG alone. The isopropyl derivative 4b,
which is bulkier than 4a, was less reactive than 4a. The reaction rates of 4
using colyophilizates with 1 were faster than those using AKG alone. The
acetylation of tert-butyl derivative 4c was not observed for 2 h. Because of
Table 1. Colyophilized lipase with modified b-CyD 1-catalyzed acylation of racemic
2 in cyclohexane
Time
(h)
Conv.
(%)
Ees
(%)a
Eep
(%)a
1
Eb
Enhancement
—
—
a
0.5
2
30
54
50
44
26
42
98
93
74
34
97
84
95
96
99
102
50
1.0
—
2
118
106
179
1.2
1.0
1.8
b
2
c
2
aDetermined by GC (cyclodextrin-b-236M-19 (0.25 mm ꢀ 25 M).
bCalculated by E ¼ ln(1 2 c)(1 2 ees)/ln(1 2 c)(1 þ eep), where c is conversion/100.

2166
K. Shioji et al.
Table 2. Colyophilized lipase AKG with 1 catalyzed acylation of racemic 4 in THF
a
Conv. Eesa
Eep
(%)
1
log P
4
a
Time Solvent (%)
%
E
Enhancement
—
a
2
THF
THF
47
48
45
34
30
46
40
40
36
76
88
74
50
42
76
60
61
55
87
94
90
97
97
91
93
93
97
34
98
1.0
2.9
1.2
3.4
2.7
1.0
1.0
1.0
2.3
0.4
1.58
b
40
c
20.04
20.01
113
94
d
—
a
b
17
46
51
b
47
108
c
aDetermined by HPLC (CHIRALPAK AD with hexane/2-propanol (97:3). (S)-4a:
[a]D25 ¼ 240.9 (c ¼ 1.5, CHCl3, 98% ee). (R)-5a: [a]2D5 ¼ 19.8 (c ¼ 1.8, CHCl3,
53% ee). (S)-4b: [a]D25 ¼ 241.4 (c ¼ 1.6, CHCl3, 98% ee). (R)-5b: [a]2D5 ¼ 49.6
(c ¼ 1.7, CHCl3, 88% ee).
the steric hindrance of tert-butyl group, the reaction was extremely slow
(conv. ¼ 2.5% for 48 h) (Scheme 2).
The decrease in the reaction rate of 4 might be caused by host–guest
complexation between substrate 4 or product 5 and 1 in solution. The com-
plexation may indeed prevent substrates as well as product inhibition in the
enzymatic reaction[15] and improve enantioselectivity by the preferential
binding of the (R)-enantiomer over the (S)-enantiomer. The ability of 4 to
undergo complexation with nicotinoyl CyD 1c was inferred by induced
1
shifts in H NMR spectroscopic studies. In the presence of each substrate 2
and 4 (20 equivalents for 1c), the upfield shift of the aromatic ring protons
of 1c was observed (Dd 0.025–0.010). However, a specific interaction of
(R)- or (S)-4 with 1 was not observed. When the mixture of lipase AKG (lyo-
philized from buffer alone) and lyophilized CyD 1c at a 1:6 weight ratio as
catalyst was carried out, the enhancement in the E-value was observed (2.2-
fold). This enhancement was smaller than that observed when lipase was
colyophilized with 1c using the same weight ratio. The lipase colyophilized
with 1c was inactivated for 3 days, which indicated that the nicotinoyl
Scheme 2.

Optical Resolution with Colyophylized Lipase
2167
group of 1c interacted with not only the substrate or product, but also the
enzyme. Therefore, the clear effect of the modified b-CyD additive is useful
in both direct interaction with enzymes and in the complexation with either
substrate or product. On the other hand, poor enhancement of the esterification
of 4 using benzoylated CyD 1b was observed. It was reported that the enzyme
activity and enantioselectivity of CyDs with varying ring size were both
enhanced. These results suggested that the changes in enzyme activity and
enantioselectivity were caused by complexation with the phenyl groups of
the PCL in the open-lid form.[16,17] Surface hydrophobicity of Pseudomonas
fluorescens lipase (PFL) was the largest among the lipases.[18] Recently, it
was shown that PFL undergoes self-assembly as a result of close interaction
between the hydrophobic areas of the lipase surface.[19] The aggregation
improves enantioselectivity by the same degree as that afforded by interfa-
cially adsorbed lipase. The partition coefficients (log P) of 1a, 1b, 1c, and
1d were determined as 0.4, 1.58, 20.04, and 20.1, respectively. Thus, in
our case, amphiphilic CyDs are considered to interact with the hydrophobic
surfaces of PFL to enhance enantioselectivity.
In summary, enantioselective transesterification of primary-alcohol-type
phosphine oxides could be accomplished using colyophilized PFL with
modified CyDs. This methodology has potential for practical use, because
special acyl moieties or donors are not required.
EXPERIMENTAL
Enzyme and Chemicals
Lipase AKG was obtained as a gift from Amano Pharmaceutical Co., Ltd., and
used as a catalyst after lyophilization with modified CyDs. b-CyD was
obtained from Wako. Diisopropyl ether was distilled prior to use. Permethyl
and peracetyl CyDs were purchased from Wako.
Synthesis
Pernicotinoyl b-CD 1c
To a solution of b-CyD (1 mmol, 1.1 g) in dry pyridine (50 ml), nicotinoyl
chloride (168 mmol, 29.9 g) was added, and the mixture was stirred at 708C
for 7 days. The reaction was quenched by the addition of water and concentrated
in vacuo. The pH of the residue was adjusted to 9 by the addition of Na2CO3
solution and then extracted with ethyl acetate. The organic layer was washed
withwateranddriedoverMgSO4. Thefiltrate wasconcentrated to givecrudeper-
nicotinoyl b-CyD and purified by recrystallizing from an ethyl acetate–hexane
solution in 63% yield. Colorless crystals, 1H NMR (400 MHz, CDCl3)
d ¼ 4.23 (t, 1H, J ¼ 9.2 Hz), 4.66 (d, 1H, J ¼ 7.6 Hz), 4.85 (d, 1H,

2168
K. Shioji et al.
J ¼ 10.8 Hz), 4.94 (dd, 1H, J ¼ 3.2 Hz), 5.07 (s, 1H), 5.70 (d, 1H, J ¼ 3.2 Hz),
6.06 (t, 1H, J ¼ 9.2 Hz), 6.98–705 (m, 2H), 7.55 (s, 1H), 7.63 (d, 1H,
J ¼ 7.2 Hz), 7.75 (d, 1H, J ¼ 8.0 Hz), 8.46 (d, 1H, J ¼ 8.0 Hz), 8.53–8.60
(m, 3H), 8.87 (s, 1H), 9.06 (s, 1H); 13C NMR (100 MHz, CDCl3) d ¼ 63.4,
70.3, 71.7, 72.8, 97.5, 123.3, 123.4, 123.8, 123.9, 124.6, 125.5, 136.8, 137.1,
137.7, 150.5, 150.8, 151.2, 153.9, 154.0, 154.3, 164.0, 164.9, 165.0. MS
(MALDI-TOF): 3366.2 [M þ Na]þ; calc. for C168H133N21O56: 3339.8.
Phosphine Oxide 4
To a solution of ethylphenylphosphinate (30 mmol), in dry THF (30 mL),
alkyl magnesium bromide (39 mmol) was added, and the mixture was
stirred at 08C for 2 h. Tert-butyllithium (36 mmol) at 2788C and gaseous for-
maldehyde (150 mmol) were added to the reaction mixture, and the mixture
was stirred at 2158C for 2 h. The reaction was quenched with aq. NH4Cl,
and the organic layer was extracted with CH2Cl2 (30 mL ꢀ 2). The extract
was dried over MgSO4 and concentrated in vacuo to give crude phosphine
oxide 4. Purification was carried out by column chromatography (hexane/
ethyl acetate) to give 4a and 4b in 76% and 68% yields, respectively.
Ethyl(1-hydroxymethyl)phenylphosphine Oxides 4a
Colorless liquid, 1H NMR (400 MHz, CDCl3) d ¼ 1.10–1.18 (m, 3H), 1.85–
2.05 (m, 1H), 2.17–2.29 (m, 1H), 4.05–4.15 (m, 2H), 7.46–7.54 (m, 3H),
7.69–7.74 (m, 2H). 13C NMR (100 MHz, CDCl3) d ¼ 5.48, 5.53, 19.37,
20.05, 60.08, 60.90, 128.74, 128.85, 129.81, 130.71, 130.93, 131.01,
132.16, 132.18. 31P NMR (162 MHz, CDCl3) d ¼ 42.49. IR (NaCl, CHCl3):
1159, 1169, 3021 cm21. UV (CHCl3): 255 nm. Found: H, 7.29; C, 57.80,
calc.: H, 7.11; C, 57.69.
Isopropyl(1-hydroxymethyl)phenylphosphine Oxides 4b
Colorless liquid, 1H NMR (400 MHz, CDCl3) d ¼ 1.90 (dd, J ¼ 9.2 Hz, 3H),
1.33 (dd, J ¼ 9.2 Hz, 3H), 2.27–2.35 (m, 1H), 4.60 (dq, J ¼ 36 Hz, 2H),
7.50–7.58 (m, 3H), 7.72–7.77 (m, 2H). 13C NMR (100 MHz, CDCl3)
d ¼ 15.46, 15.50, 15.95, 15.98, 26.28, 26.95, 59.25, 60.01, 128.71, 128.82,
129.19, 130.06, 131.13, 131.21, 132.06, 132.09. 31P NMR (162 MHz,
CDCl3) d ¼ 45.29. IR (NaCl, CHCl3): 1149, 1157, 3021 cm21. UV
(CHCl3): 257 nm. Found: H, 7.58; C, 60.76; calc.: H, 7.63; C, 60.60.
Colyophilization of Lipase with CyDs
Modified CyDs (enzyme–CyD ¼ 1:6) were suspended in a solution of lipase
AKG (5 mg) in phosphate buffer (20 mM, pH 6.0, 1 mL), sonicated for 30 s,
and lyophilized for 48 h.

Optical Resolution with Colyophylized Lipase
2169
General Procedure for Esterification of Racemic Alcohols using
Colyophilized Lipase
To a solution of racemic alcohols (0.014 mmol) in cyclohexane or THF
(0.5 mL), colyophilizates and vinyl acetate (0.042 mmol) were added, and
the mixture was stirred at 368C for an appropriate time. The reaction
mixture was filtered, and the solvent was removed under reduced pressure.
The reaction was analyzed by HPLC using a Waters 600 dual pump
equipped with a tunable absorbance detector (Waters 484) and a
CHIRALPAK AD (0.46 ꢀ 25 cm) column, and it was eluted with a
hexane/2-propanol (97:3) solvent mix including 0.1% trifluoroacetic acid.
Determination of Partition Coefficient
Solutions of modified CyDs were prepared in H2O at concentrations of
0.02 mM and 0.04 mM, and their concentrations were determined with
respect to their optical absorption, obtained using a UV/vis spectrometer.
Equal volumes (5 mL) of the aqueous CyD solutions and 1-octanol were
stirred at 368C for 1 h. The H2O phase was separated and its concentration
determined.
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