Nearly aqueous-like activity of lipoprotein lipase treated with glucose-headed surfactant in organic solvent

Article abstract of DOI:10.1016/j.molcatb.2016.10.009

In this work, we explored the activation of a lipoprotein lipase from Burkholderia species by glucose-headed surfactants (GHSs) for enhancing its catalytic activity in organic solvent. Three GHSs were prepared and then tested as the additives for inducing the activation of lipoprotein lipase. The kinetic parameters of GHS-treated lipoprotein lipase were determined for the hydrolysis or alcoholysis of p-nitrophenyl acetate. It was found that GHS-treated lipoprotein lipase was 4 to 5 orders of magnitude more active than its native counterpart in organic solvent. Such a dramatic activity enhancement was largely the result of a huge increase in the turnover frequency k_cat. Surprisingly, the k_cat values in organic solvent were one order of magnitude greater than their aqueous counterparts. As a result, the k_cat/K_m of GHS-treated lipoprotein lipase in organic solvent became comparable to the aqueous level within one order of magnitude. We thus have demonstrated for the first time that a lipase can display nearly aqueous-like activity in organic solvent. As an illustrative application of GHS-treated lipoprotein lipase, we performed the dynamic kinetic resolution of two secondary alcohols, which provided the products of high enantiopurity (98–99%ee) with high yields (90–91%).

Full text of DOI:10.1016/j.molcatb.2016.10.009

Accepted Manuscript
Title: Nearly Aqueous-like Activity of Lipoprotein Lipase
Treated with Glucose-Headed Surfactant in Organic Solvent
Author: Yeonock Oh Yoon Kyung Choi Inyeol Yun
Eungyeong Lee Kyungwoo Kim Mahn-Joo Kim
PII:
S1381-1177(16)30202-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.10.009
MOLCAB 3452
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Journal of Molecular Catalysis B: Enzymatic
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Revised date:
Accepted date:
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6-10-2016
7-10-2016
Please cite this article as: Yeonock Oh, Yoon Kyung Choi, Inyeol Yun, Eungyeong Lee,
Kyungwoo Kim, Mahn-Joo Kim, Nearly Aqueous-like Activity of Lipoprotein Lipase
Treated with Glucose-Headed Surfactant in Organic Solvent, Journal of Molecular
Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.10.009
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Nearly Aqueous-like Activity of Lipoprotein Lipase Treated with Glucose-
Headed Surfactant in Organic Solvent
Yeonock Oh,† Yoon Kyung Choi,† Inyeol Yun, Eungyeong Lee, Kyungwoo Kim, and Mahn–Joo Kim*
Department of Chemistry, Pohang University of Science and Technology, 77 Cheongam-ro, Pohang 37673,
Republic of Korea.
† These authors contributed equally.
* Corresponding author. Tel.: +82 54 279 2112; fax +82 54 279 0654
E-mail address: mjkim@postech.ac.kr (M.-J. Kim)
1

Graphical Abstract
Highlights



Glucose-headed surfactants were synthesized and applied for enhancing the activity of lipase
in organics solvent.
Glucose-headed surfactants enhanced dramatically the turnover frequency of Burkholderia
species lipoprotein lipase beyond the aqueous level.
The catalytic efficiency of Burkholderia species lipoprotein lipase in the presence of
glucose-headed surfactant in organic solvent reached the aqueous level within one order of
magnitude.

Surfactant-treated Burkholderia species lipoprotein lipase was successfully applied to the
dynamic kinetic resolution of secondary alcohols.
2

Abstract
In this work, we explored the activation of a lipoprotein lipase from Burkholderia species by glucose-
headed surfactants (GHSs) for enhancing its catalytic activity in organic solvent. Three GHSs were
prepared and then tested as the additives for inducing the activation of lipoprotein lipase. The kinetic
parameters of GHS-treated lipoprotein lipase were determined for the hydrolysis or alcoholysis of p-
nitrophenyl acetate. It was found that GHS-treated lipoprotein lipase was 4 to 5 orders of magnitude
more active than its native counterpart in organic solvent. Such a dramatic activity enhancement was
largely the result of a huge increase in the turnover frequency kcat. Surprisingly, the kcat values in
organic solvent were one order of magnitude greater than their aqueous counterparts. As a result, the
kcat/Km of GHS-treated lipoprotein lipase in organic solvent became comparable to the aqueous level
within one order of magnitude. We thus have demonstrated for the first time that a lipase can display
nearly aqueous-like activity in organic solvent. As an illustrative application of GHS-treated
lipoprotein lipase, we performed the dynamic kinetic resolution of two secondary alcohols, which
provided the products of high enantiopurity (98-99 %ee) with high yields (90-91%).
KeyWords
Biocatalysis, Lipase, Surfactant, Activation, Organic solvent
1. Introduction
Transformations based on lipase catalysis provide a useful methodology for the synthesis of
optically active compounds [1-3]. Among them, dynamic kinetic resolution (DKR) employing
a lipase and a metal-based racemization catalyst in combination [4-8] is particularly useful for
the synthesis of enantiomerically-enriched alcohols and esters [9-26]. The DKR procedures,
3

however, suffers often from the low activity of lipase employed in organic solvent. The
activity of native lipase in organic media has been known to be several orders of magnitude
lower than its activity in water. Although several useful methods employing surfactants
[27,28], ionic liquids [29-32], and inorganic salts [33-37] have been developed for enhancing
the poor enzymatic activity in organic solvent, none of them are applicable for enhancing the
activity of lipase in organic solvent to the aqueous level. We herein wish to report a new
approach employing glucose-headed surfactants (GHSs) for the dramatic activation of lipase.
It was found that a GHS-treated lipase displayed nearly aqueous-like activity in organic
solvent.
Recently, we reported that Burkholderia species lipoprotein lipase (BSLPL) was useful as
the enzyme for the DKR of secondary alcohols, particularly sterically demanding alcohols
such as diarylmethanols [38]. In this work, the commercial BSLPL, which was poorly active in
organic solvent, was treated with both ionic surfactant and dextrin for its activation before use.
This treatment enhanced the activity (kcat/Km) of BSLPL in organic solvent by a factor of 103
[39]. Such a strong activation, however, was still insufficient to reach the aqueous-level
activity in organic solvent. Further activation 6, lines by another factor of 103 in was necessary.
On the basis of the previous result that the combination of ionic surfactant with dextrin
induced the strong activation of BSLPL, we envisioned that the covalent conjugates between
ionic surfactant and sugar as the molecular mimics of the ionic surfactant-dextrin combination
might activate BSLPL more strongly. We thus synthesized GHSs 1a-c (Fig. 1) and applied
them for the further activation of BSLPL.
2. Results and Discussion
4

2.1 Synthesis of glucose-headed surfactants
The synthesis of GHSs was accomplished according to a protocol described in Scheme 1. In a typical
procedure, disubstituted benzoic acid 2 was coupled with an amino alcohol to give the corresponding
amide 3, which was then reacted with perbenzoylglucosyl bromide [40], followed by debenzoylation
to give 1. This synthesis was straightforward and provided good yields.
2.2 Activity of GHS-treated BSLPL in organic solvent
GHS-containing BSLPLs (BSLPL-1a, -1b, -1c and -1c-G) were prepared by freeze-drying
commercial BSLPL (protein content, 52%) with GHS or both GHS and glucose (G). Here, glucose
was added to prepare GHS-containing BSLPL in the more powdery form. The weight ratios between
BSLPL and 1 and between BSLPL, 1, and G were optimized by examining the catalytic performance
of each GHS-containing BSLPL preparation in the transesterification of 1-phenylethanol with
isopropenyl acetate in toluene. In case of BSLPL-GHS mixtures (BSLPL-1a , -1b, and -1c), the
optimal weight ratio was 1 : 1. In case of BSLPL-GHS-glucose mixture (BSLPL-1c-G), the optimal
weight ratio was 1 : 1 : 1. The activities of native and GHS-containing BSLPLs in water were then
measured for the hydrolysis of AcOPNP in phosphate buffer (pH 7.0) and those in organic solvent
were measured for the transesterification of p-nitrophenyl acetate (AcOPNP) with benzyl alcohol in
toluene or tert-butyl methyl ether (TBME). The kinetic parameters of each BSLPL preparation
obtained from these kinetic measurements are described in Table 1.
The kcat/Km data from Table 1 clearly indicate that GHSs enhanced dramatically the activity
of BSLPL in organic solvent (compare entries 3-8). The activity enhancements were in the range of
104-105. The highest activity was observed for BSLPL-1c-G in TBME (entry 8). In this case, the
activity of BSLPL reached within one order of magnitude of its aqueous activity (compare entries 1, 2,
and 8). To our knowledge, such a high activity of lipase in organic solvent is unprecedented. For all
5

the GHS-containing BSLPL preparations, the activity enhancements were largely the result from a
dramatic increase of kcat. Furthermore, all the kcat values of GHS-treated BSLPLs in organic solvent
were significantly larger than their aqueous counterparts (compare entries 1-2 with entries 4-8).
Particularly, the kcat values of BSLPL-1b, -1c, and -1c-G in organic solvent were one order of
magnitude greater than those in water. This is quite surprising because it implies that BSLPL is more
active in organic solvent than in water at the substrate-saturated condition. To the best of our
knowledge, such a high turnover frequency (TOF) of lipase in organic solvent is also unprecedented.
It is noted that Celite-immobilized BSLPL (protein content, 1 %) was 5 times more active than its
native counterpart in organic solvent (entry 9). The activity of Celite-immobilized BSLPL, however,
was negligibly small relative to those of GHS-treated BSLPLs [44].
The kcat values of GHS-treated BSLPLs in organic solvent, which are larger than their aqueous
counterparts, suggest that GHSs induce the activation of enzyme during the lyophilization and protect
or stabilize the activated enzyme in organic solvent. Lipases have been known to be fully activated at
the water-oil interface [41,42]. We thus assume that GHSs induce such a full activation of BSLPL by
providing microenvironments mimicking water and oil around the enzyme. GHSs have a glucose head
as the water surrogate and a branched tail as the oil surrogate. The glucose head has four hydroxyls
around its 6-membered ring so that it could provide nicely a water-mimicking hydrogen bonding
network around the enzyme. On the other hand, the branched tail has three side chains attached to a
central benzene ring, thus acting nicely as a mimic of triglyceride oil which is the ester between
glycerol and three long fatty acids.
On the other hand, the Km values of GHS-treated BSLPLs in organic solvent are unfavorably large
relative to their aqueous counterparts. Several factors could contribute to the large Km. They include
the mass transfer barrier of insoluble enzyme preparations, the higher stability of hydrophobic
substrate in organic solvent, and the inefficient binding of substrate by the interference of GHS
present in or around the active site of enzyme. At present, the negative effects of these factors are
6

inevitable and difficult to alleviate.
2.3 Kinetic and dynamic kinetic resolution of secondary alcohols with GHS-treated BSLPL
To show the utility of GHS-treated BSLPL as the catalyst, we explored the kinetic and dynamic
kinetic resolution of two representative substrates (4a and 4b). For these resolutions, we employed
BSLPL-1c-D (D = dextrin) which was comparable to BSLPL-1c-G in activity but easier to handle
owing to its more powdery nature. First, we performed the kinetic resolutions of 4a and 4b with
BSLPL-1c-D in the presence of isopropenyl acetate (IPA) in toluene at room temperature. It was
found that both resolutions (0.3 mmol of substrate and 3 mg of BSLPL-1c-D) proceeded smoothly to
46-51% completion with high enantioselectivities (E = >100) within 6h (Scheme 2). We then
performed the dynamic kinetic resolution of each substrate on the same scale with a solution
containing substrate (0.3 mmol), BSLPL-1c-D (3 mg), racemization catalyst 6 (4 mol%), IPA (1.5
equiv), K2CO3 (1 equiv), and molecular sieves (30 mg) in toluene. Both DKR reactions reached
completion within 24 h and provided the products of high enantiopurity (98-99% ee) with 90-91 %
isolated yields (Scheme 3). These results thus suggest that GHS-treated BSLPLs have great potential
as the catalysts for the kinetic and dynamic kinetic resolution of secondary alcohols.
3. Conclusions
We have for the first time demonstrated that GHSs enhanced dramatically the TOF (kcat) of a
synthetically useful lipase, BSLPL, in organic solvent beyond the aqueous level. The catalytic
efficiency (kcat/Km) of the most active GHS-treated BSLPL in organic solvent thus reached within one
order of magnitude of its aqueous counterpart. This means that the GHS-treated BSLPL displayed
almost aqueous-like activity in organic solvent. However, the GHS-treated BSLPL has unfavourably
large Km in organic solvent. Accordingly, a new strategy for reducing the Km significantly could lead
to the realization of a super-active lipase in organic solvent. Further studies toward this end are in
progress. In the meantime, the GHS-containing BSLPL preparations developed in this work should
7

find use as the highly active biocatalysts for the reactions in organic media.
1. Experimental
1.1 General procedure for the synthesis of compounds 1a-c
.
The synthesis of 1a from 2a is described as the representative. Disubstituted benzoic acid 2a
(0.3 g, 0.632 mmol), ethanolamine (46 µL, 0.758 mmol), triethylamine (222 µL, 1.580 mmol),
and 1-hydroxybenzotriazole monohydrate (HOBt) (0.103 g, 0.758 mmol) were dissolved in
anhydrous DMF (5 mL). 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
.
o
(EDCI HCl) (0.147 g, 0.758 mmol) was added in small portions at 0 C, and the resulting
solution was stirred at room temperature for overnight. After DMF had been removed by high
pressure rotary evaporation, the residue was diluted with CH2Cl2 and then washed
successively with sat’d NaHCO3 solution, 0.5N HCl solution, and brine. The organic phase
was dried over anhydrous Na2SO4. After removal of solvent, the crude product was purified by
column chromatography on silica (CH2Cl2/methanol = 15/1) to give 3a as a pale-yellowish oil
(0.305 g, 93 % yield): 1H NMR (300 MHz, CDCl3, ppm) 6.97 (d, Ј = 2.24 Hz, 2H), 6.93 (t, Ј =
5.37 Hz, 1H), 6.60 (t, Ј = 2.22 Hz, 1H), 4.15-4.10 (m, 4H), 3.85-3.75 (m, 6H), 3.74-3.63 (m,
12H), 3.62-3.58 (m, 6H), 3.57-3.48 (q, Ј=7.00 Hz, 4H), 3.02 (t, Ј=5.35 Hz, 1H), 1.19 (t,
Ј=7.00 Hz, 6H). 13C NMR (75 MHz, CDCl3, ppm): 168.5. 160.2, 136.5, 106.2, 105.4, 71.0,
70.9, 70.8, 70.0, 69.9, 68.0, 66.9, 62.5, 43.3, 15.3.
The compound 3a (0.247 g, 0.477 mmol), 2,4,6-collidine (0.9 equiv., 56 µL), and silver
trifluoromethanesulfonate (1.2 equiv., 0.147g) were dissolved in anhydrous CH2Cl2 (8.0 mL) and the
o
resuling mixture was stirred at -45 C. A solution of perbenzoylated glucosyl bromide (1.2 equiv.,
o
0.377g) in anhydrous CH2Cl2 (3.0 mL) was added into the mixture dropwise at -45 C over 30
o
minutes. The resulting mixture was stirred at -45oC for 30 minutes and then at 0 C for 3 hours. The
completion of reaction was confirmed by TLC. After pyridine (0.5 mL) had been added, the reaction
8

mixture was diluted with CH2Cl2 and filtered through Celite pad. The filtrate was sequentially washed
with 1 M aqueous Na2S2O3 solution (10 mL), 0.5 N HCl solution (10 mL), and brine (2 x 10 mL).
Then the organic layer was dried over anhydrous Na2SO4, and the solvent was removed by rotary
evaporation. The crude product was purified by column chromatography eluted with CH2Cl2/methanol
= 20/1 to give O-perbenzoylglucosylated intermediate (0.471g, 0.430 mmol, 90% yield) as a pale-
yellowish oil. A mixture of O-perbenzoylglucosylated intermediate (0.322 g, 0.294 mmol, 1.0 equiv.)
and sodium methoxide (8.0 equiv., 25 wt% in methanol) in methanol (5.0 mL) was stirred at r.t. for 6
hours. Amberlite IR-120 resin (H+ form) was added into the reaction mixture to adjust pH to 4. The
resin was removed by filtration through Celite and the filtrate was concentrated under reduced
pressure. The crude product was then purified by short-pad chromatography on silica gel
(CH2Cl2/methanol = 10/1) to obtain 1a (0.186 g, 0.273 mmol, 93% yield) as a colorless oil: 1H NMR
(300 MHz, DMSO-d6, ppm) 8.41 (t, Ј = 5.40 Hz, 1H), 7.00 (d, Ј = 2.07 Hz, 2H), 6.67 (t, Ј = 1.91 Hz,
1H), 5.02 (d, Ј = 4.37 Hz, 1H), 4.93 (dd, Ј1 = 10.52 Hz, and Ј2 = 4.54 Hz, 2H), 4.50 (t, Ј = 5.80 Hz,
1H), 4.18 (d, Ј = 7.73 Hz, 1H), 4.12 (t, Ј = 3.79 Hz, 4H), 3.88-3.83 (m, 1H), 3.76-3.73 (m, 4H), 3.70-
3.66 (m, 1H), 3.65-3.58 (m, 5H), 3.57-3.50 (m, 9H), 3.48-3.45 (m, 6H), 3.45-3.38 (m, 4H), 3.18-3.06
(m, 3H), 3.06-2.96 (m, 1H), 1.09 (t, Ј = 7.00 Hz, 6H); 13C NMR (75 MHz, DMSO-d6, ppm) 165.9,
159.5, 136.5, 105.8, 103.9, 103.2, 77.0, 76.6, 73.5, 70.0, 69.9, 69.8, 69.3, 68.9, 67.5, 65.6, 61.1, 15.2;
TOF-MS (ESI+) calcd. for C31H53N1Na1O15 702.33129, found 702.32759.
Compound 1b (0.284 g, 0.370 mmol, 90% yield, colorless oil): 1H NMR (300 MHz, DMSO-d6, ppm)
8.48 (t, Ј=5.44 Hz, 1H), 7.02 (d, Ј=2.19 Hz, 2H), 6.67 (t, Ј=2.15 Hz, 1H), 4.97 (d, Ј=4.92 Hz, 1H),
4.90 (dd, Ј1=10.47 Hz, and Ј2=4.55 Hz, 2H), 4.49 (t, Ј=5.89 Hz, 1H), 4.16-4.08 (m, 5H), 3.89-3.80
(m, 1H), 3.76-3.73 (m, 4H), 3.64-3.62 (m, 1H), 3.59-3.55 (m, 4H), 3.54-3.50 (m, 16H), 3.47-3.44 (m,
4H), 3.41 (q, Ј=7.00 Hz, 6H), 3.18 (d, Ј=5.24 Hz, 2H), 3.17-3.01 (m, 3H), 2.99-2.93 (m, 1H), 1.09 (t,
Ј=6.99 Hz, 6H). 13C NMR (75 MHz, DMSO-d6, ppm): 166.2, 159.9, 136.9, 106.3, 104.3, 103.5, 77.4,
9

77.2, 73.8, 70.5, 70.4, 70.3, 70.2, 70.1, 69.7, 69.3, 68.3, 67.9, 66.0, 61.5, 15.6.; TOF-MS (ESI+)
calcd.for C35H61N1Na1O17 790.38372, found 790.38027.
1
Compound 1c (0.035 g, 0.036 mmol, 90% yield, pale-yellowish oil): H NMR (300 MHz, CDCl3,
ppm) 7.71 (t, J=5.52 Hz, 1H), 7.27-7.17 (m, 8H), 7.09 (d, Ј=2.16 Hz, 2H), 6.59 (t, Ј=2.04 Hz, 1H),
4.52 (s, 4H), 4.26 (d, J = 7.18 Hz, 1H), 4.16-4.13 (m, 4H), 3.96(m, 2H), 3.86-3.83 (m, 4H), 3.75-3.51
(m, 30H), 3.50-3.38 (m, 4H), 3.27 (m, 1H), 2.95-2.82 (m, 2H), 2.69 (m, 1H), 1.23 (d, J=6.93 Hz, 12
13
H). C NMR (75 MHz, CDCl3, ppm): 167.4, 159.6, 148.4, 136.8, 135.4, 128.0, 126.4, 106.5, 104.4,
103.2, 76.4, 75.6, 73.2, 70.9, 70.6, 70.5, 70.4, 70.1, 70.0, 69.6, 69.2, 67.5, 62.4, 40.1, 33.8, 24.0.;
TOF-MS (ESI+) calcd.for C51H77N1Na1O17 998.50892, found 998.50050.
1.2 Preparation of GHS-containing and Celite-immobilized lipoprotein lipase.
Burkholderia species lipoprotein lipase (BSLPL) is commercially available as lipoprotein lipase-3 for
diagnostic use from Amano. We used the samples provided by Amano. The protein content of LPL
described in the technical information was 52%. Our determination of protein content using the
Bradford method [43] indicated that it contained 50% (w/w) protein. BSLPL-1a, -1b, and -1c were
prepared, respectively, by freeze-drying a water-dioxane (1:1, v/v) solution (100 μL) containing
BSLPL (52% protein, 0.25 mg) and GHS (0.25 mg). BSLPL-1c-G was prepared by freeze-drying a
water-dioxane (1:1, v/v) solution (100 μL) containing commercial BSLPL (52% protein, 0.25 mg), 1c
(0.25 mg), and glucose (0.25 mg). BSLPL-1c-D was prepared by freeze-drying a water-dioxane (1:1,
v/v) solution (100 μL) containing commercial BSLPL (52% protein, 0.25 mg), 1c (0.25 mg), and
dextrin (0.25 mg). BSLPL-Celite (protein content, 1%, w/w) was prepared by freeze-drying an
aqueous solution containing a new sample of BSLPL (22.3% protein, 0.50 mg) and Celite (10.5 mg).
1.3 Determination of enzymatic activity.
10

The kinetic parameters (Vmax and Km) of BSLPL-1b for the hydrolysis of p-nitrophenyl acetate
(AcOPNP) were determined with solutions containing AcOPNP (0.1-0.3 mM) and BSLPL-1b (2.5
μg) in 0.05 M phosphate buffer (pH 7.0, 1 mL). The rate measurement at each substrate concentration
o
was performed twice at 25 C by following the absorbance at 400 nm for the production of p-
nitrophenol with a UV-visible spectrophotometer. The Vmax and Km values were then estimated from
the non-linear curve fitting of average initial rates. The kinetic parameters (Vmax and Km) of BSLPL-
1a, -1b, -1c, and -1c-G for the alcoholysis of AcOPNP were obtained through two sets of experiments
to determine Vmax and Vmax/Km separately. The Vmax values were obtained through a set of experiments
with solutions containing AcOPNP (0.05-0.25 M), BSLPL-GHS (BSLPL 0.25 mg), and benzyl
alcohol (2.0 equiv.) in toluene (5 mL). The Vmax/Km values were determined with the solutions
containing AcOPNP (0.05-0.25 M), BSLPL-GHS (BSLPL 0.25 mg), and benzyl alcohol (0.5 M) in
toluene (1 mL). The rate measurement at each substrate concentration was performed twice as
o
follows. The solution was shaken at 250 rpm at 25 C. An aliquot was sampled periodically, filtered
through a short silica gel-pad, and analyzed by GC to determine the conversion % against time. The
kinetic parameters were then estimated from the non-linear curve fitting of average initial rates.
1.4 Kinetic resolution with GHS-treated BSLPL
In a typical procedure, isopropenyl acetate (1.5 equiv.) was added to a 4 mL-vial containing BSLPL-
1c-D (3 mg), substrate (0.3 mmol), and anhydrous toluene (0.5 M). The resulting solution was then
o
shaken at 25 C until the reaction reached 46-50% conversion. After being diluted with methylene
chloride, the reaction mixture was filtered through a celite pad, concentrated, and then analyzed by
HPLC to determine the enantiomeric excesses of remaining substrate and acetylated product. The
enantioselectivity (E) was then calculated using the equation: E = ln[1-c(1+eep)]/ln[1-c(1-eep)] where
c = ees/(ees+eep).
11

The kinetic resolution of 4a: (S)-4a (82 %ee) and (R)-5a (97% ee); 46% conversion; E = >100. The
kinetic resolution of 4b: (S)-4b (>99 %ee) and (R)-5b (97% ee); 50% conversion; E = >100.
1.5 Dynamic kinetic resolution with GHS-treated BSLPL
As the representative procedure, the DKR of 4a is described. Freshly dried and degassed toluene (600
μL) was added to a pear-shaped Schlenk flask containing 4a (0.3 mmol), [Ru]-catalyst 6 (12.6 mg, 5
mol %), K2CO3 (41.4 mg, 0.3 mmol), and molecular sieve 4 Å (30 mg) under argon. The resulting
solution was stirred at 60 oC for 30 min, then BSLPL-1c-D (3 mg) and isopropenyl acetate (49.6 μL,
0.45 mmol) were added into the flask, and the resulting solution was stirred at room temperature for
24h. The solution was diluted with methylene chloride and filtered through a celite pad. The filtrate
was concentrated and purified by silica gel column chromatography to give (R)-7a.
25
(R)-3-Chloro-1-phenyl-propyl acetate (5a): 58 mg (90% yield, 99% ee), [α]D = +59.8 (c = 1.0,
25
1
13
CHCl3) [lit.22 [α]D = +59.4 (c = 1.0, CHCl3, 98% ee)]. The H and C NMR data were in good
agreement with those reported in the literature.22
25
(R)-1-(4-Isopropyl-phenyl)-pentyl acetate (5b): 68 mg (91% yield, 98% ee), [α]D = +67.1(c
25
1
= 1.0, CHCl3) [lit.22 [α]D = +68.8 (c = 1.0, CHCl3, >99% ee)]. The H and 13C NMR data were
in good agreement with those reported in the literature.22
Acknowledgements
This work was supported by the National Research Foundation of Korea (2015R1D1A1A01059851).
We thank Amano for the generous supply of the BSLPL sample used in this work and specially Dr.
Hirose for providing the technical information.
12

Appendix A. Supplementary Data
Supplementary data (NMR spectra of surfactants and non-linear curve fitting of kinetic data)
associated with this article can be found in the online version at http://dx.doi.org/
10.1016/j.molcatb.x0xx000x
13

References
[1]
U. T. Borncheuer, R. J. Kazlauskas, Hydrolases in Organic Synthesis, second ed., Wiley-
VCH, Weinheim, 2006.
[2]
[3]
T. Matsuda, Future Directions in Biocatalysis, Elsevier Science, Amsterdam, 2007.
V. Gotor, I. Alfonso, E. Garcia-Urdiales, Asymmetric Organic Synthesis with Enzymes,
Wiley-VCH, Weinheim, 2008.
[4]
P. M. Dinh, J. A. Howarth, A. R. Hudnott, J. M. J. Williams, Catalytic racemisation of
alcohols: Applications to enzymatic resolution reactions, Tetrahedron Lett. 37 (1996)
7623—7626.
[5]
[6]
[7]
[8]
J. H. Lee, K. Han, M.-J. Kim, J. Park, Chemoenzymatic Dynamic Kinetic Resolution of
Alcohols and Amines, Eur. J. Org. Chem. (2010) 999—1015.
Y. Kim, J. Park, M.-J. Kim, Dynamic Kinetic Resolution of Amines and Amino Acids by
Enzyme–Metal Cocatalysis, ChemCatChem.3 (2011) 271—277.
H. Pellissier, Organocatalyzed Dynamic Kinetic Resolution, Adv. Synth. Catal. 353
(2011) 659—676.
O. Verho, J.-E. Bäckvall, Chemoenzymatic Dynamic Kinetic Resolution: A Powerful
Tool for the Preparation of Enantiomerically Pure Alcohols and Amines, J. Am. Chem.
Soc. 137 (2015) 3996—4009.
[9]
B. A. Persson, A. L. E. Larsson, M. Le Ray, J. E. Bäckvall, Ruthenium- and Enzyme-
Catalyzed Dynamic Kinetic Resolution of Secondary Alcohols, J. Am. Chem. Soc. 121
(1999) 1645—1650.
[10]
J. H. Choi, Y. H. Kim, S. H. Nam, S. T. Shin, M.-J. Kim, J. Park,
Aminocyclopentadienyl Ruthenium Chloride: Catalytic Racemization and Dynamic
Kinetic Resolution of Alcohols at Ambient Temperature, Angew. Chem., Int. Ed. 41
(2002) 2373—2376.
[11]
M.-J. Kim, Y. I. Chung, Y. K. Choi, H. K. Lee, D. Kim, J. Park, (S)-Selective Dynamic
Kinetic Resolution of Secondary Alcohols by the Combination of Subtilisin and an
Aminocyclopentadienylruthenium Complex as the Catalysts, J. Am. Chem. Soc. 125
(2003) 11494—11495.
14

[12]
[13]
B. Martin-Matute, M. Edin, K. Bogar, J.-E. Bäckvall, Highly Compatible Metal and
Enzyme Catalysts for Efficient Dynamic Kinetic Resolution of Alcohols at Ambient
Temperature, Angew. Chem., Int. Ed. 43 (2004) 6535—6539.
J. H. Choi, Y. K. Choi, Y. H. Kim, E. S. Park, E. J. Kim, M.-J. Kim, J. Park,
Aminocyclopentadienyl Ruthenium Complexes as Racemization Catalysts for Dynamic
Kinetic Resolution of Secondary Alcohols at Ambient Temperature, J. Org. Chem. 69
(2004) 1972—1977.
[14]
[15]
B. Martin-Matute, M. Edin, K. Bogar, F. B. Kaynak, J.-E. Bäckvall, Combined
Ruthenium(II) and Lipase Catalysis for Efficient Dynamic Kinetic Resolution of
Secondary Alcohols. Insight into the Racemization Mechanism, J. Am. Chem. Soc. 127
(2005) 8817—8825.
A. Berkessel, M. L. Sebastian-Ibarz, T. N. Müller, Lipase/Aluminum-Catalyzed
Dynamic Kinetic Resolution of Secondary Alcohols, Angew. Chem., Int. Ed. 45 (2006)
6567—6570.
[16]
[17]
P. Hoyos, M. Fernández, J. V. Sinisterra, A. R. Alcántara, Dynamic Kinetic Resolution
of Benzoins by Lipase−Metal Combo Catalysis, J. Org. Chem. 71 (2006) 7632—7637.
S. Akai, K. Tanimoto, Y. Kanao, M. Egi, T. Yamamoto, Y. Kita, A Dynamic Kinetic
Resolution of Allyl Alcohols by the Combined Use of Lipases and [VO(OSiPh3)3],
Angew. Chem., Int. Ed. 45 (2006) 2592—2595.
[18]
[19]
M.-J. Kim, Y. K. Choi, S. Kim, D. Kim, K. Han, S.-B. Ko, J. Park, Highly
Enantioselective Dynamic Kinetic Resolution of 1,2−Diarylethanols by
a
Lipase−Ruthenium Couple, Org. Lett. 10 (2008) 1295—1298.
R. M. Haak, F. Berthiol, T. Jerphagnon, J. A. Gayet, C. Tababiono, C. P. Postema, V.
Ritleng, M. Pfeffer, D. B. Janssen, A. J. Minnaard, B. L. Feringa, J. G. De Vries,
Dynamic Kinetic Resolution of Racemic β-Haloalcohols: Direct Access to
Enantioenriched Epoxides, J. Am. Chem. Soc. 130 (2008) 13508—13509.
L. K. Thalen, A. Sumic, K. Bogar, J. Norinder, A. K. A. Persson, J.-E. Bäckvall,
Enantioselective Synthesis of α-Methyl Carboxylic Acids from Readily Available
Starting Materials via Chemoenzymatic Dynamic Kinetic Resolution, J. Org. Chem. 75
(2010) 6842—6847.
[20]
[21]
X. Gang, Y. Chen. J. Wu, Y. Cheng, L. Yang, Dynamic kinetic resolution of secondary
aromatic alcohols with new efficient acyl donors, Tetrahedron: Asymmetry 22 (2011)
1373—1378.
15

[22]
H. J. Kim, Y. K. Choi, J. Lee, E. Lee, J. Park, M.-J. Kim, Ionic-Surfactant-Coated
Burkholderia cepacia Lipase as a Highly Active and Enantioselective Catalyst for the
Dynamic Kinetic Resolution of Secondary Alcohols, Angew. Chem. Int. Ed. 50 (2011)
10944—10948.
[23]
[24]
[25]
T. Ema, Y. Nakano, D. Yoshida, S. Kamata, T. Sakai, Redesign of enzyme for improving
catalytic activity and enantioselectivity toward poor substrates: manipulation of the
transition state, Org. Biomol. Chem. 10 (2012) 6299—6308.
M. Egi, K. Sugiyama, M. Saneto, R. Hanada, K. Kato, S. Akai, A Mesoporous-Silica-
Immobilized Oxovanadium Cocatalyst for the Lipase-Catalyzed Dynamic Kinetic
Resolution of Racemic Alcohols, Angew. Chem., Int. Ed. 52 (2013) 3654—3658.
C. Kim, J. Lee, J. Cho, Y. Oh, Y. K. Choi, E. Choi, J. Park, M.-J. Kim, Kinetic and
Dynamic Kinetic Resolution of Secondary Alcohols with Ionic-Surfactant-Coated
Burkholderia cepacia Lipase: Substrate Scope and Enantioselectivity, J. Org. Chem. 78
(2013) 2571—2578.
[26]
[27]
J. Cho, J. Lee, J. Park, M.-J. Kim, Highly enantioselective dynamic kinetic resolution of
alkyl aryl carbinols carrying a trimethylsilyl group with a highly active lipoprotein
lipase preparation, Tetrahedron: Asymmetry 26 (2015) 840—845.
K. Fukunaga, Y. Mine, K.-I. Samejima, M. Yoshimoto, K., Y. Sugimur, Structural effects
of amphiphiles on Candida rugosa lipase activation by freeze-drying of aqueous
solution of enzyme and amphiphile , J. Biosci. Bioeng. 96 (2003) 525—528.
Y. Mine, K. Fukunaga, K.-I. Samejima, M. Yoshimoto, K. Nakaoa, Y. Sugimur,
Preparation of gemini-type amphiphiles bearing cyclitol head groups and their
application as high-performance modifiers for lipases, Carbohydr. Res. 339 (2004)
493—501.
[28]
[29]
[30]
J. K. Lee, M.-J. Kim, Ionic Liquid-Coated Enzyme for Biocatalysis in Organic Solvent,
J. Org. Chem. 67 (2002) 6845—6847.
T. Itoh, Y. Matsushita, Y. Abe, S.-H. Han, S. Wada, S. Hayase, M. Kawatsura, S. Takai,
M. Morimoto, Y. Hirose, Increased Enantioselectivity and Remarkable Acceleration of
Lipase-Catalyzed Transesterification by Using an Imidazolium PEG–Alkyl Sulfate Ionic
Liquid, Chem Eur. J. 12 (2006) 9228—9237.
16

[31]
[32]
[33]
Y. Abe, K. Yoshiyama, Y. Yagi, S. Hayase, M. Kawatsura, T. Itoh, A rational design of
phosphonium salt type ionic liquids for ionic liquid coated-lipase catalyzed reaction,
Green. Chem. 12 (2010) 1976—1980.
K. Yoshiyama, Y. Abe, S. Hayase, T. Nokami, T. Itoh, Synergetic Activation of Lipase
by an Amino Acid with Alkyl–PEG Sulfate Ionic Liquid , Chem. Lett. 42 (2013) 663—
665.
Y. L. Khmelnitsky, S. H. Welch, D. S. Clark, J. S. Dordick, Salts dramatically enhance
activity of enzymes suspended in organic solvents, J. Am. Chem. Soc. 116 (1994)
2647—2648.
[34]
[35]
V. M. Paradkar, J. S. Dordick, Aqueous-Like Activity of α-Chymotrypsin Dissolved in
Nearly Anhydrous Organic Solvents, J. Am. Chem. Soc. 116 (1994) 5009—5010.
M. T. Ru, J. S. Dordick, J. A. Reimer, D. S. Clark, Optimizing the salt-induced
activation of enzymes in organic solvents: Effects of lyophilization time and water
content, Biotechnol. Bioeng. 63 (1999) 233—241.
[36]
M. T. Ru, K. C. Wu, J. P. Lindsay, J. S. Dordick, J. A. Reimer, D. S. Clark, Towards
more active biocatalysts in organic media: Increasing the activity of salt-activated
enzymes, Biotechnol. Bioeng. 75 (2001) 187—196.
[37]
[38]
A. L. Serdakowski, J. S. Dordick, Enzyme activation for organic solvents made easy,
Trends Biotechnol. 26 (2007) 48—54.
J. Lee, Y. Oh, Y. K. Choi, E. Choi, K. Kim, J. Park, M.-J. Kim, Dynamic Kinetic
Resolution of Diarylmethanols with an Activated Lipoprotein Lipase, ACS Catal. 5
(2015) 683—689.
[39]
[40]
E. Lee, Y. Oh, Y. K. Choi, M.-J. Kim, Aqueous-Level Turnover Frequency of Lipase in
Organic Solvent, ACS Catal. 4 (2014) 3590—3592.
P. S. Chae, S. G. F. Rasmussen, R. R. Rana, K. Gotfryd, A. C. Kruse, A. Manglik, K. H.
Cho, S. Nurva, U. Gether, L. Guan, C. J. Loland, B. Byrne, B. K. Kobilka, S. H.
Gellman, A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for
Solubilization and Stabilization of Membrane Proteins, Chem. Eur. J. 18 (2012) 9485—
9490.
[41]
B. Borgström, H. L. Brockman, Lipases, Elsevier, Amsterdam, 1984.
17

[42]
[43]
P. Woolley, S. B. Petersen, Lipases, Cambridge University, Cambridge, 1994.
M. M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72
(1976) 248—254.
[44]
For a previous study on the activities of Celite-immobilized lipases in organic solvent,
see: L. T. Kanerva, O. Sundholm, Enzymatic acylation in the resolution of methyl threo-
2-hydoxy-3-(4-methoxyphenyl)-3-(2-X-phenylthio)propionates in organic solvents,
Chem. Soc. Perkin Trans. 1 (1993) 2407—2410). In this study, 3 lipases were
immobilized on Celite. One of them displayed a reduced activity and the other two did
enhanced activities. The activity enhancements, however, were 3-fold or less.
18

Fig. 1 Glucose-headed surfactants
Scheme 1. Synthesis of glucose-headed surfactants
Scheme 2. Kinetic resolution of secondary alcohols with GHS-treated BSLPL
19

Scheme 3. Dynamic kinetic resolution of secondary alcohols with GHS-treated BSLPL
20

Table 1
Kinetic Parameters of BSLPL Preparations for the Hydrolysis and Transesterifications of p-
Nitrophenyl Acetate (AcOPNP)a
b
kcat
Km
kcat/Km
Entry
Lipase
ROH
HOH
Solvent
(kcat/Km)rel
(s-1)
(M)
(s-1/M)
1c
2
BSLPL
buffer
buffer
2.8 x 102
2.7 x 102
6.4 x 10-2
1.0 x 103
2.7 x 103
4.5 x 103
4.9 x 103
4.8 x 10-4
5.2 x 10-4
2.5 x 10-1
1.7 x 10-1
2.0 x 10-1
1.5 x 10-1
1.2 x 10-1
5.7 x 105
5.1 x 105
2.6 x 10-1
5.9 x 103
1.4 x 104
3.0 x 104
4.1 x 104
2.2 x 106
2.0 x 106
1
BSLPL-1bd HOH
3c
BSLPL
PhCH2OH toluene
4
BSLPL-1ad PhCH2OH toluene
BSLPL-1bd PhCH2OH toluene
BSLPL-1cd PhCH2OH toluene
BSLPL-1c-GePhCH2OH toluene
t-butyl
2.3 x 104
5.4 x 104
1.2 x 105
1.6 x 105
5
6
7
BSLPL-1c-GePhCH2OH
5.4 x 103
1.8 x 10-1
9.3 x 10-2
1.4 x 10-1
5.8 x 104
1.3
2.2 x 105
5.0
methyl
8
9
ether
BSLPL-
Celitef
PhCH2OH toluene
a The Vmax and Km values were obtained using the nonlinear curve fitting of average rates per 1 mg of
BSLPL (52% protein) measured in duplicate. Each kcat was then calculated using the equation Vmax
=
kcat[E]0, where kcat is the turnover frequency of single enzyme molecule and [E]0 is the total number of
enzyme molecules in solution. For the kinetic measurements the heterogeneous enzyme solutions
were shaken rapidly at 250 rpm because they displayed maximum activities above 150 rpm.
b For AcOPNP
c From ref. 39.
d The weight ratio between BSLPL and surfactant = 1 : 1. The content of water determined by the
Karl-Fisher titration was less than 4% (w/w).
e The weight ratio between BSLPL, surfactant, and glucose (G) = 1 : 1 : 1.
f This contained 1%(w/w) protein.
21