The Structural Effect of Benzoate Surfactant Tails on the Activity of Lipoprotein Lipase in Organic Solvent

Full text of DOI:10.1002/bkcs.11963

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DOI: 10.1002/bkcs.11963
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Y. Oh
KOREAN CHEMICAL SOCIETY
The Structural Effect of Benzoate Surfactant Tails on the Activity of
Lipoprotein Lipase in Organic Solvent
*
Yeonock Oh
Department of Chemistry, Pohang University of Science and Technology, Pohang 37673, Republic of
Korea. *E-mail: oyo0203@postech.ac.kr
Received October 7, 2019, Accepted December 17, 2019
Keywords: Lipase, Activation, Surfactant, Structural effect, Organic solvent
Lipases are useful as catalysts for the synthesis of optically
active compounds.1–3 In particular, they are increasingly
being used in asymmetric synthetic transformations to pro-
duce enantiopure pharmaceuticals.4 Lipases show high
activity in water, particularly at the water–oil interface.
However, most organic compounds are insoluble in aque-
ous medium. Furthermore, they are often degraded or
converted to side products by water during the reaction.
Product recovery is also difficult from water, and the ther-
modynamic equilibria of many reactions are unfavorable in
aqueous medium.5 These problems can be overcome by
using organic solvents as reaction media. Synthetic applica-
tions of lipases in organic solvents, however, suffer often
from their poor activities in nonaqueous media. It has been
known that the activity of native lipase in organic media is
several orders of magnitude lower than its aqueous counter-
part. Thus, various methods have been developed to enhance
the activity of lipase in organic solvent. One of the most
widely used methods is the use of surfactants. The Schiavon
group reported on the improvement of enzymatic activity by
poly(ethylene glycol)-type surfactants in 1985.6 The Mine
group reported that water-soluble nonionic Gemini-type sur-
factants enhanced the activities of commercially available
lipases for the transesterification of alcohols.7–9 Recently, the
Kim group reported that benzoate-based surfactants (1 and
2, Figure 1) enhanced significantly the activity of lipoprotein
lipase from Burkholderia species (BSLPL) in organic sol-
vent.10,11 Interestingly, surfactant 2 with two hydrophilic
tails was much stronger activator than 1 with two hydropho-
bic tails. Lately, I reported that benzoate surfactants 3 with a
hydrophobic ring at the end of each hydrophilic tail were
even more effective than 2 in enhancing the activity of
BSLPL.12 Furthermore, 3b was the best among them. These
results encouraged me to explore the structural effects of sur-
factant tails on the activity of BSLPL. Now I wish to report
that surfactants with a hydrophilic and a hydrophobic tail are
superior to those with only hydrophilic or hydrophobic tails
in the BSLPL-activating ability.
Methyl 3,5-dihydroxybenzoate was coupled with undecyl
bromide to obtain 5, which was then reacted with the
bromo derivative of triethyleneglycol ethyl ether to afford
6. Methyl ester 6 was hydrolyzed and then converted to the
target 4 as the potassium salt. This synthesis was straight-
forward and provided high yields in most steps.
Surfactant-coated BSLPLs containing each benzoate sur-
factant were prepared according to the protocol reported
previously.13 The activities of native and surfactant-coated
BSLPLs were measured as the specific activities in the
acetylation of 1-phenylethanol in toluene at 25 ꢀC. The
results are described in Table 1.
Native BSLPL showed very low activity in toluene (entry
1). The activity of BSLPL was enhanced by two orders of
magnitude if it was co-lyophilized with surfactant 1 with two
long alkyl chains as the tails (entry 2). Surfactant 2 with
oligoethylene glycol chains activated BSPL one order of mag-
nitude more strongly (entry 3). This implies that benzoate sur-
factants with hydrophilic tails are more powerful activators
than those with hydrophobic tails. Interestingly, surfactants 3a-
b with an aromatic ring at the end of each oligoethylene glycol
chain were even better than 2 in activating BSLPL (entries
4–5). This observation suggests that the presence of both
hydrophilic and hydrophobic parts in each tail is beneficial to
the activation pf BSLPL. And it is noteworthy that surfactant
3b induced the strongest activation (entry 5). Finally, surfactant
4 also induced the similarly strong activation of BSLPL (entry
6). Thus, the comparison between three surfactants (1, 2, and
4) in lipase–activation power reveals that surfactants with both
hydrophilic and hydrophobic tails are more powerful activators
than those with only hydrophobic or hydrophilic tails.
The results from the recent molecular dynamics simula-
tion study on the mechanism of lipase activation14 suggest
that the hydrophilic parts of surfactants contribute to the
stabilization of enzyme by interacting with the hydrophilic
surface of enzyme. On the other hand, the hydrophobic
parts of surfactants have favorable interactions with hydro-
phobic residues around the active site of enzyme, thus help-
ing the enzyme maintain active open conformation in
organic solvent. Consequently, these suggestions provide
theoretical rationale for insight into the relationship
between surfactant structure and lipase activity.
Four different surfactants 1–4 were chosen to explore the
structural effects of surfactant tails. Surfactants 1–3 were
prepared according to the literature procedures.10–12 The
synthesis of new surfactant 4 is described in Scheme 1.
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Scheme 1. Synthesis of benzoate surfactant 4
activity relationship in the activation of BSLPL. The results
suggest that the BSLPL-activating ability of surfactant
depends significantly on the hydrophilic and hydrophobic
nature of its tails. Furthermore, the tails of surfactant should
have a balance in hydrophilic and hydrophobic nature for
inducing high lipase activity in organic solvent. It is believed
that the structural characteristics of benzoate surfactants
described in the manuscript can be used as the guidelines for
the design of more efficient surfactants, leading to the prepa-
ration of highly active enzymes for use in organic solvent.
Figure 1 Structures of benzoate surfactants.
Surfactant 1 with no hydrophilic tails should have favorably
strong interactions with hydrophobic parts around the active
site of enzyme but poor interactions with hydrophilic parts of
enzyme. To the contrary, surfactant 2 have two hydrophilic
tails, thus interacting more favorably with hydrophilic parts of
enzyme. Surfactant 2 also could have interactions with hydro-
phobic parts of enzyme owing to some hydrophobic nature of
two tails although the hydrophobic interactions are relatively
weaker than their hydrophilic counterparts. These more favor-
able interactions between surfactant 2 and enzyme should
enhance enzymatic activity. Thus, BSLPL-2 (containing sur-
factant 2) becomes more active than BSLPL-1. Surfactant
4 has both hydrophilic and hydrophobic tails, which allows
for favorable interactions with both hydrophilic and hydropho-
bic parts of enzyme. As a result, BSLPL-4 displays higher
activity compared to BSLPL-2. On the other hand, surfactant
3 has both hydrophilic and hydrophobic parts in each tail for
favorable interactions with enzyme. These interactions make
BSLPL-3, particularly BSLPL-3b, as active as BSLPL-4. It
is concluded that surfactants with both hydrophilic and
hydrophobic tails are superior to those with only hydrophilic
or hydrophobic tails in the BSLPL-activating ability.
Experimental
General. All the commercially available starting materials
and reagents were purchased from Sigma-Aldrich Co. LLC.
(St. Louis, MO, USA), Acros Organics (Morris, NJ, USA),
and TCI Co., LTD (Tokyo, Japan) and used without addi-
tional purification. Lipase from Burkholderia species was
purchased from Amano Enzyme Inc. (Yokohama, Japan).
All solvents were purified by purifying system; Ultimate
Solvent System, Glass Contour from Nikko Hansen & Co.,
1
Ltd. (Osaka, Japan). H NMR and 13C NMR spectra were
recorded in CDCl3 solution at ambient temperature on a
300 MHz Fourier transform nuclear magnetic resonance
(FT-NMR) Spectroscopy; AVANCE 300, Bruker (Billerica,
MA, USA). Chemical shift are given in δ (ppm) relative to
tetramethylsilane (TMS) as internal standard. Multiplicities
were recorded as s (singlet), d (doublet), t (triplet), and m
(multiplet). Reaction conversion was determined by HPLC;
Agilent 1100 with (R, R)-Whelk-01 column. Molecular
In summary, I prepared a new benzoate surfactant 4 and
compared it with other surfactants 1–3 to see the structure–
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Table 1. Activities of BSLPL preparations in the acetylation of
(t, J = 6.20 Hz, 2H), 4.14 (t, J = 3.81 Hz, 2H), 6.66 (s, 1H),
7.17 (s, 2H). 13C NMR (75 MHz, CDCl3, ppm): δ 14.0, 15.1,
22.6, 25.9, 29.1, 29.3, 29.3, 29.5, 29.5, 31.8, 52.1, 66.9, 67.7,
68.3, 69.5, 69.8, 70.5, 70.6, 70.7, 70.8, 106.7, 107.5, 108.0,
131.8, 159.7, 160.1, 166.8.
1-phenylethanol in organic solvent.a
OAc
OH
BSLPL
preparation
+ CH3CO2C(CH3)=CH2
+ CH3COCH3
toluene, 25oC
To the solution of 6 (1.0 mmol) in ethanol (1.0 mL) were
added aq. KOH (2.0 equiv). After stirring for 3 h at r.t., the
reaction mixture was neutralized by 1 N HCl and concen-
trated under reduced pressure. The residue was extracted
with methylene chloride and the solvent was evaporated to
BSLPL
Entry Surfactant preparation
Specific
activity (U)b Enhancement
1
none
Native
BSLPL
0.38
1
1
obtain 7 (1.41 g, 97% yield): H NMR (300 MHz, CDCl3,
2
3
4
5
6
1
2
3a
3b
4
BSLPL-1
BSLPL-2
BSLPL-3a
BSLPL-3b
BSLPL-4
200
5.3 × 102
3.9 × 103
5.0 × 103
7.9 × 103
7.4 × 103
ppm): δ 0.88 (t, J = 6.60 Hz, 3H), 1.21 (t, J = 6.99 Hz,
3H), 1.27 (s, 14H), 1.42 (t, J = 7.71 Hz, 2H), 1.78 (t,
J = 7.09 Hz, 2H), 3.52–3.78 (m, 10H), 3.88 (m, 2H), 3.97
(m, 2H), 4.16 (t, J = 4.05 Hz, 2H), 6.71 (s, 1H), 7.23 (s,
2H). 13C NMR (75 MHz, CDCl3, ppm): δ 14.1, 15.0, 22.6,
25.9, 29.1, 29.3, 29.3, 29.5, 29.6, 31.9, 66.6, 67.6, 68.3,
69.5, 69.7, 70.5, 70.6, 70.7, 107.2, 107.9, 108.5, 131.2,
159.7, 160.1170.9. TOF-MS (ESI+) calcd. For
[C26H44O7 + Na]+ 491.29847, found 491.29687.
1500
1900
3000
2800
a BSLPL preparations containing BSLPL (25%, w/w), dextrin (50%,
w/w), and surfactant (25%, w/w) were lyophilized in 1:1 (v/v)
water-1,4-dioxane. The reactions were performed with solutions
containing 1-phenylethanol (0.5 M), BSLPL preparation (1 mg), and
isopropenyl acetate (1.5 equiv) in toluene at 25 ꢀC. Rate
measurements were done three times for each enzyme preparation.
b 1 U = 1 μmole of product per hour per mg of BSLPL. The error
range of the specific activity values is Æ3% for all entries.
To the solution of 7 (0.5 mmol) in tetrahydrofuran
(THF) (0.5 mL) was added KOH (0.1 M, 2.3 mL). After
stirring for 1 h at r.t., the reaction mixture was concentrated
under reduced pressure and further dried in vacuo for 24 h
1
to afford 4 quantitatively. (252 mg, 99% yield): H NMR
(300 MHz, CDCl3, ppm): δ 0.76 (t, J = 6.62 Hz, 3H), 1.15
(t, J = 7.01 Hz, 3H), 1.22 (s, 14H), 1.38 (t, J = 7.68 Hz,
2H), 1.71 (t, J = 7.08 Hz, 2H), 3.43–3.75 (m, 10H), 3.81
(m, 2H), 3.93 (m, 2H), 4.10 (t, J = 4.09 Hz, 2H), 6.68 (s,
1H), 7.15 (s, 2H). 13C NMR (75 MHz, CDCl3, ppm): δ
14.2, 14.8, 22.8, 25.4, 28.9, 29.3, 29.3, 29.6, 29.7, 32.0,
66.9, 67.5, 68.3, 69.4, 70.0, 70.6, 70.7, 70.9, 107.3, 107.9,
108.7, 131.5, 159.7, 160.2, 171.0.
mass was measured by liquid chromatography–mass spec-
trometry (LC–MS); JMS-T100 L (AccuTOF), Jeol (Tokyo,
Japan). All enzyme preparations were lyophilized by freeze
dryer; FD-1000 or FDU-1200, Eyela (Tokyo, Japan).
Synthesis of 4.
To the solution of methyl 3, 5-
dihydroxybenzoate (810 mg, 4.81 mmol), K2CO3 (2.0
equiv) and 18-crown-6 (0.05 equiv) in acetone (20 mL)
was added 1-bromoundecane (1.0 equiv) and the mixture
was stirred under reflux for 48 h. After cooled down to r.t.,
the mixture was filtrated through celite-pad to remove
insoluble materials. The filtrate was evaporated under
reduced pressure and the residue was purified by silica gel
Preparation of Surfactant-coated BSLPL Preparation.
The enzyme powder (50 mg) was dissolved in deionized
water (15 mL) and then mixed with dextrin and benzoate sur-
factant dissolved in 1,4-dioxane (15 mL) (enzyme: dextrin:
surfactant = 1: 2: 1 weight ratio). The resulting solution was
lyophilized to yield surfactant-coated BSLPL preparation.
1
chromatography to give 5 (1.02 g, 66% yield): H NMR
(300 MHz, CDCl3, ppm): δ 0.80 (t, J = 5.70 Hz, 3H), 1.19
(s, 14H), 1.35 (m, 2H), 1.68 (t, J = 6.70 Hz, 2H), 3.82 (s,
3H), 3.85–3.89 (m, 2H), 5.88 (s, 1H), 6.55 (s, 1H), 7.08 (d,
J = 10.7 Hz, 2H). 13C NMR (75 MHz, CDCl3, ppm): δ
14.1, 22.6, 25.9, 29.1, 29.3, 29.3, 29.5, 29.6, 31.9, 52.3,
68.3, 107.1, 107.7, 109.0, 131.8, 156.8, 160.4, 167.2.
To the solution of 5 (1.0 g, 3.10 mmol), K2CO3 (2.0 equiv)
and 18-crown-6 (0.05 equiv) in acetone (20 mL) was added
1-bromo-2-(2-(2-ethoxyethoxy)ethoxy)ethane (1.0 equiv) and
the mixture was stirred under reflux for 48 h. After cooled
down to r.t., the mixture was filtrated through celite-pad to
remove insoluble materials. The filtrate was evaporated under
reduced pressure and the residue was purified by silica gel
chromatography to give 6 (1.26 g, 84% yield): 1H NMR
(300 MHz, CDCl3, ppm): δ 0.88 (t, J = 5.30 Hz, 3H), 1.20
(t, J = 7.01 Hz, 3H), 1.27 (s, 14H), 1.44 (m, 2H), 1.76 (m,
2H), 3.51–3.67 (m, 10H), 3.85 (m, 2H), 3.87 (s, 3H), 3.95
Activity Measurements of BSLPL Preparations.
Isopeopenyl acetate (1.5 equiv.) was added to a 20 mL vial
containing BSLPL preparation (1 mg), 1-phenylethanol
(0.5 mmol), and anhydrous toluene (0.5 M). The resulting
solution was then shaken at 250 rpm at 25 ꢀC. An aliquot
was sampled periodically, filtered through a short silica pad,
and analyzed by HPLC to determine the conversion % against
time. The specific activity values were calculated from a slope
of conversion %-time plot. The rate measurement for each
BSLPL preparation was performed three times.
Acknowledgments. Author is grateful to the National
Research Foundation of Korea (2015R1D1A1A01059851)
for financial support of this work.
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