One-Step Synthesis of 4-Octyl Itaconate through the Structure Control of Lipase

Article abstract of DOI:10.1021/acs.joc.0c02995

4-Octyl itaconate is a novel antiviral and immunoregulatory small molecule showing great potential in the treatment of various autoimmune diseases and viral infections. It is difficult to selectively esterify the C4 carboxyl group of itaconate acid via one-step direct esterification using chemical catalysts, while the two-step route with itaconic anhydride as an intermediate is environmentally unfriendly and costly. This research investigated the one-step and green synthesis of 4-octyl itaconate through the structure control of lipase, obtaining 4-octyl itaconate with over 98% yield and over 99% selectivity. Multiscale molecular dynamics simulations were applied to investigate the reaction mechanism. The cavity pocket of lipases resulted in a 4-octyl itaconate selectivity by affecting distribution of substrates toward the catalytic site. Toluene could enhance monoesterification in the C4 carboxyl group and contribute to a nearly 100% conversion from itaconate acid into 4-octyl itaconate by adjusting the catalytic microenvironment around the lipase, producing a shrinkage effect on the channel.

Full text of DOI:10.1021/acs.joc.0c02995

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Article
One-Step Synthesis of 4‑Octyl Itaconate through the Structure
Control of Lipase
Changsheng Liu, Yilin Wang, Jiahao Liu, An’nan Chen, Juntao Xu, Renwei Zhang, Fang Wang,
Kaili Nie,* and Li Deng*
Cite This: J. Org. Chem. 2021, 86, 7895−7903
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ABSTRACT: 4-Octyl itaconate is a novel antiviral and immunoregulatory small molecule showing great potential in the treatment
of various autoimmune diseases and viral infections. It is difficult to selectively esterify the C4 carboxyl group of itaconate acid via
one-step direct esterification using chemical catalysts, while the two-step route with itaconic anhydride as an intermediate is
environmentally unfriendly and costly. This research investigated the one-step and green synthesis of 4-octyl itaconate through the
structure control of lipase, obtaining 4-octyl itaconate with over 98% yield and over 99% selectivity. Multiscale molecular dynamics
simulations were applied to investigate the reaction mechanism. The cavity pocket of lipases resulted in a 4-octyl itaconate selectivity
by affecting distribution of substrates toward the catalytic site. Toluene could enhance monoesterification in the C4 carboxyl group
and contribute to a nearly 100% conversion from itaconate acid into 4-octyl itaconate by adjusting the catalytic microenvironment
around the lipase, producing a shrinkage effect on the channel.
costly. The reported one-step synthesis under acidic conditions
led to a low yield and poor selectivity (Scheme 1). The two-
step synthesis route showed a high selectivity of 94% 4-OI with
95% yield,14 while generation of the intermediate (itaconic
anhydride), which was susceptible to hydrolysis during storage,
in the two-step route was costly. The production process of
itaconic anhydride, moreover, required an acid catalyst, a high-
temperature condition, and an extensive subsequent purifica-
tion step, thus increasing the pollution potential and
production costs (Scheme 1). Being environmentally friendly,
an efficient one-step synthesis route of 4-OI with high
regioselectivity would accelerate its medical research in
immunoregulation and application in polymer synthesis.
Development of biocatalysis may provide new insights into
the one-step synthesis of 4-OI. Compared with chemical
catalysts, lipases are well known for their high regioselectiv-
ity,15 high catalytic activity,16 and environmentally friendly
reaction conditions.17 Hence, lipases have been widely used in
INTRODUCTION
■
Itaconic acid (IA) and its derivates are important compounds
in polymers, agro-chemicals, and drug production.1 Recently,
research has demonstrated that itaconic acid has potent anti-
inflammatory, antimicrobial,2 and antiviral effects.3 The
immunity regulation of itaconic acid relies on its α,β-
unsaturated carboxylic acid to alkylate the key cysteine
residues by Michael addition.4 However, the natural form of
itaconic acid is too hydrophilic to cross the lipid bilayer.5
4-Octyl itaconate (4-OI) is a cell-permeable itaconate
derivative4 with a free α,β-unsaturated carboxylic acid near
the terminal carbon, which mimics the endogenous itaconate
better than other itaconate derivatives toward electrophilic-
ity.4,5 4-OI has great potential in treating various autoimmune
diseases,6 such as multiple sclerosis (MS),7 psoriasis,8 and
lupus erythematosus.9 Even more enticing is that 4-OI shows a
potent antiviral and anti-inflammatory activity toward SARS-
CoV2 and various human pathogenic viruses.10 4-OI has anti-
inflammation and antivirus efficacy10 to treat virtual replication
and the cytokine storm.11 Besides, 4-OI is also valuable in
renewable polymer synthesis,12 such as poly(mono-n-octyl
itaconate)13 and maleic anhydride-mono-octyl itaconate
copolymers.
Received: December 29, 2020
Published: June 4, 2021
However, the current synthesis route of 4-OI has low
selectivity and low yield or is environmentally unfriendly and
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Scheme 1. Synthesis Route of 4-OI
sustainable and green production.18 The selectivity of lipases
relies on the cavity shape and residue compositions in the
pocket,19 which may improve monoesterification in the C4
carboxyl group of IA. Sun et al. reported the selective
amidation route of phenylglycinol by Novozym 435 (CALB),
where the specific cavity shape contributed to high chemo-
selectivity for amidation (89.41%) rather than esterification
(0.21%).20 Hamberg et al. reported a site-directed mutagenesis
strategy to improve the regioselectivity of CALB toward
monoacylation from diols by changing the key residue
compositions.21 Meanwhile, solvents could influence the
catalytic microenvironment and adjust the selectivity of
enzymes, including substrate, stereo-, regio-, and chemo-
selectivity.22 Duan et al. reported that the increase of the
solvent log P could improve the regioselectivity of CALB
toward the sn-2 hydroxyl of a glycerol molecule, rather than the
sn-1 position.23 Bellot et al. also found that the increase in
solvent polarity by adding a n-hexane/2-methyl-2-butanol
equal-volume mixture improves the selectivity toward mono-
glyceride from 6 to 94% (molar percentage) in esterification
between glycerol and oleic acid by Rhyzomucor miehei lipase.24
Based on the drawbacks of acid-catalyzed synthesis of 4-OI,
a one-step green synthesis route of 4-OI based on the
regioselectivity of enzymes was developed. Meanwhile, the
catalytic microenvironment was improved to enhance this
process. Molecular dynamics (MD) simulation was utilized to
illustrate the mechanism of the high regioselectivity in this
process. Compared with the traditional route using itaconic
anhydride as the intermediate, this method reduced the
operation steps and the production costs, improved the
chemical sustainability, and provided a green, alternative way
for the synthesis of 4-OI.
RESULTS AND DISCUSSION
■
Optimization of Reaction Conditions. Esterification of
IA is a cascade process, where IA is converted to mono-octyl
itaconate (MOI) and the generated MOI is later esterified to
di-octyl itaconate (DOI) (Figure S3). The composition of
MOI and DOI in the final product was determined by reaction
kinetics, expressed as the production rate of MOI (VMOI) and
the generation rate of DOI (VDOI), as well as their ratio (Figure
S3). As shown in Figure 1a, H2SO4 catalyzed the cascade
esterification persistently without any selectivity toward MOI
and Novozym 435 (CALB) to produce the highest MOI of
42%. The optimal CALB dosage was 50 wt % to IA, yielding
82% 4-OI at 24 h (Figure 1b). A higher dosage of lipases can
provide more catalytic active sites, accelerating VMOI and VDOI
synchronously (Figure S3). As a result, the yield of MOI
increased and the monoester ratio (MER) decreased. On
extending the reaction time, the accumulation of MOI in the
reaction system accelerates VDOI and reduces VMOI (Figure S3),
reducing the MER (Figure 1c). The time course curve showed
that the highest yield of MOI (91%) appeared at 36 h. A
similar observation was found in the lipase-catalyzed
production of monoacylglycerol (MAG) from pinolenic acid
and glycerol, where the increase of lipase dosage and reaction
time increased the production of the byproduct, diacylglycerol
(DAG).25
The substance molar ratio always shows a profound effect on
the lipase-induced reaction.26 Theoretically, more octanol
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Figure 1. Optimization of various parameters in the enzymatic synthesis route. (a) Catalysts (0.01 g of lipases or 0.1 wt % sulfuric acid,
concentration of itaconic acid is 0.64 mol·L−1, concentration of 1-octanol is 6.4 mol·L−1, 24 h), (b) lipase dosage (0.01−0.1 g of Novozym 435,
concentration of itaconic acid is 0.64 mol·L−1, concentration of 1-octanol is 6.4 mol·L−1, 24 h), (c) time course (0.05 g of Novozym 435,
concentration of IA is 0.64 mol·L−1, concentration of 1-octanol is 6.4 mol·L−1, 6−72 h), and (d) substrate molar ratio (0.05 g of Novozym 435,
concentration of IA is 0.256−6.4 mol·L−1, concentration of 1-octanol is 6.4 mol·L−1, 36 h). All reactions were performed with 0.769 mmol of IA as
the substrate at 50 °C and at 800 rpm.
facilitates the production of DOI and reduces the MER (Figure
S3). Pereira et al. reported that an increase of lauric acid
enhanced the production of di- and trilaurin in the cascade
esterification of glycerol and lauric acid.27 Intriguingly, lipase-
mediated cascade esterification in this work showed an inverse
phenomenon, where more octanol resulted in less DOI and
yielded a higher MER (Figure 1d). We assume that excessive
octanol may act as a solvent to influence cascade esterification.
Computational Studies of the Selectivity toward MOI
in CALB. Unlike the acid catalyst, CALB selectively
synthetized MOI (Figure 1a). In lipase-catalyzed esterification,
the first step was the attack of the carboxyl group toward the
hydroxyl oxygen of Ser105, forming the first tetrahedral
intermediate (Figure S4).28 Interestingly, the generated MOI
would become a competitive substrate for IA, further affecting
the composition of the final product. We investigated the
movement of IA and MOI in the cavity pocket, and the
enzymatic conformation was stable after 5000 ps (Figure S5),
shown as a convergent root mean square deviation (RMSD)
value.29 Figure 2a shows that IA was more likely to form an
acyl complex with the hydroxyl oxygen of Ser105 (0.4 nm)
than MOI (1.0 nm). Furthermore, the independent gradient
model (IGM)30 showed that IA (Figure 2b) had stronger
nonbonded interactions (blue, H bonds) with Thr40, Ser105,
Gln106, Gln157, and Ile189, while MOI only had weak
nonbonded interactions (green vdW) with surrounding
residues (Figure 2c). Besides, the larger spatial size of MOI
blocks its approach toward Ser105. Finnveden et al. reported
similar observations in the esterase-catalyzed monosubstitution
of divinyl adipate (DVA),31 where the restricted space of
esterase blocked the generated monosubstituted products from
continuing to the next disubstituted reaction. Thus, IA was a
better competitor than MOI to bind the catalytic site and
proceed with the esterification process, matching the
phenomenon of MOI high accumulation in the experiment.
Computational Assay of the Effect of Octanol on
Cascade Esterification. The distribution of IA/MOI around
CALB lipase under more and fewer octanol molecules was
researched. The trajectory of RMSD in this conformation
remained steady after 5000 ps, indicating the convergence of
MD simulation results (Figure S6). With more octanol
molecules (1000 octanol molecules), the g(r) distribution of
majority IA was concentrated in 2 nm (Figure 3a), while that
of MOI appeared in 2.5 nm; as a result, IA was more likely to
enter the cavity pocket than MOI to react. However, with
fewer octanol molecules (100), IA and MOI were almost
distributed in the same place, where they had a similar
possibility to approach the cavity pocket (Figure 3b). The
distribution of competitive substrates around the lipase further
affects their reaction rate differently, agreeing with results in
Figure 1d. The enhanced diffusion of substances from the
lipase surface was also reported by Cao et al.,29 where glucose
could improve diffusion of harmful methanol molecules away
from lipases and yield a protective effect. In this work, excess
octanol (log P = 2.64) created a hydrophobic environment to
influence the microscopic distribution of IA (log P = −0.26)
and MOI (log P = 2.91) around lipases. Octanol not only
affects the reaction kinetics as the substrate but also acts as a
solvent to adjust the catalytic microenvironment of lipases.
Influence of Solvents. Inspired by octanol’s role as a
solvent, a variety of solvents with log P ranging from −1.3 to 4
were investigated in esterification of MOI. Application of
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Figure 2. Distribution of IA/MOI in the cavity pocket. (a) MD results of the distance between IA/MOI and hydroxyl oxygen of Ser105 (IA, blue;
MOI, red), (b) IGM analysis of IA, and (c) IGM analysis of MOI. (IGM, blue, represents strong nonbonded interactions, such as H bonds and
halogen bonds; green represents middle strong nonbonded interactions, such as van der Waals (vdW) interactions; and red represents steric effects,
such as ring tension and steric hindrance.)
Figure 3. g(r) Distribution of IA/MOI around the enzyme. MD results of the distance between IA/MOI and the centroid of lipase with (a) 1000
octanol molecules (IA, blue; MOI, red) and (b) 100 octanol molecules (IA, blue; MOI, red).
organic solvents in the lipase-catalyzed reaction has been
widely researched, which could improve the solubility of
hydrophobic substrates, help the recovery and reusability of
lipases, favor the synthesis over hydrolysis, improve the mass
transfer, and alter the regioselectivity of lipases.32,33 However,
solvents also affect the enzymatic activity by altering the critical
water layer and the protein structure, especially the cavity
pocket and active sites.33 Figure 4 shows that toluene (TOL,
log P = 2.68) contributed to the highest yield of 99% MOI and
the optimal toluene/octanol ratio was 1:1 (v/v) (Figure S7).
To make the process more economical, recyclability under the
optimal condition was investigated, where lipase can be reused
for 16 batches with over 90% yield of MOI (Figure S8). TOL
exerted a profound effect on production of MOI by increasing
the yield and the MER.
Mechanism of the CALB Regioselectivity on 4-OI in
Silico. This route showed a good performance in a 5 g scale-up
reaction with over 98% yield, and MOI with 94% purity can be
1
obtained (Table S1) through a series of separation. H NMR
(Figure S10), 13C NMR (Figure S11), and heteronuclear
multiple bond correlation (HMBC; Figures S2 and S12)
revealed that our product was 4-OI with over 99% selectivity
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right side, where the CC rigid bond was placed in a formed
cleft (red arrow), and leave a channel for the attack of octanol.
In the C4_Ser 105 complex, the distance between octanol and
Ser105 was 3.4 Å, meeting the necessary distance requirement
of 4 Å for a nucleophilic attack.34 The hydrogen bond with
Thr40 and hydrophobic interactions with surrounding
residues, especially Ile189, Val190, Thr138, and Gln157, help
to stabilize this conformation (Figure 5b). However, the
formation of the C1_Ser 105 complex blocks the attack of
octanol (Figure 5c) in the cavity pocket, yielding a 7 Å
distance between the hydroxyl oxygen of octanol and Ser105
(interactions with surrounding residues are shown in Figure
5d). Ferrari et al. had similar findings where the catalytic cavity
shape of CALB affected its selectivity in the peptide acylation
reactions, especially its tightness and depth.35
Furthermore, GFN-FF MD was used to analyze the C4/C1
distribution in the active site. In this stable conformation after
40 ps (Figure S9), the C4 carboxyl group of IA was “closer”
than the C1 carboxyl group to the hydroxyl oxygen of Ser105
(Figure 5f), indicating that C4 was more likely to interact with
the Ser105 residue. Figure 5e shows that C4 had stronger
nonbonded interactions (blue, H bonds) with Gly39, Gln106,
and Ser105, while C1 only had middle nonbonded interactions
with Val154 and Leu140 (green, vdW). Besides, the CC
bond could produce some vdW interactions with Thr40 and
Ile238 to stabilize this conformation. Li et al. used a similar
IGM to analyze the enzyme−peptide interaction, where H
bonds and hydrophobic and vdW interactions contribute to
the stable enzyme−peptide conformation.36 Then, the C4
carboxyl group of IA is more likely to form an acyl complex
Figure 4. Effect of organic solvent on the yield of MOI and MER
(solvent/octanol = 1:1 v/v). All reactions were performed using 0.05
g of Novozym 435, concentration of IA is 0.32 mol·L−1, and
concentration of 1-octanol is 3.2 mol·L−1 (octanol group,
concentration of 1-octanol is 6.4 mol·L−1) at 50 °C, 800 rpm, and
24 h.
(Figure S1). In this reaction, both C4 and C1 are esterified,
while esterification of C4 is dominant. The formation of the
IA_Ser105 acyl complex and the attack of octanol are
necessary phases in the esterification process (Figure S4).28
Molecular docking results (Figure 5a) showed that the
formation of the C4_Ser 105 complex could cling to the
Figure 5. Regioselectivity from the cavity pocket toward 4-OI. (a) Docking result of the C4_Ser 105 complex; (b) C4_Ser 105 complex binding
site, prepared with LigPlot +; (c) docking result of the C1_Ser 105 complex; (d) C1_Ser 105 complex binding site, prepared with LigPlot; (e)
independent gradient model (IGM) analysis (blue represents strong nonbonded interactions, such as H bonds and halogen bonds; green represents
middle strong nonbonded interactions, such as vdW interactions; red represents steric effects, such as ring tension and steric hindrance); and (f)
MD results of the distance between C1/C4 and hydroxyl oxygen of Ser105.
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with the Ser105 residue than the C1 carboxyl group and
proceed with further esterification.
(Figure 7d). The improvement effect of TOL on the MOI
selectivity was investigated on other diacids (Figure 8).
Interestingly, the side chain seems to be the key for the
specific cavity pocket of CALB and TOL to improve MER. In
the cascade esterification of succinic acid and 1-octanol, CALB
did not exhibit selectivity toward the monoester, and the MER
was lowered to less than 10% at 8 h. Besides, the addition of
TOL did not improve the MER and it even lowered MER to
almost 0 at 8 h (Figure 8b). However, for other diacids with
side chains (malic acid, 2-methylglutaric acid, 2-methylsuccinci
acid), CALB exhibited a higher MER at 8 h (87% malic acid
monoester, 62% 2-methylglutaric acid monoester, 90% 2-
methylsuccinci acid monoester). The addition of TOL further
improves the monoester process, yielding 96% malic acid
monoester at 8 h, 70% 2-methylglutaric acid monoester at 8 h,
and 92% 2-methylsuccinci acid monoester at 8 h (Figure
8a,c,d).
Reaction Dynamics. In this enzymatic synthesis process,
the solvent environment performed an important role. TOL
showed the best performance among solvents, improving the
yield of MOI from about 85 to 99% with IA/octanol = 1:10
(molar ratio) at 24 h (Figure 4). Interestingly, TOL does not
improve the production rate of all esterified products (VMOI
and VDOI) simultaneously. The addition of TOL could improve
VMOI by 44.84% and decrease VDOI to 56.87%, further
increasing the ratio between VMOI and VDOI from 88.43 to
225.20 (Table S2). Meanwhile, TOL could improve the Vmax
of the MOI production rate by 637.35% and reduce the Vmax of
the DOI production rate to 26.82% (Table S3). This opposite
effect of TOL on VMOI and VDOI might be due to the enhanced
diffusion of hydrophobic MOI from the cavity pocket to the
external hydrophobic environment by TOL, matching the
previous MD result in Figure 3a.
To further verify this hypothesis, we investigated the effect
of mixed solvents with various log P values (Table S4) on 4-OI
production (Figure 6). In a high log P environment (>2), MOI
CONCLUSIONS
■
The present research demonstrated a one-step and green
synthesis route of 4-octyl itaconate by the structure control of
the lipase, yielding outstanding performances in yield and
selectivity. The special cavity of CALB and the shrinkage effect
of the solvent exhibited a nearly 100% direct conversion of 4-
OI from IA and octanol, meeting the principles of green
engineering in reducing operating steps and saving energy
consumption. Under the optimal condition, 4-OI with over
98% yield and over 99% selectivity was obtained. Lipases can
be reused for 16 batches with over 90% yield of 4-OI. A
multiscale molecular simulation analysis was used to reveal the
catalytic mechanism. First, the cavity pocket of CALB resulted
in a 4-OI selectivity by affecting the acyl−enzyme complex
conformation and microdistribution of C1/C4 and IA/MOI.
Second, TOL could improve the 4-OI selectivity by affecting
the substrate distribution of IA/MOI around the enzyme
surface and performing a shrinkage effect. This strategy also
applies to monoesterification of other diacids with side chains.
EXPERIMENTAL SECTION
■
Figure 6. Effect of mixed solvent on the yield of MOI and MER
(mixed solvent/octanol = 1:1 v/v). All reactions were performed
using 0.05 g of Novozym 435, concentration of IA is 0.32 mol·L−1,
and concentration of 1-octanol is 3.2 mol·L−1 at 50 °C, 800 rpm, and
24 h.
Materials. IA (99%) was obtained from TCI. 1-Octanol (99%)
was obtained from Sigma-Aldrich. 4-Octyl itaconate (98%) standard
was purchased from Ark. Dimethylsulfoxide (DMSO), acetone,
butanone, tetrahydrofuran (THF), dichloromethane (DCM), trihalo-
methanes (THMS), benzene, toluene (TOL), octanol, carbon
tetrachloride (CTC), cyclohexane (CYH), n-hexane, and heptane
were of reagent grade. Novozym 435 (Candida antarctica lipase B
immobilized on macroporous acrylic resin, CALB), Lipozyme TL IM
(lipase from Thermomyces lanuginosus, immobilized on silica), and
Novozym 40086 (lipase from Rhizomucor miehei, immobilized on
acrylic resin) were purchased from Novozymes, Beijing, China.
Porcine pancreas lipase (PPL) powder was purchased from Sigma Life
Science, and Candida sp. 99-125 lipase powder was purchased from
Beijing CAT New Century Biotechnology Co., Ltd.
yield had a similar performance with over 90% yield, while the
MER was reduced. TOL shows a better performance than the
mixed solvent with similar log P values, although they have the
same effect on adjusting microdistribution of substances. TOL
may change the enzymatic conformation, especially the key
residues, to improve both the catalytic activity and selectivity.
Computational Analysis of TOL on the Cavity Pocket.
The computational results showed that TOL performed a
structure control effect of the lipase. The cavity pocket of
CALB was separated into two channels (Figure 7a) by two
“saddle” residues of ILE189 and ILE285. However, the two
channels were reduced to one in the TOL environment
(Figure 7b). TOL has a shrinkage effect on the left channel by
closing the distance between LEU144 and VAL286 residues
(Figure 7d), while it does not affect the original saddle residues
(Figure 7c) and the right channel. Thus, TOL could improve
the selectivity of CALB toward 4-OI by shrinking the cavity
Esterification of Itaconic Acid. IA (0.769 mmol) and 1-octanol
(0.769−19.225 mmol) were added into a 4 mL brown glass bottle. In
experimental groups containing organic solvents, various solvents (1.2
mL) were added to this bottle. The mixture was then preheated at 50
°C for 10 min. Catalysts (0.01−0.1 g of lipases or 0.5 wt % sulfuric
acid) were added and the reaction was conducted in a thermostatic
bath at 50 °C, 800 rpm, and 24−72 h. Samples (20 μL) were removed
for gas chromatography (GC) analysis at various times after adding 1
mL of n-hexane and centrifuging at 8000 rpm for 3 min. All reactions
were conducted at least in triplicate.
Scale-Up Experiment and Separation of Mono-octyl
Itaconate (MOI). The scale-up experiment was conducted in a 250
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Figure 7. Assay of the solvent effect on the cavity pocket. (a) Cavity pocket without TOL, (b) cavity pocket with TOL, (c) distance between
ILE189 and ILE285, and (d) distance between LEU144 and VAL286.
Figure 8. Application on other diacids. (a) malic acid, (b) succinic acid, (c) 2-methylglutaric acid, and (d) 2-methylsuccinci acid. All reactions were
performed using 0.05 g of Novozym 435 at 50 °C, 800 rpm, and 24 h. The concentration of diacids and 1-octanol is 0.64 and 6.4 mol·L−1,
respectively, in the solvent-free system; the concentration of diacids and 1-octanol is 0.32 and 3.2 mol·L−1, respectively, in the solvent system.
mL three-necked, round-bottom flask, with 2.5 g of Novozym 435, 5 g
of IA (38.5 mmol), 50 g of 1-octanol (385 mmol), and 60 mL of TOL
at 50 °C, 250 rpm, and 24 h. The reaction mixture was filtered to
separate lipases. A 1:1 saturated NaCl salt solution (V/V) was added
to remove the residual unreacted IA. The supernatant was collected
and organic solvents were removed under vacuum. The residue
containing only mono-octyl itaconate (MOI) and 1-octanol was
separated by short-path distillation37 under the following conditions:
TEvaporation wall (°C) = 30, Tcooling wall (°C) = 2, scraper speed (rpm) =
250, and pressure (Pa) = 0.1. 1-Octanol was removed as the light
phase and a white solid product was obtained.
GC Analysis. A GC-2010 Plus (Shimadzu) gas chromatograph
with an FID detector and a DB-1 chromatographic column (30 m ×
0.25 mm, 0.1 μm, Agilent) was used to analyze samples. The
temperatures of the injection port and the FID detector were set as
360 and 380 °C, respectively. The temperature programming of the
column was set at 100 °C for 0.2 min; it was first increased to 165 °C
at 8 °C·min−1 and maintained for 1 min and then increased to 340 °C
at 20 °C·min−1 and maintained for 1.92 min.17 Data including the
yield of MOI and MER are calculated as wt %. MOI yield refers to the
percentage of MOI among all reaction substances (IA, MOI, and
DOI), while MER yield refers to the percentage of MOI between IA
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esters (MOI and DOI). All of the data were the averages of triplicate
experiments.
orcid.org/0000-0001-8792-529X; Email: dengli@
mail.buct.edu.cn
NMR Identification. The structure of the purified product was
1
analyzed using H NMR and 13C NMR spectra and HMBC with a
Authors
CDCl3 solvent. All nuclear magnetic resonance (NMR) spectra were
recorded on an Avance 600 spectrometer (600.13 MHz) equipped
with a BBI probe head with a z-gradient from Brucker Biospin GmbH.
All NMR data were processed and analyzed with MestReNova. Full
1H and 13C assignments were obtained at 25 °C from standard 1D
experiments, as well as a two-dimensional (2D) correction experiment
Changsheng Liu − Beijing Bioprocess Key Laboratory and
State Key Laboratory of Chemical Resource Engineering,
College of Life Science and Technology, Beijing University of
Chemical Technology (BUCT), Beijing 100029, P. R. China
Yilin Wang − Beijing Bioprocess Key Laboratory and State
Key Laboratory of Chemical Resource Engineering, College of
Life Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
Jiahao Liu − Beijing Bioprocess Key Laboratory and State Key
Laboratory of Chemical Resource Engineering, College of Life
Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
An’nan Chen − Beijing Bioprocess Key Laboratory and State
Key Laboratory of Chemical Resource Engineering, College of
Life Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
Juntao Xu − Beijing Bioprocess Key Laboratory and State Key
Laboratory of Chemical Resource Engineering, College of Life
Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
Renwei Zhang − Beijing Bioprocess Key Laboratory and State
Key Laboratory of Chemical Resource Engineering, College of
Life Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
Fang Wang − Beijing Bioprocess Key Laboratory and State
Key Laboratory of Chemical Resource Engineering, College of
Life Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China
1
(1H, 13C HMBC). The H and 13C chemical shifts were referenced
against TMS. Chemical shifts for 1H and 13C NMR spectra are
reported in ppm (δ) relative to the residue protium in the solvent
(CDCl3: δ 7.26, 77.0 ppm, and the multiplicities are presented as
follows: s = singlet, d = doublet, t = triplet, q quartet, m = multiplet,
brs = broad single).
1H NMR (CDCl3, 600 MHz) δ 6.46 (s, 1H), 5.83 (s, 1H), 4.10 (t,
2H, J = 6.7 Hz), 3.34 (s, 2H), 1.64−1.60 (m, 2H), 1.35−1.23 (m,
10H), 0.88 (t, 3 H, J = 6.9 Hz).
13C{1H} NMR (CDCl3,150 MHz) δ 171.3, 170.6, 133.4, 130.6,
65.3, 37.3, 31.8, 29.2, 29.1, 28.5, 25.8, 22.6, 14.1.
1H NMR and 13C NMR data were identical to the data in the
literature.5
Based on NMR spectroscopy, our product was 4-OI, rather than 1-
OI, which was consistent with previous reports.4 14 4-OI had chemical
,
1
shifts in 6.46 and 5.83 ppm at H NMR, while 1-OI had chemical
shifts in 6.36 and 5.74 ppm14 at 1H NMR (Figure S1). Our products
showed over 99% selectivity of 4-OI, and the 1% chemical shifts might
be caused by DOI (Figure S1). To further identify the structure of our
product, HMBC (1H detected heteronuclear multiple bond
correlation) was conducted. The H5-C4 and H3-C4 interactions
further revealed that our product was 4-OI (Figure S2).
Assay of 4-OI Selectivity. The cavity pocket of CLAB lipase
(pdb: 5GV5) and the microenvironment jointly contributed to the
high 4-OI selectivity. A multiscale molecular simulation analysis from
perspectives of the acyl−enzyme complex structure, molecular
dynamics (MD), IA/MOI distribution, and channel change was
conducted to investigate their influence (shown in the SI).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02995
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02995.
Notes
■
sı
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Detailed experimental methods, NMR data, separation
data, and MD data; generation rate measurement;
preparation of mixed solvents; multiscale molecular
simulation analysis; IA/MOI distribution on the cavity
pocket; catalytic mechanism of lipase (N435); effect of
TOL dosage on the yield of MOI and selectivity;
composition of the reaction liquid in each step; and the
production rate of MOI/DOI in the solvent-free/solvent
system (PDF)
This study was funded by the National Key Research and
Development Program of China (2017YFB0306904) and the
National Natural Science Foundation of China (Grant nos.
21978017, 21978019, and 21978020).
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AUTHOR INFORMATION
Corresponding Authors
■
Kaili Nie − Beijing Bioprocess Key Laboratory and State Key
Laboratory of Chemical Resource Engineering, College of Life
Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China;
orcid.org/0000-0002-0613-9850; Phone: +86-010-
64414543; Email: niekl@mail.buct.edu.cn; Fax: +86-010-
64416428
Li Deng − Beijing Bioprocess Key Laboratory and State Key
Laboratory of Chemical Resource Engineering, College of Life
Science and Technology, Beijing University of Chemical
Technology (BUCT), Beijing 100029, P. R. China;
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