Lipase-catalyzed chemoselective ester hydrolysis of biomimetically coupled aryls for the synthesis of unsymmetric biphenyl esters

Article abstract of DOI:10.3390/molecules24234272

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and s

Full text of DOI:10.3390/molecules24234272

molecules
Article
Lipase-Catalyzed Chemoselective Ester Hydrolysis of
Biomimetically Coupled Aryls for the Synthesis of
Unsymmetric Biphenyl Esters
Janna Ehlert 1,2 , Jenny Kronemann 1,2, Nadine Zumbrägel 1,2 and Matthias Preller 1,2,
*
1
Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany;
Ehlert.Janna@mh-hannover.de (J.E.); jenny.kronemann@gmx.de (J.K.); nadine.zumbraegel@web.de (N.Z.)
Centre of Biomolecular Drug Research (BMWZ), Leibniz University Hannover, 30167 Hannover, Germany
Correspondence: preller.matthias@mh-hannover.de
2
*
Academic Editor: Marco Filice
Received: 30 October 2019; Accepted: 21 November 2019; Published: 23 November 2019
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Lipases are among the most frequently used biocatalysts in organic synthesis, allowing
numerous environmentally friendly and inexpensive chemical transformations. Here, we present a
biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis
using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions.
The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the
potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic
activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time,
temperature, pH, organic solvent, and water–organic solvent ratios, to improve the chemoselectivity,
and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and
increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida
rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas
fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize
the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction
conditions were transferred to the preparative scale with high yields, underlining the potential
of the presented biomimetic approach as an alternative strategy to the commonly used transition
metal-based strategies for the synthesis of diverse biphenyl esters.
Keywords: lipases; biphenyl esters; biocatalysis; chemoselectivity; biomimetic synthesis; organic
synthesis
1
. Introduction
Selective transformation of one specific functional group in a molecule, containing multiple groups
of similar reactivity is of the utmost relevance for targeted synthesis of molecules with pharmaceutical
activities, as well as materials with well-defined properties for technical applications. Particularly,
ester hydrolysis is one of the most fundamental chemical transformations. Successful strategies
for the preferential monohydrolysis of a number of different esters have been reported under often
strong basic conditions [1–3], however general applicability is limited by competing elimination
reactions under these conditions and chemoselectivity regarding a wide spectrum of multiester
compounds. No study has yet been reported for the selective transformation of biphenyl-based
esters using lipase biocatalysis. Biphenyl-based building blocks with ester functions represent an
important class of precursors that give rise to compounds with anti-infective, anti-inflammatory,
and anti-cancer potencies [4–10], as well as in technical applications, such as in solar cells, where
they are used as linkers for dyes [11] and hole transport materials [12], in metal organic frameworks
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for gas storage [13], and as catalysts [13
,
14] in lithium ion batteries [15] or rectification devices [16],
in nanocomposite thin films for use in organic light emitting diodes (OLED) [17], or even as material
in HPLC columns for the isolation of aromatic compounds [18]. Chemoselective hydrolysis and
subsequent functionalization of biphenyl ester-based compounds therefore allow the generation
of tailored molecules with increased potency and selectivity for their biological targets, improved
bioavailability (absorption-distribution-metabolism-excretion-toxicity, ADMET) profiles, or unique
magnetic or electronic properties.
Biocatalysis strategies using lipases have become an attractive tool for modern organic synthesis
in industry and academic research, due to the broad accepted substrate spectra and the usually mild
conditions that can be applied, such as low temperatures and neutral pH values [19
use reduces the risk of side reactions considering the high chemo-, regio-, and stereoselectivity of
lipases [22 23]. Given their tolerance for organic solvents, varying the ratios of water and organic
–21]. Their synthetic
,
solvent mixtures provides a means to control enzymatic reaction and selectivities. In addition, it offers
the advantage to even convert small molecules with low solubilities in aqueous medium, in order to
biocatalytically obtain the desired target products. Lipases catalyze a plethora of different reactions,
including hydrolysis, esterification, transesterification, and alcoholysis [24], the major physiological
function of lipases however is the hydrolytic cleavage of triacylglycerides for energy production, and
their ubiquitous expression and functions in diverse cellular compartments, which facilitate enzyme
activity under a variety of conditions [19
–21]. Industrial applications of lipases include the generation
of important pharmaceuticals through transesterifications and ester cleavage [19
,
25–27]. They are
additionally used for biodiesel production in food and flavor industry as well as waste water treatment
and generation of agrochemicals [24]. In organic synthesis, lipases are often used for desymmetrization
and dynamic kinetic resolution towards enantiopure compounds [28
,29]. Furthermore, lipases have
been shown to biocatalytically convert benzylic esters [30 31]. However, the conversion of symmetric
,
biphenyl-based ester building blocks to unsymmetric esters has not yet been investigated.
Here, we present the combination of eco- and cost-friendly biomimetic oxidative phenol coupling
with lipase-catalyzed monohydrolysis of biphenyl esters for the targeted synthesis of unsymmetric
biphenyl-based ester building blocks. We systematically analyzed the catalytic activities of a series of
lipases from different organisms for their potency to hydrolyze biphenyl esters. We are investigating
particularly the effect of the reaction conditions, in terms of water–organic solvent ratios, pH, type
of solvents, and reaction time, on the efficacy and chemoselectivity of the lipases, as well as their
accepted substrate spectrum. Accompanying molecular docking experiments helped to rationalize
the observed catalytic activities and chemoselectivities. Our study provides a biomimetic strategy
using lipase catalysis for the controlled synthesis of unsymmetric biphenyl ester building blocks under
mild conditions.
2
. Results
2
.1. Biomimetic Synthesis of Biphenyl Ester Building Blocks
Biphenyl esters have been reported as important building blocks for the development of
pharmaceuticals in diverse indication areas, and for use in a wide range of technical applications [4–18].
In order to allow an inexpensive, environmentally friendly, and efficient access to biphenyl
ester core moieties, we adopted a biomimetic iron(III)-catalyzed, solid-phase coupling reaction.
In nature, this type of chemical reaction is usually used by plants to metalloenzymatically produce
specific metabolites required for plant growth and protection [32
coupling of aryls and phenols have been reported for the synthesis of natural products under mild
conditions [35 36]. We obtained C–C bond formation through oxidative coupling of commercially
available 4-hydroxyphenyl acetate ( ) using iron(III) chloride on silica with 60% yield of biphenyl ester
(Scheme 1). This reaction builds mechanistically on mesomeric effects due to the ortho substitution
with the electron donating hydroxyl group. Iron(III) chloride acts as the catalyst and is reduced to
–34]. Fe(III)-catalyzed oxidative
,
1
2

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allow oxidative phenol coupling (Scheme S1). In a second step, the purified biphenyl ester
2 was
converted to biphenyl ester
3 through methylation of the free hydroxyl groups at the biphenyl scaffold
using methyl iodide with 90% yield.
Scheme 1. Biomimetic iron(III)-catalyzed synthesis route towards symmetric biphenyl esters 2 and 3.
Subsequently, the biphenyl ester building blocks
hydrolysis to yield either the unsymmetric acids and 5, or the symmetric acids
Scheme 2). To gain control over the obtained products, we tested a series of lipases, and systematically
2
and
3
were subjected to a lipase-catalyzed ester
4
6
and , respectively
7
(
varied the reaction conditions and reaction time. As a control, we synthesized biphenyl ester building
blocks without substituents at the ortho position of the biphenyl moiety, in order to examine the
accessible substrate spectrum of the lipases for differently substituted biphenyl ester building blocks.
Scheme 2. Schematic representation of lipase catalyzed ester hydrolysis of biphenyl esters. Depending
on the chemoselectivity of the lipases, biphenyl esters 2 and 3 were converted to either unsymmetric
acids 4 and 5 or symmetric acids 6 and 7, respectively.
2
.2. Chemical Synthesis of Biphenyl Ester Building Blocks without Ortho Substituents
To examine the substrate spectrum and the impact of the ortho substitution at the biphenyl core
moiety on the hydrolysis by lipases, we synthesized biphenyl esters without ortho substituents at the
core moiety. Since in the absence of ortho substitutions the iron(III)-catalyzed oxidative phenol coupling
is not applicable for the synthesis of biphenyl ester 10, we used a palladium-catalyzed Suzuki–Miyaura
cross-coupling reaction of the aryl bromide (
9) and an in situ generated phenyl boronic acid using
the XPhos-Pd-G2 catalyst and XPhos as the ligand (Scheme 3). Methyl 2-(3-bromophenyl)acetate (
9)
was synthesized from the commercially available 3-bromo phenyl acetic acid (8) by methylation using
methyl iodide with quantitative yields.

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Scheme 3. Synthesis of the biphenyl ester building block without ortho substitutions at the core moiety
10) through methylation of 3-bromo phenyl acetic acid ( ), followed by Suzuki–Miyaura cross coupling.
(
8
Lipase-catalyzed ester hydrolysis of the symmetric biphenyl ester 10 resulted exclusively in ester
cleavage of all available ester groups in the molecule and the formation of the symmetric biphenyl-based
carboxylic acid 11 (Scheme 4).
Scheme 4. Biocatalytic ester cleavage of the symmetric compound 10 using lipases gave exclusively
the symmetric acid 11. The unsymmetric product was intermediately detected only in traces.
2
.3. Activity and Chemoselectivity of Lipases on Biphenyl Ester Hydrolysis
In our attempt to optimize the biocatalytic ester hydrolysis of biphenyl-based esters, we initially
tested the hydrolysis activity of a series of nine lipases from different organisms (Table 1) on the
conversion of biphenyl esters and using a colorimetric assay. Bromothymol blue (BTB) served as
2
3
the pH indicator to determine the formation of free carboxylic acids, indicated by a color change from
blue to yellow (Figure S1). The enzymatic reactions were performed in 96-well format in phosphate
buffer, pH 7.4, and 10% DMSO (v/v) at room temperature. As a positive control, glyceryl tributyrate
(
TAG (13)) was hydrolyzed by all lipases. At the given conditions, Candida antarctica Lipase B (CALB)
was found to be able to cleave the biphenyl esters and to release the corresponding carboxylic acids
Table 1). Exchanging the organic solvent DMSO with either water-insoluble toluene or water-soluble
2
3
(
acetonitrile, we did not detect any acid formation even for CALB (Figure S1).
Table 1. Results of the colorimetric activity assay of nine lipases and their activity to hydrolyze the ester
groups in compounds 13, 2, and 3. +: color change→hydrolysis, –: no color change→no hydrolysis.
TAG
Lipase
Biphenyl Ester 2
Biphenyl Ester 3
1
3
ANL
CALB
CRL
MML
ROL
RNL
PPL
+
–
+
–
–
–
–
–
–
–
–
+
–
–
–
–
–
–
–
+
+
+
+
+
+
+
+
PCL
PFL
Lipases: Aspergillus niger Lipase (ANL), Candida antarctica Lipase B (CALB), Candida rugosa Lipase (CRL), Mucor miehei
Lipase (MML), Rhizopus oryzae Lipase (ROL), Rhizopus niveus (RNL), Porcine pancreas Lipase (PPL), Pseudomonas
cepacia Lipase (PCL), Pseudomonas fluorescens Lipase (PFL).
To assess the impact of the reaction time on the enzymatic ester hydrolysis, we monitored the
formation of the hydrolysis products during lipase catalysis via analytical LC-MS. We found that

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hydrolysis of biphenyl ester
2
, containing free hydroxyl groups at the core moiety by CALB, gave a
mixture of unsymmetric and symmetric acids
the reaction time and the pH value (Figure 1, Figure S2). Longer reaction times led to a shift of the
product ratio towards the fully hydrolyzed, symmetric acid (Figure 1). The most chemoselective
conversion with CALB towards the unsymmetric acid was determined for a reaction time of 2 h at
pH 8.0, giving a product ratio of 20%:50%:30% ( ). In order to analyze the catalytic conversion
4 and 6 already after 1 h. The ratio was dependent on
6
4
2:4:6
of other biphenyl ester building blocks and examine the substrate spectrum of the lipase regarding
ortho substitutions at the biphenyl core moiety, we performed the same analytical LC-MS analysis
using biphenyl esters
CALB led to a favorable hydrolytic ester cleavage to the symmetric acids
examined pH values (Figures S3 and S4). However, at a reaction time of 2 h, the unsymmetric product
with methoxy groups at the ortho position of the biphenyl rings was clearly detectable (13%,) while
the major product was still the symmetric acid (42%) (Figure S3). Therefore, CALB shows sufficient
3
and 10. In both cases, biocatalytic hydrolysis under the same conditions using
7
(50–75%) and 14 (90%) at all
5
6
promiscuity to accept a series of ortho substituted biphenyl esters as substrates with time and pH
dependencies regarding the chemoselective hydrolysis of the two ester groups.
Figure 1. Time dependency of the chemoselective hydrolysis of biphenyl ester
2 with free hydroxyl
groups at the core moiety obtained by analytical LC-MS and comparison of the UV-signal ratios of
◦
biphenyl esters
0% DMSO (v/v) using CALB. (
The chemical structures, retention times, and m/z values for compounds 2, 4, and 6 are given.
2
(black),
4
(blue), and
6
(green). All reactions were performed at 25 C, pH 8.0, and
1
a
) Reaction time: 3 h; (
b
) reaction time: 2 h; ( ) reaction time: 1 h.
c
Motivated by the result that the unsymmetric acid 5 with methoxy groups was also detectable,
and previous studies, which reported a maximal catalytic activity of the applied lipases at elevated
temperature and organic solvent content [37], we varied systematically the temperature and reaction
time of the lipase-catalyzed hydrolysis of biphenyl ester 3 at an increased organic solvent content of

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4
0% DMSO (v/v) (Table S1). Unfortunately, CALB rapidly led to the unwanted symmetric product 6
as shown before. The four lipases Rhizopus niveus lipase (RNL), Mucor miehei lipase (MML), Candida
rugosa lipase (CRL), and Pseudomonas fluorescens lipase (PFL) were found to be active to hydrolyze
compound
Using analytical LC-MS, we found a critical dependency of the increased DMSO content, reaction
time, and temperature on the chemoselective conversion of compound to the unsymmetric product
. Increasing the DMSO concentration to 40% (v/v) led to a reduced reaction velocity of the lipase
3 in the colorimetric lipase activity assay in the presence of the elevated DMSO concentration.
3
5
biocatalysis, indicated by the small amounts of formed acids even after 24 h at temperatures of both
◦
2
5 and 30 C (<5% of compound
5). Elevated temperatures improved the catalytic activity of the four
lipases, with reactant (
3
):unsymmetric product (
5
) ratios for the four lipases CRL, MML, RNL, and
◦
PFL of 6.7:1 ( : 13%), 3.6:1 (
5
5: 22%), 6:1 (
5: 14%), and 3.5:1 (
5
: 22%), respectively after 48 h at 37 C.
All four lipases exhibited a high chemoselectivity towards the formation of the unsymmetric acid 5,
without detectable cleavage of the second ester group (i.e., the formation of the symmetric acid
presence of 40% DMSO (v/v) after 48h at 37 C and pH 8.0) (Figure 2).
7 in the
◦
Figure 2. Chemoselective hydrolysis of the biphenyl ester
to the unsymmetric product by four lipases at increased DMSO concentration, temperature, and
reaction time, obtained by analytical LC-MS from the UV-signal ratios of compounds (black), (blue),
(green). The reaction time for all experiments was 48 h at 37 C, pH 8.0, and 40% DMSO (v/v).
Chemical structures, retention times, and m/z values of 3, 5, and 7 are given. ( ) Biocatalysis by PFL;
b) biocatalysis by MML; (c) biocatalysis by RNL; (d) biocatalysis by CRL.
3 with methoxy groups at the core moiety
5
3
5
◦
and
7
a
(
2
.4. Molecular Docking Experiments
Although various studies showed the promiscuity of lipases to allow broad substrate spectra, our
results clearly revealed that the catalytic activity and chemoselectivity of the studied lipases can be
controlled and strictly depend on the reaction conditions, particularly the pH values, the amount of
organic solvent, as well as the reaction time and temperature. The optimal reaction conditions differed

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slightly for the different lipases, but eventually led to highly selective monohydrolysis of compounds
with several ester functions and the synthesis of unsymmetric biphenyl-based ester building blocks.
These four identified lipases showed differing efficacies in converting the reactant
unsymmetric product (Figure 2). To rationalize the different efficacies of the four lipases, we performed
complementing in silico binding predictions of the biphenyl ester reactant to the four lipases CRL,
3 to the
5
3
RNL, MML, and PFL using molecular docking. Molecular docking provides structural insights into
the binding of the small molecule biphenyl esters to the lipase enzymes—in terms of their predicted
binding poses and binding affinities—which is a prerequisite for the biocatalytic conversion of the
compounds. The active site of lipases contains a highly conserved catalytic triad, consisting of a serine,
a histidine, and a negatively charged glutamate or aspartate residue [38
predicted to bind in the active site of all four lipases, in close proximity to the catalytic triad, however
with slightly different binding poses and positions (Figure 3). The ester group of in the active site
,39]. The biphenyl ester 3 was
3
shows larger variations in its orientation for the binding to CRL and RNL, while for the binding to
MML and PFL, this ester group remains mostly bound to the catalytically active serine. In accordance
with the different poses, the computed binding affinities to the four lipases varied between
−
3.23
mol (Table 2). These predicted binding affinities correlate well with the observed
biocatalytic efficacies of the lipases to convert reactant 3 to the unsymmetric biphenyl ester 5.
−
1
and
−
6.79 kcal
·
Figure 3. Predicted binding poses of biphenyl ester
3
(grey) to the active sites of the four lipases
) PFL (blue) using molecular docking.
(
a
) CRL (green), ( ) RNL (magenta), ( ) MML (cyan), and (d
b
c
Two representative conformations of the largest cluster of docking solutions are shown. The serine,
histidine, and glutamate/aspartate residues of the catalytic triad are show in sticks representation
(yellow). Hydrogen-bonding interactions are shown as dotted lines. The variation in the orientation
of the ester group in the active site is indicated by the transparent surface, as obtained from all
conformations within the cluster.

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Table 2. In silico predicted free binding energies of biphenyl ester 3 to lipases CRL, RNL, MML, and
0
PFL. ∆G values were computed with AutodockTools.
Predicted Affinity of 3 to Lipase
CRL
−3.23
RNL
−5.64
MML
PFL
0
−1
)
∆
G (kcal · mol
−6.66
−6.79
2
.5. Lipase-Catalyzed Biphenyl Ester Conversion in Preparative Scale
After successfully establishing the chemoselective hydrolysis of biphenyl esters using lipase
biocatalysis, we demonstrated the enzyme-catalyzed reaction of and at a preparative scale to be
able to isolate the unsymmetric acids and for subsequent benzylation of the free carboxylic acid
2
3
4
5
with benzyl bromide to yield the unsymmetric biphenyl esters 14 and 15 containing both a methyl
ester and a benzylic ester group (Scheme 5).
MeO
O
OMe
O
MeO
O
OH
O
MeO
O
OBn
O
Lipase
BnBr, CH CN
3
8
4 °C, 2 h
OR OR
OR OR
OR OR
2
3
: R=H
: R=Me
4: R=H
5: R=Me
14: R=H
15: R=Me
Scheme 5. Two-step strategy for the lipase-catalyzed conversion of the symmetric ester reactants
◦
to unsymmetric biphenyl esters. Conditions for the biocatalytic conversion of 2: CALB, 25 C, 2 h.
Conditions for the biocatalytic conversion of 3: PFL, 37 C, 48 h.
◦
We isolated the highly pure unsymmetric acid
biphenyl ester products 14 and 15 without purification of the intermediates gave yields of 20% and
2%, respectively. However, during the two-step synthesis of 14, a byproduct (16) was formed (40%,
5 with 41% yield. Synthesis of the unsymmetric
1
Scheme S2), which showed benzylated phenolic hydroxyl groups. Unsatisfied by the moderate yields,
we repeated the two-step reaction, while isolating the intermediate acid, and we obtained yields of
8
1% for the successful synthesis of unsymmetric biphenyl ester 15 (35% o2s).
3
. Discussion
Lipases are versatile biocatalysts for numerous targeted chemical transformations and widely used
tools in organic synthesis, however their efficiency and selectivity to hydrolyze esters with biphenyl core
moieties has not been examined so far. Here we established a biomimetic strategy for the chemoselective
synthesis of unsymmetric biphenyl-based ester building blocks by combining iron(III)-catalyzed
oxidative coupling reactions to construct the biphenyl core moiety, with selective monohydrolysis of
the biphenyl diesters using lipases. This synthetic route offers a simple, environmentally friendly, and
inexpensive access to unsymmetric biphenyl ester building blocks under mild conditions and can be
used for the synthesis of a wide range of biphenyl esters with broad technical and pharmaceutical
applications. We systematically addressed the impact of the reaction conditions on the catalytic
activity and chemoselectivity of the lipases, particularly the effect of the reaction time and temperature,
as well as the pH, water–organic solvent ratio, and type of organic solvent.
The bioinspired oxidative phenol coupling using the cheap and non-toxic iron(III) catalyst
FeCl3 6 H O offered us an efficient solid-phase homocoupling of phenol esters in one step to produce
·
2
symmetric biphenyl-based methylesters in high yields. In the second step, we used lipase biocatalysis
to chemoselectively hydrolyze one of the two equivalent ester groups. For that purpose, we profiled
a series of nine lipases from different organisms, covering bacteria, fungi, and vertebrates, for their
efficacy to catalyze the hydrolytic ester cleavage of the biphenyl esters. Five of the lipases were found
to be able to cleave the biphenyl esters under different reaction conditions with a marked dependency

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of the enzymatic activity and particularly the chemoselectivity on the reaction time, temperature,
pH value, and organic solvent content. Our results are in good agreement with previous studies,
which reported that the activity of some of the lipases was critically dependent on the temperature and
DMSO concentration [37].
The identified active lipases CALB, CRL, RNL, MML, and PFL, capable of hydrolyzing biphenyl
esters, showed a general acceptance of ortho substitutions at the biphenyl core moiety and hence,
an advantageous level of promiscuity, allowing therefore their manifold use for biocatalytic conversions
of a broad spectrum of biphenyl ester building blocks. For the biphenyl ester
2 with free hydroxyl
groups in the ortho position of the core moiety, we found optimal reaction conditions that favor the
formation of the unsymmetric product
at a moderate pH of 8, 25 C, and 10% DMSO (v/v) after a reaction time of 2 h. Biphenyl
groups at the biphenyl moiety was quickly converted to the symmetric product
4
by a factor of two over the symmetric product
6
using CALB
with methoxy
7 under the same
◦
3
conditions. However, increasing the solvent content to 40% DMSO (v/v) and elevating the reaction
temperature critically affected the activity and particularly the chemoselectivity of the four lipases CRL,
MML, RNL, and PFL to efficiently and almost exclusively produce the unsymmetric biphenyl ester 5
by monohydrolysis of solely one ester group in the biphenyl diester. The chemoselective hydrolysis
of only one ester group in the molecule was independent of the reaction time, and the overall yields
increased steadily up to 48 h. The highest yields of unsymmetric biphenyl ester were obtained with
lipases MML and PFL. The hydrolysis efficacies of the four lipases correlated well with predicted
binding affinities by complementing computational docking experiments. Lipases such as PFL and
MML featured favorable binding poses in proximity to the catalytic triad in the active site, and hence
higher binding affinities of the biphenyl esters.
We were able to successfully transfer the optimized reaction conditions to a preparative scale and
isolate the unsymmetric esters
4 and 5. Interestingly, the yields of the purified unsymmetric products
were significantly higher than the yields obtained at an analytical scale, underpinning the potential of
using lipases as biocatalysts for the selective monohydrolysis of biphenyl esters.
Biphenyl esters have been shown to represent an interesting class of building blocks for the
development of pharmaceuticals and functionalized materials for use in a wide range of technical
applications. This approach has great potential for the chemoselective conversion of a single functional
group out of multiple groups with similar reactivities in a molecule, which can be challenging
through traditional synthetic approaches, and the selective synthesis of unsymmetric biphenyls.
Our biomimetic two-step strategy offers a series of advantages compared to other synthetic routes,
such as the Suzuki–Miyaura coupling, the most frequently used synthetic approach for the synthesis
of biphenyl core moieties, including no need for aryl halides, low cost, and mild reaction conditions,
whereas the usually basic conditions of the Suzuki–Miyaura coupling include degradation of reactants,
environmentally friendly, and no transesterifications by the solvent. In addition, the present study
opens a novel facet for the effective and selective total synthesis of natural products, such as
the pharmaceutically relevant ellagitannins, which possess biphenyl ester motifs connected to a
glucopyranose, and which exhibit anti-infective, anti-inflammatory, and anti-cancer activities.
Summarizing our studies, we were able to establish a selective and efficient biomimetic strategy
towards unsymmetric biphenyl esters by combining iron-catalyzed phenol coupling and lipase
biocatalysis for the selective ester monohydrolysis under mild conditions. Varying critical reaction
parameters, including time, pH, temperature, and solvent, control the activity and chemoselectivity of
the enzymatic ester hydrolysis reaction.
4
. Materials and Methods
4
.1. Chemical Methods—General Information
Reactions with air- or moisture-sensitive reactants were performed under oxygen-free and dry
inert atmosphere (nitrogen or argon). Chemicals were purchased from ABCR, Acros, AppliChem,

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AlfaAesar, Carl Roth, Fluka, Fluorochem, Sigma Aldrich, Merck and TCI and used without further
purification. Dry solvents were freshly distilled or directly purchased from commercial suppliers and
if not otherwise stated directly used without further purification. Silica gel column chromatography
was performed under pressure with silica (60 Å, size 35–70 µM) purchased from Merck. TLC on silica
gel was performed with silica coated glass slides from Merck (spot detection with UV or staining
potassium-permanganate, ninhydrine or bromocresol green). Nuclear magnetic resonance spectra
were recorded with a Bruker DPX-400 spectrometer. 13C NMR spectra were measured without H
coupling. Chemical shifts were reported in ppm and calibrated to residual proton signal of deuterated
1
solvents (CDCl3:
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. C NMR spectra
were reported as values relative to the residual solvent signal (CDCl3: = 77.16 ppm, CD OD:
δ = 7.26 ppm, CD OD: δ = 3.31 ppm). Multiplicities are described using the following
3
13
δ
3
δ = 49.00 ppm) as internal standards. High resolution mass spectra were obtained with Micromass
LCT via loop-mode injection from a Waters (Alliance 2695) HPLC system with achieved ionization by
electrospray ionization (ESI). Calculated and found masses are given in the synthetic procedures.
4
.2. Analytical LC-MS
Data from analytical LC–MS were obtained using a Waters 2767 sample manager connected to
Waters 2545 pumps and system fluidics organizer (SFO), a Phenomenex Kinetex column (2.6
00 Å, 4.6 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C 300 Å) eluted
at 1.0 mL
SQD-2 mass detector operating simultaneously in ES and ES modes between 100 m/z and 650 m/z.
The solvent gradient ( : HPLC grade ddH O containing 0.05% formic acid; : HPLC grade CH CN
containing 0.045% formic acid) was as follows: start: 10%
µ, C ,
18
1
×
5
−1
·
min with a Waters 1998 Diode Array detector (200–600 nm), Waters 2424 ELSD, and Waters
+
-
A
B
2
3
B, 10 min 10%→90% B, 2 min 90% B, 1 min
9
0%→10% B, 2 min 10% B (total time: 15 min).
4
.3. Preparative LC-MS
Purification of compounds with preparative LC-MS was done using a Waters mass-directed
autopurification system comprising a Waters 2767 autosampler, a Waters 2545 pump system and SFO,
and a Phenomenex Kinetex Axia column (5 , C , 100 Å, 21.2 250 mm) equipped with a Phenomenex
Security Guard precolumn (Luna C 300 Å), eluting at 20 mL
µ
×
-
18
1
·
min at ambient temperature (solvents as
5
mentioned in analytical LC-MS). The post-column flow was split in a ratio 100:1 and the minority flow
was made up of HPLC grade MeOH (0.045% formic acid) to 1 mL min for simultaneous analysis
by diode array (Waters 2998), evaporative light scattering (Waters 2424), and ESI mass spectrometry
in positive and negative modes (Waters SQD-2). Detected peaks were collected into glass test tubes.
The solvent was evaporated, and the residue analyzed by NMR.
−1
·
4
.4. Dimethyl-2,2‘-(6,6‘-dihydroxy-[1,1‘-biphenyl]-3,3‘-diyl)diacetate (2)
Silica gel (3.62 g) was added to a solution of FeCl3 6 H O (1.76 g, 6.51 mmol) in diethyl ether
95 mL) and methanol (5 mL) at room temperature and stirred for 5 min. The reaction mixture
·
2
(
was concentrated under reduced pressure and the obtained yellow solid was stirred for 18 h in
◦
vacuo (1.5 mbar) at 55 C. A solution of 4-hydroxyphenyl acetic acid (
1, 61.7 mg, 0.41 mmol) in
dichloromethane (15 mL) was added at room temperature. After thorough mixing the solvent was
◦
evaporated. The remaining dark solid was stirred in vacuo (1.5 mbar) at 60 C. After 60 h, the solid
was dissolved in methanol (25 mL) and filtered through Celite. The solvent was removed in vacuo
and the crude product was purified by silica gel chromatography (first column: petrol ether—EtOAc,
1
0
7
:1; second column: petrol ether—EtOAc, 2:1) to provide a highly viscous, colorless oil of
2
(40.26 mg,
1
.122 mmol, 60%): H-NMR (400 MHz, CDCl ):
δ
(ppm) = 7.17 (dd, J = 2.39 Hz, J = 8.19 Hz, 2H),
3
13
.14 (d, J = 2.39 Hz, 2H), 6.91 (d, J = 8.19 Hz, 2H), 6.21 (bs, 2H), 3.70 (s, 6H), 3.59 (s, 4H). C-NMR
(
100 MHz, CDCl3):
δ
(ppm) = 172.73, 152.16, 132.18, 130.59, 126.71, 124.26, 117.09, 52.21, 40.18. HRMS
+
m/z calculated for C H O Na [M + Na] : 353.1003; found 353.1001.
18
18
6

Molecules 2019, 24, 4272
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0
0
0
0
4
.5. Dimethyl-2,2 -(6,6 -dimethoxy-[1,1 -biphenyl]-3,3 -diyl)-diacetate (3)
Potassium carbonate (0.11 g, 0.78 mmol) was added to a solution of biphenyl acetate 2 (0.12 g,
◦
0
.36 mmol) in dry DMF (1 mL) at room temperature. The reaction mixture was heated to 50 C and
stirred for 45 min, before it was cooled to room temperature and a solution of methyl iodide (0.05 mL
,
◦
0.85 mmol) in dry DMF (0.1 mL) was added slowly. The mixture was stirred for 14 h at 50 C, after
which it was cooled to room temperature and quenched by the addition of H O (13 mL). The organic
2
layer was separated and extracted with CH Cl . NaOH (10%, 2.6 mL) was added to the combined
2
2
organic layers and stirred for 2 h. The organic layer was separated, and the aqueous layer was
extracted with CH Cl . The combined organic layers were washed with H O, dried over Na SO , and
2
2
2
2
4
concentrated. The crude product was purified by silica gel chromatography (petrol ether—EtOAc, 2:1)
1
to yield a colorless oil of
3
(0.12 g, 90 %): H-NMR (400 MHz, CDCl ):
δ
(ppm) = 7.24 (dd, J = 2.05 Hz
,
3
J = 8.53 Hz, 2H), 7.15 (d, J = 2.05 Hz, 2H), 6.92 (d, J = 8.53 Hz, 2H), 3.76 (s, 6H), 3.69 (s, 6H), 3.59 (s, 4H)
.
13
C-NMR (100 MHz, CDCl3):
2.01, 40.35. HRMS m/z calculated for C H O Na [M + Na] : 381.1416; found 381.1314.
δ
(ppm) = 172.37, 156.20, 132.36, 129.41, 127.57, 125.68, 111.19, 55.80,
+
5
20
22
6
4
.6. Methyl-2-(3-bromophenyl)acetate (9)
-Bromo phenyl acetic acid ( , 4 g, 18.7 mmol, 1.0 eq) and dried K CO (8.84 g, 56 mmol, 3.0 eq
3
8
)
)
2
3
were dissolved in acetone (40 mL) under inert conditions. Methyl iodide (10.5 mL, 56 mmol, 3.0 eq
◦
was added dropwise and the reaction mixture was stirred for 67 h at 30 C. After cooling the reaction
to room temperature, the suspension was filtered with Celite and the solvent was removed in vacuo.
The crude product was purified by silica gel chromatography (PE:EtOAc/4:1) yielding the product
methyl-2-(3-bromophenyl)acetate (9) after evaporation as colorless oil in 100% yield (4.25 g, 18.7 mmol,
1
quant.). R (PE:EtOAc/3:1): 0.85; H-NMR (400 MHz, CD Cl):
(
δ = 7.44 (d, J = 1.5 Hz, 1H), 7.41
f
3
13
dt, J = 6.5, 2.2 Hz, 1H), 7.23–7.17 (m, 2H), 3.71 (s, 3H), 3.60 ppm (s, 2H); C-NMR (100 MHz, CD Cl):
3
δ (ppm) = 171.4, 136.1, 132.4, 130.3, 130.1, 128.0, 122.6, 52.2, 40.7.
0
0
0
4
.7. Diethyl 2,2 -([1,1 -biphenyl]-3,3 -diyl)diacetate (10)
The halogenated compound (90 mg, 0.40 mmol, 1.0 eq), XPhos-Pd-G2 (3.1 mg, 0.004 mmol,
.01 eq), XPhos (3.8 mg, 0.008 mmol, 0.02 eq), B (OH) (106.2 mg, 1.20 mmol, 3.0 eq), and KOAc
9
0
2
4
(
116.3 mg, 1.20 mmol, 3.0 eq) were dissolved in ethanol under inert conditions. The reaction mixture
◦
was stirred for 30 min at 80 C until the color changed to yellow, and then cooled down to room
temperature. K CO (272.9 mg, 2.0 mmol, 5.0 eq) and 9 (90 mg, 0.40 mmol, 1.0 eq) were added and
2
3
◦
the reaction mixture was stirred for 16 h at 80 C. After cooling to room temperature, the reaction
mixture was filtered with Celite and the solvent evaporated. The crude product was purified by silica
gel chromatography (PE:EtOAc/9:1) yielding the biphenyl ester 10 as yellowish oil (40 mg, 0.014 mmol,
1
3
4%). R (PE:EtOAc): 0.64. H-NMR (400 MHz, CDCl ):
δ
(ppm) = 7.49 (d, J = 7.6 Hz, 4H), 7.39
f
3
(
t, J = 7.8 Hz, 2H), 7.28 (d, J = 7.5 Hz, 2H), 4.17 (q, J = 7.1 Hz, 4H), 3.68 (s, 4H), 1.27 (t, J = 7.1 Hz, 6H).
13
C-NMR (100 MHz, CDCl3):
δ (ppm) = 171.5, 141.2, 134.6, 128.9, 128.2, 128.2, 1256.0, 60.9, 41.5, 14.2.
+
HRMS (ESI) m/z calculated for C H O [M + H] = 327.1591, found: 327.1619.
20
22
4
4
.8. Colorimetric Lipase Activity Screening
Lipases were purchased from Sigma Aldrich (Lipase basic kit). In every well of a 96-well microtiter
-1
plate protein (final concentration: 10 U
·
mL ) was 180
µL phosphate buffer (10 mM, pH 7.4) with
0
.01% bromothymol blue (BTB) mixed with 20 µL compound (100 mM in DMSO, CH CN or toluene).
3
The final concentration of the organic solvent in each well was 10% (v/v). For every lipase a negative
control, containing only protein and BTB in phosphate buffer, as well as a positive control using
glyceryl tributyrate (TAG (13), 100 mM in DMSO) were prepared. The plate was shaken gently for 28 h
at room temperature and the color was monitored at 0, 1, 2, 4, 24, and 28 h, respectively. The assay

Molecules 2019, 24, 4272
12 of 16
was performed in duplicates. With lipases RNL, MML, PFL, and CRL, the assay was additionally
performed in the presence of 40% DMSO (v/v).
4
.9. Analysis of Lipase Activity and Selectivity by Analytical LC-MS
In total, 1 mg lipase in 180 L phosphate buffer (50 mM, pH 7.4/8.0/9.0 (AMP-buffer)) was mixed
with 20 L compound (100 mM in DMSO) for all experiments with 10% DMSO (v/v). For experiments
µ
µ
in the presence of 40% DMSO (v/v), 80
mixtures were shaken at 25 or 37 C, and 20
µ
L compound (25 mM in DMSO) was used. The reaction
L samples were taken at the indicated time points for
L 2M
L CH CN. Analytical LC-MS measurements were performed as mentioned
◦
µ
analytical LC-MS measurements. The reaction in the samples was stopped by the addition of 5
HCl and diluted with 20
µ
µ
3
before. The product–reactant ratios were determined by comparing the UV signals from a diode array,
where the ratio of peak areas was proportional to the ratio of concentrations.
0 0 0
.10. Benzyl 2-(2 ,6-dihydroxy-5 -(2-methoxy-2-oxoethyl)-[1,1 -biphenyl]-3-yl)acetate (14) and
4
2
0
0
0
-(2 ,6-Bis(benzyloxy)-5 -(2-methoxy-2-oxoethyl)-[1,1 -biphenyl]-3-yl)acetic acid (16)
0.3 mg of CALB were dissolved in phosphate buffer (4 mL, 50 mM, pH 8.0) and biphenyl ester
15 mg, 0.045 mmol, 1.0 eq) in DMSO (450 L) was added to the mixture. The solution was stirred for
h at room temperature. To terminate the reaction 1 M HCl was added to adjust the pH to 1, and
1
2
(
µ
2
the aqueous layer was washed three times with EtOAc. The solvent was evaporated, and the residue
was directly used for benzylation without any purification steps. The crude product was dissolved in
CH CN (10 mL), and 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (1.5 mg, 0.01 mmol, 0.22 eq) and benzyl
3
◦
bromide (90
µ
L, 0.757 mmol, 16.8 eq) were added. The reaction mixture was stirred for 2 h at 84 C
.
After cooling down dH O was added and the aqueous layer was washed three times with EtOAc.
2
The combined organic layer was dried over Na SO and the solvent was evaporated. Purification
2
4
of the crude product was performed using a Büchi Reverlis Prep equipped with a 4 g silica column
Ecoflex) and PE/EtOAc gradient; 14 eluted at 5% EtOAc and 16 at 50% EtOAc. The unsymmetric ester
was obtained as brown oil with 20% yield (3.6 mg, 0.009 mmol), and the acid 16 with a 45% yield
9.2 mg, 0.019 mmol).
(
1
4
(
1
1
(
5
1
4
: R (PE:EtOAc/4:1): 0.646. H-NMR (400 MHz, CDCl ):
δ
(ppm) = 7.31 (dd, J = 10.8, 4.7 Hz, 5H), 7.22
d, J = 7.5 Hz, 4H), 7.20–7.14 (m, 2H), 5.92 (ddt, J = 15.9, 11.0, 5.6 Hz, 1H), 5.16 (dd, J = 11.0, 1.4 Hz, 3H),
.14–5.09 (m, 2H), 4.22 (dd, J = 4.2, 1.4 Hz, 4H). 13C-NMR (100 MHz, CDCl3):
(ppm) = 178.4, 134.4,
34.4, 128.7, 128.7, 125.8, 120.2, 120.2, 116.5, 116.5, 116.5. HRMS [ESI] m/z calculated for C H O
f
3
δ
24
22
6
+
[
M + Na] = 429.1314, found: 429.1311.
1
1
6
: R (PE:EtOAc/4:1): 0.132. H-NMR (400 MHz, CDCl ):
δ
(ppm) = 7.37–7.26 (m, 10H), 7.26–7.09
f
3
(
(
m, 4H), 7.06 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.28 (t, J = 5.4 Hz, 1H; OH), 5.14 (s, 2H), 5.10
13
d, J = 4.2 Hz, 2H), 3.70 (s, 3H), 3.67–3.59 (m, 4H). C-NMR (100 MHz, CDCl ):
δ
(ppm) = 172.5, 172.1,
3
1
6
5
54.1, 153.0, 133.6, 132.2, 130.3, 130.2, 128.8, 128.7, 128.7, 128.3, 128.3, 127.5, 117.9, 114.5, 72.1, 72.0,
+
6.9, 66.7, 52.3, 52.2, 40.5, 40.0. HRMS (ESI) m/z calculated for C H O [M + Na] = 519.1784, found:
3
1
28
6
−
19.1782 and calculated for C H O [M + H] = 495.1808, found: 495.1810.
31
28
6
0
0
0
4
.11. 2-(2 ,6-Dimethoxy-5 -(2-methoxy-2-oxoethyl)-[1,1 -biphenyl]-3-yl)acetic acid (5)
A total of 29.7 mg of PFL was dissolved in phosphate buffer (3.36 mL, 50 mM, pH 8.0) and
15 mg, 0.042 mmol, 1.0 eq) in DMSO (2.23 mL) was added. The solution was stirred for 48 h at 37 C.
3
◦
(
To terminate the reaction 1 M HCl was added to receive pH 1 and the aqueous layer was washed three
times with EtOAc. The solvent was evaporated, and the residue was purified with preparative LC–MS
as mentioned above. Compound
1H-NMR (400 MHz, CDCl3):
(ppm) = 7.26–22 (m, 2H), 7.15 (dd, J = 4.1, 2.3 Hz, 2H), 6.92 (dd, J = 8.4,
.1 Hz, 2H), 3.75 (d, J = 3.6 Hz, 6H), 3.69 (s, 3H), 3.62 (s, 2H), 3.59 (s, 2H). C-NMR (100 MHz, CDCl ):
5 was obtained as a colorless oil with 41% yield (5.9 mg, 0.017 mmol).
δ
13
4
3

Molecules 2019, 24, 4272
13 of 16
δ
(ppm) = 176.6, 172.4, 156.3, 156.1, 132.5, 132.3, 129.5, 129.4, 127.6, 127.3, 125.7, 125.0, 111.2, 111.2, 55.8,
+
55.8, 52.0, 40.2, 40.0. HRMS (ESI) m/z calculated for C H O [M + Na] = 367.1158, found: 367.1161.
19 20 6
0
0
0
4
.12. Benzyl 2-(2 ,6-dimethoxy-5 -(2-methoxy-2-oxoethyl)-[1,1 -biphenyl]-3-yl)acetate (15)
Firstly, (4 mg, 0.012 mmol, 1 eq) was dissolved in CH CN (3 mL). DBU (0.5 mg, 0.003 mmol,
5
3
0
8
.2 eq) and benzyl bromide (4
4 C for 2 h. After cooling down, H O was added, and the aqueous layer was washed three times
µ
L, 0.024 mmol, 2.0 eq) were added. The reaction mixture was stirred at
◦
2
with EtOAc. The combined organic layers were dried over Na SO and the solvent was evaporated.
2
4
The residue was purified using silica gel chromatography using a PE:EtOAc gradient (start: 100% PE,
CV; 50%:50% PE:EtOAc, 2 CV; 100% EtOAc, 15 eluted at PE:EtOAc 50%:50%). Compound 15 was
2
obtained as a colorless oil with 81% yield (4.1 mg, 0.009 mmol).
1
H-NMR (400 MHz, CDCl3):
δ (ppm) = 7.36–7.29 (m, 5H), 7.26–7.22 (m, 2H), 7.14 (dd, J = 13.1,
2
.3 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 5.13 (s, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 3.69 (s, 3H), 3.63 (s, 2H), 3.59
13
(s, 2H). C-NMR (100 MHz, CDCl ):
δ
(ppm) = 172.34, 171.72, 162.43, 162.01, 158.74, 156.20, 156.17,
3
1
7
32.37, 132.31, 129.44, 129.38, 128.50, 128.14, 128.09, 127.58, 127.57, 125.65, 125.58, 111.67, 111.16, 77.32,
+
7.00, 76.68, 66.52, 55.80, 55.78, 51.99, 40.47, 40.32. HRMS (ESI) m/z calculated for C H O [M + Na]
26
26
6
=
457.1627, found: 457.1624.
4
.13. Molecular Docking
All compounds were sketched in Schrödinger Maestro (Schrödinger Release 2019-3: Maestro;
Protein Preparation Wizard; Epik; Impact, Schrödinger, LLC, New York, NY, USA, 2019), and energy
minimized with Schrödinger MacroModel (Schrödinger Release 2019-3: MacroModel, Schrödinger,
-
4
−1 −1
LLC, New York, NY, USA, 2019) and the OPLS3 force field [40] to a gradient of 10 kJ mol Å .
·
Molecular docking was performed using Autodock 4.2 (MGL Tools) [41] and the Lamarckian genetic
algorithm [42]. Protein coordinates were obtained from the pdb database (PDB codes: CRL: 1TRH [43],
MML: 4TGL [44], RNL: 1LGY [45], PCL: 3LIP [46]) and preprocessed with the Protein Preparation
Wizard of the Schrödinger Suite and AutodockTools. The search space covered the catalytic center of
the proteins.
Supplementary Materials: The following supplementary materials are available online. Scheme S1: Scheme
illustrating the mechanism of the biomimetic Fe(III)-catalyzed oxidative phenolic coupling for the synthesis of
biphenyl esters. Fe(III) is reduced by electron acceptance of aromatic electrons. Radicals and charges can be
stabilized by mesomeric effects of the electron donating ortho substitution. Scheme S2: Formation of byproduct 16
during the two-step protocol. Byproduct 16 is formed at 40% yield showing that the acid group is less reactive
than phenolic hydroxyl groups when intermediate 4 is not isolated. Figure S1: Colorimetric lipase assay of nine
lipases from different organisms. A color change from blue to yellow indicates the conversion of ester groups to
free carboxylic acids. The hydrolysis activity of the lipases is markedly dependent on the organic solvent, since no
catalytic activity of any lipase was detectable with acetonitrile (CH CN) or toluene. Figure S2: UV signals and
3
ratios of compounds
the chemoselective biphenyl ester hydrolysis. Figure S3: UV signals and ratios of compounds
and (green) obtained by analytical LC–MS show that CALB converses compound directly to
signals and ratios of compounds 10 (black), 11 (green), and 12 (blue) obtained by analytical LC–MS. Table S1:
2
(black),
4
(blue), and
6
(green), obtained by analytical LC–MS reveal the pH-dependency of
(black), (blue),
. Figure S4: UV
3
7
5
7
3
Ratios of biphenyl ester 3 to asymmetric acid 5 after different reaction times and at different temperatures and
conversion with lipases CRL, MML, RNL, and PFL (40% DMSO (v/v)). Figures S5–S7: Compound characterization
data (UV absorption and mass spectra of relevant compounds).
Author Contributions: Methodology and software: J.E. and J.K.; investigation, validation, and formal analysis:
J.E., J.K., and N.Z.; supervision, project administration, and resources: M.P.; conceptualization, visualization,
writing—original draft, and writing—review and editing: J.E. and M.P.
Funding: This research received no external funding.
Acknowledgments: We are very thankful to the groups of Russell J. Cox and Oliver Plettenburg for sharing
equipment, particularly Carsten Schotte for support with the preparative LC–MS. We thank Andreas Kirschning
for helpful discussion.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

Molecules 2019, 24, 4272
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Sample Availability: Samples of the compounds are not available from the authors.
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