Hydrolases-mediated transformation of oleuropein into demethyloleuropein

Article abstract of DOI:10.1016/j.bioorg.2018.12.005

Phenolic compounds present in extra virgin olive oil have recently attracted considerable attention due to their pharmacological activities. Among them oleacein (3,4-DHPEA-EDA), structurally related to oleochantal (4-HPEA-EDA), is one of the most studied. 3,4-DHPEA-EDA has been synthesized through decarboxylation of demethyloleuropein catalyzed by Er(OTf)₃. Demethyloleuropein is extracted from black olives drupes in very limited amounts and only in particular periods of the year. The availability of demethyloleuropein could be increased by a selective hydrolysis of the methyl ester moiety of oleuropein, a secoiridoid present in large amount in olive leaves. In this work we describe a new enzymatic method for carrying out a selective hydrolysis of oleuropein via the screening of a panel of hydrolases (lipases, esterases and proteases). Among all the enzymes tested the best results was obtained using α-chymotrypsyn from bovine pancreas as biocatalyst, thus revealing a classic example of catalytic enzyme promiscuity.

Full text of DOI:10.1016/j.bioorg.2018.12.005

Accepted Manuscript
Hydrolases-mediated transformation of oleuropein into demethyloleuropein
Luca Cariati, Manuela Oliverio, Francesco G. Mutti, Sonia Bonacci, Tanja
Knaus, Paola Costanzo, Antonio Procopio
PII:
S0045-2068(18)30264-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.005
YBIOO 2669
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
16 March 2018
27 November 2018
3 December 2018
Please cite this article as: L. Cariati, M. Oliverio, F.G. Mutti, S. Bonacci, T. Knaus, P. Costanzo, A. Procopio,
Hydrolases-mediated transformation of oleuropein into demethyloleuropein, Bioorganic Chemistry (2018), doi:
https://doi.org/10.1016/j.bioorg.2018.12.005
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Hydrolases-mediated transformation of oleuropein into
demethyloleuropein
Luca Cariatia, Manuela Oliverio*a, Francesco G. Muttib, Sonia Bonaccia, Tanja Knausb, Paola
Costanzoa, Antonio Procopioa
aDipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Viale Europa –
Campus Universitario “S. Venuta” – Loc. Germaneto - 88100 (CZ), ITALY.
bVan't Hoff Institute for Molecular Sciences, HIMS-Biocat, Science Park 904, 1098 HX Amsterdam,
The Netherlands
*Dr. Manuela Oliverio. Dipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro,
Viale Europa – Campus Universitario “S. Venuta” – Loc. Germaneto - 88100 (CZ), ITALY. Email:
m.oliverio@unicz.it; Tel: +39.0961.369.4121; Fax: +39.0961.369.4237
KEYWORDS. Oleuropein, demethyloleuropein, hydrolases, biocatalysis, enzyme promiscuity.
1
1 ABBREVIATIONS
3,4-DHPEA-EDA, oleacein; 4-HPEA-EDA, oleochantal; VOO, Virgin olive oil; EC 3, hydrolases; EC
3.4.21.1, Chymotrypsin; HPLC, high performance liquid chromatography; HRMS, high resolution mass
spectrometry.

ABSTRACT. Phenolic compounds present in extra virgin olive oil have recently attracted considerable
attention due to their pharmacological activities. Among them oleacein (3,4-DHPEA-EDA), structurally
related to oleochantal (4-HPEA-EDA), is one of the most studied. 3,4-DHPEA-EDA has been
synthesized through decarboxylation of demethyloleuropein catalyzed by Er(OTf)3. Demethyloleuropein
is extracted from black olives drupes in very limited amounts and only in particular periods of the year.
The availability of demethyloleuropein could be increased by a selective hydrolysis of the methyl ester
moiety of oleuropein, a secoiridoid present in large amount in olive leaves. In this work we describe a
new enzymatic method for carrying out a selective hydrolysis of oleuropein via the screening of a panel
of hydrolases (lipases, esterases and proteases). Among all the enzymes tested the best results was
obtained using α-chymotrypsyn from bovine pancreas as biocatalyst, thus revealing a classic example of
catalytic enzyme promiscuity.
1. INTRODUCTION.
Virgin olive oil (VOO) plays a primary role in the Mediterranean diet and it represents a very important
agricultural product not only for economic reasons but also for health care [1]. Numerous evidences
suggest that the high consumption of VOO, rich in phenol and flavonoids, is responsible of the lowered
incidence of pathology such as cardiovascular disease or diabetes [2]. Recently, it has also been
demonstrated his benefic role in the incidence of Alzheimer disease and Parkinson disease [3]. The main
phenolic compounds present in VOO are secoiridoids derivates of hydroxytyrosol (3,4-DHPEA) and
tyrosol (p-HPEA) that occurs as either simple phenols or esterified with elenolic acid to form,
respectively, oleuropein (1), its derived demethyloleuropein (2), ligstroside (3), together with their
aglicones 3,4-DHPEA-EA (4) and p-HPEA-EA (5) (Figure 1) [4]. Furthermore, recently, intensive
studies carried out in order to clarify the phenol composition of VOO highlighted the presence of two
other important compounds characterized by a dialdehyde moiety linked with 3,4-DHPEA and p-HPEA,
named 3,4-DHPEA-EDA (6) and p-HPEA-EDA (7), respectively [5], that show peculiar sensory and
healthy properties [6].

Particularly, the structural similarity between 3,4-DHPEA-EDA and p-HPEA-EDA, a phenol owning an
anti-inflammatory activity similar to that reported of ibuprofen [5a], and the presence of a catechol
moiety that potentially gives greater antioxidant activity, has triggered an intensive study of the
pharmacological properties of 3,4-DHPEA-EDA. The powerful antioxidant activity of 3,4-DHPEA-
EDA against reactive oxygen and nitrogen species was proved, using in vitro cellular and non cellular
screening systems [7]. Furthermore, the anti-inflammatory action of compound 6 and its ability to
inhibit 5-lipoxigenase was demonstrated as well [8].
R
O
OGlc
O
O
HO
R1OOC
1
1
R=OH, R =Me; oleuropein (OL)
2 R= OH, R1=H; demethyloleuropein
3 R=H; R1=Me; ligstroside
CHO
R
O
R
O
CHO
O
O
O
HO
CHO
HO
H3COOC
6 R=OH; 3,4-DHPEA-EDA (oleacein)
7 R=H, p-HPEA-EDA (oleochantal)
4
5
R=OH; 3,4-DHPEA-EA (oleuropein aglycone)
R=H; p-HPEA-EA (ligstroside aglycone)
Figure 1. Chemical structure of main phenolic compounds present in VOO
Additionally to these important pharmacological properties, it has to be mentioned that compound 6 is
the predominant secoiridoid in the crushed paste and in VOO. 3,4-DHPEA-EDA content in VOO ranges
from 111 to 285 mg/kg, depending on the irrigation condition [9], while the amount of other
secoiridoids is much lower compared to 3,4-DHPEA-EDA [10]. It is presumed that demethyloleuropein
(2) act as a precursor for the formation of 3,4-DHPEA-EDA during crushing due to an increase in the
esterases and β-glucosidases activity [11]. Demethyloleuropein is an indicator of the maturation of the

olives. Indeed, his relative amount increases as soon as the maturation proceeds, while the content of
oleuropein decreases [12]. In the past ten years, we invested numerous efforts for the development of
new green methods aimed at realizing an efficient manipulation of oleuropein and its derivatives in
order to obtain more powerful pharmacologically active compounds [13]. In particular, we recently
developed a new method for the synthesis of compound 6 using demethyloleuropein (2) as substrate and
therefore mimicking the natural formation of 3,4-DHPEA-EDA in olive fruits [14]. The transformation
was carried out in water, under reflux, in the presence of Er(OTf)3 as very low toxic Lewis acid. This
synthetic protocol allowed to obtain a contemporary deglucosylation and decarboxylation affording the
desired product in only 4 hours. Despite the process accomplishes many green chemistry tasks, the use
of demethyloleuropein (2) as starting material, usually extracted from black olive fruits only in
particular periods of the year, makes this process difficult to replicate. Therefore, we decided to carry
out a synthesis of demethyloleuropein via screening of a panel of hydrolases (EC 3), using oleuropein
(1) as starting material. We demonstrate that as oleuropein is indeed the secoiridoid present in larger
amount in olive leaves, the method allows for increasing the availability of the precursor
demethyloleuropein and consequently increasing the availability of 3,4-DHPEA-EDA as well.
2. MATERIAL AND METHODS.
2.1. General
All chemicals were obtained from Aldrich Chem. Co. The HPLC were performed by RP-HPLC analysis
on a Thermo Scientific Ultimate Dionex 3000 UHPLC equipped with an Hypersil gold column (C18)
(dim.mm 250x4.6, particle size 5µm) and a mobile phase consisted of the following solvents: A (H20 +
TFA pH 2,46), B (methanol). The gradient was as follows: 0-1 min, 95% A, 5% B, 1-17 min 40% A -
60% B, 19-25 min 5% A-95% B, 27-30 min 95% A-5% B, 30-35 min 95% A-5% B Absorbance were
obtained at the wavelength of 230 nm, 254 nm and 280 nm. The instrumentation performance,
chromatograms, and initial data processing were carried out with Chromelion software. TLC were
performed using silica plates 60-F264 on alumina, commercially available from Merk. Liquid flash
chromatography was performed on a Supelco VERSA FLASH HTFP station using octadecyl-
functionalized silica gel commercially available from Sigma-Aldrich. The qualitative identification was
performed a Thermo Scientific Q Exactive (Thermo Fisher, Milan, Italy) mass spectrometer working
both in positive and negative mode at 35,000 resolving power, operating in SIM mode by flow injection

(flow rate 15 μL/min for each stock solution). Data were evaluated by Xcalibur 2.2.SP1 (Thermo Fisher
Scientific, Bremen, Germany). The mass accuracy, directly calculated from Xcalibur, is defined by the
formula Δ(ppm) = [(theoretical mass measured mass)/theoretical mass] × 1.000.000. The synthesized
13
compound is known, its 1H-NMR and C-NMR were compared with that reported in the literature [14].
Spectra were recorded on a Bruker WM 300 instrument, at 300 MHz on samples dissolved in CDCl3.
Chemical shifts are given in parts per million (ppm) from tetramethylsilane as the internal standard for
1H-NMR spectra (0.0 ppm).
2.2. Oleuropein Extraction
The MW-assisted extraction of oleuropein (1) [13a] was performed on several samples of olive leaves
from Coratina cultivar of Olea europea L., Oleaceae, dried for 48 h at 50 °C, milled, and kept at r.t.
until use. Oleuropein was purified by flash chromatography in a SUPELCO VERSA FLASH station
(eluent: CHCl3/MeOH 8:2) and characterized by 1H-NMR, 13C-NMR and HRMS, by comparison with
data reported in the literature [13a].Pure oleuropein was used as reagent for the hydrolysis reaction.
2.3. Enzymes- mediated synthesis of demethyloleuropein
Oleuropein (1), 0.55 mmol, and α-chymotripsin from bovine pancreas, 26 units, were added to a solution
(50 mL) of buffer potassium phosphate at pH 7. The reaction was shacked for 72 h, 130 rpm at 4°C.
Afterwards the reaction was stopped, the aqueous buffer was removed via lyophilization and the
reaction mixture was solubilized in methanol until reaching a concentration of 50 µg/mL and analyzed
by HPLC/MS. (See Supporting Information) The product was purified by flash chromatography and
characterized by HRMS, while 1H-NMR and 13C-NMR were compared with those reported into the
literature [14](See Supporting Information).
Demethyloleuropein (2). Pink powder. HRMS [M-H]= 525.1611 m/z (theoretic mass 525.1617 m/z)
3. RESULTS AND DISCUSSION.
We turned our attention in the use of the enzymes for carrying out the conversion of oleuropein (1) into
demethyloleuropein (2) because common organic protocols for cleavage of esters involve strong basic

or acidic conditions. The presence of two ester groups in the oleuropein (1) structure and the poor
reactivity of methyl group as leaving group make impossible to use these methods for carrying out a
selective hydrolysis of oleuropein. Although some methods for a selective hydrolysis of methyl ester are
present in the literature, they usually require either toxic solvents or expensive catalysts [15]. On the
contrary, hydrolytic enzymes - usually employed to catalyze the formation or the cleavage of esters and
amides - allow for carrying out these transformations in very green conditions [16]. Thereby, the
chemoselective hydrolysis of the methyl ester moiety of oleuropein was carried out via the screening of
a panel of hydrolases. Especially, 23 hydrolases commercially available were screened. As a first
attempt the reactions were performed using 0.092 mmol of oleuropein, run at 30°C in an orbital shaker
at 150 rpm, pH 8, in 0.5 mL of potassium phosphate buffer, for 24 hours in presence of 13 units of
enzyme (1 unit is defined as the amount of the enzyme that catalyzes the conversion of 1 µmol of
substrate per minute) (Scheme 1).
Scheme 1. Enzymatic oleuropein hydrolysis.
The solvent was removed by lyophilization and the crude was solubilized in methanol until reaching a
concentration of 1 mg/mL. The crude was analyzed by UHPLC and HRMS. Unfortunately, the
hydrolysis carried out at pH 8 led only to the formation of hydroxytyrosol due to the basic pH (data not

shown). We thought that this side-reaction was due to the basicity of the reaction medium rather than
the enzymatic activity because several reports present in the literature demonstrate that oleuropein is not
directly converted in hydroxytyrosol through the action of esterases [17]. We decided to use a lower pH
until to reach a neutral value in order to verify if under this condition the selective hydrolysis could be
obtained. The results are shown in Table 1.
We observed that compound 2 was detected in few of the biotransformations performed (entries 16, 21,
22, 23, Table 1). Therefore some enzymes showed the ability to hydrolyze the methyl ester, but a low
conversion was obtained in all of these cases. Also in this case the side reaction, namely the hydrolysis
of oleuropein in hydroxytyrosol, was the predominant reaction (all entries, Table 2). In order to
demonstrate the influence of the pH on the reaction profile, a reaction at pH 8 was carried out in absence
of enzyme (entry 24, Table 1). A remarkably hydrolysis of oleuropein into hydroxytyrosol was noticed,
confirming our previous assumption. In general, the best result for the desired enzymatic hydrolysis was
obtained with α-chymotrypsin from bovine pancreas, also known as Bos taurus α-chymotrypsin (entry
21, Table 1).
Table 1. Screening of hydrolases for oleuropein hydrolysis carried out at pH 7a.
Entry
Enzyme
Substrate (%)
Demethyloleuropeinb(%)
Hydroxytyrosol
(%)
21
1
2
3
L. Candida cylindracea
79
78
75
-
-
-
L. porcine pancreas type2
22
L. Pennicillium camemberti
AE014
25
4
5
6
L. C1AE0 Candida
79
74
59
-
-
-
21
26
41
cylindracea
L. AE77 Pseudomonas
stutzeri
L. C2AE02 Candida
cylindracea
7
8
L. AE08 Rizhopus spp.
59
78
-
-
41
22
L. AE078 Pseudomonas
florescense
9
L. AE010 Aspergillus niger
17
62
78
27
-
-
-
-
83
38
22
73
10
11
12
L. AE057 Mucor miehei
L. AE04 Achromobacter spp.
L. AE0156 Candida
antarctica B
13
L. A1AE05 Alcaligenes spp.
55
-
45

Entry
Enzyme
Substrate (%)
Demethyloleuropeinb(%)
Hydroxytyrosol
(%)
24
14
15
L. A2AEO11 Alcaligenes spp.
76
70
-
-
L. AE0158 Candida
antarctica A
30
16
17
L. P2AE012 Pseudomonas
cepacia
Pepsine porcine gastric
mucosa
65
74
8
-
27
26
18
19
20
Papaine from Papaia Latex
70
77
73
-
-
-
30
23
27
Proteinase Bacterial
Thermolysin bacillus
(Thermoproteolyticus rokko)
α-chymotrypsin bovine
pancreas
21
22
41
52
20
6
39
42
L. P1A06Pseudomonas
cepacia
23
24
L. Rhizomucor miehei
52
30
5
-
43
70
No enzymec
a
General reaction conditions: the reactions were run at 30°C in an orbital shaker at 150 rpm,
oleuropein 92 mM, phosphate buffer pH 7 (1 mL), for 24 hours in presence of 13 enzyme units. b
Conversion determined by HPLC. cReaction carried out at pH 8
Chymotrypsin (EC 3.4.21.1) is an enzyme that belongs to class of proteases, and it catalyzes
therefore the hydrolysis of peptide bonds. It is active as homodimer and each monomer is
constituted by 245 amino acid residues with MW of 25.7 kDa (PDB: 4CHA and 1YPH) [18].
The reactivity of α-chymotrypsin on an ester moiety is a classic example of catalytic enzyme
promiscuity. Indeed, several studies have already demonstrated that proteases, which can cleave
a much stronger amide bond, are capable of hydrolyzing the weaker carboxylic ester bond. In
fact, the bonds broken in the two cases (C-N, C-O) differ, but the catalytic mechanism is very
similar [19]. Therefore, chymotrypsin from bovine pancreas was selected as the lead enzyme for
trying to improve the conversion and consequently the yield. The results of the reaction
optimization study are depicted in Table 2.
No improvement was noticed prolonging the reaction time and increasing the concentration of
the biocatalyst (entry 1, Table 2). The influence of water-miscible organic cosolvents or the use
of biphasic aqueous/organic media were not investigated as oleuropein possesses an elevated
solubility in aqueous buffer at neutral or slightly basic pH. Conversely, the use of common

organic cosolvents for biocatalytic application such as DMSO may reduce enzyme activity and
complicate the isolation of the final product after the reaction.
Table 2. Oleuropein hydrolysis carried out with α-chymotrypsyn from bovine pancreasa
2(b)(%)
20(c)
10
Entry
T (°C)
Enzyme units
26
Time(h)
48
1
2
3
4
5
6
7
8
30
4
13
24
20
4
13
24
15(c)
26
24
10
4
13+13(d)
13+13+13(e)
13+13
13+13
48
20
4
72
35
4
72
35
35(f)
4
72
aGeneral reaction conditions: the reactions were run in an orbital shaker at 150 rpm, phosphate
buffer pH 7 (1 mL), oleuropein 0,092 mmol. bConversion determined by HPLC. cHydroxytyrosol
d
was detected as additional product in these samples to be 41% and 37%, respectively. Another
aliquot of the enzyme was added after 24 hours. eTwo aliquots of the enzyme were added after 24
f
and 48 hours. The reaction was performed on 0.55 mmol. The product was characterized by
HRMS while 1H-NMR and 13C-NMR were compared with those reported into the literature [14]
A reduction in terms of conversion was noticed reducing the temperature (entries 2, 3, Table 2).
However, it has to be mentioned that when the reaction was carried out at 4°C the formation of
the side product was not observed. This phenomenon is very likely due to a complete depletion
of the spontaneous chemical hydrolysis of oleuropein into hydroxytyrosol that can occur in
aqueous buffers, even at pH 7. Therefore this temperature was used for carrying out further
studies on the hydrolytic reaction of the methyl ester group. No improvement was noticed
increasing the concentration of the biocatalysis (entry 4, Table 2). The best result was achieved
in presence of 26 enzyme units portion-wise added, and running the reaction for 72 hours (entry
7, Table 2). The reason of this increased conversion is due to the fact that enzyme activity was
completely lost after 48 h incubation time in the reaction buffer. Hence, the addition of small
aliquots of enzyme during the time enabled to sustain a higher catalytic activity during the

overall course of the reaction (entries 1, 4, Table 2). However, a further increase in the enzyme
concentration, while keeping the same total reaction time (72 h), did not ameliorate the
conversion into the desired product (entry 6, Table 2). Once the optimized reaction conditions
were found, the reaction was repeated using 300 mg of oleuropein (entry 8, Table 2). The
purification of the crude mixture by flash chromatography allowed us to obtain the product with
25% of yield. Even if the yield is moderate, this procedure allowed us to obtain
demethyloleuropein in very practical green and economic fashion, and without the need for
extraction methods commonly used for its recovery. These additional extraction procedures are
expensive and require high amount of solvents and several purification steps [20]. The present
protocol combined with the catalytic decarboxylation of demethyloleuropein allows for obtaining
3,4-DHPEA-EDA in only two synthetic steps, therefore increasing the availability of this
important biophenol and allowing detailed studies on its pharmacological properties.
CONCLUSIONS. In conclusion a new alternative synthesis of demethyloleuropein via
screening of a panel of hydrolases is presented. Among all the enzymes α-chymotrypsyn from
bovine pancreas has been found as the best hydrolase, thus revealing a classic example of
enzyme promiscuity. Even if the yield is moderate the use of enzymes allowed to carry out a
controlled hydrolysis in very green conditions hence critically improving the availability of
demethyloleuropein from an agricultural waste. Moreover, the enzymatic reaction proceeded
with complete chemoselectivity at pH 7 and 4 °C. The present protocol combined with the
catalytic decarboxylation of demethyloleuropein mediated by Er(OTf)3 [14], represents the first
mixed catalytic/biosynthetic pathway for 3,4-DHPEA-EDA. Further optimization of the first
biocatalytic step through the use of immobilized α-chymotrypsyn can enhance the stability of the
enzyme in solution and consequently increase the overall yield and decrease the catalyst loading
in order to meet the requirements for the application on a large scale [21].

ASSOCIATED CONTENT
Supporting Information., HPLC analysis of reaction mixtures and HRMS, 1H-NMR and 13C-
NMR spectra of demethyl oleuropein are reported into a word file.
AUTHOR INFORMATION
Corresponding Author
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
Financial supporting by the following grants: PON a3_00359 CUP F61D11000120007
“Potenziamento Strutturale”, European Research Council (ERC StG-2014, grant agreement No
638271, BioSusAmin) and NWO (Sector Plan for Physics and Chemistry) are acknowledged
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Graphical abstract

Highlights
Demethyloleuropein was synthesized from oleuropein via the screening of a panel of
hydrolases (EC 3).

 α-chymotrypsin was the best biocatalyst, thus revealing a classical example of enzyme
promiscuity.
 The enzymatic reaction proceeded with complete chemoselectivity at pH 7 and 4°C.