Conversion of oleic acid into azelaic and pelargonic acid by a chemo-enzymatic route

Article abstract of DOI:10.3390/molecules25081882

A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in 44% isolation yield after three steps, avoiding column chromatography purifications. In the first step, the lipase-mediated generation of peroleic acid in the presence of 35% H₂O₂ is employed for the self-epoxidation of the unsaturated acid to the corresponding oxirane derivative. This intermediate is submitted to in situ acid-catalyzed opening, to afford 9,10-dihydroxystearic acid, which readily crystallizes from the reaction medium. The chemical oxidation of the diol derivative, using atmospheric oxygen as a stoichiometric oxidant with catalytic quantities of Fe(NO₃)₃·9·H₂O, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and NaCl, affords 9,10-dioxostearic acid which is cleaved by the action of 35% H₂O₂ in mild conditions, without requiring any catalyst, to give pelargonic and azelaic acid.

Full text of DOI:10.3390/molecules25081882

molecules
Article
Conversion of Oleic Acid into Azelaic and Pelargonic
Acid by a Chemo-Enzymatic Route
Elisabetta Brenna 1,
* ,
, Danilo Colombo 1, Giuseppe Di Lecce 2, Francesco G. Gatti 1
Maria Chiara Ghezzi 1, Francesca Tentori 1, Davide Tessaro 1 and Mariacristina Viola 1,†
1
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta”, Politecnico di Milano,
Via Mancinelli 7, 20131 Milano, Italy; danilo.colombo@polimi.it (D.C.); francesco.gatti@polimi.it (F.G.G.);
mariachiara.ghezzi@polimi.it (M.C.G.); francesca.tentori@polimi.it (F.T.); davide.tessaro@polimi.it (D.T.);
mariacristina.viola@polimi.it (M.V.)
2
Oleificio Zucchi S.p.A., Via Acquaviva, 26100 Cremona, Italy; dirlab@oleificiozucchi.com
*
Correspondence: mariaelisabetta.brenna@polimi.it; Tel.: +39-02-2399-3077
†
The authors are listed in alphabetical order.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Josefina Aleu
Received: 30 March 2020; Accepted: 16 April 2020; Published: 18 April 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic
acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed
at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in
44% isolation yield after three steps, avoiding column chromatography purifications. In the first
step, the lipase-mediated generation of peroleic acid in the presence of 35% H2O2 is employed
for the self-epoxidation of the unsaturated acid to the corresponding oxirane derivative. This
intermediate is submitted to in situ acid-catalyzed opening, to afford 9,10-dihydroxystearic acid,
which readily crystallizes from the reaction medium. The chemical oxidation of the diol derivative,
using atmospheric oxygen as a stoichiometric oxidant with catalytic quantities of Fe(NO3)3
·
9 H2O,
·
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and NaCl, affords 9,10-dioxostearic acid which
is cleaved by the action of 35% H2O2 in mild conditions, without requiring any catalyst, to give
pelargonic and azelaic acid.
Keywords: lipase; biocatalysis; unsaturated fatty acid; oxidative cleavage; oxidation
1. Introduction
The employment of renewable feedstocks in the chemical industry is steadily advancing to ensure
more efficient use of natural resources, reduce the dependence on fossil raw materials, and give a
contribution to achieving sustainable consumption and production patterns [1].
Fats and oils represent an important class of renewable feedstock from which the so-called
oleochemicals are obtained. They are abundant in nature, biodegradable, and have nontoxic properties.
They have long hydrocarbon chains, resembling the structure of petroleum components, but they are
also characterized by several functional groups useful for chemical modification. The major process
for transforming fats and oils into oleochemicals is hydrolysis, converting natural triglycerides into
crude glycerine and mixtures of fatty acids. The latter are then submitted to reactions involving either
the carboxylic group (to afford soaps, esters, amides, amines, and alcohols) or the reduction/oxidation
of the C=C double bonds, if present. Among these procedures developed to obtain fine chemicals,
the oxidative cleavage of unsaturated fatty acids for the production of dicarboxylic acids, hydroxy
acids, and amino acids has received great attention in the last decade [
two dicarboxylic acids prepared from oleochemicals have been commercialized, i.e., sebacic acid (
2
–
4
]. Until recently, only
),
obtained by the alkaline cleavage of castor oil [5], and azelaic acid (2), which is produced together with
1
Molecules 2020, 25, 1882; doi:10.3390/molecules25081882
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1882
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pelargonic acid (
3) by ozonolysis of oleic acid (
4) (Figure 1) [6]. Sebacic and azelaic acid are extensively
employed in the synthesis of new generation biodegradable copolymers [
occurring in wheat, rye, and barley, also finds application as an active ingredient in products for the
topical treatment of acne [ ], and for the stimulation of hair regrowth [ ]. It works by inhibiting the
7]. Azelaic acid, naturally
8
9
growth of skin bacteria causing acne, and by keeping skin pores clear. Pelargonic acid, found in nature
as ester derivatives in the oil of pelargonium, is used as an herbicide to prevent the growth of weeds
both indoors and outdoors, and as a blossom thinner for apple and pear trees [10].
Figure 1. Sebacic acid (1), azelaic acid (2), pelargonic acid (3), and oleic acid (4).
Oleic acid is the most abundant monounsaturated fatty acid in nature [11], present in a wide
range of vegetable and animal oils and fats. Several works have been published in recent years
describing alternative methods to the ozone-promoted oxidative scission, most of which are based
on metal catalysis [12]. Among them, some very effective one-pot procedures involve the use of
H2O2 as primary oxidant in the presence of tungsten derivatives: (i) methyltrioctylammonium
tetrakis(oxodiperoxotungsto)phosphate [13] (40% H2O2, 85 ◦C, yields of compounds
analysis of the crude mixture were 79% and 82%, respectively); (ii) WO3 and Na2SnO3 in t-BuOH [14
(31% H2O2, 130 ◦C, sealed glass vial, isolation yields were 89% and 65% for
and , respectively);
(iii) a new hybrid organic/inorganic polyoxotungstate in t-BuOH [15] (30% H2O2, 120 C, yield of
compounds and by GC/MS analysis of the crude mixture were 79% and 80%, respectively); iv)
H2WO4 [16] (60% H2O2, reflux, isolation yield was 60% for ); (v) Na2WO4 aqueous solution/H3PO4
aqueous solution with a suitable phase transfer catalyst in a sealed flask [17] (30% H2O2, 90 ◦C, yield
of compound by GC/MS analysis of the crude mixture was max 54%); (vi) the peroxo–tungsten
2 and 3 by GC/MS
]
2
3
◦
2
3
2
2
complex [C5H5N(n-C16H33)]3{PO4[WO(O2)2]4} as a phase-transfer catalyst/co-oxidant [18] (30% H2O2,
◦
85 C, yields of compounds
2 and 3 by GC/MS analysis of the crude mixture were 79% and 80%,
respectively), using in this case oleic acid obtained upon hydrolysis of high oleic sunflower oil by
Candida cylindracea lipase.
As for biocatalytic methods, only a few reports have appeared in the literature. Song et al.
developed [19
), followed by the oxidation of the intermediate alcohol
submitted to Baeyer–Villiger (BV) oxidation to afford ester
3 and the hydroxy acid 8, which was finally oxidized to azelaic acid (2).
,
20] a multi-step enzymatic procedure (Figure 2) starting from the hydration of oleic acid
to the ketone derivative that was in turn
. The latter was hydrolyzed to afford acid
(4
5
7
6
Figure 2. Synthesis of azelaic acid (2) from oleic acid (4) according to references [19,20].

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In [19] the possibility to obtain a regioisomer of ester
7
giving directly diacid
2
upon hydrolysis was
described, but it was not considered for further study and optimization in the following publication [20].
The same research group described the preparation of azelaic acid from ricinoleic acid [21]. The use
of linoleic acid for the biocatalyzed production of azelaic acid was reported by Hauer et al. [22,23].
A multi-enzymatic one-pot reaction was developed to convert linoleic acid into azelaic acid by
combining a 9S-lipoxygenase and 9/13-hydroperoxide lyase to obtain 9-oxononanoic acid submitted to
the final oxidation to acid
tropicalis ATCC20962 to transform nonanoic acid and its esters into azelaic acid
2
catalyzed by an alcohol dehydrogenase. In 2019 the capability of Candida
with the aid of nonane
2
addition and continuous glucose supply [24] was investigated, to improve the production yield of
diacid 2 obtained in the ozonolysis process of oleic acid.
Recently, we were involved in a project aimed at the valorization of the side-stream products
generated by an Italian plant for vegetable seed oil refining (Oleificio Zucchi, Cremona) by using
biocatalytic methods. An important step of the refining process (neutralization) is represented by the
removal of free fatty acids, producing a side-product, called soapstock, which is currently disposed
of by Zucchi in bio-digesters. The fatty acids profile of this material depends on the nature of the
vegetable oil, and, in particular, the one obtained from sunflower oil is highly enriched in oleic acid [25].
Thus, we started to investigate a novel chemo-enzymatic oxidative scission of oleic acid, to be applied
to the sunflower soapstock coming from Oleificio Zucchi for its valorization. The preliminary results
of this study were obtained while working on commercial oleic acid, as a model compound, to study
each step of the most suitable synthetic procedure more easily using a less complex starting material.
The results are herein reported.
2. Results
The enzymatic synthesis of azelaic acid reported in 2013 by Song et al. [19] (Figure 2) consisted
of the use of recombinant Escherichia coli cells expressing at the same time the genes encoding an
oleate hydratase from Stenotrophomonas maltophilia, an alcohol dehydrogenase (ADH) from Micrococcus
luteus, and a BV monooxygenase (BVMO) from Pseudomonas putida KT2440 for the transformation of
oleic acid into 9-(nonanoyloxy)nonanoic acid (
by a cell extract of E. coli expressing the esterase gene from P. fluorescens, afforded pelargonic acid
) and 9-hydroxynonanoic acid ( ). In a further development of the work [20], the oxidation of
derivative by an ADH from P. putida GPo1 completed the route to azelaic acid. As the final product
concentration in the reaction medium was only a few millimolar, likely because of the toxic effects of
pelargonic acid on the E. coli cells, the authors investigated the hydrolysis of and the subsequent
oxidation of derivative into acid by chemical methods [26]. The ester intermediate was purified
by extraction and column chromatography, hydrolyzed with sodium hydroxide in methanol/water
(4/1) at 60 ◦C to afford 9-hydroxynonanoic acid
, which was separated from pelargonic acid by column
chromatography. Finally, the oxidation of the terminal hydroxy group of derivative was achieved
7). The hydrolysis of this latter compound, mediated
(3
8
8
7
8
2
7
8
8
using NaClO2 (1.2 equiv.), 2,2,6,6-tetramethyl-piperidin-1-yl oxyl (TEMPO) (4 mol%), and NaOCl
(2 mol%) in aqueous acetonitrile. After these two steps, no purification was needed. The overall molar
yield of azelaic acid from oleic acid was 58%.
We adopted a different strategy (Figure 3), consisting of the epoxidation of oleic acid to derivative 9,
followed by the formation of threo-9,10-dihydroxystearic acid (10) due to the acid-catalyzed hydrolytic
cleavage of the oxirane ring, promoted directly in the epoxidation medium. The chemical oxidation
of the diol afforded 9,10-dioxostearic acid (11), which was submitted to oxidative cleavage to afford
a mixture of pelargonic (3) and azelaic (2) acid, through the intermediate anhydride 12. In a recent
publication [27], the oxidation of the methyl ester derivative of compound 10, using a solvent-free
procedure of dehydrogenative oxidation catalyzed by commercial 64 wt.% Ni/SiO2 in the presence of
1-decene as a scavenger, was employed to afford a mixture of the two possible regioisomeric vicinal
ketols, that were successively cleaved with formic acid/hydrogen peroxide, and afforded up to 80%
pelargonic acid and azelaic acid monomethyl ester.

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Figure 3. The synthesis of azelaic acid (
2
) from oleic acid (
4) described in this paper. (i) H2O2 35%,
◦
Novozyme 435, acetonitrile, 5 h, 50 C; (ii) NaHSO3 saturated solution, H2SO4 2 M, 3 h, r.t.; (iii)
atmospheric O2, cat. Fe(NO3)3
30 ◦C.
·
9 H2O/TEMPO/NaCl, toluene, 5 h, 100 ◦C; (iv) 35% H2O2, toluene, 3 h,
2.1. Epoxidation of Oleic Acid (4) to 8-(3-Octyloxiran-2-yl)Octanoic Acid (9)
The capability of certain lipases to catalyze the perhydrolysis (i.e., lysis by hydrogen peroxide) of
carboxylic acid esters, hence forming peroxycarboxylic acids in aqueous hydrogen peroxide solutions,
had been already patented by Clorox co. in the late eighties [28]. In 1990, an immobilized form of lipase
B from Candida antarctica (Novozyme 435) was shown [29] to catalyze the formation of peroxycarboxylic
acids directly from the corresponding carboxylic acid. In that case, the reaction was combined with the
epoxidation of alkenes. A few years later, Warwel et al. [30] described that, when unsaturated fatty
acids (or their esters) are treated with hydrogen peroxide in the presence of Novozyme 435, epoxidized
derivatives are obtained through two sequential steps. Firstly, unsaturated fatty acids are converted
into unsaturated peroxy acids by lipase-catalyzed perhydrolysis. Unsaturated peroxy or carboxylic
acids are in turn epoxidized via a classical Prileshajev reaction, which is, in this case, referred to as a
“self-epoxidation reaction” even though it proceeds predominantly via an intermolecular process.
The reaction has been widely exploited not only for the epoxidation of fatty acids and esters
but also for the derivatization of vegetable oils [31]. Typically, the reaction medium consists of an
aqueous layer containing hydrogen peroxide, an organic layer (usually a toluene solution) containing
the fatty acid derivative, and a solid phase represented by the immobilized enzyme. The main
issue of this chemo-enzymatic procedure is the deactivation of lipase. Temperature, reaction time,
concentration of H2O2 in the reaction medium, and the related concentration of peracid generated
in situ are critical parameters to be considered. Temperatures not higher than 50 ◦C, diluted H2O2
solution (max 1% w/w in the final solution) and reaction times not longer than 6 h represent the most
common experimental conditions.
We decided to perform the chemo-enzymatic epoxidation of oleic acid in a water-miscible solvent,
such as acetonitrile, for the following reasons: i) to promote the dissolution of both oleic acid and H2O2
in the same medium, and ii) to enable the in situ acid-catalyzed hydrolysis of the epoxide derivative at
the end of the reaction, after removal of the enzyme, by addition of a diluted solution of sulfuric acid.
The preliminary experiments were carried out with 0.30 mmol of commercial oleic acid (91% purity by
GC/MS, the major contaminants are palmitic and stearic acid), changing the molar ratio H2O2/oleic
acid (1.8 and 2.2), the temperature (30 ◦C and 50 ◦C), the amount of Novozyme 435 (10 mg and 30 mg),
and the solvent volume (2 mL and 6 mL). The reactions were monitored by GC/MS. The results of this
screening are reported in Tables S1 and S2 (see Supplementary Material). The following conditions were
found to be optimal for running the reaction: 0.15 M oleic acid, 0.27 M H2O2, 5 mg
435, in acetonitrile at 50 C for 5 h under stirring with final 98% conversion (GC/MS,). Epoxide
·
mL−1 Novozyme
◦
9

Molecules 2020, 25, 1882
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could be recovered from the reaction mixture at 83% isolation yield (see Supplementary Material),
starting from commercial oleic acid. The stereochemistry of the oxirane ring of derivative was
9
based on the cis configuration of oleic acid and was confirmed by comparison with literature data (see
Supplementary Material).
2.2. Acid–Catalyzed Cleavage of 8-(3-Octyloxiran-2-yl)Octanoic Acid (9) to 9,10-Didhydroxystearic Acid (10)
The ring-opening of epoxystearic acid to the corresponding threo diol derivative 10 (Figure 3)
9
was promoted by diluted sulfuric acid, and the reaction conditions were adjusted to obtain quantitative
conversion after 3 h at room temperature (Table S3 of Supplementary Material). The relative
configuration of diol 10 was established following the anti mechanism of the hydrolytic opening of
the oxirane ring of cis-epoxide 9 and was confirmed by literature data (see Materials and Methods).
The acid-catalyzed hydrolysis was then performed, without isolation of the epoxide, by addition of a
2.0 M solution of H2SO4 directly to the reaction mixture of the epoxidation step, after removal of the
enzyme by filtration and decomposition of peroxy species. This one-pot two-steps sequence led to the
spontaneous crystallization of dihydroxystearic acid 10, which was easily recovered from the reaction
medium by filtration as a pure white solid. Satisfactory isolation yields (77%) were obtained, even
when the reaction was run at a 2 g scale, starting from commercial 91% oleic acid.
2.3. Oxidation of 9,10-Dihydroxystearic Acid (10) to 9,10-Dioxostearic Acid (11)
The following procedures were tested to achieve the oxidation of either one or both the hydroxy
groups of derivative 10: (i) alcohol dehydrogenase—mediated oxidation (commercial kit from EVOXX);
(ii) chemo-enzymatic oxidation with laccase and hydroxybenzotriazol (HOBt); (iii) aerobic oxidation
with catalytic Fe(NO3)3
·
9 H2O, TEMPO, and NaCl. Only the latter was successful and diol 10 could
be converted into the corresponding dioxo derivative 11 (Figure 3). Starting from 0.50 g of diol 10
,
according to the literature [32], a loading of 1 mol% for each catalyst was enough to afford complete
conversion into the diketone in toluene solution at 100 ◦C in 5 h. After work-up (quenching with water
and extraction), the crude residue was submitted directly to the following step of oxidative cleavage.
2.4. Oxidative Cleavage of 9,10-Dioxostearic Acid (11) to Azelaic (2) and Pelargonic Acid (3)
For the final step of the synthetic procedure, we investigated the Baeyer–Villiger (BV) oxidation of
diketone 11, to prepare the corresponding anhydride 12, and hydrolyze it to azelaic (
acid ( ). A wide range of oxidants has been employed for the BV reaction, including mineral and
organic peracids. Hydrogen peroxide can be used if suitably activated by a catalyst, or in the presence
of a strong acid, or even in alkaline conditions [33 34]. -Diketones react readily with BV reagents:
2) and pelargonic
3
,
α
in inert solvents anhydrides are formed, while in alkaline or acidic media simple carboxylic acids
are generally produced in good yields [35]. In 1930 Böeseken et al. [36] prepared 9,10-diketostearic
acid (11) by oxidation of 9-octadecynoic acid with 70% nitric acid at 10–25% yield and submitted it to
the reaction with 15% excess peracetic acid in acetic acid for one day. They obtained the quantitative
conversion of the diketone into a mixture of acids 2 and 3.
Thus, we first considered the possibility to perform the BV oxidation of compound 11 with the
corresponding peroxycarboxylic acid produced by lipase-mediated perhydrolysis in the presence
of H2O2, using for preliminary experiments a sample of 11 isolated and purified by column
chromatography. We treated dioxostearic acid 11 (50 mg) with 1.6 mol of H2O2 per mol of dioxostearic
acid in the presence of 2.5 mg
mL−1 Novozyme 435 in toluene (2 mL), the same solvent employed
·
for the preceding oxidation. The cleavage was complete after 3 h at 30 ◦C, to give a mixture of 96%
pelargonic and azelaic acids, with tiny quantities of octanoic (0.3%), stearic (0.4%), and palmitic acid
(2%), with other minor components (GC/MS). To our surprise, when the blank reaction was carried out
in parallel in identical conditions without the presence of the enzyme, the same oxidative cleavage
was observed, affording a mixture containing acids
2 and 3, besides 51% of intermediate anhydride 12
(1H NMR). The presence of intermediate 12 was highlighted by NMR spectroscopy. The 13C NMR

Molecules 2020, 25, 1882
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spectrum of the crude reaction mixture showed the presence of two singlets at 169.73 and 169.67 ppm
for the carboxylic carbon atoms of the anhydride, next to those around 180 ppm which belong to acids
2
and 3
and to the COOH group of compound 12. In the 1H NMR spectrum, the triplet of the CH2
groups linked to the CO-O-CO moiety is at 2.44 ppm, a little more deshielded than the triplet of the
CH2 beside COOH in compounds 2 and 3 and in the anhydride itself, occurring at 2.35 ppm. The 1H
and 13C NMR spectra of anhydride 12 are not known in the literature, and the spectroscopic data of
lauric anhydride reported in [37] were used as reference data.
The reaction was repeated in acetonitrile and only 11% of anhydride was found in the final mixture.
When the oxidation was carried out in toluene and 2 M H2SO4 was added to the reaction mixture
during the workup procedure, after having decomposed peroxy species with NaHSO3 saturated
solution, complete hydrolysis of intermediate 12 was obtained.
After investigation of every single step, the whole procedure was performed starting from 2 g of
commercial oleic acid. Acidic hydrolysis was performed soon after epoxidation in a one-pot procedure,
to afford diol derivative 10 as a pure compound at 70% isolated yield by filtration of the first crop of
crystalline material and recovery of other product by further treatment of the mother liquors. The
oxidation to dioxoderivative 11 gave a crude compound (75% purity by GC/MS) that was submitted
directly to the last step of oxidative cleavage in toluene with only 35% H2O2, to provide a mixture of
azelaic and pelargonic acids. Diacid
described in the literature [16], and based on the solubility of compound
between ethyl acetate and hot water afforded an aqueous phase from which azelaic acid crystallised
upon cooling. After three extraction cycles, diacid could be recovered as a pure compound in 73%
yield. Pelargonic acid was isolated from the organic phase at 77% isolation yields, showing 91%
chemical purity (GC/MS). The separation of diacid from compound was also investigated by using
column chromatography, eluting with hexane–EtOAc mixtures with an increasing amount of the more
2
was recovered following a procedure, which had been already
2
in hot water. Repartition
2
3
2
3
polar solvent (see Supplementary Material), affording pure 2 and 3 in slightly higher isolation yields
(81% and 84%, respectively).
3. Discussion
Ozonolysis of alkene bonds is a useful chemical transformation which is employed not only at the
laboratory level but also at industrial scale for the rapid and effective oxidative cleavage of C=C double
bonds [38]. The primary concern related to ozonolysis chemistry is represented by the serious safety
issues connected with the reaction, and in particular with the explosive hazard due to the instability of
intermediate ozonides. The present industrial production of azelaic acid is entirely based on ozonolysis
of oleic acid, being the global azelaic acid market valued at 94 million USD in 2017 and expected to
reach 140 million USD by 2025 [39]. The market is mainly driven by growing demand for plastics and
lubricants, which hold above 70% of global azelaic acid consumption.
The growing attention towards the development of safer and more environmentally friendly
production technologies has stimulated the investigation of alternative methods for the conversion
of oleic acid into azelaic acid. Suitable references have been reported in the Introduction. We gave
a contribution to this search by investigating a chemo-enzymatic approach to achieve the target
oxidative scission.
We decided to use the in-situ peroxidation of oleic acid
4 by lipase-mediated perhydrolysis in the
presence of hydrogen peroxide 35% as a safe procedure to afford the peroxycarboxylic acid needed to
promote the epoxidation step at the beginning of the synthetic sequence. Oleic acid itself undergoes the
conversion into the reactive peroxy species, so it is possible to avoid the use of an additional carboxylic
acid that would remain in the reaction mixture as a by-product to be removed from the desired final
compound. The advantage of the proposed procedure is also that storage and manipulation of peracid
are avoided: it is generated in the reaction medium, and the excess is destroyed at the end of its use.
The best biocatalyst for this reaction is Novozyme 435 which has the advantage of being an immobilized
form of C. Antarctica B that can be recovered and re-used. Preliminary experiments were performed

Molecules 2020, 25, 1882
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starting from 1 g of oleic acid, and recovering the enzyme by filtration and washing with water and
acetonitrile. The enzyme was kept at 4 ◦C for 18 h and re-used in a subsequent reaction. After four
runs the conversion of oleic acid (GC/MS analysis) into the epoxide was 78%. The evaluation of the
enzyme reusability in these experiments can only be used as a first orientation because it is influenced
by the effects of manipulation and storage on the enzyme support, overlapping those due to hydrogen
peroxide and peracid. Long-term performance of the enzyme should be best studied under continuous
process conditions, as suggested by recent literature [40]. Very positive results on the stability of this
lipase in this type of reaction have been obtained using both a packed-bed reactor [41] and a continuous
stirred tank reactor [40]. This kind of investigation is now in progress in our research group.
We considered also the possibility to avoid the isolation and purification of some of the
intermediates of our procedure to reduce quantities of waste, solvents and separation aids. We
chose the solvent of the epoxidation reaction to telescope the first two steps of the procedure,
and perform the acid-catalyzed opening of the oxirane ring without isolation of epoxide 9. Diol
derivative 10 could be obtained as a pure crystalline compound by crystallization from acetonitrile,
after quenching the peroxy species with NaHSO3 and promoting epoxide cleavage with catalytic
H2SO4 aqueous solution. No column chromatography was needed, thus favoring the isolation yield
of diol 10 and limiting further use of solvents. Even the purification of dioxostearic acid 11 could
be avoided, and the raw material was submitted directly to the following step to afford azelaic and
pelargonic acid. The use of toluene as a solvent for both the diol oxidation and the final oxidative
cleavage reaction will be useful in the future for developing the procedure in continuous flow mode.
For the last step of the whole sequence, we discovered the unexpected capability of H2O2 35%
w/w to promote the oxidative cleavage of diketone 11 in organic solvents, either toluene or acetonitrile,
at 30 ◦C, without the addition of any catalyst. H2O2 is considered as a green oxidant, generating water
as a by-product. It is safely stored and transported, and easily available on the market at a cheap price.
4. Materials and Methods
4.1. General Methods
Chemicals and solvents were purchased from Merck (Merck Life Science S.r.l., Milan, Italy) and
used without further purification. Trimethylsilyldiazomethane 10% solution in hexane (TCI Europe
N.V.) was purchased from Zentek Srl (Milan, Italy). Novozyme 435 (Novozymes) was purchased
from Strem Chemicals Inc. (Bischheim, France). TLC analyses were performed on Macherey Nagel
1
pre-coated TLS sheets Polygram® SIL G/UV254 purchased from Chimikart s.r.l. (Naples, Italy). H and
13C NMR spectra were recorded on a 400 or 500 MHz spectrometer in CDCl3 solution at r.t. The chemical
shift scale was based on internal tetramethylsilane. GC/MS analyses were performed using an HP-5MS
column (30 m
×
0.25 mm
×
0.25 µm, Agilent Technologies Italia Spa, Cernusco sul Naviglio, Italy). The
following temperature program was employed: 50 ◦C/10 ◦C min−1/250 ◦C (5 min)/50 ◦C min−1/300 ◦C
(10 min). The samples for GC/MS were treated with MeOH and trimethylsilyldiazomethane 10% in
hexane, to derivatize carboxylic acids by transformation into the respective methyl esters.
4.2. One-Pot Two-Step Synthesis of Threo-9,10-Didhydroxystearic Acid (10)
A suspension of Novozyme 435 (240 mg) in acetonitrile (48 mL), containing oleic acid (2.2 g, 91%
purity, 7.1 mmol) and 35% H2O2 w/w (1.1 mL, 12.8 mmol) was shaken in an orbital shaker (160 rpm,
50 ◦C) for 5 h. The enzyme was removed by filtration, washed with acetonitrile, and stored at 4 ◦C to
be re-used. A saturated solution of NaHSO3 (2 mL) was added to the filtrate, followed by the addition
of 2 M H2SO4 (963
sum of two crystallization crops). H NMR (CD3OD, 400 MHz) [42]:
2.29 (2H, t with J = 7.4 Hz, CH2COOH), 1.70–1.15 (26H, m, 13 CH2), 0.97–0.82 (3H, m, CH3). 13C NMR
(CD3OD, 100.6 MHz) [42]: = 177.6, 75.29, 75.26, 35.0, 34.0, 33.9, 33.0, 30.8, 30.7, 30.6, 30.42, 30.37, 30.2,
27.04, 26.96, 26.1, 23.7, 14.4. GC/MS (EI) as a methyl ester, obtained by treatment with MeOH and
µ
L). After 15 h at room temperature, diol 10 was recovered by filtration (1.62 g, 72%,
1
δ = 3.45–3.35 (2H, m, 2CHOH),
δ

Molecules 2020, 25, 1882
8 of 11
trimethylsilyldiazomethane 10% in hexane, tr = 23.85 min: m/z (%) = 294 (M+ – 36, 1), 187 (48), 155
(100), 138 (30).
4.3. Oxidation of 9,10-Dihydroxystearic Acid (9) to 9,10-Dioxostearic Acid (10)
A mixture of diol 10 (1.55 g, 4.9 mmol), Fe(NO3)3 9 H2O (20 mg, 0.049 mmol), TEMPO (8.0 mg,
·
0.049 mmol), and NaCl (3 mg, 0.049 mmol) in toluene (45 mL) was stirred at 100 ◦C for 4–5 h. The
reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was dried
and concentrated under reduced pressure to give the crude dioxo derivative 11 (1.83 g, 75% purity by
GC/MS analysis, estimated content of compound 11 1.37 g) which was employed in the successive
1
step without further purification. H NMR (CDCl3, 400 MHz) [43]:
δ = 2.72 (4H, t with J = 7.3 Hz,
2CH2CO), 2.35 (2H, t with J = 7.4 Hz, CH2COOH), 1.69–1.49 (6H, m, 3 CH2), 1.40–1.20 (16H, m, 8 CH2),
0.92–0.84 (3H, m, CH3). 13C NMR (CDCl3, 100.6 MHz) [43]:
δ
200.3, 200.2, 180.0, 36.2, 36.1, 34.2, 31.9,
29.4, 29.3, 29.2, 29.1, 29.04, 28.96, 24.2, 23.2, 23.1, 22.8, 14.2. GC/MS (EI) as a methyl ester, obtained by
treatment with MeOH and trimethylsilyldiazomethane 10% in hexane, tr = 22.48 min: m/z (%) = 326
(M+, 1), 295 (5), 185 (100), 141 (54).
4.4. Oxidative Cleavage of 9,10-Dioxostearic Acid (11) to Azelaic (2) and Pelargonic Acid (3)
A mixture of crude dioxo derivative 11 (1.75 g, 75% purity, estimated content of comp◦ound 11
4.21 mmol) and 35% H2O2 w/w (579 µL, 6.73 mmol) in toluene (35 mL) was stirred at 30 C for 3
h. A saturated solution of NaHSO3 (750
pH = 2. The reaction mixture was then extracted with ethyl acetate. The organic phase was dried
and concentrated under reduced pressure to give a crude mixture containing 93% (GC/MS) of acids
and
, which was heated at 50 ◦C for 1 h in a 1:1 mixture of EtOAc and water. Water was separated,
and diacid crystallized upon cooling. Other two extractions of the organic phase with hot water
allowed the isolation of diacid as a pure compound. Pelargonic acid was isolated from the organic
phase showing 91% chemical purity (GC/MS).
Azelaic acid (
): 578 mg (73%, >99% chemical purity by GC/MS and NMR); 1H NMR (CDCl3,
400 MHz) [44]: δ = 2.35 (4H, t with J = 7.4 Hz, 2CH2COOH), 1.75–1.55 (4H, m, 2CH2), 1.4–1.2 (6H, m,
3CH2). 13C NMR (CDCl3, 100.6 MHz) [44]:
= 180.2, 34.2, 29.0, 28.9, 24.3. GC/MS (EI) as a methyl
µL) was added, followed by the addition of H2SO4 2 M till
2
3
2
2
3
2
δ
ester, obtained by treatment with MeOH and trimethylsilyldiazomethane 10% in hexane, tr = 13.9 min:
m/z (%) = 185 (M+ - 31, 55), 152 (100), 143 (47), 111 (63).
Pelargonic acid (
= 2.35 (2H, t with J = 7.5Hz, CH2COOH), 1.75–1.55 (2H, m, CH2), 1.4–1.2 (10H, m, 5CH2), 0.80–0.95
(3H, m, CH3). 13C NMR (CDCl3, 100.6 MHz) [45]:
= 180.5, 34.2, 31.9, 29.3, 29.20, 29.18, 24.8, 22.7, 14.2.
3
): 512 mg (77%, 91% chemical purity by GC/MS); 1H NMR (CDCl3, 400 MHz) [45]:
δ
δ
GC/MS (EI) as a methyl ester, obtained by treatment with MeOH and trimethylsilyldiazomethane 10%
in hexane, tr = 9.33 min: m/z (%) = 172 (M+, 0.5), 141 (15), 129 (18), 87 (45), 74 (100).
5. Conclusions
The chemo-enzymatic conversion of oleic acid into azelaic and pelargonic acids herein described
represents a sustainable alternative to ozonolysis, currently employed at the industrial scale. Azelaic
acid can be produced in high chemical purity in 44% isolation yield after three steps, avoiding
column chromatography purifications. Intermediate diol 10 and final azelaic acid
2 are purified by
crystallization from acetonitrile and water, respectively. The procedure shows some valuable aspects,
even if it is not a one-pot process, as those using H2O2 and tungsten derivatives already known in the
literature [13–18].
The reagents of the three steps are: (i) H2O2 35% for the epoxidation of acid
cleavage of diketone 11, and atmospheric oxygen for diol 10 oxidation, both producing H2O as a
by-product; (ii) H2O for the acid-catalyzed hydrolysis of both epoxide and anhydride 12, generating
4 and the oxidative
9
no side-product, being fully incorporated in the reacting products. The organic solvents used during
the reactions are limited to acetonitrile and toluene; water and ethyl acetate are employed for quenching

Molecules 2020, 25, 1882
9 of 11
and separation procedures. The final oxidative cleavage of dioxo derivative 11 occurs in mild conditions
and generates a very tiny quantity of oxidized impurities, thus increasing the economic value of the
process, and reducing the complexity and cost of final azelaic acid purification. Hydrogen peroxide is
itself very effective in promoting the cleavage with no need for catalysts or harsh acidic or alkaline
conditions and generating water as a side-product. The reaction medium can be either toluene or
acetonitrile. The use of an enzymatic method to produce in situ H2O2 will be considered for further
development of the process.
Studies are now in progress to apply this synthetic procedure to soapstock recovered from the
neutralization step during vegetable seed oil refining at Oleificio Zucchi. A pre-treatment step has to
be added where the lipase-mediated hydrolysis of the triglycerides, which are inevitably present in
this by-product, is carried out.
A further development of the process will also be the optimization of the entire sequence in
continuous flow mode, taking advantage of the fact the enzyme employed for the generation of the
key peroxy species is already marketed in immobilized form. Besides an expected higher productivity
value and an advantage for the process scalability study, the use of a continuous flow reactor will most
likely increase the stability of lipase, and allow for a more reliable evaluation of lipase reusability.
Supplementary Materials: The following are available online, Table S1: Effect of H2O2/oleic acid molar ratio and
temperature on the chemo-enzymatic epoxidation of oleic acid, Table S2: Effect of the amount of lipase and solvent
on the chemo-enzymatic epoxidation of oleic acid, Table S3: Effect of the amount of aqueous 2M sulfuric acid
on the ring-opening of epoxystearic acid
9, Characterization of 8-((2SR,3RS)-3-octyloxiran-2-yl)octanoic acid (9),
Recovery and re-use of Novozyme 435, Procedure for the oxidative cleavage of 9,10-dioxostearic acid (11) to give a
mixture of 9-(nonanoyloxy)-9-oxononanoic acid (12), azelaic (
separation of azelaic (2) and pelargonic acid (3).
2), and pelargonic acid (3), Column chromatography
Author Contributions: Conceptualization, E.B. and D.T.; methodology, E.B., D.T., and G.D.L.; experimental
procedure, M.V., M.C.G., F.T., and D.C.; characterization, F.G.G., M.V., M.C.G., and G.D.L.; data analysis, F.T., D.C.,
E.B.; writing—original draft preparation, E.B., F.T., D.C.; writing—review and editing, E.B., D.T. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by Fondazione Cariplo—INNOVHUB, project SOAVE (Seed and vegetable
Oils Active Valorization through Enzymes), grant number 2017-1015.
Conflicts of Interest: The authors declare no conflict of interest.
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