Synthesis and characterization of polyethylene terephthalate (PET) precursors and potential degradation products: Toxicity study and application in discovery of novel PETases

Article abstract of DOI:10.1016/j.chemosphere.2021.130005

Polyethylene terephthalate (PET) is widely used material and as such became highly enriched in nature. It is generally considered inert and safe plastic, but due to the recent increased efforts to break-down PET using biotechnological approaches, we realized the scarcity of information about structural analysis of possible degradation products and their ecotoxicological assessment. Therefore, in this study, 11 compounds belonging to the group of PET precursors and possible degradation products have been comprehensively characterized. Seven of these compounds including 1-(2-hydroxyethyl)-4-methylterephthalate, ethylene glycol bis(methyl terephthalate), methyl bis(2-hydroxyethyl terephtahalate), 1,4-benzenedicarboxylic acid, 1,4-bis[2-[[4-(methoxycarbonyl)benzoyl]oxy]ethyl] ester and methyl tris(2-hydroxyethyl terephthalate) corresponding to mono-, 1.5-, di-, 2,5- and trimer of PET were synthetized and structurally characterized for the first time. In-silico druglikeness and physico-chemical properties of these compounds were predicted using variety of platforms. No antimicrobial properties were detected even at 1000 μg/mL. Ecotoxicological impact of the compounds against marine bacteria Allivibrio fischeri proved that the 6 out of 11 tested PET-associated compounds may be classified as harmful to aquatic microorganisms, with PET trimer being one of the most toxic. In comparison, most of the compounds were not toxic on human lung fibroblasts (MRC-5) at 200 μg/mL with inhibiting concentration (IC50) values of 30 μg/mL and 50 μg/mL determined for PET dimer and trimer. Only three of these compounds including PET monomer were toxic to nematode Caenorhabditis elegans at high concentration of 500 μg/mL. In terms of the applicative potential, PET dimer can be used as suitable substrate for the screening, identification and characterization of novel PET-depolymerizing enzymes.

Full text of DOI:10.1016/j.chemosphere.2021.130005

Chemosphere 275 (2021) 130005
Contents lists available at ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Synthesis and characterization of polyethylene terephthalate (PET)
precursors and potential degradation products: Toxicity study and
application in discovery of novel PETases
a
b
b
ꢀ
ꢁ c
Milica Djapovic , Dusan Milivojevic , Tatjana Ilic-Tomic , Marija Ljesevic ,
Efstratios Nikolaivits d, Evangelos Topakas d, Veselin Maslak a
,
,
**
Jasmina Nikodinovic-Runic b
,
*
a University of Belgrade, Faculty of Chemistry, Studentski Trg 16, P.O. Box 51, Belgrade, 11158, Serbia
b Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000, Belgrade, Serbia
c University of Belgrade-Institute of Chemistry, Technology and Metallurgy, Njegoseva 12, 11000, Belgrade, Serbia
d Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou 9, 15780,
Athens, Greece
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ PET plastic precursors and possible
degradation products toxicologically
evaluated.
ꢀ Low toxicity observed in vitro on
human fibroblasts.
ꢀ Three compounds harmful to nema-
tode Caenorhabditis elegans only at
high concentration.
ꢀ Six compounds classified as toxic and
moderately toxic against Allivibrio
fischeri.
ꢀ PET dimer and trimer are used as
substrates for PET hydrolyzing
enzyme.
a r t i c l e i n f o
a b s t r a c t
Polyethylene terephthalate (PET) is widely used material and as such became highly enriched in nature.
It is generally considered inert and safe plastic, but due to the recent increased efforts to break-down PET
using biotechnological approaches, we realized the scarcity of information about structural analysis of
possible degradation products and their ecotoxicological assessment. Therefore, in this study, 11 com-
pounds belonging to the group of PET precursors and possible degradation products have been
comprehensively characterized. Seven of these compounds including 1-(2-hydroxyethyl)-4-
methylterephthalate, ethylene glycol bis(methyl terephthalate), methyl bis(2-hydroxyethyl ter-
ephtahalate), 1,4-benzenedicarboxylic acid, 1,4-bis[2-[[4-(methoxycarbonyl)benzoyl]oxy]ethyl] ester and
methyl tris(2-hydroxyethyl terephthalate) corresponding to mono-, 1.5-, di-, 2,5- and trimer of PET were
synthetized and structurally characterized for the first time. In-silico druglikeness and physico-chemical
properties of these compounds were predicted using variety of platforms. No antimicrobial properties
Article history:
Received 13 December 2020
Received in revised form
11 February 2021
Accepted 14 February 2021
Available online 17 February 2021
Handling Editor: Willie Peijnenburg
Keywords:
Polyethylene terephthalate (PET)
PET dimer
were detected even at 1000 mg/mL. Ecotoxicological impact of the compounds against marine bacteria
PET trimer
* Corresponding author.
** Corresponding author.
E-mail addresses: vmaslak@chem.bg.ac.rs (V. Maslak), jasmina.nikodinovic@
imgge.bg.ac.rs, jasmina.nikodinovic@gmail.com (J. Nikodinovic-Runic).
https://doi.org/10.1016/j.chemosphere.2021.130005
0045-6535/© 2021 Elsevier Ltd. All rights reserved.

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Toxicity
PETase
Allivibrio fischeri proved that the 6 out of 11 tested PET-associated compounds may be classified as
harmful to aquatic microorganisms, with PET trimer being one of the most toxic. In comparison, most of
the compounds were not toxic on human lung fibroblasts (MRC-5) at 200
centration (IC50) values of 30 g/mL and 50 g/mL determined for PET dimer and trimer. Only three of
these compounds including PET monomer were toxic to nematode Caenorhabditis elegans at high con-
centration of 500 g/mL. In terms of the applicative potential, PET dimer can be used as suitable substrate
for the screening, identification and characterization of novel PET-depolymerizing enzymes.
© 2021 Elsevier Ltd. All rights reserved.
mg/mL with inhibiting con-
m
m
m
1. Introduction
been described as capable of partially degrading PET to oligomers
or even monomers (Kawai et al., 2019; Qiu et al., 2020; Ru et al.,
2020). Noteworthy, all known PET hydrolases have been charac-
terized using BHET as a model substrate of choice, and all have
relatively low turnover rates, which makes their use for the effi-
cient bioremediation almost impossible. Nevertheless, biodegra-
dation and other biotechnological approaches offer a tremendous
opportunity for waste treatment and valorization and are under
intensive development. Novel PETases may be available from the
untapped microbial diversity, therefore, a more suitable substrate
allowing for the efficient screening methodology would be crucial.
Our goal was to synthetize the library of compounds that could
be seen either as PET building blocks or as products of PET degra-
dation (Fig. 1) and to assess their toxicity, as well as the potential to
be used as substrates for PET hydrolyzing enzymes. We tested the
toxicity of the compounds using the marine luminescent bacteria,
Allivibrio fischeri, healthy human lung fibroblast cell line (MRC-5)
and terrestrial nematode Caenorhabditis elegans.
Since its discovery, its first synthesis, and its patenting in 1941,
polyethylene terephthalate (PET) has become a widely used ma-
terial in a number of industrial branches ranging from packaging,
textile and fabrics, films, automotive, electronics and many more
(Malik et al., 2017; Sargent et al., 2019). The global PET resin pro-
duction reached 30.3 million tons in 2017 (www.statista.com). The
high production rate is also reflected in the PET share in the post-
consumer plastic waste (Ragaert et al., 2017). Although it can be
perceived as an ideal plastic for recycling, due to its high melting
temperature and the possibility to process it without the use of
additives, coupled with possibility to easier separate and collect the
main PET product, beverage bottles, only approximately 30% of PET
is recycled (Ragaert et al., 2017; Wierckx et al., 2015). Nowadays in
the focus of the research community are the innovative ways to use
the post-consumer waste PET as a feedstock for new materials with
higher values (Tiso et al., 2020; Zhou et al., 2019).
Due to its extensive use, PET is highly enriched in nature, but is
generally considered inert and “safe” plastic (Zimmermann et al.,
2019). It’s toxicity is usually associated with the leach of anti-
mony upon exposure to heat (Wittkowski et al., 2019). Nevertheless
‘phthalates’ have also been labeled as toxic and possible endocrine
disruptors (Sathyanarayana, 2008; Sax, 2010). More recently,
microplastics and bigger fragments of plastic are recognized as
specific problem found worldwide in oceans and terrestrial envi-
ronments (Gao et al., 2019; Zhang et al., 2020). Microplastics were
recently found in the snow of once pristine Mount Everest (Napper
et al., 2020), high mountain lakes of Alps (Pastorino et al., 2020), as
well as in the deep-marine environments (Kane and Clare, 2019).
PET debris is often eaten by fish and other marine creatures and as
such enters the food chain (Webb et al., 2013). On the other side,
plastic in agricultural soils poses a risk for drinking water supplies
(Wanner, 2021). The first evidence of microplastic in human
placenta was reported recently (Ragusa et al., 2021).
Microorganisms are often examined as a frontline against
various pollution, including the plastic one (Amobonye et al., 2020).
In 2016, bacterium Ideonella sakaiensis was reported to degrade
amorphous PET when cultured in the presence of yeast extract as
an additional carbon source (Yoshida et al., 2016). Subsequently,
PETase and mono-(2-hydroxyethyl)TPA (MHET)ase enzymes, the
ester-bond hydrolyzing enzymes of this strain have been reported
and their activity was improved via protein engineering (Austin
et al., 2018; Palm et al., 2019). It has been shown that PETase cat-
alyzes the depolymerization of PET to bis(2-hydroxyethyl)-TPA
(BHET), MHET, and terephthalic acid (TPA). MHETase converts
MHET to TPA and ethylene glycol (EG) (Austin et al., 2018). Some
known cutinases, including the ones from Humilica insolens (HiC),
Pseudomonas mendocina (PmC), and Fusarium solani (FsC), were
also shown to be catalytically active using low-crystallinity PET
films as model substrates (Kawai et al., 2019; Ronkvist et al., 2009).
On the other side, only few species of bacteria and fungi have
2. Materials and methods
2.1. Compound synthesis and structural properties assessment
Model compounds (5e11; Fig. 1A) were synthesized according
to established procedures for preparation of different esters. Two
main approaches were explored: i) SchotteneBaumann reaction for
acylation of alcohols with acyl halide in the presence of organic
bases, and ii) esterification of acids with alcohols in the presence of
DCC, known as Steglich esterification (Fig. S1). Synthetic protocols
required optimizations in terms of ratio of reactants and other re-
action conditions. All compounds were isolated as pure compounds
and were well characterized using NMR and IR spectroscopy as well
as mass spectrometry.
All chromatographic separations were performed on Silica
10e18, 60 Å, ICN Biomedicals. Standard techniques were used for
the purification of reagents. 1H and 13C NMR spectra were recorded
with Bruker Avance at 500 MHz (1H at 500 MHz, 13C at 125 MHz),
and Varian/Agilent NMR 400 MHz (1H at 400 MHz, 13C at 100 MHz).
Chemical shifts (d) are expressed in ppm and coupling constant (J)
in Hz. TMS was used as an internal standard. The following
abbreviation were used for signal multiplicities (brs ¼ broad
singlet, s ¼ singlet, t ¼ triplet, q ¼ quartet, dd ¼ doublet of doublets,
tt ¼ triplet of triplets, m ¼ multiplet). IR spectra (ATR) were
recorded with a Perkin-Elmer-FT-IR 1725X spectrophotometer,
n
values are given in cmꢁ1. Mass spectra were obtained on MS LTQ
Orbitrap XL. Melting points were determined on the Electrothermal
WRS1B apparatus and were reported uncorrected.
2.1.1. - (bis(2-Ethoxyethyl)terephthalate) (BEET)
A solution of terephthaloyl chloride 12 (10.16 g; 0.05 mol; 1 eq)
in toluene (60 mL) was added dropwise to a solution of 2-
ethoxyethanol 13 (12.10 mL; 0.125 mol; 2.5 eq) and pyridine
2

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Fig. 1. Structures of PET precursors and possible degradation products: A) Commercially available compounds: EG ¼ ethylene glycol, TPA ¼ terephthalic acid, DMTP ¼ dimethyl
terephthalate, BHET ¼ bis(2-hydroxyethyl) terephthalate. New or previously not structurally characterized compounds: BEET ¼ bis(2-ethoxyethyl) terephthalate, EGDB ¼ 1,2-
Ethylene glycol dibenzoate, M(HET)1
¼
1-(2-hydroxyethyl)-4-methylterephthalate, M2(HET)1.5
¼
ethylene glycol bis(methyl terephthalate), M(HET)2
¼
methyl bis(2-
hydroxyethyl terephtahalate), M2(HET)2.5 ¼ 1,4-Benzenedicarboxylic acid, 1,4-bis[2-[[4-(methoxycarbonyl)benzoyl]oxy]ethyl] ester and M(HET)3 ¼ methyl tris(2-hydroxyethyl
terephthalate). Compounds 9 and 11 are methyl esters of PET dimer and PET trimer, respectively. B) Schematic diagram of possible PET oligomers.
(10.10 mL; 0.125 mol; 2.5 eq) in toluene (40 mL) over 15 min. The
reaction mixture was stirred at 60 ꢂC for 5 h and the resulting
pyridinium salt was separated by filtration. The filtrate was diluted
with ethyl acetate (60 mL) and washed with 1 M HCl, saturated
NaHCO3 and brine. The organic solution was dried over anhydrous
Na2SO4, concentrated in vacuo and purified by short-path distilla-
tion, to afford a diester 5 (Fig. 1) as colorless oil (11.17 g, 72%, bp
159e162 ꢂC/0.2 mmHg) (Chase, 1963).
1H NMR (400 MHz, CDCl3): dH 8.06 (dd, J ¼ 8.2, 1.0 Hz, 4H), 7.57
(tt, J ¼ 7.0, 1.2 Hz, 2H), 7.44 (t, J ¼ 7.7 Hz, 4H), 4.67 (s, 4H). 13C NMR
(100 MHz, CDCl3): dc 166.5, 133.3, 130.0, 129.8, 128.6, 62.9. IR (ATR)
nmax: 3145, 3068, 2977, 2911,1713,1601,1481,1453,1338,1258,1119,
704.
7 - 1-(2-Hydroxyethyl)-4-methylterephthalate (M(HET)1; PET
monomer).
Monomethyl terephthalate (15): KOH (1.46 g; 26 mmol; 1 eq)
was dissolved in methanol (52.2 mL) with stirring at 80 ꢂC and
solution of dimethyl terephthalate 3 (5.00 g; 26 mmol; 1 eq) and
57.4 mL of toluene was added. The white suspension was heated at
120 ꢂC for 5 h. The mixture was filtered, and solid salt was washed
with dichloromethane. The unreacted dimethyl terephthalate was
removed in this way. The solid salt was dissolved in water (70 mL),
filtered to clarity and acidified with concentrated HCl. The precip-
itate of monomethyl terephthalate was filtered, washed with water
and dried to yield a product as a white solid (3.65 g; 78%, mp 214 ꢂC)
(Konosonoks et al., 2005; Li et al., 2015).
1H NMR (500 MHz, CDCl3): dH 8.11 (s, 4H), 4.51e4.46 (m, 4H),
3.81e3.73 (m, 4H), 3.58 (q, J ¼ 7.0 Hz, 4H), 1.23 (t, J ¼ 7.0 Hz, 6H). 13
C
NMR (125 MHz, CDCl3): dc 165.9, 134.1, 129.8, 68.4, 66.9, 64.8, 15.3.
IR (ATR) nmax: 2976, 2871, 1723, 1577, 1449, 1408, 1385, 1272, 1117,
732. HRMS (ESI): m/z [MþNa]þ calculated for C16H22O6: 333.1314;
found: 333.1311.
6 - 1,2-Ethylene glycol dibenzoate (EGDB).
To a solution of ethylene glycol 1 (6.30 mL; 0.110 mol; 1 eq) and
pyridine (17.8 mL; 0.220 mol; 2 eq) in toluene (120 mL) benzoyl
chloride (33.1 mL; 0.275 mol; 2.5 eq) was added dropwise over
15 min. The reaction mixture was heated at 120 ꢂC for 4 h and the
resulting pyridinium salt was separated by filtration. The filtrate
was diluted with ethyl acetate (80 mL) and washed with 1 M HCl,
saturated NaHCO3 and brine, successively. The organic solution was
dried over anhydrous Na2SO4. The drying agent was filtered off and
the filtrate was concentrated in vacuo. The crude product was
recrystallized from ethanol, and diester 6 was obtained as white
solid (27.60 g; 93%, mp 71 ꢂC) (Ishido et al., 1977; Ren et al., 2011).
1H NMR (400 MHz, DMSO): dH 8.05 (s, 4H), 3.87 (s, 3H), 3.63 (brs,
COOH). 13C NMR (100 MHz, DMSO):
dc 166.7, 165.8, 134.9, 133.3,
129.7, 129.5, 52.6. IR (ATR) nmax: 3428, 3014, 2959, 1721, 1505, 1438,
1410, 1340, 1259, 1127, 1019, 957.
1-(2-Benzyloxyethyl)-4-methylterephthalate (16): A solution of
dicyclohexylcarbodiimide (DCC) (0.68 g; 3.32 mmol; 1.2 eq) and
dichloromethane (5 mL) was added dropwise to a cold (0 ꢂC) sus-
pension of monomethyl terephthalate 15 (0.50 g; 2.77 mmol; 1 eq),
3

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
2-(benzyloxy)ethanol 18 (0.46 g; 3.01 mmol; 1.1 eq), 4-
dimethylaminopyridine (DMAP) (68 mg; 0.56 mmol; 0.2 eq) and
dichloromethane (20 mL). The reaction was carried out at room
temperature for 4 h. The byproduct dicyclohexylurea (DCU) was
removed by filtration and precipitate was washed three times with
ethyl acetate. The filtrate was concentrated and the residue was
purified by dry flash chromatography (SiO2; eluent: petroleum
ether/ethyl acetate ¼ 9:1), to afford 0.72 g (82%) of benzyl ether 16,
as a colorless oil.
1499, 1453, 1409, 1378, 1360, 1272, 1099, 1022, 731, 700.
4-((2-(Benzyloxy)ethoxy)carbonyl)benzoic acid (20): Potas-
sium hydroxide (0.26 g; 4.7 mmol; 1 eq) was dissolved in 2-(ben-
zyloxy)ethanol (0.71 g; 4.7 mmol; 1 eq) with stirring and gentle
heating. Then, solution of bis(2-benzyloxyethyl) terephthalate 19
(2.04 g; 4.7 mmol; 1 eq) and toluene (45.0 mL) was added, and
reaction mixture was heated at 120 ꢂC for 3.5 h. After cooling to
room temperature, the reaction mixture was filtered. The solid
ionic salt was washed with ethyl acetate. Solid product was then
dissolved in water (40 mL), acidified with HCl (conc.) to pH ¼ 2,
whereby the product separated as a colloidal precipitate. After 12 h,
the mixture was filtered, and solid product was washed with water
and dried. 4-((2-(Benzyloxy) ethoxy)carbonyl)benzoic acid 20 was
obtained as white solid (0.58 g; 42%, mp 185 ꢂC).
1H NMR (400 MHz, CDCl3): dH 8.11 (s, 4H), 7.38e7.27 (m, 5H),
4.61 (s, 2H), 4.55e4.50 (m, 2H), 3.95 (s, 3H), 3.84e3.80 (m, 2H). 13
C
NMR (100 MHz, CDCl3): dc 166.4, 165.9, 138.0, 134.1, 134.0, 129.8,
129.6, 128.6, 127.9, 127.8, 73.3, 67.9, 64.7, 52.6. IR (ATR) nmax: 3385,
3061, 3031, 2953, 2861, 1723, 1557, 1500, 1438, 1409, 1380, 1274,
1104, 1102, 878, 732, 701.
1H NMR (400 MHz, CDCl3): dH 8.20e8.13 (m, 4H), 7.39e7.27 (m,
5H), 4.63 (s, 2H), 4.57e4.52 (m, 2H), 3.86e3.81 (m, 2H). 13C NMR
(100 MHz, CDCl3): dc 170.8, 165.8, 137.9, 134.8, 133.2, 130.3, 129.9,
128.6, 128.0, 127.9, 73.3, 67.9, 64.7. IR (ATR) nmax: 3361, 3061, 3031,
3008, 2960, 2892, 2855, 1718, 1577, 1499, 1470, 1446, 1389, 1366,
1268, 1136, 1268, 1136, 1093, 1012, 987, 870, 788, 727, 690.
1-(2-Hydroxyethyl)-4-methyl terephthalate (7): A mixure of
benzyl ether 16 (0.72 g; 2.3 mmol), ethyl acetate (40 mL) and 10%
palladium on charcoal (35 mg) was exposed to hydrogenolysis at 45
psi for 3 h in Parr apparatus at room temperature. The mixture was
filtered through a plug of celite and the solvent was removed in
vacuo. PET monomer 7 was obtained as a white solid after recrys-
tallization from methanol (0.47 g, 91%, mp 87 ꢂC) (Kudrna, 1964;
Yasukawa et al., 2016).
2-((4-((2-(Benzyloxy)ethoxy)carbonyl)benzoyl)oxy)ethyl
methyl terephthalate (21): A catalytic amount of DMAP (26.6 mg;
0.22 mmol; 0.2 eq) was added into the solution of PET monomer 7
(270.0 mg; 1.20 mmol; 1.1 eq), acid 20 (328.8 mg; 1.09 mmol; 1 eq)
and dichloromethane (12 mL). The mixture was cooled in an ice
bath, and a solution of DCC (269.8 mg; 1.31 mmol; 1.2 eq) in
dichloromethane (3 mL) was added dropwise. The obtained reac-
tion mixture was stirred at 0 ꢂC for 5 min, and then at room tem-
perature for 20 h. The byproduct, dicyclohexylurea (DCU), was
removed by filtration and washed with ethyl acetate three times.
The combined organic solution was concentrated on rotavapor, and
residue was purified by dry flash chromatography (SiO2; dichloro-
methane/petroleum ether/ethyl acetate ¼ 60:40:5). Protected PET
dimer 21 was obtained as a white solid (353.5 mg, 64%, mp 79 ꢂC).
1H NMR (400 MHz, CDCl3): dH 8.12 and 8.11 (two overlaping
singlets in ratio 4:4, 8H), 7.35e7.27 (m, 5H), 4.71 (s, 4H), 4.61 (s, 2H),
4.52e4.49 (m, 2H), 3.94 (s, 3H), 3.84e3.79 (m, 2H). 13C NMR
1H NMR (400 MHz, CDCl3): dH 8.14e8.08 (m, 4H), 4.52e4.47 (m,
2H), 4.00e3.96 (m, 2H), 3.95 (s, 3H), 1.95 (brs, OH). 13C NMR
(100 MHz, CDCl3): dc 166.2, 166.0, 134.1, 133.6, 129.6, 129.5, 67.0,
61.2, 52.4. HRMS m/z [MþNa]þ calculated for C11H12O5: 247.0582;
found: 247.0586.
8 - Ethylene glycol bis(methyl terephthalate) (M2(HET)1.5).
A solution of methyl 4-(chlorocarbonyl)benzoate (17) (16.68 g;
0.084 mol; 2 eq) and dichloromethane (80 mL) was added over
30 min into the solution of ethylene glycol (2.35 mL; 0.042 mol; 1
eq), pyridine (10.2 mL; 0.126 mol; 3 eq) and dichloromethane
(100 mL) at 0 ꢂC. The reaction mixture was stirred for 12 h at room
temperature. The resulting pyridinium salt was filtered, and filtrate
was washed successively with saturated aqueous CuSO4, water,
saturated NaHCO3 and brine, then dried over Na2SO4. The organic
solution was concentrated and residue was purified by dry flash
chromatography (SiO2; eluent: dichloromethane/petroleum ether/
ethyl acetate ¼ 60:40:5), to afford of diester 8 as a white solid
(10.25 g, 63%, mp 163 ꢂC) (Fuentes et al., 2015).
(100 MHz, CDCl3): dc 166.3, 165.8, 165.7, 138.0, 134.34, 134.28, 133.7,
133.6, 129.9, 129.82, 129.79, 129.77, 128.6, 128.0, 127.8, 73.3, 67.9,
64.7, 63.1, 52.6. IR (ATR) nmax: 2933, 2857, 1725, 1649, 1503, 1451,
1409, 1373, 1278, 1102, 1021, 876, 732, 701.
1H NMR (400 MHz, CDCl3): dH 8.10 (brs, 8H), 4.70 (s, 4H), 3.94 (s,
Methyl bis(2-hydroxyethyl terephthalate (9): The catalytic
amount of 10% palladium on charcoal (16 mg) was added into the
solution of benzyl ether 21 (320.4 mg; 0.63 mmol) and 1,4-dioxane
(8 mL). The hydrogenolysis of protected dimer was performed
under hydrogen atmosphere, with rubber balloon filled with
hydrogen, at room temperature for 4 h. The reaction mixture was
filtered through a plug of celite, solvent was removed in vacuo and
the crude product was purified by dry flash chromatography. The
unreacted protected dimer was eluted using dichloromethane/
ethyl acetate ¼ 9:1, and then the deprotected dimer was eluted
with ethyl acetate. PET dimer 9 was obtained as a white solid
(230.1 mg, 87%; mp 128 ꢂC).
6H). 13C NMR (100 MHz, CDCl3):
dc 166.3, 165.7, 134.3, 133.6, 129.82,
129.77, 63.1, 52.6. IR (ATR) nmax: 3423, 3059, 2965, 1723, 1505, 1443,
1410, 1378, 1292, 1127, 1102, 977, 725. HRMS (ESI): m/z [MþNa]þ
calculated for C20H18O8: 409.0899; found: 409.0893.
9 - Methyl bis(2-hydroxyethyl terephthalate) (M(HET)2, PET
dimer).
bis(2-Benzyloxyethyl) terephthalate (19): A solution of ter-
ephthaloyl chloride 12 (1.06 g; 5.2 mmol; 1 eq) and toluene (10 mL)
was added dropwise to a solution of 2-(benzyloxy)ethanol 18
(1.98 g; 13.0 mmol; 2.5 eq), pyridine (1.1 mL; 13.0 mol; 2.5 eq) and
toluene (10 mL) during 15 min. The reaction mixture was stirred at
60 ꢂC for 5 h and the resulting pyridinium salt was separated by
filtration. The filtrate was diluted with ethyl acetate (15 mL) and
washed with 1 M HCl, saturated NaHCO3 and brine, successively.
The extract was dried over anhydrous Na2SO4, concentrated in
vacuo. The crude product was purified by dry flash chromatography
(SiO2; eluent: toluene/ethyl acetate ¼ 8:2). The product 19 was
obtained as colorless oil, which solidified after several days (2.04 g,
89%, mp 47 ꢂC).
1H NMR (400 MHz, CDCl3): dH 8.11and 8.10 (two overlapping
singlets in ratio 4:4, 8H), 4.70 (s, 4H), 4.51e4.47 (m, 2H), 4.00e3.96
(m, 2H), 3.94 (s, 3H), 1.94 (brs, OH). 13C NMR (100 MHz, CDCl3):
dc
166.3, 166.1, 165.7, 165.6, 134.3, 134.0, 133.8, 133.6, 129.9, 129.83,
129.80, 129.7, 67.2, 63.2, 63.1, 52.6. IR (ATR) nmax: 3390, 3013, 2962,
1718, 1581, 1506, 1439, 1412, 1388, 1344, 1276, 1130, 1069, 1021, 894,
871, 727. HRMS m/z [MþNa]þ calculated for C21H20O9: 439.1005;
found: 439.0992.
1H NMR (400 MHz, CDCl3): dH 8.10 (s, 4H), 7.36e7.23 (m, 10H),
4.60 (s, 4H), 4.54e4.48 (m, 4H), 3.83e3.78 (m, 4H). 13C NMR
10e1,4-Benzenedicarboxylic acid, 1,4-bis[2-[[4-(methox-
ycarbonyl)benzoyl]oxy]ethyl] ester (M2(HET)2.5).
(100 MHz, CDCl3):
73.15, 67.8, 64.5. IR (ATR) nmax: 3062, 3030, 2951, 2863, 1722, 1580,
d
c 165.7, 137.8, 133.9, 129.6, 128.4, 127.8, 127.7,
A solution of methyl 4-(chlorocarbonyl)benzoate 17 (595.8 mg;
3.0 mmol; 2 eq) and dichloromethane (5 mL) was added into the
4

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
solution of bis(2-hydroxyethyl)terephthalate
4
(381.4 mg,
from https://www.molinspiration.com/.
1.5 mmol, 1 eq), pyridine (364 ml; 4.5 mmol; 3 eq) and dichloro-
methane (10 mL) at 0 ꢂC over 15 min. The reaction mixture was
stirred at room temperature for 12 h. Resulting pyridinium salt was
separated by filtration. The filtrate was washed with saturated
aqueous CuSO4, water, saturated NaHCO3 and brine successively,
and then dried over anhydrous Na2SO4. The solvent was evaporated
under reduced pressure, and the residue was purified by dry flash
chromatography (SiO2; eluent: dichloromethane/toluene/ethyl
acetate ¼ 60:40:3). Product 10 was obtained as a white solid
(537.6 mg, 62%, mp 199 ꢂC) (Brooke et al., 2002).
2.2. Biological evaluations
2.2.1. Bacterial MIC assay
To compare planktonic antimicrobial activity, Escherichia coli
(NCTC 9001) and Staphylococcus aureus (ATCC 25923) were used as
test microorganisms in the microdilution assay in 96-well format
recommended by the National Committee for Clinical Laboratory
Standards (M07-A8). The highest concentrations of compounds
tested were 1000
mg/mL (which corresponded to 16.1 mM con-
1H NMR (400 MHz, CDCl3): dH 8.11 and 8.10 (two overlapping
singlets in ratio 4:8, 12H), 4.70 (s, 8H), 3.94 (s, 6H). 13C NMR
centration of compound 1 and 1.64 mM concentration of com-
pound 11) and the inoculum was 5 ꢃ 105 cfu/mL.
(100 MHz, CDCl3): dc 166.3, 165.7, 165.6, 134.3, 133.9, 133.6, 129.9,
129.81, 129.77, 63.2, 63.1, 52.6. IR (ATR) nmax: 3429, 3013, 2960,
1724, 1505, 1470, 1437, 1410, 1340, 1266, 1128, 1021, 838, 870, 723.
HRMS: m/z [MþNa]þ calculated for C30H26O12: 601.1322; found:
601.1314.
11 - Methyl tris(2-hydroxyethyl terephthalate) (M(HET)3, PET
trimer).
Protected PET trimer (22): A solution of DCC (198.0 mg;
0.96 mmol; 1.2 eq) and dichloromethane (2 mL) was added drop-
wise into the solution of PET dimer 9 (366.5 mg; 0.88 mmol; 1.1 eq),
acid 20 (240.1 mg; 0.80 mmol; 1 eq), DMAP (48.9 mg; 0.4 mmol; 0.5
eq) and dichloromethane (8 mL) at 0 ꢂC. Then reaction mixture was
stirred at room temperature for 24 h. DCU, as a byproduct, was
removed by filtration. The precipitate was washed three times with
ethyl acetate. The combined organic filtrate was collected and
concentrated on rotavapor. The residue was purified by dry flash
chromatography (SiO2; dichloromethane/petroleum ether/ethyl
acetate ¼ 60:40:3). The product 22 was obtained as a white solid
(268.4 mg, 48%, mp 134e136 ꢂC).
2.2.2. Aliivibrio fischeri toxicity test
The inhibitory effect on the light emission of A. fischeri (also
called Vibrio fischeri) was determined according to the ISO 11348
standard, using freeze-dried bacteria. Toxicity analysis was per-
formed with BioFix® Lumi-10 (Macherey-Nagel GmbH & Co. KG,
Duren, Germany). Freeze-dried bacteria (A. fischeri NRRL B-11177,
Macherey-Nagel GmbH & Co. KG, Duren, Germany) were recon-
stituted with the provided solution before the test. Bacteria were
incubated at 15 ꢂC with 1 mL of 2% (w/v) NaCl with different con-
centrations of PET compounds (1e11). Stock solutions of PET com-
pounds (50 mg/mL in DMSO) were diluted in 2% (w/v) NaCl up to
500
mg/mL. All samples except 1 (EG), 4 (BHET) and 5 (BEET) formed
a suspension when mixed with NaCl, hence, all of the 500
mg/mL
sample solutions were centrifuged for 5 min at 10,000 rpm. The
supernatants were transferred to a 1.5 mL tube and used for the
preparation of serial dilutions. Serial dilutions of all tested samples
were prepared by diluting each starting concentration by 50%
(500e15.625
mg/mL). The bioluminescence was monitored after
1H NMR (400 MHz, CDCl3): dH 8.115, 8.101, 8.098 (three over-
lapping singlets in ratio 4:4:4, 12H), 7.36e7.27 (m, 5H), 4.704, 4.698
(two overlapping singlets in ratio 4:4, 8H), 4.60 (s, 2H), 4.53e4.49
(m, 2H), 3.94 (s, 3H), 3.83e3.79 (m, 2H). 13C NMR (100 MHz, CDCl3)
dC 166.3, 165.8, 165.70, 165.68, 165.6, 137.9, 134.35, 134.33, 133.9,
133.65, 133.59, 129.9, 129.8, 129.78, 129.76, 128.6, 127.9, 127.8, 73.3,
67.9, 64.1, 63.19, 63.13, 52.6. IR (ATR) nmax: 3030, 2927, 2854, 1719,
1627, 1577, 1503, 1449, 1410, 1377, 1342, 1275, 1128, 1020, 876, 729.
PET trimer (11): The catalytic amount of 10% palladium on
charcoal (10 mg) was added into the solution of protected PET
trimer 22 (204.2 mg; 0.29 mmol) and 1,4-dioxane (6 mL). The
hydrogenolysis of protected PET trimer was performed under
hydrogen atmosphere, with rubber balloon filled with hydrogen, at
room temperature for 6 h. The reaction mixture was filtered
through a plug of celite, solvent was removed in vacuo and the
crude product was purified by dry flash chromatography. The
unreacted protected trimer was eluted using dichloromethane/
ethyl acetate ¼ 9:1, and then product of deprotection was eluted
with dichloromethane/ethyl acetate ¼ 1:1. PET trimer 11 was ob-
tained as a white solid (141.2 mg, 80%, mp 173 ꢂC).
15 min of incubation with the test solution.
2.2.3. Cytotoxicity assay
Cytotoxicity, as a measure of anti-proliferative effect on healthy
human lung fibroblast MRC-5 cell line (obtained from ATCC) was
determined using standard colorimetric MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
(Hansen et al., 1989). Each tested compound was added to the cells
at a concentration ranging from 25 to 250 m
g mLꢁ1 and the treat-
ment lasted for 48 h. The MTT assay was performed two times in
four replicates and the extent of MTT reduction was measured
spectrophotometrically at 540 nm using a Tecan Infinite 200 Pro
€
multiplate reader (Tecan Group Ltd., Mannedorf, Switzerland).
Cytotoxicity was expressed as the concentration of the compound
inhibiting cell growth by 50% (IC50) in comparison to control
(DMSO-treated cells).
2.2.4. Caenorhabditis elegans survival assay
C. elegans is a genetically tractable multicellular organism with
metabolically active digestive, reproductive, endocrine, sensory
and neuromuscular systems that has been widely used in toxicity
screens (Hunt, 2017; Wittkowski et al., 2019).
1H NMR (400 MHz, CDCl3): dH 8.113, 8.100 and 8.098 (three
overlapping singlets in ratio 4:4:4, 12H), 4.71 and 4.70 (two over-
lapping singlets in ratio 4:4, 8H), 4.51e4.47 (m, 2H), 4.00e3.96 (m,
2H), 3.94 (s, 3H), 1.60 (s, OH). 13C NMR (100 MHz, CDCl3): 166.4,
166.1,165.7,165.63,165.61,134.3,134.1,133.88, 133.87,133.79,133.6,
129.89, 129.88, 129.84, 129.81, 129.77, 67.2, 63.19, 63.16, 63.1, 61.4,
52.6. IR (ATR) nmax: 3425, 3014, 2964, 1717, 1581, 1506, 1469, 1439,
1411, 1389, 1343, 1271, 1129, 1070, 1021, 971, 870, 725. HRMS (ESI):
m/z [MþNa]þ calculated for C31H28O13: 631.1428; found: 631.1433.
Structural properties, logP, pKa, logD, solubility and geometry
were predicted using software available from https://chemicalize.
com/. Physico-chemical properties of PET molecules used for drug
likeness determination were predicted using software available
Caenorhabditis elegans N2 (glp-4; sek-1) was propagated under
standard conditions, synchronized by hypochlorite bleaching, and
cultured on nematode growth medium using E. coli OP50 as a food
source, as described previously (Stiernagle, 2006). The C. elegans
survival assay was carried out as described previously with some
modifications (Brackman et al., 2011; Scoffone et al., 2016). In brief,
synchronized worms (L4 stage) were suspended in a medium
containing 95% M9 buffer (3 g of KH2PO4, 6 g of Na2HPO4, 5 g of
NaCl, and 1 mL of 1 M MgSO4 ꢃ 7H2O in 1 L of water), 5% LB broth
(Oxoid, UK), and 10
mg of cholesterol (Sigma-Aldrich) per mL.
5

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Experiment was carried out in 96-well flat bottomed microtiter
range of 0.01e1 mM.
plates (Sarstedt, Germany) in final volume of 100
quotes (25 L) of this suspension of nematodes (25e35 nematodes)
were transferred to the wells of a 96-well microtiter plate where
50 L of medium were previously added. Next, 25 L solvent control
(DMSO) or 25 L of a concentrated compound solution was added
to the test wells. Final concentrations of the compounds were
500 g/mL, 250 g/mL, 125 g/mL, 62.5 g/mL made out from stock
solutions (50 mg/mL in DMSO) of each compound. Subsequently
the plates were incubated at 25 ꢂC for 2 days. The fraction of dead
worms was determined after 48 h by counting the number of dead
worms and the total number of worms in each well, using a ste-
reomicroscope (SMZ143-N2GG, Motic, Germany). The compounds
were tested at least three times in each assay and each assay was
repeated at least two times (n ꢄ 6). As negative control experiment,
nematodes were exposed to medium containing 1% (v/v) DMSO.
mL per well. Ali-
m
2.4. Statistical analysis
m
m
Descriptive statistics (mean and standard deviation, SD) were
used to describe the level of inhibition in different toxicity assays.
Differences in effective concentration between the treatment in
comparison to the untreated control were assessed using the
standard non-parametric test (ANOVA), and results were consid-
ered statistically significant at p < 0.01. Statistical analysis was
performed with STATISTICA 13.3 software.
m
m
m
m
m
3. Results and discussion
3.1. Synthesis and characterization
It is of a great importance to obtain pure oligomers of PET in
order to study their biological properties and to assess them as
platform molecules. They could be obtained either via partial hy-
drolysis of PET (top-down approach) or via synthesis step by step
(bottom up approach). Preparation of oligomers by partial hydro-
lysis of PET is less controllable process and usually followed by
prolonged and tedious chromatographic purification of the hy-
drolysis products. Synthesis of PET oligo esters, starting from
ethylene glycol (1) and terephthalic acid (2), as well as their
differently functionalized derivatives were developed over the last
60 years. Pioneering synthetic work in this field was carried out by
2.3. Application of PET dimer and trimer (9 and 11) in PETase
activity assessment
2.3.1. PET-hydrolase gene synthesis and expression
Synthetic gene of Humicola insolens cutinase (HiC) was con-
structed based on its deposited amino acid sequence (PDB ID:
4OYY) and was codon optimized for expression in Escherichia coli,
while cloned in pET26b(þ) vectors (GenScript Biotech BV, the
Netherlands). Recombinant expression vector was transferred to
E. coli BL21 (NewEngland Biolabs, Ipswich, Massachusetts, USA) and
enzyme was expressed based on
a methodology described
ꢁ
(Dimarogona et al., 2015). Briefly, overnight bacterial cultures (1%,
Zahn et al. (Penisson and Zahn, 1970; Zahn and Gleitsman, 1963),
v/v) were used to inoculate 500 mL LB medium supplemented with
followed by Haslin et al. (1980) and more recently by Brooke et al.
(2002). Possible PET oligomers can have free carboxylic group and
hydroxyl group or both of them on the ends of the chain (Fig. 1B). In
addition, these groups could be functionalized as ester and ether. It
is well known that molecules with polar groups, such as eOH and
eCOOH, usually have better solubility in water and polar solvents
and these properties could be tuned by chemical modification.
With this in mind, we determined a set of model compounds 1e11
for this study (Fig. 1A).
100 m
g mLꢁ1 ampicillin, and culture was incubated at 37 ꢂC to mid-
exponential phase (OD600 of approximately 0.6). Then cultures
were cooled down to 16 ꢂC during 1 h, when 0.2 mM isopropyl 1-
thio-b-D-galactopyranoside (IPTG) was added to the medium and
was left to incubate overnight. After that time E. coli cells were
harvested by centrifugation at 4000ꢃg for 15 min at 4 ꢂC and
resuspended in 50 mM Tris-HCl pH 8, 300 mM NaCl buffer. Cell
suspension was disrupted by sonication during four 1-min cycles
(8 s pulses and 5 s pause) at 40% amplitude using a 20 kHz high
intensity (400 W) ultrasonic processor (VC 400, Sonic & Materials,
Newtown, CT, USA). Cell debris was removed by centrifugation at
20,000ꢃg, 30 min, 4 ꢂC twice and loaded onto an immobilized
metal-ion (Co2þ) affinity chromatography (IMAC), as previously
described (Nikolaivits et al., 2016). The purity of isolated enzyme
was checked on SDS-PAGE electrophoresis (12.5% (w/v) poly-
acrylamide) and protein concentration was determined by
measuring the absorbance at 280 nm, based on the calculated
molar extinction coefficient.
Model compounds 5, 6,
8 and 10 were obtained by
SchotteneBaumann reaction, starting from acid chlorides and al-
cohols. This is typical procedure for preparation of esters, involving
the addition of acid chloride into the solution of alcohol and the
organic base. However, the yield of these esters was highly
dependent on the reaction conditions and required substantial
optimization to achieve yields of more than 70%. Diesters 5 and 6
were obtained by addition of acid chloride into the solution of
appropriate alcohol/diol and pyridine in toluene at 0 ꢂC (Fig. S1).
After usual work up products were isolated in good yields (72% and
71%). Compound 10 was identified as main side product of the
synthesis of compound 8 and both were obtained in more than 60%
yields in separate reactions (Fig. S1).
2.3.2. Enzymatic hydrolysis reactions and analysis of hydrolysis
products
Reactions containing 1 mg/mL of each substrate (BHET, 8
(M2(HET)1.5), 9 (M(HET)2) and 10 (M2(HET)2.5)) in 0.1 M sodium
phosphate buffer pH 8 were initiated by adding 0.5 mМ of HiC and
PET monomer 7, dimer 9 and trimer 11 were prepared by
coupling reaction between carboxylic acid and alcohols in the
presence of dicyclohexyl carbodiimide (DCC), well known as Steg-
lich procedure (Neises and Steglich, 1978). This mild procedure was
applied in reaction with sensitive substrates such as alcohols 18, 7
and 9, which in reaction with appropriate acids gave benzyl pro-
tected monomer 16, dimer 21 and trimer 22 in 82%, 64% and 48%
yields, respectively (Fig. S1). Deprotection of these compounds was
performed by hydrogenolysis and PET models 7, 9 and 11 were
obtained in excellent yields (80e91%). All NMR spectra showing
purity of the compounds are depicted in Fig. S2-S14.
left to incubate at either 30 ꢂC or 60 ꢂC for 24 h. After that time 0.1%
of 6 M HCl was added in each reaction and centrifuged at 5000ꢃg at
10 ꢂC. Supernatant was collected and analyzed on a SHIMADZULC-
20AD HPLC equipped with a SIL-20A autosampler. The column used
was a C-18 reverse-phase NUCLEOSIL®100-5 (Macherey-Nagel,
Germany) and the mobile phase was 20% acetonitrile, 20% 10 mM
sulfuric acid in ultrapure water at a flow rate of 0.8 mL/min.
Detection of terephthalic acid (TPA) and its derivatives took place
with a photodiode array detector Varian ProStar at 241 nm.
Quantification of TPA, MHET and BHET was performed by con-
structing calibration curves with standard concentrations in the
Molecular descriptors that characterize electronic and structural
properties of compounds have provided useful predictive infor-
mation on the toxicity of compounds (Bakire et al., 2018).
6

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Calculated structural properties, including logP, pKa, logD, solubil-
ity and geometry were determined for all 11 compounds with a
view of providing information that is important for biological
properties determination (Table 1 and Table 2). In addition, pre-
dicted physico-chemical properties of PET molecules used for drug
likeness determination were also estimated (Table 3).
permeability of potential new drug based on the molecular weight,
octanol/water partition coefficient, number of H-bond donors and
number of H-bond acceptors is known as “Rule of five” (Lipinski,
2004). Briefly, poor absorption or permeation are more likely to
occur when the molecule has molecular weight more than 500,
logP over 5, and contains more than 5 H-bond donors or 10 (2 ꢃ 5)
H-bond acceptors. The critical limit for acceptable drug-likeness is
that no more than one violation of the rule exists in molecule. We
have subjected all PET related compounds to this in silico assess-
ment and notably, only 10 (M2(HET)2.5) and 11 (M(HET)3) have
three violations each (Table 3). The rest of investigated PET com-
pounds obey the “Rule of five” and meet all criteria for good solu-
bility and permeability.
For investigated compounds with miLogP below 4 (all except 8
(M(HET)1), 10 (M2(HET)2.5)) and 11 (M(HET)3)) can be suggested
that they have favorable physicochemical profiles for oral
bioavailability (Veber et al., 2002). In addition, sufficient oral
bioavailability is expected for molecules with 10 rotatable bonds or
fewer (Veber et al., 2002), which is fulfilled in 6 different PET
compounds with 10 (M2(HET)2.5) and 11 (M(HET)3) having 18 and
20 ratable bonds, respectively (Table 3).
Topological polar surface area (TPSA) can be defined as a sum of
the surface areas occupied by the oxygen and nitrogen atoms and
the hydrogen atoms attached to them and represent the hydrogen
bonding capacity of the molecules, as such represents good pre-
dictor of the drug transport properties, drug absorption. Molecules
with TPSA <140 Å2 have good intestinal absorption, while those
with TPSA <60 Å2 show good bloodebrain barrier penetration
(Prasanna and Doerksen, 2009). Only compounds 10 (M2(HET)2.5)
and 11 (M(HET)3) do not fall into this category, and 1 (EG), 3
(DMTP) and 6 (EGDB) have the potential to cross the bloodebrain
barrier (Table 3). Having concluded that most of the compounds
have good solubility and good potential to cause effect of living cells
we have assessed them across different systems to evaluate their
toxicity.
The acid dissociation constant (pKa), the 1-octanolewater
partition coefficients (Kow) and logP as the 10-base logarithmic
measure of the Kow and solubility were estimated for all com-
pounds being useful for the environmental risk assessment of
chemicals (Table 2). For example 1-octanolewater systems display
similarities to the partition of compounds between water and the
biological membranes of microorganisms or cells, due to the
amphiphilic nature of 1-octanol, similar to a generalized lipid phase
in terms of their dielectric properties (Turner and Williamson,
2005). Correlations between the toxicity results and Kow values
have been successfully obtained for diverse classes of compounds
(Czerwinski et al., 2006), Szwej et al. (2015). Given that compounds
having ionizable groups exist in solution as a mixture of different
ionic forms, the ionization of those groups, depends on the pH of
the environment, therefore LogD (the distribution constant) is the
more appropriate descriptor for lipophilicity of ionizable com-
pounds because it accounts for the pH dependence of a molecule in
aqueous solution (Table 2). Solubility as an ability for a given sub-
stance to dissolve in a solvent and logS as the 10-based logarithm of
the solubility measured in mol/L was also predicted for each of the
compounds using H2O as a solvent (Table 2). Polarity and polariz-
ability presented in Table 1 are also useful descriptors for the pre-
diction of chemical reactivity and bioactivity of given compounds
(Tandon et al., 2020).
Compounds 1, 2 and 4 have polar groups on the ends, while
compounds 3, 5, 6, 8 and 10 have ether or ester non-polar groups on
the ends. PET oligomers 7, 9 and 11 have free OH group on the end
and methyl ester on the other side of the chain. Overall, polar
compounds among PET-associated library are 1, 2 and 4, while
majority of monoesters (7, 9 and 11) and diesters (3, 5, 6, 8 and 10)
are nonpolar (Tables 1 and 2). Compounds 8, 9 and 10, corre-
sponding to 1.5-, di- and 2.5-mer of PET, can accept 4,5 and 6
hydrogen bonds (Table 1). With logP of higher than 4, predicted for
10 (M2(HET)2.5) and 11 (M(HET)3) these compounds can be
considered hydrophobic (Table 2).
3.2. Toxicity analysis
Aquatic toxicity is required in the assessment of the toxicity of
organic chemicals to marine and freshwater organisms, therefore
A. fischeri (also called Vibrio fischeri) model system was employed in
the assessment of PET model compounds (Fig. 2). A. fischeri is a
bioluminescent, Gram-negative marine bacterium that can be
found free living and in a mutualistic association with certain
squids and fishes and has been successfully employed as model
organism in toxicity evaluations of number of chemicals (Di Nica
In the search for new potential drug candidates establishing
druglikeness is very important step. Druglikeness allows the
assessment of the pharmacokinetic profile of the tested molecules
based on the prediction of their absorption and distribution. An
effective methodology for estimation of potential solubility and
Table 1
Predicteda structural properties of PET model compounds 1e11.
PET compound
1
2
3
4
5
6
7
8
9
10
11
Atom count
10
4
0
1
18
12
0
2
24
14
0
4
32
18
0
8
44
22
0
12
1
34
20
0
7
2
28
16
0
6
1
46
28
0
11
2
50
30
0
13
68
42
0
18
72
44
0
20
Non-hydrogen atom count
Asymmetric atom count
Rotatable bond count
Ring count
0
1
1
1
2
3
3
Aromatic ring count
Hetero ring count
0
0
1
2
2
0
1
0
0
2
4
0
1
0
0.2
0
2
1
0
0.33
2
4
1
0
0.5
0
4
2
0
0.12
0
2
1
0
0.27
1
3
2
0
0.2
0
4
2
0
0.24
1
5
3
0
0.2
0
6
0
157.8
56.2
146.39
3
0
0.23
1
7
Fsp3b
Hydrogen bond donor count
Hydrogen bond acceptor count
Formal charge
0
0
0
0
0
0
0
0
Topological polar surface area (Å2)
Polarizability (Å3)
40.46
5.72
14.55
74.6
15.08
40.57
52.6
19.21
50.11
93.06
24.2
62.69
71.06
31.65
81.69
52.6
28.76
74.2
72.83
21.7
56.4
105.2
37.7
98.25
125.43
40.2
104.54
178.03
58.69
152.68
Molar refractivity (cm3/mol)
a
predicted values obtained using https://chemicalize.com/.
Fsp3 ¼ sp3 carbon atom count/total carbon atom count.
b
7

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Table 2
Predicteda pKA, LogP and solubility of PET compounds 1e11.
PETcompound
1
2
3
4
5
6
7
8
9
10
11
pKa
14.83
3.32
1.288
ndb
14.79
0.6
nd
2.60
Nd
15.1
1.29
nd
15.1
3.099
nd
15.1
4.907
LogP
pH
ꢁ1.209
logD
1.98
3.782
3.789
5.598
1.7
4.6
6.5
7.4
8
pH
ꢁ1.21
ꢁ1.21
ꢁ1.21
ꢁ1.21
ꢁ1.21
1.28
1.98
1.98
1.98
1.98
1.98
0.6
0.6
0.6
0.6
0.6
2.60
2.60
2.60
2.60
2.60
3.78
3.78
3.78
3.78
3.78
1.29
1.29
1.29
1.29
1.29
3.79
3.79
3.79
3.79
3.79
3.10
3.10
3.10
3.10
3.10
5.60
5.60
5.60
5.60
5.60
4.91
4.91
4.91
4.91
4.91
ꢁ0.32
ꢁ3.67
ꢁ4.91
ꢁ5.40
Solubility [logS]
1.7
4.6
6.5
7.4
1.14
1.14
1.14
1.14
1.14
1.14
High
ꢁ1.6
ꢁ2.03
ꢁ2.03
ꢁ2.03
ꢁ2.03
ꢁ2.03
ꢁ2.03
High
ꢁ1.71
ꢁ1.71
ꢁ1.71
ꢁ1.71
ꢁ1.71
ꢁ1.71
High
ꢁ2.77
ꢁ2.77
ꢁ2.77
ꢁ2.77
ꢁ2.77
ꢁ2.77
High
ꢁ4.06
ꢁ4.06
ꢁ4.06
ꢁ4.06
ꢁ4.06
ꢁ4.06
Moderate
ꢁ1.88
ꢁ1.88
ꢁ1.88
ꢁ1.88
ꢁ1.88
ꢁ1.88
High
ꢁ4.48
ꢁ4.48
ꢁ4.48
ꢁ4.48
ꢁ4.48
ꢁ4.48
Moderate
ꢁ4.24
ꢁ4.24
ꢁ4.24
ꢁ4.24
ꢁ4.24
ꢁ4.23
Moderate
ꢁ6.35
ꢁ6.35
ꢁ6.35
ꢁ6.35
ꢁ6.35
ꢁ6.35
Low
ꢁ6.01
ꢁ6.01
ꢁ6.01
ꢁ6.01
ꢁ6.01
ꢁ6.02
Low
0
0.39
0.39
0.39
ꢁ1.61
High
8
Intrinsic solubility
Solubility category
a
Predicted values obtained using https://chemicalize.com/.
nd ¼ not determined using the prediction software.
b
Table 3
Predicteda physico-chemical properties of PET model molecules 1e11 used for drug likeness determination.
PET compound
1
2
3
4
5
6
7
8
9
10
11
miLogP
TPSA
natoms
Mw
ꢁ0.95
1.76
74.6
12
166.13
2.28
52.61
14
194.19
4
0.83
93.07
18
254.24
2.62
71.08
22
310.35
6
3.71
52.61
20
270.28
1.55
72.84
16
224.21
4.06
105.22
28
386.36
8
3.33
125.45
30
416.38
9
5.83
157.83
42
578.53
12
5.11
178.06
44
608.55
13
40.46
4
62.07
2
nON
4
6
4
5
nOHNH
nviolations
nrotb
2
0
1
2
0
2
0
0
4
2
0
8
0
0
12
0
0
7
1
0
6
0
0
11
1
0
13
0
3
18
1
3
20
volume
62.27
138.05
173.1
223.22
291.88
244.99
198.16
334.05
359.11
495
520.06
LogP e Octanol-water partition coefficient.
TPSA e Molecular Polar Surface Area.
natoms e Number of atoms in molecule.
Mw e Molecular weight.
nON e Number of nitrogen and oxygen atoms in molecule.
nOHNH e Number of amino and hydroxyl groups.
nviolations e Number of violated Lipinski rules.
nrotb e Number of rotable bonds.
volume e Volume of molecule.
a
predicted values obtained using www.molinspiration.com.
et al., 2017). The inhibition of bioluminescence in A. fischeri is
considered more sensitive for nonspecific toxicity than some other
available bioanalytical approaches (Neale et al., 2012).
not toxic and not harmful to aquatic organisms. The NOEC (no
observed effect concentration) on fish, daphnia and algae for EG is
above 100 mg/L (European Chemicals Agency, dossier 15,973). The
EC50 for the algae Pseudokirchneriella subcapitata is 36.64 g/L
Compound 6 (EGDB) showed the highest toxicity to A. fischeri
ꢁ
with EC50 value lower than 7.81
mg/mL (Table S1). In addition to 6,
(Aruoja et al., 2014). According to Macario et al. (Macario et al.,
compound 11 (M(HET)3) with EC50 lower than 10
be considered as toxic (Passino and Smith, 1987; Ventura et al.,
2016), while compounds 2 (TPA) and 5 (BEET) with EC50 between
10 and 100 mg/mL (27.1 and 43.0, respectively) were deemed as
moderately toxic. The rest of the compounds could be considered
harmless (EC50 values higher than 125 mg/L). Compounds 1,4, 7, 8,
9, 10 have showed very low inhibition of A. fischeri biolumines-
cence, and the highest inhibition was around 30% for the com-
pounds 4, 7 and 10 (Fig. 2). Since all observed effects were lower
than 50%, EC50 could not be calculated. With the exception of
compound 9, there was no concentration-inhibition dependence
observed, and thus EC20 and EC10 values could not be calculated
either (Table S1).
m
g/mL could also
2018), the EC50 value after 15 min of A. vibrio exposure to
ethylene glycol was 87.75 g/L. In a different report, the EC50 value
after 15 min was 621 ppm, corresponding to approximately 0.62 g/L
(Docherty and Kulpa, 2005). TPA (2) also has no adverse effect in
both short and long term ecotoxicity tests on aquatic organisms.
The lowest concentration of test substance which results in a 50
percent reduction in growth rate (ErC50) in the tests with fish,
Daphnia and algae was for algae (ErC50: >19.0 mg TPA/L) (European
Chemicals Agency, dossier 15,563). Ventura et al. (2016) showed
that there was no toxic effect on V. fisheri (the EC50 could not be
determined). This differs from our results, since we have obtained
the EC50 value of 27.1 mg/L for TPA. In their study, Ventura et al.
stated that TPA is non-toxic and that EC50 could not be defined due
to very low solubility in water, and there is no report of the con-
centrations used for the test. In our study, the stock solutions of
compounds were prepared in DMSO, which could account for the
Observed results for EG (1) are in line with the REACH (Regis-
tration, Evaluation, Authorisation and Restriction of Chemicals)
registration dossier for EG and higher glycols that are considered
8

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Fig. 2. Inhibition of A. fischeri bioluminescence upon exposure to PET compounds (average the SD and comparison to the untreated control, p < 0.01).
observed difference. TPA levels in the environment have been
estimated by the US Environment Protection Agency and range
from 0.038 mg/L in marine water to 50 mg/L in a sewage treatment
plants (data available from https://www.epa.gov/risk/regional-
screening-levels-rsls). These values are also available for com-
showed acute toxicity on A. fischeri (Figs. 2 and 3). BHET (4) is also
produced and used on the large scale and no toxicity or environ-
mental hazards data are available (Qiu et al., 2020). In this study, 4
was not harmful to A. fischeri and C. elegans, while it caused 30%
decrease in MRC-5 cell proliferation at 200 mg/mL (Figs. 2 and 3).
pounds 1 (0.004
m
g/L of tap water and 0.013 mg/kg of residential
PET bottled water that contained BHET didn’t induce any cytotoxic,
genotoxic or endocrine disruptive effect (Bach et al., 2013; Mao
et al., 2005). BHET is the common model compound used in the
screen for enzymatic depolymerization of PET and it is also major
intermediate in biotechnological upcycling of PET (Allen, 2019;
Palm et al., 2019; Tiso et al., 2020).
soil) and 3 (0.019 mg/L of tap water and 0.08 mg/kg of residential
soil) and not for 4 (BHET).
The effect of compounds on pathogenic bacteria, healthy human
lung fibroblast MRC-5 cell line and nematode C. elegans was
assessed in order to gain deeper understanding and more specific
effects (Fig. 3). None of the compounds were affecting the growth of
In summary, PET trimer (11) was toxic on A. fischeri and MRC-
5 cells, PET monomer (7) was toxic on cells and nematode, while
PET dimer (9) was toxic on cells and not on other two systems.
Dimethyl terephthalate (3) was toxic on nematode and A. fischeri.
Overall, not any clear tendency in respect to the chemical features
of the compounds studied could be inferred about their toxicity
levels in three systems employed within this study. Possibly the
effect of the presence of free hydroxyl groups contributed to
toxicity of 11, 9 and 7 on different systems. Also, the most hydro-
phobic compound 11, was the most toxic in two studied systems. It
has been previously shown that increasing hydrophobicity of fu-
rans and derivatives decreased toxicity on A. fischeri (Ventura et al.,
2016). However, the results depicted in Figs. 2 and 3 show that the
hydrophobic/hydrophilic nature of these compounds is not enough
to explain all the results, and that other structural descriptors may
play the important role.
common human pathogens E. coli and S. aureus even at 1000 mg/mL
(results not shown). This result may indicate that they do not
possess any antimicrobial properties, however they can possibly be
internalized and bio-augmented by bacteria throughout the food
chain, which is a risk to be taken into consideration. Most of the
compounds were not toxic to cells at 200
for 9 (M(HET)2),11 (M(HET)3) and 7 (M(HET)1) IC50 values of 30, 50
and 100 g/mL, respectively were calculated. These three being
mg/mL (Fig. 3A), however
m
monoesters with free hydroxyl group (Fig. 1). Compound 11 (PET
trimer), although violating Lipinski ‘Rule of Five’ and as such not
predicted to have biological activity, exhibited inhibition of cell
proliferation, possibly due to the formation of suspension upon
application onto cells. 10 (M2(HET)2.5) was causing about 20e30%
cell death irrespective of the amount applied (Fig. 3A). When the
compounds were assessed using nematode model system, 7
(M(HET)1) and 3 (DMTP) were the most toxic, in the concentration
of 500
mg/mL, while most of the compounds were not toxic in
3.3. Enzymatic hydrolysis of PET model compounds
250 g/mL (Fig. 2B). Noteworthy is the fact that concentrations of
m
test compounds of 500
C. elegans model.
TPA (2) although on the Hazardous Substance List due to the fact
that can cause irritation of nose, throat and lungs upon breathing,
but widely used even as an additive to poultry feeds (Slinger et al.,
1962), did not show any adverse effects on cells and C. elegans, but
mg/mL are considered quite high in
Degradability of polymeric materials is a function of their
structures, the presence of degradative microbial population and
the environmental conditions that encourage microbial growth.
Our goal in this study was to test whether these novel synthesized
PET oligomers could be hydrolyzed by a proven PET depolymerase
and act as a substrate for screening and characterization of such
9

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
Fig. 3. Effect of PET model compounds on A) healthy human fibroblast MRC-5 cell line survival rate in comparison to DMSO treated control that was set to 100%, and B) percent
survival of C. elegans (average the SD). The results are expressed as the percent survival after 48 h of treatment and compared to the untreated control (p < 0.01).
enzymes. HiC has proven to be an efficient depolymerase having
the ability to hydrolyze various types of polyesters (Gigli et al.,
2019; Su et al., 2020; Weinberger et al., 2017). However, the ma-
jority of studies performed so far utilize enzymes of bacterial origin
for the degradation of PET (Kawai et al., 2019). HiC was used for the
first time in 2009 probably as a crude preparation provided by
Novozymes for the degradation of low crystallinity (lcPET e 7%
crystallinity) and biaxially oriented (boPET e 35% crystallinity) PET
films (Ronkvist et al., 2009). Even though HiC resulted in 97%
weight loss of lcPET after 96 h at 70 ꢂC, the degradation of boPET
was a lot lower. However, the authors reported that the hydrolysis
product was almost exclusively TPA. On the other hand, Carniel
et al. studied the synergism between HiC (Novozym©51,032 crude
enzymatic preparation) and lipase CALB aiming to increase TPA
yield and overall hydrolysis of pretreated PET-bottle films (Carniel
et al., 2017). The combination of these enzymes increased PET to
TPA yield 7.7 times leading to mole fraction of TPA up to 0.88 after
14 days of reaction at 50 ꢂC. Additionally, HiC has been used for
treatment of chemically treated PET fibers, increasing the TPA yield
from 85% to 97% (after 24 h at 50 ꢂC) by hydrolyzing residual
oligomers (Quartinello et al., 2017).
HiC was firstly tested on BHET at a concentration of 1 mg/mL. At
60 ꢂC BHET was autohydrolyzed completely after 24 h to MHET and
TPA. Nonetheless, at 30 ꢂC only a small amount was hydrolyzed at
the control reactions (approx. 0.6 mM MHET), when the enzyme
completely hydrolyzed BHET producing almost completely MHET
and a small amount of TPA (0.06 mM). Even so, BHET is a water-
soluble compound that can be hydrolyzed by various esterases/li-
pases that do not necessarily hydrolyze insoluble hydrophobic PET
(Barth et al., 2016; Ion et al., 2020; Palm et al., 2019). Consequently,
we tested the activity of HiC on the insoluble compounds 8
(M2(HET)1.5), 9 (M(HET)2) and 10 (M2(HET)2.5), which constitute
of 1.5, 2 and 2.5 PET monomers, respectively. Overall, five products
were detected with the applied method: 2 (TPA), MHET, 4 (BHET),
monomethyl TPA (MTPA) and 7 (M(HET)1), which is the methylated
MHET (Fig. 4, Table S2; Fig. S15).
Compound 10 (M2(HET)2.5) proved to be a very resilient sub-
strate to autohydrolysis and enzymatic hydrolysis, with only traces
of the products detected after 24 h (Table S2). However, comparing
the different reaction temperatures we can see that at 60 ꢂC only
TPA and methyl TPA were detected, while at 30 ꢂC mostly MHETand
MTPA with fewer amounts of BHET and M(HET)1. We suspect that,
since the enzymatic mechanism in both temperatures is the same,
TPA at 60 ꢂC was derived from the autohydrolysis of enzymatically
produced MHET. Similarly, the absence of M(HET)1 might be a
result of its autohydrolysis to methyl TPA. Considering that HiC has
a very low activity on MHET (according to the test reaction with
BHET), the enzyme probably preferentially cleaves 10 (M2(HET)2.5)
at the first main-chain ester bond from the sides of the molecule,
releasing two MTPA units and BHET, which is further converted to
In this work, we utilized the purified recombinant form of HiC in
order to limit interference from other enzymes or medium com-
ponents. Reactions were performed at pH 8 at either 30 or 60 ꢂC.
The pH was selected considering that HiC could act well at pH 8, but
also that most PET-hydrolyzing enzymes have an optimum pH at
8e9 (Nikolaivits et al., 2018). The two temperatures were chosen in
order to cover the cases of both thermophilic and mesophilic
enzymes.
10

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
cultures or agar plates, but also as an inducer for polyester-
hydrolyzing enzymes (Liebminger et al., 2007; Ribitsch et al.,
2011). Recently, ethylene glycol bis(p-methylbenzoate), resem-
bling 8 (M2(HET)1.5), has been utilized as substrate for the devel-
opment of a turbidimetric assay for high-throughput screening of
ꢁ
PET oligomer-hydrolyzing enzymes (Belisario-Ferrari et al., 2019).
These model compounds contain benzoic acid moiety and due to
that are more amenable to microbial and enzymatic attack, in
contrast to compounds 7e11, which were designed to encompass
only TPA and EG fragments, resembling more closely to realistic PET
fragments (Fig. 1), leading to the discovery of more efficient PET
depolymerases.
4. Conclusion
Microplastics have moved into virtually every crevice on Earth,
with PET being in the top of widely utilized materials. Recent efforts
to biotechnologically degrade waste PET and valorize building
blocks are evident (Tan et al., 2019; Tiso et al., 2020). In a view of
their extensive occurrence either from natural degradation of PET
waste accumulated in nature or from the (bio)industrial degrada-
tion, library of 11 compounds, building blocks or hydrolysis prod-
ucts of PET, have been synthesized and thoroughly characterized in
terms of structure and their toxicity. The structural and toxicity
data for most of these important molecules was not existing in the
available literature.
Fig. 4. Products of enzymatic reactions identified after incubation of HiC with 1 mg/mL
of each M2(HET)1.5 and M(HET)2 for 24 h at 30 ꢂC or 60 ꢂC.
MHET. Hence, compound 10 (M2(HET)2.5) can be utilized as a
substrate for screening purposes for the discovery or protein en-
gineering of highly PET-active enzymes, especially thermophilic
ones, even in a high throughput manner in a 96-well microtiter
plate format. Recently, this approach was applied for the PET
derived nanoparticles (Pfaff et al., 2021).
Different levels of toxicity are found among these closely related
compounds, across panel of tests, which may indicate different
mechanisms of toxicity with the main driving force for the
observed toxicity compounds’ lipophilicity, and thus their pene-
tration into the cell. Some of the compounds exhibited a consid-
erable level of toxicity to A. fischeri indicating their environmental
hazard. Overall PET-trimer (M(HET)3), methyl tris(2-hydroxyethyl
terephthalate), may have the highest toxicity, indicating the
longer the oligomers released the greater the risk. Our under-
standing of polymer degradation has been advanced in recent
years, but the lack of information available on the participating
microorganisms and particularly enzymatic mechanisms involved
is evident. The obtained results suggest that compounds reported
in this study, especially PET dimer, may be a more suitable substrate
for the assessment of novel PETases and, as such, should be adopted
as an alternative to BHET.
Using compounds 8 and 9, 1.5- and dimer respectively, we
noticed that the latter results in 10 times higher product concen-
tration in all cases (Fig. 4). For both compounds, there was also a
high amount of products detected at 60 ꢂC in control reactions
(absence of enzyme) compared to the control reactions at 30 ꢂC,
where no products were detected. High temperature in combina-
tion with slightly alkaline pH led to autohydrolysis of these sub-
strates. In both cases, the concentration of products detected for the
enzymatic reaction were 2-fold higher compared to the control
reactions. At 30 ꢂC autohydrolysis for both starting compounds was
zero, so we could more easily observe the enzyme’s mode of action.
For 8 (M2(HET)1.5) the main product was methylated TPA (MTPA)
(279
M(HET)1 would be released, what we noticed were the small
amounts of MHET (33 М) and TPA (4 М). These could derive from
mМ). Even though one would suspect that equal amounts of
m
m
the cleavage of the resulting M(HET)1 either at the methyl or the
ethylene glycol ester bonds.
The reaction that led to the highest product release was the one
with the compound 9 (M(HET)2), representing the PET dimer. In
this case, MTPA was the main product (2.3 mM) followed by MHET
(1.7 mM) and BHET (0.2 mM). Clearly HiC cleaves at the first main-
chain ester bond from the MTPA side releasing MTPA and BHET,
which is further converted to MHET and a small amount in TPA (42
CRediT author contribution statement
Milica Djapovic: Synthesis methodology, Investigation, Data
curation, Writing - original draft. Dusan Milivojevic: Toxicity
assessment, Data curation, Writing - editing. Tatjana Ilic-Tomic:
Investigation, Cell toxicity methodology, Writing - original draft.
m
М). The small amount of liberated M(HET)1 detected (87 mM)
ꢀ
ꢁ
Marija Ljesevic: Investigation, eco-toxicity assessment, Writing -
original draft. Efstratios Nikolaivits: Investigation, Enzymatic
degradation and HPLC methodology, Writing - review and editing.
Evangelos Topakas: Investigation, Resources, Writing e editing.
Veselin Maslak: Conceptualization, Resources, Writing - review and
editing. Jasmina Nikodinovic-Runic: Conceptualization, Funding
acquisition, Writing - review and editing.
might show a secondary first cleavage point at the center of the
molecule. Studies of the hydrolysis of compound 9 (M(HET)2) with
a another polyesterase have shown a different mode of action for
the two enzymes (Nikolaivits et al., 2020). This fact could imply that
PET dimer can be used as a substrate for various polyesterases and
potentially correlate their hydrolysis mechanism (based on product
release) with their ability to degrade PET polymer.
The first and most used PET model substrate is bis (benzoylox-
yethyl) terephthalate (3PET), resembling 10 (M2(HET)2.5), and has
been successfully utilized for the discovery of microbes and en-
zymes that had the ability to modify the surface of PET fabrics
(Fischer-Colbrie et al., 2004; Heumann et al., 2006). 3PET has also
been used as a sole carbon source for screening microbes in liquid
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
11

M. Djapovic, D. Milivojevic, T. Ilic-Tomic et al.
Chemosphere 275 (2021) 130005
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mentierter polyetherester: synthese definierter oligomerer des poly-
butylenterephthalats. Makromol. Chem. 181, 301e323.
Heumann, S., Eberl, A., Pobeheim, H., Liebminger, S., Fischer-Colbrie, G., Almansa, E.,
Cavaco-Paulo, A., Gübitz, G.M., 2006. New model substrates for enzymes
hydrolysing polyethyleneterephthalate and polyamide fibres. J. Biochem. Bio-
phys. Methods 69, 89e99.
Acknowledgements
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under grant
agreement No 870292 (BioICEP).
Hunt, P.R., 2017. The C. elegans model in toxicity testing. J. Appl. Toxicol. 37, 50e59.
Ion, S., Voicea, S., Sora, C., Gheorghita, G., Tudorache, M., Parvulescu, V.I., 2020.
Sequential biocatalytic decomposition of BHET as valuable intermediator of PET
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2021.130005.
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