Bioactive (co)oligoesters with antioxidant properties-synthesis and structural characterization at the molecular level

Article abstract of DOI:10.1039/c6ra09870c

A contemporary synthetic route, starting from the bioactive compound via the corresponding glycidyl ester and β-substituted β-lactone to (homo)- and (co)oligoesters with a bioactive moiety covalently linked as pendent groups along an oligomer backbone, was reported. The bioactive compounds were selected from antioxidants used in cosmetics. Two models of bioactive (homo)- and (co)oligoesters were synthesized via anionic ring-opening (co)oligomerization of p-methoxybenzoyloxymethylpropiolactone (p-AA-CH₂-PL) initiated by p-anisic acid sodium salt. An analytical protocol was developed for a detailed structural characterization at the molecular level of these bioactive (co)oligoesters. The molecular level structure of the obtained bioactive (homo)- and (co)oligoesters was established by electrospray ionization tandem mass spectrometry (ESI-MS/MS) supported by ¹H NMR analysis. Additionally, the results presented here are important for the analysis of designed biodegradable polymeric controlled-release systems of bioactive compounds with potential applications in cosmetology.

Full text of DOI:10.1039/c6ra09870c

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PAPER
Bioactive (co)oligoesters with antioxidant
properties – synthesis and structural
Cite this: RSC Adv., 2016, 6, 57751
characterization at the molecular level
a
b
b
ac
Magdalena Maksymiak, Tomasz Bałakier, Janusz Jurczak, Marek Kowalczuk
and Grazyna Adamus*a
A contemporary synthetic route, starting from the bioactive compound via the corresponding glycidyl ester
and b-substituted b-lactone to (homo)- and (co)oligoesters with a bioactive moiety covalently linked as
pendent groups along an oligomer backbone, was reported. The bioactive compounds were selected
from antioxidants used in cosmetics. Two models of bioactive (homo)- and (co)oligoesters were
synthesized via anionic ring-opening (co)oligomerization of p-methoxybenzoyloxymethylpropiolactone
2
(p-AA-CH -PL) initiated by p-anisic acid sodium salt. An analytical protocol was developed for a detailed
structural characterization at the molecular level of these bioactive (co)oligoesters. The molecular level
structure of the obtained bioactive (homo)- and (co)oligoesters was established by electrospray
Received 16th April 2016
Accepted 8th June 2016
1
ionization tandem mass spectrometry (ESI-MS/MS) supported by H NMR analysis. Additionally, the
DOI: 10.1039/c6ra09870c
results presented here are important for the analysis of designed biodegradable polymeric controlled-
release systems of bioactive compounds with potential applications in cosmetology.
www.rsc.org/advances
acid (p-methoxybenzoic acid) and levulinic acid (4-oxopentanoic
acid), which were found to be the main compounds in Pimpinella
1
. Introduction
Increased interest in bioactive compounds is related to the anisum and other herbs and in Dioscorea villosa as a by-product in
5
potential possibilities of using them as agents added to food, the production of diosgenin from wild yam, respectively.
cosmetics or pharmaceuticals. The term ‘bioactive substances’
The development of macromolecules with a dened struc-
encompasses all components comprising therapeutic and ture and properties, aimed specically for biomedical applica-
nutritional substances that are normally found in plants in tions, has resulted in diverse biodegradable polymers with
1
small quantities. A well-known group of bioactive agents are advanced architectures. Recently, the attachment of cosmetics
phenolic compounds which include, e.g. avonoids, phenolic to specic polymer carriers has spurred particular interest.
acids or tannins. Phenolic acids are aromatic secondary plant PHAs, mostly poly(3-hydroxybutyrate) and poly(3-hydrox-
metabolites which are widely spread throughout the plant ybutyrate-co-3-hydroxyvalerate), have been used as carriers for
kingdom. This group includes hydroxyl derivatives of cinnamic drug delivery because of their biocompatibility, biodegrad-
acid and benzoic acid. Phenolic acids can be easily absorbed in ability and controllable retarding properties which can be
the human system and offer a host of anti-aging benets. They modulated by variations in the processing and molecular
6,7
also reveal very good preservative properties, including anti- weight of the polymer composition. The synthetic analogue of
2
bacterial and antifungal effects.
PHAs can be obtained via anionic ring-opening polymerization
p-Anisic acid (benzoic acid derivative) possesses antioxidant, of b-substituted b-lactones, e.g. atactic poly(3-hydroxybutyrate)
8
anti-tumor and anti-inammatory properties; it has also been is obtained via anionic ROP of b-butyrolactone.
9
found that it has biochemical properties, thus making it suitable
b-Lactones are attractive intermediates in organic synthesis
as a cosmetic ingredient. p-Anisic acid has recently become and are useful as monomers in ROP.
3
10–12
Among the synthetic
increasingly signicant as a multi-functional raw material in both methods for their preparation, carbonylation of epoxides seems to
4
the cosmetics and food industries. Today, several antimicrobial be the most effective approach. In recent years, highly active and
fragrance ingredients are commercially available, such as, p-anisic selective catalysts for epoxide carbonylation have been discov-
13–15
ered,
which opened up access to a new source of biologically
active b-lactone monomers and turned out to have very benecial
and far-reaching consequences in the synthesis of new bioactive
polyesters, including polymeric delivery systems.
a
Polish Academy of Sciences, Centre of Polymer and Carbon Materials, 41-819 Zabrze,
Poland. E-mail: gadamus@cmpw-pan.edu.pl; Tel: +48 32 271 60 77 ext. 226
b
Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
c
Herein, we report the synthetic strategy for bioactive (co)
oligoesters based on anionic homo- and co-polymerization of b-
School of Biology, Chemistry and Forensic Science, Faculty of Science and Engineering,
University of Wolverhampton, Wolverhampton WV1 1SB, UK
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substituted b-lactones containing bioactive moieties selected anisic acid) along the oligomer chains. The structure of indi-
from compounds used as preservatives and antioxidants in vidual macromolecules of the resulting (co)oligoesters
cosmetics. The ability of these lactones to undergo anionic (including the chemical structure of their end groups) was
n
1
ring-opening oligomerization in the presence of metal-free determined using ESI-MS supported by H NMR spectroscopy.
anionic initiators has been demonstrated. The chemical structure of the individual (co)oligoester chains
These novel bioactive (co)oligoesters have a larger loading of was conrmed by ESI-MS/MS and by investigating the frag-
biologically active substances (p-anisic acid, p-AA) per polymer mentation product patterns of individual molecular ions.
macromolecule in comparison to the already reported conju-
gates of oligo(3-hydroxybutyrate) (OHB) with several selected
antioxidants.
16–18
2. Experimental part
Taking into consideration the prospective use of synthesized 2.1. Materials
new control delivery systems in cosmetics and their contact with
the skin, it was important to elucidate the structure and
homogeneity of the obtained products.
(
R,S)-b-Butyrolactone (Sigma-Aldrich Chemie GmbH, Steinheim,
Germany) was distilled over CaH
boiling at 56 C (9 mmHg) was collected. 4-Methoxybenzoylox-
ymethythylpropiolactone (p-AA-CH -PL) was synthesized by
carbonylation of the respective epoxide under ambient CO pres-
2
and the fraction
ꢀ
Previously, mass spectrometry was successfully applied for
structural characterization at the molecular level of homo-
2
19–29
polyesters, copolyesters and their degradation products.
In
30
sure. p-Anisic acid sodium salt (sodium p-anisate, 4-methox-
our recent works we reported the application of ESI-MS/MS
techniques for characterization at the molecular level of OHB
conjugates with bioactive compounds including antioxidants
used in cosmetics. The molecular structure of bioconjugates, in
which antioxidants (a-lipoic, p-coumaric, p-anisic and vanillic
acids) are covalently bonded to OHB chains as the end group,
ybenzoic acid sodium salt for synthesis, MERCK),
dimethylsulfoxide (DMSO, 99.8%, Aldrich) were used as received.
2.2. Synthesis of 4-methoxybenzoyloxymethylpropiolactone
2.2.1. General considerations. NMR spectra were recorded
16–18
1
13
has also been proven by tandem mass spectrometry.
We using a Varian Mercury spectrometer ( H NMR, 500 MHz;
C
have also demonstrated the utility of this technique for the NMR, 125 MHz) and referenced against tetramethylsilane as an
analysis of individual molecules of homo- and co-polyesters internal standard. The following abbreviations were used to
obtained by anionic ring-opening copolymerization of selected indicate multiplicity: s, singlet; d, doublet; t, triplet; br, broad
11,12
b-substituted b-lactones.
spin system; m, multiplet; coupling constants are reported in
In the present study, electrospray mass spectrometry was Hz. Analytical TLC was carried out on commercial plates coated
applied in order to obtain more detailed structural information with 0.25 mm Merck Kieselgel 60. Preparative ash silica
about bioactive (co)oligoesters containing bioactive moieties (p- chromatography was performed using Merck Kieselgel 60 (230–
Scheme 1 Synthesis of (a) 4-methoxybenzoyloxymethylpropiolactone, (b) oligo(3-hydroxy-3-(4-methoxybenzoyloxymethyl)propionate), and
(
c) oligo[(3-hydroxy-3-(4-methoxybenzoyloxy- methyl)propionate)-co-(3-hydroxybutyrate)].
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Table 1 (Homo)- and (co)oligoester characteristics by SEC
4
stirred for 3 h and then quenched with saturated NH Cl solu-
tion. The organic layer was washed with 1 M HCl, water, brine,
a
Composition [mol%]
b
ꢁ1
c
dried over anhydrous MgSO and concentrated in vacuo. The
4
Sample
(p-AA-CH
2
-HP)/HB
M
n
[g mol
]
w n
M /M
crude ester was crystallized (ethyl acetate/hexane) and isolated
as white crystals (13.5 g, 80%).
1
2
100/0
50/50
700
900
1.15
1.23
2.2.3. Procedure for carbonylation of glycidyl 4-methox-
0
a
1
ybenzoate (p-anisate). [N,N -Bis(3,5-di-tert-butylsalicydene)-1,2-
Molar composition of (co)oligoesters estimated from H NMR.
b
c
Number-average molar mass. Molar mass distribution estimated by phenylenediaminochromium]chloride (0.9 mmol, 564 mg),
SEC.
Co
30 mmol, 6.78 g) were placed in an oven-dried Schlenk tube
equipped with a magnetic stir bar under an argon atmosphere.
00 mesh). All manipulations of air- and/or water-sensitive The reaction vessel was then equipped with a latex balloon
2 8
(CO) (1.35 mmol, 462 mg) and glycidyl 4-methoxybenzoate
(
4
compounds were carried out using standard Schlenk line containing carbon monoxide and was subjected to three
techniques under an atmosphere of dry argon. All carbonylation vacuum/CO cycles. Anhydrous dimethoxyethane (30 mL) was
reactions were set up and run in a well-ventilated fume hood added via syringe and the reaction was stirred overnight. Then
equipped with a carbon monoxide detector (see MSDS for CO gas was carefully vented off. The reaction mixture was
proper handling of CO). Dimethoxyethane and methylene evaporated under reduced pressure and the residue was puri-
chloride were dried by distillation over CaH prior to use.
ed by ash column chromatography (hexane, ethyl acetate) to
2
2
.2.2. Preparation of glycidyl 4-methoxybenzoate (p-ani- give 4-methoxybenzoyloxymethylpropiolactone (4.95 g, 65%).
sate). A catalytic amount of DMF was added (ca. 0.2 mL) to
a
0
stirred suspension of 4-methoxybenzoic acid (11.4 g,
.075 mol) in 100 mL of anhydrous methylene chloride. Oxalyl
chloride (7.7 mL, 0.09 mol) was then added dropwise and the
suspension was stirred at RT until it became clear (2–3 h). The
solution was then concentrated in vacuo and acid chloride was
used directly in the next step without any further purication.
1
H NMR (CDCl , 500 MHz) d 7.99 (d, 2H, J ¼ 9 Hz), 6.93 (d,
3
1
2
2H, J ¼ 8.8 Hz), 4.86 (m, 1H), 4.69 (dd, 1H, J ¼ 13 Hz, J ¼ 3 Hz),
1
2
1
4
-Methoxybenzoyl chloride was added dropwise to a stirred 4.53 (dd, 1H, J ¼ 13 Hz, J ¼ 5 Hz), 3.87 (s, 3H), 3.62 (dd, 1H, J
2 1 2 13
solution of glycidol (6 mL, 0.09 mol) and triethylamine ¼ 16 Hz, J ¼ 6 Hz), 3.43 (dd, 1H, J ¼ 16 Hz, J ¼ 4 Hz), C NMR
(
12.5 mL, 0.09 mol) in 100 mL of anhydrous methylene chloride (125 MHz) d 40.2, 55.4, 63.4, 67.9, 113.8, 121.3, 131.8, 163.8,
ꢀ
ꢁ1
at 0 C. The reaction mixture was then allowed to reach RT, was 165.6, 166.7; IR (neat, KBr) nCO ¼ 1830 cm
.
1
Fig. 1 H NMR spectrum (in CDCl
3 2 m n 2
) of the (p-AA-CH -HP) /HB oligomer obtained via co-oligomerization of b-BL with p-AA-CH -PL initiated
by sodium p-anisate.
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ring-opening polymerization of b-substituted b-lactone con-
taining the p-anisic acid moiety, e.g. 4-methoxybenzoylox-
ymethylpropiolactone (p-AA-CH -PL). The reaction was
2
conducted in dimethylosulfoxide (DMSO) at room tempera-
ture in a glass reactor that was amed and dry argon-purged
prior to use. The monomer p-AA-CH
2
-PL (0.20 g, 0.85 mmol)
was added to reactors that contained the solvent DMSO
(
4 mL) and the required amount of initiator: p-anisic acid
ꢁ
+
3
sodium salt (sodium p-anisate, (CH OPhC(O)O Na ), 0.02 g,
0
.13 mmol). The average molar mass of the conjugates was
controlled by the monomer–initiator ratio. The progress of
polymerization was monitored by Fourier transform infrared
spectroscopy (FT-IR) based on the intensity of the signals
arising from the carbonyl groups of the monomer and olig-
ꢁ1
omer at 1837 and 1743 cm , respectively. Aer the comple-
tion of oligomerization, the resulting oligomers were
precipitated from cold hexane and dried under vacuum.
Chloroform solutions of oligomers were acidied with dilute
HCl(aq) and the mixtures were stirred vigorously for 10 min.
Aer phase separation, the organic layer was removed and
acidied again (as described above). Then the oligomer
solution was washed 10 times with 5 mL of distilled water.
Next, the solvent was evaporated and the oligomers were
dried under vacuum at room temperature for 48 h and
analyzed.
2 n
Fig. 2 ESI-mass spectrum (positive-ion mode) of the (p-AA-CH -HP)
(homo)oligoester obtained via anionic ring-opening oligomerization
p-AA-CH -PL) initiated by sodium p-anisate.
(
2
2.3. Oligomerization of 4-methoxybenzoyloxymethylpropio-
lactone in the presence of p-anisic acid sodium salt
The bioactive (homo)oligoester containing the p-anisic acid
moiety e.g. oligo(3-hydroxy-3-(4-methoxybenzoyloxymethyl)
propionate) (p-AA-CH -HP)
was prepared via anionic
2
n
Fig. 3 ESI-MS/MS spectrum of the sodium adduct of oligomers Fig. 4 ESI-mass spectra (positive ion-mode) of oligomers obtained by
containing three 3-hydroxy-3-(4-methoxybenzoyloxymethyl)propio- (co)oligomerization of b-BL with p-AA-CH -PL initiated by sodium p-
2
nate repeating units and the theoretical fragmentation pathway.
anisate and spectral expansion in the mass range m/z 660–1020.
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.4. (Co)oligomerization of 4-methoxybenzoyloxymethylpro-
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ꢀ
ꢁ1
2
in chloroform at 35 C with a ow rate of 1 mL min using an
isocratic pump (VE 1122, Viscotek), a PLgel 3 mm MIXED-E
piolactone in the presence of p-anisic acid sodium salt
(
Polymer Laboratories) ultra-high efficiency column (300 mm
The bioactive random (co)oligoester containing the p-anisic
acid moiety e.g. oligo[(3-hydroxy-3-(4-methoxybenzoylox-
ymethyl)propionate)-co-(3-hydroxybutyrate)] was prepared via
anionic ring-opening copolymerization of b-substituted
ꢂ 7.5 mm) and a Shodex SE 61 differential refractive index
detector. The injection volume was 100 mL of the sample in
chloroform (0.3% w/v). Polystyrene standards (Polymer Labo-
ratories) with narrow molar mass distributions were used to
2
b-lactone containing the p-anisic moiety (p-AA-CH -PL) with
generate a calibration curve.
b-butyrolactone (b-BL). The reaction was performed under an
argon atmosphere at room temperature. Monomers p-AA-CH2-
PL (0.20 g, 0.85 mmol) and b-BL (0.073 mL, 0.85 mmol) were
added to reactors containing the solvent: DMSO 5 mL and the
1
2.5.2. Nuclear magnetic resonance (NMR) analysis.
H
NMR spectra of the homo- and copolyesters were recorded
using a Bruker-Avance 600 MHz multinuclear magnetic reso-
nance spectrometer. The analyses were run in CDCl3 using
tetramethylsilane (TMS) as an internal standard.
3
required amount of initiator: sodium p-anisate, (CH OPhC(O)
ꢁ
+
O Na ), (0.031 g, 0.18 mmol). Progress of the copolymerization
was monitored by FT-IR spectroscopy based on the intensity of
2.5.3. Fourier transform infrared spectroscopy (FTIR)
ꢁ
1
analysis. FTIR spectra were recorded using a FTS 40A Bio-Rad
spectrometer at room temperature.
the carbonyl-group signals of the monomers at 1837 cm
ꢁ
1
(
p-AA-CH -PL) and 1830 cm (b-BL) and that of the oligomers
at 1740 cm . The resulting (co)oligoesters were puried as
2
n
ꢁ1
2.5.4. Electrospray mass spectrometry (ESI-MS ) analysis.
Electrospray mass spectrometry analysis was performed
using a Finnigan LCQ ion trap mass spectrometer (Finnigan,
San Jose, CA, USA). The obtained bioactive (co)oligoesters
were dissolved in methanol (99.8%, pure p.a.-basic, POCH
described above.
2.5. Measurements
2
.5.1. Size-exclusion chromatography (SEC) analysis. SA). The solutions were introduced into the ESI source by
ꢁ1
Number-average and weight-average molar mass and molar continuous infusion at a rate of 10 mL min
using an
mass distribution (M /M ) were determined by SEC conducted instrument syringe pump. The LCQ ESI source was operated
w
n
Table 2 Structural assignments of ions appearing in the expanded region (m/z 660–1020) of the ESI-MS spectrum of oligomers obtained by (co)
oligomerization of b-BL with p-AA-CH -PL initiated by sodium p-anisate
2
Composition
Series
Chemical structure
(p-AA-CH
2
-HP)/HB
m/z
669
755
841
(
(
(
(
(
p-AA-CH
p-AA-CH
p-AA-CH
p-AA-CH
p-AA-CH
2
2
2
2
2
-HP)
-HP)
-HP)
-HP)
-HP)
1
1
1
1
1
/HB
/HB
/HB
/HB
/HB
3
4
5
6
7
927
1013
A
(
(
(
(
p-AA-CH
p-AA-CH
p-AA-CH
p-AA-CH
2
2
2
2
-HP)
-HP)
-HP)
-HP)
2
2
2
2
/HB
/HB
/HB
/HB
1
2
3
4
733
819
905
991
B
HB
HB
HB
HB
6
7
8
9
691
777
863
949
C
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ꢀ
2
at 4.5 kV and the capillary was heated to 200 C. Nitrogen was describes the anionic ring-opening oligomerization of p-AA-CH -
used as the nebulizing gas. For the ESI-MS/MS experiments, PL initiated by sodium p-anisate which led to the bioactive
monoisotopic ions of interest were isolated in the ion trap (homo)oligoester: oligo(3-hydroxy-3-(4-methoxybenzoyloxymethyl)
and collisionally activated using the helium damping gas propionate), (p-AA-CH -HP)n (sample 1, Table 1). Whereas the
2
present in the mass analyzer as a collision gas. The RF bioactive (co)oligoester: oligo[(3-hydroxy-3-(4-methoxybenzoylox-
amplitude, which had a substantial voltage range, was set ymethyl)propionate)-co-(3-hydroxybutyrate)] (p-AA-CH
such that the peak height of the molecular ion decreased by (sample 2, Table 1) was synthesized via anionic ring-opening (co)
at least 50%. The analyses were performed in positive-ion oligomerization of p-AA-CH -PL with b-butyrolactone (b-BL) initi-
mode. ated by sodium p-anisate (Scheme 1c).
2 m n
-HP) /HB
2
The obtained bioactive (homo)- and (co)oligoesters were
1
preliminarily characterized by SEC, H NMR techniques and
n
3
. Results and discussion
ESI-MS . The results of this characterization of the obtained
products by SEC are summarized in Table 1.
The chemical structures of the bioactive (homo)- and (co)oli-
goesters obtained in this study were determined by H NMR
In recent years, much attention has been focused on the
synthesis of b-lactones by catalytic carbonylation of epox-
ides.
obtained monomers may be used on a large scale for the
synthesis of new biodegradable homo- and copolymers with
specic characteristics, e.g. for prospective applications of
1
13–15
This is a relatively simple synthetic method, thus the
1
spectroscopy. The H NMR spectrum of (p-AA-CH
2
-HP)
m
/HB
n
is
1
presented in Fig. 1. The H NMR spectrum reveals signals ascribed
to the protons of the methyl, methylene and methine groups of 3-
hydroxybutyrate and 3-hydroxy-3-(4-methoxybenzoyloxymethyl)
propionate repeating units (corresponding signals: 3, 3 , 1 and 1
as well as 2 and 2 ). Additionally, overlapping signals correspond-
30
controlled release systems of biologically active substances.
The monomer, 4-methoxy-benzoyloxymethylpropiolactone
p-AA-CH -PL), which was used in this study was prepared by
0
0
0
(
2
ing to the protons of the terminal group (derived from the incor-
carbonylation of a suitable glycidyl ester (Scheme 1a). The
resulting new b-substituted b-lactone containing covalently
attached p-anisic acid was used for the synthesis of biologically
active (homo)- and (co)oligoesters for prospective applications
in cosmetology in controlled release systems. Scheme 1b
porated initiator) and p-anisic acid embedded as side groups along
0
the p-AA-CH
spectrum.
2
-HP chain (signals 4–6 , Fig. 1) were detected in the
+
Fig. 5 ESI-MS/MS spectrum (in positive-ion mode) of a selected sodium adduct of [p-AA-(p-AA-CH
2
-HP)
1
/HB
5
+ Na] (co)oligoester at m/z 841
terminated by p-anisic acid and carboxylic end groups and containing one p-AA-CH -HP comonomer unit.
2
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.1. Molecular structural studies of the (homo)oligoester
the three types of oligomers are present in the mass spectrum of
the sample in Fig. 4. Two main sets of ions, which appear in the
mass spectrum at m/z ¼ 151 + (n ꢂ 86) + (m ꢂ 236) + 1 + 23
containing bioactive p-anisic acid moieties
n
Electrospray ion trap tandem mass spectrometry (ESI-MS ) was
applied to obtain detailed information about the structure of
the resulting (p-AA-CH
(Fig. 4) with a peak-to-peak mass increment of 236 Da (labeled
as Series A and B), were observed in the mass spectrum. The
2
-HP)
n
oligoester at the molecular level.
-HP) oligomers ob-
signals belonging to Series A and B correspond to sodium cat-
The ESI mass spectrum of (p-AA-CH
2
n
+
ionized [(p-AA-CH
2
-HP)
m
/HB
n
+ Na] (co)oligoester chains
tained here is depicted in Fig. 2. One main set of ions with
a peak-to-peak mass increment of 236 Da was observed in the
mass spectrum. The structures of the end groups and repeating
units can be inferred based on the mass assignment of singly
charged ions observed in the mass spectrum. The series of ions
terminated by p-anisic acid and carboxylic end groups and
containing one (Series A) or two (Series B) (p-AA-CH -HP)
2
comonomer units that can be arranged with different combi-
nations along the oligomer backbone. The third and less
abundant series of ions (with a lower relative intensity) labeled
Series C (see Fig. 4) was observed in the mass spectra at m/z ¼
2 1 2 7
labeled as (p-AA-CH -HP) – (p-AA-CH -HP) correspond to the
sodium cationized 3-hydroxy-3-(4-methoxybenzoyloxymethyl)
propionate oligomers terminated by p-methoxybenzoyl (p-ani-
sate, derived from the initiator) and carboxyl end groups.
Tandem mass spectrometry (ESI-MS/MS) was used for
further structural elucidation of the oligomers' structure. In this
technique, the molecular ion of interest is separated from the
other ions formed during the electrospray ionisation process
and is induced to dissociate into fragments. These can be used
for structural characterization (including end-group analysis) of
the individual polymer chains. To verify the structure of indi-
vidual ions belonging to the main series, ESI-MS/MS fragmen-
tation experiments were performed for ions selected from this
series.
151 + (n ꢂ 86) + 1 + 23. These series can be assigned to sodium
cationized oligo(3-hydroxybutyrate) terminated by p-anisate and
carboxyl end groups. Thus the mass spectrometry analysis
indicated that the (co)oligoester contained one, two or three
bioactive p-anisic moieties distributed along the oligomer
chains was synthesized (Fig. 4). The general structures of
the ions present in the ESI-MS spectra (Fig. 4) are depicted in
Table 2.
Previously published studies on the fragmentation of indi-
vidual precursor ions of polyesters (derivatives of b-hydroxy
acids) revealed that b-hydrogen rearrangement was the main
mechanism inducing fragmentation of such polyesters via ester
26,31–34
bond cleavage.
Fig. 3 shows the ESI-MS/MS spectrum of the ion at m/z 883
corresponding to the sodium adduct of the oligoester contain-
ing three 3-hydroxy-3-(4-methoxybenzoyloxymethyl)propionate
repeating units terminated by p-anisate and carboxyl end
groups. The fragmentation pathway of the ion at m/z 883, which
occurs as a result of random breakage of ester bonds along the
oligomer backbone and of ester bonds between the backbone
and the bioactive pendant groups, is shown in Fig. 3. The
product ion at m/z 731 corresponds to the oligomer formed by
the loss of p-anisic acid (152 Da) derivative from the oligomer
terminal group or from the bioactive pendant group of the p-AA-
CH -HP chain; whereas the product ion at m/z 647 corresponds
2
to the oligomer formed by the loss of 4-methoxybenzoylox-
ycrotonic acid (236 Da). A comparison of the fragmentation
spectra and the theoretical path of the ion fragmentation
showed that all theoretically predicted structures of the product
ions are shown at the spectrum, which conrms the structure of
the obtained bioactive oligoester (Fig. 3).
3
.2. ESI-MS reveals the random structure of (p-AA-CH -HP) /
2 m
HB (co)oligoesters
n
The ESI-mass spectrum (positive ion-mode) of oligomers ob-
tained by (co)oligomerization of b-BL with p-AA-CH -PL initi-
ated by sodium p-anisate (together with spectral expansion in
the range m/z 650–1050) are depicted in Fig. 4. Three series of
2
Scheme 2 Theoretical fragmentation pathway of the sodium adduct
+
of [(p-AA-CH
2
-HP)
1
/HB
5
+ Na] (co)oligoester at m/z 841 terminated
2
by p-anisic acid and carboxyl end groups containing one p-AA-CH -
singly charged ions (labeled as A, B and C) that correspond to HP comonomer unit.
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The presence of these oligomer chains can be expected at each acid (152 Da), which can be derived from the terminal group
degree of polymerization, and their amount depends on the and/or from the bioactive pendant group of the p-AA-CH -HP
2
comonomer ratio, polymerization mechanism, degree of copoly- comonomer unit. While the product ion at m/z 755 corresponds
merization (e.g. the number of comonomeric units in a macro- to the oligomer formed by the loss of the last 3-hydroxybutyrate
molecule) as well as on the comonomer composition distribution. unit in the oligoester chains in the form of crotonic acid (86 Da),
the formation of the product ion at m/z 605 is associated with
the loss of 4-methoxybenzoyloxycrotonic acid (236 Da, see
Scheme 2) if the last comonomer unit in the oligoester chain is
3
.3. Structural characterization of random (p-AA-CH -HP) /
2 m
HB (co)oligoesters with tandem ESI-MS/MS
n
p-AA-CH -HP. All theoretically predicted product ions are
2
More detailed insight into the structure of individual (co)oli- present in the ESI-MS/MS spectrum in Fig. 5, which is in good
goester chains may be obtained by using tandem ESI-MS/MS agreement with the theoretical fragmentation pathways we
spectrometry. This technique has been applied for structural proposed in Scheme 2. Moreover, such a fragmentation pathway
studies and investigation of fragmentation product patterns of indicates that the p-AA-CH -HP comonomer unit is randomly
2
individual molecular ions while allowing differentiation distributed along the oligomer chain.
between individual molecular chains of random and diblock
copolyesters. Fig. 5 presents the ESI-MS/MS spectrum (in precursor ion at m/z 819 corresponding to the sodium adduct of
positive-ion mode) of the precursor ion at m/z 841, which [p-AA-(p-AA-CH -HP) /HB + Na ] (co)oligoester chains termi-
Fig. 6 shows the ESI-MS/MS product ions' spectrum of the
33
+
2
2
2
corresponds to the sodium adduct of oligomers terminated by nated by p-anisate and carboxyl end groups, containing two HB
carboxyl and p-anisate (derived from the initiator used) end and two p-AA-CH -HP repeating units in the (co)oligoester
2
groups and containing ve 3-hydroxybutyrate repeating units chain. In this case, random breakage of ester bonds along the
and one p-AA-CH -HP unit in the (co)oligoester chain. Frag- oligomer chain and ester bonds between the chain and the
2
mentation of this precursor ion at m/z 841 (belonging to Series bioactive pendant group led to the formation of two series of
A, Fig. 4) takes place as a result of random breakage of ester product ions at m/z 733, 667, 647, 583, 581, 497, 495, 431, 411.
bonds along the oligomer chain and ester bonds between the
Thus the product ion at m/z 667 corresponds to the oligomer
chain and the bioactive pendant group (see the fragmentation formed by the loss of the p-anisic acid molecule (152 Da),
pathway in Fig. 5 and Scheme 2). Thus the product ion at m/z a derivative from the oligomer end group or from the bioactive
689 corresponds to the oligomer formed by the loss of p-anisic
+
Fig. 6 ESI-MS/MS spectrum (in positive-ion mode) of a selected sodium adduct of [p-AA-(p-AA-CH
2 2 2
-HP) /HB + Na] (co)oligoester at m/z 819
terminated by p-anisic acid and carboxylic end groups and containing two p-AA-CH -HP comonomer units.
2
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bonded p-anisic acid enabled the formation of bioactive (homo)
oligoesters having from 1 to 8 molecules of p-anisic acid and
(co)oligoesters containing from 2 to 3 molecules of p-anisic acid
bound to the polymer backbone as a bioactive terminal and
pendant groups through hydrolyzable ester linkages.
An analytical method for a detailed structural characteriza-
tion at the molecular level of bioactive (co)oligoesters has been
developed. The molecular structures of the obtained (homo)-
and (co)oligoesters were determined with the use of ESI-mass
1
spectrometry supported by H NMR analyses. Structures of the
end groups and copolymer composition were veried by ESI-
MS/MS. It was shown that fragmentation of selected ions of
bioactive (co)oligoesters proceeded via random breakage of
ester bonds along the oligomer chain as well as of ester bonds of
the bioactive pendant group. Additionally, the ESI-MS/MS
fragmentation experiments conducted for selected sodium
adducts of the obtained (co)oligoesters conrmed that the
bioactive moieties were covalently bonded to oligo(3-
hydroxybutyrate) chains as terminal and pendant groups.
Moreover, the fragmentation pathway of selected oligomers
2
indicated that the p-AA-CH -HP comonomer units were
randomly distributed along the (co)oligoester chain. Thus the
obtained results demonstrate the utility of this technique for
the analysis of individual molecules of the bioactive (homo)-
and (co)oligoesters studied here.
Novel delivery systems obtained via the elaborated synthetic
strategy contain
a larger loading of biologically active
substances per polymer macromolecule than the respective
16–18
cosmetic delivery system that we have already reported.
The
object of further work will be to carry out preliminary studies
proving the usefulness of the developed (co)oligoesters as new
controlled release systems for applications in cosmetology.
Scheme 3 Theoretical fragmentation pathway of the sodium adduct
+
of [p-AA-(p-AA-CH
2
-HP)
2
/HB
2
+ Na] (co)oligoester at m/z 819
terminated by p-anisic acid and carboxyl end groups containing two
p-AA-CH -HP comonomer units.
2
Acknowledgements
2
pendant group of the p-AA-CH -HP comonomer unit. While the
product ion at m/z 733 corresponds to the oligomer formed by the
loss of HB units in the form of crotonic acid if the HB unit is the
last comonomer unit in the oligoester chain (86 Da; see Fig. 6 and
This work was supported by the National Science Centre: project
titled “Studies on the synthesis of novel biodegradable control
release systems of bioactive compounds for perspective appli-
Scheme 3). The product ion at m/z 583 corresponds to the oligomer cations in cosmetology”, decision no. DEC-2013/09/N/ST5/
formed by the loss of the last, in the oligoester chain, p-AA-CH -HP 00890 and project titled “Low pressure catalytic synthesis of
comonomer unit in the form of 4-methoxybenzoyloxycrotonic acid novel beta-lactone monomers and their anionic ring-opening
236 Da, see Scheme 3). Such a fragmentation pathway indicates (co)polymerization leading to synthetic analogues of aliphatic
that the two p-AA-CH -HP comonomer units are randomly biopolyesters”, decision no. DEC-2012/07/B/ST5/00627.
distributed along the oligomer chain.
2
(
2
33
ESI-MS/MS experiments were also performed for ions
derived from Series C (assigned as HB in the spectral expan- References
n
sion in Fig. 4). The selected ions correspond to the sodium
1
P. M. Kris-Etherton, K. D. Hecker and A. Bonanome,
Bioactive compounds in foods: their role in the prevention
of cardiovascular disease and cancer, Am. J. Med., 2002,
adducts of p-AA-oligo(3-hydroxybutyrate) (p-AA-OHB) chains
terminated by p-anisate and carboxylic end groups. Fragmen-
tation of these ions proceeds via a similar fragmentation
pathway and was previously reported in ref. 18.
113, 71S.
2
3
C. A. Rice-Evans, N. J. Miller and G. Paganga, Structure-
antioxidant activity relationships of avonoids and
phenolic acids, Free Radicals Biol. Med., 1996, 20, 933.
S. Papageorgiou, A. Varvaresou, E. Tsirivas and
C. J. Demetzos, New alternatives to cosmetics preservation,
Cosmet. Sci., 2010, 61, 107.
4
. Conclusions
The results of the present study revealed that anionic oligo-
merization of b-substituted b-lactones containing covalently
This journal is © The Royal Society of Chemistry 2016
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View Article Online
RSC Advances
Paper
4
U. Straetmans, J. Janichen, W. Petersen, M. Kinder, 18 M. Maksymiak, R. Debowska, K. Bazela, A. Dzwigalowska,
C. Johnson and G. Reynolds, Concentrated, aqueous
solutions of p-methoxybenzoic acid for use in cosmetic and
dermatologic formulations, US 20060229291 A1, Oct 12,
A. Orchel, K. Jelonek, B. Dolegowska, M. Kowalczuk and
G. Adamus, Designing of Biodegradable and
Biocompatible Release and Delivery Systems of Selected
Antioxidants Used in Cosmetology, Biomacromolecules,
2015, 16, 3603.
2
006.
M. Sautour, A.-C. Mitaine-Offer and M.-A. Lacaille-Dubois,
The Dioscorea genus:
5
6
a
review of bioactive steroid 19 G. Montaudo and R. P. Lattimer, Mass Spectrometry of
saponins, J. Nat. Med., 2007, 61, 91.
A. J. Anderson and E. A. Dawes, Occurrence, metabolism,
Polymers, CRC Press, Boca Raton, London, New York,
Washington D.C, 2002.
metabolic role, and industrial uses of bacterial 20 A. C. Crecelius, A. Baumgaertel and U. S. Schubert, Tandem
polyhydroxyalkanoates, Microbiol. Mol. Biol. Rev., 1990, 54,
50.
mass spectrometry of synthetic polymers, J. Mass Spectrom.,
2009, 44, 1277.
4
7
8
A. Shrivastav, H.-Y. Kim and Y.-R. Kim, Advances in the 21 S. Koster, M. C. Duursma, J. J. Boon, M. W. F. Nielen,
Applications of Polyhydroxyalkanoate Nanoparticles for
Novel Drug Delivery System, BioMed Res. Int., 2013, 2013, 1.
Z. Jedli n´ ski, M. Kowalczuk, W. Gł ´o wkowski, J. Grobelny and
M. Szwarc, Novel polymerization of b-butyrolactone initiated
C. G. Koster and R. M. A. Heeren, Structural analysis of
synthetic homo- and copolyesters by electrospray
ionization on a Fourier transform ion cyclotron resonance
mass spectrometer, J. Mass Spectrom., 2000, 35, 739.
by potassium naphthalenide in the presence of a crown ether 22 M. A. Arnould, R. Vargas, R. W. Buehner and
or a cryptand, Macromolecules, 1991, 24, 349.
A. Pommier and J. M. Pons, Recent Advances in b-Lactone
Chemistry, Synthesis, 1993, 5, 441.
C.
Wesdemiotis,
Tandem
Mass
Spectrometry
9
Characteristics of Polyester Anions and Cations Formed by
Electrospray Ionization, Eur. J. Mass Spectrom., 2005, 11, 243.
10 L. R. Rieth, D. R Moore, E. B. Lobkovsky and G. W. Coates, 23 J. De Winter, O. Coulembier, P. Dubois and P. Gerbaux,
Single-Site b-Diiminate Zinc Catalysts for the Ring-Opening
Polymerization of b-Butyrolactone and b-Valerolactone to
Poly(3-hydroxyalkanoates), J. Am. Chem. Soc., 2002, 124,
Collision-induced dissociation of polymer ions : charge
driven decomposition for sodium-cationized polylactides,
Int. J. Mass Spectrom., 2011, 308, 11.
1
5239.
24 I. Osaka, M. Watanabe, M. Takama, M. Murakami and
R. Arakawa, Characterization of linear and cyclic polylactic
acids and their solvolysis products by electrospray
ionization mass spectrometry, J. Mass Spectrom., 2006, 41,
1369.
1
1 G. Adamus and M. Kowalczuk, Synthesis of Poly(2-methyl-3-
hydroxyoctanoate) via Anionic Polymerization of a-Methyl-b-
pentyl-b-propiolactone, Biomacromolecules, 2008, 9, 696.
2 G. Adamus, I. Kwiecie n´ , M. Maksymiak, T. Bałakier,
1
J. Jurczak and M. Kowalczuk, Molecular level structure of 25 T. Gruendling, S. Weidner, J. Falkenhagen and C. Barner-
novel synthetic analogues of aliphatic biopolyesters as
revealed by multistage mass spectrometry, Anal. Chim.
Acta, 2014, 808, 104.
3 Y. D. Y. L. Getzler, V. Mahadevan, E. B. Lobkovsky and
G. W. Coates, Synthesis of ß-Lactones: A Highly Active and
Kowollik, Mass spectrometry in polymer chemistry: a state-
of-the-art up-date, Polym. Chem., 2010, 1, 599.
26 Mass Spectrometry in Polymer Chemistry, ed. C. Barner-
Kowollik, T. Gr u¨ ndling, J. Falkenhagen and S. Weidner,
Viley-VCH, Weinheim, 2011.
1
1
Selective Catalyst for Epoxide Carbonylation, J. Am. Chem. 27 T. Josse, J. De Winter, P. Dubois, O. Coulembier, P. Gerbaux
Soc., 2002, 124, 1174.
and A. Memboeuf, A tandem mass spectrometry-based
method to assess the architectural purity of synthetic
polymers: case of a cyclic polylactide obtained by click
chemistry, Polym. Chem., 2015, 6, 64.
4 J. A. R. Schmidt, V. Mahadevan, Y. D. Y. L Getzler and
G. W. Coates, A Readily Synthesized and Highly Active
Epoxide Carbonylation Catalyst Based on a Chromium
Porphyrin Framework. Expanding the Range of Available ß- 28 M. Hakkarainen, G. Adamus, A. H o€ glund, M. Kowalczuk and
Lactones, Org. Lett., 2004, 6, 373.
A.-C. Albertsson, ESI-MS Reveals the Inuence of
Hydrophilicity and Architecture on the Water-soluble
Degradation Product Patterns of Biodegradable Homo- and
Copolyesters of 1,5-dioxepan-2-one and 3-caprolactone,
Macromolecules, 2008, 41, 3547.
1
5 J. W. Kramer, E. B. Lobkovsky and G. W. Coates, Practical ß-
Lactone Synthesis: Epoxide Carbonylation at 1 atm, Org.
Lett., 2006, 8, 3709.
1
6 M. Maksymiak, R. D ˛e bowska, K. Jelonek, M. Kowalczuk and
G. Adamus, Structural characterization of biocompatible 29 A. H ¨o glund, M. Hakkarainen, M. Kowalczuk, G. Adamus and
lipoic
acid–oligo-(3-hydroxybutyrate)
conjugates
by
A.-C. Albertsson, Fingerprinting the Degradation Product
Patterns of Different Polyester-ether Networks by
Electrospray Ionization Mass Spectrometry, J. Polym. Sci.,
Part A: Polym. Chem., 2008, 46, 4617.
electrospray ionization mass spectrometry, Rapid Commun.
Mass Spectrom., 2013, 27, 773.
7 M. Maksymiak, M. Kowalczuk and G. Adamus, Electrospray
1
tandem
characterization
hydroxybutyrate) conjugates, Int. J. Mass Spectrom., 2014,
59, 6.
mass
spectrometry
of p-coumaric
for
the
structural 30 T. Bałakier, W. Chaładaj, J. Jurczak, G. Adamus and
acid–oligo(3-
M. Kowalczuk, An effective protocol for the synthesis
enantiomerically pure 4-substituted oxetane-2-ones,
Tetrahedron, 2013, 69, 4990.
3
57760 | RSC Adv., 2016, 6, 57751–57761
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
31 S. M. Hunt, M. R. Binns and M. Sheil, Structural 33 G. Adamus, Molecular Level Structure of (R,S)-3-
characterization of polyester resins by electrospray mass
spectrometry, J. Appl. Polym. Sci., 1995, 56, 1589.
2 P. Rizzarelli, C. Puglisi and G. Montaudo, Matrix-assisted
Hydroxybutyrate/(R,S)-3-Hydroxy-4-ethoxybutyrate Copolyesters
with Dissimilar Architecture, Macromolecules, 2009, 42,
4547.
3
laser desorption/ionization time-of-ight/time-of-ight 34 E. Zagar, A. Krzan, G. Adamus and M. Kowalczuk, Sequence
tandem mass spectra of poly(butylene adipate), Rapid
Commun. Mass Spectrom., 2006, 20, 1683.
distribution in microbial poly(3-hydroxybutyrate-co-3-
hydroxyvalerate) co-polyesters determined by NMR and
MS, Biomacromolecules, 2006, 7, 2210.
This journal is © The Royal Society of Chemistry 2016
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