Synthesis and Structural Characterization of Bioactive PHA and - MDPI

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Synthesis and Structural Characterization of Bioactive PHA and γ-PGA Oligomers for Potential Applications as a Delivery System Iwona Kwiecien´ 1 , Iza Radecka 2 , Michał Kwiecien´ 1 and Gra˙zyna Adamus 1, * 1 2

*

Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze 41-819, Poland; [email protected] (I.K.); [email protected] (M.K.) School of Biology, Chemistry and Forensic Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton WV1 1SB, UK; [email protected] Correspondence: [email protected]; Tel.: +48-32-271-6077 (ext. 226)

Academic Editor: Naozumi Teramoto Received: 22 January 2016; Accepted: 19 April 2016; Published: 25 April 2016

Abstract: The (trans)esterification reaction of bacterial biopolymers with a selected bioactive compound with a hydroxyl group was applied as a convenient method for obtaining conjugates of such compound. Tyrosol, a naturally occurring phenolic compound, was selected as a model of a bioactive compound with a hydroxyl group. Selected biodegradable polyester and polyamide, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) and poly-γ-glutamic acid (γ-PGA), respectively, were used. The (trans)esterification reactions were carried out in melt mediated by 4-toluenesulfonic acid monohydrate. The structures of (trans)esterification products were established at the molecular level with the aid of ESI-MS2 (electrospray ionization tandem mass spectrometry) and/or 1 H NMR (nuclear magnetic resonance) techniques. Performed analyses confirmed that the developed method leads to the formation of conjugates in which bioactive compounds are covalently bonded to biopolymer chains. The amount of covalently bonded bioactive compounds in the resulting conjugates depends on the type of biopolymers applied in synthesis. Keywords: biodegradable polymers; polyhydroxyalkanoates; P(3HB-co-4HB); poly-γ-glutamic; γ-PGA; conjugates; tyrosol; mass spectrometry

1. Introduction Design of biodegradable delivery systems for bioactive substances is a rapidly developing field. The aim of designing delivery systems is modification of properties of bioactive molecules, such as solubility, stability and bioactivity, and improve the delivery efficiency. For these purposes biodegradable, biocompatible and nontoxic polymeric carriers might be applied [1–4]. Potential uses of polyhydroxyalkanoates (PHAs) in polymeric delivery systems of bioactive compounds have been evaluated in a number of studies—for example, delivery systems for steroids [5], antibiotics [6,7], or antitumor agents [8,9]. Polyhydroxyalkanoates are aliphatic polyesters produced under controlled conditions via biotechnological processes using numerous microorganisms and by varying the producing strains and substrates. The PHAs are accumulated as granules in the cell, reaching 90% levels of dry cell mass [10]. In this way, a number of polyesters which differ in monomer composition have been synthesized [11,12]. It was essential to search for different inexpensive raw materials for PHA production, and it is already known that these biopolyesters can be produced from renewable resources and from a broad range of waste and surplus materials, such as glycerol from biodiesel production, protein hydrolysates, meat and bone meal from slaughtering and rendering industries or molasses from the sugar industry [13–15].

Materials 2016, 9, 307; doi:10.3390/ma9050307

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Previously, we reported (i) one-pot synthesis in which the respective bioactive compoundoligomer conjugates were obtained through the transesterification reaction of PHA biopolyesters by selected bioactive compounds with a carboxyl group in the presence of 4-toluenesulfonic acid monohydrate and (ii) a two-step procedure of obtaining bioactive PHA conjugates, designed for bioactive compounds with hydroxyl group; in the first step, cyclic oligomers were obtained from bacterial poly(3-hydroxybutyrate) according to the method described in the literature [16]; subsequent lipase-catalyzed transesterification of the cyclic oligomers by the bioactive compounds leads to the formation of the conjugates [17]. The proposed synthetic strategy for bioactive compounds with a hydroxyl group is too complex and economically disadvantageous for industrial application. The method of coupling of specific bioactive compounds (containing a hydroxyl group) to biodegradable polymeric carriers has been presented. The elaborated method is a promising way of obtaining delivery systems for retarded release of bioactive compounds. Our hypothesis was that the (trans)esterification reaction of bacterial biopolymers with a bioactive compound with a hydroxyl group may prove to be a more simple and economically favorable method for obtaining such conjugates. Tyrosol was selected as a model of bioactive compound with a hydroxyl group. Tyrosol is a naturally occurring phenolic compound found, for example, in olive oil and wine [18,19]. Tyrosol and its derivatives show interesting biological properties, such as antioxidant [20] and anticancer effects [21] as well as preventing inflammation-induced osteopenia [22] and smoking-induced oxidative stress [23]. The tyrosol-polymer conjugates were synthesized via (trans)esterification reaction of tyrosol with selected biopolymers (such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) or poly-γ-glutamic acid (γ-PGA)) carried out in melt under an argon atmosphere mediated by 4-toluenesulfonic acid monohydrate (TSA¨ H2 O). To the best of our knowledge and according to relevant information, there are no earlier reports on the use of the (trans)esterification method for the synthesis of delivery systems of tyrosol based on such biopolymers. 2. Results It was found that the “one-pot” transesterification method developed by us for obtaining conjugates of bioactive compounds with the carboxyl group [17] could also be an implement for preparing conjugates of bioactive compounds with a hydroxyl group. The tyrosol-oligo(3HB-co-4HB) conjugates were synthesized via a transesterification reaction of Materials P(3HB-co-4HB) under an 2016, 9, 307 with tyrosol in the presence of TSA¨ H2 O (20 wt %) carried out in melt 3 of 13 argon atmosphere (Scheme 1). The results of the experiments performed are summarized in Table 1. Table 1.ofResults transesterification reaction of P(3-HB-co-4HB) by tyrosol in the presence of The amount TSA¨of Hthe 2 O was chosen based on our previous experience, wherein the relationship 4-toluenesulfonic acid monohydrate (TSA·H2O) (20 wt %). between the number average molar masses (Mn ) of the resulting conjugates and the amount of Sample Tyrosol (wt %) M n (g/mol) Mwour /Mn previous research, it was TSA¨ H2 O used in the process was experimentally determined [17]. In 1 10 1800 3.16 observed that increasing the quantity of water, which was introduced with TSA, led to the decrease of 2 1600 2.58 the molar mass of resulting oligomers20 [17]. The optimal molar masses of the oligomers was obtained 3 30 1000 by using of 20 wt % of TSA¨ H2 O; therefore, the amount of TSA¨ H2 O2.11 used in the each synthesis in 4 40 850 1.87 this study was the same and equaled the 20 wt %. From inspection of Table 1, it can be observed 5 50 950 1.76 that increasing the quantity of bioactive substance led to the decrease of molar mass of resulting The amount of tyrosol compared to the amount of biopolymer (0.25 g) used for the transesterification products. Moreover, based on these results inn Table 1), itdetermined was determined and Mw/M values were using GPCthat (gel the amount reactions, expressed in weight percent. The Mn(shown of bioactive substance used in transesterification reactions influenced the dispersity index Mw /Mn . permeation chromatography). The Mn and Mw/Mn values for P(3-HB-co-4HB)biopolymer were equal Through increasing the amount of tyrosol, a decrease of dispersity index was achieved. Mn = 250,000 g/mol; Mw/Mn = 2.5.

Scheme 1. Transesterification reaction of P(3HB-co-4HB) by tyrosol, mediated by TSA·H2O. R = CH3,

Scheme 1. Transesterification reaction of P(3HB-co-4HB) by tyrosol, mediated by TSA¨ H2 O. R = CH3 , x = 1–3HB units; R = H and x = 2–4HB units. x = 1–3HB units; R = H and x = 2–4HB units.

The structures of obtained conjugates were preliminarily characterized by GPC (gel permeation chromatography) and 1H NMR techniques (results summarized in Table 1). Further structural studies were performed with the aid of ESI-MS (electrospray ionization mass spectrometry). Tyrosol-oligo(3HB-co-4HB) conjugates with number average molar masses in the range

4 5

40 50

850 950

1.87 1.76

The amount of tyrosol compared to the amount of biopolymer (0.25 g) used for the transesterification reactions, expressed in weight percent. The Mn and Mw/Mn values were determined using GPC (gel Materials 2016, 9, 307 3 of 13 permeation chromatography). The Mn and Mw/Mn values for P(3-HB-co-4HB)biopolymer were equal Mn = 250,000 g/mol; Mw/Mn = 2.5. Table 1. Results of the transesterification reaction of P(3-HB-co-4HB) by tyrosol in the presence of 4-toluenesulfonic acid monohydrate (TSA¨ H2 O) (20 wt %). Sample

Tyrosol (wt %)

M n (g/mol)

M w /M n

1 2 3 4 5

10 20 30 40 50

1800 1600 1000 850 950

3.16 2.58 2.11 1.87 1.76

Scheme Transesterification reaction of P(3HB-co-4HB) by tyrosol, by TSA·H 2O. R = CH3, The1.amount of tyrosol compared to the amount of biopolymer (0.25 g) used formediated the transesterification reactions, expressed in weight percent. The Mn and Mw /Mn values were determined using GPC (gel permeation x = 1–3HB units; R = H and x = 2–4HB units. chromatography). The Mn and Mw /Mn values for P(3-HB-co-4HB)biopolymer were equal Mn = 250,000 g/mol; Mw /Mn = 2.5.

The structures of obtained conjugates were preliminarily characterized by GPC (gel permeation chromatography) andof1H NMR conjugates techniques (results summarized in Table 1). (gel Further structural The structures obtained were preliminarily characterized by GPC permeation 1 H NMR techniques (results summarized in Table 1). Further structural chromatography) and studies were performed with the aid of ESI-MS (electrospray ionization mass spectrometry). studies were performed with the aid with of ESI-MS (electrospray mass spectrometry). Tyrosol-oligo(3HB-co-4HB) conjugates number average ionization molar masses in the range Tyrosol-oligo(3HB-co-4HB) conjugates with number average molar masses in the range 850–1800 g/mol 850–1800 g/mol were obtained. were 1H NMR spectrum of the products obtained through the transesterification reaction of The obtained. The 1 H NMR spectrum of the products obtained through the transesterification reaction of P(3HB-co-4HB) with tyrosol mediated by TSA·H2O (Sample 2, Table 1) is presented in Figure 1. In P(3HB-co-4HB) with tyrosol mediated by TSA¨ H2 O (Sample 2, Table 1) is presented in Figure 1. In this this spectrum, signals corresponding to the protons of both comonomeric units were observed, spectrum, signals corresponding to the protons of both comonomeric units were observed, signals signals labelled 1–3 correspond to the protons of the 3-hydroxybutyrate repeating units and signals labelled 1–3 correspond to the protons of the 3-hydroxybutyrate repeating units and signals labeled 4–5 labeled 4–5 correspond to the protons of the 4-hydroxybutyrate repeating units. The signals correspond to the protons of the 4-hydroxybutyrate repeating units. The signals corresponding to corresponding to the protons of the tyrosol molecule bonded to the oligomers were labeled the protons of the tyrosol molecule bonded to the oligomers were labeled 7–10, while signals of the7–10, whileprotons signalsofof protonstyrosol of the molecule unbonded tyrosol molecule thethe unbonded were labelled 7’–10’. were labelled 7’–10’.

1 NMR Sample22from fromTable Table Figure 1. 1. The Figure TheH1 H NMRspectrum spectrum of of Sample 1. 1.

In order to obtain more detailed structural information about the obtained conjugates, the ESI-MSn technique was applied. Recently, ESI-MS has been successfully applied in the structural studies of conjugates of biodegradable oligomers and bioactive substances, such as food preservatives [24], herbicides [25,26], non-steroidal anti-inflammatory drugs [27,28], or antioxidants used in cosmetology [29–31].

In order to obtain more detailed structural information about the obtained conjugates, the ESI-MSn technique was applied. Recently, ESI-MS has been successfully applied in the structural studies of conjugates of biodegradable oligomers and bioactive substances, such as food preservatives [24], herbicides [25,26], non-steroidal anti-inflammatory drugs [27,28], or antioxidants Materials 2016, 9, 307 4 of 13 used in cosmetology [29–31]. The ESI-MS spectrum (in positive-ion mode) of the selected tyrosol-oligo(3HB-co-4HB) conjugate 3, Table 1)(in is positive-ion presented inmode) Figureof2.the Theselected spectrum consists of singly charged ions. The(Sample ESI-MS spectrum tyrosol-oligo(3HB-co-4HB) conjugate The main series of ions correspond to sodium adduct of tyrosol-oligo(3HB-co-4HB) conjugates (Sample 3, Table 1) is presented in Figure 2. The spectrum consists of singly charged ions. Thewith main tyrosol and hydroxyl end groups. The additional series of signals (however, with significant lower series of ions correspond to sodium adduct of tyrosol-oligo(3HB-co-4HB) conjugates with tyrosol and intensity) on theThe spectrum correspond to: sodium adduct of significant oligomerslower with intensity) hydroxyl visible and hydroxylvisible end groups. additional series of signals (however, with carboxyl end groups (which to: formed due to the hydrolysis of biopolyesters), on the spectrum correspond sodium adduct of partial oligomers with hydroxyl and carboxyloligomers end groups terminated by crotonate and carboxyl end groups (formed due to the partial thermal degradation of (which formed due to the partial hydrolysis of biopolyesters), oligomers terminated by crotonate biopolyesters), as well as oligomers terminated by crotonate and tyrosol end groups which are the and carboxyl end groups (formed due to the partial thermal degradation of biopolyesters), as well as result of transesterification betweenand tyrosol and formed dueresult to the partial thermal oligomers terminated by crotonate tyrosol endoligomers groups which are the of transesterification degradation of biopolyesters. between tyrosol and oligomers formed due to the partial thermal degradation of biopolyesters.

Figure2.2.ESI-MS ESI-MS spectrum mode of the conjugates obtained Figure spectrum ininpositive-ion positive-ion mode of tyrosol-oligo(3HB-co-4HB) the tyrosol-oligo(3HB-co-4HB) conjugates by transesterification reaction between P(3HB-co-4HB) and tyrosol (for 3HB units R = CH ,x= 1, 3, obtained by transesterification reaction between P(3HB-co-4HB) and tyrosol (for 3HB units R =3CH for 4HB units R = H and x = 2). x = 1, for 4HB units R = H and x = 2).

InIn order totofurther bioconjugate structure order furtherstructural structuralclarification clarificationofoftyrosol-oligo(3HB-co-4HB) tyrosol-oligo(3HB-co-4HB) bioconjugate structure 2) 2was assignment (series A,A, Figure 2),2), tandem mass spectrometry (ESI-MS used. assignment (series Figure tandem mass spectrometry (ESI-MS ) was used. 2 spectrum 2 spectrum Figure (in(inpositive-ion 935 Figure3 3presents presentsESI-MS ESI-MS positive-ionmode) mode)ofofthe theprecursor precursorion ionatatm/z m/z 935 corresponds adductofoftyrosol-oligo(3HB-co-4HB). tyrosol-oligo(3HB-co-4HB). Fragmentation ofion, thiswhich ion, proceeds which correspondstoto sodium sodium adduct Fragmentation of this proceeds via β-hydrogen rearrangement at the esterleads groups, leads to thebreakage random of breakage of ester via β-hydrogen rearrangement at the ester groups, to the random ester bonds along bonds along the 3HB-co-4HB oligomer chain accompanied by an expulsion of neutral molecules and the 3HB-co-4HB oligomer chain accompanied by an expulsion of neutral molecules and to the creation to of theproduct creationions) of product ions) of (see scheme Figure 3).toAccording to the structures the at (see scheme Figure 3). of According the structures assigned the assigned product ion product ion at m/z 831 corresponds to the oligomer by the loss of 3-hydroxybutyric acidproduct (104 m/z 831 corresponds to the oligomer formed by theformed loss of 3-hydroxybutyric acid (104 Da), the Da), product ion at m/z 815 corresponds to the oligomer by the loss of 4-ethenylphenol ionthe at m/z 815 corresponds to the oligomer formed by the lossformed of 4-ethenylphenol (120 Da), the product (120 the 797 product ion at m/z to thebyoligomer by(138 the loss tyrosol (138 ionDa), at m/z corresponds to 797 the corresponds oligomer formed the loss formed of tyrosol Da) of and the product Da) the729product ion at m/zoligomer 729 corresponds to loss the of oligomer formed by the loss of ionand at m/z corresponds to the formed by the 2-(4-hydroxyphenyl)ethyl crotonate 2-(4-hydroxyphenyl)ethyl crotonate (206 Da)(see scheme on Figure 3). (206 Da)(see scheme on Figure 3).

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Figure 3.3.ESI-MS/MS ESI-MS/MS product product ion ion spectrum spectrum of of the the sodiated sodiated tyrosol-oligo(3HB-co-4HB) tyrosol-oligo(3HB-co-4HB) conjugate conjugate at at Figure m/z 935 of this this ion. ion. m/z 935 and and theoretical theoretical fragmentation fragmentation pathway pathway of

The conjugates of tyrosol and bacterialpoly(3-hydroxybutyrate-co-4-hydroxybutyrate) The conjugates of tyrosol and bacterial poly(3-hydroxybutyrate-co-4-hydroxybutyrate) described described above contain one bioactive molecule per oligomer chain. However, conjugate of bioactive above contain one bioactive molecule per oligomer chain. However, conjugate of bioactive compounds compounds with an increased amount of biologically active moieties along the oligomer chain seems with an increased amount of biologically active moieties along the oligomer chain seems to be to be a much more interesting option. For the synthesis of such conjugates of poly-γ-glutamic acid a much more interesting option. For the synthesis of such conjugates of poly-γ-glutamic acid (γ-PGA), polymers made of D- or L-glutamic acid units connected by amide linkages [32], were used (γ-PGA), polymers made of D- or L-glutamic acid units connected by amide linkages [32], were as carriers. The γ-PGA is naturally occurring polymer that is also biodegradable, edible and used as carriers. The γ-PGA is naturally occurring polymer that is also biodegradable, edible non-toxic toward humans and the environment, produced extracellularly by several species of and non-toxic toward humans and the environment, produced extracellularly by several species bacteria of the genus Bacillus classified as GRAS (Generally Regarded As Safe) by the US Food and of bacteria of the genus Bacillus classified as GRAS (Generally Regarded As Safe) by the US Food and Drug Administration [33,34]. Poly-γ-glutamic acid has been already tested as a carrier for bioactive Drug Administration [33,34]. Poly-γ-glutamic acid has been already tested as a carrier for bioactive substances, for example paclitaxel [35], cisplatin [36], insulin [37] and heparin [38]. substances, for example paclitaxel [35], cisplatin [36], insulin [37] and heparin [38]. For preparing conjugates of tyrosol and γ-PGA, similar procedures as for conjugates of For preparing conjugates of tyrosol and γ-PGA, similar procedures as for conjugates of tyrosol-oligo(3HB-co-4HB) were applied. Esterification reactions were carried out in melt in the tyrosol-oligo(3HB-co-4HB) were applied. Esterification reactions were carried out in melt in the presence of TSA·H2O (20 wt %) (Scheme 2). Under these conditions, carboxyl groups along the presence of TSA¨ H2 O (20 wt %) (Scheme 2). Under these conditions, carboxyl groups along the γ-PGA γ-PGA chain should undergo esterification with tyrosol molecules while peptide bonds along the chain should undergo esterification with tyrosol molecules while peptide bonds along the polymer polymer chain could be hydrolyzed due to the presence of the water introduced. chain could be hydrolyzed due to the presence of the water introduced. It is known that, as a result of thermal degradation of polyamides, various products could be It is known that, as a result of thermal degradation of polyamides, various products could be obtained. Heating of polypeptides in the presence of water, leads to hydrolysis of peptide bonds obtained. Heating of polypeptides in the presence of water, leads to hydrolysis of peptide bonds [39]. [39]. The cis-eliminations are also known mechanisms of thermal decomposition of polyamides The cis-eliminations are also known mechanisms of thermal decomposition of polyamides through a through a 6-membered ring which causes NH-CH2 bond scission and leads to oligomers with 6-membered ring which causes NH-CH2 bond scission and leads to oligomers with unsaturated alkyl unsaturated alkyl and amide end groups further transformed into nitriles [40–42]. Other known and amide end groups further transformed into nitriles [40–42]. Other known products of thermal products of thermal degradation are cyclic amides, which are result of intramolecular amide degradation are cyclic amides, which are result of intramolecular amide exchange [41–43]. exchange [41–43]. Based on our previous results as well as other literature information we predicted that Based on our previous results as well as other literature information we predicted that esterification reaction of γ-PGA with tyrosol, which is carried out in melt, can lead to oligomer esterification reaction of γ-PGA with tyrosol, which is carried out in melt, can lead to oligomer with with different structures. In order to obtain detailed structural information about obtained products, different structures. In order to obtain detailed structural information about obtained products, i.e., i.e., the structures of the end groups and amount of repeating units in each conjugate oligomers, the structures of the end groups and amount of repeating units in each conjugate oligomers, the the ESI-MS technique was used. ESI-MS technique was used.


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Scheme 2. reaction of γ-PGA by tyrosol, mediated mediated by by TSA¨ TSA·H2O; (A) oligomers with Scheme 2. Esterification Esterification (A)oligomers oligomers with Scheme 2. Esterificationreaction reactionofofγ-PGA γ-PGAby bytyrosol, tyrosol, mediated by TSA·HH2O; with 2 O;(A) amino and carboxyl end groups; (B) cyclic amides and/or oligomers with unsaturated alkyl and amino and carboxyl end groups; (B) cyclic amides and/or oligomers with unsaturated alkyl and amide amino and carboxyl end groups; (B) cyclic amides and/or oligomers with unsaturated alkyl and amide end groups; (C) oligomers with unsaturated alkyl and nitriles end groups. endamide groups; oligomers with unsaturated alkyl and nitriles end groups. end(C) groups; (C) oligomers with unsaturated alkyl and nitriles end groups.

In Figure 4, ESI-MS spectrum betweentyrosol tyrosoland andγ-PGA γ-PGA In Figure 4, ESI-MS spectrumofofthe theproduct productobtained obtained in in reaction reaction between inin In Figure 4, ESI-MS spectrum of the product obtained in reaction between tyrosol and γ-PGA in thethe presence of of TSA·H 2O2O is is presented. presence TSA·H presented. the presence of TSA¨ H2 O is presented.

Figure 4. Cont.


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Figure 4. ESI-MS spectrum (in positive-ion mode) of the tyrosol-γ-PGA conjugates obtained by Figure 4. ESI-MS spectrum (in positive-ion mode) of the tyrosol-γ-PGA conjugates obtained esterification reaction between γ-PGA and tyrosol (a) and spectral expansion in the range m/z by esterification reaction between γ-PGA and tyrosol (a) and spectral expansion in the range 840–1040 (b) and m/z 480–800 (c). m/z 840–1040 (b) and m/z 480–800 (c).

Figure Figure4.4.ESI-MS ESI-MSspectrum spectrum(in (inpositive-ion positive-ionmode) mode)ofofthe thetyrosol-γ-PGA tyrosol-γ-PGAconjugates conjugatesobtained obtainedby by

esterification reaction between and (a) spectral expansion range esterification reaction between γ-PGA andtyrosol tyrosol (a)and andcan spectral expansion inthe thedegree rangem/z m/z The presence of a tyrosole molecule inγ-PGA the obtained conjugate be expected atineach of The presence of athe tyrosole molecule in conjugate can be an expected atin each 840–1040 (b) and 480–800 840–1040 (b) andm/z m/z 480–800 (c).the obtained polymerization and amount of this (c). bioactive substance increases with increase the degree degree of polymerization and the this bioactive substance increases an increase in bioactive the degree of polymerization. For amount example,ofγ-PGA dimers can be bonded with a with maximum of three The presence of a tyrosole molecule in the obtained conjugate can be expected at each degree The presence of a tyrosole molecule in the obtained conjugate can be expected at each degree of molecules polymerization. For example, γ-PGA dimers can while be bonded a maximum of threefor bioactive due to presence of three carboxyl groups, γ-PGAwith tetramer can be bonded up to ofof polymerization polymerizationand andthe theamount amountofofthis thisbioactive bioactivesubstance substanceincreases increaseswith withan anincrease increaseininthe thedegree degree five bioactive molecules to thecarboxyl presence groups, of five carboxyl groups.tetramer can be bonded for up to molecules due to presencedue of three while γ-PGA ofofpolymerization. polymerization.For Forexample, example,γ-PGA γ-PGAdimers dimerscan canbe bebonded bondedwith withaamaximum maximumofofthree threebioactive bioactive From the inspection spectrumof(Figure 4), it can be observed that a higher degree of five bioactive molecules dueoftomass the presence five carboxyl groups. molecules moleculesdue duetotopresence presenceofofthree threecarboxyl carboxylgroups, groups,while whileγ-PGA γ-PGAtetramer tetramercan canbe bebonded bondedfor forup uptoto polymerization increases the number of possible structures tyrosol-γ-PGA (see From the of mass spectrum (Figure 4), itcarboxyl can ofbe observed thatconjugates a higher degree five bioactive due totothe ofoffive groups. fiveinspection bioactivemolecules molecules due thepresence presence fivecarboxyl groups. 4b,c). of Figure polymerization increases the number possible structures of be tyrosol-γ-PGA (seeofof From the ofofmass spectrum (Figure 4), aahigher From theinspection inspection massof spectrum (Figure 4),ititcan can beobserved observedthat thatconjugates higherdegree degree The structures of the ions visible on the mass spectrum, which represent the γ-PGA oligomers Figure 4b,c).polymerization polymerization increases increases the the number number ofof possible possible structures structures ofof tyrosol-γ-PGA tyrosol-γ-PGA conjugates conjugates (see (see obtained in esterification carried outoninthe melt, were assignedwhich basedrepresent on different mechanisms of The structures of the ions visible mass spectrum, the γ-PGA oligomers Figure 4b,c). Figure 4b,c). thermalindecomposition of polyamides discussed in literature [40–44]. The proposed structures were The ofofthe visible on mass represent the oligomers Thestructures structures theions ions visible onthe the massspectrum, spectrum, which represent theγ-PGA γ-PGA oligomers obtained esterification carried out in melt, were assigned based onwhich different mechanisms of thermal placed in obtained Table 2. in esterification carried out in melt, were assigned based on different mechanisms obtained in esterification carried out in melt, were assigned based on different mechanisms decomposition of polyamides discussed in literature [40–44]. The proposed structures were placed inofof thermal thermaldecomposition decompositionofofpolyamides polyamidesdiscussed discussedininliterature literature[40–44]. [40–44].The Theproposed proposedstructures structureswere were Table 2.

Tableplaced 2. Structural assignments of the most intensive ions appearing in the expanded regions at m/z ininTable 2.2. placed Table 480–800 and 840–1040 of the ESI–MS most intensive ions appearing in the expanded regions at m/z Table 2. Structural assignments of thespectrum.

assignments ofofthe Table2.2.Structural Structural assignments themost mostintensive intensiveions ionsappearing appearingininthe theexpanded expandedregions regionsatatm/z m/z 480–800 andTable 840–1040 of the ESI–MS spectrum. Structure Ions (m/z) 480–800 480–800and and840–1040 840–1040ofofthe theESI–MS ESI–MSspectrum. spectrum.

OR

O

A

N

H2N

AA

H2H N2N OO

N

B

NN

OR

B

O

BB ORand/or and/or H

O

O O N OR OR

O

NN

OO

O

OR

C

CC

O

H O ON

C O

O

+ OR OR

OR OR

OO O

OO

++

nn

++H H

O

OR

499; 508; 628; 637; 748; 757; 766; 877; 886; 895; 997; 1006; 1015; 1024; 1126; 1135; 499; 637; 748; 757; 766; 499; 508;628; 628;637; 637;748; 748; 757; 766;877; 877;886; 886; 499;508; 508; 628; 757; 766; 1144; 1153 877;997; 886; 895; 997; 1006; 1015; 1024;1135; 895; 1015; 1024; 1126; 895; 997;1006; 1006; 1015; 1024; 1126; 1135;

OO

1126;1153 1135; 1144; 1144; 1153 1144; 1153

++H H

O

NH NH 2 2

n nN N

OO

HH

+ +

OR OO ORH OO C N n nN N

OR OR

++

+

OR O O NH 2OR H

OR OR OR

NN H OO

+

OO

H

H Hn N

OR

517; 757; 766; 775; 886; 517; 637;646; 646;757; 757;766; 766; 775; 886;895; 895;1015; 1015; 517;637; 637; 646; 775; 886; 895; 1015; 1024; 1135; 1144; 1153 1024; 1135; 1144; 1153 1024; 1135; 1144; 1153

++H H

OR OR

HH

HHn N

OR

O

OR OR

OR OR

OO

and/or

IonsIons (m/z) Ions(m/z) (m/z)

517; 637; 646; 757; 766; 775; 886; 895; 1015; ++1024; 1135; 1144; 1153

+H

n nN N

+ HOO

n

HH

O

OO

O

H

OR

OO OO O

HH H NN

OR

O

+

OR

O

Structure Structure Structure n N

OR OR

OO

O

A

O

H

++490; 610; 619; 739; 748; 859; 868; 877; 979; 490; 610; 619; 859; 868; 490; 610; 619; 739; 748; 859; 868; 490; 610;1108; 619;739; 739;748; 748; 859; 868;877; 877;979; 979; 988; 997; 1006; 1117; 1126 HH + + CC NN 877; 979; 988; 997; 1006; 1108;

HH

988; 997; 1006; 988; 997; 1006;1108; 1108;1117; 1117;1126 1126 1117; 1126

R = H or –C2H4PhOH. 2H 4PhOH. R=2=H or–C –C 2H 4PhOH. R = H or R–C HH4or PhOH.

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In order to establish the structure of tyrosol-γ-PGA conjugates, ESI-MS2 analyses were 2 analyses performed containing different conjugates, amounts ofESI-MS bioactive molecule perperformed oligomer In orderfor to selected establishmolecules the structure of tyrosol-γ-PGA were 2 chain. Figure 5 shows ESI-MS different spectra amounts of conjugates containing four molecules per for selected molecules containing of bioactive molecule perbioactive oligomer chain. Figure 5 2 oligomer chain. shows ESI-MS spectra of conjugates containing four bioactive molecules per oligomer chain. Figure 55 presents presents ESI-MS ESI-MS22 spectrum spectrum (in (in positive-ion positive-ion mode) mode) of of the the precursor precursor ion ionatatm/z m/z 1015 1015 Figure correspondingto toγ-PGA γ-PGAoligomers oligomerscontaining containingfour fourtyrosol tyrosol molecules per one oligomer chain. One corresponding molecules per one oligomer chain. One of of the possible structures of ion at m/z 1015 is shown on the scheme in Figure 5. The product ion at the possible structures of ion at m/z 1015 is shown on the scheme in Figure 5. The product ion at m/z 997 loss of of thethe water (18(18 Da). TheThe product ion ion at m/z m/z 997 corresponds correspondstotothe theoligomer oligomerformed formedbybythe the loss water Da). product at 895 corresponds to the oligomer formed by the loss of the 4-ethenylphenol (120 Da). The product ion m/z 895 corresponds to the oligomer formed by the loss of the 4-ethenylphenol (120 Da). The product at m/z 886 886 corresponds to the oligomer formed bybythe 5-membered ion at m/z corresponds to the oligomer formed theloss lossofofthe the pyroglutamic pyroglutamic acid, acid, 5-membered lactam (129 (129 Da), Da), from from the theN-terminal N-terminal end. end. The The product product ion ionat atm/z m/z 877 877 corresponds corresponds to to the the oligomer oligomer lactam formed by the loss of the tyrosol (138 Da). The product ion at m/z 868 corresponds to the oligomer formed by the loss of the tyrosol (138 Da). The product ion at m/z 868 corresponds to the oligomer formed by the loss of the γ-glutamic acid, 147 Da, (in cases when the last repeating unit is not formed by the loss of the γ-glutamic acid, 147 Da, (in cases when the last repeating unit is not esterified) esterified) from C-terminal end. from C-terminal end.

Figure 5. 5. ESI-MS/MS ESI-MS/MS product ion spectrum spectrum of of the the sodiated sodiated tyrosol-γ-PGA tyrosol-γ-PGAconjugate conjugateatatm/z m/z 1015 1015 and and Figure product ion theoretical fragmentation pathway of this ion. theoretical fragmentation pathway of this ion.

The structural verification of the ions at m/z 1015 corresponding to tyrosol-γ-PGA oligomer The structural verification of the ions at m/z 1015 corresponding to tyrosol-γ-PGA oligomer conjugates with the aid of ESI-MS2 techniques confirmed that those oligomers contained four conjugates with the aid of ESI-MS2 techniques confirmed that those oligomers contained four tyrosole tyrosole units, which are covalently bonded to γ-PGA chains. Moreover, esterified carboxyl groups units, which are covalently bonded to γ-PGA chains. Moreover, esterified carboxyl groups are are randomly distributed along the obtained conjugate chains. randomly distributed along the obtained conjugate chains. 3. Discussion 3. Discussion Previously,the thetwo-step two-step method for preparing conjugates of bioactive compound with a Previously, method for preparing conjugates of bioactive compound with a hydroxyl hydroxyl group was reported by some of us. In the first step, cyclic oligomers of PHB group was reported by some of us. In the first step, cyclic oligomers of PHB (poly(3-hydroxybutyrate)) (poly(3-hydroxybutyrate)) were obtained; in the second step, these cyclic oligomers were applied in were obtained; in the second step, these cyclic oligomers were applied in transesterification reactions transesterification reactions bioactive compounds the presence of ofenzymes. novel with bioactive compounds in with the presence of enzymes. Theinnovel application syntheticThe strategies application of synthetic strategies described here based on (trans)esterification of biopolymers with described here based on (trans)esterification of biopolymers with bioactive compounds with a hydroxyl


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group turned out to be a relatively quick, simple and solvent-free way of obtaining conjugates of biopolymers with such bioactive compounds. In this study, P(3HB-co-4HB) biopolymer was used to obtain conjugates with tyrosol via transesterification reaction mediated by TSA¨ H2 O. Similarly, as was previously established, increasing the quantity of water, which is introduced with TSA to the reaction medium, led to the decrease of molar mass of resulting oligomers [17]. Moreover, based on results shown in Table 1, it was determined that amount of bioactive substance used in transesterification reactions influenced the dispersity index Mw /Mn . Through increasing the of amount of tyrosol, a decrease of dispersity index was achieved. The transesterification of P(3HB-co-4HB) biopolyester with tyrosol leads to conjugates which contain one bioactive molecule per oligomer chain. The poly-γ-glutamic acid is a polyamide which contains carboxyl groups in each repeating unit, and these carboxyl groups allow for coupling bioactive moieties to each repeating unit. Therefore, application of the γ-PGA, as a polymeric carrier provides a possibility to increase the amount of biologically active moieties along the oligomer chain. Applying γ-PGA in our study was successful and allowed us to obtain conjugates of bioactive compounds with the γ-PGA oligomer chain. As was expected, the conjugates we synthesized contained a higher amount of biologically active moieties distributed along the γ-PGA oligomer chain. The amount of tyrosol molecules which are bonded to the γ-PGA oligomer chain increased with an increase of molar masses of γ-PGA oligomers. For example, γ-PGA dimer can be bonded with a maximum of three bioactive molecules due to the possibility of esterification by tyrosol of the three carboxyl groups present in this oligomer, while γ-PGA tetramer can be bonded up to five bioactive molecules due to presence of five carboxyl groups. The tyrosol-γ-PGA conjugates were obtained in an esterification reaction between tyrosol and γ-PGA biopolymer carried out in melt. Under these reaction conditions, the esterification between tyrosol and γ-PGA biopolymer can be accompanied by thermal decomposition of γ-PGA, which is typical for polyamides. The presence of tyrosol-γ-PGA conjugates with different structures in the products of esterification reaction between tyrosol and γ-PGA was identified using mass spectrometry. However, it is noteworthy that all types of obtained γ-PGA oligomers contain bioactive molecules bonded along oligomer chains, which was confirmed using electrospray ionization tandem mass spectrometry. The structural characterization of the tyrosol-γ-PGA with the aid of ESI-MS2 techniques allowed the structure of those conjugates at the molecular level to be established. Moreover, these studies confirmed that esterified by tyrosol carboxyl groups are randomly distributed along γ-PGA conjugate chains. Determination of the most appropriate molar mass of tyrosol-oligo(3HB-co-4HB) and tyrosol-γPGA conjugates, as well as the influence of the presence of various structures of the end groups in tyrosol-γ-PGA conjugates on properties and potential applications of this conjugates as a delivery system of tyrosol, will be investigated in further studies. 4. Materials and Methods 4.1. Materials The poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was purchased from Tianjin Green Bio-Science (Tianjin, China); the number-average molar mass, as determined by GPC, was Mn = 250,000 g/mol and the dispersity index was Mw /Mn = 2.5. The 4HB unit content was 8.8 mol % (based on the 1 H NMR spectrum). The poly-γ-glutamic acid was purchased from Natto Biosciences (Montreal, QC, Canada), Mn = 47,800 g/mol, Mw /Mn = 3.2. Additionally, chloroform, hexane and N,N-Dimethylformamide (DMF) were supplied by POCH SA (Gliwice, Poland). The 4-(2-hydroxyethyl)phenol (tyrosol) and 4-toluenesulfonic acid monohydrate were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Dialysis membrane Spectra/Por (MWCO (molecular weight cut-off): 12,000–14,000) was purchased from Carl Roth (Karlsruhe, Germany).


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4.2. Measurements Gel permeation chromatography (GPC) analyses were carried out using Viscotek VE 1122 pump (Malvern Instruments Ltd., Worcestershire, UK), Shodex SE-61 RI detector (JM Science Inc., Grand Island, New York, NY, USA), and PLgel 3 µm MIXED-E (Polymer Laboratories, Santa Clara, CA, USA) high-efficiency column (300 mm ˆ 7.5 mm). Analyses were performed at 35 ˝ C using CHCl3 as mobile phase with the 1 mL/min flow rate. The instrument was calibrated with polystyrene narrow standards. Nuclear magnetic resonance (NMR) analyses were performed using NMR Spectrometer Avance II 600 MHz Ultrashield Plus (Bruker, Rheinstetten, Germany); CDCl3 was used as the solvent and tetramethylsilane was used as the internal standard. Electrospray mass spectrometry (ESI-MSn ) analyses were performed in positive-ion mode using a Thermo LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA). Solutions of samples were introduced into the ESI source by continuous infusion at a 10 µL/min flow rate using the instrument syringe pump. Settings and conditions: spray voltage: 5.0 kV, capillary temperature: 200 ˝ C, sheath gas: nitrogen, auxiliary gas: helium. For ESI-MS/MS experiments, precursor ion was isolated by the ion trap and collisionally activated. 4.3. Synthesis of Conjugates Biopolymer (0.25 g) and an appropriate amount of bioactive substance (amount of tyrosol for synthesis of PHA-based conjugates is placed in Table 1; for synthesis of PGA-based conjugates 0.1 g of tyrosol was used) and 4-toluenesulfonic acid monohydrate (TSA¨ H2 O) (0.05 g; 20 wt % compared to biopolymer) were placed into a round bottom flask equipped with a magnetic stirring bar. The vessel, containing reagents, was placed into a Heat-On Block System (RB Radley & Co. Ltd., Essex, UK) located on a stirring hot plate. Reactions were carried out in the melt (at 167–172 ˝ C) under an argon atmosphere. The molten reagents were stirred for 90 s; then, the reaction mixture was cooled. The PHA-based conjugates were purified by added 5 cm3 of chloroform and washed 5 times with distilled water to remove any residual 4-toluenesulfonic acid. Then, the products were precipitated with cold hexane and dried under a vacuum at room temperature. The PGA-based conjugates were dissolved in DMF; solutions were dialyzed through a dialysis tubes for 72 h, precipitates settled at the bottom of the dialysis bag. Precipitated products were dried under a vacuum at room temperature. The yield of reactions was between 60% and 78%. 5. Conclusions The method for the preparation of conjugates consisting of bioactive compounds with hydroxyl group (tyrosol) and oligomers from bacterial biopolymers has been discussed in detail. The developed method of (trans)esterification of selected biopolymers (such as P(3HB-co-4HB) or γ-PGA) in the presence of tyrosol mediated by 4-toluenesulfonic acid monohydrate is occurring in melt. This one-pot synthesis method is rapid and does not use solvents, and, therefore, is promising from a scale-up perspective because of the relatively low cost of reagents and the rather simple procedure. The structural characterization at the molecular level of the trans-esterification products with the aid of ESI-MS2 and/or 1 H NMR techniques confirmed that the developed method leads to the formation of conjugates in which bioactive compounds are covalently bonded to biopolymer chains. It was shown that transesterification of P(3HB-co-4HB) with tyrosol leads to the (3HB-co-4HB) oligomers that contain one bioactive molecule covalently bonded to the oligomer chain, while esterification of γ-PGA with tyrosol results in conjugates with increased amount of biologically active moieties along the oligomer chain. Thus, unexpectedly, it was found that esterification reactions of γ-PGA with tyrosol carried out in melt in the presence of TSA¨ H2 O enabled the preparation of conjugates containing from one to seven bioactive molecule per oligomer chain, which was confirmed using electrospray ionization tandem mass spectrometry. The signals in ESI mass spectrum correspond to individual macromolecular ion of conjugates; therefore, based on the mass assignment of singly


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charged ions observed in the mass spectrum, we were able to established amount of repeating units in each conjugate oligomers (degree of oligomerization) as well as amount of bioactive molecules per oligomer chain. The developed biodegradable polymeric systems should allow for the delivery of tyrosol, thereby prolonging and improving the efficacy of this bioactive compound. The release of tyrosol should be accompanied by the formation of non-toxic degradation products of P(3HB-co-4HB) or γ-PGA carriers. Acknowledgments: This work was supported by the Polish National Science Center (Decision Nos. DEC-2013/11/N/ST5/01364 and DEC-2013/11/B/ST5/02222). Author Contributions: I.K. and G.A. were responsible for conception and design of the experiments. I.K. and M.K. carried out the practical work of the synthesis part. I.K., I.R., M.K. and G.A. were involved in the interpretation of data from performed analyses of obtained conjugates. I.K. and G.A. were the main people involved in drafting and editing of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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