Synthesis of fully bio-based poly (3-hydroxybutyrate)-oligo-2-ethyl oxazoline conjugates

This work refers to the synthesis and characterization of poly (3-hydroxybutyrate)-b-oligo (2-ethyl oxazoline) (oligoEtOx). Cationic ring-opening polymerization of 2-ethyl oxazoline yielded poly (2-ethyl oxazoline) (oligoEtOx) with a hydroxyl end. Carboxylic acid-terminated PHB was reacted with oligoEtOx via dicyclohexylcarbodiimide chemistry to obtain PHB-b-oligoEtOx conjugates. The obtained PHB-b-oligoEtOx conjugates were successfully characterized by 1H- and 13C NMR, FTIR, DSC, and size exclusion chromatography. PHB-b-oligoEtOx conjugates can be promising biologic active materials.

PHB is a crystalline polymer with melting transition (Tm) at approximately 170 °C.It can also be synthesized by the anionic ring-opening polymerization of beta-butyrolactone (Hazer, 1996;Arkin et al., 2001).
The synthetic PHB is in R, S configuration, while bacterial PHB is only in R configuration (Caputo et al., 2022).
Very recently, Becer et al. reported the synthesis of poly (2-ethyl oxazoline)-b-poly (acrylate) hybrid multiblock copolymers via a click reaction.They evaluate their self-assembly behavior into stomatocyte-like nanoparticles (Hayes et al., 2023).The multiamide structure of polyEtOx makes it a candidate to mimic peptides, and it shows an antibacterial effect against Staphylococcus aureus (Hoogenboom, 2009).
Poly (2-ethyl oxazoline) is a new class of functional peptide that mimics with potential in a variety of biological applications (Zhou et al., 2020).PolyEtOx is a thermosensitive polymer with a lower critical solution temperature (LCST), changing the aqueous solution temperature at approximately 62 °C (Christova et al., 2003;Park and Kataoka, 2007;Obeid et al., 2009;Hoogenboom and Schlaad, 2011).
Winnik et al. reported the cloud point of aqueous methyl poly(I-propyl oxazoline) with Mn 10 K g/mol.Turbidity decreases with the increasing concentration from ~48 °C to ~39 °C.
In this work, we report the synthesis of poly (3hydroxybutyrate)-oligo-2-ethyl oxazoline, fully bio-based amphiphilic polymer conjugates.Two carboxyl-terminated PHB were synthesized by refluxing PHB with adipic acid in the presence of Stannous octoate.Then, the carboxyl-terminated PHB was reacted with the hydroxyl end of oligooxazoline, which was obtained by the ring-opening cationic polymerization of 2-ethyl oxazoline.The physicochemical characterization of the PHB-oligo-2-ethyl oxazoline conjugates was carried out in detail.

Synthesis of oligo(2-ethyl oxazoline) (oligoEtOx)
2-Ethyl oxazoline was oligomerized by ring-opening cationic polymerization.A mixture of 2-ethyl oxazoline (2.01 g) and MepTs (0.20 g) as the catalyst was dissolved in acetonitrile (AcCN, 2.0 mL) in a reaction bottle.Argon was passed through the solution for 2 min.Polymerization was carried out at 100 °C for 70 min.The polymer precipitated in excess diethyl ether.It was dried under vacuum at 40 °C for 24 h (yield: 1.98 g, Mn 900 g/mol, and PDI: 1.57).

Synthesis of dicarboxylic acid-terminated PHB, PHB-COOH
A mixture of adipic acid (1.00 g), PHB (0.64 g), and Sn-oct (20 mg) was dissolved in CHCl 3 (20 mL).It was refluxed at 85 °C for 4 h.After half of the solvent was evaporated, the product was precipitated from excess methanol and dried under vacuum at 40 °C for 24 h.The yield was 0.86 g.

Characterization
1 H NMR spectra were taken with an Agilent NMR 600 MHz NMR (Agilent, Santa Clara, CA, United States) spectrometer equipped with a 3-mm broadband probe.FT-IR spectra of the substituted polymer samples were recorded using a Bruker Model, Tensor II instrument with the ATR technique in the transmissive mode and a scan rate of 4,000 to 450 cm −1 .A Viscotek GPCmax autosampler system, consisting of a pump, three ViscoGEL GPC columns (G2000H HR, G3000H HR, and G4000H HR), and a Viscotek differential refractive index (RI) detector, was used to determine the molecular weights of the polymer products.A calibration curve was generated with five polystyrene (PS) standards of molecular weight 2,960, 8,450, 50,400, 200,000, and 696,500 Da with low polydispersity.Data were analyzed using Viscotek OmniSEC Omni 01 software.Differential scanning calorimetry (DSC) was used in the thermal analysis of the obtained polymers.The DSC analysis was carried out under nitrogen using a TA Q2000 DSC instrument that was calibrated using indium (Tm = 156.6 °C) and a Q600 Simultaneous DSC-TGA (SDT) series thermal analysis system.DSC measures the temperatures and heat flows associated with thermal transitions in the polymer samples obtained.The dried polymer samples were heated from −60 °C to 220 °C under a nitrogen atmosphere.All melting endotherms (Tm) were reported as peak temperatures, while all glass transition temperatures (Tg) were reported as midpoint temperatures.Thermogravimetric analysis (TGA) was used to determine the decomposition temperature (T d ) characteristics of the polymers by measuring the weight loss under a nitrogen atmosphere over time.In these analyses, the obtained polymers were heated from 20 °C to 600 °C at a rate of 10 °C/min, and the results were determined based on the first derivative of each curve.Scanning electron microscopy (SEM)  Frontiers in Chemistry frontiersin.org03 imaging (Zeiss EVO lS10) was used for the characterization of the obtained polymers.
PHB with two carboxylic acid terminals was obtained by the reaction of an equimolar amount of adipic acid and PHB under reflux conditions at 85 °C.The characteristic signals were observed in the 1H NMR and 13C NMR spectra of the assynthesized PHB-COOH sample, which is seen in Figure 1  1 H NMR spectra of the oligooxazoline and the as-synthesized PHB-oligoEtOx-23 conjugate in CDCl 3 .

Synthesis of PHB-oligoEtOx polymer conjugates
OligoEtOx was capped with the carboxylic acid ends of PHB-COOH to produce the novel PHB-b-oligoEtOx block copolymer.The reaction pathways can be seen in Figure 2.
The reaction conditions and results are listed in Table 1.Changing the feed percentage of oligoEtOx from 14% to 41% against PHB(COOH) 2 was reacted at room temperature.The yield of the obtained block copolymer was gravimetrically determined.The polymer obtained was precipitated from the acidified diethyl ether and dried in vacuum.For further purification, it was soaked in distilled water for 24 h in order to remove the unreacted oligoEtOx residue.
Characterization of PHB-oligoEtOx conjugates was carried out by   Thermal properties of the block copolymers were measured using a differential scanning calorimeter (DSC).The oligoEtOx sample has a wide glass transition (Tm) between 10 and 76 °C and the maximum at 64 °C.In the PHB-oligoEtOx polymer conjugate, the same wide melting transition between 6 and FTIR spectrum of PHB-oligEtOx-24 compared with the pristine oligEtOx.
Frontiers in Chemistry frontiersin.org06 80 °C together with that of PHB at 128 °C was observed.The PHB homopolymer has a melting transition at 170 °C.The lower melting transition of the PHB block in the copolymer shows the plasticizing effect of oligoEtOx.Figure 6 shows the DSC curves of PHB-oligEtOx-21 and homo oligoOx.Homo oligoEtOx showed the glass transition temperature (Tg) at 10 °C.

Conclusion
A fully biodegradable amphiphilic copolymer was obtained in this work.The hydroxyl end of oligoEtOx can easily be reacted with some other reagents to obtain polyoxazoline derivatives.Water-soluble hydrophilic oligoEtOx makes the hydrophobic polymers amphiphilic, which can be useful for medical applications.Combining natural and biodegradable hydrophobic properties of PHB with hydrophilic oligoEtOx yields a novel amphiphilic natural biopolymer.
Block copolymers containing hydrophilic and hydrophobic blocks gain the unique properties of both the related blocks.These different polymer blocks can be arranged linearly or as brush-type copolymers.The insertion of the hydrophilic polymer in a block copolymer can improve the colloidal stability of the biologic active nanoparticles for biomedical applications.Therefore, the PHB-b-oligoEtOx block copolymer can be a promising biopolymer for medical applications.

FIGURE 2
FIGURE 2Reaction pathways of the synthesis of the oligoEtOx-b-PHB block copolymer.
Figure 4 shows the 13 C NMR spectra of the as-synthesized PHB-oligoEtOx-23 and −24 conjugates in CDCl 3 .

TABLE 1
Synthesis conditions and results of the PHB-b-oligoEtOx block copolymer at room temperature for 24 h.