New way of anionic ring-opening copolymerization of β-butyrolactone and ε-caprolactone: determination of the reaction course

Poly(ε-caprolactone)-block-poly(β-butyrolactone) copolymers were prepared in two-step synthesis. Firstly, poly(ε-caprolactone) (PCL) was obtained by anionic ring-opening polymerization of CL initiated with anhydrous KOH activated 12-crown-4 cation complexing agent. Reaction was carried out in tetrahydrofuran solution and argon atmosphere at room temperature. Then, β-butyrolactone (BL) and 18-crown-6 were added to the system, resulting in PCL-block-PBL copolymer, which contains after methylation hydroxyl starting group and methyl ester end group. The main product was contaminated with PCL and PBL homopolymers formed in a side reactions. 13C NMR technique was used for determination of chemical structure of polymers obtained. The course of the studied processes was proposed. MALDI-TOF technique was used to reveal the macromolecules’ architecture where several series were found. The identified series shown that mainly copolymeric macromolecules were formed with scare contribution of homopolymeric polybutyrolactone with trans-crotonate starting groups and polycaprolactone, which is congruent with the proposed reaction mechanism. Moreover, critical approach concerning previously reported PCL-block-PBL copolymer synthesis by use of NaH as initiator was also presented.


Introduction
Biodegradable polymers, such as poly(glycolide), poly(lactide), poly(β-butyrolactone) and poly(εcaprolactone) [1][2][3] have several medical application, for example as bioabsorbable materials. It is necessary for the polyesters to present different mechanical and physical properties to adjust the adequate time of their degradation. Copolymerization is attractive for modulating the basic properties of each homopolymer [4][5][6]. Particularly interesting are block copolymers, which have a larger number of applications. Blocks with different physical properties, for example a soft, amorphous segment together with a hard semicrystalline one, can be used to modulate the thermal and mechanical material behavior [4,[7][8][9]. The soft phase provides elasticity and influences on the degradation behavior, whereas the rigid phase gives mechanical strength and acts as a physical crosslinker [6]. The successive ring opening polymerization of β-butyrolactone (BL) and L-lactide (LLA) [7] or -caprolactone (CL) [8,10] results in block copolymers consisting of PBL as a softer segment and PLLA or PCL as a harder ones. However, many effective catalysts based on tin-compounds were applied for the synthesis. These compounds are toxic and polymers obtained become non useful for pharmaceutical and biomedical applications [2,6]. In the literature one may found examples of application of anionic polymerization to obtain the copolymers comprising of CL and BL blocks [11][12][13]. Till now, only one paper presents the possibility of the anionic copolymerization of BL and CL by use of low toxic sodium hydride as initiator [14]. The mentioned process was conducted in bulk containing both monomers at 70 °C. Yields of copolymers are greatly influenced by the molar ratio of monomers BL/ CL. The molar composition of PBL-block-PCL copolyesters, determined by NMR spectra showed, that incorporation of CL is favored over the incorporation of BL. These copolymers are stable up to temperatures near 200 °C. The crystallization process was studied by DSC and WAXS showing, that the amorphous PBL segments chain do not affect the crystallinity of the PCL blocks. However, the course of copolymerization proposed by authors [14] seems to be questionable.
The aim of the present work was reinvestigation of PCLblock-PBL copolymerization initiated with NaH. Moreover, new concept of block copolymers synthesis was proposed by using of anhydrous KOH as initiator in tetrahydrofuran solution at room temperature. The course of the process was proposed and discussed.

Polymerization
All syntheses were carried out at room temperature in a 50 cm 3 reactor equipped with a magnetic stirrer and a Teflon valve enabling substrates delivery and sampling under argon atmosphere. Homopolymerization of BL and CL and their copolymerization mediated with NaH were carried out in bulk at 70 °C according to literature data [14]. Polymerization of CL initiated with anhydrous KOH activated 12C4 in THF solution at room temperature was performed according to the method described previously [14]. Potassium hydroxide was obtained in the reaction of pure potassium hydride with distilled water in THF. KH (0.08 g, 2.0 mmol), THF (15.8 cm 3 ) and 12C4 (0.88 g, 4.0 mmol) were introduced into the reactor, and then water (0.036 g, 2.0 mmol) was added by use of a microsyringe. The reaction mixture was then stirred for 30 min until all hydrogen (44.7 cm 3 ) was evolved. This resulted in a fine dispersion of pure anhydrous potassium hydroxide in the ether medium. The reactors for synthesis of KOH and polymerization of BL initiated with NaH were performed in glass apparatus à 50cm 3 equipped with a magnetic stirrer and two Teflon valves. One of them enabled substrates delivery, the second was joined by elastic canula with calibrated tube filled with water and placed vertically in the vessel containing water. The envolved H 2 was collected in the tube and then it was analyzed by chromatographic method. The obtained system was used as the initiator, when CL (4.2 cm 3 , 4.6 g, 40 mmol) was introduced into the reactor. The reaction mixture was then stirred for several hours (~ 120 h). After this time relatively high conversion (90%) of the monomer was observed by SEC technique. Then, unreacted KOH was removed by centrifugation and polymer solution was introduced under argon into the second reactor containing 18C6 (0.53 g, 2.0 mmol) and BL at CL/BL equal to 1/0.5 or 1/1 molar ratio. The system was then mixed by 50 h. After total BL conversion the reaction mixture was treated with HCl/H 2 O or CH 3 I quenching agents and mixed by 30 min. Then, the mixture was placed in a 250 cm 3 separator containing H 2 O (10 cm 3 ) and CHCl 3 (100 cm 3 ). After shaking during 5 min two layers were formed, i.e. interior polymer layer and superior layer containing water and the potassium salt. The layers were separated and the superior layer was removed. After two washings with distilled water, copolymer was obtained by evaporating of chloroform and water in vacuum. The concentration of the monomers during the polymerization was monitored by SEC method. The yields of the reactions were 98-99%. All investigated processes were homogeneous.

Measurements
100 MHz 13 C NMR spectra were recorded in CDCl 3 at 25 °C on a BruckerAvance 400 pulsed spectrometer equipped with 5 mm broad band probe and applying Waltz16 decoupling sequence. Chemical shifts were referenced to tetramethylsilane serving as an internal standard. In order to obtain a good spectrum of the copolymer main chain exhibiting its microstructural details, about 3000 scans were satisfactory, but in order to observe the signals of the polymer chain ends more then 10 000 scans were necessary. Molar masses and dispersities of copolymers were obtained by means of size exclusion chromatography (SEC) on a Shimadzu Prominance UFLC instrument at 40 °C on a Shodex 300 mm × 8 mm OHpac column using tetrahydrofuran as a solvent. Polystyrenes were used as calibration standards. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra was recorded on a Shimadzu AXIMA Performance instrument. Dithranol (1,8-dihydroxy-9,10-dihydroanthracen-9-one) was used as a matrix. All data were obtained in a positive-ion linear mode, applying the accumulation of 200 scans per spectrum. The calibration of the linear-mode analysis was done using protein standards in mass range up to 8000 Da. The samples were dissolved in dichloromethane at a concentration of 2 mg/mL. The sample solutions were mixed with a matrix solution in the same solvent. Data were acquired in continuum mode until acceptable averaged data were obtained and were analyzed using a Shimadzu Biotech Launchpad program.

Results and discussion
In 2010 Monsalve et al. [14] reported the possibility of preparation of PBL-block-PCL copolymers by anionic ring-opening polymerization using NaH as initiator. BL and CL were polymerized in bulk at 70 °C in glass reactor equipped with magnetic stirrer. After removing of BL oligomers and unreacted NaH from the prepared copolymer, it was then precipitated in methanol and dried in vacuum. Basing on analysis of NMR spectrum of the product, the authors proposed, that in the first step of the process NaH exclusively deprotonates BL (Scheme 1).
In this reaction hydrogen evolves and sodium transcrotonate forms, which initiates anionic BL ring opening polymerization via carboxylate anions as propagating species. It leads to blocks of PBL of a relatively low molar mass. Then, the latter initiates the polymerization of CL being in the reaction mixture through alkoxide anions as propagating species leading to blocks of PCL with relatively higher molar mass. This suggestion was visualized by us on Scheme 2.
However, according to the literature data the above course of the process seems to be questionable. It was previously well established by Penczek et al. [16], that carboxylate anions do not initiate CL polymerization. It is in accordance with the general inability of these anions to react with esters. The only exception are esters with good leaving group. The strained ring, like in BL can be considered as an equivalent of a good leaving group. Thus, carboxylate anions initiate easily BL polymerization forming exclusively carboxylate growing species [16]. Moreover, sodium hydride appeared also to be active initiator of CL polymerization in bulk at 70 °C [17]. The authors proposed, that initiation proceeds by acyl-oxygen bond cleavage, resulting in the formation of macromolecules with aldehyde starting groups and alkoxide end groups as the growing species (Scheme 3).
Therefore, we concluded, that it is unlikely that PBLblock-PCL copolymers could be synthesized in the system containing mixture of both monomers and sodium hydride as initiator. Homopolymerization of BL and CL should be rather expected in this case.
In order to confirm this hypothesis experimentally we repeated in the first step homopolymerization of BL and Scheme 1 Anionic polymerization of β-butyrolactone initiated with NaH [11] Scheme 2 Course of ε-caprolactone polymerization through alkoxide anions as propagating species leading to PBL-block-PCL copolymer according to suggestion proposed in [11] 1 3 CL in the presence of NaH in bulk at 70 °C. The polymers obtained were dissolved in anhydrous tetrahydrofuran and treated with CH 3 I as quenching agent. The most interesting are carbon signals of terminal groups, i.e. starting and end ones shown in 13 C NMR spectra of both hompolymers (Fig. 1).
These results confirm previous data concerning homopolymerization of BL and CL initiated with NaH [14,17]. Then, we repeated polymerization of BL and CL mixture in the same conditions. 13 C NMR spectrum of the products isolated after 96 h (Fig. 2) reveals main signals derived from carbons of PBL and PCL [18] as well as the same signals of terminal groups (additional signals at 23.0, 28.9, 29.4, 34.6, 69.3 and 176.2 derive from unreacted CL [18].
It early indicated, that homopolymers were formed in this system and not expected copolymer, which should possesses exclusively trans-crotonate starting groups and methoxy end groups. Therefore, we decided to prepare CL/BL block copolymer using different method.
First step of the synthesis was PCL preparation by ringopening polymerization of CL initiated with anhydrous KOH activated by weak ligand 12-crown-4 (12C4). It allowed to obtain linear polyester with relatively high yield. Stronger ligands, i.e. 15C5 or 18C6 are inconvenient, because of high tendency of CL to the formation of cyclic macromolecules in these systems [19]. 13 C NMR analysis of polymer obtained after methylation reveals several strong signals characteristic for carbons derived from mers of CL. They are following signals which correspond well with literature data [17]: δ (in ppm) = 24.7, 25.7, 28.5, 34.3, 64.3 (OCO(CH 2 ) 5 ), 173.7 (COO). Moreover, Fig. 3 shows weak carbon signals of terminal groups, which were identified as HOOCCH 2 -(at Basing on these data the course of the process was proposed on Scheme 4. After initiation of CL polymerization by acyl-oxygen bond cleavage propagation takes place on alkoxide centers resulting in macromolecules 1, which undergo transformation willingly during polymerization to 2 by cation exchange reaction. Equlibrium between 1 and 2 is postulated to be strongly shifted to the right similarly to that, observed previously in early stage of BL polymerization with KOH [19,20]. In the second step BL was added to produce copolymer chains. The amount of BL was calculated to supply either 1/0.5 or 1/1 feed ratio of CL to BL. After BL addition, 2 behaves as macroinitiators, when strong ligand 18C6 is added to the system [21]. It is necessary to apply 18C6 rather than 12C4 to enhance the  However, macroinitiator 1 can also react with BL but in different way, i.e. by monomer deprotonation (Scheme 6), similarly to proposed earlier by Kricheldorf et al. [21] for BL polymerization initiated with t-BuOK.
Similar reaction occurs with carboxylate anions, which was known as side chain transfer reaction to monomer [22]. Both reactions lead to the formation of some PBL macromolecules with unsaturated trans-crotonate starting groups. 13 C NMR analysis of the final products confirmed the proposed course of the process. The spectrum reveals signals characteristic for carbons derived from mers of both monomers [14,17]. These are following strong signals: (1)  i.e. CH 3 CH = CHCOO-(at 122.6 and 144.7 ppm) and -CH 2 COOH (at 33.6 ppm) (Fig. 4). Table 1 shows results of polymers analysis obtained by SEC method with exemplary SEC chromatogram of PCL/ PBL (1/1) copolymer shown on Fig. 5. In order to properly evaluate the obtained data we assume that in the course of the synthesis linear block copolymers were synthesized. P S s t a n d a r d s -M a r k-H o u w i n k c o n s t a n t s -K = 1.76•10 -2 mL/g, a = 0.679.
Dispersity of the synthesized block copolymers is rather high, that may indicate the occurrence of transesterification reaction due to the polymerization in polar solvent. It is also presumably caused by cation exchange reaction, which transforms reactive alkoxide centers of growing chains to carboxylate anions, which are inert in anionic polymerization of CL. Moreover, chain transfer reaction with BL comonomer could result in higher dispersity of CL/BL copolymers. These reactions are also responsible for lowering of molar mass of the polymers obtained.
Analysis of the products by MALDI-TOF technique confirm chemical structure of macromolecules. Figure 6 shows spectrum of the products obtained at equimolar ratio of co-monomers.
Some series of MALDI-TOF signals were identified. There are several series identified that correspond to adducts of either H + , Na + or K + to the respective macromolecules.

Scheme 6 Formation of homopolymers in the side reactions
There are copolymeric macromolecules with proton adduct [BL n /CL m + H] + (signals marked with full circle), with potassium adduct [BL n /CL m + K] + (signals marked with square) and sodium adduct [BL n /CL m + Na] + (signals marked with triangle). In addition to these there are also signals of homopolymeric macromolecules of polybutyrolactone with trans-crotonate starting groups and COOH end group which form adducts with sodium ion [BL n + Na] + (signals marked with star). Finally some homopolymers of CL for macromolecules with COOH starting groups and CH 2 OH   Formation of homopolymers in the process is undesired. It is impossible to eliminate chain transfer reaction with monomer but it could be possible to minimalize it. Kricheldorf et al. [21] observed, that unsaturation of PBL decreases in less polar solvents and lower temperatures. It seems also to be reasonable, that decreasing of basicity of carboxylate active centres should be expected by use of Na + counterion in bulk or non-polar solvent. Optimalization of synthesis presented in this work needs further investigations.

Conclusions
In the present work we proposed new method of PCL-block-PBL lactones copolymers synthesis in THF solution at room temperature. The main features of this process are: • In the first step CL polymerizes with anhydrous KOH in the presence of 12-crown-4, giving homopolymer with potassium carboxylate centers after cation exchange; the latter initiates polymerization of added BL after activation by 18-crown-6. • PCL-block-PBL copolymers after methylation possess hydroxyl starting groups and ester end groups and have relatively high dispersity (M w /M n ≈2.1-2.25). • Derived samples contain small amounts of CL and BL homopolymers which can be formed in side chain transfer reaction with BL comonomer. • The proposed procedure for minimalisation of side reactions involves use of Na + counterion and/or less polar solvent and weak cation complexing ligands, which is the subject of the further studies. • Preparation of BL/CL block copolymers in the presence of NaH described previously is improbable to realize, due to inability of carboxylate anions to initiate polymerization of ε-caprolactone. In this system homopolymers of BL and CL should be formed rather, than copolymers.