Biocatalytic synthesis of δ-gluconolactone and ε-caprolactone copolymers *

The biodegradability and biocompatibility properties of ε-caprolactone homopolymers place it as a valuable raw material, particularly for controlled drug delivery and tissue engineering applications. However, the usefulness of such materials is limited by their low hydrophilicity and slow biodegradation rate. In order to improve polycaprolactone properties and functionalities, copolymerization of ε-caprolactone with δ-gluconolactone was investigated. Since enzymatic reactions involving sugars are usually hindered by the low solubility of these compounds in common organic solvents, finding the best reaction medium was a major objective of this research. The optimal copolymerization conditions were set up by using different organic media (solvent and solvents mixtures), as well as solvent free systems that are able to dissolve (completely or partially) sugars, and are nontoxic for enzymes. Native and immobilized lipases by different immobilization techniques from Candida antarctica B and Thermomyces lanuginosus have been used as biocatalyst at 80°C. Although the main copolymer amount was synthesized in DMSO:t-BuOH (20:80) medium, the highest polymerization degrees, up to 16 for the copolymer product, were achieved in solventless conditions. The products, cyclic and linear polyesters, have been characterized by FT-IR and MALDI-TOF MS analysis. The reaction product analysis revealed the formation of cyclic products that could be the major impediment of further increase of the chain length.


INTRODUCTION
Synthesis of copolymers based on bio-derived monomers by in vitro enzymatic catalysis has attracted many research interests in the past decades (Martin et al., 1992).The utilization of enzymes for synthesis of polyesters was comprehensively reviewed (Kumar et al., 2001;Matsumura, 2006).
ε-Caprolactone (ECL) represents one of the most valuable cyclic monomers in ring opening polymerization reactions of lactones catalyzed by lipases.The resulting polyester, poly-(e-caprolactone), (PCL), was intensively studied and well characterized (Gross et al., 2010).Due to its biodegradability, biocompatibility and interesting mechanical properties, PCL was used in different studies as raw material for microsphere, nanoparticles, films, fibers synthesis, but mostly as drug delivery systems (Dash & Konkimalla, 2012).However, there are some limitations of PCL applicability due to its low hydrophilicity, low melting point and slow degradation rate (Sinha et al., 2004).A possible solution to eliminate these drawbacks could be sugars derivatives that can introduce new functionalities and properties to the PCL hydrophobic backbone.
Over the last few years, enzymatic copolymerizations of PCL with various monomers/polymers such as sugars derivatives, poly(ethylene glycol), alkyl diacids, diols have been reported (Juais et al., 2010;Gross et al., 2001) and, as expected, the newly introduced functionalities suggested novel potential applications.The immobilized lipase from Candida antarctica B, commercially available as Novozyme 435, demonstrated to be most efficient among lipases from different sources that have been screened for these reactions.Sugar-derivative based polyesters were successfully synthesized, starting from alditols.The lipases were proved to be efficient when adipic acid, 1,8-octanediol and different alditols such as: mannitol, xylitol, glucitol, galactitol, erythritol and ribitol were used as raw materials (Liu et al., 2006).A different glucose derivative, isosorbide, was used as multifunctional initiator of ring-opening polymerization of ECL catalyzed by immobilized Yarrowia lipolytica lipase, yielding PCL-isosorbide oligomers with M n values up to 1068 (Barrera-Rivera & Martinez-Richa, 2009).Juais and coworkers synthesized sugar-based polyesters from the same sugar derivative (isosorbide) and an aliphatic dicarboxylic acid diester (Juais et al., 2010).Up to date, enzymatic synthesis of copolymers of ECL with sugar-derived lactones has not yet been reported.
In this study D-glucono-δ-lactone (GL), a low priced raw material, easily available by oxidation of glucose, was selected as co-monomer for the synthesis of copolymers with ECL.Immobilized lipases from various sources and different reaction media were tested to find the suitable reaction conditions.Although sol-gel entrapped lipases were proved as very efficient catalysts for esterification and transesterification reactions (Tomin et al., 2011;Ursoiu et al., 2012), they were not investigated, at our best knowledge, for polyester synthesis.
Methods.Polymerization in organic solvents.ECL (0.445 mL, 4 mmole) and Candida antarctica lipase B (50 mg, Novozyme 435) were added to GL (0.356 g, 2 mmole) dissolved in 1 mL organic medium.The reactions were performed in 4 mL Micro Reactions Vessels at 80ºC, under argon atmosphere, magnetically stirred at 300 rpm.The reactions were stopped by filtration of the enzymes.
Polymerization in bulk.To 0.356 g GL and 0.445 mL ECL, 50 to 100 mg lipase, native or immobilized, were added.The reactions were performed into a 4 mL vial at 50/80°C in Ar atmosphere.At the end of reaction 1 mL of tetrahydrofurane was added and the immobilized enzyme and unsolved GL were removed by filtration.The polymer obtained by evaporating the solvent was dried overnight in vacuum, at 60°C.
Pre-equilibration of water activity.Raw materials and immobilized lipase Novozyme 435 were equilibrated with saturated salt solutions at 25°C in separate containers.The salts chosen for this study were MgCl 2 (α w 0.225), K 2 CO 3 (α w 0.432), Na 2 SO 4 (α w 0.95) and K 2 SO 4 (α w 0.973).The equilibration was performed overnight previous to the starting of the reaction.
Lipase sol-gel immobilization.Lipase from Candida antarctica B was immobilized by sol-gel entrapment method, as described before (Corici et al., 2011).In a 4 ml glass vial, 780 μL lipase solution, 200 μL PEG 20000 (4%), 100 μL NaF (1M) and 200 μL isopropyl alcohol were mixed (magnetic stirring, 600 rot/min), and 6 mmol silane precursors were added.The resulting mixture was mixed at ambient temperature until the gel formation was observed, and the gel was kept for 24 h at 4°C for a complete polymerization.The bulk gel was washed and dried at room temperature as described elsewhere (Zarcula et al., 2010), and kept at 4°C until further use.
Products characterization.Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR).Fourier Transform Infrared (FT-IR) spectra of the samples were obtained in attenuated total reflectance (ATR) mode on a Bruker Vertex 70 (Bruker Daltonik GmbH, Germany) spectrometer equipped with a Platinium ATR, Bruker Diamond Type A225/Q.Spec-tra were collected in the range 4000-400 cm -1 with a resolution of 4 cm -1 and with 64 co-added scans.
MALDI-TOF MS analysis.MALDI-TOF MS analysis of products was carried out using Bruker BIFLEX III matrix assisted laser desorption ionization time-oflight mass spectrometry (Bruker Daltonik GmbH, Germany) at an acceleration voltage of 20 kV using 2,5-dihydroxybenzoic acid as matrix.Sample preparation, weight average (M w ) and number average (M n ) molecular numbers, and the product relative content were determined as described elsewhere (Kakasi-Zsurka et al., 2011).Within every MALDI-TOF spectrum, we estimated the relative composition of the reaction product assuming that the ionization efficiency was not significantly different for the oligomeric species present in the mixture.

Lipase-catalyzed copolymerization of ECL and δ-gluconolactone and identification of the products
Synthesis of different bio-polymers by ECL grafting/insertion was intensively studied in the last years, using the chemical route and Sn(Oct) 2 as a catalyst.Li and coworkers (2005) described the starch-g-poly(εcaprolactone) chemical synthesis by three different polymerization methods, in bulk, in toluene suspension or in suspension/bulk polymerization, at temperatures higher than 120°C.The highest PCL grafting efficiency obtained was 40% (wt), and the homopolymer synthesis could not be avoided (Li et al., 2005).
The use of lipases as biocatalysts for copolymers synthesis can lead to highly ordered repeat-unit chain sequence (Matsumura, 2006), and lipases can facilitate copolymerizations of monomers that are difficult to be achieved by traditional methods (Kumar et al., 2000).
Recently, our group firstly demonstrated the ability of lipase to use GL, together with 3-hydroxybutyric acid, as substrate for copolymer formation (Kakasi-Zsurka et al., 2011).In the present study, ECL was used as co-monomer, anticipating higher cycle reactivity due to the higher polymerization degree of PCL synthesized by lipases, compared to polyhydroxybutyrate (Mee van der et al., 2006).The possible reaction products can be linear (a) or cyclic copolymers (b), together with possible linear (c) or cyclic homopolymers of ECL (d) (Fig. 1).The homopolymer formation as co-product is explained by (i) higher solubility of ECL in non-polar organic solvents compared to the less soluble sugar derivatives, resulting in easier accessibility into the lipase catalytic site; (ii) higher reactivity of the 7-membered cycle (Mee van der et al., 2006).
The product formation was monitored by infrared spectroscopy.A shift of the bands corresponding to the carbonyl group stretching vibrations was observed during the polymerization reaction, from 1721 cm -1 in ECL and 1722 cm -1 in GL raw materials, to 1766 cm -1 in the ester product.
The products were identified based on MALDI-TOF MS spectra.A typical MALDI-TOF MS spectrum is depicted in Fig. 2, for a product obtained in solvent-less system using sol-gel immobilized lipase.The peak series which can be assigned to the sodium adduct ions of different oligomer series ([M+Na] + ) indicate the formation of linear and cyclic copolymers containing GL unit into the backbone (as depicted in Fig. 1a, b), together with ECL homopolymers (Fig. 1c, d).For example (inset of Fig. 2), the peak at m/z 1131.6 corresponds to the sodium adduct ion of a linear oligomer with n=8 and m=1 and the peak at m/z 1177.6 corresponds to the sodium adduct ion of a cyclic oligomer with n=7 and m=2 (i.e. two inserted GL units).PCL homopolymer series were also detected, e.g. the peak at m/z 725.4 demonstrates the presence of the oligomer with n=9.
The identification of the reaction products by MALDI-TOF MS demonstrates, for the first time, the lipase-catalyzed insertion of gluconolactone, as 2,3,4,5,6-pentahydroxy-caproic unit, into a PCL backbone.The formation of linear and cyclic copolymers was also confirmed by NMR analysis (data given in the Supplementary material).

Influence of the reaction medium and the water content
The reaction medium in the copolymers synthesis catalyzed by lipases represents an essential parameter, particularly when the solubility of the raw materials in the non-polar solvent is low.
Organic solvents with different partition coefficient value, logP, as well as solvent-less systems were investigated as reaction media.Since the suitable solvents for sugar dissolution such as dimethylsulfoxide, pyridine, dimethylformamide can significantly decrease the lipase activity due to the strip off the necessary water from the enzyme microenvironment (Kennedy et al., 2006), in this study mixtures of DMSO and t-BuOH were tested.Previous studies concerning syntheses of sugar esters indicated that a concentration up to 20% DMSO in the reaction media can be tolerated by lipases, with partial loss of activity (Croitoru et al., 2012).Our experiments were conducted 24 h, using Novozyme 435 as catalyst.At increasing log P values only a slight decrease of the number average molecular weight (M n ) and weight average molecular weight (M w ) was observed (Table 1), excepting at high DMSO concentration (DMSO:t-BuOH =1:4) when the above-mentioned sensitivity of lipases toward DMSO resulted in lower molecular weights.However, important differences concerning the product composition were observed.The relative content of linear (LC) and cyclic (CC) copolymers was higher than previously found in the reaction products obtained from 3HBA and GL (Kakasi-Zsurka et al., 2011).The formation of linear (LH) and cyclic (CH) homopolymers could not be avoided, possibly due to the ECL excess employed, particularly in the solvent-less systems, where ECL had the role of reaction medium as well.The relatively high amount of cyclic copolymers explains the lower polymerization degree of ECL compared to the experiments with non-sugar raw materials (Matsumura, 2006).
The presence of water is essential in lipase-catalyzed synthesis reactions, as small amounts of water are necessary to preserve the activity of the enzyme in organic solvents.Water was also proved to be an important nucleophilic reagent in the chain initiation step of ringopening polymerization reactions (Ma et al., 2009).In the studied process, water could have two opposite effects, facilitating the GL solubility but shifting the reversible process toward hydrolysis.Therefore, determination of the optimum water level was imperious.Thus, the substrates and enzyme were pre-equilibrated separately with saturated salt solutions with different water activity prior to the reaction.All experiments were carried out 24 h at 80°C under argon atmosphere.The distribution of product species and the PDI were estimated by MALDI-TOF MS analysis.As shown in Table 1B, the highest M n value was obtained at 0.95 water activity.At low water activity values only traces of cyclic homopolymers were detected, but the formation of cyclic copolymer could not be avoided, regardless of the water activity value in the reaction system.Mei et al. have found that the increase of water content of the enzyme (Novozyme 435) led to increase of the molecular weight of the synthesized ECL homopolymers (Mei et al., 2002).In our case, such a dependence could not be evidenced, because of the more complex polymerization pathway.

Influence of the lipase immobilization method
The investigation of the influence of reaction medium and water activity on the copolymerization reaction was carried out using Novozyme 435.In fact, this extremely versatile immobilized lipase has been used in the majority of studies involving lipase-catalyzed polymerization (Miletic et al., 2012).However, since the copolymerization reaction also yielded considerable amounts of homopolymers, a screening study was considered useful in order to increase the copolymer content, as well as the polymerization degree.
The results depicted in Fig. 3, estimations of the relative composition of the products based on m/z intensities from the MALDI-TOF MS spectra (Kakasi-Zsurka et al., 2011), indicate that Lipozyme TL IM and the Solgel CALB 2 lipase were more effective for the synthesis of the ECL-GL copolymer than Novozyme 435, the lipase most frequently used for polymer synthesis.Porcine pancreas lipase was not appropriate for this reaction, as copolymer could not be detected and the homopolymer polymerization degree was not higher than 5 (data not shown).Other authors reported ECL copolymer a logP-partition coefficient value (Du, 2000;Sangster, 1989,) b number average molecular weight; c weight average molecular weight; d polydispersity index, LC, linear copolymer; CC, cyclic copolymer; LH, linear homopolymer; CH, cyclic homopolymer; α w , water activity values (Adlercreutz, 2008) formation at higher molecular weights with more reactive derivatized sugars (ethyl glucoside) in the presence of different lipases, but using the sugar derivative only as initiator for the ring-opening polymerization of ECL (Bisht, 1998).The other native lipase, CALB yielded a relative content of about 30% copolymer, in 1:1 cyclic to linear ratio.The enzyme immobilization method together with the characteristics of the support material has important influence on the immobilized enzyme activity and substrate specificity.Important differences were observed for our reaction system, probably as a result of different immobilization methods and supports, leading to particular support porosities and other characteristics that influenced the immobilized enzyme activity.Using Novozyme 435 as biocatalyst, linear homopolymer synthesis was facilitated, while the copolymer content was not higher than 20% and the highest polymerization degree obtained for the copolymer was 15.
In the best experiments with sol-gel entrapped Candida antarctica lipase (Sol gel CALB 2), a polymerization degree of 13 for the linear copolymer (m/z value 1587, Fig. 2), as well as about 50% relative content of linear copolymer was obtained.Replacing 3-NH 2 PrTMOS with OcTMOS as silane precursor during the immobilization process resulted in higher M n values, but the copolymer content was lower, probably because of the increased hydrophobicity of the matrix that can influence the diffusion of the substrate to and through the lipase catalytic site.A more accurate correlation between the biocatalyst activity and structure of the support is difficult to achieve, since several characteristics that influence the enzyme conformation and mass transfer during the catalyzed process should be taken in account.A morphological characterization of the entrapped lipases, providing information about the microstructure, porosity and texture of the matrix, will be realized in our forthcoming study and hopefully will help to better elucidate the mechanism of copolymer synthesis by sol-gel immobilized lipases.
Another important outcome of the studied process is the linear/cyclic ratio in the copolymer product.At the studied conditions, it was not possible to lower the cyclic polymer content below 50%.Cyclic polymer formation was reported by other authors, as well.Wahlberg et al., using ECL and d,l-lac- tide as raw materials, detected cyclic copolymers with one or two d,l-lactide units during the initial stage of the reaction, that gradually disappeared at long reaction time (up to 696 h) (Wahlberg et al., 2003).As our experiments with immobilized enzymes were carried out at a quite low temperature (50°C), the presence of cyclic species in all formed products cannot be considered as the effect of the temperature.

CONCLUSIONS
The insertion of GL into the hydrophobic backbone of poly(ε-caprolactone) was demonstrated for the first time, leading to oligomers containing 2,3,4,5,6-pentahydroxy-caproate units.Such new copolymers can exhibit new and more useful properties compared to PCL.Among the tested lipases, sol-gel entrapped CALB has proved higher activity for the synthesis of copolymers, compared to Novozyme 435.

Figure 1 .
Figure 1.Formation of linear (a) and cyclic (b) copolymers and linear (c) and cyclic (d) homopolymers, as possible reaction products.The structures indicate the masses of the corresponding oligomers, m and n meaning the numbers of GL and ECL repeat units, respectively.178 and 114 are the masses of the GL and ECL units, respectively, and 18 is the mass of the H 2 O end-group.