ENZYMATIC SYNTHESES AND CHARACTERIZATION OF ALIPHATIC POLYESTERS VIA Yarrowia lipolytica LIPASE BIOCATALYSIS Karla A. Barrera-Rivera and Antonio Martínez-Richa Departamento de Química. División de Ciencias Naturales y Exactas. Campus Guanajuato. Universidad de Guanajuato. Noria Alta s/n. Colonia Noria Alta. Guanajuato, Guanajuato, México. 36050 Introduction Enzymatic polymerizations are a powerful and versatile approach which can compete with chemical and physical techniques to produce known materials (such as ‘commodity plastics’) and also to synthesize novel macromolecules so far not accessible via traditional chemical approaches. Enzymatic polymerizations can prevent waste generation by using efficient catalytic processes with high stereo- and regio–selectivity, prevent or limit the use of hazardous organic reagents by, for instance, using water as a green solvent, design processes with higher energy efficiency and safer chemistry by conducting reactions at low temperatures under ambient atmosphere, and increase atom efficiency by avoiding extensive protection and deprotection steps. Because of these characteristics, enzymatic polymerizations can provide an essential contribution to achieving industrial sustainability in the future. 1 Yarrowia lipolytica lipase has demonstrated to be efficient in the synthesis of different biodegradable polyesters, obtained by ring-opening polymerization of cyclic esters. Using biocatalysis with YLL, oligomeric PCL diols can be efficiently produced. These bifunctional monomers can then used to prepare biodegradable linear polyester urethanes. 2-5 Experimental Materials. ε-CL (Aldrich Chemicals Co.) was dried over calcium hydride and distilled under reduced pressure before use. Chloroform, toluene and methanol were obtained from Karal and used as received. Chloroform-d (99.8 %), was obtained from Sigma-Aldrich. Diethylene glycol, 1,3- propanediol, Lewatit VP OC 1026 and K2629 beads, stannous 2- ethylhexanoate, hexamethylenediisocianate (HDI) and 1,2-dichloroethane anhydrous 99.8 % were purchased from Sigma Aldrich and used as received. Instrumentation. Solution 1 H and 13 C-NMR spectra were recorded at room temperature on a Varian Gemini 2000. Chloroform-d (CDCl 3 ) was used as solvent. FT-IR spectra were obtained with the ATR technique on films deposited over a diamond crystal on a Perkin-Elmer 100 spectrometer with an average of 4 scans at 4 cm -1 resolution. Gel permeation chromatography (GPC-MALLS) was used to determine molecular weights and molecular weight distributions, M w /M n , of macrodiols samples. The chromatographic set- up used consists of an Alliance HPLC Waters 2695 Separation Module having a vacuum degassing facility on online, an auto sampler, a quaternary pump, a columns thermostat, and a Waters 2414 Differential Refractometer for determining the distribution of molecular weight. The temperature of the columns was controlled at 33 ºC by the thermostat. Tensile properties were measured in a MTS Synergie 200 testing machine equipped with a 100 N load cell. Type 3 dumbbell test pieces (according to ISO 37) were cut from film. A crosshead speed of 200 mm/min was used. Strain was measured from crosshead separation and referred to 12 mm initial length. Five samples were tested for each polymer composition. Synthesis of α-Hydroxyl-ω-(Carboxylic Acid) Poly(ε-Caprolactone) with Yarrowia lipolytica lipase. Vials are previously dried and purged with dry nitrogen. In a typical experiment 1 mL of ionic liquid (1-butylpyridinium tetrafluoroborate [BuPy][BF 4 ]), 1.0 g of ε-caprolactone (8.76 mmol) and 0.1 g of YLL were placed at 60°C for 24 h, , the polymer was extracted by five consecutive extractions with 5 mL toluene and the enzyme was filtered off. Toluene was removed by evaporation at reduced pressure. Polymer was crystallized from cold chloroform/methanol and dried under vacuum. Molecular weights and conversions during reaction were monitored by 1 H- NMR. Synthesis of α,ω-telechelic poly (ε-caprolactone) diols (HOPCLOH). For DEG1PCL, (10 mmol of ε-CL, 1 mmol of DEG), DEG2PCL (10 mmol of ε-CL, 0.5 mmol of DEG), DEG3PCL (of ε-CL,0.25 mmol of DEG), PCL-1,3- propanediol (1) (10 mmol ε-CL/0.5 mmol 1,3-prop/12 mg YLL-1026), PCL- 1,3-propanediol (2) (10 mmol ε-CL/0.25 mmol 1,3-prop/12 mg YLL-1026) , PCL-1,3-propanediol (4) (10 mmol ε-CL/1 mmol 1,3-prop/12 mg YLL- K2629), PCL-1,3-propanediol (5) (10 mmol ε-CL/0.5 mmol 1,3-prop/12 mg YLL-K2629) were placed in a 10 mL vial previously dried, in all cases 12 mg of immobilized YLL was used. Vials were stoppered with a teflon silicon septum and placed in a thermostated bath at 120 ºC for 6 h. No inert atmosphere was used. After the reaction was stopped, the enzyme was filtered off. PCL diols were dried at 70 ºC and vacuum for 12 h, and stored at ambient temperature in a dessicator at vacuum until used. Synthesis of PCL macrodiisocyanate. Dry PCL diol (1.5 g) and HDI in the appropriate amount and 2 mL of 1,2-dichloroethane were charged in a round bottom flask. The catalyst, stannous 2-ethylhexanoate (1% mol by PCL diol moles) was added, and stirred for 4 h at 80 ºC. The resulting slurry was poured over a leveled glass. The solution was covered by a conical funnel to protect it from dust and to avoid the excessively fast solvent evaporation, and allowed to stand at ambient temperature for 24 h. The film was then released and dried in vacuum. Samples for physical characterization were cut from films, film thickness ranged from 50-80 µm. Figure 1 . Synthesis of α-ω telechelic polycaprolactone diols (HOPCLOH). Results and Discussion α-Hydroxyl-ω-(Carboxylic Acid) Poly(ε-Caprolactone). The synthesis of α-hydroxyl-ω-(carboxylic acid) poly(ε-caprolactone) has a molecular weight of 8000 Da and a polydispersity of 1.6, monomer conversion was 100%. NMR data for PCL: 1 H NMR (200 MHz, CDCl 3 , ppm) δ 4.031(t, 2H,[CH 2 O], 3.613 (t, 2H, [CH 2 OH]), 2.363 (t, 2H, [CH 2 CO 2 H]), 2.28 (t, 2H, [CH 2 O 2 ]), 1.62 (m, 4H, [(CH 2 ) 2 ]), 1.38 (q, 2H, [CH 2 ]). 13 C NMR (200 MHz, CDCl 3 , ppm) δ 177.616 (a), 173.944 (j), 173.754 (g), 64.310 (f), 62.679 (q), 34.373 (k), 34.267 (h), 33.812 (b), 32.401 (p), 28.479 (e), 25.664 (d), 25.444 (m), 24.822 (l), 24.716 (i), 24.488 (c). IR (cm -1 ): 2945(υ CH ), 1724 (υ C=O ), 1166 (δ O–C=O ). Synthesis of α,ω-telechelic poly (ε-caprolactone) diols (HOPCLOH). The synthetic pathway is described in Figure 1. The first step was the formation of the PCL diols by the ring-opening polymerization of ε-CL and using DEG and 1,3-propanediol as initiators in the presence of immbobilized YLL. Under the same conditions, the CL and DEG were allowed to polymerization in the absence of enzyme for control. After precipitation, no corresponding copolymers could be obtained, which indicate that the lipase enzymes actually catalyze the copolymerization of CL and DEG. Results for the synthesized PCL diols are shown in Tables 1 and 2. In Figure 2 the GPC traces of the synthesized PCL-diols are shown. The stress-strain curves of the different polymers are shown in Figure 3. Characteristic values derived from these curves are presented in Table 3. Three regimes are visible. First, the behavior at low deformations can be explained as the pure elastic deformation belonging to regular elastomers. Secondly, a zone due to plastic flow is observed. This is much the same for all the polymers studied, indicating a great feasibility for shear induced crystal fragmentation. Thirdly, at strains above 600 % an upswing in some of the curves can be observed, which can be attributed to the strain induced crystallization of soft segment chains. O O + H O O O H O H O O O O n H O O O O H p O O H r O + 120 ºC YLL Polymer Preprints 2012, 53(2), 319