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Microwave assisted lipase catalyzed synthesis of biodegradable poly (pentadecanolactone)

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  • Save American Journal of Biomedical En gineer in g 2013, 3(1): 9-13 DOI: 10.5923/j.ajbe.20130301.02 Biodegradable Poly-pentadecalactone (PDL) Synthesis via Synergistic Lipase and Microwave Catalysis Anil Mahapatro1,*, Taína D. Matos Negrón2 1Bioengineering Program & Department of Industrial and M anufacturing Engineering, Wichita State University, Wichita, KS-67260, USA 2Center for M aterials Research,(CM R), Norfolk State University, Norfolk, VA-23508, USA Abstract A large nu mber of currently used synthetic biodegradable polymers in bio med ical engineering applications are polyesters based materials and thus research on the synthesis, properties, manufacturing and processing of aliphatic polyesters continues to be of great importance. Poly -ω-pentadecalactone (PPDL) a lactone based ring opening polymer has good mechanical properties and the presence of hydrolysable ester linkages along the poly mer chain making it desirable as a biodegradable material for diversified bio med ical engineering applications. In this paper we report the formation of PPDL using the synergistic effects of lipase and microwave (MW) technology. The effect of reaction time on the PPDL poly mer chain growth has been investigated. PPDL have been formed using lipase and MW irradiation at varying reaction time intervals (30-240 mins). Synergistic MW and lipase catalyzed poly merization of PPDL gave a number average molecular weight (Mn) of 24,997 g/ mol and a polydispersity index (PDI) of 1.93 in 240 mins as compared to Mn of 8,060 g/mo l and PDI of 2.17 using lipase and tradit ional heating. Thermal characterizat ion of PPDL fo rmed using MW and lipase catalysis showed that MW did not have a detrimental effect on the thermal properties of the poly mer obtained. Keywords Micro wave, Lipase, Biodegradable Poly mer, Po ly-pentadecalactone 1. Introduction Bio d eg rad ab le p o ly mers fin d v ario u s b io med ical eng in eering ap p licat ions th at co u ld b e categ o rized as temporary support device (sutures, bone fixation devices), temporary barrier (a rtific ial skin), drug delivery device (nano, micro pa rt ic les ), t is s ue engineering s caffo ld and mu lt ifu n ct ion al d ev ices (b io deg rad ab le d ru g elu t ing stents)[1]. Although numerous biodegradable poly mers find use in biomedical engineering applications a large number of cu rrent ly used s ynt het ic b iod eg rad ab le p o ly mers are p o ly est ers b ased materials [2]. Th us res earch o n t he synthesis, propert ies, manufacturing and p rocessing of aliphatic polyesters continues to be of great importance. The p rod uct ion o f bo th alip hat ic and s emi-alip hat ic polyesters fo llo ws t wo synthet ic st rateg ies (a) either rin g -o pen in g p o ly merizat io n (ROP) o f cyclic es ters (lactones or cyclic oligoesters) or (b) polycondensation of diacids and dio ls, hyd ro xyacids and/or their di-methyl esters[2]. Both of the above methodologies require use of a catalyst or an initiator. In case of biodegradable polymers for pharmaceut ical or medical applications, the potential toxicity of the catalyst plays a critical role in the choice of * Corresponding author: (Anil Mahapatro) Published online at Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved the catalyst used as a residual trace of the catalyst could lead to cytotoxicity[3].Th is has led to academic and industrial interest to look for a less toxic group of catalysts, initiators and processes for the synthesis and manufacturing of biodegradable polyesters. Microwave and enzy matic assisted polymer chemistry both individually have been gaining interest in green manufacturing as an alternative energy source and catalyst respectively[4, 5]. M icrowave assisted synthesis provides several advantages and benefits such as localized heating, reduced chemical reactions times, increased product yield and increased selectivity[6]. M icrowave assisted synthesis has expanded to a variety of biological molecu les such as peptides, oligo mers and carbohydrates[7]. Co mprehensive reviews have covered the use of microwave in o rganic chemistry[4] and in recent years in poly mer chemistry[7]. En zy me cataly zed polymerizat ions have been explored intensively since the 1990’s. En zy matic catalysts provide an alternate green catalytic substitute for the toxic metal complexes usually employed in polyester polymer synthesis[5, 8, 9]. The main advantages of enzyme catalyst are 1) h igh catalytic activ ity, 2) h igh efficiency under mild conditions (temperature, solvent), 3) bioco mpatibility, 4) reusability, and 5) stereo and regioselectiv ity[5,10]. En zy matic catalyzed poly merizat ions serve as an environmental friendly synthetic process and provide an example of green poly meric chemistry[10]. Relatively fewer reports exist on the applications of 10 Anil M ahapatro et al.: Biodegradable Poly-pentadecalactone (PDL) Synthesis via Synergistic Lipase and M icrowave Catalysis micro wave assisted enzymatic react ions, with emphasis on organic s mall molecu le t ransformations[11-16]. Enhanceme nts in the initial rate of reaction,[17, 18] p roduct yields[15, 19] and enantioselectivity[13] have been reported when using microwave heating as compared to conventional heating. However understanding of this area is poor and often controversial. Leadbeater[12] and co-workers investigated the effect of microwave irrad iation on lipase catalyzedtransesterificat ion of methyl acetoacetate in toluene. They reported minor d ifferences between conventional and micro wave heating. Rejasse et al.[20] studied the effect of micro wave heating on the stability of Candida antarctica Lipase B (CA LB) and the kinetics of butyl butyrate synthesis. They reported an increase in enzy mat ic stability in o rganic med iu m under microwave field suggesting a possible explanation for an increase in conversion rates under micro wave heating[20]. The field of microwave assisted enzy matic polymerizatio ns has been recently receiving attention for exp loit ing the synergistic benefits of lipase and microwave processed in the field of poly mer chemistry[21, 22]. Kerep and Ritter investigated the influence of MW irradiat ion on lipase catalyzed ring opening polymerization of ε-caprolactone[22]. They reported that MW assisted enzymat ic poly merizations had accelerating effects depending on the kind of boiling solvent used[22]. Recently our laboratory investigated the effects of micro wave process parameters (power, intensity, MW irrad iation time and temperature) on lipase catalyzed polymerization of capro lactone[23]. In this paper we report the formation of poly-ω-pentadecalactone (PPDL) a lactone based ring opening polymer using the synergistic effects of lipase and micro wave technology. Good mechanical properties of PPDL and the presence of hydrolysable ester lin kages along the polymer chain of PPDL have led to significant interest and consideration of PPDL as a biodegradable material for d iversified bio medical engineering applications. 2. Materials and Methods 2.1. Reagents temperature mon itored using an in situ optical probe. The reaction was set in a ‘CEM’ microwave synthesizer ‘Exp lorer’ at constant temperature of 70℃, constant power of 200W and at p redetermined reaction time periods of 30 min, 60 min, 120 min and 240 min. The temperature and power setting were determined based on previous protocols established in our laboratory[23]. Control reaction was carried out using enzyme catalyzed poly merization in a traditional o il bath for 240 min. The react ions were terminated by dissolving the residual mono mer and poly mer in chloroform and separating the insoluble enzy me by filtration using 10-15 mm g lass-fritted filters. The poly mer was then dissolved in ch loroform and precipitated using methanol. The insoluble materials was filtrated and washed three times with 5 mL port ions of fresh methanol. The polymer was dried using vacuum oven and subsequently characterized. 2.3. Characterization 2.3.1. Nuclear Magnetic Resonance (NM R) Proton (1H) NM R spectra were obtained on a Bru ker Advanced 300 MHz spectrometer at 300 and 75.13 MHz. The chemical shifts in parts per million (pp m) were referenced relative to tetramethylsilane (TM S). 2.3.2. Gel Permeation Chro matography (GPC) The molecular weights of poly mers were measured on a Viscotek GPC max 2001 TDA 360 triple-detector system at a temperature of 35 ℃ .Narro w mo lecular weight polystyrene standards obtained from Sig ma-A ldrich were used to generate the conventional calibration curve. The mobile phase utilized to study these systems was tetrahydrofuran (THF) at a flow rate of 1ml/ min. 2.3.3. Thermograv imetric Analysis (TGA ) Thermal analysis was performed on a Perkin -Elmer TGA6/DSC6 system with constant N2 flow. Weight of sample was in the range 8-10 mg. The analyses were performed at a rate of 10℃/ min fro m room temperature to 600℃ The monomer ω -pentadecalactone (PDL)[98%, mo lecula r weight 240.38], Novozy me-435 (Candida antartica Lipase B), chloro form (CHCl3), methanol (CH3OH) were purchased from Sig ma-Aldrich and used without further purification. 2.2. Microwave Assisted enzyme-catalyzed ring-opening Polymerization of PDL 3. Results and Discussion Figure 1 represents the schematic representation of MW assisted Lipase catalysis of poly-ω-pentadecalactone. The reactions were carried out at 70℃ in bulk for up to 4 hours in MW irradiat ion and a control reaction using traditional oil bath heating. All reactions were carried in solvent-free environ ment. The PDL mono mer (500 mg, 2.08 mmol) and lipase (50 mg of CA LB) was used for the reaction. These reagents consisting of an en zy me to monomer ratio of 1/ 10 wt/wt wereplaced in a 7 mL microwave (MW) reaction vials with Figure 1. Schematic representation of synergistic microwave and novozyme-435 catalyzed polymerization of poly-pentadecalactone from the cyclic monomer ω-pentadecalactone American Journal of Biomedical Engineer ing 2013, 3(1): 9-13 11 Figure 2 shows the proton NMR spectra of poly-ω-pentadecalactone at various reaction conditions with novozyme-435 as a catalyst and microwave irradiation along with the control reaction (novozy me-435 cataly zed polymerization of PDL in a traditional heated oil bath). Fro m our 1H NM R spectra we can deduce that ω-polypentadecalactone was formed at all the reaction times. The peaks assignments in 1H-NM R (CDCl3) spectra were: 4.05 (t , J 7 Hz, CHO), 3.64 (t, J 7 Hz, CH2OH), 2.31 (t, J 7 Hz, CH2 CO), 1.62 and 1.33 (22H, CH2) pp m. The chemical shifts reported are in good agreement with earlier reports of lipase catalyzed poly merization of ω-pentadecalactone using conventional heating[24]. hrs and Mn of 22,100 g/ mol (PDI 3.3) in 72 hrs respectively[24]. Ou r reported Mn of 24,994 (PDI 1.93) after only 4 hrs is much higher than those even obtained with tradit ional heating at much higher times of 72 hrs[24]. Figure 3 shows changes of the Mn over time indicating that with increase in the reaction time the Mnvalues went up fro m 13, 342 g/mo l in 30 min to 24,997 g/mo l in 240 min (4 h rs ). Figure 2. 1H NMR spectra of poly-pentadecalactone in CD3Cl at reaction intervals of 30, 60, 120 and 240 minutes and with conventional heating in an oil bath (240min OB) Table 1. Polymerization of ω-Pentadecalactone in Bulk at 70oC catalyzed by Novozyme-435 under Microwave conditions Sample PPDL- 30 min PPDL- 60 min PPDL- 120 min PPDL- 240 min PPDl- 240 min in oil bath* Mn 13,342 15,066 19,406 24,997 8,060 Mw 25,953 32,670 39,259 48,459 17,555 DPavg P DI % yield 56 1.94 62 63 2.16 41 81 2.02 61 104 1.93 56 34 2.17 20 *reaction carried out in using lipase catalysis using conventional heating in oil bath, Mn is number average molecular weight, Mw is weight average molecular weight, DPavg is average degree of polymerization and PDI is polydispersity index Table 1 summarizes the obtained mo lecular weights, PDI and % yields at different time intervals. Using synergistic lipase and MW irradiation after 240 min (4 hrs) the value ofnumber average mo lecular weight (Mn) of the poly mer obtained was 24,997 g/mo l with a polydispersity index (PDI) of 1.93. In contrast the polymer obtained using traditional heating in an oil bath for 240 min gave a Mn of 8,060 g /mol with a PDI of 2.17. This confirms that synergistic effects of MW and lipase catalysis results in a significant (three fold) increase in the Mn obtained as compared to traditional o il bath heating within the same time period. This trend is in agreement with previously reported results on ring opening polymerization (ROP) on caprolactone using synergistic MW and lipase polymerizat ion[23]. Bishht et al[24], have carried out the lipase catalyzed polymerization with lipase catalysis in traditional oil bath and have reported a Mn of 15,300 g/ mol (PDI o f 4.4) in 24 Figure 3. Effect of number average molecular weight (Mn) of the PDL formed vs reaction time for microwave assisted Novozyme-435 catalyzed p o lymerizat io n s In order to get a better understanding of polymer initiat ion and prorogation of PDL using synergistic MW and lipase catalysis the PDI and % y ield of poly merization were plotted against degree of poly merization (DPavg). Figure 4 shows variations in the PDI vsDPavg wh ich gives an indication on how the dispersity of the length of the polymer chains varies as the polymer chain (PPDL) grows. Similarly figure 5 gives an indication of the changes in % yield (rat io of polymer formed to monomer/low mo lecular weight oligomer) as the polymer chain grows (DPavg). Fro m figure 4 we can see that PDI increases initially 1.94 (30 min) to 2.16 (60min). During this time the Mn of the polymer rises fro m 13,342 (DPavg: 55) to 15,066 (DPavg: 63) (figure 3) and % yield drops fro m 62 to 41 respectively. A mechanism for the lipase-catalyzed ring opening polymerization of lactones is postulated by involving an acyl-enzy me intermediate[25]. The catalytic site of lipase resides in the serine residue and the lipase reaction proceeds via an acyl-enzy me intermediate[5, 25]. The poly merization occurs in two steps i.e ring opening of the lactone to form an oligo mer and propagation of these oligomoers to form high mo lecular weight polymer[5]. Thus during the initial stages with more lactone being ring opened the PDI increases due to which the nu mber of low mo lecular weight oligo mers present decreasing the relative % yield. As time progresses the oligo mer and other s mall poly mer chains combine to g ive high mo lecular weight poly mers. Due to the known chain selectivity of en zy mes catalysis the PDI of the polymer decreases. With format ion of high mo lecular weight poly mer the % yield increases. This is evident from Figure 4 and Figure 5 where the PDI drops fro m 2.16 to 12 Anil M ahapatro et al.: Biodegradable Poly-pentadecalactone (PDL) Synthesis via Synergistic Lipase and M icrowave Catalysis 1.93 fro m 60 min to 240 min and subsequently % yield increase fro m 41 to 56. Similar trends were reported by Bisht et al. wh ile carrying out the lipase catalyzed polymerization using traditional o il bath heating[24]. onset temperature of thermal degradation to begin at 350℃ [26]. Thus enzyme catalyzed PPDL using MW irradiation gives similar results to PPDL obtained using conventional heating[26]. Th is indicates no adverse changes in thermal properties of PPDL formed due to the synergistic MW and lipase heating. 4. Summary and Conclusions Figure 4. Effect of polydispersity index (PDI) of PDL formed vs reaction time for microwave assisted Novozyme-435 catalyzed p o lymerizat io n s In summary we have demonstrated the synergistic MW and lipase catalyzed polymerizat ion of PDL. Synergistic MW and lipase catalyzed poly merization of PPDL gave an average mo lecular weight (Mn) o f 24,997 g/ mol and a polydispersity index (PDI) o f 1.93 in 240 mins as compared to Mn of 8,060 g/ mol and PDI o f 2.17 using lipase and traditional heating. Thermal characterization of PPDL formed using MW and lipase catalysis did not have a detrimental effect on the thermal properties of the poly mer obtained. ACKNOWLEDGEMENTS We would like to acknowledge Wichita State Un iversity, the Center of Biotechnology and Bio medical Sciences (CBBS) at Norfolk State University for part ial support of the work. REFERENCES Figure 5. Effect of % yield on DPavg for microwave assisted Novozyme-435 catalyzed polymerizations [1] Buddy Ratner, Allan Hoffman, Frederick Schoen, Lemons J. Biomaterials Science: An Introduction to M aterials in M edicine. 2nd ed: Elsevier; 2004. [2] Kricheldorf HR. Syntheses of Biodegradable and Biocompatible Polymers by M eans of Bismuth Catalysts. Chemical Reviews, 109, 11, 5579-94, 2009. [3] M ahapatro A, Kalra B, Kumar A, Gross RA. Lipase-catalyzed polycondensations: Effect of substrates and solvent on chain formation, dispersity, and end-group structure. Biomacromolecules, 4, 3, 544-51, 2003. [4] Caddick S. M icrowave assisted organic reactions. Tetrahedron, 51, 38, 10403-32, 1995. [5] Gross RA, Kumar A, Kalra B. Polymer synthesis by in vitro Figure 6. T GA curves of Novozyme-435 catalyzed PPDL enzyme catalysis. Chemical Reviews, 101, 7, 2097-124, Thermal properties of enzy me-cataly zed PPDL in bulk 2001. were studied using thermogravimetric analysis (TGA). [6] Hayes BL. M icrowave Synthesis: Chemistry at the Speed of Figure 6 shows the TGA curves for PPDL synthesized at 30, Light: CEM Publishing; 2002. 60, 120 and 240 minutes along with the control reaction [7] Hoogenboom R, Schubert U, S. M icrowave-Assisted (traditional o il bath heating). The TGA curves shows a Polymer Synthesis: Recent Developments in a Rapidly weight loss with an onset temperature at above 350℃ and 10% weight loss at 380℃. No solid residue was observed Expanding Field of Research. M acromolecular Rapid Communications, 28, 4, 368-86, 2007. after 500℃. These results are similar to those reported by [8] M ahapatro A, Kumar A, Gross RA. M ild, solvent-free Letizia-Focareteet al[26], where they have reported the omega-hydroxy acid polycondensations catalyzed by American Journal of Biomedical Engineer ing 2013, 3(1): 9-13 13 Candida antarctica Lipase B. Biomacromolecules, 5, 1, 62-8, [18] Roy I, Gupta M N. Applications of microwaves in biological 2004. sciences. Current Science, 85, 12, 1685-93, 2003. [9] M ahapatro A, Kumar A, Kalra B, Gross RA. Solvent-free adipic acid/1,8-octanediol condensation polymerizations catalyzed by Candida antartica lipase B. M acromolecules, 37, 1, 35-40, 2004. [10] Kobayashi S. Recent Developments in Lipase-Catalyzed Synthesis of Polyesters. M acromolecular Rapid Communications, 30, 4-5, 237-66, 2009. [11] M augard T, Gaunt D, Legoy MD, Besson T. M icrowave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized β-galactosidase from Kluyveromyceslactis. Biotechnology Letters, 25, 8, 623-9, 2003. [12] Leadbeater NE, Stencel LM , Wood EC. Probing the effects of microwave irradiation on enzyme-catalysed organic transformations: the case of lipase-catalyzed transesterifications reactions. Organic and biomolecular chemistry, 5, 1052-5, 2007. [19] Yadav GD, Lathi PS. Synergism between microwave and enzyme catalysis in intensification of reactions and selectivities: transesterification of methyl acetoacetate with alcohols. Journal of M olecular Catalysis A: Chemical, 223, 1-2, 51-6, 2004. [20] Rejasse B, Lamare S, Legoy M D, Besson T. Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation. Organic and biomolecular chemistry, 2, 1086-9, 2004. [21] Atsushi Y, Yoshizawa-Fujita. M , Yuko T, M asahiro R. M icrowave-assisted enzymatic polymerization of PLGA copolymers and hybridization with hydroxyapatite 238th ACS National M eeting. Washington, DC,2009. [22] Kerep P, Ritter H. Influence of microwave irradiation on the lipase-catalyzed ring-opening polymerization of e-caprolactone. M acromolecular Rapid Communications, 27, 9, 707-10, 2006. [13] Carrillo-M unoz J-R, Bouvet D, Guibe-Jampel E, Loupy A, Petit A. M icrowave-Promoted Lipase-Catalyzed Reactions. Resolution of (+-)-1-Phenylethanol. The Journal of Organic Chemistry, 61, 22, 7746-9, 1996. [23] M atos TD, King N, Simmons L, Walker C, M cClain AR, M ahapatro A, et al. M icrowave assisted lipase catalyzed solvent-free polycaprolactone synthesis. Green Chemistry Letters and Reviews, 4, 1, 73 - 9, 2011. [14] Karmee SK. Application of M icrowave Irradiation in [24] Bisht KS, Henderson LA, Gross RA, Kaplan DL, Swift G. Biocatalysis. Research Journal of Biotechnology, 1, 2, 1, Enzyme-Catalyzed Ring-Opening Polymerization of 2006. ω-Pentadecalactone. M acromolecules, 30, 9, 2705-11, 1997. [15] Lin G, Lin W-Y. M icrowave-promoted lipase-catalyzed reactions. Tetrahedron Letters, 39, 24, 4333-6, 1998. [16] Zhao H, Baker GA, Song Z, Olubajo O, Zanders L, Campbell SM . Effect of ionic liquid properties on lipase stabilization under microwave irradiation. Journal of M olecular Catalysis B: Enzymatic, 57, 1-4, 149-57, 2009. [17] Parker M -C, Besson T, Lamare S, Legoy M -D. M icrowave radiation can increase the rate of enzyme-catalysed reactions in organic media. Tetrahedron Letters, 37, 46, 8383-6, 1996. [25] Kumar A, Kalra B, Dekhterman A, Gross RA. Efficient Ring-Opening Polymerization and Copolymerization of Caprolactone and Pentadecalactone Catalyzed by Candida antartica Lipase B. M acromolecules, 33, 17, 6303-9, 2000. [26] Letizia-Focarete M , Scandola M , Kumar A, Gross RA. Physical characterization of poly(ω-pentadecalactone) synthesized by lipase-catalyzed ring-opening polymerization. Journal of Polymer Science Part B: Polymer Physics, 39, 15, 1721-9, 2001.

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