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Protease-Catalyzed Oligomerization and Hydrolysis of Alkyl Lactates Involving L-Enantioselective Deacylation Step Hitomi Ohara,*,† Emiko Nishioka,† Syuhei Yamaguchi,† Fusako Kawai,‡ and Shiro Kobayashi*,‡ †
Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Center for Nanomaterials and Devices, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
‡
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INTRODUCTION Currently, poly(lactic acid) (PLA) is one of the most actively studied polymers in fundamental research as well as applications, mainly from viewpoints of “green plastics”1 and “green polymer chemistry”.2−14 Lactic acid (LA) is a typical one of bio-based renewable resources. PLA is prepared either by ringopening polymerization of lactide normally using Sn(II) catalyst15 or by dehydration polycondensation of LA using an acid catalyst.16−23 To broaden the applications of PLA materials, a new way to realize thermally more stable properties has been examined by designing stereocomplexes from PLLA and PDLA chains.24,25 In recent years, an enzyme has been widely used as catalyst to produce polyesters.3,4,7−10,12−14,26−29 Lipase-catalyzed ringopening polymerization was enantioselective for D,D-lactide.30 A lipase catalyst was also employed for ring-opening polymerization and copolymerization of lactide31−35 as well as ringopening polymerization of an O-carboxylic anhydride derived from LA.36 Very recently, we have reported new results on a lipase (Novozym 435)-catalyzed enantioselective condensation oligomerization of alkyl lactates (RLa)s as well as their hydrolysis, evidencing the first clear-cut reaction mechanism of the lipase catalysis; the mechanism involves a D-deacylating step to govern the enantioselection.37 We have also reported enantioselective depolymerizing hydrolysis of PLA by protease as well as lipase catalysts.38 In this paper, we describe a protease-catalyzed oligomerization of (RLa)s, the mechanism of which, in contrast to the Novozym 435 catalysis, is preferentially L-enantioselective, involving the enantioselection governed by a deacylation step. The enantioselection in catalysis of the lipase37 and the protease of the present study is similar in regard to the stereochemistry of the substrate in the oligomerization of D- and L-alkyl lactates and in the depolymerizing hydrolysis of PDLA and PLLA,38 respectively; protease is L-selective while lipase is D-selective.
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then kept in a screw bottle with silica gel. The bottle was preserved in a refrigerator. All the alcohols of extra-purity grade and molecular sieves 3A were obtained from Kanto Chemical Co. (Tokyo, Japan). 1,4-Dioxane, deuteriochloroform (CDCl3), and other solvents were commercially available and used without further purification. Commercially available ethyl L-lactate (EtLLa, Tokyo Chemical Ind. Co., Tokyo, Japan), and n-butyl L-lactate (BuLLa, Sigma-Aldrich Co., St. Louis, MO) were used. n-Hexyl lactates (HxLa) were prepared by esterification of LLA and DLA with n-hexanol as reported.37 EtDLa and BuDLa were prepared as reported.37 Characterization of these alkyl lactates was performed by measuring 1H NMR and ESI-TOF-MS. Oligomerization of Alkyl Lactates. A typical procedure was as follows. A mixture of 0.5 mmol of an alkyl lactate in a test tube containing 17 wt % of enzyme for the substrate, 0.5 mL of 1,4-dioxane, and 0.1 g of molecular sieves 3A was incubated with stirring by magnetic bar. Characterization of the product oligo(lactic acid)s (oligoLAs) was performed by 1H NMR and ESI-TOF-MS analyses. Hydrolysis of Alkyl Lactates. A typical run was as follows. To a mixture solution of EtLLa (0.5 mmol) and 1,4-dioxane (0.5 mL) containing n-propylbenzene (200 μL, as internal standard), distilled water (2.5 mmol) and Savinase CLEA (10 mg) were added. The mixture was homogeneous except for the catalyst and reacted at 60 °C with stirring. The consumption of EtLLa was followed by 1H NMR spectroscopy. Analytical Methods. 1H NMR measurements were recorded on a spectrometer ARX-500 (500 MHz, Bruker BioSpin GmbH, GER). ESI-TOF-MS analysis was performed by using a micrOTOF instrument (Bruker Daltoniks GmbH, GER).
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EXPERIMENTAL SECTION
Materials. Aqueous D-lactic acid (DLA, 90% solutions, HiPure 90) and L-lactic acid (LLA, 90% solutions, High Purity 90) were purchased from Purac Biochem b.v. (NLD). Savinase CLEA, Esperase CLEA, B. amyloliquefaciens protease, and Alcalase CLEA are the products of CLEA Technologies b.v. (NLD). There are cross-linked aggregate enzymes and kindly gifted by Shigematsu & Co. Ltd. (Osaka, Japan). Proleather FG-F, Umamizyme G, PROTIN SD-AY10, PROTIN SDAY10F, Protease P, and Samoase PC10F are the products of Amano Enzyme Inc. (Nagoya, Japan). Papain and Bromelain are the products of Wako Pure Chemical Ind. Lt. (Osaka, Japan). Proteinase K is the product of Nacalai Tesque Inc. (Kyoto, Japan). All the enzymes were kept in a decompressed desiccator containing silica gel for 1 day and © 2011 American Chemical Society
RESULTS AND DISCUSSION
Protease-Catalyzed Enantioselective Oligomerization. Since lipase (Novozym 435)-catalyzed oligo-condensation of alkyl D- and L-lactates (RDLa and RLLa) was shown as a perfect enantioselective reaction for D-lactates,37 we searched in this study for a catalyst having an L-enantioselectivity. In nature, proteases are known to hydrolyze proteins for L-amino acid residues.39 Therefore, a possibility of such catalysis was examined by using six different kinds of protease for (RLa)s. The reaction was carried out under various conditions. As shown in Table 1, it has been found that the reaction of (RLLa)s was induced by four proteases (codes 1−10 and 15) to give preferentially oligo(L-lactic acid)s (oligoLLAs; dimer to pentamer) (Scheme 1), although the yield is not high under the reaction conditions. Other seven proteases listed in the Experimental Section were also examined but inactive as catalyst (data not shown). Figure 1 shows the ESI-TOF-MS chart of the products of an oligoLLAs mixture (code 2), containing
Received: July 20, 2011 Revised: August 22, 2011 Published: August 26, 2011 3833
dx.doi.org/10.1021/bm201004g | Biomacromolecules 2011, 12, 3833−3837
Biomacromolecules
Note
Table 1. Protease-Catalyzed Oligomerization of Alkyl Lactate Monomers (RLa)s products reaction conditionsa b
code
enzyme
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Savinase CLEA Savinase CLEAd Savinase CLEA Savinase CLEAd Savinase CLEA Esperase CLEA Esperase CLEA Esperase CLEA B. amyloliquefaciens protease B. amyloliquefaciens protease B. amyloliquefaciens protease Alcalase CLEA Alcalase CLEA Alcalase CLEA Proleather FG-F Umamizyme G
a b
enzyme (wt %) 17 17 17 17 17 17 17 17 17 17 17 17 17 17 100 50
c
conversion (%) monomer (RLa)
temp (°C)
RLLa
RDLa
EtLa EtLa BuLa BuLa HxLa EtLa BuLa HxLa EtLa BuLa HxLa EtLa BuLa HxLa BuLa BuLa
60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 37
19 56 10 25 10 27 16 10 10 4
