Jul 16, 2013 - and the obtained CA-based polymers had relatively high molar masses .... catalyzed polymerization reactions, the release of ring strain.
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Cite this: Polym. Chem., 2013, 4, 4312
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Ring-opening polymerization of bile acid macrocycles by Candida antarctica lipase B† Satu Strandman,a I-Huang Tsai,a Robert Lortieb and X. X. Zhu*a
Received 21st May 2013 Accepted 5th June 2013 DOI: 10.1039/c3py00651d www.rsc.org/polymers
Lipase-catalyzed polymerization was explored as an alternative for Ru-catalyzed ring opening metathesis polymerization in the synthesis of polyesters bearing large functional moieties as part of the main chain. The selectivity of Candida antarctica lipase B (CALB) towards the functional groups of cholic acid (CA) was demonstrated, and the obtained CA-based polymers had relatively high molar masses, rubber-like elasticity, and glass transitions close to body temperature.
Aliphatic polyesters are commonly used as biodegradable materials in medical applications, such as sutures, stents, bone screws, tissue engineering scaffolds, and drug delivery systems.1 They are synthesized via the polycondensation of diols and diacids or hydroxy acids, or via ring-opening polymerization (ROP) of lactones or macrolactones by different mechanisms.1 Many of these methods require the use of organometallic catalysts and one possibility for reducing the metal residues in the nal product is to use enzymes to catalyze the polymerization.2,3 Due to some of the limitations of common aliphatic polyesters, such as slow biodegradation and increased acidity upon degradation that may cause changes in the solubility of the incorporated therapeutic agent in a drug delivery system4 or induce inammation at the site of an implant,5 there is growing interest towards functionalized polyesters. These polymers can exhibit tunable physico-chemical characteristics, including hydrophilicity and degradation rate, and a possibility for surface modication, such as attaching peptide ligands with a RGD (Arg-Gly-Asp) sequence to improve cell adhesion that will further trigger cell growth and proliferation.4,6 However, their synthesis oen requires protective group chemistries.
a Department of Chemistry, Universit´e de Montr´eal, C.P. 6128, Succursale Centre-ville, Montr´eal, QC, H3C 3J7, Canada. Fax: +1-514-340-5290; Tel: +1-514-340-5172 b
National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montr´eal, QC, H4P 2R2, Canada
† Electronic supplementary 10.1039/c3py00651d
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In our earlier studies, we synthesized a variety of main-chain functional polyesters and poly(ester-amide)s with tailored thermomechanical properties by entropy-driven ring-opening metathesis polymerization (ED-ROMP) of bile acid-based macrocycles, based on ring-chain equilibrium.7–10 Bile acids are amphiphilic endogenous steroids present in large quantities in the human body,11 and they are commercially available at low cost with enantiomeric purity, which makes them attractive starting compounds of natural origin for the synthesis of polymers with possible biomedical applications.12,13 They also have relatively high pKa which would suggest a moderate variation of local pH upon degradation of these materials.14 Recently, Hodge and co-workers reported the results of lipase-catalyzed polymerization of lithocholate (LCA) macrocycles yielding weightaverage molecular weights (Mw) of 14 500–49 500 g mol1.3 This work demonstrated the possibility of incorporating large functional moieties, such as steroid rings, in the polymer chain via an enzymatic reaction, but the reactivity of more complex, functional group-bearing macrocycles was not shown, and the possible presence of catalyst residues in the nal polymer was not discussed. Lipase-catalyzed reactions usually proceed via transesterication, although Hodge and coworkers also suggested the possibility of entropy-driven ring-opening polymerization (ED-ROP) for large strainless macrocycles bearing 12–84 ring atoms when the monomer concentration is high (>15 wt%).3 LCA-based monomers yielded signicantly higher Mw values (107 000–274 000 g mol1) in ED-ROMP with the help of Grubbs' catalyst.7 The large difference in molar masses seems to suggest that the enzymatic polymerization takes place via transesterication rather than via ED-ROP. However, lipasecatalyzed polymerization of simple aliphatic macrolactones without the lithocholate moiety provided high molar masses (up to 120 500 g mol1), comparable to those obtained by EDROMP, and more rigid macrocycles (cholaphanes) composed solely of lithocholic acid did not polymerize at all.3 Therefore, the structure of the cyclic monomer seems to play an important role in enzyme-catalyzed reactions.
This journal is ª The Royal Society of Chemistry 2013
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Communication Here, we investigated the Candida antarctica lipase B (CALB)catalyzed polymerization of macrocycle 1 based on a more hydrophilic and abundant bile acid, cholic acid (CA), synthesized according to an earlier procedure via ring-closing metathesis (RCM) of a corresponding diene.9 CA bears several hydroxyl groups in the steroid ring, which can act as handles for functionalization and increase the intermolecular interactions of the polymers, further affecting the thermo-mechanical properties. However, the additional hydroxyl groups may cause complications in the case of transesterication in comparison with LCA which possesses only one OH group. According to the preliminary tests, 80 C reaction temperature provided the highest molar masses in accordance with the literature reports on other monomers, which showed that the enzyme activity decreased above 90 C leading to lower conversions and molar masses.15 A high monomer concentration (30 wt%) was chosen to favor the polymerization in ringchain equilibrium,16 and it may also favor the interchain transesterication reactions, although up to a maximum concentration, above which lower conversions were reported.17 In lipase-catalyzed ROP, the reactive intermediate is an enzymeactivated monomer, acyl-enzyme, formed as a result of the cleavage of the monomer.2,17 If the nucleophile attacking the bond is a water molecule, then the cleaved monomer is released. If the nucleophile is a hydroxyl group created by ring opening, the chain grows. The balance between the chain growth and the hydrolysis comes from the amount of water present, oen contained in the enzyme, and the compared reactivity of water and the hydroxyl group. In the course of the reaction, the total concentration of water will actually diminish, as each polymer chain with one free acid and hydroxyl terminal groups corresponds to the use of one water molecule. Therefore, low water content (#0.2 wt%) is expected to lead to lower conversions, but it allows higher molar masses.15 Non-polar solvents, such as toluene, are preferred in the polymerization, as they do not remove the essential water from the enzyme or disrupt the active conformation.18 In this study, the enzyme was dried over P2O5 for 24 h under vacuum, and the water content of the reaction mixture was determined by Karl-Fischer titration aer the polymerization, being 0.13–0.19 wt%. According to the results shown in Table 1, higher lipase concentrations (10 wt%) led to lower molar masses, which is in accordance with the earlier observations on small cyclic monomers such as 3-caprolactone (3-CL), as the extent of ester hydrolysis is higher,19 suggested by the higher polydispersity at high lipase content. The lower molar mass could also stem from the fact that in the presence of more lipase, more chains grow simultaneously, which leads to faster depletion of the monomer. The conversions vary due to the purication of small polymerization batches (100 mg) by reprecipitation to remove the residual cyclic monomer and oligomers. In chemically catalyzed polymerization reactions, the release of ring strain (ROP) or increase in conformational freedom (ED-ROP) drives the reaction. In Ru-catalyzed ED-ROMP of bile acid-based macrocycles, the reactions were fast, and Mw values of up to 452 000 g mol1 and conversions of up to 98.6% could be reached in 30 min.9 In lipase-catalyzed transesterication, a
This journal is ª The Royal Society of Chemistry 2013
Polymer Chemistry Table 1 Enzymatic polymerization of cholic acid-based monomer 1 in toluene at constant temperature (80 C) and monomer concentration (0.3 g mL1)
Entry
Lipasea (wt%)
Time (h)
Yieldb (%)
Mwc (g mol1)
PDI
Tdegr ( C)d
Tge ( C)
1 2 3 4 5 6 7 8
10 10 10 5 5 5 2.5 2.5
48 24 96 24 24 72 24 48
67 30 50 47 78 51 29 49
11 300 11 500 10 100 30 400 30 200 29 900 29 800 26 300
1.94 1.75 1.93 1.20 1.35 1.35 1.32 1.30
349 349 348 347 361 344 345 346
37 37 36 35 40 36 34 40
a
Wt% (of the mass of the monomer) of CALB immobilized on polymer beads (Aldrich). b Mass yield aer purication by reprecipitation. Weight-average molecular weight determined by SEC in THF against polystyrene standards. d Degradation temperature from the onset of the thermogravimetric analysis (TGA) thermogram, estimated error 2 C. e Glass transition temperature from the onset of the differential scanning calorimetry (DSC) thermogram, estimated error 1 C. c
new ester bond involving the acyl moiety still in the form of an acyl-enzyme intermediate and a free nucleophile formed from the ester cleavage is created. In the case of macrolides, larger rings provide higher hydrophobicity favored by CALB, but the binding abilities are expected to be independent of the ring size.20 The rate of lipase-catalyzed ROP reactions can be determined by either the affinity of the enzyme for the substrate controlling the acyl-enzyme intermediate formation, or by the deacylation step modulated by the surroundings of the nucleophile alcohol at the end of the propagating chain. The long reaction times observed here (24–96 h) are coherent with both possibilities. The three ester bonds susceptible of being hydrolyzed in monomer 1 are rather hindered. This certainly impedes the interaction with the active site of the enzyme, compared to the more accessible ester in simple non-substituted lactones. The alcohol moieties resulting from the cleavage of these esters are also hindered. One would be the hydroxyl at the 3-position of the steroid ring, and the other two are two carbon atoms away from an ester. It is known that the accessibility of the propagating alcohol chain end has a strong inuence on the rate of CALB-catalyzed ROP reactions.21 A GPC chromatogram conrms that monomer 1 was converted to a polymer (Fig. 1) through enzymatic catalysis. The molar masses of polymers started to decrease aer a certain reaction time (Table 1), depending on the enzyme concentration. This could be attributed to the reversible nature of the enzymatic reaction leading to the hydrolysis of polymer chains. The sterically hindered axial 7- and 12-OH groups of the bile acid steroid ring are much less reactive than the equatorial 3-OH and hence, it is possible to modify chemically the OH only at the 3-position without protection–deprotection chemistry.22 In the literature, CALB catalyzed the acylation of 3-OH of methyl cholate with methyl esters of fatty acids with high selectivity and yield.23 In another study, cholic acid oligomers were obtained upon lipase-catalyzed homopolycondensation, which took place between the COOH group and OH at the 3-position.24 The
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selectivity of CALB towards hydroxyls has also been exploited in the lipase-catalyzed polycondensation of adipic acid with polyols occurring only on primary hydroxyls and leaving the secondary hydroxyls intact, thus avoiding the crosslinking typical for chemical catalysis,25 as well as in the polymerization of isopropyl aleuriteate and its copolymerization with 3-CL yielding linear polymers with pendent secondary hydroxyls.26 Since CALB transforms monomers into a polymer via transesterication, there is a possibility that the polymerization can also take place at these two positions. By comparing the NMR spectra of monomer 1 and polymer 5 (Fig. 2), the chemical shis of the protons at 7- and 12-positions did not change aer the polymerization, that is, the peaks of both protons stayed at 3.9 (7-a) and 4.0 (12-a) ppm. According to the simulated NMR spectra, if the hydroxyl group at 7- and 12-positions reacted during the polymerization, the chemical shis of their proton signals would change and appear at 4.3 (7-a) and 4.7 (12-a) ppm. The earlier reports on the OH group functionalization of CA showed even larger downeld shis.27 Therefore, the NMR spectra prove that the transesterication reaction did not take place at these two positions. As the ester groups of
primary hydroxyls (adjacent to (d) in Scheme 1) are less sterically hindered than the ester of secondary hydroxyl at the 3-position of the steroid ring, they are expected to be more susceptible to transesterication. Synthesizing macrocycles by metal-catalyzed reactions, such as ring-closing metathesis (RCM) or cyclodepolymerization, is a common strategy, and to the best of our knowledge, there are no reports on the catalyst residues in the polymers synthesized from them by metal-free approaches. Although the macrocyclic monomers were synthesized by ruthenium-catalyzed RCM with Grubbs' 1st generation catalyst, leaving the residual Ru content of 40–120 ppm aer treating the reaction mixture with DMSO for partial Ru removal29 and purifying the product several times by column chromatography on SiO2, the nal Ru content of enzymatically synthesized polymers was as low as 12 ppm. For comparison, a polymer based on monomer 1 synthesized by Rucatalyzed ED-ROMP (with Grubbs' 2nd gen. catalyst) contained 310 ppm of residual Ru, which is rather typical for polymers prepared by ROMP. In both cases, the polymers were puried by reprecipitation in methanol, and the difference in their colour is clearly visible (grey vs. white). In fact, without repeated precipitations, the amount of Ru in the ROMP-synthesized polymers could be even higher, more than 1000 ppm.30 Some current strategies for reducing the residual Ru content in polymers include the use of supported catalysts,31,32 smallmolecule catalyst-solubilizing agents, or scavenger-functionalized particles.30 The latter, more easily available methods could provide residual Ru levels from
