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Biomacromolecules 2008, 9, 752–757
Insights into the Initiation Process of Enzymatic Ring-Opening Polymerization from Monofunctional Alcohols Using Liquid Chromatography under Critical Conditions Matthijs de Geus,† Ron Peters,‡ Cor E. Koning,† and Andreas Heise*,†,‡ Department of Polymer Chemistry, Technische Universiteit Eindhoven, Den Dolech 2, P.O. Box 513, 5600 MB Eindhoven, The Netherlands, and DSM Research, P.O. Box 18, 6160 MD Geleen, The Netherlands Received October 18, 2007 Revised Manuscript Received November 29, 2007
Introduction The synthesis of functional polymers is a central theme of polymer chemistry. Of particular interest are telechelic polymers with defined end groups that can be further modified or reacted. A convenient way to introduce end groups into a polymer is via a functional initiator, which carries the desired functionality next to an initiation moiety (initiator approach). The feasibility of this approach has been demonstrated for basically all types of controlled polymerization techniques such as controlled radical polymerization, anionic polymerization, and ring-opening polymerization (ROP).1–3 The latter is commonly used to synthesize aliphatic polyesters, e.g., polylactides and polylactones such as polycaprolactone (PCL). The biocompatibility and biodegradability of these materials makes them interesting building blocks for biomedical applications, for example, for degradable scaffolds and drug release devices.4–6 Controlled metal mediated ROP has extensively been used for the synthesis of these materials as it provides the necessary control to tailor the molecular weight, architecture, and end-functionality in the synthesis. Mechanistically, the metal mediated ROP proceeds via a monomer insertion mechanism in which the polymer end group is activated by the metal complex (e.g., tin octanoate).7–10 A hydroxy compound added to the polymerization mixture will act as an initiator and result in the incorporation of this compound into the growing polymer chain. The feasibility of this initiator approach has been shown for many examples, including the synthesis of very complex polymer architectures.11 Enzyme mediated ROP has emerged as an important alternative method for the preparation of aliphatic polyesters in the past decade.12–14 Many examples of lipase catalyzed ROP have been reported, including various lactones and carbonates.15 Endfunctionalized polyesters have been synthesized by the initiator method in analogy to metal mediated ROP using various hydroxyl functionalized nucleophiles. Similar to the metal mediated ROP of CL, a nucleophile can be considered as initiator of the polymerization. This initiator can be water, alcohol, amine, or thiol. In several studies, the initiation by various primary alcohols has been investigated. This ranges from simple monoalcohols, like short aliphatic alcohols or benzylalcohol,16–19 to more complex polyols like sugars,20–22 and * Corresponding author. Email:
[email protected]. Telephone: 0031-(0)40 2473012. Fax: 0031-(0)40 2463966. Address: Dr. Andreas Heise, Technische Universiteit Eindhoven, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. ‡ DSM Research. † Department of Polymer Chemistry, Technische Universiteit Eindhoven.
dendritic structures,23 where regioselective initiation from only one hydroxyl group was observed. In the case of functional initiators, end-functional polymers can be synthesized in which the end group can be utilized for further reactions. Chemoselective initiation was reported by Martinelle et al. using thioalcohols as initiators. Because of the selective initiation of enzymatic ROP from the alcohol group of the initiator, thiol end-capped PCL was obtained.24,25 Recently, the enzymatic synthesis of acrylated PCL and poly(pentadecalactone) applying a hydroxethyl acrylate initiator and the subsequent radical polymerization to yield comb copolymers was reported.26,27 In our group, we applied the enzymatic polymerization of CL in the presence of bifunctional initiators carrying a group for controlled radical polymerization. Block copolymers were obtained by macroinitiation from the functional PCL.28–32 While reasonable control over the molecular weight and endfunctionalization can be achieved by the addition of a nucleophile, enzymatic ROP does not fulfill the definition of a controlled polymerization. Mechanistically, the lipase catalyzed polymerization follows a monomer activation mechanism and is prone to side reactions such as inter- and intramolecular endto-end condensation, backbiting of the terminal OH group onto an activated carbonyl, hydrolysis of polymer chains, and chain transfer between polymer chains.33–36 The extent of these reactions is partly dependent on the reaction conditions but cannot be completely suppressed. As a function of the reaction conditions, the side reactions can also influence the end group structure of the obtained polymers. End group analysis of polymers from reaction samples may provide more insight if detailed quantitative information on the presence of different polymer species can be derived. Typically, end group analysis on polymers can be achieved by mass spectrometric techniques, such as electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Both techniques have enough resolution to distinguish individual low molecular weight polymer molecules. The latter has also been applied to study enzyme catalyzed reactions.17 However, a major drawback of mass spectrometric techniques is that they can only be used in a qualitative manner because several factors affect the intensity of the MS signal, e.g., specific sample properties such as molecular weight, molecular weight distribution, end groups, and the nature of the polymer. On the other hand, techniques such as end group titration and end group modification in combination with 1H or 31P NMR spectroscopy have been applied to obtain insight into the end group functionality in enzymatic ROP. One of the most systematic studies using NMR end group analysis was reported by Kaplan, who investigated the initiation of caprolactone with methoxypoly(ethylene glycol) (MPEG) as initiator under various conditions.37 While these techniques do provide quantitative data on specific end groups, they only take linear polymer species into account and data becomes less accurate when applying higher molecular weight polymer. Additionally, they cannot provide any insight on the molecular level, i.e., how end groups are distributed over the polymer chains. In this article, we report on our systematic study of the initiation process in enzymatic ROP using liquid chromatography under critical conditions (LCCC). LCCC efficiently separates components of a mixture, differing by as little as a single end group.38 It is thus a powerful technique to obtain
10.1021/bm701158y CCC: $40.75 2008 American Chemical Society Published on Web 01/12/2008
Notes
Biomacromolecules, Vol. 9, No. 2, 2008 753
Scheme 1. Possible Products in a Ring-Opening Polymerization of CL with Candida antarctica Lipase B (CALB) in the Presence of Monofunctional Alcohol as an Initiator and Water as the Competing Nucleophilea
a
The reaction concentration and initiator structure were systematically varied.
information about the presence of cyclic and linear polymers with different end groups even when only present in low quantities. Using LCCC, we were able for the first time to directly quantitatively monitor the incorporation of monofunctional nucleophiles in enzymatic ROP under various reaction conditions, providing us with a unique insight into the initiation process of the enzymatic polymerization.
Experimental Section Materials. All chemicals were purchased from Aldrich and used without further purification unless otherwise noted. -Caprolactone (CL) was distilled over CaH2 and stored over molecular sieves. Toluene was dried over aluminum oxide and stored on molecular sieves. Molecular sieves (3 Å) were dried in an oven at 420 °C before use. Novozym 435 was obtained from Novozymes A/S and stored over P2O5 in a desiccator. The enzyme was then dried under vacuum at 50 °C, as described later in the polymerization procedure. Initiators 4 and 5 were synthesized according to literature procedures.31 Methods. Size exclusion chromatography (SEC) was performed on a Waters model 510 pump and Waters 712 WISP using PL-gel mixed D columns (300 mm × 7.5 mm, Polymer Laboratories) at 40 °C. THF was used as eluent with a flow rate of 1.0 mL/min. All samples were diluted to 1.0 mg/mL and filtered using 0.2 µm syringe filters. All samples were calibrated using universal calibration: K ) 0.00109 dL/g and a ) 0.6.36 1H NMR spectroscopy was performed using a VARIAN 400 MHz NMR at 20 °C. Samples were dissolved in CDCl3 to a concentration of ∼30 mg/mL. Data were acquired using VNMR software. MALDI-TOF-MS analysis was carried out on a Voyager DESTR from Applied Biosystems using trans-2-[3-(4-tert-butylphenyl)2-methyl-2-propenylidene]-malononitrile (DCTB) as matrix material. All spectra were recorded in the reflector mode. Samples were prepared using 1 mg/mL of polymer in THF. The ratio of polymer sample to matrix was 1:5 (w/w %). Karl Fischer coulometry was performed on a Mettler Toledo titrator DL39 with APURA CombiCoulomat fritless electrolyte. All LCCC experiments were conducted on an Agilent 1100, equipped with a quaternary pump, degasser, autosampler, column oven, and a diode array detector (DAD) with 10 mm cell (Agilent, Waldbronn, Germany). The mobile phase, 17.5% (v/v) ultrapure water with 0.1% (v/v) formic acid and 82.5% (v/v) THF, was mixed and pumped with a flow rate of 0.5 mL/min. Four Nucleosil 120-5 (Machery-Nagel) C18 columns in series (250 mm × 4 mm) were used to establish the critical separation (T ) 40 °C). The injection volume was 5 µL. Detection with a Sedex 75 ELSD system (Sedere, Vitry/Seine, France) was performed with 3.6 bar air pressure at the nebulizer and a drift tube temperature at 35 °C. The detector signal was collected with an Atlas version 8.1 data management system (Thermo Electron Cooperation, Manchester, UK). Enzymatic Polymerization of E-Caprolactone (CL). Different polymerizations were conducted using reference conditions as follows. In a 25 mL round-bottom flask equipped with a magnetic stirrer bar, 250 mg of Novozym 435 was dried in a vacuum oven at 50 °C for 16 h. The flask was backfilled with N2, and dried molecular sieves
were added to the flask. Subsequently, the flask was placed in an oil bath at 60 °C and the polymerization started by the addition of a stock solution of -caprolactone (4.10 or 0.9 M) and benzyl alcohol (47 or 10.3 mM) in toluene. Immediately after the start of the reaction, a sample was withdrawn from the reaction mixture and analyzed using Karl Fischer coulometry. At specified time intervals, further samples (∼0.15 mL) were withdrawn from the reaction mixture with a syringe for analysis. After 120 min, the reaction was stopped by removing Novozym 435 by filtration using dichloromethane as solvent. The polymer was dried under vacuum at 40 °C.
Results and Discussion As depicted in Scheme 1, three different reaction products can be expected from an enzymatic ROP in the presence of a nucleophilic initiator as a consequence of the polymerization mechanism. Generally it is agreed that in the first step the lipase activates the carbonyl bond of ester compounds by forming an enzyme–substrate complex. In analogy to lipase catalyzed ester formation of low molecular weight substrates, it is further assumed that the polymerization proceeds by attack of a nucleophile on the enzyme activated substrate.32,33 Three types of nucleophiles are available in the reaction system: (i) BA, (ii) water, and (iii) the hydroxyl end group of the polymer chain. The competition of nucleophiles in the attack on the enzyme activated carbonyl bond can thus lead to the three main products shown in Scheme 1, i.e., PCL end-capped with BA (1), PCL end-capped by water (2), and cyclic polymers by ring closure/ backbiting (3). While thorough drying of the reaction medium including the enzyme can increases the yield of polymer endcapped with the initiator by minimizing water initiation, it cannot be completely prevented because complete drying results in deactivation of the enzyme.31 The question is thus how the two nucleophiles (water and BA) compete in the initiation process and how cycle formation relates to this. To provide quantitative data on the evolution of the three different PCL species with LCCC first the critical conditions had to be established. Establishing the Critical Conditions. The separation of a given polymer sample with different end groups using LCCC can only be achieved if the individual contribution of the polymer backbone to the total retention (interactions) is eliminated, i.e., at the critical conditions.39 Hence, retention at these conditions is solely based on differences in end groups and not on molecular weight. The critical conditions of a polymer can be obtained by careful adjustment of the eluent composition, eluent flow, and temperature. While this has been done in the analysis of several other polymers like telechelic polyacrylates and poly(styrene),40–45 to our knowledge, it has never been reported for PCL. The PCL reaction samples were separated according to end groups with LCCC using the conditions described in the Experimental Section. The evaporative light scattering detection
754 Biomacromolecules, Vol. 9, No. 2, 2008
Notes
Figure 1. Quantitative LCCC separation of polymers obtained from benzyl alcohol initiated enzymatic ROP of -caprolactone and subsequent analysis of the fractions with MALDI-TOF-MS for peak assignment in the LCCC chromatogram.
(ELSD) chromatogram of a typical reaction mixture from enzymatic ROP of CL in the presence of BA exists of three well-separated peaks. Identification of the three peaks with MALDI-TOF-MS after preparative separation revealed that the linear carboxylic acid end-functionalized polymer species (2) eluted first, as these had the fewest interactions with the C18 column, followed by the linear hydroxyl-ester species (1) and finally the cyclic polymer species (3) (Figure 1). In general, the quantification method to use is UV detection, but the high background adsorption of THF in combination with the weak UV adsorption of the PCL makes UV detection unsuitable. RI detection is not possible either because no welldefined PCL standards are available. Therefore, ELSD with a universal calibration was used for quantification. To perform this ELSD calibration, a number of polymers containing different amounts of species with the different end groups were prepared. A calibration line for linear and cyclic species was made, and by using an iterative method, calibration of the ELSD signal was obtained for each individual component. Influence of the Monomer Concentration. To obtain insight into the influence of monomer concentration on the initiation process in enzymatic ROP, a simple model reaction was chosen, comprising benzyl alcohol (BA) as a monofunctional nucleophile (initiator), -caprolactone (CL), and immobilized Candida antarctica lipase B (Novozym 435). Reactions were carried out with decreasing concentration of monomer, i.e., a bulk reaction and two reactions in toluene (4.1 and 0.9 M CL). During the reaction, samples were taken and analyzed by 1H NMR, SEC, MALDI-TOF-MS, and LCCC. BA was selected because we previously observed that its incorporation into the polymer chain occurs rapidly, suggesting a good initiation efficiency.31 For the present experiments, all reaction components were dried prior to the enzymatic ROP to reduce the water concentration as much as possible (Karl Fischer coulometry: water < 0.08 mg water/g sample). Quantification of all products (1–3) as a function of time provides an insight into the competition of the nucleophiles and thus the initiation process. By combining results from LCCC and SEC, further information could be obtained into the actual processes in enzymatic
Figure 2. Chromatograms obtained from SEC (left) and LCCC (right) of enzymatic ROP performed under diluted conditions (entry b, Table 1); percentages refer to the monomer conversion at the specific time intervals. 1: BA initiated; 2: water initiated; 3: cyclics.
ROP. Figure 2 depicts the SEC and LCCC chromatograms of samples taken at various times of a reaction carried out under dilute conditions (Entry b, Table 1). The increase in molecular weight of the PCL is evident from the shift of the SEC traces, reaching 1300 g/mol after 120 min. The SEC chromatograms reveal a significant amount of “nongrowing” polymer of low molecular weight. While from SEC no further structural information can be derived, LCCC provides additional information on the evolution of polymer species 1–3. From Figures 2 and 3A, it can be seen that the concentration of cyclic species 3 is as low as 7% (w/w) after 5 min of reaction. At 15 min, the concentration of cyclic polymer species increases to 26% (w/ w), after which it remains almost constant throughout the reaction. This high concentration of cyclic structures is not unexpected for a reaction under diluted conditions. The formation of cycles under dilute conditions has been qualitatively reported previously17 and is in agreement with common theories
Notes
Biomacromolecules, Vol. 9, No. 2, 2008 755
Table 1. Results of the Characterization of the Final Polymer Product after a Reaction Time 120 min (a,b,d,e) and 60 min (c), Obtained by Enzymatic ROP of CL under Different Conditions (All Reactions Were Carried Out with a Monomer to Initiator Ratio of 1:87) entry
initiator
[CL]0
CL conversiona[%]
Mnb[g/mol]
Mw/Mnb
(a) (b) (c) (d) (e)
BA BA BA 4 5
4.1 0.9 bulk 1 1
76 59 51 70 68
2800 1300 2300 2100 3500
2.7 2.3 2.5 3.4 2.5
a CL-conversion determined with 1H NMR spectroscopy; b Average molecular weight and molecular weight distribution determined with SEC in THF at 40 °C, using a universal calibration.
Figure 4. SEC (left) and LCCC (right) chromatograms of eROP performed in bulk (entry c, Table 1); percentages refer to monomer conversion at the specific time intervals. 1: BA initiated; 2: water initiated; 3: cyclics.
Figure 3. Evolution of individual polymer species during enzymatic ROP of CL performed with BA at different concentrations (A: 0.9 M; B: 4.1 M; C: bulk). b: 1 (BA initiated), 9: 2 (water initiated), 2: 3 (cyclics).
on thermodynamic ring-chain equilibria in polycondensation reactions.46 Fractionation of the final sample at 120 min by preparative SEC and subsequent MALDI-TOF-MS analysis of the individual mass samples revealed that the “nongrowing” low molecular mass fraction in the SEC mainly consists of cyclic polymers. From the LCCC chromatograms in Figure 2, it can also be derived that after the first 5 min, only small amounts of linear BA end-capped polymers 1 are present (31% (w/w)). Most linear species contain both hydroxyl and carboxylic acid end groups (2: 62% (w/w)), i.e., are ring-opened by water. However, after 15 min, linear BA end-capped polymers become the dominant linear species, increasing to 50% (w/w) after 120 min. When the reaction was carried out in bulk, the molecular weight increase was more significant, reaching 2300 g/mol after
60 min (Figure 4). The LCCC analysis revealed that the formation of cyclic structures is largely suppressed compared to the diluted conditions (Figure 3A). As in the previous reaction, polymer species 2 (water initiated) dominates the early phase of the bulk reaction (89% (w/w)). Only after 30 min was significant end-capping with BA observed (68% (w/w) of polymer 1). After 60 min, there was no major change in the composition of the mixture; the concentration of the BA endcapped polymer 1 being much higher (73% (w/w)) than that of the water initiated species 2 (25% (w/w)). Figure 3 summarizes the results obtained from the quantification of the LCCC traces. Generally it can be observed that the concentration of cyclic structures depends on the concentration of the reaction, being lowest for the bulk reaction. Moreover, the results clearly show that in all three cases the initial efficiency of water to end cap in the enzymatic ROP is significantly higher than the efficiency of BA, which was considered to be a very good “initiator”. While this leads to a high concentration of water initiated species in the early stage of the polymerization, the concentration of BA end-capped chains surpasses the water initiated chains during the course of the reaction. A high initial water initiation was also observed by Kaplan in the presence of MPEG (2000 g/mol) by NMR analysis.37 While a slow incorporation of the relatively high molecular weight MPEG could be expected, it is surprising to see the same phenomenon even in the case of the low molecular initiator BA. These results confirm that the end-capping of PCL with BA in enzymatic ROP is a combination of initiation and transesterification, the latter caused by a nucleophilic attack of BA on an enzyme activated carbonyl bond of a polymer chain. From the present data, it is not possible to fully distinguish between the two mechanisms. Without further research, it can only be speculated that the higher water activity is caused by easier access to, or higher local concentration of, water close to the active site of the enzyme. Influence of the Initiator Structure. It can be expected that in the case of sterically hindered monofunctional alcohols, the water eficiency becomes even more dominant. Hence, we compared how the different polymer species evolved when different initiators are employed under otherwise constant reaction conditions. We chose two initiators previously used for the chemoenzymatic synthesis of block copolymers by combining enzymatic ROP with ATRP because detailed kinetic
756 Biomacromolecules, Vol. 9, No. 2, 2008
Notes
able to directly quantify this for the first time for enzymatic ROP by LCCC. The results also imply that controlling the molecular weight by monomer to initiator ratio is difficult. In the best case, end-group fidelity in enzymatic ROP can thus be achieved by thermodynamic control because high water activity will kinetically dominate the initiation process. The results of these analyses allow a deeper understanding of the kinetics of the initiator method and as such will be of great use in engineering new, functional polymers by enzymatic catalysis. Acknowledgment. . This work was conducted with financial support from the Dutch Polymer Institute (DPI). Supporting Information Available. LCCC optimization; LCCC plots of BA initiation at semidiluted conditions; LCCC and SEC plots of initiator 4. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 5. Evolution of individual polymer species during enzymatic ROP of CL performed with initiators 4 and 5 at a CL concentration of 4.1 M; b: 1 (4/5 initiated), 9: 2 (water initiated), 2: 3 (cyclics).
data on these initiators were available.31 In Figure 5, the presence of the individual polymer species is depicted as a function of reaction time for enzymatic ROP with different initiators. In both cases, water is the primary nucleophile at the initial stages of the polymerization, which corresponds with the previous observation when BA was used as a nucleophile. However, when comparing the two functional nucleophiles, the incorporation of the initiator is observed to be different. The incorporation of 4 follows the same pattern as that for BA, i.e., high water initiation at the beginning of the reaction and incorporation of 4 as the reaction proceeds, finally reaching 70% (w/w) after 120 min. On the other hand, the bulky alcohol 5 is incorporated very slowly. After 30 min, only 5% (w/w) of the chains are initiator end-capped, while 80% of the chains are water initiated. Moreover, a relatively high concentration of cyclic polymer species was formed during the initial stages of the polymerization (29% (w/w) after 5 min). This can be explained by the low efficiency of the nucleophile, hence intramolecular backbiting will be the most common process for releasing the activated substrate. Upon incorporation of the initiator, the presence of cyclic polymer species decreases. Nevertheless, incorporation of 5 is very slow; in fact, within the experimental time, an increase to only 25% (w/w) at 120 min was reached. This behavior can be explained by the size of the nucleophile 5, as it has to diffuse into the active site of the enzyme in order to release the activated substrate. Additionally, 5 contains a chiral center, which may further complicate the accessibility of this initiator into the active site. While qualitatively these results are in agreement with what we previously observed for these initiators, only the quantification with LCCC provides a full picture.
Conclusions The results of this study are significantly different from what is typically observed in chemical ROP, where under dry conditions a monofunctional alcohol is the sole nucleophile and thus a real initiator. In enzymatic ROP, a completely dry reaction medium cannot be realized because the enzyme requires traces of water to retain its activity. Our results show that under all investigated reaction conditions, water dominates the initial initiation process. Subsequently, the kinetic of initiator incorporation is dependent on the reaction conditions and the initiator structure. While this is not a new observation, we have been
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BM701158Y
