Macrocyclic Oligoesters Incorporating a Cyclotetrasiloxane Ring

Jun 10, 2015 - (1) The ability to tailor the spatial arrangement of functional groups around ring and .... pentane-1,5-diol, (1R,2R)-trans-cyclohexane...
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Macrocyclic Oligoesters Incorporating a Cyclotetrasiloxane Ring Mark B. Frampton,† Drew Marquardt,‡ Tim R.B. Jones,† Thad A. Harroun,‡ and Paul M. Zelisko*,† †

Department of Chemistry and Centre for Biotechnology and ‡Department of Physics, Brock University, St. Catharines, Ontario Canada, L2S 3A1 S Supporting Information *

ABSTRACT: Macrocyclic oligoester structures based on a cyclotetrasiloxane core consisting of tricyclic (60+ atoms) and pentacycylic (130+ atoms) species were identified as the major components of a lipase-mediated transesterification reaction. Moderately hydrophobic solvents with log P values in the range of 2−3 were more suitable than those at lower or higher log P values. Temperature had little effect on total conversion and yield of the oligoester macrocycles, except when a reaction temperature of 100 °C was employed. At this temperature, the amount of the smaller macrocycle was greatly increased, but at the expense of the larger oligoester. For immobilized lipase B from Candida antarctica (N435), longer chain length esters and diols were more conducive to the synthesis of the macrocycles. Langmuir isotherms indicated that monolayers subjected to multiple compression/expansion cycles exhibited a reversible collapse mechanism different from that expected for linear polysiloxanes.

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INTRODUCTION The design of new materials based on siloxane frameworks is gaining popularity. Of particular interest are 2D-cyclic and 3Dcubic oligosiloxanes.1 The ability to tailor the spatial arrangement of functional groups around ring and cage motifs could lead to interesting characteristics making these materials even more desirable. Linear and cyclic siloxanes are common components in the formulation of personal care products, particularly those that require intimate contact with human skin; these types of siloxanes are ideal for such applications owing to their lack of bioactivity.2 Most commonly linear and cyclic siloxanes are used in topical formulations where they impart lubricity while avoiding an oily texture, which can result if natural fatty acids and oils are used. Siloxanes have an unusual surface activity that allows them to be readily used as lubricants and antifoaming agents.2 There is considerable interest to design new siloxane− organic hybrid compounds that possess the desirable characteristics of siloxanes and organic polymers. First, there is the economic cost that must be factored into consideration. Second, and more importantly, there is the potential for superior performance of the hybrid molecule over the pure polymer.2 The incorporation of fatty acid-like domains onto a siloxane core could be expected to generate compounds possessing interesting characteristics derived from the differing physical properties of the siloxane and aliphatic components.3 Macrolactones are of interest to many fields of research and are envisioned as being useful as biologically active compounds, chelating agents, and monomers for ring-opening polymerization (ROP).4,5 As monomers for ROP, macrolactones provide the advantage of reducing the production of volatile organic compounds (VOCs) as byproducts, which are of © XXXX American Chemical Society

concern in Canada and the United States due to the associated environmental toxicity.6−9 We have been interested in applying biocatalytic and enzymatic methods in organosilicon chemistry, primarily using immobilized lipases, which owing to their regio- and chemoselectivity, high degree of tolerance to elevated temperatures, and capacity to tolerate repeated reaction cycles makes them ideal for carrying out transformations of a wide range of substrates.10,11 Of particular interest to our group is the condensation polymerization of suitable precursors to yield siloxyester-based polymers and more recently macrocyclic oligoesters. Many strategies have been developed in the quest for reliable methods to produce macrocyclic oligoesters including (1) dilution approaches composed of both high dilution and pseudohigh dilution processes, (2) distillation from reaction mixtures, (3) polymer-supported reactions, (4) cyclodepolymerization, and (5) extraction from condensation polymerization.12 Enzymatic syntheses of macrolactones/macrocyclic oligoesters derived from AA-BB type systems have been the subject of several previous reports.13−15 Sih-synthesized lactones enzymatically from diesters and diols of varying chain lengths in anhydrous organic solvents with lipases from Candida cylindracea (OF 360), Pseudomonas spp. (AK and K-10), and porcine pancreas.13 Mezoul et al. demonstrated the synthesis of polyalkylene phthalate lactones.14 The substitution pattern of the phthalate ring strongly influenced the outcome of the lactonization process; esters having an ortho relationship Received: April 17, 2015 Revised: June 9, 2015

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Maryland, U.S.A.). Toluene, pentane, and ethyl acetate were of reagent grade and were stored over 4 Å molecular sieves before use. Instrumentation. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance AV-300 (BB-PABBO probe) or AV-600 (BBO Z-gradient ATMA probehead and an inverse triple resonance gradient TXI probe) nuclear magnetic resonance spectrometers. 1H and 13C spectra were referenced to CDCl3 (7.26 and 77.16 ppm for residual CHCl3, respectively) and 29Si spectra were referenced to tetramethylsilane (TMS, 0.0 ppm). Fourier-transform infrared (FT-IR) spectra were obtained using a Mattson Research Series infrared spectrometer operating in transmission mode. Samples were prepared as thin films on KBr plates. Each spectrum consisted of 32 scans at 2 cm−1 resolution. Electrospray ionization mass spectrometry (EI-MS) was carried out using a Kratos Concept 1S High Resolution E/B mass spectrometer in negative ion mode. Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS) spectra were acquired on a Bruker Autoflex MALDI-ToF mass spectrometer in the positive ion mode. Samples were dissolved into HPLC grade acetone, sonicated, and combined with a NaCl/acetone mixture and sonicated a second time. A small sample was transferred to a stainless steel plate that was preloaded with a paste composed of dithranol/THF. Methods. Catalyst Choice. Commercially available lipases were screened for their capacity to perform the transesterification of 1,3,5,7tetrakis(methyl-9-carboxynonyl)-1,3,5,7-tetramethylcyclotetrasiloxane (2) with octane-1,8-diol (5). Ester 2 and octane-1,8-diol were combined in a 1:2 stoichiometry in toluene (50 mM) and melted to form a homogeneous mixture at 55 °C for 10 min. The enzyme (either Candida antarctica lipase B on acrylic resin, C. antarctica lipase A immobilized on Immobead 150 (CALA), Rhizomucor miehei lipase (Lipozyme), Thermomyces lanuginosa lipase immobilized on Immobead 150 (TLL), or free C. antarctica lipase B mixed with cyclodextrins (CV-CALBY, Chiral Vision)) was added at 5 wt % with respect to the total mass of the monomers. The reactions were stopped after 24 h and analyzed by 1H NMR to determine total consumption of the esters. Synthesis of Methyl-9-decenoate (1). To a stirred solution of 9decenoic acid (1.83 g, 10.8 mmol) in methanol (10.0 mL) were added 115.0 mg (6.05 × 10−4 mol) of p-TsOH. The mixture was heated to reflux for 4 h. Methanol was removed in vacuo, and the crude residue was extracted into 20 mL of diethyl ether and washed with 10 mL of 1 M KHCO3, 10 mL of brine, and subsequently dried over Na2SO4. Ether was removed in vacuo to yield 1.78 g (9.65 mmol, 90%) of a clear, colorless oil. 1H NMR (CDCl3, 300 MHz): δ 1.30 (s), 1.68 (m), 2.03 (m), 2.30 (t, J = 7.5 Hz), 3.66 (s), 4.91 (m), 4.95 (m), 5.01 (m), 5.80 (m); 13C NMR (CDCl3, 75 MHz): δ 24.9, 28.8, 28.9, 29.1, 33.7, 34.1, 51.4, 114.2, 139.1, 174.3; EI-MS (m/z): M+ 184. FTIR (KBr, cm−1): 1436, 1641, 1742, 2855, 2928, 2976, 3076. The syntheses of cyclotetrasiloxane esters 2, 3, and 4 were carried out as reported previously.24 The detailed procedures for these syntheses and spectroscopic characterization are presented in the Supporting Information. Synthesis of Macrolactone 6. A 10 mL round bottomed flask was charged with 171.1 mg (1.75 × 10−4 mol) of ester 2 and 52.2 mg (3.53 × 10−4 mol) of octane-1,8-diol and melted at 100 °C to form a homogeneous mixture. The mixture of monomers was diluted to either 25 mM with toluene. Novozym 435 (N435) was then added to the reaction mixture and stirred for 120 h. The reaction was terminated by the addition of 10 mL of CHCl3 and stirred for a further 10 min. The enzyme catalyst was removed by filtering the reaction mixture through a medium porosity glass fritted Büchner funnel; the beads were subsequently rinsed with two 5 mL volumes of chloroform, and the excess solvent was removed in vacuo. The product was isolated via column chromatography on silica gel and eluted with ethyl acetate in hexanes by increasing the polarity from 30 to 50% ethyl acetate. Macrolactone 6: 1H NMR (600 MHz, CDCl3 at 7.26 ppm): δ 0.06, 0.063, 0.07, 0.09, 0.53 (m, 8H), 1.32 (br, 64H), 1.64 (br, 16H), 2.32 (t, 8H), 4.1 (m, 8H); 13C NMR{1H} (150 MHz, CDCl3 at 77.0 ppm): δ 16.9, 17.2, 22.85, 22.97, 25.04, 25.14, 25.82, 28.46, 28.59, 28.73, 28.97, 29.00, 29.12, 29.28, 29.33, 29.36, 29.44, 32.76, 33.18,

inhibited the two-component Novozyme catalyst from producing lactones.14 Berkane et al. showed that lactonization was dependent on both monomer structure and the initial concentration of the monomers.15 The molar cyclization constants from polyalkylene succinate syntheses were in agreement with the theoretical Jacobsen-Stockmayer equilibrium values. Cyclization equilibrium constants were not significantly affected by the reaction temperature, suggesting that enthalpic contributions must also be considered in enzymatic lactonizations. In a study of pentaethylene terephthalate lactone formation, Hilker et al. concluded that a distribution of lactones was produced via enzymatic oligomerization.5 In a few cases, such as the pairing of dimethyl terephthalate/diethylene glycol, dimethyl terephthalate/propane-1,3-diol, and 2,5-pyridinodicarboxylate/diethylene glycol, mainly cyclic dimers were formed. Macrocyclic oligoesters derived from seco acids (AB-type systems) have been explored using lipases from C. antarctica,16 Pseudomonas spp.,17,18 Mucor meihei,18 and porcine pancreas.18,19 One advantage to using enzymes lies in their selectivity for producing chiral or enantioenriched lactones. Some degree of control over ring−chain equilibrium could be achieved with porcine pancreatic lipase with secondary seco acids compared to primary seco acids with the former leading to the formation of lactones and the latter giving linear polyesters.19 Diastereomeric lactones were isolated when a mixture of (±)-7-hydroxyoctanoic acid and isooctane was treated with a Pseudomonas lipase.18 A high (R)-selectivity was observed by Lobell and Schneider using lipase from Pseudomonas spp. (SAM II) and a vinyl ester with a chain length consisting of at least eight carbon units.17 Much of our work with enzymes has focused on using immobilized lipases, particularly lipase B from Candida antarctica immobilized on a cross-linked divinylbenzene resin. This enzyme preparation facilitates removal of the enzyme catalyst from the reaction medium and, to some extent, allows the enzyme to be used at elevated temperatures over repeated reaction cycles.20−23 In this paper we report a lipase-mediated process for the synthesis of novel macrocyclic oligoesters that are derived from a cyclotetrasiloxane framework. We examined the effect of five lipases, temperature, substrate concentration, and monomer structure on the formation of macrocyclic oligoesters. Three different cyclotetrasiloxane-derived esters possessing C5, C7, and C10 chain lengths, two acyclic diols, and a cyclic diol were included for study. The behavior of Langmuir monolayers and the thermal behavior for the macrocyclic oligoesters were determined.

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EXPERIMENTAL SECTION

Materials. Lipase B from Candida antarctica immobilized on Lewatit VP OC 1600 cross-linked divinylbenzene resin (Novozym435, N435), C. antarctica lipase A immobilized on Immobead 150, lipase from Thermomyces lanuginosa, lipase from Rhizomucor miehei, methyl 4-pentenoate, methyl 6-heptenoate, 9-decenoic acid, 1,3,5,7tetramethylcyclotetrasiloxane, pentane-1,5-diol, (1R,2R)-trans-cyclohexane-1,2-diol, and platinum(0) 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex (Karstedt’s catalyst, 2% Pt in xylenes) were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Octane-1,8-diol was obtained from Alpha Aesar (Ward Hill, New Jersey, U.S.A.). Candida antarctica lipase B recombinant from Pichia pastoris and stabilized with cyclodextrins was obtained from Chiral Vision (Leiden, The Netherlands). Deuterated chloroform (CDCl3, 99.9% deuterated) was obtained from Cambridge Isotope Laboratories (Andover, B

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Figure 1. Representative reaction scheme for the enzymatic synthesis of macrocyclic oligoesters. 33.20, 33.26, 34.47, 34.52, 64.20, 64.23, 173.96; 29Si{1H} (119.2 MHz, CDCl3, TMS at 0.0 ppm): −19.66, −19.98, −20.17, −20.21, −20.33, −20.39, −20.68; FTIR (cm−1): 1061, 1159, 1256, 1461, 1734, 2853, 2922; FAB-MS (m/z): M + 1142. Langmuir Isotherms. Langmuir isotherms were performed using a KSV NIMA Minitrough (50 mm × 155 mm) and Delrin barriers in a vibration-free environment. Barrier control and data acquisition were achieved using the LB measurement system provided by KSV NIMA (Biolin Scientific, Linthicum Heights, Maryland U.S.A.). The Wilhelmy balance was calibrated using a 264.9 mg calibration standard provided by the manufacturer prior to data acquisition. The surface pressure isotherms were measured using prewetted paper Wilhelmy plates at a temperature of 21.5 °C that was controlled by a circulating water chiller. Ultrapure Milli-Q water (18.2 MΩ at 25 °C) was used as the subphase. The subphase surface pressure was maintained below 0.1 mN/m prior to the application of the film; when the pressure of the subphase exceeded 0.1 mN/m, the surface was recleaned and the procedure was repeated. Langmuir monolayers were spread using 6 μL of 1 mg/mL chloroform solutions. Data acquisition commenced after a 10 min equilibration period to allow for solvent evaporation prior to compression−decompression cycling. The barriers were compressed/ decompressed at a rate of 5 mm/min; at least three cycles of the compression/expansion were performed. Between experiments the trough and barriers were thoroughly cleaned using chloroform and water for the Teflon Langmuir trough, and ethanol and water for the Delrin barriers. Differential Scanning Calorimetry (DSC). DSC thermograms were acquired using a Shimadzu DSC-60 and a TA-60WS Thermal Analyzer. Aluminum pans (34 μL max volume) were used for preparing samples and an empty aluminum pan was used as the control to which samples were compared. Samples were subjected to two heating and cooling cycles so that each sample had the same thermal memory. Samples were cooled to −150 °C from room temperature. Samples were heated at a rate of 10 °C/min to 30 °C, cooled at a rate of 10 °C/min to −150 °C; this was done twice. Thermal transitions were taken from either the second heating or cooling cycle.

reactions were hexanes (log P = 3.5) and toluene (log P = 2.5). In these two solvents, N435 achieved 58% total consumption of the methyl esters (Table 1). Previous studies have also Table 1. Effect of Solvent on the Esterification of 50 mM Ester 2 and Octane-1,8-diol Catalyzed by 5 wt % of N435a

a

solvent

log P

%conversion (NMR)

THF toluene hexanes isooctane D4

0.5 2.5 3.5 4.5 5.1

18 58 58 29 11

Reactions were stirred at 150 rpm, 60 °C for 24 h.

identified toluene as a good solvent for ester formation using enzymatic catalysis.28,29 It should be noted that structures have been presented to show that the oligoesters are formed by making linkages at esters with a [1,3]-relationship with respect to the cyclosiloxane ring. It is within reason to expect that these linkages may also have a [1,5]-relationship. We have previously indicated24 that the tetramethylcyclosiloxane ring is a mixture of geometric isomers that may in fact promote lactonization in a [1,3] or [1,5] nature. Solvents with lower or higher log P values than toluene or hexanes appeared to be less conducive to high levels of ester consumption. In the most hydrophilic solvent, THF (log P = 0.5), only 18% conversion was achieved. Hydrophilic solvents, such as THF, tend to strip water from the hydration shell of the enzyme, which not only accelerates denaturation, but also increases the likelihood of desorption of the enzyme from the immobilization support.30,31 Isooctane and D4, log P = 4.5 and 5.1, respectively, were also not ideal solvents for N435, and conversion was low at 29 and 11%, respectively. It was clear that in these instances, and due to the absence of a clear trend, that log P values were not a good indicator for predicting enzyme performance in various solvents without considering other mitigating factors. A similar conclusion was reached by Gargouri et al., who also found that log P values were a poor metric for determining solvent suitability.16 Our results showed that hexanes and toluene are ideal solvents; however, we chose to carry on with toluene, as it possesses a higher boiling point. Choosing the Appropriate Enzyme. Several immobilized fungal lipases were screened for their capacity to synthesize macrocyclic oligoesters from octane-1,8-diol and ester 2 at 60 °C over the course of 24 h in toluene (50 mM). Lipozyme, an immobilized form of Rhizomucor meihei, lipase from Thermomyces lanuginosa, and lipase A from Candida antarctica immobilized on Immobead 150 were not suitable candidates for carrying out the desired transesterification reaction in organic media. Analysis of the 1H NMR data revealed that none of the selected enzymes performed any appreciable transesterification of the methyl esters. Similarly, lipase B from C. antarctica that was not immobilized but rather stabilized by

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RESULTS AND DISCUSSION Enzymatic Activity Does Not Correlate with Solvent Partition Coefficient. Lipases are renowned for the capacity to function at lipid water interfaces, as well as in neat organic solvents. Several organic solvents, spanning a range of partition coefficient (log P) values, were screened as potential solvents for synthesizing macrocyclic oligoesters. The log P values were taken from Laane et al.,25 except those for 2,2,4-trimethylpentane (log P = 4.5), which was taken from Halling,26 and octamethylcyclotetrasiloxane (log P = 5.1), which was taken from Luu and Hutter.27 Tetrahydrofuran, toluene, hexanes, 2,2,4-trimethylpentane (isooctane), and octamethylcyclosiloxane were tested as reaction media for the synthesis of cyclotetrasiloxane-derived macrocyclic oligoesters at an initial concentration of 50 mM using 5 wt % of N435 at 60 °C (Figure 1). This temperature (60 °C) was chosen, as it was below the boiling point of the lowest boiling solvent, negating the need to deal with refluxing solvents, but still allowed for the melting of octane-1,8-diol. After 24 h, the solvents that appeared to be the most beneficial for the enzyme-mediated C

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Figure 2. Structures of macrolactone 6 and 7 derived from the cyclotetrasiloxane core.

cyclodextrins also did not mediate the transesterification of the chosen substrates. Only N435 was a suitable enzyme choice, and while it only converted 58% of the free methyl esters in the allotted time, it was chosen to serve as a platform to optimize further reaction conditions. The success of an enzyme in organic synthesis is related to how substrate molecules fit into the active site of the enzyme. Lipases have evolved to accept fatty acids of shorter (C4) or longer chain lengths (C22), and some are quite specific in their substrate preference.32 The failure of the lipases other than N435 to perform the transesterification reactions can be attributed to the geometric mismatch between the large cyclotetrasiloxane ring system, from either the siloxane ring itself or the chain length of the fatty acid ester, and the active site of each protein. This phenomenon has been explored for N435 where the transesterification of trisiloxanes with variable chain length methyl esters was concerned.33 The cyclodextrinstabilized CalB possessed low solubility in the organic medium, due to the highly hydrophilic nature of the cyclodextrin stabilizer as well as the enhanced degree of glycosylation of the

enzyme and formed large enzyme aggregates that resulted in low activity toward the chosen substrates. These observations prompted the conclusion that the best enzyme for attempting the synthesis of macrolactones would be N435. This enzyme preparation was used in the determining the optimal parameters of substrate concentration, reaction temperature, and ester structure. Lower Substrate Concentrations Favor Ring Formation. The reports in the literature which discuss the enzymemediated synthesis of polyester macrocycles typically employ monomer concentrations of >100 mM.5,15 We performed a series of experiments with the aim of determining the effect of concentration of monomers on the synthesis of one, or a small few, polycyclic macrocycles. At a concentration of 50 mM using 5 wt % of N435 for 120 h, conversion of the methyl esters was high at 95% (determined by 1H NMR). The MALDI-ToF MS spectrum indicated ion peaks with (M + Na)+ = 1166 m/z (macrolactone 6) and (M + Na)+ = 2307 m/z (macrolactone 7) with some lower intensity peaks that indicated partially lactonized species. The structures of these two macrocycles are D

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Figure 3. Distribution of acyclic and cyclic oligoesters, produced at different monomer concentrations in toluene, as determined by MALDI-ToF MS.

success of the reaction in the range that was tested. At every temperature in which the reaction was carried out, conversion was high, ranging from an average of 93% at 60 °C to a high of 96% at 80 °C (Figure 4). More importantly, the observed

presented in Figure 2. The mass ion of greatest intensity was macrolactone 6, while the intensity of the ion representing macrolactone 7 often varied between 20 and 38% with respect to the base peak. A graphical representation of the identity of each species formed in the reaction, and the relative proportion of each species, is presented in Figure 3. Reducing the monomer concentration to 25 mM had little effect on the consumption of 2, again giving 90−95% consumption with the two dominant species identified as 6 (100% intensity in the MALDI-ToF MS) and 7 (5−13% intensity in the MALDI-ToF MS). Reducing the monomer concentration further to 20 mM and then 10 mM afforded greater than 85% consumption of the ester functional groups with a similar distribution of products noted above. At concentrations below 25 mM it became apparent, by examination of the 1H NMR spectra, that the acrylic resin on which the CalB molecule was immobilized had started to degrade. The degradation of the acrylic substrate matrix in N435 had also been reported by Poojari et al.23 The degradation of the acrylic matrix was more prevalent at 5 mM where only 50% consumption of the monomers was attained after 120 h. As a result of these observations, a concentration of 25 mM was chosen for all future experiments to maximize product formation and to suppress the degradation of the N435 beads. Reaction Temperature Changes the Distribution of Products. Temperature is known to play an important role in regulating enzymatic activity. However, in some systems, the choice of monomers can dictate the optimal and maximum temperatures that permit the enzyme to retain catalytic activity. In some cases, this range can be well above the physiological maximum for any given enzyme. For instance, previous reports suggested that 70−90 °C was an optimal temperature range for ester-based polymerizations mediated by lipases.34−39 However, the addition of siloxane-containing components imparted thermal stability to the enzyme allowing transformations to be carried out at 130 °C with only a minor loss in residual activity.20,21,33 In order to determine the optimal temperature for maximizing conversion and yield of the macrocyclic oligoesters, reactions were carried out from 60 to 100 °C for 120 h. Temperature did not have a significant impact on the

Figure 4. Conversion of ester 2 as a function of temperature.

distribution of macrocyclic oligoesters favored the formation of 6 (70−73%) with evidence for 7 (10−25%) as well (Figure 5). At 100 °C the distribution of the observed products changed such that the proportion of 6 increased dramatically to 88%, resulting in a concomitant reduction in the amount of 7 to 7%. Oligoester 6 could be isolated by column chromatography in low to moderate yields (17−40%) using 15% ethyl acetate in hexanes as the eluent. The 1H NMR spectrum is presented in Figure 6 and indicates the presence of a single compound, which was confirmed by both MALDI-ToF and FAB-MS, each of which showed a molecular ion peak corresponding to (M + Na)+ at 1164 m/z (MALDI-ToF MS) and 1142 m/z (FABMS). N435 Favored Longer Chain Length Esters and Acyclic Alcohols. The effect of the ester chain length was taken into consideration. In addition to ester 2, two additional esters, 1,3,5,7-tetrakis(6-carboxyhexyl)-1,3,5,7-tetramethylcyE

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Table 2. Effect of Chain Length of Tetraesters and Diols on the N435-Catalyzed Synthesis of Macrocyclic Oligoestersa

Figure 5. Proportion of macrolactones 6 (gray bars) and 7 (white bars) produced at each of the surveyed reaction temperatures as determined from MALDI-ToF.

clotetrasiloxane tetramethyl ester (3) and 1,3,5,7-tetrakis(4carboxybutyl)-1,3,5,7-tetramethylcyclotetrasiloxane tetramethyl ester (4), were synthesized and examined as substrates. Employing the same reaction conditions used for the esterification of 2 and 5, a clear decrease in conversion was observed as the chain length of the ester was decreased (Table 2, entries 1 and 3). N435 converted 46% of 3 using octane-1,8diol, and less than 5% of 4 under the same conditions. The amount of oligoesters decreased to below 2% for ester 3; the relative proportion of oligoesters was not determined for reactions with ester 4 due to low substrate conversion. Decreased conversion resulted from an incompatibility between the cyclosiloxane ester and the active site of the enzyme. The

Conditions: 25 mM in PhMe, 100 °C, 150 rpm, 120 h, 5 wt % N435. The bracketed values are isolated yields after column chromatography.

a

*

Figure 6. 1H NMR spectrum for oligoester 6. F

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Figure 7. Langmuir π−A isotherms for a monolayer of 6: (A) three successive compression−decompression cycles (the inset is an expanded isotherm indicating the surface pressure at which monolayer collapse occurs); (B) three precompression−decompression cycles (red lines), which were followed by subsequent, complete compression−decompression cycles (black lines; the inset indicates that monolayer collapse at higher surface pressure following precompression−decompression cycling).

Langmuir Isotherms. Siloxanes exhibit unique surface properties when spread at the air−water interface due in part to their hydrophobic nature.2,40 The methyl groups on silicon direct themselves into air, while the oxygen and silicon atoms interact with the liquid environment. The flexible nature of the siloxane backbone allows the polymer chains to reorient themselves to adopt more favorable orientations when their local environment is compressed. This flexibility is facilitated by the low barrier to linearization of the silicon−oxygen bond.2 Compression of monolayers of polysiloxanes spread on an aqueous subphase has shown that film collapse occurs in two phases: first chains in random orientations lying flat on the surface of the subphase begin to adopt a coiled geometry, and second, the compressing chains overlap to form bilayer and multilayer stacks. It has been hypothesized that as few as 20 siloxane repeat units are required for a silicone polymer to adopt a fully helical structure.41 We are interested in learning how substituted cyclosiloxanes spread at the air−water interface as these properties may affect their use in potential future applications. Two-barrier compression was carried out at a rate of 5 mm/min and the subphase temperature was held constant at 21.5 °C. Surface pressure (π)-area (A) isotherms (π−A) were collected and are shown in Figure 7. Three consecutive compression−expansion cycles were carried out with little change in the overall shape of the isotherms. The π started to increase at a mean molecular area of ∼200 Å2 and steadily increased until a critical point (πc) was reached at πc = 15.3 ± 1.0 mM/m at approximately 78 Å2. The observed inflection point, where monolayer collapse occurred, possessed a different line shape than expected for long alkyl fatty acids, for example, stearic acid, which has a reported π = 55 mN/m.42 Collapse of the Langmuir film also appeared to be a much different process than that previously observed for regular linear polydimethylsiloxane (PDMS). Unlike linear PDMS, the adsorbed monolayer of 6 did not exhibit any conspicuous structural changes (bilayer, multilayer

shorter chain length esters bring the siloxane ring into a much closer proximity to the opening of the active site, which occludes entry of the diol into the active site inhibiting release the enzyme from its acylated state. This chain-length affect has been observed previously in trisiloxane-containing esters and in the synthesis of D4 octyl esters and exemplifies the importance of not only proximal, but distal, steric contributions in chemoenzymatic approaches in organic synthesis.24,33 Diol structure is known to affect conversion and the increase in molecular mass of polyesters and tends to favor the formation of higher molecular weight polyesters.30 In this study, changing the chain length of the diol from octane-1,8diol to pentane-1,5-diol (8) had little change on the average conversion of the ester when 2 was the acyl donor. The proportion of the homologous macrolactone to 6 was found at levels of 82% by MALDI-ToF MS (Table 2). Changing from an acyclic diol to trans-(1R,2R)-cyclohexane-1,2-diol (9), elicited a pronounced effect on the outcome of the reaction. First, it became clear that the cyclic diol was less able to release the acyl-enzyme intermediate and transesterification was found to only be 43%. More importantly, however, was the complete absence of cyclic species in the MALDI-ToF MS spectra. As a result of the low level of conversion, and in conjunction with the information available for the cyclic diol with ester 2, the esterification of 3 or 4 with diol 9 was not attempted. The success of the enzyme catalyst for any transformation can be put into terms of the structure of the reacting substrates. In these experiments, the cyclosiloxane offers a degree of steric bulk that must be navigated by the enzyme. For this to be successful, the chain length of the pendant ester must be carefully chosen. Here, a small changes in ester or diol chain length or geometry produced substantial decreases in conversion and production of macrolactones. This is a result of the cyclosiloxane ring being unable to bind optimally to the enzyme or by inhibiting entry into the active site by the diol. G

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Biomacromolecules

>1000 mN/m, solid.3 In the Cs−1 versus π plots for the Langmuir monolayer, the maximum value that was obtained was (Cs−1)max = 39.8 mN/m when π = 14.4 mN/m. The presence of a single maximum occurring below 50 mN/m indicated that the monolayer remained in the liquid expanded phase during compression. The nature of the phase transition was not significantly altered, despite an increase (Cs−1)max = 45.7 mN/m when π = 15.5 mN/m when the film was subjected to multiple compressions in which a surface pressure which was lower than the collapse pressure was attained. Thermal Properties. The thermal characteristics of ester 2 and oligoester 6 were determined using differential scanning calorimetry (DSC). The lactonization process may produce macrolactones in which the linkages have either a [1,3] or [1,5] relationship around the siloxane ring. The presence or absence of distinct melting or crystallization transitions would aid in confirming the nature of the linkages. The samples were heated in aluminum pans from −150 to 30 °C at 10 °C/min in order to remove any thermal memory. The DSC thermogram for 2 displayed many features of interest; first there was a Tm at −35 °C, which was followed by an enthalphy change at −19.3 °C and a third at 2.3 °C (Figure 9). The enthalpy change observed

stacks, or micelle formation) upon collapse as the overall architecture of the oligoester does not possess enough siloxane linkages to facilitate coiling or chain stacking. Furthermore, the molecule itself does not possess any strongly hydrophilic anchoring groups (carboxylic acid, esters, or amides) that would anchor one end of the molecule into the subphase and allow the hydrophobic portion of the molecules to lift from the surface of the subphase and protrude into the air−water interface. Secondary structural changes probably do not occur to any great extent as cyclosiloxane rings have a strong interaction with the surface of the aqueous subphase.43 The expansion of the barriers was performed at the same rate as the compression cycles. During the expansion, the observed inflection point was found to be slightly lower at 13.5 ± 0.7 mN/m, indicating only a marginal hysteresis in the films. Similar behavior has been observed during the reversible collapse of triphenylsilyl ether-terminated amphiphiles.44 Monomeric liquid crystals based on a siloxane framework tend to show a much greater degree hysteresis upon decompression.45 The reversible, collapsible nature of the Langmuir monolayer has been seen previously in amphiphilic silyl ether films,44 benzo[A]phenanthrene ether monolayers,46 and cross-linked and branched siloxanes films.47 Three compression cycles were performed and stopped prior to reaching the inflection point at πc = 15.3 ± 1.0 mM/m to determine the effect of compressing the monolayer on future phase transitions. The right panel of Figure 7 shows that precompression cycles lead to a small increase in πc to 17 mN/ m at 74 Å2. The reproducible nature of the isotherms upon repeated compression and decompression, both with and without preinflection cycling, indicated good dispersion of the monolayer upon barrier expansion and that an irreversible phase transition did not occur. To further characterize the nature and location of the phase transitions in the Langmuir film, the compression modulus (Cs−1) was plotted against π (Figure 8). The phase transitions of the Langmuir films are divided based on the maximum values of Cs−1. These phases have been previously defined as Cs−1 < 12.5 mN/m, gaseous; Cs−1 = 12.5−50 mN/m, liquid expanded; Cs−1 = 50−100 mN/m, liquid; Cs−1 = 100−250 mN/m, liquid condensed; Cs−1 = 250−1000 mN/m, condensed; and Cs−1 =

Figure 9. DSC thermograms of macrocyclic oligoester 6 (dotted line) and the C10D4 core 2 (solid line).

at −19.3 °C is interpreted as the reordering of the ends of the ester chains, which are distal to the point of attachment to the siloxane ring. The melting of alkyl-substituted polyhedral oligomeric silsesquioxane cages undergo a pretransition, which has been described as a solid−solid transition or rotator phase,48,49 that decreases in temperature and intensity as the alkyl chain length decreases.50 The endotherm at 2.3 °C was the solid−liquid phase transition as the remainder of the alkyl chain melted. Upon cooling, there was an observable hysteresis loop in the freezing−melting transition and two crystallization exotherms were seen, Tc1 −38 °C and Tc2 −45 °C, which are the reverse processes of the above indicated melting transitions. When the free esters are linked together in the form of macrolactones, the thermal transitions all but disappear. The only observable transition in the DSC spectrum occurred at −90 °C, and was very broad and has been interpreted as the glass transition temperature. The absence of sharp endotherms/exotherms indicates not only the transition from a glassy to an amorphous state of the macrocycle, but that the substitution pattern on the cyclosiloxane ring is likely not

Figure 8. Compression modulus (Cs−1) as a function of surface pressure (π) for 6. H

DOI: 10.1021/acs.biomac.5b00518 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

(10) Gross, R. A.; Kumar, A. J.; Kalra, B. Chem. Rev. 2001, 101, 2097−2124. (11) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. Rev. 2001, 101, 3793−3818. (12) Hodge, P. Chem. Rev. 2015, 114, 2278−2312. (13) Zhi-Wei, G.; Sih, C. J. J. Am. Chem. Soc. 1988, 110, 1999−2001. (14) Mezoul, G.; Lalot, T.; Brigodiot, M.; Maréchal, E. Polym. Bull. 1996, 36, 541−548. (15) Berkane, C.; Mezoul, G.; Lalot, T.; Brigodiot, M.; Maréchal, E. Macromolecules 1997, 30, 7729−7734. (16) Gargouri, M.; Drouet, P.; Legot, M. D. J. Biotechnol. 2002, 92, 259−266. (17) Lobell, M.; Schneider, M. P. Tetrahedron: Asymmetry 1993, 4, 1027−1030. (18) Zhi-Wei, G.; Ngooi, T. K.; Scilimati, A.; Fülling, G.; Sih, C. J. Tetrahedron Lett. 1988, 29, 5583−5586. (19) Gutman, A. L.; Oren, D.; Boltanski, A.; Bravdo, T. Tetrahedron Lett. 1987, 28, 5367−5368. (20) Frampton, M. B.; Séguin, J. P.; Marquardt, D. M.; Harroun, T. A.; Zelisko, P. M. J. Mol. Catal. B: Enzym. 2013, 85−86, 14−155. (21) Frampton, M. B.; Zelisko, P. M. Chem. Commun. 2013, 49, 9269−9271. (22) Poojari, Y.; Clarson, S. J. Biocatal. Agric. Biotechnol. 2013, 2, 7− 11. (23) Poojari, Y.; Beemat, J. S.; Clarson, S. J. Polym. Bull. 2013, 70, 1543−1552. (24) Frampton, M. B.; Jones, T. R. B.; Zelisko, P. M. RSC Adv. 2015, 5, 1999−2008. (25) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 30, 81−87. (26) Halling, P. J. Enz. Microbiol. Technol. 1994, 16, 178−206. (27) Luu, H. M.; Hutter, J. C. Environ. Health Perspect. 2001, 109, 1095−1101. (28) Robinson, G. K.; Alston, M. J.; Knowles, C. J.; Cheetham, P. S. J.; Motion, K. R. Enz. Microbiol. Technol. 1994, 16, 855−863. (29) Kumar, A. J.; Gross, R. Macromolecules 2000, 1, 133−138. (30) Poojari, Y.; Palsule, A.; Cai, M.; Clarson, S. J.; Gross, R. A. Eur. Polym. J. 2008, 44, 4139−4145. (31) Chen, B.; Hu, J.; Miller, E. M.; Xie, W.; Cai, M.; Gross, R. A. Biomacromolecules 2008, 9, 463−471. (32) Pliess, J.; Fischer, M.; Schmid, R. D. Chem. Phys. Lipids 1998, 93, 67−80. (33) Frampton, M. B.; Zelisko, P. M. Enz. Microbiol. Technol. 2014, 58−59, 87−92. (34) Frampton, M. B.; Subczynska, I.; Zelisko, P. M. Biomacromolecules 2010, 11, 1818−1825. (35) Poojari, Y.; Clarson, S. J. J. Inorg. Organomet. Polym. 2010, 20, 46−52. (36) Poojari, Y.; Clarson, S. J. Macromolecules 2010, 43, 4616−4622. (37) Poojari, Y.; Clarson, S. J. Silicon 2009, 1, 165−172. (38) Sharma, B.; Azim, A.; Azim, H.; Gross, R. A.; Zini, E.; Focarete, M. L.; Scandola, M. Macromolecules 2007, 40, 7919−7927. (39) Jiang, Z.; Liu, C.; Gross, R. A. Macromolecules 2008, 41, 4671− 4680. (40) Smith, A. L., Ed. Introduction to Silicones. In The Analytical Chemistry of Silicones; Wiley: New York, NY, U.S.A., 1991. (41) Granick, S.; Clarson, S. J.; Formoy, T. R.; Semlyen, J. A. Polymer 1985, 26, 925−928. (42) Griffith, E. C.; Adams, E. M.; Allen, H. C.; Vaida, V. J. Phys. Chem. B 2012, 116, 7849−7857. (43) Makarova, N. N.; Godovsky, Y. K.; Larkin, D. Y.; Buzin, A. I. J. Polym. Sci., Part A: Polym. Chem. 2007, 49, 120−127. (44) Devries, C. A.; Haycraft, J. J.; Han, Q.; Noor-e-Ain, F.; Raible, J.; Dussault, P. H.; Eckhardt, C. J. Thin Solid Films 2011, 519, 2430− 2437. (45) Chen, X.; Xue, Q.-B.; Yang, K.-Z.; Zhang, Q.-Z. Macromolecules 1996, 29, 5658−5663. (46) Haycraft, J. J.; DeVries, C. A.; Flores, H. G.; Lech, A.; Hagen, J. P.; Eskhardt, C. J. Thin Solid Films 2007, 515, 2990−2997.

uniform and is probably a mixture of [1,3] and [1,5] linkages around the siloxane ring which prevent the alkyl chains from packing efficiently.

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CONCLUSIONS We have examined an enzymatic process for the synthesis of macrocyclic oligoesters that are based on a cyclotetrasiloxane core. Enzyme choice, substrate concentration, reaction temperature, and solvent showed clear effects on the enzymatic process. N435 was the only candidate lipase that carried out the desired transformation. Moderately hydrophobic solvents with log P values in the range of 2−3 were more suitable than those at lower or higher log P values. Temperature had little effect on total conversion and yield except when a reaction temperature of 100 °C was employed, which gave an increased proportion of macrolactone 6. For N435, longer chain length esters and diols were more conducive to the synthesis of the macrocycles. Langmuir isotherms indicated that monolayers were reversibly collapsible after multiple compression/expansion cycles and monolayers remain in the liquid condensed phase even at maximum compression. The thermal behavior is the macrolactone suggests a potential mixture of lactones that differ in connectivity on the siloxane ring.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Synthetic procedures and additional supporting figures. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00518.

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to the Natural Sciences and Engineering Research Council (NSERC) and the Advanced Biomanufacturing Centre (ABC) at Brock University for funding of this work. The authors would also like to thank Terry Marquardt for the construction and donation of the Langmuir trough enclosure.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b00518 Biomacromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.biomac.5b00518 Biomacromolecules XXXX, XXX, XXX−XXX