Chemoenzymatic Synthesis of Narrow-Polydispersity Glycopolymers

Kitazawa, S.; Okumura, M.; Kinomura, K.; Sakakibara, T. Chem. Lett. 1990, 1733−1736. [Crossref], [CAS]. (32) . Syntheses and properties of novel vin...
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March/April 2004

Published by the American Chemical Society

Volume 5, Number 2

© Copyright 2004 by the American Chemical Society

Communications Chemoenzymatic Synthesis of Narrow-Polydispersity Glycopolymers: Poly(6-O-vinyladipoyl-D-glucopyranose) Luca Albertin,† Claudia Kohlert,† Martina Stenzel,† L. John R. Foster,‡ and Thomas P. Davis*,† Centre for Advanced Macromolecular Design, School of Chemical Engineering and Industrial Chemistry, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, NSW 2052 Sydney, Australia Received June 20, 2003; Revised Manuscript Received December 4, 2003

The glycomonomer 6-O-vinyladipoyl-D-glucopyranose was prepared via lipase catalyzed transesterification of divinyladipate with R-D-glucopyranose in dry acetonitrile and acetone. The desired 6-O regioisomer was obtained in good yield, and its structure was confirmed by correlation NMR spectroscopy. Controlled radical polymerization of the unprotected monomer was performed in protic media using both xanthate and dithiocarbamate as chain transfer agents to give poly(6-O-vinyladipoyl-D-glucopyranose) with Mn of 17 and 19 kDa (SEC) respectively and a polydispersity as low as 1.10. To the best of our knowledge, this is the first example of a narrow-polydispersity, poly(vinyl ester)-like glycopolymer. Introduction In a general sense, glycopolymers can be defined as synthetic polymers carrying carbohydrate moieties as pendant or terminal groups.1 Since the pioneering work of Horejsi et al.,2 glycopolymers have raised an ever increasing interest as artificial materials for a number of biological and biomedical uses. This is mostly due to the expectation that polymers displaying complex functionalities, similar to those of natural glycoconjugates, might be able to mimic, or even exceed, their performance in specific applications (biomimetic approach). For instance, studies have been published on the use of glycopolymers as macromolecular drugs,3-7 drug delivery systems,8-10 cell culture substrates,11,12 stationary phase in separation problems13,14 and bioassays,15 responsive16 and catalytic17 hydrogels, surface modifiers,18-21 and artificial tissues and artificial organs substrates.11 * To whom correspondence should be addressed. E-mail: camd@ unsw.edu.au. † School of Chemical Engineering and Industrial Chemistry. ‡ School of Biotechnology and Biomolecular Sciences.

Although essential, the presence of appropriate functional groups in a glycopolymer is usually insufficient to bestow it with the biological and physicochemical properties required by a given application. In fact, control of the macromolecular architecture has proved essential to enable sophisticated functions5,22 and to allow a precise correlation between these and the polymer structure. For this reason, in the past decade, more and more polymer chemists have become involved in the synthesis of novel glycopolymers via both traditional and precise polymerization techniques.1,20 Apart from polymer modification, which allows for a lesser control and reproducibility of the final material, glycopolymers have been so far prepared via ring opening, ring opening metathesis, and vinyl polymerization, the latter accounting for most of the publications in the field. To date, alkenyl-,23,24 alkynyl-,25 acryloyl-,26-30 methacryloyl-,31,32 acrylamide-,33,34 styryl-,35-37 and vinyl ether-38 derived glycopolymers have been successfully synthesized by cationic, anionic, and radical vinyl polymerization techniques. Particularly interesting is a report by Narain and Armes on the

10.1021/bm034199u CCC: $27.50 © 2004 American Chemical Society Published on Web 01/07/2004

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atom transfer radical polymerization of 2-gluconamidoethyl methacrylate and 2-lactobionamidoethyl methacrylate in water and methanol, because it describes the preparation of a low polydispersity glycopolymer without having to resort to protecting group chemistry.39,40 Recently, the first example of reversible addition-fragmentation chain transfer (RAFT) polymerization of a sugar monomer was provided by Lowe et al., who successfully polymerized the commercially available 2-methacryloxyethyl glucoside in water and in the presence of a water soluble dithioester.41 Little attention has been so far devoted to the preparation of poly(vinyl ester)like glycopolymers, although they can offer distinctive advantages in terms of environmental biodegradability42 and, whenever in vivo applications are sought after, biocompatibility of the polymer backbone. In fact, hydrolytic cleavage of the pendant groups leaves a poly(vinyl alcohol) main chain, a material already used in a number of medical applications43,44 and that does not seem to interact with cellular blood components.45 In the past few years, two groups have reported the enzyme catalyzed synthesis of carbohydrate-functionalized vinyl esters and their radical polymerization.46-49 Intrigued by the potentiality of this chemoenzymatic approach to the synthesis of poly(vinyl ester)-like glycopolymers, we decided to start a systematic investigation of both the enzyme catalyzed synthesis of unprotected glycomonomers and of their controlled radical polymerization to materials of desired architecture. In this article we present our preliminary results on the lipase catalyzed synthesis 6-O-vinyladipoyl-D-glucopyranose (6-O-VAGlu) and on its controlled radical polymerization in water and methanol. Experimental Section Materials and Methods. Unless otherwise specified, all chemicals were reagent grade. R-D-Glucopyranose, lithium bromide, aniline (Aldrich), divinyladipate (TCI), 4,4′-azobis(cyanopentanoic acid) (ACPA, Fluka), and deuterium oxide (99.9%, Cambridge Isotopes) were used as received. All solvents were HPLC grade (Asia Pacific Specialty Chemicals) and both acetonitrile and acetone were dried for 48 h on activated 4 Å molecular sieves prior to use. Flash chromatography was performed with a 76 mm O.D. glass column loaded with 200 g of silica gel (60 Å, 40-63 µm, Merk) and eluted with ethyl acetate/hexane/ethanol 7:2:1 at flow rate of 0.05 m min-1. The same eluent mixture was used for TLC analysis, which was performed on glass backed silica gel plates (60 Å, 5-17 µm, Macherey-Nagel). Following solvent evaporation, the developed plates were immersed in a freshly made aniline/orthophosphoric acid (88%)/1-butanol 3:15:52 solution for 3 s and heated at 80 °C for 30 min for spots detection. Analysis. All NMR experiments were realized with a Brucker Avance 300 MHz spectrometer (magnetic field strength of 300.2 and 75.5 MHz for 1H and 13C, respectively). Molecular weights and molecular weight distributions were measured by size exclusion chromatography (SEC) on a Shimadzu modular LC system comprising a DGU-12A solvent degasser, a LC-10AT pump, a SIL-10AD auto

Communications

injector, a CTO-10A column oven, and a RID-10A refractive index detector. The system was equipped with a 50 × 7.8 mm guard column and three 300 × 7.8 mm linear columns (Phenomenex; 500, 103 and 104 Å pore size, 5 µm particle size). N,N-Dimethylacetamide (HPLC, 0.03% w/v LiBr) was used as eluent at a flow rate of 1 mL min-1, whereas the columns temperature was maintained at 40 °C constant. 50 µL of polymer solution (5 mg mL-1) was injected. Calibration was performed with narrow polydispersity polystyrene standards (Polymer Laboratories) in the range 5 × 102-7 × 105 Da and SEC traces were elaborated with Cirrus 1.0 software (PL). LC-MS analysis was performed with a Thermo-MAT high-pressure liquid chromatography system consisting of a solvent degasser, a quaternary pump, an autoinjector, and a dual-wavelength UV-detector and equipped with a C8 Luna column (Phenomenex, 150 × 4.6 mm, 100 Å pore size, 5 µm particle size). The system was interfaced to a Thermo Finnigan LCQ Deca ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an atmospheric pressure-ionization source operated in nebulizerassisted electro-spray mode. The instrument was calibrated with caffeine (Aldrich), MRFA (tetrapeptide, Thermo Finnigan), Ultramark 1621 (Lancaster), and polypropylene glycol (Mn 2,700, Aldrich) in the mass range of 195-3822 amu. All spectra were acquired in positive ion mode over a range of m/z 100-1000 with a spray voltage of 5 kV, a capillary voltage of 35 V, a tube lens offset of -30 V, and a capillary temperature of 350 °C. Nitrogen was used as sheath gas at a flow rate of 0.5 L/min and helium as the dumping gas. 3 mg mL-1 samples in acetonitrile/ammonium formate 1 mM 25:75 v/v were prepared and 5 µL was injected for analysis. The eluent was a mixture of acetonitrile/ammonium formate 1 mM with an acetonitrile content of 25% v/v for the first 7 min that was linearly increased to 70% v/v in the following 3 min and then maintained constant for the last 5 min of analysis (flow rate 1 mL min-1). Vibrational spectra were collected in the range 500-4000 cm-1 with a Perkin-Elmer 2000 FT-IR operated in transmittance mode. Samples mulls were suspended in mineral oil (Aldrich) and placed between NaCl plates. Thermal analyses were carried out with a Perkin-Elmer DSC 7 Differential Scanning Calorimeter operated at a rate of 10 °C/min. Synthesis of 6-O-Vinyladipoyl-D-glucopyranose (2). In a typical experiment, 15.2 g (0.0835 mol) of R-D-glucose (1) and 8 g of Novozym 435 were suspended in 140 mL of acetonitrile (or acetone) containing 25 g (0.123 mol) of divinyladipate. The mixture was magnetically stirred at 250 rpm and 50 °C for 24 h before stopping the reaction by filtering off the enzyme. The filtrate was washed with 2 × 100 mL of methanol, and the collected organic phases were rotary evaporated to dryness to yield a yellow-brown syrup which partially crystallized when left overnight at room temperature. The gross product was then purified by flashchromatography, and the fractions were checked by TLC for the presence of the monoester (Rf ) 0.25). The pooled fractions were then rotary evaporated to afford the title compound as a white powder (average yield 50% with respect to glucose). Tm ) 72, 109 °C (DSC, maximum of the melting peak). ESI-MS: calcd for C14H26O9N, 352.16;

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found 352.2 (M+NH4+). FTIR (NaCl plates) ν (cm-1): 1740, 1704 (CdO); 1646 (CdC). 1H NMR (D2O) δ (ppm): 7.05 (dd, 1 H, J ) 13.9, 6.2 Hz, 13-H), 5.08 (d, 0.46 H, J ) 3.8, 1-HR), 4.85 (dd, 1 H, J ) 13.9, 1.8 Hz, 14-H cis), 4.60 (dd, 1 H, J ) 6.2, 2.0, 14-H trans), 4.52 (d, 0.54 H, J ) 7.9 Hz, 1-Hβ), 4.2 (m, 2 H, 6-H), 3.88 (m, 0.46 H, 5-H), 3.59 (m, 3-HR), 3.52 (m, 5-Hβ), 3.39 (m, 2-Hβ), 3.36 (m, 3-Hβ), 3.31 (m, 4-H), 3.12 (m, 0.52 H, 2-HR), 2.36 (m, 4 H), 1.54 (m, 4 H). 13C NMR (D2O) δ (ppm): 23.13 (C-10), 23.38 (C-9), 23.42 (C-9), 32.90 (C-10), 33.10 (C-8), 33.13 (C-8), 63.11 (C-6β), 63.19 (C-6R), 69.03 (C-5β), 69.41 (C-4), 69.47 (C4), 71.25 (C-2β), 72.43 (C-3R), 73.24 (C-5R), 73.86 (C-2R), 75.39 (C-3β), 91.97 (C-1β), 95.84 (C-1R), 99.04 (C-14), 140.95 (C-13), 173.41 (C-12), 173.43 (C-12), 175.96 (C-7), 175.99 (C-7). Synthesis of the RAFT Agents. Both 4-Cyano-4-diethylthiocarbamoylsulfanyl-4-methyl-butyric acid (5) and 2-thiopropionylsulfanyl-propionic acid methyl ester (6) were prepared according to the methods described by Rizzardo et al.50,51 5, yield 86%. 1H NMR (CDCl3) δ (ppm): 4.05 (q, 4 H, J ) 7.2 Hz), 2.62 (m, 4 H), 1.98 (s, 3 H), 1.20 (t, 6 H, J ) 7.2 Hz). 6, yield 79%. 1H NMR (CDCl3) δ (ppm): 4.62 (q, 2 H, J ) 7.2 Hz), 4.38 (q, 1 H, J ) 7.2 Hz), 3.72 (s, 3 H), 1.55 (d, 3 H, J ) 7.2 Hz), 1.39 (t, 3 H, J ) 7.2 Hz). Controlled Polymerization of 6-O-Vinyladipoyl-D-glucopyranose. In a test tube, 2.33 g (7 × 10-3 mol) of monomer was dissolved in 12 mL of distilled water and 0.52 mL of a 2.7 × 10-2 M water solution of 4,4′-azobis(cyanopentanoic acid) (1.4 × 10-5 mol) were added. The resulting solution was split into two equal portions, and each portion was transferred into a 10 mL Schlenk tube. Meanwhile, 73 mg (2.6 × 10-4 mol) of 4-cyano-4-diethylthiocarbamoylsulfanyl-4-methyl-butyric acid (5) were dissolved in 2 mL of water containing an equimolar amount of Na2CO3 to facilitate dissolution. To one of the Shlenk tubes was added 0.225 mL (3.0 × 10-5 mol) of RAFT agent solution; both tubes were then sealed with greased glass stoppers, degassed with 3 cycles of freeze-pump-thaw, and transferred to a preheated water bath (60 °C) for 48 h. The reaction was then stopped by quenching with ice cold water for 5 min. After removing the water by freeze-drying, conversion was calculated by 1H NMR through the disappearance of one of the vinyl protons (13-H) and using the glucose ring hydrocarbon protons 2-6 as internal standard. When 2-thiopropionylsulfanyl-propionic acid methyl ester 6 was used as RAFT agent, the above-reported procedure was modified as follows: anhydrous methanol (Aldrich, received under N2) was used as a solvent; 68 mg of RAFT agent were dissolved in 2 mL of methanol and 190 µL were used (3.1 × 10-4 mol); prior to freeze-drying the samples, most of the methanol was evaporated by air sparging. Prior to GPC analysis, all samples were redissolved in water and precipitated in acetone. Results and Discussion Monomer Synthesis. The lipase catalyzed synthesis of 6-O-vinyladipoyl-D-glucopyranose (6-O-VAGlu) is shown in Scheme 1. For clarity, the position numbering used in

Figure 1. 1H NMR spectrum of 6-O-vinyladipoyl-D-glucopyranose in D2O. See Scheme 1 for nuclei numbering.

NMR assignment is also reported (see the Experimental Section). Novozym 435 (a commercially available immobilized lipase) was chosen as the catalyst for the transesterification of divinyladipate with R-D-glucose because of its ability to catalyze regioselective esterification and transesterification reactions in a number of organic solvents (i.e., THF, acetone, acetonitrile, and pyridine)49,52,53 and because it can be easily recovered from the reaction medium by filtration. Similar results were obtained when acetonitrile or acetone were used as a solvent. TLC analysis of the gross product suggested the presence of two compounds, one with Rf ) 0.25 and the other with Rf ) 0.75. Reverse phase LCMS analysis confirmed that together with the main product 2 (r.t. 5.3 and 5.8 min, m/z 352.2, M+NH4+) two other byproducts formed during the acylation process. One of them (r.t. 11.5 and 11.6 min, m/z 506.2) could be bis-Ovinyladipoyl-D-glucopyranose (calcd for C22H36O12N, 506.22, M+NH4+) formed by further acylation of 2, whereas no evident assignment could be found for the other byproduct (r.t. 11.0 and 11.1 min, m/z 494.1). Interestingly, although pure R-D-glucose was used as starting material, HPLC separation indicated that all products were present as mixture of anomers in a ratio R/β ) 1:1.2, where the more polar R anomers is the one with the shorter r.t. After chromatographic purification, 2 was recovered in high purity, and its structure was confirmed by correlation NMR experiments (1H-1H COSY, HMQC, and HMBC).54 Figure 1 shows the 1H NMR spectrum of the purified product in which all three vinyl protons as well as the anomeric proton of the R and β form can be clearly distinguished. Comparison between the 13C spectrum of 2 with that of the starting R-Dglucopyranose (Figure 2) reveals that the greatest change in chemical shift has occurred for C-5 (-2.2 and -2.3 ppm for the R and β forms, respectively) and C-6 (+2.6 and +2.7 ppm for the R and β forms, respectively). Both the sign and the entity of these shifts are consistent with the studies of Y. Tsuda et al. on the positional isomers of O-acylglucopyranosides55 and confirms that the acylation took indeed place in position 6.

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Scheme 1. Lipase Catalyzed Synthesis of 6-O-vinyladipoyl-D-glucopyranose 2 and Its Controlled Radical Polymerizationa

a For clarity, the position numbering of 2 used in the NMR assignment is also reported (see the Experimental Section). Conditions: (i) divinyl adipate, Novozym 435, acetonitrile (or acetone), 50 °C, 24 h; (ii) ACPA, methanol, RAFT agent 5, 60 °C, 48 h; (iii) ACPA, water, RAFT agent 6, 60 °C, 48 h.

Scheme 2. RAFT Agents Used for the Controlled Polymerization of 6-O-VAGlu.

Figure 2. Expanded region of the 13C NMR spectrum of 2 (top) and of R-D-glucopyranose (bottom). See Scheme 1 for nuclei numbering.

Controlled Radical Polymerization. Vinyl esters are a class of relatively unreactive monomers that polymerize only with a radical mechanism and through unstable, unconjugated radical chain carriers whose propagation is very difficult to control. In fact, reversible addition-fragmentation chain transfer (RAFT) polymerization is the only technique to date available for controlling the polymerization of vinyl es-

ters,56,57 although studies on the atom-transfer radical polymerization of this type of monomers are also in progress.58 Furthermore, when the controlled polymerization of highly functionalized monomers and macromonomers is researched, RAFT offers the distinctive advantage of being relatively insensitive to the presence of functional groups;59,60 these combined characteristics suggested to us the possibility to control the polymerization of unprotected vinyl ester-like glycomonomers via RAFT in order to prepare narrow polydispersity glycopolymers. To our knowledge, this has been so far achieved only in four other cases: by Grande and co-workers for acrylamide derivatized glycomonomers and using a cyanoxyl-mediated polymerization in which the initiator must be generated in situ;33 by Armes and co-worker for two methacrylate-functionalized carbohydrates via ATRP;39,40 by Lowe et al., who successfully polymerized the commercially available 2-methacryloxyethyl glucoside in water and in the present of a water soluble RAFT agent;41 and by Fukuda and co-workers with the nitroxide mediated

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Scheme 3. Chain Equilibrium by Reversible Addition Fragmentation between the Propagating Species P•m and P•n and the Polymeric Dormant RAFT Compound 7 or 9

Table 1. Controlled Radical Polymerization of 6-O-Vinyladipoyl-D-glucopyranose (2)a run no.

solvent

1 2 3 4

H2O H2O CH3OH CH3OH

5 6

0 4.6 0 4.8

66 27 17 14

53.1 19.6 34.5 17.1

6-O-VAGlu 0.55 M, ACPA 1.1 mM, temperature 60 °C. SEC, styrene equivalents. a

c

RAFT [RAFT agent] conversion Mn agent (mM) (%)b (KDa)c Mw/Mnc 2.15 1.19 1.76 1.10 b 1H

NMR.

acetate56 (Scheme 2). From the collected data, it appears evident that both RAFT agents effectively allow for the preparation of narrow polydispersity poly(6-O-vinyladipoylD-glucopyranose), and a PDI as low as 1.10 was observed for polymer 4 (Figure 3b). The low conversions observed after 24 h are consistent with the low initial monomer concentration used (0.55 M). With respect to this, the use of water as solvent afforded the best results, even though a marked decrease in conversion was observed in the presence of 5 when compared to the control experiment. The same effect was not observed with RAFT agent 6 in methanol. Finally, the SEC traces of the reprecipitated polymers show a single, symmetric peak and seem to suggest that no significant bimolecular termination took place during the process (Figure 3). Conclusion

Figure 3. MWD of polymer 3 (a) and 4 (b) (SEC, polystyrene equivalents).

polymerization of a styrene derivative in N,N-dimethyl formamide.61 The mechanism of RAFT polymerization with thiocarbonylthio-based RAFT agents involves a series of additionfragmentation steps that lead to an equilibrium between propagating species and dormant polymeric RAFT compounds (Scheme 3).62 In the case of vinyl esters, the high reactivity and relatively little steric bulk of the propagating radical makes it a very poor homolytic leaving group, and for the process to be effective, the intermediate radical 8 in Scheme 3 must be somehow destabilized by the Z group. This can be achieved by choosing electron-donating Z groups and accounts for the experimental observation that only xanthates (Z ) OR) and dithiocarbamates (Z ) NR2) are effective RAFT agents for the polymerization of vinyl esters. Results for the controlled radical polymerization of 6-OVAGlu are summarized in Table 1, where runs 1 and 3 refer to control experiments in which no RAFT agent was added to the reaction medium. The dithiocarbamate 5 was chosen for the controlled polymerization in water because it can be easily prepared via a one step synthesis and its sodium salt is water soluble; when methanol was used as a solvent instead, the xanthate 6 was preferred since it is known to be an effective RAFT agent for the polymerization of vinyl

This preliminary report details the facile, two-steps chemoenzymatic synthesis of narrow-polydispersity poly(6O-vinyladipoyl-D-glucopyranose). The glycomonomer 6-Ovinyladipoyl-D-glucopyranose 2 was prepared in good yield (50%) from the direct acylation of D-glucose with divinyladipate in the presence of Novozym 435, an immobilized lipase B from Candida Antarctica. Radical polymerization of the unprotected monomer in the presence of the dithiocarbamate 5 or the xanthate 6 allowed to effectively reduce the polydispersity of the obtained polymer to a value as low as 1.10, albeit a decrease in conversion was observed when 5 was used. Studies are in progress to optimize the enzyme kinetics as well as to probe the livingness of the polymerization process, the final goal being the synthesis of biologically active glycopolymers with a well-defined molecular weight and in a controlled fashion. Acknowledgment. We thank Novozymes A/S for the generous donation of Novozym 435. We also thank Dr. J. Hook for assistance with NMR experiments and useful discussions. One of the authors (L.A.) acknowledges financial support from the Australian Department of Education, Training and Youth Affairs through an International Postgraduate Research Scholarship. T.P.D. acknowledges the award of an Australian Professorial Fellowship. References and Notes (1) Okada, M. Prog. Polym. Sci. 2001, 26, 67-104. (2) Horejsi, V.; Smolek, P.; Kocourek, J. Biochim. Biophys. Acta 1978, 538, 293-298. (3) Choi, S. K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 4103-4111. (4) Gordon, E. J.; Strong, L. E.; Kiessling, L. L. Bioorg. Med. Chem. 1998, 6, 1293-1299. (5) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Curr. Opin. Chem. Biol. 2000, 4, 696-703.

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