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COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11170 Received 13th September 2013, Accepted 9th October 2013
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A method for highly efficient catalytic immobilisation of glucose oxidase on the surface of silica† Yong-Kyun Sim, Jung-Woo Park, Bo-Hyeong Kim and Chul-Ho Jun*
DOI: 10.1039/c3cc47011c www.rsc.org/chemcomm
A simple, mild and convenient method has been developed for catalytic immobilisation of glucose oxidase (GOx), chemically modified to contain pendant methallylsilyl groups, on an untreated silica surface.
Development of new and efficient enzyme immobilization methods and their applications are very important in industrial use since heterogenized enzymes can be readily recovered for repeated use and sometimes show high enzymatic activities with enhanced stability compared with homogenous ones.1 Among them, heterogenisation of native enzymes via covalent bonding is an important tool in modern chemical and biological investigations because the strong bonds generated in this process prevent or reduce enzyme desorption. Common methods for covalent attachment of enzymes to solid supports, such as silica, employ reactions of native enzymes with functional group containing solid supports (e.g., NHS-esters, aldehydes, and epoxides) (Fig. 1a).1,2 Another method for covalent immobilisation of enzymes utilizes reactions of chemically functionalized enzymes with functional groups present on solid supports.3 Nearly all of these protocols require the use of solid supports that contain reactive functional groups, which are generated by reactions of the supports with large excesses of silane coupling reagents at high temperatures.1,4 Silica supports prepared in this manner contain only a small number of reactive functional groups in comparison to the large number of hydroxyl groups that are present on native silica. As a result, protocols that utilize non-modified silica should lead to higher degrees of enzyme immobilisation. We envisioned that an ideal strategy for this purpose would involve the use of direct catalytic immobilisation of an appropriately chemically modified (CM) enzyme. Enzymes containing pendant methallylsilyl groups, covalently linked through amide bonds with lysine amine groups, would be useful for this purpose because they should be capable of undergoing mild, acid-catalysed reactions with hydroxyl groups present on the silica surface.5,6
Department of Chemistry, Yonsei University, 50 Yonseiro, Seodaemun-gu, Seoul 120-749, Korea. E-mail: [email protected]; Fax: +82-2-3147-2644; Tel: +82-2-2123-2627 † Electronic supplementary information (ESI) available: Experimental details and characterization data of compounds. See DOI: 10.1039/c3cc47011c
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Chem. Commun., 2013, 49, 11170--11172
Fig. 1 Comparison of previous and the new approaches to covalent immobilisation of enzymes. (a) Conventional immobilisation using surface modified functional groups. (b) The new approach employing reaction of chemically modified GOx with non-treated silica.
The overall approach would involve initial chemical modification of an enzyme through amide bond forming reaction with the NHS-ester-functionalized methallylsilane derivative 1 (Fig. 1b). Next, immobilisation of the chemically modified GOx on nonmodified silica would be performed under mild, Lewis acidcatalysed conditions. Observations made in the study described below show that an important advantage of the new protocol is This journal is
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Communication that the methallylsilane coupling agent is more easily prepared and purified than the related alkoxysilanes employed earlier for this purpose. Immobilisation reactions of enzymes that are chemically modified with methallylsilane groups take place at much lower temperatures than those required for analogous reactions of their alkoxysilane counterparts, thus avoiding the possibility of enzyme deactivation. In addition, the reaction can be carried out in one pot-sequential process without isolation or purification, which makes the reaction simple and convenient. Owing to the ease with which it can be assayed, GOx was chosen as a model enzyme for the initial immobilisation studies.7 Specifically, the activity of this enzyme is readily determined by measuring the amount of H2O2 generated from the reaction of O2 with glucose. In addition, the degree of GOx immobilisation on silica was determined by using the Bradford assay. To explore the new immobilisation protocol, chemically modified GOx was prepared by amide bond forming reaction of amine residues in lysines of GOx with NHS-ester-functionalized methallylsilane 1 (Fig. 2). Reactions with varying amounts of NHS-ester-functionalized methallylsilane 1 (16, 32, 48 and 64 equiv.) in H2O at 0 1C for 2 h led to formation of the respective modified GOxs, GOx-CM(16), GOx-CM(32), GOx-CM(48) and GOx-CM(64) (Fig. 2a).9 Analysis of these modified enzymes by using MALDI-TOF showed that the use of 48 equiv. of 1 in this process is optimal (Fig. 2b), giving a GOx that contains ca. 9.3 equiv. of dimethallylsilyl groups (Fig. 2c). Importantly, use
Fig. 2 (a) Chemical modification of GOx with NHS-ester functionalized methallylsilane 1. (b) MALDI-TOF analysis of GOx and chemically modified GOxs. (c) Conversion of lysine amine groups. (d) Comparison of activities of native GOx with that of the chemically modified GOx-CM(48) formed using 48 equiv. of 1.
This journal is
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ChemComm of a large excess of 1 in the reaction did not lead to an increased loading of methallylsilane. In addition, the enzymatic activities of the modified GOxs are not dramatically lowered by introduction of methallylsilane groups. For example, GOx-CM(48) was observed to have 80% of the activity of native GOx (Fig. 2d). Immobilisation reactions of the GOx-CMs with silica8 were carried out in the presence of 10 mol% of Sc(OTf)3 (2, based on methallylsilyl groups) (Fig. 3a). These processes produced GOx-CM(16)@Si, GOx-CM(32)@Si, GOx-CM(48)@Si and GOxCM(64)@Si. The developed GOx grafting method, in which GOx is treated with NHS-ester-functionalized silica, was utilized as a standard for comparison purposes. Specifically, the grafted silica GOx-Graft@Si was prepared through reaction of GOx with NHS-ester-grafted silica (NHS-grafted silica), which was obtained by acid catalyzed reaction of NHS-ester-functionalized methallylsilane 1 with silica (Fig. 3b).5e,8 The results of measurements with the immobilised enzymes show that the catalytic activity (76 mM [H2O2] using 1.56 10 2 mg sample) and the degree of GOx loading (114 mg GOx per mg silica) of GOx-CM(48)@Si are much higher than those of GOx-CM(16)@Si (10 mM [H2O2]; 65 mg GOx per mg silica), GOx-CM(32)@Si (17 mM [H2O2]; 81 mg GOx per mg silica), and GOx-CM(64)@Si (59 mM [H2O2]; 73 mg GOx per mg silica) (Fig. 3c and d).10 These results demonstrate the importance of selecting the amount
Fig. 3 Schematic for the preparation of (a) GOx-CM(X)@Si through immobilisation of chemically modified glucose oxidase (GOx-CM(X)) onto non-modified silica (X = 16, 32, 48 and 64) and (b) GOx-Graft@Si through immobilisation of GOx onto NHS-ester-grafted silica. Comparisons of (c) enzyme activities and (d) enzyme loadings between GOx-CM(X)@Si and GOx-Graft@Si.
Chem. Commun., 2013, 49, 11170--11172
11171
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ChemComm of 1 in the enzyme functionalization process. The observation that the enzyme loading of GOx-CM(64)@Si is lower than that of GOx-CM(48)@Si is interesting in that it suggests that less efficient immobilisation of the silica surface takes place when unreacted 1 is present. Based on this finding, we conclude that use of 48 equiv. of 1 is ideal for the immobilisation reaction forming the immobilised GOx-CM. The results of a comparison study show that all GOx-CM@Sis have higher loading levels and catalytic activities than those of GOx-Graft@Si (activity ca. 4 mM [H2O2], see Fig. 3c; enzyme loading 21 mg GOx per mg silica, see Fig. 3d). In addition, it is noteworthy that use of the new immobilisation method involving chemical modification of GOx requires a much lower quantity of coupling reagent 1 (1.03 mg with 20 mg silica to form GOx-CM(48)@Si) than the corresponding reagent employed in conventional immobilisation of GOx (20.2 mg of 1 with 20 mg silica to form GOx-Graft@Si) (for the experimental details, see ESI†). The study described above has led to the development of an efficient method for immobilisation of glucose oxidase (GOx) on non-modified silica that involves the use of a chemically modified GOx containing NHS-ester-functionalized methallylsilanes. The GOx-immobilised silica, prepared by utilizing this method along with a small amount of the coupling reagent, displays a high enzyme loading level and high enzymatic activity. Applications of this new strategy to immobilise other bioactive molecules or enzymes are currently under study. This study was supported by the Mid-career Research Program (Grant 2011-0016830) through NRF grant funded by the MEST.
Notes and references 1 For reviews: (a) S. Hudson, J. Cooney and E. Magner, Angew. Chem., ¨der, Int. Ed., 2008, 47, 8582; (b) P. Jonkheijm, D. Weinrich, H. Schro C. M. Niemeyer and H. Waldmann, Angew. Chem., Int. Ed., 2008, 47, 9618; (c) U. Hanefeld, L. Gardossi and E. Magner, Chem. Soc. Rev., 2009, 38, 453; (d) R. A. Sheldon, Adv. Synth. Catal., 2007, 349, 1289.
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Communication 2 (a) X. Zhang, R.-F. Guan, D.-Q. Wu and K.-Y. Chan, J. Mol. Catal. B: ¨, G. Lu, Y. Wang, Y. Guo, Y. Guo, Enzym., 2005, 33, 43; (b) Y. Lu Z. Zhang, Y. Wang and X. Liu, Adv. Funct. Mater., 2007, 17, 2160; (c) A. Schlossbauer, D. Schaffert, J. Kecht, E. Wagner and T. Bein, J. Am. Chem. Soc., 2008, 130, 12558; (d) S. Libertino, F. Giannazo, V. Aiello, A. Scandurra, F. Sinatra, M. Renis and M. Fichera, Langmuir, 2008, 24, 1965. ´. Berenguer-Murcia and 3 For a review: R. C. Rodrigues, A R. Fernandez-Lafuentec, Adv. Synth. Catal., 2011, 353, 2216. 4 (a) K. B. Yoon, Acc. Chem. Res., 2007, 40, 29, and references therein; ´, M. Boualleg, J.-M. Camus, T. K. Maishal, J. Alauzun, (b) I. Karame ´ret, R. J. P. Corriu, E. Jeanneau, A. Mehdi, C. Reye ´, J.-M. Basset, C. Cope L. Veyre and C. Thieuleux, Chem.–Eur. J., 2009, 15, 11820; (c) F. Hoffmann, ¨ba, Angew. Chem., Int. Ed., 2006, M. Cornelius, J. Morell and M. Fro ´n ˜ez, F. Sanceno ´n, 45, 3216; (d) A. B. Descalzo, R. Martı´nez-Ma K. Hoffmann and K. Rurack, Angew. Chem., Int. Ed., 2006, 45, 5924; (e) G. J. A. A. Soler-Illia and P. Innocenzi, Chem.–Eur. J., 2006, 12, 4478; ( f ) A. Corma and H. Garcia, Adv. Synth. Catal., 2006, 348, 1391; (g) C. M. Crudden, M. Sateesh and R. Lewis, J. Am. Chem. Soc., 2005, 127, 10045. 5 (a) Y.-R. Yeon, Y. J. Park, J.-S. Lee, J.-W. Park, S.-G. Kang and C.-H. Jun, Angew. Chem., Int. Ed., 2008, 47, 109; (b) J.-W. Park, Y. J. Park and C.-H. Jun, Chem. Commun., 2011, 47, 4860; (c) D. H. Lee, E.-A. Jo, J.-W. Park and C.-H. Jun, Tetrahedron Lett., 2010, 51, 160; (d) S. Park, J. Pai, E.-H. Han, C.-H. Jun and I. Shin, Bioconjugate Chem., 2010, 21, 1246; (e) U.-Y. Jung, J.-W. Park, E.-H. Han, S.-G. Kang, S. Lee and C.-H. Jun, Chem.–Asian J., 2011, 6, 638. 6 For immobilisation of organic functional groups onto silica using stable alkenylsilanes, see: (a) J.-W. Park and C.-H. Jun, J. Am. Chem. Soc., 2010, 132, 7268; (b) T. Shimada, K. Aoki, Y. Shinoda, T. Nakamura, N. Tokunaga, S. Inagaki and T. Hayashi, J. Am. Chem. Soc., 2003, 125, 4688; (c) K. Aoki, T. Shimada and T. Hayashi, Tetrahedron: Asymmetry, 2004, 15, 1771; (d) Y. Maegawa, T. Nagano, T. Yabuno, H. Nakagawa and T. Shimada, Tetrahedron, 2007, 63, 11467; (e) Y. Wang, S. Hu and W. J. Brittain, Macromolecules, 2006, 39, 5675; ( f ) G. Hreczycho, M. K. Chmielewski, H. Maciejewski, T. Ratajczak and B. Marciniec, Tetrahedron Lett., 2013, 54, 3605. 7 Glucose oxidase was purchased from Aldrich. Co., and used after purification by gel-filtration. 8 Large pore sized silica balls were used as solid supports. The specification of silica is as follows; size: 10 mm, pore size: 30 nm; which was purchased from Fuji Silysia Ltd. 9 The amounts of 1 correspond to 1, 2, 3 and 4 equivalents, respectively, based on the number of lysine groups in GOx (16 determined by LC-MS/MS (Q-TOF)) measurements, see ESI†. 10 In the absence of Sc(OTf)3 (2), GOx-CM cannot be immobilised onto the silica surface.
This journal is
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Published on 09 October 2013. Downloaded by University of California - Riverside on 31/10/2014 01:47:53.
COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 11170 Received 13th September 2013, Accepted 9th October 2013
View Journal | View Issue
A method for highly efficient catalytic immobilisation of glucose oxidase on the surface of silica† Yong-Kyun Sim, Jung-Woo Park, Bo-Hyeong Kim and Chul-Ho Jun*
DOI: 10.1039/c3cc47011c www.rsc.org/chemcomm
A simple, mild and convenient method has been developed for catalytic immobilisation of glucose oxidase (GOx), chemically modified to contain pendant methallylsilyl groups, on an untreated silica surface.
Development of new and efficient enzyme immobilization methods and their applications are very important in industrial use since heterogenized enzymes can be readily recovered for repeated use and sometimes show high enzymatic activities with enhanced stability compared with homogenous ones.1 Among them, heterogenisation of native enzymes via covalent bonding is an important tool in modern chemical and biological investigations because the strong bonds generated in this process prevent or reduce enzyme desorption. Common methods for covalent attachment of enzymes to solid supports, such as silica, employ reactions of native enzymes with functional group containing solid supports (e.g., NHS-esters, aldehydes, and epoxides) (Fig. 1a).1,2 Another method for covalent immobilisation of enzymes utilizes reactions of chemically functionalized enzymes with functional groups present on solid supports.3 Nearly all of these protocols require the use of solid supports that contain reactive functional groups, which are generated by reactions of the supports with large excesses of silane coupling reagents at high temperatures.1,4 Silica supports prepared in this manner contain only a small number of reactive functional groups in comparison to the large number of hydroxyl groups that are present on native silica. As a result, protocols that utilize non-modified silica should lead to higher degrees of enzyme immobilisation. We envisioned that an ideal strategy for this purpose would involve the use of direct catalytic immobilisation of an appropriately chemically modified (CM) enzyme. Enzymes containing pendant methallylsilyl groups, covalently linked through amide bonds with lysine amine groups, would be useful for this purpose because they should be capable of undergoing mild, acid-catalysed reactions with hydroxyl groups present on the silica surface.5,6
Department of Chemistry, Yonsei University, 50 Yonseiro, Seodaemun-gu, Seoul 120-749, Korea. E-mail: [email protected]; Fax: +82-2-3147-2644; Tel: +82-2-2123-2627 † Electronic supplementary information (ESI) available: Experimental details and characterization data of compounds. See DOI: 10.1039/c3cc47011c
11170
Chem. Commun., 2013, 49, 11170--11172
Fig. 1 Comparison of previous and the new approaches to covalent immobilisation of enzymes. (a) Conventional immobilisation using surface modified functional groups. (b) The new approach employing reaction of chemically modified GOx with non-treated silica.
The overall approach would involve initial chemical modification of an enzyme through amide bond forming reaction with the NHS-ester-functionalized methallylsilane derivative 1 (Fig. 1b). Next, immobilisation of the chemically modified GOx on nonmodified silica would be performed under mild, Lewis acidcatalysed conditions. Observations made in the study described below show that an important advantage of the new protocol is This journal is
c
The Royal Society of Chemistry 2013
View Article Online
Published on 09 October 2013. Downloaded by University of California - Riverside on 31/10/2014 01:47:53.
Communication that the methallylsilane coupling agent is more easily prepared and purified than the related alkoxysilanes employed earlier for this purpose. Immobilisation reactions of enzymes that are chemically modified with methallylsilane groups take place at much lower temperatures than those required for analogous reactions of their alkoxysilane counterparts, thus avoiding the possibility of enzyme deactivation. In addition, the reaction can be carried out in one pot-sequential process without isolation or purification, which makes the reaction simple and convenient. Owing to the ease with which it can be assayed, GOx was chosen as a model enzyme for the initial immobilisation studies.7 Specifically, the activity of this enzyme is readily determined by measuring the amount of H2O2 generated from the reaction of O2 with glucose. In addition, the degree of GOx immobilisation on silica was determined by using the Bradford assay. To explore the new immobilisation protocol, chemically modified GOx was prepared by amide bond forming reaction of amine residues in lysines of GOx with NHS-ester-functionalized methallylsilane 1 (Fig. 2). Reactions with varying amounts of NHS-ester-functionalized methallylsilane 1 (16, 32, 48 and 64 equiv.) in H2O at 0 1C for 2 h led to formation of the respective modified GOxs, GOx-CM(16), GOx-CM(32), GOx-CM(48) and GOx-CM(64) (Fig. 2a).9 Analysis of these modified enzymes by using MALDI-TOF showed that the use of 48 equiv. of 1 in this process is optimal (Fig. 2b), giving a GOx that contains ca. 9.3 equiv. of dimethallylsilyl groups (Fig. 2c). Importantly, use
Fig. 2 (a) Chemical modification of GOx with NHS-ester functionalized methallylsilane 1. (b) MALDI-TOF analysis of GOx and chemically modified GOxs. (c) Conversion of lysine amine groups. (d) Comparison of activities of native GOx with that of the chemically modified GOx-CM(48) formed using 48 equiv. of 1.
This journal is
c
The Royal Society of Chemistry 2013
ChemComm of a large excess of 1 in the reaction did not lead to an increased loading of methallylsilane. In addition, the enzymatic activities of the modified GOxs are not dramatically lowered by introduction of methallylsilane groups. For example, GOx-CM(48) was observed to have 80% of the activity of native GOx (Fig. 2d). Immobilisation reactions of the GOx-CMs with silica8 were carried out in the presence of 10 mol% of Sc(OTf)3 (2, based on methallylsilyl groups) (Fig. 3a). These processes produced GOx-CM(16)@Si, GOx-CM(32)@Si, GOx-CM(48)@Si and GOxCM(64)@Si. The developed GOx grafting method, in which GOx is treated with NHS-ester-functionalized silica, was utilized as a standard for comparison purposes. Specifically, the grafted silica GOx-Graft@Si was prepared through reaction of GOx with NHS-ester-grafted silica (NHS-grafted silica), which was obtained by acid catalyzed reaction of NHS-ester-functionalized methallylsilane 1 with silica (Fig. 3b).5e,8 The results of measurements with the immobilised enzymes show that the catalytic activity (76 mM [H2O2] using 1.56 10 2 mg sample) and the degree of GOx loading (114 mg GOx per mg silica) of GOx-CM(48)@Si are much higher than those of GOx-CM(16)@Si (10 mM [H2O2]; 65 mg GOx per mg silica), GOx-CM(32)@Si (17 mM [H2O2]; 81 mg GOx per mg silica), and GOx-CM(64)@Si (59 mM [H2O2]; 73 mg GOx per mg silica) (Fig. 3c and d).10 These results demonstrate the importance of selecting the amount
Fig. 3 Schematic for the preparation of (a) GOx-CM(X)@Si through immobilisation of chemically modified glucose oxidase (GOx-CM(X)) onto non-modified silica (X = 16, 32, 48 and 64) and (b) GOx-Graft@Si through immobilisation of GOx onto NHS-ester-grafted silica. Comparisons of (c) enzyme activities and (d) enzyme loadings between GOx-CM(X)@Si and GOx-Graft@Si.
Chem. Commun., 2013, 49, 11170--11172
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Published on 09 October 2013. Downloaded by University of California - Riverside on 31/10/2014 01:47:53.
ChemComm of 1 in the enzyme functionalization process. The observation that the enzyme loading of GOx-CM(64)@Si is lower than that of GOx-CM(48)@Si is interesting in that it suggests that less efficient immobilisation of the silica surface takes place when unreacted 1 is present. Based on this finding, we conclude that use of 48 equiv. of 1 is ideal for the immobilisation reaction forming the immobilised GOx-CM. The results of a comparison study show that all GOx-CM@Sis have higher loading levels and catalytic activities than those of GOx-Graft@Si (activity ca. 4 mM [H2O2], see Fig. 3c; enzyme loading 21 mg GOx per mg silica, see Fig. 3d). In addition, it is noteworthy that use of the new immobilisation method involving chemical modification of GOx requires a much lower quantity of coupling reagent 1 (1.03 mg with 20 mg silica to form GOx-CM(48)@Si) than the corresponding reagent employed in conventional immobilisation of GOx (20.2 mg of 1 with 20 mg silica to form GOx-Graft@Si) (for the experimental details, see ESI†). The study described above has led to the development of an efficient method for immobilisation of glucose oxidase (GOx) on non-modified silica that involves the use of a chemically modified GOx containing NHS-ester-functionalized methallylsilanes. The GOx-immobilised silica, prepared by utilizing this method along with a small amount of the coupling reagent, displays a high enzyme loading level and high enzymatic activity. Applications of this new strategy to immobilise other bioactive molecules or enzymes are currently under study. This study was supported by the Mid-career Research Program (Grant 2011-0016830) through NRF grant funded by the MEST.
Notes and references 1 For reviews: (a) S. Hudson, J. Cooney and E. Magner, Angew. Chem., ¨der, Int. Ed., 2008, 47, 8582; (b) P. Jonkheijm, D. Weinrich, H. Schro C. M. Niemeyer and H. Waldmann, Angew. Chem., Int. Ed., 2008, 47, 9618; (c) U. Hanefeld, L. Gardossi and E. Magner, Chem. Soc. Rev., 2009, 38, 453; (d) R. A. Sheldon, Adv. Synth. Catal., 2007, 349, 1289.
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Chem. Commun., 2013, 49, 11170--11172
Communication 2 (a) X. Zhang, R.-F. Guan, D.-Q. Wu and K.-Y. Chan, J. Mol. Catal. B: ¨, G. Lu, Y. Wang, Y. Guo, Y. Guo, Enzym., 2005, 33, 43; (b) Y. Lu Z. Zhang, Y. Wang and X. Liu, Adv. Funct. Mater., 2007, 17, 2160; (c) A. Schlossbauer, D. Schaffert, J. Kecht, E. Wagner and T. Bein, J. Am. Chem. Soc., 2008, 130, 12558; (d) S. Libertino, F. Giannazo, V. Aiello, A. Scandurra, F. Sinatra, M. Renis and M. Fichera, Langmuir, 2008, 24, 1965. ´. Berenguer-Murcia and 3 For a review: R. C. Rodrigues, A R. Fernandez-Lafuentec, Adv. Synth. Catal., 2011, 353, 2216. 4 (a) K. B. Yoon, Acc. Chem. Res., 2007, 40, 29, and references therein; ´, M. Boualleg, J.-M. Camus, T. K. Maishal, J. Alauzun, (b) I. Karame ´ret, R. J. P. Corriu, E. Jeanneau, A. Mehdi, C. Reye ´, J.-M. Basset, C. Cope L. Veyre and C. Thieuleux, Chem.–Eur. J., 2009, 15, 11820; (c) F. Hoffmann, ¨ba, Angew. Chem., Int. Ed., 2006, M. Cornelius, J. Morell and M. Fro ´n ˜ez, F. Sanceno ´n, 45, 3216; (d) A. B. Descalzo, R. Martı´nez-Ma K. Hoffmann and K. Rurack, Angew. Chem., Int. Ed., 2006, 45, 5924; (e) G. J. A. A. Soler-Illia and P. Innocenzi, Chem.–Eur. J., 2006, 12, 4478; ( f ) A. Corma and H. Garcia, Adv. Synth. Catal., 2006, 348, 1391; (g) C. M. Crudden, M. Sateesh and R. Lewis, J. Am. Chem. Soc., 2005, 127, 10045. 5 (a) Y.-R. Yeon, Y. J. Park, J.-S. Lee, J.-W. Park, S.-G. Kang and C.-H. Jun, Angew. Chem., Int. Ed., 2008, 47, 109; (b) J.-W. Park, Y. J. Park and C.-H. Jun, Chem. Commun., 2011, 47, 4860; (c) D. H. Lee, E.-A. Jo, J.-W. Park and C.-H. Jun, Tetrahedron Lett., 2010, 51, 160; (d) S. Park, J. Pai, E.-H. Han, C.-H. Jun and I. Shin, Bioconjugate Chem., 2010, 21, 1246; (e) U.-Y. Jung, J.-W. Park, E.-H. Han, S.-G. Kang, S. Lee and C.-H. Jun, Chem.–Asian J., 2011, 6, 638. 6 For immobilisation of organic functional groups onto silica using stable alkenylsilanes, see: (a) J.-W. Park and C.-H. Jun, J. Am. Chem. Soc., 2010, 132, 7268; (b) T. Shimada, K. Aoki, Y. Shinoda, T. Nakamura, N. Tokunaga, S. Inagaki and T. Hayashi, J. Am. Chem. Soc., 2003, 125, 4688; (c) K. Aoki, T. Shimada and T. Hayashi, Tetrahedron: Asymmetry, 2004, 15, 1771; (d) Y. Maegawa, T. Nagano, T. Yabuno, H. Nakagawa and T. Shimada, Tetrahedron, 2007, 63, 11467; (e) Y. Wang, S. Hu and W. J. Brittain, Macromolecules, 2006, 39, 5675; ( f ) G. Hreczycho, M. K. Chmielewski, H. Maciejewski, T. Ratajczak and B. Marciniec, Tetrahedron Lett., 2013, 54, 3605. 7 Glucose oxidase was purchased from Aldrich. Co., and used after purification by gel-filtration. 8 Large pore sized silica balls were used as solid supports. The specification of silica is as follows; size: 10 mm, pore size: 30 nm; which was purchased from Fuji Silysia Ltd. 9 The amounts of 1 correspond to 1, 2, 3 and 4 equivalents, respectively, based on the number of lysine groups in GOx (16 determined by LC-MS/MS (Q-TOF)) measurements, see ESI†. 10 In the absence of Sc(OTf)3 (2), GOx-CM cannot be immobilised onto the silica surface.
This journal is
c
The Royal Society of Chemistry 2013
