Journal of the American Chemical Society
Communication
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Prof. Dr. Donald Hilvert and Dr. An
Vandemeulebroucke (ETH Zurich) for fruitful discussions and
Dr. Thomas Schweizer (ETH Zurich) for GPC measurements.
Moreover, the help from the following undergraduate students is
highly appreciated: Darija Dedic, Karoline Herget, and Juri Regli.
■
̈
̈
REFERENCES
■
(1) Broyer, R. M.; Grover, G. N.; Maynard, H. D. Chem. Commun.
2011, 47, 2212.
(2) Klok, H. A. Macromolecules 2009, 42, 7990.
(3) Lutz, J. F.; Borner, H. G. Prog. Polym. Sci. 2008, 33, 1.
̈
(4) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922.
(5) Grover, G. N.; Maynard, H. D. Curr. Opin. Chem. Biol. 2010, 14,
818.
Figure 4. Time-course measurements of the two-step enzymatic cascade
reaction: comparison of (1) free and (2) polymer-bound SOD and
HRP. O2•− was produced by pulse radiolysis, formation of ABTS•− was
followed spectrophotometrically at λ= 415 nm; for calculation of the
ABTS•− yield, see SI. Conditions: [ABTS2−]0 = 0.25 mM, [HRP] = 0.4
μM, [SOD] = 0.2 μM, 100 mM phosphate buffer pH 7.2, 25 °C.
(6) Gauthier, M. A.; Klok, H. A. Chem. Commun. 2008, 2591.
(7) Heredia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007, 5, 45.
(8) Gauthier, M. A.; Klok, H. A. Polym. Chem. 2010, 1, 1352.
(9) Duncan, R. Curr. Opin. Biotechnol. 2011, 22, 492.
(10) Veronese, F. M.; Mero, A.; Pasut, G. Protein PEGylation, basic
science and biological applications; Birkhauser Verlag AG: Basel,
Switzerland, 2009.
(11) Zhu, G.; Wang, P. Nanoscale Biocatalysis: Methods and Protocols;
Humana Press Inc.: Totowa, NJ, 2011; Vol. 743, p 27.
(12) Sheldon, R. A. Adv. Synth. Catal. 2007, 349, 1289.
(13) Schoffelen, S.; van Hest, J. C. M. Soft Matter 2012, 8, 1736.
(14) Pereira de Souza, T.; Stano, P.; Luisi, P. L. ChemBioChem 2009,
10, 1056.
copies of the same protein,16,21−23 this is, to the best of our
knowledge, the first report on the preparation of a polymer−
enzyme conjugate containing multiple copies of two different types
of enzymes on one and the same polymer chain. The conjugation
reaction could be quantified spectrophotometrically and
occurred under mild conditions and therefore under complete
retention of the catalytic activity of the two enzymes. The reason
why the two enzymes could be linked in two consecutive steps to
the dendronized polymer by using the same conjugation
chemistry is probably due to the different reaction behaviors of
the two enzymes (see SI). Reaction of de-PG12000-BAH-(FL)-
HyNic with HRP-4FB resulted in a polymer−HRP conjugate
which still had reactive HyNic groups that could not react with
HRP-4FB but reacted with the added SOD-4FB instead. This
behavior is probably related to the smaller size of SOD as
compared to HRP (radii of gyration: RG(HRP) = 2.65 nm,24
RG(SOD) = 1.82 nm)25 and to the differences in the isoelectric
points, pI(HRP isoenzyme C) = 9.026 and pI(SOD) = 4.95.27
Whether this methodology is applicable to other polymer−
enzyme systems is currently under investigation. Furthermore,
we are elaborating experimental conditions for a localized
enzymatic cascade reaction, i.e. encapsulated inside lipid vesicles
or immobilized on solid surfaces, for mechanistic studies and
bioanalytical applications.28−30 Our findings open the fascinating
opportunity to eventually perform cascade reactions on single
hybrid molecules in confined geometries.
(15) Guo, Y. F.; van Beek, J. D.; Zhang, B. Z.; Colussi, M.; Walde, P.;
Zhang, A.; Kroger, M.; Halperin, A.; Schluter, A. D. J. Am. Chem. Soc.
̈
̈
2009, 131, 11841.
(16) Grotzky, A.; Manaka, Y.; Kojima, T.; Walde, P. Biomacromolecules
2011, 12, 134.
(17) Solulink Company, San Diego, USA, Catalog & Reference Manual,
2008.
(18) Childs, R. E.; Bardsley, W. G. Biochem. J. 1975, 145, 93.
(19) Michel, E.; Nauser, T.; Sutter, B.; Bounds, P. L.; Koppenol, W. H.
Arch. Biochem. Biophys. 2005, 439, 234.
(20) Heredia, K. L.; Grover, G. N.; Tao, L.; Maynard, H. D.
Macromolecules 2009, 42, 2360.
(21) Tao, L.; Kaddis, C. S.; Loo, R. R. O.; Grover, G. N.; Loo, J. A.;
Maynard, H. D. Macromolecules 2009, 42, 8028.
(22) Chen, J. P.; Yang, H. J.; Hoffman, A. S. Biomaterials 1990, 11, 625.
(23) Altikatoglu, M.; Arioz, C.; Basaran, Y.; Kuzu, H. Cent. Eur. J. Chem
2009, 7, 423.
(24) Laberge, M.; Huang, Q.; Schweitzer-Stenner, R.; Fidy, J. Biophys.
J. 2003, 84, 2542.
(25) Martel, P.; Powell, B. M.; Zepp Johnston, R. A.; Petkau, A.
Biochim. Biophys. Acta 1983, 747, 78.
(26) Aibara, S.; Yamashita, H.; Mori, E.; Kato, M.; Morita, Y. J. Biochem.
1982, 92, 531.
(27) Bannister, J.; Bannister, W.; Wood, E. Eur. J. Biochem. 1971, 18,
178.
(28) Gorris, H. H.; Walt, D. R. J. Am. Chem. Soc. 2009, 131, 6277.
(29) Gorris, H. H.; Walt, D. R. Angew. Chem., Int. Ed. 2010, 49, 3880.
(30) Logan, T. C.; Clark, D. S.; Stachowiak, T. B.; Svec, F.; Frechet, J.
M. J. Anal. Chem. 2007, 79, 6592.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental protocols including polymer, linker and
conjugate synthesis as well as enzymatic activity measurements
for SOD and HRP, separately and together using pulse radiolysis,
and additional UV/vis-spectra. This material is available free of
AUTHOR INFORMATION
Corresponding Author
■
11395
dx.doi.org/10.1021/ja304837f | J. Am. Chem. Soc. 2012, 134, 11392−11395