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bioconjugates through other thiol-mediated coupling reac-
tions. Further investigations in this direction are currently
underway.
Acknowledgements
The authors gratefully acknowledge the Agence Nationale de la
Recherche et de la Technologie for nancial support to Barbara
Maret.
Scheme 5 Side-products formed by Michael addition of sulphite and
TCEP on maleimide 3.
Notes and references
1 J.-C. Rossi, L. Boiteau, H. Collet, B. M. Tsamba, N. Larcher
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2 Along with the p-methoxybenzyl thioether and S-2-
trimethylsilylethyl protecting groups, see: P. G. M. Wuts
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3 Y. Sohma and S. B. H. Kent, J. Am. Chem. Soc., 2009, 131,
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Fig. 3 (A) Size exclusion chromatograms for the Michael addition of
2c to mal-HRP. Column: Superdex 200 10/300GL; eluent: 5 mM
phosphate buffer and 150 mM NaCl pH 7.4, flow 0.5 mL minꢁ1; UV
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¨
6 J. Lukkari, M. Meretoja, I. Kartio, K. Laajalehto, M. Rajamaki,
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¨
M. Lindstrom and J. Kankare, Langmuir, 1999, 15, 3529–
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¼
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9 J. March, Advanced Organic Chemistry, Wiley, New York, 4th
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153 ꢂ 6, 80 mg mLꢁ1, 5 mM phosphate buffer pH 7.4 and
150 mM NaCl, lexc 485 nm, lem 535 nm). We also veried that
the enzyme integrity was neither altered by the presence of the
covalently attached uorescein, nor by the deprotection and
conjugation reaction conditions (Fig. 3b) through monitoring
the HRP-catalysed oxidation of o-phenylenediamine by UV
spectroscopy according to a previously reported method.19 In
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products shows that the separation of sulphite and excess TCEP 10 J. L. Kice, J. M. Anderson and N. E. Pawlowski, J. Am. Chem.
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D. J. Braydenc and D. M. Haddleton, Chem. Commun.,
ˆ
In summary, we have demonstrated that the S-sulphonate
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´
addition reaction and with the use of biomolecules. This 13 G. Miralles, P. Verdie, K. Puget, A. Maurras, J. Martinez and
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