1060 Bioconjugate Chem., Vol. 21, No. 6, 2010
Moth-Poulsen et al.
(12) Gemeiner, P., and Breier, A. (1982) Aldehydic derivatives of
bead cellulose-relationships between the matrix structure and
function in immobilization of enzymes catalyzing hydrolysis of
high molecular substrates. Biotechnol. Bioeng. 24, 2573–82.
(13) Falsey, J. R., Renil, M., Park, S., Li, S. J., and Lam, K. S.
(2001) Peptide and small molecule microarray for high through-
put cell adhesion and functional assays. Bioconjugate Chem 12,
346–53.
(14) Olivier, C. (2003) Alpha-oxo semicarbazone peptide or
oligodeoxynucleotide microarrays. Bioconjugate Chem. 14, 430–
9.
(15) Groll, J., Amirgoulova, E. V., Ameringer, T., Heyes, C. D.,
Rocker, C., Nienhaus, G. U., and Moller, M. (2004) Biofunc-
tionalized, ultrathin coatings of cross-linked star-shaped poly-
(ethylene oxide) allow reversible folding of immobilized proteins.
J. Am. Chem. Soc. 126, 4234–4239.
auxiliary reagents or heavy metal ions are needed, and in contrast
to activated ester strategies such as NHS esters (10), the reactive
species used here, the Bni group is stable for long periods of
time (years of shelf-life time). As a proof of principle, we
successfully coupled proteins to gold surfaces. By careful
selection of the end group, Bni might also be employed to attach
protein or other nucleophile-containing molecules on silicon
surfaces or on polymers.
We foresee that this concept can, in a more general way, be
used both for the attachment of nucleophiles to surfaces and
for the subsequent release of active components by an external
stimuli in the form of light. Also, we expect that this photoactive
linker molecule will allow for a simple immobilization of arrays
of DNA or proteins on surfaces integrated in advanced devices
such as microfluidic channels.
(16) Schmid, E. L., Keller, T. A., Dienes, Z., and Vogel, H. (1997)
Reversible oriented surface immobilization of functional proteins
on oxide surfaces. Anal. Chem. 69, 1979–1985.
(17) Hodneland, C. D., Lee, Y. S., Min, D. H., and Mrksich, M.
(2002) Selective immobilization of proteins to self-assembled
monolayers presenting active site-directed capture ligands. Proc.
Natl. Acad. Sci. U.S.A. 99, 5048–5052.
(18) Muir, T. W., Sondhi, D., and Cole, P. A. (1998) Expressed
protein ligation: A general method for protein engineering. Proc.
Natl. Acad. Sci. U.S.A. 95, 6705–6710.
(19) Camarero, J. A., Kwon, Y., and Coleman, M. A. (2004)
Chemoselective attachment of biologically active proteins to
surfaces by expressed protein ligation and its application for
“protein chip” fabrication. J. Am. Chem. Soc. 126, 14730–14731.
(20) Mezzasoma, L., Bacarese-Hamilton, T., Di Cristina, M., Rossi,
R., Bistoni, F., and Crisanti, A. (2002) Antigen microarrays for
serodiagnosis of infectious diseases. Clin. Chem. 48, 121–130.
(21) Perler, F. B. (1999) A natural example of protein trans-
splicing. Trends Biochem. Sci. 26, 209–211.
(22) Sigal, G. B., Mrksich, M., and Whitesides, G. M. (1998) Effect
of surface wettability on the adsorption of proteins and detergents.
J. Am. Chem. Soc. 120, 3464–3473.
(23) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click
chemistry: Diverse chemical function from a few good reactions.
Angew. Chem., Int. Ed. 40, 2004–2021.
ACKNOWLEDGMENT
D.S. would like to thank the Danish Councils for Independent
and Strategic Research for funding. K.M.P. would like to thank
The Danish Council for Independent Research for funding.
Supporting Information Available: Information on the
purification of the protein conjugate and full NMR spectra of
the irradiated mixtures. This material is available free of charge
LITERATURE CITED
(1) Wong, S. L., Khan, F., and Micklefield, J. (2009) Selective
covalent protein immobilization: strategies and applications.
Chem. ReV. 109, 4025–4053.
(2) Camarero, J. A. (2008) Recent developments in the site-specific
immobilization of proteins onto solid supports. Biopolymers 90,
450–458.
(3) Hatzakis, N. S., Engelkamp, H., Velonia, K., Hofkens, J.,
Christianen, P. C. M., Svendsen, A., Patkar, S. A., Vind, J., Maan,
J. C., Rowan, A. E., and Nolte, R. J. M. (2006) Synthesis and
single enzyme activity of a clicked lipase-BSA hetero-dimer.
Chem. Commun. 19, 2012.
(4) English, B. P., Min, W., van Oijen, A. M., Lee, K. T., Luo,
G. B., Sun, H. Y., Cherayil, B. J., Kou, S. C., and Xie, X. S.
(2006) Ever-fluctuating single enzyme molecules: Michaelis-
Menten equation revisited. Nat. Chem. Biol. 2 (2), 87–94.
(5) Wu, G., Datar, R. H., Hansen, K. M., Thundat, T., Cote, R. J.,
and Majumdar, A. (2001) Bioassay of prostate-specific antigen
(PSA) using microcantilevers. Nat. Biotechnol. 19, 856–860.
(6) MacBeath, G., and Schreiber, S. L. (2000) Printing proteins as
microarrays for high-throughput function determination. Science
289, 54851760-1763.
(7) Kuznetsova, S., Zauner, G., Aartsma, T. J., Engelkamp, H.,
Hatzakis, N., Rowan, A. E., Nolte, R. J. M., Christianen,
P. C. M., and Canters, G. W. (2008) The enzyme mechanism of
nitrite reductase studied at single-molecule level. Proc. Natl.
Acad. Sci. U.S.A. 105, 3250–3255.
(8) Camarero, J. A. (2008) Recent developments in the site-specific
immobilization of proteins onto solid supports. Biopolymers 90,
450–458.
(9) Hermanson, G. T. (2008) Bioconjugate Techniques, 2nd ed.,
Academic Press, London.
(10) Tourniera, E. J. M., Wallach, J., and Blonda, P. (1998)
Sulfosuccinimidyl 4-(N-maleimidomethyl)-1-cyclohexane car-
boxylate as a bifunctional immobilization agentOptimization of
the coupling conditions. Anal. Chim. Acta 361, 33–44.
(11) Mateo, C., Torres, R., Fernandez-Lorente, G., Ortiz, C.,
Fuentes, M., Hidalgo, A., Lopez-Gallego, F., Abian, O., Palomo,
J. M., Betancor, L., Pessela, B. C. C., Guisan, J. M., and
Fernandez-Lafuente, R. (2003) Epoxy-amino groups: A new tool
for improved immobilization of proteins by the epoxy method.
Biomacromolecules 4, 772–777.
(24) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J.,
Chang, P. V., Miller, I. A., Anderson, L., Codelli, J. A., and
Bertozzi, C. R. (2007) Copper-free click chemistry for dynamic
in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 104, 16793–16797.
(25) Tornøe, C. W., Christensen, C., and Meldal, M. (2002)
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific
copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes
to azides. J. Org. Chem. 67, 3057–3064.
(26) Brennan, J. L., Hatzakis, N. S., Tshikhudo, T. R., Dirvianskyte,
N., Razumas, V., Patkar, S., Vind, J., Svendsen, A., Nolte,
R. J. M., Rowan, A. E., and Brust, M. (2006) Bionanoconjugation
via click chemistry: The creation of functional hybrids of lipases
and gold nanoparticles. Bioconjugate Chem. 17, 1373–1375.
(27) Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless,
B. K., and Finn, M. G. (2003) Bioconjugation by copper(I)-
catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc.
125, 3192–3193.
(28) Amit, B., Ben-Efraim, D. A., and Patchornik, A. (1976) Light-
sensitive amides. the photosolvolysis of substituted 1-acyl-7-
nitroindolines. J. Am. Chem. Soc. 98, 843–844.
(29) Nicolaou, K. C., Safina, B. S., and Winssinger, N. (2001) A
new photolabile linker for the photoactivation of carboxyl groups.
Synlett 2001, 900.
(30) Pass, S., Amit, B., and Patchornik, A. (1981) Racemization-
free photochemical coupling of peptide segments. J. Am. Chem.
Soc. 103, 7674–7675.
(31) Papageorgiou, G., and Corrie, J. E. T. (2000) Effects of
aromatic substituents on the photocleavage of 1-acyl-7-nitroin-
dolines. Tetrahedron 56, 8197–8205.