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Fig. 5 Di-scFv (bottom panels) was tested on sections of human breast
(MCF7) and prostate tissue (DU145) by IHC. The brown membrane
staining can be easily detected on the cells with scFv and di-scFv proteins,
and increased binding was observed for di-scFv (bottom panels) relative to
scFv (top panels). Panel A: scFv bound to MCF7. Panel D: Di-scFv
bound to MCF7. Panel B: scFv bound to DU145. Panel E: Di-scFv bound
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demonstrated that the di-scFv had 2–4 times increased binding to
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The multivalent 1,3-dipolar cycloaddition strategy described
herein provides a general approach for modular design applica-
tions. Azide–alkyne 1,3-dipolar cycloaddition has been established
as an effective chemistry for the covalent modification of
biomolecules such as proteins,13,14 DNA,15 carbohydrates,16 virus
particles11,17 and bacterial surfaces.18 In virtually all of these cases,
a large macromolecule and a small molecule were ‘‘clicked’’
together. Since small molecules can freely diffuse, a critical reactant
concentration can be achieved, and the reaction proceeds
efficiently. As far as we are aware, azide–alkyne 1,3-dipolar
cycloaddition has not been successfully applied to the ligation of
two large macromolecules, each possessing a single reaction site."
Here we show that the efficiency of such a reaction is limited by
macromolecular reaction dynamics, which can be overcome by the
site-specific introduction of a polyfunctional small molecule.
This work was supported by National Cancer Institute NCI
grant PO1-CA47829 and National Science Foundation CHE-
0196482. The NSF CRIF program (CHE-9808183), NSF grant
OSTI 97-24412 and NIH grant RR11973 provided funding for the
NMR spectrometers used in this project.
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Notes and references
" Finn and co-workers have ligated large proteins onto viral particles, but
in this case, the virus has several reactive sites and approximately 80%
efficiency was observed. See ref. 17.
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This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 695–697 | 697