C O M M U N I C A T I O N S
Figure 3. Confirmation of reaction chemoselectivity using fluorescent
probes. (a) Samples were prepared by mixing lysozyme and chymotrypsi-
nogen A. In some cases, these proteins had been labeled with anilines using
reagent 10 (these samples are boxed in red in graphic b). The protein
mixtures were then combined with NaIO4 and fluorescent rhodamines 14a
or 14b. (b) SDS-PAGE, followed by visualization with fluorescence imaging
Figure 4. Attachment of a PEG polymer to eGFP using the oxidative
coupling strategy. (a) A single aniline group was introduced through a native
chemical ligation with 16. (b) Following exposure to 18 and NaIO4, a single
PEG conjugate (indicated by the arrow) could be seen for 17. The upper
bands in the gel arise because of protein aggregation and are independent
of the reaction conditions.
(bottom) and Coomassie staining (top), indicating the samples in which
successful labeling had occurred.
minimize sulfur oxidation.13 To date, these approaches have not
been successful with our aniline coupling strategy. We are currently
evaluating alternative oxidants to minimize these side reactions.14
Similar to the small molecule studies, a small amount (3-8%) of
protein crosslinking was detected in some instances.
The fluorescent labeling of uniquely functionalized proteins has
proven useful for reactive profiling studies.15 To explore the
feasibility of the oxidative coupling reaction for this purpose,
samples of lysozyme (6) and chymotrypsinogen A (12) were labeled
with NHS-ester 10, Figure 3a. Mixtures of the labeled and unlabeled
proteins were then exposed to fluorescent dialkylphenylenediamine
Supporting Information Available: Experimental procedures and
characterization data for all intermediates. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
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(
(
2) Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256-10257.
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M. B. J. Am. Chem. Soc. 2004, 126, 15942-15943. (f) Tilley, S. D.;
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1
4a (250 µM) and NaIO
that only the aniline-bearing proteins participated in the reaction
Figure 3b, lanes 4-6). Additional control experiments confirmed
4
(1.5 mM). SDS-PAGE analysis indicated
(
(
(
(
4) For general reviews, see (a) Wang, L.; Schultz, P. G. Angew. Chem., Int.
Ed. 2005, 44, 34-66. (b) Link, A. J.; Mock, M. L.; Tirrell, D. A. Curr.
Opin. Biotech. 2003, 14, 603-609.
that no reaction occurred in the absence of the oxidant (lane 1) or
aniline component (lanes 2-3). To test the stability of the reaction
product, a labeled sample of lysozyme was subjected to a series of
conditions for 24 h and then analyzed. No losses in fluorescence
were observed from pH 4-10, or in the presence of NaIO ,
4
reductants such as glutathione and dithionite, or nucleophilic
reagents such as hydrazine or benzyloxyamine.11
5) (a) Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1996,
1
18, 8150-8151. (b) Zhang, Z. W.; Smith, B. A. C.; Wang, L.; Brock,
A.; Cho, C.; Schultz, P. G. Biochemistry 2003, 42, 6735-6746.
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Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. (b) Sen Gupta,
S.; Kuzelka, J.; Singh, P.; Lewis, W. G.; Manchester, M.; Finn, M. G.
Bioconj. Chem. 2005, 16, 1572-1579. (c) Deiters, A.; Cropp, T. A.;
Mukherji, M.; Chin, J. W.; Anderson, J. C.; Schultz, P. G. J. Am. Chem.
Soc. 2003, 125, 11782-11783. (d) Link, A. J.; Tirrell, D. A. J. Am. Chem.
Soc. 2003, 125, 11164-11165. (e) Agard, N. J.; Prescher, J. A.; Bertozzi,
C. R. J. Am. Chem. Soc. 2004, 126, 15046-15047.
7) (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010. (b) Kiick,
K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Nat. Acad. Sci.
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As a convenient way to confirm the overall conversion of the
reaction, a native chemical ligation strategy16 was used to install a
single aniline at the C-terminus of the green fluorescent protein
(
6b,17
(
A206K eGFP) according to Figure 4.1
Upon exposure of a 50
µM solution of the aniline-labeled protein (17) to fluorescent probe
4a (250 µM) and NaIO (1 mM), 82% conversion was obtained
in 2 h, as measured by UV-vis analysis. When exposed to poly-
ethylene glycol)-substituted phenylenediamine derivative 18, 45%
conversion to the singly labeled protein conjugate was observed
1
4
11
(8) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004-2021.
(
(9) Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.;
King, D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125,
935-939.
(determined by optical densitometry after staining) (Figure 4b).
(10) (a) Bandrowski, E. Chem. Ber. 1894, 27, 480. (b) Ritter, J. J.; Schmitz,
G. H. J. Am. Chem. Soc. 1929, 51, 1587-1589. (c) Corbett, J. F. J. Chem.
Soc. B 1969, 818. (d) Blake, A. J.; Hubberstey, P.; Quinlan, D. J. Acta
Crystallogr., Sect. C: Cryst. Struct. Comm. 1996, 52, 1774-1776.
(11) See Supporting Information for mechanisms, spectra, and details.
In summary, we have developed an efficient protein modification
reaction that targets a bioorthogonal functional group under mild
reaction conditions. This strategy features excellent chemo-
selecitvity, rapid coupling rates in buffered aqueous solution, and
product stability over a wide range of conditions. Alternative
activation strategies and coupling partners are under development,
as are studies to determine the specific mechanisms by which these
reactions proceed.
(
12) We have found that the phenylenediamine group can be attached to the
protein and coupled to aniline reagents with the same chemoselectivity.
(13) For examples see (a) Chen, J. H.; Zeng, W. G.; Offord, R.; Rose, K.
Bioconjugate Chem. 2003, 14, 614-618. (b) Zhang, L. S.; Tam, J. P.
Anal. Biochem. 1996, 233, 87-93.
4 2 3 6
(14) We have already found that (NH ) Ce(NO ) can be used with similar
success, allowing improved compatibility with glycoprotein substrates.
(
15) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125,
4686-4687.
Acknowledgment. We gratefully acknowledge the Lawrence
Berkeley National Laboratory, Materials Sciences Division (U.S.
DOE Contract No. DE-AC03-76SF00098), the NIH (Grant
GM072700-01), and the Berkeley Chemical Biology Graduate
Program (NRSA Training Grant 1 T32 GMO66698) for financial
support. J.M.H. was supported by an NSF predoctoral fellowship.
(
16) (a) Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science
1994, 266, 776-779. (b) Tolbert, T. J.; Wong, C. H. J. Am. Chem. Soc.
2
000, 122, 5421-5428.
(
17) (a) Tsien, Y. Annu. ReV. Biochem. 1998, 67, 509-544. (b) Zhang, J.;
Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol. Cell Biol. 2002,
3
, 906-918.
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