Bioconjugation by native chemical tagging of C-H bonds

Article abstract of DOI:10.1021/ja407739y

A general C-H functionalization method for the tagging of natural products and pharmaceuticals is described. An azide-containing sulfinate reagent allows the appendage of azidoalkyl chains onto heteroaromatics, the product of which can then be attached to a monoclonal antibody by a click reaction. This strategy expands the breadth of bioactive small molecules that can be linked to macromolecules in a manner that is beyond the scope of existing methods in bioconjugation to permit tagging of the seemingly untaggable .

Full text of DOI:10.1021/ja407739y

Communication
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Bioconjugation by Native Chemical Tagging of C−H Bonds
Qianghui Zhou,† Jinghan Gui,† Chung-Mao Pan,† Earl Albone,‡ Xin Cheng,‡ Edward M. Suh,§
Luigi Grasso,‡ Yoshihiro Ishihara,† and Phil S. Baran*,†
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Morphotek, Inc., 210 Welsh Pool Road, Exton, Pennsylvania 19341, United States
§Next Generation Systems, Eisai, Inc., 4 Corporate Drive, Andover, Massachusetts 01810, United States
S
* Supporting Information
ABSTRACT: A general C−H functionalization method
for the tagging of natural products and pharmaceuticals is
described. An azide-containing sulfinate reagent allows the
appendage of azidoalkyl chains onto heteroaromatics, the
product of which can then be attached to a monoclonal
antibody by a “click” reaction. This strategy expands the
breadth of bioactive small molecules that can be linked to
macromolecules in a manner that is beyond the scope of
existing methods in bioconjugation to permit tagging of
the “seemingly untaggable”.
etermining the mode of action of medicinal compounds
Dby bioconjugation is an essential tool in chemical
biology.1 Bioconjugation requires a covalent and/or non-
covalent linkage of the small-molecule drug with macro-
molecular tags including antibodies, nucleic acids, and receptor-
binding proteins.2 This process of derivatization often involves
“click” chemistry,3 with the most popular method arguably
being the azide−alkyne cycloaddition:4 a functional group on
the lead compound is attached to a linker with an alkyne (or
alternatively, an azide), which is then reacted with a
macromolecular tag with a pendant azide (or alternatively, an
alkyne). Typically, only conventional functional groups can be
tagged by linkers with “clickable” units (Figure 1A),5a−e but in a
recent elegant example, Romo’s group has functionalized
activated C−H bonds (allylic C−H, benzylic C−H, and C−H
α to a heteroatom) to tag natural products.5f Although many
medicinal agents contain traditionally “taggable” functional
groups such as heteroatom−H bonds5a−c and π bonds,5e some
Figure 1. (A) Literature precedent involving the tagging of
compounds [such as camptothecin (1) and buspirone (2)]
conventional functional groups. (B) Identification of a challenge: the
present the challenge of not having any apparent chemical
handles (Figure 1B). Herein, we show the invention of a
reagent that enables the tagging of unactivated C−H bonds in
bioactive heteroarenes for use in bioconjugation. This powerful
native chemical tagging technique takes place in water and in
the absence of protecting groups.
Our laboratory has been interested in assembling a toolkit of
(fluoro)alkanesulfinate reagents for heteroaromatic systems.6
Such reagents form radicals in situ via an oxidative process,
resulting in a formal C−H functionalization. For the purpose of
bioconjugation, an alkyl linker bearing an azide moiety was
desired. Considering the mild reaction conditions involved in
this chemistry, it was conjectured that the relatively sensitive
azide would survive the heterocycle functionalization reaction.
tagging of unactivated C−H bonds. (C) Designing an azide-containing
fluoroalkylsulfinate salt that converts a heteroaromatic C−H bond into
a linker for bioconjugation.
Thus, a sulfinate reagent bearing an azide moiety, sodium
(difluoroalkylazido)sulfinate (DAAS-Na; 3), was designed
(Figure 1C). The synthesis, reactivity, and application of this
reagent (which has been commercialized through Sigma-
Aldrich) are described below.
Received: July 27, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja407739y | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
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Figure 2. (A) Synthesis and general reaction of DAAS-Na (3). (B) Formal C−H functionalization of complex natural products. (C) Formal C−H
a
functionalization of non-natural, biologically active agents that contain heteroaromatic moieties. RSM = recovered starting material. The resulting
product containing the azide linker has a carbonyl unit in lieu of the difluoromethyl group.
B
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Journal of the American Chemical Society
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Figure 3. (A) Appending a monoclonal antibody to an azide-containing bioactive agent. (B) Mass spectral data that verify the formation of drug−
a
antibody conjugates. The two indicated values refer to the mass spectral data arising from two different methods of conjugation (please see the
b
Supporting Information for details); the slight differences in mass between the two methods are deconvolution artifacts. The symbol §§ indicates
c
that the second method of bioconjugation was not conducted. The symbol ## indicates that the bioconjugation was unsuccessful.
The synthesis of this reagent begins with chlorinated
(fluoroalkylsulfonyl)pyridine 4, which was synthesized pre-
viously6c in three steps from commercially available 2-
mercaptopyridine (Figure 2A). Displacement of the chloride
with an azide, followed by removal of the pyridine using sodium
ethanethiolate, provided DAAS-Na (3) in 78% yield over two
steps. Sodium sulfinate reagent 3 was then used in a
heteroarene functionalization reaction that involves ZnCl2
camptothecin (1). While the tertiary alcohol in 1 is likely to be
too hindered for chemical derivatization, sulfinate 3 allows for
the radical functionalization of the quinoline moiety to give the
corresponding azido-containing product in 42% isolated yield.
Sceptrin (6), a cell motility inhibitor7 that can be accessed by
total synthesis,8 was also functionalized to give a single product
in 35% yield. (Notably, however, electron-rich heteroarenes
such as pyrroles, indoles, and even imidazoles facilitate the
hydrolysis of the gem-difluoro product, and therefore a ketone
product was obtained.) Papaverine (7), an antispasmodic agent,
has two sites of reactivity, giving rise to products in 16 and 17%
yields, respectively. Known pharmaceutical agents of varying
complexity can be equally functionalized using this method
(Figure 2C). A range of pyridine-containing drugs (8−16) can
be functionalized at their C2 and/or C4 positions, which are
the most susceptible sites for nucleophilic radical attack. Azine-
based pharmaceuticals (2, 17, and 18) and medicinal agents
containing other heterocyclic cores (19−22) were also
t
and TsOH·H2O as acid additives and BuOOH as an oxidant.
Although various organic solvents have been used previously
for sulfinate radical functionalizations,6b either CH2Cl2:H2O or
DMSO:H2O (2.5:1) was sufficient for the reaction of DAAS-Na
(3). These optimized reaction conditions were used throughout
the examination of the substrate scope.
The strength of this chemistry lies in the ability to append
azide-terminated linkers to heteroarene moieties in complex
natural products bearing sensitive functional groups (Figure
2B). A representative natural product example is depicted with
C
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Journal of the American Chemical Society
Communication
functionalized with DAAS-Na (3). It is notable that alcohol,
amine, amide, amidine, hydrazide, sulfonamide, and alkene
functional groups were tolerated in this reaction. The multiple
sites of C−H activation for many of these compounds allow the
exploration of structure−activity relationships for these
molecules as well as the opportunity to evaluate optimal linker
attachment with respect to bioactivity.
The main goal of this research program is realized by
appending macromolecular tags to these bioactive, small-
molecule agents. To this end, the synthesized azide-linked
medicinal agents were reacted with a dibenzylazacyclooctyne-
containing monoclonal antibody in a copper-free azide−alkyne
cycloaddition (Figure 3A).9 Two methods of bioconjugation
were employed, which we refer to as the conventional and high-
throughput methods (see Supporting Information for details).
The generated drug−antibody conjugates were verified by mass
spectrometry, confirming the feasibility of this C−H function-
alization approach to bioconjugation (Figure 3B). Future
studies will examine whether antibody−drug conjugates
generated using this strategy can exert cytotoxicity when
armed with potent cytotoxic small molecules.
Although this method of compound tagging through C−H
functionalization extends the scope of existing methods in
bioconjugation, it is not without its limitations. Reaction yields
can be low, and mixtures of regioisomeric products may be
obtained, which may render the product purification process
difficult. Also, some acid-sensitive substrates and functional
groups that are susceptible to oxidation (e.g., aliphatic
thioethers) are not tolerated. However, the mild (room
temperature or 50 °C) and convenient (open air, aqueous
solvent) reaction conditions as well as the one-step nature of
this chemical tagging method outweigh the occasional draw-
backs.
In summary, this work demonstrates an approach to the
native chemical tagging of small molecules at seemingly inert
C−H bonds. This method holds great potential for labeling and
bioconjugation of molecules that do not present functional
groups for conventional reactions. Studies demonstrating the
enhanced efficacy of these medicinal agents will be reported in
due course.
REFERENCES
■
(1) For an early review in bioconjugation targeting monoclonal
antibodies, see: Koppel, G. A. Bioconjugate Chem. 1990, 1, 13−23. For
recent reviews in enzyme profiling using bioconjugated chemical
probes, see: (b) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106,
3279−3301. (c) Carlson, E. E. ACS Chem. Biol. 2010, 5, 639−653.
(d) Bottcher, T.; Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed.
̈
2010, 49, 2680−2698.
(2) For a definition of bioconjugate chemistry, see: Meares, C.
Bioconjugate Chem. 1990, 1, 1−1.
(3) For a definition of “click” chemistry, see: Kolb, H. C.; Finn, M.
G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004−2021.
(4) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596−2599. (b) Tornøe, C. W.;
Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057−3064.
(5) (a) Peddibhotla, S.; Dang, Y.; Liu, J. O.; Romo, D. J. Am. Chem.
Soc. 2007, 129, 12222−12231. (b) Chamni, S.; He, Q.-L.; Dang, Y.;
Bhat, S.; Liu, J. O.; Romo, D. ACS Chem. Biol. 2011, 6, 1175−1181.
(c) Staub, I.; Sieber, S. A. J. Am. Chem. Soc. 2008, 130, 13400−13409.
(d) Zhou, C.-Y.; Li, J.; Peddibhotla, S.; Romo, D. Org. Lett. 2010, 12,
́
2104−2107. (e) Robles, O.; Serna-Saldívar, S. O.; Gutierrez-Uribe, J.
A.; Romo, D. Org. Lett. 2012, 14, 1394−1397. (f) Li, J.; Cisar, J. S.;
Zhou, C.-Y.; Vera, B.; Williams, H.; Rodríguez, A. D.; Cravatt, B. F.;
Romo, D. Nat. Chem. 2013, 5, 510−517.
(6) (a) Fujiwara, Y.; Dixon, J. A.; Rodriguez, R. A.; Baxter, R. D.;
Dixon, D. D.; Collins, M. R.; Blackmond, D. G.; Baran, P. S. J. Am.
Chem. Soc. 2012, 134, 1494−1497. (b) Fujiwara, Y.; Dixon, J. A.;
O’Hara, F.; Funder, E. D.; Dixon, D. D.; Rodriguez, R. A.; Baxter, R.
́
D.; Herle, B.; Sach, N.; Collins, M. R.; Ishihara, Y.; Baran, P. S. Nature
2012, 492, 95−99. (c) Zhou, Q.; Ruffoni, A.; Gianatassio, R.; Fujiwara,
Y.; Sella, E.; Shabat, D.; Baran, P. S. Angew. Chem., Int. Ed. 2013, 52,
3949−3952.
(7) Cipres, A.; O’Malley, D. P.; Li, K.; Finlay, D.; Baran, P. S.; Vuori,
K. ACS Chem. Biol. 2010, 5, 195−202.
(8) (a) Baran, P. S.; Zografos, A. L.; O’Malley, D. P. J. Am. Chem. Soc.
2004, 126, 3726−3727. (b) Baran, P. S.; Li, K.; O’Malley, D. P.;
Mitsos, C. Angew. Chem., Int. Ed. 2006, 45, 249−252. (c) O’Malley, D.
P.; Li, K.; Maue, M.; Zografos, A. L.; Baran, P. S. J. Am. Chem. Soc.
2007, 129, 4762−4775.
(9) (a) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;
Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 16793−16797. (b) Ning, X.; Guo, J.;
Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253−
2255. (c) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J.
T.; van Hest, J. C. M.; van Delft, F. L. Chem. Commun. 2010, 46, 97−
99.
ASSOCIATED CONTENT
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S
* Supporting Information
Experimental procedures and analytical data (1H and 13C
NMR, MS) for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
pbaran@scripps.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work was provided by NIH/NIGMS
(GM-106210), Morphotek, Eisai, Inc., and SIOC (postdoctoral
fellowships to Q.Z. and J.G.). The authors thank J. Eric Carlson
for providing materials to demonstrate linker attachments, as
well as Ryan Gianatassio, Alessandro Ruffoni, Darryl D. Dixon,
and Erik Daa Funder for technical contributions to the early
stages of this project.
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dx.doi.org/10.1021/ja407739y | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX