Forging isopeptide bonds using thiol-ene chemistry: Site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases

Article abstract of DOI:10.1021/ja300500a

Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We

Full text of DOI:10.1021/ja300500a

Communication
pubs.acs.org/JACS
Forging Isopeptide Bonds Using Thiol−Ene Chemistry: Site-Specific
Coupling of Ubiquitin Molecules for Studying the Activity of
Isopeptidases
Ellen M. Valkevich,† Robert G. Guenette,† Nicholas A. Sanchez,† Yi-chen Chen,†,‡ Ying Ge,‡,§
and Eric R. Strieter*,†
‡
§
†Department of Chemistry, Human Proteomics Program, and Department of Cell and Regenerative Biology, School of Medicine
and Public Health, University of WisconsinMadison, Madison, Wisconsin 53706-1322, United States
S
* Supporting Information
with information-rich acyl groups such as ubiquitin (Ub) and
Ub-like proteins.7
ABSTRACT: Chemical methods for modifying proteins
can enable studies aimed at uncovering biochemical
function. Herein, we describe the use of thiol−ene
coupling (TEC) chemistry to report on the function of
branched (also referred to as forked) ubiquitin trimers. We
show how site-specific isopeptide (Nε-Gly-L-homothiaLys)
bonds are forged between two molecules of Ub,
demonstrating the power of TEC in protein conjugation.
Moreover, we demonstrate that the Nε-Gly-L-homothiaLys
isopeptide bond is processed to a similar extent by
deubiquitinases (DUBs) as that of a native Nε-Gly-L-Lys
isopeptide bond, thereby establishing the utility of TEC in
the generation of Ub-Ub linkages. TEC is then applied to
the synthesis of branched Ub trimers. Interrogation of
these branched derivatives with DUBs reveals that the
relative orientation of the two Ub units has a dramatic
impact on how they are hydrolyzed. In particular, cleavage
of K48C-linkages is suppressed when the central Ub unit is
also conjugated through K6C, whereas cleavage proceeds
normally when the central unit is conjugated through
either K11C or K63C. The results of this work presage a
role for branched polymeric Ub chains in regulating
linkage-selective interactions.
Many reports have recently emerged describing chemical
approaches to the site-specific conjugation of Ub molecules
through native Nε-Gly-L-Lys isopeptide linkages and various
nonnative linkages.8 Indeed, some methods have elucidated
important structural distinctions for Ub dimers linked through
different lysines9 (K6, K27, K29, K33, K48, and K63) and
enabled studies that uncovered how the structure and function
of target proteins are altered upon Ub modification.10 Yet,
many of the chemical approaches designed to recapitulate the
Nε-Gly-L-Lys linkage suffer from drawbacks such as instability,
the number of synthetic manipulations required, and the use of
specialized recombinant DNA technologies for incorporating
unnatural amino acids. Development of additional methods is
therefore necessary to gain rapid access to a diverse range of Ub
modified targets.
We hypothesized TEC would provide an alternative to
known chemical approaches for forming isopeptide linkages, as
standard recombinant proteins can be employed with minimal
synthetic effort. In particular, Ub conjugation to a target protein
requires a protein harboring a Cys residue in lieu of a Lys (a
mutation introduced using site-directed mutagenesis) and Ub
bearing a small alkene such as allylamine (AA) appended to the
C-terminus. Upon TEC, an Nε-Gly-L-homothiaLys isopeptide
bond would be furnished, which is only one bond longer than
the native linkage; an alteration not expected to perturb
function (Figure 1A).11 Here, we describe the application of
TEC in the concatenation of Ub molecules to afford dimers
and trimers that exhibit similar behavior as those constructed
enzymatically. We also show how TEC can be used to reveal
biochemical details associated with a unique set of Ub
oligomers referred to as branched chains, i.e., oligomers in
which a single Ub unit is attached through multiple lysines to
other Ub units.
ddition of thiyl radicals to alkenes, termed thiol−ene
Acoupling (TEC), has the potential to serve as a powerful
method for chemically modifying proteins.1 The reasons are
manifold. For instance, bimolecular rate constants on the order
of 106 M−1 s−1 have been measured for the addition of thiyl
radicals to alkenes,2 which is ideal for carrying out reactions
with proteins at μM concentrations.3 TEC also allows for the
use of recombinant proteins and offers the potential to forge
stable thioether linkages that closely mimic amino acid side
chains.4 Despite these advantages, TEC has seen limited use in
the direct modification of Cys residues in proteins, presumably
due to the number of side reactions a thiyl radical (CysS•) can
undergo.5 Yet, there are examples of TEC with peptides and
proteins, suggesting in the presence of an alkene side reactions
based on CysS• are not competitive.4,6 Inspired by these
studies, we reasoned TEC could be exploited in the
construction of isopeptide bonds, an abundant linkage
established during the posttranslational modification of proteins
Our studies commenced with the installation of AA at the C-
terminus of Ub. To accomplish this goal, we turned to a class of
proteases referred to as Ub C-terminal hydrolases (UCHs).
UCHs promote hydrolysis of Ub variants carrying C-terminal
extensions through the formation of a Ub1−76-S-UCH acyl-
enzyme intermediate.12 Based on this enzymatic logic along
Received: January 16, 2012
Published: April 12, 2012
© 2012 American Chemical Society
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Figure 2. Representative mass spectrometric analysis of K48C-linked
Ub dimer. (A) FT-ICR MS analysis of crude reaction mixture shown
in (C); * corresponds to the mass of Ub-AA plus the phosphinate
portion of LAP (Scheme S3). (B) An individual charge state (z = 18 or
Figure 1. Site-specific conjugation of Ub molecules using TEC. (A)
Structures of Nε-Gly-L-Lys, the native isopeptide linkage, and Nε-Gly-
L-homothiaLys, the linkage forged through TEC. (B) Overview of
TEC mechanism showing the generation of K48C-linked Ub2 after
conjugating Ub-AA and UbK48C using the free-radical photoinitiator
LAP.
M
18+) of purified K48C-linked Ub dimer. The isotopic distribution
represents intact mass of full-length dimer. Red circles correspond to
the theoretical distribution of isotopic abundance. (C,D) Representa-
tive ECD spectra of a minimal trypsin digest of K48C-linked Ub dimer
indicating the installation of a Gly-Gly-AA motif at Ub residue 48. The
c ions are shown for fragments containing the first 47 residues (C) and
the first 48 residues (D).
with the reversible nature of proteases, AA was added in large
excess relative to a Ub variant harboring a C-terminal aspartate
cap (UbD77) in the presence of the yeast UCH YUH1. Under
these conditions, the AA adduct of Ub1−76 (herein termed Ub-
AA) could readily be obtained in ∼30% yield (see Supporting
Information). This procedure enables production of Ub-AA on
a milligram scale.
With a method to generate Ub-AA, we examined conditions
to carry out TEC with UbK48C obtained through site-directed
mutagenesis. After screening a series of water-soluble free-
radical initiators, the lithium acyl phosphinate (LAP)13
photoinitiator proved to be the most effective in terms of the
amount of Ub dimer formed (Figure 1B). Importantly, control
experiments indicated that formation of dimer was dependent
on the presence of each reaction component (Figure S2).
Subsequent optimization studies revealed a marked improve-
ment in the amount of dimer formed with higher LAP
concentrations. Accordingly, 0.5 mM LAP furnished milligram
quantities of the desired product after ion-exchange chroma-
tography (Figure S4).
Extensive characterization of the K48C-linked dimer was
conducted using high-resolution tandem mass spectrometry
(MS/MS) on a Fourier-transform ion cyclotron resonance
mass spectrometer (FT-ICR). Crude TEC reactions were
monitored by MS analysis of intact proteins. Peaks correspond-
ing to two distinct ionization states of the dimer (z = 17 and
18) are readily detected when TEC reactions are carried out
with all components (Figure 2A). In addition, a peak 16 Da
(Da) larger than that of the dimer was always observed, which
might be attributed to the oxidation of the thioether linkage.14
The highly accurate mass measurement of the purified K48C-
linked dimer (3.5 ppm between the experimental and
theoretical molecular weights) provides strong evidence for
the formation of the desired product (Figure 2B). To then
verify modification of position-48, electron capture dissociation
(ECD),15 a nonergodic MS/MS technique, was performed with
a minimal trypsin digest of K48C-linked dimer. Minimal digest
removes the C-terminal diGly motif leaving a Ub1−74 unit with
a Gly-Gly-AA (171 amu) appendage (Scheme S2); this greatly
simplifies ECD analysis. ECD spectra report on the extensive
fragmentation of N-terminal c ions and C-terminal z• ions
(Figure S10), and the fragmentation pattern surrounding
position-48 unambiguously verified incorporation of the desired
modification at this position (Figures 2C and D). Taken
together, SDS-PAGE and MS data argue that TEC is a
pragmatic method for site-specifically coupling two proteins
and generating K48C-linked Ub dimers.
Encouraged by these results, we investigated the application
of TEC in the synthesis of topoisomers, e.g., dimers linked
through K6C, K11C, K27C, K29C, K33C, and K63C. Using
similar conditions to those optimal for the K48C-linked dimer,
K6C-, K11C-, and K63C-linked dimers were obtained in yields
comparable to K48C (Figure S3). However, synthesis of
K29C-, K33C-, and in particular the K27C-linked dimer proved
more challenging. Specifically, high-resolution FT-ICR MS
analysis of crude reaction mixtures containing 1:1 ratios of
UbK29C/Ub-AA and UbK33C/Ub-AA showed dimers in low
abundance relative to the monomeric substrates; in similar
experiments with UbK27C, dimer formation was not observed.
Based on these results, we reasoned the addition of CysS• to
Ub-AA could be suppressed by the steric bulk surrounding the
radical species. To test this hypothesis, different concentrations
of Ub-AA were employed. For UbK29C and UbK33C, MS
analysis pointed to a clear trend in the relative amount of dimer
to monomeric substrate; i.e., higher concentrations of Ub-AA
led to an increase in the abundance of the dimer peak (Figures
S18 and S19). Conversely, UbK27C remained refractory
toward coupling even at UbK27C/Ub-AA ratios of 1:4. This
result, however, was not surprising considering residue-27 is the
least accessible according to the structure of Ub. Nevertheless,
synthesis of six out of seven topoisomers without specialized
techniques (e.g., unnatural amino acid incorporation and total
chemical synthesis) highlights the generality of TEC.
Next, we tested the function of Ub dimers forged through
TEC. To accomplish this goal, the hydrolytic cleavage of Nε-
Gly-L-homothiaLys isopeptide linkages was investigated using
isopeptidases (also referred to as deubiquitinases or DUBs).16
Several members of the DUB family of enzymes preferentially
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Figure 4. Structure and function of branched Ub3 derivatives. (A) The
three branched Ub3 derivatives synthesized in this study: K6C, K48C-;
K11C, K48C-; and K48C, K63C-linked. The central Ub unit
conjugated to two Ub molecules is shown in blue. (B) Western
blots developed with a Ub antibody (P4D1) showing the extent to
which different DUBs (IsoT, A20-OTU, and AMSH) hydrolyze the
three branched Ub3 derivatives. Hydrolysis is indicated by the
formation of dimers (Ub2) and monomers.
Figure 3. DUB-catalyzed hydrolysis of dimers forged through TEC.
(A) General scheme for the DUB-catalyzed hydrolysis of K48C- and
K63C-linked Ub dimers. (B) Silver-stained SDS-PAGE analysis of
cleavage reactions with the K48-linkage selective A20-OTU domain.
At t = 0 min, the DUB is absent from the reaction mixture. (C) Silver-
stained SDS-PAGE analysis of cleavage reactions with K63-linkage
selective AMSH.
and function in vivo remain unclear, in part, due to the inability
to identify branched linkages from tryptic digests.19 Moreover,
these oligomers display a low affinity for 26S proteasomes, and
certain Ub-binding chaperones can prevent their formation
thereby promoting protein degradation.20 To gain more insight
into the function of this class of oligomers, our established TEC
protocol was used to synthesize three branched Ub3 top-
oisomers starting from the Ub Lys-to-Cys double mutants:
K6C, K48C; K11C, K48C; and K48C, K63C (Figure 4A). This
particular set was chosen to systematically investigate the
influence of an additional Ub unit on the hydrolysis of the
K48C-linkage. Similar to the dimers, the trimers were purified
using ion-exchange chromatography and characterized by ECD
analysis of intact proteins minimally digested with trypsin
(Figures S11−S16). Western blot analysis with a Ub antibody
shows two bands for all trimers along with a faint band
corresponding to a dimer (Figure 4B). The trimer bands can be
ascribed to the presence of reduced and oxidized forms of the
Nε-Gly-L-homothiaLys linkage as both are observed by MS
analysis. A shift in electrophoretic mobility between different
forms of Ub oligomers is common as the number of units
increases, which explains why distinct bands for the reduced
and oxidized forms of the thioether linkage are not observed
with dimers.21
Functional studies with the branched trimers revealed clear
differences in the DUB-catalyzed hydrolysis of the K48C-
linkage (Figure 4B). Cleavage of each trimer was examined with
three different DUBs: IsoT, A20-OTU, and AMSH. IsoT
hydrolyzes free polyUb chains, i.e., those not conjugated to a
target protein, with little selectivity over linkage type.22 Given
the presence of a free C-terminus in each trimer we anticipated
that IsoT-catalyzed hydrolysis would rapidly furnish dimeric
and monomeric products. Indeed, IsoT efficiently processed all
three trimers as evidenced by Western blot analysis (Figure
4B). The most striking result, however, came while studying
A20-OTU-catalyzed cleavage. That is, Western blot analysis
indicated A20-OTU cleaved the K48C-linkage in K11C, K48C-
and K48C, K63C-linked trimers, whereas the same linkage
remained intact in the K6C, K48C-linked trimer (Figure 4B).
Since other nonselective DUBs such as those in the USP (Ub-
specific protease) family,23 in particular USP7, trim K6C,
K48C-linked Ub3 down to the monomer (Figure S20), the
cleave specific linkages within a Ub oligomer. For instance, the
ovarian tumor (OTU) domain-containing protein referred to as
A20 prefers K48-linkages, whereas the DUB AMSH (associated
molecule with the SH3 domain of STAM) cleaves K63-
linkages.17 In many cases, and in particular with A20 and
AMSH, linkage specificity arises from (i) the unique sequence
context of each Ub Lys residue and (ii) direct contact with all
atoms of the Lys side chain.16 Due to the discriminating
features of linkage-selective DUBs, we surmised A20-OTU- and
AMSH-catalyzed hydrolysis of K48C- and K63C-linked dimers
would provide a stringent test for the ability of Nε-Gly-L-
homothiaLys to mimic the native linkage (Figure 3A). For
direct comparison, native K48- and K63-linked dimers were
hydrolyzed alongside the dimers synthesized by TEC. Analysis
of A20-OTU-catalyzed cleavage of Nε-Gly-L-homothiaLys
indicated that while the K63C-linked dimer is not hydrolyzed
(Figure 3B, lanes 10−12), the K48C-linked native dimer is
almost completely converted to the respective monomeric units
within 1 h (Figure 3B, lanes 4−6). These results are congruent
with those obtained for dimers linked through the native
isopeptide bond (Figure 3B, lanes 1−3 and 7−9). In the case of
K63-linkage specific DUB AMSH, the K63C-linked dimers are
rapidly hydrolyzed, whereas the K48C-linked dimers are not
processed (Figure 3C, lanes 4−6 vs 10−12). It is important to
note that although there is a small amount of each thioether
dimer remaining after 1 h, the half-life of Nε-Gly-L-
homothiaLys is nearly identical to that of Nε-Gly-L-Lys.
These results indicate TEC, unlike the Cu(I)-catalyzed
azide−alkyne cycloaddition (CuAAC),18 affords fully functional
Ub dimers that can readily be obtained in two straightforward
steps from standard recombinant proteins.
Given the ease with which functional Ub dimers can be
forged, we surmised TEC could be applied to the synthesis of
branched Ub chains, as this class of oligomers is currently
unattainable using other methods such as amber stop codon
suppression because of low incorporation efficiency of
unnatural amino acids into multiple sites. Branched oligomers
have been observed as products of specific pairs of E2 Ub-
conjugating and E3 Ub-ligating enzymes, but their abundance
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2007, 46, 5226. (d) Weinrich, D.; Lin, P. C.; Jonkheijm, P.; Nguyen,
U. T.; Schroder, H.; Niemeyer, C. M.; Alexandrov, K.; Goody, R.;
Waldmann, H. Angew. Chem., Int. Ed. 2010, 49, 1252. (e) Aimetti, A.
A.; Feaver, K. R.; Anseth, K. S. Chem. Commun. 2010, 46, 5781.
(7) (a) Pickart, C. M. Annu. Rev. Biochem. 2001, 70, 503. (b) Grabbe,
C.; Husnjak, K.; Dikic, I. Nat. Rev. Mol. Cell Biol. 2011, 12, 295.
(8) (a) Fekner, T.; Li, X.; Chan, M. K. ChemBioChem 2011, 12, 21.
(b) Strieter, E. R.; Korasick, D. A. ACS Chem. Biol. 2012, 7, 52.
(9) (a) Virdee, S.; Ye, Y.; Nguyen, D. P.; Komander, D.; Chin, J. W.
Nat. Chem. Biol. 2010, 6, 750. (b) Castaneda, C. A.; Spasser, L.;
Bavikar, S. N.; Brik, A.; Fushman, D. Angew. Chem., Int. Ed. 2011, 50,
11210.
(10) (a) McGinty, R. K.; Kim, J.; Chatterjee, C.; Roeder, R. G.; Muir,
T. W. Nature 2008, 453, 812. (b) Fierz, B.; Chatterjee, C.; McGinty, R.
K.; Bar-Dagan, M.; Raleigh, D. P.; Muir, T. W. Nat. Chem. Biol. 2011,
7, 113. (c) Chen, J.; Ai, Y.; Wang, J.; Haracska, L.; Zhuang, Z. Nat.
Chem. Biol. 2010, 6, 270. (d) Hejjaoui, M.; Haj-Yahya, M.; Kumar, K.
S.; Brik, A.; Lashuel, H. A. Angew. Chem., Int. Ed. 2011, 50, 405.
(11) Chatterjee, C.; McGinty, R. K.; Fierz, B.; Muir, T. W. Nat.
Chem. Biol. 2010, 6, 267.
results with A20-OTU suggest the additional Ub unit appended
to position-6 abrogates hydrolytic cleavage by K48-linkage
selective DUBs. In the context of other linkage selective DUBs
such as AMSH, the presence of a Ub appendage at position 48
does not influence cleavage of the K63C-linkage as indicated by
the formation of Ub2 and Ub upon hydrolysis of the K48C,
K63C-linked trimer (Figure 4B). Additional work is necessary
to determine whether other branch points, e.g., K6C and K11C,
affect hydrolysis of K63C-linkages.
Our systematic examination of branched trimer topologies
suggests that branch points in a polyUb chain furnish a
regulatory mechanism for linkage-selective interactions. Con-
sistent with this analysis, K6-linkages are proposed to suppress
degradation of target proteins by 26S proteasomes.19b,24 In
principle, this could lead to the accumulation, and possibly
aggregation, of the target protein, which, in turn, would set the
stage for clearance by the lysosomal pathway.25 If the latter is
either unable or slow to process the aggregated proteins bearing
polyUb chains, then toxic levels may begin to accrue in the cell:
this is a hallmark of many neurodegenerative diseases.
Interestingly, mixed K6-, K11-, and K48-linked polyUb chains
have been observed in Tau aggregates isolated from brain tissue
of individuals with Alzheimer’s disease.26
(12) Johnston, S. C.; Riddle, S. M.; Cohen, R. E.; Hill, C. P. EMBO J.
1999, 18, 3877.
(13) Fairbanks, B. D.; Schwartz, M. P.; Bowman, C. N.; Anseth, K. S.
Biomaterials 2009, 30, 6702.
(14) (a) Vogt, W. Free Radical Biol. Med. 1995, 18, 93. (b) Xu, G.;
Chance, M. R. Chem. Rev. 2007, 107, 3514. (c) Reddie, K. G.; Carroll,
K. S. Curr. Opin. Chem. Biol. 2008, 12, 746.
(15) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem.
Soc. 1998, 120, 3265.
(16) Komander, D.; Clague, M. J.; Urbe, S. Nat. Rev. Mol. Cell Biol.
2009, 10, 550.
In summary, the work described herein showcases the utility
of TEC in the construction as well as the biochemical analysis
of dimeric and trimeric Ub conjugates. With these new tools,
future work will focus on understanding the abundance and
function of branched Ub oligomers in vivo.
(17) (a) Komander, D.; Barford, D. Biochem. J. 2008, 409, 77.
(b) Sato, Y.; Yoshikawa, A.; Yamagata, A.; Mimura, H.; Yamashita, M.;
Ookata, K.; Nureki, O.; Iwai, K.; Komada, M.; Fukai, S. Nature 2008,
455, 358.
(18) (a) Eger, S.; Scheffner, M.; Marx, A.; Rubini, M. J. Am. Chem.
Soc. 2010, 132, 16337. (b) Weikart, N. D.; Sommer, S.; Mootz, H. D.
Chem. Commun. 2012, 48, 296.
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures, characterization of products,
and structural analysis. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
(19) (a) Kim, H. T.; Kim, K. P.; Lledias, F.; Kisselev, A. F.; Scaglione,
K. M.; Skowyra, D.; Gygi, S. P.; Goldberg, A. L. J. Biol. Chem. 2007,
282, 17375. (b) Ben-Saadon, R.; Zaaroor, D.; Ziv, T.; Ciechanover, A.
Mol. Cell 2006, 24, 701.
(20) Kim, H. T.; Kim, K. P.; Uchiki, T.; Gygi, S. P.; Goldberg, A. L.
EMBO J. 2009, 28, 1867.
AUTHOR INFORMATION
Corresponding Author
strieter@chem.wisc.edu
■
Notes
(21) Dong, K. C.; Helgason, E.; Yu, C.; Phu, L.; Arnott, D. P.;
Bosanac, I.; Compaan, D. M.; Huang, O. W.; Fedorova, A. V.;
Kirkpatrick, D. S.; Hymowitz, S. G.; Dueber, E. C. Structure 2011, 19,
1053.
(22) Reyes-Turcu, F. E.; Shanks, J. R.; Komander, D.; Wilkinson, K.
D. J. Biol. Chem. 2008, 283, 19581.
(23) Faesen, A. C.; Luna-Vargas, M. P.; Geurink, P. P.; Clerici, M.;
Merkx, R.; van Dijk, W. J.; Hameed, D. S.; El Oualid, F.; Ovaa, H.;
Sixma, T. K. Chem. Biol. 2011, 18, 1550.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge Profs. S. H. Gellman, M. K.
Mahanthappa, and R. T. Raines for helpful discussions and
critical reading of the manuscript. Financial support is provided
by UW-Madison, the Greater Milwaukee Shaw Scientist
Program, and Susan G. Komen for Cure.
(24) (a) Sato, K.; Hayami, R.; Wu, W.; Nishikawa, T.; Nishikawa, H.;
Okuda, Y.; Ogata, H.; Fukuda, M.; Ohta, T. J. Biol. Chem. 2004, 279,
30919. (b) Wu-Baer, F.; Lagrazon, K.; Yuan, W.; Baer, R. J. Biol. Chem.
2003, 278, 34743. (c) Yu, X.; Fu, S.; Lai, M.; Baer, R.; Chen, J. Genes
Dev. 2006, 20, 1721.
(25) Pandey, U. B.; Nie, Z.; Batlevi, Y.; McCray, B. A.; Ritson, G. P.;
Nedelsky, N. B.; Schwartz, S. L.; DiProspero, N. A.; Knight, M. A.;
Schuldiner, O.; Padmanabhan, R.; Hild, M.; Berry, D. L.; Garza, D.;
Hubbert, C. C.; Yao, T. P.; Baehrecke, E. H.; Taylor, J. P. Nature 2007,
447, 859.
REFERENCES
■
(1) (a) Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995. (b) Hoyle,
C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49, 1540.
(2) Reddy, S. K.; Cramer, N. B.; Bowman, C. N. Macromolecules
2006, 39, 3673.
(3) Kalia, J.; Raines, R. T. Curr. Org. Chem. 2010, 14, 138.
(4) (a) Chalker, J. M.; Bernardes, G. J.; Davis, B. G. Acc. Chem. Res.
2011, 44, 730. (b) Floyd, N.; Vijayakrishnan, B.; Koeppe, J. R.; Davis,
B. G. Angew. Chem., Int. Ed. 2009, 48, 7798.
(26) Cripps, D.; Thomas, S. N.; Jeng, Y.; Yang, F.; Davies, P.; Yang,
A. J. J. Biol. Chem. 2006, 281, 10825.
(5) (a) Nauser, T.; Schoneich, C. J. Am. Chem. Soc. 2003, 125, 2042.
(b) Schoneich, C. Chem. Res. Toxicol. 2008, 21, 1175.
(6) (a) Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.Eur. J.
2009, 15, 11444. (b) Lo Conte, M.; Staderini, S.; Marra, A.; Sanchez-
Navarro, M.; Davis, B. G.; Dondoni, A. Chem. Commun. 2011, 47,
11086. (c) Wittrock, S.; Becker, T.; Kunz, H. Angew. Chem., Int. Ed.
6919
dx.doi.org/10.1021/ja300500a | J. Am. Chem. Soc. 2012, 134, 6916−6919