A General Supramolecular Approach to Regulate Protein Functions by Cucurbit[7]uril and Unnatural Amino Acid Recognition

Article abstract of DOI:10.1002/anie.202100916

Regulation of specific protein function is of great importance for both research and therapeutic development. Many small or large molecules have been developed to control specific protein function, but there is a lack of a universal approach to regulate t

Full text of DOI:10.1002/anie.202100916

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 11196–11200
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Protein Regulation
Hot Paper
AGeneral Supramolecular Approach to Regulate Protein Functions by
Cucurbit[7]uril and Unnatural Amino Acid Recognition
Wenbing Cao, Xuewen Qin, Yong Wang, Zhen Dai, Xianyin Dai, Haoyu Wang, Weimin Xuan,
Yingming Zhang, Yu Liu, and Tao Liu*
Abstract: Regulation of specific protein function is of great
importance for both research and therapeutic development.
Many small or large molecules have been developed to control
specific protein function, but there is a lack of a universal
approach to regulate the function of any given protein. We
report a general host–guest molecular recognition approach
involving modification of the protein functional surfaces with
genetically encoded unnatural amino acids bearing guest side
chains that can be specifically recognized by cucurbit[7]uril.
Using two enzymes and a cytokine as models, we showed that
the activity of proteins bearing unnatural amino acid could be
turned off by host molecule binding, which blocked its
functional binding surface. Protein activity can be switched
back by treatment with a competitive guest molecule. Our
approach provides a general tool for reversibly regulating
protein function through molecular recognition and can be
expected to be valuable for studying protein functions.
surface of proteins and inhibit their functions. However, the
aforementioned methods are limited to specific proteins or to
proteins with a specific sequence motif. A universal host–
guest molecular recognition approach for targeting the bind-
ing surface of any protein and thus reversibly regulating its
function, would be highly desirable.
Nearly all naturally occurring proteins are composed of
combinations of the 20 canonical amino acids, and achieving
high-affinity, high-specificity host–guest molecular recogni-
tion on protein surfaces with this limited set of building blocks
is challenging. We hypothesized that this challenge could be
overcome by using genetic code expansion, whereby synthetic
unnatural amino acids can be genetically encoded into
a protein in place of any naturally occurring residue via site-
directed mutagenesis with nonsense codon suppression.[11]
Genetic code expansion has previously been used to precisely
control protein function at the single-residue level by means
of photoregulation[12] or chemical decaging.[13] Despite their
power, however, these methods cannot regulate protein
function in a reversible manner. Although unnatural amino
acid containing azobenzene group provide a reversible layer
for protein function regulation, the design is difficult and may
not be applied to any given protein.[14] We reasoned that site-
specific replacement of residues in proximity to a proteinꢀs
functional surface-such as the substrate entry site for an
enzyme or the receptor binding site for a cytokine-with an
unnatural amino acid bearing a guest side chain would allow
residue-specific recognition by a host molecule and thus
permit reversible, on-demand control of the proteinꢀs func-
tion (Scheme 1).
An ideal unnatural amino acid for this purpose would
closely resemble canonical amino acids, to minimize any
deleterious effects on protein activity after mutation. Aro-
matic residues are natural guests for many host molecules,
and the interaction of these pairs have been well-docu-
mented.[15] Logsdon et al. demonstrated that among a series
of phenylalanine analogs, 4-tert-butyl-l-phenylalanine (tBuF)
and 4-(aminomethyl)-l-phenylalanine (pAMF) (Supporting
Information, Figure S1) are the best guest molecules recog-
nized by CB[7] with high affinity.[15a] CB[7] is one of the most
commonly used host molecules in biological systems with low
cytotoxicity.[4a,b,16] The reported Kd values for binding of
CB[7] to tBuFand pAMFare 0.25 mM and 0.46 mM, indicating
that CB[7] binds these residues 35 and 19 times as selectively
as phenylalanine[15a] and a few hundred to a few thousand
times as selectively as tyrosine and tryptophan.[17]
P
roteins are key regulators of biological processes, and
molecules that precisely control protein functions are of great
importance for protein functional studies. Molecular recog-
nition based on host–guest chemistry resembles protein-based
recognition, such as antibody–antigen and biotin–streptavidin
binding, and has already been used for protein modification,
regulation, and assembly.[1] For example, Finbloom et al. used
cucurbit[6]uril-catalyzed click chemistry to site-specifically
modify proteins and synthesize protein conjugates.[2] In
addition, cucurbit[7]uril (CB[7]) and PEG-modified CB[7]
can recognize N-terminal phenylalanine residues of target
proteins, thus inhibiting their function[3] and enhancing their
pharmacokinetic properties.[4] Cucurbit[8]uril can recognize
of proteins with N-terminaL l-phenylalanylglycylglycine
(FGG) motifs and methionine-terminated peptides,[5] and
this behavior has been used to regulate protein dimeriza-
tion,[6] oligomerization,[7] and ordered assembly.[8] Modified
calixarenes[9] and molecular tweezers[10] can recognize the
[*] W. Cao, Z. Dai, X. Dai, Prof. W. Xuan, Prof. Y. Zhang, Prof. Y. Liu
College of Chemistry, State Key Laboratory of Elemento-Organic
Chemistry, Nankai University
94 Weijin Road, Nankai District, Tianjin 300071 (P. R. China)
W. Cao, X. Qin, Y. Wang, Z. Dai, H. Wang, Prof. T. Liu
State Key Laboratory of Natural and Biomimetic Drugs, Department
of Molecular and Cellular Pharmacology, Pharmaceutical Sciences
Peking University
38 Xueyuan Road, Haidian District, Beijing 100191 (China)
E-mail: taoliupku@pku.edu.cn
To genetically incorporate tBuF and pAMF into proteins,
we utilized two previously reported Methanococcus janna-
schii TyrRS tRNACUA pairs.[18] Nonsense codon suppression
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202100916.
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K113tBuF (Figure S9b). Analysis of
the GST structure suggested that
the fact that K113 is farther from
the active site than the other two
mutated residues might explain why
the activity of this mutant was not
inhibited by CB[7]. Next, we deter-
mined whether a competitive guest
molecule could restore protein
activity. Indeed, we found that
addition of FGG at a concentration
of only 1.2 mM efficiently restored
the activity of GST-Q207tBuF (Fig-
ure 1b). These findings support the
Scheme 1. Illustration of the use of supramolecular host–guest chemistry to regulate protein function
precisely and reversibly. Active proteins containing a genetically encoded guest molecule (tBuF) in
proximity to the functional site could be deactivated by high-affinity binding between the guest side
chain and the biocompatible synthetic macrocycle CB[7], which is big enough to block the functional
interaction surface. Addition of a competitive guest molecule (FGG) with removal of CB[7] from the
protein, thereby restoring the activity of the protein.
efficiency was measured by recombinant expression of
a superfolder green fluorescent protein (sfGFP) mutant
bearing an amber stop codon at Y151 and subsequent
quantitative fluorescence assay. Full-length sfGFP was
expressed only in the presence of tBuF, and the fluorescence
signal in the presence of tBuF was 70 times that in its absence
(Figure S2). Incorporation of pAMF into sfGFP was detect-
able, but unfortunately it was not as efficient as incorporation
of tBuF (Figure S3); therefore, pAMF was not analyzed
further. Incorporation of tBuF was confirmed by high-
resolution mass spectrometric analysis of the purified
mutant protein. The observed mass for sfGFP-Y151tBuF
was 27,637.3 Da (Figure S4), which agreed well with the
calculated mass (27,637.0 Da). The yield of the purified
sfGFP mutant was 45 mgLÀ1. These results indicate that tBuF
could be efficiently introduced into proteins at a desired
location by means of unnatural amino acid mutagenesis.
Next, we attempted to incorporate tBuF into a model
protein, glutathione S-transferase (GST),[19] to test the utility
of our host–guest molecular recognition system for reversible
control of enzymatic activity. After inspection of the crystal
structure of GST bound to its substrate, GSH, we chose three
residues-R108, K113, and Q207, which are located at the
entrance of the substrate binding pocket-as sites for mutation
into tBuF (Figures 1a). Histag-labeled GST and the three
mutant enzymes (GST-R108tBuF, GST-K113tBuF, and GST-
Q207tBuF) were expressed in Escherichia coli BL21 and
purified by Ni-NTA chromatography. The expression yields of
the purified mutants were similar to those of wild-type GST,
which was approximately 40 mgLÀ1 (Figure S5).
To evaluate the enzyme activity, a previously reported
GST assay was adopted (Figure S6). No obvious difference
was observed between the wild-type and mutant enzymes
(Figure S7). Addition of 1 or 2 mM CB[7] had no effect on
wild-type GST activity, and neither did addition of the
competitive guest molecule FGG (Figure S8). FGG was
selected due to its excellent water solubility and biocompat-
ibility. Next, we determined the activities of the mutant
enzymes in the presence of 1 mM CB[7]. Complete inhibition
was observed for GST-Q207tBuF (Figure 1b), and nearly
complete inhibition also was observed for GST-R108tBuF
(Figure S9a); in contrast, no inhibition was detected for GST-
Figure 1. a) Crystal structure GST (cyan) bound to its substrate, GSH
(red) (PDB ID: 1u87). The mutation sites selected for incorporation of
tBuF are indicated in yellow. b) Assay of enzymatic activity of the GST-
Q207tBuF mutant (pH 6.5, phosphate-buffered saline, 258C, [GST-wt]
=3 mM, [CB[7]]=1 mM, [FGG]=1.2 mM, [GSH]=1 mM, [CDNB]
=1 mM (CDNB=1-chloro-2,4-dinitrobenzene). c) Isothermal titration
calorimetry analysis of CB[7] binding to GST-Q207tBuF (20 mM).
Assays were performed in triplicate. Data are presented as the mean
and error bars represent the standard deviation.
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PTP1B-F182tBuF, respectively (Figure S11). It is possible
that mutation into the bulky tBuF could have an impact on
protein stability and yield,[20] and therefore more positions
could be tested. After purification of the enzymes, their
activities were assessed by means of a colorimetry assay kit
that detects the release of phosphate from a peptide substrate
(Figure S12). In the absence of CB[7], all three mutants
showed activities similar to the wild-type activity (Fig-
ure S13). However, in the presence of 1 mM CB[7], the
activity of PTP1B-R47tBuF was completely inhibited (Fig-
ure 2b); whereas the activities of the other enzymes were only
slightly affected by the addition of the host molecule
(Figures S14 and S15). Again, these results are consistent
with the structural analysis showing that of the mutated
residues, R47 is closest to the substrate binding pocket.
Addition of FGG (1.2 mM) restored more than 70% of the
enzyme activity, while the FGG itself at different concen-
trations does not influence the enzyme activity (Figure S16).
To convey a more in depth understanding of the system,
different ratios of protein and CB[7], effects on Kcat and KM of
CB[7], and a titration of FGG inhibitor were studied. A
decrease of enzymatic activity was observed with increasing
concentrations of CB[7] (Figure S17). A decrease of KM and
apparent Kcat was also observed with the addition of inhibitors
(Figure S18). The enzymatic activities were restored with
increasing concentrations of competitors (Figure S19). Col-
lectively, these data indicating that the function of PTP1B-
R47tBuF could indeed be reversibly controlled by means of
our host–guest molecule recognition approach.
To further demonstrate the utility of this approach, we
extended it to the regulation of cytokines. Cytokines are
important regulatory proteins that play crucial roles in cell
signaling and cytokine engineering is a hot topic in the
therapeutic protein field. We wondered whether our host–
guest system could be used to regulate cytokine function,
which could in turn allow for regulation of cellular activities.
To evaluate this possibility, we selected the pleiotropic
cytokine tumor necrosis factor alpha (TNFa)-which induces
many cellular processes, including apoptosis and inflamma-
tion-as a model cytokine.
TNFa binds to TNFR1 (tumor necrosis factor receptor 1)
and induces cell death through the caspase pathway (Fig-
ure S20). To design a controllable TNFa, we examined the
crystal structure of a TNFa trimer and data obtained by
mutation analysis of TNFa bound to TNFR1.[21] On this basis,
we chose four residues near the binding interface for mutation
to tBuF (Figure 3a): Q21, Q25, Q31, and A145. The mutant
and wild-type proteins were obtained by recombinant expres-
sion in E. coli BL21, and their cytotoxicities to L929 cells were
evaluated. The measured bioactivities of TNFa-Q21tBuF,
TNFa-Q25tBuF, TNFa-Q31tBuF, and TNFa-A145tBuF
were, respectively, 92%, 97%, 87%, and 7% of the wild-
type bioactivity (Figure 3b). The biocompatibilities of the
host (CB[7]) and the competitive guest (FGG) were eval-
uated on cells at concentrations ranging from 0.25 to 2.5 mM
and from 0.5 to 2.5 mM, respectively. No cytotoxicity was
observed for either compound in the tested concentration
ranges (Figure S21), confirming that they are safe to use on
living cells, as has previously been reported in the litera-
Figure 2. a) Crystal structure of PTP1B (yellow) bound to an analogue
of the peptide substrate (red) (PDB ID: 1bzh). Sites selected for
incorporation of tBuF are indicated in green. b) Protein tyrosine
phosphate assay of the PTP1B-R47tBuF mutant. The activity of the
enzyme (500 nM) was assayed in the absence of CB[7], in the presence
of 1 mM CB[7], or in the presence of 1 mM CB and 1.2 mM FGG in
Tris buffer (pH 7.4). Assays were performed in triplicate. Data are
presented as the mean and error bars represent the standard
deviation.
idea that enzyme function can be efficiently and reversibly
regulated by supramolecular host–guest recognition and
binding with a single-residue resolution.
To confirm that the observed inhibitory effects were due
to specific molecular recognition, we used isothermal titration
calorimetry to measure the binding affinities between CB[7]
and two of the GST mutants. The Kd values for binding of
CB[7] to GST-R108tBuF and GST-Q207tBuF were deter-
mined to be 24.2 and 4.45 mM, respectively (Figure 1c;
Figure S10a), whereas no binding between wild-type GST
and CB[7] could be detected (Figure S10b). These findings
suggest that the observed inhibition was indeed due to specific
molecular-recognition-induced binding between the host
molecule CB[7] and the site-specifically encoded guest tBuF.
Protein tyrosine phosphorylation is an important post-
translational modification that regulates various cellular
processes, and its malfunction results in many human diseases.
Our laboratory has a long-term interest in studying protein
tyrosine phosphatases, a family of enzymes that regulate the
dephosphorylation of phosphotyrosine residues.[18b] To deter-
mine whether our tBuF-CB[7] recognition system could be
generalized to other enzymes, we selected protein tyrosine
phosphatase 1B (PTP1B) as our next target protein. PTP1B,
which has been extensively studied, is an important drug
target because it regulates numerous signaling cascades.
Upon thorough inspection of the crystal structure of PTP1B
bound to an analogue of the peptide substrate, we identified
three potential mutation sites near the binding pockets: R47,
S118, and F182 (Figure 2a). The corresponding mutant
proteins were constructed and expressed in E. coli BL21,
and expression yields were determined to be 8,15 and
6 mgLÀ1 for PTP1B-R47tBuF, PTP1B-S118tBuF, and
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Conclusion
In summary, we have developed a simple, universal
supramolecular host–guest interaction approach to reversibly
regulate protein function. Specifically, we introduced a recog-
nition element to the surfaces of several proteins by
genetically encoding the unnatural amino acid tBuF contain-
ing a guest side chain. Guided by structural information, we
installed tBuF at selected positions in proximity to each
proteinꢀs functional interface, and we found that the resulting
mutant proteins could be specifically recognized by the
biocompatible macrocyclic host molecule CB[7] with high
affinities. Using two enzymes and a cytokine as model
proteins, we showed that active proteins containing the
built-in guest residue at carefully selected sites could be
reversibly regulated by the host molecule and by a competitive
guest molecule. The components of the system showed great
biocompatibility and could be used on living cells. Therefore,
the system can serve as a general tool for reversible regulation
of protein function through molecular recognition and can be
expected to be valuable for the study of protein functions.
This proof-of-principle study opens up design and application
possibilities where host macrocycles and guest amino acid
pairs feature. Higher binding affinity and selectivity on
proteins could be accessible in the future. The development
of novel pairs would also overcome some current limitations
and allow targeting of intracellular proteins as well as
achieving reversibility with multiple cycles.
Figure 3. a) Crystal structure of TNFa trimer (PDB ID: 2tnf). Sites
selected for incorporation of tBuF are indicated in red. b) Cytotoxicity
assay of TNFa mutants. L929 cells were treated with cytokines
(2 ngmLÀ1) in the absence of CB[7], in the presence of 1 mM CB[7], or
in the presence of 1 mM CB[7] and 1.2 mM FGG, along with actino-
mycin D (1 mgmLÀ1). Cytotoxicity was measured with a CCK8 kit.
Assays were performed in triplicate. Data are presented as the mean
and error bars represent the standard deviation. c) Images of L929
cells after treatment with TNFa-Q25tBuF (2 ngmLÀ1), TNFa-Q25tBuF
(2 ngmLÀ1) with 1 mM CB[7], and TNFa-Q25tBuF (2 ngmLÀ1) with
1 mM CB[7] and 1.2 mM FGG, respectively.
Acknowledgements
This work was financially supported by the National Major
Scientific and Technological Special Project for “Significant
New Drugs Development” (2019ZX09739001), the National
Natural Science Foundation of China (21922701, 21778005
and 91853111), Beijing Natural Science Foundation
(JQ20034), the National Key Research and Development
Program of China (2016YFA0201400), Shenzhen Institute of
Synthetic
Biology
Scientific
Research
Program
(DWKF20190004) and Clinical Medicine Plus X-Young
Scholars Project (PKU2020LCXQ029), Peking University.
We thank Qian Wang at the SKLNBD Research for
assistance in ITC experiments.
ture.[16] The cytotoxicities of wild-type TNFa in the absence
and presence of CB[7] (1 mM), as well as in the presence of
both CB[7] (1 mM) and FGG (1.2 mM), were also assayed; no
obvious cytotoxicity was observed (Figure S22). Next, we
evaluated the bioactivities of the four TNFa mutants in the
presence of 1 mM CB[7]. As shown in Figure 3b, in the
presence of 1 mM CB[7], the bioactivities of TNFa-Q21tBuF,
TNFa-Q25tBuF, and TNFa-Q31tBuF were inhibited 27%,
83%, and 78%, respectively. Subsequent treatment with
1.2 mM FGG restored the activities of the Q25 and Q31
mutants to 100% and 87%, respectively (Figure 3b,c). These
findings indicate that by introducing tBuF in proximity to the
receptor binding interface, we could precisely and reversibly
regulate the bioactivity of TNFa by host–guest molecular
interactions, and could in principle be extended to other
cytokines.
Conflict of interest
The authors declare no conflict of interest.
Keywords: cytokines · enzymes · protein function ·
supramolecular chemistry · unnatural amino acids
[1] a) J. An, S. Kim, A. Shrinidhi, J. Kim, H. Banna, G. Sung, K. M.
Park, K. Kim, Nat. Biomed. Eng. 2020, 4, 1044 – 1052; b) Y. H.
Liu, Y. M. Zhang, H. J. Yu, Y. Liu, Angew. Chem. Int. Ed. 2021,
60, 3870 – 3880; c) Q. Luo, C. Hou, Y. Bai, R. Wang, J. Liu, Chem.
Rev. 2016, 116, 13571 – 13632; d) H. Qianzhu, A. P. Welegedara,
H. Williamson, A. E. McGrath, M. C. Mahawaththa, N. E.
Dixon, G. Otting, T. Huber, J. Am. Chem. Soc. 2020, 142,
Angew. Chem. Int. Ed. 2021, 60, 11196 –11200
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org 11199

Angewandte
Communications
Chemie
17277 – 17281; e) Y. M. Zhang, N. Y. Zhang, K. Xiao, Q. Yu, Y.
Liu, Angew. Chem. Int. Ed. 2018, 57, 8649 – 8653; Angew. Chem.
2018, 130, 8785 – 8789; f) S. van Dun, C. Ottmann, L. G. Milroy,
L. Brunsveld, J. Am. Chem. Soc. 2017, 139, 13960 – 13968.
[2] J. A. Finbloom, K. Han, C. C. Slack, A. L. Furst, M. B. Francis, J.
Am. Chem. Soc. 2017, 139, 9691 – 9697.
Schrader, G. Bitan, E. E. Wanker, S. Ebbinghaus, E. Sanchez-
Garcia, J. Am. Chem. Soc. 2017, 139, 5640 – 5643; d) D. Bier, S.
Mittal, K. Bravo-Rodriguez, A. Sowislok, X. Guillory, J. Briels,
C. Heid, M. Bartel, B. Wettig, L. Brunsveld, E. Sanchez-Garcia,
T. Schrader, C. Ottmann, J. Am. Chem. Soc. 2017, 139, 16256 –
16263.
[3] H. H. Lee, T. S. Choi, S. J. C. Lee, J. W. Lee, J. Park, Y. H. Ko,
W. J. Kim, K. Kim, H. I. Kim, Angew. Chem. Int. Ed. 2014, 53,
7461 – 7465; Angew. Chem. 2014, 126, 7591 – 7595.
[11] a) C. C. Liu, P. G. Schultz, Annu. Rev. Biochem. 2010, 79, 413 –
444; b) A. Dumas, L. Lercher, C. D. Spicer, B. G. Davis, Chem.
Sci. 2015, 6, 50 – 69; c) H. Xiao, P. G. Schultz, Cold Spring
Harbor Perspect. Biol. 2016, 8, a023945; d) Y. Huang, T. Liu,
Synth. Syst. Biotechnol. 2018, 3, 150 – 158.
[12] a) J. Wang, Y. Liu, Y. Liu, S. Zheng, X. Wang, J. Zhao, F. Yang, G.
Zhang, C. Wang, P. R. Chen, Nature 2019, 569, 509 – 513; b) A.
Gautier, D. P. Nguyen, H. Lusic, W. An, A. Deiters, J. W. Chin, J.
Am. Chem. Soc. 2010, 132, 4086 – 4088; c) E. Arbely, J. Torres-
Kolbus, A. Deiters, J. W. Chin, J. Am. Chem. Soc. 2012, 134,
11912 – 11915; d) J. Luo, R. Uprety, Y. Naro, C. Chou, D. P.
Nguyen, J. W. Chin, A. Deiters, J. Am. Chem. Soc. 2014, 136,
15551 – 15558; e) W. Ren, A. Ji, H. W. Ai, J. Am. Chem. Soc.
2015, 137, 2155 – 2158; f) S. B. Erickson, R. Mukherjee, R. E.
Kelemen, C. J. Wrobel, X. Cao, A. Chatterjee, Angew. Chem.
Int. Ed. 2017, 56, 4234 – 4237; Angew. Chem. 2017, 129, 4298 –
4301.
[13] a) J. Li, S. Jia, P. R. Chen, Nat. Chem. Biol. 2014, 10, 1003 – 1005;
b) J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M.
Yang, S. Jia, X. Zhang, P. R. Chen, Nat. Chem. 2014, 6, 352 – 361;
c) J. Li, P. R. Chen, Nat. Chem. Biol. 2016, 12, 129 – 137; d) M. K.
Bilyard, H. J. Bailey, L. Raich, M. A. Gafitescu, T. Machida, J.
Iglesias-Fernandez, S. S. Lee, C. D. Spicer, C. Rovira, W. W. Yue,
B. G. Davis, Nature 2018, 563, 235 – 240.
[14] a) D. G. Mohua Bose, J. Xie, E. Brustad, P. G. Schultz, J. Am.
Chem. Soc. 2006, 128, 388 – 389; b) Y. H. Tsai, S. Essig, J. R.
James, K. Lang, J. W. Chin, Nat. Chem. 2015, 7, 554 – 561; c) C.
Hoppmann, I. Maslennikov, S. Choe, L. Wang, J. Am. Chem. Soc.
2015, 137, 11218 – 11221.
[15] a) L. A. Logsdon, C. L. Schardon, V. Ramalingam, S. K. Kwee,
A. R. Urbach, J. Am. Chem. Soc. 2011, 133, 17087 – 17092;
b) S. J. Barrow, S. Kasera, M. J. Rowland, J. del Barrio, O. A.
Scherman, Chem. Rev. 2015, 115, 12320 – 12406.
[16] D. Das, K. I. Assaf, W. M. Nau, Front. Chem. 2019, 7, 619.
[17] D. M. Bailey, A. Hennig, V. D. Uzunova, W. M. Nau, Chem. Eur.
J. 2008, 14, 6069 – 6077.
[18] a) J C. Jackson, S. P. Duffy, K. R. Hess, R. A. Mehl, J. Am. Chem.
Soc. 2006, 128, 11124 – 11127; b) H. Tang, Z. Dai, X. Qin, W. Cai,
L. Hu, Y. Huang, W. Cao, F. Yang, C. Wang, T. Liu, J. Am. Chem.
Soc. 2018, 140, 13253 – 13259.
[19] Y. Liu, T. Pan, Y. Fang, N. Ma, S. Qiao, L. Zhao, R. Wang, T.
Wang, X. Li, X. Jiang, F. Shen, Q. Luo, J. Liu, ACS Catal. 2017, 7,
6979 – 6983.
[4] a) C. L. Maikawa, A. A. A. Smith, L. Zou, G. A. Roth, E. C.
Gale, L. M. Stapleton, S. W. Baker, J. L. Mann, A. C. Yu, S.
Correa, A. K. Grosskopf, C. S. Liong, C. M. Meis, D. Chan, M.
Troxell, D. M. Maahs, B. A. Buckingham, M. J. Webber, E. A.
Appel, Nat. Biomed. Eng. 2020, 4, 507 – 517; b) M. J. Webber,
E. A. Appel, B. Vinciguerra, A. B. Cortinas, L. S. Thapa, S.
Jhunjhunwala, L. Isaacs, R. Langer, D. G. Anderson, Proc. Natl.
Acad. Sci. USA 2016, 113, 14189 – 14194; c) J. M. Chinai, A. B.
Taylor, L. M. Ryno, N. D. Hargreaves, C. A. Morris, P. J. Hart,
A. R. Urbach, J. Am. Chem. Soc. 2011, 133, 8810 – 8813.
[5] Z. Hirani, H. F. Taylor, E. F. Babcock, A. T. Bockus, C. D.
Varnado, C. W. Bielawski, A. R. Urbach, J. Am. Chem. Soc.
2018, 140, 12263 – 12269.
[6] a) R. P. Bosmans, J. M. Briels, L. G. Milroy, T. F. de Greef, M.
Merkx, L. Brunsveld, Angew. Chem. Int. Ed. 2016, 55, 8899 –
8903; Angew. Chem. 2016, 128, 9045 – 9049; b) H. D. Nguyen,
D. T. Dang, J. L. van Dongen, L. Brunsveld, Angew. Chem. Int.
Ed. 2010, 49, 895 – 898; Angew. Chem. 2010, 122, 907 – 910;
c) D. A. Uhlenheuer, J. F. Young, H. D. Nguyen, M. Scheepstra,
L. Brunsveld, Chem. Commun. 2011, 47, 6798 – 6800; d) L. M.
Heitmann, A. B. Taylor, P. J. Hart, A. R. Urbach, J. Am. Chem.
Soc. 2006, 128, 12574 – 12581.
[7] D. T. Dang, J. Schill, L. Brunsveld, Chem. Sci. 2012, 3, 2679 –
2684.
[8] a) C. Hou, J. Li, L. Zhao, W. Zhang, Q. Luo, Z. Dong, J. Xu, J.
Liu, Angew. Chem. Int. Ed. 2013, 52, 5590 – 5593; Angew. Chem.
2013, 125, 5700 – 5703; b) F. Guagnini, S. Engilberge, K. O.
Ramberg, J. Perez, P. B. Crowley, Chem. Commun. 2020, 56,
360 – 363.
[9] a) H. S. Park, Q. Lin, A. D. Hamilton, J. Am. Chem. Soc. 1999,
121, 8 – 13; b) M. A. Blaskovich, Q. Lin, F. L. Delarue, J. Sun,
H. S. Park, D. Coppola, A. D. Hamilton, S. M. Sebti, Nat.
Biotechnol. 2000, 18, 1065 – 1070; c) Y. Wei, G. L. McLendon,
A. D. Hamilton, M. A. Case, C. B. Purring, Q. Lin, H. S. Park,
C. S. Lee, T. Yu, Chem. Commun. 2001, 1580 – 1581; d) J. Sun,
D. A. Wang, R. K. Jain, A. Carie, S. Paquette, E. Ennis, M. A.
Blaskovich, L. Baldini, D. Coppola, A. D. Hamilton, S. M. Sebti,
Oncogene 2005, 24, 4701 – 4709; e) R. E. McGovern, H. Fer-
nandes, A. R. Khan, N. P. Power, P. B. Crowley, Nat. Chem. 2012,
4, 527 – 533.
[10] a) S. Sinha, D. H. Lopes, Z. Du, E. S. Pang, A. Shanmugam, A.
Lomakin, P. Talbiersky, A. Tennstaedt, K. McDaniel, R. Bakshi,
P. Y. Kuo, M. Ehrmann, G. B. Benedek, J. A. Loo, F. G. Klarner,
T. Schrader, C. Wang, G. Bitan, J. Am. Chem. Soc. 2011, 133,
16958 – 16969; b) D. Bier, R. Rose, K. Bravo-Rodriguez, M.
Bartel, J. M. Ramirez-Anguita, S. Dutt, C. Wilch, F. G. Klarner,
E. Sanchez-Garcia, T. Schrader, C. Ottmann, Nat. Chem. 2013, 5,
234 – 239; c) T. Vçpel, K. Bravo-Rodriguez, S. Mittal, S.
Vachharajani, D. Gnutt, A. Sharma, A. Steinhof, O. Fatoba, G.
Ellrichmann, M. Nshanian, C. Heid, J. A. Loo, F. G. Klarner, T.
[20] W. N. Chen, G. Otting, ACS Med. Chem. Lett. 2018, 9, 109 – 113.
[21] X.-M. Zhang, I. Weber, M.-J. Chen, J. Biol. Chem. 1992, 267,
24069 – 24075.
Manuscript received: January 20, 2021
Revised manuscript received: February 10, 2021
Accepted manuscript online: February 12, 2021
Version of record online: April 12, 2021
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