A DNA-Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Article abstract of DOI:10.1002/anie.201902513

Here we show a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed.

Full text of DOI:10.1002/anie.201902513

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Accepted Article
Title: A DNA-encoded chemical library incorporating elements of
natural macrocycles
Authors: Cedric Stress, Basilius Sauter, Lukas Schneider, Timothy
Sharpe, and Dennis Gillingham
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201902513
Angew. Chem. 10.1002/ange.201902513
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A DNA-encoded chemical library incorporating elements of
natural macrocycles
Cedric J. Stress, Basilius Sauter, Lukas A. Schneider, Timothy Sharpe and Dennis Gillingham*
Abstract: Here we perform a seven-step chemical synthesis of a
DNA-encoded macrocycle library (DEML) on DNA. Inspired by
polyketide and mixed peptide-polyketide natural products, the library
was designed to incorporate rich backbone diversity. Achieving this
diversity, however, comes at the cost of custom synthesis of
bifunctional building block libraries. Our work outlines the importance
of careful retrosynthetic design in DNA-encoded libraries, while
revealing areas where new DNA synthetic methods are needed.
has reported a highly side-chain diverse DEML built on a
constant peptide scaffold (see Fig. 1A & B). Missing from the
current set is
a library that includes more hydrophobic
components in the backbone. Given this background, we
focused on the construction of an encoded natural product-like
DEML with diverse ring scaffolds. Scaffold diversity, however,
comes at a high synthetic cost since a limited number of
bifunctional hydrocarbon precursors are commercially available.
Here we report the synthesis and preliminary evaluation of a
DEML where the backbone is inspired by the polyketide and
mixed peptide-polyketide macrocyclic natural products. The
DEML contains over two thousand distinct scaffolds, which,
along with its side-chain diversity, gives a total library size of
over a million members.
Nearly all approved macrocyclic drugs are natural products or
close derivatives.[1] Developing novel macrocycle drugs is
challenging and unpredictable because they violate all or some
of Lipinski's rules for oral bioavailability.[2] A second problem is
that non-peptidic macrocycles are not amenable to chemical
optimization the way that small molecule drugs are. Macrocyclic
‘hits’ are often highly optimized and synthetically cumbersome
(because they are large and derived from natural products), with
little opportunity for chemical tuning. Macrocycle libraries to date
are typically peptide derived and have great side-chain
diversity,[3] but far less backbone diversity. Recent work,
however, suggests that the backbone is intimately involved in
binding of macrocycles to their targets.[4] In fact natural
macrocycles as a class are characterized by highly diverse
backbones rather than side-chains.[1, 5] Great strides have been
made in understanding the properties of macrocycles that
Modern drug discovery typically begins with screening diverse
compound libraries to find ‘hits’ against a given target, which are
then further optimized into lead structures for in vivo testing.[10]
However, high throughput screening (HTS) of compound
collections is costly, and throughput scales linearly with the size
of the compound collection since each compound must be
spatially separated for testing. Technologies for screening of
pooled compound libraries offer an alternative, but come with
the problem of accurate hit deconvolution after selection.[11]
Biological and biochemical selection techniques physically
connect the genotype (DNA) and phenotype (aptamer, protein).
Thus, after a functional or binding selection, the structure of best
performers can be determined by standard DNA sequencing.
The advent of next generation sequencing has further
revolutionized the field because now information on entire pools
of selected molecules can be gleaned, offering a more nuanced
interpretation of selection datasets. Selection approaches were
restricted to natural systems until Brenner and Lerner, inspired
by the success of monoclonal antibody development with phage
display libraries,[11] proposed in the early nineties that sequential
synthesis and encoding on a chimeric bead might allow
selection of wholly artificial molecules.[12] It took several years for
this concept to become practical,[11, 13] but today several library
improve their prospects as drugs, despite their seemingly
6]
unfavorable physicochemical properties. The Whitty[4,
and
Kihlberg[7] groups have independently examined large
collections of macrocycles to try and identify a set of actionable
rules for predicting oral bioavailability beyond the rule-of-five. A
key feature seems to be a macrocycle’s ability to minimize polar
surface area when exposed to non-polar solvents. This
‘chameleonic’ property is likely the source of many macrocycle’s
ability to maintain cell permeability despite their size. But these
datasets are based on approved drugs from Nature’s bounty of
macrocycles. A bigger collection of macrocycles to screen and
study would allow us to determine whether they are truly
unusual in terms of cell permeability and protein binding; or
whether our current thinking is biased by the compound set
offered by natural evolution. In this vein, a number of exciting
methods, both biological[8] and chemical,[9] have recently been
developed to create large collections of encoded macrocycles.
In particular, the Liu lab has created a backbone diverse DNA-
encoded macrocycle library (DEML),[9a-c] while the Neri lab[9d]
construction technologies have been demonstrated.[14]
A
challenge in DECL synthesis is the compatibility of the chemical
synthesis with the DNA itself and the biochemical steps
(encoding and amplification). Here we push the limits of current
chemical synthesis on DNA by performing
a seven-step
synthesis of a DNA-encoded macrocycle library (DEML) with
hydrophobic backbone elements reminiscent of polyketide and
mixed peptide-polyketide natural products (see Fig. 1C & D).
[*]
C. J. Stress, B. Sauter, L. A. Schneider, Prof. Dr. D. G. Gillingham
Department of Chemistry, University of Basel
St. Johanns-Ring 19, CH-4056 Basel (Switzerland)
E-mail: dennis.gillingham@unibas.ch
Dr. T. Sharpe
Biophysics Facility, Biozentrum, University of Basel
Klingelbergstrasse 50/70, CH-4056 Basel (Switzerland)
Supporting information for this article is given via a link at the end of
the document.
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The synthesis began with
a
Cu-catalyzed azide-alkyne
cycloaddition (CuAAC) to connect the DNA with the DE-1 sub-
library member 2 (step 1, Fig. 2B). The next step was to
introduce the key trifunctional lynchpin 3 (step 2). This proved
challenging because an -azido--amino acid proved unstable,
and azido-alanine led to elimination of the azide to create
dehydroalanine under basic conditions. These two compact
molecules were our preferred lynchpins because the aim was to
keep the out-of-the-ring diversity element as close to the
macrocyclic core as possible, since it would then have the
greatest effect on the macrocycle’s conformational space. In the
end we had to settle for compound 3, which contained an
additional methylene spacer. Saponification of the methyl ester
(step 3) then opened the necessary coupling site to install the
DE-2 sub-library. In the test synthesis we used alanine as a
coupling partner and its coupling efficiency was highest with
DMTMM (step 4). Given how critical this step was to the library
we carried out a comprehensive screen across 126 amino acids
and evaluated the reactions by LC-MS analysis. We scored the
reactions by coupling efficiency and purity, applying thresholds
of >80% conversion and ≥50% product purity for inclusion in the
DEML itself. Most amino acids exceeded these limitations,
although we could identify trends in the poorly reacting members.
In general, other than 1-aminocyclopropanecarboxylic acid and
sarcosine, α,α-disubstituted and N-methylated amino acids were
poorly coupled. Also problematic were homoprolines as well as
compounds with leaving groups in the sidechain (chloroalanine,
azidoalanine). The full list of the coupling results are shown in
the ESI. The choice of the coupling reagent had a big impact on
coupling efficiencies for the different building blocks. While
EDC/HOAt/DIPEA is often the system of choice[15] we found that
coupling with DMTMM-BF4/NMM was better in most cases,
resulting in higher conversions and cleaner products.
EDC/HOAt/DIPEA was only superior for strained cyclic
secondary amino acids and tyrosine derivatives, a problem that
likely stems from reaction of the tyrosine hydroxy group with
DMTMM-BF4/NMM. Informed by this reaction screening, in the
library synthesis itself we coupled each DE-2 according to the
optimal conditions identified in this screen. In total, after
screening and applying the quality filters, a set of 102 different
amino acids were selected for DE-2.
Figure 1. A The macrocycle library produced by the Liu lab.[9a] B The
macrocycle library from the Neri lab looks at deep side-chain diversity derived
from a single scaffold. C The library described here uses a DE-1 comprised
exclusively of carbon or ether chains and a DE-2 that is a mix of carbon
frameworks and amino acids. D Representative diversity element members.
The macrocycle synthesis on DNA. The building blocks of the
two ring scaffold diversity elements (DE-1 & DE-2) were
designed to cover a broad swath of ring structures with different
sizes, shapes, strain and polarities (see the Electronic
Supporting Information [ESI] for building blocks) Fig. 1C shows
the general design of the DEML as well as a few representative
examples of the building blocks (Fig. 1D). The synthetic starting
point of the macrocycle ring was a readily available 2-
iodoterephthalic acid derivative (1 in Fig. 2A). In four chemical
steps this precursor could be elaborated into the building blocks
(BBs) for DE-1. Before embarking on the DEML synthesis we
next carried out a full macrocycle synthesis of a single library
member on a short single-stranded DNA so that we could
optimize the chemistry at each step (Fig. 2B&C).
Prior to macrocyclization the ester and the 2-
nitrophenylsulfonyl (Nos) group needed to be removed from 6.
Both could be done in
a one-pot operation using 2-
mercaptoethanol (BME) in combination with DBU (step 5, Figure
2) to deliver the macrocyclization precursor. This seemingly
simple step turned out to be tricky and dependent on the nature
of the DE-2 moiety. In the ESI we describe a detailed
optimization study across twenty representative macrocycle
precursors with constant DE-1 and varying DE-2 (Figure S11,
Table S3). Most of the test compounds showed good
deprotection efficiencies and low sideproduct formation. The
best substrates were members with cyclic (proline derived) DE-2
elements. In contrast, amino acids with sterically hindered side-
chains like adamantyl and dicyclohexyl were poorly deprotected.
An unusual side-product was observed in certain cases,
seemingly derived from cleavage of the trifunctional linker and
the DE-2 moiety to leave the terminal amide of, for example,
compound 2 (for details see Figure S9). The azide is responsible
Figure 2. A The DE-1 sub-library is created in four chemical steps from a
readily available precursor.
B A representative library member was
synthesized completely on ssDNA to probe the compatibility with DNA. C The
DNA remains intact throughout all seven steps of the macrocycle synthesis.
Nos = 2-nitrophenylsulfonyl.
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for this side-reaction since if we synthesize a variant of 4 with an
alkyne instead of an azide, it does not occur. The Asn was also
a poor performer in the Nos deprotection because the side-chain
amide seemed to back-bite onto the backbone under the
deprotection conditions. Nevertheless these undesired reactions
affected only a small subset of the DE-2 members (particularly
methylhistidine, serine) and hence we continued without further
optimization. Macrocyclization (step 6) and the CuAAC (step 7)
both occurred in high chemical yield and delivered pure product
7, according to LC-MS analysis. As shown in the PAGE gel (Fig.
2C) the DNA shows no detectable degradation over all seven
synthetic steps.
Split-and-pool synthesis of the library. With the synthetic
route to the DEML established, the next task was to elaborate
this into the full library. In DE-1 we included alkyl, aromatic,
heteroaromatic and polyene moieties as well as combinations
thereof. Since the DE-1 building blocks needed to be
synthesized individually we limited the number of members in
this group to twenty-one (synthetic procedures and analytical
data are found in the [ESI]). As iterative cross-coupling
strategies continue to evolve,[16] we expect that DE-1 diversity in
this scaffold could be expanded further. The DE-1 members
were directly encoded by clicking each individual DE-1 with a
Figure 3. Complete library synthesis and encoding strategy. The detailed
chemical steps are outlined in Figure 2.
Prior to protein binding assays all theoretical members of our
library were analyzed for their physicochemical properties with
the RDKit (www.rdkit.org) package, which included a custom
script to interface with our library tables (described in the ESI).
We compared the results with the published guidelines for oral
unique 5'-hexynyl modified oligonucleotide (Figure
3 and
17]
macrocyclic drugs recently defined by the Whitty[4,
and
Scheme S1 for complete library assembling procedure). The
DE-1 library was pooled and split in 102 vessels and coupled
with the DE-2s. The reactions were encoded by annealing with a
partially complementary DNA strand, bearing the coding
sequence for the introduced (second) diversity element in the 5’-
overhang. After Klenow fill-in the vessels were pooled and
purified to yield the DEML precursor library with 2142 encoded
macrocycle precursors. These were deprotected with BME/DBU
and macrocyclized with DMTMM at dilute concentration (10µM).
Random sampling of several of these macrocyclizations showed
at least 60% conversion and in most cases >80% conversion.
In preparation for introduction of the last diversity element the
coding double-stranded DNA was restricted by BamHI, yielding
a 5'-overhang of four bases on the non-macrocycle bearing
strand. Despite extensive optimization, restriction yield could not
be improved beyond 80% (see Figure S14 in the ESI for further
details). The restricted DEML was split into 663 vessels and
coupled with an alkyne library. The CuAAC was performed at
2.5 M concentration and so we used large excesses of alkyne
(100 eq.), copper catalyst (200 eq.) and ascorbate (200 eq.) to
obtain full conversion. The individual reactions were
subsequently encoded by T4 ligation and Klenow fill-in with a
pre-annealed partially double-stranded DNA strand, showing a
5'- phosphorylated GATC overhang and the six-bases codon for
the DE-3 (see Figure S15a in the ESI for detailed encoding
strategy). Purification of the DEML was performed by reversed
phase column chromatography.
Kihlberg[7, 18] groups (Figure 4). Our library falls largely within the
reported guidelines since the majority of the members showed a
molecular weight below 1000 Da, a total polar surface area
(TPSA) between 150 and 250 Å2 and an estimated partition
coefficient (AlogP) between 0 and 6. Furthermore the diversity of
our macrocycle scaffolds can be seen by the ring-size
distribution. While the majority of the members consists of 17 -
23 ring atoms, the range is from 16 up to 33 ring atoms. Further
property analyses and comparison to the published guidelines
are shown in the ESI.
Figure 4.
A Ring size distribution of all 2142 macrocycle scaffolds.
B Molecular property distribution across the entire 1.4 x 106-membered DEML
library. Blue/orange indicate the region found favorable for oral bioavailability
of known macrocyclic drugs according to published guidelines.
Protein selections. After these evaluations of the DEML we
used it for selections against two human blood serum proteins,
human serum albumin (HSA) and α-1-acid glycoprotein (AGP).
HSA is the most abundant protein in human blood serum and
carries mostly acidic ligands.[19] AGP is a second, less abundant
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transport protein in human blood serum, and it carries mainly
neutral and basic molecules such as the macrocyclic polymyxins
(e. g. Colistin).[20] Protein binding assays were performed on
hydrophilic streptavidin-coated magnetic beads, which were
pretreated with biotinylated HSA and AGP respectively.[21] The
DNA of the eluted binders from the treatments was amplified by
PCR and analyzed by next generation high throughput
sequencing in comparison with library fingerprint and bead only
(dummy) selections. The resynthesized hits from HSA selections
delivered mid-high micromolar binding and were not pursued
further (full discussion in the ESI). In the case of AGP binding
several hits were selected for resynthesis (see ESI for detailed
synthetic procedures). The fourth highest hit contained the most
highly represented DE-2 in the top 100 hits, and indeed showed
a strong thermal denaturation shift in a differential scanning
fluorimetry assay. Detailed characterization by isothermal
titration calorimetry (ITC) revealed a low micromolar dissociation
constant (KD = 7 µM, Figure 5c). Intriguingly, a similar binding
constant (KD = 4 M) was determined when we tested the azide
precursor (Figure 5d), suggesting that the macrocyclic scaffold,
and not the DE-3 side-chain, was the key for good binding. This
is supported by the fact that other similar scaffolds show no
binding in DSF (ESI-21 and ESI-23). Also indicative of the
importance of the macrocycle is the fact that the uncyclized
congener of the macrocycle lost >5-fold binding potency, giving
a Kd = 23.3 ± 3.8 M (see ESI, Figure S28).
We have synthesized and characterized a macrocycle library
bearing hydrophobic backbone elements reminiscent of
macrocyclic polyketide natural products. Although only 21
members, the bifunctional hydrocarbon DE-1 sub-library
preparation was the most time-consuming and challenging part
of the synthesis, highlighting the need for continued
development of carbon-carbon bond-forming reactions
compatible with DNA encoded library construction. Although
over a million members, a better way to conceptualize this
library is that it contains 2142 macrocyclic scaffolds each with
663 side-chain variations. It is therefore encouraging that even
with such a relatively small number of scaffolds, a low
micromolar hit could be identified in a protein selection. Scaffold
diversification is a great challenge in all types of library
preparation and our results reveal this is no different for
macrocycles. While the current state of synthesis will dictate that
encoded libraries are dominated by a single core structure with
diverse side-chains, iterative synthetic strategies[16, 22] and new
carbon-carbon bond-forming methods[23] compatible with DNA
would open new areas of chemical space to DNA encoded
chemical libraries. Our results with the DEML reported here
suggest this to be a compelling pursuit for organic synthesis.
Acknowledgements
The Swiss National Science Foundation (SNF) is gratefully
acknowledged for supporting this work (Grant: CRSII2_160699).
Keywords: macrocycles • DNA encoded libraries • Lipinski rules
• DNA chemistry • chemical library
References
[1]
[2]
E. M. Driggers, S. P. Hale, J. Lee, N. K. Terrett, Nat. Rev. Drug Discov.
2008, 7, 608-624.
(a) C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug
Deliv. Rev. 2012, 64, Supplement, 4-17; (b) C. A. Lipinski, J. Pharmacol.
Toxicol. Methods 2000, 44, 235-249.
[3]
(a) Y. Li, R. De Luca, S. Cazzamalli, F. Pretto, D. Bajic, J.
Scheuermann, D. Neri, Nature Chem. 2018, 10, 441-448; (b) D. L.
Usanov, A. I. Chan, J. P. Maianti, D. R. Liu, Nature Chem. 2018, 10,
704-714; (c) T. Passioura, T. Katoh, Y. Goto, H. Suga, Annu. Rev.
Biochem. 2014, 83, 727-752.
[4]
[5]
E. A. Villar, D. Beglov, S. Chennamadhavuni, J. A. Porco Jr, D.
Kozakov, S. Vajda, A. Whitty, Nat. Chem. Biol. 2014, 10, 723.
(a) S. Collins, S. Bartlett, F. Nie, H. F. Sore, D. R. Spring, Synthesis
(Germany) 2016, 48, 1457-1473; (b) A. K. Yudin, Chem. Sci. 2015, 6,
30-49.
[6]
[7]
A. Whitty, L. A. Viarengo, M. Zhong, Org. Biomol. Chem. 2017, 15,
7729-7735.
B. Over, P. Matsson, C. Tyrchan, P. Artursson, B. C. Doak, M. A. Foley,
C. Hilgendorf, S. E. Johnston, M. D. Lee Iv, R. J. Lewis, P. McCarren,
G. Muncipinto, U. Norinder, M. W. D. Perry, J. R. Duvall, J. Kihlberg,
Nat. Chem. Biol. 2016, 12, 1065.
Figure 5. A The four top hits from AGP selection results were resynthesized.
B The TCTT codon, corresponding to the 2-chlorophenyl side-chain, was the
most strongly selected codon in the top 100 hits. C ITC reports a KD = 7 M. D
The azide precursor to the selected macrocycle shows a similar binding
constant (KD = 4 M), indicating that the macrocycle scaffold is the key binding
feature.
[8]
(a) S. S. Kale, C. Villequey, X. D. Kong, A. Zorzi, K. Deyle, C. Heinis,
Nature Chem. 2018, 10, 715-723; (b) K. Deyle, X. D. Kong, C. Heinis,
Acc. Chem. Res. 2017, 50, 1866-1874; (c) T. J. Kang, Y. Hayashi, H.
Suga, Angew. Chem. Int. Ed. 2011, 50, 2159-2161; (d) Y. Yamagishi, I.
Shoji, S. Miyagawa, T. Kawakami, T. Katoh, Y. Goto, H. Suga, Chem.
Biol. 2011, 18, 1562-1570; (e) Y. Goto, T. Katoh, H. Suga, Nat. Protoc.
2011, 6, 779; (f) C. Heinis, T. Rutherford, S. Freund, G. Winter, Nat.
Chem. Biol. 2009, 5, 502.
[9]
(a) D. L. Usanov, A. I. Chan, J. P. Maianti, D. R. Liu, Nature Chem.
2018, 10, 704-714; (b) B. N. Tse, T. M. Snyder, Y. Shen, D. R. Liu, J.
Am. Chem. Soc. 2008, 130, 15611-15626; (c) Z. J. Gartner, B. N. Tse,
This article is protected by copyright. All rights reserved.

10.1002/anie.201902513
Angewandte Chemie International Edition
COMMUNICATION
R. Grubina, J. B. Doyon, T. M. Snyder, D. R. Liu, Science 2004, 305,
1601-1605; (d) Y. Li, R. De Luca, S. Cazzamalli, F. Pretto, D. Bajic, J.
Scheuermann, D. Neri, Nature Chem. 2018, 10, 441-448.
J. Zhang, X. D. Li, X. Li, X. Huang, X. Li, J. Am. Chem. Soc. 2018, 140,
15859-15867; (k) M. H. Hansen, P. Blakskjær, L. K. Petersen, T. H.
Hansen, J. W. Højfeldt, K. V. Gothelf, N. J. V. Hansen, J. Am. Chem.
Soc. 2009, 131, 1322-1327.
[10] R. Macarron, M. N. Banks, D. Bojanic, D. J. Burns, D. A. Cirovic, T.
Garyantes, D. V. S. Green, R. P. Hertzberg, W. P. Janzen, J. W. Paslay,
U. Schopfer, G. S. Sittampalam, Nat. Rev. Drug Discov. 2011, 10, 188.
[11] R. A. Lerner, S. Brenner, Angew. Chem. Int. Ed. 2017, 56, 1164-1165.
[12] S. Brenner, R. A. Lerner, Proc. Natl. Acad. Sci. USA 1992, 89, 5381-
5383.
[15] (a) R. M. Franzini, F. Samain, M. Abd Elrahman, G. Mikutis, A. Nauer,
M. Zimmermann, J. Scheuermann, J. Hall, D. Neri, Bioconjug. Chem.
2014, 25, 1453-1461; (b) Y. Li, E. Gabriele, F. Samain, N. Favalli, F.
Sladojevich, J. Scheuermann, D. Neri, ACS Comb. Sci. 2016, 18, 438-
443.
[13] (a) G. Zimmermann, D. Neri, Drug Discovery Today 2016, 21, 1828-
1834; (b) D. Neri, R. A. Lerner, Annu. Rev. Biochem. 2018, 87, 479-502.
[14] (a) Z. J. Gartner, D. R. Liu, J. Am. Chem. Soc. 2001, 123, 6961-6963;
(b) F. Buller, L. Mannocci, Y. Zhang, C. E. Dumelin, J. Scheuermann, D.
Neri, Bioorganic and Medicinal Chemistry Letters 2008, 18, 5926-5931;
(c) L. Mannocci, Y. Zhang, J. Scheuermann, M. Leimbacher, G. De
Bellis, E. Rizzi, C. Dumelin, S. Melkko, D. Neri, Proc. Natl. Acad. Sci.
USA 2008, 105, 17670-17675; (d) S. Melkko, J. Scheuermann, C. E.
Dumelin, D. Neri, Nat. Biotechnol. 2004, 22, 568-574; (e) M. A. Clark, R.
A. Acharya, C. C. Arico-Muendel, S. L. Belyanskaya, D. R. Benjamin, N.
R. Carlson, P. A. Centrella, C. H. Chiu, S. P. Creaser, J. W. Cuozzo, C.
P. Davie, Y. Ding, G. J. Franklin, K. D. Franzen, M. L. Gefter, S. P. Hale,
N. J. V. Hansen, D. I. Israel, J. Jiang, M. J. Kavarana, M. S. Kelley, C.
S. Kollmann, F. Li, K. Lind, S. Mataruse, P. F. Medeiros, J. A. Messer,
P. Myers, H. O'Keefe, M. C. Oliff, C. E. Rise, A. L. Satz, S. R. Skinner,
J. L. Svendsen, L. Tang, K. van Vloten, R. W. Wagner, G. Yao, B. Zhao,
B. A. Morgan, Nat. Chem. Biol. 2009, 5, 647; (f) C. Zambaldo, S.
Barluenga, N. Winssinger, Curr. Op. Chem. Biol. 2015, 26, 8-15; (g) D.
R. Halpin, P. B. Harbury, PLOS Biology 2004, 2, e173; (h) Y. Li, P.
Zhao, M. Zhang, X. Zhao, X. Li, J. Am. Chem. Soc. 2013, 135, 17727-
17730; (i) F. V. Reddavide, W. Lin, S. Lehnert, Y. Zhang, Angew. Chem.
2015, 127, 8035-8039; (j) Y. Zhou, C. Li, J. Peng, L. Xie, L. Meng, Q. Li,
[16] J. Li, S. G. Ballmer, E. P. Gillis, S. Fujii, M. J. Schmidt, A. M. Palazzolo,
J. W. Lehmann, G. F. Morehouse, M. D. Burke, Science 2015, 347,
1221-1226.
[17] E. A. Villar, D. Beglov, S. Chennamadhavuni, J. A. Porco, D. Kozakov,
S. Vajda, A. Whitty, Nat. Chem. Biol. 2014, 10, 723-731.
[18] P. Matsson, B. C. Doak, B. Over, J. Kihlberg, Adv. Drug Deliv. Rev.
2016, 101, 42-61.
[19] (a) G. Rabbani, S. N. Ahn, Int. J. Biol. Macromol. 2019, 123, 979-990;
(b) M. Fasano, S. Curry, E. Terreno, M. Galliano, G. Fanali, P. Narciso,
S. Notari, P. Ascenzi, IUBMB Life 2005, 57, 787-796.
[20] M. A. K. Azad, J. X. Huang, M. A. Cooper, K. D. Roberts, P. E.
Thompson, R. L. Nation, J. Li, T. Velkov, Biochem. Pharmacol. 2012,
84, 278-291.
[21] (a) W. Decurtins, M. Wichert, R. M. Franzini, F. Buller, M. A. Stravs, Y.
Zhang, D. Neri, J. Scheuermann, Nat. Protoc. 2016, 11, 764-780; (b) M.
Wichert, N. Krall, W. Decurtins, R. M. Franzini, F. Pretto, P. Schneider,
D. Neri, J. Scheuermann, Nature Chem. 2015, 7, 241-249.
[22] I. A. Stepek, J. W. Bode, Curr. Op. Chem. Biol. 2018, 46, 18-24.
[23] J. P. Phelan, S. B. Lang, J. Sim, S. Berritt, A. J. Peat, K. Billings, L. Fan,
G. A. Molander, J. Am. Chem. Soc. 2019, 141, 3723-3732.
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