Homogeneous and Functional Group Tolerant Ring-Closing Metathesis for DNA-Encoded Chemical Libraries

Article abstract of DOI:10.1021/acscombsci.9b00199

Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA-chemical conjugates lead to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally problematic functional groups, and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Additionally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected on-DNA [i, i+4] peptide.

Full text of DOI:10.1021/acscombsci.9b00199

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Article
Homogeneous and Functional Group Tolerant Ring-
Closing Metathesis for DNA-Encoded Chemical Libraries
Olivier B.C. Monty, Pranavanand Nyshadham, Kurt Bohren, Murugesan
Palaniappan, Martin M. Matzuk, Damian W Young, and Nicholas Simmons
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.9b00199 • Publication Date (Web): 08 Jan 2020
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ACS Combinatorial Science
Homogeneous and Functional Group Tolerant Ring-Closing
Metathesis for DNA-Encoded Chemical Libraries
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Olivier B. C. Monty,ab§ Pranavanand Nyshadham,a Kurt M. Bohren,a Murugesan Palaniappan,a Martin
M. Matzuk,a Damian W. Young,*a and Nicholas Simmons*a§
[a]Center for Drug Discovery and Department of Pathology and Immunology, Baylor College of Medicine, One Baylor Plaza,
Houston, Texas 77030, United States; [b]Rice University Department of Chemistry, 6100 Main Street, Houston, TX 77005
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KEYWORDS: DNA-encoded chemistry, DNA-encoded libraries, homogeneous ring-closing metathesis, functional group-
tolerant ring-closing metathesis, stapled peptides
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ABSTRACT: Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA–
chemical conjugates leads to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that
includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally
problematic functional groups and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Addition-
ally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a
single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected
on-DNA [i, i+4] peptide.
as novel molecular frameworks for the probing of unexplored
chemical space.22 DECL productions are based on combinato-
rial chemistry, and the RCM reaction would therefore be a sig-
nificant enhancement to the technology. Stimulated by our in-
terest in combining chemical diversity and large numbers for
drug discovery, we became interested in the application of
RCM to the production of DECLs. Our work was not done in a
vacuum, however, and the precedents upon which we built need
mentioning.
Introduction
Since the conception of DNA-encoded combinatorial chemi-
cal libraries by Brenner and Lerner,1 many variations of DNA-
encoded chemical screening technologies have been developed
to aid drug discovery efforts.2 Advancements both in the pro-
duction of large quantities of synthetic DNA oligomers3 and in
high-throughput DNA-sequencing methods4 now allow for
rapid and cost-effective screens of vast DNA-encoded chemical
library (DECL) collections against biological targets.5 DECLs
are single-pot chemical libraries consisting of compounds that
each possess a covalently-attached, unique DNA sequence
“barcode,” which enables identification of binder compounds
by DNA sequencing after multiplexed target-based screens.
Compared to high-throughput compound collections and
screens, DECLs are relatively inexpensive to prepare and use,6
and, most importantly, offer the opportunity for deeper explo-
ration of chemical space5 enabled by unprecedented numbers of
compounds per DECL—millions to trillions compared to only
hundreds of thousands per HTS chemical library.7 Additionally,
DECLs have provided starting points for the development of
several clinical candidates,6,8 and are considered by many to
have become one of the pillars of drug discovery.9 However,
while large numbers is the main advantage of DECLs, the
chemical diversity achievable under its umbrella is limited by
the number of effective DNA-compatible reactions. Currently,
only a limited set of DNA-compatible, solution-phase chemical
reactions have been reported,10–18 and expanding the repertoire
of chemical reactions to more effectively sample chemical
space is a major goal within this area.
The modification of proteins using the cross-metathesis (CM)
reaction29 has been demonstrated in aqueous tert-butanol, using
a high excess (10,000 eq) of MgCl2 as a Lewis acidic masking
agent. Additionally, RCM-stapling of unprotected peptides has
been achieved in water using the water-soluble AquaMet cata-
lyst, also in the presence of MgCl2 (400 eq).30 The Mg2+ ion is
believed to act as a mild Lewis acid, masking coordinating func-
tional groups typically prevalent in biomolecules.29,31 Finally
Lu et al.32 reported the use of a 3rd generation Grubbs cata-
lyst/MgCl2/aq. tert-butanol system for the on-DNA RCM and
cross metathesis (CM) reactions. While the latter study estab-
lished that RCM could be achieved on DNA–chemical conju-
gates, the reported conditions were not adequate, in our hands,
for the use of RCM in a DECL production.
The addressed limitations of the previously reported work32
include a) the insolubility of the Ru catalyst used in aqueous
tert-butanol, b) the tendency of phase separation between high-
salt (MgCl2) aqueous solutions and tert-butanol leading to het-
erogeneity, c) the absence of pH control, d) a narrowly explored
substrate scope, and e) the formation of significant amounts of
side products arising from catalyst decomposition, which limits
the yield of the reaction. Furthermore, we demonstrate 1) the
applicability of the developed conditions to a single-substrate
DECL as a way to test for susceptibility to varying DNA se-
quences, 2) the maintenance of DNA integrity post-RCM via
sequencing analysis, and 3) the first successful stapling of an
unprotected on-DNA [i, i+4] peptide as an illustration of the ro-
bust functional group tolerance of the developed conditions.
Within the compendium of synthetic methodologies, the ring-
closing metathesis (RCM) reaction has become a mainstay for
the construction of organic molecules spanning a wide range of
structural diversity and complexity. Accordingly, it has been
applied across, medicinal, natural product and diversity-ori-
ented synthetic endeavors.19–22 The widespread popularity of
RCM is grounded in several reasons: its broad and well-studied
functional group and substrate tolerance, the commercial avail-
ability of a wide array of air-stable, tunable Ru-based cata-
lysts,23,24 its successful application in aqueous media,25,26 and its
relevance to the production of drug-like compounds27,28 as well
Results and Discussion
Our work relies on three key findings: 1) a solvent system
that allows for homogeneous reaction conditions with much
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lower catalyst loading, 2) a non-classical acidic “buffer” that
the more robust catalyst B. Additionally, a reaction concentra-
tion of 0.02 mM would be manageable during DECL produc-
tion, which is the central motivation behind our efforts.
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not only avoids basicity but also masks coordinating functional
groups—in addition to the effect of MgCl2, and 3) the use of a
decomposition-resistant catalyst, B, that minimizes side reac-
tions and maximizes conversion. Our developed reaction con-
ditions are presented in Scheme 1 and our studies towards its
development and application are discussed in the following sec-
tions.
Development of a BrØnsted Acidic “Buffer.”
To avoid catalyst degradation at basic pH37 and to enhance
the masking of coordinating functional groups, we sought to in-
clude an acidic buffering system. Despite numerous reports of
RCM in the presence of aqueous buffers,38,39 we found that even
modest amounts of common buffers (e.g., phosphate) or “non-
coordinating” buffers40,41 (e.g., 2-(N-morpholino)ethanesul-
fonic acid) inhibited reactivity. Hypothesizing the suppression
was due to undesired coordination of the buffer’s non-halide
conjugate bases to the Ru center, we considered whether the in-
clusion of large amounts of NH4Cl could induce an acidic pH
without presenting RCM-suppressing buffer anions. Although
not a buffer in the traditional sense, NH4Cl may block Lewis-
base interactions via protonation and/or hydrogen-bonding
(e.g., a 4M aqueous solution of NH4Cl has a pH of ~5). Indeed,
as illustrated in Table 1, adding NH4Cl significantly enhances
conversion (e.g. entries 2 vs 9). With a homogenous solvent
system capable of tolerating high ionic strength in hand, we op-
timized the proportions of NH4Cl and MgCl2 for both high con-
version and quality of the LC-MS trace. Ultimately a combina-
tion of 20,000 equiv. of MgCl2 and 12,000 equiv. of NH4Cl for
30 min with 10 equiv. of catalyst42 was found to be optimal (Ta-
ble 1).
Scheme 1. Reaction Conditions and Performance Summary
of This Work versus Previously Reported Work
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Application of a Decomposition-Resistant Cata-
lyst.
i) Grubbs 3rd generation catalyst A and its 2,2’-biphenyldiamine
derivative B; ii) Previously reported conditions for the on-DNA
RCM and CM reactions; iii) Our main conditions for the on-DNA
Despite these encouraging results, conversions remained lim-
ited by the formation of side products. These appeared to origi-
nate in the reduction of one or both olefins, the loss of meth-
ylene from one of the olefins and/or the isomerization of the
starting material to an unreactive internal olefin (see Supporting
Information). Unfortunately, attempts to suppress those unde-
sired reactions with standard additives such as benzoquinones36
were unsuccessful (various benzoquinones were incompatible
with NH4Cl and also degraded DNA). While several catalytic
species have been proposed as culprits behind such side reac-
tions,37,43,44 a compelling study by Fogg and coworkers45—
showing that the addition of sub-stoichiometric amounts of a
poisoning ligand limits isomerization—proved to be key to our
discovery of the adequacy of B for our purposes. Indeed, we
found that the addition of 2,2’-biphenyldiamine, C, to A, as
shown in Scheme 2(ii), enhances the percent conversion (see
Supporting Information). However, we were aware of another
study by Fogg and coworkers46 which reported the 40% equi-
librium yield of decomposition-resistant catalyst B from A in
the presence of C, at RT. We therefore suspected that the in situ
formation of B was responsible for the conversion enhancement
observed. The substitution of B for A, as shown in Scheme
1(iii), indeed proved to significantly enhance conversion be-
yond what an A-C combination could achieve (Tables 2-4).
Since catalyst B was not commercially-available at the time of
the study and given its reported synthesis requires a glove box,46
we also studied alternative reaction conditions involving cata-
lyst A instead (Scheme 2).
*
RCM and CM reactions; Average percent conversion for the in-
vestigated substrate scope (22 substrates).
Development of a Solvent System for Reaction Ho-
mogeneity.
To enhance the reproducibility of RCM on substrates with
variably-sized DNA tags at a range of scales, our initial work
focused on developing a fully homogeneous catalyst–aqueous
solvent system combination. Unfortunately, many conventional
RCM catalysts (e.g., Grubbs I, Grubbs II, Hoveyda–Grubbs I,
Hoveyda–Grubbs II, Grela) are extremely insoluble in most
aqueous alcoholic mixtures and were ultimately found to be un-
productive for on-DNA RCM under a variety of conditions.
Ammonium-functionalized RCM catalysts (e.g., Aquamet)30,33
have high solubility in aqueous solutions but were also unpro-
ductive for on-DNA RCM, presumably due to DNA–
ammonium interactions.34 Even the fast-initiating Grubbs III
catalyst A (Scheme 1) utilized in the previously reported on-
DNA RCM exhibits limited solubility in alcohols (soluble up to
t
1 mM in pure BuOH). In addition, most non-alcoholic water-
miscible solvents (e.g., 1,2-dimethoxyethane or 1,4-dioxane)
are metal-coordinating and generally suppress metathesis. In-
formed by reports of conventional RCM in ethyl acetate as sol-
vent,35 we were pleased to discover methyl acetate as a much
better solvent for both catalysts A and B (~4 mM). When a me-
thyl acetate/ethanol/water solvent mixture was used, fully ho-
mogeneous reaction mixtures were observed, even for solutions
at high ionic strengths. Due to the fixed solubility of B in methyl
acetate, a reaction concentration of only 0.02 mM was possible
to maintain adequate solvent percentages. While high dilution
favors intramolecular metathesis, it can also stress the cata-
lyst36—a trade-off we attempted to mitigate through the use of
Of note, reactions within DECLs are typically performed on
nano- to micromole scales, and mass spectrometry is the main
method of characterization. Therefore, to further corroborate
the formation of the putative cyclized product, we developed a
chemical derivatization protocol through OsO4-mediated dihy-
droxylation, which reveals the number of olefins present
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(Scheme 3). Results of the dihydroxylation of the post-RCM
reaction mixture of 1a were consistent with the formation of the
cyclized product as the major product (see Supporting Infor-
ditions (Scheme 2)—to a series of substrate scope studies (Ta-
bles 2–4). Overall, while the conditions reported by Lu et al.
exhibit comparable conversions for simple substrates, signifi-
cant differences were observed for those containing unfavora-
ble coordinating functional groups.
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mation).
Table 1 Screen for the Optimal Equivalences of MgCl2 and
NH4Cl
We first investigated the ring-size scope of the reaction (Ta-
ble 2). Although all four conditions generally produced similar
results, the tBuOH/MgCl2 system significantly underperformed
in the case of 3a. This may stem from the presence of the glycol
chain within this substrate. Of note, these reactions were com-
plete after only 30 minutes at room temperature, which is re-
markable given it can take days and high temperatures for rings
of comparable size to close in organic media.24
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Table 2. Ring Size Scope Study
aAll reactions were run with 1 nmol of 1a. b The percent conver-
sions (% conv.) were determined by LC/MS after the quenching
procedure, as described in the Supporting Information. c The MS
trace was of higher quality relative to that of Trial 6 despite a lower
conversion. d The MS trace was of higher quality relative to Trial
10; * Catalyst B instead of A; Δ Catalyst B instead of A, reaction
time 5 min; ¥ Catalyst B instead of A, reaction time 60 min; e, f The
MS signals for those were of lower quality relative to Trial 10.
Scheme 2. Alternative Reaction Conditions using Catalyst
A
a
All reactions were run with 1 nmol of each diene; b The percent
conversions (% conv.) were determined by LC/MS after quench-
ing, as described in the Supporting Information;
terms of number of member atoms; Reaction conditions: Green =
Main Reaction Conditions, Blue = Alternative #2, Black = Alter-
native #1 and Red = Lu et al.
c-e
Ring sizes in
To investigate the functional group tolerance of the four re-
action conditions, we synthesized a series of substrates differ-
entiated only by substitution at a single position (Table 3). Us-
ing substrate 4a (R = H) as benchmark, we were able to demon-
strate the remarkable functional group tolerance of our main re-
action conditions. Indeed, functional groups such as the carbox-
ylic acid of 8a or the amine of 13a—groups typically protected
in organic-phase RCM—exhibited at least half the conversion
obtained for reference compound 4a. This suggests that the in-
corporation of NH4Cl exerts an acidic masking effect, con-
sistent with other reports of RCM in the presence of acidic ad-
ditives.47,48 In contrast, the previously reported unbuffered
MgCl2/tBuOH condition exhibited significant sensitivity to the
indole of 5a, the thiophene of 7a, the carboxylic acid of 8a and
the phenol of 10a.
*The screening results used to determine the optimal equivalence
of C are provided in the Supporting Information, Table S1. Δ Av-
erage percent conversion for the investigated substrate scope (22
substrates).
Scheme 3. Confirmation of Ring Formation via Chemical
Modification.
We also tested a series of on-DNA compounds designed to
investigate other DECL-relevant substrate features (Table 4).
While we had previously observed that internal olefins were un-
reactive to our RCM conditions, we sought to assess the impact
of small substituents in β-methyl 14a and ɑ-methyl 15a. When
compared to the analogous unsubstituted 1a, 14a and 15a were
cyclized in modest conversions, suggesting a low-tolerance for
We next turned to applying these four conditions—the previ-
ously reported conditions from Lu et al. (Scheme 1, ii), our main
reaction conditions (Scheme 1, iii), and the two alternative con-
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Table 4. Additional Substrate Scope Study
Table 3. Functional Group Tolerance Study
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a, b As in Table 2; Reaction conditions: Green = Main Reaction Con-
ditions, Blue = Alternative #2, Black = Alternative #1 and Red =
Lu et al.; nd = not determined; note that no thiol-containing sub-
strate was included due to dimerization via disulfide formation be-
tween DNA conjugates.
steric-encumbrance at or near the olefin. The presence of an al-
lylic chalcogen is known to enhance RCM efficiency through a
pre-association effect.49 However, comparable conversions
were observed for alkyl 16a and related allyl ether 17a. The
proximity of coordinating groups to the olefin may also inhibit
successful cyclization. Although all four RCM conditions ef-
fected negligible cyclization on 18a and 19a—bearing a proxi-
mally-located pyridyl and sulphonamide, respectively—a mod-
est amount of cyclization was observed for 21a—exhibiting a
more distally-located pyrimidine. Homoallylic alcohols are a
potential substrate series for on-DNA RCM, as the water-com-
patible allylation of aldehydes is well-known.50 Homoallyl al-
cohols 20a and 22a were successfully cyclized, although steri-
cally encumbered 20a afforded lower conversion. Finally we
applied these conditions to the CM of 5-hexenoic acid with the
CM-favored thioether substrate51 23a. All four conditions pro-
vided the CM product in moderate to good conversion (Scheme
4).
Scheme 4. On-DNA Cross Metathesis (CM)
a, b As in Table 2; c-k Ring sizes in terms of number of member at-
oms; Reaction conditions: Green = Main Reaction Conditions,
Blue = Alternative #2, Black = Alternative #1 and Red = Lu et al.
The reaction was run with 1 nmol of 23a; A 50 mM stock solution
of the 5-hexenoic acid building block was prepared in ethanol; The
percent conversions (% conv.) were determined by LC/MS after the
quenching procedure, as described in the Supporting Information;
Reaction conditions: Green = Main Reaction Conditions, Blue =
Alternative #2, Black = Alternative #1 and Red = Lu et al.
Certain on-DNA reactions exhibit sensitivity to the length of
the DNA-tag, which may be due to differences in solubility
and/or intermolecular DNA interactions. To test the viability of
our new RCM conditions on a late-stage DECL substrate, we
prepared 56-bp dsDNA substrate 24a from 17-bp dsDNA sub-
strate 1a. Application of our main reaction conditions (Scheme
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1, iii) to 24a afforded 24b at 69% conversion, which was close
tide 26b were tested within a homogeneous time resolved fluo-
rescence (HTRF) assay for binding to the coactivator region of
ERɑ. Both 26a and 26b displayed a dose-dependent HTRF re-
sponse and significantly enhanced co-activator region binding
was observed for stapled peptide 26b (see Supporting Infor-
mation). It is noteworthy that DECL technology is analogous to
phage display libraries.54 However, our methodology is partic-
ularly useful for incorporating unnatural amino acids such as
the α-methyl-α-pentenyl amino acid required for RCM stapling.
Although highly specialized systems for incorporating unnatu-
ral amino acids for phage display have been reported,55 there
have been no reports of making stapled peptides by phage dis-
play.
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to the 78% conversion observed for 1a (Scheme 5). After an
adapted precipitation procedure to minimize chaotropic effects
(see Supporting Information), the 56-bp dsDNA was obtained
in 20% yield (quantified by Bioanalyzer electropherogram anal-
ysis). Although the 56-bp dsDNA was the dominant component
within the post-RCM reaction mixture, small amounts of larger
DNA segments were observed (see Supporting Information),
which may arise from intermolecular metathesis. These may re-
quire removal by HPLC during full-scale DECL synthesis.
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Scheme 5. Application of the Main Reaction Conditions to a
56-bp DNA Substrate and to a Single-Substrate DECL
Scheme 6. RCM Stapling of an Unprotected [i, i+4] Peptide
Connected to DNA via Two Different Linkers
i) The reaction was run with 5 nmol of 24a; ii) The reaction was
run with 5 nmol of the single-substrate DECL.
The application of newly developed on-DNA conditions to a
pilot DECL and subsequent analysis by DNA sequencing are
useful tests prior to beginning a full DECL production. To in-
vestigate the potential for DNA base-related effects, we pre-
pared a small single-substrate DECL of ~47,000 unique 56-bp
dsDNA sequences from substrate 1a. This DECL was built
through a three-cycle “split-and-pool” approach, featuring three
cycles of splitting into portions, performing 36 ligations of
unique dsDNA tags, and pooling. All DNA tags used within a
cycle were of identical molecular weight, and thus the DECL
was observed as a single ensemble mass after purification by
HPLC (see Supporting Information). The application of our
main reaction conditions to the DECL resulted in the observable
loss of MW=28 (loss of ethylene from RCM cyclization). Ad-
ditionally, sequenced samples of the DECL before and after the
RCM reaction showed no significant differences, thus ascer-
taining DNA integrity post-RCM (see Supporting Information).
Each reaction was run with 1 nmol of material; the percent conver-
sion (% conv.) was determined by LC/MS after the quenching pro-
cedure, as described in the Supporting Information. Reaction con-
ditions: Green = Main Reaction Conditions, and Red = Lu et al.
Conclusions
In summary, we have developed a RCM reaction system pro-
moting homogeneity, minimization of side reactions and mask-
ing of coordinating functional groups, and 1) studied and con-
trasted its applicability to a diverse range of substrates, 2) en-
sured its compatibility to DNA through the successful cycliza-
tion of a single-substrate DECL, and 3) demonstrated its func-
tional group tolerance through the production of the first on-
DNA stapled peptide. We believe this work will allow for the
integration of the well-documented capacity of RCM to gener-
ate chemical diversity into DECLs. Efforts to apply this work
towards the production of RCM-based DECLs and their screen-
ing are ongoing within these laboratories and will be reported
in due course.
In addition to the synthesis of small-molecule macrocycles,
RCM has also been used to prepare stapled peptides—linear
peptides conformationally constrained through an intramolecu-
lar linkage—to simulate protein–protein interactions.52 To test
the relevance of our reaction conditions for the production of
on-DNA stapled peptides, we synthesized substrates 25a and
26a through copper-catalyzed azide–alkyne cycloaddition of an
alkynylated [i, i+4] SRC stapled peptide precursor known to
bind the coactivator region of ERɑ.53 The application of our
main reaction conditions to both substrates provided the stapled
peptide in moderate conversions while, in the case of 25a, sig-
nificantly outperforming the conditions reported by Lu et al.
(Scheme 6)—the comparison was not studied for 26a. To fur-
ther corroborate the successful production of the stapled pep-
tide, adapted samples of a control, peptide 26a and stapled pep-
Experimental Procedures
Each RCM substrate was constructed from 17-bp dsDNA
DNA headpiece (see Supporting Information for structure) and
was purified by HPLC due to RCM-inhibiting effects induced
by residual chemical building block impurities. Procedural de-
tails for all four RCM methods are included in the Supporting
Information. Additionally, while reaction mixtures involving
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(3)
Kosuri, S.; Church, G. M. Large-Scale de Novo DNA
17-bp dsDNA tagged substrates were fully homogeneous, those
with longer dsDNA tags (56 bp dsDNA) tended to be slightly
cloudy. While this did not appear to affect the reaction conver-
sion, a specific precipitation procedure was required to obtain
suitable DNA recovery (see Supporting Information). For ap-
plication to substrates with DNA tags of alternative sizes (>56-
bp) or composition, adjustments may be required to ensure sub-
strate solubility.
Synthesis: Technologies and Applications. Nat. Methods 2014, 11 (5),
499–507.
(4)
Nissen, I.; Kohler, M.; Beisel, C.; Neri, D. High-Throughput
Sequencing for the Identification of Binding Molecules from DNA-
Encoded Chemical Libraries. Bioorganic Med. Chem. Lett. 2010, 20
(14), 4188–4192.
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Buller, F.; Steiner, M.; Scheuermann, J.; Mannocci, L.;
(5)
Goodnow, R. A.; Dumelin, C. E.; Keefe, A. D. DNA-
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Encoded Chemistry: Enabling the Deeper Sampling of Chemical
Space. Nat. Rev. Drug Discov. 2017, 16 (2), 131–147.
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Yuen, L. H.; Franzini, R. M. Achievements, Challenges, and
ASSOCIATED CONTENT
Supporting Information.
Opportunities in DNA-Encoded Library Research: An Academic Point
of View. ChemBioChem 2017, 18 (9), 829–836.
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Content: general procedures and reaction conditions; catalyst and
substrate synthesis information; RCM-specific experimental proce-
dures; experiments for the confirmation of RCM cyclization; DNA
sequencing results for verification of DNA integrity post-RCM;
characterization information; post-metathesis MS deconvolution
spectra.
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Dickson, P.; Kodadek, T. Chemical Composition of DNA-
Encoded Libraries, Past Present and Future. Org. Biomol. Chem. 2019,
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Finger, J. N.; et al. DNA-Encoded Library Screening Identifies
Benzo[b][1,4]Oxazepin-4-Ones as Highly Potent and Monoselective
Receptor Interacting Protein 1 Kinase Inhibitors. J. Med. Chem. 2016,
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Harris, P. A.; King, B. W.; Bandyopadhyay, D.; Berger, S.
This material is available free of charge via the Internet at
http://pubs.acs.org.”
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Neri, D.; Lerner, R. A. DNA-Encoded Chemical Libraries:
AUTHOR INFORMATION
Corresponding Author
A Selection System Based on Endowing Organic Compounds with
Amplifiable Information. Annu. Rev. Biochem. 2018, 87 (1), 479–502.
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Kowalczyk, A.; Petersen, A.; Naderi-Oboodi, G.; Orzechowski, L.;
Strebel, Q. DNA Compatible Multistep Synthesis and Applications to
DNA Encoded Libraries. Bioconjug. Chem. 2015, 26 (8), 1623–1632.
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Employing Photoredox Catalysis for DNA-Encoded Chemistry:
Decarboxylative Alkylation of α-Amino Acids. ChemMedChem 2018,
13 (20), 2159–2165.
(12) Li, J.-Y.; Huang, H. Development of DNA-Compatible
Suzuki-Miyaura Reaction in Aqueous Media. Bioconjug. Chem. 2018,
29 (11), 3841–3846.
(13) Ruff, Y.; Berst, F. Efficient Copper-Catalyzed Amination of
DNA-Conjugated Aryl Iodides under Mild Aqueous Conditions.
Medchemcomm 2018, 9 (7), 1188–1193.
(14) Li, Y.; Gabriele, E.; Samain, F.; Favalli, N.; Sladojevich, F.;
Scheuermann, J.; Neri, D. Optimized Reaction Conditions for Amide
Bond Formation in DNA-Encoded Combinatorial Libraries. ACS
Comb. Sci. 2016, 18 (8), 438–443.
(15) Du, H. C.; Simmons, N.; Faver, J. C.; Yu, Z.; Palaniappan,
M.; Riehle, K.; Matzuk, M. M. A Mild, DNA-Compatible Nitro
Reduction Using B 2 (OH) 4. Org. Lett. 2019, 21 (7), 2194–2199.
(16) Wang, X.; Sun, H.; Liu, J.; Dai, D.; Zhang, M.; Zhou, H.;
Zhong, W.; Lu, X. Ruthenium-Promoted C–H Activation Reactions
between DNA-Conjugated Acrylamide and Aromatic Acids. Org. Lett.
2018, 20 (16), 4764–4768.
(17) Xu, H.; Ma, F.; Wang, N.; Hou, W.; Xiong, H.; Lu, F.; Li,
J.; Wang, S.; Ma, P.; Yang, G.; et al. DNA‐Encoded Libraries: Aryl
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* E-mail: nsimmons@its.jnj.com.
* E-mail: Damian.young@bcm.edu.
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script. / §These authors contributed equally.
Funding Sources
This work was supported by the Welch Foundation (Grant Q-
0042), a Core Facility Support Award (RP160805) from the Cancer
Prevention Research Institute of Texas (CPRIT), grant
OPP1160866 from The Bill and Melinda Gates Foundation, and
National Institutes of Health grant P01HD087157 from The Eunice
Kennedy Shriver National Institute of Child Health and Human
Development.
ACKNOWLEDGMENT
We would like to acknowledge Kevin MacKenzie (Baylor College
of Medicine—BCM) for NMR spectroscopic assistance,
Murugesan Palaniappan (BCM) and Zhifeng Yu (BCM) for naive
sequencing assistance, Kevin Riehle (BCM) for bioinformatic
analysis assistance, John C. Faver (BCM) for cheminformatic
analysis assistance, and the Fogg Research Group (University of
Ottawa) for the generous provision of catalyst B. We would also
like to acknowledge Srinivas Chamakuri (BCM) for his
unwavering generosity and invaluable guidance.
ABBREVIATIONS
CM, cross metathesis; DECL, DNA-encoded chemical library;
DNA, deoxyribonucleic acid; ERα, estrogen receptor alpha; HPLC;
high performance liquid chromatography; HTRF; homogeneous
time-resolved fluorescence; RCM, ring-closing metathesis.
(19) Stanton, B. Z.; Peng, L. F.; Maloof, N.; Nakai, K.; Wang,
X.; Duffner, J. L.; Taveras, K. M.; Hyman, J. M.; Lee, S. W.; Koehler,
A. N.; et al. A Small Molecule That Binds Hedgehog and Blocks Its
Signaling in Human Cells. Nat. Chem. Biol. 2009, 5 (3), 154–156.
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Reactions in Total Synthesis. Angew. Chemie Int. Ed. 2005, 44 (29),
4490–4527.
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