A Mild, DNA-compatible nitro reduction using B2(OH)4

Article abstract of DOI:10.1021/acs.orglett.9b00497

A hypodiboric acid system for the reduction of nitro groups on DNA-chemical conjugates has been developed. This transformation provided good to excellent yields of the reduced amine product for a variety of functionalized aromatic, heterocyclic, and aliphatic nitro compounds. DNA tolerance to reaction conditions, extension to decigram scale reductions, successful use in a DNA-encoded chemical library synthesis, and subsequent target selection are also described.

Full text of DOI:10.1021/acs.orglett.9b00497

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
A Mild, DNA-Compatible Nitro Reduction Using B2(OH)4
Huang-Chi Du,† Nicholas Simmons,*,† John C. Faver, Zhifeng Yu, Murugesan Palaniappan,
Kevin Riehle, and Martin M. Matzuk
Center for Drug Discovery, Baylor College of Medicine, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: A hypodiboric acid system for the reduction of
nitro groups on DNA−chemical conjugates has been
developed. This transformation provided good to excellent
yields of the reduced amine product for a variety of
functionalized aromatic, heterocyclic, and aliphatic nitro compounds. DNA tolerance to reaction conditions, extension to
decigram scale reductions, successful use in a DNA-encoded chemical library synthesis, and subsequent target selection are also
described.
he DNA-encoded chemical library (DECL) screens are an
T
economic and efficient method for hit discovery.1 Recent
advances in DNA high-throughput sequencing and DNA
synthesis have enabled routine screens of large DECL
collections,2 and successful reports of DECL-based target
campaigns have spurred wide interest in the platform.1,3
However, solution-phase synthesis of DECLs is limited by
constraints imposed by DNA-integrity and DNA-solubility
concerns.4 Adaptation of common chemical reactions to a mild,
generally applicable, and partially aqueous condition is needed
to expand the repertoire of both DNA-compatible chemical
transformations5 as well as available DECL chemical space.6
Nitro-substituted, bifunctional compounds represent a
versatile set of building blocks for the synthesis of DECLsas
an incorporation of the nitro functional group for select drug−
target classes,7 as a substituent to facilitate other reactions under
DNA-compatible conditions (e.g., nucleophilic aromatic sub-
stitution8), but most importantly as a widely commercially
available set of masked, bifunctional anilines that can be unveiled
and screened directly or further functionalized in a subsequent
build cycle. As a routine transformation in drug synthesis,
numerous methods9 have been developed for nitro reduction
with metal reagents, such as iron10a or zinc,10b as well as
nonmetallic reagents, such as sodium dithionite10c and
trichlorosilane.10d However, in the context of DECL synthesis
there are limited disclosed methods. Satz and co-workers have
utilized a Raney-nickel system,5a we have described a sodium
dithionite protocol,11 and recently, Ding et al. have developed an
iron sulfate condition12 for use in the on-DNA synthesis of
benzimidazoles through an SNAr, o-amino nitro reduction, and
aldehyde condensation sequence (Figure 1A). However, to the
best of our knowledge, all of these conditions have only been
widely demonstrated in the reduction of o-aminonitroarenes,
and our attempts to develop dithionite as a general reducing
agent for nitroarenes was not successful.13 Seeking a general
condition for the synthesis of nonbenzimidazole DECLs, we
initiated studies to develop a mild and general nitro reduction
for application to a wide variety of on-DNA nitro substrates
(Figure 1, B).
Figure 1. On-DNA nitro reductions: (A) conditions from previous
work using Raney-Ni, sodium dithionite, or iron sulfate for the
reduction of ortho-amino nitroarenes; (B) this work.
We were intrigued by recent reports which utilized diboron
reagents for nitro reduction, with bis(pinacolato)diboron in
basic, alcoholic solvent at 110 °C14a or hypodiboric acid in
neutral water at 80 °C,14b conditions that might be tolerated by
DNA. We initiated our studies on substrate-functionalized
“headpiece” DNA commonly used in DECL synthesis, which
features a distal amino group for compound attachments
covalently connected to two complementary DNA strands via
a PEG-type bifurcated linker (our “headpiece” DNA is 17-bp
dsDNA with a 3′ overhang; see the Supporting Information for
the full structure). As our DECL reaction optimization and
applications were conducted on nanomole amounts of DNA at
micromolar concentrations, reaction assessments were con-
ducted by LC−MS.15 Preliminary on-DNA nitro reduction
experiments utilizing bis(pinacolato)diboron failed, likely due to
aqueous temperature limitations and poor reagent solubility.
However, upon switching to water-soluble hypodiboric
acid,16 a small amount of 2a was observed for model nitroarene
1a at ambient temperature in aqueous alcoholic solvent (Table
1, entry 1). Encouraged by this result, we began a pH screen
(Table 1, entries 2−5) which revealed a clear trend toward
enhanced conversions under increasingly basic conditions. This
is consistent with a proposed mechanism for diboron-mediated
Received: February 6, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b00497
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Table 1. Optimization of Nitro Reduction Conditions
Scheme 1. Substrate Scope of the Nitro Reduction
a
b
c
entry
buffer/base
cosolvent
2a (%)
d
1
2
3
4
5
6
7
8
pH 8.2 borate
MIPO
11
<5
28
26
81
80
78
68
40
pH 5.5 phosphate
pH 10.6 borate
pH 12 phosphate
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
MIPO
MIPO
MIPO
MIPO
CH3CN
none
e
DMA
f
9
10
11
DMF
g
DMSO
93
>95
CH3CH2OH
a
b
c
500 equiv used. 30% (v/v) cosolvent used. Conversion determined
d
e
f
by LC−MS. 1-Methoxy-2-isopropanol. Dimethylacetamide. Dime-
thylformamide. Dimethylsulfoxide.
g
nitro reduction, in which reduction is promoted by coordination
of an alkoxide to boron.14 We next set out to determine
cosolvent effects, utilizing several common, water-miscible
organic solvents. Use of acetonitrile, dimethylacetamide,
dimethylformamide, and pure water provided lowered con-
version to 2a, and although DMSO provided the aniline in
excellent conversion, high amounts of DMSO may impair
isolation of DNA (Table 1, entries 5−10). Ultimately, use of
aqueous ethanol with B2(OH)4 and NaOH cleanly provided 2a
in >95% conversion after only 2 h (Table 1, entry 11).
With this established condition in hand, we explored the
scope of this reaction with a range of nitroarene substrates
(Scheme 1). Based on our previous success, we first applied
these conditions against o-amino nitroarenes. Gratifyingly, o-
amino anilines 2a−e were cleanly formed, suggesting this
protocol could be useful for benzimidazole DECL processes. To
examine electronic effects, we investigated the formation of
electronic deficient sulfonyl-substituted anilines 2f−i, neutral
anilines 2j−l, and electron-rich methoxy-substituted anilines
2m−q. All provided the desired anilines in good to excellent
yields, although electron-rich nitro substrates were reduced at
slower rates. Notably, the urea linker used in 2k and 2l was
tolerated, as ureas may be cleaved under some basic conditions.
Substrates with functional groups susceptible to off-target
reduction, such as aldehyde 1r and nitriles 1s and 1t, underwent
nitro reduction without significant formation of reduced side
products. Halogenated nitroarenes are known to be problematic
substrates due to the formation of dehalogenated byproducts.17
Although formation of halogenated 2u−y was realized, the
desired anilines were formed in lowered yields, and significant
dehalogenation was observed for brominated 1y. Potential
coordination of a nearby group did not inhibit reduction
(methyl ester 2z and free acid 2aa), and steric effects appeared
minimal (ortho-disubstituted 2bb). Extension of these con-
ditions to nitro-substituted heterocycles was successful, with
pyridyl substrates 1cc−ee, azoles 1ff and 1gg, and electron-rich
heterocycles 1hh−jj all providing the desired amine product in
moderate to excellent yields. Finally, application to aliphatic
nitro substrates 1kk and 1ll furnished the expected amines.
Some on-DNA transformations exhibit DNA length related
effects, particularly when optimized on short DNA substrates.18
a
The conversions of nitro substrates 1 to amino products 2 are
b
shown. Conversion determined by LC−MS. On a 1,4-cyclo-
hexanedicarboxylic acid linker. Significant debromination also
observed. Product observed as the carboxylic acid.
c
d
To simulate a late-stage substrate in a DECL synthesis, we
prepared substrate 3 from nitroarene 1m, which features a 56-bp
B
DOI: 10.1021/acs.orglett.9b00497
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dsDNA tag. Application of the reduction conditions efficiently
provided the desired aniline, no evidence of DNA decom-
position was detected by LC−MS or gel electrophoresis, and the
product underwent efficient ligation with an additional DNA tag
to form 4 (Scheme 2; see the Supporting Information for full
details).
and nitro-functionalized benzoic acids were connected through
acylation, nitro-functionalized benzaldehydes were added
through reductive alkylation, and N-Boc protected diamines
and nitro-functionalized anilines were attached through a
“reverse” acylation reaction (Scheme 4). Within each well,
Scheme 4. Use of Nitro Reduction in a DECL Synthesis
Scheme 2. DNA Length, Stability, and Ligation Test
As this reaction proceeded at significantly lower temperatures
than previously reported,14 we sought to further validate
formation of the presumed on-DNA aniline product. Using
similar conditions modified for decigram scale, nitroarene
substrates 5 and 7 both provided the desired amine products in
good or better isolated yield within 5 min (Scheme 3). Thus,
Scheme 3. Nitro Reduction with B2(OH)4 on Decigram Scale
unique encoding DNA tags (codon 1) were then ligated,
followed by deprotection of N-Boc carbamates or reduction of a
nitro group to the corresponding amine. Before pooling, each
well was assessed by LC−MS to determine if the building block
coupled, if DNA ligation was completed, if the amino group was
fully formed, and if the final cycle 1 product was made in high
purity. As the deprotections were performed after the codon 1
tag ligation, the nitro reduction conditions were applied to
hundreds of DNA-linked substrates that each contained a
unique 30-bp dsDNA tag. Although some variability in
amidation efficiencies was observed (e.g., for very electron-
deficient nitro-anilines), in general, coupled nitro substrates
cleanly reduced to the amine with our hypodiboric acid
conditions (>80% conversion). However, consistent with
previous rate observations, some substrates with electron-
donating groups para to the nitro group had moderate
conversions (∼50%). Upon pooling and precipitation, post-
cycle 1 DNA recovery was excellent and similar to other in-
house DECL productions. After this material was split into
hundreds of wells, well-specific ligation of cycle 2 codons and
amine substrate functionalization by either nucleophilic
substitution of heteroaromatic dihalides, acylation of carboxyl-
functionalized aromatic halides, or reductive amination of
aldehyde-functionalized aromatic halides was performed. After
pooling, this material underwent a third cycle of splitting,
functionalization through palladium-catalyzed cross-coupling of
boronic acids and anilines, and ligation of cycle 3 codons to
ultimately provide a DECL of ∼75 million compounds (full
details are available in the Supporting Information).
further optimization of this system may be useful for applications
in which acidic/hydrogenation conditions are problematic or for
which metal contamination must be minimized.19
Having demonstrated the wide substrate scope of this
reaction, we next sought to use the nitro reduction within a
three-cycle, split-and-pool type DECL synthesis. Within this
DECL synthesis, each cycle consists of an initial split of DNA
“headpiece”-derived materials into hundreds of wells, the
attachment and/or transformation of a unique building block,
ligation of a unique encoding pair (“codon”) of DNA
oligonucleotides, and a final pooling. Thus, a three-cycle
DECL featuring hundreds of unique building blocks per cycle
will produce a collection of millions of small-molecule structures
each connected to a unique, identifying DNA sequence. Within
the first cycle, all transformations may be directly observed by
LC−MS, but later cycles have complex spectra due to pooling
and require other analysis methods.20 After DECL synthesis is
completed, adapted samples of the DECL may be amplified and
sequenced to allow analysis of the population of DNA sequences
within the sample. Altogether, evaluation of post-transformation
cycle 1 LC−MS traces, analysis of intra-DECL sequence
populations, comparison of the sequencing fidelity/amplifica-
tion efficiency to other in-house DECLs of identical DNA
architecture,21 and the discovery of structurally confirmed
DECL hits are definitive tests to determine a new trans-
formation’s DNA tolerance, susceptibility to DNA base-specific
effects, and overall suitability for practical DECL applications.
Starting from a DNA headpiece functionalized with linkers
featuring either amino or carboxyl termini that had been split
into hundreds of individual wells, N-Boc-protected amino acids
To probe for potential sequence-specific or well-specific
effects, a sample of the library was elaborated with DNA tags to
̈
enable a “naive” sequencing to assess the distribution of codons
within the unselected DECL. In DECL productions unbiased by
reaction-induced DNA damage, incomplete codon ligations, or
varying DNA recovery, cycle 1 codon populations are expected
to follow a Gaussian distribution.22 Furthermore, as only a
C
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Letter
subset of the DECL proceeded through a nitro reduction
pathway, comparison of codon populations encoding substrates
that underwent the nitro reduction versus the N-Boc
deprotection would serve as an intra-DECL control for potential
In summary, we have developed a DNA-compatible nitro
reduction for application to aromatic and aliphatic nitro groups
that maintains DNA integrity and ligation efficiency. The utility
of this methodology was demonstrated in the synthesis of a
DECL which was applied to a kinase target. This methodology
should complement existing nitro reduction approaches and be
useful in future DECL productions.
̈
nonspecific DNA damage. Gratifyingly, naive sample amplifica-
tion efficiency and the sequence fidelity of amplicons were
within expected ranges,21 no sequence-dependent population
effects were detected, codon 1 distributions were approximately
Gaussian, and the normalized populations of cycle 1 codons for
each deprotection pathway were nearly equivalent (Figure 2).22
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b00497.
Details of experimental procedures, DNA structures,
DECL construction, and selection experiments (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: nicholas.simmons@bcm.edu.
ORCID
Nicholas Simmons: 0000-0003-0766-5382
Author Contributions
†H.-C.D. and N.S. contributed equally.
Notes
Figure 2. Normalized, observed cycle 1 codon populations from the
unselected DECL split by an associated chemical deprotection method
(N-Boc deprotection or nitro reduction).22
The authors declare no competing financial interest.
Finally, this DECL was utilized in a selection23 against the
kinase PLK1, an important regulator of cell division in
eukaryotic cells.24 After a three-round selection of the DECL
against recombinant His-PLK1, amplification, and sequencing,
cheminformatics analysis25 and hit resynthesis revealed a
compound series which inhibited PLK1 kinase activity at low
nanomolar concentrations. This series contained 1,3-diamino-
benzene26 which had been produced in cycle 1 through two
independent pathways: the N-Boc deprotection of acylated 3-
(Boc-amino)aniline and the reduction of acylated 3-nitroaniline.
Enrichment of DNA encoding this building block by each
pathway was comparable at the mono-, di-, and trisynthon27
levels (Figure 3), providing additional evidence that the nitro
reduction provided the expected amine product and did not
induce pathway-specific effects on later DECL synthetic
transformations (see the Supporting Information for selection
details and n-synthon analysis).
ACKNOWLEDGMENTS
■
The authors acknowledge Kevin MacKenzie (Baylor College of
Medicine) for NMR spectroscopic assistance and Pranavanand
Nyshadham (Baylor College of Medicine) and Phil Rosner
(Baylor College of Medicine) for mass spectrometry assistance.
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
No. OPP1160866 from The Bill and Melinda Gates Foundation,
and National Institutes of Health Grant No. P01HD087157
from The Eunice Kennedy Shriver National Institute of Child
Health and Human Development.
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̈
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(9) For a recent review of nitro reduction conditions, see: Orlandi, M.;
Brenna, D.; Harms, R.; Jost, S.; Benaglia, M. Recent Developments in
the Reduction of Aromatic and Aliphatic Nitro Compounds to Amines.
Org. Process Res. Dev. 2018, 22, 430−445.
̌
́
(21) As our DECLs are built with chemically modified, intra-
molecularly joined segments of dsDNA, amplification does not proceed
at theoretical rates. Furthermore, amplification efficiency is significantly
affected by the design of adapters added to enable amplification/
sequencing/decoding (e.g., degenerate segments, primer overhangs,
etc.). Separately, DNA sequence fidelity is affected by the chosen
method of DNA sequencing and sample preparation. However, analysis
̈
of hundreds of naive/selection experiments using dozens of DECLs
that have been produced/sequenced with identical overall DNA
architectures/methods have provided an experimental baseline to
which can compare the performance of new DECLs.
(22) Assuming that equal concentrations of cycle 1 codons are present
in the library sample, the observed counts for cycle 1 codons after DNA
sequencing should follow a normal distribution centered at the average
̈
count. DNA sequencing of the naive DECL resulted in a number of
decoded molecules equal to 21674.6 times the diversity of the cycle 1
E
DOI: 10.1021/acs.orglett.9b00497
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
encoding region. Consequently, the observed average count for all c1
codons was 21674.6, and the count distribution of c1 codons roughly
followed a normal distribution with mean 21674.6. In Figure 2, counts
for c1 codons are normalized by the mean count to produce percentage
population fractions..
(23) DECL affinity “selections” are experiments designed to enrich
small-molecule binders of a biological target within a pool of DNA-
encoded compounds, and the structures of enriched binders are then
determined by DNA sequencing and bio- and cheminformatic analysis.
For a discussion of the principles and practice of DECL selection, see:
Chan, A. I.; McGregor, L. M.; Liu, D. R. Novel selection methods for
DNA-encoded chemical libraries. Curr. Opin. Chem. Biol. 2015, 26, 55−
61.
(24) Combes, G.; Alharbi, I.; Braga, L. G.; Elowe, S. Playing polo
during mitosis: PLK1 takes the lead. Oncogene 2017, 36, 4819−4827.
(25) For a discussion of our enrichment analysis method, see: Faver, J.
C.; Riehle, K.; Lancia, D. R.; Milbank, J. B. J.; Kollmann, C. S.;
Simmons, N.; Yu, Z.; Matzuk, M. M. Quantitative Comparison of
Enrichment from DNA-Encoded Chemical Library Selections. ACS
Comb. Sci. 2019, 21, 75−82.
(26) 1,3-Diaminobenzene is the deprotected structure of the cycle 1
building block; the full hit structure is not shown.
(27) The term “synthon” refers to each building block/encoding DNA
component and may be analyzed by itself (“mono-synthon”) or in
combination with components from other cycles (i.e., “di-synthon” or
“tri-synthon” within a three-cycle DECL).
F
DOI: 10.1021/acs.orglett.9b00497
Org. Lett. XXXX, XXX, XXX−XXX