Selective Chemical Functionalization at N6-Methyladenosine Residues in DNA Enabled by Visible-Light-Mediated Photoredox Catalysis

Article abstract of DOI:10.1021/jacs.0c10616

Selective chemistry that modifies the structure of DNA and RNA is essential to understanding the role of epigenetic modifications. We report a visible-light-activated photocatalytic process that introduces a covalent modification at a C(sp3)-H bond in the methyl group of N6-methyl deoxyadenosine and N6-methyl adenosine, epigenetic modifications of emerging importance. A carefully orchestrated reaction combines reduction of a nitropyridine to form a nitrosopyridine spin-trapping reagent and an exquisitely selective tertiary amine-mediated hydrogen-atom abstraction at the N6-methyl group to form an α-amino radical. Cross-coupling of the putative α-amino radical with nitrosopyridine leads to a stable conjugate, installing a label at N6-methyl-adenosine. We show that N6-methyl deoxyadenosine-containing oligonucleotides can be enriched from complex mixtures, paving the way for applications to identify this modification in genomic DNA and RNA.

Full text of DOI:10.1021/jacs.0c10616

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Selective Chemical Functionalization at N6‑Methyladenosine
Residues in DNA Enabled by Visible-Light-Mediated Photoredox
Catalysis
Manuel Nappi,† Alexandre Hofer,† Shankar Balasubramanian,* and Matthew J. Gaunt*
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ABSTRACT: Selective chemistry that modifies the structure of DNA
and RNA is essential to understanding the role of epigenetic
modifications. We report a visible-light-activated photocatalytic
process that introduces a covalent modification at a C(sp3)−H bond
in the methyl group of N6-methyl deoxyadenosine and N6-methyl
adenosine, epigenetic modifications of emerging importance. A
carefully orchestrated reaction combines reduction of a nitropyridine
to form a nitrosopyridine spin-trapping reagent and an exquisitely
selective tertiary amine-mediated hydrogen-atom abstraction at the
N6-methyl group to form an α-amino radical. Cross-coupling of the
putative α-amino radical with nitrosopyridine leads to a stable conjugate, installing a label at N6-methyl-adenosine. We show that
N6-methyl deoxyadenosine-containing oligonucleotides can be enriched from complex mixtures, paving the way for applications to
identify this modification in genomic DNA and RNA.
which to covalently modify and manipulate the N6-methyl-
adenine nucleobase; there is currently no method to directly
modify nucleic acids at N6-methyl deoxyadenosine or N6-
methyl adenosine residues, and a selective chemical trans-
formation must target the C(sp3)−H bonds of the N-
methylamine motif amidst thousands of similarly reactive
entities.
We were inspired by the cellular processing of the
methylation state in oligonucleotide sequences, wherein a
class of dioxygenases, which include ALKBH1, have been
proposed to demethylate N6mdA in mammalian DNA9,11
through the action of electrophilic enzyme-bound Fe(IV)O
intermediates (Figure 1B).18 By analogy with the correspond-
ing demethylation in RNA,19 the C−H bond of the N6−
methyl group would project toward the promiscuous Fe-bound
oxygen-centered radical facilitating selective hydrogen atom
abstraction (HAA), which is followed by an oxygen-rebound
process with the resulting Fe(III)−OH species en route to
formation of N6-hydroxymethyl-adenosine and spontaneous
expulsion of formaldehyde. We questioned whether a synthetic
process could generate a discrete α-amino radical intermediate
on the N6-methyl group of N6mdA, mimicking the enzymatic
INTRODUCTION
■
Nucleic acids display several types of C(sp3)−H bonds within
their canonical nucleotides, each with subtly different intrinsic
reactivities that are influenced by steric, inductive, and
conjugative effects imparted by the proximal chemical
environment. Beyond the core genetic information stored by
the four-letter nucleotide sequences of A, C, G, and T(U),
nucleic acids contain a second layer of molecular programming
in the form of reversible chemical modifications to its
nucleobases, the so-called epigenetic code, which introduces
further diversity to the catalogue of C(sp3)−H bonds present
in these biomacromolecules. Methylation at specific positions
of the nucleobases provides the most common means by which
genomic DNA is reversibly marked.1,2 Bacteria can methylate
A and C in their own genome,3 while in eukaryotes, DNA
methylation was thought to only occur at C,2 which has been
linked to gene regulation. Importantly, the discovery of novel
chemical methods for the selective modification of 5-
methylcytosine (5mC) and its derivatives in DNA has been
unequivocally responsible for a better understanding of its
function.4 Recently, N6-methyl deoxyadenosine (N6mdA,
Figure 1A) has been reported (somewhat controversially) in
various eukaryote genomes,5−16 and comprehension of its
biological roles remains nascent.5,10 In contrast, the presence of
N6-methylation at adenosine (m6A) in the RNA world is well-
established and has been implicated in a wide range of cellular
processes.17 Even though methyl groups within N-methyl-
amines are not traditionally reactive, the exclusivity of this
motif might underpin a site-selective chemical approach with
Received: October 6, 2020
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A

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Figure 1. Evolution of a strategy for the chemical modification of N6mdA in DNA. (A) Canonical DNA bases and N6mdA. (B) Proposed
mechanism for biochemical demethylation of N6mA via Fe-dependent enzyme-controlled hydrogen atom abstraction and oxygen-rebound. (C)
Plan for covalent modification at N6mdA via trapping of an “on-DNA” α-amino radical intermediate.
demethylation pathway, but also intercept the incipient radical
with a modular reagent, installing a covalently bound label
directly onto N6mdA-containing DNA sequences (Figure 1C).
The C−H bonds in N6mdA’s methyl group have fairly high
pair on the N6-atom and display “hydridic” character (Figure
2A).23,24 In contrast, the C−H bonds of the methyl group of
thymine, although weaker, are relatively neutral as a result of
being adjacent to the less polarizing pyrimidine heterocycle.
This subtle electronic effect may provide a sufficiently distinct
reactivity profile to enable a kinetically controlled polarity
match between an electrophilic hydrogen atom abstracting
agent and the more hydridic C−H bonds in the N6-
methylamine motif in N6mdA. The resulting C−H bond
cleavage would lead to formation of an α-amino radical on the
modified nucleotide. The intercepting reagent must react
quickly with the incipient N6mdA-derived α-amino radical and
form an open shell species more stable than its precursor. We
reasoned that deployment of a spin trapping reagent could
provide a potential solution to this challenge. Spin trapping
reagents (STRs) are highly reactive molecules, frequently used
in excess quantities to capture radicals in the form of persistent
radical products, which can enable the identification of short-
lived species in complex systems.25 Among commonly used
STRs, nitrosoarenes are particularly suitable for the inter-
ception of nucleophilic carbon-centered radicals, the properties
of which should be inherent to a N6mdA-derived α-amino
radical. Nitrosoarene-derived STRs are, however, highly
electrophilic and often display promiscuous nonradical
reactivity with nucleophiles, can undergo facile dimerization,
and readily decompose to nonproductive products. To
compound these problems, a nitrosoarene must also be
compatible with the HAA step, itself a radical reaction,
without displaying deleterious reactivity. We questioned
whether these problems might be circumvented if a process
could be designed wherein the STR was generated in situ,
consequentially linked to the chemistry required to facilitate
bond dissociation energies (BDE, ∼92−94 kcal/mol).20
A
selective reagent will need to target a strong C−H bond that is
present at extremely low effective concentration (reported
N6mdA levels in higher eukaryotes range from 5 ppm to 0.05%
for N6mdA with respect to A),9,11,12 among a plethora of
similar strength or weaker C−H bonds: for example,
deoxyribose units contain many different C−H bonds, each
with similar BDE’s,21 and the methyl group in thymidine (and
5mC) displays activated C−H bonds with lower BDE (89−90
kcal/mol).22 Moreover, such a N6mdA functionalization
strategy also necessitates a method to productively intercept
the “on-DNA” α-amino-radical to fashion a stable covalent
linkage to the oligonucleotide. The challenges associated with
addressing these problems are multifaceted: first, use of a
proximity-driven rebound mechanism thought to facilitate
enzymatic demethylation is unlikely to be feasible in a
synthetic scenario, and so the coupling step will need to be
fast in order to accommodate the likely short lifetime of the
DNA-derived α-amino-radical; second, the HAA and covalent
functionalization steps need to operate in concert while
minimizing deleterious and nonselective reactivity.
These difficulties place additional constraints on potential
chemical solutions that must already operate at low
concentrations, in aqueous solutions, and avoiding acidic or
oxidative conditions, which might damage the nucleic acid
architecture.
The C−H bonds in the methyl group of N6mdA will be
partially polarized as a result of their interaction with the lone
B
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the HAA step, thereby closely linking the proximity of this
reactive species to the incipient N6mdA-derived radical.
Accordingly, we hypothesized that a mild method to reduce
a water-soluble nitroarene might be leveraged alongside the
HAA step for the in situ generation of a nitrosoarene STR and
lead to a selective functionalization process (Figure 2B).
RESULTS AND DISCUSSION
■
Our design plan for a C(sp3)−H functionalization of N6mdA
focused on the visible light-mediated reduction of 3-nitro-
pyridine 1a, a water-soluble oxidant that could serve as a
precursor for the nitrosoarene STR (Figure 2C). We
speculated that Ru(II)(phen)3Cl2 could function as a photo-
catalyst because it displays adequate aqueous solubility and the
oxidative quenching cycle of its triplet excited-state (E[Ru-
(II)*/Ru(III)] = −0.87 V),26 accessed by visible-light
irradiation, is well matched to the reduction potential of the
nitropyridine (Ered = −0.44 V vs SCE estimated based on the
value for 4-nitropyridine);27 nitropyridine-derived radical
anion int I would be produced alongside [Ru(III)(phen)3].
Int I could undergo hydrogen atom transfer with the bulk
solvent (or perhaps N6mdA) to int II and eliminate water to
form the STR, 3-nitrosopyridine 2a.28−32
Crucially, the concentration of the 3-nitrosopyridine (2a)
would be controlled by the photochemical activity of the
catalyst, avoiding the presence of potentially deleterious
superstoichiometric levels of STR. Concurrent with the
formation of the STR, the [Ru(III)(phen)3] intermediate
(E[Ru(III)/Ru(II)] = +1.26 V)33 would be reduced by
quinuclidine 3 (Eox = +1.10 V vs SCE),34 reforming the
photocatalyst to close the cycle and, importantly, generate the
quinuclidine radical cation (int III), a powerful electrophilic
hydrogen atom abstractor. Protonated quinuclidine has a BDE
of 101 kcal/mol34 meaning that its radical cation could be
sufficiently reactive to remove a hydrogen atom from the
N6mdA-methyl group; MacMillan has shown that the
quinuclidine radical cation displays polarity-matched reactivity
for strong electron-rich C−H bonds under mild reaction
conditions via Ir-catalyzed photoredox-mediated single elec-
tron oxidation.24,33 Therefore, selective HAA from the methyl
group in N6mdA by int III will generate the desired α-amino
radical (int IV). Cross coupling of the 3-nitrosopyridine STR
2a with α-amino radical int IV would then generate a nitroxide
persistent radical (int V), which can ultimately undergo well-
established oxidative decay to fashion a covalent modification
at the N6-position of the nucleobase in the form of a N-
hydroxyformamidine linkage 4.35
Our initial studies focused on establishing a cross coupling
protocol on a representative oligonucleotide 5a
(CTTGACAG[N6mdA]CTAG, Figure 3A). Following an
extensive assessment of the reaction parameters, the explor-
atory experiments revealed that irradiation of a solution of 5a,
3-nitropyridine 1a, quinuclidine 3, and [Ru(phen)3]Cl2 with a
60 W CFL bulb for just 10 min at room temperature led to the
formation of N-hydroxyformamidine-N6mdA oligonucleotide
conjugate 6 with 16% conversion to product, as determined by
LC-MS analysis (Figure 3A). A 30% conversion to the
demethylated oligonucleotide 7 was also observed.36,37 It is
important to stress that both conjugate 6 and demethylated
oligonucleotide 7 arise from the same putative α-amino radical,
int-IV, which reflects a 46% conversion of the N6-methyl
group via the new hydrogen atom abstraction process and is
surprisingly high given the highly complex molecular frame-
Figure 2. Design plan for a N6mdA-selective functionalization
process. (A) Nitrosoarene-derived STRs; can a stable precursor,
such as a nitroarene, be used for in situ generation of reactive spin
trapping reagents? (B) Selective HAA at N6mdA. (C) Design plan for
photoredox-facilitated covalent modification of N6mdA based on the
merger of selective HAA and spin trapping via in situ generation of
nitrosoarenes.
C
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Figure 3. Visible light-mediated photoredox strategy for covalent functionalization of N6mdA. (A) Oligonucleotide conjugation via HAA and spin
trapping via an oxidative photocatalytic quenching cycle with [Ru(II)(phen)3]Cl2. (B) The use of modular nitropyridine probes in oligonucleotide
functionalization and subsequent elaboration by Huisgen cycloaddition. (C) Selectivity parameters in the oligonucleotide functionalization are
defined as “HAA selectivity” (reflecting the position of C−H bond cleavage) and “Probe selectivity” (reflecting the selectivity of reaction via
nitrosopyridine vs nitropyridine). (D) Scope of N6mdA functionalization using standard conditions detailed in panels A and B. For the reaction of
5c, Ru(bpz)3(PF6)2 was used as a catalyst.
work upon which this transformation is affected. We believe
that the constant oxidative quenching of the triplet excited
state of the metallophotocatalyst by 3-nitropyridine and high
concentration of quinuclidine prevents oxidative damage of
DNA, especially at G nucleobases. This is reflected in the
observation that the transformation using the Ru(phen)3Cl2 as
catalyst produces a cleaner reaction profile compared to the
use of other, more oxidizing photocatalysts, [Ru(II)(bpz)3]-
(PF6)2 (Figure S16, Supporting Information). The oligonu-
cleotide conjugate (6) has a half-life of approximately 12 h at
room temperature in neutral or basic solutions (pH 7−11).
We next sought to incorporate a latently reactive
functionality capable of downstream elaboration to tailored
nucleic acid fragments. A design-augmentation process
revealed an alkyne-containing, amide-linked nitropyridine 1b
could be coupled with 5a upon treatment with the
D
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Figure 4. Pull-down strategy for the enrichment of N6mdA-containing oligonucleotides. (A) A pull down procedure involving photoredox
functionalization with an alkyne-derived nitropyridine, Huisgen cycloaddition with a biotin-derived azide, immobilization on streptavidin coated
magnetic beads, oligonucleotide separation by sequential washing, and selective cleavage of N6mdA-derived oligonucleotides delivers an
enrichment of >50:1. (B) Pull down experiments using 99nt ssDNA and 99bp dsDNA in the presence and absence of salmon sperm (SS) DNA
demonstrate enrichment in complex mixtures of DNA sequences; the blue dot indicates the position of N6mdA. dsDNA displaying the methyl
group (red) of the N6mdA nucleobase (blue) in the major groove.
E
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Ru(phen)3Cl2, quinuclidine, and irradiation for 10 min (Figure
3B), forming the desired alkyne-containing oligonucleotide 8
(11% conversion to product, identified by HRMS). It is
notable that 1b is devoid of potentially competitive hydridic
C−H bonds and the amide substituent does not seem to affect
the oxidative reactivity of the 3-nitropyridine core.
Exploiting the newly installed alkyne functionality, we found
that a “click” Huisgen-cycloaddition between 8 and PEG3
biotin-derived azide 9 necessitated specific conditions for an
effective reaction; a solution of copper sulfate and sodium
ascorbate required the addition of quinuclidine (presumably to
act as a ligand for the copper-catalyst) to facilitate cyclo-
addition to the biotin-conjugated oligonucleotide 10 with 92%
conversion to product.
A series of control experiments showed that the photoredox
coupling reaction on an oligonucleotide without a N6mdA
residue (CTTGACAGACTAG, 7) formed no N-hydroxyfor-
mamidine-containing products arising from the incorporation
of a 3-nitrosopyridine unit, indicating, as expected, that
hydrogen atom abstraction does not take place in the
oligonucleotide unless the N6-methylation is present. It is
remarkable that the hydrogen atom abstraction step is so
exquisitely selective for the N6-methyl group in spite of the
vast number of similar C−H bonds in oligonucleotides
(termed “HAA selectivity”, Figure 3C). We were, however,
able to detect trace levels of oligonucleotides that had a mass
ion reflecting the inclusion of an intact 3-nitropyridine (16
mass units higher than N-hydroxyformamidine-derived oligo-
nucleotide 8). Although we were not able to elucidate the
structure of this trace-level modification, a series of control
experiments revealed that the addition of 3-nitropyridine was
taking place at G residues (Figures S18 and S19, Supporting
Information). We were able to calculate that selectivity for the
formation of the desired N6mdA-derived N-hydroxyformami-
dine linkage compared to the inclusion of 3-nitropyridine at G
was 50:1 for N6mdA per G nucleobase (termed “Probe
selectivity”, Figure 3C), a ratio which is, again, quite
remarkable given the proclivity of G nucleobases to undergo
oxidative side reactions.
procedure installs the N-hydroxyformamidine linkage, a more
labile functional group, which we believed would permit the
use of significantly milder, nucleophile-mediated, cleavage
conditions in the retrieval of the labeled oligonucleotide. This
is important because mass analysis of the photoredox reaction
mixtures had suggested that the trace products arising from
unselective functionalization at G do not contain an electro-
philic N-hydroxyformamidine linkage. Consequently, we
speculated that these off-target products of functionalization
at G could be retained on the streptavidin beads during
cleavage, thereby enhancing the selectivity observed in the
photoredox step and enrichment of the N6mdA-derived
oligonucleotide. Guided by this hypothesis, we began the
pull-down and enrichment procedure by conducting the
photoconjugation with oligonucleotide 5a and nitropyridine
1b in the presence of a distinct but, importantly, non-
methylated oligonucleotide CGTACTAGACG 11, as a means
to test whether our method could be used to enrich N6mdA-
containing oligonucleotides (Figure 4A). The N-hydroxyfor-
mamidine product 8 was formed with 10% conversion and
observed by LC-MS alongside unreacted 5a, the demethylated
oligonucleotide 7, the control oligonucleotide 11, and traces of
the G-nitropyridine functionalized oligonucleotide (50:1 probe
selectivity, N6mdA/G). Subsequent cycloaddition with biotin-
azide 9 afforded the N6mdA biotin-conjugated oligonucleotide
10. Treatment of the oligonucleotide mixture with streptavi-
din-coated magnetic beads allowed immobilization of all of the
species containing biotin (specifically, 12 plus the trace levels
of product arising from unselective reaction at G residues) and
permitted the removal of unlabeled oligonucleotides (7 and
11) via successive washing procedures. Following this, we
found that the electrophilic nature of the N-hydroxyformamide
linkage made it susceptible to reaction with aqueous hydrazine
and led to the release of an N6-(hydrazonomethyl)dA-
containing oligonucleotide 13 with a small amount of 7
(arising from the hydrolysis of 13) and trace quantities of the
other oligonucleotides that had been indiscriminately retained
by the streptavidin-coated beads. The recovery of both 13 and
7 provides direct evidence for the presence of N6mdA in the
starting sequence, and their ratio to all other oligonucleotides
gives rise to an enrichment greater than 50:1. It is particularly
important to note that the maximum theoretical enrichment
value that can be obtained as a result of the observed
photoredox probe selectivity is ∼17:1, since oligonucleotide 13
contains three G residues (probe selectivity of 50:1 N6mdA per
G residue). Therefore, the observed enrichment of >50:1
clearly demonstrates that the hydrazine cleavage procedure is
selective for N-hydroxyformamidine linkage in the N6mdA-
derived oligonucleotide conjugates versus products of reaction
at G (that are presumably retained on the beads), leading to
the observed enhanced enrichment.
Figure 3D shows a preliminary scope of the N6mdA
functionalization tactic, with the conversions to conjugates in
line with those observed in the optimization studies. Not only
did the reaction work on oligonucleotides in combination with
nitropyridines 1a and 1b but it also converted longer and self-
complementary DNA sequences (42 and 49mers) to the
desired products. Importantly, a reaction with an N6-methyl
adenosine residue in an RNA oligonucleotide was successfully
converted to the corresponding conjugate although the yields
were lower than for the DNA congeners; partial strand
decomposition was observed in the RNA oligonucleotide,
which will require further optimization of the reaction
conditions. Nevertheless, the success of this methodology on
RNA oligonucleotides has many potential applications in the
emerging field of m6A-focused epitranscriptomics.17
The versatile biochemical properties inherent to the biotin
motif provide a means to isolate the modified N6mdA-derived
oligonucleotide from other nucleic acid fragments via a
streptavidin-based pull-down procedure,38 which could enable
us to enrich N6mdA-containing oligonucleotides in complex
mixtures. Classical methods for substrate retrieval from
streptavidin pull-down protocols involve relatively harsh
reaction conditions, which are designed to denature the
protein scaffold. However, our photoredox conjugation
To simulate the complex matrix of a cellular DNA sample,
where the concentration of N6mdA with respect to dA will be
very low, we combined longer single-stranded (ss) DNA
fragments (99 nucleotides: N6mdA-containing oligonucleotide
14(m) and sequence 15 with canonical dA residue) with a 10-
fold excess of salmon sperm DNA to create complex DNA
mixtures with N6mdA/dA ratios of 1:383 (0.26%). Applying
the photoconjugation and pull-down procedure to these
samples and now using quantitative PCR (qPCR) to analyze
the enriched fractions and determine the amplifiable amount of
both initially methylated 14(m) and 15 DNA sequences after
the pull-down, we found that the N6mdA-containing ssDNA
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fragment (14(m), Figure 4B, experiment (i)) was enriched to a
level of approximately 10:1 (Figure S32, Supporting
Information). Importantly, a parallel experiment (ii) using
the same nucleic acid sequences but having the N6mdA residue
in the other sequence (15(m)) showed a similar level of
enrichment (11:1). These results clearly demonstrate that
N6mdA is required for the enrichment, the observed
enrichment is not dependent on the oligonucleotide sequence,
and the protocol is functional in complex DNA mixtures.
In double stranded DNA, the methyl group of N6mdA is
thought to project into the major groove of the double helix,
providing an additional challenge for the photoredox
functionalization of complex nucleic acid samples due to
potentially adverse steric and electronic effects that arise from
the local chemical environment (see the image in Figure 4B).
Despite this congestion, we found that on applying the
sequential photoredox conjugation, click reaction, and pull-
down procedure to a mixture of N6mdA-containing 99 base
pair dsDNA (16(m)), experiment (iii) and a nonmethylated
double stranded fragment (17) resulted in an enrichment of
4:1 (Figure S33, Supporting Information).
A parallel experiment (iv) using the same nucleic acid
sequences but having two N6mdA residues in the second
template (16 and 17(2m)) showed that the N6mdA-
containing dsDNA (17(2m)) fragment was recovered with
an enrichment of greater than 9:1, indicating a positive
cumulative effect. Furthermore, when 16(m)/17 was com-
bined with SS DNA (N6mdA/dA ratio is 1:3433, 0.03%) in
experiment (v), the N6mdA-containing oligonucleotide 16(m)
was again recovered with an enrichment of almost 4:1. The
corresponding experiment (vi) using the same nucleic acid
sequences but with two N6mdA residues in the second
template (N6mdA/dA ratio is 1:1717, 0.06%) showed an
increased enrichment of greater than 8:1 for the N6mdA-
derived ds-oligonucleotide 17(2m), again highlighting that the
presence of additional N6mdA residues enhances the output
(Figure S34, Supporting Information). Taken together, this set
of experiments demonstrates the applicability of the developed
chemistry on longer ssDNA, dsDNA, as well as in complex
samples with excess DNA, providing a proof of concept for the
enrichment of N6mdA-DNA strands and showcasing the
potential for future application with this underexplored
methylated nucleotide.
method for locating N6mdA in genomic DNA can be founded
will require a number of further challenges to be addressed,
despite the remarkable selectivity observed in this functional-
ization process. First, while the photocatalytic hydrogen atom
abstraction process proceeds with competent efficiency (46%
conversion, a combination of conjugation and demethylation,
with respect to the starting oligonucleotide), the spin trapping
of the N6mdA-derived oligonucleotide radical requires
improvement in order to increase the yield of the desired
conjugation product. Second, the already high (50:1) probe
selectivity for reaction at N6mdA per G residue could be
further increased because the effective discrimination of the
process becomes diminished as the length of the oligonucleo-
tide increases (due to the increased concentration of G
residues in complex DNA). Finally, further investigation of the
unexplored reactivity of N-hydroxyformamidines should lead
to improved chemoselectivity in the release step of the pull-
down procedure and higher final enrichment. Together, the
next phase of these studies will focus on the development of a
detection protocol for N6mdA-containing oligonucleotides that
already returns an enrichment of 4:1 for a single N6-
methylation in a 99 base pair double stranded fragment that
is part of a complex DNA matrix. Not only might this
technology provide an unequivocal answer to the controversy
surrounding the presence of N6mdA in genomic mammalian
DNA but could also lead to sequencing methods that will
further unravel the role of this epigenetic modification and
should also be amenable to targeting methylated nucleobases
in the many forms of RNA that regulate cellular function.17,39
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c10616.
■
sı
Materials and methods, experimental procedures, useful
1
information, optimization studies, H NMR spectra, 13
C
NMR spectra, and MS data (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Shankar Balasubramanian − Department of Chemistry,
University of Cambridge, Cambridge CB2 1EW, United
Kingdom; Cancer Research UK Cambridge Institute,
University of Cambridge, Cambridge CB2 0RE, United
Kingdom; School of Clinical Medicine, University of
Cambridge, Cambridge CB2 0SP, United Kingdom;
orcid.org/0000-0002-0281-5815; Email: sb10031@
cam.ac.uk
CONCLUSION
■
While the cell’s biochemical machinery is capable of regulating
the methylation state of A in nucleic acids, we have developed
a selective chemical transformation that generates and
intercepts an “on-DNA” α-amino radical to form a stable
covalent modification at N6mdA residues. Orchestrated by a
visible light-activated photoredox catalyst, a polarity-matched
hydrogen atom abstraction step at the N6-methyl group of
N6mdA generates an α-amino radical and dovetails with a
distinct, in situ reaction to form a nitrosopyridine spin trapping
reagent. Together, these features lead to a radical cross
coupling process that introduces a modular functional handle
into oligonucleotide sequences. This strategy is underpinned
by a previously unknown transformation founded on a
mechanistically unique and remarkably selective photoredox
cross coupling reaction, which targets a traditionally unreactive
and scarce motif amidst the complex scaffold of nucleic acids.
To set this work in context, the evolution of this synthetic
method toward a basic technology upon which a chemical
Matthew J. Gaunt − Department of Chemistry, University of
Cambridge, Cambridge CB2 1EW, United Kingdom;
orcid.org/0000-0002-7214-608X; Email: mjg32@
cam.ac.uk
Authors
Manuel Nappi − Department of Chemistry, University of
Cambridge, Cambridge CB2 1EW, United Kingdom;
orcid.org/0000-0002-3023-0574
Alexandre Hofer − Department of Chemistry, University of
Cambridge, Cambridge CB2 1EW, United Kingdom;
orcid.org/0000-0001-6396-3689
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c10616
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Author Contributions
†M.N., A.H., S.B., and M.J.G. devised the project. M.N. and
A.H. conducted and analyzed the experiments. M.N., A.H.,
S.B., and M.J.G. wrote the manuscript. M.N. and A.H.
contributed equally.
Notes
The authors declare the following competing financial
interest(s): S.B. is an advisor and shareholder of Cambridge
Epigenetix Ltd. A patent application is pending.
ACKNOWLEDGMENTS
■
We are grateful to the Marie Curie Foundation (H2020-
MSCA-IF-2017, ID: 789407 and H2020-MSCA-IF-2015, ID:
702462), Swiss National Science Foundation
(P2ZHP2_168448), Leverhulme Trust (RPG-2016−370)
EPSRC (EP/N031792/1), Wellcome Trust (209441/z/17/
z), Cancer Research UK program grant (C9681/A18618) and
core funding (C14303/A17197), and the Royal Society (for
Wolfson Merit Award).
(16) Musheev, M. U.; Baumgartner, A.; Krebs, L.; Niehrs, C. The
origin of genomic N6-methyl-deoxyadenosine in mammalian cells.
Nat. Chem. Biol. 2020, 16, 630−634.
(17) Zaccara, S.; Ries, R. J.; Jaffrey, S. R. Reading, writing and
erasing mRNA methylation. Nat. Rev. Mol. Cell Biol. 2019, 20, 608−
624.
(18) Martinez, S.; Hausinger, R. P. Catalytic Mechanisms of Fe(II)-
and 2-Oxoglutarate-dependent Oxygenases. J. Biol. Chem. 2015, 290,
20702−20711.
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