Exploiting Substrate Promiscuity to Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes

Article abstract of DOI:10.1021/jacs.8b04722

Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme's crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.

Full text of DOI:10.1021/jacs.8b04722

Subscriber access provided by YORK UNIV
Communication
Exploiting Substrate Promiscuity to Develop
Activity-Based Probes for TET Family Enzymes
Uday Ghanty, Jamie E. DeNizio, Monica Yun Liu, and Rahul M. Kohli
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 06 Dec 2018
Downloaded from http://pubs.acs.org on December 6, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Exploiting Substrate Promiscuity to Develop Activity-Based Probes
for TET Family Enzymes
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Uday Ghanty†,‡, Jamie E. DeNizio , Monica Yun Liu , Rahul M. Kohli
†,‡
†,‡
†,‡,*
†Department of Medicine, ‡Department of Biochemistry and Biophysics, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104.
Supporting Information Placeholder
ABSTRACT: TET enzymes catalyze the repeated
oxidation of 5-methylcytosine in genomic DNA. Due to
the challenges of track-ing reactivity within a complex
DNA substrate, chemical tools to probe TET activity are
limited, despite these enzyme’s crucial role in epigenetic
regulation. Here, building on precedents from related
Fe(II)/-ketoglutarate-dependent dioxygenases, we
show that TET enzymes can promiscuously act upon
cytosine bases with unnatural 5-position modifications.
Oxidation of 5-vinylcytosine (vC) in DNA results in the
predominant formation of a 5-formylmethylcytosine
product that can be efficiently labeled to provide an
endpoint read-out for TET activity. The reaction with 5-
ethynylcytosine (eyC), moreover, results in the
formation of a high-energy ketene intermediate that can
selectively trap any active TET isoform as a covalent
enzyme-DNA complex, even in the complex milieu of a
total cell lysate. Exploiting substrate promiscuity
therefore offers a new and needed means to directly
track TET activity in vitro or in vivo.
assays employed are of limited throughput, relying upon
indirect analysis with coupling enzymes or degradation
of DNA into nucleosides, followed by LC/MS/MS or
1
-5,7-9
TLC analysis.
Similarly, while genetic approaches
have provided functional insights, understanding when
and where TET enzymes are active has been limited by
the lack of any activity-dependent chemical probes.
TET enzymes are Fe(II)/-ketoglutarate (-KG)-
dependent dioxygenases that act via generation of a
reactive Fe(IV)-oxo intermediate that drives proton
abstraction and subsequent rearrangements that result in
10
substrate oxidation. The fact that mC, hmC, fC, and
even rarely T, can each be substrates for oxidation
11-14
suggests a degree of intrinsic promiscuity.
Tolerance
to substrate variations has been previously observed
with another Fe(II)/-KG-dependent dioxygenase,
thymine hydroxylase (TH), that acts on the free thymine
nucleobase; Stubbe and coworkers showed that various
unnatural modifications at the 5-position of uracil could
15,16
be oxidized by TH.
We reasoned that if TET
enzymes are similarly active on unnatural 5-modified
cytosine bases built into DNA, then those alternative
substrates might be useful for tracking TET activity.
Methylation of cytosine bases in a CpG context is an
important epigenetic modification involved in gene
silencing, imprinting, and cellular differentiation. While
Given the steric constraints evident in the DNA-bound
structure of a truncated, active version of human TET2
1
2
(
TET2-CS) (Figure S1), we focused on exploring TET
5
-methylcytosine (mC) represents the most prevalent
reactivity with two substituents of comparable size to
ox-mCs, 5-vinylcytosine (vC) and 5-ethynylcytosine
(eyC) (Figure 1). To this end, we first generated DNA
modification, the discovery of ten-eleven translocation
(TET) family enzymes dramatically expanded the
complexity of the epigenetic landscape (Figure 1A).
1
containing the desired modifications.
A
20-bp
TET
hydroxymethylcytosine (hmC), which can be further
oxidized to 5-formylcytosine (fC) and 5-
enzymes
can
oxidize
mC
to
5-
oligonucleotide substrate (S20) was synthesized
containing 5-iodocytosine (iC) at the 5´-end. This
terminal base was then converted to vC (S20-vC) or eyC
carboxylcytosine (caC), and these oxidized mC bases
(ox-mCs) can both serve as intermediates in DNA
(
S20-eyC), respectively, by a modified Suzuki-Miyaura
1
7,18
1
-5
cross-coupling reaction
or a modified Sonogashira
demethylation and play independent epigenetic roles.
1
9
reaction performed on-resin (Figure S2). The purified
oligonucleotides were hybridized to an unmodified
complement strand and reacted either with or without
TET2-CS, under conditions optimized for activity.
When analyzed by ESI-MS, we readily detected new
The importance of ox-mCs is evident in loss-of-function
studies that highlight a host of pathologies, including a
6
strong association with hematologic malignancies.
In spite of the significance of TET enzymes, studying
their activity has remained a significant challenge. Most
ACS Paragon Plus Environment

Journal of the American Chemical Society
between the proposed possibilities, the S20-vC product
Page 2 of 5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was incubated with Na(OAc) BH prior to labeling with
3
ARP-ONH (Figure S3A). A mass shift was no longer
2
observed, and the ESI-MS was consistent with reductive
amination (Figure S3B), which would only be possible if
5
-formylmethylcytosine is the major product. To also
probe the minor product (~5:1 ratio, Figure S3B)
identity, we synthesized 5-(1,2-dihydroxyethyl)-C-
containing DNA, which could plausibly arise via
hydrolysis of an epoxide intermediate, as previously
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15,16
observed for the reaction of 5-vinyluracil with TH;
2
D-TLC co-spotting supported the identification of the
diol as the minor product (Figure S4).
We were intrigued that, unlike mC, which can be
oxidized multiple times, S20-vC appears to only be
oxidized once, generating the aldehyde. Structural
modeling suggested that while vC can be
accommodated, the oxidation product might be sterically
excluded from further oxidation (Figure S1). Consistent
with this hypothesis, we generated a bulkier 5-
propenylcytosine containing substrate (S20-pC) and did
not observe product formation by 1D-TLC (Figure S5).
Figure 1. Promiscuity of TET activity on unnatural 5-
modified cytosine bases in DNA. (A) TET enzymes can
accept three different potential substrates (mC, hmC and
fC), generating three associated products (hmC, fC and
caC). (B) S20-vC (50 nM) was reacted with or without
TET2-CS (0.6 µM) and the purified DNA analyzed by ESI-
MS. The masses listed are consistent with the substrate
oligonucleotide strand (red), the major product (blue),
minor product (black), and the major product derivatized
with hydroxylamine aldehyde reactive probe (ARP)
Having established that TET enzymes show
promiscuity towards these unnatural 5-modified
substrates, we next aimed to exploit this reactivity in the
development of new chemical probes for TET activity.
To facilitate the generation of DNA probes, we
synthesized nucleotide triphosphate versions of 5-
vinylcytosine and 5-ethynylcytosine (Figure S6). A 90-
bp DNA template was designed containing ten central
CpG pairs (S90) and lacking other cytosines outside of
(green). In concert with reduction experiments (Figure S3),
ARP conjugation identifies the major product as 5-
formylmethylcytosine. (C) S20-eyC was similarly reacted
and analyzed by ESI-MS. The substrate oligonucleotide
mass is shown in red and the noted product mass in blue,
corresponding to 5-carboxymethylcytosine.
products, suggesting that TET2-CS is able to
accommodate these unnatural substrates for oxidation
(Figure 1B, 1C). For S20-vC, the major product showed
a mass increase consistent with a single oxidation event
and a heavier minor product, that could be consistent
with a diol. For S20-eyC, the recovered product detected
was consistent with a carboxymethyl product; however,
we consistently also noted a decreased recovery of DNA
after the reaction.
We sought to better characterize the reaction products
resulting from S20-vC oxidation. According to the TET
reaction mechanism, the Fe(IV)-oxo intermediate could
potentially abstract a proton from either carbon of the
vinyl group. Subsequent hydroxyl radical rebound and
tautomerization could generate either a 5-acetyl or 5-
formylmethyl product (Figure S3). To confirm that one
of these singly-oxidized species represented the major
product, we incubated S20-vC reaction product with an
aldehyde/ketone reactive hydroxylamine derivative
Figure 2. 5-vinylcytosine offers an endpoint assay for TET
activity. PCR using modified dCTPs was used to generate
9
0-mer DNA substrates with ten CpG modifications (S90-
xC10). The S90-xC10 substrates (50 nM) were reacted with
or without TET2-CS (0.6 µM) and then derivatized with
the fluorescent aldehyde-reactive Alexa Fluor 647
hydroxylamine (AF-647). The reactions were analyzed on a
5% TBE-PAGE gel and imaged for AF-647 fluorescence
and then stained with ethidium bromide (EtBr) to detect
total DNA. S90-fC10 without TET2-CS oxidation was used
as a positive control for AF-647 labeling.
(
ARP-ONH ) and observed a quantitative shift in the
2
major product (Figure 1B). Then, to differentiate
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
the primers. The template was amplified with modified
the species as an enzyme-DNA complex, we generated
smaller 59-bp Cy5-labeled DNA substrates containing
less CpGs (S59-eyC ; S59-eyC ). We also purified the
1
2
3
4
5
6
7
8
9
dCTPs resulting in symmetrically modified DNA (S90-
xC ) with vC or eyC at the central CpG sites. Control
1
0
3
1
DNA was also synthesized with unmodified C, mC or
fC. The S90-xC10 substrates were incubated in the
presence or absence of TET2-CS. As aldehyde-reactive
probes have proven utility with fC detection,20 the
reaction products were then incubated with the Alexa
Fluor 647 hydroxylamine (AF-647). The reactions were
run on a polyacrylamide gel and then imaged for AF-
full catalytic domains of TET1, TET2 and TET3. We
observed that all combinations of TET enzymes with
either S90-eyC10 or S59-eyC3 resulted in activity-
dependent complex formation (Figure S9A). The
complexes ran at different masses that correlated with
the sizes of each isozyme or DNA substrate. As further
validation, Proteinase K eliminated detection of the
enzyme-DNA complex (Figure S9B). When excess
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
47 fluorescence (Figure 2). Across the substrate series,
we observed AF-647 labeling of S90-vC10 treated with
TET2-CS, and not with S90-C , S90-mC , or S90-
enzyme was reacted with S59-eyC , intact DNA was
1
about equivalent to enzyme-DNA complex, suggesting
complex formation is at least as efficient as
carboxymethyl generation (Figure S10).
1
0
10
eyC . The labeling is comparable to that of an S90-fC
1
0
10
control without TET reaction, suggesting efficient
conversion. To compare reactivity, we also performed a
titration of TET2-CS against S90-xC10 substrates and
assessed substrate consumption by LC/MS/MS (Figure
S7). Consumption of vC and eyC was, respectively,
only ~1.7-fold or ~3.5-fold reduced relative to mC,
suggesting robust reactivity. In the AF-647 labeling
experiment, it is particularly notable that although mC
oxidation can generate fC among its products, under
these conditions no labeling of S90-mC was observed,
1
0
likely due to conversion to caC. The fact that vC
generates the aldehyde as the major and terminal product
makes this unnatural substrate particularly useful as a
means to assay for TET endpoint activity.
In the reaction of S90-eyC with TET2-CS (Figure 2),
1
0
we were struck by one added observation: as with S20-
eyC, the recovery of S90-eyC10 was diminished after
reaction. In studying 5-ethynyluracil, Stubbe and
colleagues had previously demonstrated that oxidation
by TH led to the generation of a highly-reactive ketene
intermediate that reacted with and covalently inhibited
1
6
TH. In a similar vein, Cravatt and colleagues employed
aryl acetylenes as activity-based probes of P450
2
1
enzymes. Recognizing these precedents, we considered
whether a ketene intermediate that could yield either a
carboxymethyl product or a stable protein-DNA
complex could explain the eyC-DNA loss (Figure 3A).
Figure 3. 5-ethynylcytosine is an activity-dependent probe
forming enzyme-DNA crosslinks. (A) Oxidation of eyC
could result in the formation of a high-energy ketene
intermediate that could either yield the carboxymethyl
product or an enzyme-DNA complex. (B) Cy5 labeled S90-
xC10 substrates (50 nM) containing C, mC or eyC were
reacted with TET2-CS (0.6 µM). The reactions were
analyzed by PAGE and the gels imaged for Cy5
fluorescence or stained for protein (Sypro Ruby). The
formation of a new species with eyC was dependent upon
activity in the presence of the co-substrate -KG. (C) Cell
extracts from HEK293T cells transfected with empty vector
(X), TET2-CS (WT), or catalytically inactive TET2 (Mut)
were incubated with S90-eyC10 substrate. The enzyme-
DNA crosslinked species are identical to those observed
when purified TET2-CS was added to extracts from cells
transfected with the empty vector (X + Enz).
To explore this hypothesis, we generated the S90-xC10
substrates with a 5´-Cy5 labeled primer and reacted
either with or without TET2-CS. The reaction products
were analyzed by SDS-PAGE and imaged first for Cy5
fluorescence to detect the DNA and then stained to
detect protein. With the S90-eyC10 alone, and not with
S90-C10 or S90-mC , we observed the formation of a
10
new species containing Cy5-fluorescence running at a
molecular weight higher than the DNA substrate or
TET2 alone (Figure 3B). Eliminating (-KG, an
essential co-substrate, from the reaction results in
disappearance of the new species, implying
a
requirement for TET activity, and formation of the
species tracked with pH and salt conditions preferred for
optimal TET reactivity (Figure S8). To better identify
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
As a demonstration of the utility of eyC in biological
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
settings, we examined the activity-dependent probe in
cell lysates. HEK293T cells were transfected with a
wild-type TET2-CS expression plasmid, a catalytically-
inactive mutant (HxD), or an empty plasmid control. 24
hrs after transfection, the cells were collected, lysed and
the total cell lysate incubated with S59-eyC under TET
This work was supported by the National Institutes of
Health R01-GM118501. J.E.D was supported by T32-
GM07729 and the NSF; M.Y.L by F30-CA196097. We
thank J. Salvino and F. Mohammed for experimental
assistance.
1
0
reaction conditions. As predicted, lysates from cells
transfected with mutant TET2 or empty vector did not
result in a new Cy5-labeled species. By contrast, cells
expressing TET2-CS yielded a new enzyme-DNA
complex (Figure 3C), whose identity was further
confirmed by both Western blotting and by comparison
to the complex formed with purified TET2-CS (Figure
S11). This complex was not formed with the HxD
mutant and required -KG (Figure 3C), demonstrating
that eyC can indeed function as an activity-based probe
even in a complex cell lysate.
REFERENCES
(1) Tahiliani, M.; Koh, K. P.; Shen, Y.; Pastor, W. A.;
Bandukwala, H.; Brudno, Y.; Agarwal, S.; Iyer, L. M.; Liu, D. R.;
Aravind, L.; Rao, A. Science 2009, 324, 930-935.
(2) Ito, S.; Shen, L.; Dai, Q.; Wu, S. C.; Collins, L. B.; Swenberg,
J. A.; He, C.; Zhang, Y. Science 2011, 333, 1300-1303.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(3) He, Y. F.; Li, B. Z.; Li, Z.; Liu, P.; Wang, Y.; Tang, Q.; Ding,
J.; Jia, Y.; Chen, Z.; Li, L.; Sun, Y.; Li, X.; Dai, Q.; Song, C. X.;
Zhang, K.; He, C.; Xu, G. L. Science 2011, 333, 1303-1307.
(4) Pfaffeneder, T.; Hackner, B.; Truss, M.; Munzel, M.; Muller,
M.; Deiml, C. A.; Hagemeier, C.; Carell, T. Angew. Chem. Int. Ed
Engl. 2011, 50, 7008-7012.
(5) Kriaucionis, S.; Heintz, N. Science 2009, 324, 929-930.
(6) Bowman, R. L.; Levine, R. L. Cold Spring Harb Perspect.
In summary, our work suggests that TET enzymes
have a broad substrate tolerance that includes unnatural
-position modifications on cytosine bases. Given the
Med. 2017, 7, 10.1101/cshperspect.a026518.
(7) Liu, M. Y.; DeNizio, J. E.; Kohli, R. M. Methods Enzymol.
5
2
016, 573, 365-385.
rapidly expanding interest in the role of ox-mC bases in
epigenetic regulation, these unnatural modified bases
offer needed chemical tools for probing TET biology.
Specifically, vC, which is associated with one major
product rather than three sequential substrate/product
pairs, offers a more facile means to detect product
formation in a high throughput manner. Also, eyC, due
to its crosslinking reactivity, can be applied to
understand when and where TET isozymes are active in
given cellular niches or to identify TET-like enzymes in
other species, such as the TET-like enzyme from the
amoeba Naegleria gruberi, which readily reacts with the
probe (Figure S12). Analogous probes may also be
useful for other Fe(II)/-KG-dependent enzymes such as
histone demethylases, which have been recently shown
(8) Crawford, D. J.; Liu, M. Y.; Nabel, C. S.; Cao, X. J.; Garcia, B.
A.; Kohli, R. M. J. Am. Chem. Soc. 2016, 138, 730-733.
9) Globisch, D.; Munzel, M.; Muller, M.; Michalakis, S.; Wagner,
(
M.; Koch, S.; Bruckl, T.; Biel, M.; Carell, T. PLoS One 2010, 5,
e15367.
(10) Lu, J.; Hu, L.; Cheng, J.; Fang, D.; Wang, C.; Yu, K.; Jiang,
H.; Cui, Q.; Xu, Y.; Luo, C. Phys. Chem. Chem. Phys. 2016, 18,
4
728-4738.
11) Hu, L.; Lu, J.; Cheng, J.; Rao, Q.; Li, Z.; Hou, H.; Lou, Z.;
(
Zhang, L.; Li, W.; Gong, W.; Liu, M.; Sun, C.; Yin, X.; Li, J.; Tan,
X.; Wang, P.; Wang, Y.; Fang, D.; Cui, Q.; Yang, P.; He, C.; Jiang,
H.; Luo, C.; Xu, Y. Nature 2015, 527, 118-122.
(12) Hu, L.; Li, Z.; Cheng, J.; Rao, Q.; Gong, W.; Liu, M.; Shi, Y.
G.; Zhu, J.; Wang, P.; Xu, Y. Cell 2013, 155, 1545-1555.
(13) Liu, M. Y.; Torabifard, H.; Crawford, D. J.; DeNizio, J. E.;
Cao, X. J.; Garcia, B. A.; Cisneros, G. A.; Kohli, R. M. Nat. Chem.
Biol. 2017, 13, 181-187.
(14) Pfaffeneder, T.; Spada, F.; Wagner, M.; Brandmayr, C.;
Laube, S. K.; Eisen, D.; Truss, M.; Steinbacher, J.; Hackner, B.;
Kotljarova, O.; Schuermann, D.; Michalakis, S.; Kosmatchev, O.;
Schiesser, S.; Steigenberger, B.; Raddaoui, N.; Kashiwazaki, G.;
Muller, U.; Spruijt, C. G.; Vermeulen, M.; Leonhardt, H.; Schar, P.;
Muller, M.; Carell, T. Nat. Chem. Biol. 2014, 10, 574-581.

22
to accept N -alkylated-Lys analogs.
Transfecting
probe-containing oligonucleotides or feeding nucleoside
2
3
analogs for genomic incorporation could offer a
powerful means to localize TET enzymes or otherwise
track epigenome dynamics in future work.
(15) Thornburg, L. D.; Lai, M. T.; Wishnok, J. S.; Stubbe, J.
Biochemistry 1993, 32, 14023-14033.
(16) Thornburg, L. D.; Stubbe, J. Biochemistry 1993, 32, 14034-
ASSOCIATED CONTENT
1
4042.
17) Lercher, L.; McGouran, J. F.; Kessler, B. M.; Schofield, C. J.;
Davis, B. G. Angew. Chem. Int. Ed Engl. 2013, 52, 10553-10558.
18) Knapp, D. M.; Gillis, E. P.; Burke, M. D. J. Am. Chem. Soc.
(
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website.
Supporting Information includes methods, synthetic
schemes, and additional substrate and product
characterization.
(
2009, 131, 6961-6963.
(19) Filichev, V. V.; Astakhova, I. V.; Malakhov, A. D.; Korshun,
V. A.; Pedersen, E. B. Chemistry 2008, 14, 9968-9980.
(20) Raiber, E. A.; Beraldi, D.; Ficz, G.; Burgess, H. E.; Branco,
M. R.; Murat, P.; Oxley, D.; Booth, M. J.; Reik, W.;
Balasubramanian, S. Genome Biol. 2012, 13, R69.
(21) Wright, A. T.; Cravatt, B. F. Chem. Biol. 2007, 14, 1043-1051.
AUTHOR INFORMATION
Corresponding Author
(22) Hopkinson, R. J.; Langley, G. W.; Belle, R.; Walport, L. J.;
*
Rahul M. Kohli: rkohli@pennmedicine.upenn.edu
Dunne, K.; Munzel, M.; Salah, E.; Kawamura, A.; Claridge, T. D. W.;
Schofield, C. J. Chem. Commun. (Camb) 2018, 54, 7975-7978.
(23) Guan, L.; van der Heijden, G. W.; Bortvin, A.; Greenberg, M.
M. Chembiochem 2011, 12, 2184-2190.
Notes
No competing financial interests have been declared.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Table of Contents Artwork
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment