Co-opting a Bioorthogonal Reaction for Oncometabolite Detection

Article abstract of DOI:10.1021/jacs.6b09706

Dysregulated metabolism is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here we report the development of a chemical approach to detect the oncometabolite fumarate. Our strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddition of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. We demonstrate hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, we show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. Our studies define a novel intersection of bioorthogonal chemistry and metabolite reactivity that may be harnessed to enable biological profiling, imaging, and diagnostic applications.

Full text of DOI:10.1021/jacs.6b09706

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Communication
Bioorthogonal oncometabolite ligation
Thomas T. Zengeya, Julie Garlick, Rhushikesh A Kulkarni, Mikayla Miley, Allison Roberts,
Youfeng Yang, Daniel Crooks, Carole Sourbier, W. Marston Linehan, and Jordan L. Meier
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b09706 • Publication Date (Web): 18 Nov 2016
Downloaded from http://pubs.acs.org on November 18, 2016
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Bioorthogonal oncometabolite ligation
Thomas T. Zengeya,[a]‡ Julie M. Garlick,[a]‡ Rhushikesh A. Kulkarni, [a] Mikayla Miley, [a] Allison M.
Roberts, [a] Youfeng Yang,[b] Daniel R. Crooks,[b] Carole Sourbier,[b] W. Marston Linehan, [b] and Jordan
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L. Meier*[a]
[a] Chemical Biology Laboratory, National Cancer Institute, Frederick MD, 21702, [b] Urologic Oncology Branch, National
Cancer Institute, Bethesda MD, 20817.
*Email: jordan.meier@nih.gov
Supporting Information Placeholder
ABSTRACT: Dysregulated metabolism is a hallmark of
many diseases, including cancer. Methods to fluoresꢀ
cently detect metabolites have the potential to enable
new approaches to cancer detection and imaging. Howꢀ
ever, fluorescent sensing methods for naturally occurring
cellular metabolites are relatively unexplored. Here we
report the development of a chemical approach to detect
the oncometabolite fumarate. Our strategy exploits a
known bioorthogonal reaction, the 1,3ꢀdipolar cycloadꢀ
dition of of nitrileimines and electronꢀpoor olefins, to
detect fumarate via fluorescent pyrazoline cycloadduct
formation. We demonstrate hydrazonyl chlorides serve
as readily accessible nitrileimine precursors, whose reacꢀ
tivity and spectral properties can be tuned to enable deꢀ
tection of fumarate and other dipolarophile metabolites.
Finally, we show this reaction can be used to detect enꢀ
zyme activity changes caused by mutations in fumarate
hydratase (FH), which underlie the familial cancer preꢀ
Figure 1. Fluorogenic detection of the oncometabolite
disposition syndrome HLRCC. Our studies define a
fumarate via a [3+2] cycloaddition reaction. (a) Schematic
novel intersection of bioorthogonal chemistry and meꢀ
for fumarate detection. (b) Fluorogenic reaction of
fumarate with previously reported hydrazonyl chloride 1
(λex = 320 nm) after 1 hour. Note that only a single diasterꢀ
omer of the pyrazoline product is shown.
tabolite reactivity that may be harnessed to enable bioꢀ
logical profiling, imaging, and diagnostic applications.
Dysregulated metabolism is a hallmark of many diseasꢀ
es, including cancer.1 In addition to providing building
blocks to support cell growth, metabolic changes can
also directly stimulate cell signaling pathways.2 For exꢀ
ample, in hereditary leiomyomatosis and renal cell canꢀ
cer (HLRCC), inactivating mutations in the fumarate
hydratase (FH) gene cause the accumulation of
fumarate.3 High levels of this “oncometabolite” disrupt
the activity of enzymes involved in epigenetic signaling,
thereby facilitating malignant transformation.4ꢀ5 Methꢀ
ods to sensitively detect fumarate have the potential to
facilitate the rapid diagnosis of HLRCC, and also identiꢀ
fy new disease settings in which fumarate may play a
signaling role. However, the current gold standard methꢀ
od for fumarate detection, mass spectrometry, requires
lengthy sample preparation, specialized equipment, and
does not have the potential to form the basis for cellular
imaging approaches.
In designing a chemical method to identify aberrant FH
activity, we noticed that fumarate formally constitutes an
electron–poor dipolarophile. Alkene dipolarophiles are
rare in nature, but are wellꢀknown in the bioorthogonal
reaction literature for their ability to undergo fluorogenic
1,3ꢀcycloadditions with nitrileimine dipoles.6ꢀ7 Although
metabolites such as fumarate have not been explored as
nitrileimine reaction partners, their similarity to previꢀ
ously reported substrates8 suggested the novel possibility
of exploiting this reactivity to monitor FH activity via
fluorogenic detection of fumarate (Figure 1).
To explore the feasibility of this approach, we first
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Scheme 1. Synthesis of nitrileimine precursors 1-5a
Table 1. Optical properties of hydrazonyl chlorides upon
reaction with fumarate dipolarophiles
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2
3
4
5
6
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8
fumarate
fluorogenicityb
Φ
fapp
λex
λem
ε (Mꢀ1
Comp.
(nm)a (nm)a cmꢀ1)a
a
320
320
350
390
390
490 21,500 0.04
490 15,000 0.05
18x
7x
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2
3
4
5
490
9,700 0.29
63x
31x
4x
9
530 13,800 0.12
590 15,300 0.01
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a
Measured values refer to the optical properties of the pyꢀ
razoline cycloadduct formed after reaction of ethyl
fumarate (10 mM) with hydrazonyl chloride (0.1 mM) in a
1:1 mixture of CH3CN/aqueous sodium phosphate (100
mM, pH 7). Quantum yields (Φfapp) were determined by
b
comparison to a diphenylanthracene standard. Relative
aReagents and conditions: i) 1:1 CH2Cl2/DMF, 100 ºC (miꢀ
crowave), 15 min, ii) CH2Cl2, Nꢀchlorosuccinimide (1 eq),
dimethyl sulfide (1 eq), ꢀ78 ºC, 3 h.
fluorescence of fumarate (10 mM) versus vehicle control
containing solutions upon addition of hydrazonyl chloride
(0.1 mM). Comp. = compound.
evaluated the reactivity of fumarate with hydrazonyl
chloride 1, a nitrileimine precursor known to form deꢀ
tectible fluorescent cycloadducts (Figure 1).7 Fumarate
is less polarizable and has a higher lying LUMO (12.2
eV) than the acrylyl dipolarophiles that have been comꢀ
monly applied in bioorthogonal protein labeling applicaꢀ
tions (LUMOethyl acrylate = 2.9 eV; Figure S1).6ꢀ7 This has
the potential to limit reactionꢀbased detection strategies,
as the rate of nitrileimineꢀalkene cycloadditions are inꢀ
versely related to the free energy gap between the niꢀ
trileimineꢀHOMO and dipolarophileꢀLUMO.9 However,
we found that incubation of 1 with fumarate in buffer
resulted in a timeꢀdependent increase in fluorescence,
suggesting the HOMOꢀLUMO gap is not limiting. Reacꢀ
tions of 1 and fumarate were complete within 60
minutes, and formation of the cycloaddition product was
verified by LCꢀMS (Figure S1). Fumarate reacted with 1
at a slower rate than ethyl acrylate, consistent with its
higher LUMO energy (Figure S1). However, both dipoꢀ
larophiles reacted to form products that display a 100
nm bathochromic shift in emission relative to the hydraꢀ
zonyl chloride starting material. This results in high sigꢀ
nalꢀtoꢀnoise, and for fumarate we observed an 18ꢀfold
increase in fluorescence at 490 nm upon reaction of 1
(Figure 1b). These studies establish the reactivity of
fumarate with nitrileimines.
these molecules by assessing each hydrazonyl chloride’s
fluorescence following reaction with ethyl fumarate, a
fumarate analogue that reacts to completion with niꢀ
trileimines due to its low energy LUMO (Figure S2).
Deletion of the 4ꢀmethoxy group from the C3 aryl unit
of 1 alters the electronic character of this ring from elecꢀ
tronꢀdonating to inductively electronꢀwithdrawing (2),
but did not greatly affect excitation or emission properꢀ
ties of the ethyl fumarateꢀcycloadduct (Table 1). Howꢀ
ever, removal of an additional 4ꢀmethoxy group from the
N3ꢀaryl ring (3) results in a fluorophore that absorbs
strongly from 330ꢀ370 nm and whose emission intensity
is considerably increased. This is consistent with previꢀ
ous studies of diaryl pyrazoline dyes,11ꢀ12 which found
that reducing pushꢀpull polarization decreases fluoroꢀ
phore Stokes shift (via a redꢀshift of excitation) and inꢀ
creases quantum yield. To build on this observation, we
explored two additional hydrazonyl chlorides, 4 and 5.
These nitrileimine precursors each contain electron
withdrawing pꢀcarboxymethyl esters in their N3ꢀaryl
ring, but form pyrazolines with vastly different electronꢀ
ics due to the presence or absence of an electronꢀ
donating methoxy group in the C3 aryl unit.13 As above,
the less polarized pyrazoline precursor 4 exhibited a reꢀ
duced Stokes shift but increased fluorescence intensity
relative to 5 upon reaction with ethyl acrylate (Table
1).13 Examination of overall trends highlighted two opꢀ
timal nitrileimine precursors: 3, which forms the brightꢀ
est pyrazolines of any hydrazonyl chloride tested, and 4,
which is strongly fluorogenic and excitable at higher
wavelengths that may be less damaging to biomolecules.
The sensitivity of fumarate detection by a nitrileimine
dipole is expected to be highly dependent on the brightꢀ
ness of the resultant pyrazoline fluorophore.10 However,
the chemical optimization of hydrazonyl chlorides for
use in fluorogenic 1,3ꢀdipolar cycloadditions has not
been described. To develop an optimized probe for FH
profiling, we synthesized a small panel of diaryl hydraꢀ
zonyl chlorides in which the electronic properties of the
auxochrome subunits were systematically varied
(Scheme 1). We characterized the optical properties of
Having identified brighter agents for dipolarophile deꢀ
tection, we next benchmarked their ability to detect onꢀ
cometabolite fumarate. In model reactions, solutions of
fumarate were treated individually with hydrazonyl
chlorides 1ꢀ5, and assessed for fluorescence. Fumarate
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Figure 3. (a) Profiling FH activity in kidney cancer cell
lines using 4 (λex = 390 nm). Profiling data for additional
kidney cancer cell lines is provided in the Supplementary
Information. (b) Correlation between FH activity measureꢀ
ments by 4 and GCꢀMS measurements of fumarate in kidꢀ
ney cell lines. Significance was analyzed by unpaired Stuꢀ
dent’s tꢀtest (ns = P > 0.05, **** = P < 0.0001).
Figure 2. Fluorogenic profiling of FH activity. (a) Strategy
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for fluorogenic detection of FH activity. (b) Detecting reꢀ
combinant FHꢀdependent conversion of malate to fumarate
using 4 (λex = 390 nm). (c) Detecting timeꢀdependent conꢀ
version of malate to fumarate using 4.
activity was monitored using a twoꢀstep protocol, first
incubating FH with malate, and then “developing” the
newly formed fumarate via reaction with 4 in a solution
of 1:1 CH3CN/sodium phosphate prior to fluorescence
analysis. The use of CH3CN in the developing buffer
was found to be critical to fumarate detection, as it inꢀ
creases hydrazonyl chloride solubility and slows comꢀ
peting hydrolysis.15 Using this approach, we found 4
was able to detect FH activity, exhibiting enzymeꢀ and
timeꢀdependent increases in fluorescence (Figure 2).
These studies establish the feasibility of using
bioorthogonal ligation chemistry to monitor FH activity.
reacted more slowly with 1ꢀ5 than ethyl fumarate, but
formed fluorophores with identical excitation and emisꢀ
sion maxima (Figure S3). This suggests the distinguishꢀ
ing carboxylates of fumarate and ethyl fumarate do not
impact the pyrazoline fluorophore π system.11,12 Conꢀ
sistent with trends observed for ethyl fumarate, we
found that hydrazonyl chlorides forming bright pyraꢀ
zolines also exhibit large fluorescence increases upon
fumarate treatment, up to 63ꢀ and 31ꢀfold in the case of
3 and 4, respectively (Table 1, right column). Detailed
analysis of fumarate reactions found 3 and 4 form apꢀ
proximately equivalent yields of pyrazoline, with the
faster cycloaddition kinetics of 4 offset by a correspondꢀ
ing increased rate of hydrolysis (Figure S4). This emꢀ
phasizes pyrazoline fluorophore brightness as a primary
determinant of the sensitivity of nitrileimineꢀmediated
fumarate detection. Optimized compound 4 was able to
detect fumarate in the presence of proteinaceous cell
extracts at concentrations as low as 100 ꢀM (>2ꢀfold
fluorescence increase; Figure S5). Importantly, neither
denatured proteomes, nor high concentrations of acetate
or glutathione, caused the fluorescence of 3 or 4 to inꢀ
crease (Figure S5ꢀ6). This indicates that fluorogenic reꢀ
actions of hydrazonyl chlorides require a dipolarophile
partner, and that abundant biological nucleophiles
should not affect background.14 These studies define 3
and 4 as sensitive reagents for the detection of metaboꢀ
lite dipolarophiles.
Finally, we sought to define the ability of our method to
identify oncometabolic HLRCC cell lines on the basis of
altered FH activity. Unfractionated extracts were preꢀ
pared from a patientꢀderived HLRCC cell line harboring
a homozygous FH mutation (UOK262EV), as well as an
isogenic variant of this cell line engineered to stably
express FH from a wildꢀtype allele (UOK262WT).16ꢀ17
Applying our optimized protocol, we found that adding
malate/4 to FHꢀdeficient UOK262EV extracts resulted
in negligible fluorescence, while applying an identical
strategy to FHꢀwild type UOK262WT proteomes caused
a strong fluorescence increase (Figure 3a). Quantitative
analysis found this signal reflects formation of ~183 ꢀM
fumarate in the UOK262WT lysates (Figure S7). Analyꢀ
sis of additional kidney cell lines observed a diverse
spectrum of FH activity (Figure 3a/S7). GCꢀMS analysis
found that cells with low FH activity harbored high enꢀ
dogenous fumarate (Figure 3b/S7). This suggests the
primary metabolic consequence of disrupted cellular FH
is accumulation of fumarate in the TCA cycle, consistent
with HLRCC’s pathology. These studies define the niꢀ
trileimineꢀalkene cycloaddition as a novel approach to
fluorescently profile endogenous cellular FH activity.
Next, we evaluated the ability of our optimized fluoroꢀ
genic reaction to profile FH activity. Since FH is a reꢀ
versible enzyme, two potential approaches to monitor its
activity can be envisioned: 1) incubating FH with
fumarate, and monitoring fumarate consumption (loss of
signal), or 2) incubating FH with malate, and observing
fumarate accumulation (gain of signal). The latter apꢀ
proach struck us as preferable, since it allows direct corꢀ
relation of FH activity with fluorescence. We first exꢀ
plored this strategy using recombinant FH. Enzyme
Bioorthogonal chemistry forms the critical underpinning
for innumerable applications;18ꢀ20 however, there are still
few examples in which bioorthogonal reactions have
been harnessed to detect endogenous biomolecules or
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enzyme activities.21ꢀ23 Here we coꢀopt a bioorthogonal
reaction to detect the oncometabolite fumarate. This
strategy unifies bioorthogonal chemistry with reactionꢀ
based sensing methods,24 by identifying contexts in
which biologically inert reactions can be harnessed to
study endogenous biology. We define the tuneable fluoꢀ
rescence and reactivity of hydrazonyl chlorideꢀderived
nitrileimines, and show these agents can be used to reꢀ
port on FH mutation in cell models of the familial canꢀ
cer syndrome HLRCC. A paradigm of hereditary cancer
disorders is that they often highlight more widespread
mechanisms of transformation.25ꢀ26 From this perspecꢀ
tive, we envision our fluorogenic probes may enable the
rapid identification of alternative stimuli and cancer conꢀ
texts that disrupt the tumor suppressive activity of FH.
Importantly, this approach is not limited to FH and
should, in theory, be applicable to study any enzyme that
produces or consumes a dipolarophile detectable by our
method. Examples of nitrileimine detectable metabolites
We thank Dr. Martin Schnermann (NCI) and Dr. Rolf Swenson (Inꢀ
tramural Probe Development Center, NHLBI) for helpful discussions.
This work was supported by the Intramural Research Program of the
NIH, National Cancer Institute (ZIA BC011488ꢀ02).
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Author Contributions
‡These authors contributed equally.
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hydroxynoneal, and prostaglandin A1 (Figure S8).
Finally, we note some limitations of our method. First,
our current probes are unable to directly detect differꢀ
ences in endogenous fumarate in FHꢀmutant and wild
type cell lines, highlighting a need to improve sensitivity
for future applications in imaging and diagnostics. Poꢀ
tential routes to address this include the use of diaryl
tetrazoles, a nonꢀlabile class of nitrileimine precursors,27
as well as mitochondrial localization tags, which may
facilitate reaction with concentrated subcellular dipolarꢀ
ophile pools.28 Sensitivity will also likely benefit from
analyzing FHꢀdeficient tissue samples, which exhibit
>10x increased accumulation of fumarate relative to the
cell lines analyzed here.3 Second, the broad scope of
nitrileimineꢀmetabolite reactivity (Figure S8) suggests
future imaging methods based on this reaction may be
limited to contexts such as HLRCC, where a single
known metabolite contributes disproportionately to
overall endogenous dipolarophile “load”. Other disorꢀ
ders associated with metabolite dipolarophiles include
hereditary tyrosinemia, glutaric aciduria, fluoroacetate
poisoning, and enoylꢀCoA hydratase deficiency.25, 29 The
discovery of new dipolarophileꢀdriven disease contexts
will also benefit from integrating these probes with LCꢀ
MS metabolomic methods, where similar chemoselecꢀ
tive reactions have been powerfully applied.30ꢀ31 Overall,
our studies define a novel intersection of bioorthogonal
chemistry and metabolite reactivity, and in so doing proꢀ
vide a chemical basis from which to pursue biological
profiling, imaging, and diagnostic methods.
ASSOCIATED CONTENT
Supporting information, including supplementary figures,
protocols, and synthetic characterization data is available
on the ACS Publications website.
(30) Carlson, E. E.; Cravatt, B. F., Nat Methods 2007, 4, 429ꢀ35.
(31) Chang, J. W.; Lee, G.; Coukos, J. S.; Moellering, R. E., Anal Chem
2016, 88, 6658ꢀ61.
ACKNOWLEDGMENT
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O
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Ar
O
N
Ar
O
O
N
1
2
O
Ar
N
Ar
Ar
N
Ar
fumarate
N
-HCl
O
N
H
Cl
O
O
(FH reporter)
hydrazonyl
chloride
fluorescent pyrazoline
cycloadduct
OCH3
nitrileimine
9
10
11
OCH3
H3CO
Cl
OCH3
H3CO
Cl
OCH3
b
12
fumarate
N
N
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12000
10000
8000
6000
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0
N
N
H
Cl
O2C
CO2
1
1
18x
increase
1 + fumarate
400
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700
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Wavelength

R1
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OMe
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O
MeO
Cl
OMe
i), ii)
+
N
R3
N
H
Cl
H2N
N
H
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OMe
OMe
Cl
OMe
N
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N
N
H
H
Cl
Cl
Cl
Cl
2
3
O
O
OMe
MeO
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MeO
N
N
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N
H
N
H
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Ar N Ar
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O
O
N
O
O
FH
O
4
O
O
O
O
O OH
O
O
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L-malate
fumarate
fluorescent product
2unit/mL + 4
1.5unit/mL + 4
1unit/mL + 4
0.5unit/mL + 4
6
b
c3 10
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FH
1000k
800k
600k
400k
200k
2 106
1 106
0
0
0
30
60
90
120
450
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600
Length of FH/malate incubation
(min)
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1
0
4 105
a
b
1
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UOK262EV
3
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8
9
3 105
2 105
1 105
0
****
R2 = 0.866
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ns
UOK262WT
UOK140
HK2
1
2
3
4
5
FH activity
(fold fluorescence)
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