Olefin Metathesis-Based Fluorescent Probes for the Selective Detection of Ethylene in Live Cells

Article abstract of DOI:10.1021/jacs.8b05191

Ethylene is an important plant hormone that is involved in a variety of developmental processes including agriculturally important ripening of certain fruits. Owing to its significant roles, a number of approaches have previously been developed to detect ethylene via molecular interactions. However, there are no current approaches for detection that are selective via a discrete homogeneous molecular interaction. Here we report two profluorescent chemodosimeters for the selective detection of the plant hormone ethylene. The approach consists of a BODIPY fluorophore with a pendant ruthenium recognition element based on a Hoveyda-Grubbs second generation catalysts. A marked increase in fluorescence is observed upon exposure to ethylene and selectivity is observed for ethylene over other alkenes, providing a unique approach toward ethylene detection. Imaging in live cells demonstrated that ethylene could be detected from multiple relevant sources.

Full text of DOI:10.1021/jacs.8b05191

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Communication
An Olefin Metathesis-Based Fluorescent Probe for
the Selective Detection of Ethylene in Live Cells
Sacha N. W. Toussaint, Ryan Calkins, Sumin Lee, and Brian W Michel
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05191 • Publication Date (Web): 03 Oct 2018
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An Olefin Metathesis-Based Fluorescent Probe for the Selective
Detection of Ethylene in Live Cells
Sacha N.W. Toussaint,† Ryan T. Calkins,† Sumin Lee,‡ Brian W. Michel*,†
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†Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80210, United States
‡Department of Chemistry, University of California, Berkeley, California, 94720, United States
Supporting Information Placeholder
detection. The affinity of many late transition metals for π systems is
well-described by the Dewar-Chatt-Duncanson model of bonding,4
which coupled with the quenching effects of these metals on
fluorescence is an attractive strategy for ethylene detection. Burstyn
and coworkers developed a silver impregnated poly(vinyl phenyl
ketone) (PVPK) film for sensing ethylene.5 It is suggested that the Ag+
ion is coordinated to both the carbonyl and aromatic ring, but upon
exposure to ethylene the Ag+-arene binding is disrupted resulting in a
decrease in fluorescence. Esser and Swager used a copper scorpionate
ABSTRACT: Ethylene is an important plant hormone that is involved
in variety of developmental processes including agriculturally
a
important ripening of certain fruits. Owing to its significant roles, a
number of approaches have previously been developed to detect
ethylene via molecular interactions. However, there are no current
approaches for detection that are selective via a discrete homogeneous
molecular interaction. Here we report two profluorescent
chemodosimeters for the selective detection of the plant hormone
ethylene. The approach consists of a BODIPY fluorophore with a
pendant ruthenium recognition element based on a Hoveyda-Grubbs
2nd generation catalysts. A marked increase in fluorescence is observed
upon exposure to ethylene and selectivity is observed for ethylene over
complex bound to
a conjugated fluorescent polymer to detect
ethylene.6 Competitive coordination to ethylene disrupts the binding
of the metal to the polymer, which results in an increase in
fluorescence. The Swager group has also adapted this system to a
chemoresistive sensor.7 Kodera and coworkers used a Ag+ ion bound
by an N,S,S-macrocyclic ligand to interact with a pendant anthracene.8
Upon binding of ethylene measureable changes were observed in the
absorbance and fluorescence spectra of the anthracene moiety. While
the coordination based approaches are attractive in terms of
developing a reversible sensor for ethylene (Figure 1), these are not
other alkenes, providing
a unique approach towards ethylene
detection. Imaging in live cells demonstrated that ethylene could be
detected from multiple relevant sources.
Ethylene is one of the five major plant hormones and is intricately
involved in complex signaling pathways controlling a disparate array of
plant phenotypes. The expression of ethylene in the cell triggers many
responses ranging from the regulation of cellular growth processes such
as inhibition of cell division and leaf abscission to environmental
stresses such as pathogen response and drought.1 However, it is most
commonly known as a ripening agent of climacteric fruit such as kiwi
fruits, apples, tomatoes, mangos, and bananas. As a consequence of its
essential role in multiple facets of plant growth, development, and post-
harvest characteristics, a number of methods for ethylene detection
have been developed. The primary means of ethylene detection rely on
gas chromatography methods or laser based systems.2 GC is the most
prevalent method and can sensitively detect ethylene in the low parts
per billion range. The ability to detect ethylene along with
advancements in genetic approaches has led to an increased
understanding of associated signaling pathways in fruit ripening over
the past decades.3 However, practical limitations imposed by many
current methods have spurred interests in developing molecular
approaches towards ethylene detection. An appropriately designed
method for detecting ethylene in solution has the potential to allow for
ethylene detection at the cellular level.
Plants detect ethylene with copper containing ethylene receptors
(ETRs) that bind to the π bond of ethylene via interactions with metal
d orbitals. Therefore it is not surprising that the few recently reported
approaches towards detection of this small and unique phytochemical
have utilized an interaction with transition metals as the basis for
Figure 1. Molecular strategies for detection of ethylene.
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viable for applications in more complex environments, such as a cell,
observed for BEP-4 and 78 for BEP-5. Interestingly, while BEP-4
shows a larger overall increase in fluorescence, it appears that BEP-5
reacts faster. For example after 1 h BEP-4 reaches 12% of its maximum,
while BEP-5 is at 30% of maximum turn-on. While a number of other
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due to either heterogeneity or a lack of selectivity. Small molecule
fluorescent chemodosimeters provide exceptional spatial and temporal
resolution, allow for better signal build up, and are well suited for
potential application in biological systems.9
Scheme 1. Synthesis and Reactivity of BEP-4 and BEP-5.
Most reaction-based fluorescent probes rely on a selective
nucleophilic attack or oxidative transformations that are viable at room
temperature. Such approaches have successfully resulted in small
molecule fluorescent probes for reactive analytes such as H2O2, ClO-,
O
H
O
H
1
HO
O
O
2
3
a
b
O2 , NO, ONOO-, H2S, formaldehyde, and biological thiols, amongst
·-
4
9
others.9 However, ethylene is particularly challenging to detect as it is
not suitably nucleophilic or otherwise reactive with other organic
substrates under ambient or biological conditions. This was indeed
noted by Burstyn and others leading to the use of transition metal-π
interactions for ethylene detection. As such, we sought to utilize a
selective reaction mediated by a transition metal to develop a
fluorescent chemodosimeter for ethylene. After evaluating a number of
strategies for selectively reacting with ethylene, olefin metathesis was
explored as an intriguing approach.
1a
2a
3a
Br
Br
Br
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c
N
N
Mes
Cl
Mes
1
O
Ru
O
Cl
O
d
e
2
4
3
N
N
N
H
O
B
N
B
F
F
It is well-established that both steric and electronic effects influence
the reactivity of olefins with metathesis catalysts.10 Indeed it is useful to
categorize olefin reactivity for predicting product outcomes of cross-
metathesis reactions. These categories range from highly active type I
olefins that undergo homodimerization rapidly such as ethylene and
allyl alcohol to type IV olefins such as trisubstituted and electron poor
olefins that do not participate in cross metathesis. This reactivity trend
poises that a ruthenium catalyst should be selective for ethylene over
other, more substituted alkenes.
We hypothesized that a Hoveyda-Grubbs 2nd generation type
complex appended with a fluorophore would provide a sensitive and
selective chemodosimeter for the detection of ethylene. The presence
of a proximal heavy metal acts to quench fluorescence from the
fluorophore providing the “off-state” and upon reacting with the
analyte the metal is displaced resulting in an increase in fluorescence.11
This approach utilizing the unique reactivity of a transition metal is in
line with other methods used for challenging small molecule analytes
including NO,12 CO13 and H2S14. Indeed this metal-displacement
approach is becoming a common strategy for chemodosimeter
design.15
F
4a
F
5a
BEP-4
ethylene
N
N
2
Mes
Cl
Mes
H
O
Ru
1
1
N
N
Cl
a-e
F
HO
Br
5
2
O
B
F
5
3
4
3
4
1b
BEP-5
Conditions: (a) 2-iodopropane, K2CO3, Cs2CO3, DMF, RT,
8 h. (b)
Ph3PMeBr, n-BuLi, THF, -78 oC to RT to 30 oC 1 h, -78 oC add 2a to RT 8 h
(c) n-BuLi, THF, -78 oC to RT, -78 oC quench w/ DMF. (d) 2,4-
dimethylpyrrole, 1 drop TFA, CH2Cl2, RT, 90 min; DDQ, 10 min; then Et3N,
BF3·Et2O, RT 2 h. (e) Grubbs 2nd Gen., CuCl, DCM, 40 oC, 1 h.
To evaluate this hypothesis, two BODIPY-based profluorescent
probes, BEP-4 and BEP-5, were prepared (Scheme 1). Starting from 4-
bromosalicylaldehyde 1a, alkylation and Wittig olefination provided
bromostyrene 3a. Lithium-halogen exchange followed by
a
dimethylformamide quench provided aldehyde 4a. Condensation of
the aldehyde with 2,4-dimethylpyrrole followed by DDQ oxidation and
chelation with BF3 provided the penultimate BODIPY 5a. Subsequent
reaction with Grubbs 2nd generation catalyst provided the BODIPY
Ethylene Probe-4 (BEP-4) (with numerical assignment arising from
the position of the bromine in the initial starting material relative to the
aldehyde). Additionally, to evaluate the influence of structural variation
on both reactivity and selectivity the analogous BEP-5 was prepared
from 5-bromosalicaldehyde 1b.
Next BEP-4 and BEP-5 were optically characterized. As expected,
the ruthenium containing complexes, BEP-4 and BEP-5 are weakly
fluorescent in toluene (BEP-4: λem = 512 nm, Φ = 0.002; BEP-5: λem
=
513 nm, Φ = 0.004. See SI figure S2) as compared to the styrene
products of a reaction with ethylene 5a and 5b ((5a: λem = 514 nm, Φ =
0.24; 5b: λem = 515 nm, Φ = 0.21. See SI figure S2). To evaluate the
reactivity of the probes to ethylene both BEP-4 and BEP-5 were
dissolved in toluene and exposed to a balloon of ethylene gas (Figure
2a and 2b). Over 60 min a 14-fold turn-on was observed for BEP-4 and
24-fold for BEP-5 at their relative λem. A maximum turn-on 113 is
Figure 2. BEP-4 and BEP-5 optical characterization in toluene. Turn-
on fluorescence response of 2 μM (a) BEP-4 or (b) BEP-5 under an
atmosphere of ethylene. λex =475 nm. Normalized emission at max λem
vs time. Error bars represent SD at each time point from three experi-
ments. . Fluorescence responses of 2 μM (c) BEP-4 or (d) BEP-5 to
allyl alcohol and biologically relevant alkene species. Bars represent
normalized integrated fluorescence intensity responses between 490
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and 630 nm with λex = 475 nm for the respective analytes (100 μM) at t
= 0, 5, 10, 15, 30, 45, and 60 min (black).
mango. The cells were then treated with 2 μM BEP-5 and imaged.
Ethylene gas resulted in a 74% increase in fluorescence and gas from a
sealed chamber holding a ripe banana and mango resulted in a 61%
increase in mean fluorescence compared to a control that was
protected from ethylene exposure (see Figure S5 and S6). Additionally,
BEP-4 and BEP-5 were able to detect ethylene from the ethylene
releasing molecule ethephon, which undergoes hydrolysis to ethylene
and is used as an agricultural ethylene source, in PC12 cells. Depending
on concentration of probe used, BEP-4 displayed a 14-22% increase in
mean fluorescence and BEP-5 showed a 25-31% increase in mean
fluorescence relative to the appropriate control (See Figure S7). Two
significant differences between mammalian cells and plants is the
presence of a cell wall and chlorophyll, which can interfere in some
imaging experiments. Chlamydomonas reinhardtii, represents a
valuable model system as it has both a carbohydrate rich cell wall and
chlorophyll. In the green algae C. reinhardtii BEP-4 was able to detect
exogenous ethylene gas and ethylene from ripe fruit (Figure 4). BEP-5
was also able to detect exogenous ethylene gas, however no statistically
significant turn-on was observed for ethylene from fruit (Figure S9).
These results are especially exciting considering that other examples of
olefin metathesis in biological systems often required significant efforts
to design compatibility.16 We hypothesize that the relatively
hydrophobic nature of BEP-4/5 drives membrane diffusion and
cellular accumulation. Additionally, the fact that these probes do not
necessitate turnover of a catalytic cycle via a prone methylidene
intermediate is a potential benefit, which may pose a challenge in other
attempts to perform catalytic olefin metathesis in aqueous
environments.17
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solvents were evaluated, characterization was conducted in toluene due
to lower vapor pressure and common use in olefin metathesis
reactions. However, it should be noted that both probes respond to
ethylene in an 80/20 mixture of water and acetone (Figure S3). Owing
to the difficulty in accurately dosing small amounts of ethylene to a
solution of probe, allyl alcohol was used as a fast-reacting
monosubstituted type I alkene benchmark. In a typical experiment a 2
μM probe solution of BEP-4 or BEP-5 would be treated with 100 μM of
isoprenol, prenol, limonene, oleic acid or allyl alcohol. This series of
alkenes serves to represent a range of alkenes species to evaluate probe
selectivity and extent of turn-on. We were delighted to observe the
probe solutions exhibit a significant increase in florescence only when
exposed to the type I alkene, allyl alcohol. Isoprenol, prenol, limonene,
and oleic acid showed little to no turn-on as compared to the control
sample. These results suggest that both BEP-4 and BEP-5 demonstrate
promising selectivity for ethylene over other potential terpene and
terpenoid compounds that would be present in plant samples.
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Turn-on in florescence is presumably due to metathesis with
ethylene or allyl alcohol and cleavage of the covalent connection
between the BODIPY and the ruthenium producing the corresponding
products 5a and 5b. To support this BEP-5 was reacted with an excess
of ethylene to return the styrene 5b. It should be noted that the control
samples for BEP-4/5 demonstrate a slow background turn-on.
Exposure of a BEP-4 solution to open atmosphere does result in
isolable aldehyde product, which is also observed in the mass spectrum
of the crude mixture. However, exclusion of moisture and oxygen does
not appear to completely ablate this effect and while exposure to a
long-wave handheld UV lamp does increase this background turn-on,
carefully limiting exposure of the probes to light does not provide a
completely stable background.
With these findings in mind we went on to evaluate the ability of the
two probes to detect an environment of ethylene. This was done by
injecting ethylene into an airtight cuvette via a gastight syringe and
observing fluorescence intensity after 60 minutes (Figure 3). Using a
series of different ethylene injections ranging from 5 μL to 500 μL
resulted in a dose dependent increase of fluorescents as seen in figure 3.
Limits of detection were calculated to be 29 μL for BEP-4 and 20 μL for
BEP-5. This corresponds to 13 ppm and 9 ppm ethylene, respectively,
in the headspace of the cuvette.
Figure 4. Confocal microscopy of BEP-4 in live Chlamydomonas rein-
hardtii cells. Pelleted cells were treated with 20 μM BEP-4 in PBS for
15 min at 37 °C, excess unreacted probe was removed, cells were resus-
pended, plated and imaged. (a) Control (b) cells diffused with eth-
ylene gas for 15 min, media removed and cells were treated with probe
as in the control (c) cells diffused with gas from banana for 1 h, media
removed and cells were treated with probe as in control.. Scale bar
represents 10 μm in all images. (d) Normalized mean fluorescence
intensities of Chlamydomonas cells with each treatment. Error bars
denote SD (n = 2). **p < 0.01. *p < 0.05.
Figure 3. BEP-4 and BEP-5 in toluene show turn-on response to eth-
ylene gas injected via gas tight syringe. Turn-on fluorescence response
of 2 μM (a) BEP-4 or (b) BEP-5. λex =475 nm, λem = 513 nm (BEP-4)
and 514 nm (BEP-5). Endpoints represent normalized fluorescence 60
min after the addition of ethylene. Error bars denote SD.
Finally, we evaluated the ability of BEP-4 and BEP-5 to detect
exogenous ethylene in live cells via confocal microscopy. Excitingly,
both BEP-4 and BEP-5 were successful in detecting ethylene in a live
cell environment with a variety of ethylene sources. HEK239T cells
were exposed directly to ethylene gas or vapor from a ripe banana and
In summary two new chemodosimeters based on Hoveyda-Grubbs
2nd generation catalysts were developed for the selective detection of
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J. N. Ethylene Sensing by Silver(I) Salt-Impregnated Luminescent Films.
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ethylene. The probes do not appear to react significantly with olefins
that are not rapidly reacting type I olefins, however a steady increase in
background fluorescence is observed. A dose dependent increase in
fluorescence upon exposure to ethylene gas is observed. Slight
structural variation provides some variance in fold turn-on and rate of
reaction with ethylene. While these initial findings are promising,
structure-activity studies to improve sensitivity by varying the ligand
identities on Ru are being investigated. Both BEP-4 and BEP-5 were
capable of detecting ethylene in a cellular environment. Efforts to apply
these probes in plant cells, such as Arabidopsis thaliana are currently
underway.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: Brian.Michel@du.edu
ORCID
Brian W. Michel: 0000-0002-4737-8196
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Financial support from the University of Denver is gratefully
acknowledged. We thank Professor Michelle Knowles and Alan
Weisgerber (University of Denver) for assistance with cell
culture and imaging. We thank Professor Krishna Niyogi and
Dr. Masakazu Iwai (University of California, Berkeley) for
providing Chlamydomonas and helpful suggestions for imaging.
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