A new fluorogenic transformation: Development of an optical probe for coenzyme Q

Article abstract of DOI:10.1021/ol0507569

(Chemical Equation Presented) A new fluorogenic transformation based on a quinone reduction/lactonization sequence has been developed and evaluated as a tool for probing redox phenomena in a biochemical context. The probe presented herein is an irreversible redox probe and is reduced selectively by biologically relevant quinols such as ubiquinol but is inert to reduced nicotinamides (e.g., NADH). The ensuing cyclization is fast and quantitative and provides a measurable optical response.

Full text of DOI:10.1021/ol0507569

ORGANIC
LETTERS
2005
Vol. 7, No. 12
2417-2420
A New Fluorogenic Transformation:
Development of an Optical Probe for
Coenzyme Q
Matthew S. Tremblay and Dalibor Sames*
Department of Chemistry, Columbia UniVersity, New York, New York 10027
sames@chem.columbia.edu
Received April 7, 2005
ABSTRACT
A new fluorogenic transformation based on a quinone reduction/lactonization sequence has been developed and evaluated as a tool for
probing redox phenomena in a biochemical context. The probe presented herein is an irreversible redox probe and is reduced selectively by
biologically relevant quinols such as ubiquinol but is inert to reduced nicotinamides (e.g., NADH). The ensuing cyclization is fast and quantitative
and provides a measurable optical response.
As part of a program aimed at the development of optical
probes for visualizing enzymatic redox processes,1 we be-
came interested in the electron transport chain as an intriguing
target for a new type of redox-active fluorogenic molecular
probe. Given the central role of redox cofactors such as
ubiquinone (Coenzyme Q), plastoquinone, and menaquinone
as mobile electron carriers in the electron transport chains
of different species, we reasoned that if the reduction of a
p-quinone core structure could be coupled to a change in
fluorescence, it would serve as a basis for the development
of such a probe.
nism. Accordingly, we proposed that reduction of a p-
quinone bearing a (Z)-R,â-unsaturated ester in the 2-position
would result in facile lactonization to form the familiar
coumarin fluorophore (Figure 1). This transformation was
Since p-quinols (reduced p-quinones) are typically unstable
to oxygen, we expected that a reasonable fluorogenic mech-
anism would involve “trapping” of the quinol component of
the redox pair. Intramolecular cyclization resulting in the
expansion of a π-system has been shown by us1b,2 and by
others3 to be an effective fluorescence “switching” mecha-
Figure 1. Fluorogenic transformation triggered by quinone reduc-
tion.
inspired in part by the biosynthesis4 and biochemical role
of R-tocopherol (vitamin E), whose chromanol ring system
is essentially a “trapped” quinol that can undergo ring-
opening oxidation to the corresponding quinone as part of
its antioxidant mechanism.5
(1) (a) Yee, D. J.; Balsanek, V.; Sames, D. J. Am. Chem. Soc. 2004,
126, 2282-2283. (b) Chen, G.; Yee, D. J.; Gubernator, N. G.; Sames, D.
J. Am. Chem. Soc 2005, 127, 4544-4545.
(2) Moreira, R.; Havranek, M.; Sames, D. J. Am. Chem. Soc. 2001, 123,
3927-3931.
10.1021/ol0507569 CCC: $30.25
© 2005 American Chemical Society
Published on Web 05/17/2005

Central to the success of this design was the speed of the
cyclization reaction. The lactonization of the quinol needed
to be fast relative to the quinone reduction so that the optical
signal correlated directly with the rate of the reduction event
and was not complicated by the kinetics of the cyclization.
Also, the tendency for quinols to reoxidize in air required
that the trapping occur quickly. Lactonization of a quinol
generated in situ by reduction of a quinone has been shown
to be spontaneous in aqueous media, but only when
facilitated by a trialkyl lock mechanism.6 This system,
however, was not suitable for our design, since the geminal
dimethyl group precludes the π-system expansion upon
cyclization. Thus, we set out to explore lactonization kinetics
of fully conjugated systems.
could be reduced quantitatively under mild conditions
(aqueous Na2S2O4 or brief catalytic hydrogenation in CHCl3)
to give the isolable quinols 4a-c.
Quinols 4a-c underwent clean lactonization to the cor-
responding coumarins 5a-c at varying rates depending on
substitution (Scheme 2a). The cyclization of quinol 4a in
Scheme 2
We synthesized a series of probe candidates based on 2,3-
dimethoxy-5-methyl-1,4-benzoquinone (the ubiquinone core)
since it has been the subject of extensive biological7 and
synthetic8 study and because we expected that an electron-
rich hydroquinone would be sufficiently nucleophilic to effect
the lactonization; benzo- and cyclohexo-fused R,â-unsatur-
ated esters were chosen to avoid olefin isomerization during
synthesis or under assay conditions (Scheme 1). The steri-
Scheme 1
cally demanding Suzuki coupling of electron-rich boronic
acids 1a,b with the appropriate aryl or vinyl bromide was
carried out using Buchwald’s conditions9 and furnished the
adducts 2a-c. Standard oxidative demethylation with cerric
ammonium nitrate gave the air-stable quinones 3a-c, which
1
a Reaction monitored by H NMR in CDCl3. b Not determined.
c Phosphate buffer (50 mM), pH ) 7.4. d Reaction was complete
upon dissolution (<15 s).
CDCl3 was slow and required elevated temperature. The
5-methyl substituent in 4a was identified as the chief
detriment to cyclization, since the desmethyl derivative 4b
cyclized nearly 2 orders of magnitude faster, now proceeding
at room temperature but still slowly. Analogues of 4b
containing activated esters were examined (see Supporting
Information), but none proved to be more promising than
the cyclohexo- derivative 4c, which cyclized an order of
magnitude faster than 4b in CDCl3. The cyclization of 4c
was further examined in different solvent systems in order
to approximate varying degrees of hydrophobicity and proton
availability10 (Scheme 2b). To our delight, 4c cyclized in only
seconds at room temperature in neutral phosphate buffer.
The 3c/4c redox pair had thus satisfied the first requirement
(3) (a) Kim, T.-H.; Swager, T. M. Angew. Chem., Int. Ed. 2003, 42,
4803-4806. (b) Zhang, S.-W.; Swager, T. M. J. Am. Chem. Soc. 2003,
125, 3420-3421.
(4) Cahoon, E. B.; Hall, S. E.; Ripp, K. G.; Ganzke, T. S.; Hitz, W. D.;
Coughlan, S. J. Nat. Biotechnol. 2003, 21, 1082-1087.
(5) Liebler, D. C.; Baker, P. F.; Kaysen, K. L. J. Am. Chem. Soc. 1990,
112, 6995-7000.
(6) Carpino, L. A.; Triolo, S. A.; Berglund, R. A. J. Org. Chem. 1989,
54, 3303-3310
(7) (a) Yu, C.-A.; Gu, L.; Lin, Y.; Yu, L. Biochemistry 1985, 24, 3897-
3902. (b) Gu, L.-Q.; Yu, L.; Yu, C.-A. Biochim. Biophys. Acta 1990, 1015,
482-492. (c) Larsen, P. L.; Clarke, C. F. Science 2002, 295, 120-123. (d)
Jonassen, T.; Davis, D. E.; Larsen, P. L.; Clarke, C. F. J. Biol. Chem. 2003,
278, 51735-51742.
(8) Ohshima, M.; Miyoshi, H.; Sakamoto, K.; Takegami, K.; Iwata, J.;
Kuwabara, K.; Iwamura, H.; Yagi, T. Biochemistry 1998, 37, 6436-
6445.
(9) Yin, J.; Rainka, M. P.; Zhang, X.-X.; Buchwald, S. L. J. Am. Chem.
Soc. 2002, 124, 1162-1163.
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Org. Lett., Vol. 7, No. 12, 2005

for a potential probe, namely, that the lactonization reaction
occur spontaneously.
Coumarin 5c showed two absorption bands (271 and 328
nm) and fluoresced weakly, but detectably, with the emission
maximum at 550 nm when excited at 330 nm in neutral
aqueous media (see Supporting Information). Since quinone
3c has no absorption bands above 300 nm and no detectable
fluorescence, the 3c/5c pair constitutes a competent optical
switch.
Having satisfied the criteria for facile cyclization and
optical switching, quinone probe 3c was then screened
against various biologically relevant reductants, and a
qualitative reduction profile was generated (Figure 2). The
Figure 3. Reduction of probe 3c monitored by absorbance and
fluorescence. Ubiquinol (UQH2, 3 equiv) was added to 3c in pH
7.4 phosphate buffer at rt, and absorbance spectra were measured
at 0, 1, 5, and 10 min. The absorbance increase at ∼270 nm is due
to formation of ubiquinone (UQ) as well as 5c; the increase at ∼330
nm is due to formation of 5c only. Inset: the same assay monitored
by fluorescent emission at 550 nm.
Figure 2. Reduction profile of 3c. DQH2 ) duroquinol; UQH2 )
ubiquinol; MQH2 ) menaquinol; NADH ) nicotinamide adenine
suggests its potential usefulness for detecting local redox
potential and electron fluxes in quinone/quinol pools such
as those found in membrane-bound electron transport
chains. Typical detection limits were in the range of
30-50 µM reductant according to the UV-vis assay and
5-10 µM according to the fluorescence assay, visualized
with 1 equiv of probe 3c. This could be improved by further
optimization of coumarin 5c’s photophysical properties;
however, the high local concentration of membrane-confined
quinone redox cofactors should render the current detection
limit satisfactory for use in intact cells or membrane
preparations.
Probe 3c was partially decomposed (accompanied by
partial reduction to 5c) by dithiothreitol and completely
decomposed by glutathione (Figure 2). The instability of
certain quinones to thiols highlights a potential limitation
of using the quinone core as a biochemical redox probe,
since thiols are important, and abundant, biological reduc-
tants. However, preliminary study of the reaction between
probe 3c and thiols indicates that the undesired reaction may
be thwarted by rapid localization of the probe into hydro-
phobic membranes where most quinone redox processes take
place.
dinucleotide hydride; RF ) riboflavin; DOP ) dopamine; Fe2+
)
FeCl2; DTT ) dithiothreitol; GSH ) glutathione. [Conditions: pH
7.4, rt, 10 min, 100 µM 3c, 1-10 equiv of reductant.]
relative reduction rate was estimated by measuring the rate
of formation of coumarin 5c by UV-vis absorption in the
presence of reductant (1-10 equiv) at rt in pH 7.4 phosphate
buffer (Figure 3); formation of 5c was corroborated by
simultaneous HPLC analysis. Coumarin 5c was detected in
the presence of all three p-quinols tested (1-3 equiv). The
relative rates of reduction by the p-quinols were proportional
to their reported midpoint potentials11 (duroquinol, E° )
∼
(+)100 mV; ubiquinol, E° ) ∼(+)60 mV; menaquinol,
E° ) ∼(-)100 mV). Since quinol 4c is immediately and
irreversibly converted to coumarin 5c, the redox couple
3c/4c is unable to equilibrate with other quinone/quinol
couples, allowing complete conversion to occur. Despite the
significantly more negative midpoint potential of NADH
(-320 mV),11 it did not reduce 3c even when present in
10-fold excess; it seems that in this case, a significant ki-
netic (i.e., mechanistic) barrier sufficiently blocks an other-
wise thermodynamically favored process. Probe 3c was
not reduced by riboflavin, dopamine, or Fe(II). The notable
selectivity of probe 3c for quinol cofactors over NADH
In summary, a new fluorogenic chemical transformation
triggered by p-quinone reduction has been developed and
incorporated into a probe that responds spontaneously and
irreversibly to reduction at room temperature in aqueous
media. This process has been coupled to the selective
detection of biologically relevant p-quinol cofactors, dem-
onstrating its potential usefulness as a molecular redox probe.
Although preliminary studies have identified some limitations
of this design, we are currently investigating alternative
(10) Leahy, D. E.; Morris, J. J.; Taylor, P. J.; Wait, A. R. Pharmacochem.
Libr. 1991, 16, 75-82.
(11) (a) Garrett, R. H.; Grisham, C. M. Biochemistry, 2nd ed.; Harcourt
College Publishers: Orlando, FL, 1999; Chapter 21, p 677. (b) Loach,
P. A. In Handbook of Biochemistry: Selected Data for Molecular
Biology, 2nd ed.; Sober, H. A., Ed.; CRC Press: Cleveland, 1970; pp J33-
J40.
Org. Lett., Vol. 7, No. 12, 2005
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applications of this probe, as well as modified designs that
circumvent these problems.
Supporting Information Available: Detailed synthetic
procedures, kinetic data, spectral and photophysical charac-
terization, and assay protocol. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. Generous support for this work was
provided by the G. Harold and Leila Y. Mathers Charitable
Foundation. D.S. is the recipient of the Pfizer Award for
Creativity in Organic Chemistry.
OL0507569
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Org. Lett., Vol. 7, No. 12, 2005