Rapid photochemical generation of ubiquinol through a radical pathway: An avenue for probing submillisecond enzyme kinetics

Full text of DOI:10.1021/jo000028t

3244
J . Org. Chem. 2000, 65, 3244-3247
approach possible. Esters based on N-hydroxypyridine-
Ra p id P h otoch em ica l Gen er a tion of
2-thione have been used in the photochemical generation
of alkyl radicals as well as nitrogen- and oxygen-based
radicals.9-16 Herein we report the extension of this
methodology with the synthesis of the carbonate ester
of ubiquinol-2 and N-hydroxypyridine-2-thione (PTOC-
Q2H2, 1).17 We present the characterization of this
compound, and demonstrate the release of ubisemi-
quinone upon photolysis of PTOC-Q2H2 in acetonitrile
and aqueous detergent solution, the formation of ubiquinol
by both disproportionation and reduction of semiquinone,
and electron transfer from the photogenerated ubiquinol
to a quinol-oxidizing enzyme, cytochrome bo3, from Es-
cherichia coli.
Ubiqu in ol th r ou gh a Ra d ica l P a th w a y: An
Aven u e for P r obin g Su bm illisecon d
En zym e Kin etics
Brian E. Schultz, Kirk C. Hansen, Charles C. Lin, and
Sunney I. Chan*
Arthur Amos Noyes Laboratory of Chemical Physics,
California Institute of Technology,
Pasadena, California 91125
Received J anuary 10, 2000
In tr od u ction
The use of photoreleasable protecting groups (“cages”)
on bioactive molecules provides a means for the rapid
initiation of bimolecular reaction chemistry in biological
systems.1,2 In this approach, the protecting group renders
an otherwise biologically active molecule inert, so that
the molecule can be mixed with an enzyme or other
biological target molecule without any reaction taking
place. Irradiation of the “caged” molecule leads to the
release of the protecting group, so that the biological
substrate is free to react with its target biomolecule.
Because the bimolecular chemistry can be initiated by a
laser pulse, the time frame over which the reaction can
be probed is determined by the photochemistry leading
to the release of substrate. This time frame can be much
shorter than the time scale of milliseconds associated
with stopped-flow and other rapid-mixing techniques. For
some caged substrates, such as carboxylic acids and
phosphates derivatized with benzoin moieties,3-6 release
of active substrate is essentially instantaneous upon
irradiation. For alcoholic substrates such as quinols,
however, release of free substrate has been limited by
chemistry that occurs after photocleavage of the cage
molecule. When protecting groups such as R-carboxy-
nitrobenzyl7 and 3′,5′-bis(carboxymethoxy)benzoin8 are
used to derivatize quinols, a carbonate linker is required.
Upon irradiation, the quinol is released as a carbonate
monoester, and the slow decarboxylation of this species
is rate-limiting in generating the free quinol.
Resu lts a n d Discu ssion
The synthesis of PTOC-Q2H2 (1) followed standard
methodology for the synthesis of N-hydroxypyridine-2-
thione esters, using a one-pot synthesis. Ubiquinol-2 was
reacted with diphosgene and pyridine in THF, and the
thallium salt of N-hydroxypyridine-2-thione was added
to generate the carbonate ester. Purification yielded
compound 1 as a mixture of two isomers, arising from
esterification of the quinol at the 1-position or at the
4-position of the quinol ring. These two isomers were
resolvable by chromatography, but their chemical and
photophysical properties were sufficiently similar that it
was deemed unnecessary to separate the two prior to the
reactivity studies. The synthesis gave an overall yield of
61%.
Photolysis of PTOC-Q2H2 in either acetonitrile or 100
mM sodium phosphate, 0.1% Brij-35 (a nondenaturing
detergent used in protein studies), pH 7.4 with a mercury
arc lamp under anaerobic conditions yielded equimolar
amounts of 2,2′-dithiobispyridine, ubiquinol-2, and ubiqui-
none-2, as determined by HPLC analysis. The structure
of the starting material and the known chemistry of
N-hydroxypyridine-2-thione esters suggested the forma-
tion of carbon dioxide as well. On the basis of prior
photochemical studies of N-hydroxypyridine-2-thione
esters,18,19 the photolysis of PTOC-Q2H2 was assumed to
follow the sequence diagrammed in Figure 1. According
to this scheme, irradiation of PTOC-Q2H2 (1) led to the
homolytic cleavage of the N-O bond to yield the 2-pyr-
idylthiyl radical (6) and the oxygen-based radical 2. Rapid
An alternative approach to the photochemical genera-
tion of quinol involves the photolysis of a precursor
molecule to generate free semiquinone, which can then
be reduced rapidly to a quinol. The use of the photolabile
protecting group N-hydroxypyridine-2-thione makes this
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* To whom correspondence should be addressed. Phone: (626) 395-
6508. Fax: (626) 578-0471. E-mail: chans@its.caltech.edu.
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(17) Abbreviations: PTOC-Q2H2, compound 1 (PTOC ) pyridine-
2-thioneoxycarbonyl).
(18) Bohne, C.; Boch, R.; Scaiano, J . C. J . Org. Chem. 1990, 55,
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Biophys. Acta 2000, 1456, 121-137.
10.1021/jo000028t CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/26/2000

Notes
J . Org. Chem., Vol. 65, No. 10, 2000 3245
F igu r e 1. Expected photolysis pathway for PTOC-Q2H2.
second-order disproportionation reaction of semiquinone.
The fit, which used an extinction coefficient of 3000 M-1
cm-1 for the semiquinone,20 gave a rate constant of 2.2
× 108 M-1 s-1 for semiquinone decay. This fit is included
in Figure 2. The high quality of the fit suggests that
disproportionation of semiquinone was the dominant if
not exclusive decay pathway for this species. Trace (b)
in Figure 2 shows an absorption transient that arose from
the photolysis of PTOC-Q2H2 in 100 mM sodium phos-
phate, 0.1% Brij-35, pH 7.4. Both the maximum yield of
semiquinone and the final concentration of photoproducts
were significantly lower than those in acetonitrile. A
second-order kinetic model did not fit the data in a
satisfactory fashion, and thus a rate constant was not
calculated. Nevertheless, for those molecules of PTOC-
Q2H2 that did photolyze, the photolysis and subsequent
reaction chemistry were complete within 400 µs.
To measure the extent to which an exogenous one-
electron reductant can increase the rate of reduction of
ubisemiquinone to ubiquinol, the photolysis of PTOC-
Q2H2 was performed in acetonitrile in the presence of
varying concentrations of 2-mercaptoethanol. 2-Mercap-
toethanol has a high solubility in both acetonitrile and
water, and it will not perform the two-electron reduction
of quinone to quinol in a kinetically facile manner.
Control experiments on the steady-state photolysis of
PTOC-Q2H2 in acetonitrile in the presence of 2-mercap-
toethanol, using a mercury arc lamp, revealed that thiols
can reduce ubisemiquinone to ubiquinol with the associ-
ated formation of disulfides. This reaction can compete
effectively with the disproportionation of semiquinone,
leading to a higher yield of quinol (data not shown).
Figure 3 shows absorption transients that monitored the
decay of ubisemiquinone following laser photolysis of
PTOC-Q2H2 in the presence of different concentrations
of 2-mercaptoethanol. In the absence of thiol, the semi-
quinone signal decayed cleanly via the disproportionation
reaction. As the concentration of thiol was increased, the
semiquinone decayed more rapidly as a result of the
reaction with the thiol. This decay was expected to follow
a rate law arising from competing first- and second-order
processes. However, the data were not adequately fit
using this model, suggesting more complex kinetics. In
light of this inability to extract fundamental rate con-
stants from the transient absorption data, the time taken
for the absorption signal to decay to (1/e) times the
amplitude of the decay curve was used as an indicator
of reaction rate. Using this criterion, the presence of 1M
2-mercaptoethanol resulted in approximately a 4-fold
increase in the rate of semiquinone decay, with a
F igu r e 2. (a) Absorption transient following photolysis of 50
µM PTOC-Q2H2 in acetonitrile, measured at 420 nm, and the
fit of the data by a second-order decay model. (b) Absorption
transient following the photolysis of 50 µM PTOC-Q2H2 in 100
mM sodium phosphate, 0.1% Brij-35, pH 7.4. For both traces
a single 308 nm pulse from a XeCl excimer laser, with a pulse
width of 25 ns and an energy of 2.1 mJ , was used for the
photolysis.
decarboxylation of 2 yielded free ubisemiquinone (3),
which disproportionated to form quinone (4) and quinol
(5). The 2-pyridylthiyl radical dimerized to form 2,2′-
dithiobispyridine (7) or reacted with starting material 1
to make 2,2′-dithiobispyridine and 2. Species 2 was not
detected in this study, but the analogous intermediates
RCOO• formed during the photolysis of other N-hydroxy-
pyridine-2-thione esters are usually not observed on
account of an extremely rapid decarboxylation reac-
tion.18,19
Formation and decay of ubisemiquinone in acetonitrile
and aqueous detergent solution following photolysis of
PTOC-Q2H2 were monitored using transient absorption
spectroscopy. Photolysis was achieved using a 308 nm
pulse from a XeCl excimer laser with a pulse width of 25
ns, and the semiquinone concentration was monitored at
420 nm, the λmax for protonated semiquinone. Trace (a)
in Figure 2 shows an absorption transient following the
irradiation of 50 µM PTOC-Q2H2 in acetonitrile. An
immediate increase in absorbance at 420 nm was fol-
lowed by a decay of the signal on a millisecond time scale.
The wavelength dependence of this decay over the
wavelength range 390 nm to 500 nm was consistent with
the spectrum of protonated ubisemiquinone reported by
Land and Swallow,20 confirming the identity of the
species responsible for the signal. The transient at 420
nm was fit by a kinetic model which assumed only a
(20) Land, E. J .; Swallow, A. J . J . Biol. Chem. 1970, 245, 1890-
1894.

3246 J . Org. Chem., Vol. 65, No. 10, 2000
Notes
F igu r e 5. Oxidation of ubisemiquinone by the 2-pyridylthiyl
radical.
On the basis of control spectra, the peak at 340 nm in
Figure 4 (a) was assigned as arising from 2-mercapto-
pyridine at a concentration of 13 µM, with the associated
photolysis of 17 µM PTOC-Q2H2. The formation of 2-mer-
captopyridine stands in contrast to the photolysis of
PTOC-Q2H2 in the absence of enzyme, in either aceto-
nitrile or detergent solution, where the disulfide was the
exclusive product formed from the N-hydroxypyridine-
2-thione moiety. We attribute the formation of the thiol
to the reaction shown in Figure 5, in which the 2-pyr-
idylthiyl radical (6) reacts with ubisemiquinone (3) to
form 2-mercaptopyridine and ubiquinone (5). This is a
thermodyamically favorable process that bypasses the
desired formation of ubiquinol (4) shown in Figure 1. The
oxidation of semiquinone by the 2-pyridylthiyl radical in
the presence of enzyme presumably arose from the
localization of PTOC-Q2H2 in the lipid phase surrounding
the individual enzyme molecules. As the 2-pyridylthiyl
radical and ubisemiquinone were in close proximity upon
photolysis of PTOC-Q2H2, limited mobility of these spe-
cies in a lipid phase would have allowed the two species
to react before they diffused away from each other.
Even with the oxidation of ubisemiquinone by the
2-pyridylthiyl radical, the fate of a small percentage of
the reducing equivalents liberated in the photolysis of
PTOC-Q2H2 could not be determined. The sink for these
reducing equivalents was most likely lipid peroxides or
amino acid residues on the enzyme molecules.
To demonstrate that the reduction of cytochrome bo3
observed during irradiation in the presence of PTOC-
Q2H2 arose from photoreleased substrate and did not
arise from direct photoreduction of the enzyme, cyto-
chrome bo3 was irradiated in the absence of PTOC-Q2H2
under the same conditions as described above. The
results of this experiment are shown as the dotted trace
(b) in Figure 4. Photoreduction of 0.06 µM heme was
calculated on the basis of the difference spectrum in the
Soret region, a significantly lower value than was ob-
served during the irradiation of enzyme in the presence
of caged substrate. Thus, the reducing equivalents in the
latter reaction arose primarily from the photoreleased
substrate.
F igu r e 3. Absorption transients following the photolysis of
50 µM PTOC-Q2H2 in acetonitrile in the presence of 2-mer-
captoethanol. The laser pulse width was 25 ns, with an energy
of 2.0 mJ . Top f bottom: 0 M, 10 mM, 100 mM, 1 M
2-mercaptoethanol. The lower absorbance with 1 M 2-mercap-
toethanol arose from a lower yield of semiquinone during
photolysis.
F igu r e 4. (a) Optical difference spectrum from the irradiation
of 25 µM PTOC-Q2H2 in the presence of 1.2 µM cytochrome
bo3 in 100 mM sodium phosphate, 0.1% Brij-35, pH 7.4 for 20
s using a mercury arc lamp. The solution contained 100 µg/
mL glucose oxidase, 50 µg/mL catalase, and 50 mM glucose to
scavenge dioxygen from the solution. The reaction was per-
formed under an argon atmosphere. (b) Optical difference
spectrum from the irradiation of 1.2 µM cytochrome bo3 in the
absence of PTOC-Q2H2. Other reaction conditions are identical
to those in (a).
characteristic decay time of approximately 80 µs. This
time scale is more rapid than that of standard turbulent
mixing (e.g., stopped-flow) by a factor of 10.
Photolysis of PTOC-Q2H2 was performed under strictly
anaerobic conditions in the presence of cytochrome bo3,
a ubiquinol-oxidizing respiratory enzyme from E. coli, to
demonstrate electron transfer from released substrate
into the enzyme. Glucose oxidase (100 µg/mL), catalase
(50 µg/mL), and glucose (50 mM) were added to the
solution to scavenge any residual dioxygen. The results
of this experiment are shown in Figure 4. The solid trace
(a) shows the photolyzed minus unphotolyzed difference
spectrum that arose from the steady-state irradiation of
25 µM PTOC-Q2H2 for 20 s in the presence of 1.2 µM
enzyme. The peaks at wavelengths greater than 390 nm
were characteristic of the reduction of the b and o3 hemes
in cytochrome bo3. The magnitude of this difference
spectrum corresponded to the reduction of 0.4 µM heme.
Assuming that the heme reduction was indicative of full
enzyme reduction (five electrons per enzyme molecule),
only 6% of the electrons available upon photolysis
reduced the enzyme.
Following photolysis of PTOC-Q2H2, the reaction of
photogenerated ubiquinol with enzyme is expected to
display second-order kinetic behavior in the concentration
regime studied in this work. However, the low yield of
ubiquinol in these experiments led to slow kinetics of
enzyme reduction, such that significant electron transfer
to cytochrome bo3 was not observed in the transient
absorbance experiments, even on a time scale as long as
100 ms.
Con clu sion
The use of N-hydroxypyridine-2-thione as a protecting
group for quinols has led to a system in which semi-
quinone can be produced on a submicrosecond time scale,

Notes
J . Org. Chem., Vol. 65, No. 10, 2000 3247
butyllithium in hexanes (0.594 mL, 1.48 mmol) was added
dropwise. After 30 min the temperature was adjusted to 0 °C
and diphosgene (0.220 g, 1.11 mmol) was added, followed by the
slow addition of dry pyridine in THF. After 1 h the temperature
was again decreased to -78 °C and the thallium salt of
N-hydroxypyridine-2-thione was added (0.486 g, 1.475 mmol).
The extent of reaction was monitored by TLC. Upon completion
of the reaction, the reaction mixture was filtered and the solvent
was removed under reduced pressure. The resulting yellow oil
was purified by flash chromatography on a silica column using
1/1 hexane/EtOAc: yield 440 mg (61%); Rf ) 0.25 (1/1 hexane/
EtOAc); 1H NMR (CDCl3, 300 MHz) δ 8.363 (m, 1 H), δ 8.204 (s,
1 H), δ 7.354 (t, J ) 7.20 Hz, 1 H), δ 7.218 (m, 1 H), δ 5.049 (q,
J ) 5.70 Hz, 2 H), δ 3.914 (s, 3 H), δ 3.857 (s, 3 H), δ 3.337 (d,
J ) 6.60 Hz, 2 H), δ 2.074 (m, 7 H), δ 1.747 (s, 3 H), δ 1.637 (s,
3 H), δ 1.565 (s, 3 H); HRMS (FAB) m/z (MH+) calcd 474.1950,
obsd 474.1938.
Stea d y-Sta te P h otolysis. Samples were placed in a quartz
cuvette and irradiated using an Oriel 66011 Hg vapor arc lamp
operating at 450 W and equipped with water-cooled Schott glass
UG11 and WG320 filters. The optical absorbance was monitored
with an HP 8452 diode array spectrophotometer. The photolysis
was judged to be complete when no further spectral changes
occurred upon continuing illumination. HPLC analysis was
performed using a Shimadzu LC-6A system equipped with a
Vydac C-18 reverse phase column.
and formation of quinol from the semiquinone can occur
on a submillisecond time scale, almost an order of
magnitude more rapidly than reagents can be mixed in
traditional rapid mixing techniques. As demonstrated
with E. coli cytochrome bo3, photolysis of PTOC-Q2H2
leads to the reduction of enzyme, with the actual reduc-
tion rate determined by the amount of substrate formed
in the photolysis reaction. Thus, as long as the biochemi-
cal system of interest can tolerate the short-term pres-
ence of radical species, and the oxidation of semiquinone
by the 2-pyridylthiyl radical is avoided, the radical-based
mechanism for photochemical generation of quinol con-
stitutes a rapid methodology for the initiation of bi-
molecular chemistry of semiquinones and quinols in
biological systems on a submillisecond time scale.
Exp er im en ta l Section
Gen er a l P r oced u r es. Anhydrous THF was prepared by
refluxing over sodium and benzophenone and was distilled prior
to use. All other solvents were of reagent grade. Glassware was
oven-dried. The thallium salt of N-hydroxypyridine-2-thione was
prepared according to the literature procedure.19 The synthesis
of ubiquinone-2 is described elsewhere.21 2-Mercaptoethanol was
distilled under argon prior to use. All other reagents were from
commercial sources and used as received. Type VII glucose
oxidase from Aspergillus niger and bovine liver catalase were
purchased from Sigma. Cytochrome bo3 from E. coli was
prepared as described previously.22
Syn th esis of 1. A solution of ubiquinone-2 (∼1.3 g) in 10 mL
of diethyl ether was treated with an excess of sodium dithionite
in 10 mL of water and vortexed for 10 min under nitrogen gas
to give ubiquinol-2. The organic layer was rinsed twice with 8
mL of water to remove any remaining reductant, and the solvent
was removed under reduced pressure overnight. The ubiquinol
that resulted (1.25 g, 3.90 mmol) was redissolved in dry THF.
The reaction mixture was cooled to -78 °C and 2.5 M n-
La ser Sp ectr oscop y. The laser setup for transient absorb-
ance spectroscopy has been described previously.23 Photolysis
was performed with a Lambda-Physik LPX201I XeCl excimer
laser (308 nm) with pulse energies of 2.0-2.2 mJ at the sample.
The probe beam came from a 75 W xenon arc lamp. When
possible, a 385 nm high-pass filter was used between the probe
lamp and the sample to minimize adventitious photolysis. An
Instruments SA 1690B double monochromator in conjunction
with a photomultiplier tube was used for signal detection.
Ack n ow led gm en t. Funding for this work came
from grant GM22432 from the National Institutes of
Health. K.C.H. is the recipient of a National Research
Service Predoctoral Award.
J O000028T
(21) Stowell, M. H. B.; Wang, G.; Day, M. W.; Chan, S. I. J . Am.
Chem. Soc. 1998, 120, 1657-1664.
(22) Musser, S. M.; Stowell, M. H. B.; Lee, H. K.; Rumbley, J . N.;
Chan, S. I. Biochemistry 1997, 36, 894-902.
(23) DiMagno, T. J .; Stowell, M. H. B.; Chan, S. I. J . Phys. Chem.
1995, 99, 13038-13047.