Caged mitochondrial uncouplers that are released in response to hydrogen peroxide

Article abstract of DOI:10.1016/j.tet.2010.01.103

Caged versions of the most common mitochondrial uncouplers (proton translocators) have been prepared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) from caged states with second order rate constants of 10 (±0.8) M^-1 s^-1 and 64.8 (±0.6) M^-1 s^-1, respectively. The trigger mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen peroxide-activated uncouplers may be useful for studying the biological process of ageing.

Full text of DOI:10.1016/j.tet.2010.01.103

Tetrahedron 66 (2010) 2384–2389
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Caged mitochondrial uncouplers that are released in response to hydrogen
peroxide
Caroline Quin b, Linsey Robertson b, Stephen J. McQuaker b, Nicholas C. Price c, Martin D. Brand a,
,
Richard C. Hartley b
*
a Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA
b Centre for the Chemical Research of Ageing, WestCHEM Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom
c Faculty of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2009
Received in revised form 21 December 2009
Accepted 27 January 2010
Available online 2 February 2010
Caged versions of the most common mitochondrial uncouplers (proton translocators) have been pre-
pared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4-
dinitrophenol (DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) from caged states
with second order rate constants of 10 (ꢀ0.8) Mꢁ1 sꢁ1 and 64.8 (ꢀ0.6) Mꢁ1 sꢁ1, respectively. The trigger
mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen
peroxide-activated uncouplers may be useful for studying the biological process of ageing.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Mitochondria
Caged
Uncoupler
Boronate
Hydrogen peroxide
Ageing
Oxidative stress
ROS
1. Introduction
biological processes leading to their production, rather than inter-
acting directly with the ROS like conventional antioxidants. Im-
Across the world, life expectancy is steadily increasing while the
birth rate is dropping, leading to an ageing population.1 Since age is
the primary risk factor for many diseases,2 understanding the
mechanisms of ageing may allow us to reduce significantly
the burden of disease and increase human healthspan. Arguably the
most convincing theory of ageing is the mitochondrial free radical
theory,3 which postulates that the generation of reactive oxygen
species (ROS) in mitochondria is an inevitable consequence of ox-
idative ATP production, and that the ROS are the primary cause of
the macromolecular damage that accumulates to limit lifespan.
However, it is agreed that the theory’s explanation of ageing is
incomplete.4 For these reasons, fluorescent probes have been de-
veloped for detecting ROS,5 and potential therapeutic antioxidants,
particularly those which target the mitochondrion,6,7 are of in-
creasing interest.
portantly, the functional molecules would moderate such processes
only when they were leading to the production of excess ROS that
could not be effectively managed by the endogenous antioxidant
system. Their selective and responsive nature should make them
useful chemical biological tools for understanding the effects of
ROS-generating processes on cellular damage and on lifespan. The
advantage of using chemical biology, rather than molecular biology,
would be that the functional molecules could be used in any cell
line or organism in a dose-dependent and reversible way. Here we
demonstrate the chemical reactivity of the first prototypes.
The electron transport chain in the mitochondrial inner mem-
brane pumps protons out of the mitochondrial matrix to generate
an electrochemical gradient of protons. Protons flowing back
through ATP synthase, then convert the energy of the proton mo-
tive force into chemical energy in the form of ATP. There is evidence
that when the potential across this membrane rises there is a sub-
stantial increase in ROS and many more evade capture by the de-
fence system.8,9 Production of excess ROS involves generation of
superoxide on the inside of the mitochondrial inner membrane by
electron-leakage from complex I and complex III of the electron
transport chain.8,10 Superoxide dismutase (SOD) rapidly converts
Here we introduce the new concept of functional molecules that
are designed to sense ROS and respond by shutting down the
* Corresponding author.
E-mail address: richh@chem.gla.ac.uk (R.C. Hartley).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.103

C. Quin et al. / Tetrahedron 66 (2010) 2384–2389
2385
this superoxide into hydrogen peroxide, which is uncharged at
physiological pH and diffuses away from the matrix of the mito-
chondrion. In the event of the hydrogen peroxide coming into
contact with iron(II) ions, hydroxyl radicals are produced that react
with biomolecules at rates close to that of diffusion causing dam-
age. Our prototype functional molecules are designed to sense
hydrogen peroxide and respond by releasing a proton-translocator
(uncoupler) that will allow protons to flow back across the mito-
chondrial inner membrane without passing through ATP synthase
(uncoupling the electron transport chain from ATP production),11
so reducing the membrane potential and stopping ROS pro-
duction (Scheme 1). Here there is an interesting parallel with
uncoupling proteins, UCP2 and UCP3, which are postulated to cause
mild uncoupling in response to oxidative stress as part of the nat-
ural antioxidant defence system;10,12 although this remains
controversial.13
Our first targets, caged uncouplers 15 and 19 were synthesized
as shown (Scheme 3). 4-Bromobenzylic alcohol 11 was protected as
the THF acetal 12. Lithiation–boronation gave arylboronate 13. This
was deprotected to give alcohol 14, which was used to displace
fluoride from 2,4-dinitro-1-fluorobenzene to yield caged un-
coupler 15. Alcohol 14 was converted into the bromide 16 by the
literature method,23 reaction with deprotonated malononitrile
gave arylboronate 17. This was then coupled with diazonium salt
18, freshly generated from 4-trifluoromethoxyaniline, to give caged
uncoupler 19.
The reaction between the caged uncouplers 15 and 19 and al-
kaline hydrogen peroxide was studied under pseudo-first order
conditions. One millilitre of a 200
coupler 15 in a 1:1 mixture of DMF and 0.14 M aqueous sodium
bicarbonate (pH 8.3) was treated at 37 ꢂC with 20
M aqueous
mM solution of the caged un-
m
hydrogen peroxide and the absorption monitored at 410 nm (the
Complex 1
e–
High membrane
potential
ACTIVE
O2
SOD
O2
uncoupler
HO
O
H2O2
pH 8.3
Remnants of
trigger
Inactive
Inactive
Trigger
Trigger
uncoupler
uncoupler
Scheme 1. Proposed mode of action of caged uncouplers.
Arylboronate esters react with hydrogen peroxide in mildly al-
kaline media to give phenols (e.g., at pH 8.3, which is the approx-
imate pH of the matrix of active mitochondria), and this has been
used to make a range of fluorescent sensors that are sensitive to,
and selective for, hydrogen peroxide.5,14,15 One mode of action in-
volves the conversion of an arylboronate into a phenol, which
fragments to release a fluorescent molecule.15 We aimed to use this
reaction to release the most commonly employed mitochondrial
absorption maximum of DNPꢁ 3 under these conditions). The av-
erage of three runs is presented in Figure 2. This gave a Alim of
M DNPꢁ 3 released or
a yield of 54% relative to the hydrogen peroxide added. The average
apparent first order rate constant was 1.85ꢃ10ꢁ3 sꢁ1, giving a sec-
ond order rate constant kDNP of 9.3 Mꢁ1 sꢁ1. Repeating the experi-
D
0.220 (ꢀ0.006), corresponding to 10.7
m
ment with 10
corresponding to 5.96
m
M hydrogen peroxide gave a
D
Alim of 0.122 (ꢀ0.01),
m
M DNPꢁ 3 released or a yield of 60% relative
uncouplers, dinitrophenol (DNP)11,16,17
1
and carbonylcyanide
to the hydrogen peroxide added. The average apparent first order
rate constant was 2.16ꢃ10ꢁ3 sꢁ1, giving a second order rate con-
stant kDNP of 10.8 Mꢁ1 sꢁ1. The overall value for kDNP is therefore 10
(ꢀ0.8) Mꢁ1 sꢁ1 and a 57 (ꢀ3)% yield of DNPꢁ 3 was obtained with
p-trifluoromethoxyphenylhydrazone (FCCP) 2 (Fig. 1).11,18,19 These
are mildly acidic compounds (pKa 4.120 and 5.8,21 respectively) that
are deprotonated in the mitochondrial matrix to form lipophilic
anions DNPꢁ 3 and FCCPꢁ 4, which can cross the inner mitochon-
drial membrane, pick up a proton, and return. Thus, arylboronate 5,
bearing a caged uncoupler, should react with the hydroperoxide ion
in the alkaline medium of the mitochondrial matrix to give a borate
ester intermediate 6. Hydrolysis to give the phenoxide ion 7 would
then be followed by fragmentation to give the active uncoupler 8
and the quinone methide side product 9, which would be trapped
rapidly by hydroxide or water to give 4-hydroxybenzyl alcohol 10
(Scheme 2).22
O
B
O
O
B
O
O
HO2
Uncoupler
Uncoupler
5
6
hydrolysis
O2N
F3CO
O
OH
NH
N
NO2
CN
CN
DNP 1
FCCP 2
Uncoupler
7
F3CO
O2N
OH
O
H2O
N
N
O
NO2
CN
Uncoupler
DNP– 3
FCCP– 4
CN
OH
10
9
8
Scheme 2. Reaction between caged uncouplers and hydrogen peroxide.
Figure 1. Uncouplers in their protonated and unprotonated forms.

2386
C. Quin et al. / Tetrahedron 66 (2010) 2384–2389
release the two most common uncoupling agents, DNPꢁ 3 and
Br
Br
FCCPꢁ 4, at a useful rate. The next stage will be to target these caged
uncouplers to mitochondria and test the mitochondrial free radical
theory of ageing.
O
cat. TsOH
HO
THFO
11
12 96%
2. Experimental
2.1. Synthesis
(i) nBuLi, THF, –78 °C
(ii)
O
i
BOPr
O
All reactions under an inert atmosphere were carried out using
oven dried or flame-dried glassware. Solutions were added via
syringe. Tetrahydrofuran and dichloromethane were dried where
necessary using a solvent drying system, PuresolvÔ, in which sol-
vent is pushed from its storage container under low nitrogen
pressure through two stainless steel columns containing activated
alumina and copper. Acetone was dried over 4 Å molecular sieves.
Triethylamine was dried by distillation over calcium hydride and
stored over potassium hydroxide. Reagents, including DNP 1, were
obtained from commercial suppliers and used without further
purification unless otherwise stated. 1H, 13C and 19F NMR spectra
were obtained on a Bruker DPX/400 spectrometer operating at 400,
100 and 376 MHz, respectively. All coupling constants are mea-
sured in hertz. DEPT was used to assign the signals in the 13C NMR
spectra as C, CH, CH2 or CH3. Mass spectra (MS) were recorded on
a Jeol JMS700 (MStation) spectrometer. Infrared (IR) spectra were
obtained on a Shimadzu FTIR-8400S spectrometer using attenuated
total reflectance (ATR) so that the IR spectrum of the compound
(solid or liquid) could be directly detected (thin layer) without any
sample preparation.
O
O
O
O
B
B
(i) MsCl, Et3N, CH2Cl2
(ii) LiBr, acetone,
Δ
Br
16 63%
RO
13 R = THF 84%
AlCl3
EtOH
NCCH2CN
14 R = H 67%
NaH
THF-DMF
O2N
Et3N
F
O
O
NO2
B
O
O
B
CN
CN
17 90%
2.2. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
(FCCP) 2
O2N
F3CO
Cl
O
A solution of NaNO2 (350 mg, 5.0 mmol) in water (5 mL) was
cooled to 0 ꢂC. This solution was added dropwise to a stirred so-
lution of 4-trifluoromethoxyaniline (886 mg, 5.0 mmol) in aqueous
hydrochloric acid (1.6 M, 35 mL) at 0 ꢂC. The mixture was stirred at
0 ꢂC for 5 min and then added dropwise into a stirred solution of
malononitrile (0.48 mL, 7.5 mmol) and NaOAc (12.5 g, 152 mmol) in
water (50 mL). A yellow precipitate formed was collected by fil-
tration and was washed with ice-cold water. The precipitate was
dissolved in Et2O, dried over MgSO4 and the solvent removed under
reduced pressure. Recrystallization from EtOAc/hexane gave FCCP 2
as a yellow amorphous solid (951 mg, 75%). Mp 145–148 ꢂC
NO2
15 21%
N2
18
NaOAc
H2O-MeOH-EtOH
O
O
B
OCF3
N
N
(decomp.). nmax (ATR): 3066 (CH), 2226 (CN), 1618 (Ar) cmꢁ1
. dH
NC CN
(CDCl3, 400 MHz): 7.53 (2H, d, J¼9.2 Hz, H-2 and H-6), 7.20 (2H, m,
H-3 and H-5). dC (CDCl3, 100 MHz): 147.89 (C), 141.66 (C), 123.48
(CH), 121.91 (CF3, q, J¼254.1 Hz), 118.75 (CH), 114.47 (C), 109.85 (C),
87.57 (C). 1H and 13C NMR in good agreement with literature.24
19 46%
Scheme 3. Synthesis of caged uncouplers 15 and 19.
respect to the hydrogen peroxide used. The stoichiometry of hy-
drogen peroxide to caged uncoupler 15 is clearly greater than 1:1. A
plausible explanation for this is that the para-quinone methide
produced by fragmentation of the probe is predominantly trapped
by hydrogen peroxide rather than water.
Similar experiments with caged uncoupler 19 gave good pseudo-
first order kinetics, a calculated second order rate constant kFCCP of
64.8 (ꢀ0.6) Mꢁ1 sꢁ1 at pH 8.3 and a 58 (ꢀ2)% yield of FCCPꢁ 4 rel-
ative to the hydrogen peroxide used. For comparison, the second
order rate constant for reaction between glutathione and hydrogen
peroxide at 37 ꢂC is only 0.87 Mꢁ1 sꢁ1, albeit at pH 7.4. Thus, we
have demonstrated that caged uncouplers 15 and 19 react with
alkaline hydrogen peroxide to release uncouplers at a biologically
appropriate rate.
2.3. 2-(40-Bromobenzyloxy)tetrahydrofuran 12
A solution of 4-bromobenzyl alcohol 11 (13.00 g, 69.5 mmol),
2,3-dihydrofuran (6.3 mL, 83.5 mmol) and para-toluene sulfonic
acid monohydrate (20 mg, 0.1 mmol) in anhydrous DCM (50 mL)
was stirred at rt for 1.5 h. The reaction mixture was then washed
with H2O (2ꢃ100 mL) and saturated aqueous NaHCO3 (100 mL) and
the organic phase dried over MgSO4 and concentrated under re-
duced pressure to afford the acetal 12 as a yellow oil (17.17 g, 96%).
nmax (ATR): 2982 (CH), 2951 (CH), 2882 (CH), 1487 (Ar) cmꢁ1
. dH
(CDCl3, 400 MHz): 7.45 (2H, d, J¼8.3 Hz, H-30 and H-50), 7.20 (2H, d,
J¼8.3 Hz, H-20 and H-60), 5.19 (1H, dd, J¼2.6 and 3.7 Hz, H-2), 4.65
(1H, d, J¼12.4 Hz, ArCHAHB), 4.42 (1H, d, J¼12.4 Hz, ArCHAHB),
3.95–3.84 (2H, m, 2ꢃH-5), 2.07–1.80 (4H, m, 2ꢃH-3 and 2ꢃH-4). dC
(CDCl3, 100 MHz): 137.44 (C), 131.41 (CH), 129.43 (CH), 121.29 (C),
103.17 (CH), 67.97 (CH2), 67.08 (CH2), 32.34 (CH2), 23.44 (CH2).
In summary, we have prepared the first examples of hydrogen
peroxide-activated caged uncouplers and demonstrated that they

C. Quin et al. / Tetrahedron 66 (2010) 2384–2389
2387
Figure 2. Hydrogen peroxide triggered release of DNP/DNPꢁ from a 200
mM solution of caged uncoupler 15 in 1:1 DMF/aqueous NaHCO3 monitored by absorbance at 410 nm. Initial
concentrations of hydrogen peroxide were 20
mM for line (A) and 10 mM for line (B).
ꢄ
ꢄ
LRMS (EIþ): 258 [Mþ , (81Br), 13%], 256 [Mþ , (79Br), 13], 171
(517 mg, 67%). nmax (ATR): 3312 (OH), 2978 (CH), 2930 (CH), 2880
(
81BrC6H4CH2þ, 100), 169 (79BrC6H4CHþ2 , 100), 71 (C4H7Oþ, 100).
(CH), 1614 (Ar) cmꢁ1
.
dH (CDCl3, 400 MHz): 7.78 (2H, d, J¼7.6 Hz, H-
ꢄ
HRMS: 258.0079 and 256.0099. C11H1381BrO2 requires Mþ
,
3 and H-50), 7.33 (2H, d, J¼7.7 Hz, H-2 and H-6), 4.66 (2H, s, ArCH2),
1.33 (12H, s, 4ꢃMe). dC (CDCl3, 100 MHz): 144.07 (C), 134.99 (CH),
ꢄ
258.0085 and C11H1379BrO2 requires Mþ , 256.0097.
126.06 (CH), 83.82 (C), 65.05 (CH2), 24.83 (CH3). LRMS (EIþ): 234
ꢄ
[Mþ
,
50%], 135 [HOCH2C6H411BOHþ, 100]. HRMS: 234.1426.
2.4. 2-[40-(400,400,500,500-Tetramethyl-100,300,200-dioxaborolan-200-yl)-
benzyloxy]tetrahydrofuran 13
ꢄ
C13H1911BO3 requires Mþ , 234.1427. 1H and 13C NMR agree with
literature.23
A solution of bromide 12 (12.00 g, 46.7 mmol) in anhydrous THF
(130 mL) was cooled to ꢁ78 ꢂC and degassed with argon for 15 min.
Under argon, nBuLi (1.68 M in hexanes, 31.7 mL, 56.0 mmol) was
added dropwise over 3 h and the mixture allowed to stir for 15 min.
2.6. 2-[40-(200,400-Dinitrophenoxymethyl)phenyl]-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane 15
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(11.4 mL,
Benzylic alcohol 14 (200 mg, 0.854 mmol) was mixed with 2,4-
dinitrofluorobenzene (0.11 mL, 4.1 mmol) and two drops of anhy-
drous triethylamine added. The reaction mixture was stirred at rt
overnight and then concentrated under reduced pressure. The
mixture was triturated with ether and the resulting pale yellow
solid recrystallized from EtOAc/hexane to afford caged uncoupler
15 as an amorphous solid (72 mg, 21%). Rf [SiO2, petroleum ether/
EtOAc (4:1)]: 0.23. Mp: 143–145 ꢂC. nmax (ATR): 2981 (CH), 2931
56.0 mmol) was added dropwise and the resulting solution allowed
to stir for 2 h. The reaction was quenched with H2O (100 mL) and
the product extracted with DCM (3ꢃ100 mL). The organic layers
were combined, washed with brine (100 mL), dried over MgSO4
and concentrated under reduced pressure to give a yellow oil. Im-
purities were removed by Kugelrohr distillation leaving acetal 13 as
the residual yellow oil, which solidified on standing (11.87 g, 84%).
Mp: 41–44 ꢂC. nmax (ATR): 2978 (CH), 2932 (CH), 2882 (CH), 1614
(CH), 2894 (CH), 1604 (Ar), 1521 (NO2), 1342 (NO2) cmꢁ1
. dH (CDCl3,
(Ar) cmꢁ1
.
dH (CDCl3, 400 MHz): 7.78 (2H, d, J¼7.7 Hz, H-30 and H-
400 MHz): 8.74 (1H, d, J¼2.8 Hz, H-300), 8.34 (1H, dd, J¼2.8 and
9.3 Hz, H-500), 7.85 (2H, d, J¼8.1 Hz, H-20 and H-60), 7.44 (2H, d,
J¼8.1 Hz, H-30 and H-60), 7.21 (1H, d, J¼9.3 Hz, H-600), 5.39 (2H, s,
ArCH2), 1.35 (12H, s, 4ꢃMe). dC (CDCl3, 100 MHz): 156.21 (C), 140.23
(C), 139.24 (C), 136.90 (C), 135.39 (CH), 128.95 (CH), 126.15 (CH),
121.96 (CH), 114.98 (CH), 84.01 (C), 71.98 (CH2), 24.86 (CH3). LRMS
(CIþ): 401 [(MþH)þ, 50%], 219 (100). HRMS: 401.1521.
C19H2211BN2O7 requires (MþH)þ, 401.1520.
50), 7.34 (2H, d, J¼7.7 Hz, H-40 and H-60), 5.20 (1H, dd, J¼1.2 and
4.2 Hz, H-20), 4.72 (1H, d, J¼12.4 Hz, ArCHAHB), 4.50 (1H, d,
J¼12.4 Hz, ArCHAHB), 3.97–3.86 (2H, m, 2ꢃH-5), 2.09–1.83 (4H, m,
2ꢃH-3 and 2ꢃH-4), 1.34 (12H, s, 4ꢃMe). dC (CDCl3, 100 MHz):
141.60 (C), 134.86 (CH), 126.99 (CH), 103.09 (CH), 83.73 (C), 68.60
(CH2), 67.02 (CH2), 32.33 (CH2), 24.84 (CH3), 23.44 (CH2). LRMS
ꢄ
(EIþ): 304 (Mþ
,
4%), 217 (75), 83 (100). HRMS: 304.1842.
ꢄ
C17H2511BO4 requires Mþ , 304.1846.
2.7. 2-(40-Bromomethylphenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane 16
2.5. [4-(40,40,50,50-Tetramethyl-10,30,20-dioxaborolan-20-yl)-
phenyl]methanol 14
Following the procedure of De Filippis et al.,23 benzylic alcohol
14 (500 mg, 2.14 mmol) was dissolved in anhydrous DCM (15 mL)
and degassed with argon for 15 min. The solution was cooled to
0 ꢂC and anhydrous triethylamine (324 mg, 3.20 mmol) was added.
Aluminium trichloride (40 mg, 0.30 mmol) was added to a stir-
red solution of acetal 13 (1.00 g, 3.29 mmol) in anhydrous EtOH
(10 mL). The mixture was stirred overnight at rt and then concen-
trated under reduced pressure. The mixture was dissolved in Et2O
and undissolved solid was removed by filtration. The filtrate was
evaporated under reduced pressure and redissolved in anhydrous
EtOH (10 mL). Aluminium trichloride (30 mg, 0.22 mmol) was
added and the solution was stirred overnight at rt. The mixture was
concentrated under reduced pressure, dissolved in Et2O and filtered
to remove undissolved solid. The solution was then concentrated
under reduced pressure to afford the benzylic alcohol 14 as an oil
Methanesulfonyl chloride (198 mL, 2.56 mmol) was added and the
solution was allowed to stir for 1 h. Organics were washed with
H2O (3ꢃ4 mL), dried over MgSO4 and concentrated under reduced
pressure. The resultant solid was dissolved in anhydrous acetone
(15 mL) and degassed with argon for 15 min. Lithium bromide
(1.90 g, 21.4 mmol) was added and the resultant solution was
heated under reflux overnight. The solution was concentrated un-
der reduced pressure and the residue dissolved in DCM (15 mL),

2388
C. Quin et al. / Tetrahedron 66 (2010) 2384–2389
washed with H2O (3ꢃ4 mL), dried over MgSO4 and concentrated
under reduced pressure to yield the target bromide 16 as an
amorphous solid (413 mg, 63%). Mp: 76–78 ꢂC (cubes from Et2O/
hexane. Lit.23 75–77 ꢂC). nmax (ATR): 2976 (CH), 2930 (CH), 2872
ꢁ57.58. LRMS (CIþ): 471 [(MþH)þ, 11B, 11%], 446 (39), 337 (40), 283
(95), 219 (91), 189 (100). HRMS: 471.1821. C23H23O3N4F311B re-
quires (MþH)þ, 471.1820.
(CH), 1613 (Ar) cmꢁ1
.
dH (400 MHz, CDCl3): 7.79 (2H, d, J¼7.7 Hz, H-
2.10. Details of kinetic experiments
20 and H-60), 7.39 (2H, d, J¼7.7 Hz, H-30 and H-50), 4.49 (2H, s, CH2),
1.34 (12H, s, 4ꢃMe). dC (100 MHz, CDCl3): 140.65 (C), 135.21 (CH),
128.30 (CH), 83.90 (C), 33.33 (CH2), 24.84 (CH3). LRMS (EIþ): 298
The stock solutions of caged uncouplers were made up by
weight in DMF (caged uncoupler 15, 1 mM; caged uncoupler 19,
10 mM). The concentration of the stock H2O2 solution was checked
by titration against KMnO4,25 and diluted accurately to a concen-
tration of 1 mM. Reactions were carried out in a 1:1 mixture of DMF
and 0.14 M aqueous sodium bicarbonate (pH 8.3).
ꢄ
ꢄ
ꢄ
[Mþ , (81Br), 4%], 296 [Mþ , (79Br), 4], 217 [Mþ ꢁBrꢄ, 100]. HRMS:
ꢄ
298.0549 and 296.0567. C13H1811B81BrO2 requires Mþ , 298.0563
ꢄ
and C13H1811B79BrO2 requires Mþ , 296.0583. 13C NMR agrees with
literature.23
Absorption measurements were made on a JASCO V550 double
beam spectrophotometer using matched quartz cuvettes of 1 cm
pathlength, with the cuvette compartment maintained at 37 ꢂC by
a circulating water bath. The spectrophotometer was calibrated
using a solution of potassium chromate in 0.05 M KOH, whose
2.8. 2-Cyano-2-[40-(400,400,500,500-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]propionitrile 17
A suspension of NaH (80% in mineral oil, 70 mg, 2.3 mmol) in
anhydrous THF (16 mL) and anhydrous DMF (1.6 mL) was stirred
at rt under argon. A solution of malononitrile (0.12 mL, 2.3 mmol)
in anhydrous THF (2 mL) was added dropwise to the solution and
hydrogen gas was evolved. The reaction mixture was stirred at rt
for 30 min under argon before a solution of bromide 16 (329 mg,
1.11 mmol) in anhydrous THF (4 mL) was added dropwise. The
reaction mixture was stirred at 25 ꢂC overnight. Saturated aque-
ous NH4Cl (40 mL) was added to quench the reaction and the
mixture was extracted with EtOAc (3ꢃ20 mL). The combined or-
ganic extracts were washed with H2O (3ꢃ20 mL), dried over
MgSO4 and concentrated under reduced pressure. Column chro-
matography [SiO2, hexane/EtOAc (4:1)] then gave the product
boronate ester 17 as an amorphous solid (281 mg, 90%). Rf [SiO2,
hexane/EtOAc (4:1)]: 0.13. Mp: 135–137 ꢂC. nmax (ATR): 2982 (CH),
absorption coefficient at 372 nm is 4830 Mꢁ1 cmꢁ1 26
For this
.
standard solution, the measured absorbance was proportional to
concentration up to an absorbance of 1.2; all measurements
reported in this paper were made with absorbance values less
than 1.0.
Comparison between the spectra of caged uncoupler 15 and
DNPꢁ 3, and between caged uncoupler 19 and FCCPꢁ 4 showed
that the appropriate wavelengths to monitor the reactions were
410 nm and 385 nm, respectively, since in each case the starting
material showed a very much smaller absorption than the
product. The appropriate absorption coefficients for the prod-
ucts (DNPꢁ 3 at 410 nm and FCCPꢁ 4 at 385 nm) under the
conditions used for the reactions were determined using a par-
allel dilution approach in which stock solutions of these com-
pounds were diluted into buffer systems where the absorption
coefficients had been published,27,28 and into the buffer system
used in the present work. Comparison of the observed absor-
bance values from these parallel dilutions allowed the appro-
priate coefficients for our experiments to be calculated
(20,500 Mꢁ1 cmꢁ1 at 410 nm for DNPꢁ 3 and 29,700 Mꢁ1 cmꢁ1 at
385 nm for FCCPꢁ 4).
2361 (CN), 1614 (Ar) cmꢁ1
. dH (400 MHz, CDCl3): 7.84 (2H, d,
J¼7.1 Hz, H-2 and H-6), 7.32 (2H, d, J¼7.2 Hz, H-3 and H-5), 3.94
[1H, t, J¼6.8 Hz, CH(CN)2], 3.26 (2H, d, J¼6.8 Hz, CH2), 1.34 (12H, s,
4ꢃCH3). dC (100 MHz, CDCl3): 135.86 (C), 135.61 (CH), 128.46 (CH),
112.19 (C), 84.00 (C), 77.30 (CH), 36.66 (CH2), 24.85 (CH3). LRMS
(CIþ): 283 [(MþH)þ, 100%]. HRMS: 283.1613. C16H20O2N211B re-
quires (MþH)þ, 283.1621.
Reactions were initiated by adding small aliquots of H2O2 to
solutions of caged uncoupler 15 or caged uncoupler 19 in the DMF/
buffer mixture, in a total volume of 1 mL. The blank reaction con-
tained DMF/buffer in place of the H2O2. Control experiments
showed that, under the conditions used, there was a slow break-
down of the caged uncouplers in the buffer; in the case of caged
uncoupler 15, this amounted to 3% of the initial compound over
a time period of 2000 s; in the case of caged uncoupler 19, this
amounted to 14% of the initial compound over a time period of
1000 s. Since the reactions were carried out with the caged un-
couplers in considerable excess over H2O2 and the uncouplers in
DMF/buffer solution were used in the blank reaction, the sponta-
neous breakdown did not affect the observed pseudo-first order
rate constants for the production of DNPꢁ 3 or FCCPꢁ 4 or the
observed yields, although it would have small effects on the cal-
culated second order rate constants. The concentrations of the
caged uncouplers and H2O2 were chosen to give convenient rates of
reaction while maintaining a reasonable approximation to pseudo-
first order conditions in order to facilitate kinetic analysis. For caged
2.9. 2-Cyano-3-[40-(400,400,500,500-tetramethyl-100,300,200-
dioxaborolan-200-yl)phenyl]-2-(4%-trifluoro-
methoxyphenyldiazo)propionitrile 19
A solution of NaNO2 (25 mg, 0.36 mmol) in H2O (0.3 mL) was
cooled to 0 ꢂC and added dropwise to a stirred solution of 4-tri-
fluoromethoxyaniline (62.8 mg, 0.355 mmol) and concd HCl
(0.2 mL) in H2O (1.5 mL) at 0 ꢂC. The resulting solution of di-
azonium salt 18 was stirred for 5 min and then added dropwise to
a stirred solution of boronate 17 (100 mg, 0.354 mmol) and NaOAc
(116 mg, 1.42 mmol), in a mixture of H2O (1.3 mL), MeOH (1.5 mL)
and EtOH (1.5 mL) at 0 ꢂC. The resultant solution was stirred at
0 ꢂC for 1 h and then at rt overnight. The precipitate was filtered
off and washed with ice-cold H2O (4 mL) to yield the caged un-
coupler 19 as an amorphous yellow solid. Further material was
obtained by concentration of the filtrate under reduced pressure
followed by extraction with DCM. Removal of solvent under re-
duced pressure followed by chromatography [SiO2, petroleum
ether/EtOAc (9:1)] gave further caged uncoupler 19 (combined
yield 77 mg, 46%). Rf [SiO2, petroleum ether/EtOAc (9:1)]: 0.34.
uncoupler 15, the concentration used was 200
concentrations 10 M and 20 M. Reactions were performed in
triplicate. For caged uncoupler 19, the concentration used was
50 M, and the H2O2 concentrations 5 M and 10 M. Reactions
were performed in triplicate at 5 M H2O2, and in duplicate at
10 M H2O2. In each case, the experimental data (absorbance vs
mM and the H2O2
m
m
Mp: 120–122 ꢂC. nmax (ATR): 2937 (CH), 2162 (CN), 1614 (Ar) cmꢁ1
.
dH (CDCl3, 400 MHz): 7.90 (2H, d, J¼9.0 Hz, H-2% and H-6%), 7.82
(2H, d, J¼8.0 Hz, H-30 and H-50), 7.36–7.39 (4H, m, H-20, H-60, H-3%
and H-5%), 3.69 (2H, s, CH2), 1.34 (12H, s, 4ꢃCH3). dC (CDCl3,
100 MHz): 153.08 (C), 147.71 (C), 135.51 (CH), 132.86 (C), 130.15
(CH), 125.81 (CH), 121.48 (CH), 120.38 (q, J¼259.2 Hz), 111.85 (C),
84.17 (C), 69.35 (C), 36.62 (CH2), 24.99 (CH3). dF (CDCl3, 376 MHz):
m
m
m
m
m
time) were fitted to a first order kinetic process using Microcal
Origin software; this gave the end-point (limiting absorbance
change) and the first order rate constant. Division of this rate

C. Quin et al. / Tetrahedron 66 (2010) 2384–2389
2389
8. Review of ROS production in mitochondria: Murphy, M. P. Biochem. J. 2009, 417,
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constant by the concentration of the caged uncoupler in excess
yielded the second order rate constant for the reaction.
9. Lambert, A. J.; Brand, M. D. Biochem. J. 2004, 382, 511–517.
10. Brand, M. D.; Affourtit, C.; Esteves, T. C.; Green, K.; Lambert, A. J.; Miwa, S.;
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11. Review of uncouplers: Skulachev, V. P. Biochim. Biophys. Acta 1998, 1363,
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Acknowledgements
The University of Glasgow, The Wellcome Trust, and the Lloyds
TSB Foundation for Scotland and the Royal Society of Edinburgh, for
studentship awards to C.Q., L.R. and S.J.M., respectively. R.F.
Schneider, State University at Stony Brook for providing a pro-
cedure for the titration of hydrogen peroxide.
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